Sustained activation of insulin receptors internalized in GLUT4 vesicles of insulin-stimulated skeletal muscle

Exposure of target cells to insulin results in the formation of ligand rece ptor complexes on the cell surface and their subsequent internalization int o the endosomal apparatus. A current view is that endocytosis of the insuli n receptor (IR) kinase results in its rapid deactivation and sorting of the IR back to the cell surface or to late endocytic compartments. We report h erein that, in skeletal muscle, in vivo stimulation with insulin induced a rapid internalization of the IR to an insulin-sensitive GLUT4-enriched intr acellular membrane fraction. After 30 min of stimulation, IR content and ty rosine phosphorylation were increased by three and nine times in that fract ion, respectively, compared with unstimulated muscles. In vitro autophospho rylation assays revealed that the kinase activity of internalized IRs was m arkedly augmented (eight to nine times) by insulin. In marked contrast with hepatic endosomes or adipocyte low-density microsomes, no IR tyrosine deph osphorylation activity was observed in GLUT4-enriched vesicles isolated fro m skeletal muscle. The activated IR was recovered in immunopurified GLUT4 v esicles after insulin stimulation. Insulin also increased tyrosine-phosphor ylated insulin receptor substrate 1 and phosphatidylinositol 3-kinase adapt er (p85) subunit contents in the intracellular membrane fraction, but these signaling molecules were not directly associated with GLUT4 vesicles. Thes e results show that, in skeletal muscle, the activated IR reaches a GLUT4-e nriched compartment where its activity is apparently sustained. We propose that compartmentalization of activated IRs to GLUT4 vesicles may play a rol e in sustaining insulin signaling at this locus in skeletal-muscle.