World Journal of Diabetes - BPG Management System

Published by Baishideng Publishing Group Inc

World Journal of DiabetesWorld J Diabetes 2015 April 15; 6(3): 367-542

ISSN 1948-9358 (online)

EDITOR-IN-CHIEFLu Qi, BostonJingbo Zhao, Aalborg

STRATEGY ASSOCIATE EDITOR-IN-CHIEFUndurti Narasimha Das, Shaker HeightsMin Du, LaramieGregory I Liou, AugustaZhong-Cheng Luo, QuebecDemosthenes B Panagiotakos, Athens

GUEST EDITORIAL BOARD MEMBERSJuei-Tang Cheng, TainanChih-Hsung Chu, KaohsiungLow-Tone (Larry) Ho, TaipeiCheng-Cheng Hsiao, KeelungYung-Hsi Kao, TaoyuanChi Feng Liu, TaipeiShing-Hwa Liu, TaipeiWayne H-H Sheu, TaichungEing-Mei Tsai, KaohsiungChin-Hsiao Tseng, TaipeiYen Tzung-Hai, TaipeiChing-Shuang Wu, KaohsiungWei-Chung Vivian Yang, TaipeiWen-Chin Yang, Taipei

MEMBERS OF THE EDITORIAL BOARD

Argentina

Justo P Castaño, CordobaEduardo Spinedi, La Plata

Australia

Sof Andrikopoulos, Heidelberg HeightsHugh Russell Barrett, PerthBernhard T Baune, TownsvilleGrant Brinkworth, AdelaideLouise Janet Maple Brown, CasuarinaMelinda Therese Coughlan, MelbourneJosephine Maree Forbes, MelbournePaul A Fournier, PerthAngela Gialamas, Adelaide Mark Douglas Gorrell, NewtownGraeme Hankey, PerthAnandwardhan A Hardikar, MelbourneMichael Horowitz, AdelaideKarin Jandeleit-Dahm, MelbourneMartha Lappas, VictoriaPeter J Little, MelbourneXin Liu, BrisbaneDianna Josephine Magliano, CaufieldRobyn McDermott, AdelaideBeverly Sara Muhlhausler, AdelaideChristopher Nolan, CanberraLuciano Pirola, MelbourneMaryam Rashidi, VictoriaKarly Calliopi Sourris, VictoriaGreg Tesch, ClaytonJack Ronald Wall, PenrithOwen Llewellyn Woodman, Bundoora

AustriaChristian Heinz Anderwald, ViennaHelmuth Martin Borkenstein, GrazWalter Hermann Hörl, ViennaAlexandra Kautzky-Willer, Vienna

Friedrich Mittermayer, ViennaMarkus Paulmichl, SalzburgStefan Pilz, GrazGuntram Schernthaner, ViennaHarald Sourij, GrazThomas Michael Stulnig, ViennaLudwig Wagner, Vienna

Belgium

Giovanni Dapri, BrusselsChristophe De Block, AntwerpEkaterine Tskitishvili, LiegeF Andre Van Assche, LeuvenLuc F Van Gaal, Antwerp

Brazil

Monica Levy Andersen, Vila ClementinoClaudia RL Cardoso, Rio de JaneiroRicardo Vitor Cohen, São PauloMarcelo Correia, Rio de JaneiroCassyano Januario Correr, CuritibaMatheus Roriz Cruz, Porto AlegreCintia Chaves Curioni, Rio de JaneiroFreddy Goldberg Eliaschewitz, Rua GoiásRodrigo Jorge, Ribeirão PretoLuciana Ansaneli Naves, Asa NorteJúlio César Voltarelli, Ribeirão PretoBernardo L Wajchenberg, PinheirosJacqueline Nelisis Zanoni, Maringá

Canada

Jean-Luc Ardilouze, Sherbrooke

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Editorial Board2011-2015

The World Journal of Diabetes Editorial Board now consists of 712 members, representing a team of worldwide experts in diabetes mellitus. They are from 56 countries, including Argentina (2), Australia (27), Austria (11), Belgium (5), Brazil (13), Canada (25), Chile (3), China (40), Cuba (1), Czech Republic (3), Denmark (16), Egypt (3), Finland (5), France (12), Germany (27), Greece (17), Hungary (4), India (28), Iran (8), Iraq (2), Ireland (3), Israel (10), Italy (56), Japan (30), Jordan (1), Kuwait (3), Lebanon (1), Malaysia (1), Malta (1), Mexico (4), Netherlands (9), New Zealand (3), Nigeria (2), Norway (2), Oman (3), Pakistan (2), Poland (7), Portugal (1), Qatar (1), Romania (2), Saudi Arabia (1), Singapore (4), Slovakia (1), South Africa (1), South Korea (15), Spain (24), Sweden (5), Switzerland (4), Thailand (4), Tunisia (1), Turkey (13), United Arab Emirates (3), United Kingdom (27), United States (213), Venezuela (1), and Yemen (1).

December 11, 2012WJD|www.wjgnet.com

World Journal of DiabetesW J D

Subrata Chakrabarti, LondonDavid Cherney, OntarioMervyn Deitel, TorontoJean-Pierre Després, QuebecDavid Joseph Hill, LondonTian-Ru Jin, TorontoArulmozhi D Kandasamy, EdmontonJennifer L Kuk, TorontoIsmail Laher, VancouverRoger S McIntyre, TorontoDavid Meyre, OntarioJoseph Fomusi Ndisang, SaskatoonRaj Padwal, AlbertaCiriaco A Piccirillo, MontrealRemi Rabasa-Lhoret, MontrealAM James Shapiro, EdmontonGuang Sun, St. John’sValerie Taylor, HamiltonCory Toth, CalgaryAndré Tremblay, MontréalVincent C Woo, WinnipegJames Roscoe Wright, CalgaryXi-Long Zheng, Calgary

Chile

Sebastian San Martin, ValparaisoArmando Rojas-Rubio, TalcaLuis Sobrevia, Santiago

China

Pang-Zeng Chang, QingdaoJie Chen, NanjingBernard Man Yung Cheung, Hong KongWilliam Chi-shing Cho, Hong KongTian-Pei Hong, BeijingQin Huang, ShanghaiPo Sing Leung, Hong KongChao Liu, NanjingJian-Kang Liu, Xi’anLie-Gang Liu, WuhanRonald Ching Wan Ma, Hong KongJin-Sheng Qi, ShijiazhuangWing Yee So, Hong KongCheuk Chun Szeto, Hong KongKathryn Tan, Hong KongCheng-Ming Wang, YangzhouCong-Yi Wang, WuhanYu Wang, Hong KongGuang-Da Xiang, WuhanBao-Feng Yang, HarbinShu-Yu Yang, FujianXi-Lin Yang, Hong KongZai-Qing Yang, WuhanShan-Dong Ye, HefeiShi-Sheng Zhou, DalianZhi-Guang Zhou, Changsha

Cuba

Luis Sarmiento-Pérez, Havana

Czech Republic

Martin Haluzik, Prague

Michal Krcma, PlzenTerezie Pelikanova, Prague

Denmark

Charlotte Brøns, GentofteJens Sandahl Christiansen, ArhusFlemming Dela, CopenhagenKristine Færch, GentofteErik L Grove, AarhusLouise Groth Grunnet, GentofteR Scott Heller, GentofteKurt Højlund, Odense CFilip K Knop, HellerupHelle Markholst, MåløvJens D Mikkelsen, CopenhagenOle Hartvig Mortensen, CopenhagenOluf Pedersen, CopenhagenEsben Thyssen Vestergaard, AarhusMilan Zdravkovic, Søborg

Egypt

Mamdouh Moawad Ali Hssan, CairoMoshira Abdel Hakim Rateb, CairoMona Farag Schaalan, Cairo

Finland

Siamak Bidel, HelsinkiGang Hu, HelsinkiThomas Kietzmann, OuluQing Qiao, HelsinkiKaroliina Wehkalampi, Helsinki

France

Jean Claude Ansquer, DijonBertrand Cariou, NantesSylvie Dejager, Rueil-MalmaisonNaim Akhtar Khan, DijonJean-Philippe Lavigne, NîmesMichel Marre, ParisMarie-Claude Morice, MassyRiccardo Perfetti, ParisGérard Said, ParisSophie Visvikis Siest, NancyDominique Simon, ParisDidier Vieau, Villeneuve d’Ascq

Germany

Ioanna Gouni Berthold, CologneChrista Buechler, RegensburgRoland Büttner, HeidelbergMichael Froehner, DresdenHammes Hans-Peter, MannheimNadj Herbach, MunichAndrea Icks, DüsseldorfThomas Jax, NeussUlrich Arthur Julius, DresdenMichael Kluge, MunichFlorian Lang, Tuebingen Matthias Laudes, KölnRalf Lobmann, Stuttgart

Rafael T Mikolajczyk, BremenAndreas Stefan Mueller, Halle (Saale)Karsten Müssig, TübingenNahid Parvizi, Neustadt am RübenbergeThomas Peter Reinehr, DattelnMichael Ristow, JenaSven Schinner, DuesseldorfPeter Egbert Hermann Schwarz, DresdenKonstantinos Stellos, TubingenOvidiu Alin Stirban, Bad OeynhausenDiego J Walther, BerlinSilvia Anette Wein, KielChristian Wrede, BerlinDan Ziegler, Düsseldorf

Greece

George P Chrousos, AthensMoses S Elisaf, IoanninaPanagiotis Georgoulias, LarissaNikolaos Kadoglou, ThessalonikiGerasimos E Krassas, KriniSpilios Manolakopoulos, AttikiNikolaos Papanas, AlexandroupolisDimitrios Papazoglou, AlexandroupolisSokratis Pastromas, AthensMelpomeni Peppa, AthensChristina Piperi, GoudiNicholas K Tentolouris, AthensKonstantinos A Toulis, SalonikaApostolos Tsapas, ThessalonikiKonstantinos Tziomalos, ThessalonikiElias Zintzaras, Thessaly

Hungary

Mária Bagyánszki, SzegedGyörgy Jermendy, BudapestKaroly Racz, BudapestGyula Soltesz, Pécs

India

Deepak Narayan Amrapurkar, MumbaiC V Anuradha, Tamil NaduSarika Arora, New DelhiPitchai Balakumar, SivakasiMuthuswamy Balasubramanyam, ChennaiSubhabrata Chakrabarti, HyderabadAbhay Sankar Chakraborti, KolkataTapan K Chaudhuri, New DelhiKanwaljit Chopra, ChandigarhMalabika Datta, DelhiDebidas Ghosh, West BengalRavinder Goswami, New DelhiPappachan M Joseph, KeralaJothydev Kesavadev, KeralaKVS Hari Kumar, LucknowAnoop Misra, New DelhiAnalava Mitra, KharagpurViswanathan Mohan, ChennaiS P Murthy, BangalorePallavi Panchu, GunturUsharani Pingali, HyderabadAmbady Ramachandran, Egmore ChennaiVadde Ramakrishna, Kadapa

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Geetha Vani Rayasam, HaryanaRajat Sandhir, ChandigarhManju Sharma, New DelhiSuman Bala Sharma, DelhiTarun Sharma, Chennai

Iran

Mohammad Abdollahi, TehranMohammad Kazemi Arababadi, RafsanjanLeila Azadbakht, IsfahanHamid Baradaran, TehranBehrooz Broumand, TehranAhmad Esmaillzadeh, IsfahanMajid Ghayour-Mobarhan, Mashhad Mohsen Janghorbani, Isfahan

Iraq

Saad Abdul-Rahman Hussain, BaghdadAbbas Ali Mansour, Basrah

Ireland

Amar Agha, DublinMark Philip Hehir, DublinGerald H Tomkin, Dublin

Israel

Michael Aviram, HaifaGal Dubnov-Raz, Tel HashomerShimon Efrat, Tel AvivRaymond Elias Farah, SafedOren Froy, RehovotSaher Hamed, HaifaArid Nakhoul, HaifaOrit Pinhas-Hamiel, Tel HashomerHaim Werner, Tel AvivMarina Shargorodsky Zimlichman, Holon

Italy

Luigi Angrisani, NapoliMoschetta Antonio, BariAntonio Aversa, RomeRoberto Baldelli, RomeGiuseppe Barbaro, RomeAlessandro Bartolomucci, ParmaGiuseppina Basta, PisaSimona Bertoli, MilanoFederico Bilotta, RomeFabio Broglio, TorinoFrancesco G Chiarelli, ChietiSergio Coccheri, BolognaMassimo Collino, TorinoMarco Aristide Comaschi, GenoaRenzo Cordera, GenovaFrancesco Dotta, SienaGagliardini Elena, BergamoStefano Fiorucci, PerugiaMaurizio Galderisi, NaplesAmalia Gastaldelli, Pisa

Ezio Ghigo, TurinCarla Giordano, PalermoPaolo Gisondi, VeronaRiccarda Granata, TurinGiorgio Iervasi, PisaClaudia Kusmic, PisaCarmelo La Rosa, CataniaFrancesco Landi, RomeMonica Rosa Loizzo, Arcavacata RendePaolo Magni, MilanoMariano Malaguarnera, CataniaMelania Manco, RomePiero Marchetti, PisaMassimo Massi-Benedetti, PerugiaAntonio Nicolucci, ImbaroLucia Pacifico, RomeStefano Palomba, CatanzaroGiampaolo Papi, CarpiRenato Pasquali, BolognaPiermarco Piatti, MilanoDario Pitocco, RomeAntonio E Pontiroli, MilanoGiulio Marchesini Reggiani, BolognaGiuseppe Remuzzi, BergamoManfredi Rizzo, PalermoRaffaella Rosso, GenoaGiuseppe Schillaci, PerugiaLeonardo A Sechi, SassariImad Sheiban, TorinoCesare R Sirtori, MilanoGiovanni Tarantino, NaplesGiovanni Targher, VeronaDonadon Valter, PordenoneAlberto Verrotti, ChietiAndrea Viggiano, NapoliGianvincenzo Zuccotti, Milan

Japan

Masato Asahina, ChibaTakuya Awata, SaitamaYuichiro Eguchi, SagaGoji Hasegawa, KyotoSatoshi Inoue, TokyoEiji Ishimura, OsakaMasayuki Iwano, NaraTakashi Kadowaki, TokyoEisuke Kagawa, HiroshimaMasahito Katahira, AichiEiji Kawasaki, NagasakiNoriyuki Koibuchi, GunmaKazuhiko Kotani, TochigiDaisuke Koya, IshikawaNorikazu Maeda, OsakaTakayuki Masaki, OitaYuji Matsuzawa, OsakaKazuaki Nishio, TokyoKenji Okumura, NagoyaMotoaki Saito, YonagoToshiyasu Sasaoka, ToyamaMichio Shimabukuro, OkinawaKohzo Takebayashi, SaitamaHiroyuki Tamemoto, TochigiTakashi Togo, YokohamaJun Udagawa, IzumoYoshinari Uehara, FukuokaTakuya Watanabe, TokyoToshihiko Yada, Tochigi

Tohru Yorifuji, Osaka

Jordan

Yousef S Khader, Irbid

Kuwait

Kamal AA Sulaiman Al-Shoumer, KuwaitIbrahim Fadil Benter, SafatAbu Salim Mustafa, Kuwait

Lebanon

Ramzi F Sabra, Beirut

Malaysia

Mafauzy Mohamed, Kota Bharu

Malta

Charles Savona-Ventura, Msida

Mexico

Manuel González-Ortiz, GuadalajaraFernando Guerrero-Romero, DurangoJesus Alberto Olivares-Reyes, Mexico CityRocío Salceda, Mexico City

Netherlands

Sander Kersten, WageningenNanne Kleefstra, ZwolleEdwin Mariman, MaastrichtDon Poldermans, RotterdamFrançois Pouwer, TilburgHan Roelofsen, GroningenHendrik-Jan Schuurman, UtrechtSuat Simsek, AlkmaarMarcel Twickler, Bergen op Zoom

New Zealand

Paul Hofman, AucklandPeter E Lobie, AucklandElaine Rush, Auckland

Nigeria

Adejuwon A Adeneye, LagosAnthonia Okeoghene Ogbera, Lagos

Norway

Akhtar Hussain, OsloOdd Erik Johansen, Hovik

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Oman

Mohammed Al Shafaee, MuscatJumana S Saleh, MuscatRadha Shenoy, Muscat

Pakistan

Shahid Hameed, IslamabadJamil A Malik, Islamabad

Poland

Marcin Baranowski, BialystokJerzy Beltowski, LublinAlicia Hubalewska Dydejczyk, KrakowMaciej Owecki, PoznańEwa Pankowska, WarsawAgnieszka Piwowar, WroclawDorota Anna Zieba, Krakow

Portugal

M Graça Pereira, Braga

Qatar

Hong Ding, Doha

Romania

Elena Ganea, BucharestAdriana Georgescu, Bucharest

Saudi Arabia

J Fernando Arevalo, Caracas

Singapore

S Thameem Dheen, SingaporeYung Seng Lee, SingaporeDaniel Ng, SingaporeRob Martinus van Dam, Singapore

Slovakia

Katarína Šebeková, Bratislava

South Africa

Md Shahidul Islam, Durban

South Korea

Huneg-Sik Choi, GwangjuKyung Mook Choi, SeoulWon Mi Hwang, SeoulEui-Bae Jeung, Chungbuk

Ju-Hee Kang, IncheonSin Gon Kim, Seongbuk-GuSung-Jin Kim, SeoulYoung-Gyu Ko, SeoulKang-Beom Kwon, ChonbukMyung Gull Lee, BucheonSoo Lim, SeongnamByung-Hyun Park, JeonbukSeungjoon Park, SeoulKun-Ho Yoon, SeoulJeesuk Yu, Cheonan

Spain

Vivencio Barrios, MadridM Lusia Bonet, Palma de MallorcaManuel Vazquez Carrera, BarcelonaMaria Luz Martinez Chantar, DerioManuel Aguilar Diosdado, CádizJavier Espino, BadajozRicardo V García-Mayor, VigoJosé Manuel Gómez-Sáez, BarcelonaOreste Gualillo, Santiago de CompostelaJ Alfredo Martínez Hernández, PamplonaEmilio Herrera, MadridAmelia Marti, PamplonaMerce Miranda, TarragonaJF Navarro-González, Santa Cruz de TenerifeAlberto Ortiz, MadridMaria Javier Ramirez, PamplonaEugenia Resmini, BarcelonaPedro Romero-Aroca, ReusJordi Salas-Salvadó, ReusGines M Salido, CaceresVictor Sanchez-Margalet, SevilleHelmut Schröder, BarcelonaCarmen Segundo, CádizRafael Simó, Barcelona

Sweden

Joanna Hlebowicz, MalmöKaj S Stenlöf, GöteborgAnn-Britt Wiréhn, LinköpingWeili Xu, StockholmShao-Nian Yang, Stockholm

Switzerland

Kaspar Berneis, ZurichPascal Bovet, LausanneLuc Tappy, LausanneChristian Toso, Geneva

Thailand

Narattaphol Charoenphandhu, BangkokArthorn Riewpaiboon, BangkokRawee Teanpaisan, Hat-YaiViroj Wiwanitkit, Bangkok

Tunisia

Khaled Hamden, Sfax

Turkey

Ugur Cavlak, DenizliTeoman Dogru, AnkaraErsin Fadillioglu, AnkaraAbdurrahman Fatih Fidan, AfyonkarahisarMuammer Karadeniz, Bornova-IzmirCevdet Kaya, IstanbulFahrettin Kelestimur, KayseriAltan Onat, IstanbulSemir Ozdemir, AntalyaMustafa Şahin, AnkaraIlker Tasci, AnkaraBelma Turan, AnkaraSerap Yalın, Mersin

United Arab Emirates

Ernest Akingunola Adeghate, Al AinMukesh M Agarwal, Al AinSamir M Awadallah, Sharjah

United Kingdom

Nisreen Alwan, LeedsAmbika P Ashraf, BirminghamChen Bing, LiverpoolFay Crawford, EdinburghTim M Curtis, BelfastUmesh Dashora, HastingsGareth Davison, BelfastPeter Raymond Flatt, ColeraineKathleen M Gillespie, BristolPeter John Grant, LeedsLorna W Harries, ExeterNigel Hoggard, AberdeenNigel Irwin, ColeraineEdward Jude, LancashireAndreas F Kolb, AberdeenStefan Marciniak, CambridgeMoffat Joha Nyirenda, EdinburghJeetesh Patel, BirminghamSnorri Bjorn Rafnsson, EdinburghThozhukat Sathyapalan, YorkshireLatika Sibal, NewcastleRajagopalan Sriraman, LincolnRamasamyiyer Swaminathan, LondonAbd A Tahrani, BirminghamG Neil Thomas, BirminghamCecil Thompson, LondonPaul Henry Whiting, Leicester

United States

Varun Agrawal, SpringfieldMohamed Al-Shabrawey, AugustaPascale Alard, LouisvilleOmar Ali, MilwaukeeJudith Aponte, New YorkBalamurugan N Appakalai, MinneapolisHwyda A Arafat, PhiladelphiaCarl V Asche, Salt LakeSanford A Asher, PittsburghAnthony Atala, Winston-SalemSami Toufic Azar, Beirut

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George Louis Bakris, ChicagoAlistair J Barber, HersheyDaniel C Batlle, ChicagoDavid SH Bell, BirminghamRita Bortell, WorcesterSebastien G Bouret, Los AngelesDavid Lloyd Brown, Stony BrookLu Cai, LouisvilleJack D Caldwell, ErieAnna C Calkin, Los AngelesRoberto A Calle, GrotonR Keith Campbell, PullmanCarlos Campos, New BraunfelsHeping Cao, New OrleansKrista Casazza, BirminghamAaron Brandon Caughey, PortlandEileen R Chasens, PittsburghMunmun Chattopadhyay, Ann ArborXiao-Li Chen, St PaulSheri Renee Colberg, NorfolkCraig Ian Coleman, HartfordRobert Russell Conley, IndianapolisColleen M Croniger, ClevelandDoyle M Cummings, GreenvilleWilliam C Cushman, MemphisPatricia Ann D’Amore, BostonPatricia Darbishire, West LafayetteGuillaume Darrasse-Jèze, New YorkRavi M Dasu, SacramentoMichael Harvey Davidson, ChicagoPrakash Deedwania, SanFranciscoHong-Wen Deng, Kansas CityTeresa P DiLorenzo, BronxScot E Dowd, LubbockSamuel C Durso, BaltimoreKrystal L Edwards, DallasAlexander M Efanov, IndianapolisAzza B El-Remessy, AugustaAmy Zhihong Fan, AtlantaMelissa Spezia Faulkner, TucsonGeorge S Ferzli, Staten IslandPaolo Fiorina, BostonJames Edward Foley, East HanoverSamuel N Forjuoh, TempleAlessia Fornoni, MiamiMartha M Funnell, Ann ArborTrudy Gaillard, ColumbusPietro Galassetti, IrvineClaudia Gragnoli, HersheyJennifer B Green, DurhamGary J Grover, PiscatawayAlok Kumar Gupta, Baton RougeWerner K Gurr, New HavenSamy L Habib, San AntonioAbdel Rahim Hamad, BaltimoreDaniel M Herron, New YorkTiffany Hilton, RochesterRaimund Hirschberg, TorranceMichael Francis Holick, BostonZhaoyong Hu, HoustonRachel Mary Hudacko, New BrunswickYasuo Ido, BostonBrian K Irons, LubbockPamela Itkin-Ansari, La JollaHieronim Jakubowski, NewarkHong-Lin Jiang, BlacksburgPing Jiao, ProvidenceShengkan Jin, PiscatawayArpita Kalla, St LouisRichard Evers Katholi, Springfield

Melina Rae Kibbe, ChicagoBhumsoo Kim, Ann ArborTomoshige Kino, BethesdaJulienne K Kirk, Winston-SalemRenu A Kowluru, DetroitLewis H Kuller, PittsburghRajesh Kumar, TempleBlandine Laferrère, New YorkSang Yeoup Lee, MayoCong-Jun Li, BeltsvilleChing-Shwun Lin, San FranciscoJulie Lin, BostonShuo Lin, Los AngelesPeter Lindgren, San DiegoJames F List, PrincetonDong-Min Liu, BlacksburgZhen-Qi Liu, CharlottesvilleGeorge William Lyerly, ConwayJian-Xing Ma, Oklahoma CityRong Ma, Fort WorthXin-Laing Ma, PhiladelphiaDavid Maggs, San DiegoKenneth Maiese, DetroitKevin C Maki, Glen EllynSridhar Mani, BronxSuresh Mathews, AuburnLauraar McCabe, East LansingSarah E Messiah, MiamiThomas O Metz, RichlandShannon A Miller, OrlandoMurielle Mimeault, OmahaRaghavendra G Mirmira, IndianapolisPrasun J Mishra, BethesdaReema Mody, GrayslakeArshag D Mooradian, JacksonvilleMohammad Reza Movahed, TucsonJames Mu, RahwayMuraleedharan G Nair, East LansingManuel F Navedo, SeattleCharles B Nemeroff, AtlantaJoshua J Neumiller, SpokaneSteven Nissen, ClevelandHirofumi Noguchi, Fort WorthCraig Nunemake, CharlottesvillePatrick J O’Connor, MinneapolisErin St Onge, ApopkaWei-Hong Pan, Baton RougeNaushira Pandya, Fort LauderdaleMichael R Peluso, CorvallisInga Peter, New YorkAxel Pflueger, RochesterGretchen A Piatt, PittsburghJohn D Piette, Ann ArborLeonid Poretsky, New YorkWalter J Pories, GreenvilleParviz M Pour, OmahaWei Qiao Qiu, BostonTeresa Quattrin, BuffaloCristina Rabadán-Diehl, BethesdaRajendra S Raghow, MemphisSwapnil Rajpathak, BronxArmin Rashidi, NorfolkMohammed S Razzaque, BostonBeverly A S Reyes, PhiladelphiaDavid Rodbard, PotomacHelena W Rodbard, RockvilleJune Hart Romeo, ClevelandRaul J Rosenthal, Fort LauderdaleJuan M Saavedra, BethesdaStephen W Schaffer, Mobile

Frank AJL Scheer, BostonRichard E Scranton, TivertonVallabh (Raj) O Shah, AlbuquerqueAziz Shaibani, HoustonJin-Xiong She, AugustaGuo-Ping Shi, BostonCarol Ann Shively, Winston-SalemAnders AF Sima, DetroitPramil N Singh, Loma LindaRajan Singh, Los AngelesJay S Skyler, MiamiDawn Smiley, AtlantaMatthew D Solomon, StanfordMark A Sperling, PittsburghRakesh K Srivastava, TylerBangyan Stiles, Los AngelesYu-Xiang Sun, HoustonSalim Surani, Corpus ChristiArthur L M Swislocki, MartinezYa-Xiong Tao, AuburnJohn A Tayek, TorranceJohn Gaylord Teeter, New HavenCarlos Marcelo Telleria, VermillionChristopher Gordon Thanos, ProvidenceRonald G Tilton, GalvestonSerena Tonstad, Loma LindaMichael Lawrence Traub, Staten IslandGuillermo E Umpierrez, AtlantaMargrit Urbanek, ChicagoVladimir N Uversky, IndianapolisGabriel I Uwaifo, Baton RougeVolker Vallon, San DiegoShambhu D Varma, BaltimoreMaria Virella, CharlestonHong-Jun Wang, BostonMark E Williams, BostonNathan D Wong, IrvineGuangyu Wu, New OrleansZhong-Jian Xie, San FrancisocoMing-Zhao Xing, BaltimoreHariom Yadav, BethesdaLijun Yang, GainesvilleRuojing Yang, RahwaySubhashini Yaturu, AlbanyJoseph Yeboah, CharlottesvilleDengping Yin, NashvilleYisang Yoon, RochesterYi-Hao Yu, New YorkKevin CJ Yuen, PortlandIan Stuart Zagon, HersheyRobert Yuk-Lung Zee, BostonCui-Lin Zhang, RockvilleJames Xuejie Zhang, RichmondSarah Zhang, OklahomaGuixiang Zhao, AtlantaYang Zhao, IndianapolisMing-Hui Zou, Oklahoma City

Venezuela

Fuad Lechin, Caracas

Yemen

Khaled Abdul-Aziz Ahmed, Ibb

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FIELD OF VISION367 Ménage-à-trois of bariatric surgery, bile acids and the gut microbiome

Raghow R

REVIEW371 Telehealth interventions to reduce management complications in type 1 diabetes: A review

Balkhi AM, Reid AM, Westen SC, Olsen B, Janicke DM, Geffken GR

380 Type 1 diabetes: A predictable disease

Simmons KM, Michels AW

391 Nociception at the diabetic foot, an uncharted territory

Chantelau EA

403 Gut microbiota and Ma-Pi 2 macrobiotic diet in the treatment of type 2 diabetes

Fallucca F, Fontana L, Fallucca S, Pianesi M

412 Some of the experimental and clinical aspects of the effects of the maternal diabetes on developing

hippocampus

Hami J, Shojae F, Vafaee-Nezhad S, Lotfi N, Kheradmand H, Haghir H

423 Endothelial and platelet markers in diabetes mellitus type 2

Kubisz P, Stančiaková L, Staško J, Galajda P, Mokáň M

432 Diabetic neuropathic pain: Physiopathology and treatment

Schreiber AK, Nones CFM, Reis RC, Chichorro JG, Cunha JM

445 New-onset diabetes mellitus after kidney transplantation: Current status and future directions

Palepu S, Prasad GVR

456 Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus

Tangvarasittichai S

MINIREVIEWS481 Birth defects in pregestational diabetes: Defect range, glycemic threshold and pathogenesis

Gabbay-Benziv R, Reece EA, Wang F, Yang P

489 Diabetic retinopathy - ocular complications of diabetes mellitus

Nentwich MM, Ulbig MW

Contents Monthly Volume 6 Number 3 April 15, 2015

April 15, 2015|Volume 6|Issue 3|WJD|www.wjgnet.com I

500 Tyrosine isomers and hormonal signaling: A possible role for the hydroxyl free radical in insulin resistance

Molnár GA, Mikolás EZ, Szijártó IA, Kun S, Sélley E, Wittmann I

508 Interplay between Rab27a effectors in pancreatic β-cells

Yamaoka M, Ishizaki T, Kimura T

517 Eating disorders in adolescents with type 1 diabetes: Challenges in diagnosis and treatment

Pinhas-Hamiel O, Hamiel U, Levy-Shraga Y

ORIGINAL ARTICLE Prospective Study

527 Perception of difficulty and glucose control: Effects on academic performance in youth with type Ⅰ diabetes

Potts TM, Nguyen JL, Ghai K, Li K, Perlmuter L

SYSTEMATIC REVIEWS534 Effect of aerobic and anaerobic exercises on glycemic control in type 1 diabetic youths

Lukács A, Barkai L

ContentsWorld Journal of Diabetes

Volume 6 Number 3 April 15, 2015

April 15, 2015|Volume 6|Issue 3|WJD|www.wjgnet.com II

ContentsWorld Journal of Diabetes

Volume 6 Number 3 April 15, 2015

FLYLEAF

EDITORS FOR THIS ISSUE

Responsible Assistant Editor: Li Xiang Responsible Science Editor: Fang-Fang JiResponsible Electronic Editor: Su-Qing Liu Proofing Editorial Office Director: Xiu-Xia SongProofing Editor-in-Chief: Lian-Sheng Ma

NAME OF JOURNAL World Journal of Diabetes

ISSNISSN 1948-9358 (online)

LAUNCH DATEApril 15, 2010

FREQUENCY Monthly

EDITORS-IN-CHIEFLu Qi, MD, PhD, Assistant Professor, Department of Nutrition, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115, United States

Jingbo Zhao, PhD, Associate Professor, Aalborg Hospital Science and Innovation Centre, Aalborg Hospital, Aarhus University Hospital, Aalborg 9000, Denmark

EDITORIAL OFFICEJin-Lei Wang, Director

Xiu-Xia Song, Vice DirectorWorld Journal of DiabetesRoom 903, Building D, Ocean International Center, No. 62 Dongsihuan Zhonglu, Chaoyang District, Beijing 100025, ChinaTelephone: +86-10-85381891Fax: +86-10-85381893E-mail: [email protected] Desk: http://www.wjgnet.com/esps/helpdesk.aspxhttp://www.wjgnet.com

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PUBLICATION DATEApril 15, 2015

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April 15, 2015|Volume 6|Issue 3|WJD|www.wjgnet.com III

AIM AND SCOPE

Editorial Board Member of World Journal of Diabetes , Shan-Dong Ye, MD, Professor, Department of Endocrionology, Anhui Provincial Hospital, No. 17, Lujiang Road, Hefei 230001, Anhui Province, China

World Journal of Diabetes (World J Diabetes, WJD, online ISSN 1948-9358, DOI: 10.4239), is a peer-reviewed open access academic journal that aims to guide clinical practice and improve diagnostic and therapeutic skills of clinicians.

WJD covers topics concerning α, β, δ and PP cells of the pancreatic islet, the effect of insulin and insulinresistance, pancreatic islet transplantation, adipose cells and obesity.

We encourage authors to submit their manuscripts to WJD. We will give priority to manuscripts that are supported by major national and international foundations and those that are of great clinical significance.

World Journal of Diabetes is now indexed in PubMed Central, PubMed, Digital Object Identifier, and Directory of Open Access Journals.

I-V Editorial Board

INDEXING/ABSTRACTING

Rajendra Raghow

Rajendra Raghow, Department of Veterans Affairs Medical Center, Memphis, TN 38104, United StatesRajendra Raghow, Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163, United StatesAuthor contributions: Raghow R solely wrote this paper.Conflict-of-interest: Rajendra Raghow declares that there is neither a conflict of interest with regard to the publication discussed in this FOV communication nor with respect to a commercial entity.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Rajendra Raghow, PhD, Professor, Department of Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104, United States. [email protected]: +1-901-5238990 Fax: +1-901-5237274Received: June 13, 2014 Peer-review started: June 14, 2014 First decision: June 27, 2014 Revised: December 26, 2014 Accepted: January 9, 2015 Article in press: January 12, 2015Published online: April 15, 2015

AbstractBariatric surgeries have emerged as highly effective treatments for obesity associated type-2 diabetes mellitus. Evidently, the desired therapeutic endpoints such as rates of weight loss, lower levels of glycated hemoglobin and remission of diabetes are achieved more rapidly and last longer following bariatric surgery, as opposed to drug therapies alone. In light of these findings, it has been suspected that in addition to

causing weight loss dependent glucose intolerance, bariatric surgery induces other physiological changes that contribute to the alleviation of diabetes. However, the putative post-surgical neuro-hormonal pathways that underpin the therapeutic benefits of bariatric surgery remain undefined. In a recent report, Ryan and colleagues shed new light on the potential mechanisms that determine the salutary effects of bariatric surgery in mice. The authors demonstrated that the improved glucose tolerance and weight loss in mice after vertical sleeve gastrectomy (VSG) surgery were likely to be caused by post-surgical changes in circulating bile acids and farnesoid-X receptor (FXR) signaling, both of which were also mechanistically linked to changes in the microbial ecology of the gut. The authors arrived at this conclusion from a comparison of genome-wide, metabolic consequences of VSG surgery in obese wild type (WT) and FXR knockout mice. Gene expression in the distal small intestines of WT and FXR knockout mice revealed that the pathways regulating bile acid composition, nutrient metabolism and anti-oxidant defense were differentially altered by VSG surgery in WT and FXR-/- mice. Based on these data Ryan et al , hypothesized that bile acid homeostasis and FXR signaling were mechanistically linked to the gut microbiota that played a role in modulating post-surgical changes in total body mass and glucose tolerance. The authors’ data provide a plausible explanation for putative weight loss-independent benefits of bariatric surgery and its relationship with metabolism of bile acids.

Key words: Vertical sleeve gastrectomy; Bile acids; Farnesoid-X-receptor; Type-2 diabetes mellitus; Gut microbiome; Bariatric surgery

© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.

Core tip: The report of Ryan et al , raises a number of questions with regard to the prevailing notion that

FIELD OF VISION

367 April 15, 2015|Volume 6|Issue 3|WJD|www.wjgnet.com

Ménage-à-trois of bariatric surgery, bile acids and the gut microbiome

Submit a Manuscript: http://www.wjgnet.com/esps/Help Desk: http://www.wjgnet.com/esps/helpdesk.aspxDOI: 10.4239/wjd.v6.i3.367

World J Diabetes 2015 April 15; 6(3): 367-370ISSN 1948-9358 (online)

© 2015 Baishideng Publishing Group Inc. All rights reserved.

mechanical restriction of the stomach and weight loss are the sole mechanisms that mediate the therapeutic effects of bariatric surgery. The authors showed that both lowering of body mass index and improved glucose tolerance after vertical sleeve gastrectomy (VSG) surgery were mechanistically linked to an altered composition of circulating bile acids and their ability to modulate farnesoid-X receptor (FXR) mediated signaling mechanisms. Additionally, it was observed that the wild type and FXR knockout mice, after receiving VSG surgery, were significantly different with respect to the make up of their gut microbiomes. Finally, the experiments revealed that the composition of gut microbiota in wild type VSG and FXR-/- VSG mice were highly correlated with their differential abilities to lose weight and acquire glucose tolerance.

Raghow R. Ménage-à-trois of bariatric surgery, bile acids and the gut microbiome. World J Diabetes 2015; 6(3): 367-370 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/367.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.367

COMMENTARY ON HOT TOPICSThe provocative mechanism proposed by Ryan et al[1], that mechanistically links bile acids, farnesoid-X receptor (FXR) nuclear receptor signaling and microbial ecology of the gut to therapeutic superiority of bariatric surgery over intensive drug therapies, opens up new avenues of clinical research that deserves serious consideration.

According to the World Health Organization (WHO), obesity associated type-2 diabetes mellitus (T2DM) and cardiovascular diseases represent a looming health-care crisis of the 21st century [WHO Global Infobase: data on overweight and obesity mean body mass index (BMI), healthy diets and physical inactivity; www.who.int/mediacenter/]. Traditionally, obesity, T2DM and their co-morbidities have been treated with anti-diabetic drugs combined with behavioral modification therapies (e.g., better nutrition and regular exercise). However, in recent years, a number of surgical interventions, that include Roux-en Y gastric bypass, vertical sleeve gastrectomy (VSG), laparoscopic adjustable gastric banding and bilio-pancreatic diversion, have emerged as highly effective treatments for obesity-associated diabetes[2].

There is a growing body of evidence to indicate that patients undergoing bariatric surgery, achieve desirable clinical endpoints more rapidly and for a longer duration compared with therapeutic benefits obtained with anti-diabetic drug therapy alone[2]. A recently completed, randomized clinical trial revealed that the superior post-surgical therapeutic benefits (e.g., improved glucose tolerance and lower BMI) of bariatric surgery over intensive drug treatment persisted for at least three years, when the study ended. Additionally, patients

receiving bariatric surgery not only achieved their main therapeutic goal of weight loss and alleviation of diabetes but also enjoyed a better quality of life, with fewer obesity-associated complications[3]. Similar beneficial effects of bariatric surgery have also been observed in rodent models of obesity[4,5]. The strikingly more effective therapeutic outcomes of surgical procedures over pharmacological and behavioral interventions in the treatment of obesity-associated diabetes represent an unsolved medical puzzle[6,7].

The hormonal and metabolic underpinnings of surgical interventions and how they differ from conven-tional drug therapy in shaping the anabolic and catabolic pathways are being actively pursued[6-8]. Obviously, much of this research is prompted by a desire to define the basic mechanisms involved in the clinical outcome of bariatric surgery. An additional motivation for such studies is the fact that bariatric surgery poses definite risks, and thus there is an urgent need for safer, less invasive therapies to assuage obesity associated diabetes and its complications. Investigations into the putative mechanisms that differentially regulate the therapeutic outcomes of bariatric surgery vs conventional drug therapies have led to somewhat conflicting findings[6,9,10]. According to the prevailing view, a reduced intake and absorption of macronutrients by surgically downsized stomach is responsible for the post-surgical insulin sensitivity and weight loss after bariatric surgery[2]. Although this scenario is generally supported by the data gathered from rodent models of obesity and in humans, this mechanism fails to explain the paradoxical finding that a substantial fraction of patients experienced remission of their diabetes long before they exhibited a significant weight loss[6,7]. This has led some investigators to posit that in addition to causing weight loss bariatric surgery also leads to other physiological changes in the gastrointestinal (GI) tract. One of the key pieces of evidence in support of this view includes post-surgical changes in circulating bile acids seen in both rodents and humans after bariatric surgery[11,12]. It has been suggested that simultaneous changes in the composition of bile acids and gut microbiota may be causally related to obesity and a loss of BMI[13,14]. A highly revealing study, carried out with human twins discordant for obesity, showed that obese and lean individuals had unique gut microbial ecology; transplantation of the fecal microbiota from the obese or lean patients conferred analogous BMI and adiposity to the germ-free mice[15]. Differences in body composition were correlated with differences in the metabolism of short chain fatty acids, branched chain amino acids, and altered bile acids and FXR signaling[15]. These tantalizing correlations notwithstanding, the underlying mechanism by which gut microbiota regulates total body weight and glucose tolerance remain to be fully elucidated.

The experiments of Ryan et al[1], reported in a recent issue of Nature, bring us a step closer to defining the molecular interactions that mediate the metabolic effects of VSG surgery in mice. Since bariatric surgery is known

Raghow R. Gut microbiome and bile acids


to enhance enterohepatic circulation of bile acids[16] that signal via nuclear FXR, Ryan et al[1] hypothesized that altered bile acid homeostasis and FXR signaling were mechanistically involved in the weight loss and glucose tolerance. To test this hypothesis, the authors compared the gene expression profiles in the distal small intestines of wild type (WT) and FXR knockout mice after VSG surgery. These analyses led to the discovery of “glutathione-mediated detoxification”, “nuclear receptors in lipid metabolism and toxicity”, “meta-pathway bio-transformation” and “type Ⅱ interferon signaling” pathways that were highly suggestive of a role of the gut microbiome in the metabolic changes elicited by VSG surgery.

The authors undertook an empirical investigation of this hypothesis. The WT and FXR-/- mice, kept on high fat diet to induce obesity, showed similar rates of body weight loss in the first week after surgery. However, the FXR-/- mice were compromised with respect to sustained (at 5 wk and after) losses of BMI and body fat. Interestingly, while the WT and FXR-/- mice consumed lower calories in the first week after surgery only the former continued to maintain their hypophagic behavior. The cumulative caloric intake by sham-operated and VSG FXR-/- mice were similar; both cohorts of animals consumed 15% higher than VSG WT mice. Thus, the authors concluded that the inability of FXR-/- animals for sustained loss of body weight and total body fat were a result of their feeding behavior. Since FXR-/- mice were also refractory to the metabolic benefits of VSG surgery, it became apparent that the metabolic outcomes of surgery were also influenced by the genotype of the mice. Nevertheless, VSG surgery had a more pronounced effect on the bacterial ecology of the gut than the absence of a functional FXR gene. Un-weighted UniFrac-based comparison of bacterial 16S rRNA data in sham-operated and VSG mice revealed that the population of Bacterioides was significantly reduced after VSG surgery in WT mice whose guts also showed greater abundance of Porphyromonadaceae and Roseburia. Genotype independent post-surgical changes in the populations of Lactobacilli and Lactococci in the guts were also observed. The abundance of Roseburia in the WT VSG correlated negatively with glucose intolerance. The abundance of Roseburia in the guts of sham operated and VSG FXR-/- was similar and was significantly lower than in WT VSG guts. Thus, the authors posited that a functional FXR signaling was involved in the mechanisms of VSG-induced weight loss, feeding behavior and insulin sensitivity.

The current study did not directly address the molecular basis of how FXR contributes to the meta-bolic consequences of VSG surgery. The authors acknowledged however[1], that in light of the inherent signaling complexity of bile acids that activate FXR as well as a G-protein coupled receptor, takeda G-protein-coupled receptor-5[16], the explanation of these data is unlikely to be straightforward. This sentiment is supported by the contradictory phenotype of FXR-/-

mice elicited in this study; thus, FXR knockout mice were resistant to some of the deleterious effects of high fat diet while remaining less responsive to the therapeutic benefits of VSG surgery. The apparently paradoxical phenotype of FXR-/- mice is likely to be caused by (1) unique tissue-specific mechanisms involved in the activation of FXR; and (2) a possible induction of compensatory mechanisms that result from a global knockout of FXR. The data contained in this paper also did not directly address the key question of how post-surgical changes in bile acids modulate the gut microbial ecology, and vice versa. Finally, a role of the gut microbiota in differentially regulating the metabolism and energetics of WT and FXR-/- mice could only be speculated upon in light of the limited information contained in this paper. It worth remembering however, that a number of earlier studies were driven by the hypothesis that obesity-associated microbiome was more efficient at extracting energy from lipids and carbohydrates[13,14]. The observed differences in the abundance of common fatty acids and related metabolites among the WT and FXR-/-

mice following VSG surgery were not sufficiently clear to warrant an unequivocal explanation. Nevertheless, the mechanism involving more efficient extraction of energy from macronutrients by the microbiota appears to be too simplistic in light of some recent data showing that changes in gut microbial ecology impinged on the signaling mechanisms underlying satiety and motility of the GI tract[17,18]. Despite these caveats, the experiments of Ryan et al[1], have broken a new ground in elucidating a functional connection of FXR signaling and microbial ecology with the metabolic consequences of bariatric surgery. These exciting findings deserve to be systematically validated and extended in humans with an aim to discover less invasive therapies to treat obesity and its complications.

REFERENCES1 Ryan KK, Tremaroli V, Clemmensen C, Kovatcheva-Datchary P,

Myronovych A, Karns R, Wilson-Pérez HE, Sandoval DA, Kohli R, Bäckhed F, Seeley RJ. FXR is a molecular target for the effects of vertical sleeve gastrectomy. Nature 2014; 509: 183-188 [PMID: 24670636 DOI: 10.1038/nature13135]

2 Buchwald H, Estok R, Fahrbach K, Banel D, Jensen MD, Pories WJ, Bantle JP, Sledge I. Weight and type 2 diabetes after bariatric surgery: systematic review and meta-analysis. Am J Med 2009; 122: 248-256.e5 [PMID: 19272486 DOI: 10.1016/j.amjmed.2008.09.041]

3 Schauer PR, Kashyap SR, Wolski K, Brethauer SA, Kirwan JP, Pothier CE, Thomas S, Abood B, Nissen SE, Bhatt DL. Bariatric surgery versus intensive medical therapy in obese patients with diabetes. N Engl J Med 2012; 366: 1567-1576 [PMID: 22449319 DOI: 10.1056/NEJMoa1200225]

4 Chambers AP, Jessen L, Ryan KK, Sisley S, Wilson-Pérez HE, Stefater MA, Gaitonde SG, Sorrell JE, Toure M, Berger J, D’Alessio DA, Woods SC, Seeley RJ, Sandoval DA. Weight-independent changes in blood glucose homeostasis after gastric bypass or vertical sleeve gastrectomy in rats. Gastroenterology 2011; 141: 950-958 [PMID: 21699789 DOI: 10.1053/j.gas-tro.2011.05.050]

5 Wilson-Pérez HE, Chambers AP, Ryan KK, Li B, Sandoval DA,



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13 Ley RE, Bäckhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut microbial ecology. Proc Natl Acad Sci USA 2005; 102: 11070-11075 [PMID: 16033867 DOI: 10.1073/pnas.0504978102]

14 Parks BW, Nam E, Org E, Kostem E, Norheim F, Hui ST, Pan C, Civelek M, Rau CD, Bennett BJ, Mehrabian M, Ursell LK, He A, Castellani LW, Zinker B, Kirby M, Drake TA, Drevon CA, Knight R, Gargalovic P, Kirchgessner T, Eskin E, Lusis AJ. Genetic control of obesity and gut microbiota composition in response to high-fat, high-sucrose diet in mice. Cell Metab 2013; 17: 141-152 [PMID: 23312289 DOI: 10.1016/j.cmet.2012.12.007]

15 Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, Muehlbauer MJ, Ilkayeva O, Semenkovich CF, Funai K, Hayashi DK, Lyle BJ, Martini MC, Ursell LK, Clemente JC, Van Treuren W, Walters WA, Knight R, Newgard CB, Heath AC, Gordon JI. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 2013; 341: 1241214 [PMID: 24009397 DOI: 10.1126/science.1241214]

16 Matsubara T, Li F, Gonzalez FJ. FXR signaling in the enterohepatic system. Mol Cell Endocrinol 2013; 368: 17-29 [PMID: 22609541 DOI: 10.1016/j.mce.2012.05.004]

17 Schéle E, Grahnemo L, Anesten F, Hallén A, Bäckhed F, Jansson JO. The gut microbiota reduces leptin sensitivity and the expression of the obesity-suppressing neuropeptides proglucagon (Gcg) and brain-derived neurotrophic factor (Bdnf) in the central nervous system. Endocrinology 2013; 154: 3643-3651 [PMID: 23892476 DOI: 10.1210/en.2012-2151]

18 Wichmann A, Allahyar A, Greiner TU, Plovier H, Lundén GÖ, Larsson T, Drucker DJ, Delzenne NM, Cani PD, Bäckhed F. Microbial modulation of energy availability in the colon regulates intestinal transit. Cell Host Microbe 2013; 14: 582-590 [PMID: 24237703 DOI: 10.1016/j.chom.2013.09.012]

P- Reviewer: Bernhardt GA, Zhang ZM S- Editor: Ji FF L- Editor: A E- Editor: Liu SQ

Stoffers D, Drucker DJ, Pérez-Tilve D, Seeley RJ. Vertical sleeve gastrectomy is effective in two genetic mouse models of glucagon-like Peptide 1 receptor deficiency. Diabetes 2013; 62: 2380-2385 [PMID: 23434938 DOI: 10.2337/db12-1498]

6 Pories WJ. Bariatric surgery: risks and rewards. J Clin Endocrinol Metab 2008; 93: S89-S96 [PMID: 18987275 DOI: 10.1210/jc.2008-1641]

7 Rubino F, Cummings DE. Surgery: the coming of age of metabolic surgery. Nat Rev Endocrinol 2012; 8: 702-704 [PMID: 23147581 DOI: 10.1038/nrendo.2012.207]

8 Stefater MA, Wilson-Pérez HE, Chambers AP, Sandoval DA, Seeley RJ. All bariatric surgeries are not created equal: insights from mechanistic comparisons. Endocr Rev 2012; 33: 595-622 [PMID: 22550271 DOI: 10.1210/er.2011-1044]

9 Campos GM, Rabl C, Peeva S, Ciovica R, Rao M, Schwarz JM, Havel P, Schambelan M, Mulligan K. Improvement in peripheral glucose uptake after gastric bypass surgery is observed only after substantial weight loss has occurred and correlates with the magnitude of weight lost. J Gastrointest Surg 2010; 14: 15-23 [PMID: 19838759 DOI: 10.1007/s11605-009-1060-y]

10 Lima MM, Pareja JC, Alegre SM, Geloneze SR, Kahn SE, Astiarraga BD, Chaim EA, Geloneze B. Acute effect of roux-en-y gastric bypass on whole-body insulin sensitivity: a study with the euglycemic-hyperinsulinemic clamp. J Clin Endocrinol Metab 2010; 95: 3871-3875 [PMID: 20484482 DOI: 10.1210/jc.2010-0085]

11 Myronovych A, Kirby M, Ryan KK, Zhang W, Jha P, Setchell KD, Dexheimer PJ, Aronow B, Seeley RJ, Kohli R. Vertical sleeve gastrectomy reduces hepatic steatosis while increasing serum bile acids in a weight-loss-independent manner. Obesity (Silver Spring) 2014; 22: 390-400 [PMID: 23804416 DOI: 10.1002/oby.20548]

12 Patti ME, Houten SM, Bianco AC, Bernier R, Larsen PR, Holst JJ, Badman MK, Maratos-Flier E, Mun EC, Pihlajamaki J, Auwerx J, Goldfine AB. Serum bile acids are higher in humans with prior gastric bypass: potential contribution to improved glucose and lipid



Amanda M Balkhi, Adam M Reid, Sarah C Westen, Brian Olsen, David M Janicke, Gary R Geffken

Amanda M Balkhi, Adam M Reid, Sarah C Westen, David M Janicke, Gary R Geffken, Department of Clinical and Health Psychology, University of Florida, Gainesville, FL 32611, United StatesAmanda M Balkhi, Adam M Reid, Brian Olsen, Gary R Geffken, Division of Medical Psychology, Department of Psychiatry, University of Florida, Gainesville, FL 32611, United StatesGary R Geffken, Department of Pediatrics, University of Florida, Gainesville, FL 32611, United StatesAuthor contributions: All the authors equally contributed to this work.Conflict-of-interest: The authors have no conflict of interest to report.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Amanda M Balkhi, MS, Department of Clinical and Health Psychology, University of Florida, PO Box 100165, 1600 S Archer Rd, Gainesville, FL 32611, United States. [email protected]: +1-352-2658873Fax: +1-352-3766270Received: August 29, 2014Peer-review started: August 30, 2014First decision: November 27, 2014Revised: December 20, 2014Accepted: January 18, 2015 Article in press: January 20, 2015Published online: April 15, 2015

AbstractType 1 diabetes is a chronic illness with a high burden of care. While effective interventions and recommendations for diabetes care exist, the intensive nature of diabetes management makes compliance difficult. This is

especially true in children and adolescents as they have unique psychosocial and diabetes needs. Despite the development of effective in-person interventions targeting improving self-management and ameliorating psychosocial difficulties there are still a number of barriers to implementing these interventions, namely time, cost, and access. Telehealth interventions allow for the dissemination of these interventions to a broader audience. Self-management and psychosocial telehealth interventions are reviewed with a special emphasis on mobile phone and internet based technology use. While efficacy has been demonstrated in a number of telehealth interventions with improved cost effectiveness over in-person interventions, many challenges remain including high participant attrition and difficulties with receiving reimbursement for services rendered. These and other challenges are discussed with recommendations for researchers and telehealth providers provided.

Key words: Telehealth; Disease management; E-health interventions; Type 1 diabetes management; Type 1 diabetes


Core tip: Type 1 diabetes is a chronic illness with a high burden of care. Despite the development of effective in-person interventions, telehealth interventions are necessary to improve access to and engagement in interventions to improve diabetes management. Mobile phone and internet based interventions appear to have the most potential to enact change. Challenges and recommendations for these telehealth interventions are provided.

Balkhi AM, Reid AM, Westen SC, Olsen B, Janicke DM, Geffken GR. Telehealth interventions to reduce management complications in type 1 diabetes: A review. World J Diabetes 2015; 6(3): 371-379 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/371.htm DOI: http://dx.doi.

REVIEW


Telehealth interventions to reduce management complications in type 1 diabetes: A review




org/10.4239/wjd.v6.i3.371

INTRODUCTIONThe prevalence of chronic diseases, such as diabetes, cancer, cardiometabolic and respiratory conditions continues to pose a challenge for often overtaxed health care systems, requiring fundamental changes in the delivery and maintenance of patient care[1-4]. Telehealth (TH), defined as any medical activity involving an element of distance and use of a telecommunications strategy[5], represents an approach which may enable patients with chronic medical conditions to seek disease specific information and support[6-9], to be followed by clinicians more frequently and away from hospital settings[10-12], reduce healthcare costs[13], and to ultimately promote improved adherence to medical regimens resulting in improvement in health outcomes[14].

BRIEF HISTORY OF THWhile TH interventions began more than 50 years ago with closed-circuit television, research into TH interventions did not truly begin to accelerate until the 1990s with a dramatic increase in TH publications through the 2000s[15,16]. Initial TH interventions primarily emphasized providing the same care that would be provided in-person through an intermediary such as a closed circuit television or telephone. A majority of interventions that subsequently developed relied on direct telephone contact by nurses or skilled health care professionals or transmission of simple self-management data via a modem[15]. The primary strength of these early TH interventions was in providing care coordination with more frequent feedback and without an in-person visit, which resulted in cost savings and improved patient health[14]. As technology advanced, TH interventions did as well, moving to material presented through video phones, home computers, pre-programmed interactive problem solving programs, and mobile phone and internet based interventions[17,18]. The flexibility and cost effectiveness of TH makes it well suited to be used in the treatment of chronic illnesses such as diabetes.

Previous interventions have shown efficacy imple-menting TH for a variety of chronic conditions, including cancer, transplant recipients, heart failure, and chronic pulmonary disease[3,7-10,12]. These interventions have shown support for TH in providing condition specific education, social support, and self management assistance. In addition, this previous work has demonstrated the wide acceptability of TH and the ability for TH interventions to reach previously underserved populations.

NEED FOR TH IN TYPE 1 DIABETES Type 1 diabetes (T1D) is one of the most common chronic diseases in pediatrics in the United States and affects

more than 151000 youth under 20 years of age[19]. Poorlycontrolled diabetes poses many serious health com-plications thus optimal T1D management during childhood and adolescence is necessary to reduce negative health outcomes and improve life expectancy[19,20]. The mana-gement of T1D is a complex and challenging task that involves integration of daily medical tasks and lifestyle modifications. While demanding, the successful intensive management of T1D is associated with improved health outcomes and protections against complications that maintain for as many as 6-10 years following intensive management[21].

Children and adolescents with T1D have unique needs that dictate different standards of care than adults[22]. Despite parental involvement in diabetes management being common, non-adherence is especially high in the transition to and within adolescence, increasing the risk of immediate and future microvascular complications[23-28]. T1D management is further complicated by the social, emotional, and psychological demands of the disease[23]. Poor psychosocial wellbeing (e.g., depression, anxiety, stress) is related to poorer short and long term health outcomes due to suboptimal disease management[29-34]. Family functioning, parent wellbeing, and family cohesion have also been identified as an important contributor to diabetes control[35-44]. Therefore, when evaluating T1D management interventions, assessing and addressing the impact of patient and parent psychosocial wellbeing while being flexible and developmentally sensitive to the needs of the patient is essential to ensure that the intervention has a lasting impact.

TH IN T1D While previous in-person diabetes interventions have successfully targeted increasing patient knowledge[45-47], improving illness perception[48,49], fostering family communication and relationships[50,51], and advancing technological accuracy and ease of management devic-es[52-54], there are several remaining challenges to in-person interventions. Primary limitations of in-person interventions include poor ease of access for rural or underserved families, increased healthcare utilization costs, and poor attendance[55,56]. Additionally, individuals at greater risk for medical regimen nonadherence are likely to also be individuals who are at greater risk for not attending medical appointments[57], making traditional clinic based recruitment and interventions potentially ineffective. TH addresses many of the limitations of previous T1D interventions by providing a unique avenue for improving the management of T1D that is engaging, cost effective, and accessible[58].

SELF MONITORING AND EDUCATION INTERVENTIONSA hallmark of many TH interventions are to focus on providing education, improving self-monitoring through

Balkhi AM et al . Telehealth and management in diabetes


electronic check ins, and establishing more frequent communication with health care providers. While traditional phone interventions have demonstrated positive improvements in glycemic control and self-efficacy[59,60], the increased availability of smartphones and the internet facilitated further innovation and development. Deploying interventions on a mobile device, especially those compatible with text messaging, also proved effective in improving glycemic control in both adults and children[59,61-64]. These results suggestthat text messaging and intervention through mobile phones are a substantial area for outreach and inter-vention. However, despite the increased ownership of mobile phones among adults (91%) and adolescents (78%), there is still a substantial portion of individuals without a mobile phone or texting ability, especially among younger adolescents[65,66]. Additionally, some interventions using text messaging or smartphone applications in children or adolescents have not shown an ability to improve glycemic control, although secondary benefits such as increased adherence, communication or knowledge are generally noted[41,67-69]. As such, while mobile based interventions are promising, continued research into maximizing desired outcomes and cost-effectiveness is necessary.

Internet based interventions have the potential to overcome this limitation of mobile phone based interventions, because of the wide spread availability of the internet for adults (85%) and teens (95%) in the United States[66,70,71]. The internet may be especially appropriate for diabetes intervention, as one study suggests that 63.6% of parents of children with T1D use the internet to seek out diabetes information on their own[72]. For a child or adolescent with T1D, diabetes psychoeducation[73], problem solving vignettes[74], and physician monitoring of HbA1c and intervention[75-77], have all recently shown moderate to strong evidence of successfully improving glycemic control when imple-mented in an online environment. Similar results have been found with adults; however, most studies rely on adults with T2D[78-80]. Despite their demonstrated efficacy and the wide spread availability of the internet, the primary challenge that continues to plague internet based interventions is the decreased engagement and participation of users over time, with participant attrition rates of 11.5%-37% reported[74,78].

Notwithstanding the challenges in self-management interventions, these interventions have demonstrated effectiveness in multiple delivery modalities including voice calls, SMS/Text messaging, email, customized web portals, and video conferencing[61,81,82]. A systematic review revealed that telemedicine solutions for diabetes care are also feasible and acceptable to patients and providers[59]. Therefore, future research is necessary to integrate the previously proven delivery strategies with new technology that is engaging to users and cost-

effective.

PSYCHOSOCIAL AND SUPPORTIVE TH INTERVENTIONSOther TH interventions have strived to improve adherence by providing psychosocial support and decreasing family conflict around T1D management. One such method of intervention developed by Grey et al[73] bundled effective psychoeducation intervention with Coping Skills Training which improved glycemic control, quality of life, social acceptance, and self-efficacy which maintained for a year after beginning the online program (which was only 5 wk in duration). Self-efficacy has also been targeted as a potential area of psychosocial intervention, with online interventions demonstrating a significant positive impact on self-care activities[83]. Individual wellbeing has also been successfully addressed with web-based Cognitive Behavioral Therapy and peer mentoring[84,85]. Taken together, the existing psychosocial interventions for patients with T1D have shown success in engaging patients and improving psychological wellbeing, but are mixed on their abilities to minimize attrition and improve objective measures of glycemic control (i.e., HbA1c).

TH interventions have also been utilized to support the family and environment of patients with T1D. These interventions have successfully improved communication, improved HbA1c levels, and quality of life suggesting that targeting those supporting the individual with T1D (i.e., nurses, physicians, and family) may also be an effective way to improve T1D health outcomes[81]. There also appears to be awareness from family members and service providers of their need to find information and support regarding T1D care and a preference for online interventions[86]. One way to reach supporting individuals and patients may be to extend interventions to build on pre-existing online networks and supports. Social networks and forums for T1D have been qualitatively examined; despite concerns regarding the quality of the information presented on these sources, it is clear that patients and family members actively use these online sources (such as Facebook and online message boards) for diabetes information and social support[6,87-90]. Most notably, in one study 84.3% of caregivers that used online forums reported that their child’s care was impacted by information they encountered online[6]. Recent data also suggests that more than half of parents within a pediatric T1D clinic use the internet to seek out T1D information[72]. This identifies pre-existing internet sources as a potentially strong source for information dissemination but also as a potential venue for the unintended spread of misinformation. While prospective studies are needed to understand the association between parents’ use of online forums and their child’s glycemic control, these may be an appropriate area for



limited, federal, and reciprocal licenses, it is clear that the current system of licensure is hindering providers’ ability to reach out to patients who live in other states and steps need to be made to resolve these concerns[95].

PATIENT ENGAGEMENT AND ATTRITIONAttrition and noncompliance in TH interventions creates yet another barrier to successful establishment of TH interventions. As stated previously, attrition rates of 11.5%-37% among internet interventions have been reported [74,78]. However, patients who complete TH interventions may also not adhere adequately to the intervention, given the lack of in-person oversight. In a study conducted by Wangberg[83], participants were requested to repeatedly view and engage with online modules targeting self-efficacy and diabetes self-care, yet only 34% logged in more than twice to interact with the modules. Similarly, a review of TH adherence found a recurring theme in suboptimal frequency of uploading and submitting blood glucose values[96]. Given these relatively high drop-out rates and problems with noncompliance, TH programs should incorporate measures to improve adherence and keep the patients engaged in treatment.

To this end, some studies have shown improved adherence when the TH interventions are tailored toward the patients unique needs by using customized messages, programs, or personalized functions within the program[97]. For instance, a program may allow patients to use a data base of pictures of foods that have predetermined carbohydrate amounts instead of requiring the patient to estimate the carbohydrates for food they consume. Including features that communicated with patient’s preexisting diabetes technology (e.g., glucose monitors, insulin pumps) and automatically upload patients’ blood glucose levels in order for parents or providers to review and provide feedback may also be helpful. Overall, these programs should be easy to access and provide immediate feedback[98]. Programs should also take demographics into account. For instance, older individuals[99], women[100], and patients with higher self-efficacy[101,102], are more likely to adhere to internet interventions. Thus, providers who are developing or implementing TH interventions should work to determine which patients are going to be appropriate for the TH intervention and how to address those groups with a history of poor adherence to TH interventions[103].

PRIVACY AND SECURITYPossibly the most common TH concern relates to the ability for TH interventions to maintain patient privacy and security in a mobile or online environment. Appropriately managing personal health information (PHI) is an important piece to maintaining patients’ confidentiality. The federal laws provide regulations for protecting PHI under the Health Information Portability and Accountability Act[104] (HIPAA). Each state also has

future intervention.

ONGOING CHALLENGES IN THWhile research suggests that TH for patients with T1D can be a useful and effective method of improving glycemic control and overall adherence, there has been a significant delay in transitioning efficacious research interventions of T1D into community treatment settings. Chief among these issues are the financial feasibility and reimbursement for services delivered by skilled staff, creating and maintaining patient involvement in TH interventions while minimizing patient attrition, and ensuring patient safety, privacy, and legal accountability.

FINANCIAL CHALLENGESA key challenge that permeates across the literature in TH is the difficulty in obtaining reimbursement for services. The literature suggests that providers’ experience of receiving reimbursement varies signi-ficantly[91-93]. As of this publication only 15 states in the United States mandate coverage for TH services with 39 states providing at least some reimbursement for TH, although dramatically less so for behavioral TH despite behavioral TH’s appropriateness[94,95]. Additionally, recent studies have suggested that private third party reimbursement is improving across the board, though the trajectory of these improvements continues to be slow[93]. For example in 2005, 58% of TH programs received reimbursement for their services while in 2012, 45% of TH programs sought reimbursement and 81% of those reported receiving it[92,93]. Obtaining grant funding to offset these costs increases the institution’s ability to build a program[92]; however, if program personnel lack information about how to obtain reimbursement for their services from third party payers, the program may be discontinued after the grant funding has remitted[91,93]. This pattern of short term growth with long term discon-tinuation is concerning and hinders the growth of TH services.

