Dominant Negative Forms of Akt (Protein Kinase B) and Atypical Protein Kinase Cλ Do Not Prevent Insulin Inhibition of Phosphoenolpyruvate Carboxykinase Gene Transcription

Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Cλ (PKCλ), which blocked insulin-induced activation of endogenous PKCλ, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKCλ, and Rac may exist for mediating the effect of insulin. Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Cλ (PKCλ), which blocked insulin-induced activation of endogenous PKCλ, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKCλ, and Rac may exist for mediating the effect of insulin. The primary role of insulin is to control the plasma glucose concentration by stimulating glucose transport into muscle and adipose cells as well as by reducing glucose output from the liver (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). These actions of insulin are mediated by activation of effectors, such as glucose transporters and glycogen synthase, or by regulation of the amount of specific protein participants in metabolic pathways (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar, 2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar, 3White M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar). Phosphoenolpyruvate carboxykinase (PEPCK), 1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit a rate controlling enzyme of gluconeogenesis, is one such protein whose expression is regulated by insulin (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Insulin inhibits gluconeogenesis in the liver; thus, in the absence of this effect of insulin, as in diabetes mellitus or long term starvation, gluconeogenesis is increased (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). Transcription of the PEPCK gene is increased by various hormonal agents including glucocorticoids and glucagon (or its second messenger, cAMP), and insulin inhibits PEPCK gene transcription induced by these stimuli (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Given that PEPCK is not known to be subject to allosteric regulation, the inhibition of gluconeogenesis by insulin in vivo is probably due to the insulin-induced decrease in the amount of PEPCK protein. Moreover, the observations that PEPCK gene expression in the liver is increased in several of diabetes 44: PubMed Scopus Google and that the PEPCK gene a M. PubMed Scopus Google Scholar, J. Google the of this enzyme in glucose in the in of insulin the by which PEPCK gene transcription is regulated The and protein kinase signaling to the regulation of the expression of various by insulin PubMed Scopus Google Scholar). A constitutively active mutant of was to inhibit transcription of the PEPCK gene induced by protein kinase J.M. J. PubMed Scopus Google Scholar). constitutively active of or of downstream effector of did not inhibit PEPCK gene transcription induced by dexamethasone and a cAMP R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, M. Granner D.K. J. PubMed Scopus Google Scholar). the with these constitutively active the that a dominant negative mutant of or a that of important in the activation of this did not insulin inhibition of PEPCK gene transcription J.M. J. PubMed Scopus Google Scholar, R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, M. Granner D.K. J. PubMed Scopus Google that is not for this effect of role of PI which is in a of the metabolic actions of insulin Biochem. J. PubMed Scopus Google PEPCK gene transcription and Biochem. J. PubMed Scopus Google that insulin-induced inhibition of PEPCK gene expression was not by a of PI to which various metabolic actions of insulin are Biochem. J. PubMed Scopus Google Scholar). a by R.M. Granner D.K. J. PubMed Scopus Google by R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, J. J. PubMed Scopus Google that the effect of insulin PEPCK gene transcription is PI The role of Akt known as protein kinase downstream effector of PI in regulation of PEPCK gene transcription is A mutant of Akt, in which phosphorylation sites replaced for the inhibition of PEPCK transcription induced by insulin J. J. PubMed Scopus Google Scholar). J.M. J. PubMed Scopus Google that Akt in the kinase or in the did not inhibit insulin-induced of PEPCK this the role of PI 3-kinase in the inhibition of PEPCK gene transcription by insulin, by dominant negative and constitutively active of the Moreover, investigated the of several