Investigations into insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in adipocytes: novel insights from in situ studies

Trafficking of the facilitative glucose transporter GLUT4 is regulated by insulin in fat and muscle cells. Under basal conditions, GLUT4 is retained intracellularly by continually cycling through the endosomal system, but translocates to the plasma membrane in response to insulin stimulation. Intracellular GLUT4-containing vesicles fall into two categories: cellugyrin-positive (sortilin-free) and sortilin-positive (cellugyrin-negative). The former are the source of GLUT4 that cycles through the plasma membrane under basal conditions while the latter are the source of GLUT4 that translocates to the cell surface upon insulin stimulation. Fusion of GLUT4-containing vesicles with the plasma membrane is mediated by formation of SNARE complexes including the plasma membrane localized t-SNAREs Syntaxin 4 and SNAP23, and the v-SNARE VAMP2 present in the GLUT4-containing vesicles. The Sec1/Munc18 (SM) protein Munc18c also plays a key role in insulin-stimulated GLUT4 translocation, although its precise role remains controversial. Munc18c binds directly to both Syntaxin 4 and VAMP2 as well as to the assembled SNARE complex through a series of different binding modes. It has been suggested that SM/Syntaxin interactions facilitate SNARE complex formation by bringing about a conformational switch to release an inhibitory effect of syntaxins’ Habc domain. In this study I have used in situ Proximity Ligation Assay (PLA) to visualize the effects of insulin stimulation on interactions between Syntaxin 4, SNAP23, VAMP2 and Munc18c in 3T3-L1 adipocytes and fibroblasts. I find that insulin treatment results in an increase of the formation of assembled Syntaxin 4/SNAP23/VAMP2 SNARE complexes as well as recruitment of Munc18c to these complexes. These studies also reveal the existence of two pools of Syntaxin 4 under basal conditions: one in complex with SNAP23 (lacking VAMP2 and Munc18c); the other in complex with Munc18c and VAMP2 (lacking SNAP23). Additionally I have used in vitro bind