Insulin stimulates glucose uptake in adipose and muscle cells via translocation of the intracellular vesicles containing GLUT4. It was largely unknown whether and/or how the signaling molecules such as PI 3-kinase and Akt regulate the mechanical movements of the GLUT4-containing vesicles. Hence, this study was performed to test the hypothesis that actin and microtubules function in translocating GLUT4 vesicles. Treatments of insulin as well as endothelin-1 (ET-1), an insulin-mimicking peptide which does not act through PI 3-kinase, induced polymerization of actin without affecting the microtubular network. By mass spectrometry, the tyrosine kinase PYK2 was identified to be tyrosine phosphorylated specifically by ET-1 but not by insulin. Expression of the carboxyl-terminal fragment (CRNK) PYK2, but not wild type nor kinase-deficient PYK2 mutants, inhibited ET-1-stimulated actin polymerization while expression of all three PYK2 constructs had no effect on insulin-stimulated actin polymerization. More importantly, expression of CRNK, but not wild type nor kinase-deficient PYK2 constructs, blocked ET-1- but not insulin-stimulated GLUT4 translocation to the plasma membrane. These suggest that ET-1 and insulin stimulate actin polymerization via distinct signaling pathways, and that the actin polymerization is required for GLUT4 vesicle translocation.

In order to test the possible involvement of microtubule in GLUT4 vesicle translocation, time lapse imaging of 3T3-L1 adipocytes expressing GLUT4-YFP and tubulin-CFP was performed. GLUT4-YFP vesicles move long-range bi-directionally on microtubules, which suggests the presence of molecular motors on the vesicles. Moreover, insulin increased the number of vesicle movements on microtubules without changing the velocities. Interestingly, the stimulatory action of insulin appears to be independent of PI 3-kinase activation. Conventional kinesin was identified as a highly expressed kinesin isotype in adipocytes. Notably, expression of dominant negative mutants but not wild type kinesin inhibited insulin-stimulated long-range GLUT4 vesicle movements and GLUT4 translocation to the plasma membrane in live and fixed cells, respectively. These data indicate that insulin signaling induces the movement of GLUT4 vesicles on microtubule which is mediated by conventional kinesin. Overall, the data presented here provide evidence supporting the hypothesis that actin and microtubule cytoskeletons are required for insulin to mobilize GLUT4 vesicles in adipocytes.