The Chaperone Balance Hypothesis: The Importance of the Extracellular to Intracellular HSP70 Ratio...

Review ArticleThe Chaperone Balance Hypothesis The Importanceof the Extracellular to Intracellular HSP70 Ratio toInflammation-Driven Type 2 Diabetes the Effect of Exerciseand the Implications for Clinical Management

Mauricio Krause1 Thiago Gomes Heck123 Aline Bittencourt12 Sofia Pizzato Scomazzon12

Philip Newsholme4 Rui Curi5 and Paulo Ivo Homem de Bittencourt Jr12

1Laboratory of Cellular Physiology Department of Physiology Institute of Basic Health SciencesFederal University of Rio Grande do Sul 90050-170 Porto Alegre RS Brazil2National Institute of Science and Technology in Hormones and Womenrsquos Health (INCT-HSM) 90035-003 Porto Alegre RS Brazil3Department of Life Sciences Regional University of Northwestern Rio Grande do Sul State (UNIJUI) 98700-000 Ijuı RS Brazil4School of Biomedical Sciences CHIRI Biosciences Curtin University Perth WA 6845 Australia5Department of Physiology and Biophysics Institute of Biomedical Sciences University of Sao Paulo Sao Paulo SP Brazil

Correspondence should be addressed to Mauricio Krause mauriciokrauseufrgsbrand Paulo Ivo Homem de Bittencourt Jr pauloivoufrgsbr

Received 9 December 2014 Accepted 12 February 2015

Academic Editor Magdalena Klink

Copyright copy 2015 Mauricio Krause et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Recent evidence shows divergence between the concentrations of extracellular 70 kDa heat shock protein [eHSP70] and itsintracellular concentrations [iHSP70] in people with type 2 diabetes (T2DM) A vital aspect regarding HSP70 physiology is itsversatility to induce antagonistic actions depending on the location of the protein For example iHSP70 exerts a powerful anti-inflammatory effect while eHSP70 activates proinflammatory pathways Increased eHSP70 is associated with inflammatory andoxidative stress conditions whereas decreased iHSP70 levels are related to insulin resistance in skeletal muscle Serum eHSP70concentrations are positively correlated with markers of inflammation such as C-reactive protein monocyte count and TNF-120572while strategies to enhance iHSP70 (eg heat treatment chemical HSP70 inducers or coinducers and physical exercise) are capableof reducing the inflammatory profile and the insulin resistance state Here we present recent findings suggesting that imbalances inthe HSP70 status described by the [eHSP70][iHSP70] ratio may be determinant to trigger a chronic proinflammatory state thatleads to insulin resistance and T2DM development This led us to hypothesize that changes in this ratio value could be used as abiomarker for the management of the inflammatory response in insulin resistance and diabetes

1 Introduction

Heat shock proteins (HSPs) are considered part of a family ofproteins known as ldquostress proteinsrdquo since their expression isinduced by a wide range of stressors such as oxidative stress[1] thermal stress [2] ischemia [3] exercise [1] metabolicstress [4] and many others The 72 kDa member of the70 kDa family of heat shock proteins HSP70 (or HSPAencoded by theHSPA1A gene in humans) is inducible during

cell stress It is the most abundant of all HSPs accountingfor 1-2 of cellular protein [5] and is plentiful in skeletalmuscle [6] Asmolecular chaperones the intracellular HSP70proteins (iHSP70) can interact with other proteins (unfoldedin nonnative state andor stress-denatured conformations)to avoid inappropriate interactions formation of proteinaggregates and degradation of damaged proteins as well ashelping the correct refolding of nascent proteins [6] Otherfunctions include protein translocation [7] antiapoptosis

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 249205 12 pageshttpdxdoiorg1011552015249205

2 Mediators of Inflammation

[8] and anti-inflammatory responses [9 10] More recentlythe HSP roles have been expanded to include control ofcell signaling [11] modulation of immune response [12]and modulation of chronic disease conditions [13] such asdiabetes obesity and insulin resistance [14 15]

The heat shock response is regulated by a family of heatshock transcription factors (HSFs) composed of four mem-bers (HSF 1ndash4) which are maintained in an inactive mono-meric form during nonstimulated conditions [16] HSF-1 isa primary regulator of heat shock response in mammaliancells and a low concentration of it has been associated witha number of human pathologies including T2DM [17] andobesity-related fatty liver disease [18] HSF-1 activation isa multistep mechanism that involves its phosphorylationtrimerization nuclear translocation and DNA binding toheat shock elements (HSE) located at the promoter regions oftargeted heat shock genes [15] nevertheless HSF-1 activationcan be negatively regulated by posttranscriptional modifica-tion such as phosphorylation in specific serine residues andphosphorylation-dependent sumoylation [19]

Heat shock proteins were long thought to be exclusivecytoplasmic proteins with functions restricted to the intra-cellular compartment However an increasing number ofobservations have indicated that theymay be released into theextracellular space (eHSP70) having a wide variety of effectson other cells [20] eHSP70 function is in general associatedwith the activation of the immune system [21] For exampleeHSP70 has been reported to stimulate neutrophil microbi-cidal capacity [22] and chemotaxis [23] and recruitment ofnatural killer (NK) cells [24] as well as cytokine productionby immune cells [12 25] In addition eHSP70 was recentlyhypothesized to be involved in the inducement of neural cellprotection under stress conditions [15]

An intriguing aspect of HSP70 physiology is its versatilityto induce antagonistic actions depending on the locationof the protein [17] For example iHSP70 exerts a powerfulanti-inflammatory effect while eHSP70 has the oppositerole inducing the activation of several proinflammatorypathways In fact chronic exposure to eHSP70 [26] inducesthe activation of several proinflammatory pathways probablyvia binding to membrane Toll-like receptors (see below)although eHSP70-peptides have also been shown to actas anti-inflammatory and immunosuppressive factors afterinternalization and antigen processing (see Borges et al 2012for review [27])

iHSP70 exerts its anti-inflammatory effect through theinteraction with the nuclear factor 120581B (NF-120581B) blockingits activation [28] NF-120581B is a ubiquitous transcription fac-tor originally discovered in B-lymphocytes that is essentialfor arming inflammatory responses to a variety of signalsimmune function endothelial cell activation and the controlof cell growth [29] iHSP70 hampers NF-120581B activation atseveral levels by impeding the phosphorylation of inhibitorof 120581B (I120581Bs) [30] by directly binding to I120581B kinase gamma(IKK120574) [31] which will result in continued binding (andinactivation of NF-120581B) thus inhibiting downstream inflam-matory signals This is corroborated by the finding thatiHSP70 binds with liver NF-120581BI120581B complex in the cytosolthus hindering transcription of TNF120572 and inducible nitric

oxide synthase (NOS2) genes [31] which are activated via aNF-120581B dependent mechanism Stress-induced elevations iniHSP70 inhibit c-Jun N-terminal kinase- (JNK-) dependentsignal transduction hence promoting cell survival [32]

iHSP70 affects apoptosis at various levels It can inhibitcaspase activation by interfering with Apaf-1 and prevent therecruitment of procaspase-9 to the apoptosome [33] HSP70also increases Bcl-2 expression and inhibits cytochrome Crelease [34]Overexpression ofHSP70 in lymphoid tumor celllines inhibited apoptosis by attenuation of caspase activation[35] The antiapoptotic effects of HSP70 have been reportedfor mouse brain tissue where HSP70 overexpression resultedin decreased infarct sizes improved neurological deficits andfewer apoptotic cells (determined by reduced DNA ladder-ing) after middle cerebral artery occlusion [36] HSP70s havealso been shown to decrease oxidative stress so that theyare part of the intracellular antioxidant machinery makingiHSP70 even more important for the inhibition of apoptosisand inflammation [37] Cyclopentenone prostaglandins (cp-PGs) which under certain circumstances may induce HSP70expression are consequently powerful anti-inflammatoryautacoids [38ndash40]

