PI 4,5-P2 stimulates glucose transport activity of GLUT4 in the plasma membrane of 3T3-L1 adipocytes

Insulin-stimulated glucose uptake through GLUT4 plays a pivotal role in maintaining normal blood glucose levels. Glucose transport through GLUT4 requires both GLUT4 translocation to the plasma membrane and GLUT4 activation at the plasma membrane. Here we report that a cell-permeable phosphoinositide-binding peptide, which induces GLUT4 translocation without activation, sequestered PI 4,5-P2 in the plasma membrane from its binding partners. Restoring PI 4,5-P2 to the plasma membrane after the peptide treatment increased glucose uptake. No additional glucose transporters were recruited to the plasma membrane, suggesting that the increased glucose uptake was attributable to GLUT4 activation. Cells overexpressing phosphatidylinositol-4-phosphate 5-kinase treated with the peptide followed by its removal exhibited a higher level of glucose transport than cells stimulated with a submaximal level of insulin. However, only cells treated with submaximal insulin exhibited translocation of the PH-domains of the general receptor for phosphoinositides (GRP1) to the plasma membrane. Thus, PI 4,5-P2, but not PI 3,4,5-P3 converted from PI 4,5-P2, induced GLUT4 activation. Inhibiting F-actin remodeling after the peptide treatment significantly impaired GLUT4 activation induced either by PI 4,5-P2 or by insulin. These results suggest that PI 4,5-P2 in the plasma membrane acts as a second messenger to activate GLUT4, possibly through F-actin remodeling.