Maturation of the Regulation of GLUT4 Activity by p38 MAPK during L6 Cell Myogenesis
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m211136200
ISSN1083-351X
AutoresWenyan Niu, Carol Huang, Zafar Nawaz, Michelle Levy, Romel Somwar, Dailin Li, Philip J. Bilan, Amira Klip,
Tópico(s)Adipose Tissue and Metabolism
ResumoInsulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin. Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin. glucose transporter 4 mitogen-activated protein kinase fetal bovine serum Triton X-100 MAPK kinase activating transcription factor 2 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(d-mannose-4-xyloxy)-2 propylamine Insulin is the major regulator of blood glucose levels in the fed state when skeletal muscle becomes the primary consumer of glucose (1Baron A.D. 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These methods have arrived at variable conclusions regarding how closely GLUT4 translocation matches the stimulation of glucose uptake by insulin (5Klip A. Ramlal T. Young D.A. Holloszy J.O. FEBS Lett. 1987; 224: 224-230Crossref PubMed Scopus (281) Google Scholar, 6Gumà A. Zierath J.R. Wallberg-Henriksson H. Klip A. Am. J. Physiol. 1995; 268: E613-E622PubMed Google Scholar, 11Kahn B.B. Simpson I.A. Cushman S.W. J. Clin. Invest. 1988; 82: 691-699Crossref PubMed Scopus (52) Google Scholar, 12King P.A. Horton E.D. Hirshman M.F. Horton E.S. J. Clin. Invest. 1992; 90: 1568-1575Crossref PubMed Scopus (159) Google Scholar, 13Goodyear L.J. Hirshman M.F. Smith R.J. Horton E.S. Am. J. Physiol. 1991; 261: E556-E561PubMed Google Scholar, 14Marette A. Richardson J.M. Ramlal T. Balon T.W. Vranic M. Pessin J.E. Klip A. Am. J. Physiol. 1992; 263: C443-C452Crossref PubMed Google Scholar, 15Ferrara C.M. Cushman S.W. Biochem. J. 1999; 343: 571-577Crossref PubMed Scopus (30) Google Scholar, 16Shimizu Y. 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Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (148) Google Scholar)). GLUT4myc can be readily detected at the cell surface of intact cells by an enzyme-linked immunosorbent-like assay (21Wang Q. Khayat Z. Kishi K. Ebina Y. Klip A. FEBS Lett. 1998; 427: 193-197Crossref PubMed Scopus (187) Google Scholar). In these cells, GLUT4myc shows insulin-regulated behavior consistent with that of GLUT4 in 3T3-L1 adipocytes (22Clark A.E. Holman G.D. Kozka I.J. Biochem. J. 1991; 278: 235-241Crossref PubMed Scopus (49) Google Scholar, 23Yang J. Holman G.D. J. Biol. Chem. 1993; 268: 4600-4603Abstract Full Text PDF PubMed Google Scholar). Thus, 90% of GLUT4myc is sequestered intracellularly in the basal state, and a significant portion translocates to the cell surface in response to insulin (24Li 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). All GLUT4myc molecules are available for recycling to the cell surface (25Foster L.J. Li D. Randhawa V.K. Klip A. J. Biol. Chem. 2001; 276: 44212-44221Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The exocytic and endocytic rates of GLUT4myc (24Li 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) mimic those reported for GLUT4 (26Satoh S. Gonzalez-Mulero O.M. Clark A.E. Kozka I.J. Holman G.D. Cushman S.W. Diabetes. 1991; 40: 85AGoogle Scholar, 27Satoh S. Nishimura H. Clark A.E. Kozka I.J. Vannucci S.J. Simpson I.A. Quon M.J. Cushman S.W. Holman G.D. J. Biol. Chem. 1993; 268: 17820-17829Abstract Full Text PDF PubMed Google Scholar). The Km of glucose uptake is similar in L6GLUT4myc to that in the parental L6 cells (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar,29Klip A. Li G. Logan W.J. Am. J. Physiol. 1984; 247: E494-E499Google Scholar). Most significantly, the 10% of total GLUT4myc present at the cell surface is still higher than the endogenous levels of GLUT1 or GLUT3 (by almost 100-fold (30Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (121) Google Scholar)), and is responsible for both basal and insulin-stimulated glucose uptake. This functional preponderance was established by the nearly complete inhibition of both basal and insulin-stimulated glucose uptake rates by the drug indinavir (30Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (121) Google Scholar, 31Rudich A. Konrad D. Torok D. Ben-Romano R. Huang C. Niu W. Garg R.R. Wijesekara N. Germinario R.J. Bilan P.J. Kilp A. Diabetologia. 