HIV-1 Vpr Induces Adipose Dysfunction in Vivo Through Recipr(4)

andincreasedratioofphospho-HSL(Ser563)toHSLinPGFofsVpr-treatedmice(n=4pergroup;P=0.04).(G)F4/80+macrophagesinPGFofVpr-Tg(×40)andsVpr-treatedmice(×20).Arrowsindicatemacrophagesandcrown-likestructures(CLSs).(H)F4/80+macrophagesandperilipininPGFofsVpr-treatedcomparedtowater-treatedmice[×20,croppedandexpandedtodemonstrateabsentperilipinstaining(arrowheads)adjacenttoCLSs].(I)IncreasedF4/80+macrophagesinVpr-TgPGF.n=9sectionsfromthreemicepergroup(P=0.003).(J)IncreasedadipocytesizeinVpr-Tgmice(P=0.04).(KandL)IncreasedF4/80+macrophages(P=0.0002)andCLSsinPGF(P=0.006)ofsVpr-treatedmice(n=9sectionsfromfourmicepergroup).(M)IncreasedadipocytesizeinsVpr-treatedmice(P=0.03).Valuesaremeans±SE.*P<0.05,**P<0.01,***P<0.001comparedtoWT.

ofPPARa-regulatedfatoxidationgenesinliverofVpr-Tgandwild-typelittermates.mRNAlevelsofPpara(Fig.7A),Cpt1a,Aox,andLcad(Fig.7B)weredecreasedinVpr-Tg.InliverofsVpr-treatedmice,therewasadecreaseinLcadmRNAandnonsignificantdecreasesinmRNAlevelsoftheotheroxidationgenes(Fig.7,CandD).

HepatosteatosisdevelopsinbothVprmousemodels

HIVinfectionisassociatedwithahighprevalenceofhepatosteatosis(20).HepaticfatwasincreasedinVpr-Tg(Fig.7,FandG),witha1.5-foldelevationoflivertriglyceridecontent(Fig.7E)andamodestin-creaseinliverweight(Fig.7H).Fatcontentandliverweightwerealso

increasedinsVpr-treatedmiceafterexposuretosVprforonly2weeks(Fig.7,F,I,andJ).

AdditionalpathogenicpathwayscontributetoVpr-inducedhepatosteatosis

Therapiddevelopmentofhepatosteatosissuggestedpathogenicpath-waysinadditiontoincreasedFFAfluxwithimpairedhepaticfatoxida-tion.DecreasedplasmaadiponectinlevelsinVpr-TgandsVpr-treatedmicepromptedinvestigationofitshepatictargetAMPK(21),becausedecreasedAMPKactivity[resultinginloweredexpressionofPPARgcoactivator1a(PGC1a)andtherebyofPPARa(22,23)]cancause

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g

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W

Protein expression

Protein expression

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Vpr-T

Vpr-T

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

Early expression of Vpr

Preadipocyteproliferation–2 d

Lentiviralinfection

Adipocytedifferentiation

12 d

A

0 d 2 d 4 d 6 d 8 d 10 d

DoxyAdipocyteinductiondifferentiationof viralmediumtransgeneaddedexpression2.6

3T3-L1rtTAVprVpr-R80A

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Relative mRNA expression

1.4

2Relative mRNA expression

Relative mRNA expression

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rtTAVprR80A

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3.0Relative mRNA expression

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D

3T3-L1

Cyclin D1

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Cyclin D1

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Cyclin D1

Vpr-R80A

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DNA content (PI)DNA content (PI)DNA content (PI)

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Fig.5.Vprblocksdifferentiationin3T3-L1preadipocytes.(A)OilRedOstainingin3T3-L1preadipocytes10daysafterdoxycycline.Timelineindicateschronologyoflentivirusinfec-tion,doxycyclineaddition(early),anddifferentiationmediumDNA content (PI)DNA content (PI)DNA content (PI)

addition.Vprpreventedlipidaccumulation,attenuatedwith

Vpr-R80A.(B)ExpressionofPref1,Pparg,andGlut4mRNAinpreadipocytesonstateddaysafterdoxycycline.Vprreducedtheexpressionofdifferentiationgenes,attenuatedwithVpr-R80A.(C)Cellcyclehistogramsofpreadipocytes24hoursafterdoxycycline(day1).VprcausedarrestatG2-M,attenuatedwithVpr-R80A.(D)VprcausedaccumulationofcyclinsD1andB1inthepreadipocytes,attenuatedwithVpr-R80A.(E)VprcausedpersistentelevationofCcnd1(cyclinD1)mRNAexpressioninpreadipocytes.3T3-L1,nolentivirus;rtTA,infectedwithcontrolvirus;Vpr,infectedwithWT-Vprvirus;Vpr-R80A,infectedwithVprR80Avirus.Valuesaremeans±SE.*P<0.05,**P<0.01,***P<0.001(comparisonbetweenrtTAandVpr);#P<0.05,##P<0.01,###P<0.001(comparisonbetweenVprandR80A).

Cyclin B1

Cyclin B1

Cyclin B1

Cyclin B1

hepatosteatosis(24).Activated(Thr172-phosphorylated)AMPKrela-tivetototalAMPKwasdecreasedinVpr-Tgliver(Fig.7K),aswasPgc1amRNA(Fig.7L).PepckmRNA,whoseexpressioninfastingliverisnormallyup-regulatedbyPGC1a(25),wasalsodecreasedinVpr-Tg(Fig.7L),explainingthelowlevelofPEPCKpromoter–drivenVprtransgeneexpressioninlivercomparedtofat(Fig.1B).

IntrahepaticapolipoproteinB–triglyceridepackagingandtransportareregulatedbyadiposedifferentiation–relatedprotein(ADRP)[forverylowdensitylipoprotein(VLDL)–triglycerideassemblyandstorage]andmicrosomaltriglyceridetransferprotein(MTP)(forVLDL-triglycerideexport).PPARapositivelyregulatesMTPtranscription(26),andMtp

knockoutinducesfattyliverbyinhibitingVLDL-triglycerideexport(27).MtpmRNAwasdiminishedinVpr-Tgliver(Fig.7L).Vpr-Tgmanifesthepaticinsulinresistanceandhyperglycemia

Hepaticinsulinsensitivity,reflectedbyinsulin-stimulatedphospho-Akt(Ser473),wasdiminishedinVpr-Tg(fig.S5A),associatedwithelevatedpostchallengeplasmaglucoselevelsinyoungandolderVpr-Tg(fig.S5,BandC).Plasmainsulinlevelswerenotdifferent(fig.S5D).FastingplasmatriglyceridelevelswereelevatedinVpr-Tg(fig.S5E),butFFAlevelswerenotdifferent(fig.S5F).

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

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