PPARγ contributes to PKM2 and HK2 expression in fatty liver

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1 Received Nov Accepted 9 Jn Published Feb DOI:.8/ncomms667 contributes to nd expression in ftty liver Gnn Pnsyuk,, Ctherine Espeillc,, Céline Chuvin,, Ludivine A. Prdelli,, Ysuo Horie, Akir Suzuki 6,7, Jen-Sebstien Annicotte 8, Lluis Fjs 8, Mrc Foretz 9, Frncisco Verdeguer 9, Mrco Pontoglio 9, Pscl Ferré, Jen-Yves Scozec, Morris J. Birnbum, Jen-Ehrlnd Ricci, & Mrio Pende, Rpidly proliferting cells promote glycolysis in erobic conditions, to increse growth rte. Expression of specific glycolytic enzymes, nmely pyruvte kinse M nd hexokinse, concurs to this metbolic dpttion, s their kinetics nd intrcellulr locliztion fvour biosynthetic processes required for cell prolifertion. Intrcellulr fctors regulting their selective expression remin lrgely unknown. Here we show tht the peroxisome prolifertorctivted receptor gmm trnscription fctor nd nucler hormone receptor contributes to selective pyruvte kinse M nd hexokinse gene expression in PTEN-null ftty liver. Peroxisome prolifertor-ctivted receptor gmm expression, liver stetosis, shift to erobic glycolysis nd tumorigenesis re under the control of the Akt kinse in PTEN-null mouse livers. Peroxisome prolifertor-ctivted receptor gmm binds to hexokinse nd pyruvte kinse M promoters to ctivte trnscription. In vivo rescue of peroxisome prolifertor-ctivted receptor gmm ctivity cuses liver stetosis, hypertrophy nd hyperplsi. Our dt suggest tht therpies with the insulin-sensitizing gents nd peroxisome prolifertor-ctivted receptor gmm gonists, thizolidinediones, my hve opposite outcomes depending on the nutritionl or genetic origins of liver stetosis. Inserm, U8, Pris 7, Frnce. Université Pris Descrtes, Fculté de Médecine, UMRS-8, Pris, Frnce. Inserm, U89, Centre Méditerrnéen de Médecine Moléculire (CM), équipe AVENIR, Nice 6, Frnce. Université de Nice-Sophi-Antipolis, Fculté de Médecine, Nice F-6, Frnce. Deprtment of Gstroenterology, Akit University Grdute School of Medicine, Akit -8, Jpn. 6 Division of Cncer Genetics, Medicl Institute of Bioregultion, Kyushu University, Fukuok 8-88, Jpn. 7 Globl COE progrm, Akit University Grdute School of Medicine, Akit, Jpn Institut de Recherche en Cncérologie de Montpellier, INSERM U896, Montpellier 98, Frnce. 9 Institut Cochin, Université Pris Descrtes, CNRS (UMR 8), Inserm, U6, Pris F-7, Frnce. INSERM, Centre de Recherches des Cordeliers, UMR-S 87, 76 Pris, Frnce. INSERM UMR86, Fculté Lennec, F 69 Lyon Cedex 8, Frnce. Howrd Hughes Medicl Institute nd The Deprtment of Medicine, University of Pennsylvni School of Medicine, Phildelphi, Pennsylvni 9, USA. Correspondence nd requests for mterils should be ddressed to M.P. (emil: mrio.pende@inserm.fr). nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.

2 nture communictions DOI:.8/ncomms667 Glycolysis genertes energy nd intermedites for mcromolecule biosynthesis. Hence the growth fctors, nbolic hormones nd oncogenic events perfected moleculr mechnisms to upregulte glycolytic enzyme ctivities nd gene expression. The phosphtidylinositol--kinse (PIK) pthwy, mster regultor of cell growth, prolifertion nd metbolism in response to insulin, growth fctors nd oncogenic insults, is potent stimultor of glycolysis by cting on different levels, from the upregultion of glucose uptke t the membrne, to the posttrnsltionl modifictions of glycolytic enzymes, to the induction of glycolytic gene expression. In the nucleus, the trnscription fctor c-myc, encoded by the MYC oncogene, directly binds nd ctivtes the mjority of glycolytic gene promoters. In ddition, the hypoxi-inducible fctor (HIF-), which cn ccumulte in cells s consequence of inctivting muttions in the Von Hippel Lindu (VHL) tumour suppressor gene, coopertes with c-myc on the trnscriptionl ctivity of the glycolytic gene promoters. Further complexity comes from the existence of multiple isozymes ctlysing specific rections in glycolysis. For instnce, four hexokinses (HK-) ctlyse the first step of glycolysis, tht is, the phosphoryltion of glucose, while four pyruvte kinses (PKM,,, PKR) ctlyse the lst step of glycolysis, tht is, the finl conversion of phosphoenolpyruvte to pyruvte. These isozymes disply differentil tissue distribution nd confer specific kinetic nd regultory properties to the glycolytic flux 7. However the moleculr mechnisms fvouring selective expression of specific isozymes remin mysterious. Pyruvte, the end product of glycolysis, cn be reduced to lctte in the cytosol during nerobic conditions or enter the mitochondri to be further oxidized during erobic respirtion. In rpidly dividing cells, lctte production is lso observed under norml oxygen conditions, phenomenon clled erobic glycolysis or Wrburg effect, which my provide growth dvntge by producing crbon intermedites s building blocks for cellulr components. Among the glycolytic isozymes, the expression of hexokinse () nd the M isoform of pyruvte kinse () my prticulrly fvour erobic glycolysis nd cell prolifertion owing to their intrcellulr locliztion nd kinetic properties. The fetures of tht my fvour erobic glycolysis re mitochondril locliztion tht llows the