Differential Contribution of Insulin Receptor Substrates 1 Versus 2 to Insulin Signaling and Glucose Uptake in L6 Myotubes
2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês
10.1074/jbc.m412317200
ISSN1083-351X
AutoresCarol Huang, Ana C.P. Thirone, Xudong Huang, Amira Klip,
Tópico(s)Protein Degradation and Inhibitors
ResumoInsulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-à-vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (siIRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70–75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells. Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-à-vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (siIRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70–75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells. Insulin receptor substrates (IRSs) 1The abbreviations used are: IRS, insulin receptor substrate; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IGF-I, insulin-like growth factor I; α-MEM, α-minimum Eagle's medium; siIRS-1, IRS-1-specific siRNA; siIRS-2, IRS-2-specific siRNA; siNR, or nonrelevant siRNA control; siRNA, small interfering RNA. mediate diverse metabolic and mitogenic effects of insulin, and dysregulation of IRS expression and activation has been observed in both insulin resistance and diabetes (1Goodyear L.J. Giorgino F. Sherman L.A. Carey J. Smith R.J. Dohm G.L. J. Clin. Invest. 1995; 95: 2195-2204Crossref PubMed Scopus (478) Google Scholar, 2Bjornholm M. Kawano Y. Lehtihet M. Zierath J.R. Diabetes. 1997; 46: 524-527Crossref PubMed Scopus (0) Google Scholar, 3Carvalho E. Jansson P.A. Axelsen M. Eriksson J.W. Huang X. Groop L. Rondinone C. Sjostrom L. Smith U. FASEB J. 1999; 13: 2173-2178Crossref PubMed Scopus (123) Google Scholar, 4Krook A. 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Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar). However, other studies observed intact insulin-stimulated GLUT4 translocation when the interaction between insulin receptor and IRS was blocked, although insulin-mediated DNA synthesis and cell growth were reduced (17Rose D.W. Saltiel A.R. Majumdar M. Decker S.J. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 797-801Crossref PubMed Scopus (124) Google Scholar, 18Morris A.J. Martin S.S. Haruta T. Nelson J.G. Vollenweider P. Gustafson T.A. Mueckler M. Rose D.W. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8401-8406Crossref PubMed Scopus (60) Google Scholar, 19Sharma P.M. Egawa K. Gustafson T.A. Martin J.L. Olefsky J.M. Mol. Cell. Biol. 1997; 17: 7386-7397Crossref PubMed Scopus (65) Google Scholar). Furthermore, IRS-1-null mice, although significantly growth-retarded, do not develop overt diabetes, suggesting that IRS-1 plays a more important role in mediating the mitogenic rather than the metabolic effects of insulin (20Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (906) Google Scholar, 21Araki E. Lipes M.A. Patti M.E. Bruning J.C. Haag III, B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1099) Google Scholar). When the insulin response of individual tissues was examined, however, phosphatidylinositol 3-kinase activation and glucose uptake were reduced in muscle (22Yamauchi T. Tobe K. Tamemoto H. Ueki K. Kaburagi Y. Yamamoto-Honda R. Takahashi Y. Yoshizawa F. Aizawa S. Akanuma Y. Sonenberg N. Yazaki Y. Kadowaki T. Mol. Cell. Biol. 1996; 16: 3074-3084Crossref PubMed Scopus (250) Google Scholar) and adipocytes (23Kaburagi Y. Satoh S. Tamemoto H. Yamamoto-Honda R. Tobe K. Veki K. Yamauchi T. Kono-Sugita E. Sekihara H. Aizawa S. Cushman S.W. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1997; 272: 25839-25844Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), although the liver preserved normal phosphatidylinositol 3-kinase and MAPK activities (22Yamauchi T. Tobe K. Tamemoto H. Ueki K. Kaburagi Y. Yamamoto-Honda R. Takahashi Y. Yoshizawa F. Aizawa S. Akanuma Y. Sonenberg N. Yazaki Y. Kadowaki T. Mol. Cell. Biol. 1996; 16: 3074-3084Crossref PubMed Scopus (250) Google Scholar). These latter results suggested that IRS-1 participates in insulin-stimulated glucose uptake in muscle and adipocytes, whereas in liver, other IRS protein(s) is responsible for insulin action. IRS-2 was identified as the alternative insulin receptor substrate in the liver and muscle of the IRS-1-null mice (10Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (767) Google Scholar, 24Patti M.E. Sun X.J. Bruening J.C. Araki E. Lipes M.A. White M.F. Kahn C.R. J. Biol. Chem. 1995; 270: 24670-24673Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Overexpression of IRS-2 in rat adipocytes increased GLUT4 translocation (25Zhou L. Chen H. Lin C.H. Cong L.-N. McGibbon M.A. Sciacchitano S. Lesniak M.A. Quon M.J. Taylor S.I. J. Biol. Chem. 1997; 272: 29829-29833Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), whereas brown adipocytes isolated from IRS-2-null mice showed reduced insulin-stimulated glucose transport and GLUT4 translocation (26Fasshauer M. Klein J. Ueki K. Kriauciunas K.M. Benito M. White M.F. Kahn C.R. J. Biol. Chem. 2000; 275: 25494-25501Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Interestingly, when stimulated with insulin ex vivo, isolated soleus muscles from the IRS-2-null and wild type mice had similar glucose uptake response, albeit with a lower basal glucose transport rate, suggesting that IRS-2 may not be required for insulin-stimulated glucose transport in skeletal muscle (27Higaki Y. Wojtaszewski J.F. Hirshman M.F. Withers D.J. Towery H. White M.F. Goodyear L.J. J. Biol. Chem. 1999; 274: 20791-20795Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Of note, the total GLUT4 expression was decreased in the IRS-2-null mice, yet the insulin-stimulated glucose uptake response was not diminished. A later in vivo study using the hyperinsulinemic-euglycemic clamp, however, showed that although IRS-1 is the major regulator of glucose transport in muscle, IRS-2 also participates in glucose metabolism in muscle, fat, and liver (28Previs S.F. Withers D.J. Ren J.-M. White M.F. Shulman G.I. J. Biol. Chem. 2000; 275: 38990-38994Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). These studies are summarized in Table I. Taken together, studies to date support the requirement of IRS-1 in insulin-stimulated glucose metabolism in muscle, whereas the precise role of IRS-2 in this tissue requires further investigation.Table IRelationship between IRS protein expression level and insulin-stimulated glucose uptake and GLUT4 translocation in muscle and adipose tissuesGlucose uptake or GLUT4 translocationTissueIRS-1IRS-2Adipocytes overexpressing wild type IRS↑ (16Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar)↑ (25Zhou L. Chen H. Lin C.H. Cong L.-N. McGibbon M.A. Sciacchitano S. Lesniak M.A. Quon M.J. Taylor S.I. J. Biol. Chem. 1997; 272: 29829-29833Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar)Adipocytes/fibroblasts expressing inactiveaExpression or activity of IRS-1 was modulated via expression of antisense ribozyme (16) or an antibody or peptide interfering with insulin receptor-IRS-1 interaction (17-19) IRS↓ (16Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar) ↔ (17Rose D.W. Saltiel A.R. Majumdar M. Decker S.J. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 797-801Crossref PubMed Scopus (124) Google Scholar, 18Morris A.J. Martin S.S. Haruta T. Nelson J.G. Vollenweider P. Gustafson T.A. Mueckler M. Rose D.W. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8401-8406Crossref PubMed Scopus (60) Google Scholar, 19Sharma P.M. Egawa K. Gustafson T.A. Martin J.L. Olefsky J.M. Mol. Cell. Biol. 1997; 17: 7386-7397Crossref PubMed Scopus (65) Google Scholar)Muscle from IRS knock-out mice↓ (22Yamauchi T. Tobe K. Tamemoto H. Ueki K. Kaburagi Y. Yamamoto-Honda R. Takahashi Y. Yoshizawa F. Aizawa S. Akanuma Y. Sonenberg N. Yazaki Y. Kadowaki T. Mol. Cell. Biol. 1996; 16: 3074-3084Crossref PubMed Scopus (250) Google Scholar)↔ (27Higaki Y. Wojtaszewski J.F. Hirshman M.F. Withers D.J. Towery H. White M.F. Goodyear L.J. J. Biol. Chem. 1999; 274: 20791-20795Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) ↓ bAs determined by hyperinsulinemic euglycemic clamp study (28Previs S.F. Withers D.J. Ren J.-M. White M.F. Shulman G.I. J. Biol. Chem. 2000; 275: 38990-38994Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar)Adipocyte from IRS knock-out mice↓ (23Kaburagi Y. Satoh S. Tamemoto H. Yamamoto-Honda R. Tobe K. Veki K. Yamauchi T. Kono-Sugita E. Sekihara H. Aizawa S. Cushman S.W. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1997; 272: 25839-25844Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar)↓ (26Fasshauer M. Klein J. Ueki K. Kriauciunas K.M. Benito M. White M.F. Kahn C.R. J. Biol. Chem. 2000; 275: 25494-25501Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar)a Expression or activity of IRS-1 was modulated via expression of antisense ribozyme (16Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar) or an antibody or peptide interfering with insulin receptor-IRS-1 interaction (17Rose D.W. Saltiel A.R. Majumdar M. Decker S.J. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 797-801Crossref PubMed Scopus (124) Google Scholar, 18Morris A.J. Martin S.S. Haruta T. Nelson J.G. Vollenweider P. Gustafson T.A. Mueckler M. Rose D.W. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8401-8406Crossref PubMed Scopus (60) Google Scholar, 19Sharma P.M. Egawa K. Gustafson T.A. Martin J.L. Olefsky J.M. Mol. Cell. Biol. 1997; 17: 7386-7397Crossref PubMed Scopus (65) Google Scholar)b As determined by hyperinsulinemic euglycemic clamp study Open table in a new tab Insulin elicits both mitogenic and metabolic responses, which include activation of the MAPK pathway and gene expression, as well as increasing Akt activity and inducing actin remodeling. The latter two participate in the regulation of insulin-stimulated GLUT4 translocation and glucose uptake (29Tsakiridis T. Vranic M. Klip A. J. Biol. Chem. 1994; 269: 29934-29942Abstract Full Text PDF PubMed Google Scholar, 30Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (504) Google Scholar, 31Tong P. Khayat Z.A. Huang C. Patel N. Ueyama A. Klip A. J. Clin. Invest. 2001; 108: 371-381Crossref PubMed Scopus (165) Google Scholar, 32Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7569-7574Crossref PubMed Scopus (312) Google Scholar, 33Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The objective of this study was to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using siRNA-mediated specific gene silencing. The transient nature of siRNA-mediated protein reduction minimizes the possibility of compensatory up-regulation of other proteins that may mask the biological consequences of eliminating IRS-1/2. In addition, we made use of the L6-GLUT4myc rat skeletal muscle cell line that stably expresses a myc-tagged GLUT4, where the expression of the GLUT4myc is constant and resistant to manipulations that may affect expression of other proteins (34Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (122) Google Scholar). This allows us to determine the importance of IRS-1/2 in the regulation of glucose uptake and GLUT4 translocation without the confounding factor of altered GLUT4 expression (34Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (122) Google Scholar, 35Wang Q. Khayat Z. Kishi K. Ebina Y. Klip A. FEBS Lett. 1998; 427: 193-197Crossref PubMed Scopus (188) Google Scholar, 36Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar), as was observed in the skeletal muscle of IRS-2-null