Title: 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. Unlike other kinases, GSK3 is constitutively active in resting cells and is inhibited by several growth factors or hormones, such as Wnt ligands, insulin, epidermal growth factor, and platelet-derived growth factor (1Shaw M. Cohen P. FEBS Lett. 1999; 461: 120-124Crossref PubMed Scopus (82) Google Scholar, 2Saito Y. Vandenheede J.R. Cohen P. Biochem. J. 1994; 303: 27-31Crossref PubMed Scopus (127) Google Scholar, 3Tsakiridis T. Tsiani E. Lekas P. Bergman A. Cherepanov V. Whiteside C. Downey G.P. Biochem. Biophys. Res. Commun. 2001; 288: 205-211Crossref PubMed Scopus (30) Google Scholar, 4Li L. Yuan H. Weaver C.D. Mao J. Farr 3rd, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (358) Google Scholar, 5Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (757) Google Scholar). In the canonical Wnt pathway, interaction between Wnt and Frizzled receptor inhibits GSK3, which results in dephosphorylation of β-catenin leading to the nuclear accumulation of β-catenin and activation of β-catenin/T cell factor target genes (4Li L. Yuan H. Weaver C.D. Mao J. Farr 3rd, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (358) Google Scholar, 6Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 7Itoh K. Antipova A. Ratcliffe M.J. Sokol S. Mol. Cell. Biol. 2000; 20: 2228-2238Crossref PubMed Scopus (84) Google Scholar, 8van Noort M. Meeldijk J. van der Zee R. Destree O. Clevers H. J. Biol. Chem. 2002; 277: 17901-17905Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 9Cliffe A. Hamada F. Bienz M. Curr. Biol. 2003; 13: 960-966Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Also, GSK3 plays an important role in the insulin signaling pathway and it phosphorylates and inhibits glycogen synthase in the absence of insulin (10Parker P.J. Caudwell F.B. Cohen P. Eur. J. Biochem. 1983; 130: 227-234Crossref PubMed Scopus (208) Google Scholar). In insulin-sensitive tissues, insulin-derived GSK3 inactivation is mediated by phosphatidylinositol 3-kinase and protein kinase B/Akt (PKB/Akt), which phosphorylate at the N-terminal serine residue of GSK3 (serine 21 and serine 9 in GSK3α and -3β, respectively) (5Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (757) Google Scholar, 11Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4374) Google Scholar, 12Sutherland C. Cohen P. FEBS Lett. 1994; 338: 37-42Crossref PubMed Scopus (192) Google Scholar). Thus, recent reports suggest a potential role for GSK3 as a negative regulator of insulin signaling (13Orena S.J. Torchia A.J. Garofalo R.S. J. Biol. Chem. 2000; 275: 15765-15772Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Consistent with this notion, one of the GSK3 inhibitors, lithium chloride (LiCl), shows insulin-like effects including increase of glucose uptake, activation of glycogen synthase, and stimulation of glycogen synthesis in various tissues (13Orena S.J. Torchia A.J. Garofalo R.S. J. Biol. Chem. 2000; 275: 15765-15772Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Summers S.A. Kao A.W. Kohn A.D. Backus G.S. Roth R.A. Pessin J.E. Birnbaum M.J. J. Biol. Chem. 1999; 274: 17934-17940Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Tabata I. Schluter J. Gulve E.A. Holloszy J.O. Diabetes. 1994; 43: 903-907Crossref PubMed Scopus (62) Google Scholar). Additionally, inhibition of GSK3 alters gene expression profiles in insulin-responsive cell lines. For example, LiCl selectively reduces expressions of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase genes, whose expression is also inhibited by insulin (16Lochhead P.A. Coghlan M. Rice S.Q. Sutherland C. Diabetes. 2001; 50: 937-946Crossref PubMed Scopus (209) Google Scholar). Furthermore, it has been demonstrated that GSK3 can phosphorylate several transcription factors, including p53, HSF-1, and C/EBPα and regulate their transcriptional activities (17Turenne G.A. Price B.D. BMC Cell Biol. 2001; 2: 12Crossref PubMed Scopus (115) Google Scholar, 18Chu B. Soncin F. Price B.D. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 1996; 271: 30847-30857Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 19Ross S.E. Erickson R.L. Hemati N. MacDougald O.A. Mol. Cell. Biol. 1999; 19: 8433-8441Crossref PubMed Google Scholar). Adipocyte determination- and differentiation-dependent factor 1 (ADD1/SREBP1c) is a bHLH-ZIP transcription factor that is involved in the stimulation of many lipogenic genes (20Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar, 21Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (845) Google Scholar, 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), such as fatty acid synthase (FAS) (23Magana M.M. Osborne T.F. J. Biol. Chem. 1996; 271: 32689-32694Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), acetyl-CoA carboxylase (ACC) (24Magana M.M. Lin S.S. Dooley K.A. Osborne T.F. J. Lipid Res. 1997; 38: 1630-1638Abstract Full Text PDF PubMed Google Scholar), and steroyl-CoA desaturase (SCD) (25Bene H. Lasky D. Ntambi J.M. Biochem. Biophys. Res. Commun. 2001; 284: 1194-1198Crossref PubMed Scopus (99) Google Scholar). In insulin-sensitive tissues including fat, liver, and muscle, insulin stimulates the expression of ADD1/SREBP1c mRNA and its target genes via phosphatidylinositol 3-kinase and PKB/Akt (26Matsumoto M. Ogawa W. Teshigawara K. Inoue H. Miyake K. Sakaue H. Kasuga M. Diabetes. 2002; 51: 1672-1680Crossref PubMed Scopus (112) Google Scholar, 27Guillet-Deniau I. Mieulet V. Le Lay S. Achouri Y. Carre D. Girard J. Foufelle F. Ferre P. Diabetes. 2002; 51: 1722-1728Crossref PubMed Scopus (101) 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, 29Fleischmann M. Iynedjian P.B. Biochem. J. 2000; 349: 13-17Crossref PubMed Scopus (170) Google Scholar). Therefore, it has been suggested that ADD1/SREBP1c plays a key role in orchestrating insulin-dependent lipid and glucose metabolism (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, 30Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (591) Google Scholar). Furthermore, several groups have investigated the post-translational modifications of ADD1/SREBP1c that affect its transcriptional activity. Brown and Goldstein (31Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2996) Google Scholar, 32Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1105) Google Scholar, 33Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (420) Google Scholar, 34Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. 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 effect of insulin on the expression of ADD1/SREBP1c target genes in cultured hepatocytes (28Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (234) Google Scholar, 36Wang D. Sul H.S. J. Biol. Chem. 1998; 273: 25420-25426Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 37Iynedjian P.B. Roth R.A. Fleischmann M. Gjinovci A. Biochem. J. 2000; 351: 621-627Crossref PubMed Google Scholar), implying that the MAP kinase pathway might not be a major player for ADD1/SREBP1c target gene expression by insulin treatment. Given the importance of GSK3 as a negative regulator of insulin signaling and ADD1/SREBP1c as a mediator of insulin-responsive gene expression, it would be important to understand the molecular mechanism between inhibition of GSK3 and increase of ADD1/SREBP1c transcriptional activity by insulin. To address this question, we examined the roles of GSK3 on the regulation of transcriptional activity of ADD1/SREBP1c. Here, we demonstrate that GSK3 inhibitors enhance the transcriptional activity of ADD1/SREBP1c and promote the expression of ADD1/SREBP1c target genes without de novo protein synthesis. Additionally, we reveal a phosphorylation event of ADD1/SREBP1c by GSK3. Taken together, these results propose a novel regulatory role of GSK3 in the insulin-dependent activation of ADD1/SREBP1c in adipocytes and hepatocytes. Reagents and Antibodies—LiCl and cycloheximide were obtained from Sigma and SB-216763 was purchased from Tocris. GSK3 antibody was purchased from BD Sciences. Polyclonal antibody against ADD1/SREBP1c was previously described (21Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (845) Google Scholar). Mammalian expression vector for GSK3β was thankfully provided from Dr. Junichi Sadoshima (New Jersey Medical School) (38Morisco C. Zebrowski D. Condorelli G. Tsichlis P. Vatner S.F. Sadoshima J. J. Biol. Chem. 2000; 275: 14466-14475Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Cell Culture—Murine 3T3-L1 preadipocytes were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum (JBI, Daegu, Korea). At confluence, differentiation of 3T3-L1 cells was induced with DMEM containing 10% fetal bovine serum, 3-isobutyl-1-methylxanthine (500 μm), dexamethasone (1 μm), and insulin (5 μg/ml) for 48 h. Culture medium was changed every other day with DMEM containing 10% fetal bovine serum and 5 μg/ml insulin. HEK293 and Rat1-IR cells were maintained in DMEM supplemented with 10% bovine calf serum, and cultured at 37 °C in a 10% CO2 incubator. FAO cells were maintained in DMEM supplemented 10% fetal bovine serum, and HepG2 cells were maintained in α-minimal essential medium (Invitrogen) supplemented 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. Transient Transfections and Luciferase Assays—HEK293 cells and HepG2 hepatocytes were transfected with several DNA constructs 1 day before confluence by the calcium phosphate method described previously (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, 39Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (296) Google Scholar). The mammalian expression vector for ADD1/SREBP1c contains 1–403 amino acids, which was engineered by in-frame fusion with the Myc His tag derived from pCDNA3.1 (Invitrogen). After being starved with low glucose DMEM containing 0.1% bovine serum albumin for 12 h, cells were treated with LiCl, NaCl, or insulin and incubated for 24 h. Total cell extracts were prepared with lysis buffer (25 mm Tris phosphate (pH 7.8), 2 mm dithiothreitol, 2 mm 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, and 1% Triton X-100), and the activities of β-galactosidase and luciferase were measured according to the manufacturer's instructions (Promega). Rat1-IR cells were transfected with the same method as described earlier (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, 39Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (296) Google Scholar). Northern Blot Analysis—Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Twenty-five μg of each RNA were denatured in formamide and formaldehyde buffer, and separated by electrophoresis on formaldehyde-containing agarose gels and transferred to nylon membranes (Schleicher and Schuell). After cross-linking, membranes were hybridized and washed as described by the manufacturer's instructions. DNA probes were labeled by random priming using the Klenow fragment of DNA polymerase I (MBI Fermentas) and [α-32P]dCTP (Amersham Biosciences). The cDNAs used as probes were: FAS, ACC1, SCD1, ADD1/SREBP1c, and 36B4. Semiquantitative Reverse Transcriptase-PCR—SuperScript First Strand Synthesis System for the reverse transcriptase-PCR kit (Invitrogen) was used for cDNA synthesis with 1 μg of total RNA. cDNAs for ADD1/SREBP1c and glyceraldehyde-3-phosphate dehydrogenase were amplified for 30 and 27 cycles, respectively, which are not saturating stages. Reverse transcriptase-PCR products were separated by 0.7% agarose gel electrophoresis and visualized by UVP BioImaging Systems (UVP). Primers used in this study are as followings: ADD1/SREBP1c up, 5′-ATCGGCGCGGAAGCTGTCG-3′, ADD1/SREBP1c down, 5′-ACTGTCTTGGTTGTTGATGAG-3′; glyceraldehyde-3-phosphate dehydrogenase up, 5′-TGCACCACCAACTGCTTAG-3′; glyceraldehyde-3-phosphate dehydrogenase down, 5′-GGATGCAGGGATGATGTTC-3′. Protein Kinase Assay—Recombinant GSK3β proteins were expressed in Escherichia coli with an N-terminal GST tag from the pGEX-4T-1 vector and purified with glutathionine-Sepharose according to the manufacturer's protocols (Amersham Biosciences). Insect cell-expressed GSK3β was kindly provided by Crystalgenomics (Daejun, Korea). Protein kinase reaction samples were incubated at 30 °C in a total volume of 20 μl containing 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 1 mm EGTA, 1mm EDTA, 2 mm dithiothreitol, 100 μm cold ATP, 0.33 μm [γ-32P]ATP (Amersham Biosciences) and each substrate, and stopped by addition of 5% phosphoric acid for peptide substrate or SDS sampling buffer for recombinant ADD1/SREBP1c proteins. Substrates were 100 μm phospho-eIF2B peptide (Peptron, Daejun, Korea) or His-tagged recombinant ADD1/SREBP1c proteins purified from E. coli. The amino acid sequence of phospho-eIF2B was RRAAEELDSRAGpSPQL (pS denoted phosphoserine) and was characterized previously (40Welsh G.I. Patel J.C. Proud C.G. Anal. Biochem. 