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Amino Acid and Insulin Signaling via the mTOR/p70 S6 Kinase Pathway

2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês

10.1074/jbc.m106703200

ISSN

1083-351X

Autores

Frédéric Tremblay, André Marette,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Amino acids have emerged as potent modulators of the mTOR/p70 S6 kinase pathway. The involvement of this pathway in the regulation of insulin-stimulated glucose transport was investigated in the present study. Acute exposure (1 h) to a balanced mixture of amino acids reduced insulin-stimulated glucose transport by as much as 55% in L6 muscle cells. The effect of amino acids was fully prevented by the specific mTOR inhibitor rapamycin. Time course analysis of insulin receptor substrate 1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity revealed that incubation with amino acids speeds up its time-dependent deactivation, leading to a dramatic suppression (−70%) of its activity after 30 min of insulin stimulation as compared with its maximal activation (5 min of stimulation). This accelerated deactivation of PI 3-kinase activity in amino acid-treated cells was associated with a concomitant and sustained increase in the phosphorylation of p70 S6 kinase. In marked contrast, inhibition of mTOR by rapamycin maintained PI 3-kinase maximally activated for up to 30 min. The marked inhibition of insulin-mediated PI 3-kinase activity by amino acids was linked to a rapamycin-sensitive increase in serine/threonine phosphorylation of IRS-1 and a decreased binding of the p85 subunit of PI 3-kinase to IRS-1. Furthermore, amino acids were required for the degradation of IRS-1 during long term insulin treatment. These results identify the mTOR/p70 S6 kinase signaling pathway as a novel modulator of insulin-stimulated glucose transport in skeletal muscle cells. Amino acids have emerged as potent modulators of the mTOR/p70 S6 kinase pathway. The involvement of this pathway in the regulation of insulin-stimulated glucose transport was investigated in the present study. Acute exposure (1 h) to a balanced mixture of amino acids reduced insulin-stimulated glucose transport by as much as 55% in L6 muscle cells. The effect of amino acids was fully prevented by the specific mTOR inhibitor rapamycin. Time course analysis of insulin receptor substrate 1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity revealed that incubation with amino acids speeds up its time-dependent deactivation, leading to a dramatic suppression (−70%) of its activity after 30 min of insulin stimulation as compared with its maximal activation (5 min of stimulation). This accelerated deactivation of PI 3-kinase activity in amino acid-treated cells was associated with a concomitant and sustained increase in the phosphorylation of p70 S6 kinase. In marked contrast, inhibition of mTOR by rapamycin maintained PI 3-kinase maximally activated for up to 30 min. The marked inhibition of insulin-mediated PI 3-kinase activity by amino acids was linked to a rapamycin-sensitive increase in serine/threonine phosphorylation of IRS-1 and a decreased binding of the p85 subunit of PI 3-kinase to IRS-1. Furthermore, amino acids were required for the degradation of IRS-1 during long term insulin treatment. These results identify the mTOR/p70 S6 kinase signaling pathway as a novel modulator of insulin-stimulated glucose transport in skeletal muscle cells. 70-kDa ribosomal S6 kinase eukaryotic initiation factor 4E-binding protein 1 mammalian target of rapamycin phosphatidylinositol insulin receptor insulin receptor substrate minimal essential medium phosphate-buffered saline Tris-buffered saline Translational control by amino acid-dependent signaling has received considerable attention in recent years (for review, see Refs. 1Van Sluijters D.A. Dubbelhuis P.F. Blommaart E.F. Meijer A.J. Biochem. J. 2000; 351: 545-550Crossref PubMed Scopus (122) Google Scholar and 2Shah O.J. Anthony J.C. Kimball S.R. Jefferson L.S. Am. J. Physiol. Endocrinol. Metab. 