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Okadaic Acid Exerts a Full Insulin-like Effect on Glucose Transport and Glucose Transporter 4 Translocation in Human Adipocytes

EVIDENCE FOR A PHOSPHATIDYLINOSITOL 3-KINASE-INDEPENDENT PATHWAY*
  • Cristina M. Rondinone
    Correspondence
    To whom correspondence should be addressed. Tel.: 46-31-601104; Fax: 46-31-825330;
    Affiliations
    Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, Sahlgrenska University Hospital, University of Goteborg, S-413 45 Goteborg, Sweden
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  • Ulf Smith
    Affiliations
    Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, Sahlgrenska University Hospital, University of Goteborg, S-413 45 Goteborg, Sweden
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  • Author Footnotes
    * This work was supported by grants from the Swedish Medical Research Council (B-3506) and the IngaBritt and Arne Lundberg Foundation. 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:July 26, 1996DOI:https://doi.org/10.1074/jbc.271.30.18148
      The effects of the serine/threonine phosphatase inhibitor, okadaic acid, and insulin on glucose transport activity, glucose transporter 4 translocation to the plasma membrane, and the signaling pathway of insulin were examined in human adipocytes. Okadaic acid consistently produced a greater increase than insulin in the rate of glucose transport, and both agents together had a partial additive effect. Both insulin alone and okadaic acid alone stimulated the translocation of glucose transporter 4 to the plasma membrane. Insulin, but not okadaic acid, stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity, and wortmannin completely inhibited the effect of insulin on glucose transport. When the cells were incubated with both agents, okadaic acid inhibited insulin-stimulated PI 3-kinase activity but did not block the association of the p85 or p110 subunits of PI 3-kinase with insulin receptor substrate 1. Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 1 was only slightly reduced (15-30%) by okadaic acid. These data demonstrate that okadaic acid exerts a full insulin-like effect independent of the activation of PI 3-kinase. Thus, PI 3-kinase lipid kinase is not essential for glucose transporter 4 translocation in human adipocytes, and different pathways exist that lead to glucose transporter 4 translocation and increased glucose transport.

