ContentslistsavailableatScienceDirect
Journal
of
Steroid
Biochemistry
and
Molecular
Biology
j ourna l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j s b m b
The
environmental
obesogen
tributyltin
chloride
acts
via
peroxisome
proliferator
activated
receptor
gamma
to
induce
adipogenesis
in
murine
3T3-L1
preadipocytes
夽
Xia
Li
a,1,
John
Ycaza
a,1,
Bruce
Blumberg
a,b,∗aDepartmentofDevelopmentalandCellBiology,2011BiologicalSciences3,UniversityofCalifornia,Irvine,CA92697-2300,UnitedStates
bDepartmentofPharmaceuticalSciences,UniversityofCalifornia,Irvine,CA,UnitedStates
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received3January2011
Receivedinrevisedform25February2011 Accepted5March2011
Keywords:
Obesogen Tributyltin TBT PPAR␥
Endocrinedisrupter Adipogenesis
a
b
s
t
r
a
c
t
Obesogensarechemicalsthatpredisposeexposedindividualstoweightgainandobesitybyincreasing
thenumberoffatcells,storageoffatsintoexistingcells,alteringmetabolicrates,ordisturbingthe
regulationofappetiteandsatiety.Tributyltinexposurecausesdifferentiationofmultipotentstromal
stemcells(MSCs)intoadipocytes;prenatalTBTexposureleadstoepigeneticchangesinthestemcell
compartmentthatfavortheproductionofadipocytesattheexpenseofbone,invivo.Whileitisknown
thatTBTactsthroughperoxisomeproliferatoractivatedreceptorgammatoinduceadipogenesisinMSCs,
thedatain3T3-L1preadipocytesarecontroversial.HereweshowthatTBTcanactivatetheRXR–PPAR␥
heterodimereveninthepresenceofthePPAR␥antagonistGW9662.WefoundthatGW9662hasa
10-foldshorterhalf-lifeincellculturethandoPPAR␥activatorssuchasrosiglitazone(ROSI),accounting
forpreviousobservationsthatGW9662didnotinhibitTBT-mediatedadipogenesis.Whentheculture
conditionsareadjustedtocompensatefortheshorthalf-lifeofGW9662,wefoundthatTBTinduces
adipogenesis,triglyceridestorageandtheexpressionofadipogenicmarkergenesin3T3-L1cellsina
PPAR␥-dependentmanner.Ourresultsarebroadlyapplicabletothestudyofobesogenactionandindicate
thatligandstabilityisanimportantconsiderationinthedesignandinterpretationofadipogenesisassays.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
The environmental obesogen model proposes that chemical exposureisapreviouslyunappreciatedriskfactorforoverweight andobesity[1].Obesogensarefunctionallydefinedaschemicals, (dietary, endogenous, pharmaceutical, or xenobiotic), which, in combinationwiththemore widelyknownandacceptedfactors ofexcesscaloricinputandreducedenergy expenditure, predis-poseanexposedindividualtosubsequentweightgainandobesity (reviewedin[2,3–6]).Obesogenscanactbyincreasingthenumber ofadipocytesorstemcellscommittedtotheadipocytelineage,or byalteringbasalmetabolicrate,shiftingenergybalancetofavorthe storageofcaloriesandbyalteringthehormonalcontrolofappetite and satiety (reviewed in [2,3–5,7]). An increasing number of
夽ArticlesubmittedforthespecialissueonEndocrinedisruptors.
∗Correspondingauthorat:DepartmentofDevelopmentalandCellBiology,2011 BiologicalSciences3,UniversityofCalifornia,Irvine,CA92697-2300,UnitedStates. Tel.:+19498248573;fax:+19498244709.
E-mailaddress:[email protected](B.Blumberg).
1 Boththeauthorscontributedequallytothiswork.
obesogenshavebeenidentifiedinrecent yearsandthis fieldof studyisexpandingrapidly.
