Increased Levels of Nuclear SREBP-1c Associated with Fatty Livers in Two Mouse Models of Diabetes

THEJOURNALOFBIOLOGICALCHEMISTRYVol.274,No.42,IssueofOctober15,pp.30028–30032,1999©1999byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PrintedinU.S.A.IncreasedLevelsofNuclearSREBP-1cAssociatedwith

FattyLiversinTwoMouseModelsofDiabetesMellitus*

(Receivedforpublication,May25,1999,andinrevisedform,June24,1999)

IichiroShimomura ,YuriyBashmakov,andJayD.Horton§

FromtheDepartmentofMolecularGenetics,UniversityofTexasSouthwesternMedicalCenter,Dallas,Texas75235-9046

Hepaticsteatosisiscommoninnon-insulin-dependentofcholesterolandfattyacidsynthesisinliver(4,5).SREBPsdiabetesandcanbeassociatedwithfibrosisandcirrho-belongtothebasichelix-loop-helix-leucinezipperfamilyofsisinasubsetofindividuals.Increasedratesoffattytranscriptionfactors(4).Unlikeothermembersofthebasicacidsynthesishavebeenreportedinliversfromrodent

modelsofdiabetesandmaycontributetothedevelop-helix-loop-helix-leucinezipperfamily,SREBPsaresynthesizedmentofsteatosis.Sterolregulatoryelement-bindingas 1150-aminoacidprecursorsboundtotheendoplasmicproteins(SREBPs)areafamilyofregulatedtranscrip-reticulumandnuclearenvelope(4).Tobeactive,theNH2-tionfactorsthatstimulatelipidsynthesisinliver.Intheterminalsegmentmustbereleasedfromthemembranebyacurrentstudies,wemeasuredthecontentofSREBPsinsequentialtwo-stepcleavageprocess(6,7).Followingthesec-liversfromtwomousemodelsofdiabetes,obeseob/obondcleavage(site-2cleavage),the 500-aminoacidNH2-ter-miceandtransgenicaP2-SREBP-1c436(aP2-SREBP-1c)minalsegmentofSREBPisreleasedfromthemembraneandmicethatoverexpressnuclearSREBP-1conlyinadiposetranslocatestothenucleus,whereitbindstoenhancerregionstissue.TheaP2-SREBP-1cmiceexhibitasyndromethatoftargetgenestoactivatetranscription.

resemblescongenitalgeneralizedlipodystrophyinhu-Todate,threeSREBPisoformshavebeenidentifiedandmans.Bothlinesofmicedevelophyperinsulinemia,hy-characterized(4,8).SREBP-1aand-1carederivedfromaperglycemia,andhepaticsteatosis.NuclearSREBP-1csinglegenethroughtheuseofalternativetranscriptionstartproteinlevelsweresignificantlyelevatedinliversfromsitesthatproducealternateformsofexon1(9).Thethirdob/obandaP2-SREBP-1cmicecomparedwithwild-typeSREBPisoform,SREBP-2,isderivedfromaseparategeneandmice.IncreasednuclearSREBP-1cproteinwasassoci-is 45%identicaltoSREBP-1a(10).Inmostculturedcelllines,atedwithelevatedmRNAlevelsforknownSREBPtar-thepredominantSREBP-1isoformisSREBP-1a(11).Incon-getgenesinvolvedinfattyacidbiosynthesis,whichled

tosignificantlyhigherratesofhepaticfattyacidsynthe-trast,mostanimaltissues,includingliver,expressSREBP-1csisinvivo.ThesestudiessuggestthatincreasedlevelsofasthepredominantSREBP-1isoform(11).MultiplelinesofnuclearSREBP-1ccontributetotheelevatedratesofevidencesuggestthatSREBP-1andSREBP-2havedifferenthepaticfattyacidsynthesisthatleadstosteatosisinrelativeeffectsontargetgenes.SREBP-1isrelativelyselectivediabeticmice.inactivatinggenesinvolvedinfattyacidsynthesis,while

SREBP-2preferentiallyactivatesgenesinvolvedincholesterol

biosynthesis(12–15).