2000; 279: 715-729Crossref PubMed Google Scholar). This pathway participates in the phosphorylation of p70S6k1and 4E-BP1, two translational modulators located downstream of mTOR (3Xu G. Marshall C.A. Lin T.A. Kwon G. Munivenkatappa R.B. Hill J.R. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 4485-4491Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 4Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 5Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar, 6Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (294) Google Scholar, 7Patti M.E. Brambilla E. Luzi L. Landaker E.J. Kahn C.R. J. Clin. Invest. 1998; 101: 1519-1529Crossref PubMed Google Scholar, 8Fox H.L. Kimball S.R. Jefferson L.S. Lynch C.J. Am. J. Physiol. 1998; 274: C206-C213Crossref PubMed Google Scholar). First discovered as a target of the immunosuppressive drug rapamycin, mTOR (also known as FRAP (FK506-binding protein-rapamycin-associated protein) or RAFT1 (rapamycin and FKBP12 (FK506-binding protein 12) targets)) is thought to act as a sensor of ambient amino acid concentrations (3Xu G. Marshall C.A. Lin T.A. Kwon G. Munivenkatappa R.B. Hill J.R. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 4485-4491Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 4Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 5Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar, 6Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (294) Google Scholar, 7Patti M.E. Brambilla E. Luzi L. Landaker E.J. Kahn C.R. J. Clin. Invest. 1998; 101: 1519-1529Crossref PubMed Google Scholar, 8Fox H.L. Kimball S.R. Jefferson L.S. Lynch C.J. Am. J. Physiol. 1998; 274: C206-C213Crossref PubMed Google Scholar). Indeed, cells deprived of amino acids show a rapid decline in the phosphorylation of p70S6k and 4E-BP1, which is rapidly reversed after amino acid re-addition in a rapamycin-sensitive manner (4Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 5Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar, 6Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (294) Google Scholar). Furthermore, amino acid starvation did not affect the activity of a mutant form of p70S6k that is resistant to the action of rapamycin (5Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar). Reciprocally, amino acid supplementation was still able to promote p70S6k phosphorylation in the presence of rapamycin in cells expressing a rapamycin-resistant mutant form of mTOR (4Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Taken together, these findings strongly suggest that amino acids signal to p70S6k via mTOR or an mTOR-controlled element. The mTOR nutrient pathway also integrates signal arising from phosphatidylinositol (PI) 3-kinase triggered by insulin or mitogenic signaling (9Rhoads R.E. J. Biol. Chem. 1999; 274: 30337-30340Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 10Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1000) Google Scholar, 11Mendez R. Myers Jr., M.G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1996; 16: 2857-2864Crossref PubMed Scopus (207) Google Scholar, 12Sharma P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This is achieved through the phosphorylation of mTOR by the serine/threonine kinase Akt (also termed protein kinase B), which requires the lipid products of PI 3-kinase, PI 3,4-bisphosphate, and/or PI 3,4,5-triphosphate for its activation (13Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1397) Google Scholar, 14Nave B.T. Ouwens M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (780) Google Scholar, 15Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. Cancer Res. 2000; 60: 3504-3513PubMed Google Scholar, 16Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (411) Google Scholar, 17Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (837) Google Scholar). Recent advances in our understanding of mTOR regulation have shown that both hormonal and nutrient signals are prerequisite inputs necessary to fully activate this pathway (5Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar, 7Patti M.E. Brambilla E. Luzi L. Landaker E.J. Kahn C.R. J. Clin. Invest. 