Article
Alteredimmunometabolismattheinterfaceofincreasedendoplasmicreticulum(ER)stressinpatientswithtype2diabetes
RajiLenin,AravindSankaramoorthy,ViswanathanMohan,andMuthuswamyBalasubramanyam1DepartmentofCellandMolecularBiology,MadrasDiabetesResearchFoundationandDr.Mohan’sDiabetesSpecialtiesCentre,
Gopalapuram,Chennai,India
RECEIVEDDECEMBER17,2014;REVISEDAPRIL30,2015;ACCEPTEDMAY26,2015.DOI:10.1189/jlb.3A1214-609R
ABSTRACT
ThemechanismofperturbedimmunefunctioninpatientswithT2DMispoorlyunderstood.RecentstudiesimplyaroleforERstressinlinkingimmune-systemalterationsandmetabolism.Here,weinvestigatedwhetherER
stressmarkersanditsdownstreameffectorsignalsarealteredinpatientswithtype2diabetesalongwith
proinflammatoryaugmentation.Inourstudy,geneandproteinexpressionofERstressmarkers(GRP-78,PERK,IRE1a,ATF6,XBP-1andCHOP)waselevatedsignificantly(P,0.05)inPBMCsfromT2DMpatientscomparedwithcontrolsubjects.ThemRNAexpressionofboththeproinflammatorycytokines(TNF-aandIL-6)andoxidativestressmarkers(p22phox,TXNIP,and
TRPC-6;P,0.05)wasalsoincreasedinPBMCsfrompatientswithT2DM.SOCS3mRNAexpressionwasreducedsignificantly(P,0.05)indiabetespatients.mRNAexpressionofmostoftheERstressmarkersfromPBMCscorrelatedsignificantlyandpositivelywith
poorglycemiccontrol,dyslipidemia,IR,andinflamma-toryandoxidativestressmarkers.ChronicERstressinPBMCsfrompatientswithT2DMwasevidentfrom
theincreasedcaspase-3activity(P,0.01),whichisanexecutionerofapoptosis.AlongwithanimpairmentofmiR-146alevels,thedownstreamtargetsofmiR-146a,viz.,IRAK1andTRAF6mRNAlevels,werealso
elevatedsignificantly(P,0.01)inpatientswithT2DM.TherewasaninverserelationshipamongmiR-146alevelsandERstressmarkers,inflammatorymarkers,andglycemiccontrol.WedemonstrateevidenceofincreasedERstressmarkerswithimpairedmiR-146alevelsandincreasedproinflammatorysignalsinpatientswithtype2diabetes.J.Leukoc.Biol.98:000–000;2015.
Introduction
Althoughtheinvolvementofstress-activatedsignalingpathwaysinthedevelopmentofdiabeticcomplicationsiswellknown,recentstudiesimplythatseveralcellularstressmechanismsconverge,eveninthegenesisoftype2diabetes,andare
accountablefortheinsulin-secretoryandinsulin-actiondefects.ThereisincreasingevidenceinsupportofaroleforERstressinmodulatinginsulinsecretionandinsulinsensitivityinvitroandinvivo[1,2].UnderERstressconditions,severalprosurvivalmechanismsareactivatedbyUPR,whichprotectsthecellbytranslationalattenuation,inductionofchaperonesynthesis,andER-associatedproteindegradation.WhenERstressisnotmitigated,andhomeostasisisnotrestored,theUPRtriggersapoptosis[3].AlthoughIRisthehallmarkoftype2diabetes,thereisnowaconsensusthatimpairedglucoseregulationcannotdevelopwithoutinsulindeficiency.Itappearsthattype2diabetesdevelopsonthebasisofnormalbut\"weak\"bcells,unabletocopewithexcessivefunctionaldemandsimposedbyIR.Thissuggeststhatatleastinitially,thebcelldysfunctionoftype2diabetescouldbemorefunctionalthanstructural.AsianIndianswithmilddysglycemiahavebeenshownrecentlytoexhibitreducedbcellfunction,regardlessofage,adiposity,insulinsensitivity,orfamilyhistory[4].Withtheconsiderationofthisandtheplausiblebcellapoptosisintype2diabeteswithprolongedhyperglycemia/hyperlipidemia,thestudyingofERstressmechanismsmeritsattention.