consumption of mitochondrilly produced ATP to strt glycolysis, reltively high ffinity for glucose nd presence of two ctlyticlly ctive domins tht double the rte of glucose-6-phosphte formtion,6. Although the other pyruvte kinse isozymes re exclusively tetrmeric enzymes, cn lso switch to less ctive dimeric form by llosteric regultion in presence of mitogenic signls, leding to the ccumultion of glycolytic intermedites tht cn be used to increse the biomss 8. Consistently, nd isozymes re reported to be predominntly expressed in highly mlignnt tumours, nd their functionl role in promoting tumour progression hs been demonstrted 7,9. Prticulrly, reltive switch from HK (lso nmed glucokinse) to, nd from to hs been observed during progressive stges of liver trnsformtion. In liver, insulin signlling through the PIK pthwy hs fundmentl role in the regultion of lipid nd glucose metbolism, nd pthophysiologicl growth. Gene deletions brogting PIK ctivity in mouse liver led to severe insulin resistnce, glucose intolernce, incresed glucose output nd defective lipid neogenesis. Conversely, muttions ctivting the PIK pthwy, including the liver-specific deletion of the phosphtidylinositol (,,)-triphosphte-lipid phosphtse nd tumor suppressor PTEN (phosphtse nd tensin homologue) gene, cused insulin hypersensitivity, improved glucose tolernce, heptomegly, stetosis in young dult mice, followed by denocrcinom formtion since 7 months of ge,. Among the PIK-dependent effectors, the serine/threonine kinse Akt is required for the regultion of heptic glucose output, lipid ccumultion, nd tumorigenesis in PTEN-null livers 6 9. In this report, we therefore performed genetic epistsis experiments to ddress the impct of PIK/Akt ctivity on liver glycolysis. Strikingly, we find tht, wheres PTEN deletion cuses generl upregultion of glycolytic gene expression, Akt selectively controls nd isozymes. We identify peroxisome prolifertorctivted receptor gmm () nucler hormone receptor s novel trnscription fctor turning on these specific glycolytic isozymes tht re frequently upregulted in pthophysiologicl growth. Results Activtion of glycolysis in PTEN-null liver. Consistent with the positive role of the PIK pthwy on the cell switch to glycolytic metbolism, PTEN-null mouse livers showed globl upregultion of glycolytic enzymes (Fig. ). The expression of multiple glycolytic enzymes tht re commonly expressed in liver, including glucokinse (GCK), glucose phosphte isomerse (GPI), glycerldehyde -phosphte dehydrogense (GAPDH), enolse (ENO), phosphoglycerte kinse (PGK) nd L-type pyruvte kinse () ws ugmented t the messenger RNA nd protein levels (Fig.,b). The increse in expression prlleled incresed enzymtic ctivity, while mitochondril enzymes such s citrte synthse nd cytochrome c oxydse were not ffected (Supplementry Fig. S). These metbolic chnges were concomitnt with liver triglyceride ccumultion (stetosis) nd hyperplsi, hypoglycemi nd insulin hypersensitivity, nd preceded the denocrcinom formtion tht ws observed since 7 months of ge, (Supplementry Fig. S). As Akt hs crucil role mediting the ction of insulin nd PIK on heptic glucose homeostsis, stetosis, hyperplsi nd tumorigenesis 6,7,9 (Supplementry Fig. S), we ddressed the effect of Akt deletion on glycolysis of PTEN-null livers. Surprisingly, the upregultion of ll the bove mentioned glycolytic enzymes ws lso observed in ;Akt / livers (Fig.,b; Supplementry Fig. S), ruling out n involvement of Akt in this generl ctivtion of glycolysis. Akt controls expression of nd in PTEN-null liver. Specific glycolytic isozymes, nmely nd hve been demonstrted to fvour erobic glycolysis nd growth 7,. nd re expressed t low levels in wild-type dult mouse livers. As shown in Figure b nd in Figure c, PTEN deletion upregulted nd mrna nd protein expression. Importntly, Akt deletion ws sufficient to reduce by 6% the expression of both isozymes t months of ge, well before tumour onset. In ddition, Akt deletion lso blunted mitochondril levels of (Fig. d), which should ffect ATP usge towrds glycolysis. Finlly, the difference in nd expression in ;Akt / livers ws ccompnied by lower levels of heptic lctte s compred with (Fig. e). Tken together, these dt indicte tht Akt promotes erobic glycolysis in PTEN-null ftty liver. Expression of nd isozymes promotes liver growth. To evlute the functionl outcome of glycolytic isozymes expression on liver pthophysiology, denovirl-medited overexpression ws chieved in vivo (Fig. ). overexpression led to % increse in liver mss in wild type, nd % increse in ;Akt / mice (Fig. b). In ddition, promoted lctte production, stetosis nd heptocyte prolifertion (Fig. c,d,e). lso upregulted triglyceride content nd lctte relese fter 6 h of overexpression in wild-type primry heptocyte cultures (Supplementry Fig. S). overexpression in vivo in wild-type mice cused 7% increse in liver mss, n effect tht ws comprble to (Fig. b). The combintion of both nd further incresed liver mss to % s compred with green fluorescent protein ()-treted control mice. Thus nd overexpression t suprphysiologicl levels is sufficient to cuse liver stetosis nd growth. In conclusion, specific pttern nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.

