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Oxidative Stress Disrupts Insulin-induced Cellular Redistribution of Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes

A PUTATIVE CELLULAR MECHANISM FOR IMPAIRED PROTEIN KINASE B ACTIVATION AND GLUT4 TRANSLOCATION*
  • Amir Tirosh
    Affiliations
    Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel 84103 and
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  • Ruth Potashnik
    Affiliations
    Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel 84103 and
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  • Nava Bashan
    Correspondence
    To whom correspondence should be addressed: Dept. of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel 84105. Tel.: 972-7-6400304; Fax: 972-7-6403240;
    Affiliations
    Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel 84103 and

    Soroka Medical Center, Beer-Sheva, Israel 84101
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  • Assaf Rudich
    Affiliations
    Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel 84103 and
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  • Author Footnotes
    * This work was supported by grants from the Israeli Ministry of Health and from the Israeli Academy of Sciences (both to N. B. and A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:April 09, 1999DOI:https://doi.org/10.1074/jbc.274.15.10595
      In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562–1569). In this study we further characterize the cellular mechanisms responsible for this observation. Two-hour exposure to ∼25 μm H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). This was associated with reduced insulin-stimulated IRS-1 and p85-associated PI 3-kinase activities in the LDM (84 and 96% inhibition, respectively). The effect of this finding on the downstream insulin signal was demonstrated by a 90% reduction in insulin stimulated protein kinase B (PKB) serine 473 phosphorylation and impaired activation of PKBα and PKBγ. Both control and oxidized cells exposed to heat shock displayed a wortmannin insensitive PKB serine phosphorylation and activity. These data suggest that activation of PKB and GLUT4 translocation are insulin signaling events dependent upon a normal insulin induced cellular compartmentalization of PI 3-kinase and IRS-1, which is oxidative stress-sensitive. These findings represent a novel cellular mechanism for the induction of insulin resistance in response to changes in the extracellular environment.
      PM
      plasma membrane
      PI
      phosphatidylinositol
      LDM
      low density microsomal fraction
      IRS
      insulin receptor substrate
      PKB
      protein kinase B
      PBS
      phosphate-buffered saline
      The information regarding the biological actions of reactive oxygen and nitrogen species has increased considerably in recent years, revealing diverse functions (
      • Palmer H.J.
      • Paulson K.E.
      ,
      • Papa S.
      • Skulachev V.P.
      ). Exposure of tissues to free radicals in a variety of experimental systems leads to apoptosis and to cell damage (
      • Papa S.
      • Skulachev V.P.
      ). Paradoxically, reactive oxygen species have been demonstrated to participate in normal cellular responses including in signal transduction pathways and in gene regulation (
      • Palmer H.J.
      • Paulson K.E.
      ,
      • Suzuki Y.J.
      • Forman H.J.
      • Sevanian A.
      ,
      • Sen C.K.
      • Packer L.
      ). H2O2, for example, has been shown to be produced intracellularly following stimulation with platelet-derived growth factor and to mediate the normal response to this growth factor (
      • Sundaresan M.
      • Yu Z.X.
      • Ferrans V.J.
      • Irani K.
      • Finkel T.
      ). Similarly, insulin was reported to activate an adipocyte membrane bound NADPH oxidase (
      • Krieger-Brauer H.I.
      • Kather H.
      ,
      • Krieger-Brauer H.I.
      • Kather H.
      ), further supporting a potential role for reactive oxygen species as second messengers. In agreement with this concept, direct exposure of cells to H2O2 has been demonstrated to result in insulinomimetic effects, as demonstrated both by mimicking the metabolic response to insulin as well as by activating components of its signal transduction machinery (
      • Czech M.P.
      • Lawrence Jr., J.C.
      • Lynn W.S.
      ,
      • May J.M.
      • de Haen C.
      ,
      • Hadari Y.R.
      • Tzahar E.
      • Nadiv O.
      • Rothenberg P.
      • Roberts Jr., C.T.
      • LeRoith D.
      • Yarden Y.
      • Zick Y.