1997; 244: 16-21Crossref PubMed Scopus (56) Google Scholar). After incubation, phosphopeptides were spotted on P-81 chromatography paper (Whatman), and washed twice with 0.5% phosphoric acid. After drying, counts/min were measured. Phosphorylated ADD1/SREBP1c proteins were loaded onto SDS-PAGE and visualized with autoradiography. Immunoprecipitation and Assay of Protein Kinases (IP Kinase Assay)—Equal amounts of total cell extract were incubated with GSKβ antibody (Transduction Laboratories). The immunoprecipitates collected with protein A-Sepharose (Sigma) were pelleted and washed twice with 1 ml of NETN buffer (20 mm Tris-HCl (pH 8.0), 1 mm EDTA, 0.5% Nonidet P-40, and 100 mm NaCl). Activity of immunoprecipitated GSK3 complexes were determined by counting counts/min or autoradiography as mentioned previously. Orthophosphate Labeling—HEK293 cells transfected with ADD1/SREBP1c expression vector were starved in low glucose DMEM with 0.1% bovine serum albumin for 24 h. After changing the culture medium to phosphate-free DMEM (Invitrogen), [32P]orthophosphate (PerkinElmer Life Sciences, Inc.) was added and cells were incubated for 2 h. After incubation, LiCl (10 mm, 1 h), SB-216763 (10 μm, 1 h), or insulin (100 nm, 30 min) were treated and total cell lysates were extracted with NETN buffer. Radiolabeled ADD1/SREBP1c proteins were obtained by immunoprecipitation with anti-ADD1/SREBP1c antibody. The precipitates were separated by SDS-PAGE (10% polyacrylamide gel) and subjected to autoradiography. Lithium Chloride Stimulates the Transcriptional Activity of ADD1/SREBP1c and Its Target Gene Expression—To investigate whether the transcriptional activity of ADD1/SREBP1c is regulated by GSK3, LiCl, an inhibitor of GSK3, was used in luciferase reporter assays. Previous studies revealed that LiCl inhibits GSK3 activity with an IC50 of 2.5 mm and a 90% loss of GSK3 activity is observed with 32 mm LiCl treatment (41Ryves W.J. Harwood A.J. Biochem. Biophys. Res. Commun. 2001; 280: 720-725Crossref PubMed Scopus (448) Google Scholar). FAS is one of the best characterized target genes of ADD1/SREBP1c and its promoter region contains the binding sites 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, 42Amemiya-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 (212) Google Scholar, 43Latasa M.J. Moon Y.S. Kim K.H. Sul H.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10619-10624Crossref PubMed Scopus (144) Google Scholar). As shown in Fig. 1A, LiCl stimulated the FAS promoter activity in the presence of ADD1/SREBP1c in a dose-dependent manner. However, it had no influence on the basal promoter activity in the absence of ADD1/SREBP1c in HEK293 cells. When 20 mm sodium chloride (NaCl) was treated, we did not observe any changes in promoter activity, suggesting that transcriptional activity of ADD1/SREBP1c is specifically modulated by LiCl. Similar results were obtained in Rat1-IR cells that express insulin receptor (Fig. 1B). Next, to investigate the alteration of target gene expression of ADD1/SREBP1c by GSK3, we performed a Northern blot analysis using insulin-responsive cells, such as differentiated 3T3-L1 adipocytes and FAO hepatocytes in the absence or presence of GSK3 inhibitor. Consistent with the results from reporter assays, LiCl treatment remarkably increased the mRNA expression of ADD1/SREBP1c target genes, such as FAS, ACC1, and SCD1 (Fig. 2, A and B). However, LiCl had little effect on the expression level of nuclear SREBP1 protein (Fig. 2C). Together, these observations indicate that inhibition of GSK3 clearly stimulates transcriptional activity of ADD1/SREBP1c and enhances its target gene expression in adipocytes and hepatocytes. Inhibition of GSK3 by SB-216763 Stimulates ADD1/SREBP1c Target Gene Expression—Because LiCl has several targets other than GSK3, such as the phosphatidylinositol signaling pathway via inhibition of inositol monophosphatase (44Zhen X. Torres C. Friedman E. Psychopharmacology (Berl.). 2002; 162: 379-384Crossref PubMed Scopus (11) Google Scholar), we performed similar experiments using a selective GSK3 inhibitor, SB-216763 (45Coghlan M.P. Culbert A.A. Cross D.A. Corcoran S.L. Yates J.W. Pearce N.J. Rausch O.L. Murphy G.J. Carter P.S. Roxbee Cox L. Mills D. Brown M.J. Haigh D. Ward R.W. Smith D.G. Murray K.J. Reith A.D. Holder J.C. Chem. Biol. 2000; 7: 793-803Abstract Full Text Full Text PDF PubMed Scopus (793) Google Scholar, 46Aiston S. Coghlan M.P. Agius L. Eur. J. Biochem. 