3 nture communictions DOI:.8/ncomms667 ARTICLE Reltive mrna expression..... Reltive mrna expression.... :b GADPH ;AKT / ;AKT / ENO ;AKT / :b ;AKT / GCK ;AKT / VDAC ;AKT / GPI ;AKT / PGK ;AKT / P-AKT (Ser7) AKT GAPDH Rtio to ctin ENO Actin. PTEN f/f ;AKT /. PTEN f/f ;AKT / GADPH ENO b ;AKT / kd ;AKT / Rtio to VDAC ;AKT /.6.. ;AKT / b ;AKT / Lctte, µmol g :b ;AKT /... kd 7 7 :b ;AKT / Figure Akt controls expression of specific glycolytic isozymes in PTEN-null liver. Reltive trnscript () nd protein (b) levels of glycolytic enzymes in the mouse livers of indicted genotypes. For protein nlysis, the rtio to β-ctin of the densitometric ssy is presented. Dt re men ± s.e.m., n = 7 (P <. () versus ; (b) versus ; -tiled, unpired Student s t test). (c) RT QPCR for nd expression from mice livers of indicted genotypes. Dt re men ± s.e.m., n = 7 (P <. () versus ; (b) versus ; -tiled, unpired Student s t test). (d) Immunoblot nlysis of in liver mitochondri extrcts from mice of indicted genotypes. The rtio to VDAC of the densitometric ssy ± s.e.m. is presented, n = (P <. () versus ; (b) versus ; -tiled, unpired Student s t test). (e) Heptic lctte levels of rndom-fed mice of indicted genotypes. Dt re men ± s.e.m., n = 8 (P <. () versus ; (b) versus ; -tiled, unpired Student s t test). of glycolytic isozyme expression correltes with the Akt-dependent stetosis, hyperplsi nd oncogenic potentil of PTEN-null liver cells. Akt controls ctivity in PTEN-null liver. Given the distinctive fetures of nd isozymes in regulting cell metbolism, we set out to determine the trnscription fctor(s) responsible for their selective Akt-dependent regultion in PTENnull heptocytes. We initilly considered the involvement of c- Myc nd HIFα trnscription fctors, two potent stimultors of glycolytic gene trnscription. Consistently, denovirl trnsduction of c-myc nd HIFα in primry heptocytes incresed nd expression in ddition to other glycolytic enzymes such s GAPDH, PGK, ENO s well s LDHA, known non-glycolytic trget of both fctors (Supplementry Fig. S). However, we filed to revel significnt regultion of c-myc nd HIFα expression t the level of mrna, totl protein nd nucler protein in the wild type, nd ;Akt / livers (Supplementry Fig. Sb,c,d). Furthermore, the HIFα- nd c-myctrget genes GAPDH, PGK, ENO, were not regulted in n Akt- dependent mnner in livers (Fig. ). These observtions would be consistent with extr mechnisms downstrem of Akt controlling nd expression. We noticed tht ft storing tissues lso contined bundnt mount of nd. This ws the cse for the epididyml dipose tissues in wild-type mice (Fig. ), nd for stetotic livers in mice (Fig. ). is mster regultor of dipogenesis nd ft storge. In liver, levels re incresed during stetosis induced by high ft diet, leptin deficiency, or the genetic deletion of PTEN. Importntly, we found tht the increse of mrna nd nucler protein nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.

4 nture communictions DOI:.8/ncomms667 V-Tg () Actin + kd Rtio:liver weight (g)/body weight (g) :b :b:c PTEN f/f ; AKT / :b :b:c:d Lctte µg per mg liver tissue + PTEN f/f ; AKT / ;AKT / Rtio:BrdU+/totl number of nuclei PTEN f/f ; AKT / Figure nd glycolytic isozyme expression promotes liver growth. () Immunoblot nlysis of nd protein expression in livers of mice, 7 dys fter trnsduction with indicted denoviruses. (b) Liver weight reltive to body weight of mice trnsduced with indicted denoviruses. Dt re mens ± s.e.m., n = 8 (P <. () versus /; (b) versus deno/; (c) versus deno/; (d) versus / ; Akt / ; -tiled, unpired Student s t test). (c) Heptic lctte levels of rndom-fed mice of indicted genotypes 7 dys fter trnsduction with denovirus. Dt re mens ± s.e.m., n = (P <. () versus ; -tiled, unpired Student s t test). (d) H&E-stined sections of nd ;Akt / livers trnsduced with or denoviruses. Br represents µm. (e) Incresed prolifertion rte of heptocytes induced by overexpression in the livers of mice of indicted genotypes. BrdU + nuclei nd totl number of nuclei were scored in the livers of denovirustrnsduced mice dy fter dministrtion of BrdU in drinking wter. Dt presented s rtio of BrdU + to totl number of nuclei ± s.e.m., n = 8 (P <. () versus ; -tiled, unpired Student s t test). levels in livers ws reduced by % nd %, respectively, in the ;Akt / genotype (Fig. b,c), correlting with the extent of stetosis (Supplementry Fig. S). The reduction of expression in ;Akt / livers ws ccompnied by comprble reduction of FoXo phosphoryltion (Supplementry Fig. S). FoXo trnscription fctor fmily members were previously shown to be Akt substrtes nd to regulte expression in ft tissues,. Interestingly, Akt deletion lso brogted expression of heptic Cyclooxygense- (Supplementry Fig. Sb), rte-limiting enzyme in prostglndin synthesis tht ws previously shown to be upregulted in PTEN-null cells 6. Although the nture of endogenous lignds is still under debte, prostglndins were proposed s nturl regultors of 7. Thus, it is possible tht Akt ctivity might ffect both expression nd ctivtion in liver. Consistently, Akt ctivity contributed to control of the mrna expression of trgets, nmely cluster of differentition 6 (CD6) nd cell deth-inducing DFFA-like effector-c (CIDE-C) (Fig. d). Chromtin immunoprecipittion from liver extrcts using ntibodies demonstrted tht the binding of to the promoter of CD6 ws dependent on Akt ctivtion (Fig. e). nd re novel trnscriptionl trgets of PPAR. By in silico nlysis, it ppered tht the promoter regions of nd genes lso contined severl PPAR response elements (PPRE). Nmely, for, they were loclized t positions,69 bps,,6 bp,,6 bp, 76 bp, 9 bp nd for t 8 bp, 8 bp, 86 bp,,97 bp,,8 bp nd bp from the trnscription strt site. Independent chromtin immunoprecipittion ssys demonstrted tht, in PTEN-deficient livers, binding to one PPRE in the promoter of (t,8 bp position) nd to one PPRE in the promoter of (t 9 bp position) ws enriched more thn tenfold over the IgG control immunoprecipittions (Fig. e). Importntly, this nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.