1998; 101: 1519-1529Crossref PubMed Google Scholar). For instance, in Chinese hamster ovary cells expressing insulin receptors, insulin-induced phosphorylation of p70S6k and 4E-BP1 is greatly increased in the presence of amino acids (5Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar). Interestingly, this occurred without any amino acid-dependent modulation of IR/IRS tyrosine phosphorylation or PI 3-kinase and Akt activation by insulin (5Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar), suggesting that both stimuli signal to mTOR via different pathways. In marked contrast, it was found that amino acids impair the ability of insulin to stimulate the tyrosine phosphorylation of IRS proteins and PI 3-kinase activity in hepatoma and muscle cells (7Patti M.E. Brambilla E. Luzi L. Landaker E.J. Kahn C.R. J. Clin. Invest. 1998; 101: 1519-1529Crossref PubMed Google Scholar). This was observed in the face of normal insulin-induced phosphorylation of Akt, p70S6k, and 4E-BP1 (7Patti M.E. Brambilla E. Luzi L. Landaker E.J. Kahn C.R. J. Clin. Invest. 1998; 101: 1519-1529Crossref PubMed Google Scholar). Whether such discrepant findings are solely a matter of cellular context remains to be established. The regulatory role of amino acids on cellular processes is not without precedent. For instance, amino acids have been shown to increase muscle protein synthesis (18Anthony J.C. Yoshizawa F. Anthony T.G. Vary T.C. Jefferson L.S. Kimball S.R. J. Nutr. 2000; 130: 2413-2419Crossref PubMed Scopus (616) Google Scholar, 19Svanberg E. Jefferson L.S. Lundholm K. Kimball S.R. Am. J. Physiol. 1997; 272: E841-E847PubMed Google Scholar), inhibit autophagic proteolysis in liver (20Shah O.J. Antonetti D.A. Kimball S.R. Jefferson L.S. J. Biol. Chem. 1999; 274: 36168-36175Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 21Shigemitsu K. Tsujishita Y. Hara K. Nanahoshi M. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 1058-1065Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 22Mortimore G.E. Poso A.R. Lardeux B.R. Diabetes Metab. Rev. 1989; 5: 49-70Crossref PubMed Scopus (169) Google Scholar), stimulate glucose-induced protein synthesis in pancreatic β-cells (3Xu G. Marshall C.A. Lin T.A. Kwon G. Munivenkatappa R.B. Hill J.R. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 4485-4491Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and facilitate multicellular clustering of rat adipocytes (8Fox H.L. Kimball S.R. Jefferson L.S. Lynch C.J. Am. J. Physiol. 1998; 274: C206-C213Crossref PubMed Google Scholar). All of these processes are dependent upon activation of a rapamycin-sensitive pathway. In addition, amino acids were shown to blunt insulin effect on whole-body and skeletal muscle glucose disposal in humans (23Tessari P. Inchiostro S. Biolo G. Duner E. Nosadini R. Tiengo A. Crepaldi G. Diabetologia. 1985; 28: 870-872Crossref PubMed Scopus (46) Google Scholar, 24Pisters P.W. Restifo N.P. Cersosimo E. Brennan M.F. Metabolism. 1991; 40: 59-65Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 25Flakoll P.J. Wentzel L.S. Rice D.E. Hill J.O. Abumrad N.N. Diabetologia. 