Activation of Protein Kinase B/Akt Is Sufficient to Repress the Glucocorticoid and cAMP Induction of Phosphoenolpyruvate Carboxykinase Gene

A rat hepatoma cell line, H4IIE, was stably transfected with a tamoxifen regulatable Akt-1 construct. Treatment of these cells with tamoxifen caused a rapid stimulation of Akt enzymatic activity that was comparable with the activity observed with the endogenous Akt after insulin stimulation. Prior studies have extensively documented that insulin can repress the glucocorticoid and cAMP-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Activation of this regulatable Akt with tamoxifen was found to mimic the dominant inhibitory effect of insulin on PEPCK gene transcription. Dose response curves to insulin and tamoxifen demonstrated that this response was very sensitive to Akt activation although the maximal response observed with tamoxifen activation was slightly less than that observed with insulin, indicating that the response to insulin may also involve other signaling cascades. The regulation of PEPCK transcription via Akt was, like that previously described for insulin, not dependent upon 70 kDa S6 kinase activity in that it was not inhibited by rapamycin. Finally, the expression of a kinase dead Akt was able to partially inhibit the ability of insulin to stimulate this response. In summary, the present results indicate that activation of Akt alone is sufficient to repress the glucocorticoid and cAMP-stimulated increase in PEPCK gene transcription. A rat hepatoma cell line, H4IIE, was stably transfected with a tamoxifen regulatable Akt-1 construct. Treatment of these cells with tamoxifen caused a rapid stimulation of Akt enzymatic activity that was comparable with the activity observed with the endogenous Akt after insulin stimulation. Prior studies have extensively documented that insulin can repress the glucocorticoid and cAMP-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Activation of this regulatable Akt with tamoxifen was found to mimic the dominant inhibitory effect of insulin on PEPCK gene transcription. Dose response curves to insulin and tamoxifen demonstrated that this response was very sensitive to Akt activation although the maximal response observed with tamoxifen activation was slightly less than that observed with insulin, indicating that the response to insulin may also involve other signaling cascades. The regulation of PEPCK transcription via Akt was, like that previously described for insulin, not dependent upon 70 kDa S6 kinase activity in that it was not inhibited by rapamycin. Finally, the expression of a kinase dead Akt was able to partially inhibit the ability of insulin to stimulate this response. In summary, the present results indicate that activation of Akt alone is sufficient to repress the glucocorticoid and cAMP-stimulated increase in PEPCK gene transcription. phosphoenolpyruvate carboxykinase hydroxytamoxifen phosphatidylinositol estrogen receptor hemagglutinin. Insulin regulates the expression of more than 100 genes (1O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (440) Google Scholar). One of the most extensively studied transcription processes regulated by insulin is the ability of insulin to repress the induction of phosphoenolpyruvate carboxykinase (PEPCK)1 mRNA by glucocorticoids and cAMP (1O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (440) Google Scholar, 2Hanson R.W. Reshef L. Annu. Rev. Biochem. 1997; 66: 581-611Crossref PubMed Scopus (640) Google Scholar). This response to insulin is important in regulating hepatic glucose output because PEPCK is the rate-limiting step in hepatic gluconeogenesis, and PEPCK activity is primarily regulated at the transcriptional level (1O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (440) Google Scholar, 2Hanson R.W. Reshef L. Annu. Rev. Biochem. 1997; 66: 581-611Crossref PubMed Scopus (640) Google Scholar). Insulin inhibition of the glucocorticoid and cAMP induction of PEPCK mRNA has been shown to be mediated via the activation of the lipid kinase, phosphatidylinositol 3-kinase (3Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Recent studies have documented that insulin activates this enzyme by stimulating the tyrosine phosphorylation of a number of endogenous substrates (including insulin receptor substrates 1, 2, and 3), and these tyrosine-phosphorylated endogenous substrates bind and activate the PI 3-kinase, resulting in the generation of the phospholipids phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (4Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar, 