Non-insulin-dependentdiabetesmellitusisacommondisorderTogaininsightintotheseparaterolesofeachSREBPiso-thataffectsapproximately5%ofthepopulation.Affectedpa-forminvivo,wepreviouslyproducedandcharacterizedtrans-tientsareusuallyobese,andtheymanifestinsulinresistance,genicmicethatoverexpressthetruncated,transcriptionallyhyperinsulinemia,andhyperglycemia.Asmanyas40%ofnon-activenuclearformsofhumanSREBP-1a,-1c,or-2inliver(13,insulin-dependentdiabeticsdevelopevidenceofhepaticsteatosis15,16).MicethatoverexpressedSREBP-1c,thepredominantor“fattyliver,”aconditionthatleadstohepaticfibrosisandSREBP-1isoform,hada4-foldincreaseintherateoffattyacidcirrhosisinasubsetofindividuals(1).Hepaticfattyacidsynthe-synthesis,asmeasuredbytheincorporationof[3H]water.Cor-sisisincreasedinrodentmodelsofhyperinsulinemiaandlikelyresponding2–6-foldincreasesweremeasuredinmRNAlevelscontributestothedevelopmentoffattylivers(2,3).forthelipogenicgenesATPcitratelyase,acetyl-CoAcarboxyl-Sterolregulatoryelement-bindingproteins(SREBPs)1areaase(ACC),fattyacidsynthase(FAS),glycerol-3-phosphatefamilyoftranscriptionfactorsthatactivatetheentireprogramacyltransferase(GPAT),malicenzyme,andglucose-6-phos-

phatedehydrogenase(Glu-6-PD)(13,17).Nochangeswere

*ThisworkwassupportedinpartbyNationalInstitutesofHealthmeasuredinthemRNAsforthecholesterolbiosyntheticen-GrantHL-20948andgrantsfromtheMossHeartFoundationandthezymes3-hydroxy-3-methylglutaryl-coenzymeA(HMG-CoA)PerotFamilyFoundation.Thecostsofpublicationofthisarticleweresynthase,HMG-CoAreductase,andsqualenesynthaseorindefrayedinpartbythepaymentofpagecharges.Thisarticlemust

thereforebeherebymarked“advertisement”inaccordancewith18theinvivoratesofcholesterolsynthesis(13,15).

U.S.C.Section1734solelytoindicatethisfact.Incontrast,micethatoverexpressednuclearSREBP-2 RecipientofaresearchfellowshipfromtheManpeiSuzukiDiabetes(nSREBP-2)had28-foldhigherratesofcholesterolsynthesisinFoundationofTokyo,Japan.vivo,andcorresponding10–12-foldincreasesinmRNAsfor§Towhomcorrespondenceshouldbeaddressed:Dept.ofMolecular

Genetics,UniversityofTexasSouthwesternMedicalCenter,5323severalenzymesinthecholesterolbiosyntheticpathway(15).HarryHinesBlvd.,Rm.L5-238,Dallas,TX75235-9046.Tel.:214-648-TheSREBP-2transgenicmicealsohad4-foldhigherratesof9677;Fax:214-648-8804;E-mail:horton06@utsw.swmed.edu.fattyacidbiosynthesisinvivo.Thisdemonstratedthatathigh1Theabbreviationsusedare:SREBP,sterolregulatoryelement-levelsofexpression,nSREBP-2iscapableofactivatingthebindingprotein;ACC,acetyl-CoAcarboxylase;FAS,fattyacidsyn-

thase;GPAT,glycerol-3-phosphateacyltransferase;Glu-6-PD,glucose-enzymecascaderequiredforfattyacidbiosynthesis,albeit6-phosphatedehydrogenase;HMG-CoA,3-hydroxy-3-methylglutarylmuchlessefficientlythanforthatofcholesterolbiosynthesis.coenzymeA;LDL,lowdensitylipoprotein;SCD,stearoyl-CoAInthecurrentstudies,wedemonstratethattheamountofdesaturase.nSREBP-1isincreasedinfattyliversfromtwodistinctanimal

30028Thispaperisavailableonlineathttp://wendang.chazidian.comDownloaded from http://wendang.chazidian.com/ by guest on March 11, 2015

IncreasedNuclearSREBP-1cinFattyLiversofDiabeticMice30029

TABLEI

Phenotypiccomparisonofwild-type,ob/ob,andaP2-SREBP-1ctransgenicmice

Eachvaluerepresentsthemean S.E.ForexperimentB,wild-typemicewerelittermatesofaP2-SREBP-1ctransgenicmice.ThemiceusedinexperimentsAandBwere13and17weeksofage,respectively.

