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Stimulation of IRS-1-associated Phosphatidylinositol 3-Kinase and Akt/Protein Kinase B but Not Glucose Transport by β1-Integrin Signaling in Rat Adipocytes*

  • Adilson Guilherme
    Affiliations
    From the Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
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  • Michael P. Czech
    Correspondence
    To whom correspondence should be addressed: Tel.: 508-856-2254; Fax: 508-856-1617;
    Affiliations
    From the Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
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  • Author Footnotes
    * This work was supported by Grant DK30648 (to M. P. C.) from the National Institutes of Health.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:December 11, 1998DOI:https://doi.org/10.1074/jbc.273.50.33119
      The signal transduction pathway by which insulin stimulates glucose transport is not understood, but a role for complexes of insulin receptor substrate (IRS) proteins and phosphatidylinositol (PI) 3-kinase as well as for Akt/protein kinase B (PKB) has been proposed. Here, we present evidence suggesting that formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation are insufficient to stimulate glucose transport in rat adipocytes. Cross-linking of β1-integrin on the surface of rat adipocytes by anti-β1-integrin antibody and fibronectin was found to cause greater IRS-1 tyrosine phosphorylation, IRS-1-associated PI 3-kinase activity, and Akt/PKB activation, detected by anti-serine 473 antibody, than did 1 nm insulin. Clustering of β1-integrin also significantly potentiated stimulation of insulin receptor and IRS-1 tyrosine phosphorylation, IRS-associated PI 3-kinase activity, and Akt/PKB activation caused by submaximal concentrations of insulin. In contrast, β1-integrin clustering caused neither a change in deoxyglucose transport nor an effect on the ability of insulin to stimulate deoxyglucose uptake at any concentration along the entire dose-response relationship range. The data suggest that (i) β1-integrins can engage tyrosine kinase signaling pathways in isolated fat cells, potentially regulating fat cell functions and (ii) either formation of IRS-1/PI 3-kinase complexes and Akt/PKB activation is not necessary for regulation of glucose transport in fat cells or an additional signaling pathway is required.
      One of the primary metabolic responses mediated by insulin is the stimulation of facilitated glucose transport in muscle and adipose tissues. It is now widely accepted that insulin regulates glucose uptake in adipocytes by recruiting GLUT4 glucose transporter proteins to the plasma membrane from an intracellular membrane pool (
      • Stephens J.M.
      • Pilch P.F
      ,
      • Czech M.P.
      ,
      • Cheatham B.
      • Kahn C.R.
      ,
      • Cushman S.W.
      • Wardzala L.J.
      ,
      • Yang J.
      • Holman G.D.
      ,
      • Stagsted J.
      • Olsson L.
      • Holman G.D.
      • Cushman S.W.
      • Satoh S.
      ). This process is initiated when insulin binds to its specific receptor at the cell surface, causing activation of intrinsic receptor tyrosine kinase activity (
      • Yu K.-T.
      • Czech M.P.
      ,
      • White M.F.
      • Kahn C.R.
      ). The activated insulin receptor phosphorylates endogenous substrate proteins, such as Shc and insulin receptor substrate (IRS)
      The abbreviations used are: IRS, insulin receptor substrate; PI, phosphatidylinositol; PKB, protein kinase B; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CHO-T cells, Chinese hamster ovary cells expressing human insulin receptors.
      1The abbreviations used are: IRS, insulin receptor substrate; PI, phosphatidylinositol; PKB, protein kinase B; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CHO-T cells, Chinese hamster ovary cells expressing human insulin receptors.
      proteins (
      • White M.F.
      • Kahn C.R.
      ,
      • Sun X.-J.
      • Wang L.-M.
      • Zhang Y.
      • Yenush L.
      • Myers Jr., M.G.
      • Glasheen E.
      • Lane W.S.
      • Pierce J.H.
      • White M.F.
      ,
      • Lavan B.E.
      • Lane W.S.
      • Lienhard G.E.
      ,
      • Lavan B.E.
      • Fantin V.R.
      • Chang E.T.
      • Lane W.S.
      • Keller S.R.
      • Lienhard G.E.
      ,
      • Myers Jr., M.G.
      • Sun X.-J.
      • White M.F.