5 nture communictions DOI:.8/ncomms667 ARTICLE GADPH CIDE-C WAT Liver kd 7 Reltive mrna expression.... :b ;AKT / TATAbp AKT / kd Rtio to TATAbp :b ;AKT / Reltive mrna expression CIDE-C :b ;AKT / 6 CD6 :b ;AKT / Fold chnge 6 CD6 ;AKT / PPRE-9bp PPRE -8bp ;AKT / ;AKT / Figure controls nd expression in PTEN-null liver. () Immunoblot nlysis of protein expression in liver nd WAT tissue of mice. (b) RT QPCR nlysis of reltive trnscript levels of in the mouse livers of indicted genotypes. Dt re men ± s.e.m., n = (P <. () versus ; (b) versus, -tiled, unpired Student s t test). (c) Immunoblot nlysis showing levels of in liver nucler extrcts. The rtio to TATA-binding nucler protein of the densitometric ssy ± s.e.m. is presented, n = (P <. () versus ; (b) versus, -tiled, unpired Student s t test). (d) Reltive trnscript levels of CD6 nd CIDE-C in the mouse livers of indicted genotypes. Dt re men ± s.e.m., n = 7 (P <. () versus ; (b) versus ; -tiled, unpired Student s t test). (e) Endogenous chromtin immunoprecipittion ssessed for CD6, nd murine promoters reltive to immunoprecipittion with nonspecific IgG. Dt re presented s n verge of fold induction over control mice of two independent experiments, n =. binding required Akt ctivity s it ws reduced in ; Akt / livers. Tken together, these findings indicte tht nd re novel nd direct trgets of, consistent with their expression in dipose tissue nd ftty liver. Pioglitzone tretment induces growth of PTEN-null liver. Our dt suggest tht my promote metbolic dpttions downstrem of PIK nd Akt pthwy fvouring heptocyte stetosis nd prolifertion. However, in other systems my lso promote cell differentition nd cell-cycle withdrwl 8. Intriguingly, PTEN expression hs been proposed s one of the possible mechnisms by which my exert nti-tumorl ctions 9. Therefore, the functionl outcome of ctivity on PTEN-negtive liver growth is difficult to predict nd is importnt to ssess, given the potentil for phrmcologicl intervention with synthetic lignds to tret metbolic diseses. To this end, ctivity ws modulted in PTEN-null livers. nd wild-type mice were dily treted for dys with the gonist pioglitzone. This reduced fed glycemi in both genotypes (Fig. ), consistent with the insulin-sensitizing systemic effect of pioglitzone. However, pioglitzone promoted 6% increse in liver/body rtio (Fig. b), nd threefold increse in BrdU-positive cells (Fig. c), exclusively in PTEN-null livers, wheres growth ws not induced in wild-type liver controls in greement with the low expression in this genotype. Strikingly, pioglitzone selectively rised the levels of nd proteins in livers with no effect on nd GAPDH levels (Fig. d). PPAR overexpression in vivo induces liver growth. To nlyse the effects of cell-utonomous ctivtion of on liver growth, wild-type, nd ;Akt / mice were in vivo trnsduced with -expressing denovirl vector tht hs pronounced tropism for liver. In line with the results of ctivtion by phrmcologicl gents, overexpression led to significnt increse in liver/whole body rtio in wild type, nd ;Akt / livers s compred with control trnsduced livers (Supplementry Fig. S6). The effect on liver hypertrophy nd stetosis ws prticulrly striking in wild type nd ;Akt / tissues, tht express low levels of endogenous (Fig. ). Induction of nd by expression ws comprble to well-estblished trget such s CD6 (Fig. b). The mssive nd selective upregultion of nd over other glycolytic enzymes ws confirmed t the level of both mrna nd protein (Fig. b,c). PKM nd isoforms re trnscribed from the sme PKM locus nd rise from lterntive splicing of PKM pre-mrna leding to inclusion of either exon 9 in the PKM mture mrna, or exon in (ref. ). Despite their similrities, the effects on erobic glycolysis nd cncer nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.

