The fusion of insulin-stimulated GLUT4-containing vesicles (GSV) with the plasma membrane (PM) of adipose or skeletal muscle cells is governed by regulated exocytosis. In all such membrane fusion events cognate t- and v-SNAREs interact in an ordered way to allow vesicles to first dock with, and then fuse with the plasmalemma. In neurons, the best-studied example of this well conserved process, the protein species responsible for directing the final (rate-limiting) fusion step is Synaptotagmin (Syt). Despite the presence of 16 higher eukaryotic isoforms of this well conserved protein family few have been characterised outwith neuronal cells. Manipulations of cultured adipocytes resulting in aberrant GSV-PM fusion are associated with reduced insulin-stimulated glucose uptake. Furthermore, it can be demonstrated that incomplete fusion occurs in adipocytes taken from insulin resistant individuals with Type-II Diabetes Mellitus. These data imply that the control of GSV fusion with the plasma membrane represents a key rate-limiting step in insulin signalling which ultimately governs insulin action and sensitivity. Whilst the v- and t-SNAREs implicated in GSV-PM fusion are well defined, the Syt compliment of insulin sensitive cells has remained hitherto unknown.
Here we demonstrate the Syt family members expressed in 3T3-L1 adipocytes, and show that Syt-11 is the predominant isoform. Syt-11 expression is upregulated as cells acquire their insulin-sensitive adipocyte phenotype, and is further upregulated when cells are treated with Rosiglitazone. We reveal that Syt-11 is found not only in the same intracellular pool as GLUT4, but also that it is present in the plasma membrane of insulin-stimulated cells. Finally, using siRNA we can demonstrate that Syt-11 depletion leads to abrogated insulin-stimulated glucose uptake. Thus we speculate Syt-11 is the species that may direct the final fusion step in the regulated exocytosis of GSVs, so may represent the link between proximal and distal events in the insulin-signalling cascade.
01 - 05 Apr 2006
European Society of Endocrinology