Preventive Effects of Chitosan Coacervate Whey Protein on Body Composition and Immunometabolic...

Research ArticlePreventive Effects of Chitosan Coacervate Whey Protein on BodyComposition and Immunometabolic Aspect in Obese Mice

Gabriel Inaacutecio de Morais Honorato de Souza12 Aline Boveto Santamarina1

Aline Alves de Santana1 Faacutebio Santos Lira3 Rachel de Laquila4 Mayara Franzoi Moreno1

Eliane Beraldi Ribeiro1 Claudia Maria da Penha Oller do Nascimento1 Bruno Rodrigues2

Elisa Esposito5 and Lila Missae Oyama1

1 Departamento de Fisiologia Disciplina de Fisiologia daNutricao Universidade Federal de Sao Paulo 04023-060 Sao Paulo SP Brazil2 Departamento de Ciencias Biologicas Laboratorio de Movimento Humano da Universidade Sao Judas Tadeu03166-000 Sao Paulo SP Brazil

3 Departamento de Educacao Fısica da Universidade Estadual Paulista Faculdade de Ciencias e Tecnologia19060-900 Presidente Prudente SP Brazil

4Departamento de Nutricao das Faculdades Integradas Coracao de Jesus-FAINC 09020-240 Santo Andre SP Brazil5 Instituto de Ciencias e Tecnologia da Universidade Federal de Sao Paulo 04023-060 Sao Jose dos Campos SP Brazil

Correspondence should be addressed to Gabriel Inacio de Morais Honorato de Souza gabrinaciogmailcomand Elisa Esposito isaespositogmailcom

Received 23 May 2014 Revised 30 July 2014 Accepted 31 July 2014 Published 17 September 2014

Academic Editor Jose Cesar Rosa Neto

Copyright copy 2014 Gabriel Inacio de Morais Honorato de Souza et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Functional foods containing bioactive compounds of whey may play an important role in prevention and treatment of obesityThe aim of this study was to investigate the prospects of the biotechnological process of coacervation of whey proteins (CWP) inchitosan and test its antiobesogenic potentialMethods CWP (100mgsdotkgsdotday) was administered in mice with diet-induced obesityfor 8 weeks The animals were divided into four groups control normocaloric diet gavage with water (C) or coacervate (C-CWP)and high fat diet gavage with water (HF) or coacervate (HF-CWP) ResultsHF-CWP reduced weight gain and serum lipid fractionsand displayed reduced adiposity and insulin Adiponectin was significantly higher in HF-CWP group when compared to the HFThe level of LPS in HF-W group was significantly higher when compared to HF-CWP The IL-10 showed an inverse correlationbetween the levels of insulin and glucose in the mesenteric adipose tissue in the HF-CWP group CWP promoted an increase inboth phosphorylation AMPK and the amount of ATGL in the mesenteric adipose tissue in HF-CWP group Conclusion CWP wasable to modulate effects possibly due to its high biological value of proteins We observed a protective effect against obesity andimproved the inflammatory milieu of white adipose tissue

1 Introduction

Over the past decades the incidence of obesity in thepopulation has increased severely and it has become a publichealth challenge Its etiology is multifactorial encompass-ing environmental dietary physical inactivity and geneticfactors Obesity is a complex disease associated with ahigh-calorie diet which contributes to the developmentof several other chronic noncommunicable diseases [1]Obesity is also associated with increased plasma endotoxin

(lipopolysaccharide-LPS) saturated fatty acids [2 3] andproinflammatory cytokines [3] all intricately involved inthe development of comorbidities such as diabetes mellitushypertension dyslipidemia and metabolic syndrome

The fat tissue is not merely an energy storage organ asit plays crucial endocrine and immune roles White adiposetissue (WAT) is an endocrine organ secreting pro- andanti-inflammatory adipokines such as tumor necrosis factoralpha (TNF-120572) and interleukin-6 (IL-6) which are impor-tant inflammatory markers that stimulate the production of

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 281097 13 pageshttpdxdoiorg1011552014281097

2 Mediators of Inflammation

several proteins and proinflammatory cytokines in differentcell types via nuclear factor 120581B activation (NF-120581B) [4] Inaddition endotoxin and saturated fatty acids act in the samepathway of NF-120581B activation Many studies have shown thatLPS (endotoxin) can activate these proteins in adipocytesthereby increasing the gene expression of proinflammatoryadipokines [5ndash7]

The main role of adipose lipolytic enzymes is to provideother tissues with FAs in case of energy demand Triglyceridestored in the lipid droplet is first hydrolyzed by the adiposetriglyceride lipase enzyme (ATGL) also known as desnutrinreleasing a diacylglycerol moiety and FA which requires anabhydrolase domain containing 5 (ABHD-5) promoter to beactivated After hydrolysis by ATGL diacylglycerols are thenhydrolyzed sequentially by hormone-sensitive lipase (HSL)and monoglyceride lipase (MGL) producing nonesterifiedfatty acids (NEFAs) and glycerol [8] Different lipases gainaccess to the lipid droplet when the proteins coating thevesicle (perilipins) are phosphorylated Perilipin A normallyprevents lipolysis of triglyceride by surrounding the lipiddroplet thus preventing the access of lipases

Whey protein (WP) has been found to be an excellentprophylactic against obesity because of the high biologicalvalue mediated by bioactive peptides These act as antimi-crobial agents antihypertensive and regulators of immunefunction reducing body fat as well as a variety of relatedbeneficial mechanisms for human health They also haveadditional functions for example they have appetite sup-pressant effects [9] stimulate muscle protein synthesis andregulate of body energy homeostasis [10] There is plentyof evidence indicating the potential of the WP in anti-inflammatory and antioxidant effects of exercise [9 11ndash13]

Chitosan complex coacervation with WP is composed ofby-products from the processing of shrimp crab (chitosan)and cheese adding an environmental benefit to the productas these by-products may be reused and not disposed of inlandfill sites or released into rivers by producers [14] In viewof the above together with the development of fractionationtechnique and whey protein preservation employing themethod may contribute to the recovery of this valuablenutrient and increase the expression of the functional prop-erties [15] Complex coacervation is defined as a colloidalseparation forming two liquid phases This process is essen-tially driven by attractive forces with opposite charges ofa biopolymer This phenomenon occurs by the formationof a system of balance between colloids and the dilutedsupernatant [16 17] Thus the purpose of the present studywas to investigate the prospect of a biotechnological processof complexation and separation of cheese whey proteins inchitosan and test their antiobesogenic potential through themodulation of inflammatory markers and lipolytic pathwaypresent in obesity

2 Methods

21 Coacervation and Characterization In this study weused sweet cheese whey (SW 1108 bag 25 kg) with 15 fatmarketed byCompanyAlibra-PRDissolving 10 g of thewhey

powder in 100mL of distilled water Chitosan was used forthe coacervation medium molar mass with 75ndash85 degreeof deacetylation and viscosity of 200ndash800 cps (Sigma-Aldrich44887-7) A concentration of 075mgmL of Chitosan wasused Chitosan was dissolved in citric acid (208mmolL) andafter this step added to the cheese whey in a proportion of1 1 under stirring at room temperature for 1 h The pH of thesolution of chitosan and WP was adjusted to 6 with NaOH(250mmolL) and solubilized at room temperature (plusmn25∘C)with stirring Solids coagulated with chitosan known ascoacervate (CWP) were collected by centrifugation (1300 g)

In order to obtain on average 30 of the protein coac-ervate 3 L cheese whey was used Thus samples of CWPwere obtained for their chemical analysis of total lipids totalprotein and lactose Finally for measurements of samplesof mineral micronutrients Ca K Mg and P of cheesewhey CWP and Chitosan 100 120583g was subjected to an opticalemission spectrometer for inductively coupled plasma (ICPOES Perkin Elmer Optima 3000DV Norwalk CT USA) Todetermine the existing protein fractions in CWP the elec-trophoretic profile with reducing buffer containing 625mMTris-HCl 20 glycerol 2 sodium dodecyl sulfate(SDS)(10) 5 120573-mercaptoethanol and bromophenol blue at pH68 was performed

22 Animals and Treatment This study was approved by theResearch Ethics Committee of the Universidade FederaldeSao Paulo Escola Paulista de Medicina (UNIFESPEPM)as the search protocol number 047310 Experimental pro-cedures are in accordance with Principles of LaboratoryAnimal Care formulated by the National Institutes of Health(National Institutes of Health Publication number 96-23revised 1996)

Forty-nine male Swiss mice twelve-week-old fromCEDEME (Centro de Desenvolvimento de ModelosExperimentais da Universidade Federal de Sao Paulo) werehoused five in a cage in a standard experimental animallaboratory and kept under controlled conditions of light(12 h light-dark cycle with lights on at 6 am) and temperature(24 plusmn 1∘C) All mice received water and food ad libitumThe animals were divided as follows control diet plus tapwater (C-W) control diet plus coacervate (C-CWP) highfat diet plus coacervate (HF-CWP) and high fat diet plustap water (HF-W) All diets were prepared according to therecommendations of the American Institute of Nutrition [18](Table 1) Coacervate (CWP) containing 100mgsdotkgsdotday wasgiven by gavage The body weight gain was recorded twice aweek