6 nture communictions DOI:.8/ncomms667 Blood glucose (mg dl ) b PTEN f/f GADPH Tubulin :b Rtio:liver weight(g)/body weight (g) progression re minly scribed to rther thn PKM (ref. 7). In -trnsduced livers, the trnscription of PKM gene ws upregulted, leding to incresed levels of both PKM nd mrnas (Fig. b). Although the formtion of both PKM splice vrints ws detected, mrna ws more bundnt thn PKM (Supplementry Fig. S6b). Tken together, our findings indicte tht cts in vivo on PKM nd gene trnscription, nd tht heptocytes re competent for production. To ddress whether glycolysis my concur to the growth-promoting ction of in liver, -trnsduced wild-type mice were treted with the glycolytic inhibitor -bromopyruvte (-BrPA). -BrPA inhibited -induced liver growth becuse of effects on heptocyte prolifertion nd stetosis (Fig. d,e,f). The liver-cell type expressing fter expression ws determined by immunostining s the heptocytes (Fig. g), ruling out the contribution of other cell types to the observed effects on expression. It ws lso independently confirmed by modulting levels in primry heptocyte cultures by denovirl trnsduction, where incresing doses of upregulted nd mrna nd protein levels (Fig. h nd i). overexpression hd similr effects on the well-estblished trgets CD6 nd P, wheres the levels of other glycolytic enzymes such s GAPDH nd PGK remined constnt. Conversely, overexpression of PPARα ws not ble to stimulte nd mrna levels in primry heptocytes (Supplementry Fig. S7). Moreover, PPARα nd PPARβ expression ws not regulted by Akt in livers (Supplementry Fig. S7b). Altogether, our dt suggest cellutonomous regultion of nd specificlly by the γ type of PPARs. PTEN f/f :b Rtio:BrdU+/totl number of nuclei kd PTEN f/f Figure Phrmcologicl ctivtion of by Pioglitzone promotes growth of PTEN-null liver. () Blood glucose level of rndom-fed mice of the indicted genotypes. Dt re mens ± s.e.m., n = 8 (P <. () versus ; (b) versus control; -tiled, unpired Student s t test). (b) Liver- to body-weight rtio of nd mice fter tretment. Dt re mens ± s.e.m., n = 8 (P <. () versus ; (b) versus control; -tiled, unpired Student s t test). (c) Incresed prolifertion rte of heptocytes fter tretment. Number of BrdU + nuclei nd totl number of heptocytes nuclei were scored in the liver of untreted nd -treted mice, dys fter dministrtion of BrdU in drinking wter. Dt presented s rtio of BrdU + to totl number of nuclei ± s.e.m., n = 8 (P <. () versus ; (b) versus control, -tiled, unpired Student s t test). (d) Immunoblot nlysis of liver extrcts of nd mice treted with or without. :b Strikingly, the ;Akt / livers showed pthologicl signs weeks fter deno- trnsduction, including bile duct hyperplsi, heptic lobule disorgniztion, nd bllooning heptocytes (Fig. g). Thus, orchestrtes gene-expression progrm downstrem the PIK/Akt pthwy, fvouring erobic glycolysis, lipogenesis nd pthophysiologicl growth. Discussion Although the role of c-myc nd HIF-α in generl regultion of glycolytic enzyme expression is well documented, our understnding of moleculr mechnisms selectively controlling specific glycolytic enzymes is limited. Regultion of nd isozymes is especilly interesting, owing to their function in erobic glycolysis nd growth. Here we provide evidence tht hs n importnt role in nd glycolytic isozyme gene trscription in PTENnull ftty liver. We propose tht these novel trgets, in ddition to other well-estblished trget genes, concur to the growth nd metbolic dpttion of liver to nutrition, insulin, PIK, Akt nd signlling: erobic glycolysis, stetosis nd heptomegly (Fig. j). Intense efforts hve been mde to evlute whether therpy with insulin-sensitizing gents, nd gonists could be beneficil ginst stetosis-ssocited humn liver diseses, though the outcome is not conclusive yet. The rtionle behind these clinicl trils is tht gonists should llevite liver stetosis, promoting lipid ccumultion in dipose tissue nd suppressing heptic inflmmtion. This strtegy my be pproprite when the origin of liver stetosis is environmentl, owing to nutritionl hbits. However, liver stetosis nd heptic expression my rise from genetic insults, s observed when PTEN is deleted nd Akt upregulted in heptocytes. In these cses, we show tht ctivtion is detrimentl, leding to mssive stetosis nd heptocyte prolifertion. Regultion of nd by heptic is observed well before tumour onset, which in PTEN-deficient livers ppers since 7 months of ge. It is tempting to speculte tht these effects my be cuslly involved in the well-estblished correltion between liver stetosis nd incresed cncer risk. Wheres my ct s tumour suppressor in colon, brest nd prostte cncers,, ctivtion promotes polyp formtion in colon cells crrying muttions in the APC gene. Interestingly, in liver, histone decetylse (Hdc) nd nucler hormone co-repressor (N-CoR) re recruited by severl nucler hormone receptors, including, to regulte trnscription in the bsence of the lignd. Loss-offunction muttions in these trnscriptionl repressors promote stetosis nd pthologicl liver growth culminting in cncer in mice nd humns 8,9. These studies in combintion with our dt suggest functionl /Hdc/N-CoR complex in the regultion of liver metbolism, which my hve importnt implictions in tumorigenesis. Methods Animl experiments. Genertion of nd Akt / mice hs been previously described,6. Mice were mintined t C with -h-drk/-hlight cycle nd hd free ccess to food. All niml studies were pproved by the Direction Déprtementle des Services Vétérinires, Préfecture de Police, Pris, Frnce (uthoriztion number 7-). Pioglitzone ws diluted in.% crboxymethyl cellulose nd ws dministered t mg kg dose by dily gvge for dys. Group of wild-type mice trnsduced with denovirus were treted with mg kg -BrPA (Sigm) freshly prepred in PBS nd djusted to ph7.. The drug ws injected intrperitonelly every dy for 7 dys. The first injection ws h fter trnsduction with denovirus. Adenoviruses. Recombinnt denovirus expressing untgged full-length humn ws generous gift from Renud Dentin (Institut Cochin, INSERM U6, UMR8). Recombinnt denovirus expressing c-myc ws generous gift from Heiko Hermeking (Institute of Pthology Ludwig-Mximilins-University Munich). Recombinnt denovirus expressing HIF-αCA mutnt ws generous gift from Gregg L. Semenz (The Johns Hopkins University School of Medicine). Recombinnt denovirus expressing untgged full-length mouse PPARα ws described previously. Recombinnt denovirus expressing V-tgged nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.

