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Platelet-derived Growth Factor Inhibits Insulin Stimulation of Insulin Receptor Substrate-1-associated Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes without Affecting Glucose Transport*

Open AccessPublished:September 25, 1998DOI:https://doi.org/10.1074/jbc.273.39.25139
      Phosphatidylinositol 3-kinase (PI3K) activation is necessary for insulin-responsive glucose transporter (GLUT4) translocation and glucose transport. Insulin and platelet-derived growth factor (PDGF) stimulate PI3K activity in 3T3-L1 adipocytes, but only insulin is capable of stimulating GLUT4 translocation and glucose transport. We found that PDGF causes serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) in 3T3-L1 cells, measured by altered mobility on SDS-polyacrylamide gel, and this leads to a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1. The PI3K inhibitors wortmannin and LY294002 inhibit the PDGF-induced phosphorylation of IRS-1, whereas the MEK inhibitor PD98059 was without a major effect. PDGF pretreatment for 60–90 min led to a marked 80–90% reduction in insulin stimulatable phosphotyrosine and IRS-1-associated PI3K activity. We examined the functional consequences of this decrease in IRS-1-associated PI3K activity. Interestingly, insulin stimulation of GLUT4 translocation and glucose transport was unaffected by 60–90 min of PDGF preincubation. Furthermore, insulin activation of Akt and p70s6kinase, kinases downstream of PI3K, was unaffected by PDGF pretreatment. Wortmannin was capable of blocking these insulin actions following PDGF pretreatment, suggesting that PI3K was still necessary for these effects. In conclusion, 1) PDGF causes serine/threonine phosphorylation of IRS-1, and PI3K, or a kinase downstream of PI3K, mediates this phosphorylation. 2) This PDGF-induced phosphorylation of IRS-1 leads to a significant decrease in insulin-stimulated PI3K activity. 3) PDGF has no effect on insulin stimulation of Akt, p70s6kinase, GLUT4 translocation, or glucose transport. 4) This suggests the existence of an IRS-1-independent pathway leading to the activation of PI3K, Akt, and p70s6kinase; GLUT4 translocation; and glucose transport.
      EGF
      epidermal growth factor
      IRS
      insulin receptor substrate
      PI3K
      phosphatidylinositol 3-kinase
      PDGF
      platelet-derived growth factor
      MAPK
      microtubule-associated protein kinase
      GLUT4
      insulin-responsive glucose transporter
      TNFα
      tumor necrosis factor α
      pY
      phosphotyrosine
      PAGE
      polyacrylamide gel electrophoresis
      PIP2
      phosphatidylinositol 3,4-bisphosphate
      PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      ATP
      adenosine triphosphate.
      Tyrosine kinase receptors such as the insulin receptor, the epidermal growth factor (EGF)1 receptor, and the platelet-derived growth factor (PDGF) receptor activate many of the same signaling cascades. The two most prominent shared pathways are the phosphatidylinositol 3-kinase (PI3K) and the microtubule-associated protein kinase (MAPK) pathways. Activation of MAPK leads to mitogenic progression and may be involved in differentiation (for review, see Ref.
      • Seger R.
      • Krebs E.G.
      ). Numerous studies using inhibitors of PI3K activity, expression of constitutively active PI3K, and microinjection of dominant negative inhibitors of PI3K have all demonstrated that PI3K is necessary for the mitogenic effects of many growth factors and is both necessary and sufficient for the metabolic effects of insulin (
      • Conricode K.M.
      ,
      • Margalet-Sanchez V.
      • Goldfine I.D.
      • Vlahos C.J.
      • Sung C.K.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Berger J.
      • Hayes N.
      • Szalkowksi D.M.
      • Zhang B.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ).
      PI3K is composed of two subunits, an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110). The p85 subunit is composed of an N-terminal Src-homology 3 (SH3) domain and 2 Src-homology 2 (SH2) domains. The SH2 domains flank the region where the p110 associates with p85. The SH2 domains interact with phosphotyrosine residues, leading to subsequent activation of the p110 catalytic subunit (for review, see Ref.
      • Kapeller R.
      • Cantley L.C.
      ). Insulin stimulation leads to the tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1/2) and the association of PI3K with the SH2 binding sites contained within these proteins. PDGF and EGF activate PI3K through its association with phosphotyrosines located in the C terminus of their receptors.
      PI3K exhibits both lipid and protein kinase activities. PI3K phosphorylates the D3 position of the inositol ring of phosphatidylinositols, causing the production of phosphatidylinositol 3,4-bisphosphate (PIP2), and phosphatidylinositol 3,4,5-trisphosphate (PIP3) (
      • Kapeller R.
      • Cantley L.C.
      ). The role of PIP2 and PIP3, the primary products of PI3K, has been elusive. Recently, a role for PIP2 and PIP3 in the activation of an Akt kinase has been discovered (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ). PI3K 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.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ,
      • 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.
      ). The insulin receptor substrate-1 (IRS-1) is an in vitro substrate for this activity (
      • 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.
      ).
      3T3-L1 adipocytes provide a means to study the metabolic effects of insulin in a cell culture system. These cells express insulin, PDGF, and EGF receptors. Each of these ligands is capable of activating MAPK in these cells, but only insulin and PDGF activate PI3K (
      • Wiese R.J.
      • Mastik C.C.
      • Lazar D.F.
      • Saltiel A.R.
      ). Despite the disparity in insulin and PDGF receptor number in these cells, PDGF and insulin activate PI3K to a similar extent. Because the activation of PI3K has been shown to be necessary for the metabolic effects of insulin, such as the translocation of the insulin-stimulated glucose transporter, GLUT4, to the cell membrane, glucose uptake, and glycogen synthesis, it has been puzzling as to how insulin, but not PDGF, causes translocation of GLUT4 and glucose transport when both of these ligands cause stimulation of PI3K (
      • Conricode K.M.
      ,
      • Margalet-Sanchez V.
      • Goldfine I.D.
      • Vlahos C.J.
      • Sung C.K.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Berger J.
      • Hayes N.
      • Szalkowksi D.M.
      • Zhang B.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ).
      In this study, we investigated the interplay between insulin and PDGF signaling cascades in 3T3-L1 adipocytes. Insulin and PDGF lead to different time courses of activation of PI3K, with insulin causing a sustained activation of PI3K and PDGF generating transient effects. PDGF pretreatment stimulates the serine/threonine phosphorylation of IRS-1, interfering with the ability of insulin to activate PI3K. Serine phosphorylation of serine 612, one of the major serine phosphorylation sites in IRS-1, may inhibit tyrosine phosphorylation of tyrosine 608, a site of IRS-1/PI3K interaction (
      • De Fea K.
      • Roth R.A.
      ). Importantly, this inhibition of IRS-1-associated PI3K activity had no effect on insulin induction of glucose transport, GLUT4 translocation, or Akt and p70s6kinase phosphorylation. These biologic effects of insulin remain PI3K-dependent, however, because they are blocked by PI3K inhibitors. These results indicate that PI3K can be activated under the direction of insulin by an IRS-1/IRS-2-independent pathway. It appears that whereas PI3K activity is necessary for the metabolic effects of insulin, IRS-1 phosphorylation is not essential.