      ). Tyrosine phosphorylation sites on IRS proteins specifically found within multiple YXXM motifs create docking sites for Src homology 2 domains, present on the p85 regulatory subunits of type I phosphatidylinositol (PI) 3-kinase and many other signaling proteins (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.-J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ,
      • Yonezawa K.
      • Ueda H.
      • Hara K.
      • Nishida K.
      • Ando A.
      • Chavanieu A.
      • Matsuba H.
      • Yokono K.
      • Shii K.
      • Fukui Y.
      • Calas B.
      • Grigorescu F.
      • Dhand R.
      • Gout I.
      • Otsu M.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Rordorf-Nikolic T.
      • VanHorn D.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ). The binding of p85 to IRS proteins activates the associated p110 catalytic subunit of PI 3-kinase, which catalyzes the phosphorylation of phosphoinositides at the D3 position of the inositol ring (
      • Toker A.
      • Cantley L.C.
      ). A key role for these 3′-phosphoinositides in membrane trafficking is suggested by studies on the requirement of a yeast PI 3-kinase denoted VPS34 for correct sorting of carboxypeptidase Y to the vacuole (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ) and by the evidence that mutant platelet-derived growth factor and colony stimulating factor receptors lacking binding sites for type I PI 3-kinase are defective in their sorting to a degradative pathway (
      • Carlberg K.
      • Tapley P.
      • Haystead C.
      • Rohrschneider L.
      ,
      • Joly M.
      • Kazlauskas A.
      • Fay F.S
      • Corvera S.
      ).
      Blockade of PI 3-kinase activity by inhibitors (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ) or by dominant inhibitory constructs of p85 (
      • Quon M.J.
      • Chen H.
      • Ing B.L.
      • Liu M.-L.
      • Zarnowski M.J.
      • Yonezawa K.
      • Kasuga M.
      • Cushman S.W.
      • Taylor S.I.
      ) ablate insulin action on glucose transport, indicating that 3′-phosphoinositide production is necessary for GLUT4 translocation. However, the role of IRS-1 proteins in insulin action on glucose transport is less clear, with some studies indicating that IRS protein tyrosine phosphorylation can be blocked without affecting glucose transport activation (
      • Morris A.J.
      • Martin S.S.
      • Haruta T.
      • Nelson J.G.
      • Vollenweider P.
      • Gustafson T.A.
      • Mueckler M.
      • Rose D.W.
      • Olefsky J.M.
      ,
      • Sharma P.M.
      • Egawa K.
      • Gustafson T.A.
      • Martin J.L.
      • Olefsky J.M.
      ). Other studies suggest that IRS protein function is required for optimal regulation of glucose transport (
      • Bruning J.C.
      • Winnay J.
      • Bonner-Weir S.
      • Taylor S.I.
      • Accili D.
      • Kahn C.R.
      ). Also it has been shown that the protein serine/threonine kinase Akt/protein kinase B (PBK), which functions as an effector of 3′-polyphosphoinositides and becomes activated by insulin (
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ), may play a role in GLUT4 regulation by insulin (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.
      ). However, evidence against this latter hypothesis has been reported (
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      ).
      It has recently been reported that integrin engagement on the surface membrane of cells also promotes activation of PI 3-kinase as well as activation of Akt kinase (
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ). In addition, we have found that α5β1-integrin cross-linking enhances IRS-1-associated PI 3-kinase activity and potentiates insulin signaling in Chinese hamster ovary cells expressing human insulin receptors (CHO-T) (
      • Guilherme A.
      • Torres K.
      • Czech M.P.
      ). In the present studies, we addressed the question of whether β1-integrin clustering in rat adipocytes also activates the IRS-1/PI 3-kinase/Akt/PKB pathway and whether activation of this signaling pathway by β1-integrin might stimulate glucose transport as does insulin. Here we show that β1-integrin engagement indeed markedly enhances IRS-1-associated PI 3-kinase activity as well as Akt/PKB activation but has no effect on glucose uptake in rat adipocytes. This result supports the hypothesis that one or more other signaling pathways, perhaps acting in conjunction with IRS/PI 3-kinase complexes, appear to be necessary to activate glucose transport in rat adipocytes.

      Acknowledgments

      We thank Dr. C. Michael DiPersio for the gift of anti-β1-integrin polyclonal antibody, Dr. Robin Heller-Harrison and Dr. Mark W. Sleeman for helpful discussions, and Jane Erickson for expert assistance in the preparation of this manuscript. The help of Niles P. Donegan in the preparation of GLUT4-containing vesicles is greatly appreciated.

