DISSECTION OF GLUT4 RECYCLING PATHWAY INTO EXOCYTOSIS AND ENDOCYTOSISIN RAT ADIPOCYTES - EVIDENCE THAT GTP-BINDING PROTEINS ARE INVOLVED IN BOTH PROCESSES

The effects of guanine nucleotides on either exocytosis or endocytosis of GLUT4 were examined in electrically permeabilized rat adipocytes b y using D-k-(62-85), a major histocompatibility complex class I-derive d peptide, Reversal of glucose transport activity that had been stimul ated with insulin was completely blocked by D-k-(62-85), Likewise, end ocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 was almost co mpletely inhibited by the peptide, Insulin-stimulated glucose transpor t activity was enhanced about 50% by D-k-(62-85), whereas the basal tr ansport activity was stimulated only slightly, Although guanosine 5'-O -(3-thiotriphosphate) (GTP gamma S) augmented glucose transport to the same extent as insulin in the absence of the peptide, glucose transpo rt stimulated by GTP gamma S was only 60% of the insulin effect in the presence of the peptide; the effect of insulin was markedly enhanced by D-k-(62-85), whereas GTP gamma S-induced glucose transport was not affected, suggesting that GTP gamma S has an effect similar to that of the peptide, In fact, endocytosis of the 35-kDa fragment of GLUT4 was markedly inhibited by GTP gamma S, Additionally, GLUT4 endocytosis wa s accelerated by GTP but was inhibited by guanosine 5'-O-(2-thiodiphos phate), These results indicate that GTP gamma S induces translocation of GLUT4 by both stimulating exocytosis and inhibiting endocytosis, Wi th respect to the dependence on GTP hydrolysis, distinct types of GTP- binding proteins are involved in exocytosis and endocytosis of GLUT4.