HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Recipr(6)

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SerumlevelsofVprwerenotcorrelatedwithitsquantitativeeffectsontissuegeneorproteinexpression,lipidkinetics,orfatmass,sug-gestingathresholdlevelwithinthetissuesandcellsatwhichVprex-ertsitsmetaboliceffectsintissues.Thislevelcannotbedeterminedfromthepresentstudies,butitisatorbelowthatwhichwasasso-ciatedwiththelowestserumVprlevelsinVpr-Tg,andthesearewithintherangeofserumVprconcentrationsinHIVpatients.SerumVprlev-elsalsowerenotcorrelatedwithHIV-1VL;ofnote,serumlevelsofNef,anotherHIVproteinthatissecretedfromHIV-infectedimmunecellsandhasparacrineeffects,arenotcorrelatedwithHIVviremia(47).ThepresenceofVprinthecirculationofoptimallytreatedHIVpatientsandinadiposetissueandliverofHIVpatientsatautopsyprovidestranslationalrelevancetothesefindings.ThecurrentdataarelimitedindemonstratingthecriticalVprmechanismsinmice—fulltranslationwouldrequirelongitudinalmetabolicmeasurementsinbloodandtissuespecimensofHIVpatientsoncARTwitharangeofserumVprlevelsandVL.Inacross-sectionalanalysis,wefoundnocorrelationbetweenserumVprlevelsandlipidkineticratesin12hy-perlipolytic,hypertriglyceridemicHIVpatientsoncARTwitharangeofVL,suggestingthatVprhasadipose-restrictedeffectsnotreflectedquantitativelyinserumVprconcentrations.WeinferthatVprintheserumofpatientsreflectsitsproductionbyHIVsequesteredinadi-posetissue,liver,orotherreservoirsbutwehavenotmeasuredVprproductionrelativetoHIVreplicationwithinthosetissuesortheki-neticsofitsextracellulartransfer.VprislikelynotthesolepathogenicfactorinHIV-associatedmetabolicdisturbances,butithasanindepen-denteffect.Confirmationofthesemechanismsinpatientscouldpavethewayfortargetedtreatmentwithsmall-moleculeinhibitorsofVpr,GRantagonists,ordualPPARg/PPARaagonists.

HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Reciprocal Effects on PPAR/GR Co-Regulation

viralVprbySDS–polyacrylamidegelelectrophoresis(PAGE)andim-munoblot(48).Thestabilityofthepeptideinaqueoussolutionwasconfirmedbydynamiclightscattering,circulardichroism,and1Hnuclearmagneticresonancespectroscopy(48).

sVprinfusions

Alzetpumps(model1002,Durect),containingaqueoussolutionofsVprorsterilewater,wereimplantedsubcutaneouslyinwild-typemicewithdeliveryrateof0.25ml/hourtoadminister5mgofsVprevery24hoursfor14days.

SerumVpr

SerumVprwasmeasuredincodedsamplesusingICE(46).Thiselectrokineticassayusesimmobilizedmonoclonalantibodies(mAbs)toisolateVprbeforeseparationandonlinedetectionbylaser-inducedfluorescenceinsamples≥1ml.Detectionlimitsweredeterminedusingmouseserumstandards“spiked”withsVpr.Liquidchromatography–MSandmatrix-assistedlaserdesorption/ionizationanalysisconfirmedthattherecoveredanalytewascomparabletotheVprusedtoraisetheanti-VprantibodyforICE.Thespecificityofthecaptureantibodywastestedusingtwo-dimensionalelectrophoresisblotsofVpr,HIVsur-faceandinternalantigens,andTcellreceptors.Theantibodydemon-stratedreactivityonlyagainstVpr.Thesensitivityoftheassaywasintherangeof1.5to2100pg/ml.Intra-andinterassaycoefficientsofvariationwere3.22and4.13%,respectively.

