Characterization of the Role of the Rab GTPase-activating Protein AS160 in Insulin-regulated GLUT4 Trafficking

Abstract

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulinregulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.Glucose transport into mammalian muscle and fat cells is an important step in insulin action and is critical for the maintenance of glucose homeostasis within the body (1). In mammalian muscle and fat cells, insulin stimulation activates a phosphorylation cascade, which in turn causes intracellular vesicles that contain the glucose transporter GLUT4, 4 to translocate to the plasma membrane (PM) and fuse (2, 3). In the basal state GLUT4 is distributed between the endosomal system, the trans-Golgi network (TGN), and a GLUT4 storage vesicle (GSV) compartment that is highly insulin-responsive (4 -6).The protein kinase Akt is activated in response to insulin and plays a critical role in GLUT4 translocation (1, 7). However, the link between the insulin signaling pathway and GLUT4 translocation is not fully understood. The insulin-dependent movement of GLUT4 vesicles to the PM is an Akt-independent process, and this is followed by an Aktdependent step likely involving the docking and fusion of vesicles with the PM (7-9). The mechanism by which Akt controls the docking and fusion of GLUT4 vesicles with the PM is not known. However, it was previously shown that a Rab GTPase-activating protein (RabGAP) known as AS160 is phosphorylated by Akt in response to insulin (10). How AS160 functions in GLUT4 trafficking and its cognate Rab proteins are not known. The role of a variety of Rab proteins in GLUT4 trafficking including Rab3d, Rab4, Rab5, and Rab11 has been examined (11-16). However, although these Rab proteins may participate in some aspects of GLUT4 trafficking, no compelling evidence for specific involvement in the insulin-regulated trafficking of GLUT4 has been found.In this study we describe four key findings that add to our understanding of GLUT4 traffic...

The Inactivation of RabGAP Function of AS160 Promotes Lysosomal Degradation of GLUT4 and Causes Postprandial Hyperglycemia and Hyperinsulinemia

“…AS160 (Akt substrate of 160 kDa) is a Rab GTPase activating protein (RabGAP), and the Arg 973 residue on human AS160 functions as a key residue to maintain its GAP activity (5,6). In 3T3-L1 adipocytes and L6 myocytes, AS160 is required for intracellular retention of GLUT4 under basal conditions (5,(7)(8)(9). Insulin stimulates the phosphorylation of AS160 via protein kinase B (PKB), and the phosphorylation of AS160 inactivates its GAP activity (10) and promotes GLUT4 translocation onto the cell surface (5,(11)(12)(13).…”

Role of 5′AMP-activated protein kinase in skeletal muscle

“…Thus, AS160 is thought to regulate the GTPase activity of Rab proteins to inhibit GLUT4 vesicle movement to, and/or fusion with, the plasma membrane. 53,54 Phosphorylation of AS160 on one or more of the eight described phosphorylation sites by Akt inhibits its GTPase activating protein activity, such that in response to insulin, the GTP form of Rab proteins is elevated leading to an increased number of GLUT4 molecules in the plasma membrane. A recent study provided evidence that AS160 is involved in both insulin-and contraction-stimulated glucose uptake in skeletal muscle.…”

Thrifty Tbc1d1 and Tbc1d4 proteins link signalling and membrane trafficking pathways

“…Tbc1d4 has GAP activity with the Rabs 2A, 8A, 8B, 10 and 14. Rabs 10 and 14 have been shown to be present on GLUT4 vesicles and therefore these may be the important target Rabs that lie between Tbc1d4 and regulated GLUT4 vesicle activity [2,3]. Lienhard's hypothesis that links Tbc1d4 activity to both insulin signalling and GLUT4 vesicle activity is shown in Figure 1.…”

“…It cannot then act on it to convert it into its non-functional GDP form. It has been shown that Tbc1d4 may be localized to GLUT4 vesicles by virtue of its binding to cargo proteins within the GLUT4 vesicles [3,4]. The change in localization of Tbc1d4 initiated by phosphorylation may involve its association with a 14-3-3 protein and subsequent dissociation from the GLUT4 vesicles and into the cytoplasm [5].…”

“…Studies in which Tbc1d4 has been knocked down by silencing RNA techniques indicate that the protein is involved in the exocytosis limb of the GLUT4 trafficking pathway [3,6]. Recent studies have highlighted the importance of docking and fusion of GLUT4 vesicles with the plasma membrane as being a highly insulin regulated site of action and possibly the rate-limiting step in the trafficking [7,8].…”