2003; 270: 2773-2781Crossref PubMed Scopus (57) Google Scholar). As shown in Fig. 3, A and B, SB-216763 evidently enhanced FAS mRNA expression in 3T3-L1 adipocytes and HepG2 cells in a dose-dependent manner, similar to LiCl. The nuclear form of the SREBP1 protein was not significantly changed by SB-216763 treatment (Fig. 3C). To clarify whether SB-216763 efficiently inhibits GSK3 kinase activity, IP kinase assays were conducted. After HepG2 cells were treated with or without GSK3 inhibitors, total cell lysates were immunoprecipitated with GSK3 antibody. Then, immune complexes were incubated with phospho-eIF2B peptide, one of the well known substrates of GSK3 (40Welsh G.I. Patel J.C. Proud C.G. Anal. Biochem. 1997; 244: 16-21Crossref PubMed Scopus (56) Google Scholar, 47Jefferson L.S. Fabian J.R. Kimball S.R. Int. J. Biochem. Cell Biol. 1999; 31: 191-200Crossref PubMed Scopus (89) Google Scholar), and GSK3 kinase activities were measured. The kinase activity of GSK3 was decreased (about 50% reduction) by LiCl or SB-216763 treatment at concentrations of 10 mm or 10 μm, respectively (Fig. 3D, lanes 3 and 4). These observations implicate that LiCl- or SB-216763-stimulated target gene expression of ADD1/SREBP1c is closely associated with the inhibition of GSK3 kinase activity. Overexpression of GSK3β Inhibits Transcriptional Activity of ADD1/SREBP1c—To confirm the effect of GSK3 on the transcriptional activity of ADD1/SREBP1c, we investigated the effects of GSK3β overexpression. Luciferase reporter assays revealed that overexpression of GSK3β decreased the transcriptional activity of ADD1/SREBP1c in a dose-dependent fashion (Fig. 4A). Furthermore, we examined the effect of ADD1/SREBP1c target gene expression in the absence or presence of GSK3β overexpression. As shown in Fig. 4B, ectopic expression of GSK3β in HepG2 cells concomitantly inhibited FAS mRNA levels, whereas SB-216763 treatment reversed GSK3β-mediated down-regulation of FAS mRNA expression. These results suggest that GSK3β plays a negative regulator for ADD1/SREBP1c. LiCl- and SB-216763-induced Target Gene Expression of ADD1/SREBP1c Does Not Require de Novo Protein Synthesis— Although it has been demonstrated that an increase of ADD1/SREBP1c mRNA expression is important for insulin-stimulated gene expression (30Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (591) Google Scholar, 48Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (626) Google Scholar), GSK3 inhibitors shows little effect on the mRNA level of ADD1/SREBP1c and protein level of nuclear SREBP1 (Figs. 2 and 3). Thus we hypothesized that the changes of the ADD1/SREBP1c target gene expression with GSK3 inhibitors can occur at the level of post-translational modification such as phosphorylation. To test the above idea, cycloheximide, an inhibitor of de novo protein synthesis (49Chesnokov V.N. Mertvetsov N.P. Biokhimiya. 1990; 55: 1276-1278PubMed Google Scholar), was treated in the absence or presence of LiCl or SB-216763. As shown in Fig. 5, A and B, cyclohexamide did not alter the induction of FAS mRNA expression by LiCl or SB-216763 treatment. These results suggest that GSK3-mediated regulation of the ADD1/SREBP1c transcriptional activity does not require additional protein synthesis, and that modulation of ADD1/SREBP1c transcriptional activity by GSK3 is presumably accomplished by post-translational modification of ADD1/SREBP1c. Lithium Chloride Does Not Completely Mimic Insulin-stimulated Gene Expression—It has been reported that LiCl mimics insulin-stimulated glucose metabolism including an increase of glycogen synthesis and glucose uptake and regulates several hepatic gluconeogenic gene expressions, 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 clearly understood whether LiCl sufficiently mediates insulin-dependent gene expression. To clarify the idea whether LiCl is able to substitute for insulin action in regulation of several gene expression, we performed a luciferase reporter assay in Rat1-IR cells in the presence