Alpha inhibits adipogenesis via beats by dre outlet uk a beta chpdrebtshdphones CorrESponDEnCE:Jk SEtHi, DEpArtmEnt oF CliniCAl BioCHEmiStry, univErSity oF CAmBriDGE, ADDEnBrookE'S HoSpitAl, Box 232, HillS roAD, CAmBriDGE CB2 2qr, uk.InDEED, impAirED ADipoGEnESiS mAy ContriButE to tHE pAtHoGEnESiS oF oBESityASSoCiAtED ConDitionS inCluDinG Insulin rESiStAnCE, HypErlipiDAEmiAAndtypE ii DiABEtES.ADipoGEnESiS involvES tHE ACtivAtion oF A CASCADE oF trAnSCription FACtorS, wHiCH CoorDinAtE tHE ExprESSion oF GEnES rESponSiBlE For ADipoCytE FunCtion.1 ADipoGEniC Stimuli inDuCE tHE rApiD ExprESSion oF tHE trAnSCription FACtorS CCAAT/EnHAnCErBinDinG protEin(C/EBp)AnD C/EBp, wHiCH StimulAtE A trAnSiEnt StAGE oF mitotiC ClonAl ExpAnSion DurinG EArly ADipoGEnESiS.1 C/EBPAnD C/EBpAlSo promotE tHE ExprESSion oF tHE mAStEr ADipoGEniC trAnSCription FACtorS C/EBPAndpEroxiSomE proliFErAtorACtivAtED rECEptorGAmmA(PpAr).ToGEtHEr tHESE ACt CoopErAtivEly to rEGulAtE tHE ExprESSion oF GEnES ASSoCiAtED witH tErminAl DiFFErEntiAtion.1 LoCAlAndEnDoCrinE FACtorS mAy rEGulAtE ADipoGEnESiS By moDulAtinG tHE ACtivity oF tHESE trAnSCriptionAl EvEntS.2In ContrASt, inHiBition oF ADipoGEnESiS By TNF mAy BE morE rElEvAnt to pAtHoloGiCAl ConDitionS.ADipoGEnESiS iS oFtEn impAirED in DiSorDErS ASSoCiAtED witH ElEvAtED proDuCtion oF tnF, SuCH AS in CACHExiA, SEpSiSAndin oBESityrElAtED Insulin rESiStAnCE.7 THE lAttEr iS BASED on tHE HypotHESiS tHAt rEStriCtion oF ADipoSE tiSSuE ExpAnDABility mAy ContriButE to oBESityrElAtED HypErlipiDAEmiAAndSuBSEquEntly lEADinG to lipotoxiCityinDuCED Insulin rESiStAnCE.8 THE AntiADipoGEniC ACtionS oF TNF HAvE BEEn primArily AttriButED to SiGnAlS trAnSDuCED By TNF rECEptor1(TnFr1), 4, 9 tHrouGH wHiCH TNF StimulAtES numErouS SiGnAllinGPAtHwAyS.THESE inCluDE tHE ACtivAtion oF SpHinGomyElinASES, CASpASES, jnk, Erk1/2AndnuClEAr FACtorkAppA B(NFB).EACH oF tHESE HAvE BEEn impliCAtED in tHE nEGAtivE rEGulAtion oF ADipoGEnESiS.9 AltHouGH it HAS BEEn SuGGEStED tHAt ACtivAtion oF NFB iS nECESSAry For TNFinDuCED AntiADipoGEnESiS, 10, 11 tHiS ConFliCtS witH tHE rECEnt FinDinG tHAt NFB ACtivity inCrEASES DurinG ADipoGEnESiS.12 HEnCE, tHE nAturE oF tHE FinAl AntiADipoGEniC SiGnAllinGPAtHwAy(S)InDuCED By tnFr1 rEmAIns inComplEtEly unDErstooD.HErE, wE ConFirm tHAt tnF inHiBits ADipoGEnEsis By prEvEntinG tHE EArly inDuCtion oF ppArAndC/EBp.HowEvEr, tHis EFFECt CoinCiDEs witH EnHAnCED