AMP-activated Protein Kinase Activation Increases Phosphorylation of Glycogen Synthase Kinase 3β and Thereby Reduces cAMP-responsive Element Transcriptional Activity and Phosphoenolpyruvate Carboxykinase C Gene Expression in the Liver
AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3β (GSK3β) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3β within 15 min. Stimulation with AICAR or the GSK3β inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3β to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3β(S9G) but not wild-type GSK3β. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3β phosphorylation were very similar. Furthermore, AMPK-mediated GSK3β phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3β is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3β by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide. AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3β (GSK3β) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3β within 15 min. Stimulation with AICAR or the GSK3β inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3β to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3β(S9G) but not wild-type GSK3β. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3β phosphorylation were very similar. Furthermore, AMPK-mediated GSK3β phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3β is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3β by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide. Hepatic PEPCK-C 2The abbreviations used are: PEPCK-C, phosphoenolpyruvate carboxykinase C; GSK3β, glycogen synthase kinase 3β; AMPK, AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; CREB, CRE, cAMP-response element; CREB, CRE-binding protein; TORC2, transducer of regulated CREB activity 2; WT, wild type; GST, glutathione S-transferase; CBP, CREB-binding protein; UAS, upstream activating sequence; ACC, acetyl-CoA carboxylase; SIK, salt-inducible kinase. 2The abbreviations used are: PEPCK-C, phosphoenolpyruvate carboxykinase C; GSK3β, glycogen synthase kinase 3β; AMPK, AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleoside; CREB, CRE, cAMP-response element; CREB, CRE-binding protein; TORC2, transducer of regulated CREB activity 2; WT, wild type; GST, glutathione S-transferase; CBP, CREB-binding protein; UAS, upstream activating sequence; ACC, acetyl-CoA carboxylase; SIK, salt-inducible kinase. expression plays a critical role in the maintenance of glucose homeostasis, and activation of AMPK in the livers of fasted mice has been shown to reduce glucose production (1Vincent M.F. Erion M.D. Gruber H.E. Van den Berghe G. Diabetologia. 1996; 39: 1148-1155Crossref PubMed Scopus (90) Google Scholar). The oral biguanide antidiabetic agent metformin and several factors including leptin and adiponectin reportedly activate AMPK, improve insulin sensitivity, and reduce gluconeogenesis in patients with type 2 diabetes mellitus (2Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 3Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar, 4Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4289) Google Scholar, 5Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3369) Google Scholar, 6Lochhead P.A. Salt I.P. Walker K.S. Hardie D.G. Sutherland C. Diabetes. 2000; 49: 896-903Crossref PubMed Scopus (335) Google Scholar, 7Cool B. Zinker B. Chiou W. Kifle L. Cao N. Perham M. Dickinson R. Adler A. Gagne G. Iyengar R. Zhao G. Marsh K. Kym P. Jung P. Camp H.S. Frevert E. Cell Metab. 2006; 3: 403-416Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 8Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1030) Google Scholar). It was also reported that stimulation with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) suppresses hepatic PEPCK-C expression and the transcriptional activity of the cAMP-responsive element (CRE), which is present in the PEPCK-C promoter, actions similar to those of insulin (9Bady I. Marty N. Dallaporta M. Emery M. Gyger J. Tarussio D. Foretz M. Thorens B. Diabetes. 2006; 55: 988-995Crossref PubMed Scopus (106) Google Scholar). AMPK achieves its downstream effects by immediate direct phosphorylation of enzyme substrates as well as long term effects on gene expression. For example, AMPK phosphorylates and inactivates acetyl-CoA carboxylase, resulting in suppression of the conversion of acetyl CoA to malonyl CoA (4Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4289) Google Scholar). This malonyl CoA reduction allows entry of fatty acids into mitochondria and their subsequent oxidation (10Winder W.W. Wilson H.A. Hardie D.G. Rasmussen B.B. Hutber C.A. Call G.B. Clayton R.D. Conley L.M. Yoon S. Zhou B. J. Appl. Physiol. 