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• W2011210199 abstract "The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here we make use of L6 myoblasts stably expressing GLUT4 with an exofacially directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic rates of the transporter. Insulin caused a rapid (t 12 = 4 min) gain, whereas hyperosmolarity (0.45m sucrose) caused a slow (t 12 = 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation followed by addition of hypertonic sucrose, the increase in surface GLUT4myc was partly additive. Unlike the effect of insulin, the GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t 12 = 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in response to hyperosmolarity was only partially blocked by inhibition of tyrosine kinases with erbstatin analog (erbstatin A) and genistein. However, neither inhibitor interfered with the ability of hyperosmolarity to block GLUT4myc internalization. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in these cells, might mediate membrane fusion of the hyperosmolarity-sensitive pool. The intracellular traffic of the glucose transporter 4 (GLUT4) in muscle cells remains largely unexplored. Here we make use of L6 myoblasts stably expressing GLUT4 with an exofacially directed Myc-tag (GLUT4myc) to determine the exocytic and endocytic rates of the transporter. Insulin caused a rapid (t 12 = 4 min) gain, whereas hyperosmolarity (0.45m sucrose) caused a slow (t 12 = 20 min) gain in surface GLUT4myc molecules. With prior insulin stimulation followed by addition of hypertonic sucrose, the increase in surface GLUT4myc was partly additive. Unlike the effect of insulin, the GLUT4myc gain caused by hyperosmolarity was insensitive to wortmannin or to tetanus toxin cleavage of VAMP2 and VAMP3. Disappearance of GLUT4myc from the cell surface was rapid (t 12 = 1.5 min). Insulin had no effect on the initial rate of GLUT4myc internalization. In contrast, hyperosmolarity almost completely abolished GLUT4myc internalization. Surface GLUT4myc accumulation in response to hyperosmolarity was only partially blocked by inhibition of tyrosine kinases with erbstatin analog (erbstatin A) and genistein. However, neither inhibitor interfered with the ability of hyperosmolarity to block GLUT4myc internalization. We propose that hyperosmolarity increases surface GLUT4myc by preventing GLUT4 endocytosis and stimulating its exocytosis via a pathway independent of phosphatidylinositol 3-kinase activity and of VAMP2 or VAMP3. A tetanus toxin-insensitive v-SNARE such as TI-VAMP detected in these cells, might mediate membrane fusion of the hyperosmolarity-sensitive pool. glucose transporter 4 o-phenylenediamine dihydrochloride HEPES-modified RPMI tetanus toxin phosphatidylinositol 3-kinase erbstatin analog vesicle-associated membrane protein tetanus toxin-insensitive VAMP green fluorescence protein enhanced GFP minimal essential medium phosphate-buffered saline trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane soluble NSF accessory protein SNAP receptor The glucose transporter 4 (GLUT4)1 is the predominant glucose transporter of muscle and adipose cells. In untreated adipocytes, GLUT4 recycles constitutively between the plasma membrane and intracellular loci (1Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 2Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar), with the steady-state distribution favoring the latter. Morphological and biochemical studies have detected GLUT4 in distinct but inter-related intracellular pools, including sorting endosomes, TGN, recycling endosomes, and specialized GLUT4 exocytic vesicles (3Lee W. Ryu J. Souto R.P. Pilch P.F. Jung C.Y. J. Biol. Chem. 1999; 274: 37755-37762Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 4Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (709) Google Scholar, 5Hashiramoto M. James D.E. Mol. Cell. Biol. 2000; 20: 416-427Crossref PubMed Scopus (93) Google Scholar, 6Aledo J.C. Lavoie L. Volchuk A. Keller S.R. Klip A. Hundal H.S. Biochem. J. 1997; 325: 727-732Crossref PubMed Scopus (60) Google Scholar). GLUT4 endocytosis occurs via clathrin-coated vesicles, assisted by the