The interplay between iHSP70 and proinflammatorycytokines at gene regulatory level has also been reportedThepromoter region of TNF120572 gene contains an HSF1 bindingsite that represses TNF120572 transcription and thus loss of thisrepressor results in sustained expression of TNF120572 [41] thusthe HSF1 knockout is associated with a chronic elevationof TNF120572 levels and increased susceptibility to endotoxinchallenge [42] Regulation of such a network in the oppositedirections has also been demonstrated TNF120572 may tran-siently repress HSF1 activation [43] Furthermore JNK1 wasunequivocally demonstrated to phosphorylate HSF1 in itsregulatory domain causing suppression of HSF1 transcribingactivity [44] while HSP70 prevented Bax activation both byinhibiting the JNKBim pathway and by interacting with Baxin UV-induced apoptosis [45] Altogether the above findingsexplain why the induction of HSP72 (HSPA1A) in vitro (byheat shock or HSP72 transgene overexpression) reduces theexpression of inflammatory genes such as TNF120572 IL-1 IL-12IL-10 and IL-18 [46]

In contrast to the above findings eHSP70 proinflam-matory actions have been demonstrated to be mediatedby MyD88IRAKNF-120581B signal transduction pathway afterboth Toll-like receptor 2 (TLR2) and TLR4 binding in aCD14-dependent manner [47 48] thus promoting innateimmune activation [49] Due to the antagonistic actions ofthe heat shock proteins within the course of an inflam-matory response it is reasonable to hypothesize that thebalance between eHSP70 and iHSP70 might determinethe outcomemdasheither the induction or the attenuation ofinflammation Since low-grade inflammation is involved inseveral chronic diseases [17] the management of HSP70expression and its location can be crucial for the control ofinflammatory-related conditions such as T2DM We hereinsuggest that the ratio of the extracellularmediumHSP70 con-centration to intracellular HSP70 contents (eHSP70iHSP70)can determine the progress of insulin resistance and theprogression of T2DM

Mediators of Inflammation 3

2 Obesity Low-Grade Inflammation andInsulin Resistance

The incidence of T2DM has increased dramatically over thelast fifty years and this is clearly associated with growingrates of obesity [50] and physical inactivity Obesity is linkedto a chronic proinflammatory state since adipose tissueexpansion and adipose associated immune cell activationresult in the release of several cytokines such as TNF120572 whichleads to the activation of serine threonine kinases JNKs andIKK [14] It is known that both JNK and IKK phosphorylateinsulin receptor (IR) substrate-1 (IRS-1) on Ser-307 leading tothe inactivation of the insulin receptor downstream response[14] Also chronic activation of IKK has been reported indiabetic patients while a reduction in IKK activity preventsthe development of insulin resistance in vitro and in vivo [51]In addition lipid oversupply and hyperglycemia can lead toincreased deposition of lipid species such as diacylglycerolsand ceramides which can also activate JNK and IKK inliver andor skeletal muscle leading to insulin resistance [52]causing sustained hyperglycemia and hyperlipemia

Hyperglycemia per se is also known to be involved ininflammation and diabetes-associated vascular complica-tions arising from reactive oxygen species generation andaction [53 54] Chronic hyperglycemia induces the pro-duction of reactive oxygen species (ROS) [55] leading toenhancement of protein oxidation DNA oxidation and lipidperoxidation The free radical gas nitric oxide (NO∙) alsoplays a role in the insulin resistant state generated by pro-inflammatory cytokines NO∙ is synthesized at high rates bythe inducible form of nitric oxide synthase (iNOS encodedby the NOS-2 gene) which plays a significant role in celldamage associated with obesity and T2DM Interestingly aphysiological concentration of this free radical is requiredto stimulate necessary functions such as muscle GLUT4expressiontranslocation [56] and insulin secretion by 120573-cells[57] However at high concentrations NO∙ compromisesinsulin-stimulated glucose transport in skeletal muscle andcan also be toxic to 120573-cells inducing death Accordingly acutetreatment with NO∙ donors results in a reduction of insulin-stimulated glucose uptake and glycogen synthesis in isolatedsoleus muscle inducing decreased IR120573 and IRS-1 activityIn vivo this treatment also promotes insulin resistance inhealthy animals by the reduction of IRS-1 levels Obeseobob mice or rats submitted to high fat diet (HFD) haveshown enhanced NOS-2 expression associated with insulinreceptor and Akt S-nitrosylation which can be dismissedby rosiglitazone treatment by virtue of its NOS-2 expressioninhibiting activity [58]

Expanded adipose tissue triggers the release of inter-leukin-6 (IL-6) in obese subjects that is associated withalterations in glucose uptake by the skeletal muscle [59]Nevertheless it is possible that there is a dual role of serumIL-6 on glucosemetabolism probably related to the exposuretime of and concentration of IL-6 Accordingly acute IL-6treatment may increase glucose uptake in C2C12 myotubesby stimulating AMP-activated protein kinase (AMPK) ina serinethreonine kinase 11- (LKB1-) dependent pathwaywhich induces downstream AS160 activation [60] while

IL-6 may induce a modest increase in the glucose infusionrate after 4 h of hyperinsulinemic-euglycemic clamp in mice[61] Moderate doses of IL-6 stimulate basal and insulin-stimulated glucose uptake in L6 myotubes and 3T3 cellsline after 2 h [61] In addition physiological concentrationsof IL-6 were reported to stimulate insulin secretion by iso-lated pancreatic islets and BRIN-BD11 clonal 120573-cells throughAMPK activation [62] However chronic treatment (24 h)with this cytokine has been demonstrated to be able to causeinsulin resistance in C2C12 murine myotube cell line dueto impairment of insulin signaling (IRSAKT cascades) in aJNK12-dependent manner [60]

Finally obesity is often associated with a vicious cyclein which adipose tissue expansion increases the levels offree fatty acids (FFA) and proinflammatory cytokines in thecirculation which together with hyperglycemia and alteredlipoprotein profiles increase the synthesis and accumulationof intramyocellular triglycerides (IMCT) [63] Sedentarybehavior and aging are conditions related to a decreasedmobilization of the IMCT resulting in an increased synthe-sis of toxic fatty-acid-delivered metabolites (FADM) Thesemetabolites cause in turn an elevation in the productionof reactive oxygen and nitrogen species (ROS and RNS)resulting in oxidativenitrosative stress mitochondrial dys-function and the activation of stress associated transcriptionfactors such as NF-120581B which is followed by increasedproduction and release of proinflammatory cytokines (egTNF120572) TNF120572 is a major driver of insulin resistance in skele-tal muscle and in addition it can also induce activation ofstress signals in pancreatic 120573-cells leading to mitochondrialdysfunction that culminates in cell failure and death [64]