1992; 35: 357-366Crossref PubMed Scopus (50) Google Scholar, 26Abumrad N.N. Robinson R.P. Gooch B.R. Lacy W.W. J. Surg. Res. 1982; 32: 453-463Abstract Full Text PDF PubMed Scopus (67) Google Scholar). Although there is accumulating evidence that the positive effect of amino acids on translational events occurred via an mTOR-dependent pathway, the mechanism whereby amino acids negatively modulate glucose transport is still obscure. Insulin stimulates glucose transport in muscle and fat cells by activation of the insulin receptor tyrosine kinase and phosphorylation of intracellular substrates of the IRS family (mainly IRS-1) (27Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Tyrosine-phosphorylated IRS proteins propagate the signal to Src homology 2 domain-containing proteins such as the p85 regulatory subunit of PI 3-kinase, which in turn activates its p110 catalytic subunit (27Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). It is believed that PI 3-kinase activation is essential for the stimulation of glucose transport by insulin (17Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (837) Google Scholar). Downstream effectors of PI 3-kinase includes Akt, which is thought to participate in the stimulation of glucose transport by insulin in muscle cells (28Wang 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,29Hajduch E. Alessi D.R. Hemmings B.A. Hundal H.S. Diabetes. 1998; 47: 1006-1013Crossref PubMed Scopus (296) Google Scholar). The goal of the present study was to test the hypothesis that amino acids impair insulin action on glucose transport by specifically activating the mTOR/p70S6k-signaling pathway in L6 muscle cells. Furthermore, we examined whether amino acids modulate glucose transport by altering component(s) of the insulin signal transduction pathway in an mTOR/p70S6k-dependent fashion. All cell culture solutions and supplements were purchased from Life Technologies, Inc. except for fetal bovine serum, which was purchased from Wisent (St-Bruno, QC, Canada). Reagents for SDS-polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad. ECL and [2-3H]Deoxyglucose were from PerkinElmer Life Sciences. [γ-32P]ATP, protein A- and G-Sepharose, and anti-mouse or anti-rabbit immunoglobulin G conjugated to horseradish peroxidase were purchased from Amersham Pharmacia Biotech. Polyclonal antibodies against IRS-1 (raised against 20 C-terminal amino acids (C-20)) and Akt (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-specific antibodies against Akt (Ser-473) and p70 S6 kinase (Thr-421/Ser-424) were from New England Biolabs (Beverly, MA). Antibodies against phosphotyrosine (4G10 clone), IRS-1 and PI 3-kinase, and Akt substrate (Crosstide) were obtained from Upstate Biotechnology (Lake Placid, NY). Human insulin was obtained from Eli Lilly (Toronto, ON, Canada). Rapamycin was purchased from Biomol (Plymouth Meeting, PA).l-α-Phosphatidylinositol was from Avanti Polar Lipids (Alabaster, AL). Oxalate-treated TLC silica gel H plates were obtained from Analtech (Newark, DE). All other chemicals were of the highest analytical grade. A line of L6 skeletal muscle cells (kind gift of Dr. Amira Klip, Hospital for Sick Children, Toronto, ON, Canada) clonally selected for high fusion potential was used in the present study. The L6 cell line was derived from neonatal rat thigh skeletal muscle cells and retains many morphological, biochemical, and metabolic characteristics of skeletal muscle. Cells were grown and maintained in monolayer culture in α-MEM containing 2% (v/v) fetal bovine serum and 1% (v/v) antibiotic/antimycotic solution (10000 units/ml penicillin, 10000 μg/ml streptomycin and 25 μg/ml amphotericin B) in an atmosphere of 5% CO2 at 37 °C. Fully differentiated L6 myotubes were deprived of serum 4 h before experimental treatments. Then cells were incubated either in amino acid-free medium (Earle's balanced salt solution (EBSS)) or in EBSS containing 1× or 2× amino acids either individually or as a mixture, as found in MEM for 1 h. The concentrations (in μm) of amino acids (1×) in MEM were as follows: Arg, 126; Cys, 100; Gln, 2000; His, 200; Ile, 400; Leu, 400; Lys, 400; Met, 100; Phe, 200; Thr, 400; Trp, 50; Tyr, 200 and Val, 400. Vehicle (0.01% Me2SO) or rapamycin (25 nm) was added during the 1-h incubation. Cells were stimulated with insulin for different times as indicated in figure legends. 