Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês

10.1074/jbc.m402697200

ISSN

1083-351X

Autores

Joseph T. Brozinick, Eric D. Hawkins, Andrew B. Strawbridge, Jeffrey S. Elmendorf,

Tópico(s)

Cellular transport and secretion

Resumo

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. Clin. Invest. 1992; 89: 1367-1374Crossref PubMed Scopus (254) Google Scholar). In the basal state, GLUT4 cycles continuously between the plasma membrane and one or more intracellular compartments, with the vast majority of the transporter residing in the cell interior (4Kandror K.V. Pilch P.F. J. Biol. Chem. 1996; 271: 21703-21708Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 5Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 6Rea S. James D. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). Activation of the insulin receptor triggers a large increase in the rate of GLUT4 vesicle exocytosis and a smaller but important decrease in the rate of internalization by endocytosis (7Yang J. Gillingham A.K. Hodel A. Koumanov F. Woodward B. Holman G.D. Am. J. Physiol. 2002; 283: E1299-E1307Crossref PubMed Scopus (31) Google Scholar, 8Satoh S. Nishimura H. Clark A. Kozka I. Vannucci S. Simpson I. Quon M. Cushman S. Holman G. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar, 9Jhun B. Rampal A. Liu H. Lachaal M. Jung C. J. Biol. Chem. 1992; 267: 17710-17715Abstract Full Text PDF PubMed Google Scholar, 10Czech M. Buxton J. J. Biol. Chem. 1993; 268: 9187-9190Abstract Full Text PDF PubMed Google Scholar). The overall insulin-dependent shift in the dynamics of GLUT4 trafficking results in a net increase of GLUT4 protein levels at the cell surface, and these plasma membrane-localized transporters subsequently facilitate the influx of glucose into the cell. Activation of GLUT4 translocation by insulin requires a phosphatidylinositol 3-kinase (PI3K) signal involving the upstream insulin receptor and insulin receptor substrate activators and the downstream protein kinase B (Akt) and C target enzymes (5Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 11Holman G.D. Sandoval I.V. J. Cell Sci. 2001; 11: 173-179Scopus (84) Google Scholar). Recently, it has also been suggested in the 3T3-L1 adipocyte cell culture system that a second pathway occurs as a consequence of Cbl tyrosine phosphorylation, although the involvement of this pathway in skeletal muscle remains controversial (12JeBailey L. Rudich A. Huang X. Ciano-Oliveira C.D. Kapus A. Klip A. Mol. Endocrinol. 2004; 18: 359-372Crossref PubMed Scopus (133) Google Scholar, 13Thirone A.C.P. Carvalheira J.B.C. Hirata A.E. Velloso L.A. Saad M.J.A. Endocrinology. 2004; 145: 281-293Crossref PubMed Scopus (50) Google Scholar, 14Ribon V. Saltiel A.R. Biochem. J. 1997; 324: 839-845Crossref PubMed Scopus (119) Google Scholar, 15Liu J. DeYoung S.M. Hwang J.B. O'Leary E.E. Saltiel A.R. J. Biol. Chem. 2003; 278: 36754-36762Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Cbl and the adaptor protein CAP (c-Cbl-associated protein (16Ribon V. Herrera R. Kay B.K. Saltiel A.R. J. Biol. Chem. 1998; 273: 4073-4080Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar)) are recruited to the insulin receptor by adaptor molecules containing pleckstrin homology and Src-homology 2 domains (17Liu J. Kimura A. Baumann C.A. Saltiel A.R. Mol. Cell. Biol. 2002; 22: 3599-3609Crossref PubMed Scopus (138) Google Scholar). Once tyrosine-phosphorylated by the receptor, Cbl can recruit the adaptor protein CrkII to lipid rafts, along with the guanyl nucleotide exchange factor C3G (18Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (484) Google Scholar). C3G, in turn, can activate the GTP-binding protein TC10, which resides in lipid rafts (19Watson R.T. Shigematsu S. Chiang S.-H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (148) Google Scholar). Recent investigation suggests a role of TC10 in the regulation of actin dynamics (20Chunqiu Hou J. Pessin J.E. Mol. Biol. Cell. 2003; 14: 3578-3591Crossref PubMed Google Scholar, 21Inoue M. Chang L. Hwang J. Chiang S.H. Saltiel A.R. Nature. 