The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes . Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N ,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antag onist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA- AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of in sulin-dose response curves indicated that this inhibition was not sufficien t to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM in hibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as me asured by surface binding, was blocked by BAPTA/AM.. While the ionophores A 23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT 4 translocation by BAPTA-AM, they did not overcome the inhibition of glucos e transport. Moreover, glucose uptake of cells pretreated with insulin foll owed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAP TA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is inde pendent of both trafficking and signal transduction. These data indicate th at Ca2+ is involved in at least two different steps of the insulin-dependen t recruitment of GLUT4 to the plasma membrane. One involves the translocati on step. The second involves the fusion of GLUT4 vesicles with the plasma m embrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally , BAPTA-AM may inhibit the activity of the facilitative transporters by bin ding directly to the transporter itself.