3 Role of iHSP70 in Insulin Sensitivity

It has been reported that iHSP72 mRNA levels are decreasedin skeletal muscle of T2DM patients which is correlatedwith the ldquostatusrdquo of insulin resistance [65] whereas heatshock-like therapies (ie whole body warming transgenicoverexpression or pharmacological mechanisms to elevateHSPA1A protein expression) protect against high-fat-diet-and obesity-induced hyperglycemia hyperinsulinemia glu-cose intolerance and insulin resistance [14 65 66] Althoughthe underlying mechanism(s) culminating in lower iHSP70expression in T2DM individuals are not fully understood theobserved reductions are likely to be a result of the concertedcontribution of (i) reduction in the rate of HSP70 proteinsynthesis due to attenuation of initiation and elongationphases of translation (ii) suppression of HSF-1 activationand binding to HSE via an increase in glycogen synthasekinase-3120573 activity (GSK-3120573) as previously suggested [67]and (iii) decreased HSF1 expression In this regard it hasbeen recently proposed that long-term inflammatory stimuliemanating from the adipose tissue of obese individuals couldrepress HSF-1iHSP70 axis because continuous activationof inflammasome NLRP3 may lead to a state of cellularsenescence which abolishes the expression and activity ofHSF-1 [68] In line with this is the observation that theexpression of both HSF-1 and iHSP70 in adipose tissue andthe liver of type III obese patients is dramatically reduced


while the activation of JNKs is reciprocally enhanced in thesame tissues [18]

As discussed above the anti-inflammatory effect ofiHSP70 is attributed mainly to its capacity of interaction withNF-120581B In fact IKK-120573 and NF-120581B activity have been found tobe increased in different obese experimentalmodels Chronicactivation of IKK has been reported in diabetic patientswhile a reduction in IKK activity prevents the developmentof insulin resistance in vitro and in vivo [51] High-fathigh-carbohydrate intake may result in oxidative stress andconsequent NF-120581B activation in obese subjects [69] Onthe other hand NF-120581B DNA binding is suppressed afterheat shock [70] and upregulation of HSPA1A can negativelyaffect NF-120581B activity in skeletal muscle [71] In additionoverexpression of HSPA1A can restrict NO production andrelease by transfected cells in amechanism dependent onNF-120581B DNA binding and subsequent iNOS gene expression [70]

Heat treatment is capable of inducing HSPA1A expres-sion in several tissues preventing various obesity-elicitedmetabolic effects at molecular level leading to improvementof glucose tolerance insulin-stimulated glucose transportand insulin signaling accompanied by the reduction in JNKand IKK120573 activities in skeletal muscle [72] and liver [14] ofHFD mice which is almost completely abolished in trans-genicHSPA1A++mice [14] Furthermore iHSP70 expressionhas been shown to decrease JNK activity irrespective of stressstimulus [73] This is crucial for insulin sensitivity sinceenhanced rate of JNK phosphorylation is associated withglucose intolerance and insulin resistance in skeletal muscleof obese mice an effect which may be attenuated by long-term (16-week) heat treatment (415∘C) and is also observedin transgenic HSPA1A++ mice [14] Heat treatment alsoinduced improvement of mitochondrial function increasingcitrate synthase and 120573-hydroxyacyl-CoA-dehydrogenase (120573-HAD) activity in soleus and extensor digitorum longus (EDL)muscle [14] Interestingly a 12-week period of heat treatmentis unable to change fasting blood glucose of obese rats but itdoes attenuate insulin levels and decrease whole body glucoseclearance induced by HFD [72]

In obese HFD animals phosphorylation of IRS1 in Tyr612and the consequent downstream phosphorylation of Akt inSer473 and activation ofAS160 have been found to be reducedin the skeletal muscle while heat treatment reverted thisresponse in a process associated with HSPA1A expression[72] Moreover JNK inhibition has been demonstrated torapidly occur in a dose-dependent manner in heat-shockedNIH 3T3 fibroblasts via interaction between JNK1 andHSPA1A [74] Finally studies have demonstrated that HSP70is able to bind to the insulin receptor enhancing its recyclingrate after heat shock [75 76] which suggests that heat shockproteins may have direct influence upon insulin receptorfunction and activity

4 Role of eHSP70 in Insulin Resistance and120573-Cell Dysfunction

In contrast to its intracellular proinsulin signaling and anti-inflammatory effect extracellular HSP70 (eHSP70) when

chronic elevated is associated with inflammatory condi-tions including T2DM [17] Other cells (eg lymphocytesmacrophages epithelial cells dendritic cells neuronal cellsand hepatocytes) have been reported to release HSP70 pro-teins via (i) active mechanisms such as vesicular secretion(classical pathway in rest conditions) (ii) lipid rafts or (iii)exosomes [77] Once in the bloodstream eHSP70 may act asa paracrine factor [77]

With respect to the extracellular compartment eHSP70can bind to TLR2 and TLR4 in a variety of cells [77] leadingto the activation of proinflammatory pathways via MyD88and TIRAP that signal downstream to NF-120581B via IRAK4TRAF6 and IKK and inducing JNK activation via MEKK47[49 78] although high-affinity binding of eHSP70 to othersurface receptors has also been described [79] The signaltriggered by eHSP70 promotes typical immunoinflammatoryresponses directed to the combat of infections and bacterialinfiltration through the production and release of NO∙ andproinflammatory cytokines such as TNF120572 and IL1120573 [80]Moreover eHSP70 responses are positively associated withclassical inflammatory parameters such as CRP fibrinogenand monocyte counts [81] being commonly found in clinicalsituations in which a danger signalization to immune systemmust be required [77] Indeed increased serum HSP70 hasbeen reported in chronic and age-related diseases [82ndash84]

Interestingly during conditions in which individuals arechronically exposed to elevated eHSP70 levels changes inthe iHSP70 content are also observed [14 65] In fact inT2DM iHSP70 is reduced in insulin-dependent tissues suchas skeletal muscle and adipose tissue [14 17 65] while theeHSP70 is elevatedThis profile is commonly found in T2DMwhere obesity is an aggravating factor for the undesirablehigh eHSP70iHSP70 ratio [17] In addition serum HSP70levels were found to be higher in long-term (gt5 years) T2DMpatients as compared to newly diagnosed ones [85]

High eHSP70-mediated stimulation of TLR24 mayseverely jeopardize insulin signaling Accordingly TLR24-dependent activation of JNKs promotes phosphorylation ofIRS-1 at Ser307 in rodents (equivalent to Ser312 in humans)leading to inhibition of Akt activation [86] and consequentlyto a reduced glucose uptake by sensitive tissues and to a stateof resistance to insulin action Moreover it has been shownthat TLR2 is central to palmitate-induced insulin resistance[87] via JNK-mediated phosphorylation of IRS12 [88] Onthe other hand loss-of-function mutation in TLR4 preventsHFD-induced obesity and insulin resistance [89]

Ser307 phosphorylation of rodent IRS-1 may also beelicited by inflammatory cytokines via IKK in a process thatcan be inhibited by cp-PGs [90] which are powerful anti-inflammatory autacoids possessing iHSP70-inducing capac-ity [40] Indeed inhibitory Ser307 phosphorylation of IRS-1is a physiologicalmechanismof feedback inhibition of insulinsignal that is under the control of both insulinIGF1 andinflammatory cytokines though via different downstreampathways [91] TLR4 expression and signaling dependent oneHSP70 is increased in obese and T2DM subjects an effectthat can explain the high basal rate ofMAPKphosphorylationand NF-120581B activation found in these patients [92ndash95] Theabove findings help to explain why inhibition or absence of


TLR4 confers protection against insulin resistance in skeletalmuscle [96] adipose tissue and liver [26 97]