mice. Materials—Phospho-specific antibodies to p38MAPK (dual phosphorylated Thr180 and Tyr182), Akt (Thr308 and Ser473), and ERK were purchased from Cell Signaling (Beverly, MA). Polyclonal anti-phosphotyrosine, anti-IRS-1, anti-IRS-2, and anti-Rat-Akt2 (immunoaffinity-purified sheep IgG) antibodies (for immunoprecipitation, immunoblotting, and immunofluorescence experiments) were from Upstate Biotechnology (Lake Placid, NY). Dr. Morris White generously provided rabbit anti-IRS-1 and anti-IRS-2 antibodies used for immunoprecipitation. Polyclonal anti-myc (A14) and anti-Akt1 (D17-goat IgG) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SiRNAs targeted against IRS-1 (siIRS-1), IRS-2 (siIRS-2), and nonrelevant control (siNR) were purchased from Dharmacon (Lafayette, CO). The sequence of the IRS-1 siRNA used was AAC AAG ACA GCU GGU ACC AGG, and the nonrelevant control X was AUU CUA UCA CUA GCG UGA CUU. The nonrelevant control siRNA sequence was screened against the expressed sequence tag gene bank data base and had no homology to the vertebrate genome. IRS-2 Smartpool® siRNA was designed by and purchased from Dharmacon. Fetal bovine serum (FBS) and calf serum were obtained from Invitrogen. Human insulin (Humulin R) was purchased from Eli Lilly Canada, Inc. (Toronto, ON). Cytochalasin B, mercuric chloride, d-glucose, 2-deoxy-d-glucose, all protease inhibitors, and o-phenylenediamine dihydrochloride (OPD reagent) were from Sigma. 2-Deoxy-d-[1,2-3H]glucose was purchased from PerkinElmer Life Sciences. Tissue Culture—L6 muscle cells stably expressing myc-tagged GLUT4 (L6-GLUT4myc cells) were described previously (34Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (122) Google Scholar, 36Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar, 37Kanai F. Nishioka Y. Hayashi H. Kamohara S. Todaka M. Ebina Y. J. Biol. Chem. 1993; 268: 14523-14526Abstract Full Text PDF PubMed Google Scholar, 38Li D. Randhawa V.K. Patel N. Hayashi M. Klip A. J. Biol. Chem. 2001; 276: 22883-22891Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). GLUT4myc myoblasts were differentiated into myotubes in α-MEM supplemented with 2% FBS, 1% antibiotics/antimycotics over 7 days. Myotube cultures contained less than 20% mononucleated cells. Cultures were deprived of serum for 5 h prior to stimulation with insulin. Transfection of siRNA—L6 myotubes were transfected with siIRS-1 or siNR using Oligofectamine Reagent from Invitrogen as per the manufacturer's instruction, with minor modifications. The differentiation medium was changed to antibiotic-free growth medium on day 3 of myotube differentiation, and on day 4, 400 nm siRNA was transfected using Oligofectamine in serum-free α-MEM. 4 h after transfection, α-MEM containing 6% FBS was added to each well to a final concentration of 2% FBS (v/v). The siRNA duplex was never removed from the medium when using the Oligofectamine transfection protocol. In some experiments, siRNA to IRS-1 was also introduced to cells using the calcium phosphate protocol required for effective IRS-2 gene silencing as described below, and equivalent IRS-1 knock-downs were achieved with either Oligofectamine or calcium phosphate-based transfection reagent, as detailed under "Results." L6 myoblasts were transfected with siIRS-2 using a calcium phosphate-based transfection reagent (CellPhect transfection kit, Amersham Biosciences), according to the manufacturer's instruction. Briefly, cells were seeded at 30% confluence in α-MEM supplemented with 10% FBS without antibiotics/antimycotics. On day 2, myoblasts were transfected with 50 nm siRNA, and the medium was changed 12–14 h later to α-MEM supplemented with 2% FBS with 1% antibiotics/antimycotics. Cells were transfected again on day 5 with 100 nm siRNA. SiRNA transfection had no deleterious effects on either cell viability or fusion