5Shoelson S.E. Sivaraja M. Williams K.P. Hu P. Schlessinger J. Weiss M.A. EMBO J. 1993; 12: 795-802Crossref PubMed Scopus (141) Google Scholar). In addition to playing a role in the regulation of PEPCK mRNA, the activation of PI 3-kinase has been found to play a role in a number of other insulin-stimulated biological responses including stimulation of glucose uptake and GLUT4 translocation, activation of the 70-kDa S6 kinase, stimulation of glycogen synthesis and in the inhibition of apoptosis (4Saltiel A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar, 5Shoelson S.E. Sivaraja M. Williams K.P. Hu P. Schlessinger J. Weiss M.A. EMBO J. 1993; 12: 795-802Crossref PubMed Scopus (141) Google Scholar, 6Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1234) Google Scholar, 7Yenush L. White M.F. Bioessays. 1997; 19: 491-500Crossref PubMed Scopus (259) Google Scholar). In contrast, activation of the 70-kDa S6 kinase and the mitogen-activated protein kinase cascade were found not to be required for the repression of PEPCK gene transcription by insulin (3Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 8Sutherland C. Waltner-Law M. Gnudi L. Kahn B.B. Granner D.K. J. Biol. Chem. 1998; 273: 3198-3204Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Recent studies have identified a number of downstream targets of the phospholipid products of the PI 3-kinase. These include a serine/threonine kinase named Akt or PKB as well as several isoforms of the protein kinase C family of serine/threonine kinases (6Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1234) Google Scholar, 10Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1194) Google Scholar, 11Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar). In addition, the PI 3-lipids also bind and activate other classes of molecules including an ADP-ribosylation exchange factor named GRP1 (12Klarlund J.K. Rameh L.E. Cantley L.C. Buxton J.M. Holik J.J. Sakelis C. Patki V. Corvera S. Czech M.P. J. Biol. Chem. 1998; 273: 1859-1862Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). To determine which biological responses can be mediated via the activation of the Akt serine/threonine kinase, we have designed a regulatable form of this enzyme (13Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). This construct encodes an Akt chimera consisting of a myristoylated form of Akt-1 fused to a mutant hormone binding unit of the estrogen receptor. Treatment of cells with the estrogen antagonist hydroxytamoxifen (HT) causes a rapid activation of this chimeric enzyme. In the present studies we have expressed this chimera in the rat hepatoma cell line H4IIE and used it to examine the ability of Akt to repress the glucocorticoid/cAMP induction of PEPCK gene expression. The present studies demonstrate that activation of Akt is sufficient to mimic this biological response of insulin. The following were purchased: [γ-32P]ATP from NEN Life Science Products; insulin from Boehringer Mannheim; 8-bromoadenosine 3′:5′-cyclic monophosphate and dexamethasone from Sigma; hydroxytamoxifen from Research Biochemicals; LY294002 from Alexis Biochemicals; rapamycin and blasticidin S from Calbiochem; polyclonal antibodies to p70/p85 ribosomal S6 protein kinase from Upstate Biotechnology; monoclonal antibodies to Akt from Transduction Laboratories; Anti-HA monoclonal antibody 12CA5 from Boehringer Mannheim; and G418 from Life Technologies, Inc. The Phoenix retroviral packaging cell line and the retroviral vectors were gifts of Dr. Garry Nolan. The pWZL-neo retroviral vector encoding MER-Akt was as described (13Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). An inactive Akt was made by site-directed mutagenesis of the important regulatory phosphorylation sites threonine 308 and serine 473 to alanine in the plasmid encoding the full-length Akt (14Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar). This plasmid was then subcloned into the retroviral vector pBMN-Z-I-Blasto (a gift from Garry P. Nolan). H4IIE cells were infected with pWZLneo/MER-Akt or pBMN-Z-I-Blasto/Akt T308A, S473A as described previously (14Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar). The former were selected by using 1 mg/ml of G418, whereas the latter were selected by using 10 μg/ml of blasticidin S. Cells were grown to confluence in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 2.5% fetal calf serum and 2.5% newborn calf serum in an atmosphere of 5% CO2, 95% air. Before treatment, cells were deprived of serum overnight in Dulbecco's modified Eagle's medium containing 1% bovine serum albumin. Cells were then incubated with hormones and inhibitors as indicated in the figure legends. The expressed MER-Akt was specifically immunoprecipitated by using a monoclonal antibody to the HA-tag present on this enzyme. The endogenous Akt was specifically precipitated by using a polyclonal antibody to the Akt-1 pleckstrin homology domain (15Krook A. Kawano Y. Song X.M. Efendic S. Roth R.A. Wallberg-Henriksson H. Zierath J.R. Diabetes. 