Finally as recently found eHSP70 is positively correlatedwith insulin resistance and inflammation in elderly peopleThis may indicate a role for eHSP70 in impairment of insulinsignaling in the skeletal muscle that occurs with advanced ageand in T2DM [98] In addition the same group has shownthat chronic exposure of 120573-cells and islets to increased con-centrations of eHSP70 results in 120573-cell death and altered cellbioenergetics a phenomenon that apparently is mediatedthrough TLR-2 and TLR-4 activation [98] Since in T1DMthere is a dramatic increase in eHSP70 and in T2DM andaging there is a slow chronic increase in the concentrationof this protein we educe that chronic exposure of pancreatic120573-cells to eHSP70 may lead to 120573-cell failure and loss offunctional integrity in vivo

5 The eHSP70 to iHSP70 Ratio TheChaperone Balance Hypothesis

Based on the previous discussion while iHSP70 is clearlyprotective antiapoptotic anti-inflammatory and associ-ated with normal insulin sensitivity eHSP70 is related toa proinflammatory response decreased expression of theanti-inflammatory iHSP70 and reduced insulin sensitivityBecause of this we suggest that the ratio of compartmentaldistributions of HSP70 between extra- and intracellularlocations may determine the outcome of the inflammationand its associated insulin resistance

In a recent study our group observed that the ratiobetween plasma eHSP70 and iHSP70 in lymphocytes fromrats submitted to different loads of acute exercise can indicatethe inflammatory status (Heck et al manuscript in prepa-ration) Accordingly assuming the ratio 119877 = [eHSP70][[iHSP70] = 1 for the controls (resting unstimulated)moderate exercise produces a shift in 119877 to up to ca 5which is paralleled by an elevation in inflammatory markersand stimulation of cell proliferation 119877 values higher than5 denote an exacerbated proinflammatory response Con-versely 119877 values between 0 and 1 indicate a predominantlyanti-inflammatory status Thus changes in the ratio betweenextra- and intracellular HSP70 emerge as a potentially newbiomarker for inflammation and as a very sensitive indicatorof inflammatory status

We applied this simple mathematical calculation to pub-lished data from studies elsewhere For instance Yang andcollaborators have investigated the correlation between thelevel of exposure to pollution and eHSP70 in steel workers[99] From the data obtained in this study we calculated119877 (plasma to lymphocyte ratio) as 55 65 and 88 respec-tively for low moderate and high exposure as comparedto controls (119877 = 10) We applied the same calculationto the data by Rodrigues-Krause and colleaguesrsquo obesity-diabetes study [17] in which HSP70 was investigated inhealthy obese (considered the controls herein) nonobeseT2DM and in obese T2DM patients In this case settlingcontrols as 119877 = 10 we get 18 for T2DM and 60 for obeseT2DMpatients in which inflammatory unbalance was foundAlthough healthy lean subjects had not been evaluated in

this work it is likely that such 119877 marks should be evenhigher if 119877 were taken in comparison with such controlsAdditionally changes in 119877 calculation seem to be validduring heat exposure as 119877 values correlated with the heatexposure of peripheral blood mononuclear cells to differenttemperatures within a physiological range Accordingly [100]heat-shocked cells concomitantly measured HSP70 at 37∘C(119877 = 100) 39∘C (119877 = 145) 42∘C (119877 = 065) and43∘C (119877 = 048) Interestingly at 45∘C in which cells areknown to trigger JNK-dependent prosurvival inflammatorypathways [74] 119877 value was calculated as 197 confirmingproinflammatory expectations Moreover in a combinedprotocol of exercise training (60min during 11 days in a tread-mill 169ndash220msdotsminus1 1 grade) and heat acclimation (rectaltemperature elevation by 1∘C for the duration of the exercisesessions at 40∘C room temperature) [101] have observed inhuman volunteers HSPA1A alterations (in plasma and totalleukocytes) that after conversion to 119877 values furnish thefollowing picture before training 119877 values were 111 at restand 051 two days after the primary test after training andacclimation 119877 values were 022 at rest and 027 two days afterthe last training session These values can be explained bythe fact that eHSP70 plasma contents raised by ca 50 inuntrained individuals evaluated 48 h after the priming testwhile under the same circumstances iHSP70 was found tobe 29-fold the remaining values On the other hand exercisetraining combined with heat acclimation evoked a 42-foldenhancement in intracellular HSPA1A contents (hence ananti-inflammatory response) which remained unaltered 48 hafter the last session while eHSP70 did not change any moreremaining near the rest of values observed in untrainedvolunteers

The above observations led us to postulate that 119877 valuesand particularly changes in 119877 values could be used as apredictor gauge for the inflammatory response that culmi-nates in insulin resistance and diabetes despite the methodused to assess intra- and extracellular HSP70 contentsNotwithstanding 119877 values may be also useful for applicationin several other inflammatory diseases and conditions besidesbeing of value in controlling exercise impact over inflam-matory status Furthermore since eHSP70 and iHSP70 aredirectly related to insulin sensitivity 119877 value application indiabetes appears to be straightforward Figure 1 summarizesthis hypothesis

6 Changing R Values The Role ofPhysical Exercise

As discussed above eHSP70 to iHSP70 ratio (represented by119877 values) may determine the fate of the insulin sensitivitytowards either its improvement (lower ratio) or its reduc-tionimpairment (higher ratio) In this regard strategiescapable of changing the HSP70 contents in the intra- orextracellular space or both compartments are likely to beused as a therapeutic strategy for the prevention or treatmentof T2DM and its efficiency could be precisely followed by theassessment of 119877 values during the course of such treatmentIn this sense physical exercise fulfills all the prerequisites itis a known modulator of eHSP70 (whose levels drop under


Stresssignalexercise

HSF-1

TLRInflammatory

pathway

Adipose tissue

expansion

Proinflammatorycytokinesadipokines

Liver

Immune cells

Skeletal muscle

Oxidative stressprotein

damage

Diabetes

eHSP70

iHSP70eHSP70

eHSP70iHSP70

eHSP70iHSP70 Insulin sensitivity On Off

HSP70 status andR values

Changing the ratio exercise

Exer

cise

Obe

sity

The chaperone balance hypothesis

Brain

iHSP70

eHSP70

Insulin

signaling

oplus

oplus

oplus

oplus

oplus

oplus

oplus

⊖

⊖

⊖

⊖

5

R10

Figure 1 The chaperone balance Adipose tissue expansion leads to chronic release of proinflammatory cytokines and adipokines Thelow-grade inflammation can induce (i) activation of NF-120581B-dependent inflammatory pathways leading to the blockage of iHSP70 and toinsulin resistance (ii) release of proinflammatory eHSP70 chronically from immune cells and (iii) ROS and RNS oxidativenitrosativestress that leads to protein damage and denaturation eHSP70 is increased as a danger signal and to combat the plasma oxidative damagehowever when chronically elevated it (i) induces further immune activation and proinflammatory response and (ii) activates TLR and theinflammatory pathway leading to the reduction of HSF-1 activation and eventually to reduced iHSP70 Lower iHSP70 causes (i) reducedinsulin sensitivity (ii) intensification of the NF-120581B activation and inflammation and (iii) reduced antioxidant antiapoptotic and anti-inflammatory capacity The long-term insulin resistance determines the onset of diabetes completing this positive feedback mechanismLower panels when the eHSP70iHSP70 ratio chronically changes in favor of eHSP70 the ldquoinsulin sensitivity buttonrdquo is switched off and 119877values ([eHSP70][iHSP70]) rise exercise induces iHSP70 expression while the release of eHSP70 responds in an opposite manner 119877 valuesbetween 0 and 1 indicate an anti-inflammatory status and between 1 and 5 indicate an optimum immunoinflammatory surveillance statuswhile 119877 values above 5 suggest an undesirable chronic proinflammatory status

exercise training) and iHSP70 (which tends to rise under thesame circumstances) In other words although acute exercisebouts signalize a ldquostressful situationrdquo to all physiologicalsystems (please see Heck et al 2011 for review [102]) leadingto augmented but just momentary eHSP70 plasma levels[103] exercise training or regular physical activity tends toreduce the stressful impact of each exercise session leading todecreased eHSP70 and enhanced iHSP70 during the course of

training For this reason exercise can also be used as a tool todecrease or maintain the normal 119877 values and consequentlyoptimum insulin sensitivity In fact improvement of glucoseuptake and storage aswell as increase in the oxidative capacityof different muscle fibers has been shown to be associatedwith increased iHSP70 expression in the skeletal muscle [1466] Moreover physical exercise has been proposed as analternative strategy for T2DM patient treatment by virtue of


its iHSP70 enhancing capacity patients submitted to acuteexercise bouts or moderate training have shown significantincrease in muscle HSP70 expression [66 104] which isdirectly associated with reduction of insulin resistance [1472]

