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

Abstract

Rab4, a member of the Rab family of Ras-related small GTP-binding proteins, has been shown to be associated with GLUT4-containing vesicles and implicated in the insulin action on glucose transport in rat adipocytes. In the present study, we investigated the insulin effects on the guanine nucleotide exchange on Rab4. In electrically permeabilized rat adipocytes, the amount of Insulin stimulates glucose transport in muscles and adipose cells by promoting translocation of glucose transporter isoform, GLUT4, from intracellular compartmment(s) to the plasma membrane (1-3), although the molecular mechanisms of the insulin action are still obscure. Recent kinetic and morphological studies have proposed a three-compartment model for the subcellular trafficking of GLUT4 (3, 4). In the model, GLUT4 molecules are associated with at least two intracellular compartments; one is the early endosome in which GLUT4 molecules seem to appear only in the presence of insulin, and the second one is a more specialized compartment where the glucose transporter molecules are sequestrated in the basal state. Although the precise nature of the latter compartment remains indistinct, after insulin stimulation, GLUT4 molecules leave the compartment, are recruited on the plasma membrane, and then enter the endosomal recycling pathway.Over the past decade, it has become apparent that intracellular vesicle traffic is regulated by a variety of GTP-binding proteins, including Rab and Arf families of Ras-related small GTP-binding proteins (5, 6), as well as trimeric GTP-binding proteins (7) and large GTP-binding proteins such as dynamin (8). In permeabilized adipocytes, nonhydrolyzable GTP analogs induce GLUT4 translocation, suggesting that GTP-binding protein(s) may be involved in insulin-stimulated GLUT4 translocation (9 -12). In this regard, Rab3D has been shown to be predominantly expressed in adipocytes and induced during differentiation of 3T3-L1 cells into adipocytes (13), although it remains to be demonstrated whether the GTPase is involved in the insulin action (14). On the other hand, Cormont et al. (15) reported that Rab4 is associated with GLUT4-containing vesicles in rat adipocytes, and insulin stimulation resulted in redistribution of the protein from the vesicle to the cytosol. Rab4, a member of the Rab family, has been demonstrated to be associated with the early endosomes (17, 18) and implicated in regulation of the recycling of cell surface receptors, such as transferrin receptor, from the early endosomes to the cell surface (19).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 ...

Rab11 is associated with GLUT4-containing vesicles and redistributes in response to insulin

“…For cardiac insulin resistance, our laboratory has recently reported the hyperphosphorylation of insulin receptor substrate-1 (IRS-1) on Ser/Thr coupled to a defective activation of PI 3-kinase [48], making it likely that Rab11 represents a downstream target of insulin-activated PI 3-kinase. Consistently, earlier work has shown that insulin stimulates the guanine nucleotide exchange on Rab4 through a PI 3-kinase-dependent signalling pathway [49].…”

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

“…Conversely, the amount of Rab4 present on adipocyte intracellular membranes was decreased upon bafilomycin A " treatment ( Figure 6B). Similar to the effects of insulin [29][30][31][32], bafilomycin A " caused solubilization of intracellular Rab4, as indicated by the lack of a proportionate increase in Rab4 on the plasma membrane ( Figure 6B) and its quantitative recovery in the cytosolic fraction ( [32] ; and results not shown). The specificity of the bafilomycin A " effect for insulin-regulatable intracellular membrane proteins was further suggested by results demonstrating no detectable differences in the SDS\PAGE patterns of Coomassie Blue-stained intracellular membrane and plasma membrane proteins derived from bafilomycin A " -treated and control 3T3-L1 adipocytes (results not shown).…”

“…Previous studies in fat cells and adipocytes in culture have demonstrated the insulin-dependent redistribution of Rab4 from intracellular membranes to the cytosol, whereas the membrane partitioning of GDI-1 remained unaltered by similar insulin treatment [23,[29][30][31][32][33]. 3T3-L1 adipocytes were treated acutely with bafilomycin A " and, following subcellular fractionation and SDS\PAGE, the levels of GDI-1 and Rab4 were assessed by immunoblotting.…”

A Requirement for Phosphatidylinositol 3-Kinase in Pseudopod Extension

“…Other likely targets include proteins that interact with or stimulate guanine nucleotide exchange activity of members of the Rab family of GTPases. Wortmannin inhibits Rab5-mediated stimulation of endocytosis (64) and blocks insulinstimulated binding of 35 S-GTP␥S to Rab4 (65). It is possible that lipid products of PI 3-kinase bind to PH domains on guanine nucleotide exchange factors for ARF and Rab family members, thereby increasing their exchange activity.…”