A Phosphatidylinositol 3-Kinase-independent Insulin Signaling Pathway to N-WASP/Arp2/3/F-actin Required for GLUT4 Glucose Transporter Recycling

Abstract

Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actinrelated protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-⌬WA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-⌬WA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.Insulin stimulates glucose uptake by skeletal muscle and adipose tissues primarily through regulation of the subcellular distribution of the glucose transporter 4 (GLUT4) 1 (1, 2). In response to insulin, a fraction of GLUT4 present in intracellular membranes is redistributed to the plasma membrane, resulting in an increase of GLUT4 on the cell surface and enhanced glucose uptake by these cells. This effect of insulin is important in maintaining glucose homeostasis in humans, and impaired insulin action can contribute to the pathogenesis of type 2 diabetes (3). The precise mechanism by which insulin directs exocytosis of GLUT4-containing membrane vesicles remains obscure. However, it is established that activation of the insulin receptor tyrosine kinase catalyzes tyrosine phosphorylation of insulin receptor substrate proteins that bind to Srchomology 2 domain-containing molecules, including the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (4, 5). This results in activation of the p110 catalytic subunit of the kinase, which then phosphorylates cellular polyphosphoinositides at the D-3 position, forming signaling molecules such as phosphatidylinositol 3,4,5-trisphosphate. Multiple studies using various pharmacologic inhibitors, overexpression of constitutively active or dominant negative mutants, and microinjection of blocking antibodies have suggested a necessary role of the p85/p110-type PI 3-kinase in insulin-stimulated GLUT4 translocation and glucose transport (1, 2, 6 -8).On the other hand, several lines of evidence suggest the requirement of PI 3-kinase-independent pathway(s) in G...

Immunopurification and Characterization of Rat Adipocyte Caveolae Suggest Their Dissociation from Insulin Signaling

“…TC-10 is a rho family GTPase that regulates the actin-based cortical cytoskeleton (59), remodeling of which has been suggested to play a part in insulin-regulated GLUT4 translocation (60,61). However, we failed to observe caveolaeassociated actin by Western blotting (Fig.…”

Regulation of endocytic traffic by Rho GTPases

“…Recent studies point instead to regulation of actin remodelling through N-WASP. Insulin stimulation causes the appearance of Arp2\3-dependent actin comet tails on GLUT4 vesicles [89] and TC10 can cause actin-dependent motility of purified endosomes in a reconstituted in itro system [87]. This suggests that TC10 may promote GLUT4 vesicle motility ; however, it is important to note that activated TC10 does not clearly stimulate comet-tail formation when expressed in adipocytes [87].…”

“…TC10 is localized to caveolin-rich lipid rafts in the plasma membrane [90] and recruits N-WASP to these sites upon insulin stimulation [89]. Dominant-negative TC10 blocks the increase in cortical actin polymerization produced by insulin stimulation [89]. Conversely, expression of constitutively active TC10 causes a loss of cortical actin [87].…”

“…The best-supported function of TC10 is in remodelling of the cortical actin cytoskeleton. TC10 is localized to caveolin-rich lipid rafts in the plasma membrane [90] and recruits N-WASP to these sites upon insulin stimulation [89]. Dominant-negative TC10 blocks the increase in cortical actin polymerization produced by insulin stimulation [89].…”

The activation of TC10, a Rho small GTPase, contributes to v-Rel-mediated transformation

“…TC10 has been reported to function in actin cytoskeleton organization (Neudauer et al, 1998;Murphy et al, 1999), nerve regeneration (Tanabe et al, 2000), and the activation of various kinases and transcription factors (Murphy et al, 1999;Murphy et al, 2001). In addition, the involvement of TC10 in the translocation of glucose transporter 4 (GLUT4) upon insulin stimulation in muscle cells and adipocytes has been extensively characterized Jiang et al, 2001;Kanzaki and Pessin, 2001;Watson et al, 2001). Furthermore, TC10 cooperates with activated Raf to transform fibroblasts and was shown to be important in Ras-mediated transformation of NIH3T3 cells (Murphy et al, 1999).…”