Exercise-induced expression of iHSP70 in skeletal musclehas a major role in restoring muscle metabolic functionalitybesides it provides cytoprotection to damaged cells Suchan iHSP70 inducing ability has been shown as a resultantin different protocols of exercise including eccentric con-centric not damaging aerobic or resistive all of which arecapable of inducing intramuscular HSP70 expression [104ndash106] Although time and intensity of the physical effort aredeterminant factors to increase of intramuscular HSPA1Aits rise may be detectable just 2 h after the onset of an acuteexercise session when HSPA1A mRNA expression peaks[107] Moreover exercise-induced iHSP70 presents a timeand intensity dependence [108]

Since iHSP70 family members promote the facilitation ofprotein transport intomitochondria allowing and improvingstructural integrity of the organelle during fast energy flowiHSP70 content has been correlated with an increase inthe oxidative capacity of muscle cells Several studies havedemonstrated the relationship between high iHSP70 levelsin skeletal muscle and increased activity of mitochondrialenzymes after short training period [109] On the contrarydecreased mitochondrial function is known to be associatedwith the accumulation of intramyocyte triglycerides (and itsbyproducts) insulin resistance and diabetes For this addi-tional reason exercise-induced iHSP70 expression can leadto improvements in metabolite oxidation and consequentlyinsulin sensitivity

As regarded above HSF-1 is negatively regulated byGSK3120573 a serinethreonine kinase that phosphorylates thisfactor on Ser303 keeping it in its inactive form in the cytosolDuring acute physical exercise GSK3120573 activity has beenfound to be nevertheless 30 decreased in Vastus lateralismuscle concomitantly with Akt phosphorylation in Ser473and glycogen synthesis (GS) activation [110] In addition adirect relation between iHSP70 and IL-6 has been reportedDuring physical exercise IL-6 can be expressed [111] andreleased [112] by the skeletal muscle and within extracellularspace binds to the IL-6 receptor in an autocrine action[113] Interestingly the ldquomyokinerdquo IL-6 has also been foundto induce HSF-1 translocation to the nucleus upregulatingheat-induced HSP70 gene protein expression and activity inhuman hepatic cells in a PI3KAktGSK3120573 dependent path-way [114] On the other hand absence of IL-6 is associatedwith decreased expression of HSPA1A in skeletal and cardiacmuscle of mice challenged with LPS although IL-6 seems notto be required for exercise-induced HSP expression [115]

The molecular mechanism(s) underlying the connectionbetween iHSP70 expression and increased energy flow are notclear yet However changes in ATPADP ratio seem to workas a signal to the activation of different kinases such asAMPKthat is capable of decreasing GSK3120573 activity [116] AMPKis also regulated by Ca2+ during muscle contraction thatactivates Ca2+calmodulin protein kinase kinase (CaMKK) in

an LKB1-dependent pathway [117]Thus AMPK activation asresult of muscle contractionmay act as a stimulus for iHSP70induction in a HSF1-dependent way

Exercise is also known to induce eHSP70 release andaccumulation into the circulation [107] While the sourceof the eHSP70 during the exercise is still in debate currentdata have suggested that hepatosplanchnic tissues should beinvolved [103] Additionally 1205721 adrenoreceptors in the liverseem to participate [118 119] in a way that is dependenton exercise intensity type duration and training status[120] In addition recent data suggests eHSP70 concentrationincreases once systemic temperature and sympathetic activityexceed minimum endogenous criteria and is likely to bemodulated by large and rapid changes in core temperature[121] However acute physical exercise is an inducer ofeHSP70 release into the blood and exercise is not a factorof maintenance of high eHSP70 indefinitely because chronicexercise (training) suppresses eHSP70 levels [101] Hence theexpected deleterious effects of long-term exposure to higheHSP70 concentrations are never attained

In response to eHSP70 macrophages can release IL-1120573 IL-6 and TNF120572 [122] all cytokines involved in insulinresistance Interestingly in trained obese Zucker rats themacrophage cytokine release profile in response to eHSP70is changed In this case the macrophages from obese Zuckerrats released less IL-1120573 and TNF120572 but more IL-6 thanmacrophages from lean animals indicating that habitualexercise improved the release of proinflammatory cytokinesby macrophages [122]

7 Concluding Remarks

Taken as a whole the above findings clearly indicate thatwhile iHSP70 is protective and anti-inflammatory being asso-ciated with normal insulin sensitivity eHSP70 chronicallyproduced in response to low-grade inflammation (but not tochronic exercise) is related to a proinflammatory responsedecreased expression of iHSP70 and reduced insulin sensi-tivity Therefore we suggest that the ratio of HSP70 contentsbetween the extra- and intracellular compartments maydictate the outcome of the inflammation and associatedinsulin resistance (Figure 1) Since exercise is able tomodulateboth eHSP70 and iHSP70 it is reasonable to predict thatexercise is the most efficient and powerful tool currentlyavailable to normalize andormaintain eHSP70iHSP70 ratioat appropriate levels thus preventing T2DMThe appropriatetype intensity duration and frequency of exercise that bestfit each condition need to be determined

The versatility of the HSP70 to induce differentinflammation-related responses according to its location(intra- versus extracellular) places this protein as a masterregulator for the fine-tuned control of the immune systemwhile iHSP70 induces inactivation of NF-120581B eHSP70induces the opposite effect Thus we suggest that theeHSP70iHSP70 ratio may represent a better marker for theimmunoinflammatory status of the whole body in exerciseas well as in many types of diseases Finally we advocatethat 119877 values could be useful not only in assessing thecourse of therapeutic approaches in obesity-induced insulin


insensitivity and diabetes but also to evaluate inflammatorystatus in inflammation-related diseases (eg atherosclerosisand other cardiovascular diseases rheumatoid arthritissepsis and obesity-related nonalcoholic fatty liver disease)as well as in exercise training directed to immunosuppressedand cardiovascular patients Hence the above propositionsmay have an important diagnostic value for such patientssince ldquoHSP70 statusrdquo determined through 119877 values may beeasily obtained from the ratio of extracellular (plasma) tointracellular (circulating blood leukocytes or mononuclearcells) HSP70 content from a blood sample

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Mauricio KrauseThiago Gomes Heck and Aline Bittencourtequally contributed to this work