      ,
      • Wilden P.A.
      • Broadway D.
      ). However, prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 resulted in impaired insulin-stimulated lipogenesis, activation of glycogen synthase, glucose transport, and GLUT4 translocation to the plasma membrane (PM)1 (
      • Rudich A.
      • Kozlovsky N.
      • Potashnik R.
      • Bahan N.
      ,
      • Rudich A.
      • Tirosh A.
      • Potashnik R.
      • Hemi R.
      • Kannety H.
      • Bashan N.
      ).
      Insulin-stimulated GLUT4 translocation has been suggested to depend upon the activation of phosphatidylinositol 3-kinase (PI 3-kinase), which in fat cells occurs in various cellular fractions (
      • Kelly K.L.
      • Ruderman N.B.
      • Chen K.S.
      ,
      • Kelly K.L.
      • Ruderman N.B.
      ). Recently, the concept that the activation of PI 3-kinase in the low density microsomal fraction (LDM) or in GLUT4 containing vesicles is necessary for the specific ability of insulin to promote GLUT4 translocation has been suggested (
      • Nave B.T.
      • Haigh R.J.
      • Hayward A.C.
      • Siddle K.
      • Shepherd P.R.
      ,
      • Ricort J.M.
      • Tanti J.F.
      • Van Obberghen E.
      • Le Marchand Brustel Y.
      ,
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand Brustel Y.
      ,
      • Heller-Harrison R.A.
      • Morin M.
      • Guilherme A.
      • Czech M.P.
      ). In addition, overexpression of a constitutively active p110 (the catalytic subunit of PI 3-kinase), which resulted in increased total and LDM PI 3-kinase activity in primary adipocytes or in 3T3-L1 adipocytes, dramatically stimulated glucose uptake and GLUT4 translocation (
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand Brustel Y.
      ,
      • Frevert E.U.
      • Kahn B.B.
      ). In a recent study we observed that impaired insulin-stimulated GLUT4 translocation following oxidation could not be attributed to defects in activation of PI 3-kinase as detected in total cell lysate (
      • Rudich A.
      • Tirosh A.
      • Potashnik R.
      • Hemi R.
      • Kannety H.
      • Bashan N.
      ). Thus, impaired compartment-specific activation of PI 3-kinase by insulin may represent a putative cellular mechanism for oxidation induced insulin resistance.
      The events leading to the specific activation of PI 3-kinase in the LDM are not clear. Recently, insulin-induced redistribution of insulin receptor substrates (IRS) was suggested to play a role in the compartment-specific activation of PI 3-kinase in 3T3-L1 adipocytes (
      • Inoue G.
      • Cheatham B.
      • Emkey R.
      • Kahn C.R.
      ). Insulin stimulation was found to induce a reduction in the amount of IRS1/2 in the LDM, while IRS tyrosine phosphorylation was elevated. The tyrosine-phosphorylated IRS in the LDM was suggested to serve as a docking molecule for PI 3-kinase, leading to a rapid translocation of the p85 subunit from the cytosol to the LDM, resulting in increased PI 3-kinase activity in this fraction (
      • Inoue G.
      • Cheatham B.
      • Emkey R.
      • Kahn C.R.
      ,
      • Clark S.F.
      • Martin S.
      • Carozzi A.J.
      • Hill M.M.
      • James D.E.
      ).
      Although the insulin signaling steps toward GLUT4 translocation distal to PI 3-kinase activation are currently not fully understood, a number of downstream targets for PI 3-kinase have been identified (
      • Hajduch E.
      • Alessi D.R.
      • Hemmings B.A.
      • Hundal H.S.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ). Among them, a serine/threonine kinase of 60 kDa (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ) termed protein kinase B (PKB) also known as RAC protein kinase or Akt (
      • Jones P.F.
      • Jakubowicz T.
      • Pitossi F.J.
      • Maurer F.
      • Hemmings B.A.
      ,
      • Coffer P.J.
      • Woodgett J.R.
      ,
      • Bellacosa A.
      • Testa J.R.
      • Staal S.P.