ParameterExperimentAExperimentB

Wild-typeob/obWild-typeaP2-SREBP-1c

No.andsex5males5males4males4malesBodyweight(g)27.0 0.654.4 1.5a27.6 0.234.2 0.9aLiverweight(g)1.1 0.104.1 0.30a1.5 0.044.6 0.51aLiverweight/bodyweight(%)4.2 0.137.5 0.29a5.4 0.1113.3 1.3aEpididymalfatweight(g)0.43 0.054.1 0.22a0.36 0.050.13 0.03aEpididymalfat/bodyweight(%)1.6 0.207.5 0.44a1.3 0.170.39 0.08aPlasmainsulin(ng/ml)0.20 0.0321.4 2.0a0.48 0.1623.2 4.1aPlasmaglucose(mg/dl)168 15332 21a160 24295 32bPlasmaFFA(mM/liter)361 37562 17a670 69454 24bTotalplasmacholesterol(mg/dl)94 0.8172 13a77 3.7155 8.9aTotalplasmatriglycerides(mg/dl)83 4.286 9.2133 13306 57bLivercholesterolcontent(mg/g)2.4 0.037.7 0.80a1.9 0.054.5 1.2Livertriglyceridecontent(mg/g)15.4 1.7154 15a5.2 1.284.0 19aap 0.001(Student’sttest)betweenwild-typeandob/obmiceoraP2-SREBP-1cmice.

bp 0.05(Student’sttest)betweenwild-typeandob/obmiceoraP2-SREBP-1cmice.

modelsofnon-insulin-dependentdiabetes.ThefirstmodelistheRNAAnalysis—ForNortherngelanalysis,equalaliquotsoftotal“obese”(ob/ob)mousethatdevelopssevereobesityandhyperin-RNAmadefromeachmouseliverwerepooled(total,10 g),denaturedsulinemia(18).Thesecondmodelutilizestransgenicmicethatwithformaldehydeandformamide,subjectedtoelectrophoresisina1%overexpressnuclearSREBP-1cexclusivelyinadiposetissueagarosegel,andtransferredtoHybondN membranes(Amersham(aP2-SREBP-1c).Thesetransgenicanimalsareoneofthreere-PharmaciaBiotech)forhybridization.Hybridizationconditionsand

cDNAprobepreparationwerecarriedoutasdescribed(15–17).Asacentlydescribedmousemodelsthatresemblegeneralizedlipo-loadingcontrol,acDNAprobeformouse -actinwaspreparedusing

dystrophyinhumans(19–21).TheaP2-SREBP-1ctransgenicreversetranscriptase-PCRandmouseliverpoly(A) RNAasatemplatemicehaveverylittleadiposetissue,apparentlyasaresultofasdescribedpreviously(16).ThePCRprimersusedwereasfollows:5 disturbedadipocytedifferentiation.Theyalsodevelopseverehy-primer,5 -CATTGAACATGGCATTGTTACCAACTGGGA-3 ,and3 perglycemia,hyperinsulinemia,andlipidaccumulationinliver.primer,5 -GCCATCTCCTGCTCAGAGTCTAGAGCAACA-3 (24).North-Despitethestrikingdifferencesintheamountsofbodyfat,bothernblotfilterswereexposedtoaFujiPhosphorImager,andtheresultingdiabeticmousemodelsmanifestincreasednSREBP-1cproteinbandswerequantifiedusingaBio-ImagingAnalyzerwithBAS1000Mac-

BASsoftware(FujiMedicalSystems,Stamford,CT).The-foldchangefor

contentinliver,correspondingelevationsinmRNAsformultipleeachmRNAwascalculatedafternormalizationbythesignalgeneratedbylipogenicgenes,andincreasedratesoffattyacidbiosynthesis.In -actin.TheRNaseprotectionassayforSCD1andSCD2mRNAtranscriptscontrast,nSREBP-2levelsandmRNAsforgenesinvolvedinwascarriedoutasdescribedpreviously(17).