1997; 46: 2110-2114Crossref PubMed Google Scholar), because the MER-Akt does not contain the pleckstrin homology domain (13Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Western blots were performed with either the monoclonal antibody to HA (for the expressed MER-Akt) or with a monoclonal antibody to Akt-1 (for the endogenous Akt). Akt kinase assays were performed with the GSK-3 peptide (sequence GRPRTSSFAEG) (16Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4467) Google Scholar), and the phosphorylation of the peptide was monitored by analyzing the samples on a 6 m urea, 40% polyacrylamide gel as described previously (13Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Total cellular RNA was isolated by using the Qiagen RNeasy Mini Kit as detailed in their manual. Primer extension analyses for the PEPCK and β-actin mRNAs were performed essentially as described (17Forest C.D. O'Brien R.M. Lucas P.C. Magnuson M.A. Granner D.K. Mol. Endocrinol. 1990; 4: 1302-1310Crossref PubMed Scopus (95) Google Scholar). In brief, oligonucleotides PC28 (5′-GGAGAGAGGCAGGGACTCTGGTGCCACC-3′) and ACT25 (5′-GGGTGTGGACCGGGACGGAGGAGCT-3′) (complementary to positions +102 to +129 and positions +42 to +67, relative to the transcription start sites in PEPCK and β-actin, respectively) (18Beale E.G. Chrapkiewicz N.B. Scoble H.A. Metz R.J. Quick D.P. Noble R.L. Donelson J.E. Biemann K. Granner D.K. J. Biol. Chem. 1985; 260: 10748-10760Abstract Full Text PDF PubMed Google Scholar, 19Nudel U. Zakut R. Shani M. Neuman S. Levy Z. Yaffe D. Nucleic Acids Res. 1983; 11: 1759-1771Crossref PubMed Scopus (1018) Google Scholar) were high pressure liquid chromatography purified, 5′-end-labeled using [γ-32P]ATP and then incubated with 10 μg of total cellular RNA. After the primer extension reaction, the products were analyzed by electrophoresis on 7.5 m urea, 6% polyacrylamide gel, visualized by autoradiography, and quantified by PhosphorImager analysis. H4IIE hepatoma cells stably expressing a tamoxifen-regulatable form of Akt-1 were developed by infecting these cells with retroviruses encoding MER-Akt (13Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar) and selecting those cells that became resistant to the drug-selectable marker encoded by the retrovirus. The amount of tamoxifen-regulatable Akt enzymatic activity expressed in these cells was compared with the amount of insulin-stimulated endogenous Akt present in these cells. To this end, cells were treated with different concentration of either insulin or tamoxifen, lysed, and either the endogenous or expressed Akt chimera were precipitated with specific antibodies to each molecule. The amount of enzymatic activity in the precipitates was then determined via the use of a synthetic peptide based on the sequence of the Akt phosphorylation site in GSK-3 (16Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4467) Google Scholar). The maximal amounts of Akt activity stimulated by insulin and tamoxifen were approximately the same and represented an approximate 30-fold increase over basal levels (Fig. 1). The stimulation could be observed with either 0.1 or 1 nm of insulin or tamoxifen, respectively (Fig. 1). Because PEPCK mRNA transcription is low in the absence of any stimulus, we studied the ability of insulin and Akt to repress the induction of PEPCK mRNA stimulated by a combination of a nonhydrolyzable cAMP analog and the synthetic glucocorticoid dexamethasone. The levels of endogenous PEPCK mRNA were monitored by primer extension analysis. As previously reported (1O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (440) Google Scholar), insulin completely inhibited the induction of PEPCK mRNA by the dexamethasone/cAMP treatment (Fig. 2). Activation of the regulatable Akt by tamoxifen was also capable of largely inhibiting the increase in mRNA caused by the dexamethasone/cAMP treatment in the cells expressing the construct encoding the chimeric MER-Akt (Fig. 2). In contrast, tamoxifen had no effect on the induction of the PEPCK mRNA in control cells not expressing the regulatable Akt (Fig. 2), demonstrating that this effect of tamoxifen was mediated via the expressed MER-Akt. To better compare the repression of PEPCK mRNA induction with tamoxifen and insulin, a dose curve was performed with different concentrations of insulin and tamoxifen (Fig. 3). As previously reported for insulin (17Forest C.D. O'Brien R.M. Lucas P.C. Magnuson M.A. Granner D.K. Mol. Endocrinol. 