Advertisement

Insulin Regulation of Glucose-6-phosphate Dehydrogenase Gene Expression Is Rapamycin-sensitive and Requires Phosphatidylinositol 3-Kinase*

Open AccessPublished:June 12, 1998DOI:https://doi.org/10.1074/jbc.273.24.14968
      Glucose-6-phosphate dehydrogenase (G6PDH) controls the flow of carbon through the pentose phosphate pathway and also produces NADPH needed for maintenance of reduced glutathione and reductive biosynthesis. Hepatic expression of G6PDH is known to respond to several dietary and hormonal factors, but the mechanism behind regulation of this expression has not been characterized. We show that insulin similarly induces expression of endogenous hepatic G6PDH and a reporter construct containing 935 base pairs of the G6PDH promoter linked to luciferase in transient transfection assays. Using well tested and structurally distinct inhibitors of Ras farnesylation, lovastatin and B581, and a specific inhibitor of mitogen-activated protein kinase kinase activation, PD 98059, we show that the Ras/Raf/mitogen-activated protein kinase pathway is not utilized for the insulin-induced stimulation of G6PDH gene expression in primary rat hepatocytes. Similarly, using well characterized inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002, we show that PI 3-kinase activity is necessary for the induction of G6PDH expression by insulin. Rapamycin, an inhibitor of FRAP protein, which is involved in the activation of pp70 S6 kinase, blocks the insulin induction of G6PDH, suggesting that S6 kinase is also necessary for the insulin induction of G6PDH expression.
      Insulin elegantly regulates a variety of metabolic responses. The actions of insulin at the cellular level are initiated by insulin binding to its plasma membrane receptor. Following ligand-induced autophosphorylation of the receptor, phosphorylation of endogenous substrates occurs to mediate the transmission of an insulin signaling pathway (
      • Kahn C.R.
      • White M.F.
      • Shelson S.E.
      • Backer J.M.
      • Araki E.
      • Cheatham B.
      • Csermely P.
      • Folli F.
      • Goldstein B.J.
      • Huertas P.
      • Rothenberg P.L.
      • Saad M.J.
      • Siddle K.
      • Sun X-J.
      • Wilden P.A.
      • Yamada K.
      • Kahn S.A.
      ). The information available to date clearly indicates that under a number of different circumstances a variety of phosphoproteins are activated by insulin (
      • Kahn C.R.
      • White M.F.
      • Shelson S.E.
      • Backer J.M.
      • Araki E.
      • Cheatham B.
      • Csermely P.
      • Folli F.
      • Goldstein B.J.
      • Huertas P.
      • Rothenberg P.L.
      • Saad M.J.
      • Siddle K.
      • Sun X-J.
      • Wilden P.A.
      • Yamada K.
      • Kahn S.A.
      ,
      • Myers M.G.
      • White M.F.
      ). Members of the IRS
      The abbreviations used are: IRS, insulin receptor substrate; G6PDH, glucose-6-phosphate dehydrogenase; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; PI, phosphatidylinositol; LUC, luciferase; PBS, phosphate-buffered saline; PEPCK, phosphoenolpyruvate carboxykinase.
      1The abbreviations used are: IRS, insulin receptor substrate; G6PDH, glucose-6-phosphate dehydrogenase; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP kinase kinase; PI, phosphatidylinositol; LUC, luciferase; PBS, phosphate-buffered saline; PEPCK, phosphoenolpyruvate carboxykinase.
      family, which includes IRS-1, IRS-2, and the recently reported IRS-3 (p60), are thought to be essential for many of insulin's biological responses (
      • Myers M.G.
      • White M.F.
      ,
      • Lavan B.E.
      • Lane W.S.
      • Lienhard G.E.
      ). IRS-1 and IRS-2 contain common functional units but are thought to regulate unique signaling pathways in part to their distinct cellular distribution. IRS-1 and IRS-2 appear in all tissue; however, IRS-2 predominates in cells of myeloid lineage.
      A great deal of study has concentrated on IRS-1, and it has been found that activated IRS-1 recognizes and binds to Src homology 2 domains of various signal transduction proteins such as PI 3-kinase, SH-PTP-2, Grb2, and Nck (
      • Skolnik E.Y.
      • Lee C-H.
      • Batzer A.
      • Vincenti L.M.
      • Zhou M.
      • Daly R.
      • Myers Jr., M.J.
      • Backer J.M.
      • Ullrich A.
      • White M.F.
      • Schlessinger J.
      ). The critical role of PI 3-kinase in diverse actions of insulin has been established in a number of independent studies. One report demonstrates that PI 3-kinase in 3T3-L1 cells is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ), whereas another shows that PI 3-kinase, while functioning upstream of Ras and Raf, is necessary for mediating insulin stimulation of c-fos transcription (
      • Yamauchi K.