cholesterolhomeostasiswereunchangedintheliversfromob/obCholesterolandFattyAcidSynthesisinVivo—TheratesofliverandaP2-SREBP-1cmice.WeproposethatincreasednSREBP-1ccholesterolandfattyacidsynthesisweremeasuredinnonfastedmiceproteinresultsinthetranscriptionalactivationofgenesrespon-using[3H]waterasdescribedpreviously(16).Thedatapresentedare

pooledresultsfromtwoindependentexperiments,eachofwhichin-

sibleforlipogenesisandthereforecontributestothefattylivercluded512–16-week-oldmalemiceoftheindicatedgenotype.phenotypeobservedindiabeticmice.TissueCholesterolandFattyAcidComposition—Therelativehepatic

fattyacidcompositionsintheindicatedlipidfractionsweremeasured

EXPERIMENTALPROCEDURESasdescribedpreviously(17).

MaterialsandGeneralMethods—Allchemicalsusedwerefrom

Sigma.[ -32P]dCTPand[ -32P]CTPwereobtainedfromAmershamRESULTS

PharmaciaBiotech.Thecontentofcholesterolandtriglyceridein

plasmaandliverwasmeasuredasdescribedpreviously(22,23).TableIshowsthephenotypiccharacteristicsofthemiceusedPlasmainsulinwasmeasuredwithamonoclonalanti-ratinsulinradio-inthecurrentstudies.InexperimentA,westudiedmaleob/obimmunoassayusingtheRatInsulinRIAkit(LincoResearch,Inc.,St.miceontheC57BL/6Jbackgroundandage-matchedwild-typeCharles,MO).PlasmaglucoseandfreefattyacidsweremeasuredusingC57BL/6Jmalemiceascontrols.InexperimentB,westudiedtheGlucose(Trinder)100kit(SigmaDiagnostics,St.Louis,MO),andaP2-SREBP-1cmicethatoverexpresshumannSREBP-1cex-theWakoNEFACtestkit(WakoChemicals,Richmond,VA),

respectively.clusivelyinadiposetissueandsex-matchedlittermatewild-

Animals—“Obese”(C57BL/6J-ob/ob),andC57BL/6Jwild-typemicetypecontrols.TheaP2-SREBP-1cmicewerederivedfromthewerepurchasedfromtheJacksonLaboratory(BarHarbor,ME).Trans-C57BL/6JandSJLstrains.Theob/obandaP2-SREBP-1cmicegenicmicethatoverexpressthetruncatedformofhumanSREBP-1chadsimilarelevationsinplasmaglucoseandinsulin,indicat-(aminoacids1–436)inadiposetissueunderthecontroloftheadipo-ingtheyhadsimilarlevelsofinsulinresistanceanddiabetes.cyte-specificaP2enhancer/promoterweredescribedpreviously(21).Livercholesterolwasincreased2–3-foldinbothdiabeticmod-TheaP2-SREBP-1cmiceareonamixedgeneticbackgroundconsisting

ofC57BL/6JandSJLstrains.Allmicewerehousedincolonycagesinaels.Livertriglyceridecontentwasmarkedlyelevatedinlivers14-hlight/10-hdarkcycleandweremaintainedonTeklad6%(w/w)fromob/ob(10-fold)andaP2-SREBP-1c(16-fold)micecom-Mouse/RatDiet7002fromHarlanTekladPremierLaboratoryDietsparedwithwild-typelevels.Thetwomodelsdifferinthatthe(Madison,WI).InexperimentA,5malewild-typeC57BL/6Jand5maleob/obmicehadmassiveperipheralfatstoresandhigherob/obmicewerestudiedat13weeksofage.ForexperimentB,4maleplasmafreefattyacidconcentrationscomparedwiththeirwild-wild-typeand4maletransgenicaP2-SREBP-1clittermatemiceweretypecontrols.Conversely,theaP2-SREBP-1cmicehadmark-studiedat17-weeksofage.Allmiceweresacrificedduringthemid-dark

cyclefollowinga1–2-hfast.edlyreducedperipheralfatstoreswithaslightreductioninImmunoblots—Pooledlivermembranesandnuclearextractsfromplasmafreefattyacidlevels,comparedwiththeirlittermateliversofmiceinexperimentsAandBwerepreparedandimmunoblotcontrols.