ExprEssion oF sEvErAl AntiADipoGEniC GEnEs tHAt ArE Also tArGEts oF tHE wnt/CAtEnin/tCEll FACtor 4(TCF4)PAtHwAy. THErEForE, wE invEStiGAtED wHEtHEr TNF inHiBitS ADipoGEnESiS By ACtivAtinG tHE CAtEnin/TCF4PAtHwAy.Our rEsults sHow tHAt tnF EnHAnCEs tHE trAnsiEnt CAtEnin/tCF4 trAnsCriptionAl ACtivity DurinG EArly AntiADipoGEnEsis. BloCkinG tHiSPAtHwAy viA StABlE knoCkDown oF CAtEnin or ovErExprESSion oF DominAntnEGAtivE TCF4 (DntCF4)AttEnuAtEs tnFinDuCED AntiADipoGEnEsis. THErEForE, wE propoSE tHAt TNF inHiBitS ADipoGEnESiS viA A CAtEnin/TCF4 DEpEnDEntPAtHwAy.Top oF pAGErEsultssHorttErm ExposurE to tnF is suFFiCiEnt to inHiBit ADipoGEnEsis in 3t3l1 CEllsto iDEntiFy tHE EArliEst ADipoGEniC EvEnts tHAt ArE impAirED By tnF wE First invEstiGAtED tHE impACt oF limitinG tnF ExposurE to tHE First 48 H oF 3t3l1 ADipoGEnEsis.FiGurE 1A sHows tHAt tnF mEDiAtEs tHE sAmE AntiADipoGEniC EFFECts wHEtHEr it is prEsEnt For tHE First 48 H oF ADipoGEnEsis or tHrouGHout tHE 8DAy ADipoGEniC proGrAmmE.THE 48 H tnF trEAtmEnt siGniFiCAntly prEvEntED tHE ExprEssion oF Ap2 tHAt oCCurrED DurinG normAl ADipoGEnEsis(FiGurE 1B).THis is ConsistEnt witH tHE prEviously rEportED EFFECts on F442A prEADipoCytEs13AndsuGGEsts tHAt tHis tnF ACtion is not CElltypE spECiFiCAndAntiADipoGEniC EFFECts oCCur EArly DurinG tHE inDuCtion pHAsE oF tHE ADipoGEnEsis.ConFluEnt 3t3l1 prEADipoCytEs wErE inDuCED to DiFFErEntiAtE As DEsCriBED in mAtEriAlsAndmEtHoDs.CElls wErE trEAtED witH or witHout tnF(2 nG/ml)EitHEr tHrouGHout tHE DiFFErEntiAtion proCEDurE(Full)Or For tHE First 48 H AFtEr inDuCtion(48 H). (A)At 8 DAysPostinDuCtion, tHE ExtEnt oF ADipoGEnEsis wAs AssEssED By stAininG For lipiD ACCumulAtion witH oil rED o.THE DAtA ArE rEprEsEntAtivEOF FivEinDEpEnDEnt ExpErimEnts. (B 3t3l1 CElls wErE inDuCED to DiFFErEntiAtE witH or witHout tnF(2 nG/ml For tHE First 48 H)AnD totAl rnA wAs ExtrACtED At 0, 2, 4, 8, 12, 16, 24, 48, 96, 144And192 HPostinDuCtion.TrAnsCript lEvEls oF Ap2(B), PPAR2(C), C/EBP(D)AnD C/EBp(E)WErE DEtErminED By usinG rEAltimE pCrAndnormAlisED to 18s rrnA.OF tHrEE inDEpEnDEnt ExpErimEnts.StAtistiCAl siGniFiCAnCE is inDiCAtED As Follows:CompArED to Control trEAtmEnt At tHE sAmE timE point(PppFull FiGurEAndlEGEnD(240K)To invEstiGAtE FurtHEr tHE tEmporAl CHArACtEristiCs oF tnFinDuCED AntiADipoGEnEsis, wE AnAlysED ExprEssion oF sEvErAl kEy ADipoGEniC trAnsCription FACtors.In Control CElls, ADipoGEniC inDuCtion siGniFiCAntly inCrEAsED tHE ExprEssion oF ppAr2 From 8 HPostinDuCtion onwArDs,AndinCrEAsED C/EBp ExprEssion wAs DEtECtED witHin 4 HPostinDuCtion(FiGurE 1CAndD).THus tHE inCrEAsED ExprEssion oF tHEsE kEy ADipoGEniC trAnsCription FACtors oCCurs EArliEr tHAn HAs BEEn prEviously rEportED. 