The licensing and credentialing rules create another barrier to TH implementation. Similar to reimbursement regulations, there is a large discrepancy in state TH licensing laws. Current laws generally refer to the physical location of the patient as the place of service, regardless of the provider’s location. Moreover, state laws generally require that providers be licensed and credentialed at the place of service, making state lines a finite barrier to service delivery [91]. In 2011, Children’sMedical Services (CMS) began to allow institutions to accept the credentialing of the provider’s home institution instead of requiring the outside institution to put the provider through their credentialing process[91]. The CMS regulations show promise for expanding the credentialing requirements and may facilitate providers’ ability to reach patients who otherwise may not have been able to receive care. While efforts have been made to extend licensing adjustments by implementing



laws for managing PHI, which is not consistent from state to state and may be more or less stringent than HIPAA regulations[95]. When state privacy laws conflict with HIPAA, the general rule is that the provider follows the more stringent law[95]. TH providers who provide services across state lines must be aware of laws in both states and must work to resolve conflicts as they arise while being prepared for conflicting laws or regulations[105]. While managing these challenges across providers, technology technicians, nurses, medical assistants, and billing personnel in two states may be manageable with practice, negotiating differences among state and federal privacy laws is likely to be increasingly more difficult with each additional state, thereby deterring providers from expanding their services and possibly hindering patients’ access to specialized mental health care[95]. In addition, though more recent technological advances and using secure or closed networks have improved security of online data transmission[91,95] regulations are not clear regarding where or how the data should be stored (e.g., online, at the providers’ institution, or at the institution of the place of service). While providers should follow good safety procedures such as a personalized login, automatic time-out setting when not in use, encrypted data storage, and encrypted data transmission, they must also deliver informed consent and ensure a patient’s understanding of the potential and ever-evolving risks of transmitting health information over the internet or mobile networks[95,105,106]. These challenges require that a provider maintain understanding not only of the HIPAA and PHI laws but also of technological capabilities and challenges to technological safety, which is a daunting task for even the most informed provider.

CONCLUSION AND FUTURE DIRECTIONS IN THTaken together, research has shown the effectiveness and promise of TH in improving several primary (i.e., glycemic control, adherence) and secondary (i.e., social support, comorbidity, and knowledge) outcomes in T1D patients. Although a previous review suggested that phone interventions appear to have more promise than internet interventions in improving targeted T1D outcomes, the reviewed literature above suggests promise in both web based and mobile interventions. Broadly, TH interventions must strive to be theory driven, integrate multiple platforms, be secure, and be user-friendly[87,107,108]. In doing so, TH interventions will expand to a broader audience and have an improved chance at reaching those most in need for TH, the underserved individuals who have difficulty accessing traditional services.

In order to improve current TH interventions, resear-chers and providers should invest in portability. As mobile computing and mobile phones reach a larger and larger share of the United States[65] TH interventions must be optimized to provide an efficient, appealing, and

interactive environment on the smaller screen of mobile phones and tablets to reduce attrition and maintain participant engagement. This is especially true for adolescents and children, of whom a large portion (74%) access the internet from their phone and an increasing portion (25%) use their mobile phones as their primary device for navigating the web[109]. Researchers who do not adjust their TH interventions to take advantage of the increased computing power and accessibly of portable technology will continue to struggle with participant engagement and attrition as fewer and fewer young individuals use a traditional desktop computer.

Researchers and clinicians should also seek to integrate their TH interventions into existing technological infrastructure both to increase participant familiarity and ease of use. By intentionally creating interventions that integrate with diabetes technology (i.e., blood glucose monitors, insulin pumps), providers improve their ability to obtain objective health information and increase participant engagement through ease in integration. Integration, especially automated or hands free integration, with these technologies also has the benefit of providing a method for providers to easily view and provide feedback without relying on patients to physically produce their blood glucose meters at appointments. This improves the quality of information that providers have to deliver care, as well as decreases the burden of appointment preparation on the patient and potentially improving compliance.

In addition to technological integration, effective TH interventions should seek to involve family members, providers, and other supporting members of the patient’s T1D management team. An effective way to do this may be by building on the preexisting support networks targeting these individuals and developing ways to improve how individuals find, interoperate, and communicate the information they find online. Recent research has demonstrated that parents of children with T1D are especially likely to use these online sources and actively incorporate them into how they care for their child’s T1D[6,72]. Providers may benefit from this predisposition of parents to search for information online by designing and implementing procedures to inform users of appropriate sources of information and increase awareness of effective TH interventions that may provide similar information. Building atop of pre-existing resources may also reduce the infrastructure cost that contributes to the short term growth and long term discontinuation of existing TH interventions.

Finally, clinical care providers are encouraged to advocate for improved legislation regarding TH. Notwithstanding substantial improvement over the last decade regarding TH reimbursement and rules, there continues to be a lack of guidelines regarding TH interventions delivered by the mental health profession and the associated technological, privacy, and security issues created by these interventions. In fact, many of the privacy and security concerns related to TH interventions may only be feasibly addressed by policy



makers and technology manufacturers. The development and national recognition of TH guidelines in conjunction with improved licensure recognition across state lines may provide increased support to mental health pro-fessionals who wish to pursue TH interventions. As a part of these guidelines, providers and researchers are encouraged to pair with technology consultants whom are informed and educated both on technological advances and advances in privacy and security laws. With proper support and the development of structured guidelines, TH interventions can grow to fit within the evolving scope of health care policy, reimbursement, and technological advancement while reducing the number of individuals who are underserved.

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P- Reviewer: Charoenphandhu N, Daltro C S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ



Kimber M Simmons, Aaron W Michels

Kimber M Simmons, Aaron W Michels, Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO 80045, United StatesAuthor contributions: Simmons KS and Michels AW contributed to this paper.Supported by Grants from the National Institute of Diabetes and Digestive Kidney Diseases, No. R01 DK032083, K08 DK095995; Juvenile Diabetes Research Foundation, the Children’s Diabetes Foundation, and the Brehm Coalition.Conflict-of-interest: The authors have no conflicts-of-interest relevant to this manuscript.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Aaron W Michels, MD, Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Mail Stop A140, 1775 Aurora Court, Aurora, CO 80045, United States. [email protected]: +1-303-7241923 Fax: +1-303-7246784Received: September 11, 2014Peer-review started: September 11, 2014First decision: November 14, 2014Revised: November 26, 2014Accepted: January 9, 2015Article in press: January 12, 2015Published online: April 15, 2015

AbstractType 1 diabetes (T1D) is an autoimmune disease cha-racterized by loss of insulin producing beta cells and reliance on exogenous insulin for survival. T1D is one of the most common chronic diseases in childhood and the incidence is increasing, especially in children less than 5 years of age. In individuals with a genetic predisposition, an unidentified trigger initiates an abnormal immune response and the development of islet autoantibodies

directed against proteins in insulin producing beta cells. There are currently four biochemical islet autoantibodies measured in the serum directed against insulin, glutamic decarboxylase, islet antigen 2, and zinc transporter 8. Development of islet autoantibodies occurs before clinical diagnosis of T1D, making T1D a predictable disease in an individual with 2 or more autoantibodies. Screening for islet autoantibodies is still predominantly done through research studies, but efforts are underway to screen the general population. The benefits of screening for islet autoantibodies include decreasing the incidence of diabetic ketoacidosis that can be life threatening, initiating insulin therapy sooner in the disease process, and evaluating safe and specific therapies in large randomized clinical intervention trials to delay or prevent progression to diabetes onset.

Key words: Autoimmunity; Autoantibodies; Diabetes prevention; Screening; Type 1 diabetes


Core tip: Type 1 diabetes (T1D), the immune mediated form of diabetes, is now a predictable disease with the measurement of islet autoantibodies. The presence of two or more antibodies defines preclinical disease as nearly everyone with multiple antibodies progresses to clinical diabetes. With improved platforms to measure islet autoantibodies, screening the general population is now a goal. Early identification of preclinical diabetes allows for less diabetic ketoacidosis, early initiation of insulin therapy, and the potential to delay or prevent diabetes onset. Clinical trials using safe and specific therapies to block disease specific immune cells are underway in T1D.

Simmons KM, Michels AW. Type 1 diabetes: A predictable disease. World J Diabetes 2015; 6(3): 380-390 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/380.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.380

REVIEW


Type 1 diabetes: A predictable disease




INTRODUCTIONType 1 diabetes (T1D) is a chronic disease caused by immune-mediated destruction of insulin producing beta cells in the pancreas[1]. The destruction of beta cells results in insulin insufficiency, and patients develop life-threatening hyperglycemia that clinically manifests with weight loss, polyuria, and polydipsia. The majority of patients who develop T1D have high-risk human leukocyte antigen (HLA) genes. Islet autoantibodies can be measured in the serum of these high-risk individuals years before the onset of any clinical symptoms, making T1D a predictable disease. Multiple prevention trials in patients with high-risk HLA genes or in patients who have measureable autoantibodies have been completed. To date, no trial has prevented the onset of T1D, but data indicates that the disease process may be delayed by administering oral insulin to induce insulin specific regulatory T-cells in the gut, resulting in decreased inflammation in the pancreas. This review summarizes the epidemiology, risk factors and pathogenesis of T1D. The review also examines the goal of screening the general population for T1D risk and preventing disease onset in individuals with preclinical disease.

EPIDEMIOLOGYT1D is one of the most common chronic diseases in childhood and is diagnosed at an increasing rate in adults. The incidence rate varies significantly by geographical region. Sweden, Finland, Norway, United Kingdom, and Sardinia have the highest incidence of T1D at an age-adjusted rate of > 20/100000 patient years. For comparison, the United States has an incidence rate of 17.8/100000 patient years in a predominantly Caucasian population. China and South America have the lowest incidence of T1D, reported as < 1/100000 patient years[2-5]. The rate of T1D diagnosis is increasing in most countries, with rates dramatically increasing in children less than 5 years of age[6]. The annual incidence of T1D is increasing globally by 2.3% per year and is estimated to be increasing by 2.7%-2.8% in non-Hispanic white youth in the United States[7]. Large registries in both Europe and the United States show that the incidence of T1D peaks between 5 to 7 years of age and again when children enter puberty[8]. Unlike most autoimmune diseases, T1D is more common in males than females. The risk of T1D development in the general population is 1:300[9]. In children who have a genetically related sibling, the risk is increased to 1:7 and is greatest in children under 5 years of age[10,11]. Offspring of mothers with T1D carry approximately 3% risk and offspring of fathers with T1D carry approximately 5% risk[12]. Genetics confer risk for development of T1D, as does seasonal variation and birth month suggesting an environmental influence on disease pathogenesis. Children born in the spring tend to be at a greater risk for developing T1D, while diagnosis is increased during climatically cold

seasons[13-16]. This is an epidemiological association that requires further investigation.

RISK FACTORSGeneticT1D is a polygenic disorder with many genes contri-buting varying amounts of genetic risk for disease development. The genes conferring risk for diabetes are generally classified as HLA and non-HLA genes. Large genome wide association studies show that over 40 genes increase susceptibility to T1D[17,18]. The major determinant of genetic susceptibility to T1D, contributing greater than 50% of the genetic risk, is conferred by genes in the HLA complex located on chromosome 6[9]. The HLA complex is divided into 3 regions: classes Ⅰ, Ⅱ, and Ⅲ. Alleles of the class Ⅱ genes, DQ and DR (and to a lesser extent DP), are the most important determinants of T1D. These class Ⅱ molecules are expressed on antigen-presenting cells (macrophages, dendritic cells, and B cells) and present antigens to CD4 T lymphocytes. DQ and DR genes are in close linkage disequilibrium on chromosome 6 with specific DQ and DR genes inherited together. The presence of the DR4/DQ8 haplotype increases the odds ratio for T1D development to approximately 11, indicating an individual with this haplotype is 11 times more likely to develop T1D than those without. Approximately 90% of all individuals with T1D have either or both the DR4/DQ8 or DR3/DQ2 haplotypes. Interestingly, HLA genes also confer protection from T1D development. Individuals who have the specific DQ6 allele (DQB1*06:02) are dominantly protected from T1D, with an odds ratio of 0.03 for disease development[19].

Of the non-HLA genes, insulin and protein tyrosine phosphatase non-receptor type 22 (PTPN22) confer risk for T1D development but to lesser degrees than HLA genes[20]. Similar to HLA class Ⅱ genes, insulin gene polymorphisms can confer both susceptibility to and protection from T1D development. At the 5’ end of the insulin gene, there are variable numbers of tandem repeats. Having more repeats correlates to more insulin message being expressed in the thymus. The thymus responds by developing central tolerance to insulin. In individuals with fewer repeats, autoreactive T-cells can persist, and the risk for T1D development is increased[21]. PTPN22 helps regulate antigen receptor signaling and T cell activation, and a single nucleotide polymorphism (arginine to tryptophan at position 620) has been associated with a number of autoimmune disorders including T1D. A gain of function polymorphism decr-eases T cell receptor signaling which confers diabetes risk. It is unknown why decreased T cell activation leads to T1D risk, but it can be hypothesized that deficient negative selection of thymic cells may be involved[22,23].

EnvironmentGenetics alone does not lead to T1D; the environment also plays a pivotal role. This is evidenced by the fact

Simmons KM et al . Type 1 diabetes: A predictable disease


that not all individuals with high-risk genes develop T1D. In fact, the majority of individuals with high-risk HLA class Ⅱ genes (DR4/DQ8 and DR3/DQ2) do not develop T1D. There are likely one or more environmental factors that trigger and perpetuate the autoimmune disease process prior to hyperglycemia and a clinical diagnosis of hyperglycemia and T1D. Large natural history studies indicate that the development of islet autoantibodies (the first laboratory evidence of beta cell autoimmunity) in high-risk individuals often occurs between 9 mo and 2 years of age[24]. This suggests that an environmental trigger is present early in life, possibly in utero.

One of the most extensively evaluated environmental triggers is viral infection. Many viruses are implicated in the development of T1D including enteroviruses such as coxsackie B virus, cytomegalovirus, congenital rubella syndrome, and rotavirus[25-32]. Enterovirus is the leading candidate for contributing to T1D development. Epidemiologic studies in Finland show that the develop-ment of beta cell autoimmunity parallels the seasonal pattern of enterovirus infection and clinical symptoms of enteroviral infection[33,34]. Enterovirus infection was strongly associated with the development of autoantibodies in the Diabetes Autoimmunity Study in the Young (DAISY) cohort[35]. Laboratory evidence of enterovirus infection is reproducibly present in individuals with new onset T1D, pregnant women whose children develop T1D, and donor pancreata of individuals with T1D[36,37]. The exact mechanism of how viruses induce autoimmunity is not clear. The molecular mimicry hypothesis proposes that because the P2-C protein sequence of enterovirus is similar to glutamic decarboxylase (GAD), which is expressed in islet cells, the immune system erroneously targets destruction of beta cells[38]. The other leading hypothesis is that viral infection activates autoreactive T cells. As evidence, Cytomegalovirus B4 has tropism for pancreatic tissue and infection results in release of beta cell antigens that are phagocytized by macrophages and presented to autoreactive T cells[39].

Another potential environmental influence relates to the north-south division of diabetes development in the world, with a higher incidence of T1D in northern climates compared to southern. The north-south hypothesis implicates that a lack of vitamin A and/or D exposure early in life predisposes individuals to the development of autoimmune diseases including T1D. Offspring of mothers supplemented with vitamin D during pregnancy and young children supplemented with vitamin D have shown a reduced risk of T1D development that may be dose responsive[40,41]. However, an analysis from the DAISY Study found that vitamin D intake and 25(OH) vitamin D levels throughout childhood were not associated with the development of islet autoantibodies or T1D development[42].

Early introduction of cow’s milk and gluten have also been extensively studied. The introduction of gluten into an infant’s diet prior to three months and after 7 mo has been associated with increased auto-antibody development[24,43]. Some studies also indicate

that breastfeeding or using elemental formula may be protective against T1D. Other environmental factors that continue to be explored include nitrosamine compounds, maternal age, pre-eclampsia, and childhood obesity. There is no evidence to suggest that vaccines increase the risk of T1D development[44]. To date, there are no causal environmental factors that trigger the develop-ment of islet autoantibodies or increase the risk of progression to clinical T1D development. However, there is a large international prospective longitudinal study, The Environmental Determinants of Diabetes in the Young, currently underway to evaluate potential environmental factors in T1D[45].

NATURAL HISTORYThree decades ago, it was hypothesized that T1D is a chronic autoimmune disorder that develops in stages, and the model remains valid today (Figure 1). In genetically predisposed individuals (those with DR4/DQ8 and/or DR3/DQ2 haplotypes) there is an environmental trigger that leads to a break in immunologic tolerance and loss of beta cell mass. Over a period of time, usually years, there is autoimmune destruction of insulin producing beta cells that is marked by the presence of serum islet autoantibodies (Figure 2). As the process continues, very likely in a relapsing and remitting manner, there is a loss of glucose stimulated insulin release, and eventually insulin deficiency such that overt hyperglycemia results and clinical T1D is diagnosed[4].

How an inciting event leads to an aberrant immune response is not completely understood. Most hypotheses focus on immunologic abnormalities in antigen pre-sentation by HLA molecules to T cells in the thymus and peripheral lymph organs. T cells are educated in the thymus to self-antigens, such as insulin, and if there are dysregulated immune processes, self-reactive T cells can escape central tolerance and exist in the periphery[46,47]. Once these cells encounter their target antigen or peptide in peripheral lymph organs, they become activated to target beta cells. Other hypotheses focus on environmental triggers leading to immune activation and targeting of beta cells. The molecular mimicry theory proposes that a viral or bacterial protein shares amino acid sequence homology with beta cells and induces immune system activation through targeting beta cell antigen that is molecularly similar to a foreign antigen[38]. Finally an infectious triggering event may allow beta cells to become more sensitive to cytokine and free radical induced inflammation[48].

Recently the network for pancreatic organ donors has been established to study the pancreata of deceased donors with islet autoantibodies (preclinical disease) or established T1D[49]. The goal is to understand mechanisms of disease pathogenesis and interactions between beta cells and the immune system[50]. What we have gleaned from the initial efforts is that islet infiltrates (insulitis) are present in a lobular pattern in the pancreas, and there is a predominance of CD8 and CD4 T cells,



Approximately 90% of individuals have two or more islet cell autoantibodies at diagnosis, and it is likely that the remaining 10% of individuals (islet autoantibody negative) have autoantibodies against antigens that have yet to be discovered. In children, IAA is usually the first antibody to develop, and the progression to T1D is 100% in children with a persistently high level of IAA[55,56]. This is in contrast to adults who tend to have higher levels of GAD at diagnosis. Islet autoantibodies can be easily measured in the serum, with the gold standard method for detecting antibodies being fluid phase radioimmunoassays (RIA)[57]. More recently, islet autoantibodies are now able to be measured from smaller volumes of serum and without the use of radioactivity using electrochemiluminescense as a detection method while maintaining similar sensitivity and specificity to RIA[58,59]. The rate at which individuals with positive islet autoantibodies progress to clinical T1D is dependent upon the age of appearance, insulin autoantibody level, and the number of autoantibodies present[55]. Hemoglobin A1c rises 1 to 1.5 years prior to diagnosis. Therefore, reduced insulin secretion and resultant hyperglycemia occur before T1D is clinically diagnosed[60,61]. Once T1D is clinically diagnosed, indivi-duals must commit to lifelong blood glucose monitoring and intensive insulin administration via multiple daily injections or an insulin pump to achieve good glycemic control. With improved diabetes management, the risk for long-term complications such as renal failure, myocardial infarctions, stroke, and lower extremity amputations has decreased over the last two decades[62]. However despite the decreasing prevalence of complications in diabetes, the need still exists to understand the underlying pathogenesis of complications such as diabetic cardiomyopathy and

B-lymphocytes, and macrophages[51,52]. Pancreata from established T1D patients also show an overall decrease in weight compared to age matched controls, potentially related to atrophy of the exocrine pancreas with the loss of beta cells[52]. The first serological evidence of an autoimmune response to beta cells is the appearance of autoantibodies to insulin (IAA), GAD, islet antigen 2, and zinc transporter 8[53]. Placental antibodies are no longer present after approximately 6 mo, so any antibodies in serum after that time reflect endogenous antibodies. If an individual develops two or more of these antibodies, they will eventually progress to clinical onset of T1D[54].


t

Primary prevention Secondary prevention Tertiary prevention

Genetic predisposition Preclinical diabetes T1D

Loss of insulin release

↓Increasing glucose

excursions

Minimal to no insulin release

↓Abnormal glucose

levels

Abnormal immune response↓

Dysregulated T cell responses and islet autoantibodies

detectable in serum

Beta

cel

l mas

s

UnknownPrecipitating

Eventmay occur in utero

or early in life

Figure 1 Stages in the development of type 1 diabetes adapted from the initial model proposed by George Eisenbarth. In genetically at risk individuals an unknown trigger, presumably environmental, initiates an autoimmune response that results in loss of beta cell mass. Before metabolic disturbances occur, islet autoantibodies (insulin, glutamic decarboxylase, islet antigen 2, zinc transporter 8) are measureable in serum. As beta cell mass decreases, potentially in a relapsing-remitting manner, there is loss of endogenous insulin release and ensuing hyperglycemia. Within this model, there are opportunities for type 1 diabetes (T1D) prevention in genotypically high risk individuals (primary prevention) and in autoantibody positive individuals (secondary prevention). Interventions to preserve remaining beta cell mass at diagnosis are also possible (tertiary prevention).

Figure 2 Insulin autoantibodies are often the first antibody to develop in young children. In contrast, adults most often are GAD65 and IA-2 autoantibody positive at diagnosis. The ZnT8 antibody is the most recently identified autoantibody with commercial testing now available. T1D: Type 1 diabetes; GAD65: Glutamic decarboxylase; IA-2: Islet antigen 2; ZnT8: Zinc transporter 8.

IA-2GAD65

Most common antibody seen in adults diagnosed with T1D

Islet autoantibodies

ZnT8Clinical antibody testing

available as of 2014

Insulin(mlAA)

Levels predict age of diabetes onsetOften first antibody to appear in

young children


novel approaches for treating complications such as neovascularization in diabetic foot disease[63,64].

SCREENING AND PREVENTIONThe American Diabetes Association recently adapted their guidelines to recommend screening for islet auto-antibodies in high-risk individuals[65]. Highly sensitive serological assays are not widely available, and all screening is recommended to be done in the setting of a clinical research study. To date, general population screening has been done through large clinical trial networks such as the National Institutes of Health sponsored TrialNet, which enroll and screen first or second degree family members of individuals with T1D. By identifying individuals with positive islet autoantibodies, the rate of diabetic ketoacidosis (DKA) at diagnosis is reduced[66]. Preventing DKA is important as altered mental status, coma, and even death can occur[67]. In fact, DKA is the most common cause of death in children with T1D[68]. Without screening, DKA at diagnosis is relatively common[69]. In the EURODIAB study, 42% of children presented in DKA (pH < 7.3) at the time of diagnosis with T1D[3]. By identifying individuals with positive autoantibodies, insulin therapy can be initiated early, and these children can enroll in studies aimed at preserving beta cell mass. In adults, maintaining endogenous insulin secretion reduces hemoglobin A1C, reduces the risk of severe hypoglycemia, decreases reliance on exogenous insulin, and decreases the rate of long-term complications[70-77]. In children, there has been very little data collected regarding residual beta cell mass beyond the first year after diagnosis[78-82]. A case-control study did show that children without severe hypoglycemia had increased residual beta cell mass compared to those children with severe hypoglycemia[83]. An effective method of preserving beta cell mass is not yet available, and the benefit of increased residual beta cell mass in children remains to be confirmed.

According to the World Health Organization’s principles of early disease detection, T1D is a condition that meets criteria for the establishment of a screening program. These principles include the condition is an important health problem, there is a recognizable latent stage of the disease, the natural history of the disease is understood, there is an adequate and accepted laboratory screening test, providers agree on who should receive treatment and there is a treatment available, there are adequate resources for diagnosis and treatment, and the cost of overall medical care would not increase[84]. Islet autoantibodies can be reliably measured in serum, with each antibody assay having a specificity of 99% when measured by radioimmunoassay in tertiary referral centers such as the Barbara Davis Center for Diabetes. The sensitivity for each autoantibody assay ranges from 70%-80%. We view these radioimmunoassays as a confirmatory test for T1D. A desired screening test needs to be reliable with high sensitivity, cost effective, and technically feasible, likely as a multiplex assay in

which all four autoantibodies are measured in a single well of an assay plate. Currently, to measure islet autoantibodies a blood draw is required with subsequent shipping of venous or capillary blood samples to a reference laboratory. This is not feasible for population wide screening due to technical requirements of sample collection and high cost. Screening large populations of infants for metabolic diseases and other congenital disorders has been successfully done using dried blood spots[85]. To establish an accepted screening program for T1D, the sensitivity and specificity of islet autoantibodies, specifically insulin autoantibody, needs to be established using a feasible collection method such as dried blood spots on filter paper, which would be a simplified collection method and more cost effective. Overall, T1D would not be over diagnosed with general population screening as diagnosis of the disorder requires both the presence of islet autoantibodies and metabolic abnormalities.

Ideally, individuals who screen positive for islet autoantibodies can be offered a treatment to prevent or delay the progression to T1D. Many secondary prevention trials have been completed with more currently underway[86]. As population based screening may be feasible in the near future, it is important to continue secondary prevention trials with the goal of delaying or preventing progression to T1D in islet autoantibody positive individuals. Patients enrolled in clinical inter-vention trials benefit from close follow up by medical professionals, early diagnosis of T1D, decreased incidence of DKA, and early initiation of insulin therapy (Figure 3).

Secondary prevention trialsAs T1D is a predictable disease with the measurement of islet autoantibodies, it logically follows that the disease should be preventable. To date, the majority of secondary prevention trials (enrolled individuals with preclinical disease) have administered different preparations of insulin to autoantibody positive individuals in an attempt to slow the progression to T1D onset[87]. The first such trial was the diabetes prevention trial-type 1 in which at risk patients were either administered subcutaneous insulin or oral insulin in randomized, doubleblinded, placebo controlled trials. Oral insulin has no metabolic effect; however, orally administered insulin does encounter mucosal gut-associated lymphoid tissue. The role of this lymphoid tissue is to provide protection from orally acquired pathogens and to keep individuals from developing reactions to ingested proteins. By administering low doses of oral insulin, insulin-specific T-regulatory cells are produced which may release cytokines that inhibit the inflammatory cascade that leads to β-cell destruction[88-90]. Relatives of patients with T1D who were 3 to 45 years of age and had high-risk HLA genes and one or more positive autoantibodies were evaluated for abnormal glucose metabolism. Those individuals who had abnormal glucose tolerance (n = 339) were administered 0.25 units/kg per day of Ultralente insulin twice daily and received an intravenous insulin infusion for four



days at the beginning of the study and then annually. There was no effect of low-dose subcutaneous insulin on delaying the progression to T1D[91]. Participants with normal glucose tolerance received 7.5 mg/kg per day of oral insulin (n = 372). An oral glucose tolerance test was completed every 6 mo during a 6-year follow up, and there was not a delay in progression to T1D. Of interest, a post-hoc analysis showed in participants with persistently high levels of IAA (≥ 80 nU/mL) there was a delay in disease onset of approximately five years[92]. Also, the rate of progression to T1D onset after stopping insulin was more rapid[93]. A follow-up oral insulin trial through TrialNet is currently enrolling participants in order to determine if oral insulin can delay the progression to T1D in individuals with high IAA levels (ClinicalTrials.gov Identifier: NCT00419562).

Another insulin intervention trial from the Belgian T1D Registry identified study participants who were insulin autoantibody positive and did not have a HLA haplotype conferring protection (DQB*0602). Study participants were given two subcutaneous injections of insulin daily for 3 years (n = 25) or observed and prospectively followed (n = 25). The participants who were treated with insulin and those who refused treatment or agreed to observation developed T1D at the same rate[94].

Many preclinical studies have suggested that administration of intranasal insulin may delay T1D development through mucosal tolerance, in which mucosal antigens have been shown to impact regulatory T cell development[95]. To translate these findings to humans, individuals with high-risk HLA haplotypes and one or more islet autoantibodies were enrolled in the Intranasal Insulin Trial (INIT-Ⅰ) (n = 38). This randomized, doubleblinded, crossover pilot study suggested that intranasal insulin protects against the development of T1D by increasing antibody formation and decreasing T cell responsiveness[96]. The INIT-Ⅱ, a randomized, doubleblinded, placebo controlled trial, is now enrolling individuals to determine if intranasal insulin can delay or prevent the progression to T1D (ClinicalTrials.gov Identifier: NCT 00336674).

However, a large study in Finland, the T1D Prediction and Prevention Trial enrolled and followed siblings of children with T1D or infants of mothers with T1D who had high-risk HLA genes for islet autoantibody development. Once two or more autoantibodies were detected (n = 264), they were randomized to intranasal insulin (n = 137) or placebo (n = 127). Interim analyses showed no benefit of intranasal insulin in delaying the onset of T1D[97]. This indicates that intranasal insulin may not be effective at delaying diabetes onset at the administered dose and timing in the disease process. Potentially insulin antigen specific therapies may need to be administered earlier in the disease course to have an impact on delaying progression to T1D.

Several trials using non-antigen specific therapies including bacille calmette-guerin injections, Ketotifen (histamine antagonist), oral cyclosporine, and nicotina-mide (B6) have been completed. No study has prevented or delayed T1D development[98-104].

Clinical trials with drugs aimed at modulating the immune response and preserving endogenous insulin secretion in patients with new-onset T1D are termed tertiary prevention trials[51]. Only recently have these drugs expanded to prevention trials in islet auto-antibody positive individuals (Figure 3). The CTLA4-Ig antibody (Abatacept) for Prevention of Abnormal Glucose Tolerance and Diabetes in Relatives At-Risk (ClinicalTrials.gov Identifier: NCT 01773707) and Anti-CD3 monoclonal antibody (Teplizumab) for Prevention Of Diabetes In Relatives At Risk For T1D mellitus (ClinicalTrials.gov Identifier: NCT01030861) are both TrialNet studies currently enrolling participants. Abatacept is a fusion antibody that binds to antigen presenting cells and blocks co-stimulation to T cells. Anti-CD3 monoclonal antibodies bind the CD3 molecule which is present on CD8 and CD4 T cells, thereby inhibiting T cell activation[105]. Both of these drugs have shown some degree of success when used in new-onset trials[106-111]. DIAPREV-IT is an antigen-based treatment currently enrolling individuals who are positive for GAD and one or more additional autoantibodies (ClinicalTrials.gov Identifier: NCT 01122446). A GAD/alum vaccine


Figure 3 The measurement of serum islet autoantibodies has made type 1 diabetes a predictable disease. Early identification of islet autoantibody positive individuals leads to improved clinical outcomes by decreasing the risk for diabetic ketoacidosis and potentially preserving beta cell mass through clinical prevention trials. T1D: Type 1 diabetes; INIT-Ⅱ: Intranasal Insulin Trial.

Screen for isletautoantibodies

Enroll autoantibody positive individuals in secondary

prevention trials

Increased clinical monitoring in autoantibody positive individuals

Oral insulin for prevention of T1D

Anti-CD3 mAb (teplizumab) for prevention of diabetes in relatives

at risk for T1D mellitus

CTLA4-lg (abatacept) for prevention of T1D in relatives at-risk

INIT-Ⅱ

Decreased rate of diabetic ketoacidosisIncreased beta cell mass at diagnosis


is given at enrollment and 1 mo later. Although GAD vaccination is safe and easily administered, new-onset intervention trials have not shown long-term preservation of endogenous insulin secretion[108,112,113].

NOVEL APPROACHES TO PREVENT T1DCurrently, insulin is the only medication approved by the United States Food and Drug Administration for the treatment of T1D. Despite T1D being a predictable chronic autoimmune disorder, there are not any therapiesto preserve endogenous insulin production. As mentioned above, many large clinical intervention trials have not slowed the progression or prevented disease onset. We believe T1D will be preventable and that safe and specific therapies targeting the immune system are needed. One such approach is to target the trimolecular complex, which consists of a self-reactive CD4 T cell, insulin, and HLA molecule[114]. It is well established that specific HLA alleles, namely HLA DQ8 which is present in approximately 60% of all T1D patients, confer significant disease risk. DQ8 is a molecular target for diabetes intervention by using small “drug-like” molecules to block antigen presentation, thereby inhibiting specific T cell activation. Preclinical studies have shown this to be a potential pathway for diabetes intervention[115]. This concept has been advanced from bench to bedside as a clinical trial in which methyldopa (Aldomet), a clinically well-established antihypertensive drug, is being investigated to block DQ8 antigen presentation. The phase 1b dose escalation trial is using personalized medicine as methyldopa is being administered to recent onset adult T1D patients with the presence of the DQ8 gene (ClinicalTrials.gov Identifier: NCT01883804). Methyldopa is orally administered, safe as it has been used clinically for the last 50 years, and currently indicated for the treatment of pregnancy induced hypertension. Furthermore, all individuals have three class Ⅱ molecules (DQ, DR, and DP), and by blocking a single class Ⅱ molecule, there are two others to permit normal immune system function.

Other approaches have targeted components of the insulin trimolecular complex including antibodies that specifically bind to an insulin peptide in the HLA molecule. Preclinical studies in an animal model of spontaneous autoimmune diabetes indicate that this approach can delay diabetes onset[116]. Efforts are currently being made to make a human antibody, which again is a very specific immune therapy for diabetes intervention. Finally, insulin antigen specific therapy has the potential to evolve with recent advances in the field of immunology. A peptide from the insulin B chain amino acids 9-23 (B:9-23) has been extensively studied in animal models and human T1D[117,118]. It is now appreciated that insulin B:9-23 is a key autoantigen in the disease process of both mice and humans, sharing an identical amino acid sequence in both species[119,120]. A mutated insulin B:9-23 peptide, but not the native peptide sequence, induced protective immune responses (regulatory T cells) and prevented

diabetes onset in preclinical animal models[121]. With a deeper understanding of how the insulin peptide binds to HLA molecules and activates T cells, an insulin vaccine again holds promise for diabetes prevention.

In conclusion, T1D is now a predicable disease with the measurement of islet autoantibodies and prevention will naturally follow. To prevent T1D, general population screening for islet autoantibodies is needed along with a safe and specific therapy for disease intervention. The genes that confer diabetes risk are now molecular targets, and tailoring therapies to specific HLA genes is personalized medicine. The future holds promise for delaying the progression and ultimately preventing diabetes.

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P- Reviewer: Ciccone MM, Faienza MF, Haidara M, Ilangumaran S, Gray SG

S- Editor: Song XX L- Editor: A E- Editor: Liu SQ



deep pressure pain perception threshold at musculus abductor hallucis was beyond 1400 kPa (equivalent to 14 kg; limit of measurement) only in every fifth case. These discrepancies of pain perception between forefoot and hindfoot, and between skin and muscle, demand further study. Measuring nociception at the feet in diabetes opens promising clinical perspectives. A critical nociception threshold may be quantified (probably corresponding to a critical number of intraepidermal nerve fibre endings), beyond which the individual risk of a diabetic foot rises appreciably. Staging of diabetic neuropathy according to nociception thresholds at the feet is highly desirable as guidance to an individualised injury prevention strategy.

Key words: Foot ulcer; Neuroarthropathy; Insensitivity to pain; Pain perception; Diabetes mellitus; Amputation; Diabetic neuropathy


Core tip: The diabetic foot is characterised by painless ulcers and/or arthropathy. Although painless diabetic neuropathy is known as the underlying condition, little is known quantitatively about the pain evoked by noxious stimuli (nociception) at the diabetic foot. Preliminary evidence shows that pinprick pain perception threshold at plantar digital skinfolds is supranormal, beyond the upper limits of measurement. It is suggested that measuring nociception at the foot in diabetes could specify the individual risk of painless ulcers and/or arthropathy and, thereby, provide the basis of an individualised graded injury prevention strategy.

Chantelau EA. Nociception at the diabetic foot, an uncharted territory. World J Diabetes 2015; 6(3): 391-402 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/391.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.391

INTRODUCTIONThe diabetic foot (also called diabetic foot syndrome,

Ernst A Chantelau

Ernst A Chantelau, Diabetic Foot Clinic, Heinrich-Heine-University Düsseldorf, 40001 Düsseldorf, GermanyAuthor contributions: Chantelau EA solely contributed to this article.Conflict-of-interest: None.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Ernst A Chantelau, Professor, Diabetic Foot Clinic, Heinrich-Heine-University Düsseldorf, Moorenstraße 5, 40001 Düsseldorf, Germany. [email protected]: +49-421-637196 Received: July 23, 2014Peer-review started: July 25, 2014First decision: September 16, 2014Revised: September 18, 2014Accepted: January 18, 2015Article in press: January 20, 2015Published online: April 15, 2015

AbstractThe diabetic foot is characterised by painless foot ulceration and/or arthropathy; it is a typical complication of painless diabetic neuropathy. Neuropathy depletes the foot skin of intraepidermal nerve fibre endings of the afferent A-delta and C-fibres, which are mostly nociceptors and excitable by noxious stimuli only. However, some of them are cold or warm receptors whose functions in diabetic neuropathy have frequently been reported. Hence, it is well established by quantitative sensory testing that thermal detection thresholds at the foot skin increase during the course of painless diabetic neuropathy. Pain perception (nociception), by contrast, has rarely been studied. Recent pilot studies of pinprick pain at plantar digital skinfolds showed that the perception threshold was always above the upper limit of measurement of 512 mN (equivalent to 51.2 g) at the diabetic foot. However,

REVIEW


Nociception at the diabetic foot, an uncharted territory




or diabetic podopathy) is characterised by painless foot ulceration and/or arthropathy in diabetes. Foot injuries and inflammations do not hurt, as illustrated on Figure 1.

Painless diabetic neuropathy (PLDN) is the underlying condition. Skin ulcers extending to the subcutaneous tissues are completely anaesthetic, while arthropathy may display faint deep dull aching upon load bearing. Nociception, that is the perception of pain originating from neural processes of encoding and processing noxious stimuli, is failing at the diabetic foot. Although insensitivity to pain at the diabetic foot is common knowledge, details are largely unknown[1,2].

Research into painlessness in diabetes is scarce. Early studies date back to Pamela Margaret Le Quesne[3] and her group almost 30 years ago. They tried to measure the pain perception (nociception) at the diabetic foot by pinching the skin with a custom made “pinchometer”. The results were inconclusive at best[4]. Other authors designed calibrated tools for assessing hypersensitivity of so-called symptomatic, i.e., painful, diabetic neuropathy (SDN, see below). To this end, pinprick pain perception, axon-reflex reaction and temperature detection of the skin were studied. These modalities represent the functions of the afferent A-delta and C-fibres (so-called small fibres; see below) whose contributions to SDN, however, remain controversial[5,6]. SDN will not be discussed here.

To date, measuring pain perception (i.e., noci-ception) at the diabetic foot was deemed futile with the excuse that “The threshold of sensation that protects normal feet from injury is difficult to define…it is extremely difficult to define a ‘significant loss of sensation’, or at what level sensory loss becomes ‘critical’..”[7]. However, there is some dissent on this issue, as expressed by Dyck et al[8]: “Sadly, the clinical assessment of decreased sensation by physicians is generally inadequate because it is not performed or is performed badly.” Undeniably, many a clinical physician or expert was biased against measuring diminished nociception in diabetic neuropathy, and for several possible reasons: (1) quantitative sensory testing (QST, see below) was considered inferior to nerve conduction velocity studies in the detection of symptomatic (painful) diabetic neuropathy; (2) Experts of QST not always knew enough of the diabetic foot, this typical complication of symmetrical diabetic neuropathy, to understand what they read. For example those reviewers, who commented on a paper: “In a group of 20 patients (...) the authors compared sensation in ulcerated vs nonulcerated feet and were unable to find any difference. Had a difference been found, one might interpret the sensory dysfunction as a pathophysiological factor in ulcer development”[9]; (3) QST findings were assumed to be poorly reproducible, an argument which was often mixed up with the natural inter-individual variability (on average 5-fold[10]). Intra-individual coefficients of variation are acceptable, for example those of pain perception thresholds are well

below 25%[10-14]; and (4) A really important obstacle against routine QST of nociceptive functions was the lack of normative data. However, since 2005, extensive reference material using a particular QST protocol was published[12,14-17], see below. Although this protocol had primarily been devised for examining hyperalgesia in non-nociceptive pain syndromes like fibromyalgia, or neuropathic pains, and not for assessing hypoalgesia[18], the author (Chantelau EA) believes that parts of it may well be applied for studying the failing nociception at the foot in diabetes. Diabetic hyperglycaemia in general does not interfere with QST[19-21]; however, subclinical or clinical hypoglycaemia makes QST virtually impossible.

PLDN: THE PRINCIPAL RISK FACTOR FOR THE DIABETIC FOOTPeripheral diabetic (poly) neuropathy is the most frequent sequel of diabetes mellitus. The nature of diabetic neuropathy has been clearly established by Martin[22,23] in 1953 and 1954, and by Catterall et al[24] in 1956. While today many people still believe that the condition is always symptomatic with spontaneous pains and dysaesthesias in the legs and feet, these authors had already noted more than half a century ago: “non-myelinated nerve-fibre degeneration occurs in a high proportion of diabetic patients, who, on clinical examination, show no evidence of ‘clinical’ diabetic neuropathy…the presence in diabetics of such nerve-fibre disturbance explains the frequent finding of persistently cold feet and their proneness to traumatic lesions”[24]. Indeed, the basic feature of diabetic neuropathy is small fibre degeneration[25] with reduced or absent nociception at the feet as the key sign (PLDN). By contrast, SDN develops secondary to PLDN, superimposes on PLDN, and affects only a minority of 15%-30% of patients[25,26]. The symptoms of SDN like spontaneous neuropathic pains and tingling in the feet may be alleviated by drug treatment; they resolve gradually while nerve degeneration[27] and PLDN progress. However, diminished nociception in PLDN does

Chantelau EA. Nociception at the diabetic foot


Figure 1 Sketch reproduced from Brand P[2].

Pain makes a healthy man fall when he begins to twist his foot.The man who feels no pain walks on without realising the damage he is doing.

not resolve, nor does PLDN respond to drug treatment. Prevalence and incidence rates of PLDN are not known. However, the prevalence of the diabetic foot, indicative of severe, end-stage PLDN, in patients with SDN is about 10%, while the incidence is about 5%-7% per year[28,29].

Most of the patients with PLDN never have complaints until they are facing a foot injury that -unexpectedly - does not hurt. To once more quote Catterall et al[24]: “Cutaneous neuropathic changes commonly start as blisters about the tips of the toes or at the site of a corn or callosity in places constantly exposed to irritation by an ill-fitting shoe. Frequently the deceptive lack of normal sensation, and particular pain sensation, leads a patient to ignore the lesion and delay treatment until secondary infection with deep penetration of the tissues or severe inflammation is present.” PLDN is associated to the duration of diabetes and to the cumulative effects of increased blood glucose (or deficient insulin function). It is symmetrical, age- and length-dependent (it starts in advanced age by affecting the endings of the longest axons in the body, in the skin of the toes[30]). Thus, deficits in nociceptive function develop in the forefeet first, and subsequently ascend to the rearfeet, the ankles and the lower legs. The hands are affected only in most severe cases.

PLDN is a disease of the small primary afferent A-delta and C-fibres: they are thin and their conduction velocity is relatively slow. (A fraction of about 15% of small fibres extending to the skin are efferents; they belong to the sympathetic nervous system and are restricted to the dermis, innervating sweat glands, blood vessels, and the arrector pili muscles. Small fibre efferents will not be discussed here). Primary C-afferents (also named group Ⅳ fibres, axon diameter < 1.5 μm, conduction velocity 0.5-2 m/s) are unmyelinated nerve fibres. Primary A-delta afferents (also named group Ⅲ fibres, axon diameter 1-6 μm, conduction velocity 5-30 m/s) consist of thinly myelinated axons whose intracutaneous endings, however, are unmyelinated[31]. The endings of A-delta and C-afferents do not carry corpuscles but are “free”; they serve as receptors for noxious thermal,

mechanical and chemical stimuli (termed nociceptors), and as receptors for innocuous thermal stimuli. In the epidermis they are densely distributed, up to 1000 per 1 cm², which is much more than in muscles, ligaments, joints and bones (periosteum). Most nociceptors are specific of one single modality, while some are polymodal. C-fibre afferents are estimated to account for 70% of all nociceptors in the skin (the remainder are A-delta nociceptors). Cutaneous A-delta nociceptor stimulation results in a short sharp, stinging “first” pain, whereas C-fibre nociceptor stimulation results in prolonged dull, burning “second” pain. In muscles, A-delta and C-fibre nociceptors evoke the same dull pain character[32], see section 4.

In PLDN, the nociceptors degenerate progressively (“die back”) by unknown molecular mechanisms. It is not clear, whether this insidious process of central-peripheral distal axonopathy[33,34] begins already in a prediabetic stage[25]. However, PLDN superimposes on the physiological age-related decline in sensory functions after the age of 50[34-36], and is aggravated by other neuropathic conditions like vitamin B12 deficiency, alcohol toxicity, end-stage renal failure, or exposition to neurotoxic substances or conditions. Clinically, PLDN remains undetected until most nociceptors have died and painlessness of a foot trauma astounds the patient (and the doctor)[24]. The vanishing of intraepidermal free nerve endings can be quantified by histomor-phology[31,37,38]; it correlates to increasing severity of diabetic neuropathy[39-41] (Figure 2), and to rising thermal and pain perception thresholds[34,38,40].

A-delta and C- fibre nociceptors and thermal receptors “die back” not only in the skin, but presumably also inside the adjacent subcutaneous tissues (equivalent to group Ⅲ and Ⅳ fibre nociceptors in muscles, fascia, ligaments and joints) of the diabetic foot. However, this remains to be established by histopathology. A-delta (group Ⅲ) fibres seem to be particularly susceptible to the neuropathic processes, as their functions deteriorate somewhat earlier than C- (group Ⅳ) fibre functions[42].

Diabetes affects small fibres prior to large ones. When large myelinated A-beta afferents (axon diameter 6-12 μmol/L, conduction velocity 30-70 m/s) become affected, the axon and the myelin sheet get damaged, and conduction velocity decreases. A-beta fibres mediate touch, deep pressure and vibration sensation by means of their corpuscular endings: Meissner’s, Merkel’s, and Pacinian corpuscles. A-beta fibres are not involved in nociception, but have a role in allodynia[43] which, however, will not be discussed here.

Diabetic foot: End-stage complication of PLDN The diabetic foot is characterised by painless ulceration and/or arthropathy. Since 60 years or so[22,23,24,44] the condition is well known as an end-stage complication of diabetic neuropathy, of PLDN rather than SDN. The basic defect has frequently been termed “loss of protective sensation”[44], which is, in fact, “loss of


12

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Figure 2 Intraepidermal nerve fibre ending density at the lower limb in relation to severity of diabetic neuropathy (adapted from Quattrini et al[40,41]). HC: Healthy controls; DC: Diabetic controls; Dmi: Mild SDN; Dmo: Moderate SDN; Dsev: Severe SDN; SDN: Symptomatic diabetic neuropathy; INEF: Intraepidermal nerve fibre ending.


needs to be ascertained”[48]. However, measuring loss of nociception (LON) as yet has no place in assessing neuropathy in diabetes[47].

Current clinical practice of diagnosing a diabetic foot/diabetic podopathyLoss of touch or vibration sensation is not diagnostic of an active diabetic foot, while painlessness of a foot injury (soft tissue or skeletal lesion) or infection in a patient with diabetes mellitus is. Three different clinical pictures of active diabetic foot lesions may be discerned, according to the dominant clinical component: (1) septic; (2) ischaemic; and (3) arthropathic. After healing of such a lesion, the foot is an inactive diabetic foot (with scars, deformities, etc.) and remains a locus minoris resistentiae for the rest of the life. LON traditionally is not assessed systematically, but is taken as matter of fact when a patient with an injured foot does not limp. The patient history of traumatic events is often unproductive as far as normal symptomatology (pain!) is concerned. However, when concurrent symptoms of trauma onset other than pain are concerned, like swelling and erythema, patient history may be very well productive.

QST The QST protocol, published by the German Research Network on Neuropathic Pain and supported by pharmaceutical companies, was designed for non-invasively diagnosing pain syndromes like spontaneous neuropathic pains, fibromyalgia or chronic lower back pain[15,16,18]. It comprises 13 somatosensory sensory tests that measure the function of large (A-beta) and small (A-delta and C-) afferent nerve fibres, and the corresponding functions of the spinothalamic tract (cold detection threshold, warm detection threshold, thermal sensory limen, paradoxical heat sensation, cold pain threshold, heat pain threshold, mechanical detection threshold, mechanical pain threshold, mechanical pain sensitivity, dynamic mechanical allodynia, wind-up ratio, vibration detection threshold, and deep pressure pain threshold). Using this protocol in healthy subjects, a pool of normative reference data from face, hand and foot had been generated[16,17].

QST of pain perception in diabetesOf all sensory modalities that are impaired by diabetes mellitus, the nociceptive pain system is the most relevant. In the following sections, the focus will therefore be directed on pain perception thresholds: elevated thresholds indicate reduced perception of noxious stimuli, and vice versa. Pain tolerance or other sensory modalities (vibration, electrical current, chemicals, touch, thermal perception) will not be addressed. Some technical features of the threshold studies considered here are outlined below, concerning thermal (cutaneous heat and cold), and mechanical (cutaneous and deep pressure)

nociception” (see below). Ulcers and arthropathy start from single injuries (mechanical, thermal or chemical skin wound, or skeletal trauma) which are subjected to persisting repetitive stress (load bearing by walking, because they do not hurt - Figure 1), whereby they enlarge enormously until they become apparent to the patient. The initial injury may also be caused by uninterrupted repetitive mechanical wear and tear (fatigue from overuse, remaining unperceived because of lack of nociception!) The ulcers become infected, and the infection spreads to the adjacent subcutaneous tissues. Bone and joint damage aggravates due to ongoing unprotected walking. When properly treated immediately (like any other acute foot injury in subjects with preserved nociception!), the initial injuries will heal uneventfully. Human experiments showed that wound healing is not impaired by PLDN[45,46]. Perforating neuropathic foot ulcers heal if they are unloaded completely[24], and if arterial blood flow is sufficient and infection is cured. The ulcers frequently break down again when repetitive stress from walking is resumed, “due to faulty and often excessive weight-bearing by the ulcerated area”[23]. Hence, the overall amputation risk is increased: the diabetic foot is the most frequent cause of amputation in industrialised countries.

CURRENT CLINICAL PRACTICE OF DIAGNOSING DIABETIC NEUROPATHYAccording to the common misconception that neur-opathy in diabetes in general is SDN, current guidelines state that the condition be diagnosed and staged semi-quantitatively according to various symptom scores (e.g., Neuropathy Symptoms Score, Neuropathy Disability Score, Michigan Neuropathy Screening Instrument, Toronto Clinical Scoring System). However, testing sensory functions is also accepted: five simple nerve tests are considered diagnostic for symptomatic neuropathy, the 10-g monofilament plus 1 of the following 4: vibration using 128-Hz tuning fork, pinprick sensation, ankle reflexes, vibration perception threshold[47]. All except vibration perception threshold are qualitative tests, all but pinprick sensation are unsuitable for diagnosing PLDN.

Vibration and touch represent A-beta fibre functions, not nociceptive functions. Nevertheless, vibration and touch sensation is used as surrogate markers of the risk of sustaining painless foot injuries (ulceration/arthropathy). To identify loss of protective sensation (LOPS) - without assessing nociception- “any of the five tests listed could be used (…). One or more abnormal tests would suggest LOPS, while at least two normal tests (and no abnormal test) would rule out LOPS”[47].

Current guidelines claim that “estimating the severity of diabetic sensorimotor polyneuropathy has not received the attention it deserves. For a given patient with diabetes it is not sufficient to simply identify patients as having sensorimotor polyneuropathy - severity also



pain perception.

Cutaneous thermal pain perception: Thermal pain perception threshold is measured by applying warm or cold stimuli to the skin, generated by appropriate thermodes (Figure 3). The temperature of the thermode is gradually increased or decreased. The subject under study is asked to indicate whether a thermal sensation becomes painful. Heat pain represents C-fibre function, and cold pain represents A-delta fibre function. In healthy subjects, heat pain perception threshold may range from approximately 41 ℃ to approximately 47 ℃ (average approximately 44 ℃), and cold pain perception threshold ranges from approximately 1 ℃ to approximately 30 ℃ (average approximately 12 ℃).

Deep pressure pain perception: Deep pressure pain perception threshold (DPPPT) may be examined, for instance, by a hand held device (algometer, Figure 4) containing a mechanical or electronic force gauge, and a plunger with a flat blunt rubber tip. The diameter of the tip can be 11 mm (surface approximately 1 cm²), or lager, as appropriate. The plunger is pressed perpendicularly onto the skin and the underlying structures, with slowly increasing stimulus ramp (50 kPa/s, equivalent to 0.5 kg/s). The subject under study communicates when his pressure sensation turns to pain.

The deep pressure pain sensation evoked by an algometer at muscle or joint is as yet not fully under-stood. For instance, the afferents involved (high and low threshold mechanosensitive units[32]), the anatomical structures subjected to algometer pressure, and the role of spatial summation (pressure area 1 cm²)[49,50], still have to be elucidated. The contribution of fascia, which is more pain-sensitive than muscle, and of periosteum, which is more pain-sensitive than bone marrow, remains to be determined. The pain quality at reaching DPPPT is dull and burning and probably more of a pressure discomfort than of a stinging or pricking[49,50]. Inside deep tissues, stimulation of the few A-delta (group Ⅲ) nociceptors simultaneously probably does not elicit a separate pricking pain sensation like a single punctuate stimulation at the skin does[49,51]. DPPPT decreases at chronically ischaemic muscle[52,53]. DPPPT at muscle (blunt stimulation) decreases with age, at variance to cutaneous pressure pain perception threshold (CPPPT, punctuate stimulation)[42]. Using a pressure algometer with 1 cm² contact area to stimulate musculus abductor hallucis, DPPPT in healthy subjects may range from approximately 200 kPa to approximately 1000 kPa (average approximately 500 kPa, equivalent to 5 kg). Algometer stimulation of a joint evokes a DPPPT of the same range.

Cutaneous pressure pain perception: Cutaneous pressure pain perception threshold (CPPPT) is examined by the use of calibrated pinprick stimulators with a sharp edge, comparable to von Frey hairs. These punctuate stimulators are filaments of 50-350 μm diameter, with calibrated bending forces (or weight loads) over a range from 8 mN to 512 mN (equivalent to 0.8 g to 51.2 g) (Figure 5).

The investigator presses each single stimulator perpendicular to the skin (until bending of the filament) and the subject under study communicates, whether a painless touch is felt, or a painful (pricking or stinging) sensation, or nothing at all. The pain threshold is determined by the method of limits, or by the forced-choice-method. The stimulators excite single nociceptors representing A-delta (rather than C-fibre) functions; the


Figure 3 MSA Thermotest® (electronic device, expensive!). Figure 4 Algometer® (electronic device, expensive!).

Figure 5 MARSTOCKnervtest® PinPrick stimulator 512 mN (fibreglass, cheap!).


pain quality evoked is that of a stinging, pricking “first” pain. CPPPT increases with age (at variance to DPPPT, see above). At the non-callous plantar skin of healthy subjects, CPPPT may range from approximately 4 mN to approximately 450 mN (average approximately 100 mN, equivalent to 10 g).

Limitations of pain threshold measurements: QST is a subjective psychophysical method, as it is based on the patient report. “A critical element in pain threshold determination is the particular sensory experience an individual considers painful. This factor will be influenced by, among other things, the subject’s pain experience history, and the instructions given by the experimenter. For instance, thresholds are likely to be different if a subject is instructed to indicate when he perceives ‘pain’ vs when he perceives ‘a sharp or burning sensation’ or ‘an uncomfortable situation’….”[54] In particular, pressure pain measurement is affected by the hardness of the skin at the site of measurement, and by the ramp rate of increasing the stimulus of hand-held algometers, amongst others.

QST of nociception at the diabetic foot Early studies of pain perception thresholds: Between 1988 and 2011, not a single study could be identified on cold pain perception threshold, or on DPPPT. However, there are at least 4 studies on CPPPT and/or heat pain perception threshold[29,55-57], all of which found significantly elevated thresholds at the active (?) diabetic foot and the contralateral foot, as compared to neuropathic patients without diabetic foot (Table 1). In neuropathic patients with and without diabetic foot, pain perception thresholds were significantly higher than in non-neuropathic (healthy or diabetic) control subjects. Lang et al[52] reported that perception thresholds for cold pain, heat pain and pinprick were elevated at legs with chronic severe foot ischaemia (rest pain with/without tissue loss); the 16 patients (8 of whom had diabetes mellitus) had polyneuropathy according to a vibration perception threshold < 4/8 (graduated tuning fork). DPPPT at ischaemic muscle, however, was lower than at healthy controls’ muscle (199 kPa vs 295 kPa[52]). In another study of non-neuropathic patients with chronic leg ischaemia, CPPPT, and DPPPT at muscle, were lower

than in control subjects but cutaneous cold and heat pain perception thresholds were not[53].

Recent pilot studies of pressure pain perception thresholds: Between 2012 and 2014, we conducted four studies on the matter[58-61] including explorative cross-sectional and follow-up studies. Our overall database comprised 123 subjects in whom pressure pain perception thresholds were measured: 43 control subjects, 23 of whom had an acute trauma of the foot skeleton (sprain, minor fracture), 59 diabetic patients with PLDN (46 of whom with a diabetic foot), and 21 diabetic patients without PLDN. Among the 46 patients with a diabetic foot, there were 33 with active painless foot ulcer, 13 with healed foot ulcer, 11 with healed Charcot foot (grade 1, inactive[62]), and 13 with acute skeletal trauma (elective surgery). CPPPT and DPPPT were studied, as well as vibration perception threshold (not reported here). According to our study protocol, CPPPT was measured only at the forefoot (plantar digital skinfold), while DPPPT was measured at the hindfoot (m. abductor hallucis) and at the forefoot (metatarsophalangeal joint) (Figure 6).

By cross-sectional comparison, CPPPT at a plantar digital skinfold (glabrous skin) was above the safety limit of measurement of 512 mN (approximately 51.2 g) in 98% of cases with a past or present painless foot ulcer or healed arthropathy (Charcot foot). This is in line with the older reports cited above. Simultaneously, DPPPT at abductor hallucis muscle was above the safety limit of measurement (1400 kPa, equivalent to 14 kg) only in about 20% of cases. The DPPPT at metatarsaophalangeal joint was above 1400 kPa in about 50% of cases (Table 2).

Serial pressure nociception studies to monitor pain thresholds after an acute foot trauma (ankle sprain, toe fracture) in otherwise healthy subjects revealed that CPPPT and DPPPT at the site of the trauma decreased slightly, indicating normal, inflammation-mediated posttraumatic hyperalgesia (Figure 7). This observation is consistent with previous reports (Martinez et al[63]:


Pinprick stimulator CPPPT

Algometer DPPPT

Algometer DPPPT

Figure 6 Pressure pain perception threshold measurements done in own studies[58-61]. CPPPT: Cutaneous pressure pain perception threshold; DPPPT: Deep pressure pain perception threshold.

Table 1 Early studies of cutaneous pain perception thresholds (pressure, heat) at the diabetic foot, using various, non-standardised methods[29,55,56,57]

Test Diabetic subjects under study Healthy Ref.

Diabetic foot Neuropathy controlsCPPPT 1001 100 0 [29]CPPPT 1001 631 - [55]CPPPT 68 30 2 [56]Heat 1001 100 0 [29]Heat 1001 100 0 [57]

Percentages of supranormal results are shown. 1Above upper limit of measurement. CPPPT: Cutaneous pressure pain perception threshold.


decrements in CPPPT on day 1-4 at an operated knee; Lang et al[53]: reduction in CPPPT and DPPPT in a chronic ischaemic leg up to 3 mo after successful endovascular revascularisation, Dominguez et al[64]: reduced DPPPT at the foot immediately after a sprain). This physiologic, inflammation-mediated posttraumatic hyperalgesia is the essential trigger of avoidance behaviour to preserve the injured limb from further noxious and innocuous impacts.

By contrast, at the diabetic foot CPPPT was extremely elevated (above the safety limit of measurement) prior to an acute foot trauma (elective surgery), and also immediately thereafter (Figure 7A); there was no posttraumatic decrement of CPPPT at the site of the trauma[61]. This suggests absence of A-delta “first” pain quality and inflammation-mediated hyperalgesia. DPPPT at the diabetic foot, being elevated prior to the surgery, showed a small posttraumatic decrement from this baseline level (Figure 7B and C) which, however, was not accompanied by clinical signs of hyperalgesia, or avoidance reaction, respectively.

These data, however, have to be interpreted with caution since our study groups were small (only 12 and 13 subjects, respectively) and heterogeneous. Furthermore, it cannot be ruled out that our study subjects may have behaved like those young healthy women, who reacted to consecutive daily measurements of DPPPT over 4 d by increased pain sensitivity[65] - a reaction that may be an exception rather than the rule[13].

CPPPT within the normal reference range may decrease slightly upon re-testing[14] but remains consistently above the safety limit of measurement in patients with diabetic foot[58-61].

Miscellaneous studies of A-delta and C-fibre sensory functions at the diabetic foot: At least 7 studies[66-72] could be ascertained, all of which reporting that the axon-reflex reaction is not much smaller in neuropathic patients with a diabetic foot than in those without. Axon-reflex reaction in the feet of neuropathic patients was consistently smaller than in the feet of non-neuropathic control subjects.

At least 11 studies[57,67,73-81] could be ascertained, all of which reporting that neuropathic patients with a diabetic foot have higher thermal detection thresholds than neuropathic patients without. Thresholds were higher in the feet of neuropathic patients than of control subjects, and were particularly high in patients with more severe, relapsing diabetic podopathy and/or Charcot foot[76-79]. One study[79] showed a lower cooling detection threshold in the active or inactive Charcot foot vs the contralateral foot, while the warming detection threshold was similar on both feet.

CONCLUSIONAt the diabetic foot, cutaneous thermal and mechanical (pinprick) nociception is reduced dramatically. CPPPT


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Figure 7 Lowered pain perception thresholds (hyperalgesia) after acute skeletal trauma of the foot in 13 healthy control subjects (sprain, toe fracture) and in 12 diabetic foot patients (elective surgery) at the injured site. Data are combined from ref. [59,61]. A: Cutaneous pressure pain perception threshold (mN) (pinprick) at plantar digital skinfold (medians); B: Deep pressure pain perception threshold (kPa) over metatarsophalangeal joint (medians); C: Deep pressure pain perception threshold (kPa) over musculus abductor hallucis (medians).

A B C

Table 2 Simultaneous measurements of pressure pain perception thresholds at the foot in subjects with and without painless diabetic neuropathy, with and without diabetic foot

Subject populations Diabetic foot1 Healthy2 PLDN3 DM-controls4

No. of subjects 46 43 13 21% of subjects with CPPPT > 512 mN at toe skinfold 98% 4% 46% 5%% of subjects with DPPPT > 1400 kPa at m. abductor hallucis 22% 0% 0% 0%% of subjects with DPPPT > 1400 kPa at metatarsophalangeal joint 50% 2% 0% 5%

1With previous or active ulcer/arthropathy; 2Non-neuropathic, healthy; 3PLDN, no ulcer; 4Diabetes, no neuropathy, no ulcer. Percent subjects with thresholds above the safety limit of measurement are shown. Adapted from ref. [58-60]. CPPPT: Cutaneous pressure pain perception threshold (512 mN upper limit of measurement); DPPPT: Deep pressure pain perception threshold (1400 kPa upper limit of measurement).


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was elevated beyond the safety limit of measurement in most of the small number of published studies. Patients with diabetic foot ulcers unanimously display a CPPPT at a digital plantar skinfold which is beyond the safety limit of 512 mN. However, details of the causal role of increased CPPPT (indicating lack of A-delta nociception) for the manifestation of diabetic podopathy remain to be established, as well as the anatomical distribution of PLDN at the foot. Concerning DPPPT at the diabetic foot, the data is less clear, as differences to control subjects often were surprisingly small. Compared to baseline DPPPT, inflammation-mediated posttraumatic DPPPT was reduced at the diabetic foot, albeit at a high level and far from indicating hyperalgesia. Pending confirmation, this may suggest that DPPPT probably does not need to be extremely elevated for a diabetic foot to develop.

There are many more open questions, for instance: what number of residual intraepidermal free nerve fibre endings corresponds to the elevation of CPPPT at the diabetic foot? What elevation of CPPPT is necessary to appreciably increase a patient’s risk of sustaining painless foot ulceration and/or arthropathy? How much pain threshold elevation, i.e., how much “LON”, is clinically meaningful? Is the nociception at healthy tissues relevant, or is it the pain perception (hyperalgesia, allodynia), that is typically caused by stimulation of inflamed tissue? Is superficial and deep tissue nociception similarly impaired by PLDN? How much are “silent” nociceptors[82] affected? Is the stinging “first” pain more reduced than the burning “second” pain? Is there a defect in posttraumatic ongoing (non-stimulated) pain? Is absolute or relative reduction of individual pain perception essential for the diabetic foot to develop? Is pain perception or pain tolerance more relevant? Do abnormalities of the central nervous system contribute to the painlessness of the diabetic foot? To what extent is sensitization, either peripheral or central, affected at the diabetic foot? Concerning the discrepancies between CPPPT (at the forefoot) and DPPPT (at muscle of hindfoot) in our own studies[58-61] - does the distal-to-proximal gradient of PLDN provide an explanation?

Measuring nociception at the diabetics' feet-what is it good for?LON is the principal pathogenic component of diabetic podopathy, as it is the principal functional component

of PLDN. Loss of pressure perception does not increase the risk of diabetic foot ulceration, whereas a history of foot ulceration (equivalent to cutaneous LON) does[83]. Measuring nociception provides the opportunity for a clinically meaningful staging of the severity of PLDN. A critical LON needs to be elaborated (probably corresponding to a critical number of intraepidermal nerve fibre endings), “to be able to inform the patient adequately about the risk of ulcer formation and to prescribe preventative measures… otherwise, sensation loss and resultant ulcers may lead to amputation”, as Assal et al[84] advocated 25 years ago. Perhaps every patient may have his/her own individual critical LON to be taken into account. Small reductions in intraepidermal nerve fibre density may be mirrored by respective increases in CPPPT, as recent work by Selim et al[85] shows. Hence, measuring CPPPT prospectively may be suitable for monitoring progression of PLDN.

