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Membrane-targeted Phosphatidylinositol 3-Kinase Mimics Insulin Actions and Induces a State of Cellular Insulin Resistance*

Open AccessPublished:May 14, 1999DOI:https://doi.org/10.1074/jbc.274.20.14306
      Phosphatidylinositol (PI) 3-kinase plays an important role in various insulin-stimulated biological responses including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between PI 3-kinase and these biological responses is still unclear. We have investigated whether targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes is sufficient and necessary to induce PI 3-kinase dependent signaling responses, characteristic of insulin action. We overexpressed Myc-tagged, membrane-targeted p110 (p110CAAX), and wild-type p110 (p110WT) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer. Overexpressed p110CAAX exhibited ∼2-fold increase in basal kinase activity in p110 immunoprecipitates, that further increased to ∼4-fold with insulin. Even at this submaximal PI 3-kinase activity, p110CAAX fully stimulated p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas, p110WT had little or no effect on these downstream effects. Interestingly p110CAAX did not activate MAP kinase, despite its stimulation of p21 ras. Surprisingly, p110CAAX did not increase basal glycogen synthase activity, and inhibited insulin stimulated activity, indicative of cellular resistance to this action of insulin. p110CAAX also inhibited insulin stimulated, but not platelet-derived growth factor-stimulated mitogen-activated protein kinase phosphorylation, demonstrating that the p110CAAX induced inhibition of mitogen-activated protein kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells. Moreover, p110CAAX stimulated IRS-1 Ser/Thr phosphorylation, and inhibited IRS-1 associated PI 3-kinase activity, without affecting insulin receptor tyrosine phosphorylation, suggesting that it may play an important role as a negative regulator for insulin signaling. In conclusion, our studies show that membrane-targeted PI 3-kinase can mimic a number of biologic effects normally induced by insulin. In addition, the persistent activation of PI 3-kinase induced by p110CAAX expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact.
      One of the major physiological functions of insulin is to stimulate glucose transport into insulin-sensitive tissues by eliciting translocation of the major insulin responsive glucose transporter, GLUT4 from an intracellular compartment to the plasma membrane (
      • Cushman S.W.
      • Wardzala L.J.
      ,
      • Suzuki K.
      • Kono T.
      ). However, the signaling events that mediate insulin-stimulated glucose transport and GLUT4 translocation are poorly understood. Binding of insulin to its receptor results in receptor autophosphorylation and activation of the receptor tyrosine kinase, followed by tyrosine phosphorylation of several intermediate proteins, such as, insulin receptor substrates: IRS-1, 2, 3, 4, and the adaptor protein, Shc (
      • Lavan B.E.
      • Fantin V.R.
      • Chang E.T.
      • Lane W.S.
      • Keller S.R.
      • Lienhard G.E.
      ,
      • Lavan B.E.
      • Lane W.S.
      • Lienhard G.E.
      ,
      • White M.F.
      • Kahn C.R.
      ). Tyrosine-phosphorylated insulin receptor substrates and the insulin receptor itself, then bind to Src homology 2 (SH2)
      The abbreviations used are: SH2, Src homology domain 2; PI, phosphoinositide; SH3, Src homology domain 3; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PCR, polymerase chain reaction; m.o.i., multiplicity of infection; PAGE, polyacrylamide gel electrophoresis; GSK3, glycogen synthase kinase 3
      1The abbreviations used are: SH2, Src homology domain 2; PI, phosphoinositide; SH3, Src homology domain 3; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PCR, polymerase chain reaction; m.o.i., multiplicity of infection; PAGE, polyacrylamide gel electrophoresis; GSK3, glycogen synthase kinase 3
      domain containing proteins, which further propagates downstream signals.
      Phosphatidylinositol (PI) 3-kinase, a dual protein and lipid kinase is one such signaling molecule (
      • Seger R.
      • Krebs E.G.
      ). It consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. Several isoforms of the regulatory subunit, p55α, p55PIK, p85α, p85β, and two isoforms of the catalytic subunit, p110α and p110β, have been identified. The p85 subunit is composed of an NH2-terminal Src homology 3 (SH3) domain and two SH2 domains (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). The SH2 domains flank the region where p110 associates with p85. The SH2 domains interact with phosphotyrosine residues leading to activation of the p110 catalytic subunit. The p110 subunit phosphorylates phosphoinositides at the 3′-position of the inositol ring to generate phosphoinositides 3-P, 3,4-P2, and 3,4,5-P3 (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). PI 3-kinase also phosphorylates proteins on serine/threonine residues (
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      ,
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ,
      • Tanti J.F.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ).
