Insulin accelerates inter-endosomal GLUT4 traffic via phosphatidylinositol3-kinase and protein kinase B

Insulin enhances plasmalemmal-directed traffic of glucose transporter-4 (GL UT4), but it is unknown whether insulin regulates GLUT4 traffic through end osomal compartments. In L6 myoblasts expressing Myc-tagged GLUT4, insulin m arkedly stimulated the rate of GLUT4myc recycling. In myoblasts stimulated with insulin to maximize surface GLUT4myc levels, we followed the rates of surface-labeled GLUT4myc endocytosis and chased its intracellular distribut ion in space and time using confocal immunofluorescence microscopy. Surface -labeled GLUT4myc internalized rapidly (t(1/2) 3 min), reaching the early e ndosome by 2 min and the transferrin receptor-rich, perinuclear recycling e ndosome by 20 min. Upon re-addition of insulin, the t(1/2) of GLUT4 disappe arance from the plasma membrane was unchanged (3 min), but strikingly, GLUT 4myc reached the recycling endosome by 10 and left by 20 min. This effect o f insulin was blocked by the phosphatidylinositol 3-kinase inhibitor LY2940 02 or by transiently transfected dominant-negative phosphatidylinositol 3-k inase and protein kinase B mutants. In contrast, insulin did not alter the rate of arrival of rhodamine-labeled transferrin at the recycling endosome. These results reveal a heretofore unknown effect of insulin to accelerate interendosomal travel rates of GLUT4 and identify the recycling endosome as an obligatory stage in insulin-dependent GLUT4 recycling.