23 Composition and Aspect of the Coacervate Figure 1(a)shows the protein profile of the coacervate by the presenceof the major proteins of larger fractions namely alpha-lactalbumin (120572-La-14 kDa) beta-lactoglobulin (120573-Lgmdash18 kDa monomer and 34 kDa dimer form) bovine serumalbumin (BSAmdash66 kD) and lactoferrin (Lacfmdash86 kDa) TheCWP has a flocculation aspect (Figure 1(b)) consisting ofproteins and micronutrients such as calcium potassiummagnesium phosphorus and sodium (Table 2)

Mediators of Inflammation 3

Table 1 Composition of the control diet and diet enriched with saturated fatty acids according to AIN-93 Coacervate was resuspended in300 120583L of water

Components () Control diet (C) High fat diet (HF)Corn starch 7207 4087Casein 140 140Soybean oil 40 40Lard mdash 312Cellulose 50 50Vitamin mix 10 10Mineral mix 35 35L-cystine 018 018Choline bitartrate 025 025Butyl hydroquinone gkg 0008 0008Energy kcalkg 38028 53628Treatment by gavage

Coacervate (CWP) (C-CWP) 100mgsdotkgsdotday (HF-CWP) 100mgsdotkgsdotdayWater (W) (C-W) 300 120583Lsdotday (HF-W) 300 120583Lsdotday

Fatty acids ()Saturated (SFA) 1712 3413Monounsaturated (MUFA) 2563 3914Polyunsaturated (PUFA) 5725 2667PUFA n3 432 637PUFA n6 5265 1998

ST CWP

Lacfr

BSA75

50

37

25

20

15 120572-La

120573-Lg monomer

120573-Lg dimer

(a) (b)

Figure 1 (a) Electrophoretic profile coacervate using 075mgml of chitosan ST standard of different molecular weights 120572-La alpha-lactalbumin (14 kDa) 120573-Lg beta-lactoglobulin monomer (18 kDa) and dimer (34 kDa) BSA bovine albumin (66 kDa) and Lacfr lactoferrin(86 kDa) (b) Aspect of CWP proteins in Chitosan freeze-dried

24 Fatty Acids Composition of Diets For total lipid extrac-tion diet samples were homogenized in chloroform andmethanol 2 1 (vv) mixed and incubated at room tem-perature for 5min Then additional volumes of 125mLchloroform and 125mL deionized H

2O were added and

finally following being vigorously homogenized for 3minsamples were centrifuged at 1000 rpm for 5min at roomtemperature The chloroform layer was dried under N

2 and

the total extract was converted into methyl esters and wasanalyzed in gas chromatography (GC) coupled with a flameionizer detector (FID) (Varian GC 3900) and fatty acidprofile was determined by calculating the retention time

using a pattern of fatty acids with known retention time(Supelco 37 Components) The addition was initiated at atemperature of 170∘C maintained for 1 minute and then aramp of 25∘Cmin to a final temperature of 240∘C whichwas maintained for 5 minutes The injector and detectorwere maintained at 250∘C and 260∘C respectively We used acolumn CP wax 52CB with a thickness of 025mm internaldiameter of 025 120583m and length of 30mwith hydrogen as thecarrier gas at a linear velocity of 22 cm sminus1

25 Oral Glucose Tolerance Test (OGTT) After 12 hoursof fasting blood was collected from the tail vein to assess


Table 2 Partial composition of micronutrients by ICP and chemical macronutrients of CWP WP and Chitosan

Nutritional compositiong100 g

CWP WP ChitosanProteins 30 35 mdashLactose 17 40 mdashLipids 04 15 mdashCa 047 055 007K 037 169 0010Mg 006 011 lt0001P 046 053 0003Na 081 132 026

basal glucose concentrationThen a glucose (Merck) solution(14 gkg) was administrated by gavage Blood samples werecollected after 15 30 45 60 and 120 minutes to measureglucose concentration using a glucose analyzer (AccuCheckRoche)

26 Experimental Procedures At the end of the experimentalperiod animals were fasted for 12 h overnight prior tobeing sacrificed by decapitation Trunk blood was collectedand immediately centrifuged (1125 g15min at 4∘C) Serumwas separated and stored at minus80∘C for later biochemicaland hormonal determination The adipose tissue depotsretroperitoneal (RET) mesenteric (MES) and epididymal(EPI) were dissected weighed immediately frozen in liquidnitrogen and stored at minus80∘C

27 Biochemical and Hormonal Serum Analyses Serum con-centrations of glucose total cholesterol triglycerides andHDL-c were measured by an enzymatic colorimetric methodusing commercial kits (Labtest Brazil) Concentrationsof insulin and adiponectin were measured using specificenzyme-linked immunosorbent assay (ELISA) kits (Miliporeand RampD Systems) LPS was determined using a commercialkit (Lonza)

28 Mesenteric Adipose Tissue TNF-120572 IL-6 and IL-10 ProteinLevel Determined by ELISA Following euthanasia mesen-teric adipose tissuewas removed homogenized into a specifictotal protein extraction buffer [1 Triton X-100 100mmTris-HCl (pH 74) 100mm sodium pyrophosphate 100mmsodium fluoride 10mm EDTA 10mm sodium orthovana-date 20mmphenylmethylsulfonyl fluoride and 01mg apro-tininmL] and centrifuged at 12000 g for 30min at 4∘CThe supernatant was saved and the protein concentrationwas determined using the BCA assay (Bio-Rad HerculesCalifornia) with bovine serum albumin (BSA) as a referenceQuantitative assessment of TNF-120572 IL-6 and IL-10 proteinswas carried out by ELISA (DuoSet ELISA RampD SystemsMinneapolis MN) following the recommendations of themanufacturer All samples were run as duplicates and themean value was reported

29 Protein Analysis by Western Blotting After euthanasiathemesenteric adipose tissuewas dissected andhomogenizedin 10mL of solubilization buffer at 4∘C [1 Triton X-100100mm Tris-HCl (pH 74) 100mm sodium pyrophosphate100mm sodium fluoride 10mm EDTA 10mm sodiumorthovanadate 20mm phenylmethylsulfonyl fluoride and01mg aprotininmL] Insoluble material was removed bycentrifugation for 30min at 9000 g in a 70 Ti rotor (BeckmanFullerton CA USA) at 4∘C The protein concentration ofthe supernatants was determined using the BCA assay (Bio-Rad Hercules CA USA) Proteins were denatured by boiling(5min) in a Laemmli sample buffer containing 100mMDTTand were run on 10 sodium dodecyl sulfate polyacrylamidegel electrophoresis in a Bio-Rad miniature slab gel apparatus

The proteins were electrotransferred from gels to nitro-cellulose membranes for sim130 h4 gels at 15 V (constant) ina Bio-Rad semidry transfer apparatus Nonspecific proteinbinding to the nitrocellulose was reduced by preincubationfor 2 h at 22∘C in blocking buffer (1 BSA 10mM Tris150mM NaCl and 002 Tween 20) The nitrocellulosemembranes were incubated overnight at 4∘C with antibodiesagainst hormone-sensitive lipase (HSL) adipose triglyceridelipase (ATGL) abhydrolase domain containing protein 5(ABHD-5) perilipin A phospho 51015840 AMP-activated proteinkinase (p-AMPK120572 1 e 2 - Thr 172) and alpha-tubulinobtained from Santa Cruz Biotechnology (Santa Cruz CAUSA) diluted 1 1000 with blocking buffer supplemented with1 BSA and then washed for 30min in blocking bufferwithout BSA The blots were subsequently incubated withperoxidase-conjugated secondary antibody for 1 h at 22∘CTo evaluate protein loading the membranes were strippedand reblotted with an anti-alpha-tubulin antibody as appro-priate Specific bands were detected by chemiluminescenceand visualizationcapture was performed by UVITEC gel-documentation system Band intensities were quantified byoptical densitometry of developed autoradiographs (ScionImage software Scion Corporation Frederick MD USA)

210 Statistical Analyses All results are presented as meanplusmn standard error of the mean (SEM) Statistical significanceswere assessed using two-way analysis of variance (ANOVA)followed by Tukeyrsquos post hoc analysis to identify significant


40424446485052545658

1 3 6 8

g

Weeks

C-WC-CWP

HF-CWPHF-W

b d

b

Figure 2 Evolution of the average gain in body mass (g) of micefor eight weeks of treatment with high fat diet (HF) or controlnormocaloric (C) associated with gavage of coacervate (CWP) orwater (W) Data submitted with an average plusmn EPM (b) C-W versusHF-W and (d) HF-CWP versus HF-W (119875 lt 005)

differences among the groups Pearsonrsquos correlation was usedto assess the associations between the analyzed variablesDifferences were considered significant for (119875 le 005) withthe StatsDirect software