Lipidkinetics

LipidkineticsweremeasuredinVpr-Tgorwild-typelittermatesandinwild-typemicereceivingsVprorwater(inthelatter,13daysafterAlzetplacement).Calorimetry(ColumbusInstruments)wasperformedbeforethekineticstudytomeasureVO2andVCO2underthesameconditionsasfortheisotopeinfusions(for4hoursafteronsetoffast-ing).Thenextday,micereceivedprimed(P)–constantIV(I)infusionsof[13C1]palmitate(P=75mmol/kg,I=75mmol/kgperhour)and[2H5]glycerol(P=140mmol/kg,I=140mmol/kgperhour)for4hours.BloodwascollectedinprechilledNa2EDTAtubesandcentrifugedat4°C,andtheplasmawasstoredat 80°C.

Plasmapalmitateisotoperatiowasdeterminedonthepentafluorobenzylderivativebynegativechemicalionization–GC/MS,withselectiveionmonitoringatmass/chargeratio(m/z)255and256(Hewlett-Packard);glycerolisotoperatiowasmeasuredontheglyceroltripropylesterde-rivativebyelectronimpact–GC/MS,withionmonitoringatm/z173to176.Standardsteady-stateequationswereusedtocalculatefluxesofpalmitateandglycerol;these,withtheconcentrationratioofplasmapalmitate(determinedbyisotopedilutionusing[2H2]palmitate)toFFA(Wako),wereusedtocalculatetotalandnetlipolysis(17).Res-piratoryexchangeratio(RER=VO2/VCO2)providedanindexofthefuelsubstrateoxidized.

Twenty-four–hourcalorimetrywasalsoperformedinwild-typemiceandVpr-Tgunderconditionsofchronicfeedingwithregularchoworhigh-fat(60%)diet.

EffectsofVpronadiposetissueandliver

Vpr-Tgandwild-typelittermateswereplacedonhigh-proteindiet(Harlan-Teklad)for3weekstoactivatethePEPCK/tTApromoter.Subgroupsofmiceweretreatedwithrosiglitazone(10mg/kgperday,intraperitoneally)orvehiclefor14days(doseanddurationopti-mizedforadiposeexpressionofPpargandGlut4mRNA).Micewere

fastedfor15hoursandeuthanizedinthemorning.Bloodandtissueswerecollected,andplasmawasimmediatelyseparated.Tissuesweresnap-frozenforRNAandproteinextractionandstoredat 80°Corfixedin10%formalinforimmunohistochemistry.

mRNAlevels

TotalRNAwasextractedfromliverusingTRIzol(Invitrogen)andfromadiposetissueusingalipidextractionkit(Qiagen)andtranscribedusingtheRNA-to-cDNAkit(AppliedBiosystems),andpolymerasechainre-action(PCR)wasperformedwiththeTaqManassay.PPARg/GRtargetgenesincluded,inadiposetissues,adiponectin(AdipoQ),adipocytepro-tein2(aP2),Cbl-associatedprotein(Cap),Glut4,Cd36,perilipin(Plin1),adipocytetriglyceridelipase(Atgl),andhormone-sensitivelipase(Hsl);inliver,carnitinepalmitoyltransferase-1a(Cpt1a),acylcoenzymeA(CoA)oxidase(Aox),long-chainacylCoAdehydrogenase(Lcad),uncouplingprotein-2(Ucp2),andmicrosomaltriglyceridetransferprotein(Mtp).Pgk1mRNAwasusedfornormalization.

Enzyme-linkedimmunosorbentassay

Plasmaconcentrationsofadiponectin(BioVendor)andaP2(Invitrogen)weremeasuredbyenzyme-linkedimmunosorbentassayfollowingthemanufacturers’protocols.

Immunoblotting

Afterextractioninradioimmunoprecipitationassay(RIPA)withphosphataseandproteaseinhibitors,proteinswereresolvedbySDS-PAGEandidentifiedaftertran …… 此处隐藏：7559字，全部文档内容请下载后查看。喜欢就下载吧 ……