Acknowledgments

Thisworkwas partially supported by grants received from theBrazilian National Council for Scientific and TechnologicalDevelopment (CNPq grants from MCTCNPq MSDECITCT-CIOTEC and CTSaude process nos 5510972007-84026262012-5 and 4023642012-0 to Paulo Ivo Homem deBittencourt Jr) CNPq (process no 5638702010-9 to RuiCuri) CNPq (process nos 4023982013-2 and 3723732013-5 to Mauricio Krause) and FAPESP (to Rui Curi) PhilipNewsholme was supported by the ldquoScience without BordersrdquoSpecial Visiting Professor Program from CNPq ThiagoGomes Heck was supported by a fellowship from CAPES-Brasılia Aline Bittencourt Sofia Pizzato Scomazzon andMauricio Krause were supported by fellowships from CNPqThe authors thank the UCD School of Biomolecular andBiomedical Science (Dublin Ireland) and the School ofBiomedical Sciences CHIRI Biosciences Curtin UniversityPerth Western Australia for their support

References

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[2] X-MYangG F Baxter R J HeadsDMYellon JMDowneyand M V Cohen ldquoInfarct limitation of the second windowof protection in a conscious rabbit modelrdquo CardiovascularResearch vol 31 no 5 pp 777ndash783 1996

[3] V Richard N Kaeffer and C Thuillez ldquoDelayed protectionof the ischemic heartmdashfrom pathophysiology to therapeuticapplicationsrdquo Fundamental and Clinical Pharmacology vol 10no 5 pp 409ndash415 1996

[4] R P Beckmann M Lovett and W J Welch ldquoExamining thefunction and regulation of hsp 70 in cells subjected tometabolicstressrdquo Journal of Cell Biology vol 117 no 6 pp 1137ndash1150 1992

[5] E G Noble K J Milne and C W J Melling ldquoHeat shockproteins and exercise a primerrdquo Applied Physiology Nutritionand Metabolism vol 33 no 5 pp 1050ndash1065 2008

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[7] W J Chirico M G Waters and G Blobel ldquo70K heat shockrelated proteins stimulate protein translocation into micro-somesrdquo Nature vol 332 no 6167 pp 805ndash810 1988

[8] C Garrido S Gurbuxani L Ravagnan and G Kroemer ldquoHeatshock proteins endogenousmodulators of apoptotic cell deathrdquoBiochemical andBiophysical ResearchCommunications vol 286no 3 pp 433ndash442 2001

[9] P I Homem de Bittencourt Jr D J Lagranha AMaslinkiewiczet al ldquoLipoCardium endothelium-directed cyclopentenoneprostaglandin-based liposome formulation that completelyreverses atherosclerotic lesionsrdquo Atherosclerosis vol 193 no 2pp 245ndash258 2007

[10] A Ianaro A Ialenti P Maffia P Di Meglio M Di Rosa andMG Santoro ldquoAnti-inflammatory activity of 15-deoxy-delta1214-PGJ2 and 2-cyclopenten-1-one role of the heat shock responserdquoMolecular Pharmacology vol 64 no 1 pp 85ndash93 2003

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[31] H-W Chen H-T Kuo S-J Wang T-S Lu and R-CYang ldquoIn vivo heat shock protein assembles with septic liverNF-kappaBI-kappaB complex regulating NF-kappaB activityrdquoShock vol 24 no 3 pp 232ndash238 2005

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[34] D Tsuchiya S Hong Y Matsumori et al ldquoOverexpressionof rat heat shock protein 70 reduces neuronal injury aftertransient focal ischemia transient global ischemia or kainicacid-induced seizuresrdquo Neurosurgery vol 53 no 5 pp 1179ndash1188 2003

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[36] Z Zheng J Y Kim H Ma J E Lee and M A YenarildquoAnti-inflammatory effects of the 70 kDa heat shock proteinin experimental strokerdquo Journal of Cerebral Blood Flow andMetabolism vol 28 no 1 pp 53ndash63 2008

[37] P C Geiger and A A Gupte ldquoHeat shock proteins are impor-tant mediators of skeletal muscle insulin sensitivityrdquo Exerciseand Sport Sciences Reviews vol 39 no 1 pp 34ndash42 2011

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[47] A Asea E Kabingu M A Stevenson and S K CalderwoodldquoHSP70 peptidembearing and peptide-negative preparationsact as chaperokinesrdquo Cell Stress Chaperones vol 5 no 5 pp425ndash431 2000

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[59] P Mitrou V Lambadiari E Maratou et al ldquoSkeletal muscleinsulin resistance in morbid obesity the role of interleukin-6 and leptinrdquo Experimental and Clinical Endocrinology andDiabetes vol 119 no 8 pp 484ndash489 2011

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[80] J Campisi T H Leem and M Fleshner ldquoStress-inducedextracellular Hsp72 is a functionally significant danger signalto the immune systemrdquo Cell Stress amp Chaperones vol 8 no 3pp 272ndash286 2003

[81] R Njemini C Demanet and T Mets ldquoInflammatory statusas an important determinant of heat shock protein 70 serumconcentrations during agingrdquo Biogerontology vol 5 no 1 pp31ndash38 2004

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[83] S K Dutta M Girotra M Singla et al ldquoSerum HSP70 a novelbiomarker for early detection of pancreatic cancerrdquo Pancreasvol 41 no 4 pp 530ndash534 2012

[84] E Dulin P Garcıa-Barreno andM C Guisasola ldquoExtracellularheat shock protein 70 (HSPA1A) and classical vascular riskfactors in a general populationrdquo Cell Stress and Chaperones vol15 no 6 pp 929ndash937 2010

[85] M Nakhjavani A Morteza L Khajeali et al ldquoIncreased serumHSP70 levels are associated with the duration of diabetesrdquo CellStress and Chaperones vol 15 no 6 pp 959ndash964 2010

[86] Y H Lee J Giraud R J Davis and M F White ldquoc-JunN-terminal kinase (JNK) mediates feedback inhibition of theinsulin signaling cascaderdquo The Journal of Biological Chemistryvol 278 no 5 pp 2896ndash2902 2003

[87] J J Senn ldquoToll-like receptor-2 is essential for the developmentof palmitate-induced insulin resistance in myotubesrdquo Journal ofBiological Chemistry vol 281 no 37 pp 26865ndash26875 2006

[88] G Solinas W Naugler F Galimi M-S Lee and M KarinldquoSaturated fatty acids inhibit induction of insulin gene tran-scription by JNK-mediated phosphorylation of insulin-receptorsubstratesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 44 pp 16454ndash164592006

[89] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[90] Z Gao D Hwang F Bataille et al ldquoSerine phosphorylationof insulin receptor substrate 1 by inhibitor kappa B kinasecomplexrdquo The Journal of Biological Chemistry vol 277 no 50pp 48115ndash48121 2002

[91] L Rui V Aguirre J K Kim et al ldquoInsulinIGF-1 and TNF-120572 stimulate phosphorylation of IRS-1 at inhibitory Ser307 viadistinct pathwaysrdquo Journal of Clinical Investigation vol 107 no2 pp 181ndash189 2001

[92] O I Vitseva K Tanriverdi T T Tchkonia et al ldquoInducibletoll-like receptor and NF-120581B regulatory pathway expression inhuman adipose tissuerdquoObesity vol 16 no 5 pp 932ndash937 2008

[93] S M Reyna S Ghosh P Tantiwong et al ldquoElevated toll-likereceptor 4 expression and signaling in muscle from insulin-resistant subjectsrdquoDiabetes vol 57 no 10 pp 2595ndash2602 2008

[94] H A Koistinen A V Chibalin and J R Zierath ldquoAberrant p38mitogen-activated protein kinase signalling in skeletal musclefrom Type 2 diabetic patientsrdquo Diabetologia vol 46 no 10 pp1324ndash1328 2003

