INSULIN-INDUCED TRANSLOCATION OF THE GLUCOSE-TRANSPORTER GLUT4 IN CARDIAC-MUSCLE - STUDIES ON THE ROLE OF SMALL-MOLECULAR-MASS GTP-BINDING PROTEINS

Subcellular fractions obtained from rat cardiac ventricular tissue wer e used to elucidate a possible functional relationship between small-m olecular-mass G-proteins and the insulin-responsive glucose transporte r GLUT4. Proteins were separated by SDS/ PAGE and transferred to nitro cellulose membranes. Incubation with [alpha-P-32]GTP revealed the pres ence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsoma l membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriche d membrane vesicles. This material was found to contain a much decreas ed amount of small G-proteins, with the exclusive presence of the 24 k Da species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decr eased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enrich ed vesicles from insulin-treated animals. Western-blot analysis of mic rosomal membranes with a panel of antisera against rab GTP-binding pro teins indicated the presence of rab4A, with a molecular mass of 24 kDa , whereas rab1A, rab2 and rab6 were not observed. rab4A was barely det ectable in GLUT4-enriched vesicles; however, insulin produced an exten sive shift of rab4A from the cytosol and the microsomal fraction to th e plasma membrane with a parallel increase in GLUT4. These data show t hat a small GTP-binding protein is co localized with GLUT4 in an insul in-responsive intracellular compartment, and strongly suggest that thi s protein is involved in the exocytosis of GLUT4 in cardiac muscle. Fu rthermore, the observed translocation of rab4A. is compatible with ins ulin-induced endosome recycling processes, possibly including the gluc ose transporters.