2003; 422: 629-633Crossref PubMed Scopus (285) Google Scholar, 22Jiang Z.Y. Chawla A. Bose A. Way M. Czech M.P. J. Biol. Chem. 2002; 277: 509-515Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 23Kanzaki M. Pessin J.E. J. Biol. Chem. 2001; 276: 42436-42444Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 24Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 25Kanzaki M. Watson R.T. Hou J.C. Stamnes M. Saltiel A.R. Pessin J.E. Mol. Biol. Cell. 2002; 13: 2334-2346Crossref PubMed Scopus (79) Google Scholar). In particular, the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) (22Jiang Z.Y. Chawla A. Bose A. Way M. Czech M.P. J. Biol. Chem. 2002; 277: 509-515Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) appears to be a downstream target of TC10. Consistent with a functional role of this actin nucleation promoting factor in insulin action, a role for actin in insulin-stimulated GLUT4 translocation has been implicated by several studies. For example, treatment with the actin-depolymerizing agent cytochalasin D or the actin monomer binding Red Sea Sponge toxins latrunculin A or B inhibited insulin-stimulated GLUT4 translocation (26Omata W. Shibata H. Li L. Takata K. Kojima I. Biochem. J. 2000; 346: 321-328Crossref PubMed Google Scholar, 27Wang Q. Bilan P.J. Tsakiridis T. Hinek A. Klip A. Biochem. J. 1998; 331: 917-928Crossref PubMed Scopus (139) Google Scholar, 28Tsakiridis T. Bergman A. Somwar R. Taha C. Aktories K. Cruz T.F. Klip A. Downey G.P. J. Biol. Chem. 1998; 273: 28322-28331Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Importantly, it has been shown that insulin elicits actin filament (F-actin) formation in 3T3-L1 adipocytes and L6 myotubes (24Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Omata W. Shibata H. Li L. Takata K. Kojima I. Biochem. J. 2000; 346: 321-328Crossref PubMed Google Scholar, 27Wang Q. Bilan P.J. Tsakiridis T. Hinek A. Klip A. Biochem. J. 1998; 331: 917-928Crossref PubMed Scopus (139) Google Scholar, 29Bose A. Cherniack A.D. Langille S.E. Nicoloro S.M.C. Buxton J.M. Park J.G. Chawla A. Czech M.P. Mol. Cell. Biol. 2001; 21: 5262-5275Crossref PubMed Scopus (57) Google Scholar, 30Emoto M. Klarlund J.K. Waters S.B. Hu V. Buxton J.M. Chawla A. Czech M.P. J. Biol. Chem. 2000; 275: 7144-7151Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 31Tsakiridis T. Vranic M. Klip A. J. Biol. Chem. 1994; 269: 29934-29942Abstract Full Text PDF PubMed Google Scholar, 32Asahi Y. Hayashi H. Wang L. Ebina Y. J. Med. Invest. 1999; 46: 192-199PubMed Google Scholar, 33Guilherme A. Emoto M. Buxton J.M. Bose S. Sabini R. Theurkauf W.E. Leszyk J. Czech M.P. J. Biol. Chem. 2000; 275: 38151-38159Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 34Marcusohn J. Isakoff S.J. Rose E. Symons M. Skolnik E.Y. Curr. Biol. 1995; 5: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 35Patki V. Buxton J. Chawla A. Lifshitz L. Fogarty K. Carrington W. Tuft R. Corvera S. Mol. Biol. Cell. 2001; 12: 129-141Crossref PubMed Scopus (103) Google Scholar, 36Nakashima N. Rose D.W. Xiao S. Egawa K. Martin S.S. Haruta T. Saltiel A.R. Olefsky J.M. J. Biol. Chem. 1999; 274: 3001-3008Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Thus, insulin signaling to polymerize cortical F-actin apparently represents a required pathway for optimal movement or fusion of GLUT4-containing vesicle membranes to the cell surface membrane. Nonetheless, data interpretation has to consider that adherent cultured cells possess significant numbers of actin stress fibers that may be more susceptible to these agents than cortical F-actin. Additionally, these cell systems differ from intact tissues from which they were originally derived in both their glucose transporter isoform expression and their responses to insulin (37Wilson C.M. Mitsumoto Y. Maher F. Klip A. FEBS Lett. 1995; 368: 19-22Crossref PubMed Scopus (77) Google Scholar, 38Ruan H. Zarnowski M.J. Cushman S.W. Lodish H.F. J. Biol. Chem. 2003; 278: 47585-47953Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Together, these data indicate that cell lines may not be completely representative of in vivo insulin signaling and the glucose transport process. There is suggestive evidence in isolated rat fat cells that actin participates in insulin-stimulated GLUT4 translocation and glucose transport (39Peyrollier K. Hajduch E. Gray A. Litherland G.J. Prescott A.R. Hundal H.S. Biochem. J. 2000; 352: 617-622Crossref PubMed Scopus (53) Google Scholar). However, the role of actin in skeletal muscle, a tissue that is responsible for ∼80% of postprandial glucose disposal (40Ferrannini E. Smith J.D. Cobelli C. Pilo G. DeFronzo R.A. J. Clin. Invest. 