TheinterplayofUPR/ERstresswithinflammation,glucose/lipidmetabolism,andenergy-controlpathwaysunderlieschronic,metabolicdiseases,includingtype2diabetes[5–7].Whereasstress-signalingpathwayshavebeenwellstudiedinpreclinicalmodels,thereisanimperativeneedtostudytheinterconnectivityofthesesignalingalterationsintheclinicaldiabetessetting.WiththeuseofsystemicmarkersandPBMCsasasurrogatecellmodel,severalofourstudiesdemonstratedtheincreasedproinflamma-tionandoxidativestressinpatientswithtype2diabetesanditscomplications[8–14].Wehavealsoshownimpairmentofan\"immunomiR\"(miR-146a)thatlinksproinflammationandIRin
1.Correspondence:Dept.ofCellandMolecularBiology,MadrasDiabetesResearchFoundation,Gopalapuram,Chennai600086,India.E-mail:diasignal@gmail.com;Twitter:http://www.twitter.com/DMDSC
Abbreviations:ATF6=activatingtranscriptionfactor6,BCL-2=Bcelllymphoma2,BMI=bodymassindex,CHOP=CCAAT/enhancer-bindinghomologousprotein,Ct=cyclethreshold,ER=endoplasmicreticulum,FPG=fastingplasmaglucose,GRP-78=glucose-regulatedprotein78,HBA1c=hemoglobinA1c,HOMA=
homeostasismodelassessment,hsa=Homosapiens,IR=insulinresistance,IRAK1=IL-1R-associatedkinase1,IRE1a=inositolrequiringenzyme1a,
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0741-5400/15/0098-0001©SocietyforLeukocyteBiologyVolume98,October2015
JournalofLeukocyteBiology1
Copyright 2015 by The Society for Leukocyte Biology.
patientswithtype2diabetes[15].WhereasacceleratinginterestintheroleofERinmetabolicdiseasehasbeenfueledbyrecentreportsshowingpathwaysthatlinkERtoinflammation[5,16],thereislackofstudiesontheseaspectsinAsianIndianswhoaremoreinsulinresistantandmorepronetodeveloptype2diabetesandcardiovasculardiseases[17].AstheremightbeatightinterconnectivityandoverlappingregulationbetweenERstressandinflammation,weplannedtoinvestigatetheextentofERstressinPBMCsinpatientswithT2DMcomparedwithcontrolsubjectsandtoanalyzetheirrelationshipwithinflammatorymarkersandglycemic/lipidcontrol.Furthermore,wewantedtocheckwhethersuchstresssignalsshowanyassociationwith
impairmentofanimmunomiR(theupstreamregulatorysignal)inpatientswithtype2diabetes.
MATERIALSANDMETHODS
Subjectrecruitment
ThestudysubjectswererecruitedrandomlyfromtheongoingepidemiologycohortsandfromDr.Mohan’sDiabetesSpecialtiesCentre,atertiarydiabetescenterinChennai,SouthIndia.Studygroupscomprisedof1)subjectswithNGT(n=35)and2)patientswithT2DM(n=35),basedonWorldHealthOrganizationcriteriaforthediagnosisofdiabetes[18].Institutionalethicalcommitteeapprovalwasobtainedforthestudy,informedconsentwas
obtainedfromallofthestudysubjects,andthestudywasconductedaspertheDeclarationofHelsinki.FastingbloodsampleswerecollectedinEDTAtubesandwerehandledseparatelyforbiochemicalandmolecularinvestigations.Agene-expressionpatternofERstressandinflammation/oxidativestressmarkerswasprobedinallofthestudysubjects.ProteinexpressionofERstressmarkers,miR-146alevels,itsgenetargets(IRAK1andTRAF6),andcaspase-3activitywasstudiedinasubsetof(n=15each)ofsubjectswithNGTandpatientswithT2DM.
Anthropometricmeasurements
Height,weight,andbloodpressureweremeasuredbyuseofstandardizedmethods.BMIwascalculatedasweight(kg)/height(m2).IRwascalculatedbyHOMA-IRwithuseofthefollowingformula:fastinginsulin(mIU/ml)3fastingglucose(mmol/l)/22.5.
Biochemicalparameters
BiochemicalanalysesweredoneonanHitachi912autoanalyzer(Mannheim,Germany)byuseofkitssuppliedbyRocheDiagnostics(Mannheim,Germany).Fastingplasmaglucose(glucoseoxidase-peroxidasemethod),serumcholesterol(cholesteroloxidase-PAPmethod),serumtriglycerides(triglycerides-PAPmethod),andHDLcholesterol(directmethod–
polyethyleneglycol-pretreatedenzymes)weremeasured.LDLcholesterolwascalculatedbyuseoftheFriedewaldformula.Glycatedhemoglobin(HbA1c)wasestimatedbyhigh-pressureliquidchromatographybyuseoftheVariantmachine(Bio-RadLaboratories,Hercules,CA,USA).
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LNA=lockednucleicacid,miR-146a=microRNA146a,miRNA=microRNA,NGT=normalglucosetolerance,p22phox=NADPHoxidase,PAP=phenolplusaminophenazone,PBMC=peripheralbloodmononuclearcells,PERK=proteinkinaseR-likeendoplasmicreticulumkinase,pNA=p-nitroanilide,snRNA=smallnuclearRNA,SOCS3=suppressorofcytokinesignaling3,SUMO=smallubiquitinmodifier,T2DM=type2diabetesmellitus,TRAF6=TNFR-associatedfactor6,TRPC-6=transientreceptorpotentialchannel6,TXNIP=thioredoxin-interactingprotein,UPR=unfoldedproteinresponse,XBP-1s=Xbox-bindingprotein1spliced,XBP-1u=Xbox-bindingprotein1unspliced
2JournalofLeukocyteBiologyVolume98,October2015PBMCisolation
Freshlycollectedperipheralbloodwascarefullylayeredonhistopaque
gradient(1077)andcentrifugedat1600rpm(500g)for30min.Thebuffy-coatinterface,representing.90%lymphocytes,wasaspiratedandwashed3timesinPBS(withpH7.4)andaliquotedforvariousexperiments.
