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, ϳ50%) as well as in inhibition of sorbitol-induced glucose transport (by ϳ35%), 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 (1, 2). 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).1 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 (3). PI 3-kinase is thought to play a role in the insulin-induced translocation of GLUT4 (4, 5); however, the mechanism by which activation of PI 3-kinase results in GLUT4 translocation remains unclear (6).The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) hypothesis was initially proposed to explain the process of neurotransmitter secretion (7,8). 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 subs...

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-⌬C8 (amino acids 1 to 202) and SNAP23-⌬C49 (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 and in vivo. Although SNAP23-⌬C49 bound to neither syntaxin4 nor VAMP2, the SNAP23-⌬C8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-⌬C8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-⌬C8 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-⌬C49 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).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, 2). 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 (3-5), with the former action likely contributing most to the insulin-induced increase in the amount of GLUT4 in the plasma membrane (6).Intracellular membrane fusion is mediated by evolutionarily conserved membrane proteins known as soluble N-ethylmaleimide-sensitive factor (NSF) 1 attachment protein receptors (SNAREs) (7,8). 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-att...

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