      • Holt K.
      • Pessin J.E.
      ). Other reports using the PI 3-kinase inhibitor wortmannin, have implicated the involvement of PI 3-kinase in insulin regulation of glycogen synthase (
      • Shepherd P.R.
      • Navé B.T.
      • Siddle K.
      ,
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ), glycogen synthase kinase-3 (
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavaré J.M.
      • Proud C.G.
      ), Glut-4-mediated glucose transport (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Okada T.
      • Kawanok Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ), membrane ruffling (
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamurea Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      ), and PEPCK expression (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.
      • Granner D.K.
      • Flier J.S.
      ,
      • Sutherland C.
      • O'Brien R.
      • Granner D.K.
      ).
      Wortmannin also blocks the insulin activation of pp70 S6 kinase, the protein responsible for the hormonal and growth factor-stimulatedin vivo phosphorylation of ribosomal protein S6 kinase (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ). Rapamycin is a potent inhibitor of insulin-stimulated phosphorylation of the ribosomal protein S6 kinase but has no effect on the stimulation of PI 3-kinase by insulin. Regulation of PEPCK expression by insulin has recently been shown to be rapamycin-insensitive (
      • Skolnik E.Y.
      • Lee C-H.
      • Batzer A.
      • Vincenti L.M.
      • Zhou M.
      • Daly R.
      • Myers Jr., M.J.
      • Backer J.M.
      • Ullrich A.
      • White M.F.
      • Schlessinger J.
      ,
      • Yang S.
      • Dickson A.J.
      ) as has the insulin regulation of glycogen synthase (
      • Shepherd P.R.
      • Navé B.T.
      • Siddle K.
      ), glycogen synthase kinase-3 (
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavaré J.M.
      • Proud C.G.
      ), gene 33 (
      • Yang S.
      • Dickson A.J.
      ), and 6-phosphofructo-2-kinase (
      • Lefebvre V.
      • Mechin M.-C.
      • Louckx M.P.
      • Rider M.H.
      • Hue L.
      ), suggesting that an insensitive pathway predominates in the insulin regulation of genes for enzymes in metabolism.
      In addition to the IRS family and the PI 3-kinase pathway, another set of proteins has been identified as a proximal target for several growth factor tyrosine kinases including the insulin receptor (
      • Pronk G.J.
      • McGlade J.
      • Pelicci G.
      • Pawson T.
      • Bos J.L.
      ,
      • Kovacina K.S.
      • Roth R.A.
      ). Once activated by phosphorylation, the Shc proteins associate with various downstream effector molecules including Grb2 and the guanine nucleotide-releasing factor (
      • Sasaoka T.
      • Draznin B.
      • Leitner J.W.
      • Langlois W.J.
      • Olefsky J.M.
      ). Grb2/SOS interaction plays a role in the insulin activation of Ras, since SOS facilitates the exchange of GDP for GTP on Ras proteins (
      • Reusch J.E-B.
      • Bhuripanyo P.
      • Carel K.
      • Leitner J.W.
      • Hsieh P.
      • DePaolo D.
      • Draznin B.
      ).
      Ras activation is required for initiation of the downstream events leading to MAP kinase activation. Studies have indicated a reasonable correlation between the activation of MAP kinase and events leading to cellular proliferation (
      • Pages G.
      • Lenormand P.
      • L'Allemain G.
      • Chambard J.C.
      • Meloche S.
      • Pouyssegur J.
      ,
      • Wang L.M.
      • Keegan A.D.
      • Paul W.E.
      • Heidaran M.A.
      • Gutkind J.S.
      • Pierce J.H.
      ). MAP kinases have also been implicated in phosphorylation of some transcription factors that could be involved in insulin-mediated gene expression. In vitro , MAP kinase phosphorylates c-Jun and TCF/ELK as well as ATF-2 to restore DNA binding activity (
      • Davis R.
      ). The role of MAP kinase in metabolic events has not been demonstrated, since no obligatory role has been established in insulin stimulation of glycogen synthesis (
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Sakaue H.
      • Hara K.
      • Noguchi T.
      • Matozaki T.
      • Kotani K.
      • Ogawa W.
      • Yonezawa K.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Sakaue M.
      • Bowtell D.
      • Kasuga M.
      ,
      • Robinson L.J.
      • Razzack Z.F.
      • Lawrence Jr., J.
      • James D.E.
      ), lipogenesis (
      • Sakaue H.
      • Hara K.
      • Noguchi T.
      • Matozaki T.
      • Kotani K.
      • Ogawa W.
      • Yonezawa K.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Wiese R.J.
      • Mastick C.C.
      • Lazar D.F.
      • Saltiel A.R.
      ), gluconeogenesis (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.
      • Granner D.K.
      • Flier J.S.
      ,
      • Sutherland C.
      • O'Brien R.
