PROTEOLYTIC CLEAVAGE OF CELLUBREVIN AND VESICLE-ASSOCIATED MEMBRANE-PROTEIN (VAMP) BY TETANUS TOXIN DOES NOT IMPAIR INSULIN-STIMULATED GLUCOSE-TRANSPORT OR GLUT4 TRANSLOCATION IN RAT ADIPOCYTES

Acute insulin stimulation of glucose transport in fat and skeletal mus cle occurs principally as a result of the hormonal induced translocati on of the GLUT4 glucose transporter from intracellular vesicular store s to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusio n, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocy tes respectively. In this study we show that proteolysis of both cellu brevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transp ort or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate gluc ose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electropor ation of adipocytes with 600 nM tetanus toxin resulted in a complete l oss of both cellubrevin and VAMP expression within 60 min. However, to xin-mediated proteolysis of both these proteins had no effect on the a bility of insulin to stimulate glucose transport which was elevated ap prox. 5-fold, an activation of comparable magnitude to that observed i n cells electroporated without tetanus toxin. The lack of any signific ant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and V AMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and V AMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to t he plasma membrane.