(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 |
|