GTPase dynamin. Thus, inhibition of clathrin-coated vesicle formation via K+depletion (7Nishimura H. Zarnowski M.J. Simpson I.A. J. Biol. Chem. 1993; 268: 19246-19253Abstract Full Text PDF PubMed Google Scholar), interference with dynamin-amphiphysin pairing (8Volchuk A. Narine S. Foster L. Grabs D. DeCamilli P. Klip A. J. Biol. Chem. 1998; 273: 8169-8176Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), or expression of GTPase-deficient dynamin (9Al-Hasani H. Kinck C.S. Cushman S.W. J. Biol. Chem. 1998; 273: 17504-17510Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Kao A.W. Ceresa B.P. Santeler S.R. Pessin J.E. J. Biol. Chem. 1998; 273: 25450-25457Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), all prevent GLUT4 internalization in adipocytes. Little is known about the traffic of this transporter in muscle cells, despite the fact that muscle represents the largest in vivo site of glucose utilization.Insulin shifts the subcellular distribution of GLUT4 resulting in a new steady state where a large fraction of GLUT4 resides at the plasma membrane of skeletal muscle (11Douen A.G. Ramlal T. Rastogi S. Bilan P.J. Cartee G.D. Vranic M. Holloszy J.O. Klip A. J. Biol. Chem. 1990; 265: 13427-13430Abstract Full Text PDF PubMed Google Scholar, 12Hirshman M.F. Goodyear L.J. Wardzala L.J. Horton E.D. Horton E.S. J. Biol. Chem. 1990; 265: 987-991Abstract Full Text PDF PubMed Google Scholar, 13Klip A. Ramlal T. Bilan P.J. Cartee G.D. Gulve E.A. Holloszy J.O. Biochem. Biophys. Res. Commun. 1990; 172: 728-736Crossref PubMed Scopus (119) Google Scholar), primary adipose cells (14James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (454) Google Scholar, 15Birnbaum M.J. Cell. 1989; 57: 305-315Abstract Full Text PDF PubMed Scopus (460) Google Scholar), L6 muscle cells in culture (16Ramlal T. Sarabia V. Bilan P.J. Klip A. Biochem. Biophys. Res. Commun. 1988; 157: 1329-1335Crossref PubMed Scopus (45) Google Scholar), and 3T3-L1 adipocytes (1Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar). Studies in adipocytes indicate that this shift occurs primarily through the stimulation of GLUT4 exocytosis (1Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 2Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar), but whether or not insulin inhibits GLUT4 endocytosis is still debatable (1Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 2Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar, 17Czech M.P. Buxton J.M. J. Biol. Chem. 1993; 268: 9187-9190Abstract Full Text PDF PubMed Google Scholar, 18Jhun 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). The contribution of exocytic and endocytic pathways to insulin action in muscle cells has not been explored.Exposing 3T3-L1 adipocytes to hyperosmolar solutions also leads to the cell surface accumulation of GLUT4. It was postulated that this accumulation results from the stimulation of GLUT4 exocytosis by signals different from those elicited by insulin (19Chen D. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 20Chen D. Fucini R.V. Olson A.L. Hemmings B.A. Pessin J.E. Mol. Cell. Biol. 1999; 19: 4684-4694Crossref PubMed Google Scholar, 21Janez A. Worrall D.S. Imamura T. Sharma P.M. Olefsky J.M. J. Biol. Chem. 2000; 275: 26870-26876Abstract Full Text Full Text PDF PubMed Google Scholar) through a so-called “alternative pathway.” However, the steady-state analysis used in those studies precluded the distinction of exocytic from endocytic traffic of GLUT4. The well-known effect of hyperosmolarity to disrupt clathrin function (22Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 121: 61-72Crossref PubMed Scopus (296) Google Scholar, 23Heuser J.E. Anderson R.G. J. Cell Biol. 1989; 108: 877-886Google Scholar) has prompted us to hypothesize that hyperosmolarity accumulates GLUT4 at the cell surface by inhibiting GLUT4 endocytosis. We have previously characterized a clone of L6 myoblasts stably expressing GLUT4 tagged with an exofacial myc epitope (GLUT4myc) (24Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar), which could be used to more accurately measure GLUT4 endocytosis in response to different stimuli.The objective of this study was to investigate