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Regulation of Glucose Transport and Glycogen Synthesis in L6 Muscle Cells during Oxidative Stress

EVIDENCE FOR CROSS-TALK BETWEEN THE INSULIN AND SAPK2/p38 MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING PATHWAYS*
  • Anne S. Blair
    Footnotes
    Affiliations
    From the Department of Anatomy and Physiology, Medical Sciences Institute/Wellcome Trust Biocenter Complex, University of Dundee, Dundee DD1 5EH, United Kingdom
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  • Eric Hajduch
    Affiliations
    From the Department of Anatomy and Physiology, Medical Sciences Institute/Wellcome Trust Biocenter Complex, University of Dundee, Dundee DD1 5EH, United Kingdom
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  • Gary J. Litherland
    Affiliations
    From the Department of Anatomy and Physiology, Medical Sciences Institute/Wellcome Trust Biocenter Complex, University of Dundee, Dundee DD1 5EH, United Kingdom
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  • Harinder S. Hundal
    Correspondence
    To whom correspondence should be addressed. Fax: 44-1382-345507;
    Affiliations
    From the Department of Anatomy and Physiology, Medical Sciences Institute/Wellcome Trust Biocenter Complex, University of Dundee, Dundee DD1 5EH, United Kingdom
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  • Author Footnotes
    * This work was supported in part by the British Diabetic Association, the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust.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.
    ‡ Recipient of a CASE studentship from the Medical Research Council and SmithKline Beecham Pharmaceuticals.
Open AccessPublished:December 17, 1999DOI:https://doi.org/10.1074/jbc.274.51.36293
      We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respectively. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H2O2) blocked the hormonal activation of both these processes. Immunoblot analyses of cell lysates prepared after an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases established that H2O2 induced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mm H2O2stimulated the activity of JNK by ∼8-fold, MAPKAP-K2 (the downstream target of p38 MAP kinase) by ∼12-fold and that of PKB by up to 34-fold. PKB activation was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 pathway, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respectively. However, of the kinases assayed, only p38 MAP kinase was activated at H2O2 concentrations (50 μm) that caused an inhibition of insulin-stimulated glucose transport and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin signaling pathways in skeletal muscle.
      A key aspect of mammalian physiology involves the regulation of blood glucose levels. Glucose homeostasis is controlled largely by the action of circulating insulin, which facilitates the disposal of blood glucose in the fed state by stimulating its uptake into target tissues, primarily skeletal muscle and fat (
      • Kahn B.B.
      ,
      • Holman G.D.
      • Cushman S.W.
      ,
      • Galuska D.
      • Ryder J.
      • Kawano Y.
      • Charron M.J.
      • Zierath J.R.
      ). Both these tissues contribute toward the lowering of blood glucose, but it is widely accepted that skeletal muscle, by virtue of its large contribution to body mass, represents the major site of insulin-mediated glucose disposal (
      • DeFronzo R.A.
      • Bonadonna R.C.
      • Ferrannini E.
      ). The stimulation in glucose uptake elicited by insulin in both skeletal muscle and fat is achieved principally by the increased recruitment of the insulin regulated glucose transporter, GLUT4, to the blood-facing membranes of these tissues from its intracellular storage pools (
      • Holman G.D.
      • Kasuga M.
      ).
      Reduced insulin sensitivity is a characteristic feature of various pathological conditions such as diabetes (
      • Kahn B.B.
      ,
      • DeFronzo R.A.
      • Ferrannini E.
      ) and hypertension (
      • Ferrari P.
      • Weidmann P.
      ). Insulin resistance in skeletal muscle and fat may result in the progressive dysfunction of important hormonal effects such as glucose and protein homeostasis, and thus it is of considerable interest to understand the molecular pathology of this process. One factor that appears to be important in the progression of insulin resistance in the above conditions, as well as during hypoxia, ischemia/reperfusion injury, and sepsis, is increased oxidant stress (
      • Baynes J.W.
      • Thorpe S.R.
      ,
      • Parik T.
      • Allikmets K.
      • Teesalu R.
      • Zilmer M.
      ,
      • Duranteau J.
      • Chandel N.S.
      • Kulisz A.
