Dual Function of PI(4,5)P₂ in Insulin-Regulated Exocytic Trafficking of GLUT4 in Adipocytes

Phosphoinositides are important signaling molecules involved in the regulation of vesicular trafficking. It has been implicated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is involved in insulin-regulated GLUT4 translocation in adipocytes. However, it remains unclear where and how PI(4,5)P₂ regulates discrete steps of GLUT4 vesicle translocation in adipocytes, especially on the exocytic arm of regulation. Here, we employed optogenetic tools to acutely control the PI(4,5)P₂ metabolism in living cells. By combination of TIRFM imaging, we were able to monitor the temporal-spatial-dependent PI(4,5)P₂ regulation on discrete steps of GLUT4 translocation in adipocytes. We found that the plasma membrane localized PI(4,5)P₂ is crucial for proper insulin signaling propagation and for insulin-stimulated GLUT4 vesicle translocation in 3T3-L1 adipocytes. Global depletion of PI(4,5)P₂ on the cell surface blunted insulin-stimulated Akt phosphorylation and abolished insulin effects in promotion of the docking and fusion of GLUT4 vesicle with the plasma membrane. Furthermore, by development of a novel optogenetic module to selectively modulate PI(4,5)P₂ levels on the GLUT4 vesicle docking site, we identified an important regulatory role of PI(4,5)P₂ in controlling of vesicle docking process. Local depletion of PI(4,5)P₂ at the vesicle docking site promoted GLUT4 vesicle undocking, diminished insulin-stimulated GLUT4 vesicle docking and fusion, but without perturbation of insulin signaling propagation in adipocytes. Our results provide strong evidence that cell surface PI(4,5)P₂ plays two distinct functions on regulation of the exocytic trafficking of GLUT4 in adipocytes. PI(4,5)P₂ not only regulates the proper activation of insulin signaling in general but also controls GLUT4 vesicle docking process at the vesicle-membrane contact sites.