downstream of PI including Akt, protein kinase and the small GTPase in this of of PI such as and are for insulin metabolic actions of insulin, including of glucose glycogen synthase, and protein are to these M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar, Biochem. J. PubMed Scopus Google Scholar). These the of PEPCK gene transcription by insulin R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, R.M. Granner D.K. J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google may not be completely and a of be used in the role of a in a that a dominant negative mutant of PI blocked this effect of insulin. Moreover, a constitutively active of PI 3-kinase mimicked the effect of insulin PEPCK gene These data that insulin-induced inhibition of PEPCK gene transcription is mediated by PI in which the Akt sequence is to a sequence or a protein kinase that is that of the enzyme PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar). The expression of such glucose protein glycogen kinase and phosphorylation of known as PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar, J. PubMed Scopus Google of which are by insulin in a PI Biochem. J. PubMed Scopus Google Scholar). that of PEPCK gene transcription by was in HL1C cells that a constitutively active mutant of Akt with the of a J. J. PubMed Scopus Google Scholar). whereas the expression of Akt-AA inhibited the insulin-induced activation of Akt as well as insulin-induced of protein M. M. Kasuga M. PubMed Scopus Google activation of glycogen M. Kasuga J. PubMed Scopus Google phosphorylation of M. Kasuga J. PubMed Scopus Google and and activation of the of cAMP and for this mutant did not affect the inhibition of PEPCK gene transcription by is that Akt-AA did not completely endogenous Akt and that the of Akt was to inhibit PEPCK gene transcription to a this is both insulin-induced Akt activation not and PEPCK gene transcription inhibited by a dominant negative mutant of PI 3-kinase in a that a small in the of Akt is not to inhibit PEPCK gene the of the is that a distinct from Akt is of to PEPCK gene whereas Akt is to inhibit the expression of this gene that Akt-AA almost completely the of transfected and endogenous by insulin. did not kinase from HL1C this of Akt may not be in these cells. Furthermore, that Akt-AA inhibited insulin-induced endogenous Akt with that known of Akt with M. M. Kasuga M. PubMed Scopus Google Scholar). Akt to the plasma or in to stimuli J.M. J. 1997; PubMed Scopus Google Scholar, J. PubMed Scopus Google that of Akt to a specific is important for its activation and Akt in from is that this not completely the of Akt in a specific the that of Akt in a of the may be to to the PEPCK gene is that of Akt that is to Akt-AA is in the cells and for the regulation of PEPCK gene J. J. PubMed Scopus Google that insulin-induced inhibition of PEPCK gene transcription is blocked in cells that stably Akt-AA J. J. PubMed Scopus Google Scholar). these did not the inhibition of insulin-induced activation of endogenous Akt by this The for this and is not The be to the used to PEPCK transcription by the of a reporter gene fused to the promoter of the PEPCK gene, J. J. PubMed Scopus Google PEPCK by The gene transcription and and insulin is known to affect PEPCK (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google and that a kinase-defective mutant of Akt in which the in the kinase is replaced by not inhibit insulin-induced Akt activation M. M. Kasuga M. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar). this and Akt inhibit actions of insulin, including the phosphorylation of PubMed Scopus Google the activation of glycogen M. Kasuga J. PubMed Scopus Google and phosphorylation and activation of and glycogen in of Akt, with Akt in cells J. J. PubMed Scopus Google Scholar, Scopus Google is that kinase-defective of Akt signaling downstream of Akt by with the endogenous enzyme for its in the of HL1C cells with did not the inhibition of PEPCK gene transcription by insulin, which is with the of a J.M. J. PubMed Scopus Google Scholar). These observations the that inhibition of Akt signaling is not to insulin of PEPCK gene of are to downstream of PI 3-kinase M. M. PubMed Google Scholar, J. J. 1996; PubMed Scopus Google Scholar, J. J. 1997; PubMed Scopus Google Scholar). that expression of inhibits of both and glucose transport in by insulin M. M. PubMed Google Scholar). that PKCλ, not is in HL1C cells and that not