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Glucagon Promotes cAMP-response Element-binding Protein Phosphorylation via Activation of ERK1/2 in MIN6 Cell Line and Isolated Islets of Langerhans

2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês

10.1074/jbc.m312483200

ISSN

1083-351X

Autores

Stéphane Dalle, Christine Longuet, Safia Costes, Christophe Broca, Omar Faruque, Ghislaine Fontès, El Habib Hani, D. Bataille,

Tópico(s)

Diabetes Treatment and Management

Resumo

By using the MIN6 cell line and pancreatic islets, we show that in the presence of a low glucose concentration, corresponding to physiological glucagon release from α cells, glucagon treatment of the β cell caused a rapid, time-dependent phosphorylation and activation of p44/p42 mitogen-activated protein kinase (ERK1/2) independently from extracellular calcium influx. Inhibition of either cAMP-dependent protein kinase (PKA) or MEK completely blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Shc-p21Ras and phosphatidylinositol 3-kinase, was observed in response to glucagon treatment. Chelation of intracellular calcium (intracellular [Ca2+]) reduced glucagon-mediated ERK1/2 activation. In addition, internalization of glucagon receptors through clathrin-coated pits formation is required for ERK1/2 activation. Remarkably, glucagon promotes the nuclear translocation of ERK1/2 and induces the phosphorylation of cAMP-response element-binding protein (CREB). Miniglucagon, produced from glucagon and released together with the mother hormone from the α cells in low glucose situations, blocks the insulinotropic effect of glucagon, whereas it does not inhibit the glucagon-induced PKA/ERK1/2/CREB pathway. We conclude that glucagon-induced ERK1/2 activation is mediated by PKA and that an increase in [Ca2+]i is required for maximal ERK activation. Our results uncover a novel mechanism by which the PKA/ERK1/2 signaling network engaged by glucagon, in situation of low glucose concentration, regulates phosphorylation of CREB, a transcription factor crucial for normal β cell function and survival. By using the MIN6 cell line and pancreatic islets, we show that in the presence of a low glucose concentration, corresponding to physiological glucagon release from α cells, glucagon treatment of the β cell caused a rapid, time-dependent phosphorylation and activation of p44/p42 mitogen-activated protein kinase (ERK1/2) independently from extracellular calcium influx. Inhibition of either cAMP-dependent protein kinase (PKA) or MEK completely blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Shc-p21Ras and phosphatidylinositol 3-kinase, was observed in response to glucagon treatment. Chelation of intracellular calcium (intracellular [Ca2+]) reduced glucagon-mediated ERK1/2 activation. In addition, internalization of glucagon receptors through clathrin-coated pits formation is required for ERK1/2 activation. Remarkably, glucagon promotes the nuclear translocation of ERK1/2 and induces the phosphorylation of cAMP-response element-binding protein (CREB). Miniglucagon, produced from glucagon and released together with the mother hormone from the α cells in low glucose situations, blocks the insulinotropic effect of glucagon, whereas it does not inhibit the glucagon-induced PKA/ERK1/2/CREB pathway. We conclude that glucagon-induced ERK1/2 activation is mediated by PKA and that an increase in [Ca2+]i is required for maximal ERK activation. Our results uncover a novel mechanism by which the PKA/ERK1/2 signaling network engaged by glucagon, in situation of low glucose concentration, regulates phosphorylation of CREB, a transcription factor crucial for normal β cell function and survival. Glucagon, produced by post-translational processing of proglucagon in the pancreatic α cells, is one of the major metabolic hormones (1Unger R.H. Orci L. Porte D. Sherwin R.S. Ellenberg Rifkin's Diabetes Mellitus, Theory and Practice. 