The Role of AMPK and mTOR in Nutrient Sensing in Pancreatic β-Cells
2007; Elsevier BV; Volume: 282; Issue: 14 Linguagem: Inglês
10.1074/jbc.m610631200
ISSN1083-351X
AutoresCatherine E. Gleason, Danhong Lu, Lee A. Witters, Christopher B. Newgard, Morris J. Birnbaum,
Tópico(s)Diet, Metabolism, and Disease
ResumoThe AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic β-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of β-cell function. Here, we show that inhibition of AMPK in β-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the β-cell. The AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic β-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of β-cell function. Here, we show that inhibition of AMPK in β-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the β-cell. The β-cell is unique compared with other mammalian cell types in that its primary function to synthesize and secrete insulin is tightly coupled to its metabolic rate. Glucose is the most potent nutrient in stimulating insulin release. Upon entry into the β-cell, glucose is rapidly metabolized, resulting in the generation of mitochondria-derived metabolic intermediates including ATP. This increase in ATP leads to closure of ATP-sensitive K+ (KATP)-channels, depolarization of the plasma membrane and opening of voltage-gated L-type Ca2+ channels. The subsequent increase in intracellular Ca2+ concentration [Ca2+]i triggers insulin exocytosis (1Henquin J.C. Diabetes. 2000; 49: 1751-1760Crossref PubMed Scopus (949) Google Scholar). The β-cell also utilizes certain key amino acids that, via mitochondrial metabolism, can further generate coupling factors that elicit an insulin secretory response (2MacDonald M.J. Fahien L.A. Brown L.J. Hasan N.M. Buss J.D. Kendrick M.A. Am. J. Physiol. Endocrinol. Metab. 2005; 288: E1-E15Crossref PubMed Scopus (202) Google Scholar, 3Newgard C.B. Matschinsky F.M. Substrate Control of Insulin Release. Oxford University Press, 2001: 125-151Google Scholar). In addition to their role as insulin secretagogues, glucose and other nutrients stimulate protein translation and β-cell growth and proliferation (4Hugl S.R. White M.F. Rhodes C.J. J. Biol. Chem. 1998; 273: 17771-17779Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 5Swenne I. Diabetologia. 1992; 35: 193-201Crossref PubMed Scopus (188) Google Scholar). While much is known regarding how the β-cell couples glucose metabolism to insulin secretion, the mechanisms by which β-cells sense metabolism of other fuels, such as amino acids, and augment glucose-stimulated insulin release are less clear. Further, it is unclear how the β-cell coordinates nutrient abundance with enhanced protein translation and cell growth. This aspect of β-cell function is particularly important under conditions of increased insulin demand, such as obesity and/or insulin resistance. Failure of β-cells to compensate for this increase in demand is a critical factor in the development of type 2 diabetes (6Ahren B. Curr. Mol. Med. 2005; 5: 275-286Crossref PubMed Scopus (118) Google Scholar). The AMP-activated protein kinase (AMPK) 2The abbreviations used are: AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; BCH, β-(+/-)-2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid; GDH, glutamate dehydrogenase; ACC, acetyl-CoA carboxylase. is a heterotrimeric serine/threonine protein kinase that is activated by various pathological and physiological stresses that result in a lowered cellular ATP/ADP + AMP ratio (7Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (963) Google Scholar, 8Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar). The AMPK protein complex consists of a catalytic α-subunit and regulatory β- and γ-subunits. AMPK activity is regulated allosterically by AMP and through phosphorylation at Thr172 in the activation loop of the α-subunit. By phosphorylation of downstream targets, AMPK acts to repress pathways that consume energy and to promote ATP-producing catabolic pathways (7Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (963) Google Scholar, 8Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar). For example, by phosphorylation of acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, AMPK shuts down the ATP-depleting processes of fatty acid and cholesterol biosynthesis, respectively, and replenishes ATP by promoting fatty