      Ras is another signaling protein downstream of IRS-1 and Shc. After insulin stimulation, tyrosine phosphorylated Shc, and IRS-1, to a lesser extent, interact with another SH2 domain containing adaptor protein-Grb2, which is pre-associated with Sos, a guanine nucleotide exchange factor that promotes the formation of the active GTP-bound state of Ras (
      • Sakaue M.
      • Bowtell D.
      • Kasuga M.
      ). Stimulated Ras then activates a cascade of protein serine/threonine kinases, which include Raf, MEK, and the MAP kinases. Although PI 3-kinase and Ras appear to be on separate pathways branching from IRS-1, both have been implicated in pathways that mediate the mitogenic actions of insulin, whereas the metabolic effects of insulin are primarily activated by PI 3-kinase dependent steps.
      Several lines of evidence indicate that activation of PI 3-kinase by insulin is required for GLUT4 translocation. For example, the PI 3-kinase inhibitors, wortmannin and LY 294002 prevent GLUT4 translocation and stimulation of glucose transport in rat and 3T3-L1 adipocytes (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). Dominant-negative mutants of the 85-kDa subunit of PI 3-kinase can also inhibit GLUT4 translocation in response to insulin (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      ,
      • Kotani K.
      • Carozzi A.J.
      • Sakaue H.
      • Hara K.
      • Robinson L.J.
      • Clark S.F.
      • Yonezawa K.
      • James D.E.
      • Kasuga M.
      ,
      • Quon M.J.
      • Chen H.
      • Ing B.L.
      • Liu M.L.
      • Zarnowski M.J.
      • Yonezawa K.
      • Kasuga M.
      • Cushman S.W.
      • Taylor S.I.
      ). However, several other observations suggest that, although necessary, PI 3-kinase activation is not sufficient to promote glucose transporter translocation. Indeed, growth factors such as platelet-derived growth factor (PDGF) can stimulate PI 3-kinase as efficiently as insulin, but have only a minor effect on GLUT4 translocation (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ,
      • Gould G.W.
      • Merrall N.W.
      • Martin S.
      • Jess T.J.
      • Campbell I.W.
      • Calderhead D.M.
      • Gibbs E.M.
      • Holman G.D.
      • Plevin R.J.
      ). Similarly, interleukin 4, which induces tyrosine phosphorylation of IRS-1 and PI 3-kinase activation, does not stimulate GLUT4 translocation in L6 myoblasts (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ). Furthermore, subcellular fractionation analyses indicates that insulin, unlike other growth factors, stimulates PI 3-kinase activity not only in the plasma membrane fraction but also in the low density microsomal compartment (
      • Kelly K.L.
      • Ruderman N.B.
      ,
      • Nave B.T.
      • Haigh R.J.
      • Hayward A.C.
      • Siddle K.
      • Shepherd P.R.
      ,
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ) and possibly even in GLUT4 containing subfractions of the low density microsomal of adipocytes (
      • Clark S.F.
      • Martin S.
      • Carozzi A.J.
      • Hill M.M.
      • James D.E.
      ,
      • Heller-Harrison R.A.
      • Morin M.
      • Guilherme A.
      • Czech M.P.
      ). Thus, it appears that insulin-mediated subcompartmentalization of PI 3-kinase may be unique and might be key to the specificity of the effect of insulin on glucose transport.
      The aim of this study was to determine whether targeting of PI 3-kinase catalytic subunit to membranous structures is sufficient to trigger signaling events downstream of PI 3-kinase. This allows us to directly study PI 3-kinase-regulated cellular processes in the absence of insulin and to determine whether PI 3-kinase activation is sufficient to trigger signaling events specific for insulin. Furthermore, it avoids potential problems associated with the use of PI 3-kinase inhibitors in elucidating the actions of this enzyme. We, and, others have recently demonstrated that increased PI 3-kinase activity induced by expression of a constitutively active p110 subunit (p110*) can induce GLUT4 translocation (
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ), but it stimulates glucose transport only partially in the absence of insulin (
      • Frevert E.U.