      • Shao Z.
      • Schumacker P.T.
      ,
      • Clerk A.
      • Fuller S.J.
      • Michael A.
      • Sugden P.H.
      ,
      • Taylor D.E.
      • Piantadosi C.A.
      ). Prolonged oxidant stress in muscle and fat has been shown to reduce insulin-stimulated glucose transport significantly and to induce a compensatory increase in basal transport, through increased synthesis of GLUT1 (
      • Kozlovsky N.
      • Rudich A.
      • Potashnik R.
      • Ebina Y.
      • Murakami T.
      • Bashan N.
      ,
      • Rudich A.
      • Kozlovsky N.
      • Potashnik R.
      • Bashan N.
      ). The mechanism by which oxidant stress causes insulin resistance, be it a nonspecific effect of chronic exposure to reactive oxygen species or the specific activation of a stress-related signaling pathway, is unknown. However, since various stresses are known to induce a cellular protective response that involves activation of various protein kinases (
      • Cohen P.
      ), it is possible that this participates in modulating insulin action.
      Insulin is known to stimulate multiple signaling pathways, but there is general acceptance that phosphoinositide 3-kinase (PI3K) plays a central role in regulating glucose transport and glycogen synthesis (
      • Holman G.D.
      • Kasuga M.
      ,
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). The hormonal activation of PI3K catalyzes the production of 3′-phosphoinositides (e.g. phosphatidylinositol 3,4,5-trisphosphate), which act as key intermediates in the activation of protein kinase B (PKB/Akt), a molecule that is regulated also by growth factors and which plays a crucial role in the control of cell proliferation, differentiation, and survival (
      • Marte B.M.
      • Downward J.
      ,
      • Kolesnick R.N.
      • Kronke M.
      ). Activated PKB is known to target glycogen synthase kinase-3 (GSK3), whose phosphorylation results in its inactivation, a step that is considered crucial for the concomitant activation of glycogen synthase (
      • Cohen P.
      • Alessi D.R.
      • Cross D.A.E.
      ,
      • Cross D.A.E.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovic M.
      • Hemmings B.A.
      ). In addition, there is growing evidence that expressing constitutively active or dominant negative PKB mutants in muscle and fat cells induce changes in glucose transport and GLUT4 translocation that are consistent with the involvement of PKB in the insulin signaling pathway regulating glucose uptake (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Tanti J.F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Vanobberghen E.
      • Lemarchandbrustel Y.
      ,
      • Hajduch E.
      • Alessi D.R.
      • Hemmings B.A.
      • Hundal H.S.
      ,
      • Wang Q.
      • Somwar R.
      • Bilan P.J.
      • Liu Z.
      • Jin J.
      • Woodgett J.R.
      • Klip A.
      ). Impaired activation of these early signaling molecules represents a potential mechanism by which insulin sensitivity may be lost in response to increased oxidant stress. This possibility is supported by recent work showing that oxidant stress inhibits the hormonal activation of IRS1, PI3K, and PKB in NIH 3T3 fibroblasts and 3T3-L1 adipocytes (
      • Hansen L.L.
      • Ikeda Y.
      • Olsen G.S.
      • Busch A.K.
      • Mosthaf L.
      ,
      • Tirosh A.
      • Potashnik R.
      • Bashan N.
      • Rudich A.
      ). However, it is noteworthy that reactive oxygen species such as H2O2 also exert insulin-like effects and can activate PKB (
      • Shaw M.
      • Cohen P.
      • Alessi D.R.
      ,
      • Ushio-Fukai M.
      • Alexander R.W.
      • Akers M.
      • Yin Q.
      • Fujio Y.
      • Walsh K.
      • Griendling K.K.
      ) and numerous other protein kinases, including components of the classical MAP kinase and stress signaling pathways (
      • Clerk A.
      • Fuller S.J.
      • Michael A.
      • Sugden P.H.
      ,
      • Shaw M.
      • Cohen P.
      • Alessi D.R.
      ,
      • Wang X.
      • Martindale J.L.
      • Liu Y.
      • Holbrook N.J.
      ,
      • Ihara Y.
      • Toyokuni S.
      • Uchida K.