how insulin and hyperosmolarity affect the recycling of GLUT4 in L6 muscle cells in culture, to examine how each stimulus modulates the endocytic and exocytic arms of GLUT4 traffic, and to compare the characteristics of the donor pools of GLUT4 in each instance. We show that both insulin and hyperosmolarity induce the subcellular redistribution of GLUT4 from intracellular loci to the plasma membrane in L6 myoblasts. Whereas insulin primarily stimulates GLUT4 exocytosis, hyperosmolarity largely prevents its endocytosis. Although inhibition of tyrosine kinases prevents significantly insulin-stimulated GLUT4 exocytosis, it does not affect the hyperosmolarity-induced block of GLUT4 internalization and only partially blocks hyperosmolarity-triggered GLUT4 accumulation at the cell surface. The results suggest that insulin draws GLUT4 from a specific pool that is affected by tetanus toxin and wortmannin. In contrast, the accumulation of GLUT4 at the cell surface caused by hyperosmolarity results from both reducing GLUT4 endocytosis and stimulating its exocytosis. Hyperosmolarity is likely to draw GLUT4 from the recycling endosomal pool and/or an “alternative pool” that is insensitive to inhibition by tetanus toxin and wortmannin.DISCUSSIONGLUT4 is a determinant of insulin sensitivity in muscle and fat cells. In the L6 skeletal muscle cell line, GLUT4 expression occurs after differentiation from myoblasts into myotubes (26Mitsumoto Y. Klip A. J. Biol. Chem. 1992; 267: 4957-4962Abstract Full Text PDF PubMed Google Scholar). We have previously reported that expression of GLUT4myc in L6 myoblasts leads to the segregation of the protein to a GLUT4-specific pool, conferring insulin sensitivity to glucose uptake (24Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar). This conclusion is based on the finding that, in L6-GLUT4myc myoblasts, the intracellular GLUT4myc compartment contains the majority of the insulin-regulatable amino peptidase but less than half of the GLUT1, and the sensitivity of glucose uptake to insulin is markedly improved. Indeed, we confirm in the present study that 90% of the GLUT4myc resides intracellularly. 2In a previous report, a shorter incubation time with 0.1% Triton X-100 resulted in incomplete cell permeabilization and underestimation of the intracellular content of GLUT4myc (24Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar). Upon insulin or hypertonic sucrose stimulation, 30% of the total cellular GLUT4myc is redistributed to the cell surface within 30 min. These results demonstrate that, as with previous observations in 3T3-L1 adipocytes, GLUT4myc is vastly retained in the intracellular pool in the basal state and is redistributed to the cell surface in response to insulin and hyperosmolarity in L6-GLUT4myc myoblasts.Insulin Stimulates GLUT4 Exocytosis and Hyperosmolarity Inhibits Its InternalizationGLUT4myc undergoes rapid internalization upon insulin removal. Half of the surface-labeled GLUT4myc is internalized within 3 min. Even in the presence of insulin, the rate of GLUT4myc internalization is not appreciably slowed down, having approximately the same t 12 of 3 min. These results suggest that insulin does not regulate GLUT4 internalization in L6-GLUT4myc myoblasts. This contrasts with observations made in fat cells where a small proportion of insulin-induced gain in surface GLUT4 appears to be due to inhibition of GLUT4 endocytosis (1Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 18Jhun 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). The 3-min half-time measured for GLUT4myc internalization in L6 myoblasts is very similar to the 4.2- or 3-min half-time for GLUT4 or insulin-regulatable amino peptidase endocytosis, respectively, reported for 3T3-L1 adipocytes in the presence of insulin (1Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 43Garza L.A. Birnbaum M.J. J. Biol. Chem. 2000; 275: 2560-2567Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).GLUT4 internalizes via clathrin-coated pits (7Nishimura H. Zarnowski M.J. Simpson I.A. J. Biol. Chem. 1993; 268: 19246-19253Abstract Full Text PDF PubMed Google Scholar, 44Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (256) Google