acid oxidation. AMPK also exerts its effect on cellular energy balance through modification of gene expression and protein translation (9Inoki K. Ouyang H. Li Y. Guan K.L. Microbiol. Mol. Biol. Rev. 2005; 69: 79-100Crossref PubMed Scopus (285) Google Scholar, 10Rutter G.A. Da Silva Xavier G. Leclerc I. Biochem. J. 2003; 375: 1-16Crossref PubMed Scopus (287) Google Scholar). Because glucose-stimulated insulin secretion from the β-cell is directly tied to the generation of metabolic intermediates, the unique sensitivity of AMPK to changes in the AMP/ATP ratio makes AMPK an attractive candidate for regulation of β-cell function. Indeed, AMPK has been implicated in the regulation of glucose and amino acid-stimulated insulin release and gene expression in the β-cell, although this is controversial (11da Silva Xavier G. Leclerc I. Salt I.P. Doiron B. Hardie D.G. Kahn A. Rutter G.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4023-4028Crossref PubMed Scopus (187) Google Scholar, 12da Silva Xavier G. Leclerc I. Varadi A. Tsuboi T. Moule S.K. Rutter G.A. Biochem. J. 2003; 371: 761-774Crossref PubMed Scopus (231) Google Scholar, 13Raile K. Klammt J. Laue S. Garten A. Bluher M. Kralisch S. Kloting N. Kiess W. Diabetologia. 2005; 48: 1798-1809Crossref PubMed Scopus (20) Google Scholar). More recently, the ability of AMPK to coordinate energy availability with protein synthesis through regulation of the mammalian Target of Rapamycin (mTOR) signaling pathway has received considerable attention in non-β-cell lines (9Inoki K. Ouyang H. Li Y. Guan K.L. Microbiol. Mol. Biol. Rev. 2005; 69: 79-100Crossref PubMed Scopus (285) Google Scholar). Currently, it is not known if AMPK modulates the mTOR signaling pathway similarly in the β-cell. AMPK regulates protein translation via at least two mechanisms: phosphorylation and activation of the eukaryotic elongation factor 2 kinase (eEF2K) and inhibition of the mTOR signaling pathway (14Browne G.J. Finn S.G. Proud C.G. J. Biol. Chem. 2004; 279: 12220-12231Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 15Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3484) Google Scholar). Activation of eEF2K leads to phosphorylation of its target eEF2 and inhibition of the elongation step of protein translation. mTOR is a serine/threonine protein kinase that regulates various cellular functions, in particular, the initiation step of protein synthesis. mTOR is activated in response to hormones and growth factors, such as insulin and insulin-like growth factor 1 (IGF-1), and via the phosphatidylinositol 3-kinase-Akt signaling pathway in the presence of amino acids. Amino acids also induce mTOR activity in the absence of additional stimuli. In particular, the branch-chained amino acid leucine is required for the effect of amino acids to activate mTOR. Once activated, mTOR phosphorylates both the eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), an inhibitor of translation, and p70 ribosomal S6 kinase (p70S6K) (15Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3484) Google Scholar). Because AMPK may represent an alternative mechanism for sensing glucose and amino acids in the β-cell, it is important to understand its downstream targets in this cell type. Moreover, the ongoing development of activators of AMPK for use as drugs in the treatment of type 2 diabetes makes this a particularly relevant issue. In this study, we asked if AMPK regulates glucose-stimulated mTOR activation and insulin secretion in β-cells lines and primary rodent islets. Because amino acids contribute to both stimulation of insulin release and mTOR activation through their metabolic breakdown, we also addressed the question of how amino acids are sensed by AMPK and whether their effect to inhibit AMPK correlates with insulin release. Materials—Dulbecco's modified Eagle's medium, fetal bovine serum, penicillin/streptomycin solution, sodium pyruvate solution, l-glutamine, Minimum Essential Media (MEM) essential, and non-essential amino acid solutions were obtained from Invitrogen (Carlsbad, CA). 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) was purchased from Toronto Research Chemicals (Toronto, On, Canada). Phenformin, oligomycin, rapamycin, and insulin were from Sigma. The phospho-acetyl CoA carboxylase (p-ACC) antibody was from Upstate (Lake Placid, NY). The phospho-rpS6 antibody has been described previously (16Summers S.A. Lipfert L. Birnbaum M.J. Biochem. Biophys. Res. Commun. 