Agnès Pernin

The three subtypes of the peroxisome proliferator-activated receptors (PPARalpha, beta/delta, and gamma) form heterodimers with the 9-cis-retinoic acid receptor (RXR) and bind to a common consensus response element, which consists of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1). As a first step toward understanding the molecular mechanisms determining PPAR subtype specificity, we evaluated by electrophoretic mobility shift assays the binding properties of the three PPAR subtypes, in association with either RXRalpha or RXRgamma, on 16 natural PPAR response elements (PPREs). The main results are as follows. (i) PPARgamma in combination with either RXRalpha or RXRgamma binds more strongly than PPARalpha or PPARbeta to all natural PPREs tested. (ii) The binding of PPAR to strong elements is reinforced if the heterodimerization partner is RXRgamma. In contrast, weak elements favor RXRalpha as heterodimerization partner. (iii) The ordering of the 16 natural PPREs from strong to weak elements does not depend on the core DR1 sequence, which has a relatively uniform degree of conservation, but correlates with the number of identities of the 5'-flanking nucleotides with respect to a consensus element. This 5'-flanking sequence is essential for PPARalpha binding and thus contributes to subtype specificity. As a demonstration of this, the PPARgamma-specific element ARE6 PPRE is able to bind PPARalpha only if its 5'-flanking region is exchanged with that of the more promiscuous HMG PPRE.

OBJECTIVE: Obesity is associated with an increased risk for cardiovascular disease. Although it is known that white adipose tissue (WAT) produces numerous proinflammatory and proatherogenic cytokines and chemokines, it is unclear whether adipose-derived chemotactic signals affect the chronic inflammation in atherosclerosis. METHODS AND RESULTS: Histological examination showed that perivascular WAT (pWAT) is in close proximity to vascular walls, particularly at sites that have a tendency to develop atherosclerosis. In rodents, the amount of pWAT is markedly increased by a high-fat diet. At a functional level, supernatant from subcutaneous and pWAT strongly induced the chemotaxis of peripheral blood leukocytes. The migration of granulocytes and monocytes was mostly mediated by interleukin-8 and monocyte chemoattractant protein-1, respectively, whereas both chemokines contributed to the migration of activated T cells. Moreover, pWAT produces these chemokines, as shown by immunohistochemistry and by explant culture. The accumulation of macrophages and T cells at the interface between pWAT and the adventitia of human atherosclerotic aortas may reflect this prochemotactic activity of pWAT. CONCLUSIONS: Human pWAT has chemotactic properties through the secretion of different chemokines, and we propose that pWAT might contribute to the progression of obesity-associated atherosclerosis.

Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.

The secreted form of the interleukin-1 receptor antagonist (IL-1Ra) is an acute-phase protein intervening in the counterregulation of inflammatory processes. We previously showed that this cytokine antagonist is upregulated in the serum of obese patients, correlating with BMI and insulin resistance. In this study, we examined the expression pattern of IL-1Ra and showed that it is highly expressed not only in liver and spleen, but also in white adipose tissue (WAT), where it is upregulated in obesity. In WAT of obese humans, IL-1Ra was also markedly increased. Moreover, human WAT explants secreted IL-1Ra into the medium, a process that could be stimulated fivefold by interferon-beta. Finally, lipopolysaccharide administration induced a long-lasting expression of IL-1Ra in mouse WAT, suggesting that adipose tissue is an important source of IL-1Ra in both obesity and inflammation. In summary, we demonstrated that WAT is one of the most important sources of IL-1Ra quantitatively, suggesting that this tissue could represent a novel target for anti-inflammatory treatment. Moreover, it can be speculated that IL-1Ra, whose production is markedly increased in WAT in obese individuals, contributes further to weight gain because of its endocrine and paracrine effects on the hypothalamus and adipocytes, respectively.