      • Granner D.K.
      ), or glucose uptake (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Okada T.
      • Kawanok Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ).
      As recently reviewed by O'Brien and Granner (
      • O'Brien R.M.
      • Granner D.K.
      ), insulin regulates the transcription of the genes for several metabolic enzymes. Glycogen synthase (
      • Cohen P.
      ), glucokinase (
      • Magnuson M.A.
      • Andreone T.L.
      • Printz R.L.
      • Koch S.
      • Granner D.K.
      ,
      • Iynedjian P.B.
      • Gjinovci A.
      • Renold A.E.
      ), PEPCK (
      • O'Brien R.M.
      • Lucas P.C.
      • Forest C.
      • Magnuson M.A.
      • Granner D.K.
      ,
      • Short J.M.
      • Wynshaw-Boris A.
      • Short H.P.
      • Hanson R.W.
      ), and fatty acid synthase (
      • Moustaid N.
      • Beyer R.S.
      • Sul H.S.
      ,
      • Stapleton S.R.
      • Mitchell D.A.
      • Salati L.M.
      • Goodridge A.G.
      ), key enzymes in glycogen synthesis, glycolysis, gluconeogenesis, and fatty acid biosynthesis, respectively, have been targets of study with regard to insulin action. In the liver, the pentose phosphate pathway is a primary pathway responsible for the insulin-influenced fate of carbohydrate, since up to 50% of the glucose 6-phosphate can be shuttled through. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49), the key rate-limiting enzyme in the pentose phosphate pathway, controls the flow of carbon through this pathway and also produces NADPH needed for maintenance of reduced glutathione and reductive biosynthesis. G6PDH has not been extensively characterized with regard to insulin action, since in most tissues and organisms it has been considered to have a “housekeeping” role (
      • Kletzien R.F.
      • Harris P.K.W.
      • Foellmi L.A.
      ). While the housekeeping role of G6PDH is not disputed, the insulin induction of G6PDH activity and mRNA (
      • Kletzien R.F.
      • Harris P.K.W.
      • Foellmi L.A.
      ,
      • Stumpo D.J.
      • Kletzien R.F.
      ,
      • Fritz R.S.
      • Stumpo D.S.
      • Kletzien R.F.
      ) has been studied bothin vivo and in vitro .
      Recently, we have cloned the promoter region of the G6PDH gene (
      • Rank K.B.
      • Harris P.K.
      • Ginsberg L.C.
      • Stapleton S.R.
      ). In transient expression studies in primary rat hepatocytes, we show that a chimeric construct containing 935 base pairs of the G6PDH promoter region responds to insulin similarly to endogenous G6PDH. Few studies have utilized primary cells in culture for elucidation of signal pathways, since they are not easily experimentally manipulated or conducive to dominant negative or overexpression approaches. They are, however, an ideal model for elucidating mechanisms utilized for gene expression, since they can be maintained in a chemically defined, serum-free medium and since the metabolic responses to hormones or nutrients generally mimic those observed in vivo . For these reasons, we have utilized primary hepatocytes in culture, a representative cell population from a primary target tissue of insulin, to investigate the role of insulin signal proteins in the induction of G6PDH expression. To determine which signal proteins are necessary for transmission of the message from the insulin receptor to the G6PDH gene, we have completed studies using well characterized inhibitors of insulin signal proteins. We show, using inhibitors of Ras farnesylation (B581 and lovastatin) as well as an inhibitor of MEK (PD 98059), that the Ras/Raf/MAPK pathway is not utilized in the insulin induction of G6PDH. Wortmannin and LY 294002, two mechanistically distinct inhibitors of PI 3-kinase, however, completely block the insulin-induced activation of G6PDH expression, demonstrating a role for PI 3-kinase in the regulation of this gene. In rat hepatocytes in culture, we find that S6 kinase is activated by insulin in a rapamycin-sensitive and -insensitive way. Results from double immunoblot analysis suggest that the rapamycin-insensitive pathway for insulin activation of S6 kinase is mediated via Akt. Unlike previous studies that have found genes of glycolysis and gluconeogenesis to be rapamycin-insensitive and possibly mediated downstream of PI 3-kinase via Akt, we find the insulin induction of G6PDH to be rapamycin-sensitive.

      DISCUSSION

      Regulation of gene expression by insulin has been an intense and complex area of study for many years. Gaps still remain between understanding the upstream signaling molecules and downstreamtrans -acting factors that are important in this regulation of gene expression by insulin (
      • O'Brien R.M.
      • Granner D.K.
      ). Genes involved in hepatic glucose homeostasis that have been shown to be directly regulated by insulin include glucokinase and PEPCK, key enzymes in glycolysis and gluconeogenesis, respectively. Limited studies to elucidate insulin regulation, however, have been done in regard to the pentose phosphate pathway, an intermediary metabolism pathway essential to carbohydrate and fatty acid metabolism. G6PDH is the key enzyme in this pathway and is the focus of this paper.
