Intracellular Segregation of Phosphatidylinositol-3,4,5-Trisphosphate by Insulin-Dependent Actin Remodeling in L6 Skeletal Muscle Cells

Abstract

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) colocalize. We now report that insulin receptor substrate-1 and the p110α catalytic subunit of PI3-K (but not p110β) also colocalize with the actin structures. Akt-1 was also found in the remodeled actin structures, unlike another PI3-K effector, atypical protein kinase C λ. 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-P₃]) 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-P₃ 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-P₃ is required for both phenomena. We propose that PI-3,4,5-P₃ leads to actin remodeling, which in turn segregates p85α and p110α, thus localizing PI-3,4,5-P₃ production on membranes trapped by the actin mesh. Insulin-stimulated actin remodeling may spatially coordinate the localized generation of PI-3,4,5-P₃ and recruitment of Akt, ultimately leading to GLUT4 insertion at the plasma membrane.

ACKNOWLEDGMENTS

This study was supported by a grant from the Canadian Institutes of Health Research (MT7307) to A. Klip. N. Patel was supported by a Banting & Best Diabetes Centre-Novo Nordisk Studentship Award. A. Rudich is the recipient of the Albert Renold Career Development Award from the European Foundation for the Study of Diabetes. Z. Khayat was supported by a Canadian Institutes of Health Research Doctoral Student Award.

We are grateful to Morris White, Michael Czech, and Tobias Meyer for generously supplying IRS-1 antibody, GFP-PH-GRP1, GFP-PH-(K273A)-GRP1, and GFP-PH-Akt cDNAs used in this study.