The peroxisome proliferator-activated receptors (PPARs) are a subgroup of nuclear receptors activated by fatty acids and eicosanoids. In addition, they are subject to phosphorylation by insulin, resulting in the activation of PPARα, while inhibiting PPARγ under certain conditions. However, it was hitherto unclear whether the stimulatory effect of insulin on PPARα was direct and by which mechanism it occurs. We now demonstrate that amino acids 1–92 of hPPARα contain an activation function (AF)-1-like domain, which is further activated by insulin through a pathway involving the mitogen-activated protein kinases p42 and p44. Further analysis of the amino-terminal region of PPARα revealed that the insulin-induced trans-activation occurs through the phosphorylation of two mitogen-activated protein kinase sites at positions 12 and 21, both of which are conserved across evolution. The characterization of a strong AF-1 region in PPARα, stimulating transcription one-fourth as strongly as the viral protein VP16, is compatible with the marked basal transcriptional activity of this isoform in transfection experiments. However, it is intriguing that the activity of this AF-1 region is modulated by the phosphorylation of two serine residues, both of which must be phosphorylated in order to activate transcription. This is in contrast to PPARγ2, which was previously shown to be phosphorylated at a single site in a motif that is not homologous to the sites now described in PPARα. Although the molecular details involved in the phosphorylation-dependent enhancement of the transcriptional activity of PPARα remain to be elucidated, we demonstrate that the effect of insulin on the AF-1 region of PPARα can be mimicked by the addition of triiodothyronine receptor β1, a strong binder of corepressor proteins. In addition, a triiodothyronine receptor β1 mutant deficient in interacting with corepressors is unable to activate PPARα. These observations suggest that the AF-1 region of PPARα is partially silenced by corepressor proteins, which might interact in a phosphorylation-dependent manner. The peroxisome proliferator-activated receptors (PPARs) are a subgroup of nuclear receptors activated by fatty acids and eicosanoids. In addition, they are subject to phosphorylation by insulin, resulting in the activation of PPARα, while inhibiting PPARγ under certain conditions. However, it was hitherto unclear whether the stimulatory effect of insulin on PPARα was direct and by which mechanism it occurs. We now demonstrate that amino acids 1–92 of hPPARα contain an activation function (AF)-1-like domain, which is further activated by insulin through a pathway involving the mitogen-activated protein kinases p42 and p44. Further analysis of the amino-terminal region of PPARα revealed that the insulin-induced trans-activation occurs through the phosphorylation of two mitogen-activated protein kinase sites at positions 12 and 21, both of which are conserved across evolution. The characterization of a strong AF-1 region in PPARα, stimulating transcription one-fourth as strongly as the viral protein VP16, is compatible with the marked basal transcriptional activity of this isoform in transfection experiments. However, it is intriguing that the activity of this AF-1 region is modulated by the phosphorylation of two serine residues, both of which must be phosphorylated in order to activate transcription. This is in contrast to PPARγ2, which was previously shown to be phosphorylated at a single site in a motif that is not homologous to the sites now described in PPARα. Although the molecular details involved in the phosphorylation-dependent enhancement of the transcriptional activity of PPARα remain to be elucidated, we demonstrate that the effect of insulin on the AF-1 region of PPARα can be mimicked by the addition of triiodothyronine receptor β1, a strong binder of corepressor proteins. In addition, a triiodothyronine receptor β1 mutant deficient in interacting with corepressors is unable to activate PPARα. These observations suggest that the AF-1 region of PPARα is partially silenced by corepressor proteins, which might interact in a phosphorylation-dependent manner. peroxisome proliferator-activated receptor human PPAR triiodothyronine receptor nuclear receptor co-repressor silencing mediator for RAR and TR mitogen-activated protein kinase chloramphenicol acetyltransferase activation function The peroxisome proliferator-activated receptors (PPARs)1 are nuclear hormone receptors, the transcriptional activity of which is mostly thought to be regulated by the binding of ligand, such as certain fatty acids and eicosanoids (1Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1893) Google Scholar, 2Wilson T.M. Wahli W. Curr. Opin. Genet. Dev. 1997; 1: 235-241Google Scholar). However, the activity of some nuclear receptors is also modulated by phosphorylation, usually resulting in transcriptional enhancement, e.g. through the increased binding to ligand or DNA (3Orti E. Bodwell J.E. Munck A. Endocr. Rev. 1992; 13: 105-128PubMed Google Scholar, 4Kuiper G.G. Brinkmann A.O. Mol. Cell. Endocrinol. 