3 Results

31 Body and Tissue Weights After six weeks of treatmentthe hyperlipidic diet promoted an increase in the bodyweight when compared to the control (C-W versus HF-W)On the other hand HF-CWP showed a lower body weightwhen compared to HF-W (Figure 2) The hyperlipid dietincreased the relative mass of epididymal and mesentericdepot and adiposity (Σ of epididymal retroperitoneal andmesenteric relative weight) when compared to control group(C-W versus HF-W) while the association with coacervatereduced these parameters (HF-W versus HF-CWP) Theretroperitoneal depot was increased in theHF-Wgroupwhencompared to C-W one (Table 3)

32 Levels of Serum Lipids Insulin Adiponectin Lipopolysac-charides and OGTT The hyperlipid diet increased thetriacylglycerol (TAG) and VLDL when compared to con-trol group (C-W versus HF-W) while the association withcoacervate reduced these parameters (HF-W versus HF-CWP) Insulin level and HOMA index were increased inthe animals fed with hyperlipidic diet (C-W versus HF-W) When associated with coacervate the hyperlipid dietpromoted an increase in the adiponectin and a decrease inLPS concentrations (HF-W versus HF-CWP) (Table 4)

The oral glucose tolerance test showed that the hyperli-pidic diet promoted an increase at 15minutes when comparedto control (HF-W versus C-W) The AUC (area underthe curve) analysis increased HF-W compared with C-W(Figure 3)

33 Concentration of IL-6 IL-10 and TNF-Alpha in theMesenteric Adipose Tissue There was a significant decrease

100

100002000030000

0

120140160180200220240260280300

B 15 30 60 90 120

(mg

dL)

(Min)

AUC

b

b

C-WC-CWP

HF-CWPHF-W

C-W

C-CW

P

HF-

CWP

HF-

W

Figure 3 OGTT and AUC (area under the curve) after eight weeksof treatment with high fat diet (HF) or normocaloric control (C)associated with gavage of coacervate (CWP) or water (W) Glycemiain time zero (basal-B) 15 30 60 90 and 120 minutes after gavage of02 g100 g body weight of glucose Data submitted with an averageplusmn EPM (b) C-W versus HF-W (119875 lt 005)

in IL-10 concentrations in the animal fed with high fat dietwhen compared to animals fed the control diet (HF-W versusC-W)The concentration of IL-6 in C-CWP group was lowerwhen compared to C-W group The IL-10TNF-120572 ratio inmesenteric tissue showed no significant differences (Table 5)

34 Expression of Proteins Involved in Lipolysis PathwayFigures 4(a) 4(b) 4(c) 4(d) and 4(e) show the data of proteinexpression of HSL ATGL Perilipin A and ABHD-5 andAMPK activity respectively in mesenteric adipose tissue

HSL protein expressions were reduced in C-CWP andHF-Wwhen compared to the C-Wgroup (Figure 4(a))Therewas an increase in ATGL in HF-CWP group when comparedto HF-W group (Figure 4(b)) Perilipin A was significantlyhigher in the HF-W group when compared to the C-Wand HF-CWP groups (Figure 4(c)) There was a significantdecrease in the protein expression of ABDH-5 in C-CWPwhen compared to C-W and HF-CWP groups (Figure 4(d))The phosphorylation of AMPK (Figure 4(e)) was higher inHF-CWP compared to HF-W group

35 Correlations A positive correlation between AUC andTAG (119903 = 086 119875 lt 005) was found in the HF-Wgroup (Figure 5(a)) Figure 5(b) shows a positive correlationbetween insulin levels and STA inHF-W(119903 = 096 119875 = 0006)group Figures 5(c) and 5(d) show an inverse correlationbetween insulin (119903 = minus085 119875 = 002) (Figure 5(c)) andglucose (119903 = minus088 119875 = 001) levels with IL-10 in themesenteric adipose tissue in HF-CWP group (Figure 5(d))

4 Discussion

Numerous procedures for isolation and recovery of WPhave been investigated and reported [19] The functional


C-W C-CWP HF-CWP HF-W0

50

100

150

HSL

(UA

)

AB

(a)

C-W C-CWP HF-CWP HF-W

D

0

50

100

150

200

250

ATG

L (U

A)

(b)


50

100

150 B D

Peril

ipin

A (U

A)

(c)


50

100

150

A

C

ABH

D-5

(UA

)

(d)


50

100

150

D

pAM

PK (U

A)

HSL120572-Tubulin

ATGL120572-Tubulin

120572-Tubulin

120572-Tubulin

120572-Tubulin

Perilipin A

ABHD-5

pAMPK

(e)

Figure 4 Protein expression of HSL (a) ATGL (b) perilipin A (c) ABHD-5 (d) and pAMPK (e) in the mesenteric adipose tissue The dataare expressed in arbitrary units (AU) Data submitted with an average plusmn EPM (A) C-W versus C-CWP (B) C-W versus HF-W (C) C-CWPversus HF-CWP and (D) HF-CWP versus HF-W (119875 lt 005)

Table 3 Total mass (g) delta on the body mass gain (g100 g body weight) and relative mass of tissue (RMT) (g100 g body weight) of micetreated with high fat diet (HF) or control normocaloric (C) associated with gavage of coacervate (CWP) or water (W)

C-W(119899 = 10)

C-CWP(119899 = 10)

HF-CWP(119899 = 19)

HF-W(119899 = 19)

RTM (g100 g)EPI

404 plusmn 017 352 plusmn 021 406 plusmn 028 493 plusmn 044

bd

MES180 plusmn 024 129 plusmn 021 172 plusmn 01 26 plusmn 036

bd

RET112 plusmn 010 082 plusmn 004 135 plusmn 012 148 plusmn 018

b

Σ adipose tissue697 plusmn 038 564 plusmn 041 714 plusmn 046 902 plusmn 055

bd

EPI epididymal MES mesenteric RET retroperitoneal Data submitted with an average plusmn EPM bC-W versus HF-W and dHF-CWP versus HF-W (119875 lt 005)


16000 18000 20000

C-W

22000 24000

100

120

140

160

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

r = 015 P = 076

17000 18000 19000 20000 21000 22000 23000

r = minus002 P = 09795

100

105

110

115

120

125

C-CWP

AUCAUC

AUCAUC

16000 18000 20000 22000 24000 26000

90

100

110

120

130

r = 042 P = 040

HF-CWP

18000 20000 22000 24000 26000 28000 30000

r = 086 P = 005

HF-W

140

150

160

170

180

(a)

00 05 10 15

5

6

7

8

9

STA

(g100

g)

4

5

6

7

8

STA

(g100

g)

8

9

10

11

12

STA

(g100

g)

r = 034 P = 049

r = minus075 P = 005 r = 096 P = 0006

C-W

Insulin (ng120583g protein) Insulin (ng120583g protein)


00 05 10 15 00 05 10 15

04 05 06 07 08 09

40

45

50

55

60

65

STA

(g100

g)

C-CWP

r = minus033 P = 066

HF-CWP HF-W

(b)

Figure 5 Continued


00 05 10 15

C-W C-CWP

HF-CWP HF-W

00 05 10 15 00 05 10 15

r = minus052 P = 028 r = 071 P = 028

r = 039 P = 051

0

100

200

300

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

0

50

100

150

0

50

100

200

150

04 05 06 07 08 09

r = minus085 P = 002

STA

(g100

g)



(c)

0

100

200

300

IL-10

(pg

mg)

C-W

r = minus048 P = 032

80 90 100 110 120

Glucose (mgdL)

80 90 100 110 120

Glucose (mgdL)60 80 100 120 140

Glucose (mgdL)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

C-CWP

8060 70 90 100 110 120

Glucose (mgdL)

r = minus003 P = 096

r = minus088 P = 001 r = minus025 P = 068

HF-CWP HF-W

0

50

100

200

150

(d)

Figure 5 Correlation between different experimental groups (a) TAG and AUC in different experimental groups (b) Insulin and STA (sumof adipose tissues MES RET and EPI) (c) Insulin and IL-10 of serum and mesenteric adipose tissue respectively and (d) glucose and IL-10of serum and mesenteric adipose tissue respectively


Table 4 Serum of triacylglycerol total cholesterol glucose insulin and adiponectin of mice treated with high fat diet (HF) or controlnormocaloric (C) associated with gavage of coacervate (CWP) or water (W)

Serum measurements C-W(119899 = 10)

C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)

Triacylglycerol (mgdL)13337 plusmn 428 12709 plusmn 485 12930 plusmn 431 14155 plusmn 394

bd

Total cholesterol (mgdL)12875 plusmn 422 12946 plusmn 476 12899 plusmn 335 15003 plusmn 600