Scholar). K+depletion and hypertonic shock are two strategies known to perturb the formation of clathrin coats (22Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 121: 61-72Crossref PubMed Scopus (296) Google Scholar, 23Heuser J.E. Anderson R.G. J. Cell Biol. 1989; 108: 877-886Google Scholar, 41Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (339) Google Scholar) by preventing the interaction between clathrin and adaptors proteins (22Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 121: 61-72Crossref PubMed Scopus (296) Google Scholar). We demonstrate here that K+ depletion mimics hyperosmolarity by causing a gain in GLUT4myc at the cell surface and preventing GLUT4myc endocytosis. These results strongly support the concept that hyperosmolarity accumulates GLUT4 at the cell surface, at least in part, through inhibition of GLUT4 endocytosis.Hyperosmolarity is a stress stimulus that activates a tyrosine kinase pathway (19Chen D. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 45Hresko R.C. Mueckler M. J. Biol. Chem. 2000; 275: 18114-18120Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), and tyrosine kinase activity is required for the surface gain in GLUT4 in 3T3-L1 adipocytes (19Chen D. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). However, it is unlikely that this effect is related to the retention of GLUT4 at the cell surface, because an inhibitor of the tyrosine kinases, erbstatin A, was unable to prevent the inhibition of GLUT4myc endocytosis by hyperosmolarity in muscle cells (Fig. 9 C). Erbstatin A and genistein prevented the hyperosmolarity-induced GLUT4 externalization by only ∼50% (Fig. 9, A and B). We propose that inhibition of GLUT4 endocytosis accounts for 50% of the GLUT4 surface accumulation, and the remaining 50% arises from the stimulation of GLUT4 exocytosis in response to hyperosmolarity. Chenet al. (19Chen D. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) reported complete prevention of hyperosmolarity-induced GLUT4 translocation by inhibition of tyrosine kinases. Therefore, it is conceivable that GLUT4 endocytosis is not blocked by hyperosmolarity in 3T3-L1 cells.Insulin and Hyperosmolarity Draw GLUT4 from Different Intracellular PoolsGLUT4 accumulated at the cell surface with very different time courses in response to insulin and hypertonic sucrose. We speculate that there may be an insulin-regulated exocytic GLUT4 pool in L6 muscle cells, which can be rapidly mobilized by insulin and that hyperosmolarity draws GLUT4 from an alternative pool of GLUT4 and/or the recycling endosomes. Supporting this concept that insulin and hyperosmolarity draw GLUT4 from different intracellular pools in L6 muscle cells is their differential sensitivity to tetanus toxin.VAMP2 and VAMP3 are expressed in muscle and fat cells (46Cain C.C. Trimble W.S. Lienhard G.E. J. Biol. Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar, 47Volchuk A. Mitsumoto Y. He L. Liu Z. Habermann E. Trimble W. Klip A. Biochem. J. 1994; 304: 139-145Crossref PubMed Scopus (69) Google Scholar, 48Volchuk A. Sargeant R. Sumitani S. Liu Z. He L. Klip A. J. Biol. Chem. 1995; 270: 8233-8240Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and reside in different GLUT4 pools in both muscle and fat cells (5Hashiramoto M. James D.E. Mol. Cell. Biol. 2000; 20: 416-427Crossref PubMed Scopus (93) Google Scholar, 40Randhawa V.K. Bilan P.J. Khayat Z.A. Daneman N. Liu Z. Ramlal T. Volchuk A. Peng X.R. Coppola T. Regazzi R. Trimble W.S. Klip A. Mol. Biol. Cell. 2000; 11: 2403-2417Crossref PubMed Scopus (98) Google Scholar,48Volchuk A. Sargeant R. Sumitani S. Liu Z. He L. Klip A. J. Biol. Chem. 1995; 270: 8233-8240Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). As shown earlier (40Randhawa V.K. Bilan P.J. Khayat Z.A. Daneman N. Liu Z. Ramlal T. Volchuk A. Peng X.R. Coppola T. Regazzi R. Trimble W.S. Klip A. Mol. Biol. Cell. 2000; 11: 2403-2417Crossref PubMed Scopus (98) Google Scholar) and confirmed here, tetanus toxin reduced the insulin-dependent GLUT4myc translocation in L6 myoblasts. Our previous study also demonstrated that the reduction can be rescued by the tetanus toxin-resistant