1994; 100: 103-107Crossref PubMed Scopus (75) Google Scholar, 5Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1716) Google Scholar, 6Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1772) Google Scholar, 7Denner L.A. Weigel N.L. Maxwell B.L. Schrader W.T. O'Malley W.B. Science. 1990; 250: 1740-1743Crossref PubMed Scopus (313) Google Scholar, 8Sugawara A. Yen P.M. Apriletti J.W. Ribeiro R.C.J. Sacks D.B. Baxter J.D. Chin W.W. J. Biol. 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White-Carrington S. Szalkowski D. Moller D.E. J. Biol. Chem. 1996; 271: 31771-31774Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Whereas the transcriptional capacity of the mostly adipocyte-specific PPARγ isoform is inhibited in certain cell types by the mitogen-activated protein kinase (MAP-K)-dependent phosphorylation of a serine residue in its amino terminus (Ser-112) (11Hu E.D. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; 274: 2100-2103Crossref PubMed Scopus (937) Google Scholar, 14Zhang B. Berger J. Zhou G. Elbrecht A. Biswas S. White-Carrington S. Szalkowski D. Moller D.E. J. Biol. Chem. 1996; 271: 31771-31774Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), we have shown recently that PPARα is phosphorylated in response to insulin, resulting in an enhanced transcriptional response (10Shalev A. Siegrist-Kaiser C.A. Yen P.M. Wahli W. Burger A.G. Chin W.W. Meier C.A. Endocrinology. 1996; 137: 4499-4502Crossref PubMed Scopus (162) Google Scholar). However, it remained unclear whether this represented a direct effect of insulin and by which mechanism insulin stimulated the activity of PPARα. The PPARα isoform is highly expressed in liver and brown adipose tissue, both of which are target tissues for insulin action (15Lemberger T. Braissant O. Juge-Aubry C. Keller H. Saladin R. Staels B. Auwerx J. Burger A.G. Meier C.A. Wahli W. Ann. N. Y. Acad. Sci. 1996; 804: 231-251Crossref PubMed Scopus (155) Google Scholar). 2A. Gorla-Bajszczak and C. A. Meier, unpublished data. PPARα binds and is activated by fibrates, certain fatty acids, arachidonic acid analogs (e.g. 5,8,11,14-eicosatetraenoic acid), and leukotriene B4 (2Wilson T.M. Wahli W. Curr. Opin. Genet. Dev. 1997; 1: 235-241Google Scholar, 15Lemberger T. Braissant O. Juge-Aubry C. Keller H. Saladin R. Staels B. Auwerx J. Burger A.G. Meier C.A. Wahli W. Ann. N. Y. Acad. Sci. 1996; 804: 231-251Crossref PubMed Scopus (155) Google Scholar). However, in transient transfection systems, PPARα exhibits strong transcriptional activity even in the absence of exogenous ligands (16Sher T. Yi H.F. McBride O.W. Gonzalez F.J. Biochemistry. 1993; 32: 5598-5604Crossref PubMed Scopus (450) Google Scholar, 17Juge-Aubry C. Pernin A. Favez T. Burger A.G. Wahli W. Meier C.A. Desvergne B. J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Although this might be due to the presence of an endogenous stimulator, this activity could also be mediated through a constitutive trans-activating domain, similar to the activation function (AF)-1 region present in some other members of the nuclear receptor family, such as the retinoic acid and estrogen receptors (18Ali S. Metzger D. Bornert J.M. Chambon P. EMBO J. 1993; 12: 1153-1160Crossref PubMed Scopus (379) Google Scholar, 19Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (849) Google Scholar, 20Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Because we have previously demonstrated that PPARα is phosphorylated in response to insulin, resulting in the stimulation of basal as well as ligand-dependent transcriptional activity, we hypothesized that PPARα might contain a phosphorylation-regulated trans-activation domain (10Shalev A. Siegrist-Kaiser C.A. Yen P.M. Wahli W. Burger A.G. Chin W.W. Meier C.A. Endocrinology. 