      Recently, the use of inhibitors of specific steps of insulin signaling pathways has permitted definition of the importance of that pathway to metabolic responses. Most of these studies have not focused on the liver, a major target organ of insulin. When liver cells have been used, they are generally transformed cells and thus may not always represent the metabolic responses observed in vivo . Inhibitors of farnesylation, a critical step in Ras processing, have been successfully used to show the role of the Ras/MAP kinase pathway in insulin signal transduction (
      • Defeo-Jones D.
      • McAvoy E.M.
      • Jones R.E.
      • Vuocolo G.A.
      • Haskell K.M.
      • Wegrzyn R.J.
      • Oliff A.
      ,
      • McGuire T.F.
      • Xu X-Q.
      • Corey S.J.
      • Romero G.G.
      • Sebti S.M.
      ). Lovastatin, a compound that has been shown to disrupt the early events of insulin mitogenic signaling (
      • McGuire T.F.
      • Xu X-Q.
      • Corey S.J.
      • Romero G.G.
      • Sebti S.M.
      ), blocks posttranslational modification of Ras (
      • Defeo-Jones D.
      • McAvoy E.M.
      • Jones R.E.
      • Vuocolo G.A.
      • Haskell K.M.
      • Wegrzyn R.J.
      • Oliff A.
      ). Unfortunately, lovastatin has been shown to have other sites of action including inhibition of hydroxymethyl-glutaryl coenzyme A reductase, the rate-limiting step of cholesterol biosynthesis. It has also been shown to block cell transformation by Raf, a signal protein downstream of Ras (
      • Cox A.D.
      • Garcia A.M.
      • Westwick J.K.
      • Kowalczyk J.J.
      • Lewis M.D.
      • Brenner D.A.
      • Der C.J.
      ), as well as inhibit both the platelet-derived growth factor and insulin activation of PI 3-kinase (
      • McGuire T.F.
      • Corey S.J.
      • Sebti S.M.
      ). For these reasons, we also chose to use a new specific inhibitor of Ras farnesylation, B581, to substantiate our studies. B581 mimics the CAAX binding site of farnesyltransferase and blocks Ras transformation and subsequent MAP kinase activation (
      • Cox A.D.
      • Garcia A.M.
      • Westwick J.K.
      • Kowalczyk J.J.
      • Lewis M.D.
      • Brenner D.A.
      • Der C.J.
      ). We show for the first time in primary hepatocytes in culture that both of these inhibitors efficiently block MAP kinase activation; however, neither inhibitor prevents insulin induction of G6PDH expression.
      In light of evidence that suggests that farnesylated Ras-GTP interacts with the catalytic subunit of PI 3-kinase and increases its activity, we tested whether or not the farnesylation inhibitors had any effect on PI 3-kinase activation by insulin (
      • Rodriquez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). Neither lovastatin nor B581 inhibited the insulin induction of PI 3-kinase, suggesting that in hepatocytes, Ras is not an upstream mediator of PI 3-kinase (data not shown).
      Mechanisms have been proposed for Ras-independent activation of MAP kinase (
      • Whitehurst C.E.
      • Owaki H.
      • Bruder J.T.
      • Rapp U.R.
      • Geppert T.D.
      ,
      • Blumer K.J.
      • Johnson G.L.
      ). MAP kinase has a number of substrates including transcription factors that may be involved in regulation of gene expression (
      • Lazar D.F.
      • Wiese R.J.
      • Brady M.J.
      • Mastick C.
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      • Cuatrecasas P.
      • Saltiel A.R.
      ,
      • Gille D.
      • Sharrocks A.D.
      • Shaw P.E.
      ,
      • Pulverer B.J.
      • Kyriakis J.M.
      • Avruch J.
      • Nikolakaki E.
      • Woodgett J.R.
      ). MEK or MAP kinase kinase, a dual specific kinase catalyzes the phosphorylation of MAP kinase on threonine and tyrosine residues causing activation. Recently, an inhibitor of MEK, PD 98059, has been described (
      • Lazar D.F.
      • Wiese R.J.
      • Brady M.J.
      • Mastick C.
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      • Cuatrecasas P.
      • Saltiel A.R.
      ). This inhibitor has been used to show the role of MEK and MAP kinase in the regulation of c-fos transcription (
      • Lazar D.F.
      • Wiese R.J.
      • Brady M.J.
      • Mastick C.
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      • Cuatrecasas P.
      • Saltiel A.R.
      ). Inhibition of MEK, however, had no effect on insulin-stimulated glucose uptake in 3T3-L1 cells; glycogen synthase in 3T3-L1 cells and M6 myocytes (
      • Lazar D.F.