RNAisolationandreal-timePCR
TotalRNAfromcellswasisolatedasdescribedpreviously[15].TheRNAqualityandconcentrationoftotalRNAweremeasuredbyuseofnanodrop.RNA(1mg)wasconvertedtocDNAbyuseof100unitsRTenzyme,40mMOligo(d)T18primer(NewEnglandBiolabs,Ipswich,MA,USA),103RTbuffer,20URNaseinhibitor(AmershamBiosciences,Piscataway,NJ,USA),and2.5mMeachdNTPSandincubatedat42°Cfor1h.Quantitativereal-timePCRwasperformedforspecificgenesbyuseofSYBRGreenmastermix(Finnzymes,Woburn,MA,USA).PCRamplificationwascarriedoutbyuseofABI-7000(AppliedBiosystems,FosterCity,CA,USA)withcycleconditions(initialcycle,50°Cfor2min;initialdenaturation,95°Cfor15min,40cyclesofdenaturation95°Cfor15s,andannealing/extensionof60°Cfor1min).TheexpressionlevelofRNAwasdeterminedbyuseof22DDCtandnormalizedbyuseofb-actin.TheprimersequencesofspecificgenesareinTable1.
Proteinexpression
Cellswerelysedbyuseofradioimmunoprecipitationassaybuffer[50mMTris-HCl(pH8.0),150mMNaCl,0.1%SDS,0.2%sodiumazide,1%TritonX-100,0.25%sodiumdeoxycholate,and13proteaseinhibitor].Inbrief,cellswere
TABLE1.Primersequencesofspecificgenes
Name
Sequence
ReferenceGRP-78ForwardCTG[19]PERKReverseForwardTTACCATGGTTCTCACTAAAATG
GAAGGCCAGCAATAGTTCCAG
[19]IRE1aReverseForwardGGAGCGTGACCACACGACCAAGATGGAGAGACCACAG
GAG
[19]XBP-1uReverseForwardACCTGGAGCAACAGAATACACCATCAC
ATTCCACTGTCACCATTG
[19]XBP-1sReverseForwardTCCTGCTTCTGATGGGTCGTAGCGGTATTGAC
CGCGACAGCCTCTG
[20]ATF6ReverseForwardGCTCCTGGCGTCCTAAGGCAACTCAGTTGGACCGGAAGGAG
TG
[19]CHOPReverseForwardCCTGTATTACCTATCATGTCGTTTCCTCACCTAATGACCAG
C
[19]TRPC-6ReverseForwardTGGTTTAATGAGGAGCTGGGCGAGAGAAGTCTCGAGCTGGG
G
[19]TNF-aReverseForwardTATCCCGGCAGGCCTGGAACAACAGCTCTTCAGCCT
AAA
[15]IL-6ReverseForwardTCAGACGCTAGCCCACAGCACCGCTCAGATCAT
TCACATCCTT
[15]SOCS3ReverseForwardTGTTTTCTGCCAGTGCC
CTT
[15]TXNIPReverseForwardGCGTCGATTCGGGACCAGC
[15]p22phoxReverseForwardGCCCCAGGACATGCCAACTTGCTGT
CAGAACTCAAGAGACA
[19]IRAK1ReverseForwardGTCTTCACAATGACCTCCTACCAGGAATGAACATCATTCTGGTACTTTGG
CGCTGTCCCCAG
[15]TRAF6ReverseForwardGGGTGCAGGGATGCT
ACCT
ReverseGCCATGAAAAGATGCAGAGGAATC
[15]b-ActinForwardTGAACAG
TCGGGTATAACGCTCAAACTAReverseCGTGTCCGCTTCCCACCCCATTCCAGGATCAAT
GT
[19]
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Leninetal.DualburdenofERstressandinflammationindiabetics
sonicatedandincubatedfor1hiniceandcentrifugedat16,000gfor5minat4°C.ThesupernatantcollectedwasquantifiedforproteinbytheBradfordmethod.Proteins(15mg)wereresolvedona10%SDS-PAGEandtransferredtopolyvinylidinefluoridemembranes.After1hblockingin5%BSAandincubationwiththeappropriateprimaryantibodiesandHRP–conjugatedsecondaryantibodies,detectionwasperformedbyuseofanECLkit(GEHealthcare,Pittsburgh,PA,USA).b-Actinwasusedasaninternalcontrol.MeandensitometrydatafromindependentexperimentswerenormalizedtocontrolbyuseofImageJsoftwareandrepresentedastheratiooftestproteinandb-actin.