analysisforendogenousSREBP-1andSREBP-2wasperformedasFig.1showsimmunoblotsofSREBP-1and-2innucleardescribedpreviously(14,16).extracts(N)andmembranes(P)fromliversofob/obmice,Downloaded from http://wendang.chazidian.com/ by guest on March 11, 2015

30030IncreasedNuclearSREBP-1cinFattyLiversofDiabeticMice

aP2-SREBP-1cmice,andtheirrespectivewild-typecontrolsFAS,GPAT,andtheNADPH-producingenzymesmalicen-afterabrief1–2-hfast.TheamountoftranscriptionallyactivezymeandGlu-6-PD.LesssignificantchangesweremeasuredinnSREBP-1was3–4-foldincreasedinliversfromob/ob(lane2)themRNAsforthegenesinvolvedincholesterolmetabolismandaP2-SREBP-1c(lane6)micecomparedwiththeirrespec-(i.e.lowdensitylipoproteinreceptor,HMG-CoAsynthase,andtivewild-typecontrols(lanes1and5).ConsistentwithpreviousHMG-CoAreductase).Similarresultswereobtainedinoneexperiments(11,17,25),thepredominantSREBP-1mRNAadditionalindependentexperiment(datanotshown).transcriptpresentwasSREBP-1casdeterminedbyanRNaseToconfirmthatthechangesmeasuredinthemRNAsfortheprotectionassay(datanotshown).ThissuggeststhatthelipogenicenzymesresultedinincreasedratesoffattyacidSREBP-1isoformmeasuredbyimmunoblotispredominantlysynthesisinvivo,weused[3H]watertodirectlymeasurenewlytheSREBP-1cisoform.IncontrasttotheincreaseinnSREBP-synthesizedcholesterolandfattyacidsinliversofwild-type1c,theamountofnSREBP-2showedverylittlechangeinlivers

fromeitherdiabeticmodel(lanes4and8versuslanes3and7,anddiabeticmice(TableII).Thefattyacidsyntheticrateswererespectively).Similarresultswereobtainedinoneotherinde-6-foldhigherpergramofliverand23-foldhigherperorganinpendentexperiment.ob/obmice.LiversfromaP2-SREBP-1cmicehadfattyacidToquantifythemRNAlevelsofknownSREBP-1ctargetsyntheticratesthatwere2.5-and5.4-foldincreasedpergramgenes,aseriesofNorthernblotanalyseswereperformedonandorgan,respectively.Incontrast,liversfromob/obandpooledsamplesoftotalRNAfromtheliversofthemicede-aP2-SREBP-1cmicehad6-and3-foldreductionsintherateofscribedinTableI.ThemRNAlevelsforseveralkeygenesincorporationof[3H]waterintodigitonin-precipitablesterolsinvolvedincholesterolsynthesisandlipogenesisareshowninpergramofliver,respectively.ThelowercholesterolsyntheticFig.2forwild-type(WT)andob/ob(ob)mice(panelA)andforratespresumablyreflectpost-transcriptionalsuppressionofwild-type(WT)andaP2-SREBP-1c(Tg)mice(panelB).BothHMG-CoAreductaseactivityowingtotheincreasedlevelofhyperinsulinemicmodelshadsimilar2.5–5-foldelevationsinhepaticcholesterol(26).

themRNAsforthelipogenicenzymesATPcitratelyase,ACC,Amajorfattyacidmodifyingenzymeinliveristhe 9de-

saturase,alsoknownasstearoyl-CoAdesaturase(SCD).Two

isoformsofSCD(SCD1andSCD2)arecurrentlyknown(27).