2-Deoxyglucose was determined as previously described (30Bedard S. Marcotte B. Marette A. Biochem. J. 1997; 325: 487-493Crossref PubMed Scopus (141) Google Scholar). Briefly, after experimental treatments, cells were rinsed once with HEPES-buffered solution (20 mm HEPES, pH 7.4, 140 mm NaCl, 5 mmKCl, 2.5 mm MgSO4, and 1 mmCaCl2) and were subsequently incubated for 8 min in HEPES-buffered solution containing 10 μm 2-deoxyglucose and 0.3 μCi/ml 2-deoxy-[3H]glucose. After the incubation in transport medium, cells were rinsed three times with ice-cold 0.9% NaCl solution and then disrupted by adding 50 mm NaOH. Cell-associated radioactivity was determined by scintillation counting. Protein concentrations were determined by the bicinchoninic acid method, and the results were expressed in pmol/min/mg. Glucose uptake values were corrected for non-carrier-mediated transport by measuring hexose uptake in the presence of 10 μm cytochalasin B (5–10% of total uptake). After experimental treatment, medium was removed, and cells were rinsed twice in ice-cold phosphate-buffered saline (PBS) and lysed in 20 mm Tris, pH 7.4, 140 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10% glycerol, 1% Nonidet P-40, 2 mm Na3VO4, and 10 mmNaF. 500 μg of lysates were immunoprecipitated with 2 μg of anti-IRS-1 coupled to protein A-Sepharose overnight at 4 °C. Immune complexes were washed twice with wash buffer I (PBS, pH 7.4, 1% Nonidet P-40, and 2 mm Na3VO4), twice with wash buffer II (100 mm Tris, pH 7.5, 500 mm LiCl, and 2 mmNa3VO4), and twice with wash buffer III (10 mm Tris, pH 7.5, 100 mm NaCl, 1 mmEDTA, and 2 mm Na3VO4). Beads were resuspended in 70 μl of kinase buffer (8 mm Tris, pH 7.5, 80 mm NaCl, 0.8 mm EDTA, 15 mmMgCl2, 180 μm ATP, and 5 μCi of [γ-32P]ATP) and 10 μl of sonicated PI mixture (20 μg of l-α-PI, 10 mm Tris, pH 7.5, and 1 mm EGTA) for 15 min at 30 °C. The reaction was stopped by the addition of 20 μl of 8 m HCl mixed with 160 μl of CHCl3:CH3OH (1:1) and centrifuged. The lower organic phase was spotted on an oxalate-treated silica gel TLC plate and developed in CHCl3:CH3OH:H2O:NH4OH (60:47:11.6:2). The plate was dried and visualized by autoradiography with intensifying screen at −80 °C. After experimental treatment, the medium was removed, cells were rinsed twice in ice-cold PBS and lysed in 50 mm HEPES, pH 7.4, 150 mm NaCl, 1 mmEDTA, 1 mm dithiothreitol, 10% glycerol, 1% Triton X-100, 2 mm Na3VO4, and 10 mmNaF. 200–300 μg of lysates were immunoprecipitated with 2 μg of anti-Akt1 coupled to protein G-Sepharose for 2 h at 4 °C. Immune complexes were washed 3 times in 25 mm HEPES, pH 7.4, 10% glycerol, 1% Triton X-100, 0.1% bovine serum albumin, 1m NaCl, 1 mm dithiothreitol, and 200 μm Na3VO4 and 2 times in kinase buffer (50 mm Tris, pH 7.4, 10 mmMgCl2, and 1 mm dithiothreitol). The reaction was started by adding 30 μl of kinase buffer (containing 8 μm ATP, 2 μCi of [γ-32P]ATP and 100 μm Crosstide) for 30 min at 30 °C. The reaction product was resolved on 40% acrylamide-urea gel and visualized by autoradiography with intensifying screen at −80 °C. IRS-1 was immunoprecipitated as described for PI 3-kinase assay. Tyrosine-phosphorylated proteins were immunoprecipitated with 2 μg of anti-phosphotyrosine (clone 4G10) coupled to protein A-Sepharose from 500 μg of cell lysate. Immune complexes were washed 3 times in PBS, pH 7.4, 1% Nonidet P-40, and 2 mmNa3VO4, resuspended in SDS sample buffer, and boiled for 5 min. Immunoprecipitates or cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Polyvinylidene difluoride membranes were then blocked for 1 h at room temperature in TBS (50 mm Tris, pH 7.4, 150 mm NaCl) containing 0.04% Nonidet P-40, 0.02% Tween 20, and 5% nonfat milk followed by overnight incubation at 4 °C with primary antibodies. The polyvinylidene difluoride membranes were then washed for 30 min followed by a 1-h incubation with either anti-mouse or anti-rabbit immunoglobulin G conjugated to horseradish peroxidase in TBS containing 2% nonfat milk. The polyvinylidene difluoride membranes were washed for 30 min in TBS, and the immunoreactive bands were detected by the enhanced chemiluminescence method. The effects of amino acids, insulin, and rapamycin were compared by analysis of variance tests followed by least mean square determination. Differences were considered to be statistically significant at p < 0.05. The inhibitory action of amino acids on glucose transport in skeletal muscle has been observedin vivo (23Tessari P. Inchiostro S. Biolo G. Duner E. Nosadini R. Tiengo A. Crepaldi G. Diabetologia. 