LDL (mgdL)6881 plusmn 175 7318 plusmn 307 7566 plusmn 164 803 plusmn 328

VLDL (mgdL)2632 plusmn 086 2542 plusmn 097 2586 plusmn 086 2892 plusmn 076

bd

HDL (mgdL)3362 plusmn 175 3671 plusmn 307 3513 plusmn 164 4083 plusmn 328

Glucose (mgdL)10027 plusmn 269 8984 plusmn 445 10080 plusmn 358 10528 plusmn 347

Insulin (ngmL)051 plusmn 003 064 plusmn 001 065 plusmn 001 075 plusmn 002

b

HOMA-IR089 plusmn 026 097 plusmn 015 112 plusmn 021 125 plusmn 019

b

Adiponectin (ngmL)355 plusmn 047 398 plusmn 026 404 plusmn 010 334 plusmn 029

d

Lipopolysaccharides (EUmL)258 plusmn 098 161 plusmn 066 149 plusmn 050 307 plusmn 097

d

Data submitted with an average plusmn EPM bC-W versus HF-W and dHF-CWP versus HF-W (119875 lt 005)

Table 5 Concentrations of IL-6 TNF-120572 IL-10 and IL-10TNF-120572 (pgmg of total protein content) in different experimental groups

C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927

TNF-12057211193 plusmn 2171 9260 plusmn 2286 5448 plusmn 827 6938 plusmn 1735

IL-1015336 plusmn 2701 11930 plusmn 2904 6505 plusmn 996 10074 plusmn 2097

b

IL-10TNF-120572137 plusmn 0027 128 plusmn 028 119 plusmn 010 145 plusmn 067

Data submitted with an average plusmn EPM aC-W versus C-CWP bC-W versus HF-W (119875 lt 005)

physical and chemical characteristics vary according to theprocedures used to obtain these proteins In our study it waspossible to recover an average OF 30 in WP (Table 2) Ifwe compare these numbers with published data (which oftenuse conventional techniques such as ultrafiltration (UF)) ourmethod seems to be a low efficiency process [19] Howevercost-benefit considerations should be taken into account Forexample the UF method is expensive not only in terms ofdeployment but also in terms of operation In addition tobeing cost-effective the coacervation process promotes theseparation of WP and obtains a low-calorie product

The emergence of food compounds with health ben-efits may eventually become a good strategy to improvepublic health In recent years functional food has attractedthe attention from scientific community consumers andfood manufacturers The list of nutraceuticals compounds(vitamins probiotics bioactive peptides and antioxidantsamong others) is extensive and scientific evidence seemsto increasingly support the concept of health promotionthrough food ingredients [20 21]

Functional foods are usually marketed as food containingingredients technologically manipulated to perform a benefitfor health [22] Our study lends support to previous studiesshowing the effectiveness of CWP as a nutraceutical ableto stabilize fat mass gain in animals fed with high fat dietOur findings agree with the WP intake benefits extensivelyreported in literature [23ndash27] As demonstrated there wasa decrease in body weight in the HF-CWP group when

compared to the HF-W group accompanied by a reductionin the adiposity

It is now well established that excessive consumption ofsaturated fat is related to the development of dyslipidemias[28 29] and this study further corroborates it as the animalsfed with high fat diet increased TAG and VLDL Studieshave demonstrated the insulinotropic effect of WP [9 1130] In this study we did not find any significant differencein blood glucose between the groups assessed Regardinginsulin and HOMA index the hyperlipidic diet showedsignificantly higher values Our results demonstrated theeffectiveness of this experimental model of obesity HoweverCWP treatment promoted improved glucose and insulintolerance A study undertaken by Huang et al [31] utilizingdifferent protein sources (cheese whey soy red meat andmilk) in obese mice (induced by high fat diet) has foundincreased adiponectin concentration and reduced insulinwhen the animals were fed with whey protein cheese In thepresent study we also detected the critical role of CWP inmodulating adiponectin as the valueswere higher for theHF-CWP group when compared to HF-W group These findingsindicate that CWP may have functional effects Yamauchi etal [32] have demonstrated the potential of adiponectin inreducing insulin resistance by enhancing fatty acid oxidationleading to a reduction in TAG content in obese diabetic rats

There is strong evidence of an immunomodulatory roleofWP [33] Products containingWPmay be beneficial in thetreatment of certain diseases Studies have shown that WP


promotes improvement in the treatment of gastrointestinalsymptoms of infant mice with rotavirus-induced diarrhea aprotective role in colorectal cancer in rats reduced release ofIL-6 in blood of rats undergoing transient ischemiaintestinalreperfusion andmay provide protective effects against exper-imentally induced breast cancer in animals [25 34 35] Mostof the more recent studies in the literature both in vivo andin vitro have focused on the possible effects of these proteinsin macrophages and lymphocytes Although most proteinsare degraded during the gastric digestion certain cheesewhey proteins such as 120573-Lg 120572-La or GMP are resistantto digestion and remain intact Such proteins can directlystimulate leukocyte after their digestive absorption Thusit is important to understand the mechanisms underlyingthe impact of these proteins on immunity stimulating anti-inflammatory processes in the body [33]

The IL-10 is a pleiotropic cytokine that controlsinflammatory processes by eliminating the proinflammatorycytokines production such as IL-1 IL-6 and IL-8 andTNF-120572 is produced mainly by monocytes macrophageslymphocytes mast cells and mature adipocytes [14 36]The IL-10TNF-120572 ratio has been considered an importantindicator of inflammatory status as low values are oftenassociated with increased morbidity and mortality risk Wedid not observe an increase of the IL-10TNF-120572 ratio in MESof group HF-CWP We believe that because of the shortperiod of the treatment the animals may not have developedtheir proinflammatory state With increased length oftreatment we believe find results more expressive Anotherpossibility is that the coacervate may have protected the micefrom a proinflammatory state triggered by the treatmentdiet leading to the counterbalance of IL-10 unnecessaryin HF-CWP group However a study involving a longertreatment period may be required to discern this possibleeffect

In this sense there is an immunomodulatory mechanismunderlying CWP most likely the IL-10 cytokine which has ahomeostaticmetabolic effect in themesenteric adipose tissueOur result suggests that IL-10 may be a positive regulator ofinsulin sensitivity and increased glucose uptakeThis mecha-nism can protect the adipose tissue against insulin resistanceAlthough the precise origin of the unchecked inflammatoryresponse in obesity is still unclear it is well known thatin obesity the overproduction of proinflammatory cytokinesaffects metabolism For example TNF-120572 contributes to theinability of cells to respond to insulin and to increasedlevels of insulin [37] and IL-10 was associated with othervariables closely linked to insulin sensitivity such as fastingand postload insulin concentrations HDL cholesterol andtriglyceride levels Besides the tissue-specific effect of CWPwe showed a systemic effect in the decrease in the LPS serumlevel

Regarding the composition of the CWP wemay highlightthe presence of 120572-La and 120573-Lg protein the major proteinpresent in the coacervate which has been proven effectivein suppressing the release of proinflammatory cytokines[27 38] The presence of these proteins is a great indica-tion that the coacervate components are able to modulatethe proinflammatory milieu promoted by hyperlipidic diet

In another study by our group mice previously treated withhigh fat diet and fed with a supplementation of CWP (gavage36mg proteinkg of body weight) showed a positive corre-lation between IL-10 and TNF-alpha in mesenteric adiposetissue retroperitoneal adipose tissue and liver tissueWe alsoobserved a positive correlation between lipopolysaccharideand IL-10 in the liver tissue Therefore pretreatment withhigh fat diet promoted metabolic alterations and inflamma-tion and CWP modulated the inflammatory milieu [14]

Evidence suggests that eating WP causes the decreasein calorie intake increased basal energy expenditure andmodulates insulin sensitivity and glucose homeostasis lead-ing to changes in lipid metabolism in adipose tissue liverand muscle [39ndash42] The AMPK and adiponectin are keymolecules to metabolic responses in different tissues [4344] they are involved in the preventive response againstnegative physiological processes caused by the consumptionof a diet high in saturated fatty acidsThe activation of AMPKby bioactive components of foods or medicines has beenregarded as goal since it may reverse the metabolic changesassociated with obesity and type 2 diabetes [45] It is alsoknown that adiponectin can activate AMPK in white adiposetissue [46] The animals treated with coacervate showed anincrease in AMPK activation associated with the decrease inHSL and increase in ATGL protein expression in mesentericadipose tissue Similar results were reported in the reviewundertaken by Bijland et al [47]

A study conducted by Gaidhu et al [43] showed thatAMPK activation stimulated by AICAR (5-aminoimidazol-4-carboxamida ribonucleotıdeo) initially promoted inhibition oflipolysis in adipocytes isolated as in vivo reflecting a decreasein free fatty acid in serum On the other hand prolongedtreatment with AICAR promoted an increase in lipolysiswhich the authors attributed to an increase in the contentof ATGL and reduced activity of HSL [48] However clear-cut conclusions are difficult to arrive at due to a lack of toolsfor manipulating assays using specific AMPK Furthermorethe overall effect of AMPK activation of lipolysis is stillcontroversialThe duration andmode of activation of AMPKmay be of particular importance when it comes to a processaimed at reducing the proinflammatory state caused byincreased lipolysis [47] In addition adiponectin can suppressthe activation of HSL without changing ATGL and ABHD-5in adipocytes in order to modulate a homeostatic control oflipolysis to avoid lipotoxicity [49]