Shifting the emphasis from amputation prevention by wound healing to amputation prevention by injury prevention: Once an active diabetic foot/podo-pathy has become manifest, a multitude of cumbersome, laborious, tedious, expensive and costly therapeutic interventions is necessary to save the foot and prevent an amputation. Injury prevention in high-risk PLDN - to prevent the diabetic foot- is certainly much more efficient. Preventing the first or recurrent ulcers[86] should be no question. Having had a first ulcer strongly predisposes to subsequent ones, most likely due to comorbidities from structural abnormalities that resulted from the (healed) first ulcer (scars, deformities, contractures, tissue loss). The same holds true for arthropathy. Hence, preventing the first ulcer/arthropathy must have absolute priority. To this end, intensive prophylactic measures are necessary in high-risk patients who should be identifiable by simple means. As a proposal, the following tentative risk-stratification is construed from various published data (assuming a “normal” average walking activity of 5000 strides per day [87]); (Table 3).

Subjects with critical LON at their feet require signals other than foot pain to control foot usage and avoid over-usage. These signals can only come from technical devices, like step counters or load monitors incorporated into footwear, that send acoustic or optical alarms when a critical load (or an equivalent number of steps) has accumulated. Then, the feet have to be rested - for an


Table 3 Percentages of subjects with active diabetic podopathy (ulcer, arthropathy) according to loss of nociception

Foot morbidity Degree of loss of nociception Ref.

None Mild/moderate Severe(CPPPT1 approximately 100 mN) (CPPPT1 < 512 mN) (CPPPT1 > 512 mN) [59]

Past/present painless ulcer 0% 0% 100% [28,59,77,86-101]Foot ulcer, incidence per year Approximately 0%-1% Approximately 6%-15% Approximately 20%-100%2 [28,77,86-101]Charcot-foot, prevalence Approximately 0% Approximately 0.05% Approximately 2% [102]

1Cutaneous pressure pain perception threshold, pinprick stimulator, upper limit of measurement 512 mN, measured at a plantar digital skinfold; 2Depending on compliance with prophylactic podiatry and/or special footwear. CPPPT: Cutaneous pressure pain perception threshold.


appropriate period of time as physiologically required to regenerate the stressed tissues - before their usage (walking) may be resumed. Moreover, feet with critical LON need special footwear to avoid rubbing of the skin while walking in order to prevent skin abrasion and blister formation.

Pain is a homeostatic emotion that drives behaviour, just as hunger and thirst are. Patients who cannot have it, either from acquired or inherited insensitivity to pain[103,104], either in the feet or elsewhere in the body, need our special protection, and much more specific than we, their caregivers, may have been willing to concede in the past. Research into nociception at the foot in diabetes must continue and expand. Chances to improve injury prevention will be missed, if this uncharted territory remains unexplored.

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P- Reviewer: Cui WP, Trohman RG S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ



Francesco Fallucca, Lucia Fontana, Sara Fallucca, Mario Pianesi

Francesco Fallucca, “In Unam Sapientiam” Foundation, University La Sapienza, 00161 Rome, Italy Francesco Fallucca, Mario Pianesi, International Study Center for Environment, Agriculture, Food, Health and Economics, 00161 Rome, ItalyLucia Fontana, Unit of Dietology and Diabetology, Sandro Pertini Hospital, 00157 Rome, ItalySara Fallucca, Department of Endocrinology and Diabetes, University Campus Bio-Medico, 00128 Rome, ItalyAuthor contributions: Fallucca F and Pianesi M conceived and designed the manuscript; Fallucca F, Fontana L and Fallucca S searched the literature and selected relevant studies; Fallucca F and Fontana L drafted the manuscript; Fallucca S and Pianesi M critically reviewed the manuscript drafts; all authors read and approved the final manuscript.Conflict-of-interest: The authors declare that they have no conflicts of interest.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Francesco Fallucca, Professor, “In Unam Sapientiam” Foundation, University La Sapienza, Largo Ettore Marchiafava 1, 00161 Rome, Italy. [email protected]: +39-06-8605616Received: August 28, 2014 Peer-review started: August 30, 2014 First decision: December 17, 2014Revised: January 10, 2015Accepted: January 30, 2015Article in press: February 2, 2015Published online: April 15, 2015

AbstractIn the past 10 years the prevalence of type 2 diabetes mellitus (T2DM) has increased hugely worldwide, driven by

a rise in the numbers of overweight and obese individuals. A number of diets have been shown to be effective for the management of T2DM: the Mediterranean diet, the vegetarian diet and the low-calorie diet. Results of studies clearly indicate, however, that the efficacy of these diets is not solely related to the biochemical structure of the individual nutrients they contain. This review discusses this point with reference to the potential role of the intestinal microbiota in diabetes. The macrobiotic Ma-Pi 2 diet is rich in carbohydrates, whole grains and vegetables, with no animal fat or protein or added sugar. In short- and medium-term trials conducted in patients with T2DM, the Ma-Pi 2 diet has been found to significantly improve indicators of metabolic control, including fasting blood glucose, glycosylated hemoglobin, the serum lipid profile, body mass index, body weight and blood pressure. The diet may also alter the gut microbiota composition, which could additionally affect glycemic control. As a result, the Ma-Pi 2 diet could be considered a valid additional short- to medium-term treatment for T2DM.

Key words: Ma-Pi 2 macrobiotic diet; Type 2 diabetes; Low-grade inflammation; Gut microbiota; Metabolic control


Core tip: Imbalances in the intestinal microbiota (dysbiosis) have been linked to diseases, including diabetes. In short- and medium-term trials conducted in patients with type 2 diabetes mellitus (T2DM), the Ma-Pi 2 diet, which is rich in carbohydrates, whole grains and vegetables, with no animal fat or protein or added sugar, has been found to significantly improve indicators of metabolic control, including fasting blood glucose, glycosylated hemoglobin, the serum lipid profile, body mass index, body weight and blood pressure. The diet may also alter the gut microbiota composition. Hence, the Ma-Pi 2 diet could be considered a valid additional short- to medium-term treatment for T2DM.

REVIEW


Gut microbiota and Ma-Pi 2 macrobiotic diet in the treatment of type 2 diabetes




Fallucca F, Fontana L, Fallucca S, Pianesi M. Gut microbiota and Ma-Pi 2 macrobiotic diet in the treatment of type 2 diabetes. World J Diabetes 2015; 6(3): 403-411 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/403.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.403

INTRODUCTIONIn the past 10 years, the prevalence of type 2 diabetes mellitus (T2DM) in Italy has increased greatly; currently, approximately 3.3 million people are diagnosed with T2DM (5.5% of the total population) and another million people are thought to be affected but as yet undiagnosed. These numbers are expected to increase in the near future, and it has been estimated that 9.0% of the population will have T2DM by 2030[1]. Similar increases are anticipated in other European countries and, globally, the number of people with diabetes is predicted to reach 592 million by the year 2035 - an increase of 55% from 2013 data[2].

This increase in the prevalence of T2DM, which accounts for approximately 90% of all diabetes cases, is clearly being driven by an increase in the numbers of individuals who are overweight or obese[3]. Indeed, the risk of diabetes appears to literally soar as the weight piles on; a rise in body mass index (BMI) from 21 kg/m2 (healthy) to 35 kg/m2 (obese) can increase the likelihood of developing the disease by a factor of 80[4]. In line with the increase in prevalence of diabetes, the number of obese adults, together with the rates of obesity-related diseases (e.g., coronary heart disease and stroke, hypertension and arthritis) and associated healthcare costs, are all expected to increase dramatically worldwide over the next 20 years[4,5].

T2DM and obesity, and their associated com-plications and costs, are therefore among the most pressing healthcare problems that society is currently facing. Despite improvements in our understanding of T2DM and the development of new diagnostic and treatment methods, we are still failing to control this epidemic. There is a crucial need to find effective, simple, pragmatic, sustainable and cheap interventions (e.g., those aimed at changing lifestyle, such as exercise and diet).

Recent reviews highlight the benefits of physical exercise in T2DM[6]. In Italy, the Italian Diabetes and Exercise Study[7-10] has contributed to this evidence and has helped provide definitions and standards used by the American College of Sports and Medicine and the American Diabetes Association (ADA). Yet, despite the accepted benefits of physical exercise for patients with T2DM, the implementation of exercise recommendations has proved difficult. Reasons for this include a lack of patient compliance, insufficient knowledge/awareness of benefits among general practitioners, diabetologists or exercise professionals, and a lack of dedicated facilities[11]. In addition, in order to be successful, exercise interventions in individuals

with T2DM tend to require intensive counseling and supervision from medical or exercise professionals.

As a result, recent attention has focused less on exercise and more on diet; dietary therapy (as a life-style intervention), with or without additional drug treatment, represents an efficacious and safe alternative for T2DM management[12]. Various cohort studies have shown that selected healthy eating patterns, mainly characterized by a higher intake of fruit and vegetables, are associated with a lower risk of diabetes[13]. In this review, we look at some of the various diets that have been shown to be effective for the management of T2DM, in particular the Ma-Pi 2 macrobiotic diet, and explore the potential role of the intestinal microbiota in T2DM.

MEDITERRANEAN DIETA Mediterranean-type diet is one involving: a high consumption of cereals, grains, fruits, vegetables, legumes, nuts; olive oil as the principal source of fat; low-to-moderate consumption of fish and poultry; relatively low consumption of red meat; and moderate consumption of wine, normally with meals[14].

Based on this definition, numerous epidemiological studies have indicated the favorable effects of this diet[15-17]. Such diets have been reported in two pro-spective studies[18,19] and one intervention study[20] to be associated with a reduced risk of diabetes. However, the role of the Mediterranean diet in weight control remains controversial[21], which suggests that the protective effect of this diet against diabetes are not based on weight control but through several of its dietary characteristics. Currently it is unclear which component of the Mediterranean diet contributes most to its favorable effects.

LOW CALORIE DIETWeight loss following a reduction in energy intake (caloric restriction) and/or an increase in energy expenditure (through exercise) improves insulin sensitivity, hyper-glycemia and other cardio-metabolic risk factors[22]. Caloric restriction (CR) is defined as a reduction in caloric intake by around 20%-40%, with adequate intakes of protein and micronutrients to avoid malnutrition[23,24]. Caloric intake can be reduced by eliminating the consump-tion of energy-dense foods (e.g., refined carbohydrates, potatoes, white bread, white rice, sweets and sweetened drinks) and by increasing the intake of a nutrient-dense foods (a wide variety of vegetables, fruits, nuts, low-fat dairy products, egg whites, wheat and soy proteins, fish and meat). The energy intake of such a diet is around 1100-2000 kcal/d, with approximately 26% of calories obtained from protein, 28% from fat and 46% from complex carbohydrates[23]. CR may be administered as a short- (1 mo-1 year) or long-term diet. CR in healthy individuals as well as those who are obese or have T2DM has been reported to lower blood glucose

Fallucca F et al . Gut microbiota, Ma-Pi 2 diet and T2DM


levels, fasting insulin values and BMI[25]. Weight loss induced by caloric restriction mediates a reduction in inflammatory markers and improves insulin resistance and sensitivity[26]. With adequate nutrition, CR can improve cardio-metabolic health, prevent T2DM and may be a powerful tool against obesity and insulin resistance.

VEGETARIAN DIETVegetarian diets typically include large amount of fruits, vegetables and legumes, nuts and soya-protein and usually aim to eliminate all animal products. Studies have shown that, generally, vegetarians residing in Western countries have a lower BMI and a higher ratio of polyunsaturated to saturated fat intake than individuals consuming a non-vegetarian diet (people eating meat and/or fish)[27,28]. Vegetarians have also been shown to have lower concentrations of low-density lipoprotein (LDL)-cholesterol[29-31], lower blood pressure[32,33] and a lower risk of diabetes[34] than non-vegetarians. Factors associated with the higher fiber content in vegetarian diets promote increased insulin sensitivity[35]. Vegetarian diets are also associated with other health benefits. According to the largest study ever conducted in the United Kingdom from the University of Oxford, the risk of hospitalization or death from heart disease was 32% lower in vegetarians than in people who ate meat and fish, with most of the difference in risk probably caused by effects on cholesterol and blood pressure[36]. In addition to a lower risk of cardiovascular disease[37,38], vegetarian diets have been reported to reduce the incidence of cancer in a low-risk population[39,40].

MICROBIOTAThe results of some studies indicate that the efficacy of a diet is not solely related to the biochemical structure

of the individual nutrients it contains and the current standard definition of macronutrients fails to capture important information[41]. This has become more obvious as knowledge of the gut microbiota[42-46] has been gained and the “food as hormone” hypothesis[47] has emerged.

The human gut microbiota weighs about 1500 g and the number of intestinal microbial cells it contains is tenfold greater than the total number of human body cells[48]. The gut microbiome, which represents the collective genomes of all gut microbiota, is 150 times greater than the human gene complement[46]. The gut microflora plays several metabolic active roles: it produces vitamins, synthesizes amino-acids, transforms bile acid, and is able to ferment non-digestible substrates and endogenous mucus, stimulating bacterial growth and producing short-chain fatty-acids (SCFA)[49], which work in the gut and at distance after absorption[49]. Moreover, the gut microbiota also causes pathogen displacement, by competing for attachment sites and nutrients, and by secreting antimicrobials[50]. The microbiota plays a fundamental role in the development of the immune system[51], as SCFA have a strong immunomodulatory activity, leading to the production and release of cytokines, chemokines and phagocytes[52].

The gut microbiota is also involved in the development of cells and tissues[53], and is responsible for the integrity of the gut barrier[53] through its involvement in glucagon-like peptide (GLP)-2 production by enteroendocrine L-cells, and the circulation of endotoxins[54]. In addition, the microbiota triggers the production of peptide YY, which regulates gut motility and the hungry sensation[55].

Dysbiosis is a bacterial imbalance in the gut resulting in a modification of the normal local distribution of microbial communities that correlates with biochemical and clinical modifications in the host: hyperglycemia and T2DM have been observed in the presence of a low percentage of bacterial Firmicutes and Clostridia species[56]. Diet and increase in bodyweight are linked to gut microbial imbalance, in both the presence and absence of obesity[57].

Recent studies have shown a link between dysbiosis and several diseases, including diabetes (Figure 1).

Obesity and T2DM are associated with chronic low-grade inflammation and endotoxemia. Lipopolysaccharide (LPS) consumption and a diet rich in fat increase adiposity, impaired glucose tolerance, and insulin resistance those raises are reversible by the consumption of a pre-probiotic rich diet[44,58,59], or a meal based on fiber and fruit, as recommended by the American Heart Association[60]. Bacterial LPS derived from Gram-negative bacteria residing in the gastrointestinal microflora could act as a trigger for the development of diabetes and obesity; in a series of experiments in mice, it was observed that high-fat feeding favors an increase in the Gram-negative to Gram-positive colonizing bacteria ratio, which has been associated with an increased intestinal permeability that precedes the development of metabolic endotoxemia, inflammation, and associated disorders (obesity and diabetes)[44]. Different nutrients have different enhancing-


Dysbiosis

Microbiota and human diseasesPubMed July 2014

Obesity (661)Diabetes (408)

Allergy (462)

Autoimmune diseases (179)

CVD (157)

Atherosclerosis (56)

Ageing (158)

ALS (2)Others???

Alzheimer (3)

Multiple sclerosis (23)

Hypertension (39)

Metabolic syndrome (182)

Liver diseases (518)

Cancer (671)

Hyperlipemia (10)

Figure 1 Articles indexed in PubMed concerning the microbiota and human diseases. Results of a research in PubMed looking for articles concerning gut microbiota that associated dysbiosis and human diseases. Number of articles for each kind of disease is indicated in brackets. CVD: Cardiovascular diseases; ALS: Amyotrophic lateral sclerosis.


ability to work in a manner similar to that of hormones. Identifying these food- and food metabolite-receptor interactions will provide new opportunities for studying the relationship between the food we eat and diseases, including obesity.

MA-PI 2 MACROBIOTIC DIETMacrobiotic diets are originally derived from an ancient Eastern philosophy of life based on two ancient Asian theories (Yin/Yang and the Five Transformations), formulated for Western culture by the Japanese philo-sopher Georges Ohsawa[76] and further updated by Mario Pianesi who created the 5 Ma-Pi diets[77]. The Ma-Pi 2 diet was specifically designed for patients with metabolic disorders, and consists of 50%-55% whole-grain (rice, millet and barley), 35%-40% vegetables (carrots, savoy cabbage, chicory, red radish, onions, parsley, cabbage) and 8%-10% legumes (adzuki beans, chickpeas, lentils and black beans), plus gomashio (roasted ground sesame seeds with unrefined sea salt), and fermented products [miso, wandadou jiangyou (soy sauce) and yanzimei (pickled ume plums)], which could have probiotic effect; seaweeds [Kunbu (Laminaria japonica, Aresch), Qundaicai (Undaria pinnatifida, Harv.), Haitai (Porphyria tenera, Kjell.) and Hiziki (Sargassum fusiforme, Harv.)] and Beicha tea (caffeine-free green tea). The daily average energy intake is in the range of 1700-2200 kcal (12% from proteins, 18% from fats and 70% from carbohydrates, mainly complex). The Ma-Pi 2 diet contains, on average, 18% saturated, 46% monounsaturated and 36% polyunsaturated fat, with no trans-fatty acids and an n-6:n-3 polyunsaturated fatty acid ratio of 5:1. It provides nutrients and phytocompounds with antioxidant, hypoglycemic and hypolipidemic effects, such as vitamin C, β carotene, magnesium (average 700 mg/d), manganese (average 16 mg/d), zinc (average 15 mg/d), chromium, phytosterols (average 326 mg/d), dietary fiber (average 50-60 g/d), inulin (average 9 g/d), polyphenols, tocotrienols, folates (more than 500 µg/d), quercetin, and prebiotic and probiotic products. The Ma-Pi 2 diet excludes all animal products, (including egg and dairy), and has no added sugars[77].

All of the ingredients used to prepare the Ma-Pi 2 diets are grown, stored and processed without the use of synthetic chemicals. The crops are from old seed varieties, which were produced using natural methods. All of the products used are labeled, providing detailed information on the origin and characteristics of the product and its supply chain (the Pianesian Transparent Label)[78]. The nutritional content of one Ma-Pi 2 diet used in a clinical study in T2DM patients is presented in Table 1[79].

Since 2001, various clinical studies have been carried out to assess the effect of the Ma-Pi 2 diet in T2DM patients.

Single-arm studies of the Ma-Pi 2 diet in patients with T2DM conducted in several countries and continents(e.g., America, Asia, Africa and Europe) have consistently

effects on endotoxemia (LPS production by microflora or for higher gut permeability) and the consequent inflammatory response[61]. Other factors may also influence our microbiota, such as age[62-64], chemical products and antibiotics[65], and prebiotics and probiotics[58,66-68].

The internationally endorsed definition of probiotics is “live microorganisms which when administered in adequate amounts confer a health benefit on the host”[69],whereas, definition for prebiotics is “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora, that confer benefits upon host well-being and health”[66]. Therefore, in order to be classified as a prebiotic, the ingredient should resist gastric acidity, hydrolysis and should be fermented by the intestinal microflora. The consumption of prebiotic foods enhances a reduction of triglyceride and postprandial glucose levels, gut permeability and inflammatory effect[66,70]. It has been observed that administering prebiotics to obese mice induces a microbiota modulation, lowering both diet-induced LPS endotoxemia, and systemic inflammation and liver inflammation[44]. The main positive effects of pro- and prebiotic administration are increases in GLP-1 and peptide YY secretion[71-73], a reduction in the complications of pregnancy[74], and improvements in metabolic diseases (obesity, T2DM, cardiovascular disease)[75].

New findings in microbiota knowledge are opening new fields of study about human health and disease. However, the research regarding the interaction between bacteria and human health is at the beginning and many aspects are still not properly understood; well-defined clinical studies are needed in order to develop the potential of this new therapeutic area.

Until now, scientists have focused primarily on identifying human genetic markers, but research has shown that other factors, such as the intestinal flora, play a role in the development of T2DM[45,46]. The results of metagenome-wide association studies in T2DM patients and non-diabetic controls[45,46] clearly indicate that T2DM is a significant factor in the observed variation in gut microbial samples, and suggest that the gut microbiota in T2DM patients features dysbiosis. These studies[45,46] indicate that a gut-microbiota-based index could be more effective and accurate in assessing and predicting the risk of T2DM than a human genome variation-based one.

Knowledge of the microbiota and the general concept of “food as hormone”, as proposed by Ryan et al[47], suggest that diet has an enormous impact on our health. In fact, circulating substrates derived from food have both direct and indirect actions and, ultimately, food may be considered a cocktail of hormones that exert their effects on target tissues, activating cell-surface or nuclear receptors. Food components also interact with the gut flora to induce indirect signals. Therefore, if we consider this new role for food, we can make dietary recommendations to promote health or treat specific diseases, taking into account that until now macronutrients have been classified according to their energy-yielding biochemical properties and not by their



shown that consumption of the diet for 3 or 6 mo resulted in statistically significant improvements from baseline in indicators of metabolic control, including fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), the serum lipid profile [including reductions in total cholesterol, LDL cholesterol and triglyceride values, and the LDL:high-density lipoprotein (HDL) cholesterol ratio], BMI, and insulin resistance, as well as body weight and blood pressure (P < 0.001) (Table 2)[79,80-84]. These results were apparent even in patients with poor glycemic control (HbA1c > 8.5%)[85]. In patients being treated with insulin, use of this agent fell during the intervention[79,85].

Similar results were obtained later in short-term (21-d) intervention studies[86,87], thus demonstrating the ability of the diet to achieve rapid metabolic control.

The first randomized, controlled trial comparing the Ma-Pi 2 diet (in 25 participants) with Italian dietary recommendation for T2DM (in 26 participants)-the MADIAB trial-was carried out in 2013[88]. After 21 d on the prescribed diet, which was administered under supervised conditions, the average daily energy intake was 1803 kcal (12% protein, 15% fat, and 73% complex carbohydrates, with 29 g/1000 kcal fiber) in the Ma-Pi 2 group and 1798 kcal (18% protein, 32% fat,

and 49% complex carbohydrates, with 20.5 g/1000 kcal fiber) in the control group (P = 0.860).

The multivariate analysis (adjusted for age, gender, BMI at baseline, and physical activity) showed that significantly higher percentage reductions in FBG, post-prandial blood glucose (PPBG), HbA1c, total cholesterol, LDL-cholesterol, LDL:HDL ratio, BMI, weight, waist and hip circumference, and insulin resistance were seen in the Ma-Pi 2 group than in the control group. Although the triglyceride levels were reduced in all subjects, the reduction detected in the controls was significantly higher than that in intervention subjects. Furthermore, all subjects in the Ma-Pi 2 group achieved FBG and PPBG target levels (< 110 mg/dL for FBG and < 140 mg/dL for PPBG) at the end of the 21-d dietary treatment[88].

Further results from this trial, presented at the 2014 ADA 74th Scientific Sessions, have also demonstrated that the Ma-Pi 2 diet is a safe strategy by which to reduce markers of insulin resistance and inflammation. In addition, a significant reduction in insulin-like growth factor-1 was observed in the Ma-Pi 2 subjects vs the controls (P < 0.001)[89].

The MADIAB trial is also currently assessing thehypothesis that the Ma-Pi 2 diet may affect the com-position of the gut microbiota to a greater extent than the control diet. The results of this analysis will be published later this year.

These results strengthen the idea that the Ma-Pi 2 diet could play an important role in the treatment of T2DM,


Table 1 Example of the average daily energy and nutrient intake for the Ma-Pi 2 diet during in one dietary intervention study[79]

Nutrient Ma-Pi 2 diet

Energy (kcal) 2174Protein (g) 66Tryptophan1 13Threonine1 35Isoleucine1 41Leucine1 73Lysine1 42Met + cystine1 34Phen + tyrosine1 78Valine1 50Total fat (g) 38Cholesterol (mg) 0Carbohydrates (g) 392Fiber (g) 54Vitamin C (mg) 164Folic acid (g) 751Vitamin B1 (mg) 3.52Vitamin B2 (mg) 1.3Vitamin B6 (mg) 5.55Niacin (mg) 25Vitamin B12 (g) 0.45Vitamin E (mg) 10Vitamin A (g) 3266Potassium (mg) 3646Manganese (mg) 16Iron (mg) 24Calcium (mg) 982Phosphorus (mg) 1632Zinc (mg) 15.4Magnesium (mg) 754Sodium (mg) 1724

1mg of amino acid per gram of protein.

Table 2 Metabolic effects of the Ma-Pi 2 dietary intervention after variable periods of time

Metabolic parameters Duration of Ma-Pi 2 intervention Effect

Body mass index 3 wk-6 mo ↓Body weight (kg) 3 wk-6 mo ↓Body fat mass (%) 3 wk-6 mo ↓Body fat free mass (%) 3 wk-6 mo ↔Fasting glucose (mg/dL) 3 wk-6 mo ↓Fasting insulin (mIU/mL) 3 wk ↓HbA1c 3 wk-6 mo ↓HOMA-R index 3 wk-6 mo ↓Triglycerides (mg/dL) 3 wk-6 mo ↓Total cholesterol (mg/dL) 3 wk-6 mo ↓HDL-cholesterol (mg/dL) 3 wk-6 mo ↔LDL-cholesterol (mg/dL) 3 wk-6 mo ↓Systolic blood pressure (mmHg)

3 wk-6 mo ↓

Diastolic blood pressure (mmHg)

3 wk ↓

Urinary pH 3 wk ↑Serum anion gap (mEq/L) 3 wk ↓Serum bicarbonate (mEq/L) 3 wk ↑Tumor necrosis factor alpha (ng/mL)

3 wk ↓

Interleukin 6 (pg/mL) 3 wk ↔Insulin-like growth factor 1 (ng/mL)

↓

Taken from the results of studies investigating the Ma-Pi 2 diet in men and women with type 2 diabetes over variable periods. ↑: Increase; ↓: Decrease; ↔: No effect; HbA1c: Glycosylated hemoglobin; HDL: High-density lipoprotein; LDL: Low-density lipoprotein.


as whole cereals, non-animal fats and protein, and the high fiber content (> 50 g/d) and probiotic presence could improve gut microbiota composition, favoring a reduction in inflammation and insulin resistance[90].

Eliminating all animal products from the diet may increase the risk of certain nutritional deficiencies in some micronutrients such as vitamin D, calcium, iron, zinc and long-chain n-3 (omega-3) fatty acids[40]. However, the Ma-Pi 2 diet content of those micronutrients seems to be adequate[77,79]. Special concern should be given to possible deficiencies of vitamin B-12[77], even though clinical evidences for deficiency in this vitamin are described only after several years of insufficient consumption[91]. The MA-PI 2 diet was especially conceived for the treatment of T2DM and studies in adults with T2DM consistently showed that a 3 or 6-mo consumption of the Ma-Pi 2 diet was nutritionally safe, at least for the evaluated time[79-83,85]. These evidences suggest that Ma-Pi 2 diet can be used as a short- and medium-term treatment, aimed to achieve a good metabolic control and that further research is needed to demonstrate the safety of this diet in the long-term.

However, currently available studies on the Ma-Pi 2 diet in patients with T2DM have a number of limitations, such as the use of relatively small sample sizes. This was due to the particular design of the studies, where subjects were required to dwell in isolated environment 24 h per day, in order to ensure a good compliance to the diets[80-85,87-89]. However the MADIAB randomized trial was adequately powered and corroborative analyses were performed to ensure consistency and robustness of the clinical trial results[88]. Other limitations in available studies on the Ma-Pi 2 diets include their short- or medium-term durations and the fact that, in some studies, participants (in both the intervention and control groups) were studied in a supervised environment[84,85,87-89]. Subject compliance to dietary recommendations for T2DM are required to obtain clinically significant changes in outcomes, and improvements obtained with dietary interventions in clinical trials are not easy to reproduce in daily life. Future studies should aim to address all of these issues.

CONCLUSIONA variety of diets have been shown to be effective for the management of T2DM, including the Mediterranean-style, vegetarian and low-calorie diets. However, the Ma-Pi 2 diet, in both uncontrolled and controlled short- and medium-term trials conducted in patients with T2DM in several countries and continents, has been found to achieve a speed of metabolic control that has not been reported in studies of other diets.

This finding could be attributable to the composition of the Ma-Pi 2 diet, which is high in whole grains, vegetables and legumes, and fermented products, with no animal products and no added sugars. These characteristics allow to improve glycemic control, reduce insulin requirements, improve insulin sensitivity, lower blood cholesterol and

triglycerides, reduce body weight control, and lower systemic blood pressure. The dietary habits may also modify the gastrointestinal microflora composition, which could influence glucose control.

Hence, the Ma-Pi 2 diet could be considered a valid additional short- to medium-term treatment for T2DM, particularly when glycemic control needs to be rapidly improved. Further research is needed to demonstrate the efficacy of this diet in the long-term management of T2DM.

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P- Reviewer: Bagyánszki M S- Editor: Gong XM L- Editor: A E- Editor: Liu SQ



of hippocampus structure and function. Although, the exact mechanism by which maternal diabetes affects the developing hippocampus remains to be defined. Multiple biological alterations, including hyperglycemia, hyperinsulinemia, oxidative stress, hypoxia, and iron deficiency occur in pregnancies with diabetes and affect the development of central nervous system (CNS) of the fetus. The conclusion from several studies is that disturbance in glucose and insulin homeostasis in mothers and infants are major teratogenic factor in the development of CNS. Insulin and Insulin-like growth factor-1 (IGF-1) are two key regulators of CNS function and development. Insulin and IGF-1 receptors (IR and IGF1R, respectively) are distributed in a highly specific pattern with the high density in some brain regions such as hippocampus. Recent researches have clearly established that maternal diabetes disrupts the regulation of both IR and IGF1R in the hippocampus of rat newborn. Dissecting out the mechanisms responsible for maternal diabetes-related changes in the development of hippocampus is helping to prevent from impaired cognitive and memory functions in offspring.

Key words: Maternal diabetes; Cognition complications; Teratogenic factor; Hippocampus


Core tip: Diabetes mellitus is the most seriously metabolic condition in pregnancy that affects the hippo-campal development and function of the offspring. Multiple biological alterations, including hyperglycemia and hyperinsulinemia are occurring in maternal diabetes and impair the neurodevelopment of the fetus. Insulin-like growth factor-1 (IGF-1) and insulin are important regulators of development of central nervous system. It has clearly showed that maternal diabetes disturb the regulation of both insulin receptors and IGF-1 receptors in the hippocampus of rat newborn. This article is a brief review of the literatures that suggests a probable

Javad Hami, Fatemeh Shojae, Saeed Vafaee-Nezhad, Nasim Lotfi, Hamed Kheradmand, Hossein Haghir

Javad Hami, Fatemeh Shojae, Saeed Vafaee-Nezhad, Nasim Lotfi, Department of Anatomy, School of Medicine, Birjand University of Medical Sciences, Birjand 97178, IranNasim Lotfi, Hossein Haghir, Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad 91857, IranHamed Kheradmand, Hazrat Rasoul Hospital, Tehran University of Medical Sciences, Tehran 19168, IranHossein Haghir, Medical Genetic Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad 91857, IranAuthor contributions: All authors contributed to this manuscript.Conflict-of-interest: All authors claim no conflict of interest.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Hossein Haghir, MD, PhD, Department of Anatomy and Cell Biology, School of Medicine, Mashdad University of Medical Sciences, Azadi Square, Mashhad 91857, Iran. [email protected]: +98-513-8002486 Fax: +98-513-8002486Received: August 27, 2014 Peer-review started: August 31, 2014 First decision: October 14, 2014Revised: December 22, 2014 Accepted: January 9, 2015 Article in press: January 12, 2015Published online: April 15, 2015

AbstractDiabetes mellitus during pregnancy is associated with an increased risk of multiple congenital anomalies in progeny. There are sufficient evidence suggesting that the children of diabetic women exhibit intellectual and behavioral abnormalities accompanied by modification

REVIEW


Some of the experimental and clinical aspects of the effects of the maternal diabetes on developing hippocampus




mechanism of how diabetes during pregnancy affects the hippocampus development in the offspring.

Hami J, Shojae F, Vafaee-Nezhad S, Lotfi N, Kheradmand H, Haghir H. Some of the experimental and clinical aspects of the effects of the maternal diabetes on developing hippocampus. World J Diabetes 2015; 6(3): 412-422 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/412.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.412

INTRODUCTIONDiabetes mellitus (DM) is a chronic common metabolic illness characterized by hyperglycaemia associated with insulin deficiency or insulin resistance. There are two major types of DM: type 1 DM (T1DM) results from chronic and progressive destruction of the beta-cells of the islets of Langerhans in the pancreas. T2DM is primarily characterized by both insulin resistance and relative insulin deficiency[1,2].

DM is one of the most common and most important metabolic condition affecting up to 7% of pregnancies. Several population studies show that the diabetes during pregnancy affects the health of both mothers and their infants[3-6].

There are two main types of DM occurs in pregnancy period: pregestational DM (PGDM) and gestational DM (GDM)[7]. GDM accounts for approximately 90% of all cases of diabetes in pregnancy. Of the remaining cases, 60% have preexisting T2DM before pregnancy, while 40% have a diagnosis of pre-conceptual T1DM[8].

In a study in the United Kingdom, it was found that PGDM occurs in approximately 1 out of 250 preg-nancies, with two-thirds of them having T1DM and the remaining one-third having T2DM[9]. At present, type 2 diabetes is the most prevalent form of diabetes affecting women of reproductive age in the developed countries, with a mean prevalence of 27.6% in United Kingdom[10]. Last data from Australia also showed that T2DM affected about 55% of pregnancies[11]. Overall, GDM complicated from 2% to 12% of all pregnancies. So, however, the incidence of T1DM appears to be relatively constant and similar to the rate in young adults throughout the world; the incidence of T2DM is specially rising in developed world[9,12]. Furthermore, the overall numbers of children born to mothers with diabetes will significantly rise in the next decades.

COMMON CONGENITAL MALFORMATIONS IN DIABETIC MOTHER INFANTSAn increasing number of evidence clearly showed that infants born to diabetic mothers are in increased risk of fetal and neonatal anomalies which results in increased infant mortality and morbidity rates. Although, the

wide spectrum of the effects of diabetes in pregnancy on the fetus development are determined by the time of onset and the severity of DM[6,13,14].

Despite considerable progress in the clinical mana-gement of pregnant women who have diabetes, the prevalence of major congenital anomalies is approximately 3 times more in infants of diabetic women in comparison to the offspring of normal mothers[12,15,16].

According to previous works, diabetes in pregnancy has been accompanied by increased risk of a wide range of fetal complications and malformations, in-cluding respiratory distress syndrome, macrosomia, organomegaly, obstetric, metabolic and hematological complications[13,14,16,17].

The overall rate of major congenital anomalies in infants born to diabetic mothers is about 6% to 13%, which is approximately 2-3 times higher than that of the normal condition. However, the prevalence of major structural malformations in the children born to mothers with diabetes are to 10 times greater in comparison to the general population. The frequencies of perinatal mortality are also five-fold higher in women with diabetes compared with normal women[12,14,16,18].

The prevalence of Minor congenital anomalies in infants born to 802 mothers with GDM, 117 mothers with PGDM, and 380 normal mothers were assessed by Hod et al[19] (1992). They found a range between 19.4% and 20.5% in prevalence of minor congenital anomalies in their infant in all groups studied without any marked difference between groups. In their study, there was no correlation between the type and prevalence of minor congenital anomalies with the severity and the time of onset of the diabetes in mothers. Neither there was any relationship between the type or prevalence of minor congenital anomalies with the type or appearance of major congenital anomalies[19].

Overall, the defects in central nervous system, heart, kidney, and skeletal system are among the commonest congenital malformations in the offspring of diabetic pregnancies[3,14,16,18].

PATHOGENESIS OF COMMON COMPLICATIONS IN DIABETIC INFANTS As yet, the exact mechanisms by which maternal diabetes alters the development of growing fetuses are not completely understood, and no distinct teratogenic mechanism has been identified to clearly explain a reason for this range of congenital anomalies observed in the infants born to diabetic mothers[20,21].

Many of the developmental effects of diabetic pregnancies on the fetus can be attributed to maternal glucose (metabolic) control. In normal pregnancies, the blood glucose level in women remains within a tight range. Consequently, the blood glucose concentration in the fetus is fairly constant as the glucose in the mother’sblood crosses the placenta freely. Nevertheless, in pregnancies complicated by DM, there are great

Hami J et al . Effects of maternal diabetes on developing hippocampus


variations in the maternal blood glucose level[12,14,22]. Pedersen et al[23] (1954) emphasized the correlation between high blood glucose concentration in pregnant women with hyperglycemia during fetal period[23], which stimulated the pancreas of the fetuses, leads to beta-cell hyperplasia and hypertrophy with increased insulin secretion and content[23,24].

Several lines of studies also emphasized the direct correlation between high blood glucose concentration in mothers as revealed by increased level of glycosy-lated hemoglobin (HbA1c) with increased frequency of congenital malformations in offspring[22,25]. Towner et al[26] (1995) found a striking correlation between pregestational elevated HbA1c in early pregnancy with the increased risk of fetal congenital malformations[26]. There is enough evidence accumulates to support this hypothesis that the tight glycaemic control in early and before pregnancy may reduce the frequency of anomalies in the infants born to diabetic mothers[22]. Therefore, the prevalence of congenital abnormalities in children born to mothers with diabetes has decreased in the last three decades probably as a result of the overall progress in monitoring of blood glucose concentrations in diabetic pregnant women[27-29].

In addition, fetal hyperinsulinemia also has been shown in the offspring of diabetic mothers. On separation of the newborn from the mother, the glucose former no longer is supported by placental glucose transfer, may develop neonatal hypoglycemia[30,31].

In earlier works, chronic fetal hyperinsulinemia in the third trimester of pregnancy has been produced in monkeys. The researchers showed that in utero hyperinsulinemia results in an organomegaly and fetal macrosomia, except for kidney and brain[32,33]. Increased plasma erythropoietin levels and body fat content was also evident in infants of diabetic mothers[34].

Insulin is a hormone that primarily functions as an anabolic hormone of fetal development and growth resulting in macrosomia and visceromegaly[14]. On the basis of in vitro and animal studies on the pancreas, the simplified hyperinsulinemia - hyperglycemia hypothesis has been expanded in earlier studies[35,36].

Ketones in the mother’s blood readily cross the placenta, but they cannot be cause fetal hyperinsu-linemia; so, they might not influence fetal growth and development[14].

In experimental diabetic rodents, fetal hyperglycemia, neonatal hypoglycemia, and disturbances in pro-staglandin and arachidonic acid metabolism have been reported to result in congenital anomalies in their offspring[14,37]. In diabetic pregnancies in rats, it is clearly established that the elevated intracellular free oxygen radicals concentrations have been accompanied by the increased risk of embryopathies[38]. Nevertheless, hyperglycemia-induced fetal malformations in rodents have been demonstrated to associate with the number of genomic DNA mutations[39]. Therefore, there are animal

studies showing that the free radical scavengers and antioxidants may decrease the risk of teratogenicity of maternal diabetes[40,41].

DIABETIC PREGNANCY AND NEURODEVELOPMENTAL SEQUELAE IN HUMAN Both PGDM and GDM can affect fetal neurodeve-lopment[42-45]. Although the untoward effects of maternal diabetes on pregnancy outcomes with respect to congenital malformations have generally been appreciated, the effect of maternal diabetes on the development of central nervous system (CNS) in the fetuses and its behavioral sequelae remains to be completely defined. A limited number of long-term follow-up studies of neurodevelopmental outcomes in diabetic pregnancies have been reported[46-50]. Nevertheless, it is demonstrated that children born to diabetic mothers are more likely to have neurodevelopmental abnormalities including impairments in learning ability, activity level, attention span, and motor functioning. Interestingly, some of these deficiencies are well-known as risk factors in children who develop schizophrenia later, but the degree of risk is variable and may be related to the severity of metabolic derangement in the mothers with diabetes[42,46,50,51].

Examination of cognitive functioning and the behavior of the children born to diabetic mothers offers the opportunity to functionally assess the CNS development[52]. Hence the assessment of the behavior and cognition in the offspring of diabetic mothers may clarify the maternal diabetes effects on the development of CNS.

The results of earlier studies clearly demonstrated that the diabetes during pregnancies may results in intellectual and behavioral functioning disturbances in the infants[45,46,49,53]. These data suggest a teratogenic effect of diabetes in pregnancy on the function of CNS in fetuses and provides the earliest indicator of postnatal CNS deficiencies reflected in intellectual and behavioral problems observed in the children of mothers with diabetes[54].

Earlier reports on the neurologic development in infants of diabetic mothers revealed serious CNS deficits, even in the absence of structural malformations. These alterations were significantly less severe when maternal diabetes was medically controlled and treated, but some alterations in cognitive function may persist throughout childhood[6,42,45,48,51].

To elucidate the effects of diabetes during pregnancy period on cognitive functioning in the children, Yamashita et al[45] (1996) studied 33 pregnant women (24 with T2DM, 6 with T1DM, and 3 with GDM). Their long term follow-up research showed that maternal diabetes significantly affects the development of intellectual functioning in infants. Although, they report no differences in IQ score among offspring of three groups[45]. In another



DIABETES IN PREGNANCY AND NEURODEVELOPMENTAL ABERRATION IN ANIMALSNeurodevelopmental assessment of the offspring born to diabetic dams have been revealed a wide spectrum of behavioral, neurochemical, cellular, and molecular impairments[69-72]. Experimental models subjected to streptozotocin - induced type 1 diabetic pregnancies developed significant deficits in cognitive behaviors[68]. Kinney et al[73] (2003) found that the only female offspring born to diabetic dams showed deficits in long-term memory and learning. These results have suggested that the in utero diabetic condition has gender-specific effects on CNS development[73].

In a study by Plagemann et al[74] (1998), alterations in catecholamines levels in the hypothalamic nuclei of newborns born to diabetic animals were evaluated. They reported an increased hypothalamic dopamine (DA) and norepinephrine (NE) concentrations in the offspring born to diabetic rats at birth. Twenty one-day-old pups born to diabetic mothers, NE levels were strikingly increased in the ventromedial hypothalamic nucleus and the lateral hypothalamic area (LHA), while DA levels were significantly elevated in the paraventricular hypothalamic nucleus and the LHA. The authors concluded that there are strikingly differences in hypothalamic catecholaminergic systems during early development in the rat newborns born to diabetic animals[74].

It has been shown that PGDM is associated with an increases risk of neural tube defects (NTDs), also known as diabetic embryopathy. The prevalence of NTDs in the offspring of diabetic mothers is 3 to 10-fold higher than that of in general population[75-77]. In earlier studies, the mitochondrial morphological changes has been shown in developing neural tubes subjected to a diabetic environment during the same time period when the NTDs are induced in diabetic pregnancies[72,78]. Altered activity of cytochrome-c oxidase also has been manifested in the rat hippocampus that have iron deficiency in their brains due to fetal hyperglycemia[79]. It has also been hypothesized that dysfunction in energy metabolism of developing brain leads to oxidative stress elevation and abnormal regulation of intracellular calcium[80-82].

NEUROPATHOPHYSIOLOGY OF DIABETIC PREGNANCYSeveral well-known biological alterations occur in mothers with diabetes that affect fetal neurodevelopment. These biological changes lead to alterations in neurotransmitter systems, synaptic membranes, neuronal integrity, and growth factors expression that have also been implicated in the development of neurodevelopmental and neurocognitive sequelae in offspring born to diabetic mothers[14,42,69,72,74].

study, Churchill et al[55] (1969), found that diabetic mothers had offspring with significantly lower mean IQs than control infants. In a cohort study of fifty infants at 1, 3, and 5 years of age born to diabetic mothers, Stehbens et al[56] (1977) reported an adverse effect of diabetes in pregnancy on CNS structure and function in their children. They also found that three infants born to diabetic mothers had major neurologic anomalies and six of them had IQs less than 80.

Rizzo et al[57] (1991) reported a strikingly correlation between diabetes in mothers during pregnancy and lower IQ in their children. In another study, the same researcher found significant correlations between second- and third-trimester regulation of glycemia and results on the Brazelton Neonatal Behavioral Assessment Scale; they reported an association between increases in maternal glucose levels with poorer infant responses[58].

Other investigators have not found any differences in cognitive scores[59-61]. Children born to diabetic mothers may sustain minor neurological damage which does not necessarily affect their scores in IQ tests[49]. For example, Persson et al[60] reported more encouraging results in 73 infants born either to mothers with T1DM or to mothers with GDM; all subjects available for follow-up at 5 years of age had normal results on neurologic examinations, as well as normal IQs, and there was no relation between maternal diabetes and IQ[60].

In a retrospective study at considerable variance with most other reported studies, Yssing et al[62] (1975) reported a 36% incidence of cerebral dysfunction or related conditions in 740 infants of diabetic pregnancies; 18% had a major cerebral disability. In a study by Haworth et al[63] (1976) on infants of diabetic pregnancies, noted an about 30% increase in the incidence of neurological and intellectual development impairments[63]. No differences in behavioral adjustment and academic achievement between children born to T1DM mothers and children of normal mothers were reported in Hadden et al[64] study.

In addition, poorer habituation performance were also found in fetuses of diabetic pregnancies when compared to fetuses born to normal mothers[65]. Since habituation reflects the fetal CNS performance, the observed habituation abilities differences between the diabetic and normal groups suggests differences in their CNS function or maturation[65,66]. Recent investigations also have been demonstrated that there is a significant correlation between diabetes during pregnancy and increased risk of some psychologic disturbances including schizophrenia in their children[67,68].

Together, these studies suggest a wide-range of teratogenic effects of maternal diabetes on the development and function of fetal CNS[67]. However, no single molecular mechanism can fully explain the effects of maternal diabetes on fetal neurodevelopment because the CNS development is complex and regulated by a number of signaling molecules and transcription factors.



The hyperglycemia is the most distinct mechanism by which these predispositions might be mediated. The defining characteristics of diabetes during pregnancy, these are clearly showed to have effects on developing CNS and to induce fetal hyperinsulinemia, chronic tissue hypoxia, decreased in fetal iron levels and increased oxidative stress[20,83-85].

Hyperinsulinemia, hypoxia, polycythemia and iron deficiencyIt is revealed that fetal polycythemia induced by DM in pregnancy is likely triggered by the chronic in utero hyperglycemia and hyperinsulinemia and develops in response to higher concentration of blood glucose in mothers[20,42]. A study by Mimouni et al[86] (1986) showed an incidence of 29.4% fetal polycythemia in infants born to diabetic mothers in comparison to 5.9% in normal conditions. One of the reasonable explanations is that in utero hyperglycemia and hyperinsulinemia produces a hypermetabolic state in the developing fetuses, which results in relative tissue hypoxia, leading to polycythemia via excess production of erythropoietin. Elevated fetal plasma erythropoietin concentrations in diabetic pregnancies suggests an increased prevalence of chronic fetal hypoxia during development. Although there are no distinct mechanisms for fetal hypoxia in diabetic pregnancies[34].

Hypoxia affects the fetal CNS development in-cluding alterations in myelination, changes in cortical connectivity, excitotoxicity, and neuronal or glial cell death[87-89].

In the hypoxia state, excess erythropoietin and hemoglobin are produced[90]. So, the developing fetuses need for iron exceeds its supply that leads to Iron mobilization from vital tissues including the fetal brain[91,92]. It is showed that human infants born to diabetic women possess brain iron content 40% less than that in normal pregnancies[93].

Several authors have reviewed the role of iron on CNS development and function[94-96]. Iron is one of the key component of the many enzymes involve in essential reduction or oxidation reactions, neuronal replication, synthesis and catabolism of neurotransmitters and myelin production[97-101]. The uptake of Iron into the brain is maximal during the rapid brain development and growth, which coincides with the peak of myelinogenesis[99-101]. In experimental animals, iron deficiency demonstrated to affect the development of neurotransmitter systems in the brain results in behavioral alteration[98,102]. Moreover, iron deficiency in fetuses is also known to manifest as increased negative emotionality and higher levels of irritability in infants and is a predictive factor in the behavioral and developmental problems at 5 age old children[103]. There are studies suggesting that iron deficiency during brain development leads to alterations in the hippocampal structure and function[104].

Moreover, in animal models, hyperinsulinemia during in utero period reduced the fetal amino acid

concentrations, including levels of the nonprotein amino acid taurine, which is showed to be involved in the development of the brain[105].

Diabetes during pregnancy as a proinflammatory milieuDiabetes during pregnancy is associated with the disbalance of pro-inflammatory pathways supported by increased circulating concentrations of inflammatory molecules[106,107]. It is demonstrated that inflammatory cytokines affect the neuronal development and metabolism of neurotransmitters[108]. In experimental models, cytokines reduced the survival of dopaminergic and serotonergic cells[109]. Earlier investigations demonstrated that cytokines, including tumor necrosis factor alpha and interleukin-6, are increased in infants exposed to diabetes in utero and have been implicated in neuronal damage[110,111]. It is also clearly showed that imbalances in the regulation of cytokines have been increasingly associated by a number of prevalent and severe neurodevelopmental disorders[112,113].

Currently, a limited number of human studies demonstrating the effects of inflammatory prenatal environment on neurobehavioural development in children[114]. Evidence from a few studies suggests that immune alterations that occur in prenatal period may persist into postnatal life[115,116]. Therefore, increased inflammatory cytokines in diabetic pregnancies may be relevant to neurodevelopment alterations exhibited by the infants born to diabetic women.

Diabetes in pregnancy and oxidative stressEnhanced oxidative stress plays important roles in embryo development and implicated in the pathogenesis of some neurodevelopmental disorders observed in infants of diabetic mothers[83-85]. Free radicals can inactivate the biological functions of proteins and lipids and potentially leading to cell death[117,118]. It is showed that GDM is associated with an increased concentration of oxidative stress, due to both defect in the antioxidant defenses and/or overproduction of free radicals[83-85]. Although, there are several studies demonstrating the presence of elevated oxidative stress in PGDM and GDM states. Some works also suggested that this milieu can be shared with the developing fetus[83-85,119].

In experimental animals, it is emphasized that oxygen radicals play crucial roles in the progression and timing of the development and differentiation of neurons, and synaptic plasticity; so, imbalance in these signals can lead to changes in development of CNS[120,121]. On the other hands, the developing CNS is particularly susceptible to increase in oxidative stress, owing to its poor antioxidant defenses and/or high oxygen consumption[122].

Oxidative stress might contribute to the pathogenesis of some of neurodevelopmental disorders via an attenuation with gamma-aminobutyric acid receptor function in the brain, decreased synaptic efficiency in hippocampal cells, and inhibition of dopamine β-hydr-oxylase[123-125]. On the other hand, there are evidence



indicating that maternal diabetes-induced NTDs are associated with some metabolic difficulties such as increase in superoxide dismutase activity and decreased in arachidonic acid level[126]. There are evidence from human models indicating a potential role of elevated oxidative stress in neurodevelopmental diseases[127-131]. Moreover, the incidence of congenital anomalies in infants of diabetic mothers has been reported to be significantly reduced by antioxidants supplements therapy including Ascorbic Acid and vitamin E. These data suggest that elevated oxidative stress is associated with the pathophysiology of fetal dysmorphogenesis[132,133].

Role of insulin-like growth factor-1 and insulin in maternal diabetes - induced neuropathiesInsulin- like growth factor-1 (IGF-1) and insulin belong to the insulin superfamily and exert profound effects in the CNS development and function. Evidence of these peptides actions on CNS comes from a wide variety of in vitro and in vivo experimental data, the latter predominantly derived from rodent studies. In several studies, the authors demonstrated that the insulin and IGF-1 have a wide spectrum of biological actions in the developing CNS including neuronal/glial cell proliferation, differentiation, survival, synaptogenesis, longevity, and neuroregeneration. Most of these biological functions are mediated via two transmembrane receptors: the insulin receptor (IR) and the IGF-1 receptor (IGF1R)[69,70,134-142].

The IR and IGF1R, which are homologus in structure, are composed of two α-subunits and two β-subunits linked by disulphide bonds. The extracellular α- subunit binds to their cognate ligand. The β-subunit comprises an extracellular domain and a cytoplasmic domain that contains intrinsic protein tyrosine kinase activity. The binding of either IGF-1 or insulin to α-subunit of their cognate receptors stimulates phosphorylation of the β subunit on serine and tyrosine residues. The autophosphorylation of the β subunit then results in the phosphorylation of cellular substrates and signal transfer cascade for IGF-1 and insulin[141,143,144].

Using ligand binding autoradiography and in situ hybridization, several line of human and animal inves-tigations have found that IR and IGF1R are irregularly distributed in developing and mature brain with the highest densities in the hippocampus, cerebral cortex, olfactory bulb, cerebellum, and hypothalamus. Interestingly, some of brain regions show a marked difference in the density of InsR and IGF1R between embryonic vs mature brain, implying a key role for these ligands in brain development[145-147].

There are evidence from several researches impli-cating defects in IR and IGF1R signaling result in congenital developmental defects seen in offspring of diabetic mothers[145,148-150]. Ramsay et al[151] (1994) in their study was found that expression of IGF-1 in the brain were declined in swine newborns born to diabetic dams when compared to that of control animals.

The hippocampal formation subserves important physiological and behavioral functions including spatial

learning and memory and is a part of brain that parti-cularly vulnerable to changes in blood glucose level[152-154]. In an investigation by Tehranipour et al[155] (2008) the effects of maternal T1DM on density of hippocampal pyramidal cells immediately after birth were examined. Those study results were clearly indicated that maternal diabetes can decreased the numerical density of pyramidal cells in the hippocampus of rat newborns, especially in CA3[155].

Interestingly, the studies By Hami et al[156] (2012) showed that there are prominent gender-and laterality- differences in expression and distribution pattern of InsR and IGF1R in the developing rat hippocampus. The authors concluded that these differences may be a probable mechanism for the control of sex and laterality differences in development and function of the rat hippocampus[156].

In another study by Hami et al[69] (2013), the effects of diabetes in pregnancy on gene expression and protein concentration of IGF1R and IR in the developing rat hippocampus at postnatal days 0, 7, and 14 were evaluated. In that study, the authors found a markedly upregulation of both IR and IGF1R expression in the hippocampus of diabetic group newborns at first postnatal day. At the same time point, they showed only slight changes in their hippocampal protein transcripts. In 7-d old rats, there was a significant decreased in IGF-1R gene expression and protein levels in the newborns born to diabetic dams. Moreover, they found a down regulation in hippocampal IGF1R transcripts in 14-d old diabetic group offspring. Two weeks after birth, the IR gene expression was significantly declined in the hippocampus of diabetic newborns. The authors claimed that maternal diabetes strongly altered the regulation of both IR and IGF1R during development of rat hippocampus[67].

CONCLUSIONThe Incidence of congenital anomalies in infants born to diabetic women is more common in comparison to children of normal population. There are multiple lines of evidence that suggest the disturbances in intellectual and behavioral functioning observed in the children of diabetic women are accompanied by modification of hippocampus structure and function. The etiology and pathogenesis of these impairments induced by diabetes during pregnancy have spurred considerable efforts for clinically and basically researches. The final goal at these investigations was to find the teratogenic factors, which may enable preventive or protective measures to be taken in pregnancies with diabetes. Nevertheless, the exact mechanism by which diabetes during pregnancy affects the CNS development remains to be defined. Until now, several biological changes are defined to occur in diabetic mothers and to affect development of CNS (i.e., disturbance in glucose and insulin homeostasis, oxidative stress, hypoxia, and iron deficiency). Moreover, the new researches on genetic



predisposition involves in teratogenicity of diabetes in pregnancy starts to define new genes and their products involved in the etiology of CNS malfunctions and malformations observed in offspring born to diabetic mothers. Hyperglycaemia and hyperinsulinemia in the mothers and their fetuses are thought to be major factors in teratogenicity of maternal diabetes on the CNS development. There are sufficient evidences for this hypothesis as concise control of glycemia in mothers reduces the incidence of anomalies exhibited with the offspring born to diabetic mothers. Recent evidence clearly indicated that maternal diabetes markedly influences the regulation of both IR and IGF1R - as two important regulators of development and function of CNS - in the developing rat hippocampus. Dissecting out the mechanisms responsible for maternal diabetes-related changes in the development of hippocampus is helping to prevent from impaired cognitive and memory functions in offspring.

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P- Reviewer: Budziszewska B, Hernandez SRZ S- Editor: Ji FF L- Editor: A E- Editor: Liu SQ



which carries a risk of vascular impairment. DM type 2 (DM2) can be characterized by the dysfunction of haemostasis manifesting by stimulated coagulation process, disorder of platelet function and decreased fibrinolytic activity. These all are the reasons why DM2 is the most common acquired thrombophilia. Endothelial dysfunction along with platelet hyperactivity are unquestionably involved in the hyperactivation of platelets and clotting factors in DM. As a natural consequence of continuous investigation, many markers of endothelial dysfunction and diabetic thrombocytopathy have been identified and considered for implementation in clinical practice. Endothelial function can be assessed by the evaluation of endothelial markers, circulating molecules synthesised in various amounts by the endothelium. These markers precede the signs of evident microangiopathy. Platelets have an ethiopathogenic relation to the microangiopathy in DM. Their increased activity was confirmed in both types of DM. Predictors of endothelial and platelet disorder could improve the screening of individuals at increased risk, thus leading to the early diagnosis, appropriate treatment, as well as to the effective prevention of the complications of DM2. In the article we deal with the mechanisms involved in the pathogenesis of endothelial and platelet functional abnormalities, endothelial and platelet markers of DM2 considered for implementation in clinical practice and possibilities of their detection.

Key words: Diabetic thrombocytopathy; Endothelial markers; Platelet markers; Endothelial dysfunction


Core tip: Number of diabetics increases, what leads to worldwide increasing diabetes-associated vascular events. Moreover, diabetes mellitus type 2 is the most common acquired thrombophilia. Therefore, to prevent life-threatening vascular complications in subjects with diabetes, mechanisms and markers of endothelial and platelet dysfunction have been investigated. In order to contribute to better management of discussed patients and to increase knowledge about their origin, in this article we tried to summarize the pathogenesis of

Peter Kubisz, Lucia Stančiaková, Ján Staško, Peter Galajda, Marián Mokáň

Peter Kubisz, Lucia Stančiaková, Ján Staško, National Centre of Haemostasis and Thrombosis, Clinic of Haematology and Transfusiology, Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital in Martin, 03659 Martin, Slovak RepublicPeter Galajda, Marián Mokáň, Department of Internal Medicine I, Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital in Martin, 03659 Martin, Slovak RepublicAuthor contributions: Kubisz P, Staško J and Stančiaková L wrote the paper; Kubisz P, Staško J, Galajda P and Mokáň M designed and performed the research with obtaining the results, on the basis of which the review was partially written; all authors had read and approved the final version of the manuscript.Supported by Projects CEPV I (ITMS 26220120016), CEVYPET (ITMS 26220120053), APVV 0222-11 and Vega 1/0016/12.Conflict-of-interest: The authors declare no conflict of interest.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Peter Kubisz, MD, DSc, Professor, National Centre of Haemostasis and Thrombosis, Clinic of Haematology and Transfusiology, Comenius University in Bratislava, Jessenius Faculty of Medicine and University Hospital in Martin, Kollarova Str. N. 2, 03659 Martin, Slovak Republic. [email protected] Telephone: +421-43-4203232Fax: +421-43-4132061Received: September 1, 2014 Peer-review started: September 1, 2014 First decision: November 27, 2014Revised: January 12, 2015 Accepted: January 30, 2015Article in press: February 2, 2015Published online: April 15, 2015

AbstractDiabetes mellitus (DM) is an extremely common disorder

REVIEW


Endothelial and platelet markers in diabetes mellitus type 2




endothelial and platelet dysfunction and to characterize possible predictors of abnormalities of endothelium and platelets, as well as methods of their detection.

Kubisz P, Stančiaková L, Staško J, Galajda P, Mokáň M. Endothelial and platelet markers in diabetes mellitus type 2. World J Diabetes 2015; 6(3): 423-431 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/423.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.423

INTRODUCTIONDiabetes mellitus (DM) is an extremely common disorder which carries a risk of vascular impairment. In 1970s the attention has shifted towards haemostatic mechanisms with particular emphasis on the relationship between platelets and the vessel wall[1].

The term “endothelial dysfunction” is usually defined as a disorder of the endothelial capacity to maintain vascular homeostasis[2]. Endothelial dysfunction along with platelet hyperactivity are unquestionably involved in the hyperactivation of platelets and clotting factors in DM[3]. It is an early sign of vascular damage in DM type 2 (DM2) but late in DM1. Endothelial dysfunction occurs even in normoalbuminuric (NAU) patients with DM2[4]. Endothelial function can be assessed by the evaluation of endothelial “markers”, circulating molecules synthesised in various amounts by the endothelium[5]. In fact, markers precede the signs of evident microangiopathy[4].

Microalbuminuria conventionally represents albumin excretion rate 30-300 mg in a 24 h urine collection. It is associated with endothelial dysfunction and serves as a predictor of DM at levels out of the characterized reference range[4,6].

DM2 can be characterized by the dysfunction of haemostasis manifesting by stimulated coagulation process, disorder of platelet function and decreased fibrinolytic activity. These all are the reasons why DM2 is the most common acquired thrombophilia[7].

Markers of activated haemostasis, for instance prothrombin activation fragment 1 + 2 (F1 + 2) and thrombin-anti-thrombin complexes, plasma levels of fibrinogen, factor Ⅶ, factor Ⅷ, factor XI, factor XII, kallikrein, and von Willebrand factor (vWF) are elevated in DM[3]. Impairment of fibrinolytic system because of imbalance between regulators of plasminogen represents the typical sign of thrombophilia present in diabetes[7]. Increased amount of platelet aggregates in circulation, increased aggregation of platelets after addition of platelet agonists, increased platelet contractility, and the presence of elevated plasma levels of their contents, for example beta-thromboglobulin (β-TG), platelet factor 4 (PF4), and thromboxane B2 (TXB2), show platelet hyperreactivity in DM[3].

Eighty percent of individuals with DM are dying because of thrombosis[3]. The most effective tool to preserve dysfunction of the endothelium and vascular

impairment in DM is the management of hyperglycae-mia[4].

This review will characterize mechanisms responsible for the development of endothelial and platelet functional abnormalities, endothelial and platelet markers of DM2 considered for implementation in clinical practice and possibilities of their detection.

MECHANISMS OF ENDOTHELIAL DYSFUNCTION IN DM2 The pathogenesis of endothelial dysfunction in both DM1 and DM2 is multifactorial[4].

Altered cell signalization The alteration of the insulin-mediated activation of nitric oxid synthase derived from endothelium is important factor of endothelial damage in the setting of DM and insulin resistance (IR). On the other hand, the activation of insulin-signalling cascade is associated with the expression of endothelin-1 (ET-1), a vasoconstrictor and mitogenic substance, and proinflammatory molecules as intercellular adhesion molecule 1 (ICAM-1)[2].

Increased oxidative stressReactive oxygen species and circulating markers of oxidative stress are elevated in DM, IR and obesity[2].

Pro-inflammatory activation of the endotheliumSystemic inflammation present in DM can impair the function of the endothelium and lead to atherosclerosis. Individuals with diabetes or obesity have elevated circulating levels of markers of inflammation, e.g., C-reactive protein, tumor necrosis factor α, interleukin 6 (IL-6), and ICAM-1. Moreover, elevated levels of pro-inflammatory substances predict vascular complications in diabetics[2].

Activation of protein kinase CActivation of protein kinase C beta can be the cause of the association between inflammation, endothelial damage, and IR in DM[2].

Mitochondrial dysfunctionRecent works relationship between endothelial dysfunction and impaired mitochondrial biogenesis in subjects with DM[2].

ENDOTHELIAL MARKERS OF DM2vWFvWF represents a marker of the activation of the endothelium[8]. It is synthesised, stored and secreted both by megakaryocytes/platelets and by endothelial cells[9]. vWF takes part in the processes of platelet adhesion, platelet aggregation and acts as a plasma carrier for factor VIII, thus enabling its stability in the circulation. Its role in platelet adhesion is particularly

Kubisz P et al . Endothelial and platelet markers in diabetes


important under conditions of high shear stress[10] (Figure 1).

According to the results of the Framingham Off-spring Study there was strong evidence that vWF is an independent risk factor for DM (relative risk = 1.37, 95%CI: 1.07-1.75; P = 0.01)[12]. vWF level is already increased in NAU individuals, while in a few studies vWF level was increased firstly in microalbuminuric (MAU) individuals[8]. For instance, Galajda et al[13] have found significant increase of vWF in DM2 subjects without vascular impairment comparing to healthy individuals (1.33 ± 0.39 IU/mL vs 1.01 ± 0.27 IU/mL, P < 0.01)[13].

On the contrary, in the study of Kubisz et al[8] circulating vWF levels did not significantly increase in diabetic subgroups when comparing with the controls. Since then, many studies have confirmed no or no independent link between vWF and DM[14,15]. This can be explained by the fact that the association between vWF and DM relates to the concentrations of the proinflammatory cytokine IL-6. Thus, vWF as a product of the acute phase response is associated with the risk of DM complications, but the relation between vWF and DM is probably indirect[14,16].

Thrombomodulin Plasma (soluble) thrombomodulin (TM) is a marker of endothelial damage[8]. TM represents major substance of the protein C anticoagulant system[17].

It was suggested that the elevation of plasma TM concentration in patients with DM2 could be the consequence of widespread vascular damage in diabetic patients with incipient nephropathy[18]. Actually, vWF and TM levels are increased in group of subjects with DM2 and endothelial dysfunction[19].

Widespread endothelial dysfunction is present in diabetic nephropathy. Hence, plasma vWF and TM represent valuable markers of endothelial dysfunction potentially useful in early confirmation of diabetic

microvascular complications[20]. Surprisingly, our recent study found soluble TM more sensitive marker than vWF in individuals diagnosed DM2 with both, the normo- and microalbuminuria (TM P < 0.0001 in NAU, P < 0.005 in MAU)[8]. Galajda et al[13] reported on the contrary to the previous studies, when comparing the DM2 patients without vasculopathy and control group, the concentrations of TM as calcium-independent marker of endothelial injury to be similar. This is the reason to assume that only elevated concentrations of intracellular calcium (Cai)-depending endothelial and platelet indicators can predict the dysfunction of the endothelium[13].