      • Tsichlis P.N.
      ). PI 3-kinase is both necessary and sufficient for insulin-dependent phosphorylation and activation of PKB, although the exact mode of activation has not been fully elucidated (reviewed in Ref.
      • Alessi D.R.
      • Cohen P.
      ). It was suggested that binding of phosphatidylinositol 3,4,5-trisphosphate or phosphatidylinositol 3,4-bisphosphate to the pleckstrin homology domain of PKB and its translocation to the membranes are necessary for PKB phosphorylation on Thr308 and Ser473 by its upstream kinases PDK1 and PDK2, respectively. In the search for its relevant biological activity, PKB was found to mediate some of insulin's effects, such as the inhibition of glycogen synthase kinase-3 (
      • van Weeren P.C.
      • de Bruyn K.M.
      • de Vries Smits A.M.
      • van Lint J.
      • Burgering B.M.
      ,
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ), the stimulation of glucose and amino acids uptake (
      • Hajduch E.
      • Alessi D.R.
      • Hemmings B.A.
      • Hundal H.S.
      ), and protein synthesis (
      • 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.
      ). Several lines of evidence strongly support a crucial role for PKB in mediating GLUT4 translocation. In agreement with this notion, insulin activation of PKB precedes the hormonal effect on glucose transport (
      • Tanti J.F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Van Obberghen E.
      • Le Marchand Brustel Y.
      ). Moreover, overexpression of a constitutively active form of PKB resulted in enhanced glucose transport and GLUT4 translocation (
      • 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.
      ,
      • Tanti J.F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Van Obberghen E.
      • Le Marchand Brustel Y.
      ,
      • Cong L.N.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.A.
      • Taylor S.I.
      • Quon M.J.
      ,
      • Kohn A.D.
      • Barthel A.
      • Kovacina K.S.
      • Boge A.
      • Wallach B.
      • Summers S.A.
      • Birnbaum M.J.
      • Scott P.H.
      • Lawrence Jr., J.C.
      • Roth R.A.
      ,
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ), while inhibition of PKB activity by transfecting rat adipocytes with a dominant PKB-inactive mutant, significantly inhibited insulin-stimulated GLUT4 translocation (
      • Cong L.N.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.A.
      • Taylor S.I.
      • Quon M.J.
      ). The respective role of the different isoforms of PKB in mediating insulin-stimulated GLUT4 translocation in various cell types is as yet unclear.
      Although the involvement of IRS, PI 3-kinase, and PKB cellular redistribution and activation in the normal response to insulin is increasingly recognized, their relevance for the understanding of the cellular mechanisms leading to insulin resistance is largely unclear. In this study we report that impaired insulin-stimulated GLUT4 translocation induced by oxidative stress is associated with disruption of insulin-induced IRS-1 and PI 3-kinase intracellular trafficking and with inhibition of PKBα and PKBγ activation. This represents a novel cellular mechanism for the understanding of insulin resistance in response to a change in the extracellular environment.

      DISCUSSION

      This study was aimed at investigating the cellular mechanisms by which oxidative stress disrupts insulin action in 3T3-L1 adipocytes. The data presented demonstrate that while certain insulinomimetic effects of micromolar H2O2 concentrations could be observed, oxidative stress impaired the compartment-specific activation of PI 3-kinase, IRS-1 redistribution, and PKB activation induced by insulin. These provide a putative mechanism for impaired insulin-stimulated GLUT4 translocation and glucose transport activity following oxidative stress.
      The concept of signal transduction events taking place in specific cellular compartments has been suggested as a plausible explanation for understanding the specificity of hormones and growth factors action. In isolated adipocytes and in 3T3-L1 adipocytes, the activation of PI 3-kinase in the LDM has been suggested as necessary for the specific ability of insulin to induce GLUT4 translocation to the PM, resulting in enhanced glucose transport activity (
      • Nave B.T.
      • Haigh R.J.
      • Hayward A.C.
      • Siddle K.
      • Shepherd P.R.
      ,
      • Ricort J.M.