1997; 82: 219-225Crossref PubMed Scopus (61) Google Scholar, 11Munday M.R. Campbell D.G. Carling D. Hardie D.G. Eur. J. Biochem. 1988; 175: 331-338Crossref PubMed Scopus (213) Google Scholar). Recently, as a molecular mechanism underlying AMPK-mediated PEPCK-C gene suppression, the phosphorylation of TORC2 (transducer of regulated CREB activity 2), a co-activator of cAMP-responsive element binding (CREB) protein, by AMPK was reported (12Koo S.H. Flechner L. Qi L. Zhang X. Screaton R.A. Jeffries S. Hedrick S. Xu W. Boussouar F. Brindle P. Takemori H. Montminy M. Nature. 2005; 437: 1109-1111Crossref PubMed Scopus (773) Google Scholar, 13Conkright M.D. Canettieri G. Screaton R. Guzman E. Miraglia L. Hogenesch J.B. Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 14Iourgenko V. Zhang W. Mickanin C. Daly I. Jiang C. Hexham J.M. Orth A.P. Miraglia L. Meltzer J. Garza D. Chirn G.W. McWhinnie E. Cohen D. Skelton J. Terry R. Yu Y. Bodian D. Buxton F.P. Zhu J. Song C. Labow M.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12147-12152Crossref PubMed Scopus (308) Google Scholar, 15Shaw R.J. Lamia K.A. Vasquez D. Koo S.H. Bardeesy N. Depinho R.A. Montminy M. Cantley L.C. Science. 2005; 310: 1642-1646Crossref PubMed Scopus (1522) Google Scholar). TORC2 phosphorylated by AMPK or SIK binds to 14-3-3, and this complex is transported from inside the nucleus to the cytoplasm (16Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates III, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar, 17Bittinger M.A. McWhinnie E. Meltzer J. Iourgenko V. Latario B. Liu X. Chen C.H. Song C. Garza D. Labow M. Curr. Biol. 2004; 14: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Thus, by reducing the association with TORC2 and CREB in the nucleus, CRE transcriptional activity and PEPCK-C expression are suppressed. On the other hand, it has been revealed that CRE transcriptional activity is regulated not only by nuclear TORC2 but also by the phosphorylation of CREB. For example, forskolin stimulates cAMP production and enhances PEPCK-C gene expression by stimulating the protein kinase A-mediated phosphorylation of CREB at Ser-133 and by promoting subsequent recruitment of the coactivator CREB-binding protein (CBP) to the promoter (18Zhou X.Y. Shibusawa N. Naik K. Porras D. Temple K. Ou H. Kaihara K. Roe M.W. Brady M.J. Wondisford F.E. Nat. Med. 2004; 10: 633-637Crossref PubMed Scopus (114) Google Scholar, 19Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1755) Google Scholar). Furthermore, CREB has been regarded as one of the potential nuclear substrates of GSK3β, which were identified by in vitro phosphorylation and overexpression studies (20Fiol C.J. C.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 2002; PubMed Scopus Google Scholar, Chen J. E. S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, it has been that GSK3β phosphorylates CREB at to a on CREB activity, and that CREB is involved in the activation of PEPCK-C expression Chen J. E. S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. X. D. Sutherland C. Biochem. J. 2005; PubMed Scopus Google Scholar). kinase phosphorylates and inactivates GSK3β, suppression of CRE transcriptional activity is likely to be by GSK3β phosphorylation at The of CREB or TORC2 transcriptional activity and the gene expression of In this study we that AMPK activation GSK3β phosphorylation and markedly in the we to PEPCK-C gene suppression is by increased GSK3β we present evidence a critical role of GSK3β phosphorylation in the suppression of hepatic inhibitor Compound C and Akt inhibitor were from and were from Cell to were from and were from other were of was a or TORC2 in as T. M. H. K. M. Y. H. K. M. Y. Y. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of an or of AICAR the was by of the of the was in of and 2 The was at at The was and the protein was a protein assay Cell HepG2 were from the HepG2 were in and The were in a of at and by were from mice by the were in and and was and the were into or The was the as that that was of and were from and from and and from The PEPCK-C promoter to was into a HepG2 in a were with with an For the CREB or TORC2 were with binding CREB expression or and UAS, and an was used to a protein that the binding at the of a protein of The protein was transported into the nucleus by the nuclear in the binding The of the binding upstream from the promoter was from activity from was the of of was to the of and the was J. Takemori H. N. Katoh Y. Okamoto M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of were with or forskolin or AICAR for and to by the promoter of PEPCK-C, CRE, CREB, or TORC2 were as expression with the activity of the were and by and of of the wild-type GSK3β was to a to the was with of and the was The was into used to wild-type and GSK3β(S9G) were by of the expression the and the as H. Y. H. M. H. Y. M. T. M. Y. M. N. M. Y. H. T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). wild-type and of wild-type and GSK3β(S9G) were and as reported H. Y. H. M. H. Y. M. T. M. Y. M. N. M. Y. H. T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). as a and the gene was as reported H. Y. H. M. H. Y. M. T. M. Y. M. N. M. Y. H. T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). was as H. Y. H. M. H. Y. M. T. M. Y. M. N. M. Y. H. T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In HepG2 were in as shown Cell was by at for 15 at and the resulting was used for and For of protein were by and to in a of and The were with in with and with by with The were by with the HepG2 were with of CREB. were from HepG2 in as Cell were for at with and for with of protein The were with of in for and on AICAR Stimulation the of GSK3β and in was into fasted and the Ser-9 phosphorylation of hepatic GSK3β was at the reported AICAR stimulation increased phosphorylation of GSK3β phosphorylation was also markedly increased by AICAR whereas the of GSK3β protein expression was in the livers of fasted This within 15 of injecting AICAR, at and for 2 Thus, AICAR stimulation was revealed to not only but also GSK3β, markedly AMPK of PEPCK-C and in at the of gene expression P.A. M. Sutherland C. Diabetes. 2001; PubMed Scopus Google Scholar, K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.M. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Biochem. 1997; PubMed Scopus Google Scholar, C. P. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the gene downstream from the PEPCK-C gene promoter was markedly increased by forskolin in HepG2 cells, and this was suppressed by AICAR or insulin stimulation the for the PEPCK-C is reportedly that of CRE, we a assay a the CRE upstream from the gene markedly CRE transcriptional activity, whereas AICAR and the AMPK inhibitor Compound C the GSK3β inhibitor also very strongly inhibited the in CRE activity Furthermore, to that the of AICAR is by increased AMPK activity, the wild-type and of AMPK were gene The were by in the of AMPK overexpression increased whereas a suppressed it in the of AMPK was revealed to CRE transcriptional activity of of CREB and TORC2 in the and of CRE both AICAR and repressed activity we the to which of or is involved in the and suppression of the CRE transcriptional activity, a transactivation assay in HepG2 cells. the binding of the to CREB were with a the the of CREB and the of TORC2, the transactivation of CREB and TORC2 were from the and the of and This of the transactivation potential of the of its binding and shown in the transcriptional activity of CREB was inhibited by both AICAR and SB415286, whereas that of TORC2 was repressed by AICAR significantly and by These results that AMPK inhibit the of both CREB and TORC2, whereas the activity of only CREB. The of on the of phosphorylation of TORC2 also this On the other hand, TORC2 is to be phosphorylated and by a of the AMPK of phosphorylates TORC2 and its nuclear (16Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates III, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). was the in activity was In addition, TORC2 was strongly phosphorylated in Thus, as shown with the and of TORC2, CREB to be for the effects of and AICAR, whereas TORC2 to be for of the effects of AICAR stimulation and SIK of TORC2 and by AMPK and and reportedly TORC2 and phosphorylated TORC2 is transported from within the nucleus to the cytoplasm (12Koo S.H. Flechner L. Qi L. Zhang X. Screaton R.A. Jeffries S. Hedrick S. Xu W. Boussouar F. Brindle P. Takemori H. Montminy M. Nature. 