An Inhibitor of p38 Mitogen-activated Protein Kinase Prevents Insulin-stimulated Glucose Transport but Not Glucose Transporter Translocation in 3T3-L1 Adipocytes and L6 Myotubes
1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês
10.1074/jbc.274.15.10071
ISSN1083-351X
AutoresGary Sweeney, Romel Somwar, Toolsie Ramlal, Allen Volchuk, Atsunori Ueyama, Amira Klip,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 μm). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane. The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 μm). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane. phosphatidylinositol 3-kinase insulin receptor substrate-1 protein kinase B mitogen-activated protein kinase MAP kinase kinase activating transcription factor-2 interleukin-1 phosphate-buffered saline polyacrylamide gel electrophoresis phenylmethylsulfonyl fluoride The phenomenon of insulin-stimulated glucose transporter (GLUT) translocation from an intracellular location to the plasma membrane has been demonstrated in several fat and muscle cell systems since its initial report in 1980 (1Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 2Baldwin S.A. Barros L.F. Griffiths M. Biosci. Rep. 1995; 15: 419-426Crossref PubMed Scopus (23) Google Scholar, 3Bilan P.J. Mitsumoto Y. Ramlal T. Klip A. FEBS Lett. 1992; 298: 285-290Crossref PubMed Scopus (66) Google Scholar). Whether increased plasma membrane glucose transporter content can fully account for insulin-stimulated increases in glucose uptake is still being debated (4Zierler K. Diabetologia. 1998; 41: 724-730Crossref PubMed Scopus (23) Google Scholar). It has been proposed that translocation of GLUTs might only account for as little as 30% of insulin-stimulated glucose transport (4Zierler K. Diabetologia. 1998; 41: 724-730Crossref PubMed Scopus (23) Google Scholar), with the majority of insulin-stimulated glucose transport being due to changes in the intrinsic activity of GLUTs (5Moyers J.S. Bilan P.J. Reynet C. Kahn C.R. J. Biol. 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Biochem. 1995; 227: 597-611Crossref PubMed Scopus (130) Google Scholar). Three families of these proline-directed serine threonine protein kinases, which are activated by dual phosphorylation on threonine and tyrosine residues, have been identified (19Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Crossref PubMed Scopus (1662) Google Scholar). One of these is the p38 MAPK (also known as reactivating kinase, stress-activated protein kinase, and CSAID-binding protein) family, activation of which has been best characterized in response to stressors (such as UV light and hyperosmolarity) and cytokines (such as interleukin-1 or tumor necrosis factor-α) (20Paul A. Wilson S. Belham C.M. Robinson C.J. Scott P.H. Gould G.W. Plevin R. Cellular Signalling. 1997; 9: 403-410Crossref PubMed Scopus (285) Google Scholar). We have shown that insulin can phosphorylate p38 in L6 myotubes (21Taha C. Tsakiridis T. McCall A. Klip A. Am. J. Physiol. 1997; 273: E68-E76PubMed Google Scholar, 22Tsakiridis T. Taha C. Grinstein S. Klip A. J. Biol. Chem. 1996; 271: 19664-19667Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and insulin has been shown to stimulate p38 activity in hepatoma cells (23Sutherland C. Tebbey P.W. Granner D.K. Diabetes. 1997; 46: 17-22Crossref PubMed Scopus (37) Google Scholar), yet not in skeletal muscle (24Goodyear L.J. Chang P.Y. Sherwood D.J. Dufresne S.D. Moller D.E. Am. J. Physiol. 