Oneofthemorewell-understoodobesogensistheorganotin, tributyltin(TBT). Weandothers haveshownthatTBT exposure leadstoincreaseddifferentiationofpre-adipocytesinvitro[8,9], increaseddepositionoffatinvivo[8]anddifferentiationof multi-potentstromalstemcells(MSCs)intoadipocytesinvitro[10,11]. TBTandtherelatedcompoundtriphenyltinarehighaffinity ago-nistsfortwonuclearreceptorsthatareimportantforadipogenesis: theperoxisomeproliferatoractivatedreceptorgamma(PPAR␥)and the9-cisretinoicacidreceptor(RXR)[8,9].PrenatalexposuretoTBT alteredcellfateintheMSCcompartmenttofavorthedevelopment ofadipocytesattheexpenseofthebonelineage[10].Inaccordwith itsmolecularactivity,weshowedthatTBTincreased adipogene-sisandadipogeniccommitmentinMSCsbyactivatingPPAR␥and thatblockingPPAR␥actionwiththepotentandselective antago-nistGW9662stronglyinhibitedadipogenesis[10].Whileithasnot yetbeendemonstratedthatTBTactsthroughPPAR␥intheinvivo exposuremodel,itisclearthatPPAR␥activationisrequiredfor MSCstoentertheadipogenicpathway(reviewedin[12]).
However,incontrasttowhatisknownabouttheroleofPPAR␥in MSCs,thesituationinmurine3T3-L1pre-adipocytesislessclear.
0960-0760/$–seefrontmatter© 2011 Elsevier Ltd. All rights reserved.
Fig.1. TheRXR–PPAR␥heterodimerispermissiveforRXRactivationinthepresenceofGW9662.Cos-7cellsweretransfectedwiththeindicatedreceptorexpressionplasmids togetherwithMH100x4-tk-lucreporterandCMX--galactosidasetransfectioncontrolin96-wellplatesandtreatedwithadilutionseriesoftheindicatedtestcompounds orwithadilutionseriesofGW9662andaconstantamountofROSI(1.1M)orTBT(120nM).(AandB)ActivationofCMX-GAL-PPAR␥byROSIorTBT.(CandD)Activation ofCMX-GAL-PPAR␥+L-RXR␣byROSIorTBT.Dataareexpressedasrelativelightunitsnormalizedto-galactosidasetransfectioncontrolandrepresenttheaverage±SEM oftriplicates.Experimentswereperformedatleast3times.
AtleastonegrouphasshownthatGW9662 isunabletoinhibit TBT-mediatedadipogenesisinthesecellsandtheyconcludedthat adipogenesisin3T3-L1cellsmightnotbedependentonPPAR␥,or anyothernuclearreceptorforthatmatter[13].Spiegelmanand col-leaguesshowedthatPPAR␥activityisrequiredforadipogenesisin 3T3-L1cellsusingtheverylowaffinityPPAR␥antagonistbisphenol Adiglycidylether(BADGE)[14].Theysubsequentlydemonstrated thatwhilePPAR␥itselfwasrequired(togetherwithafunctional AF2activationdomain),theabilityofPPAR␥ tobeactivatedby ligandappearedtobedispensableforadipogenesis;although,the presenceofanendogenousPPAR␥ligandcouldnotbeexcluded [15].Since3T3-L1cellsareverycommonly usedandimportant modelforadipocytedifferentiation,wesoughttounderstandthese discrepanciesanddeterminewhetherPPAR␥activitywasrequired fortheinductionofadipogenesisbyTBT.
Thereareatleastfourpossiblereasonstoexplainthe observa-tionthatTBTcouldcauseadipogenesisin3T3-L1cellsbutthatthis inductioncouldnotbeblockedbytreatmentwithGW9662[13]. ThefirstandmostobviousisthattheprocessisnotPPAR␥mediated ashasbeensuggestedbyotherinvestigators[13].Weconsidered thispossibilityunlikelyduetothewell-establishedrequirement for PPAR␥in the adipogenesisof 3T3-L1 cells [14–16] and our resultsinMSCs[10].AsecondpossibilityisthattheRXR–PPAR␥
ashasbeendescribedfor9-cisretinoicacid,orthesynthetic rex-inoidLG100268incellcultureandinPPAR␣knockoutmice[17]. Noneofthesepossibilitieshaspreviouslybeenexplored.