2005; 437: 1109-1111Crossref PubMed Scopus (773) Google Scholar). shown in hepatic TORC2 was markedly stimulation with forskolin and 2 of and Stimulation of with AICAR in a in the of TORC2, phosphorylation and or 2 and of and and or 2 and of whereas stimulation with a GSK3β SB415286, not a in to the or of forskolin and of On the other hand, TORC2 was strongly phosphorylated in of with AICAR stimulation and of These results that whereas AICAR stimulation TORC2 is likely to In was phosphorylated by AICAR but not by of of GSK3β AICAR-induced of of PEPCK-C of GSK3β on Ser-9 its In AICAR-induced suppression of the PEPCK-C gene GSK3β we of the GSK3β(S9G) and of from for revealed of GSK3β(S9G) and with GSK3β of Furthermore, was by GSK3β whereas GSK3β(S9G) was not of HepG2 were with of in of HepG2 with an GSK3β a In HepG2 cells, forskolin enhances PEPCK-C transcriptional activity, and with AICAR blunted this of In wild-type cells, PEPCK-C transcriptional activity was increased as with that in and of to forskolin and AICAR were similar of in of cells, PEPCK-C transcriptional and were both significantly increased of as with that in wild-type cells. In addition, it be that AICAR-induced suppression of PEPCK-C transcriptional activity was in wild-type cells, but this was in the and of Thus, it was revealed that expression of GSK3β(S9G) PEPCK-C in HepG2 cells, which phosphorylation of Ser-9 of GSK3β is involved in the mechanism underlying AMPK or AICAR-induced PEPCK-C gene suppression. AMPK but CREB (Ser-129) in HepG2 of AMPK a on GSK3β, HepG2 were with the AMPK activator AICAR, and of CREB and GSK3β were with the were with 2 AICAR for phosphorylation of GSK3β was significantly whereas CREB phosphorylation was In the were with the AMPK inhibitor Compound phosphorylation of GSK3β at Ser-9 was and that of CREB at was slightly increased Thus, AMPK activation phosphorylation of GSK3β a mechanism that was by the AMPK inhibitor Compound that an to this to of the time-courses of AICAR-induced phosphorylation of GSK3β at CREB phosphorylation at phosphorylation at and Akt phosphorylation at The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3β phosphorylation were very similar C and of These actions were within of and were for min. of Ser-133 was by AICAR stimulation in HepG2 not The GSK3β AMPK to CREB We also in only AICAR activation of AMPK, was phosphorylation of GSK3β of CREB. in the of AICAR induced phosphorylation of and that of These results that AICAR phosphorylation of GSK3β in the of protein kinase activation and that AICAR the of GSK3β in with its effects on for GSK3β and Akt are for AMPK-mediated GSK3β HepG2 were with AICAR with or with Akt inhibitor and AICAR-induced GSK3β were inhibited by with Akt inhibitor Akt phosphorylation on was increased in a Thus, GSK3β phosphorylation is likely to be by the Akt pathway. AICAR kinase reportedly phosphorylates CREB at and phosphorylation of CREB its for a GSK3β has been to CREB activity in with Ser-133 cAMP the CREB transcriptional CREB-binding protein is a nuclear protein that binds to the protein kinase of CREB and activate N. Goodman R.H. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google we the was regulated AICAR increased the and in HepG2 as reported It was revealed that AICAR markedly decreased this This that the of CREB for was regulated by suppression of CREB phosphorylation in to AICAR Hepatic gluconeogenesis is regulated to glucose homeostasis, and several such as insulin and are involved in this PEPCK-C has been regarded as the enzyme the of hepatic but studies on PEPCK-C gene mice that PEPCK-C as an of hepatic including those of and glycogen P. M. R. C. M.A. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, P. M. P. M.A. Diabetes. 2003; PubMed Scopus Google Scholar, P. J. R.A. L. Metab. 2005; Scopus Google Scholar). The promoter of the PEPCK-C gene the CRE which was shown to an important role in PEPCK-C gene expression J.M. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Biochem. 1997; PubMed Scopus Google Scholar, M. N. F. Y. P. Kahn A. Thorens B. S. B. Diabetes. 2005; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, Liu Mol. Cell. Biol. 10: PubMed Scopus Google Scholar). and increased reportedly PEPCK-C gene expression by stimulating protein kinase A-mediated phosphorylation of CREB at Ser-133 P. G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. F. Hedrick S. R. A. D. G. Yoon C. P. B. Montminy M. Nature. 