HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Recipr(3)

27November2013

Vol5Issue213213ra164

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andPlin1werealsodecreasedinVpr-TgIF(Fig.3B).ExpressionofAtgl,aGR-regulatedgeneresponsiblefortriglyceridelipolysis,wasincreased,whereasthatofHsl,anotherGR-regulatedgenethatregulatesdiglycer-idelipolysis,wasdecreased,inVpr-TgIF(Fig.3C).

SimilarresultswerenotedinPGF,withsomedifferences.mRNAlevelsofPparg(Fig.3D),AdipoQ,andAp2(Fig.3E)werediminishedinVpr-Tgmice;rosiglitazonemodestlyincreasedPpargmRNAexpres-sioninwild-typebutnotinVpr-Tgmice.Vpr’seffectontheotherPPARgtargetsinPGFwasmoreselective:mRNAlevelsofCapweredecreased,butthoseofGlut4,Cd36,Lpl,andPlin1werenotdifferentfromwildtype(Fig.3E).mRNAexpres-sionofbothAtglandHslwasincreasedinVpr-TgPGF(Fig.3F).

TodemonstratethatcirculatingVprcanleadtoadiposegeneexpressionchangessimilartothosecausedbyVprexpressedwithinadiposecells,wecomparedPPARgandGRtargetgenemRNAlevelsinPGFofsVpr-treatedandvehicle(water)–treatedmice.TreatmentwithsVprfor2weeksde-creasedthemRNAlevelsofthePPARgtargets(Fig.3G)andincreasedthemRNAlevelsoftheGR-regulatedlipolyticgenes(Fig.3H),similartothechangesobservedinVpr-TgPGF.

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

usedadoxycycline-regulatedlentiviralex-pressionsystemin3T3-L1cells,inducingWT

eitherearly(duringpreadipocyteprolif-Vpr-Tg2eration)orlate(afteradipocytedifferen-1.5

tiation)Vprexpression.1.5*

Vprexpressioninproliferatingpre-1.01.0adipocytescompletelyblockeddifferenti-1**

ation(Fig.5A),associatedwithdecreased0.50.5

expressionofgenesoftheadipocytede-velopmenttranscriptionalcascade,from0.00.00

Pref1/Dlk1(earlyproliferationgene)throughPPARATGL HSLAdipoQ aP2 Glut4 CAP CD36 LPL Perilipin

Pparg2(earlydifferentiationgene)toGlut4WT

DEF(latedifferentiationgene)(Fig.5B).ThisWT + Rosi

WTearlydifferentiationblock(proximaltoVpr-Tg

Vpr-Tg

Pparg2expression)inpreadipocytessug-Vpr-Tg + Rosi

gestedaVpreffectdistinctfromPPARg2.082

co-repression.FlowcytometryrevealedthatVprexpressioninducedcellcycleblockat

1.0G2-M;cellcyclearrestwasattenuated,and4*1*

preadipocytedifferentiationgeneexpres-sionandlipidaccumulationwerepartially

00.00restoredwithexpressionofVprR80A,a

AdipoQ aP2 Glut4 CAP CD36 LPL PerilipinATGL HSLPPARmutantthatbluntsVpr’scellcyclearrest

function(Fig.5Candfig.S3).AssociatedVehicleGHwiththeG2-MblockwasadysregulatedsVpr

increaseincyclinD1expression(which1.52

normallyoccursattheG1-Stransition)withsomeincreaseincyclinB1(Fig.5D)anda

1.0

sustainedelevationofCcnd1mRNAlevels******

1*(encodingcyclinD1)(Fig.5E)intheVpr-0.5

expressingcells.

