CHARACTERIZATION OF 2 DISTINCT INTRACELLULAR GLUT4 MEMBRANE POPULATIONS IN MUSCLE-FIBER - DIFFERENTIAL PROTEIN-COMPOSITION AND SENSITIVITY TO INSULINS

A major objective For the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 gluc ose carriers in the muscle fiber. In this report, we provide functiona l and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin: thus, one showed a marked decreas e in GLUT4 levels but the other did not. They also showed a markedly d ifferent protein composition as detected by quantitative vesicle immun oisolation analysis. The GLUT4 pool showing no response to insulin con tained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin. w hereas only VAMPS was found in the insulin-recruitable GLUT4 pool. SDS -PAGE and further silver staining of the immunoprecipitates revealed d iscrete polypeptide bands associated to the insulin-sensitive pool, an d all these polypeptide bands were found in the insulin-insensitive po pulation. Furthermore, some polypeptide bands were exclusive to the in sulin-insensitive population. The presence of cellubrevin and SCAMP pr oteins, endosomal markers, suggest that the insulin-insensitive GLUT4 membrane population belongs to an. endosomal compartment. In addition, Re favor the view that the insulin-sensitive GLUT4 membrane pool is s egregated from the endosomal GLUT4 population and is undergoes exocyto sis to the cell surface in response to insulin. Intracellular GLUT4 me mbranes obtained from skeletal muscle contain cellubrevin, and VAMP2 a nd GLUT4-vesicles from cardiomyocytes also contain cellubrevin. This s uggests that vSNARE proteins are key constituents of GLUT4 vesicles. T he presence of the tSNARE protein SNAP25 in skeletal muscle membranes and SNAP25 and syntaxin 1A and syntaxin 1B in cardiomyocyte plasma mem branes further suggest a role of the SNAREs in GLUT4 trafficking in mu scle.