affect the inhibition of PEPCK gene transcription by insulin, this mutant almost completely inhibited insulin-induced activation of to in the regulation of the protein J. J. J. PubMed Scopus Google Scholar, R.M. PubMed Scopus Google Scholar). this that expression of inhibits the insulin-induced activation of protein kinase in various including HL1C cells. and in These with that a constitutively active mutant of PKCλ, not the effect of insulin PEPCK gene that not in this effect of small GTPase downstream effector of PI insulin-induced of 1997; PubMed Scopus Google Scholar). of a dominant negative mutant of Rac did not the effect of insulin PEPCK gene that the Rac not to this of insulin. insulin not of in HL1C not the of expression of in the is to signaling endogenous that insulin-induced of is completely blocked in and by expression of to or that in the not is that the concentration of in HL1C cells was to inhibit the endogenous that a constitutively active mutant of PI 3-kinase inhibits the of PEPCK gene transcription and that a dominant negative mutant of this kinase insulin-induced inhibition of PEPCK gene dominant negative of Akt, PKCλ, and Rac did not affect the ability of insulin to inhibit transcription of the PEPCK gene. data suggest that a downstream effector of PI 3-kinase distinct from Akt, PKCλ, and Rac the effect of insulin PEPCK gene Given that various including 1996; PubMed Scopus Google kinase J. 1996; PubMed Scopus Google and kinase M. J. PubMed Scopus Google to downstream of PI 3-kinase in insulin-induced or the of specific inhibition of such PEPCK gene transcription The primary role of insulin is to control the plasma glucose concentration by stimulating glucose transport into muscle and adipose cells as well as by reducing glucose output from the liver (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). These actions of insulin are mediated by activation of effectors, such as glucose transporters and glycogen synthase, or by regulation of the amount of specific protein participants in metabolic pathways (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar, 2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar, 3White M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar). Phosphoenolpyruvate carboxykinase (PEPCK), 1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphoinositide; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; MOI, multiplicity of infection; pfu, plaque-forming unit a rate controlling enzyme of gluconeogenesis, is one such protein whose expression is regulated by insulin (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Insulin inhibits gluconeogenesis in the liver; thus, in the absence of this effect of insulin, as in diabetes mellitus or long term starvation, gluconeogenesis is increased (1Kruszynska Y.T. Olefsky J.M. J. Invest. Med. 1996; 44: 413-428PubMed Google Scholar). Transcription of the PEPCK gene is increased by various hormonal agents including glucocorticoids and glucagon (or its second messenger, cAMP), and insulin inhibits PEPCK gene transcription induced by these stimuli (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). Given that PEPCK is not known to be subject to allosteric regulation, the inhibition of gluconeogenesis by insulin in vivo is probably due to the insulin-induced decrease in the amount of PEPCK protein. Moreover, the observations that PEPCK gene expression in the liver is increased in several of diabetes 44: PubMed Scopus Google and that the PEPCK gene a M. PubMed Scopus Google Scholar, J. Google the of this enzyme in glucose in the in of insulin the by which PEPCK gene transcription is regulated The and protein kinase signaling to the regulation of the expression of various by insulin PubMed Scopus Google Scholar). A constitutively active mutant of was to inhibit transcription of the PEPCK gene induced by protein kinase J.M. J. PubMed Scopus Google Scholar). constitutively active of or of downstream effector of did not inhibit PEPCK gene transcription induced by dexamethasone and a cAMP R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, M. Granner D.K. J. PubMed Scopus Google Scholar). the with these constitutively active the that a dominant negative mutant of or a that of important in the activation of this did not insulin inhibition of PEPCK gene transcription J.M. J. PubMed Scopus Google Scholar, R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, M. Granner