      DISCUSSION

      We and others have previously shown that PI3K activity, as well as proper targeting of this enzyme complex, are necessary for insulin-stimulated glucose transport (
      • Conricode K.M.
      ,
      • Margalet-Sanchez V.
      • Goldfine I.D.
      • Vlahos C.J.
      • Sung C.K.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Berger J.
      • Hayes N.
      • Szalkowksi D.M.
      • Zhang B.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Nave B.T.
      • Haigh R.J.
      • Hayward A.C.
      • Siddle K.
      • Shepherd P.R.
      ,
      • Sharma P.M.
      • Egawa K.
      • Gustafson T.A.
      • Martin J.L.
      • Olefsky J.M.
      ). PDGF can stimulate PI3K activity in 3T3-L1 adipocytes but does not lead to stimulation of glucose transport (
      • Conricode K.M.
      ,
      • Margalet-Sanchez V.
      • Goldfine I.D.
      • Vlahos C.J.
      • Sung C.K.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). In addition, PDGF treatment can lead to serine/threonine phosphorylation of IRS-1 (
      • Ricort J.-M.
      • Tanti J.-F.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ). In the current study, we have taken advantage of these findings to study the role of PDGF on the mechanisms controlling insulin-stimulated glucose transport. The major findings of these experiments are that PDGF can cause serine/threonine phosphorylation of IRS-1, as measured by altered mobility on a SDS-polyacrylamide gel. This effect of PDGF is inhibited by preincubation with wortmannin, indicating that the p110 subunit of PI3K is responsible for IRS-1 phosphorylation. We find that this serine/threonine phosphorylation of IRS-1 leads to a marked decrease in the ability of IRS-1 to associate with PI3K following insulin stimulation. Surprisingly, this marked decrease in insulin-stimulated IRS-1-associated PI3K activity did not lead to any measurable impairment of several downstream signaling effects of insulin, such as stimulation of GLUT4 translocation, glucose transport, and Akt and p70s6kinase phosphorylation. These results lead to several conclusions and interpretations.
      Insulin leads to extensive tyrosine phosphorylation of IRS-1, which promotes association with the SH2 domains of the p85 subunit of PI3K, leading to stimulation of PI3K activity (
      • Backer J.M.
      • Myers M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Mirapaipeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ). IRS-1 can be serine/threonine-phosphorylated in vitro by the p110 subunit (
      • 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.
      ), as well as MAPK (
      • De Fea K.
      • Roth R.A.
      ), and in vivo, tumor necrosis factor α (
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari A.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      ) and PDGF (
      • Ricort J.-M.
      • Tanti J.-F.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ) can cause serine/threonine phosphorylation of IRS-1. Serine phosphorylation of IRS-1 has been shown to prevent its tyrosine phosphorylation by the insulin receptor (
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari A.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      ) and, consequently, IRS-1 association with PI3K and subsequent PI3K activity (
      • Mothe I.
      • Van Obberghen E.
      ). We explored the functional consequences of these effects. We found that PDGF treatment of 3T3-L1 adipocytes led to serine/threonine phosphorylation of IRS-1, as monitored by mobility shift on SDS-polyacrylamide gel. This effect of PDGF can be inhibited by treatment with the PI3K inhibitors wortmannin and LY294002, but it is not greatly affected by treatment with the MEK inhibitor PD98059. These results indicate that although serine kinases, such as MAPK, can phosphorylate IRS-1 in vitro, (
      • De Fea K.
      • Roth R.A.
      ) in vivophosphorylation following stimulation with PDGF is mediated by the p110 subunit of PI3K or a serine/threonine kinase downstream of PI3K activity.
      More importantly, we studied the functional consequences of PDGF-induced IRS-1 serine/threonine phosphorylation. We found that prior treatment with PDGF caused a decrease in insulin-stimulated IRS-1 tyrosine phosphorylation and a marked impairment in the ability of IRS-1 to associate with PI3K following insulin stimulation. Cells that were pretreated with PDGF showed an 80–90% decrease in insulin stimulatable PI3K activity, precipitated with either anti-phosphotyrosine or IRS-1 antibodies. This inhibition presumably results from a PDGF-induced modification of IRS-1 preventing PI3K from binding to IRS-1. De Fea et al. (
      • De Fea K.
      • Roth R.A.