7 nture communictions DOI:.8/ncomms667 ARTICLE ; AKT / cm Reltive mrna expression / / DKO/ DKO/ CD6 PKM GCK ;AKT / kd GADPH Tubulin Rtio:liver weight (g)/ body weight (g) Reltive mrna expression :b +BrPA..... Rtio:BrdU+/totl number of nuclei CD :b +BrPA GAPDH µg TG/mg liver PGK :b +BrPA HE ; AKT / + P GAPDH Actin ; AKT / + kd 7 PIK AKT Stetosis growth PTEN, Figure Rescue of PPAR expression in liver cuses hypertrophy. () Mcroscopic view of nd ;Akt / mice 7 dys fter denovirl trnsduction of or. (b) Reltive trnscript levels nd (c) immunoblot nlysis of protein levels in livers of mice trnsduced with or denoviruses for 7 dys. Dt re mens ± s.e.m., n = (P <. () versus ; -tiled, unpired Student s t test). (d) Liver to body weight rtio of mice trnsduced with denoviruses expressing or nd treted with BrPA. Dt re mens ± s.e.m., n = (P <. () versus ; (b) versus control; -tiled, unpired Student s t test). (e) Heptocyte prolifertion fter overexpression is inhibited by BrPA tretment. Number of BrdU + nuclei nd totl number of heptocyte nuclei were scored in the livers of mice trnsduced with indicted denoviruses treted for dy before scrifice with BrdU in drinking wter. Dt re presented s rtio of BrdU + to totl number of nuclei ± s.e.m., n = 7 (P <. () versus ; (b) versus control; -tiled, unpired Student s t test). (f) Triglyceride concentrtion in liver tissue of mice trnsduced with or denoviruses, nd treted with BrPA. Dt re mens ± s.e.m., n = (P <. () versus ; (b) versus control; -tiled, unpired Student s t test). (g) H&E stining nd immunohistochemistry using nti- ntibody of liver sections of ;Akt / mice dys fter denovirl trnsduction with or. Br represents µm. (h) RT QPCR nlysis of reltive trnscript levels of glycolytic enzymes in primry heptocytes h postinfection with MOI of denoviruses expressing or. Dt re mens ± s.e.m., n = (P <. () versus ; -tiled, unpired Student s t test). (i) Immunoblot nlysis of protein levels of glycolytic enzymes in primry heptocytes trnsduced with different doses of or denovirus collected h postinfection. (j) Proposed model of contribution to metbolic chnges nd pthophysiologicl growth in PTEN-null livers. full-length humn ws custom-generted t GeneCust. Recombinnt denovirus expressing untgged humn ws cquired from Excellgen. Viruses were generted through homologous recombintion between linerized trnsfer vector pad-trck nd the denovirl bckbone vector pad-esy. Viruses were purified by the CsCl method nd dilysed ginst PBS buffer contining 7% glycerol. For in vivo trnsduction, 9 of denovirl infectious prticles were diluted in.9%ncl nd dministered retro-orbitlly, using 6G needle in totl volume of µl per mouse, nd mice were killed 7 or dys postinfection. Adenovirus coding for ws used s control in ll experiments. Metbolic studies in mice. At months of ge, glucose tolernce test ws performed in mice fter n overnight fsting ( h). Mice were intrperitonelly injected with g kg glucose, nd blood ws collected from the til vein for determintion of glucose levels t,,, nd min by Glucotrend glucometer (Roche Dignostics). Insulin tolernce test ws performed t months of ge. Overnight-fsted mice were intrperitonelly injected with U kg insulin (Actrpid), nd the glucose concentrtion in whole blood from the til vein ws mesured t,, nd 6 min. Fsting insulin levels were mesured by ELISA ssy (Crystl chem). Immunohistochemistry nd morphometric nlysis. Liver tissue ws fixed overnight in phosphte-buffered % formlin nd embedded in prffin, sectioned in µm, nd stined with eosin/hemtoxylin. Immunohistochemistry ws performed using nti-brdu ntibody (Roche), nti-β-ctenin (Clbiochem) nd nti-xp (Cell Signlling Technology) ntibodies. For -bromo- deoxyuridine (BrdU) incorportion, mice were fed with BrdU ( mg ml, Sigm-Aldrich) dissolved in drinking wter for either for dys ( tretment) or lst h (denovirl trnsduction) before killed. The results were expressed s the rtio of BrdU-positive nuclei nd totl number of nuclei in t lest res of, µm for nti-brdu stining nlysed. nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.