      • Odaka H.
      • Tanaka T.
      • Ikeda H.
      • Hiai H.
      • Seino Y.
      • Yamada Y.
      ). It is likely that the activation of these pathways plays an important role in mediating some of the biological and possibly pathological responses to environmental stresses. Indeed, in some cell types, activation of the p38 MAP kinase pathway has been implicated with some of the adverse complications associated with raised blood glucose levels and diabetes (
      • Igarashi M.
      • Wakasaki H.
      • Takahara N.
      • Ishii H.
      • Jiang Z.Y.
      • Yamauchi T.
      • Kuboki K.
      • Meier M.
      • Rhodes C.J.
      • King G.L.
      ). Whether stress kinases, such as JNK and p38 MAP kinase, participate in modulating insulin action in response to oxidative stress in skeletal muscle is unknown currently. In this study we show that acute oxidative stress activates both JNK and the p38 MAP kinase pathway in cultured rat muscle cells and that activation of the latter results in the inhibition of insulin-stimulated glucose transport.

      DISCUSSION

      The present study has shown that subjecting muscle cells to acute oxidative stress (using H2O2) results in a dramatic loss in insulin-stimulated glucose transport and glycogen synthesis. Interestingly, the loss in insulin sensitivity can be reversed rapidly, suggesting that H2O2 is likely to modulate insulin action by altering the activity of signaling molecules that mediate the biological effects of the hormone. This notion is supported by recent work showing that fibroblasts and 3T3-L1 adipocytes subjected to oxidative stress show a marked decline in insulin sensitivity that stems from a disruption in the activation of IRS1, PI3K, and PKB (
      • Hansen L.L.
      • Ikeda Y.
      • Olsen G.S.
      • Busch A.K.
      • Mosthaf L.
      ,
      • Tirosh A.
      • Potashnik R.
      • Bashan N.
      • Rudich A.
      ). However, our findings indicate that such a mechanism is unlikely to explain the inhibition in insulin-stimulated glucose transport and glycogen synthesis in L6 myotubes as both processes were inhibited by H2O2 concentrations (50 μm) that had no apparent effect on PKB activation by insulin (Fig. 5 b). This latter finding implies that the upstream insulin signaling events are fully functional. Furthermore, at higher (1 mm) concentrations, H2O2acts as an insulin mimetic, in that it activates both PKB α and γ and inhibits GSK3, a finding that is in line with similar observations reported using HEK 293 cells (
      • Shaw M.
      • Cohen P.
      • Alessi D.R.
      ). However, despite the changes in PKB and GSK3 activity that take place in the presence of 1 mmH2O2, insulin fails to elicit any stimulation in glucose uptake or glycogen synthesis, signifying that the loss in insulin action in L6 cells is likely to occur by a mechanism different to that reported in fat cells (
      • Tirosh A.
      • Potashnik R.
      • Bashan N.
      • Rudich A.
      ).
      In addition to its insulin-like effects on PKB and GSK3, H2O2 also activated two separate stress signaling pathways, JNK and p38 MAP kinase. Activation of p38 has been suggested to be important in mediating some of the harmful effects associated with hyperglycaemia in smooth muscle cells (
      • Igarashi M.
      • Wakasaki H.
      • Takahara N.
      • Ishii H.
      • Jiang Z.Y.
      • Yamauchi T.
      • Kuboki K.
      • Meier M.
      • Rhodes C.J.
      • King G.L.
      ), and we believe that this pathway may also participate in the regulation of insulin-stimulated glucose transport in L6 myotubes during acute oxidative stress. This tenet is based on three separate lines of evidence. First, the loss in insulin-stimulated glucose transport was elicited at a H2O2 concentration (50 μm) that also induced activation of the p38 MAP kinase pathway. At this H2O2 concentration, none of the other kinases assayed showed any detectable changes in phosphorylation and/or activity. Second, we were able to restore insulin-stimulated glucose transport within 30 min of removing H2O2 from the extracellular bathing solution. The ability to recover the insulin response correlated with the rapid dephosphorylation of p38 MAP kinase observed during the post-wash period. Finally, inhibiting the H2O2-induced activation of p38 MAP kinase, using SB 202190 and SB 203580, prevented the loss in insulin-stimulated glucose transport caused by H2O2. In contrast, SB 202190 and SB 203580 failed to inhibit JNK activation, consistent with previous reports showing that, at micromolar concentrations, both compounds target p38 MAP kinase selectively (for review see Ref.