[95] M R Dasu S Devaraj S Park and I Jialal ldquoIncreased Toll-Like Receptor (TLR) activation and TLR ligands in recentlydiagnosed type 2 diabetic subjectsrdquo Diabetes Care vol 33 no4 pp 861ndash868 2010

[96] M S Radin S Sinha B A Bhatt N Dedousis and R MOrsquoDoherty ldquoInhibition or deletion of the lipopolysaccharidereceptor Toll-like receptor-4 confers partial protection againstlipid-induced insulin resistance in rodent skeletal musclerdquoDiabetologia vol 51 no 2 pp 336ndash346 2008

[97] M Saberi N-B Woods C de Luca et al ldquoHematopoieticcell-specific deletion of toll-like receptor 4 ameliorates hepaticand adipose tissue insulin resistance in high-fat-fed micerdquo CellMetabolism vol 10 no 5 pp 419ndash429 2009

[98] M Krause K Keane J Rodrigues-Krause et al ldquoElevated levelsof extracellular heat-shock protein 72 (eHSP72) are positivelycorrelated with insulin resistance in vivo and cause pancreatic

120573-cell dysfunction and death in vitrordquo Clinical Science vol 126no 10 pp 739ndash752 2014

[99] X Yang J Yuan J Sun et al ldquoAssociation between heat-shockprotein 70 gene polymorphisms and DNA damage in periph-eral blood lymphocytes among coke-oven workersrdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 649 no 1-2 pp 221ndash229 2008

[100] C Hunter-Lavin E L Davies M M F V G Bacelar M JMarshall S M Andrew and J H H Williams ldquoHsp70 releasefrom peripheral blood mononuclear cellsrdquo Biochemical andBiophysical Research Communications vol 324 no 2 pp 511ndash517 2004

[101] F D CMagalhaes F T Amorim R L F Passos et al ldquoHeat andexercise acclimation increases intracellular levels of Hsp72 andinhibits exercise-induced increase in intracellular and plasmaHsp72 in humansrdquo Cell Stress and Chaperones vol 15 no 6 pp885ndash895 2010

[102] T G Heck C M Scholer and P I H de Bittencourt ldquoHSP70expression does it a novel fatigue signalling factor fromimmune system to the brainrdquo Cell Biochemistry and Functionvol 29 no 3 pp 215ndash226 2011

[103] M A Febbraio P Ott H B Nielsen et al ldquoExercise induceshepatosplanchnic release of heat shock protein 72 in humansrdquoJournal of Physiology vol 544 no 3 pp 957ndash962 2002

[104] M A Febbraio and I Koukoulas ldquoHSP72 gene expression pro-gressively increases in human skeletalmuscle during prolongedexhaustive exerciserdquo Journal of Applied Physiology vol 89 no 3pp 1055ndash1060 2000

[105] H S Thompson P M Clarkson and S P Scordilis ldquoTherepeated bout effect and heat shock proteins intramuscularHSP27 and HSP70 expression following two bouts of eccentricexercise in humansrdquo Acta Physiologica Scandinavica vol 174no 1 pp 47ndash56 2002

[106] J Liu D Zhang X Mi et al ldquop27 suppresses arsenite-induced Hsp27Hsp70 expression through inhibiting JNK2c-Jun- and HSF-1-dependent pathwaysrdquoThe Journal of BiologicalChemistry vol 285 no 34 pp 26058ndash26065 2010

[107] R C Walsh I Koukoulas A Garnham P L Moseley MHargreaves and M A Febbraio ldquoExercise increases serumHsp72 in humansrdquo Cell Stress amp Chaperones vol 6 no 4 pp386ndash393 2001

[108] E Fehrenbach A M Niess K Voelker H Northoff and F CMooren ldquoExercise intensity and duration affect blood solubleHSP72rdquo International Journal of Sports Medicine vol 26 no 7pp 552ndash557 2005

[109] K Bender P Newsholme L Brennan and P MaechterldquoThe importance of redox shuttles to pancreatic 120573-cell energymetabolism and functionrdquo Biochemical Society Transactionsvol 34 no 5 pp 811ndash814 2006

[110] K Sakamoto M F Hirshman W G Aschenbach and LJ Goodyear ldquoContraction regulation of Akt in rat skeletalmusclerdquo Journal of Biological Chemistry vol 277 no 14 pp11910ndash11917 2002

[111] M H S Chan A L Carey M J Watt and M A Feb-braio ldquoCytokine gene expression in human skeletal muscleduring concentric contraction evidence that IL-8 like IL-6 isinfluenced by glycogen availabilityrdquo The American Journal ofPhysiologymdashRegulatory Integrative and Comparative Physiologyvol 287 no 2 pp R322ndashR327 2004

[112] C MacDonald J F P Wojtaszewski B K Pedersen B Kiensand E A Richter ldquoInterleukin-6 release from human skeletal


muscle during exercise relation to AMPK activityrdquo Journal ofApplied Physiology vol 95 no 6 pp 2273ndash2277 2003

[113] C Keller A Steensberg A K Hansen C P Fischer PPlomgaard and B K Pedersen ldquoEffect of exercise trainingand glycogen availability on IL-6 receptor expression in humanskeletal musclerdquo Journal of Applied Physiology vol 99 no 6 pp2075ndash2079 2005

[114] S J Wigmore K Sangster S J McNally et al ldquoDe-repressionof heat shock transcription factor-1 in interleukin-6-treatedhepatocytes is mediated by downregulation of glycogen syn-thase kinase 3120573 and MAPKERK-1rdquo International Journal ofMolecular Medicine vol 19 no 3 pp 413ndash420 2007

[115] K A Huey and B M Meador ldquoContribution of IL-6 to theHsp72Hsp25 and120572120573-crystallin responses to inflammation andexercise training in mouse skeletal and cardiac musclerdquo Journalof Applied Physiology vol 105 no 6 pp 1830ndash1836 2008

[116] S H Choi Y W Kim and S G Kim ldquoAMPK-mediated GSK3120573inhibition by isoliquiritigenin contributes to protecting mito-chondria against iron-catalyzed oxidative stressrdquo BiochemicalPharmacology vol 79 no 9 pp 1352ndash1362 2010

[117] J M Santos S B Ribeiro A R Gaya H-J Appell and JA Duarte ldquoSkeletal muscle pathways of contraction-enhancedglucose uptakerdquo International Journal of SportsMedicine vol 29no 10 pp 785ndash794 2008

[118] J D Johnson J Campisi C M Sharkey S L Kennedy MNickerson and M Fleshner ldquoAdrenergic receptors mediatestress-induced elevations in extracellular Hsp72rdquo Journal ofApplied Physiology vol 99 no 5 pp 1789ndash1795 2005

[119] G I Lancaster and M A Febbraio ldquoExosome-dependenttrafficking of HSP70 a novel secretory pathway for cellularstress proteinsrdquo Journal of Biological Chemistry vol 280 no 24pp 23349ndash23355 2005

[120] G I Lancaster and M A Febbraio ldquoMechanisms of stress-induced cellular HSP72 release implications for exercise-induced increases in extracellular HSP72rdquo Exercise ImmunologyReview vol 11 pp 46ndash52 2005

[121] O R Gibson A Dennis T Parfitt L Taylor P W Watt andN S Maxwell ldquoExtracellular Hsp72 concentration relates toa minimum endogenous criteria during acute exercise-heatexposurerdquoCell Stress andChaperones vol 19 no 3 pp 389ndash4002014