      INTRODUCTION

      Insulin plays a key role for the regulation of metabolism in many mammalian cells, principally liver, muscle, and adipose cells (
      • White M.F.
      • Kahn C.R.
      ,
      • Kahn C.R.
      ). The ability of insulin to increase glucose transport into muscle and fat cells is mediated by the translocation of a specific glucose transporter, GLUT4,
      The abbreviations used are: GLUT
      glucose transporter
      IRS-1
      insulin receptor substrate
      PI
      phosphatidylinositol
      BSA
      bovine serum albumin
      SDS
      sodium dodecyl sulfate
      PAGE
      polyacrylamide gel electrophoresis.
      from intracellular vesicles to the cell surface (
      • Cushman S.W.
      • Wardzala L.J.
      ,
      • Suzuki K.
      • Kono T.
      ,
      • James D.E.
      • Strube M.
      • Mueckler M.
      ). The pathways mediating this translocation are poorly understood. The initial mechanism of insulin action involves its binding to specific cell surface receptors, leading to the autophosphorylation and activation of an intrinsic tyrosine kinase associated with the β receptor subunit. A major target for the insulin receptor kinase is insulin receptor substrate 1 (IRS-1) (
      • Myers M.G.
      • White M.F.
      ,
      • Myers M.G.
      • Sun X.J.
      • White M.F.
      ,
      • Sun X.-J.
      • Crimmins D.L.
      • Myers M.G.
      • Miralpeix M.
      • White M.F.
      ). In its tyrosine-phosphorylated form, IRS-1 acts as a docking protein that forms a signaling complex with other proteins with Src homology 2 domains, thus initiating divergent signaling cascades (
      • Kahn C.R.
      ,
      • Myers M.G.
      • Sun X.J.
      • White M.F.
      ,
      • Sun X.-J.
      • Rothenberg P.
      • Kahn C.R.
      • Backer J.M.
      • Araki E.
      • Wilden P.A.
      • Cahill A.
      • Goldstein B.J.
      • White M.F.
      ,
      • Keller S.R.
      • Lienhard G.E.
      ). One such target protein is the phosphatidylinositol (PI) 3-kinase, a heterodimeric enzyme consisting of an 85-kDa regulatory subunit with Src homology 2 domains capable of binding to phosphorylated IRS-1 and a 110-kDa catalytic subunit (
      • Kapeller R.
      • Cantley L.C.
      ) that phosphorylates the inositol ring of phosphatidylinositol and its phosphorylated derivatives (
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ). On the basis of experiments with the PI 3-kinase inhibitors, wortmannin, and LY 294002 (
      • Kanai F.
      • Ito K.
      • Todaka M.
      • Hayashi H.
      • Kamohara S.
      • Ishii K.
      • Okada T.
      • Hazeki O.
      • Ui M.
      • Ebina Y.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Clark J.F.
      • Young P.W.
      • Yonezawa K.
      • Fasuga M.
      • Holman G.D.
      ,
      • Cheatam B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ) and with a dominant negative mutant regulatory subunit of this enzyme (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ), PI 3-kinase has been implicated as one of the key signal transducers in insulin-stimulated glucose uptake and GLUT4 translocation.
      Okadaic acid is a tumor promoter, originally isolated from the sea sponge, that potently inhibits the activity of protein phosphatases 1 and 2A (
      • Bialojan C.
      • Takai A.
      ). It rapidly stimulates protein phosphorylation in intact cells (
      • Haystead T.A.
      • Sim A.T.R.
      • Carling D.
      • Honnor R.C.
      • Tsukitani Y.
      • Cohen P.
      • Hardie D.G.
      ) and also partially stimulates glucose uptake in rat and 3T3-L1 adipocytes (
      • Haystead T.A.
      • Sim A.T.R.
      • Carling D.
      • Honnor R.C.
      • Tsukitani Y.
      • Cohen P.
      • Hardie D.G.
      ,
      • Lawrence Jr., J.C.
      • Hiken J.F.
      • James D.E.
      ,
      • Jullien D.
      • Tanti J.F.
      • Heydrick S.T.
      • Gautier N.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ). However, okadaic acid also blocks insulin-stimulated glucose transport, translocation of glucose transporters, and PI 3-kinase activation in rat adipocytes (
      • Lawrence Jr., J.C.
      • Hiken J.F.
      • James D.E.
      ,
      • Jullien D.
      • Tanti J.F.
      • Heydrick S.T.
      • Gautier N.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Corvera S.
      • Jaspers S.
      • Pascari M.
      ).
      In this study, we examined the effects of insulin and okadaic acid on glucose transport, protein tyrosine phosphorylation, and PI 3-kinase activation in human adipocytes. We demonstrate that okadaic acid exerts a full insulin-like effect in terms of increasing glucose transport activity through the translocation of GLUT4 to the plasma membrane. Furthermore, this full insulin-like effect of okadaic acid is independent of PI 3-kinase activation. The effects of insulin and okadaic acid are also in part additive, demonstrating the presence of multiple pathways for glucose transport activation and GLUT4 protein translocation.