      • Wiese R.J.
      • Brady M.J.
      • Mastick C.
      • Waters S.B.
      • Yamauchi K.
      • Pessin J.E.
      • Cuatrecasas P.
      • Saltiel A.R.
      ); or inhibition of PEPCK expression in H4IIE cells (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.
      • Granner D.K.
      • Flier J.S.
      ). Consistent with these results, treatment of hepatocytes with PD 98059 had no effect on the insulin stimulation of G6PDH expression.
      Thus, the evidence available to date suggests that the MAP kinase pathway is not required for insulin mediation of metabolic pathways in any cell type. Similarly, evidence is accumulating to show a role for PI 3-kinase in insulin regulation of metabolic processes. Wortmannin and more recently LY 294002 have been used to indicate the importance of PI 3-kinase in insulin-stimulated glucose uptake in adipocytes (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavaré J.M.
      • Proud C.G.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ). The effects of insulin on glycogen synthase (
      • Shepherd P.R.
      • Navé B.T.
      • Siddle K.
      ,
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ) or glycogen synthase kinase-3 (
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavaré J.M.
      • Proud C.G.
      ) are also blocked by inhibiting PI 3-kinase activity with wortmannin. In hepatoma cells, wortmannin has also been effectively used to block the actions of insulin on gene 33 expression (
      • Yang S.
      • Dickson A.J.
      ), yet in this same study, wortmannin was ineffective in altering the response of PEPCK mRNA to insulin. Other recent studies, however, using wortmannin or LY 294002 show that PEPCK expression is sensitive to inhibition of PI 3-kinase activity (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.
      • Granner D.K.
      • Flier J.S.
      ,
      • Sutherland C.
      • O'Brien R.
      • Granner D.K.
      ). Our studies substantiate the role of PI 3-kinase in the regulation of metabolic processes, since insulin-induced expression of G6PDH is sensitive to both wortmannin and LY 294002.
      The signal transduction pathways that lie between PI 3-kinase and potential IRE sequence-binding proteins remain to be identified. One protein that lies downstream of PI 3-kinase is pp70 S6 kinase. This protein has been shown to be responsible for insulin-stimulated phosphorylation of ribosomal protein S6 in vivo (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ). Wortmannin, a potent inhibitor of PI 3-kinase, also blocks the activation of pp70 S6 kinase. Rapamycin, an immunosuppressant, blocks pp70 S6 kinase activation without affecting PI 3-kinase activity. In 3T3-L1 cells, glycogen synthase is inhibited by rapamycin (
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ); however, insulin regulation of glycogen synthase in PC-12 cells is rapamycin-insensitive (
      • Shepherd P.R.
      • Navé B.T.
      • Siddle K.
      ). The insulin regulation of glycogen synthase kinase-3 (
      • Cross D.A.E.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ,
      • Welsh G.I.
      • Foulstone E.J.
      • Young S.W.
      • Tavaré J.M.
      • Proud C.G.
      ), gene 33 (
      • Yang S.
      • Dickson A.J.
      ), PEPCK (
      • Sutherland C.
      • O'Brien R.
      • Granner D.K.
      ), and most recently 6-phosphofructo-2-kinase (
      • Lefebvre V.
      • Mechin M.-C.
      • Louckx M.P.
      • Rider M.H.
      • Hue L.
      ) have all also been shown to be rapamycin, insensitive. The accumulation of these data to date suggest that a rapamycin-insensitive pathway may be responsible for the regulation of metabolic genes. We show that in hepatocytes, a rapamycin-insensitive pathway for activation of S6 kinase does exist through Akt; however, the insulin regulation of G6PDH gene expression is rapamycin-sensitive. Akt is a serine/threonine kinase that functions downstream of PI 3-kinase and may regulate pp70 S6 kinase (
      • Franke T.F.
      • Yang S.
      • Chan T.O.
      • Datta K.
      • Kaziauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Kohn A.D.
      • Takeuchi F.
      • Roth R.A.
      ). Controversy exists over the exact mechanism by which Akt is activated. The generation of the lipid products phosphoinositide 3,4-bisphosphate and phosphoinositide 3,4,5-triphosphate by PI 3-kinase has been suggested to play a role in not only the translocation of Akt to the membrane but also in modulation of its activity (
      • Frech M.
      • Andjelkovic M.
      • Ingley E.
      • Reddy K.K.
      • Falck J.R.
      • Hemmings B.A.
      ,
      • Wijkander J.
      • Holst L.S.
      • Rahn T.
      • Resjo S.
      • Castan I.
      • Manganiello V.
      • Belfrage P.
      • Degerman E.
      ). Another report, however, suggests that the lipid products of PI 3-kinase are required for interaction of Akt with a specific kinase, 3-phosphoinositide-dependent protein kinase 1, that regulates its activity (
      • Cohen P.