8 nture communictions DOI:.8/ncomms667 Subcellulr frctiontion nd western blot. Mitochondri were isolted s previously described. Briefly, mg of snp-frozen liver tissue ws homogenized in IBc buffer complemented with complete protese inhibitors (Roche). After centrifugtion t 6 g for min, the superntnt contining the mitochondri ws centrifugted t 7,g for min. The pellet of mitochondri ws wshed three times with IBc buffer nd, fter the finl centrifugtion, the mitochondri were lysed in extrction buffer ( mm Tris HCl (ph 8,), % glycerol, 8 mm NCl,,7 mm KCl, % NP-, mm NF, mm EDTA, mm DTT) complemented with complete protese inhibitors (Roche). Nucler extrcts were prepred using NE-PER Kit (Pierce), ccording to mnufcturer s recommendtions, using mg of snp-frozen liver tissue s strting mteril. Totl protein extrcts were prepred s described before. Proteins were resolved by SDS PAGE, trnsferred to polyvinylidene difluoride membrne nd incubted with nti-phospho Akt (Ser7), nti-p, nti-, nti-, nti-akt, nti-c-myc, nti-lmin A/C, nti-phosphofoxo/ (Thr/), nti-foxo, nti-phosphogskβ (Ser9), nti-gskβ (Cell Signlling), nti- ( generous gift of Benoit Viollet, Cochin Institut, Pris), nti-tubulin nd nti-β-ctin (Sigm), nti-vdac nd nti-cide-c (Millipore), nti-, nti-tatabp, nti- nd nti-enolse (Snt Cruz), nti-gapdh nd nti-v (Abcm), nti-hifα (Novus) ntibodies. Heptic metbolites nlysis. TG levels were determined using Triglycerides FS Kit (Disys) ccording to the mnufcturer s instructions. mg of powdered liver tissue or pelleted primry heptocytes were used for cetone extrction. Lctte concentrtions were ssyed in liver extrcts of rndom-fed mice s described previously. Briefly, metbolites were extrcted in 9% perchloric cid from clmpfrozen liver tissues, neutrlized using potssium hydroxide, nd ssyed using spectrophotometric enzymtic procedure. Lctte levels fter denovirus-medited overexpression were determined using Lctte regent (Trinity Biotech) using mg of liver tissue or cell culture medium superntnts. Liver tissue extrcts before mesurement were filtered using K Amicon Ultr columns (Millipore). RT QPCR. Totl RNA ws isolted from mg of snp-frozen liver tissue using RNAesy Lipid Tissue Midi or Mini Kit (Qigen). Totl RNA from heptocytes ws isolted using RNAesy Mini Kit (Qigen). Single-strnd complementry DNA ws synthesized from µg of totl RNA with rndom hexmer primers nd SuperScript II (Invitrogen). Rel-time quntittive PCR (RT QPCR) ws performed on Tqmn instrument (Applied Biosystem) using SYBR Green PCR Mster Mix (Applied Biosystem). The reltive mounts of the mrnas studied were determined by mens of the CT method, with pinin, ctin or cyclophilin s reference genes nd control-treted smples s the invrint controls. Primers used for nlysis re listed in Supplementry Tble S. For PKM isoform rtio nlysis, microlitre of the complementry DNA ws mplified using DremTq Green PCR Mster Mix (Ferments) in totl volume of microlitres. Five microlitres of PCR product ws digested by FstDigest EcoNI (Ferments) nd the products nlysed on grose gel. An EcoNI site is present in exon 9 of mouse PKM, wheres it is bsent in exon of. Primers used for mplifiction of PKM isoform identifiction re shown in Supplementry Tble S. Chromtin immunoprecipittion. Rndom-fed mice were used for experiment with two mice of the sme genotype used for preprtion in ech smple for ech independent experiment. Chromtin ws prepred from freshly dissected liver, s previously described 6,7. Chromtin ws sonicted in SBAR buffer ( mm HEPES, mm NCl, mm EDTA, % Triton,.% sodium deoxycholte nd.% SDS with complete protese inhibitors (Roche) nd incubted with ntibody to (Snt Cruz, sc-796) or IgG s control. Next, immunocomplexes were recovered with Protein A sephrose beds (Amershm). After wshing, DNA protein complexes were eluted nd cross-linking ws reversed by heting the smples t 6 C for 6 h. DNA ws then purified using Qigen PCR purifiction kit. Rel-time quntittive PCR (RT QPCR) ws performed using Tqmn instrument (Applied Biosystem) ccording to the mnufcturer s instructions, using SYBR Green PCR Mster Mix (Applied Biosystem). The reltive mounts of the immunoprecipitted DNA were determined by mens of the CT method, with input DNA vlues for ech smple s the control. The enrichment over IgG control of more thn tenfold ws considered s cutoff. The results re presented s verge of fold induction over wild type () for the PPREs confirmed in independent experiments. The loction of puttive PPREs within promoter region of nd genes ws determined using MtInspector softwre (Genomtix) nd kb frgment of DNA sequence upstrem the trnscription strt. Numerous potentil PPREs were locted upstrem first exon of nd genes: :,69 bp;,6 bp;,6 bp; 76 bp; 9 bp; :,8 bp;,8 bp;,86 bp;,97 bp;,8 bp; bp. Confirmed PPREs for nd promoters re :,8 bp; : 9 bp. The primer pirs flnking puttive PPREs used for mplifiction hve been designed using PrimerBlst softwre nd re listed in Supplementry Tble S. Heptocyte culture. Primry heptocytes were isolted from wild-type C7BL/6J mice by collgense perfusion method s described previously. cells were plted in 6-well pltes in Willim s medium (Invitrogen) supplemented by % FBS, INS ( µg ml ), units ml penicillin, g ml streptomycin, nd nm dexmethsone (Sigm). After ttchment, cells were trnsduced with denoviruses expressing, PPARα,, c-myc or HIF-αCA in serum-free Willim s medium for h in - MOI dose s indicted. Subsequently, medium ws chnged nd cells were mintined in serum-free Willim s medium complemented with INS nd dexmetsone. References. Vnder Heiden, M. G., Cntley, L. C. & Thompson, C. B. 