mutant VAMP2 but not by the toxin-resistant mutant VAMP3, suggesting that VAMP2 but not VAMP3 is required for GLUT4 vesicle fusion with the plasma membrane in response to insulin (40Randhawa V.K. Bilan P.J. Khayat Z.A. Daneman N. Liu Z. Ramlal T. Volchuk A. Peng X.R. Coppola T. Regazzi R. Trimble W.S. Klip A. Mol. Biol. Cell. 2000; 11: 2403-2417Crossref PubMed Scopus (98) Google Scholar). We now show that expression of tetanus toxin does not alter GLUT4myc externalization caused by hyperosmolarity. These results support the concept that insulin and hyperosmolarity recruit GLUT4 from different intracellular pools, one requiring VAMP2 and another one that is tetanus toxin-insensitive. Thus, neither VAMP2 nor VAMP3 are the fusogenic v-SNARE for the incorporation of GLUT4 vesicles from recycling endosomes into the plasma membrane. It is conceivable that a tetanus toxin-insensitive VAMP such as TI-VAMP (49Galli T. Zahraoui A. Vaidyanathan V.V. Raposo G. Tian J.M. Karin M. Niemann H. Louvard D. Mol. Biol. Cell. 1998; 9: 1437-1448Crossref PubMed Scopus (257) Google Scholar) could mediate fusion of the recycling endosome with the plasma membrane in muscle cells. Indeed, TI-VAMP was detected in L6-GLUT4myc myoblasts, and its localization was partially distinct from that of VAMP2 or VAMP3. The possible role of TI-VAMP in fusion of the hyperosmolarity-drawn pool is under investigation.The action of insulin and hyperosmolarity on GLUT4myc externalization was partly additive. A previous study failed to observe an additive effect in 3T3-L1 adipocytes where the cells were pretreated with hyperosmolar solution prior to exposure to insulin (20Chen D. Fucini R.V. Olson A.L. Hemmings B.A. Pessin J.E. Mol. Cell. Biol. 1999; 19: 4684-4694Crossref PubMed Google Scholar). We also failed to observe any additive effect when we incubated L6-GLUT4myc myoblasts in the same manner. The lack of additivity under these latter conditions may be due to the inhibition of insulin signaling by hyperosmolarity at the level of Akt, as reported previously (20Chen D. Fucini R.V. Olson A.L. Hemmings B.A. Pessin J.E. Mol. Cell. Biol. 1999; 19: 4684-4694Crossref PubMed Google Scholar). In contrast, treating L6 muscle cells with insulin, followed by the addition of hyperosmolar solution, caused a further increase in the surface GLUT4myc compared with the effect of either stimulus alone. Hypertonic sucrose must draw GLUT4myc from an alternative GLUT4 pool and/or recycling endosomal pool to account for the appearance of additional GLUT4myc at the cell surface, and as shown in Fig. 7, also retains GLUT4myc at the cell surface. Hence, hyperosmolarity causes a compounded gain of GLUT4 molecules at the cell surface.Lastly, insulin and hyperosmolarity engage different signals in their action. PI3K activation is required for insulin-dependent GLUT4 translocation in fat and muscle cells (29Tsakiridis T. McDowell H. Walker T. Downes P. Hundal H.S. Vranic M. Klip A. Endocrinology. 1995; 136: 4315-4322Crossref PubMed Google Scholar, 30Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (998) Google Scholar, 50Khayat Z. Tong P. Yaworsky K. Bloch R. Klip A. J. Cell Sci. 2000; 113: 279-290Crossref PubMed Google Scholar, 51Kanai F. Ito K. Todaka M. Hayashi H. Kamohara S. Ishii K. Okada T. Hazeki O. Ui M. Ebina Y. Biochem. Biophys. Res. Commun. 1993; 195: 762-768Crossref PubMed Scopus (265) Google Scholar, 52Quon 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: 5403-5411Crossref PubMed Scopus (143) Google Scholar, 53Haruta T. Morris A.J. Rose D.W. Nelson J.G. Mueckler M. Olefsky J.M. J. Biol. Chem. 1995; 270: 27991-27994Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In contrast, the hyperosmolarity-induced accumulation of GLUT4 at the cell surface is not prevented by the PI3K inhibitor wortmannin in 3T3-L1 adipocytes (19Chen D. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). 