miR-146aexpressionandquantitativereal-timePCR
First-strandcDNAwassynthesizedfrom10ngofthetotalRNAbyuseofafirst-strandcDNAsynthesiskitandmiRNA-specificRTprimersetsfortargetmiRNA(hsa-miR-146a)andendogenouscontrolU6snRNA(hsa;Exiqon,Woburn,MA,USA).cDNAsynthesiswascarriedat50°Cfor30minand85°Cfor5min.cDNAsynthesisfortheanalysisoftargetgenes(mRNA)wasperformedwith40unitsRTenzyme,40mMOligo(d)T18primer(NewEnglandBiolabs),103RTbuffer,and20URNaseinhibitor(AmershamBiosciences),and2.5mMeachdNTPSandprimersofspecificgeneswasincubatedat42°Cfor1handheatinactivationofenzymedoneat85°Cfor10min.Quantitativereal-timePCRwasperformedbyuseofthemercuryLNAmiRNAPCRsystemandSYBRGreenmastermixandwithLNA-basedprimersetsfortargetmiRNAs(hsa-miR-146a)andU6snRNA(Exiqon).cDNAwasdiluted1:10withnuclease-freewater,asperthemanufacturer’sinstruction,and4mleachcDNAwasusedforquantitativereal-timePCRinanABI-7000machine(AppliedBiosystems)withappropriatecycleconditions.The
expressionlevelofmiRNAwasdeterminedbyuseof22DDCtandnormalizedtoU6snRNA.Forreal-timemeasurementofmRNAs,SYBRGreenJumpStartReadyMix(Sigma-Aldrich,St.Louis,MO,USA)wasusedforexpressionlevelofb-actinandrespectivetargetgenes.TheexpressionlevelofmRNAwasdeterminedbyuseof22DDCtandnormalizedtob-actin.
Caspase-3activityassay
Caspase-3activitywasdeterminedbycolorimetricassaybyuseofthecaspase-specific,peptide-containingaminoacidsequenceAsp-Glu-Val-AspthatisconjugatedtothecolorreportermoleculepNA(R&DSystems,Minneapolis,MN,USA).Thecleavageofthepeptidebythecaspasesreleasesthe
chromophorepNA,whichisquantifiedspectrophotometricallyat405nm.Cellsharvestedaftertreatmentwerelysed,and10mgproteinwasaliquotedfromeachsampleintoa96wellplate.DTT(0.5ml)wasaddedtoallofthewells,followedbyadditionof50ml23reactionbufferand3.5mlCaspase-3colorimetricsubstrate.Theplatewasincubatedat37°Cfor1handreadat405nmbyuseofamicroplatereader.Caspase-3activitywasexpressedasmean6SEofOD.
Statisticalanalysis
AllanalysesweredonebyuseofWindows-basedStatisticalPackagefor
SocialSciences(SPSS,Version16.0;Chicago,IL,USA).Datawereexpressedeitherasmeans6SDormeans6SE.ComparisonsbetweengroupswereperformedbyuseofunpairedStudent’st-test.Pearsoncorrelationanalysiswascarriedouttodeterminetherelationofgene-expressionsignatureswithotherriskvariables.Logisticregressionanalysiswasusedtodeterminetheassociationofindependentmolecularsignatures.P,0.05wasconsideredstatisticallysignificant.
RESULTS
Table2presentstheclinicalandbiochemicalcharacteristicsofthestudysubjects.TherewerenosignificantdifferencesinageandBMIbetweenthestudygroups.Fastingplasmaglucose(P=0.014),glycatedhemoglobin(HbA1c;P=0.001),HOMA-IR(P=0.002),totalcholesterol(P=0.005),serumtriglycerides
www.jleukbio.orgTABLE2.ClinicalcharacteristicsofthestudygroupsParameter
NGTSubjects(n=with35)T2DMPatients(nwith=35)ValuePAge,Gender,yr
44.76647.3670.175BMI,kg/mM:F2ratioFPG,23.917/1824.619/160.196HbA1c,mg/dl0.014HOMA-IR
%5.577662615157620.001Total0.002Serumcholesterol,1401.760.45.437.866790.005HDLtriglycerides,mg/dl12060.676374617261.515862.71638790.013LDLcholesterol,cholesterol,mg/dlmg/dl
mg/dl10841667.931
12237665.234
0.0240.011
Valuesaremeans6
SD.
(P=0.013),andLDLcholesterol(P=0.011)weresignificantlyhigherinpatientswithT2DMcomparedwithsubjectswithNGT.HDLcholesterol(P=0.024)wassignificantlylowerinpatientswithtype2diabetescomparedwithsubjectswithNGT.