      • Tanti J.F.
      • Van Obberghen E.
      • Le Marchand Brustel Y.
      ,
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand Brustel Y.
      ,
      • Heller-Harrison R.A.
      • Morin M.
      • Guilherme A.
      • Czech M.P.
      ,
      • Clark S.F.
      • Martin S.
      • Carozzi A.J.
      • Hill M.M.
      • James D.E.
      ,
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ). Using the subcellular fractionation protocol on sucrose cushion, the LDM fraction has been shown to contain intracellular membranes comprising recycling endosomes, the Golgi apparatus, intracellular GLUT4 storage vesicles (
      • Simpson I.A.
      • Yver D.R.
      • Hissin P.J.
      • Wardzala L.J.
      • Karnieli E.
      • Salans L.B.
      • Cushman S.W.
      ,
      • Martin S.
      • Tellam J.
      • Livingstone C.
      • Slot J.W.
      • Gould G.W.
      • James D.E.
      ,
      • James D.E.
      • Pilch P.F.
      ), as well as cytoskeleton components (
      • Clark S.F.
      • Martin S.
      • Carozzi A.J.
      • Hill M.M.
      • James D.E.
      ). Our findings of insulin-stimulated IRS-1 redistribution from the LDM to the cytosol, and of increased PI 3-kinase and tyrosyl-phosphorylated IRS-1 in this fraction following insulin, are in close agreement with previous reports (
      • Inoue G.
      • Cheatham B.
      • Emkey R.
      • Kahn C.R.
      ,
      • Clark S.F.
      • Martin S.
      • Carozzi A.J.
      • Hill M.M.
      • James D.E.
      ,
      • Heller-Harrison R.A.
      • Morin M.
      • Czech M.P.
      ). Whether the IRS-1-PI 3-kinase complex in the LDM directly interacts with the GLUT4 vesicles during the normal insulin signaling toward GLUT4 translocation is currently a subject of debate. Heller-Harrison et al. (
      • Heller-Harrison R.A.
      • Morin M.
      • Guilherme A.
      • Czech M.P.
      ) reported a direct interaction that was demonstrated by insulin-stimulated IRS-1-associated PI 3-kinase activity in isolated GLUT4 vesicles. Yet, other reports challenged this view (
      • Kelly K.L.
      • Ruderman N.B.
      ,
      • Clark S.F.
      • Martin S.
      • Carozzi A.J.
      • Hill M.M.
      • James D.E.
      ). Clark et al. (
      • Clark S.F.
      • Martin S.
      • Carozzi A.J.
      • Hill M.M.
      • James D.E.
      ) suggested that IRS-1 and PI 3-kinase could be recovered in cytoskeleton components that were nonsoluble by nonionic detergents, as opposed to the GLUT4 protein. The role of such interaction in the normal response to insulin is suggested by the demonstration that PI 3-kinase is necessary for insulin-induced actin rearrangement (
      • Martin S.S.
      • Rose D.W.
      • Saltiel A.R.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ,
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ) and by the observation that disruption of the actin filament network by cytochalasin D or by latrunculin B impairs insulin-stimulated GLUT4 translocation and glucose transport activity (
      • Wang Q.
      • Bilan P.J.
      • Tsakiridis T.
      • Hinek A.
      • Klip A.
      ). Whether the impaired insulin-induced PI 3-kinase and IRS-1 redistribution in 3T3-L1 adipocytes exposed to oxidative stress (Figs. 1 and 2, respectively) represent a defect in their interaction with GLUT4 vesicles or cytoskeleton elements could not be determined by this study. Yet, these observations demonstrate that insulin stimulated PI 3-kinase translocation to and activation in the LDM, possibly through its interaction with tyrosyl phosphorylated IRS-1, are oxidation sensitive steps that may be essential for GLUT4 translocation. To the best of our knowledge, this is the first demonstration that changes in the extra cellular environment induce insulin resistance by impairing IRS-1 and/or PI 3-kinase trafficking, without affecting proximal insulin signaling events as detected in total cell lysates.
