Regulatory Role of Glycogen Synthase Kinase 3 for Transcriptional Activity of ADD1/SREBP1c

Abstract

Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3β down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism. Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3β down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism. Glycogen synthase kinase 3 (GSK3) 1The abbreviations used are: GSK3, glycogen synthase kinase 3; ACC1, acetyl-CoA carboxylase 1; ADD1/SREBP1c, adipocyte determination- and differentiation-dependent factor 1; FAS, fatty acid synthase; PKB/Akt, protein kinase B; SCD1, steroyl-CoA desaturase 1; MAP kinase, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; IP, immunoprecipitation.1The abbreviations used are: GSK3, glycogen synthase kinase 3; ACC1, acetyl-CoA carboxylase 1; ADD1/SREBP1c, adipocyte determination- and differentiation-dependent factor 1; FAS, fatty acid synthase; PKB/Akt, protein kinase B; SCD1, steroyl-CoA desaturase 1; MAP kinase, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; IP, immunoprecipitation. is a serine/threonine kinase that has been implicated in multiple cellular processes, including the Wnt and insulin signaling pathways. 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Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar) have elegantly revealed that cholesterol-dependent proteolytic cleavage of SREBP family proteins is tightly controlled by SREBP cleavage-activating protein and Insigs. In addition, Kotzka et al. (35Kotzka J. Muller-Wieland D. Koponen A. Njamen D. Kremer L. Roth G. Munck M. Knebel B. Krone W. Biochem. Biophys. Res. Commun. 1998; 249: 375-379Crossref PubMed Scopus (72) Google Scholar) reported that ADD1/SREBP1c is phosphorylated at the N-terminal end of the protein through the MAP kinase pathway, and such phosphorylation of ADD1/SREBP1c stimulates its transcriptional activity (35Kotzka J. Muller-Wieland D. Koponen A. Njamen D. Kremer L. Roth G. Munck M. Knebel B. Krone W. Biochem. Biophys. Res. Commun. 1998; 249: 375-379Crossref PubMed Scopus (72) Google Scholar). However, PD98059, an inhibitor of MAP kinase pathway, did not antagonize the of insulin the expression of ADD1/SREBP1c target genes in D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (234) Google Scholar, D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, P.B. Roth R.A. M. A. Biochem. J. 2000; PubMed Google Scholar), implying that the MAP kinase pathway might not be a for ADD1/SREBP1c target gene expression by insulin the of GSK3 as a negative regulator of insulin signaling and ADD1/SREBP1c as a of insulin-responsive gene expression, it would be important to the between inhibition of GSK3 and increase of ADD1/SREBP1c transcriptional activity by this we the roles of GSK3 the of transcriptional activity of ADD1/SREBP1c. we that GSK3 inhibitors the transcriptional activity of ADD1/SREBP1c and the expression of ADD1/SREBP1c target genes de novo protein Additionally, we a phosphorylation of ADD1/SREBP1c by these results a role of GSK3 in the insulin-dependent activation of ADD1/SREBP1c in adipocytes and hepatocytes. and and and GSK3 ADD1/SREBP1c J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (845) Google Scholar). expression for GSK3β C. D. G. P. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Cell to in Dulbecco's modified Eagle's with of cells with and insulin for other with and and cells in with and at in a cells in and cells in at in a and cells and with several 1 by the (22Kim J.B. Sarraf P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Investig. 1998; 101: 1-9Crossref PubMed Scopus (612) Google Scholar, J.B. T. Spiegelman B.M. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). expression for ADD1/SREBP1c which by with the with glucose for cells with or insulin and for cell with and and the activities of and to the cells with the as (22Kim J.B. Sarraf P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Investig. 