      • Kahn B.B.
      ). In contrast, our membrane localized form of the p110 subunit of PI 3-kinase resulted in activation of downstream mitogenesis effects in COS-7 cells (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ). Since gene transfer in 3T3-L1 adipocytes by conventional methods is inefficient, in the current experiments we utilized, adenovirus-mediated, high efficiency gene transfer procedures (
      • Frevert E.U.
      • Kahn B.B.
      ,
      • Katagiri H.
      • Asano T.
      • Ishihara H.
      • Inukai K.
      • Shibasaki Y.
      • Kikuchi M.
      • Yazaki Y.
      • Oka Y.
      ,
      • Sharma P.M.
      • Egawa K.
      • Gastafson T.A.
      • Martin J.L.
      • Olefsky J.M.
      ), and created an adenoviral vector containing the p110-α subunit of PI 3-kinase incorporating a CAAX box at the COOH terminus in order to target the p110 subunit to cellular membranes. Our studies showed that expression of the membrane-targeted p110 subunit of PI 3-kinase in 3T3-L1 adipocytes were sufficient to induce PI 3-kinase dependent downstream signaling events, including glucose transport.

      DISCUSSION

      In this study, we have used adenoviral-mediated gene transfer to assess whether targeting of the catalytic p110 subunit of PI 3-kinase to cellular membranes by incorporating a CAAX box at the COOH terminus (p110CAAX) is sufficient to induce PI 3-kinase dependent signaling responses, characteristic of insulin action, in 3T3-L1 adipocytes. We show that when appropriately targeted, even modest levels of PI 3-kinase are sufficient to trigger full activation of the downstream serine/threonine kinases, Akt and p70 S6 kinase, and also causes stimulation of glucose transport equal to the effect of insulin. Surprisingly, insulin-mediated glycogen synthase activity was completely blocked in cells expressing p110CAAX. Furthermore, p110CAAX stimulated serine/threonine phosphorylation of IRS-1, and inhibited IRS-1 associated PI 3-kinase activity. Another major finding is that the membrane-localized PI 3-kinase activity was sufficient to mimic insulin-induced formation of GTP-bound p21 ras. Last, we found that expression of p110CAAX led to inhibition of insulin-mediated MAP kinase activation, whereas PDGF-mediated MAP kinase activation was unaffected. These results lead to several predictions and conclusions.

      p110CAAX Mimics Insulin Actions

      We demonstrate that membrane-targeted p110 (p110CAAX) promotes insulin-independent PI 3-kinase activity and is sufficient to maximally stimulate glucose uptake, in a wortmannin-sensitive manner. The level of 2-deoxyglucose uptake achieved in response to p110CAAXexpression was comparable to that seen in insulin-stimulated, control adipocytes, whereas, non-targeted wild-type p110α (p110WT) had only a slight effect on 2-deoxyglucose uptake. Since, the p110 subunit of PI 3-kinase contains a COOH-terminal membrane targeting farnesylation sequence, it seems likely that the overexpressed p110CAAX protein results in increased PI 3-kinase activity predominantly in membrane fractions, similar to insulin stimulation of endogenous PI 3-kinase. This implies that glucose uptake is not merely a function of the amount of PI 3-kinase present, but that its appropriate membrane localization is critical as well. This conclusion is quite consistent with other kinds of studies in the literature. For example, PDGF, as well as other growth factors, can stimulate PI 3-kinase in 3T3-L1 adipocytes, equally well as insulin, but only insulin leads to glucose transport stimulation (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ,
      • Gould G.W.
      • Merrall N.W.
      • Martin S.
      • Jess T.J.
      • Campbell I.W.
      • Calderhead D.M.
      • Gibbs E.M.
      • Holman G.D.
      • Plevin R.J.
      ). These findings suggested that insulin induced subcompartmentalization of PI 3-kinase is necessary for metabolic signaling. Consistent with this, Frevert and Khan (
      • Frevert E.U.
      • Kahn B.B.