of myoblasts to form myotubes. Double elimination of IRS-1 and IRS-2 was achieved using the calcium phosphate transfection protocol described above. Fluorescence Microscopy—L6-GLUT4myc myotubes grown on 25-mm-diameter glass coverslips were deprived of serum for 5 h and treated with 0–100 nm insulin for 10 min at 37 °C. Myotubes were fixed with paraformaldehyde immediately at 4 °C after insulin stimulation and permeabilized in 0.1% (v/v) Triton X-100 for 3 min to preserve actin morphology. Actin filaments were labeled with rhodamine-coupled phalloidin, and immunostaining of IRS-1 or IRS-2 in fixed and permeabilized myotubes was carried out as described previously (39Patel N. Rudich A. Khayat Z.A. Garg R. Klip A. Mol. Cell. Biol. 2003; 23: 4611-4626Crossref PubMed Scopus (63) Google Scholar). Primary antibody (IRS-1 and IRS-2 purchased from Upstate Biotechnology) was used at 1:500 dilution. Cells were examined with a Zeiss LSM 510 laser scanning confocal microscope. Acquisition parameters were adjusted to exclude saturation of the pixels. For quantification, such parameters were kept constant among the various conditions compared. Determination of 2-Deoxyglucose Uptake—2-Deoxyglucose uptake measurements were carried out as described previously (36Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar) for 5 min in HEPES-buffered saline containing 10 μm 2-[3H]deoxyglucose (0.5 μCi/ml) in the absence of insulin. Glucose transport was stopped with ice-cold 0.9% NaCl containing 1 mm HgCl2. Nonspecific uptake was determined in the presence of 10 μm cytochalasin B and subtracted from all experimental values. Immunoprecipitation of IRS-1 and IRS-2—L6 myotubes were stimulated with insulin for 5 min (for IRS-1 studies) or 3 min (for IRS-2 studies), times at which phosphorylation of each isoform peaks (13Ogihara T. Shin B.C. Anai M. Katagiri H. Inukai K. Funaki M. Fukushima Y. Ishihara H. Takata K. Kikuchi M. Yazaki Y. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 12868-12873Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). IRS-1 and IRS-2 were immunoprecipitated from L6 myotubes as described previously (34Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (122) Google Scholar, 40Somwar R. Sweeney G. Ramlal T. Klip A. Clin. Ther. 1998; 20: 125-140Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, 500 μg or 1 mg of protein of whole cell lysates in Triton X-100 were incubated with IRS-1- or IRS-2-specific antibodies at 4 °C overnight under constant rotation, and the antibody-antigen complex was pulled down by protein A/G-Sepharose beads. The pelleted protein was resolved by 7.5% SDS-PAGE, and tyrosine phosphorylation of IRS-1 or IRS-2 was detected by immunoblotting with monoclonal anti-phosphotyrosine antibody. Immunoprecipitation of Akt1 and Akt2—L6 myotubes were stimulated with 100 nm insulin for 10 min. We have determined previously that 10 min of stimulation yields maximal phosphorylation of Akt in these cells (44Niu W. Huang C. Nawaz Z. Levy M. Somwar R. Li D. Bilan P.J. Klip A. J. Biol. Chem. 2003; 278: 17953-17962Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Immunoprecipitation of Akt1 and Akt2 was performed from 200 μg of cell lysates in Triton X-100 containing phosphatase and protease inhibitors (44Niu W. Huang C. Nawaz Z. Levy M. Somwar R. Li D. Bilan P.J. Klip A. J. Biol. Chem. 2003; 278: 17953-17962Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The antibody-antigen complex was pulled down by protein A/G-Sepharose beads. The pelleted proteins were resolved by 10% SDS-PAGE, and serine and threonine phosphorylation of Akt1 or Akt2 was detected by immunoblotting using anti-phospho-Akt-Ser473 or -Thr308 antibody. Reverse Transcription-PCR of Insulin Receptor Isoforms—Isolation of total RNA and reverse transcription-PCR of insulin receptor from L6 myotubes were performed as described previously (41JeBailey L. Rudich A. Huang X. Di Ciano-Oliveira C. Kapus A. Klip A. Mol. Endocrinol. 