1998; 101: 1-9Crossref PubMed Scopus (612) Google Scholar, J.B. T. Spiegelman B.M. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). with to the of in and and by and to and and as by the by the of and used as FAS, ACC1, SCD1, ADD1/SREBP1c, and for the used for synthesis with 1 of for ADD1/SREBP1c and for and which not by and by used in this as ADD1/SREBP1c ADD1/SREBP1c GSK3β proteins in with an N-terminal the and with to the GSK3β by kinase at in a of 1 and and by of acid for or for ADD1/SREBP1c or ADD1/SREBP1c proteins E. acid of and Biochem. 1997; PubMed Scopus Google Scholar). and with ADD1/SREBP1c proteins and with and of of cell with with protein and with 1 of 1 and of GSK3 by or as cells with ADD1/SREBP1c expression in glucose with for the to and cells for LiCl 1 1 or insulin and cell with ADD1/SREBP1c proteins by with by and to the of ADD1/SREBP1c and the transcriptional activity of ADD1/SREBP1c is regulated by GSK3, an inhibitor of GSK3, used in revealed that LiCl inhibits GSK3 activity with an of and a of GSK3 activity is with LiCl A.J. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). is one of the target genes of ADD1/SREBP1c and its the for ADD1/SREBP1c (22Kim J.B. Sarraf P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Investig. 1998; 101: 1-9Crossref PubMed Scopus (612) Google Scholar, M. H. T. N. H. Y. F. Y. K. J. K. T. R. S. S. N. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Kim Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). in LiCl stimulated the activity in the of ADD1/SREBP1c in a However, it the activity in the absence of ADD1/SREBP1c in chloride we did not in that transcriptional activity of ADD1/SREBP1c is by results in cells that insulin receptor to the of target gene expression of ADD1/SREBP1c by GSK3, we a insulin-responsive such as adipocytes and in the absence or of GSK3 Consistent with the results LiCl the mRNA expression of ADD1/SREBP1c target genes, such as FAS, ACC1, and and However, LiCl the expression level of nuclear protein these that inhibition of GSK3 stimulates transcriptional activity of ADD1/SREBP1c and its target gene expression in adipocytes and hepatocytes. of GSK3 by ADD1/SREBP1c LiCl has several other GSK3, such as the phosphatidylinositol signaling pathway via inhibition of C. E. 2002; PubMed Scopus Google Scholar), we a GSK3 D.A. L. D. Brown M.J. D. A.D. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Coghlan L. Eur. J. Biochem. 2003; PubMed Scopus Google Scholar). in and enhanced mRNA expression in adipocytes and cells in a to nuclear of the protein not by efficiently inhibits GSK3 kinase kinase cells with or GSK3 inhibitors, cell with GSK3 with one of the of GSK3 Biochem. 1997; PubMed Scopus Google Scholar, J.R. J. Biochem. Cell Biol. 1999; PubMed Scopus Google Scholar), and GSK3 kinase activities kinase activity of GSK3 by LiCl or at of or 3 and that or target gene expression of ADD1/SREBP1c is with the inhibition of GSK3 kinase activity. of GSK3β of the of GSK3 the transcriptional activity of ADD1/SREBP1c, we investigated the effects of GSK3β revealed that overexpression of GSK3β the transcriptional activity of ADD1/SREBP1c in a Furthermore, we the of ADD1/SREBP1c target gene expression in the absence or of GSK3β in expression of GSK3β in cells inhibited mRNA of mRNA expression. results suggest that GSK3β plays a negative regulator for ADD1/SREBP1c. and of ADD1/SREBP1c de it has been demonstrated that an increase of ADD1/SREBP1c mRNA expression is important for gene expression M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (591) Google Scholar, I. Y. S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar), GSK3 inhibitors shows the mRNA level of ADD1/SREBP1c and protein level of nuclear and we that the of the ADD1/SREBP1c target gene expression with GSK3 inhibitors can at the level of post-translational such as the an inhibitor of de novo protein synthesis Google Scholar), in the absence or of LiCl or in and did not the of mRNA expression by LiCl or results suggest that of the ADD1/SREBP1c transcriptional activity not require protein synthesis, and that of ADD1/SREBP1c transcriptional activity by GSK3 is by post-translational of ADD1/SREBP1c. has been reported that LiCl glucose metabolism including an increase of glycogen synthesis and glucose and regulates several gene including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (16Lochhead P.A. Coghlan M. Rice S.Q. Sutherland C. Diabetes. 