The Rho family GTPase Rac1 has been implicated in the regulation of glucose uptake in myoblast cell lines. However, no evidence for the role of Rac1 has been provided by a mouse model. The purpose of this study is to test the involvement of Rac1 in insulin action in mouse skeletal muscle. Intravenous administration of insulin indeed elicited Rac1 activation in gastrocnemius muscle, suggesting the involvement of Rac1 in this signaling pathway. We then examined whether insulin‐stimulated translocation of the facilitative glucose transporter GLUT4 from its storage sites to the skeletal muscle sarcolemma depends on Rac1. We show that ectopic expression of constitutively activated Rac1, as well as intravenous administration of insulin, caused translocation of GLUT4 to the gastrocnemius muscle sarcolemma, as revealed by immunofluorescent staining of a transiently expressed exofacial epitope‐tagged GLUT4 reporter. Of particular note, insulin‐dependent, but not constitutively activated Rac1‐induced, GLUT4 translocation was markedly suppressed in skeletal muscle‐specific rac1 ‐knockout mice compared to control mice. Immunogold electron microscopic analysis of endogenous GLUT4 gave similar results. Collectively, we propose a critical role of Rac1 in insulin‐dependent GLUT4 translocation to the skeletal muscle sarcolemma, which has heretofore been predicted solely by cell culture studies.—Ueda, S., Kitazawa, S., Ishida, K, Nishikawa, Y., Matsui, M., Matsumoto, H., Aoki, T., Nozaki, S., Takeda, T., Tamori, Y., Aiba, A., Kahn, C. R., Kataoka, T., Satoh, T. Crucial role of the small GTPase Rac1 in insulinstimulated translocation of glucose transporter 4 to the mouse skeletal muscle sarcolemma. FASEB J. 24, 2254–2261 (2010). www.fasebj.org

Insulin is a hormone that regulates the blood glucose level by stimulating various physiological responses in its target tissues. In skeletal muscle and adipose tissue, insulin promotes membrane trafficking of the glucose transporter GLUT4 from GLUT4 storage vesicles to the plasma membrane, thereby facilitating the uptake of glucose from the circulation. Detailed mechanisms underlying insulin-dependent intracellular signal transduction for glucose uptake remain largely unknown. In this article, I give an overview on the recently identified signaling network involving Rab, Ras, and Rho family small guanosine triphosphatases (GTPases) that regulates glucose uptake in insulin-responsive tissues. In particular, the regulatory mechanisms for these small GTPases and the cross-talk between protein kinase and small GTPase cascades are highlighted.

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the translocation of the glucose transporter GLUT4 from intracellular storage sites to the plasma membrane. The small GTPase Rac1 has been implicated in this insulin signalling, but the mechanism whereby Rac1 stimulates GLUT4 translocation remains obscure. In the present study, we examined the role of the small GTPase RalA downstream of Rac1 in skeletal muscle fibres isolated from genetically modified mice. A dominant-negative mutant of RalA, when ectopically overexpressed, significantly reduced GLUT4 translocation in response to insulin or either one of constitutively activated mutants of Rac1 and its upstream regulators, including the guanine-nucleotide-exchange factor FLJ00068, the protein kinase Akt2 and phosphoinositide 3-kinase. Constitutively activated Rac1 also failed to induce GLUT4 translocation in mouse skeletal muscle fibres in which the expression of RalA was abrogated by specific siRNA molecules. Furthermore, we applied a novel approach to detect the activated form of RalA in situ by immunofluorescence microscopy of mouse skeletal muscle fibres, demonstrating that constitutively activated mutants of Rac1 and its upstream regulators as well as insulin indeed cause the activation of RalA. Notably, this RalA activation was remarkably impaired in rac1-deficient skeletal muscle fibres. Taken together, these results provide evidence that RalA is indeed activated and involved in the regulation of GLUT4 translocation in response to insulin downstream of Rac1 in mouse skeletal muscle.