WeshowherethattheRXR–PPAR␥heterodimercanbe acti-vatedbyTBTintransienttransfectionassaysinCOS7cells,evenin thepresenceofsaturatingamountsofGW9662;although,GW9662 completelyblocksactivationthroughPPAR␥.Usingstandardassay conditions,GW9662wasunabletoblockadipogenesisinducedby TBTorbyROSI.Wefoundthatthehalf-lifeofGW9662isextremely shortincellculture(∼2h)comparedwiththePPAR␥agonistROSI (>24h).Whenthecellcultureregimenwasalteredtoensurean ade-quatesupplyofGW9662,wefoundthatGW9662stronglyinhibited theadipogenicactivitiesofROSIandTBTasmeasuredbycell mor-phology,triglycerideaccumulationandexpressionofadipogenic genes.Therefore,PPAR␥activityisrequiredforTBT-induced adi-pogenesisin3T3-L1cellsandweconcludethatligandstabilityis animportantconsiderationwhendesigningandinterpretingthe resultsofadipogenesisassays.
2. Results
2.1. TheRXR-PPARheterodimerispermissiveforRXRactivation
ItwaspreviouslyshownthattheRXR–PPAR␥heterodimeris permissive for RXR activation in the absence of a PPAR␥ lig-and and that activation of this heterodimerwas synergistic in thepresenceofbothRXRandPPAR␥ligands[18].However,the responsivenessoftheRXR–PPAR␥heterodimerinthepresenceof aPPAR␥antagonistwasunknown.Wetestedthepermissiveness of the RXR–PPAR␥ heterodimer by employing chimeric recep-torsthat enabled ustodistinguishwhetherPPAR␥or RXRwas beingactivatedintheheterodimer.Thissystemreliesonthe het-erodimerizationinterfaceintheligandbindingdomainsofRXRand itsheterodimericpartners[19]andutilizesafusionbetweenthe GAL4DNA-bindingdomainandthePPAR␥ligandbindingdomain (GAL-PPAR␥)together witha constructcontainingonly the lig-andbindingdomainofRXR␣(L-RXR␣)[20].WhenGAL-PPAR␥is transientlytransfectedintocellstogetherwithaGAL4-dependent reportergene,itisactivatedbythePPAR␥ligandROSIasexpected andthisactivationisstronglyinhibitedbyGW9662(Fig.1a). Sim-ilarly,TBTactivatesGAL-PPAR␥andthisactivationisblockedby GW9662 in a dose-dependent manner(Fig.1b). WhenL-RXR␣
aloneistransfectedwiththereporter,itisnotresponsiveto lig-andssincethereisnoDNA-bindingdomain(SupplementalFig.1). However,whenL-RXR␣isco-transfectedwithGAL-PPAR␥andthe GAL4-reporter,theheterodimerisresponsivetothePPAR␥ligand ROSI(Fig.1c),toTBT(Fig.1d)andtotheRXRactivatorAGN195203 (SupplementalFig.1).GW9662effectivelyblocksactivationbyROSI onGAL-PPAR␥inthepresenceofGAL-L-RXR␣(Fig.1c).Incontrast, GW9662isalmostcompletelyunabletoblockactivationbyTBTon GAL4-PPAR␥+L-RXR␣(Fig.1d).ThissuggeststhattheRXR–PPAR␥
heterodimeris permissiveforRXRactivationevenwhenPPAR␥
activationisblockedbyGW9662,therebyprovidingonepossible explanationfor theinabilityof GW9662toblockTBT-mediated adipogenesis.