      • Cohen P.
      ). Moreover, our finding that both SB 202190 and SB 203580 prevent the H2O2-induced phosphorylation of p38 MAP kinase is fully consistent with reports showing that these compounds inhibit the agonist-induced phosphorylation and activity of p38 (
      • Cohen P.
      ,
      • Frantz B.
      • Klatt T.
      • Pang M.
      • Parsons J.
      • Rolando A.
      • Williams H.
      • Tocci M.J.
      • O'Keefe S.J.
      • O'Neill E.A.
      ). Collectively, the above findings support the view that the oxidant-induced activation of the p38 MAP kinase pathway plays a role in preventing the hormonal stimulation in glucose transport in skeletal muscle cells.
      The concept that there may be some element of “cross-talk” between the p38 MAP kinase pathway and the insulin signaling pathway is not without precedent. Evidence exists in the literature showing that insulin can, depending on cell type, stimulate or inhibit the activity of p38 MAP kinase (
      • Moxham C.M.
      • Tabrizchi A.
      • Davis R.J.
      • Malbon C.C.
      ,
      • Heidenreich K.A.
      • Kummer J.L.
      ), and more recently it was reported that incubation of both 3T3-L1 adipocytes and L6 myotubes with SB 203580 inhibited insulin-stimulated glucose transport in these two cell lines (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ). In the latter study the inhibitor was purported to induce a reduction in the functional activity of glucose transporters in the plasma membrane rather than blocking their acquisition from intracellular stores in response to insulin. However, we have been unable to observe any inhibition in the insulin-stimulated influx of glucose following pre-incubation of muscle cells with either SB 202190 (Fig. 6 b) or SB 203580 (data not shown). The precise reason for this discrepancy remains unclear, but it may result plausibly from differences in experimental design and the duration of cell pre-treatments.
      Previous work has reported that JNK activation in response to anisomycin (an environmental stress agent) can stimulate glycogen synthase in a manner similar to insulin in skeletal muscle (
      • Moxham C.M.
      • Tabrizchi A.
      • Davis R.J.
      • Malbon C.C.
      ). The stimulation in glycogen synthase elicited by anisomycin was attributed to an activation of PP-1 (the phosphatase that dephosphorylates and stimulates glycogen synthase; Ref.
      • Dent P.
      • Lavoinne A.
      • Nakielny S.
      • Caudwell F.B.
      • Watt P.
      • Cohen P.
      ) and a parallel inactivation of GSK3. In our study, however, although incubation of muscle cells with 1 mm H2O2 caused JNK activation and an inhibition of GSK3, we did not detect any stimulation in glycogen synthase activity or glycogen synthesis. Moreover, in the presence of H2O2, insulin fails to stimulate glycogen synthase. One possible explanation for this is that H2O2, unlike anisomycin, either inhibits or fails to activate PP-1. If so, then increased oxidant levels may also interfere with the hormonal regulation of the phosphatase pathway, by an unknown mechanism. It is highly unlikely that the p38 MAP kinase pathway participates in the regulation of glycogen synthase or PP-1 given that, unlike their effects on glucose transport, neither SB 202190 nor SB 203580 suppressed the H2O2-induced loss in insulin-stimulated glycogen synthesis.
      With one exception (
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      • Lee A.D.
      • Hansen P.A.
      • Holloszy J.O.
      ), work from a number of laboratories, including our own, has suggested that PKB is likely to be an integral component of the insulin signaling pathway regulating glucose transport (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Tanti J.F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Vanobberghen E.
      • Lemarchandbrustel Y.
      ,
      • Hajduch E.
      • Alessi D.R.
      • Hemmings B.A.
      • Hundal H.S.
      ,
      • Wang Q.
      • Somwar R.
      • Bilan P.J.
      • Liu Z.
      • Jin J.
      • Woodgett J.R.
      • Klip A.
