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• W2137934800 abstract "Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, ∼50%) as well as in inhibition of sorbitol-induced glucose transport (by ∼35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4. Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, ∼50%) as well as in inhibition of sorbitol-induced glucose transport (by ∼35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4. Insulin stimulates glucose transport into muscle and adipose tissue by inducing the translocation of vesicles containing the glucose transporter GLUT4 from the intracellular compartment to the plasma membrane (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 (770) Google Scholar). This process is thought to be a major contributor to the mechanism by which insulin reduces the blood concentration of glucose. The binding of insulin to its receptor on the surface of target cells results in receptor autophosphorylation and receptor-mediated tyrosine phosphorylation of several additional proteins, including insulin receptor substrates 1–4 (IRS1 to IRS4). 1The abbreviations used are: IRS, insulin receptor substrate; PI, phosphoinositide; NSF,N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; v- and t-SNAREs, vesicle and target membrane SNAREs; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; m.o.i., multiplicity of infection; pfu, plaque-forming unit; PBS, phosphate-buffered saline. The phosphorylated IRS proteins then bind other proteins, such as phosphoinositide (PI) 3-kinase, SHP-2, and GRB2, that contain SRC homology 2 (SH2) domains (3White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). PI 3-kinase is thought to play a role in the insulin-induced translocation of GLUT4 (4Clarke J.F. Young P.W. Yonezawa K. Kasuga M. Holman G.D. Biochem. J. 1994; 300: 631-635Crossref PubMed Scopus (334) Google Scholar, 5Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (418) Google Scholar); however, the mechanism by which activation of PI 3-kinase results in GLUT4 translocation remains unclear (6Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (188) Google Scholar). The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) hypothesis was initially proposed to explain the process of neurotransmitter secretion (7Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2628) Google Scholar, 8Söllner T. Bennett M.B. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1582) Google Scholar). According to this hypothesis, the docking and fusion of synaptic vesicles at the plasma membrane are initiated by the interaction of proteins, known as v-SNAREs (synaptobrevin/VAMP), located on the vesicle surface with corresponding proteins, known as t-SNAREs (syntaxin, SNAP-25), located on the target membrane. Membrane fusion is subsequently mediated by the cytosolic proteins α-, β-, and γ-SNAP (soluble NSF attachment protein) and NSF (N-ethylmaleimide-sensitive fusion protein), which binds SNAP and hydrolyzes ATP. This hypothesis was subsequently applied to vesicle transport from the intracellular compartment to the plasma membrane in cells other than neurons. Thus, SNARE proteins have been implicated in the insulin-induced translocation of GLUT4-containing vesicles in adipocytes. Members of the VAMP/synaptobrevin family of proteins were first shown to localize in GLUT4 vesicles in rat adipocytes (9Cain C.C. Trimble W.S. Lienhard G.E. J. Biol. Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar). Such VAMP/synaptobrevin proteins were identified as VAMP2 and cellubrevin/VAMP3 in 3T3-L1 adipocytes (10Volchuk 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, 11Tamori 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). Syntaxins 2, 4, and 5 are also expressed in 3T3-L1 adipocytes (12Timmers 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). In addition, with the use of neurotoxins that cleave VAMP2 and cellubrevin/VAMP3, these proteins were shown to function as v-SNAREs in the trafficking of GLUT4 vesicles to the plasma membrane (11Tamori 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, 13Macaulay S.L. Hewish D.R. Gough K.H. Stoichevska V. Macpherson S.F. Jagadish M. Ward C.W. Biochem. J. 1997; 324: 217-224Crossref PubMed Scopus (44) Google Scholar). Syntaxin4 is also implicated in GLUT4 translocation in adipocytes. Microinjection of the cytoplasmic domain of syntaxin4 (14Cheatham B. Volchuk A. Kahn C.R. Wang L. Rhodes C.J. Klip A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15169-15173Crossref PubMed Scopus (162) Google Scholar,15Olson A.L. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (209) Google Scholar) or of antibodies to syntaxin4 (16Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), or expression of the same cytoplasmic domain with a vaccinia virus vector (15Olson A.L. Knight J.B. Pessin J.E. Mol. Cell. Biol. 1997; 17: 2425-2435Crossref PubMed Scopus (209) Google Scholar), resulted in inhibition of insulin-induced GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. Furthermore, the SNARE complexes bound by recombinant NSF and α-SNAP in proteins solubilized from rat adipocyte membranes contained syntaxin4 but not syntaxin2 (12Timmers 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). These studies thus suggest that syntaxin4 functions as a t-SNARE in the docking and fusion of GLUT4 vesicles at the plasma membrane in adipocytes. However, although VAMP2 and VAMP3 (v-SNAREs) as well as syntaxin4 (t-SNARE) appear to play essential roles in GLUT4 translocation in these cells, the mechanism by which insulin might regulate these SNARE components remains unknown. In Saccharomyces cerevisiae, four members of the Sec1 protein family (Sec1p (17Novick P.J. Field C. Schekman R. Cell. 1980; 21: 205-215Abstract Full Text PDF PubMed Scopus (1264) Google Scholar, 18Aalto M.K. Ruohonen L. Hosono K. Käranen S. Yeast. 1991; 7: 643-650Crossref PubMed Scopus (60) Google Scholar), Slp1p (19Wada Y. Kitamoto K. Kanbe T. Tanaka K. Anraku Y. Mol. Cell. Biol. 1990; 10: 2214-2223Crossref PubMed Scopus (95) Google Scholar), Vps45p (20Piper R.C. Whitters E.A. Stevens T.H. Eur. J. Cell Biol. 1994; 65: 305-318PubMed Google Scholar, 21Cowles C.R. Emr S.D. Horazdovsky B.F. J. Cell Sci. 1994; 107: 3449-3459PubMed Google Scholar), and Sly1p (22Dascher C. Ossig R. Gallwitz D. Schmitt H.D. Mol. Cell. Biol. 1991; 11: 872-885Crossref PubMed Scopus (280) Google Scholar, 23Ossig R. Dascher C. Trepte H.-H. Schmidtt H.D. Gallwitz D. Mol. Cell. Biol. 1991; 11: 2980-2993Crossref PubMed Scopus (149) Google Scholar)) participate in vesicle transport. Sec1p-related proteins have also been identified in the nervous system of Caenorhabditis elegans (UNC-18) (24Hosono R. Hekimi S. Kamiya Y. Sassa T. Murakami S. Nishiwaki K. Miwa J. Taketo A. Kodaira K. J. Neurochem. 1992; 58: 1517-1525Crossref PubMed Scopus (152) Google Scholar, 25Gengyo-Ando K. Kamiya Y. Yamakawa A. Kodaira K. Nishiwaki K. Miwa J. Hori I. Hosono R. Neuron. 1993; 11: 703-711Abstract Full Text PDF PubMed Scopus (96) Google Scholar) and Drosophila melanogaster(ROP) (26Salzberg A. Cohen N. Halachmi N. Kimchie Z. Lev Z. Development. 1993; 117: 1309-1319PubMed Google Scholar). In mammals similar proteins, known as Munc18 (27Hata Y. Slaughter C.A. Südhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (591) Google Scholar), n-Sec1 (28Pevsner J. Hsu S.-C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1445-1449Crossref PubMed Scopus (353) Google Scholar), and rbSec1 (29Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (224) Google Scholar), were identified as syntaxin-binding proteins in brain tissue. A Sec1p homolog, known variously as muSec1 (30Katagiri H. Terasaki J. Murata T. Ishihara H. Ogihara T. Inukai K. Fukushima Y. Anai M. Kikuchi M. Miyazaki J. Yazaki Y. Oka Y. J. Biol. Chem. 1995; 270: 4963-4966Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), Munc18b (31Tellam J. McIntosh S. James D.E. J. Biol. Chem. 1995; 270: 5857-5863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and Munc18–2 (32Hata Y. Südhof T.C. J. Biol. Chem. 1995; 270: 13022-13028Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), was also shown to be expressed in several nonneural mammalian tissues. Mutations in UNC-18 result in accumulation of acetylcholine-containing secretory vesicles in neurons as well as in abnormalities in the development of the nervous system in C. elegans (24Hosono R. Hekimi S. Kamiya Y. Sassa T. Murakami S. Nishiwaki K. Miwa J. Taketo A. Kodaira K. J. Neurochem. 