2.2. ThePPARantagonistGW9662isunabletoblock adipogenesismediatedbyTBTorROSI
ConsideringthattheRXR–PPAR␥heterodimerispermissivefor RXRactivationin thepresence ofGW9662, we nexttestedthe effectsofTBTandROSIonadipogenesisof3T3-L1cellsinthe pres-enceorabsenceofGW9662(Fig.2)usingstandardadipogenesis assayconditions[8].Adipogenesiswasinferredbystainingthe cul-tureswiththetriglyceride-specificdye,OilRedO.Inaccordwith publishedresults[13] we wereunable toblockthe adipogenic
Fig.2.GW9662doesnotinhibitadipogenesismediatedbyROSIorTBTin 3T3-L1cells.3T3-L1cellsweredifferentiatedintomatureadipocytesbytheaddition ofanadipogeniccocktail(MDI)at2dayspost-confluencefor2days,followedby treatmentwith100nMROSIorTBTinthepresenceorabsenceof1MGW9662. After7daysoftreatment,cellswerefixed,stainedwithOilRedOandtheresults quantifiedusingImageJ.6individualpictureswerequantifiedandaveragedfor eachsampleanddataareshownasmeans±SEMfortriplicatesamples.Dataare expressedasaverageareafractionforaveragesof6sampleswithn=3replicates.
effectsofTBT byco-treatingthecultures withGW9662 (Fig.2). Importantly,however,GW9662didnotblocktheabilityofROSIto inducedifferentiationeither(Fig.2),anessentialcontrolthatwas absentfromthepreviouslypublishedstudy[13].Thissuggested thateitherpublishedstudiesfromotherlaboratories demonstrat-ingthatROSIactsthroughPPAR␥toinduceadipogenesisin3T3-L1 cells[8,9,14]areincorrect,orthattheremightbeadifferencein therelativestabilityofROSIandTBTcomparedwithGW9662that couldaccountfortheinabilityofGW9662toovercometheeffects ofROSIorTBT.
2.3. GW9662hasaveryshorthalf-lifeincellculture
TotestthehypothesisthatGW9662mightbeshort-livedincell culture,wenexttestedthehalf-lifeofthevariouscompoundsin 3T3-L1cellsusingmassspectrometricanalysis.Wetreated3T3-L1 cellswith1MROSIorGW9662,collectedsamplesofmediaorcell pelletsatdifferenttimepointsandanalyzedthelevelsofROSIor GW9662intheextractsbymassspectrometry.Fig.3showsthat whereasROSIhadarelativelylonghalf-lifeinthesupernatantand inthecellpellet(∼26and∼54hrespectively),GW9662hasavery shorthalf-lifeinboththemediaandinthecellpellet(∼1.9and
∼2.2hrespectively).Therefore,whileGW9662issufficiently per-sistenttoblockPPAR␥activityintherelativelyshorttransfection experimentsshowninFig.1(lessthan24htreatmentwithligands), itisunlikelytopersistlongenoughincellculturetoinhibit adi-pogenicdifferentiationoverthe7daylengthoftheadipogenesis assay.Thus,ifonewishestoblockPPAR␥activityin adipogene-sisassays,thesupplyofGW9662mustbereplenishedatregular intervalstomaintainsufficientlevelstoblockPPAR␥activity.
2.4. Underappropriateconditions,antagonizingPPARblocks TBT-andROSI-mediatedadipogenesisin3T3-L1cells
Fig.3.GW9662hasaveryshorthalf-lifecomparedwithrosiglitazone.3T3-L1cells weretreatedwith1MROSIor1MGW9662andsamplestakenat0,1,2,4,8,12, 24,36and48h.Afterextractionofsupernatantsorcellpellets,ethylacetatesoluble materialswererecoveredandanalyzedbytandemmassspectrometryand normal-izedtoaninternaldexamethasonestandard.Dataareexpressedmeans±SEMfor triplicatesamples.
thedifferentiationprocess.3T3-L1cellswereseededinto12-well plates,allowedtoreachconfluency,theninducedtodifferentiate witha standard cocktail containinginsulin, isobutylmethylxan-thine,and dexamethasonefortwo days,followed bytreatment withDMSOsolventcontrol,50 or100nM TBT,or100nMROSI. SomewellswerestainedwithOilRedOafter7days,whereas oth-erswereextractedfortriglycerideanalysisorRNApreparedfor geneexpressionanalysis.Fig.4ashowsthatundertheseconditions, 500nMGW9662stronglyinhibitedadipogenesisinducedby50or 100nMTBTor100nMROSI.Theseresultsweconfirmedby quan-titativeanalysisofOilRedOstaining(Fig.4b)andbyquantitation oftriglyceridesfromextractedcells(Fig.4c).