      Insulin is known to induce both the tyrosine phosphorylation of IRS-1, as well as its phosphorylation on serine/threonine residues. The role and interrelation of these two processes is not fully understood. While the propagation of early steps in the insulin signal appears to depend on tyrosine phosphorylation (
      • White M.F.
      • Kahn C.R.
      ), serine/threonine phosphorylation may be involved in its termination (
      • Tanti J.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari R.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      ). Several serine/threonine kinases which are normally activated in response to insulin stimulation have been suggested to phosphorylate IRS-1, potentially representing an inherent shut-down mechanism for the insulin signal. These include ERK 1/2 MAP kinases (
      • De Fea K.
      • Roth R.A.
      ), GSK3 (
      • Eldar-Finkelman H.
      • Krebs E.G.
      ), PKB (
      • Paz K.
      • Seger R.
      • Hemi R.
      • LeRoith D.
      • Kannety H.
      • Zick Y.
      ), and certain PKC isoforms (
      • De Fea K.
      • Roth R.A.
      ). Moreover, activation of insulin stimulated serine/threonine kinase(s) was suggested to play a role in the translocation of IRS-1 from the LDM fraction to the cytosol (
      • Inoue G.
      • Cheatham B.
      • Emkey R.
      • Kahn C.R.
      ). In this study we demonstrate that the activation of the serine/threonine kinase PKB was dramatically inhibited by oxidative stress (Figs. 3 and 4). Insulin induced activation of the ERK1/2 MAP kinase was also impaired (unpublished results). Interestingly, insulin induced serine/threonine phosphorylation of IRS-1 in the LDM was markedly reduced by oxidation (Fig. 2A), and was associated with inhibition of its translocation from the LDM to the cytosol. These results are consistent with the notion that serine/threonine phosphorylation of LDM IRS-1 may be needed for its normal cellular redistribution. Nevertheless, insulin stimulated tyrosine phosphorylation of IRS-1 was also impaired in the LDM following oxidation (Fig. 2B), and was associated with reduced PI 3-kinase activity in this fraction (Fig. 1 and 2). Thus, the oxidation-induced reduction in tyrosine phosphorylated IRS-1 in the LDM may represent the cause rather than the effect of alterations in serine/threonine phosphorylation of LDM IRS-1. Moreover, reduced tyrosine-phosphorylated IRS-1 in the LDM appears to represent the limiting factor for the translocation and activation of PI 3-kinase in this fraction.
      It is well established that PKB activation by insulin is dependent on PI 3-kinase (
      • Alessi D.R.
      • Cohen P.
      ). Yet whether insulin stimulation of PI 3-kinase in the LDM is required for the activation of the various known isoforms of PKB is largely unknown. Thus, the effect of oxidative stress on the basal and insulin-stimulated activity of PKBα, PKBβ, and PKBγ was evaluated. In 3T3-L1 adipocytes, similar to observations in L6 myotubes (
      • Hajduch E.
      • Alessi D.R.
      • Hemmings B.A.
      • Hundal H.S.
      ), most of the insulin-stimulated PKB activation could be attributed to PKBα and PKBγ (Fig. 4). Surprisingly, insulin barely affected PKBβ activity, which was demonstrated as the major PKB isoform regulated by insulin in primary rat adipocytes (
      • Calera M.R.
      • Martinez C.
      • Liu H.
      • El Jack A.K.
      • Birnbaum M.J.
      • Pilch P.F.
      ). Oxidative stress profoundly impaired both insulin-stimulated PKBα and PKBγ activities (Fig. 4), resulting in an overall reduction of total cellular PKB activity. The striking similarity between the effect of oxidation on PKB (Figs. 3C and 4) and on glucose uptake (Fig.6 A) suggests a potential cause and effect relationship between the impairment in both insulin stimulated PKB activation and GLUT4 translocation.
      The ability to activate PKB through wortmannin-insensitive pathway(s) following oxidative stress was evaluated to exclude a direct inhibitory effect of oxidation on either PKB or its immediate upstream kinases PDK1 and PDK2 (
      • Alessi D.R.
