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OPENACCESS
Article
PreadipocyteswereisolatedfromiWATof4-6weeksoldmalemice.TheiWATwas?nelymincedanddigestedwith1mg/mlcolla-
genase(C6885,Sigma)inserumfreeDMEM.Themincedfatwasthenincubatedfor20-30minat37Cwithgentleagitation.To
neutralizethecollagenase,FBSwasaddedto10%,themixturewas?lteredthrougha100mm?lterandcentrifugedat1500rpm
for5min;thesupernatantwasaspiratedandthepelletwaswashedandthenthecellsculturedin10cmtissuecultureplatesin
DMEMmediaincluding15%FBS.Afteronepassage,thepreadipocytesweregrowntocon?uenceandthentwodayslaterinduced
todifferentiatewith500mM3-isobutyl-1-methylxanthine,250nMdexamethasone,1mg/mLinsulinand1mMtroglitazonefor4days,
followedbyinsulinfor3days.Cellswereusedforexperimentsat8or9daysaftertheinitiationofdifferentiation.
Primaryhepatocyteswereisolatedfrom6weekoldmalemiceasdescribedpreviously(Smetsetal.,2002).Brie?ymicewereanes-
thetizedandamidlinelaparotomyperformed.Theinferiorvenacavawascannulatedandperfusedatarateof2.5ml/min.Theportal
veinwassectioned,andthesolutionallowedto?owthroughtheliver.Theliverwasperfusedsequentiallywithcalcium-freeHEPES-
phosphatebuffer(pH7.4)followedby40mg/mlofLiberaseTM(5401127001,Roche)solution.ThentheliverwaswashedinHEPES
phosphatebuffer(Calsuim-free).Hepatocyteswereremovedbymechanicaldissociationofthelivertissue,?lteredthroughsterile
100mmmeshnylon(Scienti?cs,Frederick,MD),washedtwicebycentrifugationat50gfor5min.Deadcellswereremovedby
36%percolldensitygradientcentrifugationat100xgfor10min.CellswereresuspendedwithWilliam’smediumE(12551-032,
LifeTechnologies)supplementedwith10%offetalbovineserum(FBS),GlutaMax(35050-061,LifeTechnologies),and1%peni-
cillin/streptomycinandplatedincollagen-coatedplates.Fivehlater,thecellsweretreatedwith10mMisoproterenolfor4h,and
thenthemediawascollectedandFGF21secretionwasmeasuredbyELISAasdescribedabove.
Calciummobilizationassay
ForFura2-AMexperiments,PPDIVscellswerepreincubatedwith0.5mmol/LFura2-AM(F1221,Thermo?sher)for15minat37C
beforeimaging.Cellswerewashedtwicewithmodi?edHBSS(13HBSSwith2g/lglucose,pH7.4,madefrom103HBSS
(14065,GIBCO))andimagedinthedarkatroomtemperature.ImageswereacquiredunderaZeissObserverZ1microscopeequip-
pedwitha40x/1.3NAobjectiveandPhotometricsEvolve512EMCCD.Fura2dualexcitationratioimaginginvolved2excitation?lters,
ET340xandET380xfor340and380nmexcitation,andanHQ535/45memission?lter.Exposuretimeswere200ms,andimages
weretakenevery30s.ImagingdatawereanalyzedwithMeta?uor7.7software(MolecularDevice).Fluorescenceimageswereback-
ground-correctedbydeductingthebackground(regionswithnocells)fromtheemissionintensitiesofcellsloadedwithFura2-AM.
The340/380-nmemissionratiowascalculatedatdifferenttimes.Traceswerenormalizedbysettingtheemissionratioto1beforethe
additionofdrugs.
QUANTIFICATIONANDSTATISTICALANALYSIS
Statisticalsigni?cancewasdeterminedusingeitheraStudent’sttest(whentheexerpimentaldesignedcontainedonlyonevariable)
ortwo-wayANOVAwithaHolm-Sidakposthocanalysistoadjustformultiplecomparisonswhentheexperimentaldesignwastwo
factorial.Samplesizes,experimentalreplicates,andspeci?cstatisticaltestusedaredescribedintheFigureLegends.Amultiplicity
adjustedsigni?cancethresholdofp%0.05wasusedthroughoutthestudy.Errorbarsindicates.e.m.
CellReports35,109331,June29,2021e4 |
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