Role of SNAP23 in Insulin-induced Translocation of GLUT4 in 3T3-L1 Adipocytes
2000; Elsevier BV; Volume: 275; Issue: 11 Linguagem: Inglês
10.1074/jbc.275.11.8240
ISSN1083-351X
AutoresMasatoshi Kawanishi, Yoshikazu Tamori, Hideki Okazawa, Satoshi Araki, Hiroaki Shinoda, Masato Kasuga,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoBoth syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-ΔC8 (amino acids 1 to 202) and SNAP23-ΔC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro andin vivo. Although SNAP23-ΔC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-ΔC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-ΔC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-ΔC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-ΔC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2). Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-ΔC8 (amino acids 1 to 202) and SNAP23-ΔC49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro andin vivo. Although SNAP23-ΔC49 bound to neither syntaxin4 nor VAMP2, the SNAP23-ΔC8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-ΔC8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-ΔC8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-ΔC49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2). N-ethylmaleimide-sensitive factor soluble NSF-attachment protein receptor vesicle and target SNARE, respectively synaptosome-associated 23-kDa protein wild type glutathione S-transferase hemagglutinin phosphate-buffered saline multiplicity of infection plaque-forming unit A primary function of insulin is to stimulate the transport of glucose into target tissues, prominent among which are skeletal muscle, cardiac muscle, and adipose tissue. Insulin achieves this effect by inducing the translocation of GLUT4 glucose transporters from an intracellular vesicular compartment to the plasma membrane. Under basal conditions, GLUT4 cycles slowly between this intracellular compartment and the plasma membrane (1.Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, 2.Kandror K.V. Pilch P. Am. J. Physiol. 1996; 271: E1-E14Crossref PubMed Google Scholar). However, activation of insulin receptors triggers a large increase in the rate of exocytosis of GLUT4-containing vesicles and a smaller decrease in the rate of GLUT4 internalization by endocytosis (3.Holman G.D. Kozka I.J. Clark A.E. Flower C.J. Saltis J. Habberfield A.D. Simpson I.A. Cushman S.W. J. Biol. Chem. 1990; 265: 18172-18179Abstract Full Text PDF PubMed Google Scholar, 4.Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 5.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. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar), with the former action likely contributing most to the insulin-induced increase in the amount of GLUT4 in the plasma membrane (6.Jhun 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).Intracellular membrane fusion is mediated by evolutionarily conserved membrane proteins known as solubleN-ethylmaleimide-sensitive factor (NSF)1 attachment protein receptors (SNAREs) (7.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar, 8.Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar). SNARE proteins that contribute to neuronal exocytosis include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the plasma membrane proteins synaptosome-associated 25-kDa protein and syntaxin1A. These proteins readily assemble into a stable ternary complex; however, disassembly of this complex can be reversibly induced by the ATPase NSF in conjunction with soluble cofactors termed SNAPs (soluble NSF-attachment proteins) (9.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2603) Google Scholar). The formation of the SNARE complex is thought to be a critical step in the pathway leading to membrane fusion.The insulin-stimulated trafficking of GLUT4 vesicles in adipocytes shares several features with the regulated pathway of synaptic vesicle exocytosis. Thus, members of the synaptobrevin (VAMP) family of proteins were shown to localize to GLUT4 vesicles in rat adipocytes (10.Cain C.C. Trimble W.S. Lienhard G.E. J. Biol. Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar). These proteins were identified as VAMP2 and cellubrevin in 3T3-L1 adipocytes (11.Volchuk 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 were subsequently shown to be essential for insulin-stimulated GLUT4 translocation in these cells (12.Tamori Y. Hashiramoto M. Araki S. Kamata Y. Takahashi M. Kozaki S. Kasuga M. Biochem. Biophys. Res. Commun. 