p38 Mitogen-activated Protein Kinase Plays an Inhibitory Role in Hepatic Lipogenesis
2006; Elsevier BV; Volume: 282; Issue: 7 Linguagem: Inglês
10.1074/jbc.m606742200
ISSN1083-351X
AutoresYan Xiong, Qu Fan Collins, Jie An, Edgar Lupo, Hui Yu Liu, Delong Liu, Jacques Robidoux, Zhenqi Liu, Wenhong Cao,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoHepatic lipogenesis is the principal route to convert excess carbohydrates into fatty acids and is mainly regulated by two opposing hormones, insulin and glucagon. Although insulin stimulates hepatic lipogenesis, glucagon inhibits it. However, the mechanism by which glucagon suppresses lipogenesis remains poorly understood. In this study, we have observed that p38 mitogen-activated protein kinase plays an inhibitory role in hepatic lipogenesis. Levels of plasma triglyceride and triglyceride accumulation in the liver were both elevated when p38 activation was blocked. Expression levels of central lipogenic genes, including sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase, hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl pyrophosphate synthase, and cytochrome P-450-51, were decreased in liver by fasting and in primary hepatocytes by glucagon but increased by the inhibition of p38. In addition, we have shown that p38 can inhibit insulin-induced expression of key lipogenic genes in isolated hepatocytes. Our results in hepatoma cells demonstrate that p38 plays an inhibitory role in the activation of the SREBP-1c promoter. Finally, we have shown that transcription of the PGC-1β gene, a key coactivator of SREBP-1c, was reduced in liver by fasting and in isolated hepatocytes by glucagon. This reduction was significantly reversed by the blockade of p38. Insulin-induced expression of the PGC-1β gene was enhanced by the inhibition of p38 but suppressed by the activation of p38. Together, we have identified an inhibitory role for p38 in the transcription of central lipogenic genes, SREBPs, and PGC-1β and hepatic lipogenesis. Hepatic lipogenesis is the principal route to convert excess carbohydrates into fatty acids and is mainly regulated by two opposing hormones, insulin and glucagon. Although insulin stimulates hepatic lipogenesis, glucagon inhibits it. However, the mechanism by which glucagon suppresses lipogenesis remains poorly understood. In this study, we have observed that p38 mitogen-activated protein kinase plays an inhibitory role in hepatic lipogenesis. Levels of plasma triglyceride and triglyceride accumulation in the liver were both elevated when p38 activation was blocked. Expression levels of central lipogenic genes, including sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase, hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl pyrophosphate synthase, and cytochrome P-450-51, were decreased in liver by fasting and in primary hepatocytes by glucagon but increased by the inhibition of p38. In addition, we have shown that p38 can inhibit insulin-induced expression of key lipogenic genes in isolated hepatocytes. Our results in hepatoma cells demonstrate that p38 plays an inhibitory role in the activation of the SREBP-1c promoter. Finally, we have shown that transcription of the PGC-1β gene, a key coactivator of SREBP-1c, was reduced in liver by fasting and in isolated hepatocytes by glucagon. This reduction was significantly reversed by the blockade of p38. Insulin-induced expression of the PGC-1β gene was enhanced by the inhibition of p38 but suppressed by the activation of p38. Together, we have identified an inhibitory role for p38 in the transcription of central lipogenic genes, SREBPs, and PGC-1β and hepatic lipogenesis. Hepatic lipogenesis is essential for maintaining energy balance (1.Cooper A.D. Ellsworth J.L. Hepatology. 1996; I: 92-130Google Scholar). Disorders of hepatic lipogenesis may lead to fatty liver, dyslipidemia, type II diabetes mellitus, and complications such as atherosclerosis (2.Accili D. Diabetes. 