1990; 4: 1302-1310Crossref PubMed Scopus (95) Google Scholar), the inhibition of PEPCK mRNA transcription was exquisitely sensitive to insulin, with 0.1 nm insulin causing almost a maximal response. The response to tamoxifen was also quite sensitive with 1 nm tamoxifen also causing an almost maximal response. The maximal inhibition observed with insulin was greater than that observed with tamoxifen (Fig. 3). Because previous studies have demonstrated that Akt can also activate the 70-kDa S6 kinase (20Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1894) Google Scholar), we sought to determine this enzyme was playing a role in the Akt regulation of PEPCK Cells were stimulated with tamoxifen in the or absence of an of the activation of the 70-kDa S6 kinase J. J. Nature. PubMed Scopus Google J.R. V. J. PubMed Scopus Google Scholar). was found to have a on the ability of Akt to PEPCK mRNA, to that observed with insulin (Fig. that rapamycin completely the activation of the 70-kDa S6 kinase by insulin and MER-Akt as monitored by a in the of the 70-kDa S6 kinase (Fig. As rapamycin had no effect on the activation of the enzymatic activity of either the endogenous Akt or the chimeric MER-Akt (Fig. In contrast, a specific of the PI 3-kinase J. Biol. Chem. Full Text PDF PubMed Google Scholar), completely inhibited the ability of insulin and Akt to repress PEPCK mRNA transcription (Fig. This inhibition with the ability of this to the activation of the enzymatic activity of the endogenous and the expressed MER-Akt by insulin and tamoxifen, respectively (Fig. of the PI 3-kinase LY294002 and the S6 kinase rapamycin on Akt enzymatic activity and the 70-kDa S6 kinase cells were treated with either LY294002 or nm rapamycin for 1 the addition of either insulin, or tamoxifen After an of the cells were lysed, and a of the total was used to the in S6 kinase by Western The of the cell were immunoprecipitated with either an antibody to the endogenous Akt or a monoclonal antibody to the HA-tag on the expressed Akt The were for Akt enzymatic activity by using the GSK-3 peptide and then analyzed for Akt by Western a Western of the precipitates and C a GSK-3 The S.E. of different shown in Finally, to the role of endogenous Akt in the insulin-stimulated repression of PEPCK mRNA, we expressed a kinase dead Akt in the H4IIE cells. This mutant Akt has had regulatory phosphorylation sites D.R. M. B. P. Cohen P. Hemmings B.A. EMBO J. 1996; PubMed Scopus Google Scholar) 308 and serine to alanine and is inactive D.R. M. B. P. Cohen P. Hemmings B.A. EMBO J. 1996; PubMed Scopus Google Scholar). The expression of this kinase dead Akt partially inhibited the ability of insulin to repress the dexamethasone/cAMP induction of PEPCK mRNA (Fig. Recent studies have documented that insulin activates PI 3-kinase, and this enzymatic activity to be required for insulin to several of biological responses including the regulation of the PEPCK mRNA (3Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, A.R. Am. J. Physiol. 1996; 270: E375-E385PubMed Google Scholar, 5Shoelson S.E. Sivaraja M. Williams K.P. Hu P. Schlessinger J. Weiss M.A. EMBO J. 1993; 12: 795-802Crossref PubMed Scopus (141) Google Scholar, 6Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1234) Google Scholar, 7Yenush L. White M.F. Bioessays. 1997; 19: 491-500Crossref PubMed Scopus (259) Google Scholar, 8Sutherland C. Waltner-Law M. Gnudi L. Kahn B.B. Granner D.K. J. Biol. Chem. 1998; 273: 3198-3204Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). the step after activation of PI 3-kinase, which is for the regulation of PEPCK mRNA, is not A number of serine/threonine kinases have been shown to be by the PI 3-kinase These include in cell Cantley L.