1996; 271: E403-E408PubMed Google Scholar). A group of pyridinyl imidazole compounds were recently reported to bind to and potently inhibit p38 MAP kinase. One of these, SB203580, specifically inhibited the α and β isoforms of p38 MAP kinase with an in vitro IC50 of ∼0.6 μm, while having no effect on the activity of 12 other closely related or prominent intracellular kinases (25Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1977) Google Scholar). In KB cells, SB203580 prevented the stimulation of glucose transport elicited by the potent activator of p38 MAP kinase anisomycin and by IL-1, but had no effect on insulin-like growth factor 1-stimulated glucose transport (26Gould G.W. Cuenda A. Thomson F.J. Cohen P. Biochem. J. 1995; 311: 735-738Crossref PubMed Scopus (91) Google Scholar). SB203580 also inhibited anisomycin-stimulated glucose transport in 3T3-L1 adipocytes (27Barros L.F. Young M. Saklatvala J. Baldwin S.A. J. Physiol. 1997; 504: 517-525Crossref PubMed Scopus (77) Google Scholar), with an IC50 of <1 μm. In the present study we assessed the effect of SB203580 and its inactive structural analogue, SB202474, on insulin-stimulated glucose transport. We report that SB203580 inhibits uptake of 2-deoxyglucose or 3-O-methylglucose in muscle and fat cells without interfering with translocation of GLUTs. This effect was not due to direct binding of SB203580 to GLUTs nor to a nonspecific effect on other signaling molecules. The results suggest that SB203580 attenuates the ability of insulin to stimulate the intrinsic activity of glucose transporter molecules recruited to the plasma membrane. All cell culture solutions and supplements were obtained from Life Technologies, Inc. (Burlington, ON, Canada). 3T3-L1 cells were a kind gift from Dr. G. Holman (University of Bath, United Kingdom). L6 cells transfected with Myc-tagged GLUT4 were kindly provided by Dr. Y. Ebina (University of Tokushima, Japan). Human insulin (Humulin) was obtained from Eli Lilly Canada Inc. (Toronto, ON, Canada). Protein A- and protein G-Sepharose were from Pharmacia (Uppsala, Sweden). Polyclonal anti-GLUT1 and anti-GLUT4 glucose transporter antiserum was from East Acres Laboratories (South Bridge, MA). Polyclonal antibodies to Akt1 (C-20) and p38 MAP kinase and monoclonal antibody to Myc were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Akt1 substrate peptide (Crosstide), monoclonal anti-phosphotyrosine, polyclonal anti-IRS-1, and Ak+2 and Ak+3 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). [γ-32P]ATP (6000 Ci/mmol) and enhanced chemiluminescence (ECL) reagents were purchased from Amersham (Oakville, ON, Canada). 2-d-Deoxy-[3H]glucose and 3-O-[3H]methylglucose were purchased from NEN (Mississauga, ON, Canada). ATF-2 peptide was from New England Biolabs (Mississauga, ON, Canada). Fluorescein isothiocyanate-conjugated donkey anti-rabbit and horseradish peroxidase-conjugated sheep anti-rabbit and anti-mouse antiserum were from Jackson Immunoresearch (Baltimore Pike, PA). ProLong antifade mounting solution was from Molecular Probes (Eugene, OR). Purifiedl-α-phosphatidylinositol (PI) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Oxalate-treated TLC Silica Gel H plates (250 μm) were from Analtech (Newark, DE).o-Phenylenediamine dihydrochloride (OPD reagent) was from Sigma. Okadaic acid was from Biomol (Plymouth Meeting, PA). SB203580 and SB202474 were from Calbiochem (La Jolla, CA). All electrophoresis and immunoblotting reagents were purchased from Bio-Rad (Mississauga, ON, Canada). All other reagents were of the highest analytical grade. 3T3-L1 cells were grown in monolayer culture in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) calf serum and 1% (v/v) antibiotic solution (10,000 units/ml penicillin and 10 mg/ml streptomycin) in an atmosphere of 5% CO2 at 37 °C. 3T3-L1 fibroblasts were differentiated into adipocytes as described previously (28Volchuk A. Wang Q. Ewart H.S. Liu Z. He L. Bennett M.K. Klip A. Mol. Biol. Cell. 1996; 7: 1075-1082Crossref PubMed Scopus (126) Google Scholar). L6 cells from a spontaneously fusing subclone of the original L6 muscle cells and L6 cells stably overexpressing GLUT4 tagged with a Myc epitope were grown and differentiated into