      ) recently demonstrated that the phosphorylation of IRS-1 on serine 612 causes a conformational change in IRS-1 preventing tyrosine phosphorylation of tyrosine 608, a site of IRS-1/PI3K interaction. The PDGF-induced inhibition of IRS-1-associated PI3K activity was greater than the reduction in insulin-stimulated IRS-1 tyrosine phosphorylation, indicating either that the phosphotyrosines involved in PI3K association were inhibited to a greater extent than overall IRS-1 phosphorylation or that some other biochemical event, such as a conformational change, in addition to decreased tyrosine phosphorylation, is responsible for the block in IRS-1/PI3K association. Surprisingly, this striking inhibition of insulin-stimulated IRS-1-associated PI3K activity had no measurable influence on the downstream insulin signaling events that we examined. For example, it is known that insulin stimulation of GLUT4 translocation and glucose transport is PI3K-dependent (
      • Conricode K.M.
      ,
      • Margalet-Sanchez V.
      • Goldfine I.D.
      • Vlahos C.J.
      • Sung C.K.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Berger J.
      • Hayes N.
      • Szalkowksi D.M.
      • Zhang B.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ), but PDGF pretreatment had no effect on the subsequent ability of insulin to stimulate translocation of GLUT4 proteins to the cell surface or enhance glucose transport activity. On the other hand, even with PDGF pretreatment, insulin stimulation of GLUT4 translocation and glucose transport activity was still completely inhibited by wortmannin and LY294002. This indicates that these effects of insulin remain completely dependent on PI3K activity and eliminates the possibility that a PI3K-independent mechanism, such as that induced by exercise or osmotic shock (
      • Sakaue H.
      • Ogawa W.
      • Takata M.
      • Kuroda S.
      • Kotani K.
      • Matsumoto M.
      • Sakaue M.
      • Nishio S.
      • Ueno H.
      • Kasuga M.
      ,
      • Chen D.
      • Elmendorf J.S.
      • Olson A.L.
      • Li X.
      • Earp H.S.
      • Pessin J.E.
      ), is responsible for the preserved insulin effect under the conditions of PDGF pretreatment.
      Our results show that PDGF treatment inhibits insulin-stimulated IRS-1-associated PI3K activity but does not affect the ability of insulin to stimulate glucose transport, even though glucose transport remains PI3K-dependent. In essence, these results dissociate the function of IRS-1 from stimulation of glucose transport. There are several possible interpretations for these results. The simplest interpretation is that insulin stimulation of IRS-1 tyrosine phosphorylation, with its subsequent binding to and activation of PI3K, is not important for the action of insulin to stimulate glucose transport. Alternatively, it is possible that insulin stimulates glucose transport by at least two parallel pathways, which may be interacting, or redundant. If one of these pathways involves IRS-1, then the blockade of this input is counterbalanced by the alternate pathway. In this event, the alternate pathway would also involve PI3K stimulation, because our results show that under all circumstances, insulin stimulation of glucose transport is strictly PI3K-dependent. A third possibility is that only a small fraction of IRS-1-associated PI3K is necessary for full stimulation of glucose transport. Thus, although only a small amount of IRS-1-associated PI3K activity remains after prolonged PDGF pretreatment, it is possible that this amount of activity, particularly if it is sublocalized to a critical intracellular compartment, can sustain full transport activation. However, if this were the case, one would expect to observe an inhibition of transport at submaximal insulin concentrations. In other words, the dose-response curve for insulin-stimulated glucose transport should be shifted to the right. This is the case, because in the absence of PDGF pretreatment, a low concentration of insulin will lead to a greater amount of IRS-1-associated PI3K activity than will a maximal amount of insulin under conditions of PDGF treatment. As shown in Fig. 4 B, PDGF had no effect on the insulin/glucose transport dose-response curve. On the other hand, it remains possible that a small amount of properly compartmentalized PI3K activity allows glucose transport to be stimulated in a permissive way and that some other insulin-regulatable input into the stimulatory process provides the gating function leading to a dose response.
      The literature bearing on the role of IRS-1 in insulin-stimulated glucose transport is somewhat inconsistent. Some studies argue for a significant role, whereas other papers are more consistent with the view that IRS-1 plays only a minor role, if any, in this particular biologic effect of insulin. For example, IRS-1 knockout mice show marked growth retardation but are only mildly insulin resistant and are not diabetic (