      DISCUSSION

      The salient finding of our study with human adipocytes is that okadaic acid exerts a full insulin-like effect on both glucose transport activity and translocation of GLUT4 proteins to the plasma membrane. However, the insulin-like effects occur in the absence of any measurable increase in PI 3-kinase activity. In fact, the maximal effect of okadaic acid alone on glucose transport was consistently greater than that of insulin alone. The combination of insulin and okadaic acid produced an additive effect on both glucose transport activity and GLUT4 content in the plasma membranes, suggesting the presence of different pathways to activate glucose transport and GLUT4 translocation.
      These findings differ from those recently reported with rat and mouse adipocytes (
      • Lawrence Jr., J.C.
      • Hiken J.F.
      • James D.E.
      ,
      • Corvera S.
      • Jaspers S.
      • Pascari M.
      ,
      • Hess S.L.
      • Suchin C.R.
      • Saltiel A.R.
      ) as well as our own unpublished experiments. In these cells, okadaic acid alone elicits only a small increase in glucose transport, and when combined with insulin, okadaic acid markedly inhibits the effect of insulin (
      • Hess S.L.
      • Suchin C.R.
      • Saltiel A.R.
      ). Thus, there seem to be species differences in the responsiveness to the okadaic acid-related pathway to increase glucose transport. This concept is further supported by the recent finding that okadaic acid also seems to exert a full insulin-like effect on glucose transport in human skeletal muscle, although no additive effect with insulin was demonstrated (
      • Carey J.O.
      • Azevedo J.L.
      • Morris P.G.
      • Pories W.J.
      • Dohm L.
      ).
      In agreement with previous studies in rat adipocytes (
      • Jullien D.
      • Tanti J.F.
      • Heydrick S.T.
      • Gautier N.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ), okadaic acid alone did not increase tyrosine phosphorylation of the insulin receptor or IRS-1 in human adipocytes and altered the mobility of IRS-1, probably due to a hyperphosphorylation involving serine/threonine sites as also demonstrated in animal cells (
      • Tanti J.-F.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ). Okadaic acid also produced a partial inhibition (15-30%) of the insulin-stimulated tyrosine phosphorylation of IRS-1. However, this reduction in tyrosine phosphorylation was not associated with an inhibition of the binding of the p85 or p110 subunits of PI 3-kinase to IRS-1. Taken together, the effects of insulin on the upstream signaling pathway, including tyrosine phosphorylation of the insulin receptor as well as the binding of the PI 3-kinase subunits to IRS-1, were not impaired by okadaic acid. This is consistent with the finding that the effect of insulin on glucose transport activity and GLUT4 translocation was also unimpaired. However, a surprising finding was that the activation of PI 3-kinase lipid kinase by insulin was markedly reduced by okadaic acid, in spite of the normal insulin signaling, including the binding of p85 and p110 to IRS-1. These results lead to the conclusion that either PI 3-kinase lipid kinase is not necessary for glucose transport activation or that cellular PI 3-kinase activity is redundant and that only a small activation is necessary to elicit the full insulin effect or that the association of PI 3-kinase with IRS-1 is by itself an important factor. This latter possibility is supported by recent findings showing that the association of PI 3-kinase with the signaling complex appears to be the most important factor rather than the absolute level of phosphorylated phosphoinositides (
      • Sun X.-J.
      • Rothenberg P.
      • Kahn C.R.
      • Backer J.M.
      • Araki E.
      • Wilden P.A.
      • Cahill A.
      • Goldstein B.J.
      • White M.F.
      ,
      • Myers Jr., M.G.
      • Grammer T.C.
      • Wang L.M.
      • Sun X.J.
      • Pierce J.H.
      • Blenis J.
      • White M.F.
      ).
      The finding that okadaic acid alone exerts a full insulin-like effect on glucose transport activity and GLUT4 translocation in the absence of any measurable increase in PI 3-kinase activity clearly supports the presence of an alternative and PI 3-kinase-independent pathway to stimulate glucose transport. The molecular mechanisms of this putative pathway are currently unclear. PI 3-kinase is a dual specificity kinase with both lipid and serine kinase activity (
      • 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.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Duckworth B.C.
      • Hou W.-M.
      • Schaffhausen B.
      • Cantley L.C.
      ). However, it is unlikely that the PI 3-kinase serine kinase plays a critical role since wortmannin, which inhibits the effect of insulin but not that of okadaic acid, also inhibits the PI 3-kinase serine kinase activity (
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ). This finding does not exclude a critical role of other serine kinases in mediating the insulin-like effect of okadaic acid alone. Interestingly, serine phosphorylation of the p85 and p110 subunits of PI 3-kinase markedly impairs the PI 3-kinase lipid kinase activity (
      • 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.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Duckworth B.C.
      • Hou W.-M.
      • Schaffhausen B.
      • Cantley L.C.
      ). Such an effect of okadaic acid may thus explain the inhibition of the PI 3-kinase activity in the presence of insulin.