1996; 220: 740-745Crossref PubMed Scopus (61) Google Scholar, 13.Martin L.B. Shewan A. Millar C.A. Gould C.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Furthermore, syntaxin isoform 4 was shown to contribute to the translocation of GLUT4 in 3T3-L1 adipocytes (14.Olson A. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (207) Google Scholar). In addition, SNAP23, a widely expressed isoform of SNAP25 (15.Ravichadran V. Chawla A. Roche P.A. J. Biol. Chem. 1996; 271: 13300-13303Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), was shown to be present in the plasma membrane of 3T3-L1 adipocytes (16.Araki S. Tamori Y. Kawanishi M. Shinoda H. Masugi J. Mori H. Niki T. Okazawa H. Kubota T. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 234: 257-262Crossref PubMed Scopus (85) Google Scholar) and to be colocalized with syntaxin4 in these cells (17.Tamori Y. Kawanishi M. Niki T. Shinoda H. Araki S. Okazawa H. Kasuga M. J. Biol. Chem. 1998; 273: 19740-19746Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, SNAP23 in adipocytes, like SNAP25 in neurons, may function in the exocytosis of intracellular vesicles.We have now investigated the possible role of SNAP23 as a functional plasma membrane t-SNARE in insulin-stimulated GLUT4 translocation. Our data demonstrate that SNAP23 is required for insulin-induced GLUT4 translocation to the plasma membrane and that it mediates the formation of a complex between syntaxin4 and VAMP2.DISCUSSIONWe have shown that the SNAP23 mutant SNAP23-ΔC8, which binds to syntaxin4 but not to VAMP2, inhibits the translocation of GLUT4, but not that of GLUT1, to the plasma membrane in 3T3-L1 adipocytes. This inhibition of GLUT4 translocation is likely attributable to prevention of the formation of a ternary SNARE complex among SNAP23, syntaxin4, and VAMP2 at the plasma membrane of these cells. Thus, SNAP-23 appears to function as a t-SNARE in the translocation of GLUT4 in 3T3-L1 adipocytes.SNAP23 was identified as a widely expressed homolog of SNAP25 (15.Ravichadran V. Chawla A. Roche P.A. J. Biol. Chem. 1996; 271: 13300-13303Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Although SNAP25 is essential for exocytotic membrane fusion in neurons, this t-SNARE does not appear to be expressed in most nonneural tissues, with the exception of pancreatic islets of Langerhans (34.Sadoul K. Lang J. Montecucco C. Weller U. Regazzi R. Catsicas S. Wollheim C.B. Halban P.A. J. Cell Biol. 1995; 128: 1019-1028Crossref PubMed Scopus (229) Google Scholar, 35.Jacobsson G. Bean A.J. Scheller R.H. Junttiberggren L. Deeney J.T. Berggren P.O. Meister B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12487-12491Crossref PubMed Scopus (194) Google Scholar), adrenal chromaffin cells (36.Roth D. Burgoyne R.D. FEBS Lett. 1994; 351: 207-210Crossref PubMed Scopus (100) Google Scholar), and anterior pituitary cells (37.Aguado F. Majo G. Ruiz-Montasell B. Canals J.M. Casanova A. Marsal J. Blasi J. Eur. J. Cell Biol. 1996; 69: 351-359PubMed Google Scholar). Although Jagadish et al. (38.Jagadish M.N. Fernandez C.S. Hewish D.R. Macaulay S.L. Gough K.H. Grusovin J. Verkuylen A. Cosgrove L. Alafaci A. Frenkel M.J. Ward C.W. Biochem. J. 1996; 317: 945-954Crossref PubMed Scopus (46) Google Scholar) detected SNAP25 mRNA and protein in fat cells and 3T3-L1 adipocytes by sensitive methods, Timmer et al. (39.Timmer K.I. Clark A.E. Omatsu-Kanbe M. Whiteheart S.W. Bennett M.K. Holman G.D. Cushman S.W. Biochem. J. 1996; 320: 429-436Crossref PubMed Scopus (55) Google Scholar) and Wong et al. (40.Wong P.P.C. Daneman N. Volchuk A. Lassam N. Wilson M.C. Klip A. Trimble W.S. Biochem. Biophys. Res. Commun. 1997; 230: 64-68Crossref PubMed Scopus (60) Google Scholar) did not detect SNAP25 in adipocytes. In contrast, SNAP-23 was shown not only to be expressed in 3T3-L1 adipocytes (16.Araki S. Tamori Y. Kawanishi M. Shinoda H. Masugi J. Mori H. Niki T. Okazawa H. Kubota T. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 234: 257-262Crossref PubMed Scopus (85) Google Scholar) but also to be colocalized in these cells with syntaxin4 (17.Tamori Y. Kawanishi M. Niki T. Shinoda H. Araki S. Okazawa H. Kasuga M. J. Biol. Chem. 1998; 273: 19740-19746Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and VAMP2 (41.Rea S. Martin L.B. McIntosh S. Macaulay S.L. Ramsdale T. Baldini G. James D.E. J. Biol. Chem. 1998; 273: 18784-18792Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), both of which are implicated as important mediators of the translocation of GLUT4 (12.Tamori Y. Hashiramoto M. Araki S. Kamata Y. Takahashi M. Kozaki S. Kasuga M. Biochem. Biophys. Res. Commun. 1996; 220: 740-745Crossref PubMed Scopus (61) Google Scholar, 13.Martin L.B. Shewan A. Millar C.A. Gould C.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 14.Olson A. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (207) Google Scholar). These observations thus suggest that SNAP23 plays an important role as a plasma membrane t-SNARE, functioning together with syntaxin4 and VAMP2 in the translocation of GLUT4 in 3T3-L1 adipocytes. Furthermore, the observation that botulinum neurotoxin E, which cleaves SNAP25 but not SNAP23, does not markedly inhibit insulin-induced GLUT4 translocation in 3T3-L1 adipocytes (42.Macaulay S.L. Rea S. Gough G.H. Ward C.W. James D.E. Biochem. Biophys. Res. Commun. 1997; 237: 388-393Crossref PubMed Scopus (20) Google Scholar) is consistent with the hypothesis that SNAP23, but not SNAP25, functions as a t-SNARE in the translocation of GLUT4 in these cells.SNAP25 is hydrolyzed by botulinum neurotoxin E between Arg180 and Ile181 and is also cleaved by botulinum neurotoxin A between Gln197 and Arg198 (43.Schiavo G. Santucci A. Dasgupta B.R. Mehta P.P. Jontes J. Benfenati F. Wilson M.C. Monteccuco C. FEBS Lett. 1993; 335: 99-103Crossref PubMed Scopus (374) Google Scholar). Removal of the nine COOH-terminal residues of SNAP25, which yields a fragment corresponding to that generated by neurotoxin A, reduces the extent of the interaction between SNAP25 and VAMP2 but not of that between SNAP25 and syntaxin1 (29.Chapman E.R. An S. Barton N. Jahn R. J. Biol. Chem. 1994; 269: 27427-27432Abstract Full Text PDF PubMed Google Scholar). Neither of these neurotoxins hydrolyzes SNAP23 (31.Chen F. Foran P. Shone C.C. Foster K.A. Melling J. Dolly J.O. Biochemistry. 1997; 36: 5719-5728Crossref PubMed Scopus (49) Google Scholar, 42.Macaulay S.L. Rea S. Gough G.H. Ward C.W. James D.E. Biochem. Biophys. Res. Commun. 1997; 237: 388-393Crossref PubMed Scopus (20) Google Scholar). We therefore constructed two SNAP23 mutants. SNAP23-ΔC8 lacks the eight COOH-terminal amino acids of the full-length protein and corresponds to the fragment of SNAP25 generated by neurotoxin A, whereas SNAP23-ΔC49 lacks the COOH-terminal coiled-coil region. The observation that SNAP23-ΔC8 retained the ability to bind syntaxin4 but was not able to bind VAMP2 is consistent with the binding properties of the SNAP25 fragment lacking the nine COOH-terminal residues of the full-length protein. However, whereas the binding of SNAP23-ΔC49 to syntaxin4 was markedly reduced compared with that of the wild-type protein, the binding affinity of the NH2-terminal half of SNAP25 for syntaxin1 was similar to that of the full-length protein (29.Chapman E.R. An S. Barton N. Jahn R. J. Biol. Chem. 1994; 269: 27427-27432Abstract Full Text PDF PubMed Google Scholar). This difference between SNAP23 and SNAP25 may result from the fact that SNAP25 contains two putative coiled-coil domains, which are thought to be important for association with syntaxins in its NH2-terminal region, whereas SNAP23-ΔC49 contains only one such domain; the COOH-terminal coiled-coil region of SNAP23 may thus be important for the interaction with syntaxin4. Although both overexpressed SNAP23-WT and SNAP23-ΔC8 expressed in the plasma membrane of 3T3-L1 adipocytes formed a complex with endogenous syntaxin4, only SNAP23-WT interacted to a substantial extent with VAMP2 localized on GLUT4 vesicles. This observation is consistent with our in vitro data showing that only the complex of SNAP23-WT with syntaxin4, not that of SNAP23-ΔC8 with syntaxin4, bound to GST-VAMP2. It is therefore likely that SNAP23-ΔC8 inhibited the translocation of GLUT4 in 3T3-L1 adipocytes by interfering with formation of a ternary SNARE complex consisting of SNAP23, syntaxin4, and VAMP2.Recent electron microscopic, spectroscopic, and x-ray crystallographic data indicate that SNAREs form a rod-shaped complex consisting of a coiled-coil of four α helices, one contributed by the v-SNARE (VAMP) and three by the t-SNAREs (one by syntaxin and two by SNAP25). The syntaxin and SNAP25 helices are knit together by many nonpolar interactions that hold VAMP in position in the four-helix coiled coil; the binary t-SNARE complex (syntaxin and SNAP25) forms a cradle-like structure to which the v-SNARE (VAMP) appears to bind (44.Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1900) Google Scholar). On the basis of these observations, it is likely that the coiled coil of three α helices formed by syntaxin4 and SNAP23-ΔC8 cannot associate with the α helix of VAMP2 in 3T3-L1 adipocytes. A synthetic peptide corresponding to the 24 COOH-terminal amino acids of SNAP23 inhibits insulin-induced GLUT4 translocation to the surface of permeabilized 3T3-L1 adipocytes (41.Rea S. Martin L.B. McIntosh S. Macaulay S.L. Ramsdale T. Baldini G. James D.E. J. Biol. Chem. 1998; 273: 18784-18792Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). However, this peptide did not interfere with the formation in vitro of a functional SNARE complex comprising syntaxin4, SNAP23, and VAMP2, as revealed by surface plasmon resonance (41.Rea S. Martin L.B. McIntosh S. Macaulay S.L. Ramsdale T. Baldini G. James D.E. J. Biol. Chem. 1998; 273: 18784-18792Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In contrast, our results indicate that SNAP23 functions in the translocation of GLUT4 by promoting the interaction between syntaxin4 and VAMP2.GLUT4 appears to be localized to at least two intracellular compartments: the endosomal compartment and a post-endocytotic compartment (13.Martin L.B. Shewan A. Millar C.A. Gould C.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In contrast, GLUT1 is predominantly targeted to the recycling endosomal compartment (45.Martin S. Rice J.E. Gould G.W. Keller S.R. Slot J.W. James D.E. J. Cell Sci. 1997; 110: 2281-2291Crossref PubMed Google Scholar, 46.Martin S. Tellam J. Livingstone C. Slot J.W. Gould G.W. James D.E. J. Cell Biol. 1996; 134: 625-635Crossref PubMed Scopus (180) Google Scholar, 47.Livingstone C. James D.E. Rice J.E. Hanpeter D. Gould G.W. Biochem. J. 1996; 315: 487-495Crossref PubMed Scopus (129) Google Scholar). It must, however, be added that several studies show that GLUT1 and GLUT4 largely co-localize in 3T3-L1 adipocytes (32.Calderhead D.M. Kitagawa W. Tanner L.I. Holman G.D. Lienhard G.E. J. Biol. Chem. 1990; 265: 13801-13808Abstract Full Text PDF PubMed Google Scholar, 48.El-Jack A.K. Kandor K.V. Pilch P.F. Mol. Biol. Cell. 1999; 10: 1581-1594Crossref PubMed Scopus (64) Google Scholar). Further investigations are needed to clarify the degree of the colocalization of intracellular GLUT1 and GLUT4 in adipocytes. Our observation that overexpression of SNAP23-ΔC8 inhibited translocation of GLUT4 but not that of GLUT1 suggests that SNAP23 may regulate the post-endocytotic compartment but not the endosomal compartment. Consistent with these data, introduction of antibodies to SNAP23 or a synthetic peptide corresponding to the 24 COOH-terminal amino acids of SNAP23 into 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation but not that of GLUT1 (41.Rea S. Martin L.B. McIntosh S. Macaulay S.L. Ramsdale T. Baldini G. James D.E. J. Biol. Chem. 1998; 273: 18784-18792Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Furthermore, the observations that syntaxin4 (14.Olson A. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (207) Google Scholar) and VAMP2 (13.Martin L.B. Shewan A. Millar C.A. Gould C.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) participate in the translocation of GLUT4, but not that of GLUT1, suggest that the SNARE complex comprising syntaxin4, SNAP23, and VAMP2 in 3T3-L1 adipocytes contributes to regulated exocytosis in these cells. Possible reasons why SNAP23-ΔC8 did not completely inhibit insulin-induced glucose transport in 3T3-L1 adipocytes may be explained by its lack of effect on insulin-induced translocation of GLUT1 or to GLUT4 proteins, which were not inhibited by the SNAP23-ΔC8 and translocated to plasma membrane.Analysis of neuronal SNARE proteins (syntaxin1, SNAP25, and VAMP2) has revealed that, whereas VAMP2 binds weakly to syntaxin1 in the absence of SNAP25, the presence of SNAP25 greatly increases the affinity of the interaction between these two proteins (27.McMahon H.T. Südhof T.C. J. Biol. Chem. 1995; 270: 2213-2217Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 28.Pevsner J. Hsu S.C. Braun J.E.A. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 30.Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Nieman H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar). Similarly, we have shown that SNAP23 also increases the extent of the interaction between VAMP2 and syntaxin4 in 3T3-L1 adipocytes. However, another in vitro study concerning SNAP23 reports that SNAP23 proteins did not potentiate VAMP2 binding to syntaxin4 (49.Foster L.J. Yeung B. Mohtashami M. Ross K. Trimble W.S. Klip A. Biochemistry. 1998; 37: 11089-11096Crossref PubMed Scopus (113) Google Scholar). Although the reason for the difference is not clear, we confirmed by in vivo (Fig. 1) and in vitro (Fig. 3) studies that SNAP23 potentiates the association between VAMP2 and syntaxin4. The syntaxin4-binding protein Munc18c inhibits the interaction between syntaxin4 and SNAP23 (16.Araki S. Tamori Y. Kawanishi M. Shinoda H. Masugi J. Mori H. Niki T. Okazawa H. Kubota T. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 234: 257-262Crossref PubMed Scopus (85) Google Scholar) as well as that between syntaxin4 and VAMP2 (50.Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), suggesting that this protein may negatively regulate the association between syntaxin4 and SNAP23 in 3T3-L1 adipocytes. Dissociation of Munc18c from syntaxin4 may result in the formation of the syntaxin4-SNAP23-VAMP2 complex and thereby lead to fusion of GLUT4 vesicles with the plasma membrane.In conclusion, we have shown that SNAP23, acting together with syntaxin4 and VAMP2, mediates the translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. The mechanism by which insulin regulates the formation of the syntaxin4-SNAP23-VAMP2 complex remains to be determined. A primary function of insulin is to stimulate the transport of glucose into target tissues, prominent among which are skeletal muscle, cardiac muscle, and adipose tissue. Insulin achieves this effect by inducing the translocation of GLUT4 glucose transporters from an intracellular vesicular compartment to the plasma membrane. Under basal conditions, GLUT4 cycles slowly between this intracellular compartment and the plasma membrane (1.Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar, 2.Kandror K.V. Pilch P. Am. J. Physiol. 1996; 271: E1-E14Crossref PubMed Google Scholar). However, activation of insulin receptors triggers a large increase in the rate of exocytosis of GLUT4-containing vesicles and a smaller decrease in the rate of GLUT4 internalization by endocytosis (3.Holman G.D. Kozka I.J. Clark A.E. Flower C.J. Saltis J. Habberfield A.D. Simpson I.A. Cushman S.W. J. Biol. Chem. 1990; 265: 18172-18179Abstract Full Text PDF PubMed Google Scholar, 4.Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar, 5.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. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar), with the former action likely contributing most to the insulin-induced increase in the amount of GLUT4 in the plasma membrane (6.Jhun 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). Intracellular membrane fusion is mediated by evolutionarily conserved membrane proteins known as solubleN-ethylmaleimide-sensitive factor (NSF)1 attachment protein receptors (SNAREs) (7.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar, 8.Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar). SNARE proteins that contribute to neuronal exocytosis include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the plasma membrane proteins synaptosome-associated 25-kDa protein and syntaxin1A. These proteins readily assemble into a stable ternary complex; however, disassembly of this complex can be reversibly induced by the ATPase NSF in conjunction with soluble cofactors termed SNAPs (soluble NSF-attachment proteins) (9.Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2603) Google Scholar). The formation of the SNARE complex is thought to be a critical step in the pathway leading to membrane fusion. The insulin-stimulated trafficking of GLUT4 vesicles in adipocytes shares several features with the regulated pathway of synaptic vesicle exocytosis. Thus, members of the synaptobrevin (VAMP) family of proteins were shown to localize to GLUT4 vesicles in rat adipocytes (10.Cain C.C. Trimble W.S. Lienhard G.E. J. Biol. Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar). These proteins were identified as VAMP2 and cellubrevin in 3T3-L1 adipocytes (11.Volchuk 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 were subsequently shown to be essential for insulin-stimulated GLUT4 translocation in these cells (12.Tamori Y. Hashiramoto M. Araki S. Kamata Y. Takahashi M. Kozaki S. Kasuga M. Biochem. Biophys. Res. Commun. 1996; 220: 740-745Crossref PubMed Scopus (61) Google Scholar, 13.Martin L.B. Shewan A. Millar C.A. Gould C.W. James D.E. J. Biol. Chem. 1998; 273: 1444-1452Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Furthermore, syntaxin isoform 4 was shown to contribute to the translocation of GLUT4 in 3T3-L1 adipocytes (14.Olson A. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (207) Google Scholar). In addition, SNAP23, a widely expressed isoform of SNAP25 (15.Ravichadran V. Chawla A. Roche P.A. J. Biol. Chem. 1996; 271: 13300-13303Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), was shown to be present in the plasma membrane of 3T3-L1 adipocytes (16.Araki S. Tamori Y. Kawanishi M. Shinoda H. Masugi J. Mori H. Niki T. Okazawa H. Kubota T. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 234: 257-262Crossref PubMed Scopus (85) Google Scholar) and to be colocalized with syntaxin4 in these cells (17.Tamori Y. Kawanishi M. Niki T. Shinoda H. Araki S. Okazawa H. Kasuga M. J. Biol. Chem. 1998; 273: 19740-19746Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, SNAP23 in adipocytes, like SNAP25 in neurons, may function in the exocytosis of intracellular vesicles. We have now investigated the possible role of SNAP23 as a functional plasma membrane t-SNARE in insulin-stimulated GLUT4 translocation. Our data demonstrate that SNAP23 is required for insulin-induced GLUT4 translocation to the plasma membrane and that it mediates the formation of a complex between syntaxin4 and VAMP2. DISCUSSIONWe have shown that the SNAP23 mutant SNAP23-ΔC8, which binds to syntaxin4 but not to VAMP2, inhibits the translocation of GLUT4, but not that of GLUT1, to the plasma membrane in 3T3-L1 adipocytes. This inhibition of GLUT4 translocation is likely attributable to prevention of the formation of a ternary SNARE complex among SNAP23, syntaxin4, and VAMP2 at the plasma membrane of these cells. Thus, SNAP-23 appears to function as a t-SNARE in the translocation of GLUT4 in 3T3-L1 adipocytes.SNAP23 was identified as a widely expressed homolog of SNAP25 (15.Ravichadran V. Chawla A. Roche P.A. J. Biol. Chem. 1996; 271: 13300-13303Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Although SNAP25 is essential for exocytotic membrane fusion in neurons, this t-SNARE does not appear to be expressed in most nonneural tissues, with the exception of pancreatic islets of Langerhans (34.Sadoul K. Lang J. Montecucco C. Weller U. Regazzi R. Catsicas S. Wollheim C.B. Halban P.A. J. Cell Biol. 1995; 128: 1019-1028Crossref PubMed Scopus (229) Google Scholar, 35.Jacobsson G. Bean A.J. Scheller R.H. Junttiberggren L. Deeney J.T. Berggren P.O. Meister B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12487-12491Crossref PubMed Scopus (194) Google Scholar), adrenal chromaffin cells (36.Roth D. Burgoyne R.D. FEBS Lett. 1994; 351: 207-210Crossref PubMed Scopus (100) Google Scholar), and anterior pituitary cells (37.Aguado F. Majo G. Ruiz-Montasell B. Canals J.M. Casanova A. Marsal J. Blasi J. Eur. J. Cell Biol. 1996; 69: 351-359PubMed Google Scholar). Although Jagadish et al. (38.Jagadish M.N. Fernandez C.S. Hewish D.R. Macaulay S.L. Gough K.H. Grusovin J. Verkuylen A. Cosgrove L. Alafaci A. Frenkel M.J. Ward C.W. Biochem. J. 1996; 317: 945-954Crossref PubMed Scopus (46) Google Scholar) detected SNAP25 mRNA and protein in fat cells and 3T3-L1 adipocytes by sensitive methods, Timmer et al. (39.Timmer K.I. Clark A.E. Omatsu-Kanbe M. Whiteheart S.W. Bennett M.K. Holman G.D. Cushman S.W. Biochem. J. 1996; 320: 429-436Crossref PubMed Scopus (55) Google Scholar) and Wong et al. (40.Wong P.P.C. Daneman N. Volchuk A. Lassam N. Wilson M.C. Klip A. Trimble W.S. Biochem. Biophys. Res. Commun. 1997; 230: 64-68Crossref PubMed Scopus (60) Google Schola
Referência(s)