Lipolysis does seem to play a crucial physiological roleby recruiting a source of energy mobilized in times of stressandor energy deprivation Moreover the very significantreduction in lipolysis is clearly harmful as demonstratedin the clinical domain by the syndromes resulting fromdeficiencies in the lipolytic apparatus [50] Given that it isreasonable to question whether the inhibition of lipolysis viaHSL induced by CWP is helpful or harmful since enhancedlipolytic activity and concomitant increase of free fatty acidin the circulation is clearly deleterious and leads to severalcomorbidities To clearly determine this however an analysisof free fatty acid and endogenous glycerol concentrationswould need to be carried out


Another interesting finding was the significantly higherprotein expression of perilipin A (52) in HF-W groupwhich also refers to larger deposits of triglycerides since thisprotein is primarily anchored around the droplets of neutrallipids in adipocytes This is in line with studies showing thatincreased protein expression perilipin A leads to increasedstorage of triglycerides by reducing its hydrolytic rate [51]

Finally there is plenty of evidence suggesting that theintake of WP may lower consumption of calories increasebaseline energy expenditure and improve insulin sensitivityand glucose homeostasis thus leading to changes in lipidmetabolism in adipose tissue liver and muscle [39ndash42]

5 Conclusion

CWP were able to promote nutritional and physiologicalimprovements in HF-CWP group such as reduction in bodymass and decreased serum lipid levels followed by decreasedserum insulin and LPS In addition intervention with CWPresulted in higher adiponectin contents and attenuated pro-cesses that would lead to glucose intoleranceTherefore CWPcould play a beneficial role in some way in modulatinglipolysis in animals treated with hyperlipidic diet

Abbreviations

ABHD-5 Abhydrolase domaincontaining 5

AUC Area under the curveCWP CoacervateC-CWP Control diet plus coacervateC-W Control diet plus tap waterHF-CWP High fat diet plus coacervateHF-W High fat diet plus tap waterHSL Hormone-sensitive lipaseLPS LipopolysaccharideMGL Monoglyceride lipaseNEFAs Nonesterified fatty acidp-AMPK120572 1 e 2 - Thr 172 Phospho 51015840 AMP-activated

protein kinaseTAG TriacylglycerolOGTT Oral glucose tolerance testATGL Triglyceride lipase enzymeWP Whey protein

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

All authors read and approved the final paper

Acknowledgments

The authors thank the staff of the LAMEROA Laboratoryof USP for analytical assistance of diets This work was

supported by FAPESP (Grants no 200953801-5) CAPESand CNPQ

References

[1] M F Gregor and G S Hotamisligil ldquoInflammatory mecha-nisms in obesityrdquo Annual Review of Immunology vol 29 pp415ndash445 2011

[2] M L Kueht B K McFarlin and R E Lee ldquoSeverely obese havegreater LPS-stimulated TNF-120572 production than normal weightAfrican-American womenrdquo Obesity vol 17 no 3 pp 447ndash4512009

[3] P H C Eilers M S Westerterp-plantenga T Kooistra HBiology and M S W Maastricht ldquoLeptin and the proinflam-matory state associated with human obesityrdquo Journal of ClinicalEndocrinology ampMetabolism vol 89 no 4 pp 1773ndash1778 2004

[4] M H Fonseca-Alaniz J Takada M I C Alonso-Vale and FB Lima ldquoAdipose tissue as an endocrine organ from theory topracticerdquo Jornal de Pediatria vol 83 no 5 pp S192ndashS203 2007

[5] M J Song K H Kim J M Yoon and J B Kim ldquoActivationof Toll-like receptor 4 is associated with insulin resistance inadipocytesrdquo Biochemical and Biophysical Research Communica-tions vol 346 no 3 pp 739ndash745 2006

[6] A Chait and F Kim ldquoSaturated fatty acids and inflammationwho pays the tollrdquo Arteriosclerosis Thrombosis and VascularBiology vol 30 no 4 pp 692ndash693 2010

[7] J Park S C Sung A H Choi et al ldquoIncrease in glucose-6-phosphate dehydrogenase in adipocytes stimulates oxidativestress and inflammatory signalsrdquo Diabetes vol 55 no 11 pp2939ndash2949 2006

[8] M E F Vazquez-Vela N Torres and A R Tovar ldquoWhiteadipose tissue as endocrine organ and its role in obesityrdquoArchives of Medical Research vol 39 no 8 pp 715ndash728 2008

[9] W L Hall D J Millward S J Long and L M MorganldquoCasein and whey exert different effects on plasma aminoacid profiles gastrointestinal hormone secretion and appetiterdquoBritish Journal of Nutrition vol 89 no 2 pp 239ndash248 2003

[10] T G Anthony B J McDaniel P Knoll P Bunpo G LPaul and M A McNurlan ldquoFeeding meals containing soy orwhey protein after exercise stimulates protein synthesis andtranslation initiation in the skeletal muscle ofmale ratsrdquo Journalof Nutrition vol 137 no 2 pp 357ndash362 2007

[11] J Shi E Tauriainen E Martonen et al ldquoWhey protein isolateprotects against diet-induced obesity and fatty liver formationrdquoInternational Dairy Journal vol 21 no 8 pp 513ndash522 2011

[12] D Rusu R Drouin Y Pouliot S Gauthier and P E PoubelleldquoA bovine whey protein extract can enhance innate immunityby priming normal human blood neutrophilsrdquo Journal ofNutrition vol 139 no 2 pp 386ndash393 2009

[13] B Tranberg L I Hellgren J Lykkesfeldt et al ldquoWhey proteinreduces early life weight gain in mice fed a high-fat dietrdquo PLoSOne vol 8 no 8 Article ID e71439 2013

[14] M F Moreno G I de Morais Honorato de Souza A C LHachul et al ldquoCoacervate whey protein improves inflammatorymilieu in mice fed with high-fat dietrdquo Nutrition amp Metabolismvol 11 article 15 2014

[15] J Hidalgo and P M T Hansen Selective Precipitation of WheyProteins with Carboxymethylcellulose Public Health 1970

[16] V B Tolstoguzov ldquoFunctional properties of food proteins androle of protein-polysaccharide interactionrdquo Food Hydrocolloidsvol 4 pp 429ndash468 1991


[17] C L Cooper P L Dubin A B Kayitmazer and S TurksenldquoPolyelectrolyte-protein complexesrdquoCurrent Opinion in Colloidand Interface Science vol 10 no 1-2 pp 52ndash78 2005

[18] P G Reeves F H Nielsen and G C Fahey Jr ldquoAIN-93 purifieddiets for laboratory rodents final report of the American Insti-tute ofNutrition adhocwriting committee on the reformulationof the AIN-76A rodent dietrdquo Journal of Nutrition vol 123 no11 pp 1939ndash1951 1993

[19] J N de Wit Lecturerrsquos Handbook on Whey and Whey ProductsEuropean Whey Products Association (Belgium) 2001

[20] L Chen G E Remondetto and M Subirade ldquoFood protein-based materials as nutraceutical delivery systemsrdquo Trends inFood Science and Technology vol 17 no 5 pp 272ndash283 2006

[21] R Caillard R Guillet-Nicolas F Kleitz and M SubiradeldquoTabletability of whey protein isolatesrdquo International DairyJournal vol 27 no 1-2 pp 92ndash98 2012

[22] M Niva ldquoldquoAll foods affect healthrdquo understandings of func-tional foods and healthy eating among health-oriented FinnsrdquoAppetite vol 48 no 3 pp 384ndash393 2007

[23] G Bounous andPGold ldquoThebiological activity of undenatureddietary whey proteins role of glutathionerdquo Clinical and Inves-tigative Medicine vol 14 no 4 pp 296ndash309 1991

[24] M M Iskandar N Dauletbaev S Kubow N Mawji and L CLands ldquoWhey protein hydrolysates decrease IL-8 secretion inlipopolysaccharide (LPS)-stimulated respiratory epithelial cellsby affecting LPS binding to Toll-like receptor 4rdquo British Journalof Nutrition vol 110 no 1 pp 58ndash68 2013

[25] RHakkak S KorourianM J J Ronis JM Johnston and TMBadger ldquoDietary whey protein protects against azoxymethane-induced colon tumors in male ratsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 10 no 5 pp 555ndash558 2001

[26] C F Rosaneli A E Bighetti M A Antonio J E Carvalhoand V C Sgarbieri ldquoEfficacy of a whey protein concentrate onthe inhibition of stomach ulcerative lesions caused by ethanolingestionrdquo Journal of Medicinal Food vol 5 no 4 pp 221ndash2282002

[27] S Pal V Ellis and S Dhaliwal ldquoEffects of whey protein isolateon body composition lipids insulin and glucose in overweightand obese individualsrdquo British Journal of Nutrition vol 104 no5 pp 716ndash723 2010