TranscriptionalanalysisrevealedthatthegeneexpressionofERstressmarkers(GRP-78,XBP-1u,XBP-1s,PERK,IRE1a,ATF6,andCHOP)waselevatedsignificantlyinPBMCsfromT2DMcomparedwithcontrolsubjects(Fig.1).ThemRNAexpressionofboththeproinflammatorycytokines(TNF-aandIL-6)andoxidativestressmarkers(p22phox,TXNIP,andTRPC-6)wasalsoincreasedinPBMCsfrompatientswithT2DMcomparedwithcontrolsubjects(Fig.2).Interestingly,themRNAexpressionofSOCS3wasseenreducedinPBMCsfromT2DMcomparedwithNGT(Fig.2).Table3illustratesthePearsoncorrelationanalysisofERstressgeneswithotherclinicalandbiochemical/molecularvariablesinthestudy.Interestingly,mRNAexpressionofmostoftheERstressmarkersfromPBMCssignificantlyandpositivelycorrelatedwithpoorglycemiccontrol,dyslipidemia,IR,andinflammatoryandoxidativestressmarkers(Table3).LogisticregressionwasperformedbyuseofdiabetesasthedependentvariableandtheERstressmarkersastheindependentvariable(Table4).ExpressionlevelsofGRP-78(b,1.04,P,0.001),PERK(b,1.05,P=0.010),IRE1a(b,1.02,P,0.001),ATF6(b,1.03,P,0.001),XBP-1u(b,1.04,P,0.001),XBP-1s(b,1.02,P,0.002),
Figure1.Relativegene-expression(mean6SE)dataofERstress
markers,viz.,GRP-78,XBP-1u,XBP-1s,PERK,ATF6,IRE1a,andCHOP.*P,0.05comparedwithNGT;**P,0.01comparedwithNGT.
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stressandinflammationasafunctionofcaspase-3activity.
Caspase-3activitywasalsoincreasedsignificantlyinPBMCsfromT2DMcomparedwithNGT(Fig.4C).Interestingly,themiR-146alevelswerenegativelycorrelatedtofastingplasmaglucose,HBA1c,IR,triglycerides,aswellasproinflammatory/oxidativegenemarkersandcaspase-3activity(Table5).
DISCUSSION
Ourstudypresentsthefollowingclinicallyrelevantfindings.1)DifferentarmsoftheERstressmarkers(bothatthegeneandproteinlevels)wereincreasedinPBMCsfrompatientswithtype2diabetesandcorrelatepositivelywithglycemicandlipidlevels,IR,andinflammatoryandoxidativestressmarkers.2)Forthefirst-time,wehaveshowninterconnectivitybetweendecreasedmiR-146alevelsandERstress/proinflammationinpatientswithtype2diabetes.3)OurstudyalsodemonstratesERstress
susceptible,proinflammatory,andproapoptoticphenotypesofPBMCsinpatientswithtype2diabetes,asisevidentfrom
increasedcaspase-3activityandTRPC-6genesignatures,alongwiththeotherstress-signalingalterations.
EmergingdatashowthatoneofthecentralelementsintheactivationofinflammatorypathwaysisERstress[5,21].Thismolecularpathwayisshowntointerferewiththefunctionofthemajorinsulintargettissues—hypothalamus,liver,adiposetissues,muscle,andthepancreaticbcells[22].Particularly,secretorycells,suchaspancreaticbcellsandadipocytes,facethechallengeofincreasingproteinsynthesis,severalfoldsduringacuteorchronicglucolipotoxicstresses.MonocyteshavebeenshownvulnerableandfunctionallyimpairedunderERstressinpatientswithtype2diabetes[23].Sageetal.[24],havealsoshownthatcomparedwithhealthycontrols,individualswithmetabolicsyndromehadelevatedmRNAlevelsofgenesindicativeofERstress.ConsistentwiththeliteratureinthattheIRE1a–XBP-1pathwayisanearlytriggerofUPR[5],wesawtranscriptionalup-regulationofIRE1aandXBP-1(bothXBP-1uandXBP-1s)in
Figure2.Relativegene-expression(mean6SE)dataofinflammatory/oxidativestressmarkers,viz.,TNF-a,IL-6,SOCS3,TXNIP,p22phox,andTRPC-6.*P,0.05comparedwithNGT;**P,0.01comparedwithNGT.
andCHOP(b,1.03,P=0.042)showedsignificantassociationwithtype2diabetesevenafteradjustingforage.However,thisstatisticalsignificancewaslostwhenthedataareadjustedforTNF-aandIL-6.Thisimpliesatightsignalingcross-talkandinterconnectivitybetweenproinflammationandERstressintype2diabetes.