1985; 76: 357-364Crossref PubMed Scopus (202) Google Scholar) and is regarded as a major peripheral site of insulin resistance in diabetes (41Defronzo R.A. Gunnarsson R. Bjorkman O. Olsson M. Wahren J. J. Clin. Invest. 1985; 76: 149-155Crossref PubMed Scopus (896) Google Scholar), has not been addressed. This is partially due to the difficulty in both working with skeletal muscle preparations, and in visualizing the cellular distribution of GLUT4 and the organization of cortical F-actin in a setting comprised mainly of contractile machinery. The isolated rat epitrochlearis muscle, a small flat muscle in the rat fore limb, has been extensively utilized for investigation of the regulation of glucose transport (42Henriksen E.J. Holloszy J.O. Acta Physiol. Scand. 1991; 143: 381-386Crossref PubMed Scopus (61) Google Scholar). In the present study we have used this preparation to address the role the actin cytoskeleton plays in the glucose transport process in skeletal muscle. We demonstrate for the first time that disruption of cortical F-actin in an intact muscle inhibits both insulin-stimulated GLUT4 translocation and glucose transport. Additionally, early insulin signal propagation remained intact in epitrochlearis muscle made insulin-resistant by cortical F-actin disassembly. Consistent with a more distal signal transduction disturbance, the loss of actin membrane skeleton was associated with the inability of insulin to spatially regulate N-WASP in the isolated epitrochlearis muscle preparation. The subsequent report provides a detailed account of these studies. Animals—Specific pathogen-free male Wistar rats weighing 100–125 g were obtained from Charles River Laboratories (Boston, MA). Upon arrival, rats were housed four to a cage in a temperature-controlled animal room maintained on a 12:12-h light-dark cycle. The rats were fed ad libitum National Institutes of Health standard chow and water. Muscle Preparation and Incubation—Rats in the postprandial state were anesthetized with 5 mg/100 g of body weight sodium pentobarbital. Epitrochlearis muscles were dissected out, blotted on gauze, and transferred to 25-ml Erlenmeyer flasks containing 2 ml of Krebs-Henseleit buffer (KHB) with 0.1% bovine serum albumin (BSA), 32 mm mannitol, and 8 mm glucose. The suitability of this muscle for incubation studies has been extensively described previously (42Henriksen E.J. Holloszy J.O. Acta Physiol. Scand. 1991; 143: 381-386Crossref PubMed Scopus (61) Google Scholar). The flasks were incubated in a shaking water bath maintained at 30 °C for 1 h and were continuously gassed with 95% O2/5% CO2. Muscles were initially incubated in the presence or absence of latrunculin A or B (20 μm) for various periods of time prior to incubation under basal conditions (no additions), or stimulation with insulin (13.3 nm). The muscles were then transferred to flasks containing 2 ml of KHB with 0.1% BSA, 40 mm mannitol, 2 mm pyruvate, and the same additions as in the previous incubation, and used for measurement of glucose transport, for photolabeling of surface GLUT4, or frozen and used for Western blotting/signaling assays. The flasks were incubated for 10 min at 30 °C to wash out glucose, and the gas phase in the flasks was maintained at 95% O2/5% CO2. Following the wash step the muscles were used for measurement of glucose transport or GLUT4 photolabeling as described below. Measurement of Glucose Transport Activity—Glucose transport activity was measured using 2-deoxyglucose