2001; PubMed Scopus Google Scholar, D. K. T. R. C. Montminy M.R. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar, P. J. Biochem. Cell Biol. PubMed Scopus Google Scholar). Recently, the coactivator of CREB TORC2 was reported to be important for the of CRE transcriptional activity and PEPCK-C gene expression (12Koo S.H. Flechner L. Qi L. Zhang X. Screaton R.A. Jeffries S. Hedrick S. Xu W. Boussouar F. Brindle P. Takemori H. Montminy M. Nature. 2005; 437: 1109-1111Crossref PubMed Scopus (773) Google Scholar). TORC2 is reportedly phosphorylated by AMPK and SIK, and phosphorylated TORC2 binds to protein and is from the nucleus to the cytoplasm (16Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates III, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). of nuclear TORC2 to the suppression of CRE transcriptional activity U. J. D. S. C. W. Biochem. J. 2007; PubMed Scopus Google Scholar). Thus, PEPCK-C gene suppression is likely to be by TORC2 in this study we mechanism to PEPCK-C gene suppression. This mechanism GSK3β phosphorylation and subsequent CREB We for the that AMPK activation GSK3β phosphorylation in not only such as HepG2 but also in the This phosphorylation in the was induced 15 of AICAR and for at 2 In addition, suppression of AMPK with the inhibitor Compound C GSK3β phosphorylation induced by the Akt inhibitor AICAR-induced GSK3β it that AMPK directly phosphorylates GSK3β. AICAR stimulation not activate not but Akt Thus, the molecular mechanism be identified from several be For example, AMPK activation may the activity of or the complex Science. 2005; PubMed Scopus Google Scholar, A. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). is that the of Akt or GSK3β is suppressed by AMPK a study also found Akt to be AICAR in Song L. Biochem. 2006; PubMed Scopus Google Scholar). of other be involved in the effects of AICAR it was reported that AICAR the Ser-9 phosphorylation of GSK3β induced by with a activator of protein kinase C the studies are to this of at an and Ser-9 in and it of of GSK3β such as reportedly not only hepatic glycogen but also glucose production with decreased of PEPCK-C in G.W. K. W. P. E. L. C. Diabetes. 2002; PubMed Scopus Google and mice H. J. 2006; PubMed Scopus Google Scholar). the molecular underlying the of PEPCK-C gene expression by GSK3β a previous study that GSK3β phosphorylates CREB at (20Fiol C.J. C.H. J. Biol. Chem. Full Text PDF PubMed Google we the that GSK3β phosphorylation to PEPCK-C gene suppression. AICAR stimulation induced GSK3β phosphorylation and CREB in HepG2 with very similar time-courses In addition, AMPK stimulation PEPCK-C promoter activity in HepG2 cells, and the GSK3β inhibitor SB-415286 and AICAR both strongly suppressed CRE Importantly, AICAR-induced suppression of PEPCK-C promoter activity was significantly by the of These strongly that inactivation of GSK3β is for PEPCK-C promoter and CRE activity We also and SB-415286 suppressed CREB only slightly those of TORC2 and Stimulation of with not the of TORC2 to the or of forskolin These results GSK3β inactivation to directly inhibit CREB. The of Ser-133 phosphorylation in stimulating CREB activity has been Cell. PubMed Scopus Google Scholar, M. U. 2007; 12: PubMed Scopus Google Scholar). of CREB at Ser-133 also a for phosphorylation by GSK3β at 2002; PubMed Scopus Google Scholar). of by strongly in 2002; PubMed Scopus Google Scholar). We the suppression of phosphorylation by AICAR stimulation to These results CREB and to the GSK3β may also a role in cAMP as stimulation of CREB activity on the phosphorylation of that in by this agent activate AMPK and Akt GSK3β. These results that actions to AMPK AICAR may be by the actions of this on Akt and GSK3β. AMPK and Akt in AMPK is in with decreased and AMPK and to promoting On the other hand, Akt that such as and Akt may with AMPK the to by Thus, the effects of AICAR on AMPK and Akt may the that been to their activating effects on In AMPK activation GSK3β phosphorylation in Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3β by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as and Thus, it is very likely that the suppression of PEPCK-C expression. We that TORC2 phosphorylation by AMPK is but the plays an role in PEPCK-C gene suppression. with