Intracellular segregation of phosphatidylinositol-3,4,5-trisphosphate by insulin-dependent actin remodeling in L6 skeletal muscle cells

Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular pool to the cell surface through a mechanism that is dependent on phosphatidylinositol (PI) 3-kinase (PI3-K) and cortical actin remodeling. Here we test the hypothesis that insulin-dependent actin filament remodeling determines the location of insulin signaling molecules. It has been shown previously that insulin treatment of L6 myotubes leads to a rapid rearrangement of actin filaments into submembrane structures where the p85 regulatory subunit of PI3-K and organelles containing GLUT4, VAMP2, and the insulin-regulated aminopeptidase (IRAP) collocallize. We now report that insulin receptor substrate-1 and the p110alpha catalytic subunit of PI3-K (but not p110beta) also collocalize with the actin structures. Akt-1 was also found in the remodeled actin structures, unlike another PI3-K effector, atypical protein kinase Clambda. Transiently transfected green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of general receptor for phosphoinositides-1 (GRP1) or Akt(ligands of phosphatidylinositol-3,4,5-trisphosphate [PI-3,4,5-P-3]) migrated to the periphery of the live cells; in fixed cells, they were detected in the insulin-induced actin structures. These results suggest that PI-3,4,5-P-3 is generated on membranes located within the actin mesh. Actin remodeling and GLUT4 externalization were blocked in cells highly expressing GFP-PH-GRP1, suggesting that PI-3,4,5-P-3 is required for both phenomena. We propose that PI-3,4,5-P3 leads to actin remodeling, which in turn segregates p85alpha and p110alpha, thus localizing PI-3,4,5-P-3 production on membranes trapped by the actin mesh. Insulin-stimulated actin remodeling may spatially coordinate the localized generation of PI-3,4,5-P-3 and recruitment of Akt, ultimately leading to GLUT4 insertion at the plasma membrane.