1998; 246: 76-81Crossref PubMed Scopus (49) Google Scholar). The anti-AMPKα antibody used recognizes the N terminus of both the α1 and α2 subunits of AMPK and has been described previously (17Mu J. Brozinick Jr., J.T. Valladares O. Bucan M. Birnbaum M.J. Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Infrared-labeled secondary antibodies for use with the Odyssey Infrared Imaging System (LICOR Biosciences, Lincoln, NE) were purchased from Rockland Inc. (Gilbertsville, PA). All other antibodies were from Cell Signaling Technology, Inc (Beverly, MA). Amino Acid Composition of Buffers—Krebs-Ringer bicarbonate buffer (KRBH: 115 mm NaCl, 2.5 mm CaCl2, 1.2 mm KH2PO4, 5 mm KCl, 25 mm NaHCO3, 1.2 mm MgCl2, 10 mm HEPES, pH 7.4, 0.1% bovine serum albumin) was supplemented with MEM amino acids solution, MEM non-essential amino acids solution, and l-glutamine. For these experiments, the 1× concentration of amino acids was defined as the following in mm: l-arginine 0.73, l-cystine 0.2, l-glutamine 2.0, l-histidine.HCl.H2O 0.2, l-isoleucine 0.4, l-leucine 0.4, l-lysine HCl 0.5, l-methionine 0.1, l-valine 0.4, l-phenylalanine 0.2, l-threonine 0.4, l-tryptophan 0.05, l-tyrosine 0.2, l-alanine 0.01, l-asparagine 0.01, l-aspartic acid 0.01, l-glutamic acid 0.01, glycine 0.01, l-proline 0.01, l-serine 0.01. For this study, we have defined physiological concentrations of leucine and glutamine as 0.4 mm and 0.2 mm, respectively, based on reported plasma concentrations of leucine and glutamine (0.119 mm and 0.338 mm, respectively) in fed mice (18Bolea S. Pertusa J.A. Martin F. Sanchez-Andres J.V. Soria B. Pflugers Arch. 1997; 433: 699-704Crossref PubMed Scopus (44) Google Scholar). Cell Culture and Treatment—MIN6 cells (kindly provided by Prof. Jun-Ichi Miyazaki, Osaka University, Osaka, Japan) were used between passages 28 and 40 at ∼80% confluence. MIN6 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 25 mm glucose supplemented with 15% fetal bovine serum, 1 mm sodium pyruvate, 50 μm β-mercaptoethanol, 100 μg/ml streptomycin, 100 units/ml penicillin, equilibrated with 5% CO2, 95% air at 37 °C. Prior to treatment, MIN6 cells were cultured overnight in DMEM containing 3 mm glucose and 10% fetal bovine serum. The medium was then removed, and the cells were washed one time with KRBH and incubated in KRBH for 2 h equilibrated at 5% CO2, 95% air at 37 °C. Cells were then stimulated with 3 or 30 mm glucose and/or amino acids with or without drugs for 30 min. For the experiments with insulin (Figs. 3 and 4) 200 nm insulin was added at the same time as glucose, amino acids and/or drugs were added. Medium (500 μl) was removed and assayed for insulin released by radioimmunoassay (Linco Research, St. Charles, MO). The cells were then lysed in ice-cold buffer containing 140 mm NaCl, 10 mm Tris, pH 7.4, 200 mm NaF, 10% glycerol, 1% Nonidet P-40, 1× Complete protease inhibitor mixture (Roche Applied Science, Mannheim, Germany) and 1× phosphatase inhibitor mixture 1 and 2 (Sigma). 5 μl of lysate was removed for analysis of total protein by BCA assay (Pierce). Some studies were performed with the cell line 832/13, derived as described (19Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (717) Google Scholar) from INS-1 rat insulinoma cells (20Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar). When using these cells, culture conditions, and insulin secretion assay procedures were as previously described (19Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (717) Google Scholar).FIGURE 4AMPK activity correlates with inhibition of mTOR signaling. MIN6 cells were serum, glucose, and amino acid starved for 2 h. KRBH was then replaced with KRBH containing insulin (200 nm), 1× amino acids, and 3 or 30 mm glucose as indicated. During the 30 min of stimulation, cells were also treated with Vehicle (ethanol), AICAR (2 mm), Phenformin (10 mm), or Oligomycin (1 μm). A, cells were processed for immunoblotting as described previously. The Western blot is representative of three separate experiments. B, the level of p70S6K phosphorylation compared with total p70S6K was quantitated using LICOR software. Error bars indicate ± S.D. *, p < 0.001 for the effect of 3 versus 30 mm glucose.