1992; 58: 1517-1525Crossref PubMed Scopus (152) Google Scholar, 25Gengyo-Ando K. Kamiya Y. Yamakawa A. Kodaira K. Nishiwaki K. Miwa J. Hori I. Hosono R. Neuron. 1993; 11: 703-711Abstract Full Text PDF PubMed Scopus (96) Google Scholar). The n-Sec1 protein was also shown to inhibit the association of syntaxin1 with SNAP-25 (33Pevsner J. Hsu S.-C. Braun J.E. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (524) Google Scholar), and DrosophilaROP plays a negative role in neurotransmitter release in vivo (34Schulze K.L. Littleton J.T. Salzberg A. Halachmi N. Stern M. Lev Z. Bellen H.J. Neuron. 1994; 13: 1099-1108Abstract Full Text PDF PubMed Scopus (161) Google Scholar). Munc18b and Munc18c, the latter identified from a 3T3-L1 adipocyte cDNA library, are abundant Munc18 isoforms in these cells (31Tellam J. McIntosh S. James D.E. J. Biol. Chem. 1995; 270: 5857-5863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). We have now investigated the function of Munc18 proteins in the insulin-induced translocation of GLUT4 vesicles in 3T3-L1 adipocytes. Polyclonal antibodies to SNAP-23, syntaxin4, and Munc18c were generated as described previously (35Araki 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). Rabbit polyclonal antibodies specific for the COOH-terminal portion of GLUT1 also were prepared as described previously (36Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar). Mouse monoclonal antibodies to GLUT4 were kindly provided by D. E. James (University of Queensland, Australia). Rabbit polyclonal antibodies generated in response to the cytoplasmic region of syntaxin2 and to full-length Munc18b were kindly provided by M. K. Bennett (University of California, Berkeley) and V. M. Olkkonen (National Public Health Institute, Helsinki, Finland), respectively. Mouse monoclonal antibody to c-MYC (monoclonal antibody 9E10) was purchased from Oncogene Science, Inc. Mouse full-length Munc18c cDNA and SNAP-23 cDNA were obtained as described previously (35Araki 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). Rat full-length Munc18a cDNA was obtained by reverse transcription and the polymerase chain reaction with specific oligonucleotide primers (5′-GAACGCCATGGCCCCCATTG-3′ (sense) and 5′-GATTTTAACTGCTTATTTCTTCG-3′ (antisense)) and total RNA from rat brain. Mouse full-length Munc18b cDNA was amplified by the polymerase chain reaction with specific oligonucleotide primers (5′-GATGGCGCCCTTGGGGCTG-3′ (sense) and 5′-GTCAGGGCAGGGCCACACC-3′ (antisense)) from a mouse fat cell cDNA library (CLONTECH). Munc18a, Munc18b, and Munc18c were tagged with a human c-MYC epitope by adding (at the cDNA level) a 14-amino acid sequence (AEEQKLISEEDLLK) at their COOH termini. Complementary DNAs encoding rat syntaxins 1a, 2, 3, and 4 were kindly provided by R. H. Scheller (Stanford University, Stanford, CA). 3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS). Adipogenesis was induced by treatment with insulin, dexamethasone, and isobutylmethylxanthine as described previously (37Frost S.C. Lane M.D. J. Biol. Chem. 1985; 260: 2646-2652Abstract Full Text PDF PubMed Google Scholar), and the cells were subjected to experiments after 8–13 days. COS cells were also maintained in DMEM supplemented with 10% FBS. Samples in Laemmli sample buffer (65 mm Tris, 3% (w/v) SDS, 10% (v/v) glycerol, bromphenol blue (0.025 mg/ml)) containing 5% (v/v) 2-mercaptoethanol were boiled for 5 min and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were then transferred electrophoretically to a nitrocellulose membrane in the presence of a solution containing 20 mm Tris, 230 mm glycine, 20% (v/v) methanol, and 0.02% SDS. The membrane was