ThemajorsubstratesforbothSCDisoformsarepalmitic(16:0)

andstearic(18:0)acids,whichareconvertedtopalmitoleic

(16:1)andoleic(18:1)acids,respectively(27).Wehaveprevi-

ouslyreportedthatthemRNAforSCD1wasincreasedin

transgenicmicethatoverexpressnuclearSREBP-1a,-1c,or-2

inliver(17).TheSCD1isoformistheonlySCDisoformmRNA

foundinwild-typeliver(28).However,inliversfrom

SREBP-1aand-2transgenicmice,themRNAforSCD2was

FIG.1.ImmunoblotanalysisofSREBP-1andSREBP-2inmem-alsoexpressed(17).

branesandnuclearextractsfromliversofwild-typeC57BL/6JTodeterminewhetherSCD1and/orSCD2mRNAwasele-(WT)andob/ob(ob)mice(A),andwild-type(WT)andtransgenicvatedinliversofthetwodiabeticmousemodels,weemployedaP2-SREBP-1clittermate(Tg)mice(B).Liversfrommiceineach

groupdescribedinTableI(experimentsAandB)werepooled,andanRNaseprotectionassaytodetectthemRNAforeachgenealiquots(30 gofprotein)ofthemembranesandnuclearextractswere(http://wendang.chazidian.comnes1and2showtheprotectedbandsfortheSCD1subjectedto8%SDS-PAGE.ImmunoblotanalysiswascarriedoutusingandSCD2mRNAin5 goftotalRNAfromepididymalfatpads5 g/mlrabbitanti-mouseSREBP-1IgG(lanes1,2,5,and6)orfromwild-typemice.InasmuchasbothSCD1andSCD2mRNASREBP-2IgG(lanes3,4,7,and8)astheprimaryantibodyand0.25 transcriptsarepresentinwhitefat,thisRNAwasusedasasecondaryg/mlhorseradishantibody.peroxidase-coupledFilterswereexposeddonkeytoReflectionanti-rabbitTMNEF496IgGasfilmthepositivecontrol(http://wendang.chazidian.comparedwiththeirrespectivewild-typefor15s(lanes1,2,5,and6)and30s(lanes3,4,7,and8).PandNlevels,theSCD1mRNAwas3.5-foldhigherinob/obmousedenotetheprecursorandcleavednuclearformsofSREBP,

respectively.livers(lanes3and4)and2-foldhigherinaP2-SREBP-1c

mouseFIG.2.AmountsofvariousmRNAsasmeasuredbyblothybridizationinliversofwild-typeC57BL/6J(WT)andC57BL/6J(ob/ob)(ob)mice(A),andwild-type(WT)andtransgenicaP2-SREBP-1c(Tg)mice(B).ThemiceusedinthisexperimentaredescribedinTableI.TotalRNAisolatedfromliversofindividualmicewaspooled,and10- galiquotsweresubjectedtoelectrophoresisandblothybridizationwiththeindicated32P-labeledcDNAprobe.ThedriedfilterswereexposedtoReflectionTMNEF496filmwithintensifyingscreensat 80°Cfor3–36h.TheamountofradioactivityineachbandwasquantifiedbyexposingthefilterstoaFujiPhosphorImagerandusingaBio-ImagingAnalyzerwithBAS1000MacBASsoftware(FujiMedicalSystems,Stamford,CT).The-foldchangeineachmRNAfromob/obmice(A)andaP2-SREBP-1cmice(B),relativetothatoftherespectivewild-typemice,wascalculatedaftercorrectionforloadingdifferenceswith -actin.Thesevaluesareshownbeloweachblot.Downloaded from http://wendang.chazidian.com/ by guest on March 11, 2015

IncreasedNuclearSREBP-1cinFattyLiversofDiabeticMice30031

TABLEII

Invivosynthesisofcholesterolandfattyacidsinliversfromwild-type,ob/ob,andaP2-SREBP-1ctransgenicmice

Eachvaluerepresentsthemean S.E.fromtwoindependentstudieseachofwhichhad512–16-week-oldmalemiceoftheindicatedgenotype.Themiceweremaintainedonachowdietandstudiednonfasted.Allmicewereinjectedwith[3H]waterintraperitoneally,and1hlatertheliverwasremovedformeasurementofitscontentof3H-labeleddigitonin-precipitablesterolsandfattyacidsasdescribedunder“ExperimentalProcedures.”