      Bernal, D., Brooks, J., Bruening, J., Towery, H. H., White, M. F., and Yenush, L. (1997) Diabetes 46, Suppl. 1, 5A

      ,
      • Araki E.
      • Lipes M.A.
      • Patti M.E.
      • Bruning J.C.
      • Haag B.
      • Johnson R.S.
      • Kahn C.R.
      ,
      • Yamauchi T.
      • Tobe K.
      • Tamemoto H.
      • Ueki K.
      • Kaburagi Y.
      • Yamamoto-Honda R.
      • Takahashi Y.
      • Yoshizawa F.
      • Aizawa S.
      • Akanuma Y.
      • Sonenberg N.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ). These findings are consistent with the formulation that, with respect to metabolic signaling, alternate or redundant pathways exist. Furthermore, fat cells derived from IRS-1 knockout animals show either normal glucose transport (

      Bernal, D., Brooks, J., Bruening, J., Towery, H. H., White, M. F., and Yenush, L. (1997) Diabetes 46, Suppl. 1, 5A

      ) or a 40–50% decrease in glucose transport (
      • Araki E.
      • Lipes M.A.
      • Patti M.E.
      • Bruning J.C.
      • Haag B.
      • Johnson R.S.
      • Kahn C.R.
      ,
      • Yamauchi T.
      • Tobe K.
      • Tamemoto H.
      • Ueki K.
      • Kaburagi Y.
      • Yamamoto-Honda R.
      • Takahashi Y.
      • Yoshizawa F.
      • Aizawa S.
      • Akanuma Y.
      • Sonenberg N.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Lavan B.E.
      • Lienhard G.E.
      ), despite a complete absence of IRS-1 function. In some studies using IRS-1 knockout mice or cells derived from these animals (