Thrombin-activatable fibrinolysis inhibitorThrombin-activatable fibrinolysis inhibitor (TAFI) removes C-terminal lysine part from fibrin and inhibits plasminogen activation. Taking all the facts into the consideration, TAFI represents a substantial link between coagulation and fibrinolytic processes[7]. Positive correlation between TAFI and F1 + 2 in MAU group (r = 0.427, P = 0.001) as well as TAFI and fibrinogen in the NAU group confirmed activation of TAFI in the course of stimulated coagulation[6].

TAFI antigen levels and activity are significantly (P < 0.05) increased in diabetics when comparing with healthy subjects. Inverse and significant correlation of TAFI antigen and D-dimers was found in diabetic subjects supporting the function of TAFI in diabetes-induced inhibition of fibrinolytic activity[6,7,21]. Elevated levels of TAFI may also be important in endothelial injury in MAU individuals[6,8].

A significantly elevated plasma level of TAFI was present in diabetics with microalbuminuria when compared with diabetics with normoalbuminuria (P ≤ 0.001)[8]. Progression of DM2 therefore contributes to marked TAFI mediated downregulation of fibrinolytic activity. It is also suggested that fibrinolysis inhibition can be mediated by TAFI in early stages of diabetes


Platelet

GPⅡb/Ⅲa GPⅡb/Ⅲa

vWFAggregation

Adhesion

Gplb bindinginduces CpⅡb/ⅢaexpressionGpIb GpIb

Endothelium Endothelium

Subendothelialcolagen receptorsexposed at injury site

Endothelium

Platelet

GPⅡb/Ⅲa GPⅡb/Ⅲa

vWF

vWF vWF

Figure 1 The role of von Willebrand factor in the platelet adhesion to the damaged vessel wall and the subsequent platelet aggregation. Adapted from: Trombose[11]. vWF: Von Willebrand factor; GP: Glycoprotein.


and body mass index (r = 0.43, P < 0.05) in the MAU patients and between PAI-1 and triglycerides (TAG) (r = 0.67, P = 0.01) in NAU subjects, proving that obesity, dyslipidaemia and decreased fibrinolytic activity are related to each other[6,7]. Subjects with DM2 and hyperinsulinaemia non-diabetics had significantly increased PAI-1 levels which correlated with the C-peptide (r = 0.519, P < 0.001), TAG (r = 0.685, P < 0.001), body mass index (r = 0.607, P < 0.001) and with levels vWF and soluble TM[25].

Moreover, increased PAI-1 levels are an independent risk factor for DM2. It was proposed that a a decrease of PAI-1 level may be linked with a decrease in trans-formation to DM2. On the other hand, the finding of elevation of PAI-1 preceding DM2 thus being, in the preexistence of IR, independent of glycaemia gives unquestionable evidence that abnormality of fibrinolytic system characterized with increased PAI-1 and tPA antigen occurs nearly at the beginning of the development of metabolic disorders and is considered a risk factor for future DM and metabolic complications[7].

Tissue factor pathway inhibitor Tissue factor pathway inhibitor (TFPI) acts as an inhibitor of tissue factor (TF)-initiated coagulation. TF binds to the activated factor X, subsequently TFPI-Xa complex binds to the TF/factor Ⅶa (FⅦa) complex and regulates its action[26].

Enhanced TFPI activity was confirmed in individuals with DM and mainly in those with microalbuminuria. Increases in TFPI reflect endothelial dysfunction or impaired binding of TFPI to endothelial cells by glucosa-minoglycans because TFPI is predominantly synthesized in endothelial cells[26]. It may be the first consequence of increased glycosylation, and thus the deterioration of the antithrombotic potential of endothelial cells. Mentioned increase in TFPI activity does not compensate the procoagulant state associated with increased thrombinogenesis (excess of F1 + 2), and inversely correlates with the internal thrombin potential[27,28]. Plasma levels of TF, TFPI and FⅦa are significantly increased in DM2[26].

Total TFPI (t-TFPI) has a poor anticoagulant effect. On the contrary, free form of TFPI (f-TFPI) has an increased anticoagulant capacity. Moreover, the study of Morange et al[29] showed that f-TFPI has a strong correlation with endothelial markers and t-TFPI has increased relation to parameters indicating cardiovascular risk. Normal values of f-TFPI varying according to gender, as well as informative normal values of t-TFPI can be seen in Table 1.

tPA tPA is a serine protease which converts plasminogen to plasmin, the form active in the process of fibrinolysis, thus being the most valuable initiator of fibrinolytic process. tPA is synthesised particularly in the endothelial cells, smooth muscle cells, monocytes and megakar-yocytes[6,7]. Quick tPA release to the circulation occurs

defined by microalbuminuria in DM2 subjects without macrovascular complications. It seems that discussed process is independent from the level of plasminogen activator inhibitor-1 (PAI-1)[6,8].

Elevated circulating levels of TAFI were present not only in individuals diagnosed DM2, but also in patients with obesity, IR and arterial hypertension[8]. On the contrary, increased total cholesterol level probably downregulates TAFI[6].

PAI-1 PAI-1 represents one of the most significant and quick natural inhibitors of tissue plasminogen activator (tPA) and urokinase-type plasminogen activator[7].

PAI-1 is produced in vascular and metabolic tissues[7]. Therefore, up-regulation of PAI-1 occurs predominantly in obese patients and presence of endothelial dysfunction[6].

PAI-1 is released to plasma predominantly from the endothelial cells[7]. This is the cause of the elevation of PAI-1 concentrations in individuals with DM2 and hypertension with endothelial dysfunction[6,22]. The inflammatory cytokines, as well as metabolic components are activators of PAI-1 production in the endothelial tissue[7,23]. Insulin has inhibiting influence on the PAI-1 synthesis in endothelial cells. Significant decrease of PAI-1 level in DM2 individuals with endothelial impair-ment on long-term insulin therapy was confirmed[7,24]. However, the mechanism of insulins action depend upon the metabolism in hepatic cells and endothelium[7].

PAI-1 overproduction belongs to the most typical signs of thrombophilia in DM2 leading to the dysfunction of fibrinolytic system[7]. Plasma levels of PAI-1 are significantly increased in both cohorts (MAU and NAU) compared with the controls (P < 0.0001 in both subgroups)[8].

When comparing with other parameters, PAI-1 is the most complex one associated with IR. Level of PAI-1 highly correlates with parameters of the IR syndrome in healthy individuals, patients with IR, DM2, or subjects with coronary artery disease. Positive correlation was found between PAI-1 concentrations


Table 1 The most important endothelial markers of diabetes mellitus type 2 with the method used for their detection

Endothelial markers Method of detection Normal values

PAI-1 antigen ELISA 5-40 ng/mLTAFI antigen ELISA 5.8-10.0 μg/mL

(100-172 nmol/L)t-TFPI antigen EIA 70 (40-110) ng/mLf-TFPI (men) EIA 16 ± 4 ng/mLf-TFPI (women) EIA 16 ± 3 ng/mLTM antigen EIA 10-50 ng/mLtPA antigen ELISA 1-20 ng/mLvWF antigen EIA Less than 1.4 IU/mL (140%)

EIA: Enzyme immunoassay; ELISA: Enzyme-linked immunosorbent assay; PAI-1: Plasminogen activator inhibitor 1; TAFI: Thrombin-activatable fibrinolysis inhibitor; f-TFPI: Free tissue factor pathway inhibitor; t-TFPI: Total tissue factor pathway inhibitor; TM: Thrombomodulin; tPA: Tissue plasminogen activator; vWF: Von Willebrand factor.


following the Cai - dependent stimulus. Minority of tPA is present in plasma in the form of complex binding PAI-1[7].

tPA is present mainly on the endothelial surface and may be released to circulation following its injury[7]. tPA levels are elevated in subjects with DM and metabolic syndrome. Increased tPA concentrations were proposed as a risk factor of DM in healthy subjects, because total tPA antigen levels were elevated in DM, but the level of free tPA and the tPA activity not. Therefore, the elevated total tPA is a facade masking the dysfunctional fibrinolysis in DM2[6]. Moreover, an elevated tPA antigen concentration is a substantial feature of the IR syndrome and also relates to the inflammation. tPA antigen levels have strong correlations with IR, and can serve as helpful markers of diabetes[14]. The increased tPA levels correlate with vWF and soluble TM in individuals with various locations of atherosclerotic lesions. This supports the position of tPA as an indicator of endothelial injury[7].

The tPA levels were described to be increased in DM2 in an early phase of disease while tPA levels in DM1 are increased later in presence of vascular complications. Dependence between tPA levels and PAI-1 and IR is higher in the phase of complications than dependence between tPA and presence of complications in DM2. There is a correlation between tPA and PAI-1 found in patients with DM2 and the principle cause of the tPA increase is formation of the tPA/PAI-1 complexes[7].

As it was mentioned earlier, insulin can inhibit the endothelial production of PAI-1 with the following decrease of tPA/PAI-1 complexes levels but it does not influence the tPA release from endothelium and tPA plasma levels. This can be explanation of the absent correlation between tPA and PAI-1 found in DM2 patients treated with insulin[7,25].

E-selectin E-selectin (CD62E), expressed by endothelium, is rapidly enhanced by proinflammatory molecules[30].

At first the adhesion molecules were considered to be endothelial markers because of their general elevation in various vasculopathies[25]. It was confirmed that individuals with increased E-selectin had a significantly higher probability of the development of DM2[4]. Moreover, in the study of Thorand et al[15] soluble E-selectin and ICAM-1 significantly preceded DM2. In particular, soluble E-selectin remained an independent marker of diabetes after exclusion of inflammation, levels of insulin and hemoglobin A1c in both men and women[15,30].

ICAM-1 and vascular cell adhesion molecule 1 ICAM-1 and vascular cell adhesion molecule 1 (VCAM-1) are synthesised in the endothelium and leukocytes. Increased concentrations of these cell adhesion molecules (CAMs) are early markers of endothelial dysfunction, cardiovascular disease and DM2[30].

Increased concentrations of E-selectin, ICAM-1,

and VCAM-1 raise the relative risk of DM by 1.5- to 7.5-fold[30]. Taking everything into account, elevated concentrations of CAMs representing an increased probability of the development of diabetes highlight the link between dysfunction of the endothelium and IR[15].

Vascular endothelial growth factor Vascular endothelial growth factor (VEGF) is a growth factor derived from endothelial cells, stimulating their proliferation, differentiation and survival, mediating endothelium-dependent vasodilatation, inducing increased permeability in the microcirculation and parti-cipating in other supporting functions. VEGF enhances the glomerular permeability to macromolecules and leads to profound proteinuria including albuminuria. Described action has probably a role in the development of endothelial dysfunction in diabetes[8]. The beneficial effects of VEGF-A (principal member of VEGF family) inhibition in the early phases of diabetic glomerulopathy was also confirmed[31].

Highly increased plasma VEGF levels were measured in individuals with retinopathy and nephropathy when compared with the control group. Widespread endothelial dysfunction occurs at the beginning of the development of diabetic nephropathy and precedes biochemical finding of the disorder of renal functions since the significantly increased VEGF level was present already in NAU diabetics without manifestations of microvascular impairment. Because VEGF levels were significantly elevated in the NAU cohort and had a tendency to be significant in the MAU patients, VEGF can be concluded as an indicator of diabetic endotheliopathy. VEGF ought to be evaluated only along with further markers representing the dysfunction of the endothelium, particularly soluble TM[8]. It is assumed that VEGF may be a more sensitive indicator of renal changes than microalbuminuria[8,32]. On the other hand, since there were no marked differences in VEGF concentrations in individuals with DM with physiological renal functions and at the beginning of the development of diabetic nephropathy, VEGF was not confirmed as a reliable marker of the progression in DM2 patients[8].

ET-1ET-1 is considered to be the most potent vasoconstrictor form of endothelin and is produced by the endothelial cells. During prolonged periods of stress caused by hyperglycaemia and IR in the course of DM2, there is a shift in the balance with more vasoconstrictors being produced compared to vasodilating agents. This leads to endothelial dysfunction and affects the permeability of the glomerular filtration barrier leading to microalbuminuria[33].

Even experimental studies suggested that ET-1 drives development of glomerulosclerosis and podocyte loss[34]. ET-1 is really significantly associated with early development of the endothelial dysfunction in the



glomerulus[33].

MECHANISMS OF DIABETIC THROMBOCYTOPATHY Primary dysfunction of megakaryocyte-platelet system One of the manifestations of DM is the production of subpopulation of large and hyperreactive platelets in the bone marrow. Thrombocytopoesis is increased[35,36]. IR in plateletsIn obesity and DM2 increased aggregability of platelets, as well as decreased sensitivity to antiplatelet effect of insulin was confirmed[36].

Influence of endothelial dysfunctionSubstances produced by the endothelium, represented by nitric oxide, prostacyclin and adenosine normally inhibit platelets. In diabetic patients due to the hyperglycaemia and oxidative stress the capacity of endothelium to produce prostacyclin and nitric oxide is decreased. In the cases of microangiopathy consumption of platelets in the sites of damaged endothelium causes paradoxically the decrease in the subpopulation of large activated platelets[36].

Changes of the activity of membrane pumps and increase of Cai in platelets[36] Platelet markers of DM2: Platelets have an ethiopathogenic relation to the microangiopathy in DM. Their increased activity was confirmed in both types of

DM[36,37]. The structure of the platelet with components involved in its physiological function and markers of platelet activity are illustrated in Figure 2.

Mean platelet volumeMean platelet volume (MPV) is the parameter informing about the platelet size and activation Bigger platelets indicate more activity[38]. Patients with DM have significantly higher MPV due to the elevated alpha-granule content in cytoplasm as the sign of dysfunction of megakaryocyte-platelet system[36,38]. Thus, platelets can be a useful tool to evaluate complications in DM[38]. Further studies concluded that MPV is strongly and independently associated with the presence and severity of diabetes and higher MPV is the manifestation of early diabetic thrombocytopathy in both types of DM without vascular complications[39,40].

Soluble P-selectin P-selectin (CD62P) is a highly glycosylated membrane glycoprotein constitutively expressed in endothelial cells and megakaryocytes and present in the membrane of Weibel-Palade bodies of endothelium and in α granules of platelets[36,41]. It is a marker of platelets’ activation. Its increased levels in patients with arterial hypertension, lower extremity peripheral artery disease and coronary artery disease correlate with levels of β-TG, and they do not have a relation to vWF and TM[42-44]. Increased levels of P-selectin occured already in early stages of DM1 and DM2 without vascular complications. Elevated levels of P-selectin were also found in subjects diagnosed IR and impaired glucose tolerance[45-47].

Elevated synthesis of P-selectin and ICAM-1 in diabetic human retina and choroid was confirmed. This is the reason why circulating levels of soluble adhesion molecules have been proposed as biomarkers playing a significant role in the pathogenesis of macrovascular and microvascular dysfunction and neuropathy of DM[48]. Galajda et al[45] found in patients with DM2 significantly elevated P-selectin that paradoxically did not correlate with levels of PF4, but they correlated with vWF. This can propose also its function in the endothelial dysfunction in patients with DM2[45].

P-selectin was significantly increased in patients with DM suffering myocardial infarction than in control group or those without myocardial infarction. It indi-cates contribution of elevated levels of P-selectin to the atherosclerosis leading to coronary heart disease in diabetics[49]. Thus, detection of P-selectin level could be useful marker of possible cardiovascular event in individuals with DM[50].

PF4PF4 is a basic protein found exclusively in α granules of platelets. After release it is able to modulate haemostasis. PF4 has both procoagulant and anticoagulant properties. It has also angiostatic effect - it inhibits proliferation and migration of endothelial cells[36].


Alpha granule

Dense granule

Canalicularsystem

vWFfibrinogenTSPPF4

PDGFβ-TGfactor V

Fibrinogen

GP Ⅱ

b/Ⅲ

a

GPⅡb/ⅢaADP ATPcalcium ++serotonin

Exposedcolagen

GPIb/Ⅸ/Ⅴ

vWF

Area of denuded endotheliumBasement menbrane Vascular endothelial cell

Figure 2 The structure of the platelet and its most important functions, including the secretion of stored products, as well as the binding of specific surface glycoproteins to the endothelium (bottom), and other platelet (left side). Adapted from: Trombose[11]. ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; β-TG: Beta-thromboglobulin; GP: Glycoprotein; PDGF: Platelet-derived growth factor; PF4: Platelet factor 4; TSP: Thrombospondin; vWF: Von Willebrand factor.


In patients with both types of DM the increased levels of PF4 and β-TG as the manifestation of degranulation of activated platelets without influence on their lifespan were confirmed. In DM2 these levels are increased already in the early stage of the disease without the presence of vascular complications[36].

In the study of Galajda et al[13] the correlation between vWF concentrations and calcium dependent substances as C-peptide (r = 0.680, P < 0.001) and PF4 (r = 0.613, P < 0.01) and no correlation with calcium-independent markers of endothelial damage, as TM (r = 0.287, P = 0.196) or TFPI (r = 0.296, P = 0.181) in diabetics was confirmed[13]. In advanced cases of DM associated with retinopathy and nephropathy increased levels of PF4, β-TG, P-selectin, Cai in platelets, as well as their increased aggregability were confirmed[36]. Moreover, patients with diabetes type 2 treated with sulphonylurea preparates and individuals treated 2-3 mo with insulin did not differ by endothelial and platelet parameters (vWF, TM and PF4)[7].

β-TG β-TG is a protein with 50% structural homology with PF4 acting as a leukocyte chemoattractant. Measurement of β-TG is considered to be a golden standard in the detection of activation of platelets in patients with normal renal function. Its levels are increased in renal insufficiency and correlate with levels of PF4[39].

Glycoprotein V (CD42d)Glycoprotein V (GPV) is exclusively expressed in platelets and megakaryocytes, where it is non-covalently bound to the complex GPIb/GPIX as the part of the receptor for vWF and thrombin[51].

According to the fact that platelets are the only source of GPV it can be considered as a marker of specific activation of platelets by thrombin[51].

TXB2Metabolism of arachidonic acid with incorporation of its metabolites to membrane phospholipids of platelets in DM is upregulated. Production of TXA2 in platelets is increased. Plasma levels of its stable catabolite TXB2 produced in liver, kidneys, platelets and lungs are also

increased and correlate with raised levels of β-TG and PF4[52].

Increased synthesis of TXA2 indicates that “resting” platelets of diabetics in general tend to be activated. TXA2 is a vasoconstrictor and prothrombotic factor with the risk of atherothrombogenesis present in DM[53].

Moreover, synthesis of TXB2 has inverse correlation with the minimal level of arachidonic acid inevitable to 50% platelet aggregation. As the consequence, platelet aggregates in the circulation of patients with DM2 than in the healthy subjects. Platelets of diabetics therefore exhibit the signs of hyperactivity. This fact may also be very important for the arise of vascular disease in DM[52].

Thrombospondin-1This molecule represents a multifunctional glycoprotein synthesised in a wide range of cell types, to be more exact in platelets (increased expression), vascular smooth muscle cells and various kinds of renal cells[53]. Unlike PF4 and β-TG, after release of thrombospondin (TSP) from activated platelets it binds to platelet surface, so levels of TSP-1, PF4 and β-TG usually do not correlate with each other. Last but not at least, its levels are influenced by hepatopathy. These are the reasons why analysis of TSP is not considered to be the golden standard in detection of platelet activity. Therefore, TSP is evaluated as the sign of activation of the whole vascular system[36].

TSP-1 with antiangiogenic and proatherogenic properties is involved in diabetic vasculopathy. The endothelial dysfunction in DM was confirmed in many studies, and TSP-1 surely may contribute to this impaired function because it shows antiproliferative and apoptotic effect on the endothelium. It was confirmed that TSP-1 level has a relation with renal damage and vascular impairment[53].

DETECTION OF MARKERS OF DM2Informative normal values of the most important endothelial and platelet markers with the method used for their detection are documented in Tables 1[36,54-56] and 2[36].

Size of the platelet up to 7 μm and its volume more than 10 fl contribute to increased MPV[36].

Levels of soluble P-selectin are measured using enzyme immunoassay method and their normal values vary between laboratories and are dependent on the commercial kit used for the detection[36].

Values of β-TG 50 ng/mL and more are considered to be pathological[36].

Plasma levels of VEGF, F1 + 2 and TXB2 can also be detected using enzyme-linked immunosorbent assay[6,8,57].

CONCLUSION Number of patients with DM2 is increasing incredibly and is expected to rise even in the future. Researchers


Table 2 The most important platelet markers of diabetes mellitus type 2 with the method used for their detection

Platelet markers Method of detection Normal values

βTG EIA/RIA Less than 40 ng/mLGPV EIA 80 (40-160) ng/mLMPV Electric impedance or

laser diffraction6.5 (4.5-8.5) fl

PF4 EIA/RIA Less than 10 ng/mLTSP-1 EIA 40 (10-90) ng/mL,

respectively 20-300 ng/mL

βTG: Beta-thromboglobulin; EIA: Enzyme immunoassay; GPV: Glycoprotein V; MPV: Mean platelet volume; PF4: Platelet factor 4; RIA: Radioimmunoassay; TSP-1: Thrombospondin 1.


all over the world have made strong efforts to increase knowledge and improve understanding of mechanisms of abnormalities of endothelium and platelets unquestionably involved in the pathogenesis of this challenging health problem. As a natural consequence of continuous investigation, many markers of diabetic thrombocytopathy and endothelial dysfunction have been identified and are considered for implementation in clinical practice. These substances may improve the screening of high-risk individuals and lead to the early diagnosis, appropriate treatment, as well as to the effective prevention of the complications of DM2. However, these conclusions have to be confirmed by further research.

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P- Reviewer: Pistrosch F, Rezzani R, Taguchi I S- Editor: Ji FF L- Editor: A E- Editor: Liu SQ



neuropathy, improving glycemic control as a prophylactic therapy and using medications to alleviate pain. First line drugs for pain relief include anticonvulsants, such as pregabalin and gabapentin and antidepressants, especially those that act to inhibit the reuptake of serotonin and noradrenaline. In addition, there is experimental and clinical evidence that opioids can be helpful in pain control, mainly if associated with first line drugs. Other agents, including for topical application, such as capsaicin cream and lidocaine patches, have also been proposed to be useful as adjuvants in the control of diabetic neuropathic pain, but the clinical evidence is insufficient to support their use. In conclusion, a better understanding of the mechanisms underlying diabetic neuropathic pain will contribute to the search of new therapies, but also to the improvement of the guidelines to optimize pain control with the drugs currently available. Key words: Diabetes; Neuropathic pain; Hyperglycemia; Anticonvulsants; Antidepressants


Core tip: Diabetic neuropathic pain is a common complication of diabetes and the most common form of neuropathic pain. In this review, we will discuss the various factors that may contribute to the pathogenesis of diabetic neuropathic pain, including metabolic, vascular, autoimmune and oxidative stress-related mechanisms. In addition, we will review the possibilities of pain treatment, taken into consideration the first line drugs clinically used, the antidepressants and anticonvulsants, but also other options such as opioids, tapentadol and drugs for topical use, such as lidocaine and capsaicin cream.

Schreiber AK, Nones CFM, Reis RC, Chichorro JG, Cunha JM. Diabetic neuropathic pain: Physiopathology and treatment. World J Diabetes 2015; 6(3): 432-444 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/432.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.432

Anne K Schreiber, Carina FM Nones, Renata C Reis, Juliana G Chichorro, Joice M Cunha

Anne K Schreiber, Carina FM Nones, Renata C Reis, Juliana G Chichorro, Joice M Cunha, Department of Pharmacology, Biological Sciences Sector, Federal University of Parana, Curitiba 81540-970, BrazilAuthor contributions: All authors contributed equally to this work.Conflict-of-interest: None.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Dr. Joice M Cunha, Professor, Department of Pharmacology, Biological Sciences Sector, Federal University of Parana, Rua XV de Novembro, 1299 - Centro, Curitiba 81540-970, Brazil. [email protected]: +55-41-33611720Fax: +55-41-32662042Received: August 29, 2014Peer-review started: August 30, 2014First decision: November 14, 2014Revised: November 26, 2014Accepted: February 4, 2015Article in press: February 9, 2015Published online: April 15, 2015

AbstractDiabetic neuropathy is a common complication of both type 1 and type 2 diabetes, which affects over 90% of the diabetic patients. Although pain is one of the main symptoms of diabetic neuropathy, its pathophysiolo-gical mechanisms are not yet fully known. It is widely accepted that the toxic effects of hyperglycemia play an important role in the development of this complication, but several other hypotheses have been postulated. The management of diabetic neuropathic pain consists basically in excluding other causes of painful peripheral

REVIEW


Diabetic neuropathic pain: Physiopathology and treatment




INTRODUCTIONAccording to the International Diabetes Federation, 382 million people worldwide are currently affected by diabetes[1], one of the leading causes of neuropathy[2]. The distal symmetrical polyneuropathy (DSPN) is the commonest clinical form of diabetic neuropathy, affecting more than 90% of the patients[3]. Generally, DSPN affects the toes and distal foot, but slowly progresses proximally to involve the feet and legs in a stocking distribution. It is also characterized by a progressive loss of nerve fibers affecting both the autonomic and somatic divisions, thereby diabetic retinopathy and nephropathy can occur[3]. Foot ulceration and painful neuropathy are the main clinical consequences of DSPN, linked with higher morbidity and mortality[4]. Frequently, patients look for medical help only when pain appears[5], a symptom that affects 10% to 26% of this population[6,7].

Diabetic neuropathic pain (DNP) is characterized by tingling, burning, sharp, shooting, and lancinating or even as electric shock sensations[3,8]. It is usually considered moderate to severe and often worse at night, causing sleeping disturbs. The pain can be constant and accompanied of cutaneous allodynia, which can substantially affect the quality of life of patients, impacting the ability to perform daily activities and having a negative influence on mood. The pain may also be a reason of withdrawal of recreational and social activities and may be associated with depression[3,9,10].

The pathogenesis of DNP is not fully understood. Several theories have been proposed to explain the pain related to the diabetic neuropathy, such as changes in the blood vessels that supply the peripheral nerves; metabolic and autoimmune disorders accompanied by glial cell activation, changes in sodium and calcium channels expression and more recently, central pain mechanisms, such as increased thalamic vascularity and imbalance of the facilitatory/inhibitory descending pathways[3]. Additionally, several risk factors are associated with DNP including worsening glucose tolerance, older age, longer diabetes duration, drinking alcohol and cigarette smoking[10].

Currently, only three agents are approved in the United States for the treatment of DNP: duloxetine, a selective serotonin and norepinephrine reuptake inhibitor, pregabalin, an anticonvulsant, and the dualeffect drug tapentadol, an opioid receptor agonist and norepinephrine reuptake inhibitor[11]. However, as pain relief is unsatisfactory for most patients, several pharmacological interventions have been used based on preclinical and/or clinical evidence, as well as an inference of mechanism of action.

PHYSIOPATHOLOGY OF NEUROPATHIC PAIN IN DIABETESAlthough there is a great advance in understanding

the pathophysiological mechanisms leading to the development of diabetic complications, there is not yet a plausible hypothesis to explain why some patients develop the painful form of disease while others do not. In general, researchers seek to elucidate neuropathy underlying mechanisms as a bigger event, and include pain and other sensorial manifestations as direct consequences of neuropathy. However, interestingly, pain intensity normally is not associated with neuropathy severity, and can occur even in the absence of nerve injuries[12,13]. In this review, it will be addressed the pathophysiological mechanisms currently believed to promote the DNP.

In this sense, the mechanisms that lead to DNP are not fully understood, although there is a consensus that toxic effects of hyperglycemia represent an important factor for the development of this complication[14,15]. Nonetheless, other factors besides hyperglycemia should not be discarded[16], and will be discussed as follows.

Polyol pathway hyperactivityMetabolic disorders are the primary cause of diabetic neuropathy. Hyperglycemia, induced through decreased of insulin secretion or insulin resistance, is responsible for the enhanced of the polyol pathway activity. The ratelimiting first enzyme of this pathway aldose reductase catalyzes the formation of sorbitol from glucose, with the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+. Sorbitol is further oxidized to fructose by sorbitol dehydrogenase, which is coupled with the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. It is described that during hyperglycemic states, the affinity of aldose reductase for glucose is higher, generating intracellular osmotic stress due to accumulation of sorbitol, since sorbitol does not cross cell membranes. Interesting, the nerve damage following the diabetic state seems not to be due to this osmotic stress since it has been reported insignificant sorbitol concentrations in the nerves of diabetic patients[1719]. However, the current accepted hypothesis states that polyol pathway hyperactivity is pathogenic primarily by increasing the turnover of cofactors such as NADPH and NAD+, which leads to a decrease in the reduction and regeneration of glutathione, as well as to an increase of advanced glycation end products (AGEs) production and activation of diacylglycerol and protein kinase C (PKC) isoforms. Depletion of glutathione could be the primary cause of oxidative stress and be related to the accumulation of toxic species[19]. In fact, aldose reductase inhibitors are effective in preventing the development of diabetic neuropathy in animal models, but they have demonstrated disappointing results and doselimiting toxicity in human trials[20].

Oxidative and nitrosative stressAs already mentioned, the polyol pathway activation could be the primary cause of oxidative stress associated

Schreiber AK et al . Diabetic neuropathic pain


with diabetes. However, oxidative stress could be also initiated by autoxidation of glucose and their metabolites, increased intracellular formation of AGEs, increased expression of the receptor for AGEs and its activating ligands, altered mitochondrial function, activation of PKC isoforms and overactivity of the hexosamine pathway[2123]. Moreover, there is mounting evidence that oxidative stress is caused by enhanced free radical formation due to glucose metabolism itself and/or deficits in antioxidant defense and it may play a major role among the putative pathogenic mechanisms of diabetic neuropathy. It seems that, in addition to oxidative stress, reactive nitrogen species, especially the peroxynitrite also play an important role in the development of diabetes and its complications[2426]. Although it has been clearly demonstrated significant changes in oxidative status in animal models of diabetes[27], tissue concentrations of known carbonyl compounds are nearly negligible and plasma ET1, nitric oxide, catalase and glutathione levels did not differ in neuropathic diabetic patients when compared to nonneuropathic diabetic ones[28]. In line with this observation, clinical results have been contradictory for antioxidants as alpha lipoic acid, ranging from little benefit[29,30] to interesting advantages[31,32].

Microvascular changesDNP is frequently associated with microvascular impairment[33,34]. In clinical and preclinical studies, it was found that peripheral perfusion is reduced, not only in the nervous tissue[35,36], but also in the skin[37], being an important physiological evidence of microvasculature alteration. As a result, nerve ischemia occurs, caused by raise in wall thickness and hyalinization of the basal lamina of vessels that nurse peripheral nerves[38,39], together with luminal reduction[38]. These alterations are caused by plasma protein scape of capillary membrane to endoneurium, promoting swelling and augmented interstitial pressure in the nerves, accompanied by higher capillary pressure, deposition of fibrin and thrombus development[40]. Hyperglycemia per se can evoke nerve hypoxia, especially in sensory nerves, altering their electrical stability[41]. Apparently controversial data from clinical studies described that diabetic patients suffering from the DNP presented higher levels of intravascular oxygen and augmented blood flow in the lower limbs than painless patients. Nevertheless, authors still consider a hypoxic state inside the endoneurium[42]. Alternatively, a potential sympathetic dysfunction can be the cause of higher blood flow[43].

As a result of nerve ischemia, both diabetic patients and animals have shown a progressive nerve loss in proximal and distal segments[44,45], resulting in reduction of intraepidermal nerve fiber density[12]. Consequently, axonal degeneration and regeneration also occurs, but more frequently in patients that do not experience pain. Besides axonal retraction and regeneration, another structural modification related to hyperglycemia is myelin sheath alteration. The observed demyelinization

can be related to Schwann cells altered capacity to support normal myelin sheath[46].

It is also important to point out that endothelial function in patients with DNP is also altered. The vaso dilatation induced by acetylcholine (i.e., the endotheliumdependent response) in dermal vessels of diabetic patients was reduced in comparison with healthy volunteers. In addition, vasoconstriction mediate by the sympathetic system (i.e., endotheliumindependent response) was also defective, what can also be implicated in the pathophysiology of diabetic neuropathy and then, in the DNP[47].

It is believed that one potential cause of the microvascular changes described above may be the oxidative stress, since the treatment with antioxidant agents can maintain regular perfusion, restoring sensory transmission in type 1 diabetes model[48].

Channels sprouting Damaged nerve endings are believed to contribute to pain in DNP[49,50]. The most accepted hypothesis states that disturbed action potentials can be produced by damaged nerve endings, being interpreted by central nervous system (CNS) as pain or dysesthesias[51]. Changes in ion channel expression in peripheral fibers are direct consequences of nerve injury, leading to hyperexcitability[52], that is far linked with neuropathic pain[53].

In this regard, upregulation of voltagegated sodium channels (Nav) has been widely demonstrated in neuropathic pain models[54,55]. These channels are involved in generation and transmission of action potential, and can be classified into sensitive (TTXS) or resistant (TTXR) to tetrodotoxin[56]. There are several reports that the TTXS channels Nav1.3, which primary function relays during the embrionary development[57], and Nav1.7, that is constitutively expressed in peripheral sensory neurons[58], are both upregulated in the dorsal root ganglion (DRG) of diabetic animals[5961]. Nav1.3 expression was also found increased in small and large diameter DRG neurons of diabetic rats presenting allodynia[62]. Considering TTXR sodium channels, Nav1.8 and Nav1.9 are normally expressed in peripheral nociceptive neurons[63], playing an important role in the generation of electrical activity in DRG[64]. Intriguingly, DRGs of allodynic diabetic rats showed a reduction of Nav1.8 expression in the following days after diabetes induction[62], and this reduction lasted 6 mo post diabetes induction[61], indicating that other sodium channels may play an important role in DNP. The same reduction was detected for Nav1.6, another TTXS channel, normally present at Ranvier nodes[60,62].

In addition, an increase of Nav1.6, Nav1.7 and Nav1.8 phosphorylation is another feature observed in the diabetic state, which leads to augmentation in their activity. Thus, both abnormal expression and function of TTXR and TTXS sodium channels is linked to abnormal activity of nociceptive fibers[60]. In this way, Sun et al[65] showed that TTXS and TTXR sodium



and selectively reverses hyperalgesia in a preclinical model of type 2 diabetes[80].

Resting membrane potential can also be altered by K+ voltage dependent channels (Kv), also participating in the electrical properties of neurons[81]. Regarding the currents generated by activation of these Kv channels in primary afferents there are two main types: rapidly inactivating Atype currents (IA), and slowly inactivating currents (IK)[82,83]. It was verified that the total density of Kv currents as well as the mRNA of IA subunits of KV 1.4, 3.4, 4.2 and 4.3 were reduced in large and medium size DRG neurons of diabetic rats[84]. So, this down regulation can increase neuronal excitability and peptide release[83], which might also participate in hyperexcitability of peripheral nerves of diabetic subjects.

Microglial activationIt is becoming increasingly recognized that glial cells play an important role in the pathogenesis of many diseases of the nervous system, including chronic pain states[85]. Glia comprises both macroglia (including astrocytes, radial cells and oligodendrocytes) and microglia cells, which are mainly responsible for maintain homeostasis, form myelin, and provide support and protection for neurons from both central and peripheral nervous system[85]. Normally, microglial cells comprise less than 20% of spinal glial cells but in response to dorsal root ganglia and spinal cord after nerve injury there is a robust proliferation at spinal level[86]. Activation of microglia occurs right after peripheral nerve injury, lasting for less than 3 mo, and is responsible for a production of several inflammatory mediators as cytokines, chemokines, and cytotoxic substances such as nitric oxide and free radicals, prompting to a pro inflammatory milieu[83]. Diabetes has impact on all glial cells of the spinal cord since persistent microglial activation was observed in streptozotocininduced diabetic rats lasting from 4 wk[87,88] to 6 or 8 mo[61,89]. This microglial activation has been associated with sensorial changes and upregulation of Nav1.3 sodium channels in the DRG[61], possible through p38 mitogen activated protein kinase dependent mechanism[90,91]. Conversely, diabetes is associated with a reduction in glial fibrillary acidic protein (i.e., glial fibrillary acidic protein) immunoreactive astrocytes in the spinal cord, which may affect the functional support and role of astrocytic cells in the nervous tissue, such as the clearance of neurotransmitters within the synaptic cleft[92]. Considering the potential of microglial activation in driving spinal sensitization, in the near future, drugs that target these cells may become an important therapeutic alternative in chronic pain control.

Central sensitization As already demonstrated in different neuropathic pain states, DNP may be a consequence of both peripheral and CNS changes[93,94]. It was well described that during DNP, primary afferents are sensitized, inducing dorsal horn hyperactivity and neuroplastic changes in

currents are increased in small neurons in the DRG of diabetic animals, being this related not only with sensory disturbances, but also with the rise of efficiency of conductance in polymodal C fibers, which in turn, facilitates nociceptive transmission.

In a recent in vitro study, it has been described that hyperglycemia evokes higher TTXR Na+ currents in a time and concentrationdependent manner, demonstrating a straight relationship between glucose levels and biophysical changes[66]. In DNP patients, it was reported an increase in nodal Na+ currents when compared to painless diabetic patients, what can also contribute to hyperexcitability in peripheral nerves[67].

A new concept proposed by Hoeijmakers et al[68], links the beginning of pancreatic beta cells failure and DNP with genetic disruptions on Nav1.7 channels. Sinceboth pancreatic beta cells and peripheral neurons express Nav1.7 channels, a susceptible genetic background could facilitate generation of Nav1.7 mutations, leading to gainoffunction that evokes beta cell lesions,and thereafter, diabetes and hyperexcitability in neurons[69]. According to these authors, this theory could explain why some patients have neuropathy before diabetes onset[68].

Another interesting finding related to sodium channels modulation is the increased levels of methylglyoxal in type 2 diabetes DNP patients, when compared with those painless[70,71], and in complicationfree type 1 diabetic patients[72]. This glycolytic metabolite can activate nerve endings through transient receptor potential cation channel subfamily A member 1 activation in the DRG[73], and also change the Nav1.7 and Nav1.8 function through posttranslational changes[70]. In line with this clinical observation, in preclinical models methylglyoxal was found to reduce nerve conduction velocity, to elevate calcitonin generelated peptide release from sensory nerves and to induce thermal and mechanical sensibility[70]. In addition, in diabetic states methylglyoxal is also involved in the formation of AGEs[74].

Calcium channels can also be misregulated in a diabetic condition, leading to an enhanced calcium influx in sensory neurons[75], what can deflagrate both substance P and glutamate release[76]. It was verified in two different animal models of insulin dependent diabetes that high voltage activated Ca2+ current amplitudes were increased in small diameter neurons[75,77] and the activity of Ttype channels (Cav3.2) is also augmented in small diameter fibers[78], what could be normalized by molecular knockdown of this calcium channel[79]. However, there was no translation of these results to patients in clinical trials[80]. A possible future target for pharmacological intervention over calcium channels has been proposed by Orestes and colleagues (2013), which observed that glycosilation of Cav3.2 augments the current density, accelerates kinetics, and also increases the number of channels on neuron membrane, which can be directly involved in DNP. Interestingly, the deglycosylation treatment with neuraminidase inhibits native calcium currents in nociceptors and completely



central sensory neurons[93]. The common occurrence of allodynia in DNP patients supports the idea that CNS pain processing is altered[95].

Among the factors that can lead to the hyperactivity of spinal neurons in diabetic neuropathy is the increased glutamate release from primary afferents in the spinal cord[96,97]. Moreover, spinal NMethylDaspartate (NMDA) receptor expression is augmented in this condition[98], generating increased and more frequent excitatory post synaptic currents in the lamina Ⅱ[97]. Additionally, it has been described that cAMP response elementbinding protein signaling, which directly regulates NMDA receptors activity[99] is enhanced in DNP[98,100]. Thus, it is plausible that augmented NMDA expression and glutamate release might contribute to spinal cord hyperactivity. On the other hand, GABAB receptors seem to be downregulated in the spinal cord in diabetic neuropathy[98]. Activation of GABAB receptors results in inhibition of NMDA receptor activity through a direct inhibition of voltagesensitive Ca2+ channels[101] and opening of inwardly rectifying K+ channels[102]. Furthermore, GABAB receptor activation also causes downregulation of NMDA receptors at the spinal level in diabetic rats[98]. Considering the importance of central sensitization in the hypersensitivity associated with DNP, strategies that aim to control spinal neurons hyperexcitability are very useful in pain control in this condition, as will be discussed bellow.

Brain plasticityFunctional changes in pain processing areas in the CNS, besides the spinal cord, have been ultimately linked with DNP[103], in a tight relation to increased peripheral input[93,104]. Among these areas, marked changes in the thalamus, cortex and rostroventromedial medulla (RVM) have been reported in DNP patients and or experimental models.

The ventral posterolateral nucleus (VPL) of the thalamus is the main receiving area of nociceptive stimuli that is processed in the spinal cord[105]. Projection neurons reach the thalamus trough the spinothalamic tract (STT), which represents a major ascending nociceptive pathway. It has been demonstrated that in diabetic rats, these neurons present increased spontaneous activity, enlargement of the receptive field and augmented responses to mechanical noxious and innocuous stimuli. The hyperexcitability of STT neurons probably accounts to hypersensitivity to external stimuli and spontaneous pain[93], increased in primary afferents activity[93,104] and to plastic changes in spinal neurons[93].

In addition, in studies that assessed brain imaging in diabetic rats it was reported increased activity not only at VPL, but also in different thalamic nuclei that control sensorymotor aspects[106]. In diabetic patients, a recent study showed increased activation of diverse brain areas, including medial thalamus after application of noxious thermal stimuli in feet[107]. Moreover, it has been described that DNP patients has a marked reduction in the levels of Nacetylaspartate (NAA) levels in the

thalamus compared to painless diabetic individuals[108]. It is important to point out that patients with brain disorders in which neuronal loss or dysfunction are involved have consistently decreases in brain NAA concentrations[109]. Other clinical finding related to thalamus alterations in diabetic patients is that subjects with painful type 1 diabetic neuropathy presented increased thalamus blood flow, when compared with those without pain, which was considered to reflect higher neuronal activity[110]. Taking account the thalamus relevance in the nociceptive pathway, it is plausible to suggest that the alterations reported in this area might contribute to the development and/or maintance of DNP[108,110].

Likewise, in a model of type 1 diabetes, increased glutamate transmission was reported in the anterior cingulate cortex a brain area involved in the processing of the affectivemotivational dimension of pain[111]. The consequence of higher stimulation of this area by glutamate is suggested to be a sustained negative perception of affective component of pain[111].

Changes in the endogenous pain control system have also been described in preclinical and clinical studies of DNP. The RVM is a structure that receives direct influences of periaqueductal gray matter, which is, in turn, affected by other structures, such as amygdala and hypothalamus[112]. Three different populations of cells have been describe within the RVM: activation of ON cells act in a pronociceptive way, while activation of OFF cells has the opposite effect[113] and neural cells which activation is still contradictory and remains to be better clarified[114,115]. In diabetic animals, there is evidence of a reduction on the OFF cells and increase on the ON cells population. In addition, basal activity is augmented in ON cells, and reduced in OFF cells, in a resultant misbalance between pain facilitatory and inhibitory descending modulation in diabetic animals[103]. After noxious mechanical stimulation in the periphery, there was no difference between diabetic and control ON cells activity. Thus, the mechanical hyperalgesia detected in diabetic rats could be associated with OFF cells impairment and consequently reduction on descending inhibitory tone[103].

Some studies have also addressed the levels of the main neurotransmitters of the endogenous pain control system in different areas of the CNS in diabetic rats, but they have shown discrepant results. While some researchers found reduced release of norepinephrine in the spinal cord in diabetic rats[116], others have described opposite findings[117]. There is also evidence of diminished norepinephrine levels in supra spinal areas, such as brainstem and thalamus, but higher concentration in the cortex of diabetic animals[118]. Additionally, impaired spinal opioidinduced release of serotonin (5HT) has been demonstrated in diabetic rats, and this finding may be related to opioid hyporesponsiveness in experimental DNP[119]. Increased norepinephrine and 5HT levels in the spinal cord, as well as, augmented expression of norepinephrine and 5HT in RVM neurons



was also demonstrated in diabetic rats[117]. Considering the facilitatory role of serotoninergic and noradrenergic descending modulation during chronic pain, these changes may probably account for enhanced pain during diabetic neuropathy[117]. There is also clinical evidence for misbalance between excitatory and inhibitory neurotransmitters in the CNS of diabetic patients with positive symptoms of neuropathy. In this regard, it has been found reduced levels of GABA and higher levels of glutamate in the posterior insula of diabetic patients, as well as a higher glutamate/GABA ratio within the thalamus[120]. These changes may contribute to pain development in DNP, but further studies are necessary to determine their clinical significance.

TREATMENT OF DNPDNP continues to represent a therapeutic challenge as its pathophysiology is not yet fully understood and pain relief is still unsatisfactory. The pharmacological treatments, with exception to those targeted to the glycemic control, are symptomatic, not focused on the pathophysiological mechanisms, limited by side effects[3,121] and by the development of tolerance[121].

A wide variety of drugs, used alone or in combination, has been shown to significantly reduce neuropathic pain compared with placebo in randomized controlled trials, but pain relief remains inadequate for most patients[122]. Generally, in clinical trials, treatment is considered successful if patients would obtain 50% of reduction in the pain level[123125] associated with some additional beneficial effects on sleep, fatigue, depression and quality of life[125]. Thus, the management of this condition basically consists of excluding other causes of painful peripheral neuropathy, improving glycemic control as a prophylactic therapy and using medications to alleviate pain[126].

Despite of multimodal and multidisciplinary approaches to the treatment, the primary pathway is pharmacologically based[127]. Three different agents have regulatory approval in the United States for the treatment of DNP: pregabalin, duloxetine and tapentadol[11,128]. However, as pain relief is still suboptimal and challenging for clinicians[95], drugs from various pharmacological classes have been used and some of them are included in this review.

AnticonvulsantsPregabalin was the first anticonvulsant to receive approval from the Food and Drug Administration (FDA) for the treatment of postherpetic neuralgia, DNP[129,130] and neuropathic pain after spinal cord injury[131]. Pregabalin is a GABA analogue that selectively binds to presynaptic voltagegated calcium channels containing the α2δ subunit in the brain and spinal cord, causing inhibition of the release of excitatory neurotransmitters[128,132]. Moreover, α2δ1 subunits are responsible for increasing the functional expression of these channels, as a consequence of increased trafficking. Thus, the analgesic

action of pregabalin is also proposed to be the result of impaired trafficking of α2δ1 subunit with a consequent diminished expression of functional calcium channels[133].

Several clinical trials evaluating pregabalin in DNP showed efficacy in the management of this condition[3,

134,135] with a number needed to treat (NNT) of 6.3[125]. In addition to its analgesic effects, pregabalin presents anxiolytic activity[132,135] and it has a beneficial effect on sleep and quality of life[132], contributing, therefore, to improve the general condition of the patients. The side effects include dizziness, somnolence, peripheral edema, headache and weight gain[3].

Some guidelines have also recommended gabapentin to treat DNP[136]. Besides pregabalin, gabapentin is the only other anticonvulsant drug that demonstrated efficacy in the treatment of this condition[128] with an NNT of 5.8[137]. Gabapentin and pregabalin have a similar mechanism of action and the first is licensed for neuropathic pain in the United Kingdom, but not in the United States[128]. Some clinical trials have suggested that gabapentin and pregabalin present better analgesic efficacy than tricyclic antidepressants or opioids[138] and other important aspects of these drugs include their tolerability and lack of serious toxicity[139].

AntidepressantsAntidepressants represent the first line drugs in DNP management. Duloxetine, a serotonin and norepinephrine reuptake inhibitor, is rated level A for efficacy and is approved in the United States for the treatment of this condition. Additionally, some clinical trials have pointed out the effectiveness of duloxetine in other chronic pain conditions, such as fibromyalgia and chronic musculoskeletal pain[140,141].

Results from a metaanalysis that included randomized, double blind, placebo controlled studies in patients with DNP showed the superiority of duloxetine over placebo in reduction of pain severity and in Patient Global Impression of Improvement/Change, as well as efficacy similar to gabapentin and pregabalin[142]. Moreover, in a 2wk openlabel randomized trial in diabetic patients poorly responsive to gabapentin, duloxetine was able to reduce the pain score to levels similar to those achieved with pregabalin[143,144]. Furthermore, analgesic efficacy of duloxetine in the treatment of DNP is maintained over a 6mo period[145], reinforcing its importance as a treatment option for this condition. The NNT for duloxetine varies from 1.3 to 5.1 in DNP patients[146,147], which experience more frequently nausea, somnolence and dizziness as side effects[146].

Venlafaxine is also a selective serotonin and noradrenaline reuptake inhibitor, that predominantly inhibits serotonin reuptake at low doses and noradrenaline at higher doses[148]. Venlafaxine was also shown to be effective in reducing pain intensity in diabetic neuropathic patients[149], with an NNT between 2.2 and 5.1 and a number needed to harm (NNH) of 9.6, to minor adverse effects, and of 16.2, for major adverse effects[150].

Tricyclic antidepressants can also be an alternative to



treat DNP[151]. Amitriptyline was shown to be as effective as gabapentin in a direct metaanalysis study[152] and as duloxetine in a randomized, doubleblind, crossover trial[153]. Likewise, nortriptyline was reported as being as effective as gabapentin in attenuating neuropathic pain in a doubleblind crossover trial enrolling diabetic patients[154].

Tricyclic antidepressants are estimated to have an NNT of 1.3[151,155], and an NNH from 4.2 to 10.7[151] in DNP. The most common side effects related to the use of these drugs are dry mouth, postural hypotension, arrhythmias, cognitive impairment, constipation and urinary retention, which are more frequently observed after amitriptyline than nortriptyline treatment[155].

OpioidsOpioids are recommended to be used as second or third line treatment for DNP[3,151]. One multicenter, randomized, placebocontrolled study reported the tramadol effectiveness to significantly improve scores on physical and social functioning ratings in patients with DNP, but beneath some side effects such as nausea, constipation, headache and somnolence[156]. Morphine was also shown to be effective in reducing mean daily pain scores related to diabetic neuropathy and postherpetic neuralgia[128,157]. Moreover, results of clinical trials indicated that diabetic neuropathic patients experienced a significant reduction in pain intensity and an improvement on quality of life during oxycodone treatment, compared to placeboexposed group[158,159]. Besides, oxycodone improved gabapentin but not the pregabalin effectiveness in promoting DNP relief[160,161].

The clinical evidence for the effectiveness of opioids in the control of DNP is corroborated by some preclinical data, which have reported the effectiveness of morphine[162,163] and buprenorphine[164] in reducing thermal or mechanical hypersensitivity in DNP animal models.

There is also evidence that the antihyperalgesic effect of opioids is improved by the association with some drugs, such as the antidepressants amitriptyline, moclobemide and reboxetine[165]. In line with this idea, new molecules that integrate additional mechanisms to the opioid receptor agonism have been shown efficacy in reducing nociceptive behavior in animal models of DNP, such as the cebranopadol, a nociceptin/orphanin FQ peptide and opioid receptor agonist[166], and the muopioid receptor agonist and norepinephrine reuptake inhibitor, tapentadol[162,167]. The latter was approved by FDA for DNP treatment since 2012[11]. Tapentadol has been shown to be effective in the management of different types of chronic pain, including osteoarthritis knee pain, low back pain and DNP, with a tolerable safety profile[168,169]. Specifically concerning DNP, a randomizedwithdrawal, placebocontrolled trial reported reduction of at least 30% in pain intensity in about 50% of the patients that received tapentadol[170]. Similar data were obtained in a recent clinical trial in diabetic

neuropathic patients with moderate to severe pain, which experienced nausea (21.1%) and vomiting (12.7%) as side effects[171].

Others agents The drugs discussed below are currently associated to the pharmacological treatments already described according to the patients’ symptoms and needs in order to achieve a better relief of pain in DNP conditions. However, further studies are necessary, specially controlled clinical trials, to determine the more efficacious, safe and successful combinations to be applied in the management of DNP[128].

Capsaicin topical cream: Topical agents may be associated with fewer and less clinically significant adverse events than systemic agents[172]. In addition, the possibilities of drug interactions are markedly reduced with the use of local treatments, which represent good options for patients with multiple medical problems[128]. The capsaicin cream has been shown to be effective in the treatment of neuropathic conditions[150] and is approved for topical relief of neuropathic pain[128]. Capsaicin is the pungent component of hot chilli peppers[11] and an agonist of the transient receptor potential vanilloid 1. This receptor is a ligandgated, nonselective cation channel, predominantly expressed on unmyelinated C nerve fibers[173], which, after repeated exposure to topical capsaicin, are depleted of their content of substance P and other neurotransmitters[173,174]. The C fibers depletion and desensitization reduce painful stimuli transmission from peripheral nerves to the central nervous system[173]. Some clinical trials have demonstrated the effectiveness of lowconcentration (from 0.025% to 0.075%) capsaicin cream in DNP[11,174,175]. Higher concentrations are not indicated because of desensitization of nociceptive sensory nerve endings, which may increase the risk of skin injuries in DNP patients[173,174]. Some adverse effects include itching, stinging, erythema, transient burning sensation and initial pain at the application site, that diminishes with repeated use[132,175], leading many patients to withdraw from the treatment[128,173].

Lidocaine patch: Lidocaine patches act as peripheral analgesics with minimal systemic absorption and are used in combination with other analgesic drugs[132,172]. Lidocaine blocks sodium channels and counteracts the hyperexcitability of peripheral nociceptors that contributes to neuropathic pain[132,176]. The blockade reduces ectopic discharges and raises the peripheral sensory neuron discharge threshold[176]. The few DNP clinical trials that compared topical lidocaine with other relevant interventions suggested that the effects in pain reduction are comparable to other drugs, such as capsaicin, gabapentin, amitriptyline[172] and pregabalin[172,174]. Adverse events include local irritation[172], contact dermatitis and itching[132].

Alpha lipoic acid: The benefit provided by alpha



lipoic acid (ALA) in the treatment of DNP possibly is due to its direct effects on the neuropathy, by reducing the oxidative stress, which has been defined as an important factor in the physiopatology of the diabetic neuropathy[122]. Its antioxidant and antiinflammatory actions may contribute to an allround improvement of diabetic neuropathy symptoms[135]. In some clinical trials that evaluated ALA effect in diabetic patients, pain was not a primary end point. However, they have shown a moderate benefit in terms of pain reduction[132]. In a randomized doubleblinded trial, ALAtreated patients reported a greater reduction in neuropathic pain when compared to placebotreated subjects[122]. Compared to several drugs currently in use for DNP treatment, ALA has fewer side effects[30], being nausea and vomiting the most common[132].

Isosorbide dinitrate spray: Isosorbide dinitrate is a nitric oxidedependent vasodilator with effects on both arteries and veins[177]. The improvement of pain and burning sensation could be associated with the increased generation of nitric oxide, improving microvascular blood flow[178]. In a clinical trial with diabetic patients, the isosorbide dinitrate spray reduced overall neuropathic pain and burning sensation in about 50% of the patients, which also reported improvement in their quality of life, with improvements in sleep, mobility and mood[178,179].

Final considerations about DNP treatment: Besides the fact of many diabetic complications can be reduced with improved blood glucose control and other lifestyle interventions[132,150], such as quit smoking and reducing alcohol consumption[150], the efficacy of these measures, as well as the pharmacological treatments on DNP are not predictable. The medications rated as level A based on their efficacy are able to reduce pain and improve some aspects of patients’ quality of life, but are not able to fully eliminate pain or prevent/revert the neuropathy. Even their combination does not result in satisfactory pain control, being the best improvement in pain, restricted to 50% of relief for the majority of the patients. Considering the available pharmacological options, DNP treatment has to be based mainly on patients’ symptoms, pain level and tolerance of side effects.

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P- Reviewer: Georgescu A, Li CJ, Panchu P S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ



family history for diabetes mellitus, polycystic kidney disease, and previously diagnosed glucose intolerance. Modifiable risk factors for NODAT include obesity and the metabolic syndrome, hepatitis C virus and cytomegalovirus infection, corticosteroids, calcineurin inhibitor drugs (especially tacrolimus), and sirolimus. NODAT affects graft and patient survival, and increases the incidence of post-transplant cardiovascular disease.The incidence and impact of NODAT can be minimized through pre- and post-transplant screening to identify patients at higher risk, including by oral glucose tole-rance tests, as well as multi-disciplinary care, lifestyle modification, and the use of modified immunosuppressive regimens coupled with glucose-lowering therapies including oral hypoglycemic agents and insulin. Since NODAT is a major cause of post-transplant morbidity and mortality, measures to reduce its incidence and impact have the potential to greatly improve overall transplant success.

Key words: Cyclosporine; Graft; Kidney; New-onset diabetes; Tacrolimus; Transplantation


Core tip: New-onset diabetes after kidney trans-plantation (NODAT) is detrimental to patient and graft survival. Early diagnosis through the identification of modifiable and non-modifiable risk factors for NODAT and appropriate screening, accompanied by good glycemic control that involves a multidisciplinary care approach will help result in good short- and long-term kidney transplant outcomes.

Palepu S, Prasad GVR. New-onset diabetes mellitus after kidney transplantation: Current status and future directions. World J Diabetes 2015; 6(3): 445-455 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/445.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.445

Sneha Palepu, G V Ramesh Prasad

Sneha Palepu, G V Ramesh Prasad, Division of Nephrology, University of Toronto, St. Michael’s Hospital, Toronto ON M5C 2T2, CanadaAuthor contributions: Palepu S and Prasad GVR contributed to this paper. Conflict-of-interest: Both authors (Sneha Palepu and G V Ramesh Prasad) have no conflict of interest to declare.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: G V Ramesh Prasad, MBBS, MSc, MA, FRCPC, FACP, FASN, Associate Professor of Medicine, Division of Nephrology, University of Toronto, St. Michael’s Hospital, 61 Queen Street East, 9th Floor, Toronto ON M5C 2T2, Canada. [email protected]: +1-416-8673722 Fax: +1-416-8673709 Received: August 25, 2014Peer-review started: August 25, 2014First decision: October 14, 2014Revised: November 14, 2014Accepted: January 9, 2015Article in press: January 9, 2015Published online: April 15, 2015

AbstractA diagnosis of new-onset diabetes after transplantation (NODAT) carries with it a threat to the renal allograft, as well as the same short- and long-term implications of type 2 diabetes seen in the general population. NODAT usually occurs early after transplantation, and is usually diagnosed according to general population guidelines. Non-modifiable risk factors for NODAT include advancing age, African American, Hispanic, or South Asian ethnicity, genetic background, a positive

REVIEW


New-onset diabetes mellitus after kidney transplantation: Current status and future directions




INTRODUCTIONKidney transplantation is widely considered to be the therapy of choice for patients with end-stage renal disease (ESRD) because of its ability to provide the maximum amount of replacement of renal function and a consequent improvement in patient morbidity and mortality. Among the various complications experienced by kidney transplant recipients (KTR), new-onset diabetes after transplantation (NODAT) is a well-known condition by which an organ transplant recipient such as a KTR develops diabetes mellitus (DM) at some point after the transplant procedure. The purpose of this review is to discuss the epidemiology and management of this common and important post-transplant disorder. A special emphasis will be placed on studies performed during the past ten years, with the intent of providing management recommendations for both diabetes and transplant professionals.

A diagnosis of NODAT carries with it a threat to the renal allograft, as well as the same short- and long-term implications of type 2 DM, but these occur at an accelerated pace[1]. DM can both cause and expedite the course of cardiovascular disease (CVD) as well as the failure of multiple organ systems[2]. According to the World Health Organization (WHO), the number of patients worldwide with DM has doubled from 170 million to 340 million over the past ten years. In addition, the WHO predicts that DM will be the seventh leading cause of death by 2030. This increase in incidence of DM has extended across many population groups, including perhaps transplant recipients.

Over the past decade, concomitantly with DM, there has also been a corresponding increase in the number of patients seeking kidney transplants. According to the 2010 United States Renal Data System Annual Data Report, the annual number of kidney transplants in that country increased from 13425 to 17350 over the decade between 1998 and 2008[3]. Thus, there is also a larger KTR population at increased risk for NODAT. Although type 2 DM is an important cause of ESRD and consequently an important contributor to clinical consequences in incident KTR cohorts, we focus here on NODAT, as an entity distinct from pre-existing DM in a patient receiving a kidney transplant.

INCIDENCE AND PREVALENCE OF NODATThe prevalence of NODAT after kidney transplantation has increased[4], and varies between 2% and 53% based on several estimates[5]. One reason for the wide variation in reported prevalence may be due to the challenges faced in making the diagnosis. Until about a decade ago, there was no consensus definition for NODAT. Previously, it was defined by fasting or random blood glucose levels of varying thresholds, or perhaps quite naively, by the need for insulin or oral

hypoglycemic agents in the post-transplant period[6]. This issue was addressed by the development of the 2003 Consensus Guidelines, developed by the American Diabetes Association (ADA) and the WHO[7]. According to these guidelines, NODAT is present if the patient has symptoms of DM, casual plasma glucose ≥ 11.1 mmol/L, or 8-h fasting plasma glucose ≥ 7.0 mmol/L. In 2010, the definition for DM was revised by the ADA to include the 2-h oral glucose tolerance test (OGTT), with a 2-h value of ≥ 11.1 mmol/L being indicative of DM. The general principle behind standardized NODAT incidence reporting is that the diagnostic criteria should reflect what is used in the general population. Hopefully, these developments will allow for more consistent reporting of NODAT in the future, leading in turn to more precise estimates of incidence rates.

It is well established that NODAT usually occurs early after kidney transplantation. One long-term study[8] conducted between 1976 and 2004 in 1580 Egyptian KTR demonstrated a diagnosis of NODAT in 18.2% patients overall, in whom 52.4% were diagnosed by 6 mo and 11.5% between 6 and 12 mo. A French study of 527 KTR indicated a median time-to-onset of 1.6 mo, with an incidence of 7% over 2 years[9]. Incidence estimates in the United States are 9.1% at 3 mo, 16% at 12 mo, and 24% at 36 mo[10]. The incidence may be increasing even in children[11]. Most studies reporting NODAT prevalence do not include OGTT data, as a result of which significant underestimation of incidence may occur. Variation in studies with regards to length of follow-up, intensity of routine screening, and degree of use of standardized definitions will all have an impact on incidence estimates. Furthermore, glycemic control improves in some patients with increasing time post-transplant, as a result of immunosuppressive medication dose reduction and other interventions. This in turn affects prevalence estimates and interpretation of incidence rates. Addition of HbA1c testing may also modify reported incidence rates. However, HbA1c testing has not been routinely employed at most trans-plant centres.

RISK FACTORS FOR NODATThe risk factors specific to NODAT have been well-delineated. These can be broadly classified into non-modifiable and modifiable risk factors. These are summarized in Table 1.

NON-MODIFIABLE RISK FACTORS FOR NODATNon-modifiable risk factors include advancing age, black race, genetic background, a positive family history for DM, and previously diagnosed glucose intolerance[12]. Polycystic kidney disease is also believed to be a risk factor. All of these risk factors can usually be identified

Palepu S et al . New-onset diabetes after kidney transplantation


before transplantation. Since the ESRD population continues to age, it is

possible that the incidence of NODAT will increase as a result of more elderly patients being transplanted for their ESRD. Age is considered to be the strongest risk factor for NODAT[13]. It was long ago shown that patients above the age of 45 may be at higher risk for NODAT[14]. A later study estimated that risk at about 2.9-fold[15]. The incidence of NODAT may increase by as much as 50% for every 10-year increase in age[16]. Age over 60 has been associated with a risk of 2.6-fold[10]. However, age per se is not a precluding factor to kidney transplantation[17] and the risk for NODAT does not typically dissuade transplant centres from performing transplants in older patients.

Besides African-American or Hispanic descent[12], KTR of South Asian origin may also be at higher risk for NODAT[18]. The relative risk for NODAT has been estimated at 1.68 for blacks and 1.35 for Hispanics compared to Caucasians[10]. Using the 2003 consensus criteria, blacks are considered to be at a two-fold risk for NODAT[19]. The increased risk for NODAT, at least in blacks, is believed to be at least partly due to variation in the pharmacokinetics of various immunosuppressive agents[1]. Variability in dosing requirements based on ethnicity has been demonstrated recently for tacrolimus, with increased amounts of this diabetogenic agent needed in East Asians[20]. It is unclear at this time, however, if the diabetogenic effects of immunosuppressive medication are influenced by ethnicity, although this has been speculated[7].

Genetic predisposition to NODAT includes traditional associations with the alleles human leukocyte antigen A28, A30, B27, and Bw42. A number of genetic associations with NODAT have been identified in the last decade. For example, there has been a reported association with the R325W polymorphism in the SLC30A8 zinc transporter gene of pancreatic islets, with the R/r inheritance being associated with higher risk[21]. Another study from the same group suggested the association of TCF7L2 gene variants with NODAT[22]. This

association with TCF7L2 has also been demonstrated by another group[23]. Other associations of specific gene variants with NODAT include KCNQ1[24], NFATc4[25], adiponectin 276G/T[26], and mitochondrial haplotype H[27]. While all of these associations are interesting, it remains to be seen if such findings can be replicated in different populations, and whether they can be demonstrated to be independent risk factors for NODAT in prospectively conducted studies. Furthermore, the expense and inconvenience of such assessments greatly limits the ability to study these and other gene candidates further.

In the past decade, NODAT has also been associated with autosomal dominant polycystic kidney disease (ADPKD)[28,29], as well as autosomal recessive polycystic kidney disease[30]. ADPKD in particular is a common disease and cause of ESRD, particularly in Caucasians, and so it remains to be definitively proven as a risk factor for NODAT since both ADPKD and NODAT are common in KTR. A plausible mechanism for the association also needs to be developed.