We nexttested the effects of TBT or ROSI onexpression of adipogenicgenesin thepresence or absenceof GW9662 using quantitativerealtimeRT-PCR(QPCR).BothTBTandROSIelicited significantincreasesintheexpressionofadipogenicmarkergenes suchasaP2(FABP4)(Fig.5a),adiponectin(Fig.5b),leptin(Fig.5c) and perilipin (Fig. 5d). In all cases, GW9662 strongly inhibited TBT-orROSI-mediatedincreasesinadipogenicgeneexpression, althoughitdidnotreachstatisticalsignificanceinallinstances(e.g., perilipinandadiponectinforTBT+GW9662).Takentogether,these resultsindicatedthatGW9662isabletoblockadipogenesis, triglyc-erideaccumulationandtheexpressionofadipogenicmarkergenes inducedbyeitherROSIorTBT.
3. Discussion
Manyinvestigationsintoadipocytedifferentiationutilize pre-adipocyte cell lines such as 3T3-L1, or 3T3-F442A that are alreadycommittedtotheadipocytelineage,butwhichremainas fibroblastsintheabsenceofappropriateadipogenicstimulation (reviewedin[12]).PPAR␥isoneofthekeygenesinadipocyte differ-entiationandisconsideredbymanytobethe“masterregulator”of adipogenesis[12,16]anditisreasonabletohypothesizethat treat-ingsusceptiblecellswithPPAR␥ligandswillinduceadipogenesis (reviewedin[7]).Asinterestintheidentityofchemicalobesogens
andtheirmodesofactionincreases,itbecomescritically impor-tanttohave well-definedmethods todeterminewhich cellular pathwaysarebeingperturbedbyobesogenaction.Ourstudy re-examinedthemolecularmechanismsthroughwhichTBTinduced adipogenesisin3T3-L1cellssincetherehadbeenanongoing con-troversyaboutwhetherTBTregulatesadipogenesisinthesecells throughaPPAR␥-dependentmechanism[13].
OneimportanttestforwhetherachemicalactsthroughPPAR␥
istheabilityoftheadipogeniceffecttobeblockedbyinhibiting thefunctionofPPAR␥usingspecificantagonistssuchasGW9662 [21]orT0070907[22].WhileithasbeenshownthatthePPAR␥
antagonistT0070907canblocktheabilityofanadipogeniccocktail (containingisobutylmethylxanthine,insulinanddexamethasone) toinduceadipogenesisin3T3-L1cells[22],relativelyfewstudies haveexaminedtheeffectsofGW9662ontheinductionof adipo-genesisinthesecells [13],particularlyinresponsetotreatment withcandidateobesogens.Incontrast,manystudieshavelinked theinductionofadipogenesis-relatedgenesinmatureadipocytes, witharequirementforPPAR␥activationthatcanbeblockedby GW9662[23–26].
Spigelmanandcolleaguesdemonstratedthatthesynthetic com-poundbisphenolAdiglycidyletherwasanantagonistofPPAR␥at highlevels(∼100M)andcouldinhibittheinductionof adipoge-nesisin3T3-L1cellsbyROSI[14].Morerecently,thesamegroup showedthata mutatedPPAR␥thatisunabletobeactivatedby ligandsstillsupportedadipogenesisin3T3-L1cells,whichcould beinterpretedtosuggestthatligandactivationofPPAR␥,perse,is notrequiredforadipogenesis[15].Theywerecarefultonotethat thelevelofPPAR␥proteinmightbesufficienttoobviatetheneed forligandactivationsincethisreceptorhasasignificantamount ofligand-independentactivationin transfectionassays andthat theremightbeanendogenous,albeitunidentifiedligandforPPAR␥
[15].TheydidshowthattheAF-2transcriptionalactivationdomain ofPPAR␥wasrequiredforadipogenesis,suggestingthatPPAR␥is actingasatranscriptionalactivator,irrespectiveofwhetherthis isdependentonthepresenceofligand[15].Thisleavesopenthe questionwhetheractivationofPPAR␥isrequiredforadipogenesis in3T3-L1cells.