1985; 28: 870-872Crossref PubMed Scopus (46) Google Scholar, 24Pisters P.W. Restifo N.P. Cersosimo E. Brennan M.F. Metabolism. 1991; 40: 59-65Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 25Flakoll P.J. Wentzel L.S. Rice D.E. Hill J.O. Abumrad N.N. Diabetologia. 1992; 35: 357-366Crossref PubMed Scopus (50) Google Scholar, 26Abumrad N.N. Robinson R.P. Gooch B.R. Lacy W.W. J. Surg. Res. 1982; 32: 453-463Abstract Full Text PDF PubMed Scopus (67) Google Scholar). To test whether such effect can be reproduced in vitro, L6 myocytes were either incubated in an amino acid-free medium or in a 1× or 2× amino acids mixture for 1 h (a 1× amino acid mixture was defined as the concentration of amino acids found in MEM (See “Experimental Procedures”)). After experimental treatments, glucose transport was measured in cells that were stimulated or not with insulin (100 nm). Increasing the concentrations of amino acids had no significant effect on basal glucose transport (Fig. 1A). However, the effect of a submaximal concentration of insulin (100 nm) on glucose transport was inhibited by as much as 30 and 55% in myocytes treated with 1× or 2× amino acid mixtures as compared with cells deprived of amino acids, respectively (Fig.1A). We next determined if the effect of amino acids could be observed over a wide range of insulin concentrations. Amino acid-treated cells were exposed to increasing doses of insulin, and glucose transport was determined. As depicted in Fig. 1B, the suppressive effect of amino acids was noticeable at 5 nm insulin and could still be observed at maximal doses of the hormone (0.5–1 μm). These results clearly indicate that acute exposure (1 h) to amino acids impairs the ability of insulin to stimulate glucose transport in muscle cells. To identify which amino acids were responsible for insulin resistance, we measured glucose transport in cells treated with individual amino acids at the concentration found in 2× amino acid mixtures. It was found that Cys, His, Leu, Met, Thr, and Tyr were inhibitory, whereas the remaining (Arg, Glu, Ile, Lys, Phe, Trp, and Val) amino acids failed to inhibit insulin-stimulated glucose uptake (Fig. 1C). It is noteworthy that the inhibitory influence of amino acids were neither limited to a particular chemical group (e.g. branched-chain, cationic, neutral, or polar) nor to a particular amino acid transport system (e.g. A, ASC, or L). Interestingly, glutamine, an amino acid known to activate the hexosamine biosynthetic pathway and to cause insulin resistance in rat adipocytes (31Marshall S. Garvey W.T. Traxinger R.R. FASEB J. 1991; 5: 3031-3036Crossref PubMed Scopus (146) Google Scholar, 32Traxinger R.R. Marshall S. J. Biol. Chem. 1989; 264: 20910-20916Abstract Full Text PDF PubMed Google Scholar), was without effect in L6 muscle cells. This indicates that build-up of hexosamines is unlikely to be involved in the acute inhibition of insulin-stimulated glucose transport by amino acids in muscle cells. Amino Acids Impair Insulin-stimulated Glucose Transport via themTOR Pathway—We next tested the hypothesis that amino acids impair insulin-stimulated glucose transport via the activation of mTOR. The involvement of the mTOR pathway in amino acid-induced insulin resistance was evaluated by using rapamycin, a highly specific inhibitor (33Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3937) Google Scholar) that forms a complex with FKBP12 (FK506-binding protein 12), which binds and inactivates mTOR (34Abraham R.T. Wiederrecht G.J. Annu. Rev. Immunol. 