myotubes as described previously (29Mitsumoto Y. Klip A. J. Biol. Chem. 1992; 267: 4957-4962Abstract Full Text PDF PubMed Google Scholar). For transport studies cells were treated with trypsin and seeded in 12-well plates (2.5-cm diameter well) and maintained in growth medium as described above except supplemented with 2% fetal bovine serum. Cells were maintained under the same conditions in 6-well plates for preparation of whole cell lysates and in 10-cm diameter dishes for immunoprecipitations. Cells treated with insulin or SB203580 as indicated were lysed in 1 ml of lysis buffer containing 137 mm NaCl, 20 mm Tris-HCl, 1 mm MgCl2, 1 mm CaCl2, 100 μm Na3VO4, 2 mmPMSF, 10% glycerol (v/v), and 1% Nonidet P-40 (v/v) (pH 7.5). Cell lysates were then centrifuged for 5 min at 12,000 rpm to remove cell debris and nuclei. Antiphosphotyrosine antibody (2 μg/condition) was then added to supernatants for overnight incubation under rotation at 4 °C, followed by addition of 30 μl (10% w/v) of protein A-Sepharose for 1 h. The immunoprecipitation pellets were washed three times with PBS containing 0.1% Nonidet P-40 and 100 μm Na3VO4, solubilized in 30 μl of 2 × Laemmli sample buffer, boiled for 5 min, and separated by 10% SDS-PAGE. Anti-p38 MAP kinase polyclonal antibody (1:1000 dilution) was added to the polyvinylidene difluoride membrane, followed by goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase (1:5000 dilution) and protein was visualized by the enhanced chemiluminescence method. Immunoprecipitation of p38 MAP kinase and Akt isoforms and analysis of their kinase activity was performed in a similar fashion to that described previously for Akt1 (30Somwar R. Sumitani S. Taha C. Sweeney G. Klip A. Am. J. Physiol. 1998; 275: E618-E625PubMed Google Scholar). For both assays, cells were lysed with lysis buffer containing 50 mm HEPES, pH 7.6, 150 mm NaCl, 10% glycerol (v/v), 1% Triton X-100 (v/v), 30 mm Na4P2O7, 10 mm sodium fluoride, 1 mm EDTA, 1 mm PMSF, 1 mm benzamidine, 1 mmNa3VO4, 1 mm dithiothreitol, and 100 nm okadaic acid. Polyclonal anti-p38 MAP kinase antibody (2 μg/condition) precoupled to a mixture of protein A- and protein G-Sepharose (20 μl (100 mg/ml) each per condition) beads was added to 200 μg of total protein from cell lysates. Anti-Akt1, -Akt2, and -Akt3 antibodies were also precoupled to a mixture of protein A- and protein G-Sepharose (2 μg/condition) and added to 200 μg of total protein. Antibody coupled beads were washed twice with ice-cold PBS and once with ice-cold lysis buffer before use. Proteins were immunoprecipitated by incubating with the antibody bead complex for 2–3 h under constant rotation (4 °C). Immunocomplexes were isolated and washed 4 times with 1 ml of wash buffer (25 mm HEPES, pH 7.8, 10% glycerol (v/v), 1% Triton X-100 (v/v), 0.1% bovine serum albumin, 1 m NaCl, 1 mm dithiothreitol, 1 mm PMSF, 1 μm microcystin, and 100 nm okadaic acid) and twice with 1 ml of kinase buffer (50 mm Tris/HCl, pH 7.5, 10 mm MgCl2, and 1 mm dithiothreitol). The complexes were then incubated under constant agitation for 30 min at 30 °C with 30 μl of reaction mixture (kinase buffer containing 5 μm ATP, 2 μCi of [γ-32P]ATP plus 2 μg of ATF-2 as substrate in p38 MAP kinase assays and 100 μm Crosstide as substrate in Akt assays). Following the reaction, 30 μl of the supernatant was transferred onto Whatman p81 filter paper and washed 4 times for 10 min with 3 ml of 175 mm phosphoric acid and once with distilled water for 5 min. Filters were air-dried and then subjected to liquid scintillation counting. L6 myotubes were deprived of serum for 5 h with α-minimal essential medium, 0.1% fetal bovine serum (v/v), and 25 mm glucose (serum-deprivation medium) while 3T3-L1 adipocytes were deprived of serum by incubation in Dulbecco's modified Eagle's medium for 2 h before experimental manipulations. 