Finally, previously diagnosed glucose intolerance is a risk factor for NODAT[13]. Glucose intolerance may have been diagnosed at the time of pregnancy, or when corticosteroids were used as part of the therapy for the primary renal disease or a related or unrelated systemic disease. In such cases, even though DM may have resolved with cessation of steroid therapy, the risk for DM may persist lifelong. To our knowledge, these risks have not been evaluated prospectively. Also, donor factors for NODAT considered “non-modifiable” include male gender and deceased, as opposed to living donor kidneys. However, these risk factors are unlikely to ever be evaluated prospectively, and so will not be discussed further.

MODIFIABLE RISK FACTORS FOR NODATThere are many more risk factors for NODAT that may be considered modifiable, at least on a theoretical basis. The most significant risk factors are as follows.

Obesity has detrimental effects to transplantation for a variety of reasons. Obesity has been estimated to increase the risk for NODAT, with a relative risk of 1.73[13]. The risk for NODAT increases linearly for every 1 kg above 45 kg[13]. While obesity in the context of transplantation is traditionally understood as body mass index > 30 kg/m2, body fat percentage may also be a useful marker[31]. It remains undetermined, however, whether it is pre-transplant weight that increases the risk for NODAT, or whether it is the weight gain that occurs soon after transplantation that is the cause. At least one study indicates that is the former[32]. Nonetheless, longer-term studies may be needed to demonstrate the association of weight gain with NODAT[13]. Adiponectin is a hormone that is reduced with increasing adiposity. Increased adipose tissue is also associated with inflammation. NODAT has been associated with reduced


Table 1 Risk factors for new-onset diabetes after transplantation

Non-modifiable Modifiable

Advanced age ObesityAfrican American, Hispanic, or South Asian descent

Sedentary lifestyle

Genetic, e.g., HLA B27 Metabolic syndromeAdult polycystic kidney disease Viral infections, e.g., HCV,

cytomegalovirus Previous glucose intolerance, e.g., during pregnancy, steroid therapy for renal or non-renal disease

Corticosteroids

Male donor Calcineurin-inhibitors (tacrolimus > cyclosporine)

Deceased donor SirolimusAcute rejection

HCV: Hepatitis C virus; HLA: Human leukocyte antigen.


due to its demonstrated superior efficacy and safety[44], despite the risk for diabetes. However, it is possible to use much lower doses presently[44]. Deficiency of calcineurin leads to decreased insulin production. CNI inhibit the uptake of glucose molecules by cells due to a reduction in the number of glucose transporter type 4 (GLUT-4) receptor molecules on the cell membrane surface of adipocytes[45]. GLUT-4 is an insulin-regulated protein present primarily in adipose tissue and striated muscle, enabling the translocation of glucose into the cell cytoplasm[46]. Thus, reduction in GLUT-4 leads to hyperglycemia. Tacrolimus also reduces glucokinase activity in pancreatic islets, thereby suppressing glucose-induced insulin release[47]. Although both impaired insulin release and increased insulin resistance are both believed to be mechanisms for CNI-induced NODAT, the former may be more important. Islet cell damage in the form of cytoplasmic swelling, vacuolization, and altered insulin staining can be demonstrated as a result of CNI therapy[48]. CNI also cause reduced insulin gene expression[49].

Sirolimus is another immunosuppressive agent used in transplantation that has been contextualized to NODAT. The association of sirolimus with NODAT has been believed to be due to its combination with CNI, and so conversion from a CNI to sirolimus as the main immunosuppression has been perceived as beneficial[50]. However, some studies seem to indicate that sirolimus is a risk factor for NODAT. These include both large database studies[51] and smaller single-centre reviews[52]. The effect may be mediated by hypertriglyceridemia[53]. Sirolimus may also inhibit pancreatic beta cell proliferation. At best, sirolimus is neutral with respect to NODAT risk[13].

The potential pathogenic mechanisms for drug-induced NODAT are summarized in Table 2.

CLINICAL SIGNIFICANCE OF NODATRTR who develop NODAT are most likely to be at the same risk for developing the short- and long-term complications of diabetes as people with type 2 diabetes. However, there have been few prospective, long-term, or interventional studies with adequate statistical power to support this statement. It is clear, however, from smaller studies that NODAT has an adverse effect on important transplant- and patient-related outcomes.

An older study has demonstrated that after 12 years of post-transplant follow-up, graft survival in patients with NODAT was only 48% compared to 70% in those who did not develop NODAT, with NODAT predicting a relative risk of graft loss of 3.72[54]. In the shorter term, graft survival was shown to be reduced by 17% after 3 years and 34% after 4 years in those with DM compared to those without DM[55]. In one prospective study, more than 1400 KTR underwent an OGTT at 10 wk post-transplant and were followed for a median of 6.7 years. Impaired glucose tolerance was found to be associated

adiponectin and increased C-reactive protein levels[33], indicating that these are also possible mechanisms for the association seen.

The metabolic syndrome is often associated with obesity. When a definition for metabolic syndrome such as the National Cholesterol Education Program Adult Treatment Panel Ⅲ is used, which does not contain diabetes as a mandatory component, the presence of metabolic syndrome has been associated with an increased risk for NODAT. In the Patient Outcomes in Renal Transplantation study[34], metabolic syndrome was independently associated with NODAT (HR = 3.46, 95%CI: 2.40-4.98, P < 0.0001).

Hepatitis C virus (HCV) infection has been associated with DM in the general population, particularly over the age of 40[35]. Just like DM, HCV also causes ESRD by causing glomerular disease, and HCV can be acquired through blood contamination in hemodialysis units. As a result, it is not uncommon for HCV-infected patients to be considered for kidney transplantation when there is no evidence for hepatic dysfunction and viral titres are sufficiently low or undetectable. The one-year incidence of NODAT was found to be 25.6% in those who were HCV-positive, compared to 14.4% in those who were negative[10]. Another study found an adjusted Odds ratio for NODAT approaching 4.0 in those with HCV[36]. The risk for NODAT with HCV may be exacerbated by the use of tacrolimus[37]. Cytomegalovirus (CMV) infection has also been considered as a risk factor for NODAT, with the mechanism being impaired insulin release[38]. Unlike HCV however, CMV is much more easily treated in the post-transplant setting and so receives considerably less attention as a risk factor.

Corticosteroids remain a mainstay of post-transplant immunosuppression and are a part of most medication regimens. The risk for NODAT with steroids relates both to the dose used and the duration of therapy[13]. Steroids induce gluconeogenesis and glycolysis, increa-sing both fasting and post-prandial glucose levels. Glycogenesis is decreased. Insulin resistance, to which KTR may be predisposed, is an important effect of steroid therapy. They also impair pancreatic beta-cell function[39]. While a maintenance dose of 5 mg/d of prednisone is typically used for KTR, higher doses (such as 1 mg/kg per day) are used in the early post-transplant phase. Higher doses are also used as bolus therapy when acute rejection of the transplant occurs. There is a dose dependent relationship between steroid dose and glucose levels[40].

Tacrolimus and cyclosporine are widely used calcineurin-inhibitor (CNI) drugs in KTR. Most patientsreceive one or the other drug as part of their immu-nosuppressive drug regimen. Between these two, tacrolimus is considered to be more diabetogenic by about 50%[10,41]. Unlike in the case of steroids, this effect is not believed to be dose-dependent[16,42], although this is controversial[43]. Tacrolimus is being increasingly preferred as the CNI of choice at most transplant centres



with a 40% greater mortality risk[56]. This increased risk was not seen with impaired fasting glucose (IFG). In a prospective, single-centre study of 201 consecutive KTR in Norway, the 8-year major adverse cardiac event rate was 7% in those without DM, 21% in those with DM before transplantation, and 20% in those with NODAT[57]. Perhaps due to low statistical power, NODAT was not associated with mortality in this study. In another single centre cohort of over 1800 KTR, NODAT was associated with a hazard ratio of 1.80 for mortality[58]. The main cause of mortality is CVD[58].

An important distinction to be made is if NODAT leads to patient mortality independent of transplant graft function. In one study[59], an association was detected between NODAT and death with a functioning graft, but there was no impact on graft survival when censored for death. It is easier to demonstrate associations when NODAT is combined with pre-existing DM[60]. In order to identify the unique contribution of NODAT to mortality, independent of graft function, longer follow-up is required.

NODAT also imposes a significant cost to health care systems. In the United States, NODAT was estimated to cost more than $12000 in the first post-transplant year, and over $19000 in the year after this[41]. This cost is most likely related to the treatment for, and morbidity associated with DM.

Regardless of the implications of NODAT for cardio-vascular risk, graft function, or mortality, a diagnosis of NODAT carries great significance for the individual patient. Transplant patients typically require three immunosuppressive medications, to which prophylactic antibiotics, antihypertensive agents, and others are often added. Patients with NODAT are prescribed oral hypoglycemic agents and sometimes insulin in addition to all of these. They are also subject to new dietary restrictions, which will often be superimposed on those mandated by a chronic kidney disease state (such as potassium restriction). Hospitalization may occur for both hyperglycemia and hypoglycemia[61]. All of these have an important impact on quality of life. Studies assessing this aspect of NODAT implications are few. NODAT has been associated in older studies with

peripheral neuropathy and diabetic nephropathy[54], as well as more infections[62], including severe infections[63].

PREVENTION AND MANAGEMENT OF NODATPrevention of NODATSome common preventative strategies for NODAT are summarized in Table 3. Preventing NODAT has the potential to prevent many of the short- and long-term complications of NODAT. Screening is an important part of any preventative strategy. Screening for NODAT risk prior to transplantation, as well as the risk for cardiac events, will allow for better informed consent and also help to guide post-transplant management. One recommendation is to obtain a fasting glucose level in all transplant candidates, with a subsequent OGTT performed if IFG is detected[64]. A pre-transplant OGTT may be justified if the fasting blood glucose (FBG) is as low as 5.1 mmol/L[65]. Even if the OGTT is normal, a pre-transplant random BG > 6.0 mmol/L is associated with a NODAT risk of over 25%, and > 7.2 mmol/L with a risk exceeding 50%[66]. Use of OGTT may be justifiable in all transplant candidates if the population is at particularly high risk, such as a multi-ethnic population. More sophisticated testing such as HOMA-IR assessment is not feasible routinely. HbA1c testing has not been evaluated as a pre-transplant screening strategy. If a transplant candidate is determined to be at high risk for NODAT, this should be discussed with the candidate before the transplant, and if appropriate, their proposed post-transplant immunosuppressive strategy discussed as well.

Screening for NODAT has also been employed in the post-transplant setting. Self-testing of blood glucose in the afternoon during the early post-transplant phase has been associated with an increased rate of detection of NODAT[67]. An OGTT performed at 10 wk post-transplant may help to predict longer-term hyperglycemia[68]. One difficulty with OGTT in large post-transplant clinics is that it may interfere with CNI blood level measurements, which are strictly timed. It is reasonable to measure fasting glucose at least monthly, and random blood glucose at least twice weekly for the first few months after transplantation. The HbA1c can also be checked. Since the published incidence and prevalence rates published in the literature are too variable to be helpful to individual transplant centres, it behooves every transplant centre to properly estimate its own incidence and prevalence rates, particularly in the early post-transplant period. Making an early diagnosis of NODAT is important because preventative measures can enhance the KTR’s chances for a better quality of life and also prolong their graft survival[12].

As with those at-risk for DM in other populations, lifestyle modification is likely to have a favorable impact on NODAT incidence. Recommendations for weight loss in obese patients prior to transplantation may be beneficial for preventing NODAT, but remains difficult


Table 2 Potential pathogenic mechanisms for drug-induced new-onset diabetes mellitus after transplantation

Immunosuppressive drug Mechanism for new-onset diabetes after transplantation

Corticosteroids Increased gluconeogenesisIncreased insulin resistanceReduced glycogenesisDecreased insulin releaseImpaired pancreatic beta cell function

Calcineurin-inhibitors(cyclosporine, tacrolimus)

Reduced glucose uptakeDecreased insulin releaseReduced insulin gene expressionDirect pancreatic beta cell toxicity

Sirolimus Hypertriglyceridemia? Decreased pancreatic beta cell proliferation

?: Requires further investigation.


to enforce due to the predisposition for malnutrition in patients on dialysis. Nonetheless, safe and closely supervised dietary and exercise recommendations for dialysis patients should be encouraged, particularly in those identified to be at higher risk for NODAT. Prompt attention from allied health professionals such as nurse practitioners and dieticians may help prevent NODAT from being established when hyperglycemia is detected.

The role of pharmacotherapy in the prevention of NODAT has also been investigated. Since early post-transplant hypomagnesemia is common, magnesium oxide supplementation has been investigated and has been associated with a reduction in FBG[69]. The use of statins in the post-transplant period has been associated with a reduced incidence of NODAT[70]. However, rosuvastatin has not been associated with improved insulin sensitivity in non-diabetic KTR[71], and so this pleiotropic effect of statins remains to be established[72]. Although angiotensin-converting enzyme inhibitors have also been associated with reduced NODAT incidence[70], their beneficial effect has not been demonstrated prospectively. Similarly, metformin has not been tested as a preventative strategy.

Since immunosuppressive medication remains the most readily available tool at the disposal of transplant clinicians to reduce the incidence of NODAT, much attention has been given to the reduction in exposure to existing immunosuppressive drugs such as corticosteroids and tacrolimus. Such reduction is often facilitated through the testing of newer pharmacological therapies. In a large, randomized controlled trial of corticosteroid withdrawal at 7 d post-transplant vs no withdrawal, a trend was noted towards better glycemic control in the withdrawal arm[73]. However, there was no difference in NODAT rates[73]. Despite this, fewer patients in the corticosteroid withdrawal arm required insulin for NODAT at 5 years[73]. On the other hand, a large retrospective study involving more than 25000 KTR reported significant benefits of steroid avoidance at initial hospital discharge when compared to a steroid-containing regimen with respect to NODAT at three years[74]. Corticosteroid withdrawal has also been shown to be beneficial when combined

with tacrolimus reduction[75]. Other studies have shown that complete corticosteroid withdrawal has no additional benefit beyond only dose reduction[40].

Although tacrolimus has a 50% greater association with NODAT than cyclosporine[10], it is the preferred CNI in many transplant programs for other reasons such as graft function in large clinical trials[44]. Reduced exposure to tacrolimus has also been associated with a similar incidence of NODAT to cyclosporine monitored using the 2-h instead of trough level[76]. In the randomized DIRECT study[77], which also used 2-h cyclosporine monitoring, a marginal increase in the composite safety endpoint of NODAT and IFG (33.6% vs 26.0%, P = 0.046) was noted with tacrolimus. Sometimes cyclosporine is switched to tacrolimus late after transplantation for reasons such as the development of other side effects, or rejection. In such instances, the risk of impaired glucose metabolism does not appear to be increased[78]. In addition, either tacrolimus or cyclosporine may be switched to sirolimus in order to reduce the burden of CNI nephrotoxicity. However, conversion to sirolimus has been associated with worsening insulin sensitivity[79]. The use of alemtuzumab, which is an anti-lymphocyte induction agent, has been associated with a reduced risk for NODAT[80]. Belatacept, which is a recently introduced selective costimulation blocker in kidney transplantation, has been associated with a reduced incidence of NODAT compared to cyclosporine when studied as a prespecified endpoint in two Phase Ⅲ studies[81]. Both alemtuzumab and belatacept are induction therapies and so cannot be used for this purpose if a higher risk for NODAT is detected post-transplant.

Management of NODATManagement strategies for NODAT are summarized in Table 3. Even if attempts to prevent NODAT are meticulous, it is likely that NODAT will occur in at least a proportion of KTR. The goals for management at this juncture include adequate glycemic control, perhaps with complete resolution of hyperglycemia without pharmacotherapy, and minimizing the short- and long-term complications of hyperglycemia. At our centre, non-fasting blood tests including glucose measurements


Prevention strategies Management strategies

Identification of risk factors (Table 1) with pre-transplant counseling Regular blood glucose monitoring with appropriate follow-upPre- and post-transplant screening: random blood glucose, fasting blood glucose, 2-h oral glucose tolerance test with appropriate follow-up

Multi-disciplinary care

Lifestyle modification: weight control, diet, exercise (subject to dialysis-imposed restrictions)

Lifestyle modification: weight control, diet, exercise

Rapid corticosteroid reduction or avoidance Rapid corticosteroid reductionSelective calcineurin-inhibitor use (e.g., cyclosporine instead of tacrolimus) Conversion of cyclosporine to tacrolimus? Newer immunosuppressive agents (e.g., alemtuzumab, belatacept) Oral hypoglycemic agents: metformin, sulfonylureas, meglitinides,

dipeptidyl peptidase-4 antagonists (alone and/or in combination)? Magnesium oxide Insulin? Statins Monitoring for complications

Table 3 Management strategies for new-onset diabetes after transplantation

?: Requires further investigation.


are obtained twice weekly for the first three months and weekly for the next three months. Fasting blood tests including glucose are obtained at least monthly. Our centre also employs an intensive multidisciplinary approach in the post-transplant clinic setting that includes a nurse practitioner with specialized expertise in diabetes prevention and management. This health care professional selectively reviews all clinic patients identified as having NODAT or being at high risk for NODAT. In addition, KTR have access to a dietician and pharmacist at all times in the clinic. Reading material about NODAT is also readily available. While such resource-intensive measures are probably helpful to individual patients, their overall effectiveness at a population level remains to be demonstrated. At other centres, adoption of a healthy diet and exercise program coupled with weight control strategies has demonstrated improvement in glycemic control over 6 mo[82]. One challenge in this regard will be multi-ethnicity of KTR[83] and possible language barriers. Collaboration between nephrologists and endocrinologists will help in the delivery of optimal care.

When antihypertensive medication is necessary for blood pressure control, it is reasonable to avoid or minimize the use of beta-blockers and thiazide diuretics in the absence of a compelling indication in those deemedat risk for NODAT. While clinical trials of antihypertensive agents in KTR are especially rare, there have also been no prospective studies of antihypertensive medications in KTR with NODAT as a pre-specified endpoint.

It is tempting for clinicians to alter transplant-related immunosuppression once NODAT has developed, with the view of optimizing glycemic control and perhaps avoiding the use of diabetes-specific medication. Reducing corticosteroid exposure carries an enhanced risk of transplant rejection[84]. Switching from tacrolimus to cyclosporine once hyperglycemia or NODAT has developed is controversial. Cyclosporine possesses a side effect profile somewhat different from tacrolimus that includes hypertrichosis and gingival hyperplasia. Nonetheless, this conversion has been attempted as rescue therapy from NODAT in small studies, with demonstration that glycemic control can be improved[85-87]. However, acute rejection remains a risk[88]. Specific features related to NODAT in patients that can predict a successful conversion have not yet been identified.

If it is clear that lifestyle modification alone will beinsufficient to control hyperglycemia, then pharmaco-therapy targeting glucose metabolism should be initiated. In the very early post-transplant phase, whencorticosteroids are being rapidly tapered, additional pharmacotherapy may not be required if the hyper-glycemia is mild. The choice between insulin and oral hypoglycemic agents depends on the severity, timing, and expected duration of hyperglycemia[49]. Insulin therapy is safe[50] particularly when graft function is not yet established or is unstable. Since corticosteroids are typically administered in the morning in KTR, a combination of intermediate and short-acting insulin administered several

times during the day and corresponding to mealtimes may be required. In less urgent instances, oral hypoglycemic agents can be commenced without resorting to insulin firsthand.

The choice of oral hypoglycemic agent is dictated by the level of renal function. No agent is specifically contraindicated in KTR, and there are no significant drug interactions with CNI or other immunosuppressive drugs. Pharmaceutical approaches generally mirror those utilized in the general population, with no studies specific to KTR available. In addition, the target HbA1c for KTR has not been defined[49]. Metformin improves insulin sensitivity, which is often affected in NODAT. The safety of metformin in KTR with sufficient renal function has been formally demonstrated[88]. Since the estimated GFR in KTR with well-functioning grafts often exceeds 50 mL/min per 1.73 m2, metformin can be safely used for most patients. A theoretical disadvantage to using metformin in KTR would be gastrointestinal upset, to which KTR are already prone by virtue of immunosuppressive medications such as mycophenolate mofetil. However, metformin may counter the post-transplant weight gain associated with corticosteroid administration, but this has not been demonstrated. Keeping these in mind, metformin is often employed as a first-line agent in KTR with NODAT.

Sulfonylureas, which enhance insulin secretion, are also widely used in KTR. Their use is again dictated by level of renal function, and unlike metformin, they may exacerbate post-transplant weight gain. Newer sulfonylureas like glipizide and gliclazide may be used in KTR, with appropriate monitoring for hypoglycemia. Amongst the thiazolidinediones, which are selective agonists of the peroxisomal proliferator-activated receptor gamma, rosiglitazone has been shown to improve glucose tolerance, insulin sensitivity, and even endothelial function in a small group of KTR[89]. Its short-term efficacy has also been shown by other groups[90]. Thiazolidinediones should again be used with extreme caution if graft function is severely impaired. Congestive heart failure is a significant concern, and many KTR already have impaired cardiac function. Furthermore, cyclosporine may promote a sodium-retentive state. As a result, thiazolidinediones are usually not used.

Amongst the meglitinides, which are short-acting drugs that induce insulin secretion, repaglinide has been shown to be safe in KTR[91]. Repaglinide can also be used in severe renal graft dysfunction. Dipeptidyl peptidase-4 (DPP-4) antagonists have been employed in KTR. These drugs inhibit DPP-4, which is responsible for the rapid degradation of numerous substrates including the glucagon-like peptide 1, whose role is to increase pancreatic insulin secretion. Among the DPP-4 antagonists, sitagliptin has been shown to be both safe and efficacious in KTR[92,93]. A theoretical risk in KTR is an increased risk of infections, to which they may already be prone by virtue of being in an immunosuppressed state.

It is reasonable to employ rationally selected



combinations of two or three of the above drugs before proceeding to insulin therapy. However, the initiation of insulin should not be delayed unnecessarily, since prolonged hyperglycemia may result in hospitalization from intravascular volume depletion and serious comorbidities such as infections. Opportunistic infections of many kinds typically occur in the first 6 mo post-transplant, which corresponds to the phase when NODAT usually occurs.

FUTURE DIRECTIONSRenal transplant survival has significantly improved over the last 50 years. Death with graft function, often as a result of CVD, has become a major cause of graft loss. As a result, increasing attention is being given to cardiovascular risk factors such as DM, within which NODAT is a significant component. Intensive screening for NODAT should be the norm in all transplant centres. Efforts to combat NODAT have to be balanced against the risks for graft rejection. Large clinical trials of newer immunosuppressive agents in kidney transplantation are few, but the inclusion of NODAT as a prespecified endpoint will help to better understand not only the most important contributing risks for NODAT, but also identify those interventions that are most likely to result in a lower NODAT incidence. Correspondingly, clinical trials that address treatment strategies for diabetes post-transplant have received very little attention. This is indeed unfortunate given the magnitude of NODAT prevalence and its financial implications for society, not to mention the profound impact of NODAT on transplant recipients. Until such trials are performed, multidis-ciplinary care focused on intensive glucose control in keeping with general population guidelines, along with management of other cardiovascular risk factors should remain the standard.

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78 Luan FL, Zhang H, Schaubel DE, Miles CD, Cibrik D, Norman S, Ojo AO. Comparative risk of impaired glucose metabolism associated with cyclosporine versus tacrolimus in the late posttransplant period. Am J Transplant 2008; 8: 1871-1877 [PMID: 18786231 DOI: 10.1111/j.1600-6143.2008.02328.x]



79 Teutonico A, Schena PF, Di Paolo S. Glucose metabolism in renal transplant recipients: effect of calcineurin inhibitor withdrawal and conversion to sirolimus. J Am Soc Nephrol 2005; 16: 3128-3135 [PMID: 16107580]

80 Shah T, Kasravi A, Huang E, Hayashi R, Young B, Cho YW, Bunnapradist S. Risk factors for development of new-onset diabetes mellitus after kidney transplantation. Transplantation 2006; 82: 1673-1676 [PMID: 17198258]

81 Vanrenterghem Y, Bresnahan B, Campistol J, Durrbach A, Grinyó J, Neumayer HH, Lang P, Larsen CP, Mancilla-Urrea E, Pestana JM, Block A, Duan T, Glicklich A, Gujrathi S, Vincenti F. Belatacept-based regimens are associated with improved cardiovascular and metabolic risk factors compared with cyclosporine in kidney transplant recipients (BENEFIT and BENEFIT-EXT studies). Transplantation 2011; 91: 976-983 [PMID: 21372756 DOI: 10.1097/TP.0b013e31820c10eb]

82 Sharif A, Moore R, Baboolal K. Influence of lifestyle modification in renal transplant recipients with postprandial hyperglycemia. Transplantation 2008; 85: 353-358 [PMID: 18301331 DOI: 10.1097/TP.0b013e3181605ebf]

83 Prasad GV, Vangala SK, Silver SA, Wong SC, Huang M, Rapi L, Nash MM, Zaltzman JS. South Asian ethnicity as a risk factor for major adverse cardiovascular events after renal transplantation. Clin J Am Soc Nephrol 2011; 6: 204-211 [PMID: 20884776 DOI: 10.2215/CJN.03100410]

84 Pascual J, Zamora J, Galeano C, Royuela A, Quereda C. Steroid avoidance or withdrawal for kidney transplant recipients. Cochrane Database Syst Rev 2009; 21: CD005632 [DOI: 10.1002/14651858.CD005632.pub2]

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P- Reviewer: Cardoso CRL, Ray S S- Editor: Song XX L- Editor: A E- Editor: Liu SQ



insulin resistance, dyslipidemia, β-cell dysfunction, impaired glucose tolerance and ultimately leading to T2DM. Chronic oxidative stress, hyperglycemia and dyslipidemia are particularly dangerous for β-cells from lowest levels of antioxidant, have high oxidative energy requirements, decrease the gene expression of key β-cell genes and induce cell death. If β-cell functioning is impaired, it results in an under production of insulin, impairs glucose stimulated insulin secretion, fasting hyperglycemia and eventually the development of T2DM.

Key words: Insulin resistance; Dyslipidemia; Type 2 diabetes mellitus; Oxidative stress


Core tip: Oxidative stress is underling in the development of cardiovascular disease, type 2 diabetes mellitus (T2DM) and diabetic complications. Increased oxidative stress appears to be a deleterious factor leading to insulin resistance, dyslipidemia, β-cell dysfunction, impaired glucose tolerance and ultimately leading to T2DM.

Tangvarasittichai S. Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus. World J Diabetes 2015; 6(3): 456-480 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/456.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.456

INTRODUCTIONAerobic life uses oxygen to oxidize (metabolism) food substrates (carbon- and hydrogen-rich) to obtain the heat energy and chemical essential for life. When we oxidize molecules with oxygen, the oxygen molecule itself becomes reduced and forms intermediates.

Surapon Tangvarasittichai

Surapon Tangvarasittichai, Chronic Disease Research Unit, Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, ThailandAuthor contributions: Tangvarasittichai S and his assistances performed the literatures review search and wrote the manuscript; Tangvarasittichai S designed essential ideas, drawing figures and sequencing of this review manuscript, also provided references and edited the manuscript, addressed the responses to reviewers’ concerns and contributed to the edition of the manuscript.Conflict-of-interest: The author declares that there are no conflicts of interest.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Dr. Surapon Tangvarasittichai, Associate Professor, Chronic Disease Research Unit, Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, 99 Moo 9 Tambon Tha Pho, Muang, Phitsanulok 65000, Thailand. [email protected]: +66-08-96388382 Fax: +66-08-55966300Received: September 3, 2014 Peer-review started: September 4, 2014 First decision: November 14, 2014Revised: December 25, 2014 Accepted: January 9, 2015Article in press: January 12, 2015Published online: April 15, 2015

AbstractOxidative stress is increased in metabolic syndrome and type 2 diabetes mellitus (T2DM) and this appears to underlie the development of cardiovascular disease, T2DM and diabetic complications. Increased oxidative stress appears to be a deleterious factor leading to

REVIEW


Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus




In eukaryotic cells, reactive oxygen species (ROS) are always produced as the consequence of regular physiological metabolism[1]. These ROS (pro-oxidants) productions are counter-balanced by cellular antioxidant defense mechanisms in the normal physiological conditions. ROS define as diverse chemical that have reactive properties are capable to accommodate or donate electrons (e-) to the broad range of biological molecules. Normally, the production and neutralization of ROS are balance with antioxidants in a living system and does not cause any oxidative damage, determines as physiological state[2]. The imbalance between these prooxidants and antioxidants in the living organism system to determine as oxidative stress state, brings to cellular disruption and damage[3]. The free-radical can attack polyunsaturated fatty acids oxidation in physiological systems known as lipid peroxidation. Lipid peroxidation is an autocatalytic free radical mediated destructive process whereby poly-unsaturated fatty acids in cell membranes undergo degradation to form lipid hydroperoxides[4,5]. By-products of lipid peroxidation such as conjugated dienes and malondialdehyde (MDA) are increased in the patients with obesity, metabolic syndrome and type 2 diabetes mellitus (T2DM). Car-bohydrates, lipids, proteins and DNA are the targets of oxidative stress modification biomolecules generally as the principal of ROS induced cellular damage. Therefore, these ROS modified biomolecules are used as oxidative stress markers both in vivo and in vitro measurement. Recent study suggests that ROS may act as the mechanical link of salt sensitive hypertension, over nutrition and high fat diet, metabolic syndrome and T2DM animal models[6]. ROS levels are increased in obesity, especially in abdominal obesity which is the major component of metabolic syndrome and it can be reduced by weight loss[7]. Many studies demonstrated that increased oxidative stress is associated with insulin resistance pathogenesis by insulin signals inhibition and adipokines dysregulation[8,9]. In animal studies, oxidative stress enhances insulin resistance. The evidence suggested that angiotensin Ⅱ (Ang Ⅱ) infused rats required the increased glucose load to maintain normal glucose levels during hyperinsulinemic clamp to stimulate ROS production[10]. Thus, ROS may also contribute and accelerate the insulin resistance development in insulin-targeted organs of the over nutrition and the excess salt individuals.

In the large general population studies demonstrated that insulin resistance is multifactorial[11,12] and the genetic component[11,13,14]. Insulin resistance most often precedes in many years before the onset of T2DM. Insulin resistance and the consequence of declined of insulin secretion are the principle of the T2DM patho-genesis[11,12,15,16]. The late complications of diabetes have been associated and implicated in their etiology with oxidative stress[17-19]. The influence of oxidative stress on insulin resistance, dyslipidemia, abnormal lipoprotein production and the pathophysiology of T2DM by using in vivo, in vitro and animal models data on these effects

were also included in this review.

ROSOxygen exists in air known as oxygen molecule (O2) or dioxygen. Oxygen on the surface of earth appeared in significant amounts approximately 2.5 × 109 years ago. It was created by the photosynthetic activity of plants and microorganisms (blue green algae). Increased atmospheric oxygen concentration was followed by the ozone layer formation in the stratosphere. Both oxygen and ozone layer were filters against the solar ultraviolet radiation reaching surface of the Earth. In eukaryotic cells, ROS is produced as the consequence of the normal aerobic physiological metabolism[1]. These ROS levels are counter-balanced with the cellular antioxidants in the normal physiological conditions. ROS define as diverse chemical that have reactive properties are capable to accommodate or donate electrons (e-) to the broad range of biological molecules. These species includeinstability radicals arise from an unpaired e-. Existence of the presence of oxygen and the aerobic organisms on the earth is possible[20].

O2 + e + H+ → HO2• (hydroperoxyl radical)

HO2• → H+ + O2•

- (superoxide radical)O2•

- + 2H+ + e → H2O2 (hydrogen peroxide)H2O2 + e → OH- + OH• (hydroxyl radical)

However, these molecules are also played an adverse role in the biological systems as oxidative stress. At the steady state of the living systems, oxygen metabolism always produce oxygen-derived free radicals such as superoxide O2

•-, hydroxyl OH•, alkoxyl RO•, peroxyl RO2

•, peroxynitrite ONOO- and oxygen-derived non-radicals such as hydrogen peroxide H2O2, hypochlorous acid HOCl and hypobromous acid HOBr. Both free radicals and non-radicals groups are the important factors of the oxidative stress mediated cellular damages[21]. Normally, the neutralization of ROS productions by cellular antioxidant defense mechanisms are determine as the physiological state and do not cause any oxidative damage[2]. The imbalance of the ROS production and antioxidants defense system in the living systems caused oxidative stress brings to cellular function disruption and damage[3].

This imbalance occurs due to over production of ROS and reduction of the antioxidant defense mechanisms. The electron transport chain in mitochondrial, peroxisomes and cytochrome P450 system are the most important sources of ROS production (involves in O2

•- production)[22]. Moreover, various enzymes can be accelerated ROS production such as cyclooxygenases[23,24], xanthine oxidase[25], uncoupled nitric oxide synthases (NOS)[26-28] and NADPH oxidases[29]. Drugs such as doxorubicin[30,31], cisplatin, acetaminophen[32-34] and nimesulide[35]. Heavy metals (Fe, Cd, Pb, Hg) as the toxic substances [36-39], acrolein, chloroform, carbon tetrachloride[40], tertiary butyl hydroperoxide[41-44], environmental pollutants (oxides of

Tangvarasittichai S. Oxidative stress and T2DM


nitrogen, SO2, CO2), xenobiotics, UV irradiation and the other factors induce ROS overproduction.

In metabolic disorders assist the increased ROS production in the physiological system such as obesity, insulin resistance and diabetes mellitus[45-48]. In Figure 1 summarized of obesity and metabolic syndrome elevate in oxidative stress. Superoxide radical (O2

•-), hydroxyl radical (OH•) and hydrogen peroxide (H2O2) are the three major ROS in physiological organisms[49]. Superoxide radical (O2

•-) acts as the parent ROS molecules caused from the one electron reduction of oxygen molecule by electron transport chain enzymes in mitochondrial such as enzymes in cytochrome P450, cyclooxygenase and NADPH oxidase. Various reactions of enzymes and non-enzymes system further convert these ROS molecules to hydroxyl radical (OH•), peroxynitrite ion (ONOO-) and hyperchlorous acid (HOCl). For example superoxide dismutase converts O2

•- to H2O2 by the dismutase reaction[50,51].

Elevated ROS molecules caused the cellular ma-cromolecules damage such as lipids[52], proteins[53] and nucleic acids[54]. In the anti-oxidants system of the living system, possess own antioxidant defense mechanisms[55] includes enzymes and non-enzyme molecules such as SOD, catalase (CAT) and glutathione peroxidases (GPx). Enzyme SOD catalyzes O2

•- conversion to H2O2, while CAT converts H2O2 to H2O and O2. For reduction of two peroxide molecules use non-enzymatic glutathione (GSH; reduced and oxidized forms), reduced glutathione (GSH) and GPx catalyze to produce oxidized glutathione (GSSG) and water[56]. Various enzymes play the important combination roles in the series of antioxidant defense systems such as glutathione reductase, glutathione S-transferase, and glutathione disulfide (GSSG).

ROS production is identified as endogenous and exogenous source. UV exposure and xenobiotic agents has been shown to generate these ROS[57]. In fact, dietary is the major source of these oxidant compounds, especially in animal fat as the source of high lipid peroxides[58]. ROS may also be derived from the general biochemical reactions in living organism to generate ROS as by-products or end products. In the transition heavy metals such as iron (Fe2+) and copper (Cu+) are pose the oxidative stress production, especially in

Fe2+ may cause autooxidation to cause O2•- generation

and/or interaction with H2O2 can generate OH• via the Fenton and Harber Weiss reactions[59]. Fenton chemical reaction may also causes lipid peroxides generation and propagation[60].Auto oxidation of Fe2+:

Fe2+ + O2 → Fe3+ + O2•-

Fenton reaction:H2O2 + Fe2+ → Fe3+ + OH- + OH•

Haber-Weiss reaction:

The major cellular oxidative stress is come from mitochondrial respiration. Heart, brain, kidney, liver and skeletal muscle are the effective oxygen consumption organs is converted oxygen to O2

•-, approximate releasing 0.1%-0.2% while the liver is converted oxygen to O2

•-, approximately releasing 2%[61]. Electron transport chain complex of the mitochondrial has been sourced to O2

•- generation and have been estimated upto 107 ROS molecules per mitochondria per day[62].

In the enzymatic systems of xanthine oxidase generated via xanthine dehydrogenase, which utilize oxygen molecule as e- acceptor during catabolism of xanthine. Xanthine oxidase is the generator of O2

•-, H2O2

[63] and OH• producer[64], highly expressed in epithelial, injured and diseased tissues as shown in Figure 2. Xanthine oxidase has been involved to peroxynitrite (OONO-) and nitric oxide (NO) productions through nitrite reduction[65,66]. Intracellular nitric oxide synthases (NOS) catalyze L-arginine to form citrulline and NO. Endothelial NOS and neuronal NOS are activated by calcium-induced calmodulin binding to produce NO levels[67]. Inducible NOS (iNOS) has also calmodulin bound molecule. It may rapid and chronic expression in many cell types such as smooth muscle cells, hepatocytes and macrophages. INOS is induced by the many inflammatory cytokines


FeH2O2 + O2•- O2 + OH- + OH•

Obesity/MetS

Oxidative stress ↑

Inflammation

Dyslipidemia

T2DM

Hyperinsulinemia insulin resistance

Atherosclerosis

Figure 1 Summarized of obesity and metabolic syndrome elevate in oxidative stress. T2DM: Type 2 diabetes mellitus; MetS: Metabolic syndrome.

NAD(P)H

NAD(P)+GRed

NAD(P)H oxidase

GSSG

GSH GPxH2O

O2

SOD

Xanthine

Oxidase

Hypoxanthinexanthine

Xanthineuric acid

Mitochondria

Fe2+

Fe3+

O2•-

Lipid peroxidation

DNAdamage

•OH

H2O2

O2 O2•-

Figure 2 Increased oxidative stress by xanthine oxidase. NADH: Nicotinamide adenine dinucleotide.


the chain-breaking antioxidant (vitamin E) agent is added to terminate the chain reaction. The three stages of lipid peroxidation are initiation, propagation and termination. Hydroxyl radical (•OH), alkoxyl radical (RO•), peroxyl radical (ROO•), and HO2

• species can abstract the first hydrogen atom of polyunsaturated fatty acid but not H2O2 or O2

•-[70]. Variety of lipid hydroperoxides and cyclic peroxides are the end products of the chain reaction. Lipid peroxides are stable molecules in the physiological temperatures. Lipid peroxides decomposition is catalyzed by transition heavy metals. For example, iron ion-active complexes present in circulating can participate in the Fenton reaction to promote lipid peroxide decomposition. Hemoglobin and the cytochromes molecules can also facilitate peroxide decomposition, although they do not directly catalyze Fenton chemistry. However, hemeproteins can release chelatable iron that can participate in Fenton chemistry[71]. Ferritin and hemosiderin are effective at stimulating lipid peroxidation and catalase is weakly effective, caused problems to use catalase as a probe for H2O2 in lipid peroxidation systems[72].

Reduced heavy metal [Fe+2, Cu+] react with lipid peroxides (LOOH) to alkoxyl radical or Cu+ react with LOOH to alkoxyl radical.

LOOH + Mn+ → LO• + M(n+1)+ + OH-

In the reaction oxidized-heavy metals [Fe+3, Cu+2] slowly react with LOOH to produce alkoxyl and peroxyl radicals. Both peroxyl and alkoxyl radicals initiate the chain reaction by reducing hydrogen atoms (Figure 3). The fixed oxidation metals ions can affect the rate of lipid peroxidation (Ca2+, Pb2+ and Al3+ ions). Lipid peroxidation accelerates by the iron salts stimulation result in the membrane structure changes and important implications for environmental toxicology[73].

Rawls et al[74] demonstrated that singlet O2• is

formed during the lipid peroxidation degradation and might contribute to cause more initiation in the chain

[tumor necrosis factor-α (TNF-α), interleukin-6 and growth factors] regulation at the transcriptional level, results in micromolar NO production[67]. INOS can poduce O2

•- and OONO- when lower in L-arginine substrate[68].

LIPID PEROXIDATIONFats and oils oxidized with characteristic changes in texture, color, taste and odor. This process, known as rancidity, was chemically defined in the 1940s as an autoxidative free-radical chain reaction[69]. The most powerful oxidant formed in biological systems is hydroxyl radical. It can attack any biological molecule. The initiation step of lipid peroxidation occurred when hydroxyl radicals attack to polyunsaturated fatty acids, to cause the free-radical polyunsaturated fatty acids oxidation in biological systems. Lipid peroxidation is autocatalytic lipid hydroperoxides radical production mediated poly-unsaturated fatty acids in cell membranes destruction and degradation process[4,5]. Conjugated dienes and MDA, by-products of lipid peroxidation are increased in the circulation of obesity, metabolic syndrome and T2DM patients.

First-peroxidation chain initiation, results from the attack by any species to reduce a hydrogen atom from methylene (-CH2-) group of polyunsaturated fatty acid or membrane. Because one hydrogen atom contains one electron, reduction leaves an unpaired electron on the carbon of -CH-, double bond in the fatty acid weakens the C-H bonds on the carbon atom adjacent to the other double bond and facilitates it removal. Then, the polyunsaturated fattyacid chains in lipids membrane are sensitive to cause lipid peroxidation. The carbon-centered radical forms a conjugated diene by the molecular rearrangement (Figure 3), which combines with oxygen to form a peroxyl radical that able to reduce a hydrogen atom from another fatty acid to start a chain reaction. Peroxidation continues to use up the polyunsaturated fatty acid substrate unless


Fatty acid with 3 double bond

RH

R•

R•

RO2•

ROOH

Fragmentation to aldehyde(including malondialdehyde and polymerization products)

H•

Molecularrearrangement

O2 oxygenuptakes

O

O H•

O

O

H

Hydrogen abstraction

Conjugated diene with UV absorbance at 234 nm

Peroxylradical: abstracted H+ from another fatty acid causing an autocatalytic chain reaction

Lipid hydroperoxide

Cyclic peroxide

Cyclic endoperoxide

Figure 3 The chain reaction of lipid peroxidation.


reaction. Initiation in the first-chain initiation should be used as lipid peroxide decomposition reactions to start the new chain reaction. Iron ions and ferrous ions are free radicals[55], can act in electron transfer reactions with oxygen molecule. Then, the presence of iron ions can promote the hydroxyl radicals formation by Fenton reaction. Bielski et al[75] demonstrated that the •OH radical production in any source can initiate lipid peroxidation reaction.

LH + OH• → L• + H2O

Superoxide-dependent Fenton reaction (superoxide resulting H2O2 and reducing Fe3+ to Fe2+) did not demonstrate any substantial involvement of the hydroxyl radical in liposomal peroxidation systems as detected by the scavengers action[76]. Hydroxyl radicals in the systems can be measured by spin trapping[77] or deoxyribose degradation measurements[76] but do not contribute to the lipid peroxidation rate[76]. The addition of iron ion in any preparations can stimulate peroxidation reaction by lipid hydroperoxide degradation to generate peroxyl (LO2

•) and alkoxyl (LO•) radicals.

The rate constant of the reaction when ferrous ions are reacted as 1.5 × 103 /mol/L per second[78], which is higher than the rate reaction constant of ferrous ions with H2O2 reaction (76 /mol/L per second)[79]. The iron ions stimulate lipid peroxidation by the lipid degradation reactions from the present of abundant hydroperoxide.

Iron or copper in a biological system attach to biological molecules at the specific location of OH radicals formation to cause lipid, protein and DNA damage. On lipid membrane, the propagation step of lipid peroxidation reactions does not proceedes further until the reaction reach the protein portion. Thus, lipid peroxidation in vivo causes proteins membrane damage[80,81]. This damage has more biologically important than those lipids membrane damage. Cells also contain mechanisms for recognizing and removing oxidative modified proteins[80,81].

OXIDATIVE STRESSOxidative stress occurs at the molecular level as the cellular event when increased ROS overwhelm the antioxidant defense capabilities systems. Oxidative stress was defines as the increasing ROS production, vary in intensities, the different cellular locations and may be occurred either acutely or chronically[82]. Oxidative damage to macromolecules including carbo-hydrates, proteins, lipids and DNA typically viewed as increased ROS induced cellular damage to cause the irreversible macromolecules modifications. Therefore, the by-products of these oxidative modified biomo-lecules are used as oxidative stress biomarkers in vivo and in vitro. Many research studies demonstrated the association of oxidative stress and the pathogenesis

of insulin resistance via insulin signals inhibition and adipocytokines dysregulation[8,9]. Oxidative stress biomarkers included MDA[83], 4-hydroxy-2-nonenal and isoprostanes species[84], protein carbonyls, 3-nitrotyrosine, hydroperoxides, protein oxidation products[85], glycation end products, carbohydrate modifications[86] and 8-hydroxy-2′-deoxyguanosine (8-OH-dG), an oxidized DNA product[84].

Assaying lipid peroxidationThe lipid peroxidation contributes to the pathogenesis of atherosclerosis. It is occurred in the blood vessel walls and does not occur from low density lipoproteins (LDL) in circulation[87,88]. LDL can enter to the blood vessel walls. The modified LDL (oxidized LDL) may escape from the scavenger recognition receptors and back to the circulation. Therefore, this circulating LDL peroxidation is a potentially useful biomarker of lipid peroxidation in circulation. Indeed, this assay is used for the demonstration of in vivo antioxidants inhibit the effects of lipid peroxidation[89,90].

Thiobarbituric acid-reactive substanceMDA from the oxidative polyunsaturated fatty acids (PUFA) degradation is determined by the reaction of thiobarbituric acid (TBA) with MDA to generate the stable end product of MDA-TBA adduct[91-95]. This MDA free radical has been demonstrated as a causative of the atherosclerosis pathogenesis[96,97], aging[98], cancer[99] and Alzheimer’s disease[100,101]. Serum MDA levels have been used as the lipid peroxidation biomarker and indicator of free radical damage[37,83,102]. MDA, the three-carbon dialdehyde, can exist in many forms in the aqueous circulation. This method was used the reaction of MDA with TBA and heated under acidic conditions but the TBA can react with many chemical species such as proteins, phospholipids, aldehydes, amino acid and nucleic acids[103,104]. One MDA molecule reacts with TBA two molecules to form a stable pink to red chromophore that absorbs maximally at 532 nm[105] or fluorescence detection. This chromophore is termed thiobarbituric acid reacting substances. Elevated MDA levels in T2DM patients are associated with cardiovascular disease risk[83].

IsoprostanesThe most valuable of lipid peroxidation biomarker in the biological system is the isoprostanes, elevated from the PUFA peroxidation[106-113]. Isoprostanes identified as free form and the most are esterified to lipids in circulation. Isoprostanes can be analyzed by mass spectrometry techniques, so that can easily be detected in human body fluids[108,109,112,113]. Isoprostanes appear to turn over rapidly in metabolized and excreted[108,109]. Isoprostanes and their metabolites detection in urine may be the useful biomarker for lipid peroxidation[113]. Isoprostanes assay have focused on the F2-isoprostanes measurement, which elevate from the arachidonic acid peroxidation[109]. Elevation of F2-isoprostanes levels have


Fe2LOOH LO• + LO2

• + H2Ocomplexes


been shown in conditions of the cardiovascular disease, diabetes development[114,115], cigarette smoking[111,116,

117], hyperhomocysteinaemia[118] and hypercholestero-laemia[110,119]. F2-isoprostane levels have also been shown to decrease by antioxidants supplementation both in animal models and humans subjects[120-124].

Oxidative stress in metabolic syndromeThe components of metabolic syndrome consist with abdominal obesity, dyslipidemia, hypertension and diabetes[125,126]. It is the major modern lifestyle com-plication cause from physical inactivity and overeating and associated with the increased risk of cardiovascular diseases, hypertension and T2DM that summarized in Figure 4.

Over nutrition and oxidative stress: In metabolism of glucose through glycolysis and tricarboxylic acid (TCA) cycle to generate nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) as the electron donors. In over nutrition, the excessive glucose occur and a large amount of glucose is oxidized in the glycolysis and TCA cycle to increase NADH and FADH2 generation in electron transport chain of mitochondrial and increased superoxide generation[127]. The excessive of free fatty acids (FFAs) leads to increase FFA-oxidation and acetyl coenzyme A (CoA) oxidation in TCA cycle generate the NADH and FADH2 electron donors as glucose oxidation results in mitochondrial ROS overproduction[127]. Furthermore, NADPH oxidase in the plasma membrane can convert oxygen molecule to superoxide radical and involve in ROS nutrient-based generation. In adipocytes, ROS is generated by in fused

with FFAs, treatment with NADPH oxidase inhibitor can block this ROS generation. This indicates that NADPH oxidase involves in fatty acids ROS generation[8]. Palmitate can activate diacylglycerol synthesis and protein kinase C (PKC) leading to activate NADPH oxidase[128]. Thus, over accumulated fat result in the increased fatty acids oxidation and lead to activate NADPH oxidase (in local or remotely cells) to cause ROS over production in over nutrition or obesity (Figure 4). Conversely, calorie restriction may be associated with normal physiological system[129] and may involve in normal cellular redox state[130]. In aged animals models treated with antioxidant agents or hypocaloric diets led to ameliorate in oxidative stress status and tissue function[131,132]. Treatment with resveratrol, a polyphenol reduced atherosclerosis and diabetes development[133]. These studies demonstrate that nutrition is associated with increased or decreased redox status and over nutrition result to increase oxidative stress to contribute pathogenesis of atherosclerosis, cancer and other diseases.

Oxidative stress in adipose tissue: Increased fat accumulation in human has been associated with oxidative stress biomarkers[134]. Similarly, obese mice were significantly higher oxidative stress levels in circulation[8]. Moreover, lipid peroxidation and H2O2 levels were increased in adipose tissue[8]. These mean that adipose tissue may the major source of ROS production and can be released to the circulation potentially affecting various distance organs functions and damage (Figure 4).

Increased NADPH oxidase expression in adipose


Figure 4 Summarized the increasing reactive oxygen species in obesity, metabolic syndrome and salt sensitive hypertension. FFA: Free fatty acid; MetS: Metabolic syndrome; HT: Hypertension; IGT: Impaired glucose tolerance.


Oxidative stress

MetS-salt-sensitive HT

CVD MetS

Diabetes (chronic hyperglycemia)

IGT(post prandial hyperglycemia)

Over nutritionDecreased physical activity

GlucoseFFAOrgan-celluar overload

AdipocyteMuscleLiver ↓Insulin resistance

Sodium balance ↓Renin-angiotensin system

Endothelial cells↓

Endothelial cells dysfunction

Insulin secretion ↑(β-cells)HyperinsulinemiaInsulin activty ↓

tissue associated with increased oxidative stress levels. Increased mRNA expression was found in adipose tissue of obese mice[8]. Increased ROS generation in lipid accumulation and further elevating ROS generation with FFA treatment were found in 3T3-L1 adipocytes cultured[8]. These ROS generation processes can be blocked by NADPH oxidase inhibitors, apocynin or diphenyleneiodonium. Many studies suggest that NADPH oxidase induces adipocytes ROS production[8]. Moreover, obese mice ameliorated hyperinsulinemia, hypertriglyceridemia, hyperglycemia and hepatic steatosis by supplementation with apocynin[8]. These data demonstrate that NADPH oxidase increase ROS production in obesity and metabolic syndrome may play the important roles in the atherosclerosis, T2DM and cancer pathogenesis. Adipose tissue tries to increase antioxidant enzymes levels to against ROS over production. However, these antioxidant enzymes activity and expression are decreased in adipose tissue[8,135-137]. Then, increased ROS-production enzymes and decreased antioxidant enzymes may cause oxidative stress in obese and metabolic syndrome.

Oxidative stress and salt-sensitive hypertension: As in mention above, ROS levels are increased in obesity and can be ameliorated by weight loss[7]. Obese rats induced by refined sugar or high fat diet leading to ROS overproduction and increase oxidative stress[6,138]. Many research evidences suggest that metabolic syndrome was associated with the salt-sensitive hypertension. ROS play the roles as mechanical link of metabolic syndrome and salt-sensitive hypertension[125,126], which itself leads to ROS overproduction[139-142]. Salt restriction in hypertensive obesity was more effective reduction in blood pressure than in hypertensive non-obesity patients, and weight loss in obesity and salt sensitive hypertensive patients caused the successful of blood pressure reduction[143]. Salt-sensitive hyper-tensive patients were significantly more prevalent in metabolic syndrome patients than without metabolic syndrome[144]. Oxidative stress in abdominal adipocytes due to increase adipocytokines secretion such as TNF-α, angiotensinogen, non-esterified fatty acids[126]. Interestingly, infused Ang Ⅱ-rats disturbed sodium balance to cause ROS overproduction in salt-sensitive rats[139-141]. Moreover, in salt-sensitive hypertensive patients are also increased 8-isoprostane levels[142]. Thus, ROS may the underling pathogenesis of diseases in metabolic syndrome, obese and non-obese intake excessive salt as the salt-sensitive hypertensive patients.

In high-renin patients (non-modulating salt sensitive hypertension) had elevated the homeostasis model assessment of insulin resistance (HOMA-IR) levels[145]. Insalt-sensitive hypertensive non-obesity patients had significantly lower insulin sensitivity than in non-salt-sensitive hypertensive patients[146]. Insulin resistance caused salt-sensitive hypertensive obesity and/or metabolic syndrome patients[125]. Increased renal ROS overproduction may increase the salt sensitive

hypertension[147]. Then, increased renal oxidative stress may contribute to cause salt-sensitive hypertension development. Moreover, ROS overproduction in vascular endothelial cells suppresses the NO-dependent vaso-dilation[148] and may play the role in the salt-sensitive hypertension development.

Oxidative stress in type 2 diabetesMany research studies demonstrated that T2DM patients have increased ROS production-induced higher oxidative damage in the circulation and also have reduced antioxidant defenses mechanisms[149-152]. Increased ROS production in T2DM patients is thought to activate many detrimental pathways including hexosamine pathways, advanced glycation end-products (AGEs) formation, and PKCβ1/2[127]. Hyperglycemia condition can induce oxidative stress by several mechanisms such as glucose autoxidation, polyol pathway, AGE formation and PKCβ1/2 kinase. Elevated free fatty acids, leptin and other circulating factors in T2DM patients may also contribute to cause ROS overproduction. Figure 5 demonstrates the association of increased ROS production with atherosclerosis and sources of ROS generations in T2DM patients.

Glucose autoxidationHyperglycemia due to cause increased glucose metabolism leading to increase NADH and FADH2 overproduction, which are used by the electron transport chain of mitochondria to generate ATP[153]. NADH overproduction can cause the higher proton gradient production in mitochondria. These electrons are transferred to oxygen to produce higher superoxide[154]. The NADH dehydrogenase of the complex Ⅰ ubiquinone oxidoreductase and complex Ⅲ cytochrome c reductase are the two main site of superoxide production via the electron transport chain[155].

The polyol pathwayOxidative stress increased in circulation of T2DM patients from the polyol pathway. ROS was generated by two enzymes: (1) Aldose reductase in the reaction use NADPH to change glucose to sorbitol. Sorbitol production is a minor reaction in normal physiological conditions. However, 30%-35% of glucose in T2DM conditions is metabolized by polyol pathway[156]. In the condition of sorbitol overproduction, the availability of NADPH is reduced this reflect to reduce glutathione regeneration and NOS synthase activity to cause increased oxidativestress[153]; and (2) Sorbitol dehydrogenase in the second step oxidizes sorbitol to fructose concomitant with NADH overproduction. Increased NADH may be used by NADH oxidases to increase superoxide production[157] include in mitochondrial over superoxide production.

PKCβ1/2Many structures and biochemical components changed in the circulation of T2DM patients were caused from PKCβ1/2 activation via diacylglycerol leading to cause dysfunction in endothelial contractility and permeability,



hemodynamics (retinal blood flow) changes, extra-cellular matrix protein synthesis, VEGF production and intracellular signaling in the vascular[128,158,159].

Non-enzymatic glycationGlycation end-product is the binding of ketone or aldehyde groups of glucose with the free amino groups of proteins leading Schiff bases formation without enzymes, then to form the Amadori product and rearrangements of the structure to the irreversible AGEs in the final[160,161]. AGEs has been demonstrated in atherosclerotic lesions and their tissue of T2DM patients and increased AGEs levels associated with severity of the diseases[162]. Moreover, binding of AGEs to specific cell surface receptor for AGE can activate intracellular redox signaling and subsequent to activate the expression of redox-sensitive transcription factors and inflammatory mediator[163-165].

InflammationOxidative stress is the major factor underlying in the CVD, insulin resistance and T2DM pathogenesis. These may explain by the presence of the inflammation conditions. Now, inflammation recognized as the onemanifestation of oxidative stress[166] and can be gene-rate the inflammatory mediators including adhesion molecules and interleukins to induce oxidative stress[166]. The concept of atherosclerosis is an inflammatory disease now well established. This chronic inflammation may be involved in the insulin resistance and T2DM pathogenesis[167]. Recent clinical research indicates that sub-clinical inflammation may impact in the development and progression of diabetic complications[165,168]. Moreover, excessive FFA and glucose induce inflammation effect through oxidative stress and reduced antioxidants[169]. Interestingly, the subclinical pro-inflammatory state

observed in many pathogenesis conditions such as atherosclerosis, aging, T2DM and cancer, is caused from mitochondrial ROS overgeneration[170].

Other sources of oxidative stress in diabetesNon-esterified FFAs are elevated in T2DM patients[171]. These excessive FFAs enter the citric acid cycle to generate acetyl-CoA to receive NADH overproduction to cause mitochondrial superoxide over production. In humans, infused FFA has been shown increased lipid peroxidation by elevated isoprostanes marker levels[172,173]. Adipocytokine, leptin is secreted from the adipocytes to act on the central nervous system to decrease food intake. It reflects all effects on the vascular smooth muscle cells, endothelial cells, macrophages and monocytes[174]. Leptin levels are increased and associated with cardiovascular disease in T2DM patients[175-177]. In culture of endothelial cells incubated with leptin to cause ROS production[178,179].

AntioxidantsRegulation of the cellular redox status is depends on the rate of ROS counterbalance and elimination from the enzymatic and/or non-enzymatic antioxidants. Superoxide is converted by SOD to H2O2 and O2 molecule. There are 3 isoforms of SOD such as cytosolic Cu/Zn SOD (SOD1), mitochondrial Mn-SOD (SOD2) and extracellular SOD (SOD3). Catalase, the heme metalloenzyme is expressed in peroxisomes, mitochondria, cytoplasm and nucleus. H2O2 is catalyzed by catalase to oxygen and water[180]. While glutathione peroxidase the selenoprotein, was found in both intracellular and extracellular. Gluta-thione peroxidase has a highly sensitive function for lipid peroxides degradation, converses H2O2 to water by using the thiol group of glutathione[181]. Their H2O2


Atherosclerosis

Diabetes

Glucose ↑ FFA ↑

Auto oxidation Polyol pathway AGEs

Endothelial celldysfunction

oxLDL ↑

Cytokinesadipokinesprostanoids

Insulin resistance

β-cell dysfunction

Oxidative stressROS

Inflammation Monocyte activation Cytokines VSMC migration/proliferation

Figure 5 Summarizes the reactive oxygen species associations with atherosclerosis and sources of reactive oxygen species production in type 2 diabetes. oxLDL: Oxidized low density lipoprotein; FFA: Free fatty acids; AGEs: Advanced glycation end-products; VSMC: Vascular smooth muscle cells; ROS: Reactive oxygen species.


detoxification plays the important roles to prevent lipid peroxidation production and regulation of the cellular redox status[182]. The glutathione system, thioredoxin peroxidase is key enzyme to regulate the cellular levels of thiol/disulfide while the production of antioxidant enzymes is regulated by the redox-cellular transcription factors[183]. For example, the expressed transcription factor NF-E2 related factor in the cytosolic is interrupted binding with Keap-1 as the responsible to increase oxidative stress and translocate to the nucleus for initiation of the transcription of the various antioxidant enzymes[184] as the strategy to develop many class of antioxidant, anti-inflammatory, and anticancer agents. Reduction in non-enzymatic antioxidants, thiol glutathione and thioredoxin are the major dysregulation of the cellular redox status[185]. The cellular redox status is reflected by the reduction of glutathione (GSH), oxidized glutathione (GSSG) ratio (or GSH:GSSG ratio), ascorbic acid, tocopherols and methionine and cysteine amino acids. Exogenous herbal antioxidants compounds in dietary foods include flavanoids, anthocyanins and polyphenolics act as ROS scavenging[186,187]. The direct interaction of ROS with non-enzymatic antioxidants is based on chemical structure properties. In free radicals participate in 1e- oxidation while non-radical species was 2e- oxidation. For example, O2

•- and OH• radicals react with the ascorbic acid and thiols. While the OH• more activity and instability react with methionine and tocopherols. H2O2 and the non-radical may react with thiols and methionine, and the OONO- discriminate to react with thiols, ascorbic acid, tocopherols and methionine[188].

Oxidative stress induces insulin resistanceOxidative stress plays the major role in the association with the insulin resistance pathogenesis by insulin signals disruption and adipocytokines dysregulation[8,9]. In rat models, oxidative stress enhances insulin resistance. The evidence suggested that Ang Ⅱ infused rats required the increased glucose infusion to maintain euglycemia during hyperinsulinemic clamp to stimulate ROS production[10]. For this example, Ang Ⅱ-infused rats were caused insulin resistance from the suppression on insulin-induced glucose uptake in skeletal muscle and increased in oxidative stress biomarkers in this animal experiment. In experimental model, superoxide dismutase and tempol can reduce the insulin resistance. Many evidences indicated that ROS overproduction may induce insulin resistance and confirmed by the supplementation of antioxidant tempol to cause insulin resistance amelioration in Ren-2 transgenic rats[189]. Insulin-target organs of the obese and diabetic KKAy mice were stimulated and caused ROS over production (skeletal muscle, liver and adipose tissue)[8] and to cause insulin resistance in these organs. High fat-fed mice found ROS overproduction in liver and adipose tissue of these obese mices to induce insulin resistance[190]. Many research studies suggested that antioxidant agents decreased plasma insulin, glucose, triglycerides levels and ameliorate insulin resistance in KKAy mice

with no weight loss[8]. Antioxidant coenzyme Q10 supplementation can ameliorate the increased insulin levels in circulation of SHR/cp rats[191]. As mention above, in over nutrition, the excessive glucose occur and a large amount of glucose is metabolized in the glycolysis and TCA cycle leading to increased NADH and FADH2 production in electron transport chain of mitochondrial and increased superoxide production[127]. In aged animals models treated with antioxidant agents or hypocaloric diets led to ameliorate in oxidative stress status and tissue function[131,132].

Insulin resistanceIn general population, insulin resistance precede in many years before onset of T2DM and it is also multi-factorial[11,12] such as genetic component[11,13]. Insulin resistance and reduction in insulin production are the major characteristics of the T2DM pathogenesis[11,12,14-16]. Modern lifestyle, physical inactivity, abdominal obesity and excessive of adipokines can cause insulin resistance[11,15]. In early stage, normal glucose tolerance is preserved by compensation hyperinsulinemia. About 25% of non-diabetic subject cause insulin resistance in the same ranges that found in T2DM patients[12]. Insulin resistance continuous increases and/or decreases in insulin secretory compensation responses, the deterioration into impaired glucose tolerance occurred. Increased glucose, FFA and insulin levels lead to ROS overproduction, increased oxidative stress and activate stress transduction factor pathways. This can cause insulin activity inhibition and secretion to accelerate the onset of T2DM as shown in Figure 6.

Oxidative stress has been demonstrated the implication and association in the late complications of diabetes mellitus[17,18] as in the schematic of Figure 5. Many studies have demonstrated ROS overproduction and increased oxidative stress to insulin resistance[192-194]. Both in vitro studies and in animal models demonstrated that α-lipoic acid (LA), antioxidant agent increase insulin sensitivity[194-196]. In clinical trials, supplementation with vitamin C, vitamin E, glutathione increases insulin sensitivity in both insulin-resistance and T2DM pa-tients[197,198]. LA act as insulin sensitizer agent, it increased insulin sensitivity about approximately 25% and approximately 20% higher than metformin and rosiglitazone, respectively[199,200]. Oral supplementation with LA formulation for 6 wk decreased circulating fructosamine levels[201] and increase insulin sensitivity[202] in T2DM patients and the other studies have confirmed 2.5 mmol/L of LA to cause GLUT4 activation and translocation[203-205].

Because insulin resistance occurred before chronic hyperglycemia development[12], that difference from insulin resistance in the pre-diabetic state result from oxidative stress activation by increased glucose levels. However, obesity demonstrated the strong association with insulin resistance. In this regard, the mediator of oxidative stress-induced insulin resistance of the pre-diabetic state might be from the adipocyte-derived



factor such as TNF-α[206], leptin[207], FFAs[208-210] and resistin[211]. However, the FFAs elevations are associated with insulin resistance and obesity[208,209,212]. Many studies found that increased FFA levels decrease insulin sensitivity, as in the Randle hypothesis[210] and insulin-signaling inhibition[212]. The increased fasting FFA levels are significantly correlated with decreased reduced/oxidized glutathione ratio in T2DM patients[190]. Elevated FFA concentrations cause mitochondrial dysfunction such as uncouplers of oxidative phosphorylation in mitochondria[213] and increased superoxide produc-tion[214]. These caused the exacerbated situation from FFAs induce oxidative stress and reduce intracellular glutathione caused impaired endogenous antioxidant defenses[190,215,216]. Supplementation with glutathione improves insulin sensitivity and β-cell function by the restoration of redox status in T2DM patients and healthy subjects[217].

FFA mediated the nuclear factor-κB (NF-κB) activation, as the consequence of FFAs increased ROS overproduction and glutathione reduction[216,218-220] and also linked to FFA-activated PKC-θ [221] to caused NF-κB activation[222]. Vitamin E supplementation inhibits the FFA-induced NF-κB activation[216] indicated that FFAs act as pro-inflammatory agent effects the alteration of the cellular redox status.

The HOMA-IR was proposed by Matthews et al[223] that can be used to estimate insulin resistance and insulin sensitivity in individuals. HOMA-IR is easy to calculate and no more laborious technique. HOMA-IR method derives from the mathematic calculation from fasting plasma insulin and glucose concentrations.