      REFERENCES

        • Stephens J.M.
        • Pilch P.F
        Endocr. Rev. 1995; 16: 529-546
        • Czech M.P.
        Annu. Rev. Nutr. 1995; 15: 441-471
        • Cheatham B.
        • Kahn C.R.
        Endocr. Rev. 1995; 16: 117-142
        • Cushman S.W.
        • Wardzala L.J.
        J. Biol. Chem. 1980; 255: 4758-4762
        • Yang J.
        • Holman G.D.
        J. Biol. Chem. 1993; 268: 4600-4603
        • Stagsted J.
        • Olsson L.
        • Holman G.D.
        • Cushman S.W.
        • Satoh S.
        J. Biol. Chem. 1993; 268: 22809-22813
        • Yu K.-T.
        • Czech M.P.
        J. Biol. Chem. 1984; 259: 5277-5286
        • White M.F.
        • Kahn C.R.
        J. Biol. Chem. 1994; 269: 1-4
        • Sun X.-J.
        • Wang L.-M.
        • Zhang Y.
        • Yenush L.
        • Myers Jr., M.G.
        • Glasheen E.
        • Lane W.S.
        • Pierce J.H.
        • White M.F.
        Nature. 1995; 377: 173-177
        • Lavan B.E.
        • Lane W.S.
        • Lienhard G.E.
        J. Biol. Chem. 1997; 272: 11439-11443
        • Lavan B.E.
        • Fantin V.R.
        • Chang E.T.
        • Lane W.S.
        • Keller S.R.
        • Lienhard G.E.
        J. Biol. Chem. 1997; 272: 21403-21407
        • Myers Jr., M.G.
        • Sun X.-J.
        • White M.F.
        Trends Biochem. Sci. 1994; 19: 289-293
        • Backer J.M.
        • Myers Jr., M.G.
        • Shoelson S.E.
        • Chin D.J.
        • Sun X.-J.
        • Miralpeix M.
        • Hu P.
        • Margolis B.
        • Skolnik E.Y.
        • Schlessinger J.
        • White M.F.
        EMBO J. 1992; 11: 3469-3479
        • Yonezawa K.
        • Ueda H.
        • Hara K.
        • Nishida K.
        • Ando A.
        • Chavanieu A.
        • Matsuba H.
        • Yokono K.
        • Shii K.
        • Fukui Y.
        • Calas B.
        • Grigorescu F.
        • Dhand R.
        • Gout I.
        • Otsu M.
        • Waterfield M.D.
        • Kasuga M.
        J. Biol. Chem. 1992; 267: 25958-25966
        • Rordorf-Nikolic T.
        • VanHorn D.
        • Chen D.
        • White M.F.
        • Backer J.M.
        J. Biol. Chem. 1995; 270: 3662-3666
        • Toker A.
        • Cantley L.C.
        Nature. 1997; 387: 673-676
        • Schu P.V.
        • Takegawa K.
        • Fry M.J.
        • Stack J.H.
        • Waterfield M.D.
        • Emr S.D.
        Science. 1993; 260: 88-91
        • Carlberg K.
        • Tapley P.
        • Haystead C.
        • Rohrschneider L.
        EMBO J. 1991; 10: 877-883
        • Joly M.
        • Kazlauskas A.
        • Fay F.S
        • Corvera S.
        Science. 1994; 263: 684-687
        • Okada T.
        • Kawano Y.
        • Sakakibara T.
        • Hazeki O.
        • Ui M.
        J. Biol. Chem. 1994; 269: 3568-3573
        • Cheatham B.
        • Vlahos C.J.
        • Cheatham L.
        • Wang L.
        • Blenis J.
        • Kahn C.R.
        Mol. Cell. Biol. 1994; 14: 4902-4911
        • Quon M.J.
        • Chen H.
        • Ing B.L.
        • Liu M.-L.
        • Zarnowski M.J.
        • Yonezawa K.
        • Kasuga M.
        • Cushman S.W.
        • Taylor S.I.
        Mol. Cell. Biol. 1995; 15: 5403-5411
        • Morris A.J.
        • Martin S.S.
        • Haruta T.
        • Nelson J.G.
        • Vollenweider P.
        • Gustafson T.A.
        • Mueckler M.