2-Deoxyglucose uptake measurements were carried out as described previously (31Somwar R. Sweeney G. Ramlal T. Klip A. Clin. Therap. 1998; 20: 125-140Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, following all stimulations and incubations with inhibitors cell monolayers were washed twice with HEPES-buffered saline (140 mm NaCl, 20 mm Na-HEPES, 2.5 mm MgSO4, 1 mm CaCl2, 5 mm KCl, pH 7.4) and any remaining liquid was aspirated. Cells were then incubated for 5 min in HEPES-buffered saline containing 10 μm unlabeled 2-deoxyglucose and 10 μmd-2-deoxy-[3H]glucose (1 μCi/ml for L6 and 0.5 μCi/ml for 3T3-L1) in the absence of insulin. The reaction was terminated by washing three times with ice-cold 0.9% NaCl (w/v). Nonspecific uptake was determined in the presence of 10 μm cytochalasin B. 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 (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar). 3-O-Methylglucose uptake was measured in a similar fashion with the following differences: 50 μm 3-O-methylglucose (4 μCi/ml) was added to HEPES-buffered saline and uptake allowed to occur for 30 s, a period over which 3-O-methylglucose uptake is known to be linear. After this time, cell monolayers were washed three times with 1 mm HgCl2 in saline solution before lysis with 0.05 n NaOH. Differentiated 3T3-L1 adipocytes, grown on glass coverslips in 6-well dishes, were treated with SB203580 plus or minus insulin as described in the figure legends. Plasma membrane lawns (sheets) were prepared as described previously (33Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar) with slight modifications. Following the various treatments, the cells were placed on ice and washed twice in ice-cold PBS. Hypotonic swelling buffer (23 mm KCl, 10 mm Na-HEPES, 2 mmMgCl2, 1 mm EGTA, pH 7.5) was added in three quick rinses. Five ml of breaking buffer (70 mm KCl, 30 mm Na-HEPES, 5 mm MgCl2, 3 mm EGTA, 1 mm dithiothreitol, 0.5 mm PMSF, 1 μm pepstatin A, pH 7.5) were added to each well, and the solution was aspirated up and down using a 1.0-ml pipette to promote cell breakage. The coverslips were washed three times in breaking buffer and incubated with cold 3% paraformaldehyde in breaking buffer for 10 min on ice, followed by three washes in PBS. Excess fixative was quenched with 50 mmNH4Cl/PBS for 5 min, followed by three washes with PBS at room temperature. The lawns were subsequently blocked by a 1-h incubation in 5% goat serum in PBS at room temperature, then incubated with rabbit anti-GLUT4 antiserum (1:150) for 30 min at room temperature and washed three times in PBS. Fluorescein isothiocyanate-conjugated donkey anti-rabbit antiserum (1:50) was added for 30 min then rinsed out with four washes with PBS and the coverslips mounted with ProLong Antifade mounting solution. Confocal images were obtained using a Leica TCS 4D laser confocal fluorescence microscope with a 63X objective. All images shown were collected under identical gain settings. Quantification was made using NIH Image software. Subcellular fractionation was carried out essentially as described previously (34Bashan N. Burdett E. Guma A. Sargeant R. Tumiati L. Liu Z. Klip A. Am. J. Physiol. 1993; 264: C430-C440Crossref PubMed Google Scholar). Cells were homogenized in ice-cold 255 mm sucrose, 0.5 mm PMSF, 1 μmpepstatin A, 10 μm E-64, 1 mm EDTA, and 20 mm Na-HEPES (pH 7.4) and the homogenate centrifuged at 19,000 × g for 20 min. The resulting supernatant was centrifuged at 41,000 × g to pellet the crude plasma membranes; the supernatant from this step was centrifuged at 180,000 × g for 75 min to yield the low density microsomes. Crude plasma membranes were further purified by layering on a sucrose cushion (1.12 m sucrose, 1 mm EDTA, and 20 mm Na-HEPES, pH 7.4) and centrifuged at 100,000 × g in a Beckman SW-55 rotor for 60 min. The band at the