D.K. J. PubMed Scopus Google that is not for this effect of insulin. The role of PI which is in a of the metabolic actions of insulin Biochem. J. PubMed Scopus Google PEPCK gene transcription and Biochem. J. PubMed Scopus Google that insulin-induced inhibition of PEPCK gene expression was not by a of PI to which various metabolic actions of insulin are Biochem. J. PubMed Scopus Google Scholar). a by R.M. Granner D.K. J. PubMed Scopus Google by R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, J. J. PubMed Scopus Google that the effect of insulin PEPCK gene transcription is PI The role of Akt known as protein kinase downstream effector of PI in regulation of PEPCK gene transcription is A mutant of Akt, in which phosphorylation sites replaced for the inhibition of PEPCK transcription induced by insulin J. J. PubMed Scopus Google Scholar). J.M. J. PubMed Scopus Google that Akt in the kinase or in the did not inhibit insulin-induced of PEPCK this the role of PI 3-kinase in the inhibition of PEPCK gene transcription by insulin, by dominant negative and constitutively active of the Moreover, investigated the of several downstream of PI including Akt, protein kinase and the small GTPase in this of insulin. of PI such as and are for insulin metabolic actions of insulin, including of glucose glycogen synthase, and protein are to these M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar, Biochem. J. PubMed Scopus Google Scholar). These the of PEPCK gene transcription by insulin R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, R.M. Granner D.K. J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google may not be completely and a of be used in the role of a in a that a dominant negative mutant of PI blocked this effect of insulin. Moreover, a constitutively active of PI 3-kinase mimicked the effect of insulin PEPCK gene These data that insulin-induced inhibition of PEPCK gene transcription is mediated by PI in which the Akt sequence is to a sequence or a protein kinase that is that of the enzyme PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar). The expression of such glucose protein glycogen kinase and phosphorylation of known as PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar, J. PubMed Scopus Google of which are by insulin in a PI Biochem. J. PubMed Scopus Google Scholar). that of PEPCK gene transcription by was in HL1C cells that a constitutively active mutant of Akt with the of a J. J. PubMed Scopus Google Scholar). whereas the expression of Akt-AA inhibited the insulin-induced activation of Akt as well as insulin-induced of protein M. M. Kasuga M. PubMed Scopus Google activation of glycogen M. Kasuga J. PubMed Scopus Google phosphorylation of M. Kasuga J. PubMed Scopus Google and and activation of the of cAMP and for this mutant did not affect the inhibition of PEPCK gene transcription by is that Akt-AA did not completely endogenous Akt and that the of Akt was to inhibit PEPCK gene transcription to a this is both insulin-induced Akt activation not and PEPCK gene transcription inhibited by a dominant negative mutant of PI 3-kinase in a that a small in the of Akt is not to inhibit PEPCK gene the of the is that a distinct from Akt is of to PEPCK gene whereas Akt is to inhibit the expression of this gene that Akt-AA almost completely the of transfected and endogenous by insulin. did not kinase from HL1C this of Akt may not be in these cells. Furthermore, that Akt-AA inhibited insulin-induced endogenous Akt with that known of Akt with M. M. Kasuga M. PubMed Scopus Google Scholar). Akt to the plasma or in to stimuli J.M. J. 1997; PubMed Scopus Google Scholar, J. PubMed Scopus Google that of Akt to a specific is important for its activation and Akt in from is that this not completely the of Akt in a specific the that of Akt in a of the may be to to the PEPCK gene is that of Akt that is to Akt-AA is in the cells and for the regulation of PEPCK gene J. J. PubMed Scopus Google that insulin-induced inhibition of PEPCK gene transcription is blocked in cells that stably Akt-AA J. J. PubMed Scopus Google Scholar). these did not the inhibition of insulin-induced activation of endogenous Akt by this The for this and is not The be to the used to PEPCK transcription by the of a reporter gene fused to the promoter of the PEPCK gene, J. J. PubMed Scopus Google PEPCK by The gene transcription and and insulin is known to affect PEPCK (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google and that a kinase-defective mutant of Akt in which the in the kinase is replaced by not inhibit insulin-induced Akt activation M. M. Kasuga M. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar). this and Akt inhibit actions of insulin, including the phosphorylation of PubMed Scopus Google the activation of glycogen M. Kasuga J. PubMed Scopus Google and phosphorylation and activation of and glycogen in of Akt, with Akt in cells J. J. PubMed Scopus Google Scholar, Scopus Google is that kinase-defective of Akt signaling downstream of Akt by with the endogenous enzyme for its in the of HL1C cells with did not the inhibition of PEPCK gene transcription by insulin, which is with the of a J.M. J. PubMed Scopus Google Scholar). These observations the that inhibition of Akt signaling is not to insulin of PEPCK gene of are to downstream of PI 3-kinase M. M. PubMed Google Scholar, J. J. 1996; PubMed Scopus Google Scholar, J. J. 1997; PubMed Scopus Google Scholar). that expression of inhibits of both and glucose transport in by insulin M. M. PubMed Google Scholar). that PKCλ, not is in HL1C cells and that not affect the inhibition of PEPCK gene transcription by insulin, this mutant almost completely inhibited insulin-induced activation of to in the regulation of the protein J. J. J. PubMed Scopus Google Scholar, R.M. PubMed Scopus Google Scholar). this that expression of inhibits the insulin-induced activation of protein kinase in various including HL1C cells. and in These with that a constitutively active mutant of PKCλ, not the effect of insulin PEPCK gene that not in this effect of small GTPase downstream effector of PI insulin-induced of 1997; PubMed Scopus Google Scholar). of a dominant negative mutant of Rac did not the effect of insulin PEPCK gene that the Rac not to this of insulin. insulin not of in HL1C not the of expression of in the is to signaling endogenous that insulin-induced of is completely blocked in and by expression of to or that in the not is that the concentration of in HL1C cells was to inhibit the endogenous that a constitutively active mutant of PI 3-kinase inhibits the of PEPCK gene transcription and that a dominant negative mutant of this kinase insulin-induced inhibition of PEPCK gene dominant negative of Akt, PKCλ, and Rac did not affect the ability of insulin to inhibit transcription of the PEPCK gene. data suggest that a downstream effector of PI 3-kinase distinct from Akt, PKCλ, and Rac the effect of insulin PEPCK gene Given that various including 1996; PubMed Scopus Google kinase J. 1996; PubMed Scopus Google and kinase M. J. PubMed Scopus Google to downstream of PI 3-kinase in insulin-induced or the of specific inhibition of such PEPCK gene transcription of PI such as and are for insulin metabolic actions of insulin, including of glucose glycogen synthase, and protein are to these M.F. Diabetologia. 1997; 40 Suppl. 2: 2-17Crossref PubMed Scopus (460) Google Scholar, 4Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar, Biochem. J. PubMed Scopus Google Scholar). These the of PEPCK gene transcription by insulin R.M. Granner D.K. J. 1996; PubMed Scopus Google Scholar, R.M. Granner D.K. J. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google may not be completely and a of be used in the role of a in a that a dominant negative mutant of PI blocked this effect of insulin. Moreover, a constitutively active of PI 3-kinase mimicked the effect of insulin PEPCK gene These data that insulin-induced inhibition of PEPCK gene transcription is mediated by PI Akt in which the Akt sequence is to a sequence or a protein kinase that is that of the enzyme PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar). The expression of such glucose protein glycogen kinase and phosphorylation of known as PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar, J. PubMed Scopus Google of which are by insulin in a PI Biochem. J. PubMed Scopus Google Scholar). that of PEPCK gene transcription by was in HL1C cells that a constitutively active mutant of Akt with the of a J. J. PubMed Scopus Google Scholar). whereas the expression of Akt-AA inhibited the insulin-induced activation of Akt as well as insulin-induced of protein M. M. Kasuga M. PubMed Scopus Google activation of glycogen M. Kasuga J. PubMed Scopus Google phosphorylation of M. Kasuga J. PubMed Scopus Google and and activation of the of cAMP and for this mutant did not affect the