exposed for 1 h to blocking buffer (80 mmNa2HPO4, 20 mmNaH2PO4 (pH 7.4), 100 mm NaCl, 5% (w/v) nonfat dried milk) and then incubated with primary antibody diluted in a solution containing 20 mm Tris-HCl (pH 7.4), 100 mm NaCl, 1% (w/v) bovine serum albumin, 20% FBS, and 0.05% Nonidet P-40. After washing with PBST solution (80 mm Na2HPO4, 20 mmNaH2PO4 (pH 7.4), 100 mm NaCl, 0.1% Triton X-100), the membrane was incubated for 60 min with horseradish peroxidase-conjugated donkey antibodies to rabbit immunoglobulin G (IgG) (Amersham Pharmacia Biotech) or goat antibodies to mouse IgG (Promega) diluted in PBST (1:3000) and was then washed twice in PBST. Immune complexes were visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). Subcellular fractionation of 3T3-L1 adipocytes was performed as described previously (38Simpson I.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnieli E. Salans L.B. Cushman S.W. Biochim. Biophys. Acta. 1983; 763: 393-407Crossref PubMed Scopus (330) Google Scholar) with minor modifications. Briefly, cells were scraped and homogenized in TES buffer (20 mm Tris-HCl (pH 7.4), 1 mm EDTA, 225 mm sucrose), and the homogenate was centrifuged at 16,000 × g. The resulting pellet was layered on top of a 1.12 m sucrose cushion and centrifuged at 101,000 ×g, after which the plasma membrane fraction was collected from the interface of the two solutions. Immunoprecipitation of syntaxins 2 or 4 or of Munc18c from extracts of the plasma membrane fraction (prepared with a solution containing 50 mmHepes-NaOH (pH 7.4), 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride) was performed with the corresponding specific antibodies. With the use of Lipofectin (Life Technologies, Inc.), COS cells were transiently transfected with full-length cDNAs encoding SNAP-23 or c-MYC epitope-tagged Munc18a, -b, or -c that had been cloned into the expression vector pcDL-SRα (35Araki 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, 39Takebe Y. Seiki M. Fukisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). The cells were solubilized with lysis buffer (25 mm Hepes-NaOH (pH 7.4), 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mmphenylmethylsulfonyl fluoride), and the resulting extracts were incubated, with constant agitation, at 4 °C for 1 h with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) conjugated with 2 to 3 μg of glutathione S-transferase (GST) fusion proteins containing the cytoplasmic portions of syntaxins 1a, 2, 3, or 4. The beads were washed three times with ice-cold lysis buffer, and proteins bound to the beads were then eluted with 20 μl of Laemmli sample buffer and subjected to SDS-PAGE and immunoblot analysis with either a monoclonal antibody to c-MYC (for Munc18a, -b, or -c) or antibodies to SNAP-23. Recombinant adenovirus vectors were generated by cloning cDNAs into pAxCAwt (40Kanegae Y. Lee G. Tanaka M. Nakai M. Sakai T. Sugano S. Saito I. Nucleic Acids Res. 1995; 23: 3816-3821Crossref PubMed Scopus (598) Google Scholar), which contains the CAG promoter (41Niwa H. Yamamura K. Miyazaki J. Gene ( Amst .). 1991; 108: 193-199Crossref PubMed Scopus (4597) Google Scholar), and cotransfection into 293 cells with DNA-TPC, as described previously (42Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (787) Google Scholar). Protein-encoding viruses were screened by immunoblot analysis and cloned by limiting dilution. Adenovirus vectors were propagated by a standard procedure and then purified and titrated as described previously (43Kanegae Y. Makimura M. Saito I. Jpn. J. Med. Sci. Biol. 1994; 47: 157-166Crossref PubMed Scopus (429) Google Scholar). Eight to 11 days after induction of differentiation, 3T3-L1 adipocytes were infected for 2 h at the indicated multiplicity of infection (m.o.i.), in plaque-forming units (pfu) per cell, as determined by limiting dilution assay in 293 cells. The adipocytes were subjected to various experiments about 48 h after infection. 