Oxidative stress and β-cells dysfunctionIncreased circulating glucose levels stimulate the β-Cells

function by sensing and secreting of insulin in appropriate amount[224] and as the target of oxidative stress. The processes are complex and depend on many factors[16]. The critical glucose metabolism in mitochondrial is the importance linking stimulus the insulin secretion[224-226]. Therefore, mitochondria damage and markedly blunt insulin secretion is also occur by the ability of oxidative stress (H2O2)[226]. Many studies in T2DM patients have suggested that chronic exposure to high glucose and/or high FFA levels impaired β-cells function and β-cells dysfunction[16,227]. Because β-Cells are lower in antioxidant enzymes levels (superoxide dismutase, catalase and glutathione peroxidase) and higher sensitive to oxidative stress[228]. Oxidative stress exposure to β-cells activated the increased p21 cyclin-dependent kinase inhibitor production, decreased insulin mRNA, ATP and calcium flux reductions in mitochondria and cytosol to cause apoptosis[226]. Glucose or methyl succinate can stimulate insulin secretion and inhibit by response to K+ within 30 min[226]. The results indicate that mitochondria in β-cells involved in the processes of glucose induced insulin secretion are affected by increased oxidative stress. Lipid peroxidation, oxidative stress products exposed to islets, inhibited insulin secretion and also caused glucose oxidation[229]. Conversely, antioxidants can protect β-cell against the toxicity of oxidative stress, AGEs production and inhibit NF-κB activation[230-234]. These antioxidants are N-acetyl cysteine (NAC), α-phenyl-tert butylnitrone, aminoguanidine and zinc. Recent research study evaluated β-cells function after over expression of glutamine. Hexosamine over production resulted from the deterioration of insulin signaling of glucose-stimulated insulin secretion. Fructose-6-phosphate amidotransferase is the rate-limiting enzyme increase in hexosamine pathway[235], coincident with increased


Hepatic glucose, FFA production ↑

Organ glucose, FFA uptake ↓

Glucose transport (uptake) ↓

Hyperglycemia

Hyperinsulinemia

Insulin activity ↓

Insulin resistance

Organ response to insulin ↓

Insulin receptor deficit

Glucose-inducedinsulin secretion ↓

Impaired β-cell function

Insulin deficiency

Figure 6 Insulin resistancedevelopment and consequence of β-cell dysfunction. FFA: Free fatty acid.


H2O2 production[235] that can ameliorate by NAC supplementation.

β-cells glucose-induced toxicityWest[19] demonstrated that insulin secretion in T2DM patients improved by the reduction of hyperglycemia with diet, insulin or sulfonylureas. On the other hand, in healthy normal, high glucose infused as a clamp reduces insulin secretion[236]. In the study of long term culture of HIT-T15 and/or βTC-6 cells demonstrated that increased glucose levels cause decreased insulin secretion, insulin mRNA and decreased binding of transcription factors[237,238]. Thus, glucose toxicity, the concept of the condition of hyperglycaemia itself can decrease insulin secretion which implies the irreversible damage to cellular components of β-cells[239]. Generally in β-cells, excessive glucose oxidation and metabolism will always cause to ROS over production. Superoxide dismutase and catalase are normally as the detoxified antioxidant enzymes. β-Cells are low amount of these antioxidant enzymes and also low in glutathione peroxidase, a redox-regulating enzyme[240]. Then, hyperglycaemia condition leads to increase ROS production and accumulation in β-cells and subsequent of cellular components damage. Pancreas duodenum homeobox-1 is an insulin promoter activity regulator was loss leading to β-cell dysfunction[240]. Supplementation with NAC and/or aminoguanidine can ameliorate the glucotoxic effects on insulin gene activity[230], reduced insulin levels and increased insulin mRNA and insulin sensitivity[230].

β-cells lipid-induced toxicityLipotoxicity to β-cells concept, elevation of non-esterified fatty acids concentrations in diabetic and non-diabetic obese patients, result of the enhanced adipocyte lipolysis. In the presence of the excessive fatty acid oxidation in β-cells is caused increased long-chain acyl CoA accumulation leading to inhibite β-cells function[241]. This process is as an integral part of the normal insulin secretory function. This long-chain acyl CoA can inhibit the insulin secretory function by opening β-cell K+-sensitive ATP channels. In the second mechanism, in long-term culture of β-cells formulas with FFAs can effect the potential reduction on mitochondrial membrane and uncoupler proteins-2 over expression to cause the K+-sensitive ATP channels opening which lead to decreased ATP production and insulin secretion[242,243]. Third mechanism, β-cells apoptosis might possess from triglyceride or fatty acid induced ceramide synthesis and/or nitric oxide production. Thus, impaired insulin secretion and β-cell dysfunction strongly associated with the FFA-stimulated ROS overproduction[244].

β-cells combined glucose/lipid toxicityElevation of glucose and FFA levels are the major characteristic of T2DM patients. This combination is the major β-cells toxicity and require the maximize protection. In culture cells of islets or HIT cells were

exposed to high concentrations of glucose and FFA levels. There was decrease in insulin-gene activity and insulin mRNA[245]. In the study of islets co-culture with high glucose and palmitate levels caused impaired insulin signaling of the glucose-stimulated insulin secretion[244]. Recent studies have confirmed that β-cells lipotoxicity is the concurrent status as the amplifying effect mediated by glucose toxicity in hyperglycemia condition[246,247].

DyslipidemiaInsulin resistance and T2DM are characterized by dyslipidemia one major risk factor for cardiovascular disease. Lipid triad is the complex metabolic milieu associated with dyslipidaemia[248] comprise with hyper-triglyceridemia, low levels of high-density lipoprotein cholesterol (HDL-C) and the appearance of small, dense, LDL (sdLDL) - and caused excessive post prandial lipemia[249,250]. Diabetic dyslipidemia caused from the disturbance of lipid metabolism, an early event cardiovascular complications development and was preceded in T2DM patients by several years[249-253]. Indeed, insulin resistance status in both with and without T2DM patients was display qualitatively similar lipid abnormalities[250]. The different components of diabetic dyslipidemia are closely linked to each other metabolically[249-253] and are initiated by the elevation of triglyceriderich very LDL (VLDL) from hepatic over production[249,251]. It is the key importance mechanisms to elucidate the over production of VLDL involved in diabetic dyslipidemia[249].

In insulin resistance state, decrease insulin function and lack of insulin inhibits lipolysis leads to increase FFAs generation of and lower lipoprotein lipase activity. This occurs after meal consumption, generates a chylomicron remnant rich in TG[254], caused elevated hepatic FFAs and VLDL TG-rich particles secretion. These processes affects HDL-C metabolism through the interchange with TG-rich lipoproteins via cholesteryl ester transfer protein to produce HDL particles containing high TG concentrations. These HDL-TG particles were hydrolyzed with hepatic lipase to TG and HDL. This HDL becomes smaller and less antiatherogenic activity, easily to remove from the circulation by the kidneys. Moreover, insulin resistance in T2DM patients associated with endothelial dysfunction led to increase risk of CVD[255]. The most atherogenic subfractions of sdLDL are elevated in circulation of obesity individuals, as a key feature in association with elevated triglyceride and low HDL cholesterol. Elevated sdLDL concentrations are also founded in abdominal obesity subjects and demonstrated greater myocardial risk.The mechanisms are related to excess accumulation of abdominal adipose tissues, elevated total cholesterol and LDL-C and related to high saturated-fat consumption, weight gain and obesity.

Dyslipidemia is commonly occurred in T2DM patients and might play the major role in accelerated



HDL-C can be used as markers of insulin levels, insulin resistance and CVD risk factor[258,263]. The highest % sensitivity and % specificity cut-off points corresponding to the TC/HDL-C, TG/HDL-C ratios and non-HDL-C are 3.58, 2.48 and 130.4, respectively[258]. Because of TC/HDL-C, TG/HDL-C ratios and non-HDL-C are easily calculated and ordered with every lipid profiles available to the clinician and no costs addition. The cut-off value of these ratios in Tangvarasittichai et al[258] study was lower than the results from Western populations[266-268]. Then, insulin resistance was significantly predicted by these markers. For atherosclerotic risk assessment in obesity, metabolic syndrome and T2DM patients requires more attention to lipid screening.

Development of T2DM from insulin resistanceInsulin resistance often occurs with T2DM but is insufficient for the T2DM development. β-cells dysfunction are important event for the T2DM development and progression. In early stage of insulin resistance, β-cells increase the secretory function try to compensate and control hyperglycemia. In Pima Indian population study caused acute insulin response dysfunction or decreased β-cell responses was found during the normal glucose tolerance state in individuals who eventually progressed from normal glucose tolerance to impaired glucose tolerance or T2DM when compared with individuals who persisted in the state of normal glucose tolerance[269]. There was evidence of early defects in glucose disposal by decreased insulin sensitivity before the develop-ment of glucose intolerance state, although output of circulating glucose did not increase until the progression from impaired glucose tolerance to T2DM revealed. Interestingly, individuals who demonstrated transient glucose intolerance but were able to recover and to reach normal glucose tolerance and did not show the early secretory defect observed in progressed individuals[269]. β-cells failure or dysfunction occurred as the results of the combination of increased oxidative stress, glucose and lipids accumulation to cause glucotoxicity and lipotoxicity to β-cells to progress increased apoptosis and loss of the insulin granule secretory components expression[270].

T2DMThe World Health Organization updated the prevalence of T2DM estimated by the year 2025 those 30.3 million people in the United States and total of 380 million people worldwide will be diagnosed as DM[271]. By the year 2050, those 45.6 million Americans will be diagnosed as DM[272]. T2DM is associated with obesity, sedentary lifestyle and lack of exercise in the aging population. There are a number of gene abnormalities related to T2DM, that showed significant differences exist in the abnormalities gene associated with T2DM among the various ethnic populations, such as African Americans, Asians and Europids[273,274]. The contribution of any one of these genes to T2DM is small and total

macrovascular atherosclerotic disease and increased CVD risk in T2DM patients[256]. Dyslipidemia in T2DM patients as lipids triad is characterized by increased insulin levels, hypertriglyceridemia, low HDL-C levels and increased sdLDL-particles (independent of LDL-cholesterol) and increased TG-rich remnant lipoprotein (TGRLs) concentrations[257,258]. In this manner, low HDL-C levels associated with hyperinsulinemia or insulin resistance and insulin signaling for insulin-mediated glucose disposal[259] characterized by higher fasting plasma glucose and insulin levels. Then, these major changes associated with the insulin resistance syndrome are increased TGRLs and decreased HDL-C levels. Thus, in dyslipidemia, using the lipoprotein concentration ratios are associated with insulin resistance and increased CVD risk conditions. Lipoprotein ratios might be useful to identify insulin resistance individuals even different in fasting glucose or insulin levels. Obesity, metabolic syndrome, and T2DM may also show the same dysli-pidemia characteristic[12,257,259] and measuring TG, HDL-C, TC/HDL-C and TG/HDL-C ratio in circulation may also use as insulin resistance estimation. For example, these TG, HDL-C, TC/HDL-C and TG/HDL-C ratio are independently associated with insulin levels, insulin resistance and CVD risk[258,260,261].

Lipoprotein ratios: In description above, the major change is increased TGRLs and decreased HDL-C levels are associated with insulin resistance syndrome. Insulin plays the important role in TG metabolism, in normal condition TGRLs particles reduces synthesis by the distinct pathways when compared with VLDL particles synthesis[249,258]. Insulin fails to suppress VLDL particles synthesis[262]. Insulin resistance is significantly associated with increased lipid synthesis in the liver, increased FFAs flow to the liver and decreased VLDL particles clearance resulting in increased VLDL levels in the circulation[251]. Thus, dyslipidemia (as lipoprotein ratios) may associate with insulin resistance and increased CVD risk. On this basis, waist circumference, LDL-C, TG levels, insulin resistance and the CVD risk are estimated[263]. The major features of dyslipidemia are determined by hypertriglyceridemia, low HDL-C levels and slightly high or normal LDL-C levels with altered composition. Hypertriglyceridemia is indicate as elevated atherogenic chylomicron and VLDL remnant and associated with increased CVD risk[264,265]. These phenomenons demonstrated the problems of VLDL and HDL levels but not the LDL levels and concurrent with increased insulin levels. Low HDL-C level is associated with the hyperinsulinemia and/or insulin resistance and insulin signaling for insulin-mediated glucose disposal[259]. All of these features are associated with coronary heart disease risk in obesity, metabolic syndrome and T2DM patients. The TC/HDL-C, TG/HDL-C ratios and non-HDL-C (as TC - HDL-C) were used as surrogate markers for insulin levels and insulin resistance estimation. In Tangvarasittichai et al[258] study suggests that TC/HDL-C, TG/HDL-C ratios and non-



aggregate of all described genes accounts for < 15% of the predisposition[273,275]. It is typically diagnosed in patients older than 30 years with overweight or obesity and positive in family history of T2DM. However, insulin resistance may occur and develop in many years before diagnosed as T2DM[276]. Figure 7 summarized the etiology of the T2DM pathogenesis.

Patients are diagnosed as T2DM when plasma glucose levels reach at the diagnostic criteria (Table 1). These T2DM patients are at high risk for microvascular complications (e.g., nephropathy, retinopathy and neuropathy) and macrovascular complications (e.g., peripheral vascular disease, cerebrovascular disease and cardiovascular disease). T2DM patients with good controlled plasma glucose levels demonstrated to delay the progression of microvascular and macrovascular complications[271,277].

MANAGEMENT OF DYSLIPIDEMIA AND HYPERGLYCEMIA IN T2DM PATIENTSFasting serum lipids profile should be determined annually in T2DM patients as in the recommendation by the American Diabetes Association (ADA)[278]. ADA recommended for the satisfied lipids profile level as low-risk by LDL-C < 100 mg/dL (2.6 mmol/L), triglycerides < 150 mg/dL (1.7 mmol/L) and HDL-C > 50 mg/dL (1.3 mmol/L)[276].

TreatmentLifestyle interventions: The American Diabetes Asso-ciation and the American Heart Association recommend that increased physical activity and lifestyle modifications should be advised for all T2DM patients[278,279]. Combination with such interventions included nutrition therapy or supplementation, weight loss and non-smoking. These have been help T2DM patients to receive better controlled their lipid concentrations. Nutrition interventions and supplementations should be designed according to the condition of T2DM individuals such as diabetes status,

age, other comorbidities and avoidance to intake transfat, saturated fat, cholesterol and should increase intake of fiber (fiber in oats, legumes, citrus), omega-3 fatty acids and plant stanols/sterols[278]. Glycemic control can also modify circulating triglycerides levels, especially in T2DM patients with hypertriglyceridemia and poor glycemic control[278].

Pharmacological interventions of dyslipidemiaThere are many pharmacological classes available for dyslipidemia treatment.

Statins: Statins inhibit enzyme 3-hydroxy-3-methyl-glutaryl CoA reductase suppress cholesterol synthesis and increase number and activity of LDL-receptor. Statins are effective drug for lowering LDL-cholesterol, raising HDL-C and reducing TG levels. There are seven pharmaceutical forms of statins including lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin and pitavastatin available in the market. Statins also have the other pharmacodynamic actions such as vascular inflammation reduction, immune suppression, improved endothelial function, platelet aggregability, enhanced fibrinolysis, antithrombotic action, increase neovascularization in ischemic tissue and stabilization of atherosclerotic plaques[280].

FibratesFibrates control the lipid metabolism by mediated through peroxisome proliferator-activated receptors-α activation, stimulation of β-oxidation of fatty acids in peroxisomes and mitochondria to cause lowering fatty acid and triglycerides levels in circulation. The first drug of this class is Clofibrate. Eventually, the revolution in lipid-lowering drugs research discover of many other fibrate drugs such as fenofibrate, bezafibrate, gemfibrozil and ciprofibrate. These drugs demonstrated the adverse effect to cause hepatomegaly and tumor formation in the liver of rodents. Then, they had restricted for the widely


TNF-α ↑NF-κB ↑Adiponectin ↓

FFA ↑

Liver Muscle, organs

Obesity and abdominal obesityOxidative stress ↑

Insulin resistance

Impaired glucose tolerance

Diabetes mellitusGenes Candidate/susceptibility

Environmental factors Overnutrition High energy intake High fat, carbohydrate, etc . Physical inactivity

Genes

Figure 7 Summarized the etiology of the type 2 diabetes mellitus patho-genesis. FFA: Free fatty acid; NF-κB: Nuclear factor-κB; TNF-α: Tumor necrosis factor α.

Table 1 Type 2 diabetes mellitus and glucose levels for diagnostic criteria1

Glucose management test Range Diagnosis

Fasting plasma glucose (mg/dL) (at least 8 h fast)

≥ 126 Diabetes mellitus

100-125 Impaired fasting glucose ≤ 99 Normal

2-h oral glucose tolerance test of 75 g glucose load (mg/dL) WITH

≥ 200 Diabetes

Random screening with common symptoms of diabetes (polyuria,

140-199 Impaired glucose tolerance

polydipsia, weight loss, etc.) ≤ 139 NormalHemoglobin A1c (%) ≥ 6.5 Diabetes

5.7-6.4 Prediabetes/high risk ≤ 5.7 Normal

1All tests in diabetes range must be repeated after 24 h, to be confirmed diagnosis.


use in humans. Gemfibrozil and fenofibrate are Food and Drug Administration (FDA)-approved for lipid lowering drugs due to milder effect on peroxisome proliferation.

Nicotinic acidLong term study of the coronary drug project demon-strated that niacin is the effective drug to increase HDL-C levels and reduced CVD events[281] in a non-diabetic subjects. Niacin cause adverse effects on the glycemic control levels in T2DM patients. In high doses treatment with niacin may increase blood glucose levels. The modest doses of 750-2000 mg/d of niacin are significantly increased HDL-C levels and decreased LDL-C, triglyceride levels and accompanied with modest changes in glucose levels for diabetes therapy[282,283]. However, there is no evidence for the CVD outcomes reduction with niacin supplementation in T2DM patients.

Antihyperglycemic drugs: The standard care for T2DM patients is mainly in controlled blood glucose levels by using glycemic lowering drugs and concomitant with controlled diet and increased physical activity. With proper controlled and managed these contributors such as circulating glucose levels, hemoglobin A1c, lifestyle modifications, these can be effectively controlled and reduced the progression and complications disease. In general, only approximately 50% to 60% of T2DM patients have achieved their glycemic goals[284]. There are many reasons for poor control of T2DM includingmedication efficacy, adverse effects, access to medi-cations and health care education, poor adherence, lack of lifestyle changes and no physical activity. Now a day, more pharmacologicals for T2DM treatment have been approved for use. There are 12 classes of antihy-perglycemic drugs FDA-approved in the United States[285]

such as sulfonylureas, meglitinides, thiazolidinediones, dipeptidyl peptidase-4 (DPP-4) inhibitors, biguanides, sodium glucose transporter 2 inhibitors, α-gluco-sidase inhibitors, amylin analogues and glucagon-like peptide-1 (GLP-1) receptor agonists. These are insulin analogues. Metformin is one of the most commonly prescribed medications for T2DM management. Met-formin treatment ameliorate the insulin resistance especially in liver and skeletal muscle but less effect in adipose tissue[286,287], decreased inflammatory response, improved glycemic control[288,289] and enhance β-cell function in T2DM patients by increased insulin sensitivity and glucotoxicity reduction[290]. Metformin reduces fatty acid oxidation in adipose tissue[291], increased GLUT4 translocation in muscle and adipose tissues by activated enzyme adenosine monophosphate kinase and reduced gluconeogenesis in liver[292-295]. There are many developed non-conventional drugs to improve glycemic control such as Cycloset is used together with diet and exercise to treat type 2 diabetes. Cycloset is not for treating type 1 diabetes. Welchol is a non-absorbed, polymeric form, lipid-lowering and glucose-lowering agent for oral administration. Welchol is a high-capacity bile acid-binding molecule. Afrezza Inhalation Powder is

the FDA approved the inhalation form of insulin. The new drug is not a substitute for long-acting insulin and use as the combination with conventional long-acting insulin drug for both types of diabetes and many drugs are in the late clinical trials state.

There are new medications and treatments were identified from the FDA, they are in the clinical trials or waiting for approval treatment in dyslipidemia, obesity and T2DM[296]. Recent research study reports that metformin treatment cause metabolic effects to increase GLP-1 concentration in the circulation[297,298]. GLP-1 is an incretin generated from the transcription product of the proglucagon gene. Incretin is a signaling polypeptide contained with 30-amino acid. GLP-1 secretion by ileal L-cells is not depend on the presence of nutrients in the small intestine and responsible for stimulated insulin secretion to limit glucose elevations with the higher efficacy at high glucose levels[276,299]. Elevated GLP-1 secretion might possibly cause increased glucose absorption in the distal segments of small intestine.

Incretins are the gastrointestinal hormone secreted from the intestine and stomach responsible for oral food intake and stimulated the secretion of insulin during meals in healthy peoples[276]. Two major incretin molecules are (1) GLP-1; and (2) Glucose-dependent insulinotopic peptide knows as gastric inhibitory polypeptide (GIP) and to neutralize stomach acid to protect the small intestine and no therapeutic efficacy in T2DM. GLP-1 has lower glucose levels by stimulated insulinproduction and increased glucose metabolism in adipose tissue and muscle. GLP-1 promote the pancreatic β-cells proliferation, reduce apoptosis, increase cardiac chronotropic, inotropic activity, decreases glucagon secretion, reduces glucose production, increase appetite suppression for food intake reduction and slow gastric emptying[271,276,299]. GLP-1 is degraded by enzyme DPP-4 and this enzyme does not inhibit by metformin[298]. The prevention of GLP-1degradation by DPP-4 is one method to increase the effects of GLP-1. DPP-4 inhibitor drugs inhibit the glucagon secretion which in turn increases secretion of insulin to decrease blood glucose levels and decreases gastric emptying. The FDA-approved the DPP-4 inhibitor drugs including sitagliptin (Januvia), alogliptin (Nesina), saxagliptin (Onglyza), linagliptin (Tradjenta), anagliptin, vildagliptin, teneligliptin, gemigliptin and dutogliptin. The adverse effects are dose-dependent to cause headache, vomiting, nausea, nasopharyngitis, hypersensitivity and other conditions. Other side effects of exenatide (GLP-1 agonist) note for abdominal pain, acid stomach, diarrhea, altered renal function, weight loss, dysgeusia, belching and cause pruritus, urticaria and rash reactions at the injection site.

CONCLUSIONIn this present review has described the detrimental effects from chemicals and biochemicals reaction, metals, medications, over nutrition, obesity and diseases in oxidative stress, insulin resistance development and



the progression of T2DM and the progression of diabetic complications and organ dysfunctions. Oxidative stress played underling associated with the pathogenesis of diseases, leading to increases risk of insulin resistance, dyslipidemia, elevated blood pressure, metabolic syndrome, inflammation and endothelial dysfunction. This reviewed support the oxidative stress contribution of the multifactorial etiology of oxidative stress and insulin resistance in the whole body. ROS act as the signal transduction factor and plays the important role in oxidative stress-mediated downstream signaling pathways and enhances the cell death. Furthermore, risk for several chronic diseases development associated with oxidative stress and metabolic syndrome including T2DM, hypertension, arthritis, congestive heart failure, chronic renal failure, cancer and Alzheimer’s. These diseases may be substantially reduced by dietary modifications, increased physical activity and antioxidant drugs ameliorated oxidative stress. The therapeutic approaches target on oxidative stress may delay or prevent the progression and onset of diseases. Then, antioxidants supplementation may curtail the progression and onset of the metabolic disease complications. Antioxidant interventions, an importance goal of future clinical investigations should be implementation and to improve oral bioavailability targeted to the oxidant overproduction site. Lifestyle change remains the best prevention and therapeutic approach to oppose the increasing epidemic of cardiovascular diseases, obesity, hypertension, dyslipidemia and T2DM. Finally, the connection between oxidative stress, insulin resistance, dyslipidemia, inflammation, life style, atherosclerosis and diabetes as demonstrated in the schematic in Figure 8.

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Figure 8 Connection between life style, oxidative stress, insulin resistance, inflammation and atherosclerosis. FFA: Free fatty acid; NO: Nitric oxide; MNC: Mononuclear cells; PMN: Polymorpho nuclear cells; CRP: C-reactive protein; MIF: Migration inhibitory factor.

Oxidativestress

Lipolysis ↑ Obesity

FFA ↑

Endothelial NO

AtherosclerosisVasodilatory reserve

Excess calories physical activity

Hyperglycemia(diabetes)

Inflammation inMNC/PMN cells↑ CRP, ↑ MIF

Glucose transport ↓ Hyperinsulinemia

Insulin resistant

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P- Reviewer: Sicari R, Soare A S- Editor: Ji FF L- Editor: A E- Editor: Liu SQ



Rinat Gabbay-Benziv, E Albert Reece, Fang Wang, Peixin Yang

Rinat Gabbay-Benziv, E Albert Reece, Fang Wang, Peixin Yang, Department of Obstetrics, Gynaecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United StatesE Albert Reece, Peixin Yang, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United StatesAuthor contributions: Gabbay-Benziv R and Wang F collected the data and wrote the manuscript; Reece EA contributed to the writing of the manuscript; Yang P designed the aim of the editorial and wrote the manuscript; all authors reviewed the manuscript, made their revision and finally approved it for publication. Conflict-of-interest: None of the authors have a conflict of interest.Supported by NIH R01DK083243, R01DK101972 and R56 DK095380 (to Yang P), R01DK103024 (to Yang P and Reece EA) and Basic Science Award (1-13-BS-220), American Diabetes Association (to Yang P).Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Peixin Yang, PhD, Department of Obstetrics, Gynaecology and Reproductive Sciences, University of Maryland School of Medicine, BRB11-039, 655 W. Baltimore Street, Baltimore, MD 21201, United States. [email protected] Telephone: +1-410-7068402 Fax: +1-410-7065747Received: August 29, 2014 Peer-review started: August 30, 2014 First decision: November 27, 2014Revised: December 9, 2014 Accepted: January 9, 2015 Article in press: January 12, 2015Published online: April 15, 2015

Abstract Currently, 60 million women of reproductive age (18-44 years old) worldwide, and approximately 3 million American women have diabetes mellitus, and it has been estimated that this number will double by 2030. Pregestational diabetes mellitus (PGD) is a significant public health problem that increases the risk for structural birth defects affecting both maternal and neonatal pregnancy outcome. The most common types of human structural birth defects associated with PGD are congenital heart defects and central nervous system defects. However, diabetes can induce birth defects in any other fetal organ. In general, the rate of birth defects increases linearly with the degree of maternal hyperglycemia, which is the major factor that mediates teratogenicity of PGD. Stringent prenatal care and glycemic control are effective means to reduce birth defects in PGD pregnancies, but cannot reduce the incidence of birth defects to the rate of that is seen in the nondiabetic population. Studies in animal models have revealed that PGD induces oxidative stress, which activates cellular stress signalling leading to dysregulation of gene expression and excess apoptosis in the target organs, including the neural tube and embryonic heart. Activation of the apoptosis signal-regulating kinase 1 (ASK1)-forkhead transcription factor 3a (FoxO3a)-caspase 8 pathway causes apoptosis in the developing neural tube leading to neural tube defects (NTDs). ASK1 activates the c-Jun-N-Terminal kinase 1/2 (JNK1/2), which leads to activation of the unfolded protein response and endoplasmic reticulum (ER) stress. Deletion of the ASK1 gene, the JNK1 gene, or the JNK2 gene, or inhibition of ER stress by 4-Phenylbutyric acid abrogates diabetes-induced apoptosis and reduces the formation of NTDs. Antioxidants, such as thioredoxin, which inhibits the ASK1-FoxO3a-caspase 8 pathway or ER stress inhibitors, may prevent PGD-induced birth defects.

MINIREVIEWS


Birth defects in pregestational diabetes: Defect range, glycemic threshold and pathogenesis




Key words: Pregestational diabetes; Birth defects; Glycemic threshold; Diabetic embryopathy; Range of defects


Core tip: Pregestational diabetes is a rising problem with gravid impact on adverse pregnancy outcomes. This review concentrates on diabetes-induced birth defects and the underlying mechanism of diabetic embryopathy derived from animal studies. The main defects associated with pregestational diabetes are in the cardiovascular and central nervous systems, and are linearly related to maternal glycemic control. Animal studies reveal oxidative stress and stress kinase signalling-induced apoptosis as key factors in pathogenesis. However, many questions remain unanswered, and the rate of congenital defects in human diabetic pregnancies is still high. The cause of diabetic embryopathy warrants further investigations.

Gabbay-Benziv R, Reece EA, Wang F, Yang P. Birth defects in pregestational diabetes: Defect range, glycemic threshold and pathogenesis. World J Diabetes 2015; 6(3): 481-488 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/481.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.481

INTRODUCTIONMajor structural or genetic birth defects affect approximately 3% of births[1], and are the leading cause of infant mortality in the United States[2]. The pathogenesis of most birth defects is unknown[3,4]. Pregestational diabetes (PGD) is one of the leading known causes with up to a nine-fold increase in birth defects, compared with the rate seen in nondiabetic pregnancies[5]. Although the risk for birth defects in PGD pregnancies can be significantly reduced by appropriate pregestational counselling and meticulous control of maternal glycemic levels[6], it cannot be equalized to the background risk partially because at least 40% of diabetic pregnancies are usually unplanned[6,7].

The pathophysiology of maternal diabetes induced birth defects is complex, however, clearly relates to maternal glucose levels. The mechanism is not entirely understood, but animal studies have shown it to be associated with decreased cell proliferation and increased cell apoptosis due to high oxidative stress. The second major change seen in animal models of PGD is altered gene expression causing deviation from the normal developmental process.

PGD can affect almost any organ. However, most congenital defects associated with diabetes occur in the cardiovascular, central nervous and musculoskeletal systems[8-10].

In this review we will discuss how PGD increases the risk of birth defects, the range of defects seen in diabetic

pregnancies and the relationship of these defects to maternal glucose control. As the risk of birth defects is well established in type 1 and type 2 diabetes mellitus, while it is still controversial in gestational diabetes[11], we will focus this review on PGD only.

RANGE OF DEFECTSPGD is associated with a wide range of anomalies in almost any fetal organ. Based on available literature, women with PGD have 2- to 9-fold higher risk for having babies with birth defects, with a prevalence of birth defects of 2.7%-18.6%, compared with the healthy population[5,12-17], having a prevalence of birth defects of 2%-3%.

Although any birth defect can be associated with PGD, more anomalies are seen in the cardiovascular, central nervous and skeletal systems[9,10,18]. Rare abnormalities almost exclusively associated with diabetes include caudal regression syndrome with femoral shortening and sacral agenesis. Caudal regression syndrome consists of a spectrum of congenital anomalies of the lower spine and hips, is associated with genitourinary and lower limb defects, and is one of the few syndromes in which the presence of maternal diabetes has always to be considered as the cause, if not confirmed[19]. Although very rare, some studies found this syndrome to be 200-600 times more prevalent in infants of diabetic mothers compared with nondiabetic mothers[16]. Be that as it may, it can still be seen also in non-diabetic mothers as described in a case report by Versiani et al[20] who reported 2 cases of caudal dysplasia sequence - one woman had PGD while the other had gestational diabetes.

Previous studies assessed the prevalence of con-genital anomalies in the diabetic population. Eidem et al[21] Studied the risk of congenital anomalies related to type 1 diabetes. They compared major congenital anomalies (excluding minor anomalies as defined by the EUROCAT system) between women with pregestational type 1 diabetes and controls. All women registered at the Norway national delivery registry from 1999 to 2004 were included. Anomalies were registered in 5.7% (91/1583) offspring of women with type 1 diabetes, compared with 2.9% in the background population. The risk for cardiovascular anomalies was 3 fold higher in the diabetic group (3.2% vs 0.94%, respectively) with ventricular septal defect (VSD) (12/1583) and patent ductus arteriosus (10/1583) being the most prevalent birth defects in the cardiovascular system and the most common compared with all other birth defects.

Garne et al[22] used the same EUROCAT classification to describe birth defects in 18 population-based EUROCAT registries of congenital anomalies, which included births between 1990 to 2005, and compared 669 diabetes cases with 92976 non-diabetes cases. The authors showed significantly increased odds ratios for neural tube defects (anencephaly and encephalocele, but not spina bifida) and several subgroups of congenital heart defects. Other birth defects found in increased odds ratios in

Gabbay-Benziv R et al . Birth defects in pregestational diabetes


infants of pregnancies complicated by pregestational diabetes were anotia, omphalocele and bilateral renal agenesis. Multiple congenital anomalies were present in 13.6% of the diabetes cases and 6.1% of nondiabetes cases. The odds ratio for caudal regression sequence was very high (26.4, 95%CI: 8.98-77.64), but only 17% of all caudal regression cases occurred in pregnancies complicated by PGD.

Most recently, Correa et al[23] reviewed the association of type 1 and type 2 PGD with 39 birth defects using the National Birth Defects Prevention Study. The authors examined databases from 10 birth defects surveillance systems in the United States looking for isolated (one or more major defects in same organ system or with known sequence) or multiple birth defects (2 or more major, unrelated defects) from births between 1997 to 2003. They found an association between PGD and 11 cardiac defects and 7 non cardiac defects.

Galindo et al[12] prospectively followed 126 women with pregestational diabetes in a single tertiary centre in Spain. They reported 17 offspring with congenital anomalies (13.5%). Although chromosomal abnormalities are not generally associated with PGD, one fetus had trisomy 21 and atriventricular septal defects. Eight fetuses were listed with major birth defects (6.3%), 35.3% of fetuses had cardiovascular defects, and geni-tourinary defects accounted for another 23.5%. They noted positive correlation between high levels of haemoglobin A1c (HgA1c) > 7% at early pregnancy and major congenital anomalies. Despite this study including only small number of women, the prospective nature of it allows strength in addressing its results.

Wender-Ozegowska et al[16] studied a group of 198 diabetic women and found 17 pregnancies that resulted in infants with birth defects (8.6%). The most common defects were in the cardiovascular system (5.5%), with 7/198 (3.5%) of cases being atrioventricular septal defects (3 cases of VSD and 3 cases of atrial septal defect). Wren et al[17] focused their research on cardiovascular birth defects. They followed 609 diabetic pregnancies and found that 3.6% of the women delivered infants with cardiovascular defects, the most common being transposition of great arteries, truncus arteriosus and tricuspid atresia. Janssen et al[18] studied 1511 PGD cases, with congenital birth defects prevalence of 7.2%. The most common defects occurred in the cardiovascular system, NTDs, cleft lip/palate and skeletal defects.

Although many studies discuss diabetes associated congenital anomalies, the leading type of birth defects and the exact rate of diabetes associated birth defects are difficult to determine. The literature varies widely in what is considered the “leading” type of anomaly, as well as the rate of anomalies, because of suboptimal coding of the diabetic pregnancies according to White’s classification, differences in anomaly coding and reporting bias. Moreover, the recognition of a birth defect may not always occur in the immediate neonatal period, thus, may not be entered into a registry. On the other

hand, over documentation of diabetes related birth defects, compared with birth defects in the nondiabetic population, may occur because of the known association of defects to diabetes, or the relatively high transfer of infants of diabetic pregnancies to neonatal intensive care units, both leading to a more thorough neonatal examination.

Based upon the available data, it seems that the major organ systems affected by PGD are the cardio-vascular and the central nervous systems, which could be related to their embryonic origin in the neural crest. However, PGD can increase the rate of any other birth defects described above. Therefore, all pregnant women with PGD are advised to undergo a detailed anatomical scan and full fetal echocardiography to screen for possible birth defects, regardless of their having type 1 or type 2 diabetes mellitus or glycemic control at the time of pregnancy.

GLYCEMIC THRESHOLD FOR BIRTH DEFECTSPericonceptional HgA1c is used as a surrogate marker for glycemic control, and is almost linearly related to PGD-induced birth defects. Previous studies have shown that stringent glucose control prior to or at early pregnancy, during organogenesis, can significantly reduce the incidence of birth defects[12,24-29]. However, there are still questions to be answered. Hanson et al[25] studied 532 type 1 PGD women and compared their malformation rate to 222 nondiabetic women. The rate of malformations did not differ significantly between the diabetic and the control groups (4.3% vs 2.4%) although significant different was find in levels of first trimester HgA1c. The median value of HgA1c was 7.7% in the diabetic and 5.3% in the control group (P < 0.001). However, when higher levels of HgA1c were evaluated (HgA1c greater than 10.1% - equal to 8 SD above the normal mean control value), there was statistically significant higher occurrence rate of congenital malformation (P < 0.01).

Wender-Ozegowska et al[16] tried to determine the cut-off for first trimester glycemic levels for prediction of congenital anomalies. They used whole day glycemic profile as well as HgA1c to assess glycemic control. Their cohort included 198 diabetic pregnancies and 4700 nondiabetic pregnancies. The rates of malformation were 8.6% and 3.6%, respectively. They determined the cut-off of value of HgA1c that could prediction congenital malformations as 9.3% (measured up to 16 wk).

Other studies demonstrated the connection between poor glycemic control before or early at pregnancy: Ylinen et al[29] found that the mean HgA1c for women whose infants had congenital malformations was higher than that measured for women with healthy infants prior to 15 gestational weeks (9.5% vs 8.0%, res-pectively). Mironiuk et al[27] compared an occurrence rate of congenital malformations in newborn to mothers with



it difficult to differentiate PGD-induced anomalies from others encountered in nondiabetic pregnancies. Third, most studies use HgA1c to reflect the level of glycemic control, but HgA1c only measures a woman’s average glycemic control over a 3-mo period. Therefore, it does not necessarily reflect a woman’s level of glycemic control during organogenesis and embryogenesis. Moreover, because it is an average measurement, the same level of HgA1c may reflect completely different glycemic patterns, one being constant around the average, and the other with larger fluctuations lower and above from the mean HgA1c value[35]. The effects of such fluctuations (i.e., episodes of acute hyperglycemia and hypoglycemia with “normal” HgA1c) are yet to be determined. Fourth, given the complicated metabolic nature of diabetes, coupled with metabolic changes that are normal during pregnancy, other factors may influence the developing embryo. Finally, lower prenatal detection rates of fetal anomalies (due to obesity, lack of prenatal care, etc.) in diabetic women may lead to statistic skewing when studying infants with congenital anomalies born to PGD mothers[36,37].

DOES DIABETES TYPE AFFECT THE RATES OF CONGENITAL ANOMALIES? Diabetes can be classified by the mechanism (type 1, type 2 or gestational diabetes) or alternatively, by White’sclassification[38]. Rates of diabetes, both type 1 and types 2 are increasing all over the world[39,40] causing an increase in the incidence of maternal diabetes in pregnancy[41]. Although hyperglycemia is a common mechanism for teratogenicity, differences in disease characteristics, such as age of onset, ethnicity, obesity and duration of disease, may affect the disease impact on the perinatal outcome and the rate of congenital anomalies. Current literature does not specify the risk for anomalies for any type of diabetes separately, as some studies include only type 1 diabetes[25,42], others only type 2 diabetes[32] and only a few compare the outcomes between them[43,44].

Gonzalez-Gonzalez et al[44] retrospectively compared the outcomes of type 1 diabetes (n = 904), type 2 diabetes (n = 516), gestational diabetes (n = 3188) and control (n = 115996) deliveries in Ontario, Canada. Congenital anomalies were most frequently observed in women with type 1 diabetes and gestational diabetes, with risk approaching 1.5- to 2-times that of controls. They found a total of 44 anomalies (6.1%) among all women with diabetes, most commonly in the cardiovascular system. Adjusted OR for anomalies was 3.5, 1.7, 2.5 and 1.9 for type 1, type 2, gestational diabetes and control deliveries, respectively. Peticca et al[45] found similar results in a multicentre study in Italy. They prospectively compared 504 type 1 PGD to 164 type 2 PGD pregnancies. The rate of birth defects was significantly higher in women with type 1 diabetes (5.9%), compared with in women with type 2 diabetes

type 1 diabetes (n = 170), newborns of healthy mothers (n = 26368) and mothers with GDM (n = 56). They demonstrated that type 1 diabetes was associated with congenital malformations (11.2%, 1.8% and 2.2%, respectively) and that the risk of major birth defects was directly proportional to the level of maternal blood glucose control during the first trimester. These studies all support the idea that lack of glycemic control leads to congenital malformations, but do not indicate what HgA1c value should be maintained to reduce the risk for infant anomalies. Moreover, Shields et al[30] tried to find cut-off value associated with congenital anomalies but failed to do so, and Lucas et al[31] proved the association of PGD with congenital anomalies only when HgA1c was combined with other factors such as diabetic classification (White’s classification) and maternal vascular complications. The Atlantic-Diabetes in Pregnancy (DIP) trial compared PGD pregnancy outcomes in the same population after a change in pre-pregnancy care policy and improved glycemic control throughout pregnancy. In the Atlantic DIP study, the first trial conducted from 2005-2007, included 104 diabetic pregnancies (87% type 1 PGD). The authors reported two pregnancies with birth defects that had HgA1c values of 6.6% and 5.4% at early pregnancy. The second trial, conducted from 2008-2010, included 168 pregnancies, with more women affected by type 2 PGD (81/168, 48%) and lower HgA1c values (7.3% compared to 6.9%); however, despite of presumed better prenatal care and the lower HgA1c - rate of malformation reported was not changed between the trials. The authors did not report the type of birth defects seen in either of the two studies[32,33].

Other studies have demonstrated a linear relation-ship between HgA1c and major congenital defects. Greene et al[34] showed an increase in PGD-induced birth defects correlating to the level HgA1c. Their risk for major malformation was 3.0% when HgA1c taken at first trimester was less than or equal to 9.3% and 40% with HgA1c was greater than 14.4% (RR = 13.2; 95%CI: 4.3-40.4). Todorova et al[35] studied 124 pregnancies complicated by pregestational diabetes. The mean values of HgA1c were significantly higher in pregnancies complicated by fetal malformations (n = 19/15.3%) than those values measured in pregnancies without fetal malformations [9.01% (SD ± 1.53) vs 8.06% (SD ± 1.64) P = 0.022, respectively].

In conclusion, it is clear that glycemic control is associated with a reduced risk of congenital anomalies. However, the recommended threshold of HgA1c for pregestational diabetic women planning pregnancy is still not known. Reaching a consensus as to what HgA1c level a diabetic pregnant woman should strive for has remained elusive for many reasons. First, differing definitions for major and minor anomalies, and taking into account the various forms of diabetes, limit the ability to derive conclusions from the published data. Second, PGD pregnancies can have the same spectrum of anomalies as nondiabetic pregnancies which makes



(2.0%). Difficulties in glycemic control, increased disease severity and frequent episodes of ketoacidosis and hypoglycaemia were postulated as explanations for this difference. Other studies have found the opposite results[46,47]. Clausen et al[47] compared 389 type 1 and 146 type 2 diabetics delivering in the United Kingdom. Pregnancies affected by type 2 diabetes had worse perinatal outcomes with congenital abnormalities (12.3% in type 2 vs 4.4% in type 1; P = 0.002) accounting for most of this difference. In 2005, Clausen et al[47] showed similar results with almost doubled number of congenital anomalies among pregnant women with type 2 diabetes (6.6%) compared to type 1 diabetes (2.9%)[46]. Both studies cited a delay in antenatal care, suboptimal glycemic control prior to conception, inadequate folate supplementation and maternal obesity, as possible reasons to explain the worse outcomes in pregnancies complicated by type 2 diabetes. Another explanation could be the tendency toward oral glycemic agents in type 2 diabetes, which may lead to less than optimal glycemic control compared to the use of insulin only in type 1 diabetes. Conversely, Roland et al[48], showed a high rate of congenital anomalies in women with type 1 diabetes and type 2 diabetes, compared with the nondiabetic population, with no significant difference between them. Jensen et al[43] found similar results.

Over the past years, there has been a great improvement in perinatal outcomes for women with type 1 diabetes. However, with rising incidence of obesity and type 2 diabetes, it seems that type 2 diabetes has become a more prominent concern. Women with type 2 diabetes tend to be under less stringent control of their glucose values, either because they use oral glycemic agents, have lower compliance with management strategies or have a lower prevalence of pre-pregnancy counselling, as sometimes type 2 diabetes is regarded as a “lesser” problem compared with type 1 diabetes. Moreover, lack of folate supplementation, obesity with lower ultrasound detection rates and other demographic differences between the women with type 1 or type 2 diabetes may account for the differences showed by different studies comparing the types of diabetes. There is no doubt that pre-pregnancy care and good glycemic control is equally important in type 2 and type 1 diabetes.

PATHOGENESIS OF DIABETES INDUCED BIRTH DEFECTSMost data regarding the pathophysiology of diabetes associated birth defects originates from animal studies. Our research group, as well as others, have shown that maternal diabetes triggers multiple cellular stress responses and subsequent aberrant signal transduction pathways[49-51]. All these may contribute to gene dysregulation and apoptosis in the affected organs of the developing embryo leading to structural birth defects. Studies from animal models have shown that maternal

diabetes increases the production of cellular reactive oxygen species and simultaneously impairs endogenous cellular antioxidant capacity, leading to an overall oxidative stress in the embryo[50,52-55]. Maternal diabetes also appears to increase the expression of inducible nitric oxide synthase (iNOS)[56], whose enzymatic activity catalyzes the reaction of superoxide with nitric oxide to produce reactive nitrogen species. Work in animal models indicates that reactive nitrogen species create a severe form of oxidative stress, nitrosative stress, which is responsible for the activation of cellular stress signalling. Our laboratory has shown that over expression of an antioxidant enzyme, superoxide dismutase 1 (SOD1), in SOD1 transgenic mice, mitigates maternal diabetes-induced oxidative stress and reduces embryonic malformations in diabetic pregnancies[57-59]. Likewise, studies indicate that eliminating the iNOS gene in iNOS knockout mice reduces the incidence of embryonic malformations caused by maternal diabetes[60]. Therefore, our work and that of others has shown that oxidative and nitrosative stress mediates the teratogenicity of maternal diabetes in the developing embryo.

In our studies, maternal diabetes-induced oxidative stress activates the c-Jun-N-terminal kinase 1/2 (JNK1/2)[49,50,61-63]. Deletion of either JNK1 or JNK2 gene ameliorates maternal diabetes-induced NTDs formation[49,50], supporting the hypothesis that activation of the cellular stress kinases, JNK1/2, mediates the adverse effect of maternal diabetes on neural tube closure. In the absence of JNK1 or JNK2, maternal diabetes-induced apoptosis in the neuroepithelial cells are blunted[49,50]. Thus, JNK1/2 activation induced by maternal diabetes transmits the pro-apoptotic signal emanating from oxidative stress under diabetic conditions[49,50].

JNK1/2 belongs to the mitogen-activated protein (MAP) kinase family, whose activation follows a three-tier cascade: MAP three kinases activate MAP kinase kinases, which in turn trigger JNK1/2 phosphorylation. Subsequent studies have revealed the upstream kinases in diabetic embryopathy, including apoptosis signal-regulating kinase 1 (ASK1)[64]. ASK1 is a three kinase that leads to JNK1/2 activation. We have observed that ASK1 gene deletion abolishes maternal diabetes-induced JNK1/2 activation, as well as activation of four major transcription factors downstream of JNK1/2. Similar to our findings in the JNK1 and JNK2 gene deletion studies, ASK1 gene deletion blocks maternal diabetes-induced apoptosis in the developing neural tube, and consequently reduces the number of embryos with NTDs[64]. Thus, the ASK1-JNK1/2 pathway, which is activated by oxidative stress, appears to play a causal role in the induction of diabetic embryopathy.

Recently, we have worked to understand how cellular ASK1-JNK1/2 kinase signalling relays its pro-apoptotic signals to the nucleus[64]. We have observed that ASK1 activation increases the activity of Forkhead transcription factor 3a (FoxO3a), and that FoxO3a



induces TNFR1 associated death domain (TRADD)[64]. TRADD up-regulation triggers caspase 8 activation, which, in turn, activates the caspase cascade and leads to apoptosis[64]. Both germline deletion and conditional deletion of the FoxO3a gene significantly reduce maternal diabetes-induced apoptosis and NTDs formation, underscoring the potential importance of FoxO3a in the induction of diabetic embryopathy[64]. We have successfully inhibited the whole stress pathway, ASK1-JNK1/2-FoxO3a-TRADD-caspase 8, using an endogenous ASK1 inhibitor, thioredoxin. Thioredoxin treatment ameliorates NTDs formation in embryos of diabetic dams and cultured embryos[64]. These studies reveal the ASK1 initiated stress signalling with the activity of a transcription factor and pro-apoptotic gene expression.

We and others have shown that JNK1/2 activation leads to endoplasmic reticulum (ER) stress[50]. Under ER stress, the unfolded protein response (UPR) is activated, and prolonged UPR activation induces apoptosis[65,66]. Maternal diabetes in vivo and high glucose in vitro induce ER stress and activate the UPR pathway[50]. Treatment with an ER chemical inhibitor, 4-Phenylbutyric acid, reduces high glucose-induced JNK1/2 activation, neuroepithelial cell apoptosis and NTD formation[50]. Deletion of either the JNK1 or the JNK2 gene abrogates maternal diabetes-induced UPR and ER stress[50]. These findings support a reciprocal causation between JNK1/2 and ER stress in diabetic embryopathy. Other studies have found that activation of the inositol-requiring enzyme 1 alpha, one of the major UPR arms, activates ASK1 and subsequently leads to JNK1/2 activation and apoptosis[67,68], which support our hypothesis that ASK1 plays a major role in diabetic embryopathy.

CONCLUSIONWhile the risk for major congenital defects has been well established in women with pregestational diabetes, many questions remained unanswered. For example, it is still unknown why some organs display vulnerability to glucose teratological effects more than others, or why meticulous control of maternal blood glucose does not reduce the rate of birth defects to the background risk of non-diabetic population, or why some women with pregestational diabetes will have normal pregnancies even when their levels of HgA1c are way above the accepted threshold for birth defects.

The key to understanding the true relationship between PGD and birth defects lies in a basic under-standing of the pathogenesis of congenital defects. Based on our research, the oxidative stress-triggered ASK1 pathway mediates the pro-apoptotic effect of maternal diabetes and high glucose in vitro by inducing pro-apoptotic gene expression and causing UPR and ER stress. The endogenous ASK1 inhibitor, thioredoxin, and the ER stress inhibitor, a Food and Drug Administration approved drug, may be potential therapeutics against maternal diabetes-induced structural birth defects.

We believe further research to evaluate glucose teratological effects on different embryonic organs, via animal models that can encompass the diversity seen in human diabetes, is needed. Research should utilize type 1 and type 2 models, as well as models of chronic and acute glycemia and how these two conditions may affect organogenesis. Our hope is that, by combining all known clinical data with current and future basic and translational science studies, we will be able to establish better ways to care for the diabetic mother and prevent the mal-effects of maternal diabetes on the developing embryo.

ACKNOWLEDGMENTSWe would like to thank Dr. Julie A Wu from the University of Maryland, School of Medicine, for editing assistance in preparing the manuscript.

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P- Reviewer: Ren AG S- Editor: Ji FF L- Editor: A E- Editor: Liu SQ



nations by 20% and in developing countries by 69% until the year 2030. Due to the expected rise in diabetic patients, the need for ophthalmic care of patients (i.e. , exams and treatments) will also increase and represents a challenge for eye-care providers. Development of optimized screening programs, which respect available resources of the ophthalmic infrastructure, will become even more important. Main reasons for loss of vision in patients with diabetes mellitus are diabetic macular edema and proliferative diabetic retinopathy. Incidence or progression of these potentially blinding complications can be greatly reduced by adequate control of blood glucose and blood pressure levels. Additionally, regular ophthalmic exams are mandatory for detecting ocular complications and initiating treatments such as laser photocoagulation in case of clinical significant diabetic macular edema or early proliferative diabetic retinopathy. In this way, the risk of blindness can considerably be reduced. In advanced stages of diabetic retinopathy, pars-plana vitrectomy is performed to treat vitreous hemorrhage and tractional retinal detachment. In recent years, the advent of intravitreal medication has improved therapeutic options for patients with advanced diabetic macular edema.

Key words: Laser photocoagulation; Diabetic macular edema; Diabetic retinopathy; Intravitreal injection; Prevention


Core tip: Diabetic retinopathy is a potentially blinding complication of diabetes mellitus. In patients with diabetes, regular retinal exams are essential. While laser photocoagulation is effective, if performed in time, advanced stages of diabetic retinopathy need to be treated by vitreo-retinal surgery and have limited visual prognosis. Even though new therapeutic options such as intravitreal medical therapy and sutureless pars-plana vitrectomy have improved ophthalmic care of patients

Martin M Nentwich, Michael W Ulbig

Martin M Nentwich, Michael W Ulbig, Department of Ophthalmology, Ludwig-Maximilians University, 80336 Munich, GermanyAuthor contributions: Nentwich MM and Ulbig MW contributed to this paper.Conflict-of-interest: None.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Dr. Martin M Nentwich, Department of Ophthalmology, Ludwig-Maximilians University, Mathildenstr 8, 80336 Munich, Germany. [email protected]: +49-89-440053811Fax: +49-89-440054569Received: July 17, 2014Peer-review started: July 17, 2014First decision: November 27, 2014Revised: January 3, 2015Accepted: January 18, 2015Article in press: January 20, 2015Published online: April 15, 2015

AbstractIn industrialized nations diabetic retinopathy is the most frequent microvascular complication of diabetes mellitus and the most common cause of blindness in the working-age population. In the next 15 years, the number of patients suffering from diabetes mellitus is expected to increase significantly. By the year 2030, about 440 million people in the age-group 20-79 years are estimated to be suffering from diabetes mellitus worldwide (prevalence 7.7%), while in 2010 there were 285 million people with diabetes mellitus (prevalence 6.4%). This accounts for an increase in patients with diabetes in industrialized

MINIREVIEWS


Diabetic retinopathy - ocular complications of diabetes mellitus




with diabetes, interdisciplinary care of these patients is essential. Good metabolic and blood pressure control is indispensable for reducing the risk of ophthalmic complications.

Nentwich MM, Ulbig MW. Diabetic retinopathy - ocular complications of diabetes mellitus. World J Diabetes 2015; 6(3): 489-499 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/489.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.489

INTRODUCTIONDiabetic retinopathy is a potentially blinding complication of diabetes mellitus. Reasons for loss of vision are diabetic maculopathy and complications of proliferative diabetic retinopathy (PDR) such as vitreous hemorrhage, tractional retinal detachment, and neovascular glaucoma. By 2030 developing countries will face an increase by 69% and industrialized countries by 20% of the number of patients with diabetes compared to 2010[1]. For Africa more than 18 million, according to some estimations even 24 million, diabetic patients are predicted for the year 2030[1,2].

Probability of retinal complications increases with increasing duration of disease. In up to 50% of patients with type 1 diabetes and 30% of those with type 2 diabetes potentially visionthreatening retinal changes develop over time, while early retinal changes are not noticed by the patients[3].

Diabetic retinopathy is the most common microvascular complication of diabetes mellitus and affects between 3%4% of people in Europe, while the relative risk for developing diabetic retinopathy is higher in type 1 diabetes compared to type 2[46]. Diabetes mellitus is responsible for about 15% of all cases of legal blindness (best corrected visual acuity less than 0.02) in Germany[7]. It is the main cause of blindness within the workingage population in industrialized nations[4].

While retinal changes are rarely seen in patients with type 1 diabetes before adolescence, about one third of patients have signs of diabetic retinopathy at time of initial diagnosis of diabetes mellitus. The risk of PDR is higher in type 1 diabetes than in type 2, while diabetic macular edema is more commonly found in type 2 diabetes (prevalence after 15 years of disease: type 1 vs type 2 = 15% vs 25%)[8].

Kramer et al[9] reported in a recent study, that in patients with type 1 diabetes progression of diabetic retinopathy and development of nephropathy each increase the risk for incidence of the other. This association was independent of established risk factors for microvascular complications and the authors suggested a shared etiologic basis of these two complications of diabetes mellitus[9]. Another group also found proliferative diabetic retinopathy to be an independent marker of longterm nephropathy in patients with type 1 diabetes[10].

Other studies indicated association of the presence of diabetic retinopathy and increased overallmortality and cardiovascular events both in type 1 and type 2 diabetes[11].

Therefore an interdisciplinary approach of physicians, endocrinologists, and ophthalmologists is needed for optimal care.

PATHOGENESIS OF DIABETIC RETINOPATHYMicroangiopathy due to hyperglycemia in patients with diabetes mellitus results in vascular leakage, which causes diabetic macular edema on one hand, and capillary occlusion on the other hand. Capillary occlusion then again causes retinal ischemia and increased levels of vascular endothelial growth factor (VEGF) which are responsible for the development of neovascularization and the proliferative stage of diabetic retinopathy.

More recently new pathways which may be involved in the pathogenesis of diabetic retinopathy have been identified, such as inflammation, nerve growth factor autophagy and epigenetics. A detailed discussion of all these pathways would go beyond the scope of this minireview about clinical aspects of diabetic retinopathy, however some aspects should be addressed.

Biochemical alterations such as oxidative stress, activation of protein kinase C and formation of advanced glycation end products have been detected as a response of the retina to hyperglycemia[12]. Also kinin B1 and B2 are thought to increase vascular permeability, infiltration of leukocytes and inflammation. Especially kinin B1, which is almost nonexistent in normal tissue, is upregulated in the retina of diabetic patients. These findings may be important for developing new therapeutic strategies aiming at antagonizing kinin receptors or at inhibiting kallikreins[13].

Recent investigations showed that the whole retinal neurovascular system is impaired by diabetes mellitus resulting in loss of neurovascular coupling, neurodegeneration and neuroinflammation, which can be detected even before the advent of vascular damage[14]. Clinically, reduced dark adaption, impaired colour and/or contrast vision and visual field defects are found during functional examinations of diabetic patients[15].

Diabetic retinopathy tends to deteriorate during hormonal changes such as adolescence and pregnancy[16].

CLASSIFICATION AND PATHOPYSIOLOGY OF DIABETIC RETINOPATHYDuring fundoscopy, which should be performed after dilation of the pupil (mydriasis) to allow visualization of the entire retina, presence and grade of diabetic retinopathy can be assessed clinically. Typical changes seen in early diabetic retinopathy (nonPDR) are micro

Nentwich MM et al . Diabetes mellitus: Ocular complications


aneurysms, retinal hemorrhages, and exudates (Figure 1). In the beginning, these alterations are often found slightly temporal to the central area of the macula (Figure 2). These are caused by deranged vascular integrity and loss of pericytes. In the later course of disease, intraretinal microvascular anomalies may develop, which represent dilated and therefore during fundoscopy visible retinal capillaries, and indicate possible risk of neovascularization and proliferative diabetic retinopathy.

Proliferative diabetic retinopathy is caused by increased intraocular VEGFlevels due to retinal ischemia because of capillary occlusion of retinal vessels. Proliferation may

grow at the optic disc (neovascularization at the disk) or elsewhere in the retina (neovascularization elsewhere ) into the vitreous (Figure 3). These newly formed vessels leak on fluorescein angiography and may cause vitreous hemorrhage and finally tractional retinal detachment (Figures 4 and 5). Tractional retinal detachment results in separation of the neurosensory retina from the retinal pigment epithelium. Detached sections of the retina cause relative visual-field defects (scotoma) and loss of visual acuity in cases with macular involvement.

Further, neovascularization on the iris in the anterior segment of the eye may evolve as anterior segment sequel of ischemia (Figure 6). These new blood vessels grow towards the anterior chamber angle and may obstruct the trabecular meshwork, thus increasing outflow resistance of aqueous humor. This results in increased intraocular pressure followed by optic atrophy (neovascular glaucoma).

CLASSIFICATION AND PATHOPHYSIOLOGY OF DIABETIC MACULOPATHYDiabetic maculopathy may develop in the nonproliferative and in the proliferative stage of diabetic retinopathy. While complications of untreated proliferative diabetic


Figure 3 Proliferative diabetic retinopathy with neovascularization at disk. Figure 4 Advanced proliferative diabetic retinopathy with neovascularization and limited vitreous hemorrhage.

Figure 5 Advanced proliferative diabetic retinopathy with tractional retinal detachment.

Figure 1 Non-proliferative diabetic retinopathy. Wide-field fundus photo of a 65-year-old female patient (right eye) showing several retinal hemorrhages.

Figure 2 Non-proliferative diabetic retinopathy. Color fundus photo of a 51-year-old male patient with micro-aneurysms and lipid exudates.


domain OCT). Vitrectomy is needed in patients with symptomatic tractional macular edema in order to release traction by surgical removal of the vitreous.

Apart from edema, diabetic maculopathy may also present as occlusion of perifoveal capillaries resulting in distinct visual impairment in the absence of edema on fundoscopy. In these cases, fluorescein angiography is needed to confirm the diagnosis by visualizing capillary occlusion in around the fovea.

Table 1 summarizes the different types of diabetic maculopathy (adapted from Nentwich et al[19]).

CLINICAL DIAGNOSICSDilated fundoscopy is the most important clinical investigation in patients with diabetes during screening examinations by an ophthalmologist. Dilation of the pupil is necessary in order to enable a proper stereoscopic view, which is needed for evaluation of macular edema, and to allow visualization of the peripheral retina.

Fluorescein angiography was introduced by Novotny et al[20] in the 1960s into the clinical ophthalmic practice and allows evaluation of the retinal vascular status. Fluorescein, a fluorescent dye, is injected intravenously and distributes throughout the body. In the eye, fluorescence is activated by blue light of 490 nm. During fluorescein angiography vascular leakage, capillary occlusion, ischemic areas of the retina and neovascularization can be seen (Figure 9A and C). It provides information on the area of leakage as well as of the location of nonperfused parts of the retina. In patients with suspected diabetic maculopathy, fluorescein angiography is mandatory for excluding ischemic maculopathy and guiding possible focal laser photocoagulation.

Wide-field fundus photography (2-laser wavelength nonmydriatic 200° ultrawidefield scanning laser ophthalmoscopy) has been introduced recently. Widefield images and traditional color fundus photography

retinopathy, such as vitreous hemorrhage and tractional retinal detachment involving the macula may cause the most severe loss of vision in diabetic retinopathy, diabetic maculopathy is the main cause of visual impairment in patients with type 2 diabetes[17].

Diabetic macular edema is caused by a disruption of the inner bloodretinal barrier, formed by the retinal vascular endothelium, due to hyperglycemia, increased levels of growthfactors, inflammation and cytokines. This also causes impairment of pericytes and leads to consecutive exudation of fluid, proteins and lipids by para cellular and trans cellular transport mechanisms[18]. Clinically, areas of thickened retina are often demarcated by surrounding yellowish lipid exudates (Figure 7). Diabetic macular edema may be limited or affect a large retinal area and it may also involve the macula (centerinvolving) or spare the central area (noncenterinvolving) (Figure 8).

Traction caused by vitreous attachment to the fovea may result in macular thickening, as well. Vitreous traction can excellently be documented by highresolution optical coherence tomography (OCT, spectral


Figure 6 Neovascularization of the iris. These neovascular vessels may block the trabecular meshwork and cause neovascular glaucoma.

Table 1 Types of diabetic maculopathy[19]

Focal Localized edemaLipid exudatesIntraretinal hemorrhagesFocal hyperfluorescence in late fluorescein angiography

Clinically significant without foveal thickening (non-center-involving) (sight-threatening)

Edema within 500 μm around the foveolaExudates within 500 μm around the foveola accompanied by edemaEdema ≥ 1 optic-disk diameter within one optic-disk diameter around the foveola

Clinically significant with foveal thickening (center-involving) Ill-defined edema, which may be cystoidExudatesIntraretinal hemorrhagesOrigin of leakage often not clearly identifiable by fluorescein angiography

Tractional Due to vitreous traction to the foveaThickened posterior hyaloid membraneOCT visualizes vitreal traction

Ischemic maculopathy (occlusion of the perifoveal capillaries) Loss of vision without any clearly visible cause on fundoscopyFluorescein angiography needed for diagnosisDifficult to diagnose by fundoscopy onlyEdema may be present or absent

OCT: Optical coherence tomography.


in mydriasis seem to correlate with regard to the classification of diabetic retinopathy and visualization of diabetic macular edema according to recent studies (Figure 10A)[2123]. This widefield imaging technique can be combined with fluorescein angiography and provides information about peripheral retinal ischemia or peripheral neovascularization (Figure 10B)[24].

Probably the most important new imaging technique, which was introduced in ophthalmologic practice, is OCT. Recently, resolution and recording speed have been greatly improved by spectraldomain OCT technique (SDOCT). OCT provides data on retinal volume and configuration of the macular region. In diabetic macular edema SDOCT is a very valuable examination technique comparing followup visits with baseline data, as modern OCT-devices use eye-tracking for finding the same imaging position during followup examinations (Figure 11)[25].

IMPORTANCE OF PREVENTIONIn early diabetic retinopathy, laser photocoagulation can be used effectively for preventing loss of vision[26]. However, early diabetic retinopathy is not noticed by patients because of the absence of visual loss in early diabetic retinopathy. Thus, regular retinal exams with dilation of the pupil are necessary in patients with

diabetes mellitus in order to detect sightthreatening changes timely and enable ophthalmologists to perform treatment.

Patients with type 1 diabetes should undergo retinal exams starting at the age 11 and/or after 5 years of diagnosis on a yearly basis. In case of retinal changes, shorter followup intervals are recommended.

In type 2 diabetes, the first retinal exam should be performed immediately after first diagnosis of diabetes, because the previous duration of the disease is unknown. Annual followup examinations are recommended in the absence of retinal changes, otherwise shorter intervals are recommended.

Pregnancy bears an increased risk of worsening of diabetic retinopathy due to hormonal changes. During pregnancy diabetic retinopathy may start in about 10% of cases and may worsen in an even higher percentage in case of preexisting diabetic retinopathy at time of conception[27]. In case of proliferative diabetic retinopathy before or shortly after conception, panretinal laser photocoagulation should be performed as retinal changes may worsen in one out of two women in this population. This risk can be reduced by half by thorough retinal laser photocoagulation. Therefore all females with diabetes, who are planning to get pregnant, should undergo fundoscopy prior to conception and then every three months during pregnancy to enable early intervention, if needed. Additionally good metabolic control should be achieved before conception[28].

On the other hand, the presence of diabetic retinopathy is no indication for cesarean section per se, as no study has indicated so far, that Valsalvamaneuvers during vaginal delivery bear an increased risk of vitreous hemorrhages[29].

The German Diabetes Society recommends scheduling of followup examinations depending on the grade of diabetic retinopathy as summarized in Table 2 (Adapted from[30]).

