Abstract: | The actin cytoskeleton–regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. Rac1 protein expression and insulin-stimulated PAKThr423 phosphorylation were decreased in muscles of high fat–fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.Insulin increases glucose uptake in skeletal muscle by stimulating translocation of GLUT4 from intracellular compartments to the plasma membrane and transverse tubuli (1–4). Skeletal muscle accounts for up to 75% of postprandial glucose disposal in humans (5), and normal insulin action in skeletal muscle is therefore crucial for maintaining glucose homeostasis.The Rho family GTPase Rac1 has been shown to regulate insulin-stimulated GLUT4 translocation and glucose transport in cultured muscle cells (6–8). Insulin activates Rac1, which leads to reorganization of the cortical actin cytoskeleton. Downregulation of Rac1 by small interfering RNA prevents this process (7,9) and also abolishes insulin-stimulated glucose uptake and GLUT4 translocation in L6 myoblasts (6,7). In addition, expression of a constitutively active Rac1 increases GLUT4 translocation to the same level seen after maximal insulin stimulation in this cell line (6).Even though cultured muscle cell lines are powerful tools to understand intracellular mechanisms, they differ from mature skeletal muscle in the expression and reliance of various proteins in the regulation of insulin-stimulated glucose uptake (10). Cultured muscle myoblasts, although able to fuse into myotubes, do not reach the same end-stage differentiation (e.g., do not have cross striations and do not develop transverse tubules) as muscles in vivo and therefore do not fully mature into a system that mimics fully developed skeletal muscles (11,12). Furthermore, the location, expression, and insulin-stimulated GLUT4 translocation are very different in cultured cells compared with mature muscle and may not require the same trafficking steps (2,3,13,14). As a consequence, it is imperative to investigate the role of Rac1 in insulin-stimulated glucose uptake in fully matured skeletal muscle in order to understand its role in glucose metabolism. Furthermore, the importance of skeletal muscle Rac1 on whole-body glucose homeostasis has not been determined.Rac1 activates p21-activated kinase (PAK) by facilitating autophosphorylation of PAK on threonine 423 (p-PAKThr423), and this pathway induces actin remodeling of the actin cytoskeleton (15). Accordingly, disruption of the actin cytoskeleton by actin-depolymerizing agents, such as latrunculin B, inhibits insulin-stimulated GLUT4 translocation in L6 myotubes (16,17). Dynamic rearrangement of the actin cytoskeleton is thus necessary for insulin to induce GLUT4 translocation in these cells (18).These findings also apply to mature skeletal muscle, since latrunculin B inhibits insulin-stimulated glucose uptake in rat epitrochlearis muscle (19). Furthermore, Ueda et al. (20) recently showed that Rac1 is activated by insulin in mouse skeletal muscle and that insulin-stimulated GLUT4 translocation is decreased in muscle-specific Rac1 knockout (KO) mice. PAK1 was also recently shown to be implicated in the regulation of insulin-stimulated GLUT4 translocation in mouse skeletal muscle (21). However, GLUT4 translocation does not always mimic glucose uptake, and numerous studies have reported experimental conditions where GLUT4 translocation and transport can be clearly dissociated (22–27), suggesting that GLUT4 translocation is not always an adequate measure of the functional end point, glucose uptake. Thus, the involvement of Rac1 and its downstream signaling in insulin-stimulated glucose uptake in mature skeletal muscle has not yet been investigated, and Rac1-dependent signaling has not been characterized in animal or human models of insulin resistance.A decreased ability to rearrange the cortical actin cytoskeleton in response to insulin has been proposed as a central defect in insulin-resistant muscle cells (28–30). Although exposure to insulin resistance–inducing agents decreased Rac1 activation and GLUT4 translocation (7), only small reductions in Akt signaling were observed in L6 myotubes (8). It is therefore possible that Rac1 is a major regulator of glucose uptake in mature skeletal muscle, and its dysregulation might contribute to the phenotype of muscular insulin resistance and type 2 diabetes (T2D). In the current study, we hypothesized that activation of Rac1 and its downstream target, PAK, is crucial for insulin-induced glucose uptake in mature skeletal muscle and for maintaining whole-body glucose homeostasis. We further hypothesized that Rac1-dependent signaling is downregulated in insulin-resistant states. |