2004; 53: 1633-1642Crossref PubMed Scopus (148) Google Scholar). Lipogenesis in liver includes de novo synthesis of fatty acids and cholesterols. As a major site for synthesis of fatty acids, the liver converts excess carbohydrates into fat storage in the fed state. Fatty acids synthesized in the liver are converted into triglycerides and secreted as very low density lipoproteins, which transport fatty acids to the storage sites in adipocytes. Cholesterols synthesized in the liver are also transported to other tissues via very low density lipoproteins as essential building materials for steroid hormones and cellular membranes. However, excess production of fatty acids and cholesterols from the liver may contribute to a variety of lipid disorders. The lipogenic process in the liver is primarily regulated by central lipogenic transcription factors, sterol regulatory element-binding proteins (SREBPs) 2The abbreviations and trivial names used are: SREBP, sterol regulatory element-binding protein; TG, triglyceride; FAS, fatty acid synthase; FPS, farnesyl pyrophosphate synthase; SB, SB203580; RT, reverse transcription; HFD, high fat diet; siRNA, small interfering RNA; dn, dominant negative.2The abbreviations and trivial names used are: SREBP, sterol regulatory element-binding protein; TG, triglyceride; FAS, fatty acid synthase; FPS, farnesyl pyrophosphate synthase; SB, SB203580; RT, reverse transcription; HFD, high fat diet; siRNA, small interfering RNA; dn, dominant negative. (reviewed in Refs. 3.Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; 109: 1125-1131Crossref PubMed Scopus (3710) Google Scholar and 4.Eberle D. Hegarty B. Bossard P. Ferre P. Foufelle F. Biochimie (Paris). 2004; 86: 839-848Crossref PubMed Scopus (992) Google Scholar). The SREBPs are basic helix-loop-helix-leucine zipper-containing transcription factors (5.Hua X. Sakai J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1995; 270: 29422-29427Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Among three known SREBPs, SREBP-1a and -1c are encoded by the same gene; SREBP-1c lacks the N-terminal exon compared with SREBP-1a (6.Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (246) Google Scholar). SREBP-2 is encoded by a separate gene (7.Miserez A.R. Cao G. Probst L.C. Hobbs H.H. Genomics. 1997; 40: 31-40Crossref PubMed Scopus (90) Google Scholar). Although SREBPs share similar lipogenic function, SREBP-1c is primarily involved in fatty acid synthesis, whereas SREBP-2 is mainly involved in cholesterol synthesis (8.Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. 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Chem. 2003; 278: 36652-36660Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). SREBP-1c and -2 are predominant isoforms in the liver and major regulators of hepatic lipogenesis (13.Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 1997; 99: 838-845Crossref PubMed Scopus (635) Google Scholar). SREBPs regulate lipogenesis by stimulating expression of their target genes, such as liver pyruvate kinase, acetyl CoA carboxylase, fatty acid synthase (FAS), Spot14, diacylglycerol acyltransferase, and glycerol-3-phosphate acyltransferase (reviewed in Ref. 4.Eberle D. Hegarty B. Bossard P. Ferre P. Foufelle F. Biochimie (Paris). 2004; 86: 839-848Crossref PubMed Scopus (992) Google Scholar). The function of SREBPs is primarily regulated through protein cleavage and gene expression. The "inactive" precursor form of SREBPs are integral membrane proteins of endoplasmic reticulum. Upon sterol deprivation, they are cleaved through two sequential steps to release the N terminus, which is translocated into the nucleus to activate the transcription of target genes (6.Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (246) Google Scholar, 14.Lee S.J. Sekimoto T. Yamashita E. Nagoshi E. Nakagawa A. Imamoto N. Yoshimura M. Sakai H. Chong K.T. Tsukihara T. Yoneda Y. Science. 2003; 302: 1571-1575Crossref PubMed Scopus (171) Google Scholar, 15.Nagoshi E. Yoneda Y. Mol. Cell. Biol. 2001; 21: 2779-2789Crossref PubMed Scopus (56) Google Scholar). Transcription of SREBP genes in the liver is principally regulated by insulin and glucagon (16.Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5987-5992Crossref PubMed Scopus (535) Google Scholar, 17.Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (452) Google Scholar, 18.Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (231) Google Scholar). Although the mechanism of SREBP gene transcription by these hormones has been intensively studied, there are still significant gaps in our understanding. Recently, peroxisome proliferator-activated receptor γ co-activator-1β (PGC-1β) has been shown to interact with SREBP-1c as a coactivator in regulating the transcription of lipogenic genes (19.Lin J. Yang R. Tarr P.T. Wu P.H. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. Newgard C.B. Spiegelman B.M. Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). However, the signaling pathway from hormones to the SREBP promoters, such as the identity of specific kinases, remains largely unknown. p38 mitogen-activated protein kinase (p38) is one member of the mitogen-activated protein kinase superfamily. It is a cellular sensor of many stresses caused by various stimuli (reviewed in Refs. 20.Rincon M. Conze D. Weiss L. Diehl N.L. Fortner K.A. Yang D. Flavell R.A. Enslen H. Whitmarsh A. Davis R.J. Immunol. Cell Biol. 2000; 78: 166-175Crossref PubMed Scopus (27) Google Scholar, 21.Dong C. Davis R.J. Flavell R.A. Annu. Rev. Immunol. 2002; 20: 55-72Crossref PubMed Scopus (1367) Google Scholar, 22.Zarubin T. Han J. Cell Res. 2005; 15: 11-18Crossref PubMed Scopus (1220) Google Scholar). Essentially, any significant change in extracellular environment can activate p38 and set in motion certain protective mechanisms, such as activation and production of heat shock proteins, immune responses, and apoptosis. We and others have recently shown that p38 plays an important role in the control of energy balance in brown adipocytes, muscle cells, and hepatocytes (23.Cao W. Medvedev A.V. Daniel K.W. Collins S. J. Biol. Chem. 2001; 276: 27077-27082Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 24.Cao W. Daniel K.W. Robidoux J. Puigserver P. Medvedev A.V. Bai X. Floering L.M. Spiegelman B.M. Collins S. Mol. Cell. Biol. 2004; 24: 3057-3067Crossref PubMed Scopus (429) Google Scholar, 25.Fan M. Rhee J. St.-Pierre J. Handschin C. Puigserver P. Lin J. Jaeger S. Erdjument-Bromage H. Tempst P. Spiegelman B.M. Genes Dev. 2004; 18: 278-289Crossref PubMed Scopus (247) Google Scholar, 26.Akimoto T. Pohnert S.C. Li P. Zhang M. Gumbs C. Rosenberg P.B. Williams R.S. Yan Z. J. Biol. Chem. 2005; 280: 19587-19593Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 27.Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 28.Collins Q.F. Xiong Y. Lupo Jr., E.G. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In this study, we investigated the role of p38 in the control of hepatic lipogenesis. Our results show that p38 is activated in liver by fasting and in isolated hepatocytes by glucagon. Suppression of p38 in liver or isolated hepatocytes led to elevated expression of lipogenic genes and increased triglyceride levels in both plasma and liver. Together our results support a critical role for p38 in the regulation of hepatic lipogenesis. Chemicals, Antibodies, and Plasmids−SB203580 (SB) was from Calbiochem. Glucagon and Percoll were from Sigma. Antibodies against p38 and phosphorylated p38 were from Cell Signaling Technology. Antibodies against SREBP-1 were from Santa Cruz Biotechnology. The SREBP-1c promoter construct (pBP1c, 1.3 kb) was a kind gift from Drs. Michael S. Brown and Joseph L. Goldstein (29.Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1407) Google Scholar). The constructs for dominant-negative p38α (p38(AF)) and MKK6E were kindly provided by Dr. Jiahua Han. Animal Experiments−To examine the role of p38 in lipid metabolism, C57BL/6 mice (10 mice/group) were fed with normal (standard) chow diet or high fat diet (Research Diets catalog number D12330: 58.0 kcal% fat, 16.0 kcal% protein, and 26 kcal% carbohydrate) as noted. Six weeks later, some mice were treated with SB (30 mg/kg body weight/day through gastric gavages) or the vehicle solution for another 2 weeks as noted. SB and vehicle were administered one dose/day. At completion of these treatments, blood samples were collected for measurements of lipids, and livers were harvested for quantification of triglyceride (TG) and analyses of target proteins and mRNAs. To further evaluate the role of p38 in expressions of lipogenic genes, C57BL/6 mice (6-8 weeks old) were fasted for 24 h in the presence or absence of SB (12.4 mg/kg body weight via immunoprecipitation) as noted. SB was administered as previously described (27.Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The fed mice were used as a control. Livers were collected for the measurements of lipogenic genes. Preparation, Transfection, and Viral Infection of Primary Hepatocytes−Primary hepatocytes from C57BL/6 mice fed with normal chow diet at the regular schedule were prepared as previously described (30.Seglen P.O. J. Toxicol. Environ. Health. 1979; 5: 551-560Crossref PubMed Scopus (113) Google Scholar). Briefly, under anesthesia with pentobarbital (immunoprecipitation, 30 mg/kg body weight), livers were perfused with Hanks' balanced salt solution (Invitrogen) at 5 ml/min for 8 min followed by continuous perfusion with serum-free Williams' medium containing collagenase (Worthington, type II, 130 units/ml) (Invitrogen), HEPES (10 mm), and NaOH (0.004 n) at 5 ml/min for 12 min. Hepatocytes were harvested and purified with Percoll as described previously (31.Kedderis G.L. Argenbright L.S. Miwa G.T. Toxicol. Appl. Pharmacol. 