as described in detail previously (43Brozinick J.T. Birnbaum M.J. J. Biol. Chem. 1998; 273: 14679-14682Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Following the above incubations, muscles were blotted and transferred to flasks containing 1.5 ml of KHB with 1 mm 2-deoxy-[1,2-3H]glucose (1.5 mCi/mmol) and 39 mm [1-14C]mannitol (8 μCi/mmol) and the same additions as in the previous incubation. The flasks were incubated at 30 °C for 20 min and were continuously gassed with 95%O2/5%CO2. After the incubations, the muscles were frozen between tongs cooled to the temperature of liquid nitrogen, and stored at –80 °C until they were processed for measurement of 2-deoxyglucose transport. Frozen muscles were dissolved in 0.5 ml of 1 n KOH and were then neutralized with 0.5 ml of 1n HCl. The samples were mixed, and aliquots of the supernatant were counted for radioactivity in a liquid scintillation counter. Photoaffinity Labeling of Isolated Epitrochlearis Muscles—Isolated muscles were photolabeled as described previously (44Ryder J.W. Yang J. Galuska D. Rincon J. Bjornholm M. Krook A. Lund S. Pedersen O. Wallberg-Henriksen H. Zierath J.R. Holman G.D. Diabetes. 2000; 49: 647-654Crossref PubMed Scopus (164) Google Scholar). Briefly, muscles were incubated in flasks containing 400 μm Bio-LC-ATB-BMPA for 4 min and then exposed to UV light in a Rayonet Photochemical reactor (Southern New England Ultraviolet, Branford, CT) for 4 min. Following the labeling, the muscles were trimmed of their tendons, blotted, and frozen between tongs cooled to the temperature of liquid nitrogen. Muscles were kept stored at –80 °C until processed. Crude total membranes were prepared from the photolabeled muscles and solubilized, and GLUT4s were immunoprecipitated with streptavidin beads (Pierce) as described previously (44Ryder J.W. Yang J. Galuska D. Rincon J. Bjornholm M. Krook A. Lund S. Pedersen O. Wallberg-Henriksen H. Zierath J.R. Holman G.D. Diabetes. 2000; 49: 647-654Crossref PubMed Scopus (164) Google Scholar). Briefly, biotinylated GLUT4 was immunoprecipitated overnight from Thesit (Roche Applied Science)-solubilized crude membranes (equivalent amounts of protein) with 50 μl of streptavidin beads, and the beads were washed sequentially with 1% Thesit/PBS (4×), 0.1% Thesit/PBS (3×), and PBS (1×). Subsequent to immunoprecipitation, the immunocomplex was released from the streptavidin beads with Laemmli sample buffer and then subjected to SDS-PAGE. Resolved proteins were transferred to polyvinylidene difluoride membranes, blocked in 5% milk/Tris-buffered saline with 0.1% Tween 20 (TBST), and incubated with either affinitypurified GLUT4 (courtesy of Dr. Sam Cushman) or GLUT1 antiserum (courtesy of Dr. Larry Sliecker). Membranes were washed in TBST and incubated with alkaline phosphatase-coupled secondary antibody. Photolabeled GLUT4 or GLUT1 was detected via enhanced chemifluorescence and quantified by comparison to a plasma membrane standard that was run on each gel. Tissue Lysate Preparation—Isolated muscles were incubated under the experimental conditions as described above. Following incubation, the muscles were trimmed of their tendons, blotted, and frozen between tongs cooled to the temperature of liquid nitrogen. Muscles were kept stored at –80 °C until processed. Lysates were prepared from the incubated muscles essentially as described previously (43Brozinick J.T. Birnbaum M.J. J. Biol. Chem. 1998; 273: 14679-14682Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Briefly, frozen muscles were homogenized in ice-cold lysis buffer (50 mm HEPES, pH 7.2, 2 mm EDTA, 30 mm sodium pyrophosphate, 1% Triton X-100, 10% glycerol, 10 mm NaF, 150 mm NaCl, 2 mm NaVO3, 5 μg/ml leupeptin, 1.5 mg/ml benzamidine, 0.5 mg/ml pepstatin A, 2 μg/ml aprotinin, 1 mm 4-(2-aminoethyl)benzene-sulfonylfluoride hydrochloride (Pefabloc, Roche Applied Science) and 10 μg/ml antipain) and mixed end over end for 30 min at 4 °C. Lysates were then spun at 20,000 × g for 30 min, and protein was determined on the supernatant via the bicinchoninic method (Pierce) using