14, 15 TNF trEAtmEnt ComplEtEly prEvEntED tHE inDuCtion oF BotH PpAr2AnD C/EBp(FiGurE 1CAndD).ADDitionAlly, tnF supprEssED tHE ExprEssion oF C/EBp to BElow prEinDuCtion lEvEls, rEDuCinG it By ApproximAtEly 60% At 8 H postmDi(FiGurE 1D).In ContrAst, tnF DiD not AltEr tHE ExprEssion oF C/EBp, An inDuCEr oF C/EBpAndppAr(FiGurE 1E).THEsE DAtA sHow tHAt tnF mEDiAtEs its AntiADipoGEniC EFFECts EArly in tHE ADipoGEniC proGrAmmE.TnF EnHAnCEs tHE trAnsiEnt ExprEssion oF AntiADipoGEniC mEDiAtors DurinG tHE EArly stAGEs oF AntiADipoGEnEsisto invEstiGAtE How tnF supprEssEs C/EBpAndppAr2 ExprEssion, wE AnAlysED tHE ExprEssion oF trAnsCription FACtors known to inHiBit tHEsE ADipoGEniC GEnEs.GAtA2AndGAtA3 supprEss ppAr By DirECtly BinDinG to its promotEr,AndForCEDExprEssion oF GAtA2or GAtA3 inHiBits ADipoGEnEsis.16 In Control CElls, BotH GATA2AndGATA3 mRNAs wErE DownrEGulAtED witHin 2 HPostinDuCtion.HowEvEr, nEitHEr oF tHEsE mrnAs sHowED AltErED ExprEssion in tnFtrEAtED CElls(FiGurE 2AAndB).SimilArly, tnF stimulAtEs tHE sustAinED ExprEssion oF CmyC DurinG AntiADipoGEnEsis in tA1 prEADipoCytEs17AndForCED ExprEssion oF CmyC prEvEnts ADipoGEnEsis By inHiBitinG tHE ExprEssion oF C/EBp.18 ADDitionAlly, BotH CyClin D1AnD ppArinHiBit ADipoGEnEsis By supprEssinG PPAR.19, 20 FiGurEs 2C sHow tHAt tHEMrna expression oF CmyC, CyClin D1AnD PPAR wAs siGniFiCAntly uprEGulAtED FollowinG ADipoGEniC inDuCtion,AndtHis ExprEssion wAs FurtHEr EnHAnCED By TNF trEAtmEnt.At lAtEr timEpoints(4 DAys), only CyClin D1 ExprEssion rEmAinED ElEvAtED in TNFtrEAtED CElls(FiGurE 2D).RnA sAmplEs wErE CollECtED DurinG 3t3l1 ADipoGEnEsisAndAntiADipoGEnEsis As DEsCriBED in FiGurE 1.ExprEssion oF GAtA2(A), GATA3(B), CmyC(C), CyClin D1(D)AnD ppAr(E)WAs DEtErminED By usinG rEAltimE pCrAndnormAlisED to 18s rrnA.GEnE ExprEssionAndstAtistiCAlly siGniFiCAnt DiFFErEnCEs ArE rEportED As DEsCriBED in FiGurE 1Full FiGurEAndlEGEnD(112K)TNF ACtivAtES CAtEnin/TCF SiGnAllinG DurinG AntiADipoGEnESiSIntriGuinGly, CmyC, CyClin D1AnD PpAr ArE tArGEt GEnES oF tHE CAnoniCAl Wnt SiGnAllinGPAtHwAy, wHiCH itSElF HAS BEEn SHown to inHiBit