WeandothershaveshownthatorganotinssuchasTBT[8,13] andtriphenyltin[9,27]bindtoPPAR␥andRXRs[28]with nanomo-laraffinity.Moreover,organotins induceadipogenesisin3T3-L1 cells [8,9,13,27] and in adipose-derived[10] and bone-marrow derived[11]MSCs.OnlyinthecaseoftheadiposederivedMSCs hasitbeenconvincinglyshownthatPPAR␥activationisrequired fortheadipogenicactivityofTBTandROSI[10].Onegrouphas suggestedthatTBTactsthroughanon-PPAR␥mediatedpathway toinduceadipogenesisin3T3-L1cells[13].However,considering thehighaffinitybindingofTBTtoRXRandPPAR␥,itseemsmore likelythatTBTisactingthroughoneofthesereceptorstoinduce adipogenesis.
WesoughttoaddresstheissueofwhetherTBTinduces adipo-genesisby a PPAR␥-dependent orPPAR␥-independentpathway bytestingalternativepossibilities.WefoundthattheRXR–PPAR␥
Fig.4. EffectofTBT,ROSIandGW9662onthedifferentiationof3T3-L1cells.(A)3T3-L1wasdifferentiatedintomatureadipocytesbytheadditionofanadipogeniccocktail for2days,followedbysupplementationwith50or100nMTBTor100nMROSI(toppanels)orthesameligandsplus500nMGW9662.CellswerestainedwithOilRedO7 daysafterthedifferentiationcocktailwasadded.Representativepictureswerepresentedforeachtreatment.(B)LipidaccumulationwasquantifiedbyImageJsoftwareand averageareafractionwaspresented.Datawereshownasmeans±SEMfortriplicatesamples.6individualpictureswerequantifiedandaveragedforeachsample.Dataare expressedasaverageareafractioninn=3replicates±SEM(n=6perwell).(C)Cellulartriglycerideswereextracted,quantitatedandtheresultsnormalizedtocellularDNA content.Dataareexpressedasmgtriglyceride/mgcellularDNAandarepresentedasmeans±SEMfortriplicatesamples.
GW9662wasabletoblockadipogenesiselicitedbyROSIorTBT (Fig.4)aswellastheinductionofPPAR␥targetgenessuchasaP2 (FABP4),adiponectin,leptinandperilipin(Fig.5).Takentogether, weinterpretthesedatatoindicatethatTBTactsthroughPPAR␥
in 3T3-L1 cells to induce adipogenesis and the expression of PPAR␥targetgenes.WeconcludethatPPAR␥activityisrequired for adipogenesis in 3T3-L1 cells and that ligandstability is an important consideration when designing and interpreting the resultsofadipogenesisassays.
Our results have important implications for the design and interpretationofadipogenesisassaysthatseektotestthepathways throughwhichcandidateobesogensmightact.IfGW9662isnot supplementedintothemediumfrequentlyenough,itispossible to make the erroneous inference that the candidate chemicals areactingthroughanon-PPAR␥-dependentpathway.Wesuggest that in order to avoid such mistakes, it is essential to utilize ROSI(oranotherstrongPPAR␥activator)asapositivecontrolin adipogenesisassays.Anotherindispensablecontrolmustbethat GW9662(orsomeotherPPAR␥antagonist)canblocktheinduction ofadipogenesisbyROSIbeforeconcludingthattheantagonistdoes ordoesnotinhibittheactivityofanothercandidatecompound.