      • Alessi D.R.
      • Cross A.E.
      ). Whatever the mechanism, it does appear clear that the activation of PI 3-kinase is necessary for Akt activation.
      Our data tend to support the hypothesis that PI 3-kinase is a point of divergence in insulin signaling (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ) and that the direction of divergence may be tissue-specific. This divergence results in rapamycin-sensitive (pp70 S6 kinase) and -insensitive (Akt) regulation of metabolic gene expression. With respect to G6PDH regulation by insulin, the rapamycin-sensitive pathway is utilized.
      In conclusion, based on the inhibitor data, the activation of PI 3-kinase is required for regulation of G6PDH expression by insulin. Unlike studies on many other insulin-regulated metabolic genes, this regulation is also rapamycin-sensitive. The Ras/MAP kinase pathway does not appear to be involved in insulin regulation of the expression of G6PDH in liver. Controversy does exist as to the specificity of inhibitors and the validity of results obtained with inhibitors. In our studies, however, two structurally distinct inhibitors of Ras and PI 3-kinase were utilized as well as inhibitors of other signal proteins in the pathways to alleviate a concern over nonspecific interactions of these compounds. The concentration-dependent effect of these inhibitors on their respective target proteins in this system was also shown, attesting to the specificity of the inhibitor. Much work is still needed in the identification of downstream signal proteins of PI 3-kinase including transcription factors that bind to the insulin regulatory region of G6PDH and other genes. This will undoubtedly aid in the further delineation of the signal pathway(s) utilized in the regulation of metabolic processes.

      REFERENCES

        • Kahn C.R.
        • White M.F.
        • Shelson S.E.
        • Backer J.M.
        • Araki E.
        • Cheatham B.
        • Csermely P.
        • Folli F.
        • Goldstein B.J.
        • Huertas P.
        • Rothenberg P.L.
        • Saad M.J.
        • Siddle K.
        • Sun X-J.
        • Wilden P.A.
        • Yamada K.
        • Kahn S.A.
        Rec. Prog. Horm. Res. 1993; 48: 291-360
        • Myers M.G.
        • White M.F.
        Annu. Rev. Pharmacol. Toxicol. 1996; 36: 615-658
        • Lavan B.E.
        • Lane W.S.
        • Lienhard G.E.
        J. Biol. Chem. 1997; 272: 11439-11443
        • Skolnik E.Y.
        • Lee C-H.
        • Batzer A.
        • Vincenti L.M.
        • Zhou M.
        • Daly R.
        • Myers Jr., M.J.
        • Backer J.M.
        • Ullrich A.
        • White M.F.
        • Schlessinger J.
        EMBO J. 1993; 12: 1929-1935
        • Cheatham B.
        • Vlahos C.J.
        • Cheatham L.
        • Wang L.
        • Blenis J.
        • Kahn C.R.
        Mol. Cell. Biol. 1994; 14: 4902-4911
        • Yamauchi K.
        • Holt K.
        • Pessin J.E.
        J. Biol. Chem. 1993; 268: 14597-14600
        • Shepherd P.R.
        • Navé B.T.
        • Siddle K.
        Biochem. J. 1995; 305: 25-28
        • Yamamoto-Honda R.
        • Tobe K.
        • Kaburagi Y.
        • Ueki K.
        • Asai S.
        • Yachi M.
        • Shirouzu M.
        • Yodoi J.
        • Akanuma Y.
        • Yokoyama S.
        • Yazaki Y.
        • Kadowaki T.
        J. Biol. Chem. 1995; 270: 2729-2734
        • Cross D.A.E.
        • Alessi D.R.
        • Vandenheede J.R.
        • McDowell H.E.
        • Hundal H.S.
        • Cohen P.
        Biochem. J. 1994; 303: 21-26
        • Welsh G.I.
        • Foulstone E.J.
        • Young S.W.
        • Tavaré J.M.
        • Proud C.G.
        Biochem. J. 1994; 303: 15-20
        • Okada T.
        • Kawanok Y.
        • Sakakibara T.
        • Hazeki O.
        • Ui M.
        J. Biol. Chem. 1994; 269: 3568-3573
        • Kotani K.
        • Yonezawa K.
        • Hara K.
        • Ueda H.
        • Kitamurea Y.
        • Sakaue H.
        • Ando A.
        • Chavanieu A.
        • Calas B.
        • Grigorescu F.
        EMBO J. 1994; 13: 2313-2321
        • Gabbay R.A.
        • Sutherland C.
        • Gnudi L.
        • Kahn B.B.
        • O'Brien R.
        • Granner D.K.
        • Flier J.S.
        J. Biol. Chem. 1996; 271: 1890-1897
        • Sutherland C.
        • O'Brien R.
        • Granner D.K.