ConsistentwiththemRNAresults,therewasalsoan
augmentedproteinexpressionofERstressmarkers(GRP-78,XBP-1,PERK,IRE1a,andCHOP)inPBMCsfromT2DMcomparedwithNGT(Fig.3).AsourearlierstudiesrevealedalinkbetweenimpairedmiR-146aandproinflammationintype2diabetes[15],wealsoprobedtheexpressionlevelsofmiR-146inthisstudy.ThemiR-146aexpressionlevelsweredecreasedsignificantly(P,0.001)inpatientswithtype2diabetes
comparedwithNGT(Fig.4A).Wenextexaminedtheexpressionof2confirmedgenetargetsofmiR-146a,viz.,IRAK1andTRAF6,andfoundbothofthemincreasedsignificantlyinT2DM
comparedwithNGT(Fig.4B).Ascaspase-3activationisanearlymarkerofapoptosis,wealsoestimatedthechronicburdenofER
TABLE3.Pearsoncorrelation(r)ofERstressgeneswithothervariablesinthestudyGRP-78
VariableBMIFPGHbA1cHOMA-IRCHOLESTRIGLYHDLLDLTNF-aIL-6SOCS3TXNIPp22phoxTRPC-6
r0.0860.4170.5520.3960.2240.2120.2510.1940.4730.2660.3760.2970.3280.481
P0.5290.0020.0010.0060.1260.1270.050.160.0010.1800.0400.1290.0360.011
r0.0810.4950.5310.5750.1440.30820.3600.3780.2700.3890.2360.2010.4610.401
XBP-1u
P0.5880.0170.0010.0020.3870.0390.0110.0110.2200.1460.1030.1270.0100.028
r0.2140.4730.1590.3400.1650.37820.3190.4280.4700.2060.3200.1390.4120.319
XBP-1s
P0.1960.0200.1300.0180.1400.0430.0120.0260.0120.1040.0230.0850.0320.016
r0.1850.2010.3010.1390.5680.43420.1410.3020.2400.2630.2400.2670.2290.281
ATF6
P0.2080.1160.0380.1100.0010.0020.3270.0440.1770.2150.2180.1220.1290.124
r0.0210.5120.3700.2320.3990.34220.1240.2090.3990.3660.2190.2660.4100.213
IRE1a
P0.8850.0090.010.1310.010.0190.3810.1680.0200.0240.1590.1960.0190.112
r0.1870.3170.3830.3930.5260.42120.2380.2880.5270.5300.2540.3780.4260.434
CHOP
P0.2090.1040.0080.0200.0010.0030.1010.0550.0120.0210.1960.0430.0210.022
CHOLES,cholesterol;TRIGLY,triglycerides.
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Leninetal.DualburdenofERstressandinflammationindiabetics
TABLE4.AssociationregressionofERstress(bwith)analysis
diabetesbyuseoflogistic
Unadjusted
AdjustedforageforAdjustedTNF-aAdjustedforIL-6VariablebPbPbPbPGRP-78XBP-1u1.040.0012.67XBP-1s1.040.0011.061.060.9901.06PERK1.020.0021.040.0041.030.0021.050.0671.040.070ATF61.051.490.0591.030.0750.071IRE11.030.0101.040.0011.650.9951.200.997CHOP
a1.020.0021.030.0081.03
0.0010.042
1.020.0011.05
0.0020.090
1.020.9900.0541.07
0.0631.020.322
1.011.17
0.1450.990
PBMCsfrompatientswithtype2diabetes.Inarecentstudy,ERstressmarkersIRE1a,GRP-78,andXBP-1swereshowntobeincreasedsignificantlyinadiposetissueofobese,pregnantwomenandwomenwithgestationaldiabetesmellitus[25].AsXBP-1sactivitycanbemodulatedbypost-translationalmodifica-tions,includingSUMOylation[26]andacetylation/deacetylation[27],thephysiologicandpathologicrelevanceoftheseregula-toryeventsinthecontextofmetabolicdiseaseswarrantsfurtherstudies.Importantly,ourstudynotonlyobservedincreasedmRNAandproteinlevelsofERstressmarkersinPBMCsfrompatientswithtype2diabetesbutalsodemonstratedthe
associationofthesesignatureswithproinflammatorymarkers,poorglycemiccontrol,andIR.TheexpressionofERstress
markershasbeenfoundtobeincreasedsignificantlyinhumanisletcellsfromtype2diabetessubjects,ascharacterizedbyincreasedexpressionandnucleartranslocationofCHOP[28].Postmortemstudiesalsoreporteda30–60%decreaseinbcellmassintype2diabetes[29–32]asaresultofincreasedapoptosis[29].WorkingwithMIN6bcells,isletsisolatedfromdb/dbmice,aswellaspancreassectionsofhumanswithtype2
diabetes,Laybuttetal.[33]haveshownERstresscontributingtobcellapoptosisintype2diabetes.Interestingly,Asian
Indianswithmilddysglycemia(prediabetes)havebeenshownrecentlytoexhibitreducedbcellfunctionregardlessofage,adiposity,insulinsensitivity,orfamilyhistory[4].AlthoughweusedPBMCsasasurrogatecellmodel,ourstudyimpliesaroleofincreasedERstressandproapoptoticsignalingthatis
reflectiveofslowbcelldeteriorationinthenaturalhistoryoftype2diabetes.