[28] S Zhao J Wang X Song X Zhang C Ge and S Gao ldquoImpactof dietary protein on lipid metabolism-related gene expressionin porcine adipose tissuerdquo Nutrition and Metabolism vol 7article 6 2010

[29] D E C Cintra A V Costa M D C G Peluzio S L P MattaM T C Silva and N M B Costa ldquoLipid profile of rats fedhigh-fat diets based on flaxseed peanut trout or chicken skinrdquoNutrition vol 22 no 2 pp 197ndash205 2006

[30] A H Frid M Nilsson J J Holst and I M E Bjorck ldquoEffectof whey on blood glucose and insulin responses to compositebreakfast and lunch meals in type 2 diabetic subjectsrdquo TheAmerican Journal of Clinical Nutrition vol 82 no 1 pp 69ndash752005

[31] X-FHuang Y Liu G L Rahardjo P LMclennan L C TapsellandW A Buttemer ldquoEffects of diets high in whey soy redmeatand milk protein on body weight maintenance in diet-inducedobesity in micerdquo Nutrition and Dietetics vol 65 no 3 pp S53ndashS59 2008

[32] T Yamauchi J Kamon H Waki et al ldquoThe fat-derived hor-mone adiponectin reverses insulin resistance associated withboth lipoatrophy and obesityrdquoNature Medicine vol 7 no 8 pp941ndash946 2001

[33] D Rusu R Drouin Y Pouliot S Gauthier and P E PoubelleldquoA bovine whey protein extract stimulates human neutrophilsto generate bioactive IL-1Ra through a NF-120581B- and MAPK-dependent mechanismrdquo Journal of Nutrition vol 140 no 2 pp382ndash391 2010

[34] F M Wolber A M Broomfield L Fray M L Cross and DDey ldquoSupplemental dietary whey protein concentrate reducesrotavirus-induced disease symptoms in suckling micerdquo Journalof Nutrition vol 135 no 6 pp 1470ndash1474 2005

[35] M Yamaguchi and M Uchida ldquo120572-lactalbumin suppressesinterleukin-6 release after intestinal ischemiareperfusion vianitric oxide in ratsrdquo Inflammopharmacology vol 15 no 1 pp43ndash47 2007

[36] C E Juge-Aubry E Somm A Pernin et al ldquoAdipose tissue is aregulated source of interleukin-10rdquo Cytokine vol 29 no 6 pp270ndash274 2005

[37] J M Han S J Patterson M Speck J A Ehses and MK Levings ldquoInsulin inhibits IL-10-mediated regulatory T cellfunction implications for obesityrdquo The Journal of Immunologyvol 192 no 2 pp 623ndash629 2014

[38] M Yamaguchi and M Uchida ldquo120572-Lactalbumin suppressesinterleukin-6 release after intestinal ischemiareperfusion vianitric oxide in ratsrdquo Inflammopharmacology vol 15 no 1 pp43ndash47 2007

[39] M B Zemel ldquoRole of calcium and dairy products in energypartitioning and weight managementrdquoThe American Journal ofClinical Nutrition vol 79 no 5 pp 907Sndash912S 2004

[40] B Lonnerdal ldquoNutritional and physiologic significance ofhuman milk proteinsrdquo The American Journal of Clinical Nutri-tion vol 77 no 6 pp 1537Sndash1543S 2003

[41] J-C J Bouthegourd S M Roseau L Makarios-Lahham PM Leruyet D G Tome and P C Even ldquoA preexercise 120572-lactalbumin-enriched whey protein meal preserves lipid oxi-dation and decreases adiposity in ratsrdquo American Journal ofPhysiology Endocrinology and Metabolism vol 283 no 3 ppE565ndashE572 2002

[42] L McAllan P D Cotter H M Roche R Korpela and KN Nilaweera ldquoBioactivity in whey proteins influencing energybalancerdquo Journal of Metabolic Syndrome vol 1 article 107 2012

[43] M P Gaidhu N M Anthony P Patel T J Hawke and RB Ceddia ldquoDysregulation of lipolysis and lipid metabolism invisceral and subcutaneous adipocytes by high-fat diet Role ofATGL HSL and AMPKrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol 298 no 4 pp C961ndashC971 2010

[44] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[45] B B Zhang G Zhou and C Li ldquoAMPK an emerging drug tar-get for diabetes and the metabolic syndromerdquo Cell Metabolismvol 9 no 5 pp 407ndash416 2009

[46] P Huypens E Quartier D Pipeleers and M van de CasteeleldquoMetformin reduces adiponectin protein expression and releasein 3T3-L1 adipocytes involving activation of AMP activatedprotein kinaserdquo European Journal of Pharmacology vol 518 no2-3 pp 90ndash95 2005

[47] S Bijland S J Mancini and I P Salt ldquoRole of AMP-activatedprotein kinase in adipose tissuemetabolism and inflammationrdquoClinical Science vol 124 no 8 pp 491ndash507 2013

[48] M P Gaidhu S Fediuc N M Anthony et al ldquoProlongedAICAR-inducedAMP-kinase activation promotes energy dissi-pation in white adipocytes novel mechanisms integrating HSLandATGLrdquo Journal of Lipid Research vol 50 no 4 pp 704ndash7152009


[49] L Qiao B Kinney J Schaack and J Shao ldquoAdiponectin inhibitslipolysis in mouse adipocytesrdquoDiabetes vol 60 no 5 pp 1519ndash1527 2011

[50] S Santamarina-Fojo ldquoThe familial chylomicronemia syn-dromerdquo Endocrinology and Metabolism Clinics of North Amer-ica vol 27 no 3 pp 551ndash567 1998

[51] D L Brasaemle B Rubin I A Harten J Gruia-Gray A RKimmel and C Londos ldquoPerilipin A increases triacylglycerolstorage by decreasing the rate of triacylglycerol hydrolysisrdquoTheJournal of Biological Chemistry vol 275 no 49 pp 38486ndash38493 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


several proteins and proinflammatory cytokines in differentcell types via nuclear factor 120581B activation (NF-120581B) [4] Inaddition endotoxin and saturated fatty acids act in the samepathway of NF-120581B activation Many studies have shown thatLPS (endotoxin) can activate these proteins in adipocytesthereby increasing the gene expression of proinflammatoryadipokines [5ndash7]

The main role of adipose lipolytic enzymes is to provideother tissues with FAs in case of energy demand Triglyceridestored in the lipid droplet is first hydrolyzed by the adiposetriglyceride lipase enzyme (ATGL) also known as desnutrinreleasing a diacylglycerol moiety and FA which requires anabhydrolase domain containing 5 (ABHD-5) promoter to beactivated After hydrolysis by ATGL diacylglycerols are thenhydrolyzed sequentially by hormone-sensitive lipase (HSL)and monoglyceride lipase (MGL) producing nonesterifiedfatty acids (NEFAs) and glycerol [8] Different lipases gainaccess to the lipid droplet when the proteins coating thevesicle (perilipins) are phosphorylated Perilipin A normallyprevents lipolysis of triglyceride by surrounding the lipiddroplet thus preventing the access of lipases

Whey protein (WP) has been found to be an excellentprophylactic against obesity because of the high biologicalvalue mediated by bioactive peptides These act as antimi-crobial agents antihypertensive and regulators of immunefunction reducing body fat as well as a variety of relatedbeneficial mechanisms for human health They also haveadditional functions for example they have appetite sup-pressant effects [9] stimulate muscle protein synthesis andregulate of body energy homeostasis [10] There is plentyof evidence indicating the potential of the WP in anti-inflammatory and antioxidant effects of exercise [9 11ndash13]

Chitosan complex coacervation with WP is composed ofby-products from the processing of shrimp crab (chitosan)and cheese adding an environmental benefit to the productas these by-products may be reused and not disposed of inlandfill sites or released into rivers by producers [14] In viewof the above together with the development of fractionationtechnique and whey protein preservation employing themethod may contribute to the recovery of this valuablenutrient and increase the expression of the functional prop-erties [15] Complex coacervation is defined as a colloidalseparation forming two liquid phases This process is essen-tially driven by attractive forces with opposite charges ofa biopolymer This phenomenon occurs by the formationof a system of balance between colloids and the dilutedsupernatant [16 17] Thus the purpose of the present studywas to investigate the prospect of a biotechnological processof complexation and separation of cheese whey proteins inchitosan and test their antiobesogenic potential through themodulation of inflammatory markers and lipolytic pathwaypresent in obesity

2 Methods

21 Coacervation and Characterization In this study weused sweet cheese whey (SW 1108 bag 25 kg) with 15 fatmarketed byCompanyAlibra-PRDissolving 10 g of thewhey

powder in 100mL of distilled water Chitosan was used forthe coacervation medium molar mass with 75ndash85 degreeof deacetylation and viscosity of 200ndash800 cps (Sigma-Aldrich44887-7) A concentration of 075mgmL of Chitosan wasused Chitosan was dissolved in citric acid (208mmolL) andafter this step added to the cheese whey in a proportion of1 1 under stirring at room temperature for 1 h The pH of thesolution of chitosan and WP was adjusted to 6 with NaOH(250mmolL) and solubilized at room temperature (plusmn25∘C)with stirring Solids coagulated with chitosan known ascoacervate (CWP) were collected by centrifugation (1300 g)