crystalline BSA as a standard. Akt Western Blotting—Wistar epitrochlearis lysate protein samples (100 μg) were prepared for SDS-PAGE by the addition of 2× Laemmli sample buffer and boiled for 5 min. The samples, along with molecular weight markers (Amersham Biosciences) were loaded on a 7.5% SDS-PAGE gel. Resolved samples were transferred to polyvinylidene difluoride membrane (Bio-Rad). The membranes were rinsed in water and blocked in 5% nonfat dry milk in Tween Tris-buffered saline, pH 7.5 (TTBS), for 1 h. The membranes were then rinsed in TTBS and incubated overnight in affinity-purified anti-phospho-Akt (recognizing phospho-serine 473) (Cell Signaling, Beverly, MA). Following this step, the membranes were rinsed in TTBS and incubated in horseradish peroxidase-coupled goat anti-rabbit antibody for 2 h. The membranes were then rinsed in TTBS, and resolved bands were detected via enhanced chemifluorescence (Amersham Biosciences). PI3K Kinase Assays—For phosphatidylinositol 3-kinase (PI3K) assays, 1 mg of lysate protein was immunoprecipitated overnight with 4 μg of α-IRS-1 antibody (UBI, Lake Placid, NY), and captured with protein G-agarose beads. The immunocomplexes were washed and incubated with 50 μl of the final wash buffer containing 20 μg of phosphatidylinositol, 100 mm MgCl2, 880 μm ATP, and 30 μCi of [γ-33P]ATP (PerkinElmer Life Sciences). The lipid products were resolved by TLC and quantified on an Amersham Biosciences PhosphorImager. Immunofluorescent Labeling of Fixed Tissues—Following fixation in 2% paraformaldehyde/PBS for 2 h, tissues were washed with PBS and stored at 4 °C. A small section was excised from each tissue and incubated in 0.2% Triton X-100/0.05% Tween 20/PBS for 30 min at 25 °C. The sections were then rinsed three times in 0.05% Tween 20/PBS and blocked in 5% donkey serum/0.05% Tween 20/PBS (for caveolin-3 and dystrophin labeling) or 2% BSA/0.05% Tween 20/PBS (for actin and N-WASP labeling) for 60 min at 25 °C. Sections were then incubated overnight at 4 °C in mouse IgM anti-human F-actin (Serotech Oxford, UK), anti-N-WASP (Santa Cruz Biotechnology, Santa Cruz, CA), anti-dystrophin (Neomarkers, Inc., Fremont, CA), or anti-caveolin 3 (BD Biosciences) antibodies diluted 1:50 in blocking buffer. Samples were then washed extensively in 0.05% Tween 20/PBS. Sections were incubated for 45 min at 25 °C in 1:50 rhodamine-conjugated donkey anti-mouse IgM (for actin labeling), 1:50 fluorescein isothiocyanate-conjugated donkey antigoat IgG (for caveolin-3 labeling), 1:50 fluorescein isothiocyanate-conjugated donkey anti-mouse IgG (for dystrophin labeling), and 1:50 rhodamine-conjugated donkey anti-rabbit IgG (for N-WASP labeling). All conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Following secondary antibody incubation, samples were washed extensively with 0.05% Tween 20/PBS, rinsed with ddH2O, mounted to slides in Vectashield (Vector Laboratories, Inc., Burlingame, CA), and examined via confocal microscopy (Zeiss LSM 510 NLO Confocal Microscope). All images were taken in the same focal plane of the section and under identical microscopic parameters. Images shown are representative of three to five fields from each sample. Statistical Analysis—The data were analyzed by analysis of variance to test the effect of treatment (insulin and latrunculin) on muscle glucose uptake, GLUT4 translocation, plasma membrane GLUT1 content, Akt phosphorylation, and PI3K kinase activity. When a significant F ratio was obtained, a Fisher's post-least significant difference post hoc test was employed to identify statistically significant differences (p < 0.05) between the means. Disruption of F-Actin Inhibits Insulin-stimulated Glucose Uptake—Previous studies (26Omata W. Shibata H. Li L. Takata K. Kojima I. Biochem. J. 2000; 346: 321-328Crossref PubMed Google Scholar, 27Wang Q. Bilan P.J. Tsakiridis T. Hinek A. Klip A. Biochem. J. 1998; 331: 917-928Crossref PubMed Scopus (139) Google Scholar, 31Tsakiridis T. Vranic M. Klip A. J. Biol. Chem. 1994; 269: 29934-29942Abstract Full Text PDF PubMed Google