1996; 137: 4499-4502Crossref PubMed Scopus (162) Google Scholar). In the present study, we examined the hypothesis that PPARα contains a ligand-independent transcriptional activation domain that might be subject to regulation by phosphorylation. We describe a novel AF-1 function within the A/B domain of PPARα, which harbors two consensus MAP-K sites, which are phosphorylated in response to insulin and both of which are necessary to mediate the insulin-induced transcriptional activation of PPARα. Intriguingly, these MAP-K sites are distinct from the inhibitory MAP-K site described for PPARγ. Moreover, our results demonstrate that the transcriptional activation of PPARα by insulin may involve the dissociation from co-repressor proteins, which may be related to the nuclear receptor co-repressor (NCoR) and the silencing mediator for RAR and TR (SMRT) (21Hörlein A. Näär A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Söderström M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1712) Google Scholar, 22Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1712) Google Scholar). Hence, the insulin-induced phosphorylation of PPARα and γ is mediated through distinct phosphorylation motifs, resulting in transcriptional activation or repression, respectively. A fusion construct linking the GAL4-DNA-binding domain (amino acids 1–147) upstream of the amino-terminal region (amino acids 1–92) of hPPARα was constructed by cloning the cDNA corresponding to the first 92 amino acids of hPPARα amplified by polymerase chain reaction using thePfu DNA polymerase (Life Technologies, Inc.) into theBamHI and PstI sites of the pm-GAL4-BD expression plasmid (CLONTECH, Stehelin AG, Basel, Switzerland), resulting in the vector pm-GAL4BD-hPPARα. The expression plasmid pSG5-hPPARα was kindly provided by Dr. F. Gonzalez (NCI, National Institutes of Health, Bethesda, MD) (16Sher T. Yi H.F. McBride O.W. Gonzalez F.J. Biochemistry. 1993; 32: 5598-5604Crossref PubMed Scopus (450) Google Scholar). The final construct was verified by automated sequencing (ABI 373, Perkin-Elmer). Site-directed mutagenesis was performed by polymerase chain reaction amplification of the entire pm-GAL4BD-hPPARα1–92 or pSG5-hPPARα plasmids with Pfu DNA using sense and antisense primers containing the desired mutation: S77A mutation (AGC → GCC), S76A (TCG → GCG), S12A (TCC → GCC), S21A (AGC → GCC) according to the manufacturer's protocol (QuickChange site-directed mutagenesis, Stratagene, Basel, Switzerland). The presence of the desired mutation and the absence of spurious mutations in the amplified cDNA was assessed by automated sequencing. The reporter plasmid pG5-chloramphenicol acetyltransferase (CAT) (CLONTECH, Stehelin AG, Basel, Switzerland) contains five consensus GAL4 binding sites (UASG 17-mer (×5)). As a positive control, the plasmid pM3-VP16, encoding a fusion protein of the GAL4 BD and the VP16 activation domain, was used. The reporter plasmid pBL-1xPPRE-MEp-CAT8+, containing the natural PPAR-response element from the malic enzyme promoter, was described previously (17Juge-Aubry C. Pernin A. Favez T. Burger A.G. Wahli W. Meier C.A. Desvergne B. J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The pSV2-TRβ1 expression vector encoding for the TR β1 was described previously (23Meier C.A. Parkison C. Chen A. Ashizawa K. Meier-Heusler S.C. Muchmore P. Cheng S.Y. Weintraub B.D. J. Clin. Invest. 1993; 92: 1986-1993Crossref PubMed Scopus Google Scholar). The mutant was by site-directed This mutant was shown previously to be deficient in its with corepressor (21Hörlein A. Näär A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Söderström M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1712) Google Scholar). The human cell was using the as described previously with of the expression plasmid for the vector or the fusion protein with of the reporter plasmid (17Juge-Aubry C. Pernin A. Favez T. Burger A.G. Wahli W. Meier C.A. Desvergne B. J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, Gorla-Bajszczak A. Pernin A. T. Wahli W. Burger A.G. Meier C.A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). cell were with insulin of the MAP-K Switzerland) as described in the activity was as described by the of into as by Gorla-Bajszczak A. Pernin A. T. Wahli W. Burger A.G. Meier C.A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The results were by a and activity was to the protein as by the were with of or or mutant were in and (Life Technologies, Basel, Switzerland) by with for was for as described previously (10Shalev A. Siegrist-Kaiser C.A. Yen P.M. Wahli W. Burger A.G. Chin W.W. Meier C.A. Endocrinology. 1996; 137: 4499-4502Crossref PubMed Scopus (162) Google Scholar). were in and and the fusion were with a Dr. AG, Basel, Switzerland). were by As described were with of or the were in and the fusion were by using of the were to a for at in binding was by the for in in A (CLONTECH, Stehelin AG, Basel, Switzerland) was to a of for 12 The was with by five of in The Dr. AG, Basel, was and was for at the was as described were revealed by as described by the Switzerland). In order to the that the previously described constitutive activity of the hPPARα is due to an function in the amino terminus of this we a fusion construct linking the GAL4 domain to the first 92 amino acids of hPPARα The transcriptional response of this construct was