The small ubiquitin-related modifier (SUMO) conjugating enzyme Ubc9 has been shown to upregulate GLUT4 in L6 myoblast cells, although the mechanism of action has remained undefined. Here we investigated the physiological significance of Ubc9 in GLUT4 turnover and subcellular targeting by adenovirus vector-mediated overexpression and by small interfering RNA (siRNA)-mediated gene silencing of Ubc9 in 3T3-L1 adipocytes. Overexpression of Ubc9 resulted in an inhibition of GLUT4 degradation and promoted its targeting to the unique insulin-responsive GLUT4 storage compartment (GSC), leading to an increase in GLUT4 amount and insulin-responsive glucose transport in 3T3-L1 adipocytes. Overexpression of Ubc9 also antagonized GLUT4 downregulation and its selective loss in GSC induced by long-term insulin stimulation. By contrast, siRNA-mediated depletion of Ubc9 accelerated GLUT4 degradation and decreased the amount of the transporter, concurrent with its selective loss in GSC, which resulted in attenuated insulinresponsive glucose transport. Intriguingly, overexpression of the catalytically inactive mutant Ubc9-C93A produced effects indistinguishable from those with wild-type Ubc9, suggesting that Ubc9 regulates GLUT4 turnover and targeting to GSC by a mechanism independent of its catalytic activity. Thus, Ubc9 is a pivotal regulator of the insulin sensitivity of glucose transport in adipocytes.

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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....

The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum, while DIF-1 and its analogs have been shown to possess antiproliferative activity in vitro in mammalian tumor cells. In the present study, we investigated the effects of DIF-1 and its analogs on normal (nontransformed) mammalian cells. Without affecting the cell morphology and cell number, DIF-1 at micromolar levels dose-dependently promoted the glucose uptake in confluent 3T3-L1 fibroblasts, which was not inhibited with wortmannin or LY294002 (inhibitors for phosphatidylinositol 3-kinase). DIF-1 affected neither the expression level of glucose transporter 1 nor the activities of four key enzymes involved in glucose metabolism, such as hexokinase, fluctose 6-phosphate kinase, pyruvate kinase, and glucose 6-phosphate dehydrogenase. Most importantly, stimulation with DIF-1 was found to induce the translocation of glucose transporter 1 from intracellular vesicles to the plasma membranes in the cells. In differentiated 3T3-L1 adipocytes, DIF-1 induced the translocation of glucose trasporter 1 (but not of glucose transporter 4) and promoted glucose uptake, which was not inhibited with wortmannin. These results indicate that DIF-1 induces glucose transporter 1 translocation and thereby promotes glucose uptake, at least in part, via a inhibitors for phosphatidylinositol 3-kinase ⁄ Akt-independent pathway in mammalian cells. Furthermore, analogs of DIF-1 that possess stronger antitumor activity than DIF-1 were less effective in promoting glucose consumption, suggesting that the mechanism of the action of DIF-1 for stimulating glucose uptake should be different from that for suppressing tumor cell growth.Abbreviations DIF-1, differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one]; DIF-3, differentiation-inducing factor-3 [1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one]; DMEM-HG, DMEM containing a high concentration of glucose; DMEM-LG, DMEM containing a low concentration of glucose; EtOH, ethanol; GLUT, glucose transporter; LDM, low-density microsome; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one; 8-MIBMX, 8-methoxymethyl-3-isobutyl-1-methylxanthine; 2-MIDIF-1, 2-methoxy isomer of DIF-1; PDE1, calmodulin-dependent cyclic nucleotide phosphodiesterase; PI3K, phosphatidylinositol 3-kinase; PM, plasma membrane; Tg, thapsigargin; THPH, 1-(2,4,6-trihydroxyphenyl)hexan-1-one.