      • Cohen P.
      ,
      • Cohen P.
      • Alessi D.R.
      • Cross D.A.
      ). As demonstrated in COS-7 and NIH 3T3 cells (
      • Matsuzaki H.
      • Konishi H.
      • Tanaka M.
      • Ono Y.
      • Takenawa T.
      • Watanabe Y.
      • Ozaki S.
      • Kuroda S.
      • Kikkawa U.
      ), heat shock treatment of 3T3-L1 adipocytes induced phosphorylation and activation of PKB through wortmannin-insensitive mechanisms (Fig.5, A and B, respectively). Following oxidative stress, this activation of PKB remained intact, supporting the notion that the impaired insulin-stimulated PKB activation induced by oxidation may be a consequence of the reduced ability of insulin to activate PI 3-kinase in the LDM. This, in turn, may offer the possibility that activation of PKB is also an insulin signaling event dependent on normal cellular compartmentalization of PI 3-kinase. In support of this concept is the observation that membrane-localized p110 was found to be sufficient to activate PKB in COS-7 cells (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ).
      This paper presents evidence that while micromolar concentrations of H2O2 inhibits acute metabolic response to insulin, they also induce a certain insulinomimetic effect. Exposure to H2O2 for 2 h resulted in wortmannin-sensitive increase in basal glucose transport and in PKB activities, as well as in basal PI 3-kinase activity in the LDM (Figs. 6 A, 3 C, and 1 B, respectively). These seemingly opposing effects suggest several cellular targets for H2O2 along the multistep insulin signaling network. While one or more targets may be activated by H2O2, other can eventually become rate-limiting for additional insulin stimulation.
      The potential relevance of findings presented herein may be in the understanding of the cellular mechanisms leading to peripheral insulin resistance in various conditions. Increased oxidative stress has been suggested by various mechanisms to occur in diabetic or prediabetic individuals (
      • Salonen J.T.
      • Nyyssonen K.
      • Tuomainen T.P.
      • Maenpaa P.H.
      • Korpela H.
      • Kaplan G.A.
      • Lynch J.
      • Helmrich S.P.
      • Salonen R.
      ,
      • Wolff S.P.
      • Jiang Z.Y.
      • Hunt J.V.
      ,
      • Nourooz-Zadeh J.
      • Tajaddini Sarmadi J.
      • McCarthy S.
      • Betteridge D.J.
      • Wolff S.P.
      ) and to contribute to the pathogenesis of diabetic late complications (
      • Giugliano D.
      • Ceriello A.
      • Paolisso G.
      ), as well as to peripheral insulin resistance (
      • Nourooz-Zadeh J.
      • Rahimi A.
      • Tajaddini-Sarmadi J.
      • Tritschler H.
      • Rosen P.
      • Halliwell B.
      • Betteridge D.J.
      ,
      • Paolisso G.
      • D'Amore A.
      • Volpe C.
      • Balbi V.
      • Saccomanno F.
      • Galzerano D.
      • Giugliano D.
      • Varricchio M.
      • D'Onofrio F.
      ). Reduced GLUT4 expression and impaired translocation in response to insulin stimulation were reported in adipocytes of insulin-resistant individuals (
      • Caro J.F.
      ). The impaired insulin response was suggested to represent both receptor and postreceptor mechanisms. This study offers a potential mechanism by which oxidative stress can contribute to the development of impaired insulin-stimulated GLUT4 translocation in adipocytes of diabetic subjects.

      Acknowledgments

      We thank the S. Daniel Abraham International Center for Health and Nutrition, Ben-Gurion University of the Negev, Beer-Sheva, Israel, Dr. D. R. Alessi from the University of Dundee, United Kingdom, for providing anti PKB β and γ antibodies, and to Dr. R. Seger from the Weizmann Institute of Sciences, Rehovot, Israel, for anti C-terminal PKB antibodies. We are indebted to Dr. M. J. Charron, from the Albert Einstein College of Medicine, for critical review of the manuscript.

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