        J. Biol. Chem. 1995; 270: 15501-15506
        • Chung J.
        • Kuo C.J.
        • Crabtree G.R.
        • Blenis J.
        Cell. 1992; 69: 1227-1236
        • Price D.J.
        • Grove J.R.
        • Calvo V.
        • Avruch J.
        • Bierer B.E.
        Science. 1992; 257: 973-977
        • Yang S.
        • Dickson A.J.
        Biochem. J. 1995; 310: 375-378
        • Lefebvre V.
        • Mechin M.-C.
        • Louckx M.P.
        • Rider M.H.
        • Hue L.
        J. Biol. Chem. 1996; 271: 22289-22292
        • Pronk G.J.
        • McGlade J.
        • Pelicci G.
        • Pawson T.
        • Bos J.L.
        J. Biol. Chem. 1993; 268: 5748-5753
        • Kovacina K.S.
        • Roth R.A.
        Biochem. Biophys. Res. Commun. 1993; 192: 1303-1311
        • Sasaoka T.
        • Draznin B.
        • Leitner J.W.
        • Langlois W.J.
        • Olefsky J.M.
        J. Biol. Chem. 1994; 269: 10734-10738
        • Reusch J.E-B.
        • Bhuripanyo P.
        • Carel K.
        • Leitner J.W.
        • Hsieh P.
        • DePaolo D.
        • Draznin B.
        J. Biol. Chem. 1995; 270: 2036-2040
        • Pages G.
        • Lenormand P.
        • L'Allemain G.
        • Chambard J.C.
        • Meloche S.
        • Pouyssegur J.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323
        • Wang L.M.
        • Keegan A.D.
        • Paul W.E.
        • Heidaran M.A.
        • Gutkind J.S.
        • Pierce J.H.
        EMBO J. 1992; 11: 4899-4908
        • Davis R.
        J. Biol. Chem. 1993; 268: 14553-14556
        • Sakaue H.
        • Hara K.
        • Noguchi T.
        • Matozaki T.
        • Kotani K.
        • Ogawa W.
        • Yonezawa K.
        • Waterfield M.D.
        • Kasuga M.
        J. Biol. Chem. 1995; 270: 11304-11309
        • Sakaue M.
        • Bowtell D.
        • Kasuga M.
        Mol. Cell. Biol. 1995; 15: 379-388
        • Robinson L.J.
        • Razzack Z.F.
        • Lawrence Jr., J.
        • James D.E.
        J. Biol. Chem. 1993; 268: 26422-26427
        • Wiese R.J.
        • Mastick C.C.
        • Lazar D.F.
        • Saltiel A.R.
        J. Biol. Chem. 1995; 270: 3442-3446
        • O'Brien R.M.
        • Granner D.K.
        Physiol. Rev. 1996; 76: 1109-1161
        • Cohen P.
        Biochem. Soc. Trans. 1993; 21: 555-567
        • Magnuson M.A.
        • Andreone T.L.
        • Printz R.L.
        • Koch S.
        • Granner D.K.
        Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4838-4842
        • Iynedjian P.B.
        • Gjinovci A.
        • Renold A.E.
        J. Biol. Chem. 1988; 263: 740-744
        • O'Brien R.M.
        • Lucas P.C.
        • Forest C.
        • Magnuson M.A.
        • Granner D.K.
        Science. 1990; 249: 533-537
        • Short J.M.
        • Wynshaw-Boris A.
        • Short H.P.
        • Hanson R.W.
        J. Biol. Chem. 1986; 261: 9721-9726
        • Moustaid N.
        • Beyer R.S.
        • Sul H.S.
        J. Biol. Chem. 1994; 269: 5629-5634
        • Stapleton S.R.
        • Mitchell D.A.
        • Salati L.M.
        • Goodridge A.G.
        J. Biol. Chem. 1990; 265: 18442-18446
        • Kletzien R.F.
        • Harris P.K.W.
        • Foellmi L.A.
        FASEB J. 1994; 8: 174-181
        • Stumpo D.J.
        • Kletzien R.F.
        Eur. J. Biochem. 1984; 144: 497-501
        • Fritz R.S.
        • Stumpo D.S.
        • Kletzien R.F.
        Biochem. J. 1986; 237: 637-640
        • Rank K.B.
        • Harris P.K.
        • Ginsberg L.C.
        • Stapleton S.R.
        Biochim. Biophys. Acta. 1994; 1212: 90-92
        • Stapleton S.R.
        • Stevens G.J.
        • Teel J.F.
        • Rank K.B.
        • Berg E.A.
        • Wu J-Y.
        • Ginsberg L.C.
        • Kletzien R.F.
        Biochimie. 1993; 75: 971-976
        • Xu X-Q.
        • McGuire T.F.
        • Blaskovich M.A.
        • Sebti S.M.