InadditiontotheIRE1a-andPERK-dependentUPRapoptoticpathways,ERstresscaninitiateotherproapoptoticevents,includingrelocalizationofBCL-2familymembers,cleavageofER-specificcaspases,p53activation,anddisruptionofcellularcalciumhomeostasis[34,35].CHOPcouldbeanimportantplayerofERstress-mediatedbcelldeathandmaypromotetheprogressionoftype2diabetes[36].CHOPispositivelycontrolledbythePERK–ATF4axis,andCHOPhasbeenshowntopromotethetranscriptionofBCL-2-interactingmediatorandthedown-regulationofBCL-2expression,contributingtotheinductionofapoptosis[37].Inthiscontext,theincreasedCHOPgeneandtheproteinexpressionpatternseenintype2diabetespatientsinourstudyareimportantobservations.IncreasedTRPC-6mRNA
www.jleukbio.orgexpressioninPBMCsfrompatientswithtype2diabetesinourstudyimpliesaroleforincreasedcalciumlevelsinPBMC
dysfunctionandERstress.AlterationsinCa2+homeostasishavebeendemonstratedindiabetesandassociatedcomplications[38,39]andarelinkedtoERstress.LossofCa2+homeostasisasaresultofimproperTRPCactivationcouldleadtoERstressresponsesandevenapoptosis[40].AlthoughithasbeenknownthatactivationoftheCHOPpathwayoftheUPRcancauseapoptosis,themolecularmechanismslinkingCHOPtodeathexecutionpathwaysarepoorlyunderstood.OurresultshereshowthatapoptosismightbeexecutedbytheactivationofTRPC-6andsubsequentincreaseincytosolicCa2+loadandincreasedactivationofcaspase-3inPBMCsfrompatientswithtype2diabetes.
TheincreasedTNF-aandIL-6gene-expressionpatternsinT2DMobservedinourstudyimplythattheseproinflammatorysignalsareimportantcomponentsinthepathogenesisof
type2diabetes.WehavealsoobservedadecreaseintheSOCS3gene-expressionlevelsinT2DMcomparedwithNGT,whichisinaccordancewithourearlierstudy[13].ThephysiologicroleoftheSOCSproteinsismostlikelytopreventun-controlledcytokinesignalinginthecellbynegativefeedback.However,thismechanismseemstobecompromisedinglucose-intolerantsubjects,astheyexhibitlowlevelsofSOCS3,
despiteincreasedtranscriptionofTNF-aandIL-6.Itisclearfrominvitroandinvivostudiesthatchronicandevenacutehyperglycemiacanpromoteasignificantincreaseincirculatingbiomarkersofinflammation,includingIL-6andTNF-a,
whichplayaroleinalteringmetabolichomeostasis.Activated
Figure3.Representativeproteinblots(A)andcumulativedata(mean1SE)onproteinexpressionofERstressmarkers(B).*P,0.05comparedwithNGT;**P,0.01comparedwithNGT.
Volume98,October2015
JournalofLeukocyteBiology5
Figure4.GeneexpressionofmiR-146a(A),mRNAlevelsofIRAK1andTRAF6(B),andcaspase-3activity(C)inthestudysubjects.Valuesaremean1SE.*P,0.05comparedwithNGT;**P,0.01comparedwithNGT.
macrophagesthatproduceproinflammatorycytokines,suchasTNF-a,IL-1b,andIL-6,arethoughttocontributetoIRinmuscleandadiposetissues[17,41,42].Interestinglyinourstudy,proinflammationseeninpatientswithtype2diabetesislinkedtoERstressaswellasmiRNAregulation.WeshowedearlierimpairedmiR-146alevelstobelinkedtoproinflamma-tionandIRintype2diabetes[15].miR-146awasfoundtobeinducibleuponstimulationwithLPSinaNF-kB-dependentmannerandtotargettheTRAF6andIRAK1genes[43].Inourstudy,increasedERstressmarkersinpatientswithtype2diabeteswerenegativelycorrelatedtomiR-146alevelsandpositivelycorrelatedtomiRNAtargetgenesandproinflam-matorymarkers.AfewERstress-induciblemiRNAshavebeenidentifiedandshowntohindertranslationofvarioussecretorypathwayproteins[44,45],suggestingthatmiRNAsplay
integralrolesintheUPR.Althoughfurthermechanisticstudiesareneeded,ourresultssuggestthattheregulatorycircuitofmiR-146aregulationofinflammationhasbeenlostinpatientswithtype2diabetes,anditcouldhaveacausallinkoriginatingfromincreasedERstress.
