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A Role for Kinesin in Insulin-stimulated GLUT4 Glucose Transporter Translocation in 3T3-L1 Adipocytes

2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês

10.1074/jbc.m010785200

1083-351X

Masahiro Emoto, Stephen E. Langille, Michael Czech,

Metabolism, Diabetes, and Cancer

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin. Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin. glucose transporter 4 DMEM, Dulbecco's modified Eagle's medium phosphate-buffered saline 1,4-diazabicyclo[2.2.2]octane cytoplasmic linker protein Physiological glucose homeostasis in humans is largely dependent on the actions of the hormone insulin, particularly its ability to inhibit glucose output from the liver and enhance glucose transport into fat and muscle cells. Insulin exerts this latter effect primarily through a process whereby sequestered intracellular GLUT4 glucose transporter proteins are rapidly redistributed to cell surface membranes in which they can catalyze glucose uptake into cells (1Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 2Suzuki K. Kono T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2542-2545Crossref PubMed Scopus (768) Google Scholar, 3Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 4Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). In both the basal and insulin-stimulated states, GLUT4 proteins appear to cycle between intracellular membrane and plasma membrane locations (5Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar). However, in the basal state most of the GLUT4 is diverted to intracellular perinuclear membranes, and the exocytosis rate is slow. Insulin stimulates exocytosis of GLUT4 by a mechanism that requires the p85/p110 type phosphatidylinositol 3-kinase, which is recruited to protein phosphotyrosines in response to activation of the insulin receptor tyrosine kinase (6Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (997) Google Scholar, 7Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 8Sharma P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Insulin also appears to significantly inhibit GLUT4 endocytosis (5Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 9Czech M.P. Buxton J.M. J. Biol. Chem. 1993; 268: 9187-9190Abstract Full Text PDF PubMed Google Scholar, 10Jhun B.H. Rampal A.L. Liu H. Lachaal M. Jung C.Y. J. Biol. Chem. 1992; 267: 17710-17715Abstract Full Text PDF PubMed Google Scholar). However, the precise mechanism whereby insulin signals to the GLUT4 membrane trafficking machinery remains obscure.It is known that other membrane systems, such as lysosomes (11Harada A. Takei Y. Kanai Y. Tanaka Y. Nonaka S. Hirokawa N. J. Cell Biol. 1998; 141: 51-59Crossref PubMed Scopus (274) Google Scholar), mitochondria (12Tanaka Y. Kanai Y. Okada Y. Nonaka S. Takeda S. Harada A. Hirokawa N. Cell. 1998; 93: 1147-1158Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar), Golgi membranes (13Burkhardt J.K. Echeverri C.J. Nilsson T. Vallee R.B. J. Cell Biol. 1997; 139: 469-484Crossref PubMed Scopus (552) Google Scholar, 14Burkhardt J.K. Biochim. Biophys. Acta. 1998; 1404: 113-126Crossref PubMed Scopus (75) Google Scholar), and pigment granules (15Rogers S.L. Tint I.S. Fanapour P.C. Gelfand V.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3720-3725Crossref PubMed Scopus (188) Google Scholar), are localized within cells by molecular motors. For example, membrane vesicles containing melanin appear to be driven along microtubules over relatively long distances from their perinuclear location in unstimulated melanocytes to the cell periphery upon elevation of cAMP levels (16Reilein A.R. Tint I.S. Peunova N.I. Enikolopov G.N. Gelfand V.I. J. Cell Biol. 1998; 142: 803-813Crossref PubMed Scopus (72) Google Scholar). The complex motor dynein drives movements along microtubules in the minus direction toward the perinuclear microtubule organizing center, whereas kinesin motors drive movements toward the plus growing ends of microtubules (17Hirokawa N. Science. 