Disruption of Cortical Actin in Skeletal Muscle Demonstrates an Essential Role of the Cytoskeleton in Glucose Transporter 4 Translocation in Insulin-sensitive Tissues

Abstract

Cell culture work suggests that signaling to polymerize cortical filamentous actin (F-actin) represents a required pathway for the optimal redistribution of the insulin-responsive glucose transporter, GLUT4, to the plasma membrane. Recent in vitro study further suggests that the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) mediates the effect of insulin on the actin filament network. Here we tested whether similar cytoskeletal mechanics are essential for insulin-regulated glucose transport in isolated rat epitrochlearis skeletal muscle. Microscopic analysis revealed that cortical F-actin is markedly diminished in muscle exposed to latrunculin B. Depolymerization of cortical F-actin with latrunculin B caused a time- and concentration-dependent decline in 2-deoxyglucose transport. The loss of cortical F-actin and glucose transport was paralleled by a decline in insulin-stimulated GLUT4 translocation, as assessed by photolabeling of cell surface GLUT4 with Bio-LC-ATB-BMPA. Although latrunculin B impaired insulin-stimulated GLUT4 translocation and glucose transport, activation of phosphatidylinositol 3-kinase and Akt by insulin was not rendered ineffective. In contrast, the ability of insulin to elicit the cortical F-actin localization of N-WASP was abrogated. These data provide the first evidence that actin cytoskeletal mechanics are an essential feature of the glucose transport process in intact skeletal muscle. Furthermore, these findings support a distal actin-based role for N-WASP in insulin action in vivo. Cell culture work suggests that signaling to polymerize cortical filamentous actin (F-actin) represents a required pathway for the optimal redistribution of the insulin-responsive glucose transporter, GLUT4, to the plasma membrane. Recent in vitro study further suggests that the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) mediates the effect of insulin on the actin filament network. Here we tested whether similar cytoskeletal mechanics are essential for insulin-regulated glucose transport in isolated rat epitrochlearis skeletal muscle. Microscopic analysis revealed that cortical F-actin is markedly diminished in muscle exposed to latrunculin B. Depolymerization of cortical F-actin with latrunculin B caused a time- and concentration-dependent decline in 2-deoxyglucose transport. The loss of cortical F-actin and glucose transport was paralleled by a decline in insulin-stimulated GLUT4 translocation, as assessed by photolabeling of cell surface GLUT4 with Bio-LC-ATB-BMPA. Although latrunculin B impaired insulin-stimulated GLUT4 translocation and glucose transport, activation of phosphatidylinositol 3-kinase and Akt by insulin was not rendered ineffective. In contrast, the ability of insulin to elicit the cortical F-actin localization of N-WASP was abrogated. These data provide the first evidence that actin cytoskeletal mechanics are an essential feature of the glucose transport process in intact skeletal muscle. Furthermore, these findings support a distal actin-based role for N-WASP in insulin action in vivo. Type II diabetes (noninsulin-dependent diabetes mellitus) is a major disease in the world today, afflicting over 90 million Americans. In its earliest phases, the major feature of this disease is resistance of skeletal muscle to insulin, which is of considerable importance, because it is responsible for the disposal of the majority of an exogenous glucose load (1Defronzo R.A. Jacto E. Jequier E. Maeder E. Wahren J. Felber J.P. Diabetes. 1981; 30: 1000-1007Crossref PubMed Scopus (1408) Google Scholar). Insulin stimulation of glucose transport into skeletal muscle, as well as adipose tissue and heart, occurs via the GLUT4 1The abbreviations used are: GLUT4, glucose transporter 4; Bio-LC-ATB-BMPA, 4,4′-O-[2-[2-[2-[2-[2-[6(biotinylamino)hexanoyl]amino]ethoxy]ethoxy]ethyoxy]-4-(1-azi-2,2,2,-triethyl)benzoyl]amino-1,3-propanediyl]bis-d-mannose; PI3K, phosphatidylinositol 3-kinase; N-WASP, neural Wiskott-Aldrich syndrome protein; KHB, Krebs-Henseleit buffer; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TBST, Tris-buffered saline with Tween; TTBS, Tween Trisbuffered saline; t-tubule, transverse tubular; PIP3, phosphatidylinositol 3,4,5-trisphosphate; IRS, insulin receptor substrate. -facilitated glucose transporter, one of a family of glucose transporters in mammalian tissues (2Kayano T. Burant C.F. Fukumoto H. Gould G.W. Fan Y. Eddy R.L. Byers M.G. Shows T.B. Seino S. Bell G.I. J. Biol. Chem. 1990; 265: 13276-13282Abstract Full Text PDF PubMed Google Scholar, 3Kahn B.B. J. 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Although of the cortical of which further is to this work evidence that the of cortical F-actin is in the insulin-regulated of GLUT4 to the cell between the insulin-stimulated glucose transport and the loss of insulin-stimulated GLUT4 translocation in latrunculin muscle is to the transport of the glucose transporter at in for the of glucose in this muscle the that insulin not plasma membrane levels it to this transporter for this In the is also evidence that insulin and the of GLUT4 transporters G. R. T. A. A. Klip A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. S. C. G. T. D. J. C. Klip A. A. Diabetes. PubMed Scopus Google Scholar). the ability of insulin to the of the basal of GLUT4 in the plasma membrane not on cortical that a of GLUT4 protein at the and this and in the of actin as in GLUT4 B.B. Cushman Biochem. 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Cell Sci. 2001; PubMed Google Scholar). data for the first in intact skeletal muscle that N-WASP to insulin, and this is on cortical actin In data for the first that actin cytoskeletal mechanics are for insulin-stimulated GLUT4 translocation in intact skeletal muscle. In cell surface actin was revealed not to for insulin signal or a of insulin-stimulated glucose transport, because cortical F-actin effect on Akt phosphorylation, or of insulin-stimulated glucose transport. In contrast, the loss of a actin filament at the cell surface insulin-stimulated N-WASP and filament These data are with a cortical F-actin is essential in insulin action GLUT4 translocation and glucose transport. the of cytoskeletal mechanics on signal and GLUT4 translocation, with the further of for of cortical actin provide into the of insulin action and the of insulin Holman for of the GLUT4 photolabeling