      ) showed that co-expression of both the catalytic p110α subunit and the inter-SH2 region of the p85 regulatory subunit of PI 3-kinase in 3T3-L1 adipocytes led to a much higher level of PI 3-kinase activity than seen with insulin stimulation alone, but it had only a partial effect to stimulate glucose transport without insulin. In addition, when PI 3-kinase was activated by thiophosphorylated peptides, corresponding to the phosphotyrosine binding motif of the p85 subunit of PI 3-kinase, only a minor effect on Glut 4 translocation was observed (
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux P.E.
      • Gibbs E.M.
      • Lienhard G.E.
      ). Tantiet al. (
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ) co-transfected rat adipose cells by electroporation with epitope-tagged Glut 4 and with either a constitutively active (p110*) or a kinase inactive form of p110kd (
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ). Co-transfection with the active version of p110* resulted in stimulation of epitope-tagged Glut 4 translocation, similar to insulin, and these workers also found that the p110* was localized to the same intracellular compartment as the endogenous PI 3-kinase. Taken together with our current results, these studies support the conclusion that active PI 3-kinase is sufficient to stimulate glucose transport activity, only if it is targeted to the proper subcellular membranous compartment.
      We further examined other targets of insulin action which are thought to be downstream of PI 3-kinase. Akt is a serine/threonine kinase that is activated by insulin. It is activated by a dual mechanism involving the binding of PI-(3,4)-P2 to its PH domain, as well as by serine/threonine phosphorylation by one or more Akt kinases, which may, themselves, be stimulated by the lipid products of PI 3-kinase (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ). Several lines of evidence suggest that Akt functions downstream of PI 3-kinase, e.g. insulin stimulated Akt kinase activity is inhibitable by wortmannin, a PI 3-kinase specific inhibitor, and PDGF receptor mutants that fail to activate PI 3-kinase, also fail to activate Akt. Overexpression of a constitutively active Akt in 3T3-L1 adipocytes results in increased glucose uptake and Glut 4 translocation in the absence of insulin (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ). Consistent with these findings, our data show that Akt activation is dependent on PI 3-kinase activity, and that insulin and p110CAAX induced Akt activation is inhibitable by wortmannin, indicating that Akt activation is dependent on PI 3-kinase enzymatic function.
      We also determined whether PI 3-kinase-mediated Akt activation would lead to p70 S6 kinase stimulation, since it has been shown that p70 S6 kinase is stimulated by constitutively active Akt (
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      ) and blocked by inhibitors of PI 3-kinase (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Weng Q.P.
      • Andrabi K.
      • Klippel A.
      • Kozlowski M.T.
      • Williams L.T.
      • Avruch J.
      ). Indeed, we found that overexpression of the membrane-targeted p110CAAXled to activation of p70 S6 kinase, which was completely inhibitable by wortmannin (Fig. 4). Our results are supported by the study of Wenget al. (
      • Weng Q.P.
      • Andrabi K.
      • Klippel A.
      • Kozlowski M.T.
      • Williams L.T.
      • Avruch J.
      ), which showed that transfection of a constitutively active form of PI 3-kinase (p110*) into 293 cells resulted in a 20–30-fold increase in cellular PI 3-kinase activity, that resulted in activation of p70 S6 kinase by phosphorylation at Thr-252. Wortmannin resulted in selective dephosphorylation at Thr-252 concomitant with inhibition of p70 S6 kinase activity. Furthermore, Klippel et al. (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ) reported that in COS-7 cells, expression of membrane-localized p110 is sufficient to trigger downstream responses, characteristic of insulin action, including stimulation of Akt and p70 S6 kinase. Their study further adds that these responses can also be triggered by expression of p110*, that is cytosolic, but exhibits a high specific activity. However, they observed maximum activation of downstream responses in cells expressing the membrane-localized p110. Thus, insulin treatment activates and targets PI 3-kinase to specific membrane compartments, and this action is mimicked by p110CAAX, which is sufficient to trigger downstream responses characteristic of insulin action, including stimulation of Akt, p70 S6 kinase, and glucose transport.
      The role of PI 3-kinase in Ras-mediated signaling is unclear. An association between p21 ras and PI 3-kinase was first demonstrated by co-immunoprecipitation in insulin and insulin-like growth factor-1 stimulated, Ras-transformed epithelial cells by Sjolander et al. (
      • Sjolander A.
      • Yamamoto K.
      • Huber B.E.
      • Lapetina E.G.