2003; (in press)Google Scholar), a rather selective inhibitor of glucose influx through GLUT4 but not GLUTs 1, 3, and 8 (32Murata H. Hruz P.W. Mueckler M. AIDS. 2002; 16: 859-863Crossref PubMed Scopus (202) Google Scholar, 33Hruz P.W. Murata H. Qiu H. Mueckler M. Diabetes. 2002; 51: 937-942Crossref PubMed Scopus (86) Google Scholar). Hence, L6-GLUT4myc cells are uniquely suitable to make direct comparisons between glucose uptake through GLUT4 and GLUT4 translocation. The molar expression of GLUT4myc in L6-GLUT4myc cells is 5–10 fold higher than that of endogenous GLUT4 in skeletal muscle (30Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (121) Google Scholar). Using these cells we have observed that insulin-dependent GLUT4 translocation and stimulation of glucose uptake can be segregated in time (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar), by their temperature sensitivity (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar), their susceptibility to inhibition by wortmannin (34Somwar R. Niu W. Kim D.Y. Sweeney G. Ramlal T. Klip A. J. Biol. Chem. 2001; 276: 46079-46087Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and most strikingly, by specific inhibitors of p38 MAPK. The latter (pyridinylimidazoles SB203580 and SB202190 and chemically distinct aza-azulenes A291077 and A304000) reduced the insulin response of glucose uptake without interfering with GLUT4 translocation in L6-GLUT4myc myotubes and 3T3-L1 adipocytes without directly inhibiting glucose transporters (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar, 35Sweeney 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 (273) Google Scholar, 36Somwar R. Koterski S. Sweeney G. Sciotti R. Djuric S. Berg C. Trevillyan J.M. Scherer P.E. Rondinone C.M. Klip A. J. Biol. Chem. 2002; 277: 50386-50395Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). L6-GLUT4myc muscle cells undergo differentiation from myoblasts to multinucleated myotubes through multiple cell fusions (37Yaffe D. Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 477-483Crossref PubMed Scopus (799) Google Scholar, 38Klip A. Logan W.J. Li G. Biochim. Biophys. Acta. 1982; 687: 265-280Crossref PubMed Scopus (77) Google Scholar). In search for information on the mechanisms responsible for the segregation of GLUT4 translocation and glucose uptake in myotubes, we examined the maturation of the insulin response of GLUT4 translocation and glucose uptake during myogenesis. We report that, in the myoblast stage, insulin-stimulated glucose uptake is lower than in myotubes and is not sensitive to inhibition of p38 MAPK. Yet, translocation of GLUT4myc is similar in both stages of cellular differentiation. Moreover, insulin stimulates p38 MAPK and its upstream activators MKK3/6 in myotubes but slightly if at all in myoblasts. These results further correlate the p38 MAPK pathway to GLUT4 activation, because both events mature during L6 cell differentiation from myoblasts into myotubes. SB203580 was purchased from Calbiochem (La Jolla, CA). Cytochalasin B, O-phenylenediamine dihydrochloride, and 2-deoxyglucose were obtained from Sigma (St. Louis, MO). 2-Deoxy-d-[3H]glucose was purchased from ICN (Irvine, CA). Monoclonal anti-myc (9E10) antibody and antibodies to p38 α (C-20), p38 β MAPK (K-16), Akt1, and Akt2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to Akt3 and Crosstide (Akt peptide substrate) were purchased from Upstate Biotechnology (Lake Placid, NY). ATF2 recombinant protein, specific antibodies to phosphorylated p38 MAPK (Thr-180 and Tyr-182), phosphorylated MKK3/6 (Ser189/207) and phospho-Akt (S473), and anti-pan p38 MAPK antibodies were purchased from Cell Signaling (Beverly, MA). All other reagents were purchased at reagent grade quality. For selective experiments, wild-type L6 myoblasts grown and differentiated into myotubes as previously reported (39Tsakiridis T. Vranic M. Klip A. J. Biol. Chem. 1994; 269: 29934-29942Abstract Full Text PDF PubMed Google Scholar) were used where indicated. Otherwise, L6 myoblasts stably expressing GLUT4myc, created (20Kishi K. Muromoto N. Nakaya Y. Miyata I. Hagi A. Hayashi H. Ebina Y. Diabetes. 1998; 47: 550-558Crossref PubMed Scopus (148) Google Scholar) and characterized (40Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (501) Google Scholar, 41Ueyama A. Yaworsky K.L. Wang Q. Ebina Y. Klip A. Am. J. Physiol. 