3A small inhibition was seen in one study using wortmannin concentrations of 100 nm or higher, which might have inhibited other pathways (21Janez A. Worrall D.S. Imamura T. Sharma P.M. Olefsky J.M. J. Biol. Chem. 2000; 275: 26870-26876Abstract Full Text Full Text PDF PubMed Google Scholar). Here we demonstrate that PI3K activity is not required for GLUT4myc externalization caused by hyperosmolarity in L6 myoblasts.Current models suggest that in 3T3-L1 adipocytes the increased amount of GLUT4 at the surface in response to insulin is due to recruitment of GLUT4 from a putatively static exocytic pool and from a continuously recycling pool (54Rice J.E. Livingstone C. Gould G.W. Biochem. Soc. Trans. 1996; 24: 540-546Crossref PubMed Scopus (11) Google Scholar, 55Fletcher L.M. Tavaré J.M. Biochem. Soc. Trans. 1999; 27: 677-683Crossref PubMed Scopus (7) Google Scholar, 56Foran P.G. Fletcher L.M. Oatey P.B. Mohammed N. Dolly J.O. Tavare J.M. J. Biol. Chem. 1999; 274: 28087-28095Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Our results suggest that in muscle cells a major effect of insulin is to cause GLUT4myc exocytosis from a specific pool (likely specialized vesicles), which is distinct from the pool mobilized by hyperosmolarity and from the basal-state recycling pool. Fig. 10 illustrates the pathways of intracellular GLUT4 traffic highlighted by this study.In summary, the majority (90%) of GLUT4myc is located intracellularly in L6 myoblasts in the basal state. In the presence of insulin, 70% of GLUT4myc resides intracellularly and 30% recycles back to the cell surface with a t 12 of ∼4 min. Under hyperosmolar stress, 65% of the GLUT4myc remains intracellularly and 35% recycles with a t 12 of ∼20 min. Insulin and hyperosmolarity draw GLUT4 from different pools. Insulin recruits GLUT4 primarily from a specialized pool that requires the participation of PI3K and VAMP2. Hyperosmolarity accelerates GLUT4 exocytosis from an alternative pool and/or the recycling pool that are PI3K-independent and tetanus toxin-insensitive, and it also retains the GLUT4 molecules at the cell surface by blocking their endocytosis. This latter action significantly contributes to the accumulation of GLUT4 at the cell surface by hyperosmolarity. The possible interconnections among the specialized, alternative, and recycling pools require further investigation. The glucose transporter 4 (GLUT4)1 is the predominant glucose transporter of muscle and adipose cells. In untreated adipocytes, GLUT4 recycles constitutively between the plasma membrane and intracellular loci (1Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 2Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar), with the steady-state distribution favoring the latter. Morphological and biochemical studies have detected GLUT4 in distinct but inter-related intracellular pools, including sorting endosomes, TGN, recycling endosomes, and specialized GLUT4 exocytic vesicles (3Lee W. Ryu J. Souto R.P. Pilch P.F. Jung C.Y. J. Biol. Chem. 1999; 274: 37755-37762Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 4Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (709) Google Scholar, 5Hashiramoto M. James D.E. Mol. Cell. Biol. 2000; 20: 416-427Crossref PubMed Scopus (93) Google Scholar, 6Aledo J.C. Lavoie L. Volchuk A. Keller S.R. Klip A. Hundal H.S. Biochem. J. 1997; 325: 727-732Crossref PubMed Scopus (60) Google Scholar). GLUT4 endocytosis occurs via clathrin-coated vesicles, assisted by the GTPase dynamin. Thus, inhibition of clathrin-coated vesicle formation via K+depletion (7Nishimura H. Zarnowski M.J. Simpson I.A. J. Biol. Chem. 1993; 268: 19246-19253Abstract Full Text PDF PubMed Google Scholar), interference with dynamin-amphiphysin pairing (8Volchuk A. Narine S. Foster L. Grabs D. DeCamilli P. Klip A. J. Biol. Chem. 1998; 273: 8169-8176Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), or expression of GTPase-deficient dynamin (9Al-Hasani H. Kinck C.S. Cushman S.W. J. Biol. Chem. 1998; 273: 17504-17510Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Kao A.W. Ceresa B.P. Santeler S.R. Pessin J.E. J. Biol. Chem. 1998; 273: 25450-25457Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), all prevent GLUT4 internalization in adipocytes. Little is known about the traffic of this transporter in muscle cells, despite the fact that muscle represents the largest in vivo site of glucose utilization. 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