interface was resuspended in homogenization buffer and pelleted at 40,000 × g for 20 min to yield purified plasma membranes. All fractions were resuspended in homogenization buffer to a final concentration of 2–10 mg/ml and stored at −80 °C. The movement of Myc-tagged GLUT4 to the cell surface was measured by an antibody-coupled colorimetric assay (35Wang Q. Khayat Z. Kishi K. Ebina Y. Klip A. FEBS Lett. 1998; 427: 193-197Crossref PubMed Scopus (187) Google Scholar) as follows. Quiescent L6 GLUT4myc cells treated as indicated in the legend for Table II were washed once with PBS, fixed with 3% paraformaldehyde in PBS for 3 min at room temperature, and the fixative was immediately neutralized by incubation with 1% glycine in PBS at 4 °C for 10 min. The cells were blocked with 10% goat serum and 3% bovine serum albumin in PBS at 4 °C for at least 30 min. Primary antibody (anti-c-Myc, 9E10) was then added into the cultures at a dilution of 1:100 and maintained for 30 min at 4 °C. The cells were extensively washed with PBS before introducing peroxidase-conjugated rabbit anti-mouse IgG (1:1000). After 30 min at 4 °C, the cells were extensively washed and 1 ml of o-phenylenediamine dihydrochloride reagent (0.4 mg/ml o-phenylenediamine dihydrochloride and 0.4 mg/ml urea hydrogen peroxide in 0.05m phosphate/citrate buffer) was added to each well for 10 min at room temperature. The reaction was stopped by addition of 0.25 ml of 3 n HCl. The supernatant was collected and the optical absorbance was measured at 492 nm.Table IEffect of addition of SB203580 to assay buffer during measurement of 2-deoxy-d-glucose uptake2-Deoxyglucose uptake in 3T3-L1 adipocytes2-Deoxyglucose uptake in L6 muscle cellspmol/mg/minControl5.65 ± 0.543.36 ± 0.14SB2035805.38 ± 0.173.48 ± 0.05Insulin32.18 ± 2.786.31 ± 0.15Insulin + SB20358027.41 ± 1.426.18 ± 0.193T3-L1 adipocytes and L6 myotubes were treated with or without 100 nm insulin for 30 min prior to measurement of 2-deoxy-d-glucose uptake as described under "Experimental Procedures." 2-Deoxyglucose uptake was measured in the presence or absence of SB203580 (10 μm) during the 5-min assay period. Results shown are the mean ± S.E. of three replicates within one representative of three individual experiments. Each condition was assayed in triplicate. Open table in a new tab Table IIEffect of SB203580 on 2-deoxyglucose transport and GLUT4 translocation in L6 GLUT4myc myotubesEffect of SB203580 preincubation on 2-deoxyglucose UptakeEffect of SB203580 on GLUT4myc translocation to plasma membraneControl1.0 ± 0.031.0 ± 0.04SB2035800.91 ± 0.031.03 ± 0.01Insulin1.67 ± 0.052.04 ± 0.05Insulin + SB2035801.35 ± 0.092.08 ± 0.03L6 myotubes transfected with Myc-tagged GLUT4 were treated with or without 10 μm SB203580 for 20 min followed by 100 nm insulin for 30 min in the same medium, where indicated. Cells were rinsed then 2-deoxyglucose uptake or Myc epitope exposure at the cell surface were measured as described under "Experimental Procedures." Results are expressed relative to basal values and represent mean ± S.E. of three individual experiments within which each point was assayed in triplicate. Open table in a new tab 3T3-L1 adipocytes and L6 myotubes were treated with or without 100 nm insulin for 30 min prior to measurement of 2-deoxy-d-glucose uptake as described under "Experimental Procedures." 2-Deoxyglucose uptake was measured in the presence or absence of SB203580 (10 μm) during the 5-min assay period. Results shown are the mean ± S.E. of three replicates within one representative of three individual experiments. Each condition was assayed in triplicate. L6 myotubes transfected with Myc-tagged GLUT4 were treated with or without 10 μm SB203580 for 20 min followed by 100 nm insulin for 30 min in the same medium, where indicated. Cells were rinsed then 2-deoxyglucose uptake or Myc epitope exposure at the cell surface were measured as described under "Experimental Procedures." Results are expressed relative to basal values and represent mean ± S.E. of three individual experiments within which each point was assayed in triplicate. 