      ). Subsequently, Ras was shown to bindin vitro to the p110 subunit of PI 3-kinase by Rodriguez-Viciana et al. (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). However, the relative position of PI 3-kinase with respect to Ras is confusing. Conflicting data exists suggesting that PI 3-kinase could be upstream, downstream, or independent of Ras. These alternate results are perhaps related to cell-type differences. Rodriguez-Viciana et al. (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ) reported that a point mutation of the p110 subunit of PI 3-kinase at the Ras-GTP binding site elevated PI 3-kinase activity in COS cells, and the interaction of Ras-GTP, but not Ras-GDP, with PI 3-kinase led to an increase in its enzymatic activity (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). These data suggest that Ras is upstream of PI 3-kinase. However, our data are consistent with the idea that PI 3-kinase is upstream and can activate Ras (Fig. 6). We find that membrane-targeted activated PI 3-kinase activates p21 ras, resulting in increased formation of p21 ras GTP, equal to the effect of insulin. This interpretation is in agreement with earlier data from our own laboratory, in which we reported that microinjection of dominant-negative PI 3-kinase, or PI 3-kinase inhibitory antibodies, into rat fibroblasts inhibited insulin-induced Fos induction, which was rescued by activated (T-24) Ras (
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ). Similarly, studies by Hu et al. (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ) suggest that Ras is downstream of PI 3-kinase because transfection of constitutively active PI 3-kinase resulted in Fos induction, which was blocked by both dominant-negative Ras and Raf. They also found elevated levels of GTP-bound Ras in cells transfected with constitutively active PI 3-kinase.

      Cellular Insulin Resistance Induced by p110CAAX

      Interestingly, we found that p110CAAX did not mimic all of insulin's actions, and, in some cases led to a decrease in insulin signaling indicating a partial, and selective insulin resistant state. For example, we found that p110CAAX did not mimic the effect of insulin to stimulate glycogen synthesis. Not only did p110CAAX expression fail to enhance basal glycogen synthase activity, but it completely inhibited the ability of insulin to stimulate glycogen synthesis. Activation of glycogen synthase by insulin involves a coordinated response, including phosphorylation induced inactivation of glycogen synthase kinase 3 (GSK3) and activation of protein phosphatase 1, by phosphorylation of its G subunit (pp1G) (
      • Shulman R.G.
      • Bloch G.
      • Rothman D.L.
      ). It has been suggested that GSK3 is a downstream target of Akt, which, in turn, is dependent on PI 3-kinase activity. Constitutively active Akt inhibits insulin's ability to stimulate glycogen synthesis in 3T3-L1 adipocytes (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Ueki K.
      • Yamamoto-Honda R.
      • Kaburagi Y.
      • Yamauchi T.
      • Tobe K.
      • Burgering B.M. Th.
      • Coffer P.J.
      • Komuro I.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Brady J.J.
      • Bourbonais F.J.
      • Saltiel A.R.
      ), and our data also show that activation of Akt by the membrane-localized p110CAAX is not sufficient to cause glycogen synthase activation in 3T3-L1 adipocytes. In theory, activated PI 3-kinase and Akt should inactivate GSK3 by phosphorylation leading to stimulation of glycogen synthase activity, whereas we, and others, show that p110CAAX or constitutively active Akt inhibits insulin effects on this enzyme (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Ueki K.
      • Yamamoto-Honda R.
      • Kaburagi Y.
      • Yamauchi T.
      • Tobe K.
      • Burgering B.M. Th.
      • Coffer P.J.
      • Komuro I.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Brady J.J.
      • Bourbonais F.J.
      • Saltiel A.R.
      ). However, it has been shown recently that GSK3 expression is either very low, or absent in 3T3-L1 adipocytes (
      • Benjamin W.B.
      • Pentyala S.N.
      • Woodgett J.R.
      • Hod Y.
      • Marshak D.
      ,
      • Cross D.A.
      • Watt P.W.
      • Shaw M.
      • van der Kaay J.
      • Downes C.P.
      • Holder J.C.
      • Cohen P.
      ,
      • Moule S.K.
      • Welsh G.I.
      • Edgell N.J.
      • Foulstone E.J.
      • Proud C.G.
      • Denton R.M.