inhibition of PEPCK gene transcription by insulin. is that Akt-AA did not completely endogenous Akt and that the of Akt was to inhibit PEPCK gene transcription to a this is both insulin-induced Akt activation not and PEPCK gene transcription inhibited by a dominant negative mutant of PI 3-kinase in a that a small in the of Akt is not to inhibit PEPCK gene the of the is that a distinct from Akt is of to PEPCK gene whereas Akt is to inhibit the expression of this gene that Akt-AA almost completely the of transfected and endogenous by insulin. did not kinase from HL1C this of Akt may not be in these cells. Furthermore, that Akt-AA inhibited insulin-induced endogenous Akt with that known of Akt with M. M. Kasuga M. PubMed Scopus Google Scholar). Akt to the plasma or in to stimuli J.M. J. 1997; PubMed Scopus Google Scholar, J. PubMed Scopus Google that of Akt to a specific is important for its activation and Akt in from is that this not completely the of Akt in a specific the that of Akt in a of the may be to to the PEPCK gene is that of Akt that is to Akt-AA is in the cells and for the regulation of PEPCK gene J. J. PubMed Scopus Google that insulin-induced inhibition of PEPCK gene transcription is blocked in cells that stably Akt-AA J. J. PubMed Scopus Google Scholar). these did not the inhibition of insulin-induced activation of endogenous Akt by this The for this and is not The be to the used to PEPCK transcription by the of a reporter gene fused to the promoter of the PEPCK gene, J. J. PubMed Scopus Google PEPCK by The gene transcription and and insulin is known to affect PEPCK (2O'Brien R.M. Granner D.K. Biochem. J. 1991; 278: 609-619Crossref PubMed Scopus (246) Google Scholar). and that a kinase-defective mutant of Akt in which the in the kinase is replaced by not inhibit insulin-induced Akt activation M. M. Kasuga M. PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar). this and Akt inhibit actions of insulin, including the phosphorylation of PubMed Scopus Google the activation of glycogen M. Kasuga J. PubMed Scopus Google and phosphorylation and activation of and glycogen in of Akt, with Akt in cells J. J. PubMed Scopus Google Scholar, Scopus Google is that kinase-defective of Akt signaling downstream of Akt by with the endogenous enzyme for its in the of HL1C cells with did not the inhibition of PEPCK gene transcription by insulin, which is with the of a J.M. J. PubMed Scopus Google Scholar). These observations the that inhibition of Akt signaling is not to insulin of PEPCK gene of are to downstream of PI 3-kinase M. M. PubMed Google Scholar, J. J. 1996; PubMed Scopus Google Scholar, J. J. 1997; PubMed Scopus Google Scholar). that expression of inhibits of both and glucose transport in by insulin M. M. PubMed Google Scholar). that PKCλ, not is in HL1C cells and that not affect the inhibition of PEPCK gene transcription by insulin, this mutant almost completely inhibited insulin-induced activation of to in the regulation of the protein J. J. J. PubMed Scopus Google Scholar, R.M. PubMed Scopus Google Scholar). this that expression of inhibits the insulin-induced activation of protein kinase in various including HL1C cells. and in These with that a constitutively active mutant of PKCλ, not the effect of insulin PEPCK gene that not in this effect of insulin. The small GTPase downstream effector of PI insulin-induced of 1997; PubMed Scopus Google Scholar). of a dominant negative mutant of Rac did not the effect of insulin PEPCK gene that the Rac not to this of insulin. insulin not of in HL1C not the of expression of in the is to signaling endogenous that insulin-induced of is completely blocked in and by expression of to or that in the not is that the concentration of in HL1C cells was to inhibit the endogenous protein. that a constitutively active mutant of PI 3-kinase inhibits the of PEPCK gene transcription and that a dominant negative mutant of this kinase insulin-induced inhibition of PEPCK gene dominant negative of Akt, PKCλ, and Rac did not affect the ability of insulin to inhibit transcription of the PEPCK gene. data suggest that a downstream effector of PI 3-kinase distinct from Akt, PKCλ, and Rac the effect of insulin PEPCK gene Given that various including 1996; PubMed Scopus Google kinase J. 1996; PubMed Scopus Google and kinase M. J. PubMed Scopus Google to downstream of PI 3-kinase in insulin-induced or the of specific inhibition of such PEPCK gene transcription