In order to obtain on average 30 of the protein coac-ervate 3 L cheese whey was used Thus samples of CWPwere obtained for their chemical analysis of total lipids totalprotein and lactose Finally for measurements of samplesof mineral micronutrients Ca K Mg and P of cheesewhey CWP and Chitosan 100 120583g was subjected to an opticalemission spectrometer for inductively coupled plasma (ICPOES Perkin Elmer Optima 3000DV Norwalk CT USA) Todetermine the existing protein fractions in CWP the elec-trophoretic profile with reducing buffer containing 625mMTris-HCl 20 glycerol 2 sodium dodecyl sulfate(SDS)(10) 5 120573-mercaptoethanol and bromophenol blue at pH68 was performed

22 Animals and Treatment This study was approved by theResearch Ethics Committee of the Universidade FederaldeSao Paulo Escola Paulista de Medicina (UNIFESPEPM)as the search protocol number 047310 Experimental pro-cedures are in accordance with Principles of LaboratoryAnimal Care formulated by the National Institutes of Health(National Institutes of Health Publication number 96-23revised 1996)

Forty-nine male Swiss mice twelve-week-old fromCEDEME (Centro de Desenvolvimento de ModelosExperimentais da Universidade Federal de Sao Paulo) werehoused five in a cage in a standard experimental animallaboratory and kept under controlled conditions of light(12 h light-dark cycle with lights on at 6 am) and temperature(24 plusmn 1∘C) All mice received water and food ad libitumThe animals were divided as follows control diet plus tapwater (C-W) control diet plus coacervate (C-CWP) highfat diet plus coacervate (HF-CWP) and high fat diet plustap water (HF-W) All diets were prepared according to therecommendations of the American Institute of Nutrition [18](Table 1) Coacervate (CWP) containing 100mgsdotkgsdotday wasgiven by gavage The body weight gain was recorded twice aweek

23 Composition and Aspect of the Coacervate Figure 1(a)shows the protein profile of the coacervate by the presenceof the major proteins of larger fractions namely alpha-lactalbumin (120572-La-14 kDa) beta-lactoglobulin (120573-Lgmdash18 kDa monomer and 34 kDa dimer form) bovine serumalbumin (BSAmdash66 kD) and lactoferrin (Lacfmdash86 kDa) TheCWP has a flocculation aspect (Figure 1(b)) consisting ofproteins and micronutrients such as calcium potassiummagnesium phosphorus and sodium (Table 2)






ST CWP

Lacfr

BSA75

50

37

25

20

15 120572-La

120573-Lg monomer

120573-Lg dimer

(a) (b)



2O were added and


2 and
















40424446485052545658

1 3 6 8

g

Weeks

C-WC-CWP

HF-CWPHF-W

b d

b



3 Results





100

100002000030000

0

120140160180200220240260280300

B 15 30 60 90 120

(mg

dL)

(Min)

AUC

b

b

C-WC-CWP

HF-CWPHF-W

C-W

C-CW

P

HF-

CWP

HF-

W






4 Discussion




50

100

150

HSL

(UA

)

AB

(a)


D

0

50

100

150

200

250

ATG

L (U

A)

(b)


50

100

150 B D

Peril

ipin

A (U

A)

(c)


50

100

150

A

C

ABH

D-5

(UA

)

(d)


50

100

150

D

pAM

PK (U

A)

HSL120572-Tubulin

ATGL120572-Tubulin

120572-Tubulin

120572-Tubulin

120572-Tubulin

Perilipin A

ABHD-5

pAMPK

(e)



C-W(119899 = 10)

C-CWP(119899 = 10)

HF-CWP(119899 = 19)

HF-W(119899 = 19)

RTM (g100 g)EPI


bd


bd


b


bd



16000 18000 20000

C-W

22000 24000

100

120

140

160

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

r = 015 P = 076

17000 18000 19000 20000 21000 22000 23000

r = minus002 P = 09795

100

105

110

115

120

125

C-CWP

AUCAUC

AUCAUC

16000 18000 20000 22000 24000 26000

90

100

110

120

130

r = 042 P = 040

HF-CWP

18000 20000 22000 24000 26000 28000 30000

r = 086 P = 005

HF-W

140

150

160

170

180

(a)

00 05 10 15

5

6

7

8

9

STA

(g100

g)

4

5

6

7

8

STA

(g100

g)

8

9

10

11

12

STA

(g100

g)

r = 034 P = 049

r = minus075 P = 005 r = 096 P = 0006

C-W



00 05 10 15 00 05 10 15

04 05 06 07 08 09

40

45

50

55

60

65

STA

(g100

g)

C-CWP

r = minus033 P = 066

HF-CWP HF-W

(b)

Figure 5 Continued


00 05 10 15

C-W C-CWP

HF-CWP HF-W

00 05 10 15 00 05 10 15

r = minus052 P = 028 r = 071 P = 028

r = 039 P = 051

0

100

200

300

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

0

50

100

150

0

50

100

200

150

04 05 06 07 08 09

r = minus085 P = 002

STA

(g100

g)



(c)

0

100

200

300

IL-10

(pg

mg)

C-W

r = minus048 P = 032

80 90 100 110 120

Glucose (mgdL)

80 90 100 110 120

Glucose (mgdL)60 80 100 120 140

Glucose (mgdL)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

C-CWP

8060 70 90 100 110 120

Glucose (mgdL)

r = minus003 P = 096

r = minus088 P = 001 r = minus025 P = 068

HF-CWP HF-W

0

50

100

200

150

(d)





C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)


bd




bd




b


b


d


d



C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927



b





















5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















ST CWP

Lacfr

BSA75

50

37

25

20

15 120572-La

120573-Lg monomer

120573-Lg dimer

(a) (b)



2O were added and


2 and
















40424446485052545658

1 3 6 8

g

Weeks

C-WC-CWP

HF-CWPHF-W

b d

b



3 Results





100

100002000030000

0

120140160180200220240260280300

B 15 30 60 90 120

(mg

dL)

(Min)

AUC

b

b

C-WC-CWP

HF-CWPHF-W

C-W

C-CW

P

HF-

CWP

HF-

W






4 Discussion




50

100

150

HSL

(UA

)

AB

(a)


D

0

50

100

150

200

250

ATG

L (U

A)

(b)


50

100

150 B D

Peril

ipin

A (U

A)

(c)


50

100

150

A

C

ABH

D-5

(UA

)

(d)


50

100

150

D

pAM

PK (U

A)

HSL120572-Tubulin

ATGL120572-Tubulin

120572-Tubulin

120572-Tubulin

120572-Tubulin

Perilipin A

ABHD-5

pAMPK

(e)



C-W(119899 = 10)

C-CWP(119899 = 10)

HF-CWP(119899 = 19)

HF-W(119899 = 19)

RTM (g100 g)EPI


bd


bd


b


bd



16000 18000 20000

C-W

22000 24000

100

120

140

160

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

r = 015 P = 076

17000 18000 19000 20000 21000 22000 23000

r = minus002 P = 09795

100

105

110

115

120

125

C-CWP

AUCAUC

AUCAUC

16000 18000 20000 22000 24000 26000

90

100

110

120

130

r = 042 P = 040

HF-CWP

18000 20000 22000 24000 26000 28000 30000

r = 086 P = 005

HF-W

140

150

160

170

180

(a)

00 05 10 15

5

6

7

8

9

STA

(g100

g)

4

5

6

7

8

STA

(g100

g)

8

9

10

11

12

STA

(g100

g)

r = 034 P = 049

r = minus075 P = 005 r = 096 P = 0006

C-W



00 05 10 15 00 05 10 15

04 05 06 07 08 09

40

45

50

55

60

65

STA

(g100

g)

C-CWP

r = minus033 P = 066

HF-CWP HF-W

(b)

Figure 5 Continued


00 05 10 15

C-W C-CWP

HF-CWP HF-W

00 05 10 15 00 05 10 15

r = minus052 P = 028 r = 071 P = 028

r = 039 P = 051

0

100

200

300

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

0

50

100

150

0

50

100

200

150

04 05 06 07 08 09

r = minus085 P = 002

STA

(g100

g)



(c)

0

100

200

300

IL-10

(pg

mg)

C-W

r = minus048 P = 032

80 90 100 110 120

Glucose (mgdL)

80 90 100 110 120

Glucose (mgdL)60 80 100 120 140

Glucose (mgdL)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

C-CWP

8060 70 90 100 110 120

Glucose (mgdL)

r = minus003 P = 096

r = minus088 P = 001 r = minus025 P = 068

HF-CWP HF-W

0

50

100

200

150

(d)