1999; 277: E572-E578Crossref PubMed Google Scholar) as described previously, were used throughout the study. L6-GLUT4myc cells were maintained in minimal essential medium-α supplemented with 10% FBS in a humidified atmosphere of air and 5% CO2 at 37 °C. For experiments with myoblasts only, L6 cells were seeded in medium containing 10% FBS and used at confluence, 2 days after seeding. L6 cells were differentiated in medium supplemented with 2% FBS into myotubes within 7 days after seeding. Cells were serum-depleted for 3–4.5 h prior to all experimental manipulations. Inhibitors were administered in Me2SO, and the maximum concentration of the vehicle did not exceed 0.05% (v/v). This concentration of vehicle was without effect on any of the parameters measured. Following serum depletion, cells were treated with 10 μm SB203580 for 20 min before the addition of insulin at the indicated concentrations and times as described in the figure legends. Following treatments, cells were rinsed and immediately used for measurement of 2-deoxyglucose uptake in the absence of inhibitors as described previously (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar). For the insulin-stimulated time-course analysis, cells were grown in six-well plates and treated as indicated, and then 2-deoxyglucose uptake was measured for 30 s. Nonspecific uptake was determined in the presence of 10 μm cytochalasin B, and this value was subtracted from all other values. Cell-associated radioactivity was determined by lysing the cells with 0.05 n NaOH, followed by liquid scintillation counting. Total cellular protein was determined by the Bradford method. GLUT4myc levels at the cell surface of intact myoblasts or myotubes were measured by an antibody-coupled colorimetric assay as described (24Li 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) using the anti-myc monoclonal 9E10 as the primary antibody and a donkey anti-mouse IgG conjugated to horseradish peroxidase as the secondary antibody. Briefly, cells in six-well plates were incubated as indicated, lysed on ice with 300 μl of 2× Laemmli sample buffer per well supplemented with 7.5% β-mercaptoethanol (v/v), protease, and phosphatase inhibitors (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar). Lysates were passed 5 times through a 27-gauge syringe and heated for 15 min at 65 °C. 50-μg aliquots of total protein were resolved by 10% SDS-PAGE to detect phosphorylation of p38 MAPK, MKK3/6, or Akt by immunoblotting using the corresponding phospho-specific antibodies at 1:500 dilutions. Anti-p38 α and anti-p38 β MAPK antibodies were used at 1:1000 and 1:200 dilutions, respectively. Goat anti-rabbit IgG conjugated to horseradish peroxidase was used as secondary antibody at a 1:7000 dilution. Proteins were detected by the Enhanced Chemiluminescence method according to the manufacturer's instructions (PerkinElmer Life Sciences, Boston, MA). Immunoblots were exposed to x-ray film to produce bands within the linear range, then quantified using National Institutes of Health (NIH) Image software. Immunoprecipitation of p38 MAPKα or β from 500 μg (total protein) of TX-100 detergent cell lysates containing phosphatase and protease inhibitors was performed overnight at 4 °C was performed as described (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar). Protein concentration of the lysates was determined by the bicinchoninic acid method. p38 MAPK immunocomplexes were incubated for 30 min at 30 °C in 50 μl of kinase buffer supplemented with 2 μg of ATF2 recombinant protein and 200 μm ATP per condition. Reactions were continuously mixed on a platform shaker and were stopped by the addition of 25 μl of 2× Laemmli sample buffer and heating for 30 min at 65 °C. Samples were sedimented (12,000× g), and then 50 μl of the supernatant was resolved by 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes to detect phosphorylation of ATF2, using phospho-specific ATF-2 antibody. Immunoprecipitation of Akt1, Akt2, or Akt3 from 200 μg (total protein) of TX-100 detergent cell lysates containing phosphatase and protease inhibitors overnight at 4 °C was performed as described (40Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (501) Google Scholar). Akt