Consideringtheincreasinginterestintestingchemicalsfortheir abilitytoinduceadipogenesis,ourresultswillbebroadly applica-bletothestudyofcandidateobesogensandtheirmechanismsof action.
4. Methods
4.1. Transfectionassays
pCMX-GAL4 and fusion constructs to nuclear receptor LBD [GAL4-hPPAR␥] have beenpreviously described [8]. The useof L-RXR␣asaspecificprobetoassesstheactivationofRXR, indepen-dentlyofitsheterodimericpartnerswaspreviouslyreported[20]. TransfectionswereperformedinCos7cellsessentiallyasdescribed elsewhereusingMH100-x4-TK-Lucasreporterandnormalizedto pCMX--galactosidasecontrols[29].Briefly,Cos7cellswereseeded at5000cellsperwellin96-welltissuecultureplatesin10%fetal bovineserum/DMEMandtransfectedfor8hwith11g/plateof DNA/calciumphosphateprecipitatemix
Fig.5.QuantitativerealtimeRT-PCRanalysisofgeneexpression.3T3-L1cellsweredifferentiatedintomatureadipocytesbytheadditionofanadipogeniccocktailfor2 daysfollowedbytreatment50nmTBTor100nMROSIinthepresenceorabsenceof500nMGW9662whichwasaddedevery8hduringthedifferentiationprocess.RNA wascollectedfromthecellsonday7,followedbyrealtimeRT-PCRforgeneexpressionanalysis.Datawerepresentedasmeanfoldinduction±SEMcomparedwithDMSO vehiclefortriplicatesamples.*P<0.5,**P<0.01and***P<0.001.Experimentswererepeatedatleasttwice.
5g/mLinsulin,5g/mLholo-transferrin,5g/mLselenium,0.5% definedlipidmix(Invitrogen),0.12%w/vdelipidatedbovineserum albumin(Sigma))plusligandsforanadditional24hbeforeassays forluciferaseandß-galactosidaseactivity{Grun,2006#569}.All transfectiondatapointswereperformedintriplicate,andall exper-imentswererepeatedatleastthreetimes.
4.2. 3T3-L1cellculture
3T3-L1cellsweremaintainedinDMEMsupplementedwith10% FBS,2mMl-glutamine,50U/mLpenicillinand50g/mL
strepto-mycin.Fordifferentiation,3T3-L1cellswereplatedata density of5000cells/cm2on12-wellplatesandcultureduntil2dayspost confluence.Atthistime,theywereswitchedtoadifferentiation mediumcontaining10%FBS,MDI(5g/mLinsulin,0.25M dex-amethasoneand0.5mM3-isobutyl-1-methylxanthine),8g/mL biotin, 8g/mL pantothenate 50U/mL penicillin and 50g/mL streptomycin.After2furtherdaysofincubation,cellswere main-tainedinDMEM/10%FBS,biotin,pantothenate,penicillin, strepto-mycincontainingTBTorROSIattheconcentrationsindicatedin thefigurelegends.Forantagonistassays,GW9662wasaddedto 500nMintothemediumevery8hfor7days.Cellswereharvested forsubsequentassays7daysafteradditionofthedifferentiation cocktail.Allexperimentswererepeatedatleastthreetimes.
4.3. Triglycerideassays
3T3-L1cultureandinductionofadipogenesiswereperformedin 12-wellplatesasdescribedabove.After7daysofdifferentiation, cellswerewashedwithPBS,theaqueousmediumaspiratedand
totallipidextractedbyadding1mLofhexanes:isopropanol(3:2 v/v)totheplatesandshakingfor1h.Theorganiclayerwas col-lectedtoglass13mm×100mmtesttubes,driedunderastream ofargongasand solubilizedin300Lofchloroformcontaining 1% Triton-X100.Samples were dried againunder argon, resus-pendedin200Lofpurewaterandincubatedat37◦Cfor1hto ensuredissolution.Triglycerideswerequantifiedusingthe Infin-ityTriglyceridekit(ThermoScientific)andnormalizedtocellular DNAcontent.ForDNAextraction,cellswerehomogenizedinbuffer containing0.1MNaOH,0.1MNaCl,20mMEDTA,followedby cen-trifugation.100Lofsupernatantwascollectedandmixedwith 1mLof10mMMOPS(freeacid),pH4.6,andDNAconcentration wasdeterminedbyA260measurement.