3T3-L1 adipocytes were treated with 100 nminsulin for 5 min then IRS-1 was immunoprecipitated and tyrosine phosphorylation of IRS-1 and association of the p85 subunit of PI 3-kinase was determined essentially as described previously (31Somwar R. Sweeney G. Ramlal T. Klip A. Clin. Therap. 1998; 20: 125-140Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and then electrotransferred onto polyvinylidene difluoride membranes. To detect tyrosine-phosphorylated IRS-1 the upper part of the blot was probed with anti-phosphotyrosine antibody (monoclonal, 1:5000 dilution) and protein detected by the enhanced chemiluminescence method using sheep anti-mouse immunoglobulin conjugated to horseradish peroxidase as secondary antibody. The lower part of the blot was probed with anti-p85 antibody (polyclonal, 1:1000 dilution) and protein detected using anti-rabbit IgG conjugated to horseradish peroxidase. To determine PI 3-kinase activity, cell extracts were prepared exactly the same way as for IRS-1 immunoprecipitation and PI 3-kinase activity was measured on IRS-1 immunoprecipitates as described previously (36Tsakiridis T. McDowell H.E. Walker T. Downes C.P. Hundal H.S. Vranic M. Klip A. Endocrinology. 1995; 136: 4315-4322Crossref PubMed Google Scholar). Briefly, the ability of PI 3-kinase associated with IRS-1 to convert phosphatidylinositol to phosphatidylinositol 3-phosphate was detected by separation of these lipids by thin layer chromatography (TLC). Detection and quantitation of [32P]phosphatidylinositol 3-phosphate on the TLC plates were done using a Molecular Dynamics PhosphorImager System (Sunnyvale, CA). 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. Activation of p38 MAP kinase involves dual phosphorylation on tyrosine and threonine. Insulin stimulated phosphorylation of p38 MAP kinase in 3T3-L1 adipocytes measured by phosphotyrosine immunoprecipitation followed by Western blotting with anti-p38 MAP kinase antibody (Fig.1 A). We have previously shown a similar result in L6 myotubes (22Tsakiridis T. Taha C. Grinstein S. Klip A. J. Biol. Chem. 1996; 271: 19664-19667Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). SB203580, which interacts with the ATP-binding domain of p38 MAP kinase (37Young P.R. McLaughlin M.M. Kumar S. Kassis S. Doyle M.L. McNulty D. Gallagher T.F. Fisher S. McDonnell P.C. Carr S.A. Huddleston M.J. Seibel G. Porter T.G. Livi G.P. Adams J.L. Lee J.C. J. Biol. Chem. 1997; 272: 12116-12121Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar), did not affect the basal or insulin-stimulated level of p38 phosphorylation. An in vitro kinase assay was then used to measure the ability of p38 MAP kinase to phosphorylate one of its known natural substrates, ATF-2. Insulin caused an approximately 5-fold increase in p38-mediated ATF-2 phosphorylation and this effect was inhibited by SB203580 (Fig.1 B). Fig.2 A shows a dose response of the effect of SB203580 on the uptake of 3H-labeled 2-deoxyglucose into 3T3-L1 adipocytes. The compound was given to cells for 20 min prior to addition of insulin. Treatment with the hormone alone for 30 min caused an increase in glucose uptake of over 4-fold (control, 7.4 ± 0.7 pmol/min/mg protein: insulin, 26.9 ± 4.1 pmol/min/mg protein (p < 0.05)). Pretreatment with a range of concentrations of SB203580 from 1 nm to 0.1 mm prevented stimulation by insulin in a dose-dependent fashion. The IC50 for this effect was calculated to be 0.75 μm. Fig. 2 Aalso shows that, over the same range of concentrations, SB203580 had no effect on basal 2-deoxyg
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