[122] J J Garcia LMartin-CorderoM D HinchadoM E Bote andE Ortega ldquoEffects of habitual exercise on the eHsp72-inducedrelease of inflammatory cytokines by macrophages from obesezucker ratsrdquo International Journal of SportsMedicine vol 34 no6 pp 559ndash564 2013

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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[8] and anti-inflammatory responses [9 10] More recentlythe HSP roles have been expanded to include control ofcell signaling [11] modulation of immune response [12]and modulation of chronic disease conditions [13] such asdiabetes obesity and insulin resistance [14 15]

The heat shock response is regulated by a family of heatshock transcription factors (HSFs) composed of four mem-bers (HSF 1ndash4) which are maintained in an inactive mono-meric form during nonstimulated conditions [16] HSF-1 isa primary regulator of heat shock response in mammaliancells and a low concentration of it has been associated witha number of human pathologies including T2DM [17] andobesity-related fatty liver disease [18] HSF-1 activation isa multistep mechanism that involves its phosphorylationtrimerization nuclear translocation and DNA binding toheat shock elements (HSE) located at the promoter regions oftargeted heat shock genes [15] nevertheless HSF-1 activationcan be negatively regulated by posttranscriptional modifica-tion such as phosphorylation in specific serine residues andphosphorylation-dependent sumoylation [19]

Heat shock proteins were long thought to be exclusivecytoplasmic proteins with functions restricted to the intra-cellular compartment However an increasing number ofobservations have indicated that theymay be released into theextracellular space (eHSP70) having a wide variety of effectson other cells [20] eHSP70 function is in general associatedwith the activation of the immune system [21] For exampleeHSP70 has been reported to stimulate neutrophil microbi-cidal capacity [22] and chemotaxis [23] and recruitment ofnatural killer (NK) cells [24] as well as cytokine productionby immune cells [12 25] In addition eHSP70 was recentlyhypothesized to be involved in the inducement of neural cellprotection under stress conditions [15]

An intriguing aspect of HSP70 physiology is its versatilityto induce antagonistic actions depending on the locationof the protein [17] For example iHSP70 exerts a powerfulanti-inflammatory effect while eHSP70 has the oppositerole inducing the activation of several proinflammatorypathways In fact chronic exposure to eHSP70 [26] inducesthe activation of several proinflammatory pathways probablyvia binding to membrane Toll-like receptors (see below)although eHSP70-peptides have also been shown to actas anti-inflammatory and immunosuppressive factors afterinternalization and antigen processing (see Borges et al 2012for review [27])

iHSP70 exerts its anti-inflammatory effect through theinteraction with the nuclear factor 120581B (NF-120581B) blockingits activation [28] NF-120581B is a ubiquitous transcription fac-tor originally discovered in B-lymphocytes that is essentialfor arming inflammatory responses to a variety of signalsimmune function endothelial cell activation and the controlof cell growth [29] iHSP70 hampers NF-120581B activation atseveral levels by impeding the phosphorylation of inhibitorof 120581B (I120581Bs) [30] by directly binding to I120581B kinase gamma(IKK120574) [31] which will result in continued binding (andinactivation of NF-120581B) thus inhibiting downstream inflam-matory signals This is corroborated by the finding thatiHSP70 binds with liver NF-120581BI120581B complex in the cytosolthus hindering transcription of TNF120572 and inducible nitric

oxide synthase (NOS2) genes [31] which are activated via aNF-120581B dependent mechanism Stress-induced elevations iniHSP70 inhibit c-Jun N-terminal kinase- (JNK-) dependentsignal transduction hence promoting cell survival [32]

iHSP70 affects apoptosis at various levels It can inhibitcaspase activation by interfering with Apaf-1 and prevent therecruitment of procaspase-9 to the apoptosome [33] HSP70also increases Bcl-2 expression and inhibits cytochrome Crelease [34]Overexpression ofHSP70 in lymphoid tumor celllines inhibited apoptosis by attenuation of caspase activation[35] The antiapoptotic effects of HSP70 have been reportedfor mouse brain tissue where HSP70 overexpression resultedin decreased infarct sizes improved neurological deficits andfewer apoptotic cells (determined by reduced DNA ladder-ing) after middle cerebral artery occlusion [36] HSP70s havealso been shown to decrease oxidative stress so that theyare part of the intracellular antioxidant machinery makingiHSP70 even more important for the inhibition of apoptosisand inflammation [37] Cyclopentenone prostaglandins (cp-PGs) which under certain circumstances may induce HSP70expression are consequently powerful anti-inflammatoryautacoids [38ndash40]

The interplay between iHSP70 and proinflammatorycytokines at gene regulatory level has also been reportedThepromoter region of TNF120572 gene contains an HSF1 bindingsite that represses TNF120572 transcription and thus loss of thisrepressor results in sustained expression of TNF120572 [41] thusthe HSF1 knockout is associated with a chronic elevationof TNF120572 levels and increased susceptibility to endotoxinchallenge [42] Regulation of such a network in the oppositedirections has also been demonstrated TNF120572 may tran-siently repress HSF1 activation [43] Furthermore JNK1 wasunequivocally demonstrated to phosphorylate HSF1 in itsregulatory domain causing suppression of HSF1 transcribingactivity [44] while HSP70 prevented Bax activation both byinhibiting the JNKBim pathway and by interacting with Baxin UV-induced apoptosis [45] Altogether the above findingsexplain why the induction of HSP72 (HSPA1A) in vitro (byheat shock or HSP72 transgene overexpression) reduces theexpression of inflammatory genes such as TNF120572 IL-1 IL-12IL-10 and IL-18 [46]

In contrast to the above findings eHSP70 proinflam-matory actions have been demonstrated to be mediatedby MyD88IRAKNF-120581B signal transduction pathway afterboth Toll-like receptor 2 (TLR2) and TLR4 binding in aCD14-dependent manner [47 48] thus promoting innateimmune activation [49] Due to the antagonistic actions ofthe heat shock proteins within the course of an inflam-matory response it is reasonable to hypothesize that thebalance between eHSP70 and iHSP70 might determinethe outcomemdasheither the induction or the attenuation ofinflammation Since low-grade inflammation is involved inseveral chronic diseases [17] the management of HSP70expression and its location can be crucial for the control ofinflammatory-related conditions such as T2DM We hereinsuggest that the ratio of the extracellularmediumHSP70 con-centration to intracellular HSP70 contents (eHSP70iHSP70)can determine the progress of insulin resistance and theprogression of T2DM



































HSF-1

TLRInflammatory

pathway

Adipose tissue

expansion


Liver

Immune cells

Skeletal muscle


damage

Diabetes

eHSP70

iHSP70eHSP70

eHSP70iHSP70




Exer

cise

Obe

sity


Brain

iHSP70

eHSP70

Insulin

signaling

oplus

oplus

oplus

oplus

oplus

oplus

oplus

⊖

⊖

⊖

⊖

5

R10






















Acknowledgments


References







































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































HSF-1

TLRInflammatory

pathway

Adipose tissue

expansion


Liver

Immune cells

Skeletal muscle


damage

Diabetes

eHSP70

iHSP70eHSP70

eHSP70iHSP70




Exer

cise

Obe

sity


Brain

iHSP70

eHSP70

Insulin

signaling

oplus

oplus

oplus

oplus

oplus

oplus

oplus

⊖

⊖

⊖

⊖

5

R10






















Acknowledgments


References







































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































Acknowledgments


References







































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















The Chaperone Balance Hypothesis: The Importance of the Extracellular to Intracellular HSP70 Ratio...

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Transcript of The Chaperone Balance Hypothesis: The Importance of the Extracellular to Intracellular HSP70 Ratio...