2004; 18: 359-372Crossref PubMed Scopus (133) Google Scholar). The primers used to amplify insulin receptor were: CAT TCA GGA AGA CCT TCG AGG and TCT GGG GAG TCC TGA TTG CAT. Measurement of GLUT4myc Translocation in L6 Myotubes—Cell surface myc-tagged GLUT4 was quantified by an antibody-coupled colorimetric assay as validated previously (35Wang Q. Khayat Z. Kishi K. Ebina Y. Klip A. FEBS Lett. 1998; 427: 193-197Crossref PubMed Scopus (188) Google Scholar). Briefly, after a 20-min incubation with insulin at indicated concentrations, confluent myotubes were exposed to anti-myc antibody (1:100) for 60 min, fixed with 4% paraformaldehyde for 10 min, and then incubated with peroxidase-conjugated goat anti-rabbit IgG (1:750) for 45 min, all at 4 °C. Cells were washed six times with phosphate-buffered saline, and 1 ml of OPD reagent was added for 30 min at room temperature. The reaction was stopped with 0.25 ml of 3 n HCl. The supernatant was collected, and absorbance was measured at 492 nm. Nonspecific IgG binding, as measured by a peroxidase-conjugated anti-rabbit IgG and omitting the primary antibody, was subtracted from all experimental values. Detection of Cellular Protein Expression and Protein Phosphorylation—Whole cell lysates were made from myotubes after incubation with insulin (0–100 nm) for 10 min (as described previously (42Sweeney G. Somwar R. Ramlal T. Volchuk A. Ueyama A. Klip A. J. Biol. Chem. 1999; 274: 10071-10078Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar)). We have determined previously that 10 min of stimulation yields maximal phosphorylation of Akt and p38MAPK and is also the time shown previously to yield robust ERK phosphorylation in these cells (43Pirola L. Bonnafous S. Johnston A.M. Chaussade C. Portis F. Van Obberghen E. J. Biol. Chem. 2003; 278: 15641-15651Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). 40 μg of lysates was resolved by SDS-PAGE and transferred onto polyvinylidene difluoride filters, which were incubated with primary antibodies overnight at 4 °C under constant agitation (dilutions: polyclonal anti-IRS-1 or anti-IRS-2, 1:1,000; anti-Akt, anti-ERK, and anti-p38MAPK, 1:500–1:1,000; anti-phospho-Akt-Ser473 or -Thr308, antiphospho-ERK, and anti-phospho-p38MAPK, 1:500), followed by a 1-h incubation with horseradish peroxidase-conjugated secondary antibody (1:15,000 goat anti-rabbit antibody, 1:7,500 sheep anti-mouse antibody). Protein was visualized by the enhanced chemiluminescence method and scanned within the linear range using ImageJ software. Insulin-stimulated Tyrosine Phosphorylation of IRS-2 Is More Insulin-sensitive Than That of IRS-1—We first compared the insulin sensitivity of IRS-1 tyrosine phosphorylation with that of IRS-2. L6-GLUT4myc myotubes were incubated with increasing concentrations of insulin (0.5–100 nm) for 3–5 min, and the results were expressed as a percentage of the response observed at 100 nm insulin. As shown in Fig. 1, insulin-stimulated tyrosine phosphorylation of IRS-2 is more sensitive at low concentrations of insulin than is IRS-1, with an EC50 of 0.5 nm for IRS-2 versus 5–8 nm for IRS-1. A similar difference of insulin sensitivity of IRS-1 and IRS-2 tyrosine phosphorylation was observed in myoblasts (results not shown). Hence, any possible presence of myoblasts in the cultures would be unlikely to be responsible for the marked difference in insulin-sensitivity of IRS-1 and IRS-2. Akt Phosphorylation Is More Insulin-sensitive Than Phosphorylation of p38MAPK or ERK—Akt, p38MAPK, and ERK have been shown to regulate GLUT4 translocation, glucose uptake, and mitogenic responses, respectively. However, with the exception of Akt, it
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