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Studies with Isolated Rat Islets of Langerhans—Pancreatic islets of Langerhans were isolated from male Wistar rats (250–275 g) by perfusion of the pancreatic duct and in situ collagenase (Liberase RI) digestion, as previously described (21Jensen M.V. Joseph J.W. Ilkayeva O. Burgess S. Lu D. Ronnebaum S.M. Odegaard M. Becker T.C. Sherry A.D. Newgard C.B. J. Biol. Chem. 2006; 281: 22342-22351Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Insulin secretion from islets was measured as described (21Jensen M.V. Joseph J.W. Ilkayeva O. Burgess S. Lu D. Ronnebaum S.M. Odegaard M. Becker T.C. Sherry A.D. Newgard C.B. J. Biol. Chem. 2006; 281: 22342-22351Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) in the presence or absence of 1 mm AICAR and following preincubation of 20 islets/condition in triplicate in HEPES balanced salt solution-SAB (HBSS; 114 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 1.16 mm MgSO4, 20 mm HEPES, 2.5 mm CaCl2, 25.5 mm NaHCO3, 0.2% bovine serum albumin; pH 7.2) containing 3 mm glucose for 45 min. Suppression of AMPK Activity by Adenovirus-mediated Expression of a Mutant Form of AMPK—Ad-AMPK-α2-K45R, a dominant inhibitory AMPK was prepared as described previously (17Mu J. Brozinick Jr., J.T. Valladares O. Bucan M. Birnbaum M.J. Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar, 22Yin W. Mu J. Birnbaum M.J. J. Biol. Chem. 2003; 278: 43074-43080Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). 832/13 cells were grown in 6-well plates to 90% confluence, and Ad-AMPK-α2-K45R or AdCMV-βGAL viruses were added at 50 m.o.i. in 2 ml of medium for 2 h. 24 h after viral treatment, cells were incubated in SAB containing 3 mm glucose for 2 h. The buffer was then removed and replaced with fresh SAB containing either 3 or 12 mm glucose for an additional 2 h. Where indicated, AICAR was added to the SAB/glucose at a concentration of 1 mm for the last 30 min of preincubation time and throughout the 2-h secretion period. Insulin secretion was measured by radioimmunoassay as described (19Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 49: 424-430Crossref PubMed Scopus (717) Google Scholar). Immunoblot Analysis—Cells were washed once with ice-cold PBS and then lysed by the addition of ice-cold buffer containing 140 mm NaCl, 10 mm Tris, pH 7.4, 200 mm NaF, 10% glycerol, 1% Nonidet P-40, 1× Complete protease inhibitor mixture (Roche Applied Science) and 1× phosphatase inhibitor mixtures 1 and 2 (Sigma). The lysates were then centrifuged for 10 min at 10,000 rpm at 4 °C. Protein concentrations were determined using the BCA protein assay kit. Equivalent amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by transfer to nitrocellulose membranes (Whatman, Florham Park, NJ). Detection was performed either using ECL reagents from Amersham Biosciences or a LICOR Odyssey Infrared Imager as described in the figure legends. Blots imaged using the Odyssey Infrared Imager were probed using infrared-labeled secondary antibodies (Rockland, Inc, Gilbertsville, PA) following instructions supplied by the manufacturer (LICOR Biosciences, Lincoln, NE). Quantitation on blots scanned using the Odyssey was performed using the LICOR software. AMPK Activity—AMPK was immunoprecipitated from 100 μg of MIN6 or 832/13 cell lysate by addition of α-AMPKN antibody (17Mu J. Brozinick Jr., J.T. Valladares O. Bucan M. Birnbaum M.J. Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar) for 1 h, rocking at 4 °C. 20 μl of protein A-agarose beads were added to samples, which were rocked for another 2 h at 4 °C. The beads were pelleted by centrifugation for 1 min at 13,000 rpm, 4 °C, and washed three times with lysis buffer followed by one wash with kinase reaction buffer (50 mm HEPES, pH 7.5, 1 mm dithiothreitol, 0.02% Brij-30). The kinase reaction was conducted at 30 °C for 20 min in buffer containing 50 mm HEPES, pH 7.5, 1 mm dithiothreitol, 0.02% Brij-30, 25 mm MgCl2, 0.2 mm SAMS peptide (HMRSAMSGLHLVKRR), 0.3 mm AMP, 0.1 mm ATP with 0.25 μCi/μl[γ-32P]ATP. The reactions were stopped by placing samples on ice for 1 min followed by centrifugation at 13,000 rpm for 1 min, and 18 μl of each reaction was spotted onto P81 paper. The P81 paper was washed three times with 1% phosphoric acid and once with acetone. Incorporated [γ-32P]ATP was measured by counting on a Packard liquid