C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)


bd




bd




b


b


d


d



C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927



b





















5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























40424446485052545658

1 3 6 8

g

Weeks

C-WC-CWP

HF-CWPHF-W

b d

b



3 Results





100

100002000030000

0

120140160180200220240260280300

B 15 30 60 90 120

(mg

dL)

(Min)

AUC

b

b

C-WC-CWP

HF-CWPHF-W

C-W

C-CW

P

HF-

CWP

HF-

W






4 Discussion




50

100

150

HSL

(UA

)

AB

(a)


D

0

50

100

150

200

250

ATG

L (U

A)

(b)


50

100

150 B D

Peril

ipin

A (U

A)

(c)


50

100

150

A

C

ABH

D-5

(UA

)

(d)


50

100

150

D

pAM

PK (U

A)

HSL120572-Tubulin

ATGL120572-Tubulin

120572-Tubulin

120572-Tubulin

120572-Tubulin

Perilipin A

ABHD-5

pAMPK

(e)



C-W(119899 = 10)

C-CWP(119899 = 10)

HF-CWP(119899 = 19)

HF-W(119899 = 19)

RTM (g100 g)EPI


bd


bd


b


bd



16000 18000 20000

C-W

22000 24000

100

120

140

160

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

r = 015 P = 076

17000 18000 19000 20000 21000 22000 23000

r = minus002 P = 09795

100

105

110

115

120

125

C-CWP

AUCAUC

AUCAUC

16000 18000 20000 22000 24000 26000

90

100

110

120

130

r = 042 P = 040

HF-CWP

18000 20000 22000 24000 26000 28000 30000

r = 086 P = 005

HF-W

140

150

160

170

180

(a)

00 05 10 15

5

6

7

8

9

STA

(g100

g)

4

5

6

7

8

STA

(g100

g)

8

9

10

11

12

STA

(g100

g)

r = 034 P = 049

r = minus075 P = 005 r = 096 P = 0006

C-W



00 05 10 15 00 05 10 15

04 05 06 07 08 09

40

45

50

55

60

65

STA

(g100

g)

C-CWP

r = minus033 P = 066

HF-CWP HF-W

(b)

Figure 5 Continued


00 05 10 15

C-W C-CWP

HF-CWP HF-W

00 05 10 15 00 05 10 15

r = minus052 P = 028 r = 071 P = 028

r = 039 P = 051

0

100

200

300

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

0

50

100

150

0

50

100

200

150

04 05 06 07 08 09

r = minus085 P = 002

STA

(g100

g)



(c)

0

100

200

300

IL-10

(pg

mg)

C-W

r = minus048 P = 032

80 90 100 110 120

Glucose (mgdL)

80 90 100 110 120

Glucose (mgdL)60 80 100 120 140

Glucose (mgdL)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

C-CWP

8060 70 90 100 110 120

Glucose (mgdL)

r = minus003 P = 096

r = minus088 P = 001 r = minus025 P = 068

HF-CWP HF-W

0

50

100

200

150

(d)





C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)


bd




bd




b


b


d


d



C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927



b





















5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















50

100

150

HSL

(UA

)

AB

(a)


D

0

50

100

150

200

250

ATG

L (U

A)

(b)


50

100

150 B D

Peril

ipin

A (U

A)

(c)


50

100

150

A

C

ABH

D-5

(UA

)

(d)


50

100

150

D

pAM

PK (U

A)

HSL120572-Tubulin

ATGL120572-Tubulin

120572-Tubulin

120572-Tubulin

120572-Tubulin

Perilipin A

ABHD-5

pAMPK

(e)



C-W(119899 = 10)

C-CWP(119899 = 10)

HF-CWP(119899 = 19)

HF-W(119899 = 19)

RTM (g100 g)EPI


bd


bd


b


bd



16000 18000 20000

C-W

22000 24000

100

120

140

160

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

r = 015 P = 076

17000 18000 19000 20000 21000 22000 23000

r = minus002 P = 09795

100

105

110

115

120

125

C-CWP

AUCAUC

AUCAUC

16000 18000 20000 22000 24000 26000

90

100

110

120

130

r = 042 P = 040

HF-CWP

18000 20000 22000 24000 26000 28000 30000

r = 086 P = 005

HF-W

140

150

160

170

180

(a)

00 05 10 15

5

6

7

8

9

STA

(g100

g)

4

5

6

7

8

STA

(g100

g)

8

9

10

11

12

STA

(g100

g)

r = 034 P = 049

r = minus075 P = 005 r = 096 P = 0006

C-W



00 05 10 15 00 05 10 15

04 05 06 07 08 09

40

45

50

55

60

65

STA

(g100

g)

C-CWP

r = minus033 P = 066

HF-CWP HF-W

(b)

Figure 5 Continued


00 05 10 15

C-W C-CWP

HF-CWP HF-W

00 05 10 15 00 05 10 15

r = minus052 P = 028 r = 071 P = 028

r = 039 P = 051

0

100

200

300

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

0

50

100

150

0

50

100

200

150

04 05 06 07 08 09

r = minus085 P = 002

STA

(g100

g)



(c)

0

100

200

300

IL-10

(pg

mg)

C-W

r = minus048 P = 032

80 90 100 110 120

Glucose (mgdL)

80 90 100 110 120

Glucose (mgdL)60 80 100 120 140

Glucose (mgdL)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

C-CWP

8060 70 90 100 110 120

Glucose (mgdL)

r = minus003 P = 096

r = minus088 P = 001 r = minus025 P = 068

HF-CWP HF-W

0

50

100

200

150

(d)





C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)


bd




bd




b


b


d


d



C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927



b





















5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















16000 18000 20000

C-W

22000 24000

100

120

140

160

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

TAG

(mg

dL)

r = 015 P = 076

17000 18000 19000 20000 21000 22000 23000

r = minus002 P = 09795

100

105

110

115

120

125

C-CWP

AUCAUC

AUCAUC

16000 18000 20000 22000 24000 26000

90

100

110

120

130

r = 042 P = 040

HF-CWP

18000 20000 22000 24000 26000 28000 30000

r = 086 P = 005

HF-W

140

150

160

170

180

(a)

00 05 10 15

5

6

7

8

9

STA

(g100

g)

4

5

6

7

8

STA

(g100

g)

8

9

10

11

12

STA

(g100

g)

r = 034 P = 049

r = minus075 P = 005 r = 096 P = 0006

C-W



00 05 10 15 00 05 10 15

04 05 06 07 08 09

40

45

50

55

60

65

STA

(g100

g)

C-CWP

r = minus033 P = 066

HF-CWP HF-W

(b)

Figure 5 Continued


00 05 10 15

C-W C-CWP

HF-CWP HF-W

00 05 10 15 00 05 10 15

r = minus052 P = 028 r = 071 P = 028

r = 039 P = 051

0

100

200

300

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

0

50

100

150

0

50

100

200

150

04 05 06 07 08 09

r = minus085 P = 002

STA

(g100

g)



(c)

0

100

200

300

IL-10

(pg

mg)

C-W

r = minus048 P = 032

80 90 100 110 120

Glucose (mgdL)

80 90 100 110 120

Glucose (mgdL)60 80 100 120 140

Glucose (mgdL)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

C-CWP

8060 70 90 100 110 120

Glucose (mgdL)

r = minus003 P = 096

r = minus088 P = 001 r = minus025 P = 068

HF-CWP HF-W

0

50

100

200

150

(d)





C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)


bd




bd




b


b


d


d



C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927



b





















5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 05 10 15

C-W C-CWP

HF-CWP HF-W

00 05 10 15 00 05 10 15

r = minus052 P = 028 r = 071 P = 028

r = 039 P = 051

0

100

200

300

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

0

50

100

150

0

50

100

200

150

04 05 06 07 08 09

r = minus085 P = 002

STA

(g100

g)



(c)

0

100

200

300

IL-10

(pg

mg)

C-W

r = minus048 P = 032

80 90 100 110 120

Glucose (mgdL)

80 90 100 110 120

Glucose (mgdL)60 80 100 120 140

Glucose (mgdL)

IL-10

(pg

mg)

0

50

100

150

IL-10

(pg

mg)

IL-10

(pg

mg)

0

50

100

150

C-CWP

8060 70 90 100 110 120

Glucose (mgdL)

r = minus003 P = 096

r = minus088 P = 001 r = minus025 P = 068

HF-CWP HF-W

0

50

100

200

150

(d)





C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)


bd




bd




b


b


d


d



C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927



b





















5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















C-CWP(119899 = 11)

HF-CWP(119899 = 17)

HF-W(119899 = 17)


bd




bd




b


b


d


d



C-W(119899 = 6)

C-CWP(119899 = 7)

HF-CWP(119899 = 12)

HF-W(119899 = 12)

IL-632969 plusmn 4364 13208 plusmn 2410

a21580 plusmn 4970 24161 plusmn 2927



b





















5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























5 Conclusion


Abbreviations








Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Preventive Effects of Chitosan Coacervate Whey Protein on Body Composition and Immunometabolic...

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Transcript of Preventive Effects of Chitosan Coacervate Whey Protein on Body Composition and Immunometabolic...