Scholar, 45Emoto M. Langille S.E. Czech M.P. J. Biol. Chem. 2001; 276: 10677-10682Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 46Tsakiridis T. Vranic M. Klip A. Biochem. J. 1995; 309: 1-5Crossref PubMed Scopus (63) Google Scholar) have reported that the actin-depolymerizing agent cytochalasin D and the actin monomer sequestering agents latrunculin A or latrunculin B can inhibit insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes and in primary adipocytes. Consistent with this effect on preventing GLUT4 mobilization to the cell surface, exposure of epitrochlearis muscles to increasing concentrations of latrunculin B for 2 h markedly reduced the ability of insulin to stimulate glucose uptake (Fig. 1, solid bars). Basal glucose transport was not affected at any concentration of latrunculin B tested (Fig. 1, open bars). Muscles exposed to 20 μm latrunculin B for 0.5, 1, or 2 h displayed a time-dependent decrease in insulin-stimulated glucose uptake (Fig. 2). Parallel studies performed with latrunculin A demonstrated a similar profile of impaired insulin-stimulated glucose transport (data not shown). Given the clear effect of 20 μm latrunculin B for 1 h on insulin-stimulated glucose transport, subsequent analyses were performed with this treatment parameter. Before evaluating the effect of latrunculin B on cortical F-actin, we studied the molecular architecture of the epitrochlearis muscle by using confocal immunofluorescence microscopy. To locate the position of the cell surface sarcolemma and the transverse tubular (t-tubule) membranes we used a specific antibody against caveolin-3, because the caveolae formed by homomultimers of this protein are located both at the sarcolemma and within the t-tubules (47Gabella G. J. Ultrastruct. Res. 1978; 65: 135-147Crossref PubMed Scopus (52) Google Scholar). As shown in Fig. 3A (panel 1), both the sarcolemma (closed arrows) and t-tubule (open arrows) are labeled. To further clarify the observed staining pattern, we collected images of epitrochlearis muscle labeled with dystrophin, a protein that is part of a large oligomeric complex named the dystrophin-glycoprotein complex (DGC) (48Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1119) Google Scholar, 49Campbell K.P. Kahl S.D. Nature. 1989; 338: 259-262Crossref PubMed Scopus (612) Google Scholar, 50Yoshida M. Ozawa E. J. Biochem. 1990; 108: 748-752Crossref PubMed Scopus (452) Google Scholar, 51Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Crossref PubMed Scopus (219) Google Scholar) that bridges across the sarcolemma and connects the extracellular matrix and the actin cytoskeleton. Our analyses revealed, as expected, marked cell surface sarcolemma membrane dystrophin staining (Fig. 3A, panel 2). Rhodamine-labeled phalloidin staining (data not shown) and anti-F-actin immunofluorescence (Fig. 3B) demonstrated that the muscles contained polymerized actin underneath and juxtaposed to the inner face of the sarcolemma membrane (Fig. 3B, panels 1 and 2). In parallel to the reduction in insulin-stimulated glucose transport activity, latrunculin B treatment resulted in a disruption of this cortical F-actin structure (Fig. 3, panels 3 and 4).Fig. 2Time dependence of latrunculin B on 2-deoxyglucose transport in isolated rat epitrochlearis muscles. Wistar epitrochlearis muscles were removed and incubated in the presence or absence of 13.3 nm insulin and 20 μm latrunculin B. Numbers in parentheses indicate number of observations. Values are the means ± S.E. *, significantly different from corresponding basal. †, significantly different from corresponding condition minus latrunculin B (p < 0.05). ‡, significantly different from corresponding 0.5-h incubation condition (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Effect of latrunculin B on cortical actin staining in isolated rat epitrochlearis muscles. Wistar epitrochlearis muscles were removed and incubated in the presence or absence of 20 μm latrunculin B for 1 h. Muscles were then incubated in the presence or absence of 13.3 nm insulin and fix

Referência(s)