scintillation CA1600 counter. Statistical Analysis—Data are expressed as means ± S.E. as indicated in the figure legends. Statistically significant differences between groups were analyzed using analysis of variance (JMP Start Statistics). p < 0.05 was considered statistically significant. Glucose stimulates phosphorylation of p70S6K in MIN6 cells (23Gomez E. Powell M.L. Greenman I.C. Herbert T.P. J. Biol. Chem. 2004; 279: 53937-53946Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). However, the relative contributions of a direct effect of glucose as opposed to the potential autocrine effects of secreted insulin on mTOR activity have not been determined. Therefore, we first examined glucose-stimulated mTOR activity in MIN6 cells and determined what component of that stimulation was caused by glucose alone. MIN6 cells were starved of serum, glucose, and amino acids for 2 h followed by exposure to glucose at increasing concentrations for 30 min. As shown in Fig. 1, glucose in the presence of a 1× concentration of amino acids dose-dependently increased p70S6K phosphorylation with maximal stimulation at 30 mm glucose. The 1× amino acid mixture contains a complete complement of amino acids plus glutamine (2 mm) and is equivalent to the concentration of amino acids present in MEMα medium. Phosphorylation of ribosomal protein S6 (rpS6), the downstream target of p70S6K, correlates with p70S6K activation. To determine what portion of the glucose-stimulated mTOR activity is directly caused by glucose, MIN6 cells were treated with either diazoxide or verapamil, which block insulin release by inhibiting the influx of extracellular Ca2+. Diazoxide binds to the KATP-channel maintaining it in an open conformation whereas verapamil acts directly on the Ca2+ channel to block Ca2+ influx (24Doyle M.E. Egan J.M. Pharmacol. Rev. 2003; 55: 105-131Crossref PubMed Scopus (200) Google Scholar). Both drugs completely inhibited insulin release from cells stimulated with 30 mm glucose and almost completely eliminated glucose-mediated p70S6K phosphorylation (Fig. 2A). Exposure of vehicle-treated MIN6 cells to 30 mm glucose resulted in a 2.5-fold increase in p70S6K phosphorylation compared with 3 mm glucose (Fig. 2, B and C). In cells treated with diazoxide, p70S6K phosphorylation was completely abrogated with no significant difference between the 3 and 30 mm glucose treatments (Fig. 2, B and C). However, although verapamil blocked glucose-stimulated insulin release, the mobility shift in p70S6K induced by glucose was not completely inhibited. These results suggest that whereas most of the mTOR response to glucose is probably mediated by released insulin a small, direct effect of glucose may exist.FIGURE 2A portion of glucose-stimulated mTOR activity is independent of insulin secretion. MIN6 cells were serum, glucose, and amino acid starved in KRBH for 2 h. Diazoxide (100 μm) or verapamil (100 μm) was added during the last hour of preincubation. Cells were then stimulated for 30 min with KRBH containing the indicated glucose concentration in the presence of vehicle (dimethyl sulfate), diazoxide, or verapamil. A, insulin released during the 30 min of stimulation. Error bars represent the mean ± S.E. of three separate experiments. B, cell extracts were processed for immunoblotting as described under "Experimental Procedures." C, quantitation of p70S6K phosphorylation compared with total p70S6K using LICOR software. The error bars represent the mean ± S.D. of three separate experiments. A, *, p < 0.001 for the effect of 3 versus 30 mm glucose; C, *, p < 0.001 for the effect of 3 versus 30 mm glucose.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the direct effect of glucose to activate the mTOR pathway could have been obscured by the simultaneous release of insulin, it was necessary to develop conditions that distinguish the direct contributions of glucose from those of insulin. Therefore, we next asked if stimulating MIN6 cells with glucose in the presence of a saturating concentration of insulin (200 nm) would allow us to discern specifically the direct effect of glucose on activation of the mTOR pathway. As shown in Figs. 2 and 3, increasing the glucose concentration from 3 to 30 mm enhances both p70S6K and rpS6 phosphorylation. The effect of added insulin was indicated by an increase in phosphorylation