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9 nture communictions DOI:.8/ncomms667 ARTICLE 8. Altiok, S., Xu, M. & Spiegelmn, B. M. PPARgmm induces cell cycle withdrwl: inhibition of EF/DP DNA-binding ctivity vi down-regultion of PPA. Genes Dev., (997). 9. Teresi, R. E. et l. Incresed PTEN expression due to trnscriptionl ctivtion of PPARgmm by Lovsttin nd Rosiglitzone. Int. J. Cncer 8, 9 98 (6).. Snyl, A. J. et l. Pioglitzone, vitmin E, or plcebo for nonlcoholic stetoheptitis. N. Engl. J. Med. 6, ().. Dvid, C. J., Chen, M., Assnh, M., Cnoll, P. & Mnley, J. L. HnRNP proteins controlled by c-myc deregulte pyruvte kinse mrna splicing in cncer. Nture 6, 6 68 (9).. Geschwind, J. F., Ko, Y. H., Torbenson, M. S., Mgee, C. & Pedersen, P. L. Novel therpy for liver cncer: direct intrrteril injection of potent inhibitor of ATP production. Cncer Res. 6, 99 9 ().. Srrf, P. et l. Loss-of-function muttions in PPAR gmm ssocited with humn colon cncer. Mol. Cell, (999).. Grommes, C., Lndreth, G. E., Schlegel, U. & Henek, M. T. The nonthizolidinedione tyrosine-bsed peroxisome prolifertor-ctivted receptor gmm lignd GW78 induces poptosis nd limits migrtion nd invsion of rt nd humn gliom cells. J. Phrmcol. Exp. Ther., 86 8 ().. Lefebvre, A. M. et l. Activtion of the peroxisome prolifertor-ctivted receptor gmm promotes the development of colon tumors in C7BL/6J- APCMin/+ mice. Nt. Med., 7 (998). 6. Sez, E. et l. Activtors of the nucler receptor PPARgmm enhnce colon polyp formtion. Nt. Med., 8 6 (998).. Girnun, G. D. et l. APC-dependent suppression of colon crcinogenesis by PPARgmm. Proc. Ntl Acd. Sci. USA 99, 7 76 (). 8. Knutson, S. K. et l. Liver-specific deletion of histone decetylse disrupts metbolic trnscriptionl networks. EMBO J. 7, 7 8 (8). 9. Bhskr, S. et l. Hdc is essentil for the mintennce of chromtin structure nd genome stbility. Cncer Cell 8, 6 7 ().. Hermeking, H. et l. Identifiction of CDK s trget of c-myc. Proc. Ntl Acd. Sci. USA 97, 9 ().. Kelly, B. D. Cell type-specific regultion of ngiogenic growth fctor gene expression nd induction of ngiogenesis in nonischemic tissue by constitutively ctive form of hypoxi-inducible fctor. Circ. Res. 9, 7 8 ().. Knuf, C. et l. Peroxisome prolifertor-ctivted receptor-lph-null mice hve incresed white dipose tissue glucose utiliztion, GLUT, nd ft mss: role in liver nd brin. Endocrinology 7, (6).. Frezz, C., Cipolt, S. & Scorrno, L. Orgnelle isoltion: functionl mitochondri from mouse liver, muscle nd cultured fibroblsts. Nt. Protoc., 87 9 (7).. Espeillc, C. et l. S6 kinse is required for rpmycin-sensitive liver prolifertion fter mouse heptectomy. J. Clin. Invest., 8 8 ().. Ferre, P. & Willimson, D. H. Evidence for the prticiption of sprtte minotrnsferse in heptic glucose synthesis in the suckling newborn rt. Biochem. J. 76, 8 (978). 6. Verdeguer, F. et l. A mitotic trnscriptionl switch in polycystic kidney disese. Nt. Med. 6, 6 (). 7. Annicotte, J. S. et l. The CDK-pRB-EF pthwy controls insulin secretion. Nt. Cell Biol., 7 (9). Acknowledgements We re grteful to the members of INSERM-U8 for support, nd to Dvid Sbtini, Stefno Fumglli, Benoit Viollet, Fbienne Foufelle, Renud Dentin, Pscl Pineu, Isbelle André-Schmutz, Olivier Dnos nd Anne Dejen for helpful discussions nd shring regents. We thnk Sophie Berissi, Dominique Chretien nd Sylvie Fbreg for technicl support. This work ws supported by grnts from the Europen Reserch Council, from Fondtion de l Recherche Medicle (DEQ6796) nd from Fondtion Schlumberger pour l Eduction et l Recherche to M.P., nd from the Assocition pour l Recherche sur le Cncer to M.P. nd J.-E.R. L.A.P received fellowship from the Région Provence-Alpes-Cote-d Azur nd INSERM, C.E. from Ministere de Recherche et Technologies nd from Fondtion de l Recherche Medicle. Author contributions G.P. designed, performed most of the experimentl work nd nlysed dt with contribution of C.E. nd C.C. M.J.B., Y.H. nd A.S. provided expertise nd the mouse lines. M.F. provided denovirl vectors. J.-S.A., L.F., F.V., M.Po. shred regents nd expertise, helped with in vivo ChIP experiments. L.A.P., J.-E.R., P.F. performed the lctte metbolite mesurements nd enzymtic ssys. J.-Y.S. performed pthophysiologicl nlysis. M.Pe. conceived, directed the study nd wrote the mnuscript. All uthors discussed the results nd commented on the mnuscript. Additionl informtion Supplementry Informtion ccompnies this pper t nturecommunictions Competing finncil interests: The uthors declre no competing finncil interests. Reprints nd permission informtion is vilble online t reprintsndpermissions/ How to cite this rticle: Pnsyuk, G. et l. contributes to nd expression in ftty liver. Nt. Commun. :67 doi:.8/ncomms667 (). License: This work is licensed under Cretive Commons Attribution-NonCommercil- NoDerivtive Works. Unported License. To view copy of this license, visit cretivecommons.org/licenses/by-nc-nd/./ nture communictions :67 DOI:.8/ncomms667 Mcmilln Publishers Limited. All rights reserved.