immunocomplexes were incubated in 30 μl of kinase buffer supplemented with Crosstide (150 μg/condition), 5 μmATP, and 2 μCi of [γ-32P]ATP per condition. Reactions were sedimented by centrifugation for 30 s. 25 μl of the supernatants from each reaction were transferred to a Whatman P81 phosphocellulose filters and washed four times for 5 min each with 1% phosphoric acid (v/v) and once with double-distilled water. Filters were allowed to air dry before liquid scintillation counting (40Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (501) Google Scholar). Statistical analysis was performed using either unpaired Student's t test or analysis of variance test (Fischer, multiple comparisons) as indicated in the figure legends. L6 myoblasts and myotubes were treated in parallel with 100 nm insulin for 2–15 min at 37 °C, followed by determination of 2-deoxyglucose uptake or GLUT4myc translocation. In these experiments, insulin continued to be present during the 30-s uptake of [3H]2-deoxyglucose (see “Experimental Procedures”). In this way, the time courses of insulin-stimulated GLUT4myc translocation and glucose uptake could be effectively compared at each time of insulin incubation. In myoblasts, the maximal -fold stimulation of glucose uptake by insulin (15-min time point) was lower than the -fold stimulation in GLUT4myc translocation (1.6 ± 0.1-fold compared with 2.4 ± 0.1, respectively, Fig.1A). On the other hand, in myotubes insulin-stimulated both responses more than 2-fold by 15 min (2.2 ± 0.1-fold for glucose uptake compared with 2.4 ± 0.1, for translocation, Fig. 1B). Interestingly, the magnitude of the GLUT4myc translocation response was comparable in myoblasts and myotubes, but the full response of insulin-stimulated glucose uptake was not realized in the myoblasts. In myoblasts, there was no difference in the rate of stimulation of glucose uptake by insulin and GLUT4myc translocation at the early times of insulin addition. The estimated t1/2 of glucose uptake was 3.3 min and that of the arrival of GLUT4myc at the plasma membrane also was 3.3 min. In myotubes, GLUT4myc rapidly translocated to the plasma membrane with a t1/2 of 2.0 min. In contrast, the stimulation of glucose uptake in L6 myotubes was delayed by a lag of about 2 min before a significant response could be measured. Thus, maximal stimulation of glucose uptake is reached only between 10 and 15 min in the myotubes and is significantly delayed (t1/2 of 5 min) with respect to the response in myoblasts. L6-GLUT4myc myoblasts or myotubes were preincubated with the pyridinylimidazole SB203580 (10 μm) or Me2SO vehicle only for 20 min. This was followed by treatment with insulin (100 nm) for an additional 20 min in the presence of SB203580. Uptake of 2-deoxyglucose was then measured in the absence of the inhibitor. Insulin caused a significant increase in glucose uptake in myoblasts (1.7 ± 0.1-fold above basal,p < 0.001; Fig.2A) and myotubes (2.2 ± 0.2-fold above basal, p < 0.001, Fig. 2B). Preincubation with SB203580 did not have a significant effect on either basal or insulin-stimulated glucose uptake in myoblasts (Fig.2A). Similarly, preincubation with SB203580 did not affect the basal rate of glucose uptake in myotubes. However, it reduced the stimulation of glucose uptake by 65% (insulin: 2.2 ± 0.2-fold, insulin plus SB203580: 1.4 ± 0.1-fold,p < 0.001, Fig. 2B). It is unlikely that pyridinylimidazoles act by directly binding and inhibiting GLUT4myc, because both basal and insulin-stimulated rates of glucose uptake are mediated by GLUT4myc in these cells (30Huang C. Somwar R. Patel N. Niu W. Torok D. Klip A. Diabetes. 2002; 51: 2090-2098Crossref PubMed Scopus (121) Google Scholar, 31Rudich A. Konrad D. Torok D. Ben-Romano R. Huang C. Niu W. Garg R.R. Wijesekara N. Germinario R.J. Bilan P.J. Kilp A. Diabetologia. 