A Synthetic Peptide Corresponding to the Rab4 Hypervariable Carboxyl-terminal Domain Inhibits Insulin Action on Glucose Transport in Rat Adipocytes

Abstract

The present study was conducted to examine the involvement of Rab4, a low molecular weight GTP-binding protein, in the action of insulin on glucose transport. A synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain, Rab4-(191-210), was successfully transferred into rat adipocytes by electroporation and inhibited insulin-stimulated glucose transport by about 50% without affecting the basal transport activity. In contrast, synthetic peptides corresponding to the Rab3C and Rab3D carboxyl-terminal hypervariable domain had little effect on insulin action on glucose transport. The Rab4-(191-210) peptide also reduced insulin-induced GLUT4 translocation from the intracellular pool to the plasma membrane. Furthermore, the Rab4-(191-210) peptide reduced both insulin-induced glucose transport and GLUT4 translocation in the presence of a major histocompatibility complex class I antigen-derived peptide, D k -(62-85), which is a potent inhibitor of GLUT4 internalization, suggesting that the peptide inhibited exocytotic recruitment of GLUT4-containing vesicles. The Rab4-(191-210) peptide also inhibited GTP␥S-stimulated glucose transport. In addition, insulin-stimulated glucose transport was inhibited by the addition of anti-Rab4 antibody. These results suggest that Rab4 protein plays a crucial role in insulin action on GLUT4 translocation, especially in exocytotic recruitment by the hormone of the glucose transporter to the plasma membrane from the intracellular retention pool.Insulin stimulates glucose uptake in skeletal/cardiac muscles and adipocytes by promotion of translocation of glucose transporter isoform, GLUT4, from the intracellular pool to the plasma membrane (1-3). Although the precise mechanism(s) of insulin-induced GLUT4 redistribution is still unclear, several lines of evidence indicate that GTP-binding protein(s) is involved in the insulin action. Thus, nonhydrolyzable GTP analogues induce GLUT4 translocation (4, 5) and stimulate glucose transport (6, 7) in permeabilized adipocytes. Furthermore, by dissecting the recycling pathway of GLUT4 into exocytosis and endocytosis, we have demonstrated recently that GTP␥S 1 stimulates exocytotic fusion of GLUT4-containing vesicles, whereas the nucleotide inhibits endocytosis of the glucose transporter (8). Our previous study also indicated that the number of GLUT4 molecules in recycling pool is not large in the basal state and the rate-limiting step of insulin-induced redistribution of GLUT4 is the exocytotic recruitment from the intracellular retention pool to the plasma membrane (8).The Rab family proteins of Ras-related small GTP-binding proteins have been implicated in regulation of intracellular vesicular traffic (9). Given that exocytotic movement of vesicles is regulated by the Rab family protein(s), GTP␥S may promote exocytosis of GLUT4-containing vesicles by stimulation of the Rab protein(s) associated with the vesicles, although other candidates (e.g. trimeric GTP-binding proteins) cannot be ruled out. In this regard, Baldini et al....

Insulin Stimulates Guanine Nucleotide Exchange on Rab4 via a Wortmannin-sensitive Signaling Pathway in Rat Adipocytes

“…5), similar to that of the inhibition by wortmannin of glucose transport activity (29,30). These results, together with our previous observations (16), provide further evidence that activation of Rab4 may be one of possible mechanism(s) by which insulin promotes GLUT4 translocation.…”

“…In a previous report, to elucidate the physiological significance of Rab4 in the insulin action, we incorporated into rat adipocytes a synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain and showed that the peptide specifically inhibited glucose transport and GLUT4 translocation stimulated by insulin or GTP␥S 1 (16), providing evidence that Rab4 plays a critical role in the insulin-induced GLUT4 translocation. It remains to be clarified, however, whether or not Rab4 lies downstream of the insulin receptor and functions as a signaling component of insulin activation of glucose transport.…”

Vesicle-associated Membrane Protein 2 Plays a Specific Role in the Insulin-dependent Trafficking of the Facilitative Glucose Transporter GLUT4 in 3T3-L1 Adipocytes

“…However, it has been suggested that the mode of action of GTP␥S may differ from that observed with insulin (38). Hence, it was of interest to determine if the VAMP2 inhibitory effect on GLUT4 translocation was a general effect or somewhat specific to insulindependent movement.…”

An adipocentric view of signaling and intracellular trafficking

“…In mammalian cells, there are more than 50 different rab GTPases described that are localized to different subcellular compartments with distinct regulatory functions mediating specific membrane traffic steps (for reviews see References [159][160][161][162]). Among this large family, Rab4 has been shown to associate with GLUT4 containing vesicles and implicated in insulin action in adipocytes [163,164]. Rab4 appears to undergo an insulin-stimulated redistribution from GLUT4 vesicles to the cytosol, where it associates with the GDP-dissociation inhibitor GDI-I [153,165].…”