Inhibition of Insulin-induced GLUT4 Translocation by Munc18c through Interaction with Syntaxin4 in 3T3-L1 Adipocytes

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, 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...

Protein kinase-ζ interacts with munc18c: role in GLUT4 trafficking

“…The CHO cells, like 3T3-L1 adipocytes, possess GLUT4 storage vesicles which translocate to the plasma membrane in response to insulin stimulation. Overexpression of munc18c FL significantly inhibited both glucose uptake and translocation of myctagged GLUT4 to the plasma membrane, which accorded well with the published literature [11][12][13]. Co-expression of PKCζ constructs that associated with munc18c, but not a construct unable to bind munc18c, alleviated the inhibitory effect of munc18c on glucose uptake.…”

“…Several proteins have been proposed to act to regulate the interaction between syntaxin-4 and VAMP-2. These proteins are synip [10] and munc18c [11][12][13]. Munc18c, also known as munc18-3, has two homologues, munc18a (munc18-1) and munc18b (munc18-2).…”

“…Munc18a is found in neurons where it inhibits the association of VAMP-1 and SNAP-25 with syntaxin-1 [14]. Likewise, munc18c binds to syntaxin-4, and as this association is stronger than that between VAMP-2 and syntaxin-4 [11][12][13], it acts as a clamp preventing the GLUT4 vesicle binding to the plasma membrane in the basal state. When stimulated by insulin this clamp is removed by an unknown mechanism and the GLUT4 vesicle docks with the plasma membrane.…”

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

“…In this pathway TI-VAMP (VAMP7) appears to be the primary v-SNARE required, consistent with the presence of at least two-independent GLUT4 storage vesicle populations [26]. The cognate plasma membrane t-SNAREs for insulin-stimulated GLUT4 translocation appears to be the syntaxin 4 and the SNAP23 isoforms, based upon antibody blocking studies and expression of dominant-interfering mutants and blocking peptides [47][48][49]. However, involvement of these t-SNAREs has not been as extensively investigated and further studies are still required to determine the specific function steps of these isoforms in the translocation process.…”

“…Munc18c was originally identified as one of three mammalian isoforms of the sec1 protein in yeast [52]. In all systems to date, over expression of the appropriate Munc18 protein isoform was found to inhibit membrane vesicle fusion, suggesting that this protein function as a fusogenic inhibitor [48,[53][54][55][56]. Structural studies indicated that the Munc18 proteins induces a closed conformational state of syntaxin thereby rendering it unable to bind to VAMP or SNAP consistent with in vitro binding assays [57,58].…”