2003; (in press)Google Scholar), but only the stimulated uptake was reduced by the drug. Moreover, SB203580 was not present during the transport assay. Furthermore, SB203580 did not inhibit glucose uptake when added only to the glucose transport solution in glucose uptake assays lasting up to 30 min (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar, 35Sweeney 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 (273) Google Scholar), confirming that the inhibition is due to an event different from direct inhibition of GLUT4. Nonetheless, to ascertain that the myc epitope is not related to the sensitivity to SB203580, the selective inhibition of insulin-stimulated glucose uptake by SB203580 in myotubes visà vis was confirmed in wild-type L6 muscle cells (TableI). This result conforms to our previous observation that SB203580 reduces the insulin-dependent portion of glucose uptake in wild-type L6 myotubes (35Sweeney 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 (273) Google Scholar) and further establishes that maturation into myotubes is required for this susceptibility to SB203580 to manifest. Hence, glucose uptake in L6-GLUT4myc cells obeys similar regulation as in parental, untransfected cells.Table IDifferential effect of SB203580 on insulin-stimulated glucose uptake in wild-type L6 myoblasts and myotubesMyoblastsMyotubesBasalInsulinBasalInsulinpmol/min/mg protein(−) SB20358012.46 ± 1.3619.91 ± 2.002.8 ± 0.375.60 ± 0.17(+) SB20358013.57 ± 2.2619.43 ± 2.513.0 ± 0.164.20 ± 0.201-ap ≤ 0.05 compared to the corresponding insulin-stimulated sample in the absence of SB203580.Wild-type L6 muscle cells at the stage of myoblasts or myotubes were pretreated without or with 10 μm SB203580 for 20 min, followed by treatment without or with 100 nm insulin for 20 min in the corresponding absence or presence of SB203580. Cultures were rinsed three times with HEPES-buffered saline solution without additions and uptake of 2-deoxyglucose was then determined in the absence of SB203580 or insulin. Results are in pmol/min/mg of protein.1-a p ≤ 0.05 compared to the corresponding insulin-stimulated sample in the absence of SB203580. Open table in a new tab Wild-type L6 muscle cells at the stage of myoblasts or myotubes were pretreated without or with 10 μm SB203580 for 20 min, followed by treatment without or with 100 nm insulin for 20 min in the corresponding absence or presence of SB203580. Cultures were rinsed three times with HEPES-buffered saline solution without additions and uptake of 2-deoxyglucose was then determined in the absence of SB203580 or insulin. Results are in pmol/min/mg of protein. Insulin increased GLUT4myc at the cell surface by 2.3 ± 0.2-fold in myoblasts and 2.5 ± 0.2-fold in myotubes (Fig. 2, Cand D). In contrast to the stimulation of glucose uptake, SB203580 did not affect GLUT4myc density at the cell surface of basal or insulin-stimulated myotubes (Fig. 2D). This suggests that pretreatment with SB203580 reduces the glucose transport activity of GLUT4myc molecules that had been fully inserted into the plasma membrane, consistent with previous observations (28Somwar R. Kim D.Y. Sweeney G. Huang C. Niu W. Lador C. Ramlal T. Klip A. Biochem. J. 2001; 359: 639-649Crossref PubMed Scopus (127) Google Scholar, 35Sweeney 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 (273) Google Scholar). As shown in Fig. 1 above, the time course and maximal response to insulin of glucose uptake in myotubes differs from that in myoblasts. The myotubes responded with an initial delay but then achieved a higher stimulation of glucose uptake than the myoblasts. Given that SB203580 lowered the maximal glucose uptake response in myotubes (see Fig. 2), we examined how inhibition of p38 MAPK may affect the time course of insulin-stimulated glucose uptake in myotubes. Preincubation of L6-GLUT4myc myotubes with SB203580 prior to determination of the insulin-stimulated time course of glucose uptake reduced the maximal stimulation of glucose uptake to the same levels as in insulin-stimulated myoblasts (Fig. 3). This observation is in keeping with the lack of inhibition of insulin action by SB203580 in myoblasts. However, the lag in stimulation was not prevented (Fig. 3). These tantalizing results raise the possibility that an event downstream of p38 MAPK is responsible for the maximal stimulation of glucose uptake and that other factors upstream or parallel to p38 MAPK contribute to the delay in stimulation of glucose uptake observed in myotubes relative to myoblasts (see “Discussion”). Activation of p38 MAPK by diverse stimuli leads to its phosphorylation by upstream kinases on tyrosine and threonine residues in the TGY motif of its regulatory doma
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