3T3-L1 cells were deprived of serum by incubation in 12-well plates with DMEM for 2 h. The cells were then incubated with 100 nminsulin for 20 min or 600 mmd-sorbitol for 40 min in 0.45 ml of KRH buffer (25 mm Hepes-NaOH (pH 7.4), 120 mm NaCl, 5 mm KCl, 1.2 mmMgSO4, 1.3 mm CaCl2, 1.3 mm KH2PO4). Glucose transport was initiated by the addition of 0.05 ml of KRH buffer containing 2-deoxy-d-[1,2-3H]glucose (final concentration, 0.05 mm; 0.25 μCi) to each well, and after 5 min, transport was terminated by washing cells three times with ice-cold KRH buffer. The cells were solubilized with 0.5% SDS, and the incorporated radioactivity was measured by liquid scintillation counting. Translocation of GLUT1 or GLUT4 to the plasma membrane was measured by the plasma membrane lawn assay as described previously (44Kotani K. Carozzi A.J. Sakaue H. Hara K. Robinson L.J. Clark S.F. Yonezawa K. James D.E. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 209: 343-348Crossref PubMed Scopus (144) Google Scholar). In brief, 3T3-L1 cells cultured on coverslips were washed in phosphate-buffered saline (PBS) and treated with poly-l-lysine (0.5 mg/ml) in PBS. Cells were incubated in a hypotonic solution (0.33 KHMgE buffer, comprising 30 mm Hepes-NaOH (pH 7.5), 70 mm KCl, 5 mm MgCl2, and 3 mm EGTA) and then disrupted by placement under an ultrasonic microprobe in KHMgE buffer containing 0.1 mm phenylmethylsulfonyl fluoride and 1 mm dithiothreitol. Sonicated cells were fixed in 2% paraformaldehyde and then incubated with rabbit polyclonal antibodies to GLUT1 and mouse monoclonal antibodies to GLUT4. After washing three times with PBS, the coverslips were incubated with tetramethylrhodamine isothiocyanate-conjugated antibodies to rabbit IgG and fluorescein isothiocyanate-conjugated antibodies to mouse IgG. The cells were washed with PBS, mounted in 90% glycerol in PBS containingp-phenylenediamine (1 mg/ml), and examined with a fluorescence microscope (Axiophot; Zeiss, Jena, Germany). To investigate the functional roles of Munc18 isoforms, syntaxins, and SNAP-23 in GLUT4 translocation in 3T3-L1 adipocytes, we examined the affinity of Munc18 isoforms and SNAP-23 for GST fusion proteins containing the cytoplasmic portions of syntaxins 1a, 2, 3, or 4. Interaction with syntaxin5 was not assessed because this protein appears to function as a t-SNARE in transport from the endoplasmic reticulum to the Golgi (45Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar). SNAP-23 as well as Munc18a, -b, and -c tagged at their COOH termini with a human c-MYC epitope were transiently expressed in COS cells, and the corresponding cell extracts were incubated with glutathione-Sepharose beads containing immobilized GST-syntaxin fusion proteins. After washing, proteins eluted from the beads were analyzed by SDS-PAGE and immunoblotting with antibodies to c-MYC (for the three Munc18 isoforms) or to SNAP-23. Munc18a, which is identical to n-Sec1 and Munc18–1, bound to the GST fusion proteins containing syntaxins 1a, 2, and 3, but there was no detectable binding of Munc18a to syntaxin4 (Fig.1). Munc18b showed a pattern of binding similar to that of Munc18a. In contrast, Munc18c showed a marked interaction with syntaxins 2 and 4, interacted to a much lesser extent with syntaxin1, and exhibited no detectable binding to syntaxin3. Although it is not clear whether the Munc18 isoforms expressed in COS cells bind directly or indirectly to the recombinant GST-syntaxin proteins, these results show that Munc18c is the only Munc18 isoform that interacts substantially with syntaxin4 in vitro. The observation that SNAP-23, an adipocyte homolog of SNAP-25 (35Araki 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), interacted markedly with syntaxins 1a and 4 and slightly with syntaxin2 is consistent with our previous results obtained with the yeast two-hybrid system (35Araki 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). Given that syntaxins 2 and 4 (8Söllner T. Bennett M.B. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1582) Google Scholar) and Munc18b and -c (31Tellam J. McIntosh S. James D.E. J. Biol. Chem. 1995; 270: 5857-5863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) are the major syntaxin and Munc18 isoforms in 3T3-L1 adipocytes, it is likely that Munc18b interacts with syntaxin2 and that Munc18c interacts with syntaxins 2 or 4 in these cells. We next examined the physiological interactions among Munc18b or -c, syntaxins 2 or 4, and SNAP-23 in 3T3-L1 adipocytes. Our polyclonal antibodies generated in response to a synthetic COOH-terminal peptide of Munc18c are highly specific and do not recognize Munc18a or Munc18b overexpressed in COS cells, as assessed by immunoblot analysis or immunoprecipitation (data not shown). These antibodies detected a major protein of 67 kDa, corresponding to the predicted size of Munc18c, on immunoblot analysis of a detergent extract of the plasma membrane fraction of 3T3-L1 adipocytes (Fig.2 A). In contrast, we did not detect Munc18b in 3T3-L1 adipocytes by immunoblot analysis with antibodies specific for this Munc18 isoform (data not shown). Previous studies have detected Munc18b mRNA by Northern blot analysis (31Tellam J. McIntosh S. James D.E. J. Biol. Chem. 1995; 270: 5857-5863Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and Munc18b protein by immunoblot analysis (16Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) in these cells. This discrepancy is probably attributable to a difference in the efficacy of the antibodies used in the present and previous (16Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) studies; we were able to detect Munc18b overexpressed in COS cells. Polyclonal antibodies generated in response to GST fusion proteins containing the cytoplasmic portions of rat syntaxins 2 or 4 or to a GST fusion protein containing mouse SNAP-23 detected proteins of the predicted molecular sizes in the detergent extracts of plasma membranes from 3T3-L1 adipocytes (Fig. 2, B–D). Next, we confirmed that both syntaxins 2 and 4 were immunoprecipitated from detergent extracts of the plasma membrane fraction of 3T3-L1 adipocytes with the corresponding specific antibodies (Fig. 2, B andC). Despite the interaction of Munc18c with both syntaxins 2 and 4 in the in vitro binding assay (Fig. 1), Munc18c was coimmunoprecipitated with syntaxin4 but not with syntaxin2 (Fig.2 A). In contrast, SNAP-23 was coimmunoprecipitated with both syntaxin2 and syntaxin4 (Fig. 2 D), consistent with the results of the in vitro binding assay (Fig. 1). When the immunoprecipitation was performed with antibodies to Munc18c, syntaxin4 (Fig. 2 C), but not syntaxin2 (Fig. 2 B) or SNAP-23 (Fig. 2 D), was coprecipitated with Munc18c. In summary, Munc18c was detected in the plasma membrane of 3T3-L1 adipocytes and was associated with syntaxin4 but not with syntaxin2. SNAP-23 associated with both syntaxins 2 and 4 in the plasma membrane of 3T3-L1 adipocytes, although it was not associated with syntaxin4 complexed with Munc18c. To examine the function of Munc18b and Munc18c in 3T3-L1 adipocytes, we prepared recombinant adenoviruses that were expressed with an efficiency of >95% in these cells as assessed by β-galactosidase staining (46Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar). Both c-MYC epitope-tagged Munc18b and -c were overexpressed in 3T3-L1 adipocytes with the use of this adenovirus-mediated gene transfer system. Both proteins were expressed to similar extents in an m.o.i.-dependent manner (Fig.3, A and C). Moreover, the amount of overexpressed Munc18c bound to syntaxin4 also increased in an m.o.i.-dependent manner and was maximal at an m.o.i. of 15–30 pfu/cell (Fig. 3 C); overexpressed Munc18b did not bind syntaxin4 (Fig. 3 A). Overexpression of Munc18b had no significant effect on glucose transport," @default.
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• W2137934800 title "Inhibition of Insulin-induced GLUT4 Translocation by Munc18c through Interaction with Syntaxin4 in 3T3-L1 Adipocytes" @default.
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• W2137934800 doi "https://doi.org/10.1074/jbc.273.31.19740" @default.
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