GenotypeofmiceLiverweight

gIncorporationof[3H]waterintoDigitonin-precipitablesterolsFattyacids mol/h/g mol/h/organ mol/h/g mol/h/organ

Wild-type

ob/ob

Wild-type

aP2-SREBP-1c

a1.10 0.044.06 0.13a1.42 0.073.01 0.22a1.0 0.140.15 0.01a0.73 0.090.24 0.06a1.2 0.190.60 0.04a1.06 0.120.69 0.146.9 0.543 2.3a11 2.428 4.5a7.6 0.7175 12a16 3.287 16ap 0.05(Student’sttest)betweenob/oboraP2-SREBP-1cmiceandtheirrespectivewild-typecontrols.

livers(lanes7and8)aftercorrectionforlevelsofthecontrol

mRNAencoding -actin.ThemRNAforSCD2wasnotdetected

inliversfromwild-type,ob/ob,oraP2-SREBP-1cmiceatex-

posuresupto36hat 80°C(lanes5and6andlanes9and10).

Thisresultisconsistentwithpreviousobservationsthat

nSREBP-1coverexpressioninliverandculturedcellsresultsin

increasedmRNAlevelsforSCD1butnotSCD2(12,13).

TodeterminewhethertheincreasedSCD1mRNAresulted

inincreasedhepaticmonounsaturatedfattyacidcontent,we

measuredtherelativefattyacidcompositionsofvariouslipid

fractionsfromliversofthemicedescribedinTableI.TableIII

showstherelativefattyacidcompositionsintotallipidextracts

aswellasthethreemajorlipidclassesafterfractionation.

Liversfromob/obandaP2-SREBP-1cmicehadsimilar2–3-

foldincreasesinthepercentageof16:1and18:1intotallipid

extracts.Therefore,approximately50%ofthetotalfattyacids

intheliversfromthediabeticmicearemonounsaturated(pri-

marilyoleicacid).Thesedifferencesareprimarilyaresultof

changesinfattyacidcompositioninthetriglycerideandcho-

lesterylesterfractions.

DISCUSSION

Thepurposeofthecurrentstudieswasseveralfold:1)to

determinewhethertheamountsofnSREBPswerealteredin

fattyliversfromobeseandlipodystrophicmousemodelsof

non-insulin-dependentdiabetes,2)tocorrelatethechangesin

nSREBP-1ccontentwiththerelativemRNAlevelsofknown

lipogenicSREBP-1ctargetgenes,and3)todeterminewhether

increasednSREBP-1cexpressioncontributestothedevelop-

mentoffattyliverinbothmousemodelsofdiabetes.

Previousstudiesinfastedandrefedmicesuggestedthat

nSREBP-1cwasregulatedinparallelwiththeamountsof

mRNAencodinglipogenicenzymes(29).Fastedmicehad

markedlyreducedlevelsofhepaticnSREBP-1candnSREBP-2.

Refeedingahighcarbohydrate/lowfatdietledtoa4-fold“over-

shoot”intheamountofnSREBP-1ccomparedwithpre-fasted

levels,whilenSREBP-2returnedonlytopre-fastedlevels(29).

ThepatternofregulationofnSREBP-1ccloselyparalleledthe

changesinmRNAsforlipogenicgenes,whereasthechangesin

nSREBP-2proteinparalleledthechangesinmRNAlevelsfor

genesencodingenzymesofcholesterolsynthesis(29).These

studiessuggestedthatnSREBP-1ccontributedtothefasting

andrefeedingresponsethathasbeenreportedforlipogenic

enzymesinliver(30).Thisresponsehasbeenpreviouslyattrib-

utedtothedirecteffectsofingestedglucoseorthesecondary

effectoftheelevatedinsulinthatoccursinresponsetoingested

glucose(30).

Inasmuchaslipogenesisisincreasedinliversfromob/ob

mice(3),wehypothesizedthatincreasedlevelsofnSREBP-1c

couldcontributetothisprocess.Indeed,wefoundmarkedly

elevatedlevelsofnSREBP-1cproteininliversfromob/obmice.