      There is much evidence to support a key role of PI 3-kinase activity in eliciting the stimulating effect of insulin on glucose uptake. Various studies have shown that the effect of insulin on this enzyme is reduced in states of insulin resistance (
      • Heydrick S.J.
      • Jullien D.
      • Gautier N.
      • Tanti J.F.
      • Giorgetti S.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Folli F.
      • Saad M.J.A.
      • Backer J.M.
      • Kahn C.R.
      ,
      • Goodyear L.J.
      • Georgino F.
      • Sherman L.A.
      • Carey J.
      • Smith R.J.
      • Dohm G.L.
      ), that the inhibition of PI 3-kinase by wortmannin and LY 294002 leads to the blockade of insulin-stimulated glucose transport (Fig. 2) (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatam B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ), and that the p110 subunit of the PI 3-kinase presents homology with the Vsp34 gene product that is involved in vesicular trafficking in yeast (
      • Schu P.V.
      • Kaoru T.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emry S.D.
      ). In addition, it has been shown that the ability of insulin to stimulate the recruitment of GLUT4-containing vesicles to the plasma membrane is associated with the recruitment of p85/p110 PI 3-kinase to the glucose transporter-carrying vesicles and potential participation of the lipid products of this enzyme in the trafficking of these vesicles (
      • Cheatam 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.
      ,
      • Kanai F.
      • Ito K.
      • Todaka M.
      • Hayashi H.
      • Kamohara S.
      • Ishii K.
      • Okada Y.
      • Hazeki O.
      • Ui M.
      • Ebina Y.
      ). However, a recent study demonstrated that PI 3-kinase activation is necessary but not by itself sufficient to stimulate glucose transport and GLUT4 translocation in 3T3-L1 adipocytes in response to insulin (
      • Herbst J.J.
      • Andrews G.C.
      • Contillo L.G.
      • Singleton D.H.
      • Genereux P.E.
      • Gibbs E.M.
      • Lienhard G.E.
      ).
      Our results show that wortmannin blocks the insulin- but not the okadaic acid-stimulated glucose transport in human adipocytes. In a similar way, it was recently shown that wortmannin does not interfere with the stimulatory effects of contraction and hypoxia on glucose transport in muscle while the effect of insulin is inhibited (
      • Yeh J.-I.
      • Gulve E.A.
      • Rameh L.
      • Birnbaum M.J.
      ). Taken together, these results suggest that the effect of okadaic acid on glucose transport is independent of PI 3-kinase activation and that the ability of wortmannin to block insulin-stimulated glucose transport is specific for the insulin-signaling system and not for the mechanisms responsible for the movement of transporters to the cell surface.
      The GLUT4 isoform is the principal glucose transporter isoform in human adipose cells (
      • Kozka I.J.
      • Clark A.E.
      • Reckless J.P.D.
      • Cushman S.W.
      • Gould G.W.
      • Holman G.D.
      ). The GLUT1 content is only 10% of that of GLUT4. Since GLUT1 also has low affinity for the glucose, this isoform makes only a minor contribution to the total glucose transport activity in human fat cells (
      • Kozka I.J.
      • Clark A.E.
      • Reckless J.P.D.
      • Cushman S.W.
      • Gould G.W.
      • Holman G.D.
      ). Neither insulin nor okadaic acid altered the subcellular distribution of GLUT1.
      Our results also clearly indicate that different pathways exist in human adipocytes for GLUT4 translocation and activation of glucose transport. This may explain the additive effect of insulin and okadaic acid on GLUT4 content in the plasma membranes. It is clear that insulin alone does not promote the complete redistribution of GLUT4 proteins from the intracellular pool to the plasma membranes even in highly insulin-responsive rat adipocytes (
      • Satoh S.
      • Nishimura H.
      • Clark A.E.
      • Kozka I.J.
      • Vannucci S.J.
      • Simpson I.A.
      • Quon M.J.
      • Cushman S.W.
      • Holman G.D.
      ). Although it may be due to the continued recycling of GLUT4 even in the presence of insulin (
      • Satoh S.
      • Nishimura H.
      • Clark A.E.
      • Kozka I.J.
      • Vannucci S.J.
      • Simpson I.A.
      • Quon M.J.
      • Cushman S.W.
      • Holman G.D.
      ), recent studies with skeletal muscle have also suggested the presence of different intracellular pools of GLUT4 susceptible to different stimuli (
      • Yeh J.-I.
      • Gulve E.A.
      • Rameh L.
      • Birnbaum M.J.
      ). Measurements of the redistribution of the intracellular pool of GLUT4 transporting proteins following the exposure to insulin and/or okadaic acid are necessary to define the mechanisms for the additive effect of these agents. Unfortunately, we were unable to obtain enough human tissue to quantify the number of intracellular GLUT4 proteins. The possibility that okadaic acid acts through the inhibition of GLUT4 endocytosis is highly unlikely since kinetic experiments with 3-O-methylglucose uptake have shown that the half-time for the stimulating effect of okadaic acid on glucose transport is similar to that of insulin (∼3 min, data not shown). Irrespective of mechanisms, the finding that the okadaic acid-stimulated pathway is PI 3-kinase-independent, but additive to that of insulin, makes it a potentially important therapeutic target in diabetes and other insulin-resistant states.

      Acknowledgments

      We thank Birgitta Karlsson-Svalstedt and Aino Johansson for skillful technical assistance.

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