      ). If this is correct, then agents that activate PKB should enhance glucose uptake in a manner similar to insulin. However, no increase in glucose transport was observed (Fig. 1 a) under circumstances when both PKBα and PKBγ were activated 34- and 12-fold, respectively (Fig. 3, a and b) by H2O2. We speculated that one explanation for this observation was that, like insulin, the effects of H2O2 might be inhibited downstream of PKB by a mechanism that involved the parallel activation of the p38 MAP kinase pathway. Indeed, when activation of the p38 MAP kinase pathway was inhibited using SB 202190, H2O2 stimulated glucose transport to a level comparable to that seen in muscle cells treated with insulin. Importantly, the increase in glucose transport elicited by H2O2 was sensitive to wortmannin, consistent with the observation that H2O2activates PKB in L6 myotubes in a PI3K-dependent fashion. These findings therefore support the view that PKB is a key component of the insulin-signaling pathway regulating glucose transport.
      Taken together, our findings indicate the existence of an important biochemical link between the p38 MAP kinase pathway and the insulin-signaling cascade that regulates glucose transport in skeletal muscle cells. Activation of the former in response to oxidative stress modulates the hormonal regulation of glucose transport at a point downstream of PKB. Such a mechanism may be significant physiologically and might help explain the reduced insulin sensitivity that is often observed after bouts of strenuous muscle exercise (
      • Kirwan J.P.
      • Hickner R.C.
      • Yarasheski K.E.
      • Kohrt W.M.
      • Wiethop B.V.
      • Holloszy J.O.
      ,
      • Asp S.
      • Daugaard J.R.
      • Richter E.A.
      ), during which there is increased production of reactive oxygen species (for review, see Ref.
      • Sen C.K.
      ) and a stimulation of the p38 MAP kinase pathway (
      • Goodyear L.J.
      • Chang P.Y.
      • Sherwood D.J.
      • Dufresne S.D.
      • Moller D.E.
      ,
      • Widegren U.
      • Jiang X.J.
      • Krook A.
      • Chibalin A.V.
      • Bjornholm M.
      • Tally M.
      • Roth R.A.
      • Henriksson J.
      • Wallberg-Henriksson H.
      • Zierath J.R.
      ). Precisely how activation of the p38 pathway modulates insulin-stimulated glucose transport remains poorly understood, but possible downstream effectors include: MAPKAP-K2/K3 (
      • Rouse J.
      • Cohen P.
      • Trigon S.
      • Morange M.
      • Alonso-Liamazares A.
      • Zamanillo D.
      • Hunt T.
      • Nebreda A.R.
      ,
      • Clifton A.D.
      • Young P.R.
      • Cohen P.
      ),p38-regulated/activatedkinase (PRAK) (
      • New L.
      • Jiang Y.
      • Zhao M.
      • Liu K.
      • Zhu W.
      • Flood L.J.
      • Kato Y.
      • Parry G.C.
      • Han J.
      ), MAP kinase interacting kinases (MNK1/2) (
      • Waskiewicz A.J.
      • Flynn A.
      • Proud C.G.
      • Cooper J.A.
      ), and themitogen- and stress-activatedkinase (MSK1) (
      • Deak M.
      • Clifton A.D.
      • Lucocq L.M.
      • Alessi D.R.
      ). However, we believe that MSK1 is unlikely to be involved, based on preliminary data showing that the protein kinase C inhibitor Ro 318220, which also inhibits strongly the stress-mediated activation of MSK1 but not that of MAPKAP-K2 (
      • Deak M.
      • Clifton A.D.
      • Lucocq L.M.
      • Alessi D.R.
      ), fails to halt the H2O2-induced loss in insulin-stimulated glucose uptake (data not shown). No inhibitors exist currently that inactivate MAPKAP-K2/K3, MNK1/2, or PRAK specifically, but assessing their potential role as modulators of insulin action during oxidant stress remain important issues for future study.

      ACKNOWLEDGEMENT

      We are grateful to Sir Philip Cohen (MRC Protein Phosphorylation Unit, University of Dundee) for providing reagents and antibodies for analyses of PKB, GSK3, and MAPKAP-K2.

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