ADipoGEnESiS.21, 22, 23 WE nExt invEstiGAtED Wnt10BMrna expression.UnDEr Control ConDitions, tHErE wAs A trAnsiEnt pEAk in wnt10B mrnA lEvEls, wHiCH suBsEquEntly DroppED to BElow prEADipoCytE lEvEls(FiGurE 3A).In ContrAst, wnt10B lEvEls DurinG tnFinDuCED AntiADipoGEnEsis wErE nEitHEr ElEvAtED nor mAintAinED in tHE First 48 H oF tHE proGrAmmE.InstEAD, wnt10B trAnsCript lEvEls wErE siGniFiCAntly supprEssED By tnF trEAtmEntAndonly BEGAn to rEturn to normAl AFtEr tnF wAs rEmovED.ExprEssion oF wnt10B(A)WAs DEtErminED As DEsCriBED in FiGurEs 1And2. (BAndC)3T3L1 prEADipoCytEs wErE Also trAnsFECtED witH tHE TCF rEportEr TopFlAsH As DEsCriBED in MAtEriAlsAndMEtHoDs.THrEE DAys posttrAnsFECtion, CElls wErE inDuCED to DiFFErEntiAtE in tHE ABsEnCE or prEsEnCE oF tnF(2 nG/ml).LuCiFErAsE ACtivity wAs AssAyED 12 H(B)Or 48 H(C)PostinDuCtion.OF FivE(B)Or Four(C)InDEpEnDEnt ExpErimEnts, witH EACH ExpErimEnt CArriED out in DupliCAtE. (D, EAndF)ConFluEnt 3t3l1 prEADipoCytEs wErE inDuCED to DiFFErEntiAtE witH or witHout tnF(For tHE First 48 H AFtEr inDuCtion)AndCytoSoliC protEin wAS ExtrACtED At tHE inDiCAtED timESPostinDuCtion. ExprESSion oF CAtEnin, AP2,AnD ppArwAS tHEn AnAlySED By WEStErn BlottinG.TimE 0 H protEIns wErE ExtrACtED BEForE DiFFErEntiAtion wAs inDuCED.THE p85 suBunit oF pi3k wAs usED As A loADinG Control.OF tHrEE inDEpEnDEnt ExpErimEnts. (G)CAtEninMrna expression DurinG ADipoGEnEsisAndtnFinDuCED AntiADipoGEnEsis.TotAl rnA wAs ExtrACtED As DEsCriBED in FiGurE 1. (H)3T3L1 prEADipoCytEs ExprEssinG CAtEnin s45A or An Empty vECtor wErE inDuCED to DiFFErEntiAtE witH MDI. THE ExtEnt oF ADipoGEnESiS At 8 DAySPostinDuCtion wAS ASSESSED By StAininG For lipiD ACCumulAtion witH oil rED OFull FiGurEAndlEGEnD (243K)WE nExt invEStiGAtED wHEtHEr tHE TNFinDuCED uprEGulAtion oF CmyC, CyClin D1AnD PpAr rEprESEntED A morE SpECiFiC EFFECt oF TNF on CAtEnin/TCF trAnSCriptionAl ACtivity uSinG A TCFDEpEnDEnt luCiFErASE rEportEr, TOPFLASH. TNF EnHAnCED TOPFLASHDEpEnDEnt luCiFErASE ACtivity At 12 HPostinDuCtion oF ADipoGEnESiS (FiGurE 3B) But not At 48 HPostinDuCtion (FiGurE 3C).THis is ConsistEnt witH tHE EArly But trAnsiEnt EnHAnCEmEnt oF wnttArGEt GEnEs DurinG AntiADipoGEnEsis(FiGurE 2C to invEstiGAtE wHEtHEr tnF mEDiAtEs its EFFECts on tCFDEpEnDEnt GEnE ExprEssion viA stABilisAtion oF CAtEnin, CytosoliC protEin lEvEls wErE ExAminED. AS SHown in FiGurE 3D, TNF DiD not AFFECt tHE lEvElS oF CytoSoliC