2001; 50: 937-946Crossref PubMed Scopus (209) Google Scholar). However, it has not been LiCl insulin-dependent gene expression. the LiCl is to for insulin in of several gene expression, we a in cells in the or absence of LiCl of insulin or LiCl stimulated the of by ADD1/SREBP1c and and of LiCl and insulin effects of LiCl and insulin mRNA expression adipocytes LiCl effects the expression level of ADD1/SREBP1c, insulin stimulated ADD1/SREBP1c expression these results that LiCl not insulin at for the activation of ADD1/SREBP1c, that LiCl and insulin might have effects to ADD1/SREBP1c activity. GSK3β ADD1/SREBP1c in and in inhibition of GSK3 by LiCl or the transcriptional activity of ADD1/SREBP1c de novo protein synthesis, we to GSK3 is to phosphorylate ADD1/SREBP1c GSK3 has GSK3α and in their and have J.R. EMBO J. PubMed Scopus Google Scholar, S.E. K. E. J.R. Biophys. PubMed Scopus Google Scholar). Because GSK3β is in adipocytes and we used GSK3β in the the that ADD1/SREBP1c is a of we GSK3β proteins E. or in vitro kinase with GSK3β proteins E. ADD1/SREBP1c phosphorylated as as GSK3β implying that ADD1/SREBP1c is a of at in vitro the results GSK3β proteins cells Furthermore, to the of GSK3β we the kinase activity of GSK3β is inhibited by LiCl or of LiCl a of kinase activity of of the kinase activity of GSK3β in vitro to GSK3β phosphorylates ADD1/SREBP1c in GSK3β by IP, and kinase with ADD1/SREBP1c as in ADD1/SREBP1c phosphorylated by GSK3β in and implying that ADD1/SREBP1c is a for In addition, we in in the absence or of LiCl or Consistent with the in vitro kinase the phosphorylation level of ADD1/SREBP1c protein by LiCl and it has been reported that insulin might serine phosphorylation of ADD1/SREBP1c (22Kim J.B. Sarraf P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Investig. 1998; 101: 1-9Crossref PubMed Scopus (612) Google Scholar). also that phosphorylation of ADD1/SREBP1c by insulin and insulin implying that GSK3 might phosphorylate ADD1/SREBP1c in resting Thus, it that GSK3 and other would be involved in the phosphorylation of ADD1/SREBP1c to insulin-dependent gene expression. ADD1/SREBP1c is a of key transcription factors that regulate genes involved in and fatty acid metabolism. of the SREBP family have been in several and ADD1/SREBP1c by a is a gene with to Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; PubMed Scopus Google Scholar). the SREBP the level of ADD1/SREBP1c is controlled by insulin and which with lipid and glucose metabolism M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (591) Google Scholar, I. Y. S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). a insulin the transcriptional activity of ADD1/SREBP1c at the level of its transcription and post-translational modifications (22Kim J.B. Sarraf P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Investig. 1998; 101: 1-9Crossref PubMed Scopus (612) Google Scholar, M. Ogawa W. Teshigawara K. Inoue H. Miyake K. Sakaue H. Kasuga M. Diabetes. 2002; 51: 1672-1680Crossref PubMed Scopus (112) Google Scholar, 28Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (234) Google Scholar, J. Muller-Wieland D. Koponen A. Njamen D. Kremer L. Roth G. Munck M. Knebel B. Krone W. Biochem. Biophys. Res. Commun. 1998; 249: 375-379Crossref PubMed Scopus (72) Google Scholar, I. Y. S. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, M. R. Goldstein J.L. Brown M.S. I. 