at serine 473 (Ser473) of Akt, a kinase known to be downstream of the insulin receptor (Fig. 3). In the presence of 200 nm insulin, Akt phosphorylation was maximal and did not increase further with the addition of glucose, indicating that activation of the insulin receptor was saturating. However, in the presence of saturating insulin, increasing the glucose concentration resulted in enhanced phosphorylation of p70S6K and rpS6 (Fig. 3). These results demonstrate the presence of a direct (i.e. without intervening insulin secretion) effect of glucose on mTOR that can be assessed by analysis in the presence of a saturating concentration of insulin. We next asked whether a reduction in AMPK signaling mediates glucose-stimulated activation of the mTOR pathway in the β-cell. MIN6 cells, preincubated as described previously, were exposed to 3 or 30 mm glucose plus 200 nm insulin with or without a 1× amino acid mixture for 30 min. During the 30 min of stimulation, AICAR, phenformin, or oligomycin was added as indicated to activate AMPK (Fig. 4). The drugs chosen to activate AMPK do so via three distinct mechanisms. AICAR is phosphorylated inside the cell to the AMP analogue, ZMP. Phenformin is a biguanide widely used to treat type 2 diabetes. Its method of AMPK activation is unclear but seems to be dependent on changes in the AMP:ATP ratio (25Owen M.R. Doran E. Halestrap A.P. Biochem. J. 2000; 348: 607-614Crossref PubMed Scopus (1659) Google Scholar). Lastly, oligomycin depletes cellular ATP by inhibiting mitochondrial ATP synthase. Three surrogate measures of cellular AMPK activity were employed: phosphorylation on threonine 172 of the catalytic subunit; and phosphorylation of its two substrates ACC and eEF2. As expected, pharmacological activation of AMPK led to an increase in these parameters, though the three drugs differed in their relative potencies (Fig. 4A). Again, as anticipated, incubation in a low concentration of glucose also led to activation of AMPK. Surprisingly however, amino acids completely suppressed the activation of AMPK by glucose depletion. This effect was overcome by exposure of cells to pharmacological activators of AMPK (Fig. 4A). Amino acids not only suppressed AMPK activity, but also activated mTOR signaling, as indicated by increases in phosphorylation of p70S6K and rpS6. AICAR, phenformin or oligomycin antagonized this activation of mTOR and their potencies paralleled their abilities to stimulate AMPK phosphorylation. Activation of AMPK by AICAR partially blocked the phosphorylation of p70S6k and rpS6 at both 3 and 30 mm glucose, whereas phosphorylation of these proteins was completely blocked by oligomycin or phenformin. The ability of three independent activators of AMPK to inhibit mTOR signaling suggests that the latter is a result of AMPK activation. Amino acids play an important role in regulating initiation of translation in addition to their function as precursors for protein synthesis (26Hara 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 (1132) Google Scholar, 27Kimball S.R. Prog. Mol. Subcell. Biol. 2001; 26: 155-184Crossref PubMed Scopus (82) Google Scholar). The presence of amino acids, in particular, leucine, is essential for activation of mTOR in response to glucose or insulin. Also, amino acid mixtures as well as certain individual amino acids can stimulate insulin release from the β-cell. The effect of amino acids to stimulate both initiation of protein synthesis via mTOR activation and insulin release has been attributed in part to their mitochondrial metabolism and an increase in cellular ATP level (2MacDonald M.J. Fahien L.A. Brown L.J. Hasan N.M. Buss J.D. Kendrick M.A. Am. J. Physiol. Endocrinol. Metab. 2005; 288: E1-E15Crossref PubMed Scopus (202) Google Scholar, 28McDaniel M.L. Marshall C.A. Pappan K.L. Kwon G. Diabetes. 2002; 51: 2877-2885Crossref PubMed Scopus (104) Google Scholar). As shown in Fig. 4, we observed that the addition of amino acids to the media substantially reduced the effect of glucose to inhibit phosphorylation of AMPK and AMPK targets. As shown in Fig. 5A, MIN6 cells treated with either 0.25× or 1× amino acids for 30 min following a 2-h period of serum, glucose, and amino acid starvation, exhibited a dose-dependent decrease in AMPK phosphorylation as well as phosphor
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