1988; 93: 403-412Crossref PubMed Scopus (21) Google Scholar). The viability of hepatocytes was examined with trypan blue exclusion. Cells with viability >95% were used. Hepatocytes were inoculated into collagen-coated 6-well plates (5 × 105/well) in Williams' medium. Cells were incubated overnight before any experimentation. MKK6E and p38-AF-FLAG (dominant negative p38α) were introduced into primary hepatocytes with Lipofectamine 2000 according to the manufacturer's manuals (Invitrogen). For adenoviral infection, 50 active viral particles/cell in 1 ml of Williams' medium with 2% fetal bovine serum were used to incubate with cells for 6 h followed by incubation with fresh medium containing 10% fetal bovine serum (32.Lerin C. Montell E. Nolasco T. Clark C. Brady M.J. Newgard C.B. Gomez-Foix A.M. Diabetes. 2003; 52: 2221-2226Crossref PubMed Scopus (15) Google Scholar). At 36 h post-infection, levels of p38 and expression of genes in hepatocytes were detected by immunoblottings with appropriate antibodies and TaqMan real-time reverse transcription (RT)-PCR, respectively. Culture and Transfection of Hepatoma Cell Line−The Hepa1c1c7 mouse hepatoma cell line was purchased from the American Type Culture Collection (Manassas, VA) and cultured with minimal essential α medium in the presence of 10% fetal bovine serum and antibiotics. Transient transfection of these cells was performed using Lipofectamine 2000 (Invitrogen) according to instructions from the manufacturer. Immunoblotting−Tissue or whole-cell lysates were prepared by homogenization and sonication followed by the addition of 2× Laemmli sample buffer. Aliquots (5-10 μg protein/well) were resolved with mini-Tris-glycine gels (4-20%) (Invitrogen) and transferred to nitrocellulose membranes. Levels of p38 and SREPB-1 were detected with a 1:1000 dilution of each specific antiserum (catalog numbers 9211S and 9212 from Cell Signaling Technology and catalog number sc-8984 from Santa Cruz Biotechnology) followed by a 1:10,000 dilution of goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase (RPN5783, Amersham Biosciences). Fluorescent bands were visualized with a Typhoon phosphorimaging device (Molecular Dynamics). RNA Isolation and TaqMan Real-time RT-PCR−Total RNAs from liver were prepared by using RNA purification kits from Qiagen. Real-time RT-PCR TaqMan probes and reaction agents were purchased from Applied Biosystems. Reactions were performed according to manuals from the manufacturer. All results were normalized to levels of the GADPH gene. Catalog numbers for the probes are: SREBP-1 (Mm00550338_m1)), SREBP-2 (Mm01306293_m1), FAS (Mm-00662319_m1), PGC-1β (Mm00504720_ m1), HMG CoA reductase (Mm01282491-g1), farnesyl pyrophosphate synthase (FPS) (Mm00830315_g1), and CYP51 (Mm0049968_m1). Measurement of SREBP-1c Promoter Activity−The SREBP-1c promoter was introduced into Hepa1c1c7 hepatoma cells together with the expression vector for β-galactosidase via transient transfection (24.Cao W. Daniel K.W. Robidoux J. Puigserver P. Medvedev A.V. Bai X. Floering L.M. Spiegelman B.M. Collins S. Mol. Cell. Biol. 2004; 24: 3057-3067Crossref PubMed Scopus (429) Google Scholar) and stimulated as noted. SREBP-1c promoter activity was measured by luciferase assays and normalized to the internal control of transfection, β-galactosidase. Measurements of mRNA Degradation−Transcription of lipogenic genes in hepatocytes were activated by insulin and dexamethasone for 20 h and subsequently blocked by treatment with amanitin as described previously (33.Xu J. Teran-Garcia M. Park J.H. Nakamura M.T. Clarke S.D. J. Biol. Chem. 2001; 276: 9800-9807Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Cells were then treated with either SB or vehicle solution as noted. Levels of representatives of lipogenic genes (SREBP-1c and PGC-1β) were quantified with TaqMan real-time RT-PCR. Measurements of Liver Triglyceride Content−Livers were homogenized in a buffer containing 18 mm Tris (pH 7.4), 300 mmd-mannitol, 50 mm EGTA, and 1 mm phenylmethylsulfonyl fluoride. Triglycerides in the lysates were extracted using a chloroform/methanol protocol as previously described (34.Shimabukuro M. Koyama K. Chen G. Wang M.-Y. Trieu F. Lee Y. Newgard C.B. Unger R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4637-4641Crossref PubMed Scopus (616) Google Scholar). Levels of triglycerides were quantified by using triglyceride reagents from Sigma (catalog number T2449-10ML) and normalized to protein concentration. Lipid droplets in liver slides were visualized with Oil Red O staining. Inhibition of p38 Leads to Elevation of Plasma Lipids and Fat Accumulation in Liver−During our study on the stimulatory role of p38 in hepatic gluconeogenesis (27.Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), we unexpectedly observed that p38 might inhibit hepatic lipogenesis. To fully characterize this observation, we examined the effect of p38 inhibition on levels of plasma lipids and liver TG content in mice. As shown in Fig. 1A, although plasma levels of total cholesterol (T-Chol), high density lipoprotein cholesterol (HDLChol), and TGs were not significantly changed after high fat diet (HFD) for 8 weeks, hepatic TG content was significantly increased by HFD (p < 0.05) (Fig. 2A). Levels of TG in both plasma and liver were significantly increased by the inhibition of p38 in the mice under either normal chow or high fat diet. (Figs. 1A and 2A). Both the number and size of the lipid droplets in the liver were increased by HFD, and these increases were further significantly aggravated by the inhibition of p38 (Fig. 2B). Levels of phosphorylated p38 in the liver were also enhanced by HFD, but this effect was blocked by SB (Fig. 2C). It was noted that treatment of animals with SB did not significantly influence either levels of plasma non-esterified fatty acids and insulin or body weight and food consumption (Fig. 1, B-E). Together, these results show that the blockade of p38 can elevate TG levels in both plasma and liver, suggesting an inhibitory role for p38 in hepatic lipogenesis.FIGURE 2The inhibition of p38 enhances fat accumulation in liver. Mice were treated in the same way as described in Fig. 1. A, levels of TG content in liver were quantified with a standard method. *, p < 0.05 compared with ND with no SB treatment. #, p < 0.01 compared with -SB under HFD. B, lipid content in liver tissue was visualized with Oil Red O staining. C, levels of p38 phosphorylation in liver were detected by immunoblotting with antibodies against total or phospho-p38. Results shown represent means ± S.D. of two independent experiments. #, p < 0.05 comparing HFD to either ND or HFD + SB.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Blockade of p38 Can Increase Transcript Levels of Lipogenic Genes in Fasted Liver−To investigate the possible role of p38 in suppression of hepatic lipogenesis, we chose to use fasting mice as models, because it is well known that hepatic lipogenesis is normally inhibited by fasting and p38 is activated in the liver during fasting (27.Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). As expected (Fig. 3A), levels of lipogenic gene transcripts, including SREBP-1c, FAS, hydroxy-3-methylglutaryl coenzyme A reductase, FPS, and CYP51 were decreased by fasting, and these decreases were significantly, although not completely in some cases, reversed by the inhibition of p38. SREBP-2 transcripts were not reduced by fasting but were significantly elevated upon the blockade of p38. Levels of both SREBP-1c and FAS transcripts in fed mice were also increased by SB. Phosphorylation of p38 in the liver was increased by fasting and inhibited by SB (Fig. 3B) as expected (27.Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). These results indicate that activation of p38 in liver is related to the reduction of transcript levels of central lipogenic genes, SREBP-1c, and its downstream target genes. p38 Plays a Suppressive Role in Transcription of Lipogenic Genes in Primary Hepatocytes−Lipogenesis is inhibited during fasting to protect the substrate supply for glucose production in liver (16.Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5987-5992Crossref PubMed Scopus (535) Google Scholar). The predominant suppressor of lipogenesis is glucagon, which is also the major promoter of hepatic glucose production and is always significantly elevated in the blood during fasting (35.Ruderman N. Aoki T. Cahill G. Gluconeogensis: Its Regulation in Mammalian Species. John Wiley & Sons, Inc., New York1976: 515-532Google Scholar). Glucagon is a cAMP-producing hormone that can activate p38 in primary hepatocytes (27.Cao W.H. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. Daniel K. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 36.Spector M.S. Auer K.L. Jarvis W.D. Ishac E.J. Gao B. Kunos G. Dent P. Mol. Cell. Biol. 1997; 17: 3556-3565Crossref PubMed Scopus (98) Google Scholar). To determine whether p38 mediates glucagon suppression of hepatic lipogenesis, levels of key lipogenic gene transcripts and phosphorylation of p38 in primary hepatocytes were examined in the presence or absence of p38 blockade. As predicted, levels of SREBP-1c, SREBP-2, FAS, HMG CoA-R, and FPS transcripts were inhibited by glucagon (Fig. 4A). This inhibition was significantly reversed by the blockade of p38 with either SB or overexpression of dominant negative p38α (dn-p38α). To determine the role of p38 in insulin-induced transcription of key lipogenic genes, primary hepatocytes were stimulated by insulin in the presence of a p38 inhibitor or an activator. As shown in Fig. 4B, levels of key lipogenic genes SREBP-1c and FAS were elevated by insulin as expected, and this elevation was further enhanced by the inhibition of p38 with SB but prevented by the activation of p38 with MKK6E. Furthermore, levels of the SREBP-1 protein (membrane-bound) were significantly increased by all p38 inhibitors, including SB, dn-p38α, and small interfering RNA against p38α (siRNA-p38α) but decreased by glucagon (Fig. 4C). The nuclear form of SREBP-1 was also slightly elevated by the inhibition of p38. The scrambled siRNA had no effect. (Note: cells were not deprived of cholesterol in these experiments.) Phosphorylation of p38 in isolated hepatocytes was also stimulated by glucagon and blocked by either SB or siRNA-p38α (Fig. 4D). Our results indicated that the inhibition of p38 did not affect the stability of SREBP-1c mRNA (supplemental Fig. 1). Together, these results support the notion that p38 is an inhibitor of lipogenic gene expression in isolated hepatocytes. p38 Is an Inhibitor of the SREBP-1c Promoter−To further define the role of p38 in hepatic lipogenesis, we examined the role of p38 in activation of the SREBP-1c promoter, which was introduced into Hepa1c1c7 hepatoma cells via transient transfection. Because insulin is known to stimulate transcription of the SREBP-1c gene (17.Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (452) Google Scholar, 37.Amemiya-Kudo M. Shimano H. Yoshikawa T. Yahagi N. Hasty A.H. Okazaki H. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Sato R. Kimura S. Ishibashi S. Yamada N. J. Biol. Chem. 2000; 275: 31078-31085Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 38.Chen G. Liang G. Ou J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11245-11250Crossref PubMed Scopus (431) Google Scholar, 39.Cagen L.M. Deng X. Wilcox H.G. Park E.A. Raghow R. Elam M.B. Biochem. J. 2005; 385: 207-216Crossref PubMed Scopus (125) Google Scholar), activity of the SREBP-1c promoter was stimulated with insulin in the presence or absence of a p38 inhibitor. As shown in Fig. 5A, the SREBP-1c promoter was stimulated by insulin as expected, and this stimulation was further enhanced by the inhibition of p38. Similarly, the SREBP-1c promoter was suppressed by glucagon as expected, and the suppression was significantly reversed by the inhibition of p38 with either SB or siRNA (Fig. 5B). Together, these results further support an inhibitory role for p38 in the transcription of the central lipogenic gene, SREBP-1c. p38 Can Inhibit Transcription of the PGC-1β Gene in Both Liver and Isolated Hepatocytes−PGC-1β was recently shown to be a critical coactivator of lipogenic gene expression through its interaction with SREBP-1c (19.Lin J. Yang R. Tarr P.T. Wu P.H. Handschin C. Li S. Yang W. Pei L. Uldry M. Tontonoz P. Newgard C.B. Spiegelman B.M. Cell. 2005; 120: 261-273Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). To examine whether transcription of the PGC-1β gene is a target for p38 in the suppression of hepatic lipogenesis, we examined levels of PGC-1β transcripts in livers from fed or fasted mice with or without treatment with SB. As shown in Fig. 6A, levels of PGC-1β transcripts in liver were decreased by fasting; this decline was significantly, although not completely, reversed by the blockade of p38. To more directly study the effect of p38 on levels of PGC-1β transcripts, primary hepatocytes were treated with glucagon in the presence or absence of p38 inhibition. As shown in Fig. 6B, levels of PGC-1β transcripts were reduced by glucagon, but the reduction was completely prevented by the blockade of p38 with either SB or dn-p38α. Similarly, insulin-induced expression of the PGC-1β gene was also enhanced by the inhibition of p38 but suppressed by the activation of p38 (Fig. 6C). To determine the possible role of p38 in the degradation of PGC-1β mRNA, the transcription of the PGC-1β gene was first stimulated by insulin and dexamethasone and then blocked by amanitin follo
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