      Bernal, D., Brooks, J., Bruening, J., Towery, H. H., White, M. F., and Yenush, L. (1997) Diabetes 46, Suppl. 1, 5A

      ,
      • Araki E.
      • Lipes M.A.
      • Patti M.E.
      • Bruning J.C.
      • Haag B.
      • Johnson R.S.
      • Kahn C.R.
      ), it has been suggested that IRS-2 might compensate for the loss of IRS-1 activity, although this is not the case in all studies (
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ). This is unlikely to provide an explanation for our results, because IRS-2-associated PI3K activity would have been detected in the anti-phosphotyrosine immunoprecipitates. It is interesting to note that in fat cells derived from IRS-1 knockout mice, in which IRS-1 is absent and IRS-2 is negligible, only a 50% reduction in maximal insulin-stimulated glucose transport is observed, with no change in insulin sensitivity, as detected by the insulin dose-response curve. From these studies, Kaburagi et. al. (
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ) conclude that an IRS1/2-independent but PI3K-dependent pathway must exist. They suggest that this alternate pathway might involve IRS-3 (
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ). However, signaling through IRS-3 is unlikely to explain our results, because tyrosine-phosphorylated IRS-3 (pp60) cannot be detected in 3T3-L1 cells (Ref.
      • Kaburagi Y.
      • Satoh S.
      • Tamemoto H.
      • Yamamoto-Honda R.
      • Tobe K.
      • Veki K.
      • Yamauchi T.
      • Kono-Sugita E.
      • Sekihara H.
      • Aizawa S.
      • Cushman S.W.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      and data not shown). Interestingly, when endogenous IRS-1 was decreased with an antisense ribozyme (
      • Quon M.J.
      • Butte A.J.
      • Zarnowski M.J.
      • Sesti G.
      • Cushman S.W.
      • Taylor S.I.
      ), a shift in the insulin/glucose dose-response curve was observed, whereas no change in maximal responsiveness occurred. In contrast, in fat cells derived from IRS-1 knockout mice, a decrease in maximal responsiveness was noted with no change in the dose-response curve (
      • Araki E.
      • Lipes M.A.
      • Patti M.E.
      • Bruning J.C.
      • Haag B.
      • Johnson R.S.
      • Kahn C.R.
      ). Other studies, in which the interleukin-4 receptor and GLUT4 were overexpressed in L6 myoblasts, show that stimulation with interleukin-4 had no effect on glucose transport, despite the fact that interleukin-4 strongly stimulated tyrosine phosphorylation of IRS-1 and its association with PI3K (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ). Similarly, Krook et al. (
      • Krook A.
      • Moller D.E.
      • Dib K.
      • O'Rahilly S.
      ) have shown that the expression in CHO cells of two insulin receptor mutants (Arg1174 → Gln,Pro1178 → Leu) can mediate IRS-1 phosphorylation but fail to stimulate glycogen synthesis. Thus, the studies by Isakoff et. al. (
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ) and Krook et. al. (
      • Krook A.
      • Moller D.E.
      • Dib K.
      • O'Rahilly S.
      ) are quite consistent; both show that IRS-1 phosphorylation, with PI3K activation, is not sufficient to initiate metabolic signaling, indicating that an additional insulin-derived metabolic input is necessary.
      In other studies, Morris et al. (
      • Morris A.J.
      • Martin S.S.
      • Haruta T.
      • Nelson J.G.
      • Vollenweider P.
      • Gustafson T.A.
      • Mueckler M.
      • Rose D.W.
      • Olefsky J.M.
      ) microinjected IRS-1 inhibitory antibodies and peptides into 3T3-L1 cells and found no inhibitory effect on insulin-stimulated GLUT4 translocation, despite the fact that other insulin actions, such as cytoskeletal rearrangement and DNA synthesis, were inhibited by these reagents. Similarly, Sharmaet al. (
      • Sharma P.M.
      • Egawa K.
      • Gustafson T.A.
      • Martin J.L.
      • Olefsky J.M.
      ) used an adenovirus system to overexpress the IRS-1 PTB and SAIN domains in 3T3-L1 cells. These domains behaved as competitive inhibitors of insulin receptor/IRS-1 interactions, leading to a markedly decreased IRS-1 phosphorylation and IRS-1-associated PI3K activity. Despite this inhibition of IRS-1/PI3K, insulin-stimulated Akt activation, GLUT4 translocation, and glucose transport were normal in PTB or SAIN domain-expressing 3T3-L1 adipocytes (
      • Sharma P.M.
      • Egawa K.