Vprexpressioninduced48hoursafter

0.00additionofdifferentiationmediumdid

PPAPAdipoQ aP2 Glut4 CAP CD36 LPL PerilipinATGL HSL

notblockdifferentiation(fig.S4);however,

Fig.3.AlteredexpressionofPPARg-andGR-regulatedgenesinPGFandIFofVpr-TgandPGFofb-adrenergic–stimulatedlipolysiswasaccel-sVpr-treatedmice.(A)ReducedPpargmRNAinIFofVpr-Tg(P=0.01);rosiglitazonehadnoeffect.(B)erated,asrevealedbyincreasedfreefattyacidReducedAdipoQ(P=0.02),Ap2(P=0.009),Glut4(P=0.0003),Cap(P=0.03),Cd36(P=0.005),Lpl(P=(FFA)release(Fig.6,AandB)withasimilar0.01),andPlin1(P=0.003)mRNAinIFofVpr-Tg.(C)IncreasedAtgl(P=0.04)andreducedHsl(P=0.01)trendforglycerolrelease(Fig.6,AandC).mRNAinIFofVpr-Tg.(D)ReducedPpargmRNAinPGFofVpr-Tg(P=0.001);rosiglitazoneincreasedmRNA

Theseresultsindicatedthatwhereascell

expressioninWT(P=0.01)butnotinVpr-Tg.(E)ReducedAdipoQ(P=0.01),Ap2(P=0.03),Cap(P=0.01),

cyclearrestisapathogenicmechanisminGlut4(P=0.95),Cd36(P=0.22),Lpl(P=0.90),andPlin1(P=0.30)mRNAinPGFofVpr-Tg.(F)Increased

Atgl(P=0.05)andHsl(P=0.002)mRNAinPGFofVpr-Tg.(G)ReducedmRNAofPparg(P=0.03),AdipoQpreadipocytes,theeffectsofVprinmature(P=0.04),Ap2(P=0.001),Glut4(P=0.02),Cap(P=0.02),Cd36(P=0.02),Lpl(P=0.02),andPlin1(P=0.02)adipocytesinvolvedifferentmechanisms,inPGFofsVpr-treatedmice.(H)IncreasedAtgl(P=0.01)andHsl(P=0.02)mRNAinPGFofsVpr-treatedpossiblyPPARgco-repressionorGRco-activation.Totestthis,weperformedchro-mice.n=8pergroupforallexperiments.Valuesaremeans±SE.*P<0.05,**P<0.01,***P<0.001.

matinimmunoprecipitation(ChIP)assays

Macrophageinfiltrationisincreasedandadipocytemorphologyin3T3-L1cellswithlentivirusexpressioninduced72hoursafterinitia-tionofadipocytedifferentiation,andquantifiedVpr’seffectonPPARg-isalteredinPGFofVpr-TgandsVpr-treatedmice

Macrophagenumberwasincreased(Fig.4,GandI)andadipocytesregulated(AdipoQandAp2)orGR-regulated(Hsl)geneexpression.Thewerelarger(Fig.4J)inPGFofVpr-Tgmice.Macrophages,CLSs,andlevelsofAdipoQandAp2promoterDNAimmunoprecipitatedbyanti-adipocytesizewerealsoincreasedinPGFofsVpr-treatedmice(Fig.4,PPARgantibodyafterrosiglitazonetreatmentwerelowerintheVprG,K,L,andM).PerilipinstainingwasdiminishedadjacenttoCLSs,comparedtothecontrol(rtTA)condition;incontrast,thelevelofindicatingincreasedadipocytecelldeathinsVpr-treatedmice(Fig.4H).HslpromoterDNAimmunoprecipitatedbyanti-GRantibodyafter

dexamethasonetreatmentwashigherintheVprcondition(Fig.6D).

Vprexpressedin3T3-L1preadipocytesinhibitsdifferentiation

ExpressionofPPARa-regulatedgenesisdown-regulatedinviacellcycleblock,whereasVprexpressedinmature

liverofbothVprmousemodelsadipocytesinduceshyperlipolysis

ToassessdifferentialeffectsofVpronpreadipocytescomparedtoBecausefatoxidationisbluntedinHIVpatients(17)andVpr-exposedmicematureadipocytes(bothofwhicharepresentinadiposedepots),weduringtheearlyfastingperiod,wequantifiedthemRNAexpression

A

mRNA fold difference

mRNA fold difference

mRNA fold difference

WTBWT + RosiVpr-Tg

Vpr-Tg + Rosi

C

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