CAtEnin DurinG tHE FirSt 48 HPostinDuCtion.HowEvEr, At lAtEr timE points(>48 H), CAtEnin protEin lEvEls wErE mAintAinED in TNF trEAtED CElls(FiGurE 3EAndF).In ContrAst, tnF DiD not AltEr tHE ExprEssion oF CAtEnin mrnA DurinG AntiADipoGEnEsis(FiGurE 3G).HowEvEr, tHE ConstitutivE ExprEssion oF A stABlE CAtEnin mutAnt(CAtEnin s45A)24 Also inHiBitED ADipoGEnEsis(FiGurE 3H).THis suGGEsts tHAt tnFinDuCED stABilisAtion oF CAtEnin mAy ContriButE to tHE AntiADipoGEniC ACtions oF tnF. CAtEnin knoCkDown EnHAnCES 3T3L1 ADipoGEnESiS AnDAttEnuAtEs tnFinDuCED AntiADipoGEnEsisTo ADDrESS tHE rolE oF CAtEnin in TNFinDuCED AntiADipoGEnESiS, wE GEnErAtED A 3T3L1 CEll linE in wHiCH ExprESSion oF CAtEnin iS StABly knoCkED Down By ECtopiC ExprESSion oF A SHort HAirpin RNA (SHrnA)SpECiFiC to CAtEnin.THE ExprEssion oF CAtEnin mrnAAndprotEin wAs siGniFiCAntly rEDuCED in tHEsE CElls(FiGurE 4AAndB).THE ADipoGEniC potEntiAl oF tHis CEll linE AppEArED similAr to Control prEADipoCytEs, witH BotH DiFFErEntiAtinG ComplEtEly in rEsponsE to Full inDuCtion CoCktAilAndwitH no morpHoloGiCAl DiFFErEnCEs AppArEnt At DAy 8(FiGurE 4C).HowEvEr, wHEn suBjECtED to suBmAximAl inDuCtion, tHE CAtEnin knoCkDown prEADipoCytEs ExHiBitED EnHAnCED lipoGEnEsis(FiGurE 4C)AndConSiStEntly ExprESSED HiGHEr lEvElS oF PpAr2AnD C/EBp(FiGurE 4DAndE).SurprisinGly, CAtEnin knoCkDown CElls Also ExprEssED morE ppAr2 mrnA wHEn inDuCED witH Full mDi(FiGurE 4D).THEsE rEsults inDiCAtE tHAt knoCkDown oF CAtEnin EnHAnCEs ADipoGEnEsis in 3t3l1 prEADipoCytEs. FurtHErmorE, tHEy SuGGESt tHAt CAtEnin mAy normAlly limit PpAr2AnD C/EBpExprESSion in mAturE 3T3L1 ADipoCytES.3T3L1 prEADipoCytEs wErE trAnsFECtED witH vECtors EnCoDinG SHrnAs AGAInst FirEFly luCiFErAsE(Control)Or murinE CAtEnin(SHrnA). (A) TotAl RNA wAS ExtrACtED From ConFluEnt ControlAndCAtEnin SHrnA 3T3L1 prEADipoCytESAndExprESSion oF CAtEnin mRNAWAs DEtErminED By usinG rEAltimE pCrAndnormAlisED to 18s rrnA. (B)CytosoliC protEin was ExtraCtED From ConFluEnt 3t3l1 prEaDipoCytEs ExprEssinG Control or CatEnin sHrnaAndExprEssion oF CatEnin protEin was analysED by wEstErn blottinG.P85 was usED as a loaDinG Control.THE rEsult sHown is rEprEsEntativE oF Four inDEpEnDEnt ExpErimEnts. (C ControlAndCatEnin sHrna CElls wErE inDuCED to DiFFErEntiatE witH sErum only(CosmiC CalF sErum CCs), Insulin(Ins), IBMX or MDI. THE ExtEnt oF ADipoGEnESiS At 8 DAySPostinDuCtion wAS ASSESSED By oil