Whereasithasbeenrecognizedthatinflammationplaysacentralroleintype2diabetes,ourresultsraisethe
possibilitythatERstressisattheintersectionofinflamma-tionandmetabolism.Overthepastseveralyears,this
concepthasbeensupportedbygenetic,experimental,andclinicalevidence[1,5,19,45].AcausalroleforATF5andmiR-17,mediatedbyTXNIP[46,47],hasbeendemonstratedtolinkERstressandinflammation,asthesemoleculesareregulatedbykeyregulatorsoftheERstressresponse,PERKandIRE1a.Inthepresentstudy,weobservedincreasedTXNIPgeneexpressioninpatientswithtype2diabetes,anditcorrelatedwellwiththedistalERstressmarkers,suchasCHOP.Oneofourearlierstudiesalsodelineatedaroleof6
increasedTXNIPgeneexpressionintype2diabetes,as
TXNIPgeneexpressionwaspositivelyassociatedwithproteinoxidationandgene-expressionpatternsofIL-6,TNF-a,andp22phoxinpatientswithtype2diabetes[13].Recently,
Iwasakietal.[16]havealsodescribedamolecularpathwaylinkingERtoIL-6production.WiththeuseofDNAmicroarrayandnetworkanalysesofmacrophages,they
showedcompellingevidencethatATF4,whichisinvolvedintheERstressresponse,playedanessentialroleinIL-6
expressioninducedbyvariousmetabolicstresses,includingERstress.
AlthoughweusedPBMCsasasurrogatecellmodel,ourstudyresultsofdoubleburdenofincreasedERstress(withproapoptoticsusceptibility)andproinflammationfrompatientswithtype2diabetescouldbeextrapolatedtotheunderlyingcausesofbcelldysfunction.Thisisimportant,becauseoftherecent
genome-wideassociationstudies,inwhichT2DMsusceptibilitygenesarerelatedtobcelldysfunctionandlossofbcellmass[48]andthefactthatbcelldysfunctionhasbeenevidenteveninyouth-onsettype2diabetes[49]andinsubjectswithprediabetes[4].Becauseofthecross-sectionalnatureofourwork,ourstudycouldnotrevealanycausalrelationshipbetweenERstressandtype2diabetes,andforthis,weneedprospectivefollow-upstudies.However,accumulatingliterature[50–52]givesdirec-tionality,inthatERstresstargetingwouldbebeneficialforprevention,aswellastreatmentstrategiesrelatedtotype2
diabetes.Asearlierinitiationofinsulintherapyintype2diabeteshasbeenshownrecentlytofacilitate“bcellrest,”andpreservebcellmassandfunction[53],furtherstudiesshoulddelineatewhetherthiscouldoccurbyreductionofERstress.
Toconclude,theinterconnectivitybetweenERstressandproinflammationinourstudy,alongwithdecreasedmiR-146alevelsinpatientswithtype2diabetes,isanewandunique
JournalofLeukocyteBiologyVolume98,October2015www.jleukbio.org
Leninetal.DualburdenofERstressandinflammationindiabetics
TABLE5.Associationstressmarkers,ofmiR-146aandinwithflammation
clinicalparameters,ER
miR-146a
GenesrValuePValueBMIFPG20.213HbA1c20.1450.012HOMA20.3430.011CHOLESIR20.4610.001TRIGLY20.5000.117HDL20.3010.012LDL20.4670.124GRP-7820.2510.227PERK20.1700.01ATF620.3890.006IRE120.4300.010XBP-1ua20.3120.147XBP-1s20.2290.047CHOP20.353TNF-20.3410.046IL-6a20.4090.012SOCS320.3620.014p22phox20.5710.003TXNIP20.1030.210TRPC-620.2960.010IRAK20.1320.127TRAF620.3010.041Caspase-3
20.2990.01520.3920.378
0.0230.047
observationofclinicalsignificance.Whereasananti-inflammatorystrategystillappearstobeanimportantcompo-nent,ourstudyexposesanupstreamroleofERstressand
miRNA-mediatedregulationattheintersectionofinflammationandmetabolismandpointsoutERstresspathwayasanattractivetargetforimmunometabolicdiseases,includingtype2diabetes.
AUTHORSHIP
M.B.designedthestudy,providedcriticalresearchmaterials,assistedwithanalysisoftheresults,andcomposedandeditedthemanuscript.V.M.assistedintheclinicalcharacterizationandeditedthemanuscript.R.L.andA.S.performedtheexperiments,analyzedthedata,andinterpretedtheresults.
ACKNOWLEDGMENTS
TheauthorsacknowledgegrantsupportfromtheDepartmentofBiotechnology(DBT)andIndianCouncilofMedicalResearch(ICMR),GovernmentofIndia,andtheseedmoneygrantfromMadrasDiabetesResearchFoundation(MDRF)IntramuralRe-searchFunding(MIRF).Theauthorsalsoacknowledgefinancialassistance(SeniorResearchFellowship)fromtheCouncilofScientificandIndustrialResearch(CSIR),NewDelhi,India.
DISCLOSURES
Theauthorsdeclarenoconflictofinterests.
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ofinsulinKEYWORDS:
UPR•
inflammation•
miR-146a•
PBMCs•
apoptosis
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