        • Rose D.W.
        • Olefsky J.M.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8401-8406
        • Sharma P.M.
        • Egawa K.
        • Gustafson T.A.
        • Martin J.L.
        • Olefsky J.M.
        Mol. Cell. Biol. 1997; 17: 7386-7397
        • Bruning J.C.
        • Winnay J.
        • Bonner-Weir S.
        • Taylor S.I.
        • Accili D.
        • Kahn C.R.
        Cell. 1997; 88: 561-572
        • Franke T.F.
        • Yang S.I.
        • Chan T.O.
        • Datta K.
        • Kazlauskas A.
        • Morrison D.K.
        • Kaplan D.R.
        • Tsichlis P.N.
        Cell. 1995; 81: 727-736
        • Burgering B.M.T.
        • Coffer P.J.
        Nature. 1995; 376: 599-602
        • Alessi D.R.
        • Andjelkovic M.
        • Caudwell B.
        • Cron P.
        • Morrice N.
        • Cohen P.
        • Hemmings B.A.
        EMBO J. 1996; 15: 6541-6551
        • Kohn A.D.
        • Summers S.A.
        • Birnbaum M.J.
        • Roth R.
        J. Biol. Chem. 1996; 271: 31372-31378
        • Kitamura T.
        • Ogawa W.
        • Sakaue H.
        • Hino Y.
        • Kuroda S.
        • Takata M.
        • Matsumoto M.
        • Maeda T.
        • Konishi H.
        • Kikkawa U.
        • Kasuga M.
        Mol. Cell. Biol. 1998; 18: 3708-3717
        • King W.G.
        • Mattaliano M.D.
        • Chan T.O.
        • Tsichlis P.N.
        • Brugge J.S.
        Mol. Cell. Biol. 1997; 17: 4406-4418
        • Guilherme A.
        • Torres K.
        • Czech M.P.
        J. Biol. Chem. 1998; 273: 22899-22903
        • Guilherme A.
        • Klarlund J.K.
        • Krystal G.
        • Czech M.P.
        J. Biol. Chem. 1996; 271: 29533-29536
        • Clancy B.M.
        • Czech M.P.
        J. Biol. Chem. 1991; 265: 12434-12443
        • Kandror K.V.
        • Coderre L.
        • Pushkin A.V.
        • Pilch P.F.
        Biochem. J. 1995; 307: 383-390
        • Takada Y.
        • Ylanne J.
        • Mandelman D.
        • Puzon W.
        • Ginsberg M.H.
        J. Cell Biol. 1992; 119: 913-921
        • Kornberg L.-J.
        • Earp H.S.
        • Turne C.E.
        • Prockop C.
        • Juliano R.L.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8392-8396
        • Miyamoto S.
        • Akiyama S.K.
        • Yamada K.M.
        Science. 1995; 267: 883-885
        • Tanti J.-F.
        • Gremeaux T.
        • Grillos S.
        • Calleja V.
        • Klippel A.
        • Williams L.T.
        • VanObberghen E.
        • LeMarchand-Brustel Y.
        J. Biol. Chem. 1996; 271: 25227-25232
        • Martin S.S.
        • Haruta T.
        • Morris A.J.
        • Klippel A.
        • Williams L.T.
        • Olefsky J.M.
        J. Biol. Chem. 1996; 271: 17605-17608
        • Frevert E.U.
        • Kahn B.B.
        Mol. Cell. Biol. 1997; 17: 190-198
        • Heller-Harrison R.A.
        • Morin M.
        • Czech M.P.
        J. Biol. Chem. 1995; 270: 24442-24450
        • Heller-Harrison R.A.
        • Morin M.
        • Guilherme A.
        • Czech M.P.
        J. Biol. Chem. 1996; 271: 10200-10204
        • Nave B.T.
        • Haigh R.J.
        • Hayward A.C.
        • Siddle K.
        • Shepard P.R.
        Biochem. J. 1996; 318: 55-60
        • Isakoff S.J.
        • Taha C.
        • Rose E.
        • Marcusohn J.
        • Klip A.
        • Skolnik E.Y.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10247-10251
        • Jiang T.
        • Sweeney G.
        • Rudolf M.T.
        • Klip A.
        • Traynor-Kaplan A.
        • Tsien R.Y.
        J. Biol. Chem. 1998; 273: 11017-11024