        • Romero G.
        Arch. Biochem. Biophys. 1996; 326: 233-237
        • Cohen P.
        • Alessi D.R.
        • Cross A.E.
        FEBS Lett. 1997; 410: 3-10
        • Chomczynski P.
        • Sacchi N.
        Anal. Biochem. 1987; 162: 156-160
        • Fourney R.M.
        • Miyakoshi J.
        • Day R.S.
        • Paterson M.C.
        Focus. 1988; 10: 5-6
        • Kletzien R.F.
        • Prostko C.R.
        • Stumpo D.J.
        • McClung J.K.
        • Dreher K.L.
        J. Biol. Chem. 1985; 260: 5621-5626
        • Cleveland D.W.
        • Lopata M.A.
        • MacDonald R.J.
        • Cowan N.J.
        • Rutter W.J.
        • Kirschner M.W.
        Cell. 1980; 15: 95-105
        • Stapleton S.R.
        • Garlock G.
        • Foellmi-Adams L.
        • Kletzien R.F.
        Biochem. Biophys. Acta. 1997; 1355: 259-269
        • Tobe K.
        • Kadowaki T.
        • Hara K.
        • Gotoh Y.
        • Kosako H.
        • Matsuda S.
        • Tamemoto H.
        • Ueki K.
        • Akanuma Y.
        • Nishida E.
        • Yazaki Y.
        J. Biol. Chem. 1992; 267: 21089-21097
        • Serunian L.A.
        • Auger K.R.
        Methods Enzymol. 1991; 271: 77-79
        • Defeo-Jones D.
        • McAvoy E.M.
        • Jones R.E.
        • Vuocolo G.A.
        • Haskell K.M.
        • Wegrzyn R.J.
        • Oliff A.
        Mol. Cell. Biol. 1991; 11: 2307-2310
        • McGuire T.F.
        • Xu X-Q.
        • Corey S.J.
        • Romero G.G.
        • Sebti S.M.
        Biochem. Biophys. Res. Commun. 1994; 204: 399-406
        • Lazar D.F.
        • Wiese R.J.
        • Brady M.J.
        • Mastick C.
        • Waters S.B.
        • Yamauchi K.
        • Pessin J.E.
        • Cuatrecasas P.
        • Saltiel A.R.
        J. Biol. Chem. 1995; 270: 20801-20807
        • DeClue J.E.
        • Vass W.C.
        • Papageorge A.G.
        • Lowy D.R.
        • Willumsen B.M.
        Cancer Res. 1991; 51: 712-717
        • McGuire T.F.
        • Corey S.J.
        • Sebti S.M.
        J. Biol. Chem. 1993; 268: 22227-22230
        • Cox A.D.
        • Garcia A.M.
        • Westwick J.K.
        • Kowalczyk J.J.
        • Lewis M.D.
        • Brenner D.A.
        • Der C.J.
        J. Biol. Chem. 1994; 269: 19203-19206
        • Rodriquez-Viciana P.
        • Warne P.H.
        • Vanhaesebroeck B.
        • Waterfield M.D.
        • Downward J.
        EMBO. 1996; 15: 2442-2451
        • Whitehurst C.E.
        • Owaki H.
        • Bruder J.T.
        • Rapp U.R.
        • Geppert T.D.
        J. Biol. Chem. 1995; 270: 5594-5599
        • Blumer K.J.
        • Johnson G.L.
        Trends Biochem. Sci. 1994; 19: 236-240
        • Gille D.
        • Sharrocks A.D.
        • Shaw P.E.
        Nature. 1992; 358: 414-417
        • Pulverer B.J.
        • Kyriakis J.M.
        • Avruch J.
        • Nikolakaki E.
        • Woodgett J.R.
        Nature. 1991; 353: 670-674
        • Clarke J.F.
        • Young P.W.
        • Yonezawa K.
        • Kasuga M.
        • Holman G.D.
        Biochem. J. 1994; 300: 631-635
        • Franke T.F.
        • Yang S.
        • Chan T.O.
        • Datta K.
        • Kaziauskas A.
        • Morrison D.K.
        • Kaplan D.R.
        • Tsichlis P.N.
        Cell. 1995; 81: 727-736
        • Kohn A.D.
        • Takeuchi F.
        • Roth R.A.
        J. Biol. Chem. 1996; 271: 21920-21926
        • Frech M.
        • Andjelkovic M.
        • Ingley E.
        • Reddy K.K.
        • Falck J.R.
        • Hemmings B.A.
        J. Biol. Chem. 1997; 272: 8474-8481
        • Wijkander J.
        • Holst L.S.
        • Rahn T.
        • Resjo S.
        • Castan I.
        • Manganiello V.
        • Belfrage P.
        • Degerman E.
        J. Biol. Chem. 1997; 272: 21520-21526