or absence of LiCl and/or insulin. Treatment of insulin or LiCl stimulated the transactivation of FAS promoter by ADD1/SREBP1c (Fig. 6A, lanes 4 and 6), and simultaneous treatment of LiCl and insulin showed additive effects (Fig. 6A, lane 8). Likewise, cotreatment of LiCl and insulin increased FAS mRNA expression on 3T3-L1 adipocytes (Fig. 6B). But LiCl had minor effects on the expression level of ADD1/SREBP1c, whereas insulin dramatically stimulated ADD1/SREBP1c expression (Fig. 6C). Together, these results implicate that LiCl does not completely mimic insulin effects, at least, for the activation of ADD1/SREBP1c, suggesting that LiCl and insulin might have cooperative effects to stimulate ADD1/SREBP1c activity. GSK3β Phosphorylates ADD1/SREBP1c in Vitro and in Vivo—Because inhibition of GSK3 by LiCl or SB-216763 increased the transcriptional activity of ADD1/SREBP1c without de novo protein synthesis, we decided to examine whether GSK3 is directly able to phosphorylate ADD1/SREBP1c protein. GSK3 has two isoforms, GSK3α and -β share 98% homology in their catalytic domain and have similar substrate specificity (50Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1151) Google Scholar, 51Plyte S.E. Hughes K. Nikolakaki E. Pulverer B.J. Woodgett J.R. Biochim. Biophys. Acta. 1992; 1114: 147-162Crossref PubMed Scopus (334) Google Scholar). Because GSK3β is highly expressed in adipocytes and hepatocytes, we used GSK3β in the following experiments. To test the possibility that ADD1/SREBP1c is a novel substrate of GSK3β, we purified recombinant GSK3β proteins from E. coli or insect cells. When in vitro kinase assays were conducted with recombinant GSK3β proteins from E. coli, ADD1/SREBP1c was strongly phosphorylated as well as GSK3β itself, implying that ADD1/SREBP1c is a putative substrate of GSK3β, at least, in vitro (Fig. 7A). We obtained the same results using GSK3β proteins purified from insect cells (Fig. 7B). Furthermore, to validate the effect of purified recombinant GSK3β protein, we examined whether the kinase activity of recombinant GSK3β is directly inhibited by LiCl or SB-216763. Treatment of LiCl caused a significant reduction of kinase activity of GSK3β, whereas treatment of SB-216763 completely abolished the kinase activity of GSK3β in vitro (Fig. 7C). Next, to test whether GSK3β phosphorylates ADD1/SREBP1c in vivo, endogeneous GSK3β was obtained by IP, and IP kinase assays were carried out with ADD1/SREBP1c as substrate. As shown in Fig. 7D, ADD1/SREBP1c was clearly phosphorylated by endogenous GSK3β in both HepG2 hepatocytes and 3T3-L1 adipocytes, implying that ADD1/SREBP1c is a novel substrate for GSK3. In addition, we performed in vivo orthophosphate labeling in the absence or presence of LiCl or insulin. Consistent with the in vitro kinase assay (Fig. 7), the basal phosphorylation level of ADD1/SREBP1c protein was decreased by LiCl and SB-216763 treatment (Fig. 8A). Previously, it has been reported that insulin might induce 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). We also observed that total phosphorylation of ADD1/SREBP1c was moderately increased by insulin (Fig. 8B, lane 3), and insulin treatment partially rescued LiCl-mediated reduction (Fig. 8B, lane 5), implying that GSK3 might phosphorylate ADD1/SREBP1c in resting status. Thus, it appears that GSK3 and other kinase(s) would be involved in the regulatory phosphorylation of ADD1/SREBP1c to mediate insulin-dependent gene expression. ADD1/SREBP1c is a member of key transcription factors that regulate genes involved in cholesterol and fatty acid metabolism. Three isoforms of the SREBP family have been identified in several mammalian species. SREBP-1a and ADD1/SREBP1c are generated by using alternative promoters from a single gene. SREBP-2 is derived from a different gene with 47% similarity to SREBP-1 (52Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2002; 109: 1125-1131Crossref PubMed Scopus (3776) Google Scholar). Among the three SREBP isoforms, the transcript level of ADD1/SREBP1c is sensitively controlled by insulin and nutritional status, which are associated with lipid and glucose metabolism (30Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (591) Google Scholar, 48Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (626) Google Scholar). As a major anabolic hormone, insulin enhances the transcriptional activity of ADD1/SREBP1c at the level of its own 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, 26Matsumoto 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, 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, 48Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (626) Google Scholar, 53Matsuda M. Korn B.S. Hammer R.E. Moon Y.A. Komuro R. Horton J.D. Goldstein J.L. Brown M.S. Shimomura I. Genes Dev. 2001; 15: 1206-1216Crossref PubMed Scopus (266) Google Scholar). With this regulatory mechanism, ADD1/SREBP1c induces expression of several lipogenic genes, which leads to fatty acid and triglyceride synthesis and regulates expression of several genes involved in glucose metabolism (28Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (234) Google Scholar, 30Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (591) Google Scholar, 54Stoeckman A.K. Towle H.C. J. Biol. Chem. 2002; 277: 27029-27035Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 55Foretz 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), suggesting that ADD1/SREBP1c plays a critical role in insulin-stimulated gene expression for whole body energy homeostasis. Indeed, ADD1/SREBP1c-specifc knockout mice reveal not only a severe decrease of hepatic and plasma triglyceride with reduced fat mass but also elevation of plasma glucose levels (56Liang G. Yang J. Horton J.D. Hammer R.E. Goldstein J.L. Brown M.S. J. Biol. Chem. 2002; 277: 9520-9528Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). Several lines of recent evidence indicate that GSK3 is involved in insulin resistance, which is defined as the status where peripheral tissues cannot respond to insulin and is closely associated with obesity and type II diabetes. Inhibition of GSK3 improves insulin resistance not only by an increase of glucose disposal rate in Zucker diabetic rats but also by inhibition of gluconeogenic genes such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in hepatocytes (16Lochhead P.A. Coghlan M. Rice S.Q. Sutherland C. Diabetes. 2001; 50: 937-946Crossref PubMed Scopus (209) Google Scholar, 57Cline G.W. Johnson K. Regittnig W. Perret P. Tozzo E. Xiao L. Damico C. Shulman G.I. Diabetes. 2002; 51: 2903-2910Crossref PubMed Scopus (206) Google Scholar, 58Ring D.B. Johnson K.W. Henriksen E.J. Nuss J.M. Goff D. Kinnick T.R. Ma S.T. Reeder J.W. Samuels I. Slabiak T. Wagman A.S. Hammond M.E. Harrison S.D. Diabetes. 2003; 52: 588-595Crossref PubMed Scopus (393) Google Scholar). Furthermore, selective GSK3 inhibitors potentiate insulin-dependent activation of glucose transport and utilization in muscle in vitro and in vivo (58Ring D.B. Johnson K.W. Henriksen E.J. Nuss J.M. Goff D. Kinnick T.R. Ma S.T. Reeder J.W. Samuels I. Slabiak T. Wagman A.S. Hammond M.E. Harrison S.D. Diabetes. 2003; 52: 588-595Crossref PubMed Scopus (393) Google Scholar). GSK3 also directly phosphorylates serine/threonine residues of insulin receptor substrate-1, which leads to impairment of insulin signaling (59Greene M.W. Garofalo R.S. Biochemistry. 2002; 41: 7082-7091Crossref PubMed Scopus (86) Google Scholar, 60Eldar-Finkelman H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9660-9664Crossref PubMed Scopus (283) Google Scholar). In this study, we identified a novel role of GSK3 in the regulation of ADD1/SREBP1c transcriptional activity. Treatment of GSK3 inhibitors remarkably enhanced the transcriptional activity of ADD1/SREBP1c and stimulated the expression of its target genes such as FAS, SCD1, and ACC1, which are known to be key players for fatty acid metabolism as well as insulin-sensitive genes, in adipocytes and hepatocytes (Figs. 1, 2, 3). Accordingly, we observed that the levels of ADD1/SREBP1c target gene expression by GSK3 inhibitors were similar to those by insulin (Fig. 6), implicating that stimulation of ADD1/SREBP1c by insulin appears to be mediated, at least partly, by GSK3 inhibition. We also observed that insulin augmented the effects of the GSK3 inhibitor on the transcriptional activity of ADD1/SREBP1c. In addition to the role of GSK3 in insulin signaling, these results might provide a clue to explain why expression of several lipogenic genes is reduced in fat tissues of insulin-resistant animals (61Lan H. Rabaglia M.E. Stoehr J.P. Nadler S.T. Schueler K.L. Zou F. Yandell B.S. Attie A.D. Diabetes. 