4.4. OilRedOstainingandanalysis
CellswerewashedtwicewithPBSandfixedin10%formalin inPBSfor15min,rinsedwith60%isopropylalcohol,andstained with0.3%OilRedOin60%isopropanolfor30min.Cellswerethen rinsedtwicewith60%isopropylalcoholandmountedin30% glyc-erol.Lipidaccumulationwasquantifiedaspreviously described [10]usingImageJ(version1.36b;WayneRasband).Data repre-sentmean±SEMfromn=3wellspertreatmentandn=6pictures perwell.
4.5. AnalysisofmRNAbyreal-timereversetranscriptase polymerasechainreaction
DNase-treatedRNAusingSuperScriptTMIIIRNaseH−Reverse Tran-scriptase(Invitrogen)afterDNaseIdigestion(Ambion,Austin,TX) followingthemanufacturerrecommendedprotocol.Real-timePCR wasperformedin theDNA Engine Opticon ThermalCycler[MJ Research (Watertown,MA/Bio-Rad Laboratories(Hercules,CA)]. QuantitativePCRanalysesfortargetgenes(listinginSupplemental Table1)wereperformedwithFastStartSYBRGreenQPCRMaster Mix(Roche,Nutley,NJ)and100nMofprimerschosenwithusing PerlPrimer(v1.1.14Copyright©2003–2006OwenMarshall). Rela-tivequantificationofthetargetgenetranscriptincomparisonwith
-actin(housekeepinggene) expressionlevelinthesame sam-ple,wasmadefollowingthePfafflmethod(2010)and modified aspreviouslydescribed[10].
4.6. Massspectrometricanalysis
3T3-L1cellswereseededinto10cMdishesandgrownto80% confluency in DMEM+10% FBS. Media was then aspirated and replacedwith15mLoffreshmediumcontaining1MofROSIor GW9662.After5min,a1mLaliquotofmediawastakenandfrozen at−80◦CasT=0h.PlateswereincubatedinaCO
2 incubatorat 37◦Cin5%CO
2inairfor1,2,4,8,12,24,36or48h.Samplesfor analysisofmediaweretakenfromthesameplateatsuccessive intervals.Forcellularfractions,platesweretreatedinparallelin thesamemanner.Attheendofeachtimepoint,themediumwas aspirated,thecellstrypisinizedandstoredat−80◦Cuntilall sam-pleswereready.Triplicatesamplesof300Lofmediaorcellpellets resuspendedin300LofdistilledH2Owerespikedwith dexam-ethasoneat1M(asaninternalstandard)andextractedwithan equalvolumeofethylacetate.Aftervigorousvortexingfor2min, thephaseswereseparatedbycentrifugationandtheorganicphase removedtoanautosamplervial.Thesampleswereevaporatedto drynessinaSpeedVacconcentrator,thenresuspendedin100Lof 50%methanolindistilledH2O.SampleswereanalyzedonaWaters QuattroPremierXEtriplequadrupoletandemmassspectrometer andvaluesobtainedforROSIorGW9662normalizedto dexam-ethasonetocorrectforextractionefficiency.Allexperimentswere repeatedatleasttwice.
Acknowledgements
SupportedbyagrantfromtheNIHR01-ES015849to(B.B.).J.Y. wassupportedby fellowshipsfromtheUniversity of California ToxicSubstancesResearchandTrainingProgram,theUCIMinority BiomedicalResearchSupportProgram(NIHGM-55246)andNSF GK-12Grant0638751.J.Y.wishestothankDr.FelixGrünforhelp withthedesignandimplementationofmassspectrometric mea-surements.WethankA.JanesickandR.Kaighforcommentsonthe manuscript.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.jsbmb.2011.03.012.
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