assessed in transient transfection in using a reporter that the addition of the first 92 amino acids of hPPARα to the the constitutive transcriptional activity as with an stimulation by the strong transcriptional whether the previously described stimulation of the basal transcriptional activity of PPARα by insulin might involve this novel AF-1 domain, similar were performed in the presence of insulin for As shown in with insulin the activity of the amino terminus of PPARα effect was on the and Because we have shown recently that insulin the phosphorylation of PPARα in adipose and we also assessed the effect of insulin on PPARα in the presence of the of the MAP-K pathway Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). with the hypothesis that the stimulation of the amino terminus of PPARα by insulin a phosphorylation this effect Because the results described suggest that insulin the basal transcriptional activity of PPARα through the phosphorylation of a MAP-K site in its amino we examined the of PPARα from for conserved MAP-K Whereas MAP-K are present within the first 92 amino acids of sites were highly conserved across serine serine 21, and serine the residue to a MAP-K motif described in which shown to be phosphorylated by MAP-K and results in an of transcription. Hence, we the corresponding of the construct for the analysis of these sites and the of the MAP-K sites within the amino terminus of PPARα, we performed transient transfection in to that the serine to mutations at positions 12 and were both to the stimulatory effect of insulin, similar mutations of MAP-K site not the transcriptional stimulation by The transcriptional activity of the mutant with its expression as assessed in not These results are compatible with a in which insulin the AF-1 region of PPARα through the phosphorylation of 12 and In order to the hypothesis that insulin the phosphorylation of serine 12 and 21, we performed phosphorylation in with the and mutant in are the phosphorylated fusion with an resulting in a at corresponding to the molecular of the fusion protein as well as a which is to to a These demonstrated a increased phosphorylation of in response to insulin, which is to the enhancement However, the single S12A and S21A were phosphorylated by insulin, the mutant not an in its phosphorylation with These demonstrate that 12 and are both phosphorylated by insulin and that other to the phosphorylation of the amino terminus of PPARα. In order to that the response of the and S21A was not due to the altered expression of these proteins, we performed with an As shown in the S12A and were expressed at similar as the the S21A mutant was In order to the of the and in the of the both were to As shown in the hPPARα was activated by insulin in a analysis of the a and not response to insulin analysis of can the phosphorylation of 12 and such as the or dissociation of or corepressor proteins. we hypothesized that the phosphorylation might in the dissociation of a corepressor such as or resulting in transcriptional and The of this is that the addition of a strong binder of such co-repressor an stimulation on the AF-1 region of PPARα. In order to this we with (amino acids 1–92) or in the presence of of which is to strongly interact with corepressor (21Hörlein A. Näär A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Söderström M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1712) Google Scholar, 22Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1712) Google Scholar). the addition of was to the transcriptional activity of the AF-1 domain of PPARα in a and to a corresponding to that with insulin However, to the that might other a similar was performed with a mutant to be deficient in its with corepressor proteins, such as and As shown in this mutant its capacity to activate PPARα, compatible with the that for corepressor proteins, resulting in a of the AF-1 domain of PPARα. The human PPARα was previously shown to ligand-independent transcriptional activity, which can be modulated by insulin (10Shalev A. Siegrist-Kaiser C.A. Yen P.M. Wahli W. Burger A.G. Chin W.W. Meier C.A. Endocrinology. 