These recommended examination intervals will result in an increased workload for ophthalmologists due to the rising number of diabetic patients. Studies examined the effect of an extension of followup intervals and found no increased risk of progression to sight


Figure 7 Clinically significant diabetic macular edema without involvement of the fovea. A: Fundus photo; B: Fluorescein angiogram depicting leakage of perifoveal retinal blood vessels.

A B

Figure 8 Center-involving diabetic macular edema with subfoveal edema and numerous lipid exudates.


threatening diabetic retinopathy in patients with good metabolic control (HbA1c < 8%) and no known diabetic retinopathy[3135]. By adequate selection of patients, the number of necessary screening examinations could be reduced by 40% according to one study and even by 59% in case a special mathematical algorism is used[31,33]. However, feasibility of these strategies remains to be proven in routine clinical practice and until then

ophthalmic screening intervals as indicated above are recommended.

GENERAL MEDICAL THERAPY AND INTERVENTIONSBlood glucose and blood pressure controlGood metabolic and bloodpressure control are essential for successful ophthalmic care of patients with diabetes.

In type 2 diabetes, a reduction of HbA1c from 7.9% to 7.0% resulted in a decline in the frequency of laser treatments needed[36]. In type 1 patients, improved blood glucose control with a reduction of HbA1c values from 9.1% to 7.1% reduced the risk of developing diabetic retinopathy within 6.5 years by 76%, the risk of progression of diabetic retinopathy by 54%, and the risk of developing proliferative diabetic retinopathy by 47%[37]. Therefore an HbA1c level of about 7% should be aimed for from the ophthalmologist’s point of view[19], for the individual patient a bespoke treatment regime may be needed.

Rapid improvement of metabolic control may result in temporary worsening of diabetic retinopathy (“earlyworsening”) in patients with longlasting disease and


A B

Figure 10 Wide-field picture of the right eye of a 65-year-old female patient. A: On scanning-laser-ophthalmoscope-Imaging some micro-aneurysms and lipid exudates can be seen; B: Fluorescein angiogram shows leakage from micro-aneurysms and extensive areas of retinal non-perfusion.

Figure 11 Spectral-domain optical coherence tomography of a female patient with center-involving diabetic macular edema. On the left side of the picture, an infra-red image shows the exact location of the OCT-scan on the right. The OCT-scan visualizes intraretinal edema with thickening of the fovea. OCT: Optical coherence tomography.


Figure 9 Fluorescein angiogram of a 49-year-old female patient. A: Fluorescein angiogram of the right eye 50 s after intravenous injection of fluorescein dye. Here, leaking micro-aneurysms in the macula can be seen; B: Fluorescein angiogram of the left eye 25 s after intravenous injection of fluorescein dye. Leakage from neovascular blood vessels causes spots of increased fluorescence at the optic disk and temporal to the fovea; C: Fluorescein angiogram of the temporal part of the left eye 30 s after intravenous injection of fluorescein dye. Areas of retinal non-perfusion can be seen as reason for neovascularization.

A B C

high HbA1c levels. Thus, retinal exams should be performed every three months during the first year after initiation of an improved antidiabetic treatment[5,38]. However, in the longterm, positive effects of good metabolic control outweigh these initial problems[37,39]. Also a slow decrease of blood sugar levels, which would be a therapeutic challenge, does not have any advantages in the long term and is not recommended from the ophthalmologist’s point of view[19].

Additionally, optimizing blood pressure helps to reduce the necessity of laser photocoagulation and the risk of loss of vision[40]. While some studies suggested a protective effect of ACE inhibitors, according to the results of studies available to date, reduction of blood pressure itself seems to be more important than the type of blood pressure lowering medication[4143]. Levels of about 140/80 mmHg should be aimed for.

Acetylsalicylic acid and smokingAcetylsalicylic acid has neither been shown to have any positive effect on diabetic retinopathy or diabetic macular edema, nor to be harmful for patients with diabetic retinopathy Therefore acetylsalicylic acid should be recommended for cardiovascular reasons in patients with early nonproliferative diabetic retinopathy, in case there are no contraindications[44].

Data on the effect of smoking on diabetic retinopathy is heterogeneous. While some studies did not find an association of smoking with diabetic retinal changes, others identified smoking in a multiple regression analysis as risk factor for any grade of diabetic retinopathy[4547]. However, results of a recent experimental study suggest that nicotine expedites diabetesinduced retinal changes[48]. Additionally, patient characteristics such as selective mortality among smokers and patients with proliferative diabetic retinopathy at baseline may provide some explanation for the apparently absent association, reported in some studies[47]. Cessation of smoking should therefore be recommended from the ophthalmologist’s point of view.

OPHTHALMIC THERAPEUTIC OPTIONSDiabetic macular edemaUntil the advent of antiVEGF medication, focal and grid laser photocoagulation was the standard of care

for clinical significant diabetic macular edema (CSME) as well as for diffuse diabetic macular edema.

In patients with focal CSME without involvement of the fovea, focal laser photocoagulation of microaneurisms and localized areas of leakage reduces the relative risk of moderate loss of vision by 50% (from 24% to 12%) as demonstrated by the ETDRS Group (Figure 12)[49]. In case of persisting areas of leakage two to three months after initial focal laser photocoagulation, repeated laser treatment should be considered[26].

Apart from the direct effect by coagulating microaneurysms, focal laser photocoagulation is thought to increase oxygenation of the retina by improving diffusion of oxygen from choroidal vessels and to reestablish the bloodretinal barrier by thermal stimulation of the retinal pigment epithelium and of endothelial cells of retinal capillaries[50].

Grid laser photocoagulation of diffuse diabetic macular edema with involvement of the fovea (“centerinvolving”) has a limited functional prognosis[49]. Therefore, grid laser photocoagulation has been replaced by intravitreal antiVEGF therapy, which provides superior functional results[51].

VEGF is associated with a breakdown of bloodretinal barrier causing leakage and retinal edema[52]. Intravitreal application of antiVEGF medication enables high local concentrations in the vitreous and a low systemic exposure. However, repeated intravitreal injections of antiVEGF medications are necessary in patients with centerinvolving diabetic macular edema. The average number of injections is around 7 in the first year of treatment and 4 in the second year as indicated by recent studies[53,54]. Intravitreal antiVEGF therapy is generally safe with regard to sideeffects of medication, although the incidence of systemic thromboembolic events varies among trials[55]. As the intravitreal application of medication is a surgical procedure, it is associated with the possible risk of postoperative infection, which is called endophthalmitis in case of severe infection of the inner eye and its internal structures. For this reason several recommendations of ophthalmologic societies


Table 2 Recommended timing of retinal examinations in patients with type 2 diabetes[6]

Patient characteristics Timing of retinal examination

Initial diagnosis of type 2 diabetes SoonNo diabetic retinopathy Once a yearPresence of symptoms such as During the next few days Loss of vision New difficulties during reading Altered color perception New, moving dark spots in the eyePresence of diabetic retinopathy Depending on the severity of

retinopathy, e.g., every 3-6 mo

Figure 12 Color fundus photo of a 53-year-old male patient several months after focal laser photocoagulation because of clinical significant diabetic macular edema. While the laser scars and some remaining dot-hemorrhages are visible, lipid exudates and retinal edema have disappeared.


have been published concerning prevention of postinjection endophthalmitis. Recent studies reported very low rates of post-injection endophthalmitis of less than 1 in 8000 injections performed in an operating theater setting after diligent disinfection of the conjunctiva with povidoneiodine, using sterile gloves and wearing facemasks[56,57]. Other studies report somewhat higher rates of infectious endophthalmitis after intravitreal injection, both after procedures performed in an office-setting and in a theatersetting[58]. The effect of meticulous povidoneiodine prophylaxis on the conjunctival bacterial load and contamination of needles used for injection prophylaxis has been shown in several studies[5962].

Apart from antiVEGF medication intravitreal steroids have been evaluated for the treatment of diabetic macular edema. Compared to antiVEGF medication, steroids have additional antiinflammatory effects and sustainedrelease devices can lengthen the intervals between retreatments[63]. In Europe, a nonabsorbable implant containing 190 μg fluocinolone acetonide has been approved as secondline treatment of chronic diabetic macular edema not responding to other therapeutic options. Also approval of an absorbable dexamethasone implant for the treatment of diabetic macular edema is expected by the end of 2014. While

intravitreal steroids are effective in reducing diabetic macular edema, patients need to be informed about possible progression of cataract and increased intraocular pressure[6467].

Proliferative diabetic retinopathyIn early proliferative diabetic retinopathy, panretinal laser photocoagulation is effective in reducing the risk of visual loss, while surgery needs to be performed in very advanced proliferative diabetic retinopathy[68]. The suggested screening intervals of patients with diabetes are intended to enable the treating ophthalmologist to detect proliferation amendable for laser treatment early. Panretinal laser photocoagulation should be performed when retinal neovascularization is detected during fundoscopy or via fluorescein angiography in order to prevent complications of proliferative diabetic retinopathy such as vitreous hemorrhage or tractional retinal detachment (Figure 13)[68]. Panretinal laser photocoagulation aims at eliminating nonperfused parts of the retina, thus reducing ischemia, intravitreal VEGFlevels, and the stimulus for proliferation. The efficacy of panretinal laser photocoagulation in reducing the risk of loss of vision has already been shown more than 30 years ago[68]. However, patients need to be informed about sideeffects of panretinal laser treatments such as constriction of visual fields and reduced dark-adaption due to loss of rod function. These sideeffects may also interfere with the patients’ driving ability.

Parsplana vitrectomy is performed in advanced cases of proliferative diabetic retinopathy, the presence of extensive tractional membranes, vitreous hemorrhage, and tractional retinal detachment (Figure 14). About ten years ago trans conjunctival sutureless 23 gauge vitrectomy started to replace conventional 20 gauge technique and offers comparable safety and efficacy as well as reduced surgerytimes and faster rehabilitation of patients[69].

Combination of diabetic macular edema and proliferative diabetic retinopathyIn patients with diabetic macular edema and proliferation, treatment of macular edema should be performed before panretinal laser photocoagulation in order to prevent worsening of macular edema due to peripheral panretinal laser photocoagulation.

CONCLUSIONIn patients with diabetes, regular retinal exams are essential. While laser photocoagulation is effective, if performed in time, advanced stages of diabetic retinopathy need to be treated by vitreoretinal surgery and have limited visual prognosis. Even though new therapeutic options such as intravitreal medical therapy and sutureless parsplana vitrectomy have improved ophthalmic care of patients with diabetes, interdisciplinary care remains essential. Good metabolic and blood pressure control is indispensable for reducing the risk of


Figure 13 Wide-field picture of the right eye of a 48-year-old male patient after pan-retinal laser photocoagulation because of proliferative diabetic retinopathy. The peripheral laser scars can be seen in the picture, while neovascularization have regressed.

Figure 14 Proliferative diabetic retinopathy with extensive fibro-vascular membranes.


ophthalmic complications.

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P- Reviewer: Mitra A, Sui RF S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ



Gergő A Molnár, Esztella Zsóka Mikolás, István András Szijártó, Szilárd Kun, Eszter Sélley, István Wittmann

Gergő A Molnár, Esztella Zsóka Mikolás, István András Szijártó, Szilárd Kun, Eszter Sélley, István Wittmann, 2nd Department of Medicine and Nephrological Center, University of Pécs, H-7624 Pécs, HungaryAuthor contributions: Molnár GA and Wittmann I designed the research; Mikolás EZ, Szijártó IA, Kun S and Sélley E performed the research; Molnár GA and Wittmann I wrote the paper.Supported by The European Union and the State of Hungary and co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 National Excellence Program.Conflict-of-interest: All authors have nothing to declare.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: István Wittmann, MD, PhD, Professor of Medicine, Head of the Department, 2nd Department of Medicine and Nephrological Center, University of Pécs, Pacsirta Str. 1, H-7624 Pécs, Hungary. [email protected]: +36-72-536050 Fax: +36-72-536051Received: September 3, 2014 Peer-review started: September 4, 2014 First decision: November 19, 2014Revised: December 5, 2014 Accepted: January 9, 2015Article in press: January 12, 2015Published online: April 15, 2015

AbstractOxidative stress processes play a major role in the development of the complications associated with diabetes and other diseases via non-enzymatic glycation, the hexosamine pathway, the polyol pathway and diacylglycerol-protein kinase C. Oxidative stress may lead to the production of hydroxyl free radicals, which

can attack macromolecules, such as lipids, nucleic acids or amino acids. Phenylalanine (Phe) can be enzymatically converted to the physiological para-tyrosine (p-Tyr); however, a hydroxyl free radical attack on Phe may yield meta- and ortho-tyrosine (m- and o-Tyr, respectively) in addition to p-Tyr. Hence, m- and o-Tyr may be regarded as markers of hydroxyl free radical-induced damage. Their accumulation has been described; e.g. , this accumulation has been found in the urine of patients with diabetes mellitus (DM) and/or chronic kidney disease, in cataract lenses, in vessel walls, in irradiated food and in amniotic fluid, and it may serve as an indicator of oxidative stress. The use of resveratrol to treat patients with type 2 DM led to a decrease in the urinary excretion of o-Tyr and concomitantly led to an improvement in insulin signaling and insulin sensitivity. Literature data also suggest that m- and o-Tyr may interfere with intracellular signaling. Our group has shown that erythropoietin (EPO) has insulin-like metabolic effects on fat cells in addition to its ability to promote the proliferation of erythroid precursor cells. We have shown that the supplementation of cell culture medium with m- and o-Tyr inhibits erythroblast cell proliferation, which could be ameliorated by p-Tyr. Additionally, in vivo , the o-Tyr/p-Tyr ratio is higher in patients with renal replacement therapy and a greater need for EPO. However, the o-Tyr/p-Tyr ratio was an independent determinant of EPO-resistance indices in our human study. The o-Tyr content of blood vessel walls inversely correlates with insulin- and acetylcholine-induced vasodilation, which could be further impaired by artificial oxidative stress and improved by the use of antioxidants. In rats that receive o-Tyr supplements, decreased vasorelaxation is detected in response to insulin. Additionally, o-Tyr supplementation led to the incorporation of the unnatural amino acid into cellular proteins and caused a decrease in the insulin-induced phosphorylation of endothelial nitric oxide synthase. Our data suggest that m- and o-Tyr may not only be markers of oxidative stress; instead, they may also be incorporated into cellular proteins, leading to resistance to insulin, EPO and acetylcholine.

MINIREVIEWS


Tyrosine isomers and hormonal signaling: A possible role for the hydroxyl free radical in insulin resistance




Key words: Acetylcholine; Insulin resistance; Hormone resistance; Oxidative stress; Para-tyrosine; Ortho-tyrosine; Meta-tyrosine; Erythropoietin


Core tip: Hydroxyl-free radical-derived amino acids, such as meta- and ortho-tyrosine (m- and o-Tyr, respectively) are regarded as free radical markers, but they may also be taken up and incorporated into blood vessel walls, erythroid precursors and endothelial cells. These pathological amino acids can induce vascular insulin and acetylcholine, as well as cellular erythropoietin resistance.

Molnár GA, Mikolás EZ, Szijártó IA, Kun S, Sélley E, Wittmann I. Tyrosine isomers and hormonal signaling: A possible role for the hydroxyl free radical in insulin resistance. World J Diabetes 2015; 6(3): 500-507 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/500.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.500

OXIDATIVE STRESS IN THE OLD PERSPECTIVESOxidative stress plays a role in the pathogenesis of many diseases, such as diabetes mellitus (DM), chronic kidney disease (CKD) and inflammatory diseases, as well as in the development of the complications associated with these diseases. Oxidative stress, free radicals and reactive oxygen species (ROS) are tightly connected to DM in several ways. Hyperglycemia may lead to a shift in cellular metabolism toward the polyol pathway, which leads to an oxidative shift in the NADPH/NADP ratio. NADPH is in turn required for antioxidant defense, e.g., for the reduction of oxidized glutathione by glutathione reductase. Furthermore, hyperglycemia activates the diacylglycerol-protein kinase C intracellular signaling pathway, which can activate NADPH oxidases; this oxidation leads to the translocation of nuclear factor κB into the nucleus and then to the transcription of proinflammatory cytokines, which results in increased oxidative stress. Additionally, hyperglycemia increases the rate of non-enzymatic glycation, which produces advanced glycation end-products (AGEs) that can bind to their receptors of AGE (RAGE), and the AGE-RAGE interaction also leads to inflammation and oxidative stress. DM involves the enhancement of not only non-enzymatic glycation but also enzymatic glycation via the hexosamine pathway; this enhanced glycation may also result in a proinflammatory response and oxidative stress. However, this interplay is complex; the ROS arising from oxidative stress may also contribute to the activation of the abovementioned pathways and reactions and can thus generate a vicious circle[1,2].

DETECTION OF OXIDATIVE STRESSThe study of oxidative stress processes is therefore important, albeit somewhat cumbersome. Per their definition, free radicals are highly reactive molecules with a very short lifetime; therefore, detecting these molecules requires spin traps and an electron spin resonance device[3,4], but the sensitivity of this method is usually too low for many diseases. Because of the high reactivity of free radicals, they can react with macromolecules and yield oxidation products, some of which are more chemically stable molecules. These products may include lipid peroxidation products (such as malonyldialdehyde derivatives), nucleobase products (such as 8-hydroxydeoxyguanosine), so-called advanced oxidation products or amino acid derivatives[5,6].

DETECTION OF HYDROXYL FREE RADICALS BY STABILE L-PHENYLALANINE DERIVATIVESL-Phenylalanine (Phe) is a highly abundant essential amino acid in proteins of the human body, and Phe plays a role in forming peptides and proteins; Phe also gives rise to the semi-essential amino acid L-para-tyrosine (p-Tyr) and its derivatives, such as 3,4 dihydroxy-phenylalanine (DOPA), and derivatives thereof[7]. p-Tyr is mainly produced via the enzymatic reactions catalyzed by the Phe hydroxylase enzyme, especially in the kidney and liver[8]. p-Tyr synthesis becomes impaired in patients with renal failure (e.g., CKD); therefore, the serum levels of p-Tyr in these patients are lower than those in patients/controls with normal renal function [CKD: 28 (26-34), DM + CKD: 32 (29-39) μmol/L vs controls: 56 (36-57) μmol/L][9]. However, other isomers of tyrosine, namely meta- and ortho-tyrosine (m-Tyr and o-Tyr, respectively) also exist in humans. These amino acids cannot be formed enzymatically in humans; instead, they are stable products of the reaction between the hydroxyl free radical and Phe. Additionally, p-Tyr may be formed non-enzymatically, via the hydroxyl radical, but the enzymatically produced p-Tyr is much more abundant than the free radical-derived p-Tyr. Therefore, p-Tyr is regarded as the physiologic isoform, whereas m- and o-Tyr are considered to be free radical markers[10-14].

All four aromatic amino acids (p-Tyr, m-Tyr, o-Tyr and Phe) can be readily detected in the nanomolar range via their autofluorescence and reverse-phase high performance liquid chromatography[9,15-22], as well as by gas chromatography combined with mass spectrometric detection[23] or by ultra-performance liquid chromatography combined with mass spectrometry[24].

ABUNDANCE OF THE HYDROXYL FREE RADICAL MARKERS M- AND O-TYRIn vitro measurements[25] and in silico calculations[26] have shown that in free radical reactions, the three isoforms (p-,

Molnár GA et al . Tyrosine isomers and hormone resistance


m- and o-Tyr) are not produced stoichiometrically (i.e., 1:1:1), and their concentrations and ratios may vary from tissue to tissue in vivo. These isoforms, along with others, have been detected in cataractous lenses by our group and others[16,27], in the brain after acute oxidative stress[28], in the serum/plasma of diabetic patients with/without CKD by our group[9], after the administration of thyroid hormone[29], after ischemia-reperfusion injury[14], in the hair of the Homo tirolensis and mummies[30], in irradiated food[31], in urine specimens[9,24] and in amniotic fluid[32]. In earlier publications, we have shown that m-Tyr, o-Tyr and DOPA accumulate in the non-water soluble proteins of cataractous lenses during aging and in patients with DM[16], and we also showed that the urinary excretion of o-Tyr increases in patients with type 2 DM and/or CKD [CKD: 122 (94-183), DM: 641 (272-499), DM+CKD: 403 (258-651) nmol/d vs controls: 24 (0-35) nmol/d][9]. Using the so-called fractional excretion values,

which show the renal handling of o-Tyr, we have found active urinary secretion or in loco renal synthesis of o-Tyr in the kidney of diabetic patients [DM: 125 (69-140), DM + CKD: 112 (69-187)% vs controls: 8 (4-12)%][9].

In patients with stroke, the urinary excretion of o-Tyr is associated with the total urinary albumin excretion; immunoreactive albumin excretion; and most signifi-cantly, urinary non-immunoreactive albumin excretion[17].

DECREASED URINARY EXCRETION OF O-TYR IS ASSOCIATED WITH AN IMPROVEMENT IN INSULIN RESISTANCE IN TYPE 2 DIABETESIn a human study, we demonstrated the protective effect of the polyphenolic compound resveratrol in patients with type 2 DM; a short-term administration of resveratrol led to a decrease in urinary o-Tyr excretion (Figure 1), an increase in the phosphorylation of the insulin signaling molecule protein kinase B (or Akt), (Figure 2) and an amelioration of the calculated marker of insulin resistance (homeostasis model assessment-insulin resistance) (Figure 3)[18].

ARE M- AND O-TYR JUST MARKERS OR ALSO MAKERS?All of the abovementioned papers focused on m- and o-Tyr as markers of hydroxyl free radical damage. However, the last of our abovementioned studies (on the in vivo effects of resveratrol) raised the possibility that these molecules may not only be markers; instead, they may also play a role in the development of pathological states[18]. Independent of our results, Ruggiero et al[33,34] showed that m- and o-Tyr can inhibit tumor cell growth via influencing the mitogen-activated protein kinase/extracellular signal regulated kinase (ERK) and


Figure 1 Changes in urinary ortho-tyrosine:creatinine excretion after resveratrol treatment. For each participant, the value measured at baseline was subtracted from that measured at week 4 (i.e., Δ o-Tyr:creatinine ratio), and then the resulting values were averaged within the respective groups. The values are the means, with the standard deviations represented by vertical bars. aMean values were significantly different (P = 0.043). Comparisons were performed using ANOVA and Bonferroni post hoc tests; P < 0.05 was regarded as statistically significant. Republished with permission of Cambridge University Press from Brasnyó et al[18]. o-Try: Ortho-tyrosine.

Δ o-

Tyr/

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Figure 2 Increase in protein kinase B phosphorylation in platelets upon resveratrol treatment. Values are the means, with the standard deviations represented by vertical bars. aMean values were significantly different for baseline vs week 4 within the resveratrol group (P = 0.032). Comparisons were performed using ANOVA and post-hoc tests; P < 0.05 was regarded as statistically significant. Republished with permission of Cambridge University Press from Brasnyó et al[18]. pAkt: Protein kinase B phosphorylation.

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Figure 3 Decrease in homeostasis model of assessment for insulin resistance upon resveratrol treatment. For each participant, the value measured at baseline was subtracted from that measured at week 4 (i.e., Δ HOMA-IR), and then the resulting values were averaged within the respective groups. Values are the means with the standard deviations represented by vertical bars. aMean values were significantly different for the resveratrol group vs the placebo group (P = 0.044), comparisons were performed using ANOVA and Bonferroni post hoc tests; P < 0.05 was regarded as statistically significant. Republished with permission of Cambridge University Press from Brasnyó et al[18]. HOMA-IR: Homeostasis model of assessment for insulin resistance.


phosphorylation of STAT5 and ERK1/2 (Figure 8). This finding indicates that the unnatural isoforms, o- and m-Tyr, can be incorporated into cellular proteins and interfere with the hormonal signaling of EPO[21].

This finding is consistent with a clinical observation of our group that the ratio of the pathological o-Tyr to the physiological p-Tyr (o-Tyr/p-Tyr ratio) is higher in patients who receive renal replacement therapy compared with controls. Additionally, the o-Tyr/p-Tyr ratio in the blood was higher in the individuals receiving hemodialysis and EPO replacement than in those patients receiving hemodialysis who did not require EPO replacement or in patients receiving peritoneal dialysis and requiring markedly lower EPO doses. Furthermore, the plasma o-Tyr/p-Tyr ratio was an independent predictor of the EPO dose and EPO-resistance indices in these patients. This finding is another indirect demonstration that o-Tyr may interfere with EPO signaling and lead to EPO resistance[22].

VASCULAR INSULIN RESISTANCE ACCORDING TO THE O-TYR CONTENT OF THE VESSEL WALLIn a further set of experiments, we analyzed the o-Tyr levels in the blood vessel walls of rodents and found that the o-Tyr content of blood vessels decreases toward the peripheral vasculature (i.e., thoracic aorta > abdominal aorta > femoral artery) (Figure 9A). The o-Tyr content could be increased by treatment with hydrogen peroxide and aminotriazole or by aortic banding, and it could be inhibited using superoxide dismutase and catalase (Figure 9B).

We have shown that vascular segments with higher o-Tyr content show lower vasorelaxation in response to insulin; i.e., the insulin-induced vasorelaxation is lowest in the thoracic aorta, higher in the abdominal aorta and the highest in the femoral artery (Figure 10).

Furthermore, the insulin-induced relaxation could be increased by an antioxidant (superoxide dismutase and catalase) treatment in the thoracic aorta. By contrast, pro-oxidant therapy further diminished the vasorelaxation in an ERK1/2-dependent manner[19].

VASCULAR ACETYLCHOLINE RESISTANCE ACCORDING TO THE O-TYR CONTENT OF THE VESSEL WALLIn the same set of experiments, the vasorelaxation in response to acetylcholine was also tested, and we also found an inverse relationship between the vessel wall o-Tyr content and the vasorelaxation in response to acetylcholine; i.e., the blood vessels with high o-Tyr content (see Figure 9A) show less vasorelaxation in response to Ach (Figure 11, previously unpublished data).

Based on these experiments, we subjected rats

the signal transducer and activator of transcription (STAT) pathway. In plants, m-Tyr inhibits cell growth and plant root growth and may be considered a natural herbicide[35,36]. This finding raises the possibility that the unnatural isoforms, m- and o-Tyr, might affect cellular function in mammals and plants and may interfere with hormonal signaling.

INSULIN-LIKE EFFECT OF ERYTHROPOIETIN ON GLUCOSE METABOLISMIn a subsequent study, we have shown that under hyperglycemic circumstances, erythropoietin (EPO) exerts insulin-like effects on the uptake of isotope-labeled glucose by 3T3-L1-type fat cells (Figure 4) and can lead to the translocation of glucose transporters transporters (GLUTs) from their intracellular pools to the membrane (Figure 5). EPO also improves glucose metabolism in streptozotocin-induced diabetic rats[37].

M- AND O-TYR ARE INCORPORATED INTO CELLULAR PROTEINS AND LEAD TO ERYTHROPOIETIN RESISTANCEIn further studies, we showed that the administration of m- and o-Tyr to erythroblasts inhibited erythroblast proliferation in a time- and concentration-dependent manner. Increasing doses of p-Tyr could overcome the inhibition by m- and o-Tyr, suggesting potential competition among the structural isoforms (Figure 6).

Erythroblasts grown in cell culture medium supple-mented with m- or o-Tyr incorporated the Tyr isoforms into their cellular proteins (Figure 7).

Supplementing erythroblast cells with o- or m-Tyr inhibited the EPO-dependent increase in the rate of


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Figure 4 Effects of treatment with r-mo-erythropoietin on the rate of [3H]-deoxy-D-glucose uptake in 3T3-L1 adipocytes cultured in either normal (5 mmol/L) or high (25 mmol/L) glucose medium (insulin: n = 8 and n = 6; r-mo-erythropoietin: n = 6 and n = 5, respectively). aP < 0.05 vs control in the same medium; cP < 0.05 between the two media; Comparisons with control were performed using a one-sample t-test; pairwise comparisons between 5 and 25 mmol/L glucose-cultured cells were performed using a paired samples t-test. Republished with permission of Georg Thieme Verlag KG Stuttgart, New York from Mikolás et al[37]. EPO: Erythropoietin.



Figure 5 GLUT4 translocation (red fluorescence) after a 30 min treatment with insulin (400 nmol/L) or r-mo-erythropoietin (40 ng/mL) in 3T3-L1 adipocytes cultured in high glucose (25 mmol/L) or normal glucose (5 mmol/L) medium compared with that in untreated cells (Control). Nuclei are shown with blue fluorescence. The GLUT4 translocation was apparent after both the erythropoietin (EPO) and insulin treatments, whereas it was not present in untreated cells. Representative images are shown from n = 3 independent experiments. Republished with permission of Georg Thieme Verlag KG Stuttgart, New York from Mikolás et al[37].

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Figure 6 Effect of para-, ortho-, or meta-tyrosine supplementation on the proliferation of cells cultured in medium with or without erythropoietin. A: The time-dependent increase in cell count in medium supplemented with p-Tyr (open bars), o-Tyr (black bars) or m-Tyr (grey bars) (aP < 0.05 vs p-Tyr cultured cells on the same day; n = 10); B: The alterations in the cell counts after incubation for 3 d in medium containing o- or m-Tyr and the indicated additional amount of para-tyrosine (bP < 0.001 vs p-Tyr-containing medium; n = 5). The results are expressed as the ratio of the protein content of erythropoietin (EPO) and non-EPO (control) cells (aP < 0.05 vs p-Tyr cultured cells; n = 10). Comparisons were performed using ANOVA and Bonferroni’s post-hoc test. Republished with permission of S Karger AG, Basel from Mikolás et al[21]. p-Tyr: Para-tyrosine; o-Tyr: Ortho-tyrosine; m-Tyr: Meta-tyrosine.

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to vehicle or o-Tyr supplementation for 4 wk. By the end of the 4 wk, we showed decreased vasorelaxation in response to insulin in the o-Tyr-supplemented rats

compared with the controls. Additionally, the endothelial cells that received o-Tyr supplementation incorporated o-Tyr into their cellular proteins. In these cells, the


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Figure 7 Relative meta- and ortho-tyrosine content (i.e., ratios of meta- and ortho-tyrosine/total tyrosine) of the cellular proteins of TF-1 cells (A and B, respectively). bP < 0.001 vs p- and m-Tyr cultured cells; dP < 0.001 vs o-Tyr cultured control cells; fP < 0.001 vs p- and o-Tyr cultured erythroblasts; n = 10. Comparisons were performed using ANOVA and Bonferroni’s post-hoc test. Republished with permission of S Karger AG, basel from Mikolás et al[21]. p-Tyr: Para-tyrosine; o-Tyr: Ortho-tyrosine; m-Tyr: Meta-tyrosine.

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Figure 8 Densitometric analyses of the phosphorylation of signal transducer and activator of transcription 5 (A: n = 3); densitometric analysis of extracellular signal regulated kinase 1 (B: n = 4) and extracellular signal regulated kinase 2 (C: n = 4). The data are expressed as the percent of untreated (control) cells. aP < 0.05; erythropoietin (EPO) vs control (one-sample t-test); cP < 0.05 vs para EPO (one-way ANOVA). Republished with permission of S Karger AG, basel from Mikolás et al[21].

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Figure 9 The ortho-tyrosine levels of consecutive arterial segments isolated from rats (A). There were significant differences (aP < 0.05) in the o-Tyr levels among the thoracic aorta (n = 7), abdominal aorta (n = 7), and the femoral artery (n = 9) of rats. The changes in the o-Tyr levels in the consecutive arterial segments of rats after H2O2 + AT treatment (i.e., high oxidative state), SOD + CAT treatment (i.e., low oxidative state), and aortic banding were compared with those of the control vessels (B). Control: untreated, control vessels (thoracic: n = 7; abdominal: n = 7; femoral: = 9); H2O2 + AT: hydrogen peroxide- and aminotriazole-incubated vessels (aminotriazole is an inhibitor of catalase) (thoracic: n = 5; abdominal: n = 5; femoral: n = 4); SOD + CAT: superoxide dismutase- and catalase-incubated vessels (thoracic: n = 5; abdominal: n = 5; femoral: n = 5); aortic banding (thoracic: n = 5; abdominal: n = 4; femoral: n = 4). Data are the mean ± SEM. The o-Tyr levels are relative to the phenylalanine (Phe) levels (Panels A and B) and are expressed as the percentage of the control vessels (Pane B). cP < 0.05; NS, P > 0.05 (ANOVA). Republished with permission of Informa Healthcare from Szijártó et al[19]. o-Tyr: Ortho-tyrosine; NS: Non-significant; SEM: Standard error of the mean.


insulin-induced increase in endothelial nitric oxide synthase phosphorylation was diminished compared with that in the control cells[20] (data not shown).

CONCLUSIONOur results, which are consistent with previous findings, indicate that m- and o-Tyr are valuable markers of oxidative stress and other types of stress in patients or experimental animals with DM. Furthermore, the results suggest that these unnatural amino acids may also perform a pathogenic role, i.e., interfere with the signaling of three distinct hormones: insulin (vascular and perhaps metabolic signaling), acetylcholine and erythropoietin (also has metabolic effects). This inhibition may be even more pronounced in patients who have high levels of the pathological amino acids m- or o-Tyr (e.g., in DM) and simultaneously have lower levels of physiological p-Tyr (e.g., patients with impaired kidney function). This finding, together with the effect in which p-Tyr overcomes the inhibitory effect of m- and o-Tyr, raises the possibility that the physiological amino acid p-Tyr could be a therapeutic tool in hormone resistance in states with increased oxidative stress (e.g.,

in DM).

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Figure 10 The dose–response curve of the insulin-induced relaxation of consecutive arterial segments isolated from rats. The relaxation in response to insulin was the greatest in the femoral artery (n = 7) and was less pronounced in the abdominal aorta (n = 7) and even less in the thoracic aorta (n = 7). The relaxation is depicted as a function of the logarithm of the insulin dose. Data are the mean ± SEM. aP < 0.05; NS, P > 0.05 (Extra sum-of-squares F test). Republished with permission of Informa Healthcare from Szijártó et al[19]. NS: Non-significant; SEM: Standard error of the mean.

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Figure 11 Acetylcholine-induced vasorelaxation in the abdominal aorta and femoral artery. Data are the means ± SEM. aP < 0.05; NS, P > 0.05 (Extra sum-of-squares F test). NS: Non-significant.


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P- Reviewer: Tsuruya K S- Editor: Ji FF L- Editor: A E- Editor: Liu SQ



Mami Yamaoka, Toshimasa Ishizaki, Toshihide Kimura

Mami Yamaoka, Toshimasa Ishizaki, Toshihide Kimura, Department of Pharmacology, Oita University Faculty of Medicine, Oita 879-5593, JapanAuthor contributions: Yamaoka M, Ishizaki T and Kimura T developed the concept and wrote the paper.Supported by JSPS KAKENHI Grant, Nos. 19790636, 21790876, 23791039, and 26461342; the joint research program of the Institute for Molecular and Cellular Regulation, Gunma University, Takeda Science Foundation, Novo Nordisk Pharma, Oita Broadcasting System Cultural Foundation, and Research Fund at the Discretion of the President, Oita University.Conflict-of-interest: The authors declare no competing financial interests.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Toshihide Kimura, Associate Professor, Department of Pharmacology, Oita University Faculty of Medicine, 1-1 Idaigaoka, Hasama, Yufu, Oita 879-5593, Japan. [email protected]: +81-97-5865722Fax: +81-97-5865729Received: August 25, 2014Peer-review started: August 25, 2014First decision: December 17, 2014Revised: December 24, 2014Accepted: February 4, 2015 Article in press: February 9, 2015Published online: April 15, 2015

AbstractThe small GTPase Rab27a is a member of the Rab family that is involved in membrane trafficking in various kinds of cells. Rab27a has GTP- and GDP-bound forms, and their interconversion regulates intracellular signaling pathways. Typically, only a GTP-bound GTPase binds its specific effectors with the resulting downstream signals controlling specific cellular functions. We previously

identified novel Rab27a-interacting proteins. Surprisingly, some of these proteins interacted with GDP-bound Rab27a. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in the secretory process. In pancreatic β-cells, GTP-bound Rab27a regulates insulin secretion at the pre-exocytotic stages via its GTP-specific effectors such as Exophilin8/Slac2-c/MyRIP and Slp4/Granuphilin. Glucose stimulation causes insulin exocytosis. Glucose stimulation also converts Rab27a from its GTP- to its GDP-bound form. GDP-bound Rab27a interacts with GDP-specific effectors and controls endocytosis of the secretory membrane. Thus, Rab27a cycling between GTP- and GDP-bound forms synchronizes with the recycling of secretory membrane to re-use the membrane and keep the β-cell volume constant.

Key words: Insulin; Exocytosis; Endocytosis; Rab27a; IQGAP1; Coronin 3; Glucose; Small GTPase


Core tip: The small GTPase Rab27a is a member of the Rab family that is involved in membrane trafficking in pancreatic β-cells. GTP-bound Rab27a regulates insulin secretion at pre-exocytotic stages via its GTP-specific effectors such as Exophilin8/Slac2-c/MyRIP and Slp4/Granuphilin. Glucose stimulation causes insulin exocytosis. Glucose stimulation also converts Rab27a from its GTP- to its GDP-bound form. GDP-bound Rab27a interacts with GDP-specific effectors and controls endocytosis of the secretory membrane. Thus, Rab27a cycling between GTP- and GDP-bound forms synchronizes with the recycling of secretory membrane to re-use the membrane and keep the β-cell volume constant.

Yamaoka M, Ishizaki T, Kimura T. Interplay between Rab27a effectors in pancreatic β-cells. World J Diabetes 2015; 6(3): 508-516 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/508.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.508

MINIREVIEWS


Interplay between Rab27a effectors in pancreatic β-cells




INTRODUCTIONDiabetes mellitus is defined as chronic hyperglycemia due to relative insulin deficiency. Impairment of secretory activity in pancreatic β-cells plays an important role in the pathogenesis of this disease. In particular, decreased output in the early phase of glucose-induced insulin release precedes the onset of type 2 diabetes mellitus[1,2]. Insulin secretion from pancreatic β-cells is finely tuned for glucose homeostasis by multiple physiological factors such as nutrients, hormones and neurotransmitters. Some of these factors induce or enhance insulin release whereas others decrease it, thereby enabling sophisticated glucose regulation. One characteristic of this regulation in pancreatic β-cells is the control of secretion by several nutrients. Glucose, the most important insulin secretagogue, stimulates insulin secretion mainly by the generation of ATP via glucose metabolism, although there appear to be other signaling pathways involved that have not been fully identified.

The insulin secretory process comprises insulin synthesis and its packaging into secretory granules, granule transport in the cytoplasm, interaction with the cell membrane, exocytosis as a result of an increase in cytoplasmic Ca2+, endocytosis and retrograde transport. The present study reviews recent progress in our understanding of the roles of the small GTPase Rab27a and its effectors in the secretory process.

SMALL GTPASE CYCLESmall GTPases are GTP-binding proteins with molecular masses ranging from 20 to 30 kDa. These proteins, comprising Ras, Rho, Rab, Ran and Sar/Arf, are expressed in almost all eukaryotic cells and participate in a wide variety of cellular functions including proliferation, cytoskeletal rearrangement and intracellular transport[3]. Small GTPases have GTP- and GDP-bound forms, and their interconversion regulates intracellular signaling pathways (Figure 1). Typically, only the GTP-bound small GTPase binds its specific effectors and the resulting downstream signals control specific cellular functions[4]. Therefore, GTP- and GDP-bound small GTPases are considered as active and inactive forms, respectively. The activation of small GTPases is modulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and GDP-dissociation inhibitors (GDIs). Small GTPases localize in the cytosol as a GDP-bound form under unstimulated conditions. Cell stimulation recruits GDP-bound small GTPases to the vicinity of the plasma membrane and converts them to the GTP-bound form through the action of GEFs[4,5]. These GTP-bound forms interact with their specific effectors and transduce signals. GAPs promote the intrinsic GTPase activity of small GTPases and induce the conversion of the GTP- to the GDP-bound form[4,6]. GDIs form a complex with GDP-bound small GTPases and induce their intracellular redistribution from the

plasma membrane to the cytosol[7-9].

RAB27AThe Rab family, which consists of more than 60 members, regulates membrane trafficking[10-12]. Rab27 is a Rab-family member that controls vesicle transport in various kinds of cells[13]. There are two isoforms of Rab27; Rab27a and Rab27b. Mutations in Rab27a have been reported to cause Griscelli syndrome[14]. This disorder results in pigment dilution in the skin and the hair. The same symptoms are observed in ashen mice with a natural mutation in Rab27a[15]. To date, three forms of Griscelli syndrome have been reported. Mutations in Myosin Va[14,16-18], Rab27a[14,15], and its effector Slac2-a/melanophilin[19,20] cause type 1 (GS1), type 2 (GS2), and type 3 (GS3) Griscelli syndrome, respectively. These proteins play an important role in melanosome transport in melanocytes. Peripheral melanosome distribution is regulated by the molecular motor protein myosin Va. GTP-bound Rab27a localizes on the surface of melanosomes where it interacts with Slac2-a/melanophilin. Since Slac2-a/melanophilin binds myosin Va, this complex functions as a linker protein between the melanosome and the motor protein[10,21,22]. GTP-bound Rab27 also interacts with another effector protein Slp2-a, thereby regulating docking of the melanosome to the plasma membrane[23].

Griscelli syndrome also results in immunodefici-ency[14,17]. Exocytosis of granzyme-A-containing granules is decreased in ashen mice cytotoxic T lymphocytes[24,25]. Although both Rab7 and Rab11 are also present on the surface of these granzyme-A-containing granules, Rab27a is the main regulator of granule exocytosis[26]. Indeed, GTP-bound Rab27a interacts with phosphatidylinositol 4,5-bisphosphate (PIP2) via its effector protein Munc13-4 and regulates the docking of the granules to the synapse. These results suggest that Rab27a regulates the secretion of lytic granules in cytotoxic T lymphocytes.

Rab27b, a closely related isoform of Rab27a, also participates in intracellular transport and secretion. Rab27b and its effector proteins including Slac2-c and Slp4-a are expressed in parotid acinar cells[27]. In these cells, GTP-bound Rab27a interacts with both Slac2-c and Slp4-a. Amylase release was inhibited when streptolysin O-permeabilized cells were treated with anti-Slac2-c antibody[28]. Amylase release was also inhibited when the Rab27b/Slp4-a complex was dissociated with a GST-Slp4-a-linker[29]. Isoproterenol (IPR) stimulation promoted intracellular redistribution of Slac2-c from the cytosol to a luminal site[30]. Since Slac2-c potentially interacts with Myosin Va, it is possible that the Rab27b/Slac2-c complex regulates F-actin-dependent intracellular transport of secretory vesicles. In contrast, the intracellular distribution of Slp4-a was not changed in the presence or absence of IPR stimulation[30]. The Rab27b/Slp4-a complex may regulate docking of the secretory vesicles to the membrane[31].

Amylase is also released from pancreatic acinar

Yamaoka M et al . Rab27a effectors in pancreatic β-cells


cells in which Rab27b is localized on the surface of zymogen granules[32]. Rab27b-Q78L, a constitutively active Rab27b mutant, enhances amylase release. In contrast, Rab27b-N133I, a dominant negative mutant, inhibits amylase release. These results suggest that Rab27b regulates amylase release in pancreatic acinar cells. Exophilin7/Slp1/JFC1 is a GTP-bound Rab27a interacting protein. The number of zymogen granules was increased in the pancreatic acinar cells of fasted Exophilin7/Slp1/JFC1-KO mice[33]. Moreover, amylase release was promoted in pancreatic acinar cells from Exophilin7/Slp1/JFC1-KO mice that were treated with carbamylcholine chloride or cholecystokinin-8, which mimic fed conditions. Thus, Rab27b may be involved in amylase release via Exophilin7/Slp1/JFC1 in pancreatic acinar cells.

Rab3a, which has the highest homology to Rab27, is highly expressed in synaptic vesicles[27]. In PC12 cells, Rab27a and Rab3a are co-localized on the surface of dense-core granules[34]. Silencing of both Rabs caused a significantly greater decrease in the number of these granules docked to the plasma membrane compared to silencing of either Rab alone. Since, Rab27a and Rab3a interact with the same effector proteins, these Rabs must cooperatively regulate the docking step of dense-core granules. In contrast, each of these Rabs has a specific function in pancreatic β-cells[35]. Rab3a-GAP inhibited the dot-like distribution of Rab3a in the insulin-secreting β-cell line MIN6. In contrast, the distribution of Rab27a was not changed in Rab27a-GAP expressing

cells. Furthermore, whereas Rab3a is localized on insulin granules as a GTP-bound form, the Rab27a on these granules is present as both GTP- and GDP-bound forms. Moreover, these Rabs direct unique kinetic and functional properties of the exocytic pathway.

RAB27A IN PANCREATIC β-CELLSRab27a is highly expressed in pancreatic β-cells and is involved in insulin secretion[36]. In ashen mice, blood glucose levels after a glucose load were higher, and insulin secretion in response to high glucose was lower than that in control mice. Interestingly, the decrease in insulin secretion was specific to glucose stimulation. These data suggest that Rab27a signaling in pancreatic β-cells is glucose-specific.

Insulin granules that are synthesized in pancreatic β-cells are eventually released by exocytosis via a series of stages (Figure 2). Granules that are transported in the cytoplasm attach to the inner surface of the cell membrane (docking). The contents of both docked and undocked granules are eventually released following elevation of intracellular Ca2+ levels. The granules are categorized into three types[37,38]. Residents are granules that are pre-docked to the plasma membrane before fusion. Passengers are granules that fused without stably docking. Visitors are granules that remain near the plasma membrane for some time before fusion. GTP-bound Rab27a regulates insulin secretion by modulating the transport and docking steps of insulin granules via its effector proteins (Table 1)[39-41]. To date, Exophilin8/Slac2-c/MyRIP, Slp4/Granuphilin, Exophilin7/Slp1/JFC1 and Exophilin1/Rabphilin3A are known to act as GTP-dependent Rab27a effectors in pancreatic β-cells. Exophilin8/Slac2-c/MyRIP possesses a potential


Exophilin8/Slac2-c/MyRIP

Rab27aGTP

Noc2Slp4/Granuphilin

Insulin

Plasma membranePassengersVisitors, Residents

Pancreatic beta-cell

Translocation

Docking

Fusion

?

Figure 2 Schematic model of GTP-bound Rab27a function in pancreatic β-cells. Insulin-containing granules transported in the cytoplasm attach to the inner surface of the cell membrane (docking). The contents of both docked and undocked granules are eventually released following elevation of intracellular Ca2+ levels. GTP-bound Rab27a regulates insulin secretion by modulating the granule transport and docking steps via its effector proteins. Residents are granules that are pre-docked to the plasma membrane before fusion. Passengers are granules that fused without stably docking. Visitors are granules that remain near the plasma membrane for some time before fusion.

GDP

GTPase

GDIs

GAPs

PiGTP

GDP

GEFs

GTP

GTPase

Effectors

Cellular function

ON

OFF

Figure 1 Small GTPase GTP/GDP cycle. Small GTPases localize in the cytosol as a GDP-bound form under unstimulated conditions. Cell stimulation recruits GDP-bound small GTPases to the vicinity of the plasma membrane and converts these proteins to the GTP-bound form through GEF activity. The GTP-bound form interacts with its specific effectors and thereby transduces signals. GAPs promote the intrinsic GTPase activity of small GTPases and induce conversion of the GTP- to the GDP-bound form. GDIs form a complex with GDP-bound small GTPases and induce their intracellular redistribution from the plasma membrane to the cytosol. GEFs: Guanine nucleotide exchange factors; GAPs: GTPase-activating proteins; GDIs: GDP-dissociation inhibitors.


pancreatic β-cells.

GDP-DEPENDENT EFFECTORS OF RAB27AWe have identified novel Rab27a-interacting proteins. Surprisingly, some of these proteins interacted with GDP-bound Rab27a (Table 2)[52,53]. Since GDP-bound GTPases have been considered to be an inactive form, these proteins might be suspected to be regulators of Rab27a GTP/GDP cycles. Indeed, protrudin, which was identified as a GDP-bound small GTPase interacting protein, forms a complex with GDP-bound Rab11 via its GDI consensus sequence and regulates neurite formation[54]. Therefore, protrudin is thought to be a GDI that regulates GTPase cycles. In contrast, the GDP-bound Rab27a interacting proteins that we identified do not contain any GDI consensus sequences. Moreover, these proteins interact with GDP-bound Rab27a and transduce downstream signals in a similar manner to classical GTP-dependent effectors of small GTPases. We consider that these proteins are GDP-dependent effectors of Rab27a[40,41].

Coronin 3We identified coronin 3 as a GDP-bound Rab27a interacting protein[52]. Coronins have been purified from precipitated actin of Dictyostelium discoideum cells[55]. To date, more than ten coronin subfamilies have been reported. Coronin 3 is a ubiquitously expressed coronin that shares a central domain with other coronin family members that contains five WD40 repeats, which are known to form β-propeller structures and to mediate protein-protein interactions (Figure 3). Initially, these repeats were thought to form a five-bladed β-propeller structure. However, recent studies of coronin structure demonstrated that the N-terminal region of coronin forms two additional blades (not identified from the sequence). Therefore, coronin 3 is now considered to possess a seven-bladed β-propeller structure[56,57]. It was noted that this propeller structure of coronin 3 is a GDP-bound Rab27a binding site[52]. Oligomerization of coronins and their interaction with F-actin are mediated by the C-terminal 30-40 amino acids, which form a coiled-coil structure. These interactions modulate actin assembly and participate in various cellular functions. Human coronin 3 also modulates F-actin through binding to Arp2/3[57], a key protein in the formation of a branched actin filament network[57-59]. These direct and indirect modulations of F-actin must play a crucial role in the cellular function of coronins, because they are conserved in eukaryote cells[60]. Both F-actin-binding and -bundling activities of coronin 3 were promoted by its interaction with GDP-bound Rab27a[61]. In contrast, GDP-bound Rab27a did not affect the interaction between coronin 3 and Arp2/3. GDP-bound Rab27a regulates F-actin bundling by modulating a direct effect of coronin 3 on F-actin (Figure 3).

myosin binding site. Although Exophilin8/Slac2-c/MyRIP functions as a linker protein between GTP-bound Rab27a and Myosin Va in melanocytes[42,43], this effector may form a different complex in pancreatic β-cells. Indeed, some reports suggest that there are several conditions under which Exophilin8/Slac2-c/MyRIP does not interact with Myosin Va[44]. Slp4/Granuphilin and Exophilin1/Rabphilin3A interact with Myosin Va in pancreatic β-cells[45]. Moreover, they are linked to a different subset of insulin granules. Further studies are required to investigate the regulation of the transport of insulin granules to the plasma membrane.

Slp4/Granuphilin was identified as a molecule that associates with insulin granules in pancreatic β-cells[46]. This molecule forms a complex with Syntaxin1a and Munc18-1 and regulates the docking step of insulin granules in the insulin secretion pathway[47]. The number of insulin granules docked to the plasma membrane was decreased in pancreatic β-cells from Slp4/Granuphilin-KO mice[48]. Interestingly, insulin secretion was promoted in these mice. These results suggest that Slp4/Granuphilin regulates the docking state and inhibits the fusion of the insulin granule membrane and the plasma membrane in unstimulated pancreatic β-cells (Figure 2). Exophilin7/Slp1/JFC1 also tethers insulin granules to the plasma membrane[49]. There seem to be multiple docking states of insulin granules in pancreatic β-cells.

Noc2 is a potential Rab27a effector. Noc2 displays 78% similarity to the N-terminus of Exophilin1/Rabphilin3A[50]. Ca2+ triggered insulin secretion from pancreatic islets of Noc2-KO mice was impaired, but was restored by treatment with the G-protein inhibitor pertussis toxin[51]. Although Noc2 is involved in insulin secretion through G-protein Gi/o signaling, its role in pancreatic β cells has not been identified. A yeast two-hybrid experiment identified zyxin as a Noc2-interacting protein. Because zyxin has been reported to bind the actin-binding protein α-actinin in fibroblasts[50], the interaction between Noc2 and zyxin may regulate insulin secretion by modulating actin dynamics in


Table 1 GTP-bound Rab27a effectors in pancreatic β-cells

GTP-bound Rab27a effectors Interacting proteins Function

Exophillin8/Slac2-c/MyRIP Myosin Va, Actin, PKA TranslocationSlp4/Granuphilin Munc18-1,

Syntaxin1a,Myosin VaDocking

Noc2 Munc13-1 -Exophilin7/Slp1/JFC1 - DockingExophilin1/Rabphilin3A Myosin Va Translocation

Table 2 GDP-bound Rab27a effectors in pancreatic β-cells

GDP-bound Rab27a effectors Interacting proteins Function

Coronin 3/coronin 1c F-actin Actin bundlingEndocytosis

IQGAP1 GTP-bound Cdc42 ScaffoldGTP-bound Rac1


Coronins regulate membrane internalization in some types of cells through F-actin remodeling. In pancreatic β-cells, silencing of coronin 3 inhibited the internalization of both FM4-64 and phogrin[52]. Internalization of these molecules was also inhibited when Rab27a-coronin 3 binding was inhibited by a dominant negative mutant of coronin 3. Moreover, the inhibition of F-actin binding to coronin 3 had the same effect[61]. Thus, coronin 3 regulates the endocytosis of secretory membranes via the modulation of actin assembly in pancreatic β-cells.

Endocytosis is a complex process that involves cargo sorting, membrane invagination, vesicle scission and vesicle targeting (Figure 4). The uptake of an antibody against the extracellular domain of phogrin was inhibited and phogrin was located near the plasma membrane in MIN6 cells expressing a dominant negative mutant of coronin 3[62]. Interestingly, phogrin staining near the plasma membrane remained when the cells were treated with acid wash, suggesting that the anti-phogrin antibody near the plasma membrane is separated from the plasma membrane. Thus the formation of GDP-bound Rab27a-coronin 3 complexes regulates the retro-grade transport of internalized secretory membrane, at a stage after scission from the plasma membrane.

Insulin secretagogue glucose stimulation induced the intracellular redistribution of both Rab27a and coronin 3 from the cytosol to the plasma membrane[62]. These redistributions were inhibited in Rab27a-silenced and Rab27a-Q78L-expressing MIN6 cells. Glucose-induced translocation of coronin 3 is due to its interaction with GDP-bound Rab27a. It has been reported that coronin 3 forms a closed conformation through an intramolecular interaction between its C- and N-termini[59]. Therefore, the glucose-dependent binding of its N-terminus to GDP-Rab27a may shift coronin 3 to the open conformation, which enables this molecule to interact with F-actin[41].

IQGAP1We identified IQGAP1 as another GDP-bound Rab27a interacting protein[53]. IQGAP1, an effector for Cdc42 and Rac1, is a member of the IQGAP family[63]. IQGAP1 regulates cell-cell contacts and cell migration[64-70]. In pancreatic β-cells, IQGAP1 interacts with vesicle-tethering exocysts under basal conditions. Moreover, this IQGAP-exocyst complex is dissociated by GTP-bound Cdc42[71]. GTP-bound Cdc42 regulates insulin secretion by modulating F-actin bundling[72,73]. Active Cdc42 also interacts with SNARE proteins such as VAMP2 and Syntaxin1a, and promotes the fusion step in insulin secretion[74]. Since glucose converts Cdc42 from the GDP- to the GTP-bound form[75], the IQGAP1-vesicle tethering exocyst complex is dissociated by glucose. It has been reported that vesicle-tethering to the plasma membrane is not a prerequisite for, but instead temporarily inhibits glucose-induced membrane fusion[37]. This finding raises the possibility that the IQGAP1-exocyst complex may inhibit subsequent fusion events. IQGAP1 also binds A kinase anchoring protein 79 and functions as a scaffold protein[76].

IQGAP1 binds GDP-bound Rab27a through its RasGAP related domain (Figure 3)[53]. This domain lacks GAP activity and forms part of the GTP-bound Cdc42 and Rac1 interacting site[63,70,77]. Moreover, IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Cdc42[53]. IQGAP1 is distributed in the vicinity of the plasma membrane in both glucose-stimulated and - unstimulated cells. In contrast, glucose-induced redistribution of Rab27a and its binding protein coronin 3 was inhibited in both IQGAP1-silenced cells and in cells expressing the dominant negative mutant Cdc42-T17N. Since endocytosis of secretory membrane was also inhibited in these cells, these data indicate that activated Cdc42-bound IQGAP1, to which GDP-bound Rab27a binds,


Figure 4 Schematic model of GDP-bound Rab27a function in pancreatic β-cells. Endocytosis is a complex process that involves cargo sorting, membrane invagination, vesicle scission and vesicle targeting. GDP-bound Rab27a modulates F-actin assembly and regulates the retrograde transport of the internalized secretory membrane, at the stage after scission from the plasma membrane. Scission is indicated by the dynamin step.


F-actinGDP

Rab27a Coronin 3IQGAP1

Clathrin

Dynamin 2 Plasma membrane

Insulin

Pancreatic beta-cellF-actin

Coronin 3

N WD C

β-propeller structure GDP

Rab27a GTP

Cdc42IQGAP1

CHD NTR IQR GRD CT

Plasma membrane

Figure 3 Interplay between Rab27a, coronin 3 and IQGAP1. GDP-bound Rab27a simultaneously binds coronin 3 and IQGAP1, resulting in the formation of a trimeric complex. WD: Five WD40 repeats; CHD: Calponin homology domain; NTR: N-terminal repeats; IQR: IQ repeats; GRD: RasGAP related domain; CT: Carboxy terminus of IQGAP1.


recruits endocytic machinery including coronin 3 and regulates endocytosis of secretory membrane.

In summary, IQGAP1 functions at pre-exocytotic stages via interaction with the exocyst complex[71] and this complex is dissociated by GTP-bound Cdc42. Our results indicate that IQGAP1 also plays a crucial role in the control of endocytosis via interaction with GTP-bound Cdc42 and GDP-bound Rab27a[53]. Based on the combined data, we propose a model where IQGAP1 functions as a scaffold protein and is a key molecule for membrane recycling.

IQGAP1 also interacts with GTP-bound Rac1[63,77]. Interestingly, IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Rac1[53]. Moreover, endocytosis of secretory membrane was inhibited in MIN6 cells expressing the dominant negative Rac1-T17N mutant. These results suggest that Rac1 also recruits endocytic machinery and regulates endocytosis of secretory membrane. Cdc42 and Rac1 display some different characteristics in pancreatic β-cells. The most important difference is the timing of the glucose-induced conversion from the GDP- to the GTP-bound form. Glucose stimulation causes a shift from GDP-bound Cdc42 to GTP-bound Cdc42 within 3 min. In contrast, the glucose induced conversion of GDP- to GTP-bound Rac1 requires 20 min[73,75]. These findings raises the possibility that Cdc42 regulates rapid endocytosis whereas Rac1 regulates subsequent, prolonged endocytosis, a pattern that may be associated with the biphasic release of insulin in response to glucose.

CONCLUSIONIn the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytotic stages via its GTP-specific effectors (Figure 5). Glucose stimulation causes insulin exocytosis. Glucose stimulation also converts Rab27a

from its GTP- to its GDP-bound form, which interacts with IQGAP1, recruits coronin 3, and controls endocytosis of the secretory granule membrane. A long-term overexpression of a dominant negative coronin 3 mutant caused β-cell death (unpublished data), suggesting that the membrane recycling system controlled by Rab27a may be necessary for β-cell survival. Thus, we consider that Rab27a GTP/GDP cycling synchronizes with the recycling of secretory membrane to re-use the membrane and to keep the β-cell volume constant. It raises the possibility that a pharmacological agent that modulates the recycling system may become a new therapeutic option for the treatment of β-cell dysfunction in diabetes. Further studies are required to investigate whether Rab27a is involved in the pathogeneses of diabetes mellitus.

Typically, small GTPases are predominantly present in the GDP-bound form under unstimulated conditions and are converted to the GTP-bound form by stimulation. In contrast, glucose stimulation causes a shift of Rab27a in pancreatic β-cells from its GTP- to its GDP-bound form[52]. The same conversion also occurs in thrombin stimulated platelets[78]. These findings suggest that specific Rab27a-GAPs are activated by these stimulations. Two candidate Rab27a-GAPs, EPI64A and EPI64B, have been reported[79]. In melanocytes, EPI64A has the higher GAP activity and functions as the main Rab27a-GAP. In pancreatic acinar cells, EPI64B regulates amylase secretion by modulating Rab27a GTP/GDP cycles[80]. Further studies are needed to identify and characterize Rab27a-GAPs in pancreatic β-cells.

ACKNOWLEDGMENTSWe thank all members of our laboratory for helpful suggestions.

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Figure 5 Rab27a GTP/GDP cycling synchronizes with the recycling of secretory membrane. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytotic stages via its GTP-specific effectors. Glucose stimulation causes insulin exocytosis. Glucose stimulation also converts Rab27a from its GTP- to its GDP-bound form. GDP-bound Rab27a interacts with IQGAP1, recruits coronin 3, and controls endocytosis of the secretory granule membrane. GAPs: GTPase-activating proteins.


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P- Reviewer: Elia E S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ



Orit Pinhas-Hamiel, Uri Hamiel, Yael Levy-Shraga

Orit Pinhas-Hamiel, Uri Hamiel, Yael Levy-Shraga, Pediatric Endocrinology and Diabetes Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, Ramat-Gan 52621, IsraelOrit Pinhas-Hamiel, Uri Hamiel, Yael Levy-Shraga, Sackler School of Medicine, Tel-Aviv University, Juvenile Diabetes Center, Maccabi Health Care Services, Ramat-Gan 52621, IsraelAuthor contributions: Pinhas-Hamiel O, Hamiel U and Levy-Shraga Y performed literature review, wrote the paper, and conceived the three level models and created the graphs.Conflict-of-interest: None.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Dr. Orit Pinhas-Hamiel, Pediatric Endocrine and Diabetes Unit, Edmond and Lily Safra Children’sHospital, Sheba Medical Center, Tel Hashomer, Ramat-Gan 52621, Israel. [email protected] Telephone: +972-3-5305015Fax: +972-3-5305055Received: August 27, 2014Peer-review started: August 28, 2014First decision: November 27, 2014Revised: December 9, 2014Accepted: January 15, 2015Article in press: January 19, 2015Published online: April 15, 2015

AbstractEating disorders (ED) are characterized by a persistent disturbance of eating that impairs health or psychosocial functioning. They are associated with increased rates of medical complications and mortality. Insulin omission is a unique purging behavior available to individuals with type 1 diabetes mellitus (T1DM). The standard treatment regimen for T1DM requires a major focus on food and

eating patterns. Moreover, intensive insulin therapy is associated with increasing body weight. These factors, combined with the psychological burden of chronic disease management and depression, may contribute to ED. The comorbidity of ED in T1DM patients is associated with poorer glycemic control and consequently higher rates of diabetes complications. Early recognition and adequate treatment of ED in T1DM is essential.

Key words: Type 1 diabetes; Eating disorders; Insulin omission


Core tip: Intentional insulin omission for the purpose of preventing weight gain is a unique behavior available to individuals with type 1 diabetes mellitus (T1DM). It is classified as either an inappropriate compensatory feature of bulimia nervosa or as a purging disorder component of other specified feeding or eating disorder (ED). Its prevalence increases with age, affecting up to 40% of young adult females with T1DM. The comorbid of ED in T1DM patients is associated with higher rates of short and long-term diabetes complications. A high index of suspicion is needed since ED behaviors are often well hidden and denied. Treatment involves a complex interplay of psychosocial, dietary and medical aspects and requires a multidisciplinary team.

Pinhas-Hamiel O, Hamiel U, Levy-Shraga Y. Eating disorders in adolescents with type 1 diabetes: Challenges in diagnosis and treatment. World J Diabetes 2015; 6(3): 517-526 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/517.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.517

DEFINITIONS OF EATING DISORDERSEating disorders (ED) are characterized by a persistent

MINIREVIEWS


Eating disorders in adolescents with type 1 diabetes: Challenges in diagnosis and treatment




disturbance of eating that impairs health or psychosocial functioning[1,2]. Based upon the fifth edition of the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders (DSM-Ⅴ), which was released in May 2013, ED are divided into eight mutually exclusive categories[3]. These categories are based upon observed symptoms and include anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder, avoidant/restrictive food intake disorder, pica, rumination disorder, other specified feeding or eating disorder (OSFED) and unspecified feeding or eating disorder. Some diagnoses include a dimensional component that enables clinicians to specify the severity of illness[4-6].

In patients with type 1 diabetes mellitus (T1DM), intentional insulin omission or reduction for the purpose of preventing weight gain is recognized as either an inappropriate compensatory feature of bulimia nervosa or as a purging disorder, a component of OSFED. The latter is relevant when the recurrent purging behavior to influence weight or shape (e.g., insulin omission) occurs in the absence of binge eating.

The two categories: “eating disorders not otherwise specified (EDNOS)” and “disturbed eating behavior (DEB)”, compromised a wide spectrum of eating disorder pathologies, at a frequency or severity that does not merit a formal ED diagnosis. Though these categories are not included in DSM-Ⅴ, they appear in the current review, due to their use by several studies involving individuals with diabetes.

ARE ADOLESCENTS WITH T1DM AT INCREASED RISK TO DEVELOP ED?This question should be answered for the specific ED studied, according to the tools used for assessment and the prevalence rates in the general population.

Diagnostic tools of ED in persons with T1DMThe diagnosis of ED in individuals with T1DM is difficult, since eating behaviors are often well hidden and denied. The use of different questionnaires for assessing prevalence rates of ED makes comparisons across studies difficult. Furthermore, as Markowitz et al[7] pointed out, that general diagnostic questionnaires for detecting ED are not appropriate for individuals with T1DM for two main reasons. Firstly, these questionnaires do not identify eating disorder behaviors that are unique to T1DM, such as insulin omission[7]. Secondly, such questionnaires may inflate the prevalence of eating problems in those with T1DM, because behaviors that are considered disturbed, such as particular concern about diet, reduced intake of certain food groups, and eating when not hungry, are integral to diabetes care[8].

Prevalence rates of ED among persons with T1DM Prevalence rates of ED among persons with TIDM vary according to the different ED categories and the populations studied. Some studies examined prevalence

rates of AN and BN only, while others examined the prevalence of insulin omission only, and some reported the prevalence of a combination of all ED together. Some studies reported the prevalence of full-threshold diagnoses of ED, whereas others also included subclinical ED. Reported prevalence rates differ also according to characteristics of the study populations, such as age range, and whether males were included or only females.