      ). Therefore, a role for GSK3 in our results is problematic. Perhaps the low (or absent) expression of GSK3 explains why p110CAAX does not stimulate glycogen synthase by itself. An alternate pathway for glycogen synthase activation involves pp1, which has been suggested to be downstream of the IRS-1/Shc-MAP kinase pathway by some investigators (
      • Dent P.
      • Lavoinne A.
      • Nakielny S.
      • Caudwell F.B.
      • Watt P.
      • Cohen P.
      ), but a number of reports have indicated that this is not the case (
      • Lawrence J.C.
      • Roach P.J.
      ). In addition, earlier results show that the MEK inhibitor PD098059 does not lead to a decrease in insulin stimulation of glycogen synthesis (
      • Sharma P.M.
      • Egawa K.
      • Huang Y.
      • Martin J.L.
      • Huvar I.
      • Boss G.R.
      • Olefsky J.M.
      ). Thus, a role for MAPK in the regulation of glycogen synthesis seems unlikely. Another possibility is that IRS-1 directly or through its interacting proteins, but independent of PI 3-kinase, might be involved in the inhibition of glycogen synthase activity. Indeed, we find that membrane-targeted p110CAAX serine/threonine phosphorylates IRS-1, which is inhibitable by wortmannin. This in turn prevents IRS-1 tyrosine phosphorylation and downstream signaling (Fig.7).
      Although the precise mechanisms underlying the p110CAAX induced resistance are unknown, the current results provide some interesting insights. First, despite the fact that p110CAAX stimulated Ras activation, it had no effect to stimulate MAP kinase phosphorylation, indicating a blockade of MAP kinase activation at a site downstream of Ras. Furthermore, in p110CAAX expressing cells, insulin had no effect to stimulate MAP kinase phosphorylation, compared with a robust stimulation in control cells. Since insulin is thought to stimulate MAP kinase activation by activation of Ras (
      • Sakaue M.
      • Bowtell D.
      • Kasuga M.
      ), these findings also point to a post-Ras blockade of the MAP kinase pathway. On the other hand, p110CAAX expression did not inhibit PDGF-stimulated MAP kinase phosphorylation, and this is consistent with the interpretation that PDGF can lead to MAP kinase activation through a Ras-dependent as well as a non-Ras dependent pathway (
      • Duckworth B.C.
      • Cantley L.C.
      ), and we would propose that expression of p110CAAX inhibits only the Ras-dependent input into MAP kinase. These findings also demonstrate that the p110CAAX induced inhibition of MAP kinase and insulin signaling is specific, and not due to some toxic or nonspecific effect on the cells.
      Taken together, our results are consistent with the view that p110CAAX expression inhibits the actions of insulin at a step distal to Ras activation, leading to inhibition of MAP kinase, and, possibly, glycogen synthase activation. Importantly, the cellular insulin resistance induced by p110CAAX in these cells is not global. Thus, p110CAAX expression stimulated AKT as well as p70 S6 kinase phosphorylation, and insulin had a further effect when added to p110CAAX expressing cells. This would argue that this model of cellular insulin resistance is rather unique, in that some of the insulin signaling pathways are inhibited, whereas, others are intact. The fact that persistent activation of PI 3-kinase leads to desensitization of subsequent downstream events is reminiscent of the fact that hyperinsulinemia (either in vitro orin vivo) will also lead to a state of cellular insulin resistance. However, hyperinsulinemia-induced insulin resistance affects all of insulin's actions, whereas, persistent PI 3-kinase activation selectively inhibits specific insulin signaling. Since insulin's biologic effects are pleiotrophic with engagement of multiple divergent signaling pathways, further study of these cells may enhance our understanding of which signaling pathways connect to which biologic effects.
      In summary, our studies show that PI 3-kinase activity can mimic a number of biologic effects normally induced by insulin, but that membrane targeting of this enzyme is necessary for activation of these events. In addition, the persistent activation induced by p110CAAX expression leads to desensitization of specific signaling pathways. Interestingly, the state of cellular insulin resistance is not global, in that some of insulin's actions are inhibited, whereas others are intact.

      Acknowledgments

      We thank Dr. Christopher B. Newgard for providing the adenovirus plasmids and James G. Nelson for providing differentiated 3T3-L1 adipocytes. We thank Matt Hickmann for help in establishing the glycogen synthase assay.

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