Toaddresswhetherthisobservationisspecifictoob/obmiceorFIG.3.ChangesintheamountsofmRNAsforSCD1andSCD2inliversfromwild-type(WT),C57BL/6J(ob/ob)(ob),andtrans-genicaP2-SREBP-1c(Tg)mice,asmeasuredbytheRNasepro-tectionassay.TotalRNAwasisolatedfromepididymalfatofC57BL/6Jmice,and5- galiquotsweresubjectedtoanRNaseprotec-tionassayforSCD1(lane1)andSCD2(lane2)asdescribedunder“ExperimentalProcedures.”WhitefatcontainsmRNAtranscriptsforbothSCD1andSCD2,andwasthususedasapositivecontrol.TotalRNAisolatedfromliversofmicedescribedinTableIwaspooled,and5- galiquotswerehybridizedwith32P-labeledcRNAprobeforSCD1(lanes3and4andlanes7and8)orSCD2(lanes5and6andlanes9and10).AfterRNasedigestion,theprotectedfragmentswereseparatedbygelelectrophoresisandexposedtofilmat 80°Cfor6h.whetheritrepresentsamoregeneralconsequenceofhyperin-sulinemia/hyperglycemia,westudiedaphenotypicallydiffer-entmodelofinsulin-resistantdiabetes,theaP2-SREBP-1ctransgenicmouse.Thesemiceexhibitedsimilar3–4-foldele-vationsinhepaticnSREBP-1c.TheincreasednSREBP-1clev-elsinbothmodelswereassociatedwithincreasedmRNAsformultiplelipogenicenzymesandincreasedratesoffattyacidsynthesisandtriglycerideaccumulationinliver.Thepredom-inantfattyacidsynthesizedwasoleicacid,presumablyasaconsequenceoftheincreaseinSCD1mRNA.Similarresultswerereportedinmiceinwhomthephosphoenolpyruvatecar-boxykinasepromoterwasusedtooverexpressnSREBP-1cinliver(13).ThesetransgenicmiceexhibitedincreasedmRNAlevelsforgenesinvolvedinfattyacidsynthesisandelevatedfattyacidsyntheticratesinvivointheabsenceofhyperinsu-linemiaorhyperglycemia.TheseobservationssuggestthatthemRNAchangesmeasuredinthediabeticmiceareadirectresultofincreasednSREBP-1clevelsinliver.NuclearSREBP-2levelswereunchangedinliversfromthetwodiabeticmousemodels,andnosignificantchangesweremeasuredinthemRNAsforgenesinvolvedincholesterolho-meostasis.ThesestudiessupportpreviouscellcultureandinvivostudiesshowingthattheisoformsofSREBP-1

preferen-Downloaded from http://wendang.chazidian.com/ by guest on March 11, 2015

30032IncreasedNuclearSREBP-1cinFattyLiversofDiabeticMice

TABLEIII

Fattyacidcompositionofliversfromwild-type,ob/ob,andaP2-SREBP-1cmice

Liversamplesfromindividualmiceofeachgenotypewereextracted,andthemajorclassesoflipidswereseparatedonsilicacolumns.Thelipidfractionsweremethyl-esterifiedandquantifiedbygas-liquidchromatographyasdescribedunder“ExperimentalProcedures.”EachvaluerepresentsthemeanfromthemicedescribedinTableI.Standarderrorsofthemeanswerealllessthan15%ofthemeanandareomittedforclarity.Boldvaluesdenotealevelofstatisticalsignificanceofp 0.05betweenwild-typeandob/oboraP2-SREBP-1cmice.

GenotypeofmiceFattyacid

16:016:118:018:118:218:320:4

%oftotal

Totalfattyacids

Wild-type212.11118211.110ob/ob227.12.9528.51.51.3Wild-type231.91217130.616aP2-SREBP-1c245.64.5467.11.44.4Cholesterylesters

Wild-type394.65.322131.22.1ob/ob237.11.5576.81.50.1Wild-type383.45.6367.71.52.6aP2-SREBP-1c266.11.6556.21.60.6Triglycerides

Wild-type224.11.931282.21.5ob/ob219.21.251101.90.5Wild-type245.62.141161.52.1aP2-SREBP-1c247.51.3509.31.61.1Phospholipids

Wild-type291.01910160.29.8ob/ob252.419199.60.59.7Wild-type251.51214120.417aP2-SREBP-1c231.517168.80.416tiallyactivateenzymesinvolvedinlipogenesis,whereas6.Wang,X.,Sato,R.,Brown,M.S.,Hua,X.,andGoldstein,J.L.(1994)Cell77,SREBP-2isprimarilyresponsibleforthetranscriptionalregu-53–62

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