1996; 137: 4499-4502Crossref PubMed Scopus (162) Google Scholar). The that insulin also the phosphorylation of PPAR in as well as in the that insulin might PPARα, its transcriptional In the present we demonstrate that the amino-terminal 92 amino acids of PPARα contain a trans-activation domain, which is further activated by insulin, we demonstrate that the effect of insulin on the AF-1 region is on MAP-K and the phosphorylation of two serine at positions 12 and 21, and we present that the phosphorylation of the amino terminus of PPARα may in the dissociation of corepressor proteins, which may in a further transcriptional enhancement of this The that nuclear hormone receptors contain a ligand-independent and a ligand-dependent domain well demonstrated for the and receptors (18Ali S. Metzger D. Bornert J.M. Chambon P. EMBO J. 1993; 12: 1153-1160Crossref PubMed Scopus (379) Google Scholar, 20Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, S. S. H. Chambon P. EMBO J. 1993; 12: PubMed Scopus Google Scholar). However, the activity of the domain is regulated by the binding of ligand resulting in the of proteins, the AF-1 region is thought to be by molecular that are C.K. Rosenfeld M.G. Curr. Opin. Biol. 1997; PubMed Scopus Google Scholar). The presence of a strong AF-1 region in the A/B of hPPARα shown in the present at in the basal activity with this PPAR isoform in transient transfection experiments. the present demonstrate that this activity is subject to regulation through phosphorylation of two MAP-K Because we have previously shown that PPARα is a in stimulated with insulin, it is that the these two is of This is by our that the PPARα is also activated by insulin and that this 12 and 21, compatible with the results with the 1–92 fusion Although not the a response to However, this effect is not mediated by the amino-terminal 92 amino acids, as by with a PPARα mutant the amino terminus not This stimulation by insulin may be mediated through other of PPARα or through insulin for or the transcriptional is also of to that of with insulin for the expression of the PPARα that to insulin trans-activation by PPARα the phosphorylation of a to this hormone might in the of PPARα activity in liver the expression of PPARα J.D. S. O. J. 1994; PubMed Scopus Google Scholar). In contrast to PPARα, the which was recently shown to be phosphorylated in its amino terminus in response to of the MAP-K is inhibited phosphorylation of a serine residue at as assessed by its (11Hu E.D. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; 274: 2100-2103Crossref PubMed Scopus (937) Google Scholar). examined in systems or in the of the amino terminus of PPARγ can also be stimulated by phosphorylation. Hence, it that at in adipose tissue, which the PPARα and γ at the phosphorylation by insulin on PPARα and the the is This with ligand binding and in DNA a novel mechanism for of PPAR (17Juge-Aubry C. Pernin A. Favez T. Burger A.G. Wahli W. Meier C.A. Desvergne B. J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The phosphorylation sites in PPARα are highly conserved across and they are distinct from the homologous MAP-K site shown to be phosphorylated in Hence, the of insulin on PPARα and are by the presence of distinct phosphorylation The phosphorylation-dependent in transcription are as is the for the Because the AF-1 domain as well as the phosphorylation sites within the domain of PPARα, it is to that the altered phosphorylation results in the of corepressor proteins. However, the interact in a ligand-dependent with the domain of nuclear receptors, some with AF-1 may T. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar, S.A. S.Y. M.J. O'Malley J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the corepressor proteins, such as and interact with of the nuclear receptors in a ligand-independent manner. our that can activate the amino terminus of PPARα is of the are to strongly interact with and resulting in a for corepressor proteins. Because this effect is by a mutation in the of it is to that the amino terminus of PPARα with a corepressor protein that is to partially the activity of However, whether the phosphorylation of 12 and AF-1 by the dissociation of corepressors is a of Although PPARγ was shown to interact with in this involved the region of the amino terminus Lazar M.A. Dev. 1997; PubMed Scopus Google Scholar). some demonstrate that an the transcriptional of PPARγ by insulin, at a the phosphorylated amino terminus of PPARγ and R.M. K. Heinzel T. Torchia J. T.M. R. M. S. J. C.K. Glass C.K. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and the of direct of with the amino terminus of PPARα were and C. A. Meier, unpublished data. These that the the AF-1 region of PPARα and are under the or novel corepressor are which have the to interact with the AF-1 region as well as the motif of nuclear In we have demonstrated that PPARα contains a strong ligand-independent AF-1 domain, which can be further activated through the phosphorylation of two serine residues, which are distinct from the inhibitory phosphorylation site present in In addition, the AF-1 region of PPARα can be well activated by corepressor proteins, that such might the amino terminus of PPARα. can be that the phosphorylation of the AF-1 domain of PPARα might in the dissociation of such corepressor proteins, resulting in transcriptional