(PMODTechnologies,Zurich,Switzerland)toquantifyregion-speci?cg lucoseuptake.PET/CTimagesweregeneratedusingthe multimodal 3DvisualizationmodalityinInveonResearchWorkplace. InVivoRad ioactiveGlucoseUptake Invivoradioactiveglucoseuptakeassayswereper formedsimilarlyasdescribed(Markanetal.,2014).Allmiceusedforinvivo radioactive glucoseuptakewerechow-fed12-14weekoldmaleswithwild-ty pelittermatesusedasacontrol.Miceweresinglyhousedaweekprior toinvi voradioactiveglucoseuptake.Onthedayofuptakeassay,micewerefastedfo r5hrandweretransferredtoaradioactivepro- cedureroomtoacclimate1.5 hrpriortoinitiatingtheassay.FGF21orvehiclewasaddedtoInsulin/Deoxy -D-glucose,2-[1,2-3H(N)](Per- kinElmer)mixturefora?naldoseof1mg/k gBWofFGF21,0.25UIns/kgBW,and1.25mCi/gBW.Afterinjection,micewerepl acedbackin cagesandsacri?cedbydecapitation90minpost-injection.Tis sueswerecollectedandimmediatelysnapfrozeninliquidnitrogen. Tissue swerethenprocessedtodeterminelevelsofradioactivephosphorylated2DG .Tissueswereweighedandplacedin400mLof 1MNaOHandincubatedat85Cfor 10mintohomogenize.Sampleswerethenneutralizedwith400mLof1MHCl.200m Lofhomog- enizedsampleswerethenaddedtoduplicatesolutionso f1mLicecold7%perchloricacidorduplicatesolutionsof1mLS omogyi buffer.Somogyibufferwaspreparedimmediatelybeforeusebycombi ning500mL0.3NZnSOwith500mL0.3NBa(OH).Samples 42 werespunat13,0 00xgfor5minat4C.800mLofsupernatantwasaddedto10mLofBio-SafeIIScin tillation?uid(RPI)andradioactivity measuredonascintillationcounte rincountspermin(c.p.m.).Phospho-2DGconcentrationinsampleswasmeasu redbyconverting c.p.m.measurementstodisintegrationspermin(d.p.m.) basedonthescintillationcounteref?ciency.Averaged.p.m.measuredfrom
Somogyibufferwassubtractedfromaveraged.p.m.fromperchloricacidsol utionsforeachtissueandnormalizedtotissueweight. WesternBlotAnalys is Forwesternblotanalysis,snap-frozentissueswerehomogenize doniceinlysisbuffercontaining10mMTris-HCl,pH7.4,5mM
EDTA,5mMEGTA,150mMNaCl,10%glycerol,1%NP-40,0.5%TritonX-100andprot easeinhibitors.Sampleswerecentrifuged for5minat0.5xgat4Cand infranatantcollected.Anappropriatevolumeof6XLaemmlibufferwa saddedandallsamplesincu- batedat100Cfor10minandthenbr ie?yplacedonice.ProteinconcentrationwasdeterminedbyBradf ordassayandthenequal quantityofsampleresolvedbySDS-PAGE.Protei nsweretransferredtoaPVDFmembranebeforebeingprobedwiththespeci?ed
antibodies.Antibodyinformation:b-actin(Sigma,#A5316),phosph o-ERK1/2(CellSignaling,#9101),totalERK1/2(CellSignaling, # 9102),Myc(Millipore,#05-724)andb-klotho(R&DSystems,#AF261 9). PrimaryWhiteAdipocyteIsolationandTreatment Primarywhiteadip ocyteswereisolatedfromwhitefatdepotsof4-day-oldC57BL/6J pups(n=14).Subcutaneousfatpadswere dissectedanddigested (2%BSA(Sigma,FFAfree)and0.1%collagenase(Invitrogen)inHB SS(GIBCO)).Digestedtissuewas thenplacedintoaprewarmedsha ker(37C)andallowedtoshakeat150rpmfor1h,followedbycen trifugationat1000xgfor 3minat4C.Thesupernatantwasdiscardedand thepelletwaswashedandcentrifugedthreetimeswithpreadipocytegrowthm edia (DMEM(highglucose,nopyruvateSigmaD5796),10%fetalbov ineserum(FBS;GIBCO),1XPen-Strep(GIBCO),1Xnonessential ami noacids(GIBCO),1XGlutamax(GIBCO),1MHEPES(GIBCO),and0.1m M2-mercaptoethanol(Ameresco)).Afterthe?nal wash,thepreadip ocytegrowthmediawasremovedandthepelletwasresuspendedin freshpreadipocytegrowthmediaat2mL perpup.Theresuspendedc ellswhere?lteredthrougha100mmnylon?lterandcellswerepl atedin6wellplatesat2mLperwell. Finally,cellswereincuba tedandallowedtogrowfor2daysat37Cinacellincubator. F ordifferentiation,cellswerereseededintonew6-wellplatesco ntainingfreshpreadipocytegrowthmedia.Brie?y,mediawas aspir atedfromeachwellandcellswerewashedwithroomtemperature1XPBS3times. Next,300mLoftrypsin(GIBCO)wasadded toeachwellandcellswereco llectedandputintofreshpreadipocytegrowthmediaandseededi ntonew6-wellplates.Cellswere allowedtogrowuntilcon?uent( about5days;mediawaschangedonce).Cellswerethendifferentiated usingdifferentiationmedia (DMEM,10%FBS,1XPen-Strep,5mg/ml insulin(Sigma),1mMdexamethasone(Sigma),and0.5mMIBMX(Sig ma))for2days. Followingdifferentiationintomatureadipocytes ,cellswereputinmaintenancemedia(DMEM,10%FBS,1XPen-Stre p,and 5mg/mlinsulin)untilusedforexperiment. Matureadipocyt esweretreatedwitheithervehicleorFGF21(1mg/ml)preparedinserumfreem aintenancemediaonatimecourse of0,0.25,0.5,1,2,4,8,16,and24h.Follo wingtreatments,matureadipocyteswereplacedoniceandmediawascollecte dfromeach well.Cellswerewashedthreetimeswithicecold1XPBSandwerely sedusing250mL/wellWATlysisbuffer(10mMTris-HCL(pH7.4; FisherScient i?c),5mMEDTAandEGTA(Sigma),150mMNaCl(Sigma),10%glycerol(Sigma),1% NP-40(Sigma),0.5%TritonX-100 (Sigma),andcompleteproteaseandphosph ataseinhibitorcocktails(Roche)).Celllysateswerecollectedandputoni ce.Allsamples werestoredina20Cfreezeruntiluse(2days).Adiponecti nlevelsweremeasuredinthemediafromtreatedcellsusingacommer- cially availableELISA(EMDMillipore). QUANTIFICATIONANDSTATISTICALANALYSIS Dataarepresentedasthemean±SEM;p<0.05wasconsideredsigni?cant.Student’sttestwasusedtocomparetwoindependent groups.Two-wayANOVAswithSidakposthoccorrection(GraphPadPrism)wereusedformultiplegroupanalyses. e4CellMetabolism25,935–944.e1–e4,April4,2017 |
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