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Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of recent that GSK3 is involved in insulin which is as the tissues to insulin and is with and of GSK3 insulin not by an increase of glucose in also by inhibition of genes such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in (16Lochhead P.A. Coghlan M. Rice S.Q. Sutherland C. Diabetes. 2001; 50: 937-946Crossref PubMed Scopus (209) Google Scholar, K. W. P. E. L. C. Diabetes. 2002; 51: PubMed Scopus Google Scholar, J.M. D. I. T. M.E. Diabetes. 2003; PubMed Scopus Google Scholar). Furthermore, GSK3 inhibitors insulin-dependent activation of glucose and in in vitro and in J.M. D. I. T. M.E. Diabetes. 2003; PubMed Scopus Google Scholar). GSK3 also phosphorylates serine/threonine of insulin receptor which to of insulin signaling Garofalo R.S. 2002; PubMed Scopus Google Scholar, H. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). In this study, we a role of GSK3 in the of ADD1/SREBP1c transcriptional activity. of GSK3 inhibitors enhanced the transcriptional activity of ADD1/SREBP1c and stimulated the expression of its target genes such as FAS, SCD1, and ACC1, which to be key for fatty acid metabolism as as insulin-sensitive genes, in adipocytes and we that the of ADD1/SREBP1c target gene expression by GSK3 inhibitors to by insulin that stimulation of ADD1/SREBP1c by insulin to be at by GSK3 also that insulin the effects of the GSK3 inhibitor the transcriptional activity of ADD1/SREBP1c. In to the role of GSK3 in insulin these results might a to expression of several lipogenic genes is in tissues of H. M.E. F. A.D. Diabetes. 2003; PubMed Scopus Google Scholar). with the of GSK3 and its activity in tissues or of and H. S.A. M.M. Diabetes. 1999; PubMed Scopus (226) Google Scholar, S.E. S. P. L. Diabetes. 2000; PubMed Scopus Google Scholar), that an increase of GSK3 might be with metabolism by insulin these it that transcriptional activity of ADD1/SREBP1c might be by GSK3 leading to the expression of lipogenic genes in tissues of and Roth et al. G. Kotzka J. Kremer L. S. C. Krone W. Muller-Wieland D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated that MAP kinase phosphorylates at serine in vitro and of this serine residue to of to insulin and platelet-derived growth However, the for ADD1/SREBP1c not and inhibitors of the MAP kinase pathway did not ADD1/SREBP1c target gene expression D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (234) Google Scholar, D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, P.B. Roth R.A. M. A. Biochem. J. 2000; PubMed Google Scholar). we suggest that ADD1/SREBP1c can be phosphorylated by GSK3, which is a kinase of phosphatidylinositol 3-kinase in the insulin signaling pathway. in vitro and in we that GSK3 is to phosphorylate ADD1/SREBP1c and the level of ADD1/SREBP1c phosphorylation by the GSK3 inhibitor that GSK3 might negative phosphorylation of ADD1/SREBP1c in resting Additionally, we that GSK3 with the of ADD1/SREBP1c in the not Therefore, it is that inactivation of GSK3 might be an important that stimulates the transcriptional activity of ADD1/SREBP1c. the phosphorylation of ADD1/SREBP1c by we acid a we that ADD1/SREBP1c phosphorylated at serine Furthermore, by ADD1/SREBP1c we that the N-terminal end of ADD1/SREBP1c phosphorylated by GSK3β we at target of GSK3β in the N-terminal of the ADD1/SREBP1c the of phosphorylation has not been the phosphorylation of ADD1/SREBP1c by GSK3β and the roles of in the transcriptional activity of ADD1/SREBP1c. these data suggest that GSK3 plays a role in the activity of ADD1/SREBP1c and that of ADD1/SREBP1c target gene expression might be with the of Consistent with this inhibitors of GSK3, LiCl and ADD1/SREBP1c target gene expression by its transcriptional activity. Therefore, GSK3 to negatively regulate the transcriptional activity of ADD1/SREBP1c by ADD1/SREBP1c and this is GSK3 is in the of the insulin signaling pathway. and for the with