rED O StAininG (C)Or ExtraCtion oF total rna FollowED by rEaltimE pCr analysis oF ppar2(D)AnD C/EBp(E)Mrna expression. (F ControlAndCAtEnin SHrnA CEllS wErE inDuCED to DiFFErEntiAtE witH MDI in tHE prESEnCE oF tHE inDiCAtED ConCEntrAtionS oF TNF, wHiCH wErE mAintAinED in tHE ADipoGEniC mEDiA For tHE FirSt 48 HPostinDuCtion only. THE ExtEnt oF ADipoGEnESiS At 8 DAySPostinDuCtion wAS ASSESSED By oil rED O StAininG (F)Or ExtraCtion oF total rna FollowED by rEaltimE pCr analysis oF http://www.teamskyuser.co.uk/beats-by-dr-dre-pro.html ppar2(G)AnD C/EBp(H)Mrna expression.OF tHrEE inDEpEnDEnt ExpErimEnts.The results in(C)And(F) ArE rEprESEntAtivEOF tHrEE inDEpEnDEnt ExpErimEnts. For EACH inDuCtion trEAtmEnt, StAtiStiCAlly SiGniFiCAnt DiFFErEnCES BEtwEEn ControlAndCAtEnin knoCkDown CEllS ArE rEportED AS FollowS: PppFull FiGurEAndlEGEnD(353K)THE rEquirEmEnt For CAtEnin in TNFinDuCED AntiADipoGEnESiS wAS invEStiGAtED By inDuCinG ControlAndCAtEnin knoCkDown CEllS to DiFFErEntiAtE witH MDI in tHE prESEnCE oF A rAnGE oF ConCEntrAtionS oF TNF. FiGurE 4F SHowS tHAt 2 nG/ml TNF ComplEtEly prEvEntED lipiD ACCumulAtion in BotH ControlAndCAtEnin knoCkDown CEllS.However, 1ng/ml tnf was less effective at inhibiting lipid accumulation in catenin knockdown cells. AnAlySiS oF PpAr2AnD C/EBpmRNA ExprESSion ConFirmED tHAt TNF inHiBitED tHEir ExprESSion in A ConCEntrAtionDEpEnDEnt mAnnEr (FiGurE 4GAndH).However, in catenin knockdown cells, the concentrationdependent expression of c/ebp(Figure 4h)Was rightshifted.This indicates that tnf may regulate c/ebp expression in a catenindependent manner.Catenin knockdown promotes apoptosis during tnfinduced antiadipogenesisa striking observation made during our experiments was that catenin knockdown preadipocytes were more sensitive to tnfinduced cell death during adipogenic treatment. THiS oBSErvAtion wAS ConFirmED By SuBSEquEnt ASSESSmEnt oF ADHErEntAndviABlE CEll numBErS (At 24 h).In control cells, tnf had no significant effect on cell viability during antiadipogenesis(FiGurE 5AAndB).In contrast, in catenin knockdown cells, tnf significantly reduced the number of viable cells(FiGurE 5AAndB), suggesting that TNFinduced cytotoxicity may be enhanced in the absence of catenin.Consistent with this, tnfinduced dna fragmentation was markedly enhanced in the catenin knockdown cells(FiGurE 5CAndD).

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