Prevalence of AN: A meta-analysis of controlled studies that defined ED in females according to DSM Ⅲ-R or DSM Ⅳ criteria reported the prevalence of AN in T1DM subjects was not significantly different from that of controls[9].

Prevalence of BN: This same meta-analysis showed a significantly higher prevalence of BN among the females with T1DM than those without diabetes[9].

Overall prevalence of AN, BN and EDNOS: In a recently published meta-analysis[10] of six studies of adolescents[11-16], 7.0% of the 825 individuals with T1DM had ED, compared with 2.8% of the 2282 individuals without T1DM.

Prevalence of DEB: In a meta-analysis[10] of five studies[11,15-18], a higher proportion of adolescents with T1DM than without T1DM were classified as having DEB (39.3% vs 32.5%). The prevalence rate of DEB increased significantly with weight and age, from 7.2% in the underweight group to 32.7% in the obese group, and from 8.1% in the youngest age-group (11-13 years) to 38.1% in the oldest age-group (17-19 years)[19].

Prevalence of insulin omission in T1DM: Insulin omission is a unique purging behavior available to individuals with T1DM. As presented in Table 1, insulin omission for weight loss is identified mainly in females; its prevalence increases with age, affecting up to 40% of young adult females with T1DM.

PREDISPOSING FACTORS FOR DEVELOPING EDWe suggest a three level model to describe the development of disturbed eating in adolescents with T1DM. This model comprises a hierarchically arranged continuum of potential effects on health problems (Figure 1). The first circle in the model, the premorbid status, includes a tendency to overweight, and factors relating to personality and family characteristics and dynamics. The second circle comprises factors arising at diagnosis of diabetes, such as the age of diabetes onset and satisfaction from weight loss. The third circle comprises factors associated with the chronic management of diabetes, such as recurrent hypoglycemic episodes,

Pinhas-Hamiel O et al . Eating disorders in type 1 diabetes


strict insulin treatment and carbohydrate counting.

FIRST CIRCLE - PREMORBID FACTORSIncreased body weight and a tendency to gain weight It has been suggested that the single question “Have you ever been overweight?” may be sufficient to screen for those with T1DM who are at a high risk for disordered eating attitudes/behaviors[20]. Indeed, adolescent girls with T1DM who reported ever being overweight endorsed more disordered eating attitudes and behaviors[21]. In addition, a higher body mass index percentile among teenagers and adults with T1DM, particularly among females, one to two years prior to the onset of ED, was found to be associated with disordered eating[22]. Furthermore, youth at risk for disordered eating have been shown to have poorer

diet quality, including a higher intake of total fat and saturated fat[23].

Low self-esteem and body dissatisfaction Higher levels of weight dissatisfaction tended to be associated with unhealthy weight control/disordered eating among adolescent females[24]. Low self-esteem predicts eating disturbances in the general population. Lower self-esteem was associated with disturbed eating behavior in girls with T1DM[25]. Of note, low self-esteem and body dissatisfaction could also arise along the course of the disease. In a longitudinal study the development of DEB in girls with T1DM, was predicted by lower self-esteem related to physical appearance, and lower global self-esteem[26].

Personality characteristicsPerfectionism has been associated with attitudinal aspects of eating disorders such as weight preoccupation. Borderline personality characteristics were related to insulin omission[27]. Adolescent girls with T1DM and an ED showed deficits in self-regulation and narcissistic gain from illness[28], and tended to blame themselves for the situation[29].

Comparing adolescents with ED and adolescents with T1DM and ED, those with T1DM were less pathologically compromised, scored lower on several affective areas and had a lower prevalence of depression and anxiety[30].

Family characteristics and dynamics Several factors involving family characteristics and dynamics are associated with an increased risk for ED. Firstly, in many families of adolescent girls with T1DM,


Table 1 Prevalence rates of insulin omission according to age range and gender

Prevalence of insulin omission (%)

Age range (yr)

Number and sex

Ref.

2 9-13 101 females Colton et al[12]

26.24.5

11-19 390 females380 males

Wisting et al[19]

11 12-19 361 females Jones et al[11]

14 12–19 356 females Rodin et al[61]

101


Neumark-Sztainer et al[24]

34 16-22 91 females Rydall et al[46]

40 18-30 59 females Stancin et al[74]

110


Philippi et al[75]


Increased BMI

Personality factors

Age of T1DM onset

Family factors

Weight loss at diagnosis

Intensive insulin treatment

Recurrent hypoglycemia

Insulinomission

Weight gain

Carb/calories counting

Strict eating

Bingeeating

Depression

Ⅲ

Ⅱ

Ⅰ

Eatingdisorders

Figure 1 Three level models describing the development of eating disorders in adolescents with type 1 diabetes mellitus. The outer circle delineates the premorbid status. The middle circle delineates weight changes at diagnosis and with initiation of treatment. The inner circle includes factors associated with the management of T1DM that predispose to eating disorders. Dashed arrows imply that the specific factor extends on all levels. T1DM: Type 1 diabetes mellitus; BMI: Body mass index.

Weight gain atinitiation of insulin

proposed by Goebel-Fabbri et al[38,39] suggests that the increased weight gain causes negative feelings. Fear of further weight gain can lead to insulin restriction for caloric purging or weight regulation[40].

Dietary restraintDiabetes management imposes dietary restraint. This may lead to the yearning and craving of “forbidden foods”, and result in binging, without administration of the appropriate insulin dose[41].

Hypoglycemic episodesThe delicate balance between dietary regimen, insulin and exercise that is integral to T1DM management can result in recurrent hypoglycemic episodes. Hypoglycemia is accompanied by intense hunger and by eating sweetened foods and drinks that are normally forbidden. Patients may subsequently feel guilty about consuming these foods and restrict eating, which may result in yet another hypoglycemic episode. This vicious cycle of dietary restriction, over-eating and guilt is similar to that experienced by individuals with bulimia[42].

Chronic disease and depression The risk of significant depressive symptoms in individuals with T1DM has been assessed as about double that of the general population[43]. Depression may increase the susceptibility for developing disturbed eating behavior. Indeed, among girls with T1DM, those diagnosed with depression scored higher on the Eating Disorder Examination than did those without depression; 75% and 45%, respectively[44]. Furthermore, anxiety disorders and eating disorders co-occur in the general population, and 21% of children and adolescents with T1DM were reported to screen positively for anxiety[45].

CLINICAL SIGNS OF ED AMONG INDIVIDUALS WITH T1DMThe diagnosis of subclinical or clinical eating disorders in individuals with T1DM is not easily established. Due to the frequent concealment and denial of eating disorder behaviors, a high index of suspicion is needed. In addition to the predisposing characteristics described above, a number of clinical signs should alert health care providers to the possibility of ED.

Poor glycemic control Among females with T1DM, mean HbA1c levels were shown to be significantly higher in those with disturbed eating than among those with non-disturbed eating[46]. HbA1c level of patients who confessed intentional under-dosing of insulin was 9.0% ± 1.6% compared with 7.8% ± 1.2% of compliant patien[47]. Similarly, in a nationwide population-based study, conducted in Norway, a significantly higher HbA1c was documented in patients reporting insulin restriction (9.0% ± 1.7%) than in nonrestrictors (8.3% ± 1.2%)[19] In a meta-

interactions within the family center around food and weight. The prevalence of ED was found to be higher in families in which parents tended to make negative comments about eating or weight[31]. Secondly, mothers who engaged in dieting and binge-eating themselves were more likely to have daughters with disturbed eating behavior. Maternal weight and shape concerns and impaired mother-daughter relationships significantly predicted eating disturbances in girls with T1DM, accounting for 57% of the variance[32]. Finally, family dysfunctioning has been identified as an important risk factor for eating disorders among the general population. Specifically, among girls with T1DM, less support from, poorer communication with, and less trust in their relationships with their parents, were reported among those with eating disturbances than among those without[33]. Families of girls with ED more often reported having infrequent family meals than did families without ED[34].

SECOND CIRCLE - FACTORS ASSOCIATED WITH DISEASE ONSETAge of diabetes onsetThe age of onset of T1DM that is most closely related to the subsequent development of a severe eating disorder such as AN and BN was studied in 53 women with ED compared with 49 with T1DM with T1DM who had no eating disorder - related problem[35]. It was demonstrated that for diabetes onset age between 7 to 18 years, the density of the “eating disorder” group was higher, but for the younger and older onset ages the densities were lower. Thus the development of T1DM in preadolescence or adolescence seems to place girls at risk for the subsequent development of AN or BN. Similarly, frequent insulin reduction or omission was reported in type 1 diabetic patients with later disease onset (mean age at onset between 8 and about 17 years)[36]. It was suggested that later age at diabetes onset, in particular during pubertal age with hormonal changes and gain in weight and fat mass may be associated with a greater risk for ED especially in female patients.

Weight issuesThe cycle of weight loss at disease onset and subsequent weight gain with the initiation of insulin treatment is a risk factor for the development of ED in susceptible patients[11].

THIRD CIRCLE - FACTORS ASSOCIATED WITH THE CHRONIC MANAGEMENT OF DIABETESIntensive insulin treatmentIntensive insulin treatment conveys an increased risk of weight gain[37]. A model of disordered eating and T1DM



analysis, eating problems, including both DEB and ED, were found to be associated with poorer glycemic control[10]. Using a computer model of data mining, we recently showed that adolescent females with intentional insulin omission were discriminated by HbA1c > 9.2% and by more than 20% of HbA1c measurements above the 90th percentile[48].

Recurrent episodes of diabetic ketoacidosis Intentional insulin omission has long been recognized as a cause of recurrent episodes of recurrent episodes of diabetic ketoacidosis (DKA) in adolescents with T1DM[49]. Thus, any recurrent episode of DKA in established diabetes should arouse suspicion[50].

Recurrent hypoglycemic episodes Recurrent hypoglycemic episodes may occur among individuals with T1DM and ED, mainly among those participating in binge eating and self-induced vomiting. Deliberately inducing hypoglycemia to justify eating sweets and high carbohydrate meals were described in individuals with T1DM. Eighteen percent of a cohort of males and females with T1DM, aged 10-22 years, reported intentional overdosing of insulin[47]. The most common reason (49%) stated was the desire for uncontrolled binge eating, followed by self-destructive behavior in stressful situations.

Other characteristics and signs of ED Other characteristic and clinical signs of ED are: frequently missed medical appointments, refusal to be weighed, preoccupation with appearance, a tendency to vegetarianism, and the calculating of caloric values and weighing of foods. Apparently, due to the availability of insulin omission as a weight loss strategy, females with diabetes are less likely to use other unhealthy weight control behaviors like vomiting, laxatives or diuretics,

and skipping meals or fasting, compared to their peers without diabetes[51].

DIAGNOSISSeveral questionnaires were developed specifically for adolescents with T1DM. The revised Diabetes Eating Problem Survey (DEPS-R) is a 16-item diabetes-specific self-report measure of disordered eating that was designed specifically to identify patients with T1DM who are at risk for early intervention for disordered eating behaviors[7]. The mSCOFF is a simple 5 item screening tool, with demonstrated reliability and validity, which can easily be used during a follow-up visit[52]. Using data mining methods, a clinical prediction model that provides a decision support system for the detection of intentional insulin omission for weight loss in adolescent females with T1DM was developed[48]. The model is based on identifying a pattern of HbA1c levels indicative of intentional insulin omission. After a period of apparently stable HbA1c levels, the onset of insulin omission is characterized by both high HbA1c levels and wide fluctuations between clinical visits. A typical example of the course of HbA1c levels in a patient who developed ED is depicted in Figure 2.

MORBIDITY AND MORTALITYED result in poor metabolic control and cause short and long-term complications.

Short term complicationsInsulin omission is associated with recurrent events of DKA, and disturbed eating behavior is associated with recurrent episodes of severe hypoglycemia[53]. Data from the diabetes patienten verlaufsdokumentation study, including 52215 T1DM males and females aged 8 to 30 years, revealed significantly higher rates of severe hypoglycemic episodes in adolescents with AN, BN or EDNOS, than with no ED: 12.1, 18.0, 12.9 and 5.7, respectively[54]. Moreover, rates of hypoglycemia with coma, and DKA with hospitalization were higher, and the duration of hospital stay longer, among those with eating disorders[54].

Long term complications Long-term complications have been shown to be markedly increased among individuals with both T1DM and ED[54]. Data from the DPV study revealed that both hypertension and dyslipidemia were more common in persons with T1DM and either EDNOS or BN than in those with T1DM without ED[54].

Of T1DM patients who developed serious micro-vascular complications, 21% had a probable clinical ED, 47% had a history of DEB, and 48% had a history of insulin omission. The development of two or more serious complications was associated with the presence of a probable clinical eating disorder[55].

Different ED are associated with varying prevalence


HbA

1c (

%)

Age (yr)2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

10.5

10.0

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

90

75

50

25

10

Figure 2 Typical example of the course of HbA1c levels in a patient, with intentional insulin omission for weight loss, plotted on the HbA1c percentile reference curves. HbA1c levels were around the 10th percentile from age 10 to 14 years. From age 18 the pattern changed and was characterized by sharp increases to levels above the 90th percentile, sharp decreases and multiple measurements above the 90th percentile.


rates of morbidities. For example, data from the DPV study revealed a 2.5-fold higher risk for retinopathy in T1DM patients with BN than in those without ED. Patients with EDNOS also had a higher risk but without statistical significance, whereas those with AN had no increased risk for retinopathy[54].

The duration of ED may also affect diabetes complications. The duration of severe insulin omission was the factor most closely associated with the development of retinopathy and nephropathy in T1DM females[56].

As seen in Table 2, higher rates of retinopathy[46] and nephropathy[57] were documented in young TIDM patients with ED than in those without ED.

MortalitySeveral studies documented increased relative risk of death among patients with ED. Mortality rates per 1000 person years were reported as 2.2 in girls with T1DM, 7.3 in girls with ED and 34.6 in girls with both T1DM and an ED[58]. During an 11-year period, self-reported insulin restriction at baseline increased the relative risk of death by 3.2 times in women, mean age 45 years, mean diabetes duration 28 years; those with ED were younger when they died (aged 44 vs 58 years, P < 0.01)[57]. In a 12-year follow-up of a cohort of 14 women with T1DM and ED (12 with AN), 5 died (36%)[59]. The median age of the cohort was 37 years (range 25-46), with a median duration of diabetes of 26 years (14-33). The age of death of the 5 patients was 30 years (25-42).

PREVENTIONPromoting the understanding and awareness among healthcare providers of factors involved in the deve-lopment of ED in young vulnerable individuals with T1DM may help prevent these disorders[60]. These factors include the weight gain with the initiation of insulin treatment[11], the dietary restraint necessitated by diabetes management, and recurrent hypoglycemic episodes. Diabetes treatment should therefore afford flexibility where possible, and non-depriving approaches to eating[61]. As current clinical practice includes a focus on measuring body weight, health care professionals should be sensitive to preoccupation with body weight. Interventions aimed at increasing self-esteem and body acceptance, as well as family-based interventions aimed

to improve family management of obesity and diabetes, may also help to diminish the risk of developing ED in individuals with diabetes[62].

Girls aged between 10 and 12 with a diagnosis of T1DM for at least 1 year, attended two group sessions, each of 4-h duration[63]. The content of the sessions targeted perfectionism, media literacy, self-esteem. The program was found to have impact on self-efficacy relating to diabetes management. As lower T1DM regimen adherence is associated with lower self-efficacy in adolescents, it was concluded that a brief interactive program had a favorable impact on protective factors for disordered eating.

Among college-age women at highest risk for an eating disorder an internet-based intervention, has successfully reduced weight/shape concerns and prevented eating disorders[64]. Specifically guided discussion group resulted in reduction in weight/shape concern. We did not find similar reports among adolescent girls with T1DM, however it is possible that online interventions may be successfully used to reduce eating disorder risk factors and preventing eating disorders in this group.

TREATMENTTreatment of ED in patients with T1DM is challenging, especially since the clinical improvement achieved with insulin treatment is associated with weight gain. Compliance to the therapeutic regimen may therefore be poor.

Treatment should mirror the three circle model presented above, and address the factors predisposing to the development of ED (Figure 3).

Treating the first circle - premorbid variablesIncreased body weight and a tendency to gain weight should be addressed by prudent dietary management: Since a tendency to gain weight was found to be associated with ED[22], particular attention should be given to the constellation of excess weight and preoccupation with food.

Low self-esteem, body dissatisfaction and personality variables should be addressed by individual psychological treatment or group therapy: The concurrence of eating behavior disorder and mood disorder in patients with T1DM supports the importance of treating depression as a means of


Table 2 Prevalence rates of retinopathy and nephropathy among type 1 diabetes mellitus patients with eating disorders

Complication Average T1DM duration (yr)

T1DM patients with severe ED (%)

T1DM patients with moderate ED (%)

T1DM patients without ED (%)

Retinopathy[46] 11 ± 4 86 42 24Nephropathy[46] 11 ± 4 43 20 18Nephropathy[57] 28 25 10Foot problems[57] 28 25 12

ED: Eating disorders; T1DM: Type 1 diabetes mellitus.


treating ED. If depression and anxiety are suspected, a psychiatric consultation is needed. Psychiatrists have a distinct role in the diabetes care of children under their care, specifically in working through issues associated with the burden of the disease[65]. The impact of anti-depressant and anti-anxiety medications on recovery from eating disorders in adolescents with T1DM has not been studied systematically.

The effect of psychoeducation on adolescents with T1DM and ED is controversial. A psychoeducation approach with six weekly group sessions was associated with reductions in dieting, in body dissatisfaction and with preoccupation with thinness and eating; the reduction in disturbed eating behaviour was maintained for at least 6 mo[66]. In another study, a six week intervention was as effective as a wait list control group[67].

Group cognitive-behavioral therapy intervention has been shown to improve glycemic control, well-being and diabetes related stress among poorly controlled adult T1DM patients[68], and among patients with T1DM and depression[69]; however, it was not studied in patients with insulin omission.

As family dynamics may influence the development of ED[34], family education is an important component of the treatment. However, we could not find data on the impact of family intervention as a treatment modality for adolescents with T1DM and ED.

Treating the second circle - factors associated with disease onsetCurrent T1DM management involves carbohydrate counting, which may lead to excessive preoccupation with food and diets. The cycle of weight loss at disease

onset, and subsequent weight gain with the initiation of insulin treatment, is a risk factor for the development of ED in susceptible patients[11]. Dietary treatment should focus on healthy choices and low calorie alternatives with a high satiety index.

Treating the third circle - factors associated with the chronic management of diabetesIntensive insulin treatment, dietary restraint, and hypoglycemic episodes should be addressed by the diabetes management team: As good metabolic control is associated with weight gain, changes in target blood glucose levels should be gradual. Setting higher than standard target blood glucose ranges (preprandial 120 to 150 mg/dL and postprandial < 200 mg/dL) may yield more benefit in the long run. In contrast, achieving excellent control may result in marked weight gain. Moreover, since low glucose target levels are associated with an increased risk of recurrent hypoglycemic episodes, which may result in additional increased calorie intake, setting higher target levels may be a better initial objective. In girls who were skipping insulin dosing at almost every meal, starting with adequate insulin injection at a single meal is sometimes helpful as a first step. Caregivers should be aware that patients often switch from one disturbed behavior to another, ie from insulin omission to restrictive eating or binge eating; thus, a prescribed diet should be flexible.

Treatment with an insulin pump was postulated as a means of enabling better physiological insulin delivery to adolescents with T1DM than achieved with multiple daily injections. Patients with insulin pumps require less insulin and thus gain less weight. Furthermore, the occurrence of hypoglycemic episodes is decreased, and


Individual psychological treatment

Family treatment

Group treatment

Dietary guidance for healthy eating

Realistic blood glucose targets

Flexible dietary regimen

Prevention of hypoglycemia

Treating depression and anxiety

ⅢⅡ

Ⅰ

Hospitalization

Figure 3 Three level models describing treatment modalities of eating disorders in adolescents with type 1 diabetes mellitus. The outer circle addresses treatment for premorbid variables. The middle circle delineates. The inner circle includes factors associated with the management of T1DM that predispose to eating disorders. T1DM: Type 1 diabetes mellitus.


thus the need to consume extra calories. A recently published multicenter study demonstrated a decrease in disturbed eating behaviors, as assessed by the DEPS-R, in youth with T1DM, 6 mo after pump initiation[70]. Moreover, among adolescent girls with T1DM and ED, the mean HbA1c level was significantly lower in those who were treated by insulin pumps than in those who were treated with multiple daily injections (9.07% ± 1.33% vs 10.40% ± 2.01%; P = 0.04)[71].

Failure of outpatient treatment, the presence of a severe psychopathological state and poor glycemiccontrol are key elements in the decision for hospi-talization. Information is limited regarding the long-term outcomes of hospitalized adolescents with ED. Significantly lower levels of HbA1c and lower psychological test scores related to eating disorder psychopathology, depressiveness and anxiety were reported for 9 patients who were hospitalized, compared to 10 who were not[72]. At 3-year follow-up, 78% of the hospitalized patients in that study no longer fulfilled any criteria for clinical or subclinical ED.

Thus, treatment of ED in individuals with T1DM involves a complex interplay of psychosocial, dietary and medical aspects and requires a multidisciplinary team. It is important to realize that adolescents with T1DM and ED tend to have lower motivation to change their eating habits[73]. Furthermore, they are characterized by giving up easily when confronted with frustration and with low levels of accomplishment. These personality characteristics may explain the high dropout rate from therapy (79%) among patients with T1DM and ED[73].

CONCLUSIONDeliberate insulin omission as a weight loss strategy is recognized as either an inappropriate compensatory feature of bulimia nervosa or as a purging disorder, a component of OSFED. About one-third of individuals with T1DM intentionally omit insulin. Diagnosis of eating disorders in individuals with T1DM is difficult, since eating disorder behaviors are often well hidden. Weight loss that is related to deteriorated glycemic control and recurrent DKA should raise suspicion. Early diagnosis is essential, as the combination of eating disorders and diabetes is associated with increased morbidity and mortality.

ACKNOWLEDGMENTSWe thank Ms. Cindy Cohen for her editorial assistance.

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P- Reviewer: Codoner-Franch P, Marchesini G S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ



Tiffany M Potts, Jacqueline L Nguyen, Kanika Ghai, Kathy Li, Lawrence Perlmuter

Tiffany M Potts, Jacqueline L Nguyen, Kathy Li, Lawrence Perlmuter, Department of Psychology, Rosalind Franklin University of Medicine and Science - College of Health Professions, North Chicago, IL 60064, United StatesKanika Ghai, Pediatric Endocrinology, Advocate Children’s Hospital, Park Ridge, IL 60068, United StatesAuthor contributions: Potts TM and Perlmuter L designed the research project; Ghai K provided the patient population and the testing space; Potts TM performed the research; Potts TM, Nguyen JL, Li K, and Perlmuter L analyzed the data; Potts TM, Nguyen JL, Li K and Perlmuter L wrote the paper. Ethics approval: The study was reviewed and approved by the Rosalind Franklin University of Medicine and Science Institutional Review Board and the Advocate Health Care Institutional Review Board.Clinical trial registration: Not available.Informed consent: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.Conflict-of-interest: There are no conflicts of interest related to this study.Data sharing: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: Lawrence Perlmuter, PhD, Professor, Department of Psychology, Rosalind Franklin University of Medicine and Science - College of Health Professions, 3333 Green Bay Road, North Chicago, IL 60064, United States. [email protected] Telephone: +1-847-5788754Fax: +1-847-5788765Received: August 6, 2014Peer-review started: August 6, 2014First decision: August 28, 2014Revised: October 7, 2014Accepted: February 4, 2015 Article in press: February 9, 2015

Published online: April 15, 2015

AbstractAIM: To investigate whether perceptions of task difficulty on neuropsychological tests predicted academic achievement after controlling for glucose levels and depression.

METHODS: Participants were type 1 diabetic adolescents, with a mean age = 12.5 years (23 females and 16 males), seen at a northwest suburban Chicago hospital. The sample population was free of co-morbid clinical health conditions. Subjects completed a three-part neuropsychological battery including the Digit Symbol Task, Trail Making Test, and Controlled Oral Word Association test. Following each task, individuals rated task difficulty and then completed a depression inventory. Performance on these three tests is reflective of neuropsychological status in relation to glucose control. Blood glucose levels were measured immediately prior to and after completing the neuropsychological battery using a glucose meter. HbA1c levels were obtained from medical records. Academic performance was based on self-reported grades in Math, Science, and English. Data was analyzed using multiple regression models to evaluate the associations between academic performance, perception of task difficulty, and glucose control.

RESULTS: Perceptions of difficulty on a neuro-psychological battery significantly predicted academic performance after accounting for glucose control and depression. Perceptions of difficulty on the neuro-psychological tests were inversely correlated with academic performance (r = -0.48), while acute (blood glucose) and long-term glucose levels increased along with perceptions of task difficulty (r = 0.47). Additionally, higher depression scores were associated





Perception of difficulty and glucose control: Effects on academic performance in youth with type Ⅰ diabetes

Prospective Study

ORIGINAL ARTICLE

with poorer academic performance (r = -0.43). With the first regression analysis, perception of difficulty on the neuropsychological tasks contributed to 8% of the variance in academic performance after controlling for peripheral blood glucose and depression. In the second regression analysis, perception of difficulty accounted for 11% of the variance after accounting for academic performance and depression. The final regression analysis indicated that perception of difficulty increased with peripheral blood glucose, contributing to 22% of the variance. Most importantly, after controlling for perceptions of task difficulty, academic performance no longer predicted glucose levels. Finally, subjects who found the cognitive battery difficult were likely to have poor academic grades.

CONCLUSION: Perceptions of difficulty on neurological tests exhibited a significant association with academic achievement, indicating that deficits in this skill may lead to academic disadvantage in diabetic patients.

Key words: Type 1 diabetes; Adolescents; Perception of difficulty; Academic performance; Glucose control


Core tip: The objective of the current study was to investigate the association between perceptions of difficulty and academic performance in adolescents with type 1 diabetes. Perceptions of difficulty are reflected in executing cognitive activities as well as in the task of glucose regulation. Glucose control needs to be understood, not so much from a biological perspective but as an effortful process that is also reflected in academic challenges. The problem of anergia observed generally in patients with diabetes seems to broadly affect a variety of tasks and challenges. Thus, the novelty of the study is in showing that the regulation of glucose in diabetic patients is another example of the broad challenges confronted in the life of patients with diabetes.

Potts TM, Nguyen JL, Ghai K, Li K, Perlmuter L. Perception of difficulty and glucose control: Effects on academic performance in youth with type Ⅰ diabetes. World J Diabetes 2015; 6(3): 527-533 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/527.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.527

INTRODUCTIONType 1 diabetes is one of the most common chronic diseases of childhood. In the United States, type 1 diabetes affects approximately 1 in 300 children and is usually diagnosed between the ages of 5 and 11 years[1,2]. The course of this autoimmune version of diabetes is characterized by ongoing beta cell destruction

and the need for exogenous insulin[3]. Since the central nervous system (CNS) is dependent on a supply of glucose for normal neural functioning, reduced glucose concentrations may result in temporary and/or enduring impairments in CNS function[4]. Dysregulation of the CNS may have negative effects on neuropsychological functioning, such as impaired memory, attention, motor skills, and executive functioning[5].

Adolescents with type 1 diabetes face many challenges that can impact metabolic control along with physiological and psychological complications. The need for adherence to metabolic control and compliance with regimen is effortful. Beyond the demands for medical compliance, adolescents are often burdened by depression[6] as well as anergia[7] and thus may be at academic disadvantage[8].

Along with academic adversity, individuals with type 1 diabetes may be challenged by self-evaluative factors such as the perception of control, which affects behavioral and social functioning[9,10]. The literature has described three prominent types of control: perceived control, perceived confidence (self efficacy), and perceived difficulty[11]. Perceived difficulty, a subjective experience, can be defined as the extent to which individuals believe that performance on a task or behavior would range from easy to difficult[11]. If adolescents believe they are substandard at a task because of its difficulty, they are likely to devalue the activity and decrease effort expenditure[12].

Perceived difficulty has also been shown to relate to an individual’s beliefs regarding how much effort is needed to succeed at a task and whether success is possible[13]. The effort expended in performing a task is predicted to increase proportionally with the level of perceived task difficulty[14]. Indeed, effort has been associated with academic performance. Studies[15] have found that self-reported grades are positively correlated with effort. Moreover, adherence to regimen[16] and metabolic control[17] also reflect the expenditure of effort. Therefore, poor glucose control may be positively correlated with perceptions of task difficulty and inversely associated with academic performance[18]. Indeed, self-control and willpower are both functions of available glucose levels[19]. Thus, glucose control and effort are two highly related processes that need to be differentiated.

Perceptions of difficulty rely on executive functioning skills, such as problem solving and decision-making. These skills require an individual to make a decision regarding the degree of difficulty associated with the task and whether he or she will be successful in carrying out the task. Since problem solving and decision-making skills contribute to academic performance, these executive functioning skills may be impaired in diabetic individuals[20]. A meta-analytic finding indicated that children with type 1 diabetes performed slightly, yet significantly, lower on attention/executive functioning tasks than non-diabetic individuals[21]. This finding, in turn, was associated with poor glucose control and

Potts TM et al . Perception of difficulty, glucose, and academics


hypoglycemic episodes as well[21]. To open the question of effort, three brief but well

validated neuropsychological tests were administered[22] and participants rated the difficulty of each test. Secondly, academic performance and depression levels were evaluated and analyzed. Since long-term glucose control (HbA1c) is associated with cognition[18], HbA1c levels from within the past three months were collected from medical records. Acute glucose levels immediately prior to and following neuropsychological testing were also collected and evaluated. These procedures enabled the distinction between the assessment of effort-associated processes supporting academic and neuropsychological processes vs those putatively supporting glucose control.

MATERIALS AND METHODSParticipantsThis study was designed as a cross-sectional, pro-spective study. Participants were type 1 diabetic patients who attended a diabetes clinic in a northwest suburban Chicago hospital. Adolescents were approached to participate in the study during their regularly scheduled doctor’s appointment. Adolescents were excluded from participation if they had co-morbid clinical health conditions, such as asthma, anemia, vascular disease,kidney disease, chronic tiredness, depression, psychiatric history, or obesity. Parents of the patients filled out consent forms that outlined the procedure and risks of participation. The subjects also signed an assent form. The study measures were carried out in a quiet, separate room for each participant individually. Pre-doctoral psychology students administered the assessments. Participants were provided modest monetary compensation for participation in the study. There were a total of 39 participants, consisting of 23 females and 16 males. The population was comprised of 71.8% Caucasian, 20.5% Hispanic, 5.1% Asian, and 2.6% African American. The mean age of the participants was 12.5 years (SD = 2.73). This study was approved by the Institutional Review Board at Rosalind Franklin University as well as the hospital’s review board. There is no conflict of interest related to this study.

MaterialsGlucose control: Blood glucose measurements were performed immediately prior to administering and again after completing the neuropsychological battery with glucose meters (One Touch, Blood Glucose Monitoring System). This procedure was used to obtain an average peripheral blood glucose measurement. A single HbA1c level was recorded from the medical records retrieved from the previous three months, prior to testing. Neuropsychological battery: The neuropsychological battery included the Digit Symbol task (Wechsler Intelligence Scale for Children-Forth Edition), Trail Making test Parts A and B, and Controlled Oral Word

Association (COWA) in that order. This three-part neuropsychological battery was selected because the tests are brief, well researched, psychometrically sound, and are commonly used[23]. Performance on these tests is reflective of neuropsychological status in diabetic patients in relation to glucose control[22].

The Digit Symbol task of the Wechsler Intelligence Scale for Children-Fourth Edition (DS; WISC-Ⅳ)[24] was used to assess psychomotor performance, attention, memory, and perceptual organization. It consists of Coding and Incidental Learning. In Coding, the individual was presented with several rows of a series of numbers (1 through 9). Each number was paired with a corresponding unique symbol. The individual was asked to fill in as many symbols as possible in boxes directly under the corresponding number and was allowed 120 s to complete this task. In Incidental Learning, individuals were asked to carry out two tasks: to recall as many digit-symbol pairs as possible (Pairing) and to recall as many symbols as possible without the corresponding numbers (Free Recall). Internal consistency of the Digit Symbol Coding task is 0.85.

The Trail Making Test[23] was used to assess attention, visual-motor tracking, and speed of information processing. It consists of two parts: in Part A, individuals were asked to draw a line that accurately connected the correct sequence of numbers as quickly as possible. In Part B, individuals were asked to draw a line that accurately connects the correct sequence of numbers and letters in an alternating pattern as quickly as possible. Reliability for Part A is 0.78 and for Part B 0.67.

The COWA test[25] assessed verbal functions. Individuals were asked to generate as many words as possible that began with the letter S in 60 s and then the letter F in 60 s as the examiner recorded these. Test-retest reliability is 0.88 and inter-scorer reliability was almost perfect.

Rating of task difficulty: After completing three tasks (the Digit Symbol task of the Wechsler Intelligence Scale for Children-Fourth Edition, Trail Making test Part A and B, and Controlled Oral Word Association test), participants were asked to rate the level of perceived difficulty of each task on a scale from 1-5, where 1 = very easy and 5 = very difficult. These evaluations were summed and averaged to develop a task difficulty measure.

Academic performance: Participants self reported academic grades in several courses during the current school year: Math, Science, and English. For the purposes of tabulation, grades were converted into a scale score from 1-5, where 1 = F, 2 = D, 3 = C, 4 = B, and 5 = A. Grades were then averaged for each participant.

Depression: To avoid possible reactive effects[26], participants were lastly administered the Children’sDepression Inventory (CDI)[27] which is a 27 item self report inventory used to measure depressive symp-



that higher levels of difficulty perceived on the neuropsychological test was inversely associated with academic performance after taking into account peripheral blood glucose and depression. The result of this analysis indicated that the perception of difficulty on the neuropsychological tasks contributed to 8% of the variance in academic performance after controlling for peripheral blood glucose and depression. Thus, stronger ratings of laboratory task difficulty were associated with poorer academic performance.

After controlling for academic performance and depression, a regression analysis was conducted with peripheral blood glucose as the dependent variable to test if perceptions of task difficulty were associated with glucose levels (Table 4). The predictors in the regression analysis were entered in the following order: (1) academic performance; (2) CDI; and (3) perceptions of difficulty on the neurological tests with peripheral blood glucose as the dependent variable. Results indicated that the first predictor (higher academic performance) was inversely related to peripheral blood glucose, contributing to 14% of the variance. The second predictor (CDI) showed that the levels of depression had insignificant effects on peripheral blood glucose. The third predictor (perception of difficulty) showed that the higher level of difficulty perceived on the test accounted for the higher peripheral blood glucose level. Perception of difficulty accounted for 11% of the variance after accounting for academic performance and depression. This indicated that higher perceptions of task difficulty were associated with higher blood glucose levels while depression seemed to play no significant role in this model. Individuals, who perceived the neuropsychological battery as challenging, may also experience difficulty controlling glucose levels.

The purpose of the final analysis (Table 5) was to determine whether academic performance would predict glucose levels after controlling for perceptions of task difficulty. The order of predictors was: (1) perception of difficulty; (2) academic performance; and (3) CDI with peripheral glucose levels as the dependent variable. The first predictor (perception of difficulty) showed that perception of difficulty increased with peripheral blood glucose, contributing to 22% of the variance. Most importantly, after controlling for perceptions of task difficulty, academic performance no longer predicted glucose levels. Additionally, it was found that the level of depression was not associated with peripheral glucose levels. Results from Table 5 indicated that peripheral blood glucose was positively correlated with the perception of task difficulty.

DISCUSSIONThe general purpose underlying this research was to critically evaluate the various explanations for the problems that diabetic adolescents experience in the performance of cognitive tasks. In addition to elevations

tomatology in youth aged 7-17. Respondents indicated which of three options best represented their mood in the context of everyday life: absence of symptom = 0; mild symptom = 1; and definite symptom = 2. The CDI provides a total score on five domains of depression. The reliability coefficient for the total inventory was 0.86[27].

Data was collected and entered into Statistic Package for Social Science (SPSS) Version 13. Zero order correlation tables were examined to assess the degree of relationship between the variables. P-values of less than 0.05 were considered statistically significant. Multiple regression analyses were used to assess outcome variables.

RESULTSBasic description of the sample population is presented in Table 1. The primary results (Table 2) indicated that perceptions of difficulty on the neuropsychological tests were inversely correlated with academic per-formance (r = -0.48), while acute (blood glucose) and long-term glucose levels (HbA1c) increased along with perceptions of task difficulty (r = 0.47). Finally, higher depression scores were associated with poorer academic performance (r = -0.43).

To evaluate the relationship of difficulty on neuro-psychological tests and academic performance after controlling for peripheral glucose levels and depression, a multiple regression analysis was conducted (Table 3). The predictors in the regression analysis were entered in the following order: (1) peripheral blood glucose; (2) CDI; and (3) perception of difficulty with academic performance as the dependent variable. Results from the first predictor indicated that as peripheral blood glucose increased, academic performance decreased. The second predictor (CDI) indicated that increasing depression was associated with poorer academic performance after controlling for peripheral blood glucose. Lastly, the third predictor (perception of difficulty) showed


Table 1 Characteristics of participants

Mean SD

Disease duration (mo) 48.05 41.08BMI (kg/m2) 21.18 3.85Peripheral blood glucose (mm/dL) 190.99 89.27HbA1c (%) 8.39 1.63Digit symbol coding 55.38 14.7Digit symbol paring 11.44 5.16Digit symbol free recall 7.38 1.41Trails A (s) 39.67 19.02Trails B (s) 92.74 64.84COWA 20.26 6.62CDI 7.23 5.54Perception of difficulty1 10.15 2.95Grades2 4.1 0.77

1Range 1-20; 2Range low to high; 1-5. BMI: Body mass index; HbA1c: Hemoglobin A1c; COWA: Controlled Oral Word Association; CDI: Child depression inventory.


in blood glucose, there are two alternatives, namely anergia[7] and depression[6]. Since these processes each exacerbate or contribute to the diminution of effort, it is possible that the actual mediator of compromised cognition can be found in reduced effort. Thus, the primary purpose of this introductory study was to assess the contribution of effort[14] to both metabolic control and reduced cognition in these patients.

Through a series of multiple regression analyses, the results showed that after controlling for levels of effort, the role of metabolic control was compromised with respect to cognition. A key assumption in this process is that metabolic control is per se an effortful process. If effort is viewed as the primary process, metabolic control remains as an adjunctive player in cognition.

To examine this model, we turn to the results of the multiple regression analyses presented earlier. That is, adolescents who perceived the neuropsychological battery as more difficult were more likely to have lower grades in Math, Science, and English. Thus, perceiving the battery as difficult may indicate a broad weakness in executive functioning skills, such as problem solving and decision-making. Furthermore, increased levels of depression also negatively contributed to academic performance. As a result, academic performance was

poorer in adolescents with elevated peripheral blood glucose, which in itself may be a reflection of the difficulty or effort necessary for exercising metabolic control.

This study provided insight into how academic performance may be associated with self-ratings of difficulty on neuropsychological tasks after controlling for blood glucose levels and depression. Further, the study showed how peripheral blood glucose levels can be derived from perceptions of difficulty based on neuropsychological testing after controlling for academic performance and depression. Each of these observations will be discussed serially.

Previous findings[28,29] have suggested a relationship between glucose control and academic performance. Greater difficulty in maintaining strong glucose control was associated with poorer academic performance. The list of predictors of academic performance included a neuropsychological battery that assessed psychomotor performance, attention, memory, speed of information processing, and verbal functioning.

Participants who perceived the neuropsychological battery as difficult were also more likely to exhibit poor grades (Table 4). However, this finding may not be surprising, since performance on the neuropsychological battery and academic performance share some conceptual space. Thus, a basic question in this study is whether the commonly observed relationship between glucose levels and cognitive functioning represents a basic physiological relationship between glucose and neuropsychological functioning? Alternatively, is the index of glucose control merely a reflection of the basic challenge between compliance with regimen, an effort-demanding task, and the challenge intrinsic to the cognitive task?

After controlling for appraisals of task difficulty, blood glucose levels seem to no longer account for cognitive


Table 2 Correlations among the variables

Academic performance HbA1c Peripheral blood glucose CDI Perceptions of difficulty

Academic performance1 - -0.26 -0.37a -0.43b -0.48b

HbA1c - 0.42b 0.03 0.34a

Peripheral blood glucose2 - 0.22 0.47b

CDI3 - 0.27

1Low scores indicate poor grades; 2Low score-lower glucose levels; 3Higher scores more depression. aP < 0.05; bP < 0.01. HbA1c: Hemoglobin A1c; CDI: Child depression inventory.

Table 3 Linear multiple regression analysis for variables predicting academic performance1

β R2 change

Peripheral blood glucose2 -0.37 0.14a

Child depression inventory3 -0.36 0.12a

Perceptions of difficulty on neurological tests -0.33 0.08a

1Low scores indicate poor grades; 2Low score-lower glucose levels; 3Higher score more depression. aP < 0.05. β: Standardized coefficient beta.

β R2 change

Academic performance2 -0.37 0.14b

Child depression inventory3 0.08 0.01Perceptions of difficulty on neurological test 0.38 0.11b

Table 4 Linear multiple regression analysis for variables predicting peripheral blood glucose1

1Low score-lower glucose levels; 2Low scores indicate poor grades; 3Higher scores more depression. bP < 0.01. β: Standardized coefficient beta.

Table 5 Linear multiple regression analysis for variables predicting peripheral blood glucose1

β R2 change

Perceptions of difficulty on neurological test 0.47 0.22b

Academic performance2 -0.18 0.03Child depression inventory3 0.05 0.00

1Low score-lower glucose levels; 2Low scores indicate poor grades; 3Higher scores more depression. bP < 0.01. β: Standardized coefficient beta.


performance once task difficulty is factored into the equation (Table 5). Finally, by eliminating the influence of perceptions of difficulty in maintaining blood glucose control and complying with medical regimen, peripheral blood glucose and academic performance were no longer significantly related.

This may be the first study to illustrate how perceptions of task difficulty on neuropsychological tests are associated with poor academic performance in diabetic patients. While several studies have reported that individuals with diabetes exhibit relatively poor academic performance, the present study identifies how perceptual evaluative factors diminish academic performance.

Thus, careful disease management is needed to prevent or effectively treat potentially life-threatening complications such as hypoglycemia, diabetic ketoa-cidosis, and hyperglycemia as well as effort.

Limitations to the study include a relatively small sample size as well as the use of a single item to assess perceptions of task difficulty. However, this method of assessing perceptions of difficulty with one item was also utilized in a previous study[11]. In their study, Rogers and colleagues assessed self-efficacy, perceived control and perceived difficulty with the use of a single item Likert scale question for each construct[11]. Therefore, single item indicators may be regarded as similarly robust as multiple item indicators[30,31]. Lastly, academic performance was assessed by participant self-report. Systematic biases may influence the validity of self-reported grades, but self-reported grades generally predict outcomes as effectively as actual or objectively culled grades[32].

In conclusion, this study demonstrated that per-ceptions of task difficulty can predict academic difficulty in a sample of diabetic adolescents. Task difficulty was also associated with the relationship between glucose levels and academic performance. Hence, perceptions of difficulty can be viewed as a generalized executive function. When individuals perceive a task as more difficult, this may promote academic disadvantage along with increased challenges in controlling glucose. Overall, such information may be useful in training and counseling students in enhancing academic success and performance as well as in assistance with disease control.

COMMENTSBackgroundType 1 diabetes is one of the most common chronic diseases of childhood. In the United States, type 1 diabetes affects approximately 1 in 300 children and is usually diagnosed between the ages of 5 and 11 years. Research frontiersAdolescents with type 1 diabetes face many challenges that can impact metabolic control along with physiological and psychological complications. Cognitive functioning is an important aspect of life and can be impaired in individuals with diabetes. Academic performance in diabetic adolescents may also be compromised due to fluctuations in glucose.Innovations and breakthroughsAlong with academic adversity, individuals with type 1 diabetes may be

challenged by self-evaluative factors such as the perception of control, which affects behavioral and social functioning. Perceived difficulty has also been shown to relate to an individual’s beliefs regarding how much effort is needed to succeed at a task and whether success is possible. Perceptions of difficulty rely on executive functioning skills, such as problem solving and decision-making. Since problem solving and decision-making skills contribute to academic performance, these executive functioning skills may be impaired in diabetic individuals. The current study sought to examine the role of effort on glucose and academic performance. ApplicationsThis may be the first study to illustrate how perceptions of task difficulty on neuropsychological tests are associated with poor academic performance in diabetic patients. While several studies have reported that individuals with diabetes exhibit relatively poor academic performance, the present study identifies how perceptual evaluative factors diminish academic performance.TerminologyDiabetes mellitus is a major health problem in the United States affecting approximately 29.1 million people. There are two major classifications of diabetes: insulin dependent diabetes (type 1) and non-insulin dependent diabetes (type 2). Diabetes is a group of metabolic diseases that either cause defects in insulin action, insulin secretion, or a combination of both, resulting in hyperglycemia. Perceived difficulty, a subjective experience, can be defined as the extent to which individuals believe that performance on a task or behavior would range from easy to difficult. Peer-reviewThe study addresses a relevant scientific question and is well-conducted. The results are significant indicating that deficits in perception of difficulty may lead to academic disadvantage in diabetic patients. The manuscript is interesting and may provide important information for the reader of the journal.

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30 Herman AO, Fairchild DG, Hefner JE. Single-Question Screening Can Help Identify Teens with Substance Misuse. New England Journal of Medicine Physician’s First Watch, 2014. Cited 2014-08-04. Available from: URL: http: //www.jwatch.org/fw109105/2014/07/29/single-question-screening-can-help-identify-teens-with

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P- Reviewer: Akbulut S, Cerwenka HR, Das UN, Hassan M, Hortobagyi T

S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ



diabetes. Only peer-reviewed articles in English were included published in the last 15 years. It was selected from 1999 to 2014. Glycemic control was measured with HbA1c. Studies with an intervention lasting at least 12 wk were included if the HbA1c was measured before and after the intervention.

RESULTS: A total of nine articles were found, and they were published between the years of 2002-2011. The sample size was 401 diabetic youths (166 males and 235 females) with an age range of 10-19 years except one study, in which the age range was 13-30 years. Study participants were from Australia, Tunisia, Lithuania, Taiwan, Turkey, Brazilia, Belgium, Egypt and France. Four studies were aerobic-based, four were combined aerobic and anaerobic programs, and one compared aerobic exercise to anaerobic one. Available studies had insufficient evidence that any type of exercise or combined training would clearly improve the glycemic control in type 1 diabetic youth. Only three (two aerobic-based and one combined) studies could provide a significant positive change in glycemic control.

CONCLUSION: The regular physical exercise has several other valuable physiological and health benefits that justify the inclusion of exercise in pediatric diabetes treatment and care.

Key words: Type 1 diabetes mellitus; Glycemic control; Exercise; Aerobic; Anaerobic


Core tip: Diabetic patients should be aware that exercise interferes with the glucose homeostasis. Anaerobic exercise can increase glycemia, whereas the aerobic exercise may cause a decrease during the exercise and post-exercise. By evaluating the long-term effect of exercise on glycemic control in type 1 diabetic youths

Andrea Lukács, László Barkai

Andrea Lukács, László Barkai, Faculty of Health Care, University of Miskolc, 3515 Miskolc-Egyetemváros, HungaryLászló Barkai, Velkey László Center for Child Health, 3501 Miskolc, HungaryAuthor contributions: Both authors contributed to this manuscript.Conflict-of-interest: No conflict of interest stated by any authors.Data sharing: This manuscript does not describe basic or clinical research, therefore data sharing statement is not relevant.Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/Correspondence to: László Barkai, MD, PhD, DSc, Faculty of Health Care, University of Miskolc, 3515 Miskolc-Egyetemváros, Hungary. [email protected]: +36-46-565111Fax: +36-46-366961Received: July 29, 2014 Peer-review started: July 30, 2014 First decision: October 14, 2014Revised: January 2, 2015 Accepted: January 15, 2015Article in press: January 19, 2015Published online: April 15, 2015

AbstractAIM: To evaluate the long-term effect of aerobic and/or anaerobic exercise on glycemic control in youths with type 1 diabetes.

METHODS: Literature review was performed in spring and summer 2014 using PubMed/MEDLINE, Google Scholar, Scopus, and ScienceDirect with the following terms: aerobic, anaerobic, high-intensity, resistance, exercise/training, combined with glycemic/metabolic control, glycated haemoglobin A1c (HbA1c) and type 1

SYSTEMATIC REVIEWS


Effect of aerobic and anaerobic exercises on glycemic control in type 1 diabetic youths




according to the major metabolic pathway involved in energy utilization (aerobic or anaerobic), we found insufficient evidence in the latest literature that any type of exercise or combined aerobic and anaerobic training would clearly improve the glycemic control. The regular physical exercise has several other valuable benefits that justify the inclusion of exercise in pediatric diabetes treatment and care.

Lukács A, Barkai L. Effect of aerobic and anaerobic exercises on glycemic control in type 1 diabetic youths. World J Diabetes 2015; 6(3): 534-542 Available from: URL: http://www.wjgnet.com/1948-9358/full/v6/i3/534.htm DOI: http://dx.doi.org/10.4239/wjd.v6.i3.534

INTRODUCTIONPhysiological, social and emotional benefits of regular exercise and physically active lifestyle are well documented mostly in a healthy population[1-4], but they are also important for diabetic patients regardless of the type of diabetes[5-8]. For type 1, successful diabetes management is based on individualized insulin therapy, adjusted diet and regular exercise[9].

Exercise used as prevention and therapy in diabetes is not a new concept in the literature. Even the ancient Ayurvedic physician, Susruta Shamita (born around 600 B.C.), noted the reduction in the sweetness of urine from diabetic patients with excecise, and included moderate exercise within his treatment regimens[10]. In 1919, Allen proved that exercise reduced blood glucose level and improved acutely the tolerance to a carbohydrate load[11]. After the discovery of insulin in 1921[12], Lawrence demonstrated that exercise has an effect on insulin requirements and glucose uptake[13]. Joslin believed in “troika” (group of three) in the treatment of diabetes, symbolizing insulin, diet and exercise correlation[14]. Regular exercise helps to improve overall health and fitness, and reduces risk factors for vascular complications. Diabetic youths with regular exercise have improved blood lipid profile, and increased insulin sensitivity, primarily in the skeletal muscles, which leads to a reduced need for insulin[15-17]. The American Diabetes Association recommends all levels of physical exercise including leisure activities, recreational sport and competitive sport for youths with type 1 diabetes mellitus (T1DM) if they are in good blood glucose control and have no long-term complications[18]. Until the sympathetic nervous and endocrine systems control the blood glucose level at the physiological levels during and after physical exercise in healthy subjects[19], the regulation is external in diabetic patients considering many internal influences. Youths with T1DM should be aware that exercise interferes with the glucose homeostasis, although there are individual differences in blood glucose response due to type, duration and intensity of the exercise, the pre-exercise level of counterregulatory hormones, and blood glucose

concentrations[20,21]. Several studies examined the role of physical activity and exercise in the treatment of T1DM and considered it an important component. The evidence for improvement in glycemic control is equivocal. Some studies suggest a positive effect[22-27], whereas others fail to show this effect[28-34]. Scientific questions remain concerning what is the exact effect of different types of exercise on glycemic control in youths with T1DM.

In this systematic literature review, we evaluated the latest studies examining the long-term effect of aerobic and/or anaerobic exercise on glycemic control in youths with T1DM. We also made distinguishing between concepts of physical activity, physical fitness, and aerobic-anaerobic exercise. Finally, we considered clinical applications and future directions.

ConceptualisationPhysical activity: Physical activity encompasses body movement produced by skeletal muscles which requires energy consumption[35]. Physical activity can range from sports to any other lifestyle activities including school and out-of-school activities, weekend activities where youths play, are active and expend energy. There is evidence that behavioural patterns of physical activity in childhood are maintained throughout adulthood[36,37]. This term is often used interchangeably with regular exercise and physical fitness in studies, although they are different concepts[38].

Exercise: Exercise has been defined as any form of body movement that results in an increase in metabolic demand with the intention of developing one or more components of physical fitness. It is generally planned, structured and systematic[35]. Regular exercise improves physical fitness. Exercise is characterised by five com-ponents: type [dynamic (isotonic) or static (isometric), and both of them can be performed aerobically or anaerobically on the basis of energy utilization][39], intensity (degree of effort that individual puts into exercise), duration (the length of each training session), repetition (number of times individual performs a complete movement of a given exercise) and frequency (number of exercise sessions per week)[40].

Fitness: Fitness refers to the “possession of adequate levels of strength, endurance, and mobility to provide for successful participation in occupational effort, recreational pursuits, familial obligation, and that is consistent with a functional phenotyp expression of the human genotype”[41]. Physical fitness determines cardiovascular, respiratory, and musculosceletal systems in order to perform physically demanding activities such as exercise, sport, or work. Fit youth has normal body fat, good immune system and, in general, is in a physiologic state of well-being.

Aerobic-anaerobic exercise: Whether an activity is aerobic or anaerobic depends primarily on its intensity and duration. Most physical exercises are characterised

Lukács A et al . Glycemic control and type of exercise


by both static and dynamic contractions as well as aerobic and anaerobic metabolism. Thus, exercise is classified by their dominant features.

Aerobic exercise includes any type of exercise, typically those performed at moderate levels of intensity for extended periods of time that maintains an increased heart rate. Activities such as cycling, swimming, jogging, rowing, cross-country skiing, and aerobic dancing require oxygen to produce ATP. Regular aerobic exercise improves maximal oxygen consumption and overall endurance performances. Anaerobic exercise is used to promote strength, power, and speed. Generally, anaerobic exercise has a short duration and high intensity activity. Unlike aerobic exercise, it does not depend on exogenous oxygen. Activities such as heavy weightlifting, all types of sprint (running, cycling, or swimming) or any hard exercises require anaerobic metabolism. During exercise various forms of energy sources are utilized markedly depending on the intensity and duration of exercise, but the activity is classified typically based on the predominantly used system. The anaerobic energy system is used for resistance training and increasing speed[42]. During high intensity (anaerobic) exercise almost the entire metabolic fuel source is glucose, whereas during low intensity (aerobic) exercise fat utilization increases and glucose oxidation decrease[43]. Both types of exercise increase the mechanical efficiency of the heart (cardiac adaptation), changes in morphology and functionality of the left ventricle[44].

Measuring the intensity of exercise using maximal heart rate: Maximal heart rate (HRmax) is one of the most commonly used values in clinical settings and physiology. HR increases nearly linearly with the increase in the intensity of exercise; and this is the simplest physiological response to measure. As maximal exercise intensity is approached, HR begins to plateau even as the exercise workload continues to increase. This HRmax is a reliable value and it remains constant for a longer period[45]. To estimate HRmax for children, formula 208 - 0.7 × age in years is applied[46,47]. There are several more and less accurate types of estimation existing for determining the exercise intensity[48]. Aerobic training zone is around between 60%-70% of the HRmax. Between 70%-85% of HRmax, the youths are in the mixed zone, and above 85% of HRmax the anaerobic metabolism will come to the fore.

Effects of different types of exercise on blood glucose level in type 1 diabetes patientsDifferent types of exercise produce different effects on blood glucose level. If the carbohydrate intake and the insulin dosage are not in line with the exercise the patient does, it will result in metabolic disturbances[49]. The most common problems are the evaluation of pre-exercise level of blood glucose, evaluation of the intensity and duration of the expected exercise, and to

take into consideration the time of day when the patient exercises, because of the body’s different physiological insulin need[49]. Exercise functions like insulin, so the balance between the insulin therapy and diet could be facilitated if the daily schedule for exercise and the exercise parameters are consistent[50], although this goal is almost impossible to obtain in the real life for youths. Findings from exercise training studies support the concept that moderate intensity aerobic workload increases the risk of hypoglycemia during the exercise and several hours after the exercise. High-intensity training with anaerobic utilization may increase the blood glucose level due to the release of adrenaline and noradrenalin in the blood, which then stimulates the liver to release glucose faster than normal. The exercise-induced rise in glucose level is followed by hypoglycemia after hours of finishing the exercise as counterregulatory hormone levels decrease[21,51-53]. Both aerobic and anaerobic exercise training regimens improve glucose uptake and insulin sensitivity[21,54]. School-aged children engage a combination of moderate- and high-intensity sessions in their everyday sport activities. Their exercise is often spontaneous and unplanned. Thus, a different plan is needed for each type of exercise to optimize blood glucose level, but problems with the management are well recognised. Patients differ in tolerance to exercise and insulin requirements and it is impossible to give precise guidelines suitable for everyone with T1DM, therefore experiences with regular blood glucose testing are inevitable. Continuous glucose monitoring may assist the active diabetic participants to follow the changes in blood glucose level and give information about the individual response to the exercise[21,54]. There are several guidelines to discuss safe sport participation in children and adolescents with T1DM[5,55-59]. These guidelines can be adjusted to own measurements and awareness of the usual responses to a particular type of exercise.

MATERIALS AND METHODSStudy eligibilityA systematic review was performed in spring and summer 2014 using PubMed/Medline, Google Scholar, Scopus, and ScienceDirect with the following terms: aerobic, anaerobic, high-intensity, resistance, exercise/training, combined with glycemic/metabolic control and type 1 diabetes. Only peer-reviewed articles in English were included published in the last 15 years with at least five subjects per group. Studies were selected from 1999 to 2014 that measured the long-term effect of aerobic and/or anaerobic exercise on glycemic control in type 1 diabetic children, adolescents and young adults without diabetes complications. Glycemic control was measured with glycated haemoglobin A1c (HbA1c). As the HbA1c provides an average of blood glucose control over a 12-wk period, we included studies with an intervention lasting at least 12 wk if the HbA1c was



glycemic control, exercise three times a week (one hour) for three months had no significant impact on glycemic control, but exercise four times a week (one hour) for 6 mo resulted in a significant improvement[60]. Neither guided or self-directed home-based aerobic exercise program nor supervised Pilates exercise had a significant effect on glycosylated haemoglobin after a 12-wk session[62,63].

Study investigating anaerobic exercise: We found only one study that measured the efficacy of resistance (anaerobic) and aerobic exercise in parallel. Both groups were trained three times a week (40 min) for 12 wk under supervision by a physical trainer and an endocrinologist. Interestingly, the HbA1c increased in the aerobic group significantly after 12 wk, whereas a non-significant reduction was observed in the resistance training group[64].

Studies investigating combined exercise program: Four randomised controlled trials examined the effect of combined (aerobic and anaerobic) exercise on glycemic control[30,65-67]. Supervised combined aerobic and resis-tance training twice a week (70 min) for 20 wk resulted in no significant change in glycemic control[65]. In a 12-wk supervised training followed by a 12-wk unsupervised training, HbA1c level remained unchanged regardless of the baseline HbA1c level[30]. There were two trials that explored the effect of supervised combined exercise on glycemic control for a longer duration (6 mo)[66,67]. The Egyptian study found significant improvements in HbA1c level in both training groups exercising three times a week and once a week. However, patients exercising three times a week produced significantly greater improvements[66]. In the Heyman’s study, the participants completed a two-hour supervised training and a one-hour unsupervised training a week including aerobic and strength exercises. The author could not prove a significant effect on glycated hemoglobin after a 6-mo session[67].

DISCUSSIONThe primary aim of diabetes treatment and care is to achieve as stable glycemic control as possible in order to prevent or delay the long-term complications[69]. Regular exercise is recommended for diabetic youths for several psychological and health benefits[7,70]. It is evidence-based that regular exercise has a preventive and curative role in type 2 diabetes, but its physiological role in type 1 diabetes is not fully explored. Exercise physiology in diabetes is described thoroughly by Robertson et al[7] and Riddell et al[53,59]. Another issue that remains unresolved is related to the type of exercise: aerobic or anaerobic workout is most beneficial for glycemic control. We evaluated the studies from the previous 15 years that investigated the long-term effect of exercise on glycemic control in youths with T1DM according

measured before and after the intervention. The flow diagram of study selection is shown in Figure 1.

Statistical analysisThis manuscript does not describe basic or clinical research using biostatistics for raw data, therefore biostatistics statement is not relevant.

RESULTSA total of nine articles was evaluated from nine different countries including Australia[30], Tunisia[60], Lithuania[61], Taiwan[62], Turkey[63], Brazilia[64], Belgium[65], Egypt[66] and France[67], and they were published between the years of 2002-2011. The list of articles is presented in Table 1. Across included studies the total sample size was 401 (166 males and 235 females) with an age range of 10-19 years except one study, in which the age range was 13-30 years[64]. Four studies were aerobic-based[60-63], four were combined aerobic and anaerobic programs[30,65-67], and one compared aerobic exercise to anaerobic one[64]. The Turkish study[63] conducted a Pilates program that is considered low-impact aerobic workout[68]. All studies had diabetic control group except one[61].

Effects of different types of exercise on glycemic control in youths with T1DMStudies investigating aerobic exercise: Studies measuring the effect of aerobic exercise on glycemic control presented variable results[60-63]. Only one study recognised a significant improvement in HbA1c after 12-wk exercise (girls swimming twice a week for 45 min a week)[61]. In a study investigating the impact of the frequency of the supervised aerobic training on


Articles identified from electronic search n = 685

Excluded at first screening (title) n = 456

Abstracts screened n = 229

Addition articles from reference lists n = 2

Excluded at second screening: Based on abstract n = 128

Duplicates n = 56

Total articles included n = 9 with total sample size of 401

Excluded at final screening n = 38

Total articles evaluated n = 47

Figure 1 Flow diagram illustrating selection of the included studies.


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T2DM also enunciated that either form of exercise appears to have comparable effects on glycemic control. A meta-analysis by Tonoli et al[74] with type 1 diabetic participants explored a tendency for improvement in glycaemic control due to aerobic or combined training, but they could not confirm this statistically. Individual studies on aerobic training had no significant evidence, but “in total” demonstrated a reduction in HbA1c, although they carefully interpret their results because of insufficient data on these topics[74]. Neither Kennedy’s meta-analysis in children and adults[71] nor Kavookjian’s systematic review in the adult population[75] revealed evidence for statistically significant glycemic benefits of exercise. Kennedy et al[71] suggest that HbA1c may not be a sensitive indicator of glycaemic control, and that improvement in glycaemic variability may not be reflected in this measure. Tight metabolic control is very important for diabetic individuals, and the regular physical exercise is part of the diabetes management. Available studies provide insufficient evidence that any type of exercise or combined training would clearly improve the glycemic control in type 1 diabetic young patients. Long-term exercise-induced glycemic benefits are based on the continuous effect of each successive bout of exercise. Glycemic variability can be detected by continuous glucose monitoring that could help individuals to learn their own response to different type of physical exercise; and it might be a more appropriate marker to explore the exercise-induced beneficial effect than HbA1c[71]. Youths with T1DM are recommended to be engaged in a sport they like, and do it regularly as much as possible at the same period of the day. Having the appropriate exercise physiology knowledge, they can accumulate own experiences regarding exercise in addition to insulin requirement and dietary program that may result in stable glucose levels. The insulin-diet-exercise adjustment must be personalized and discussed with the patient’s endocrinologist. The regular exercise is associated with numerous health benefits and chronically ill youths enjoy the same benefits as the healthy counterparts[1,70].

There are miscellaneous results regarding the long-term effect of various forms of exercise on glycemic control. Meta-analyses also suffered from the insufficient randomised controlled trials with greater sample sizes to examine the short- and long-term effect of glycemic control on different exercise modalities in youths with T1DM. Patients and health professionals may want more information they currently received, but there are concerns that the results would be overestimated. Different forms of exercise generate different skills in youths. They need a comprehensive form of exercise to help their healthy many-sided physical development. Diabetic patients should be aware that glycemic control is influenced by multiple factors (initial blood glucose level, insulin absorption, time of the day), and shows individual responses to exercise. It is advisable that young patients choose some kind of sports or physical exercise they like and be engaged with it for a longer

time. Clinical treatment and management should be adjusted to the exercise form and the diabetic patients should acquire how to change nutrition and insulin dose according to their daily physical exercise. Patients who can monitor themselves intensively around periods of activity can learn how to keep glucose levels in an acceptable range.

Although current studies have insufficient evidence of the beneficial effect of any type of exercise on glycemic control, any type of regular physical exercise has several other valuable physiological and health benefits that justify the inclusion of exercise in pediatric diabetes treatment and care.

ACKNOWLEDGMENTSThis research was partially carried out with the con-tribution of the Workgroup for Health Sciences, Center of Excellence of Applied Materials Science and Nano-Technology at the University of Miskolc.

COMMENTSBackgroundRegular exercise is recommended for diabetic youth for several psychological and health benefits, but its physiological role in type 1 diabetes is not fully explored. So far, there are only few studies evaluating the long-term effect of different types of exercise on glycemic control.Research frontiersIn this systematic literature review, the authors evaluated the latest studies examining the long-term effect of aerobic and/or anaerobic exercise on glycemic control in youths with type 1 diabetes. They also made distinguishing between concepts of physical activity, physical fitness, and aerobic-anaerobic exercise. Innovations and breakthroughsMost studies found no significant improvement in glycemic control regarding the aerobic and combined exercise intervention. The only one study evaluating anaerobic exercise observed a non-significant reduction in glycemic control. It might be supposed that longer duration (at least 6 mo) and more frequent (more than twice a week) exercise has a positive effect on glycemic control, but it is not proved yet. Applications Although current studies have insufficient evidence of the beneficial effect of any type of exercise on glycemic control, any type of regular physical exercise has several other valuable physiological and health benefits that justify the inclusion of exercise in pediatric diabetes treatment and care.TerminologyAerobic exercise includes any type of exercise, typically those performed at moderate levels of intensity for extended periods of time that maintains an increased heart rate. Anaerobic exercise is used to promote strength, power, and speed. Generally, it has a short duration and high intensity activity. Unlike aerobic exercise, it does not depend on exogenous oxygen. Peer-reviewThe manuscript covers an important topic in type 1 diabetes management. The contribution that the manuscript provides is an overview of the dearth of scientific evidence to support exercise intervention in this population.

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P- Reviewer: Faulkner MS, Sanlioglu AD S- Editor: Ji FF L- Editor: Wang TQ E- Editor: Liu SQ




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World Journal of Diabetes - BPG Management System

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