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), the 70-kDa S6 kinase B. L. L. J. Kahn Mol. Biol. PubMed Scopus Google Scholar), the family of Akt kinases J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1194) Google Scholar) and at several isoforms of protein kinase including and (6Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1234) Google Scholar, 11Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar). with inhibitors of the and the 70-kDa S6 kinase have shown that it is that these play a role in the ability of insulin to PEPCK gene expression (3Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 8Sutherland C. Waltner-Law M. Gnudi L. Kahn B.B. Granner D.K. J. Biol. Chem. 1998; 273: 3198-3204Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In the present we have the ability of the serine/threonine kinase Akt to PEPCK To a line of H4IIE hepatoma cells was which expressed a tamoxifen-regulatable These cells a rapid and increase in Akt enzymatic activity after tamoxifen stimulation. The amount of expressed activity after tamoxifen stimulation as with an to be comparable with that of the endogenous Akt-1 stimulated with insulin. In addition, these cells also to have an insulin-stimulated at approximately the levels of the J. and R.A. the total amount of expressed Akt activity in these cells is the same or then the total amount of endogenous Akt present in these cells after insulin stimulation. The activation of the regulatable Akt with tamoxifen treatment was found to be sufficient to repress the increase in PEPCK This response was observed with a dose of tamoxifen, in with the with the insulin stimulation. like the insulin regulation of PEPCK mRNA, the response to tamoxifen was not inhibited by an of the activation of the 70-kDa S6 kinase J. J. Nature. PubMed Scopus Google Scholar, J.R. V. J. PubMed Scopus Google Scholar). The response to tamoxifen required the of the because cells not expressing this construct no response to the The maximal response observed with tamoxifen was less then the maximal response observed with insulin, that the response to insulin may in be mediated a combination of signaling it is that this chimeric Akt does not as as the endogenous The that of a kinase inactive Akt at the ability of insulin to PEPCK mRNA is with a role for the endogenous Akt in the insulin response. the expression of a kinase inactive Akt is not studies have that expression of a kinase inactive Akt with the activation of endogenous Akt Z. D. Cantley L.C. D.R. U. S. A. 1997; PubMed Scopus Google Scholar, H. S. Birnbaum M. R. R. D. M. 1997; PubMed Scopus Google Scholar), we have observed no is that the kinase inactive Akt with the endogenous Akt for downstream substrates of the enzyme. The inhibition observed with this kinase inactive Akt is with the inhibition of insulin-stimulated glucose uptake previously observed after expression of a kinase inactive Akt H. Y. L. M.A. M.J. Mol. Endocrinol. 1997; 11: PubMed Google Scholar). the present a role for Akt in the insulin repression of the increase in PEPCK gene transcription. This the of Akt in insulin-stimulated biological is also with a that of a Akt can the basal expression of a construct containing a insulin response sequence from the of the factor binding gene S. A. S. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). These also that this Akt regulated the expression of a sequence from the of the PEPCK The present this previous because it for the that transcription of the endogenous PEPCK gene can also be regulated via In addition, we that Akt can repress the induction of PEPCK transcription by dexamethasone and the present studies demonstrate that these of Akt with Akt enzymatic comparable with those by insulin. the of the of this J.M. D. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) also reported the results of studies the ability of expressed Akt to a construct containing the of the PEPCK In these studies the found that a Akt inhibit the cAMP increase in the expression of the construct by approximately The expression of this construct was inhibited by insulin In addition, these not any inhibition of the insulin effect on this construct after the expression of different Akt These results were to indicate that Akt was not in the insulin inhibition of PEPCK gene expression. Because these studies it was to determine Akt enzymatic activity was expressed with the form of the enzyme relative to was stimulated by insulin. In addition, because a of the cells expressed these the could not determine the Akt inhibited the insulin-stimulated activation of the endogenous Recent studies have shown that inhibition of endogenous Akt by these Akt is P.C. J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, as demonstrated in the present a very low level of Akt enzymatic activity is sufficient to repress the PEPCK gene transcription. The of the insulin response that the expression of the Akt almost completely inhibit the activation of the endogenous the role of Akt in a biological response is to via the use of of Akt