Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport.

Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.