1998; 279: 516-526Crossref Scopus (1361) Google Scholar). Movements of these membranes over shorter distances seem to require actin filaments (18Rodionov V.I. Hope A.J. Svitkina T.M. Borisy G.G. Curr. Biol. 1998; 8: 165-168Abstract Full Text Full Text PDF PubMed Google Scholar, 19Rogers S.L. Gelfand V.I. Curr. Biol. 1998; 8: 161-164Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Based on these observations, it has been suggested that the microtubule and actin filament networks are highly integrated in discharging their organelle localization functions (20Wu X. Jung G. Hammer III, J.A. Curr. Opin. Cell Biol. 2000; 12: 42-51Crossref PubMed Scopus (100) Google Scholar). Recent findings in our laboratory have implicated a role of the cytoskeleton in the mechanism of insulin-stimulated GLUT4 translocation in cultured 3T3-L1 adipocytes (21Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). These studies revealed that the microtubule protein α-tubulin and the intermediate filament protein vimentin are present in the preparations of GLUT4-containing membranes and confirmed their association with vesicles containing GLUT4 by electron microscopy. Disruption of the intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide caused the dispersion of perinuclear GLUT4. These and other findings (21Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) are consistent with the hypothesis that the molecular motor dynein directs the movements of GLUT4-containing membranes to the minus ends of microtubules in the juxtanuclear region of cultured adipocytes.The major aim of the present studies was to test the hypothesis that optimal insulin-mediated GLUT4 translocation to the cell periphery requires the microtubule motor kinesin in the context of an intact microtubule and F-actin cytoskeleton. Consistent with this concept, both nocodazole and colchicine were found to inhibit GLUT4 translocation markedly as well as disrupt microtubules in 3T3-L1 adipocytes. Insulin action on GLUT4 was also dependent on intact F-actin. We took further advantage of the ability of the neuronal microtubule-associated protein hTau40 to partially inhibit the function of kinesins when it is expressed heterologously in cultured cells as described by Mandelkow and co-workers (22Ebneth A. Godemann R. Stamer K. Illenberger S. Trinczek B. Mandelkow E. J. Cell Biol. 1998; 143: 777-794Crossref PubMed Scopus (652) Google Scholar, 23Trinczek B. Ebneth A. Mandelkow E.M. Mandelkow E. J. Cell Sci. 1999; 112: 2355-2367PubMed Google Scholar). The hTau40 protein localized to microtubules in 3T3-L1 adipocytes and delayed the initial appearance of GLUT4 at the cell surface membrane in response to insulin. The data are consistent with the hypothesis that insulin-stimulated GLUT4 movements to the cell periphery involve one or more kinesins. Physiological glucose homeostasis in humans is largely dependent on the actions of the hormone insulin, particularly its ability to inhibit glucose output from the liver and enhance glucose transport into fat and muscle cells. Insulin exerts this latter effect primarily through a process whereby sequestered intracellular GLUT4 glucose transporter proteins are rapidly redistributed to cell surface membranes in which they can catalyze glucose uptake into cells (1Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 2Suzuki K. Kono T. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2542-2545Crossref PubMed Scopus (768) Google Scholar, 3Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 4Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). In both the basal and insulin-stimulated states, GLUT4 proteins appear to cycle between intracellular membrane and plasma membrane locations (5Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar). However, in the basal state most of the GLUT4 is diverted to intracellular perinuclear membranes, and the exocytosis rate is slow. Insulin stimulates exocytosis of GLUT4 by a mechanism that requires the p85/p110 type phosphatidylinositol 3-kinase, which is recruited to protein phosphotyrosines in response to activation of the insulin receptor tyrosine kinase (6Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (997) Google Scholar, 7Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 8Sharma P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Insulin also appears to significantly inhibit GLUT4 endocytosis (5Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 9Czech M.P. Buxton J.M. J. Biol. Chem. 1993; 268: 9187-9190Abstract Full Text PDF PubMed Google Scholar, 10Jhun B.H. Rampal A.L. Liu H. Lachaal M. Jung C.Y. J. Biol. Chem. 1992; 267: 17710-17715Abstract Full Text PDF PubMed Google Scholar). However, the precise mechanism whereby insulin signals to the GLUT4 membrane trafficking machinery remains obscure. It is known that