2003; 52: 688-700Crossref PubMed Scopus (111) Google Scholar). Compared with lean mice, the levels of GSK3 and its enzyme activity are elevated in fat tissues or skeletal muscles of obese and diabetic mice models (62Eldar-Finkelman H. Schreyer S.A. Shinohara M.M. LeBoeuf R.C. Krebs E.G. Diabetes. 1999; 48: 1662-1666Crossref PubMed Scopus (226) Google Scholar, 63Nikoulina S.E. Ciaraldi T.P. Mudaliar S. Mohideen P. Carter L. Henry R.R. Diabetes. 2000; 49: 263-271Crossref PubMed Scopus (327) Google Scholar), proposing that an increase of GSK3 might be associated with abnormal energy metabolism by disrupting insulin action. Under these circumstances, it appears that transcriptional activity of ADD1/SREBP1c might be repressed by increased GSK3 activity, leading to the reduced expression of lipogenic genes in fat tissues of obese and diabetic mice. Previously, Roth et al. (64Roth G. Kotzka J. Kremer L. Lehr S. Lohaus C. Meyer H.E. Krone W. Muller-Wieland D. J. Biol. Chem. 2000; 275: 33302-33307Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) demonstrated that MAP kinase phosphorylates SREBP-1a at serine 117 in vitro and mutation of this serine residue to alanine abolishes responsiveness of SREBP-1a to insulin and platelet-derived growth factor. However, the same for ADD1/SREBP1c was not confirmed and inhibitors of the MAP kinase pathway did not inhibit insulin-stimulated ADD1/SREBP1c target gene expression (28Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Biochem. J. 2000; 350: 389-393Crossref PubMed Scopus (234) Google Scholar, 36Wang D. Sul H.S. J. Biol. Chem. 1998; 273: 25420-25426Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 37Iynedjian P.B. Roth R.A. Fleischmann M. Gjinovci A. Biochem. J. 2000; 351: 621-627Crossref PubMed Google Scholar). Rather, we suggest that ADD1/SREBP1c can be phosphorylated by GSK3, which is a downstream kinase of phosphatidylinositol 3-kinase in the insulin signaling pathway. With in vitro and in vivo assays, we showed that GSK3 is able to phosphorylate ADD1/SREBP1c (Fig. 7), and the basal level of ADD1/SREBP1c phosphorylation was drastically decreased by the GSK3 inhibitor (Fig. 8), clearly indicating that GSK3 might induce negative phosphorylation of ADD1/SREBP1c in resting cells. Additionally, we observed that GSK3 was colocalized with the mature form of ADD1/SREBP1c in the nucleus (data not shown). Therefore, it is likely that inactivation of GSK3 might be an important regulatory step that stimulates the transcriptional activity of ADD1/SREBP1c. To identify the phosphorylation residue(s) of ADD1/SREBP1c by GSK3β, we performed phosphoamino acid analysis. As a result, we observed that ADD1/SREBP1c was predominantly phosphorylated at serine residues (Supplemental Materials Fig. 1). Furthermore, by using different truncated ADD1/SREBP1c recombinant proteins, we observed that the N-terminal end of ADD1/SREBP1c was phosphorylated by GSK3β (Supplemental Materials Fig. 2). Although we identified at least six putative target sites of GSK3β in the N-terminal portion of the ADD1/SREBP1c protein, the exact site(s) of phosphorylation has not been identified yet. We are currently investigating the phosphorylation site(s) of ADD1/SREBP1c by GSK3β and the functional roles of those sites in regulating the transcriptional activity of ADD1/SREBP1c. Taken together, these data suggest that GSK3 plays a role in regulating the activity of ADD1/SREBP1c and that insulin-mediated induction of ADD1/SREBP1c target gene expression might be associated with the regulation of GSK3. Consistent with this view, inhibitors of GSK3, both LiCl and SB-216763, stimulate ADD1/SREBP1c target gene expression by altering its transcriptional activity. Therefore, GSK3 appears to negatively regulate the transcriptional activity of ADD1/SREBP1c by phosphorylating ADD1/SREBP1c and this repression process is probably relieved when GSK3 is suppressed in the presence of the insulin signaling pathway. We thank Yun Sok Lee and Jun-Jae Chung for critically reading the manuscript. Download .pdf (.09 MB) Help with pdf files