1996; 14: 483-510Crossref PubMed Scopus (572) Google Scholar). In cells deprived of amino acids for 1 h, neither basal nor insulin-stimulated glucose transport was affected by the addition of rapamycin to the medium (Fig.2, lane 1 versus 2and lane 3 versus 4). Strikingly, the inhibitory effects of amino acids on insulin-stimulated glucose transport (Fig. 2, compare lanes 3 versus 7) were completely prevented by treatment with rapamycin (Fig. 2, lanes 7 versus 8). These results indicate that the inhibitory amino acids (Fig.1C) contained in the 2× amino acids mixture decrease insulin-stimulated glucose transport by activating the mTOR pathway. Furthermore, the observation that rapamycin regulates glucose transport only in medium enriched in amino acids strengthened the hypothesis that rapamycin specifically acts on mTOR to antagonize amino acid-dependent signaling. In an attempt to understand how amino acids induced insulin resistance, we next examined the activation of key signaling proteins involved in insulin action. Among them, IRS-1 and its associated PI 3-kinase activity play a prominent role in insulin-stimulated glucose transport in muscle (35Higaki 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, 36Kido Y. Burks D.J. Withers D. Bruning J.C. Kahn C.R. White M.F. Accili D. J. Clin. Invest. 2000; 105: 199-205Crossref PubMed Scopus (422) Google Scholar). L6 cells were incubated in media containing either no amino acids or a 2× amino acid mixture with or without rapamycin for 1 h and stimulated with insulin during the last 5 min. IRS-1 was immunoprecipitated from cell lysates, resolved by SDS-polyacrylamide gel electrophoresis, and incubated with anti-phosphotyrosine antibody. As shown in Fig.3A, insulin-induced tyrosine phosphorylation of IRS-1 was not affected by amino acids or rapamycin treatments. Similarly, the amount of p85 regulatory subunit of PI 3-kinase recovered in IRS-1 immune complexes was strongly increased by insulin and was not affected by amino acids and/or rapamycin (Fig.3B). Consistent with the lack of effect of amino acids on IRS-1 tyrosine phosphorylation and p85 recruitment, we observed no effects of amino acids or rapamycin on basal or insulin-induced PI 3-kinase activity in IRS-1 precipitates (Fig. 3C). It has been recently reported that dephosphorylation and/or relocalization of PI 3,4,5-triphosphate reduced Akt phosphorylation in response to insulin despite the fact that PI 3-kinase activity was intact (37Yang C. Watson R.T. Elmendorf J.S. Sacks D.B. Pessin J.E. Mol. Endocrinol. 2000; 14: 317-326Crossref PubMed Scopus (45) Google Scholar, 38Nakashima N. Sharma P.M. Imamura T. Bookstein R. Olefsky J.M. J. Biol. Chem. 2000; 275: 12889-12895Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Therefore, although incubation with amino acids failed to affect PI 3-kinase activity, it was of interest to evaluate the modulation of Akt phosphorylation and activity by amino acids and/or rapamycin. Using a phospho-specific antibody, we found that insulin robustly enhanced the phosphorylation state of Akt in cells incubated with or without amino acids and/or rapamycin (Fig. 4A). In accordance with Akt phosphorylation data, insulin-stimulated Akt kinase activity was unaffected by either amino acids or rapamycin (Fig.4B). Since the acute burst (5 min) of PI 3-kinase activity in response to insulin was not impaired by amino acids (Fig. 3), we sought to determine whether there was any relationship between insulin-stimulated PI 3-kinase activity and mTOR/p70S6k activation over time. L6 cells were placed in 2× amino acid medium for 1 h and treated with insulin for the last 5, 15, or 30 min of incubation. It was found that IRS-1-associated PI 3-kinase is maximally activated after 5 min of insulin stimulation and progressively decrease, reaching ∼30% of maximal activity after 30 min (Fig. 5A). In contrast to PI 3-kinase, th

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