other membrane systems, such as lysosomes (11Harada A. Takei Y. Kanai Y. Tanaka Y. Nonaka S. Hirokawa N. J. Cell Biol. 1998; 141: 51-59Crossref PubMed Scopus (274) Google Scholar), mitochondria (12Tanaka Y. Kanai Y. Okada Y. Nonaka S. Takeda S. Harada A. Hirokawa N. Cell. 1998; 93: 1147-1158Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar), Golgi membranes (13Burkhardt J.K. Echeverri C.J. Nilsson T. Vallee R.B. J. Cell Biol. 1997; 139: 469-484Crossref PubMed Scopus (552) Google Scholar, 14Burkhardt J.K. Biochim. Biophys. Acta. 1998; 1404: 113-126Crossref PubMed Scopus (75) Google Scholar), and pigment granules (15Rogers S.L. Tint I.S. Fanapour P.C. Gelfand V.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3720-3725Crossref PubMed Scopus (188) Google Scholar), are localized within cells by molecular motors. For example, membrane vesicles containing melanin appear to be driven along microtubules over relatively long distances from their perinuclear location in unstimulated melanocytes to the cell periphery upon elevation of cAMP levels (16Reilein A.R. Tint I.S. Peunova N.I. Enikolopov G.N. Gelfand V.I. J. Cell Biol. 1998; 142: 803-813Crossref PubMed Scopus (72) Google Scholar). The complex motor dynein drives movements along microtubules in the minus direction toward the perinuclear microtubule organizing center, whereas kinesin motors drive movements toward the plus growing ends of microtubules (17Hirokawa N. Science. 1998; 279: 516-526Crossref Scopus (1361) Google Scholar). Movements of these membranes over shorter distances seem to require actin filaments (18Rodionov V.I. Hope A.J. Svitkina T.M. Borisy G.G. Curr. Biol. 1998; 8: 165-168Abstract Full Text Full Text PDF PubMed Google Scholar, 19Rogers S.L. Gelfand V.I. Curr. Biol. 1998; 8: 161-164Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Based on these observations, it has been suggested that the microtubule and actin filament networks are highly integrated in discharging their organelle localization functions (20Wu X. Jung G. Hammer III, J.A. Curr. Opin. Cell Biol. 2000; 12: 42-51Crossref PubMed Scopus (100) Google Scholar). Recent findings in our laboratory have implicated a role of the cytoskeleton in the mechanism of insulin-stimulated GLUT4 translocation in cultured 3T3-L1 adipocytes (21Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). These studies revealed that the microtubule protein α-tubulin and the intermediate filament protein vimentin are present in the preparations of GLUT4-containing membranes and confirmed their association with vesicles containing GLUT4 by electron microscopy. Disruption of the intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide caused the dispersion of perinuclear GLUT4. These and other findings (21Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) are consistent with the hypothesis that the molecular motor dynein directs the movements of GLUT4-containing membranes to the minus ends of microtubules in the juxtanuclear region of cultured adipocytes. The major aim of the present studies was to test the hypothesis that optimal insulin-mediated GLUT4 translocation to the cell periphery requires the microtubule motor kinesin in the context of an intact microtubule and F-actin cytoskeleton. Consistent with this concept, both nocodazole and colchicine were found to inhibit GLUT4 translocation markedly as well as disrupt microtubules in 3T3-L1 adipocytes. Insulin action on GLUT4 was also dependent on intact F-actin. We took further advantage of the ability of the neuronal microtubule-associated protein hTau40 to partially inhibit the function of kinesins when it is expressed heterologously in cultured cells as described by Mandelkow and co-workers (22Ebneth A. Godemann R. Stamer K. Illenberger S. Trinczek B. Mandelkow E. J. Cell Biol. 1998; 143: 777-794Crossref PubMed Scopus (652) Google Scholar, 23Trinczek B. Ebneth A. Mandelkow E.M. Mandelkow E. J. Cell Sci. 1999; 112: 2355-2367PubMed Google Scholar). The hTau40 protein localized to microtubules in 3T3-L1 adipocytes and delayed the initial appearance of GLUT4 at the cell surface membrane in response to insulin. The data are consistent with the hypothesis that insulin-stimulated GLUT4 movements to the cell periphery involve one or more kinesins. We thank Dr. Mandelkow for kindly providing the human Tau40 cDNA construct and Jane Erickson for excellent assistance with preparation of this manuscript.