      • Gustafson T.A.
      • Martin J.L.
      • Olefsky J.M.
      ).
      To further explore the mechanisms of these effects, we examined additional targets of insulin action that are thought to be downstream of PI3K. Akt, a serine/threonine kinase downstream of PI3K, is activated following insulin or PDGF stimulation (
      • Burgering B.M.T.
      • Coffer P.J.
      ). Akt is activated by a dual mechanism involving the binding of PIP3 to the Akt PH domain (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ), as well as serine/threonine phosphorylation by one or more Akt kinases, which may themselves be stimulated by the lipid products of PI3K (
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.J.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ). Furthermore, recent data indicate that activation of Akt may be necessary for GLUT4 translocation (
      • Tanti J.-F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ). The expression of a constitutively active Akt construct in rat adipocytes (
      • Tanti J.-F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ) or 3T3-L1 adipocytes (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ) increases GLUT4 translocation and glucose uptake. Consistent with these findings, we show that PDGF pretreatment of cells does not impair the ability of insulin to activate Akt. Despite the fact that insulin-stimulated IRS-1-associated PI3K activity is markedly impaired by PDGF pretreatment, the effect of insulin to stimulate Akt in the presence of PDGF is still entirely inhibited by wortmannin and LY294002, indicating that Akt activation is dependent on PI3K. These results support a role for Akt as a PI3K-dependent upstream activator of GLUT4 translocation. Similarly, insulin-induced p70s6kinase phosphorylation was unaffected by PDGF pretreatment, demonstrating that the pathway from PI3K to p70s6kinase was still intact. Taken together, our data suggest that an insulin-regulated pathway for PI3K stimulation, which is relatively independent of IRS-1/IRS-2, must exist and that this pathway is fully sufficient to activate Akt and p70s6kinase, as well as stimulate glucose transport.
      It has been suggested that the ability of insulin, but not PDGF, to stimulate GLUT4 translocation is due to unique subcompartmentalization of PI3K following insulin treatment. Insulin stimulation leads to the localization of PI3K activity to the low-density microsomal, cytosolic, and plasma membrane fractions, whereas PDGF stimulation results only in PI3K activity in the plasma membrane fraction (
      • Ricort J.-M.
      • Tanti J.-F.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Nave B.T.
      • Haigh R.J.
      • Hayward A.C.
      • Siddle K.
      • Shepherd P.R.
      ). Our results are fully consistent with this idea, but they further indicate that a pathway independent of, or in addition to, IRS-1, is fully capable of affecting this unique subcompartmentalization of PI3K, such that activated PI3K in this compartment can fully stimulate Akt phosphorylation, p70s6kinase phosphorylation, GLUT4 translocation, and glucose transport.
      We propose the existence of another insulin-stimulated protein that interacts with and activates PI3K independent of IRS-1. IRS-2 has been shown to substitute for IRS-1 in adipocytes when expression of IRS-1 is compromised (

      Bernal, D., Brooks, J., Bruening, J., Towery, H. H., White, M. F., and Yenush, L. (1997) Diabetes 46, Suppl. 1, 5A

      ,
      • Araki E.
      • Lipes M.A.
      • Patti M.E.
      • Bruning J.C.
      • Haag B.
      • Johnson R.S.
      • Kahn C.R.
      ). However, because phosphotyrosine antibodies would precipitate IRS-2/PI3K activity, this is an unlikely explanation for our results. Thus, PI3K may be capable of interacting with another insulin receptor substrate, such as IRS-4 or some other protein. Alternatively, cross-talk between insulin receptor signal transduction and G-protein coupled receptors may be an explanation for our observations. The βγ-subunits of G-proteins activate a PI3K that is wortmannin-sensitive (
      • Tang X.
      • Downes C.P.
      ) and leads to Akt activation (
      • Tilton B.
      • Andjelkovic M.
      • Didichenko S.A.
      • Hemmings B.A.
      • Thelen M.
      ). Future experiments are necessary to determine the route of insulin-stimulated PI3K activity following PDGF treatment.

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