5th Ed. Elsevier Science Publishing Co., Inc., New York1997: 115-139Google Scholar, 2Mojsov S. Heinrich G. Wilson I.B. Ravazzola M. Orci L. Habener J.F. J. Biol. Chem. 1986; 261: 11880-11889Abstract Full Text PDF PubMed Google Scholar, 3Unger R.H. Orci L. de Groot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 1337-1353Google Scholar, 4Cryer P.E. Horm. Res. 1996; 46: 192-194Crossref PubMed Scopus (11) Google Scholar). Glucagon is released from α cells in the interprandial state in a situation (hypoglycemia) that strongly differ from that in which incretins (3Unger R.H. Orci L. de Groot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 1337-1353Google Scholar, 4Cryer P.E. Horm. Res. 1996; 46: 192-194Crossref PubMed Scopus (11) Google Scholar), such as GIP or GLP-1, are released, that is during the early postprandial period preparing the β cell to the soon-coming glucose wave (5Vilsboll T. Krarup T. Madsbad S. Holst J.J. Regul. Pept. 2003; 114: 115-121Crossref PubMed Scopus (343) Google Scholar). Glucagon regulates the rate of glucose production in liver through both gluconeogenesis and glycogenolysis and consequently adjusts, in concert with insulin, blood glucose levels according to the needs of the organism (3Unger R.H. Orci L. de Groot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 1337-1353Google Scholar, 4Cryer P.E. Horm. Res. 1996; 46: 192-194Crossref PubMed Scopus (11) Google Scholar). To achieve its intracellular effects, glucagon binds to a glycoprotein receptor that spans the plasma membrane (1Unger R.H. Orci L. Porte D. Sherwin R.S. Ellenberg Rifkin's Diabetes Mellitus, Theory and Practice. 5th Ed. Elsevier Science Publishing Co., Inc., New York1997: 115-139Google Scholar, 3Unger R.H. Orci L. de Groot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 1337-1353Google Scholar, 6Jelinek L.J. Lok S. Rosenberg G.B. Smith R.A. Grant F.J. Biggs S. Bensch P. Kuijper J.L. Sheppard P.O. Sprecher C.A. O'Hara P.J. Foster D. Walker K.M. Chen L.H.J. McKernan P.A. Kindsvogel W. Science. 1993; 259: 1614-1616Crossref PubMed Scopus (366) Google Scholar, 7Birnbaumer L. Birnbaumer M. J. Recept. Signal. Transduct. Res. 1994; 15: 213-252Crossref Scopus (94) Google Scholar, 8Cypess A.M. Unson C.G. Wu C.R. Sakmar T.P. J. Biol. Chem. 1999; 274: 19455-19464Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The glucagon receptor (Gcgr) 1The abbreviations used are: Gcgr, glucagon receptor; CREB, cAMP-response element-binding protein; ERK1/2, extracellular signal-regulated kinase 1/2; PKA, cAMP-dependent protein kinase; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate/acetoxymethyl ester; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; PI, phosphatidylinositol; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; GPCRs, G protein-coupled receptors; VDCC, voltage-dependent calcium channel. 1The abbreviations used are: Gcgr, glucagon receptor; CREB, cAMP-response element-binding protein; ERK1/2, extracellular signal-regulated kinase 1/2; PKA, cAMP-dependent protein kinase; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate/acetoxymethyl ester; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; PI, phosphatidylinositol; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; GPCRs, G protein-coupled receptors; VDCC, voltage-dependent calcium channel. belongs to the class II (or B) family of heptahelical transmembrane G protein-coupled receptors (GPCRs) and is positively coupled to adenylate cyclase through the heterotrimeric Gs protein (6Jelinek L.J. Lok S. Rosenberg G.B. Smith R.A. Grant F.J. Biggs S. Bensch P. Kuijper J.L. Sheppard P.O. Sprecher C.A. O'Hara P.J. Foster D. Walker K.M. Chen L.H.J. McKernan P.A. Kindsvogel W. Science. 1993; 259: 1614-1616Crossref PubMed Scopus (366) Google Scholar, 7Birnbaumer L. Birnbaumer M. J. Recept. Signal. Transduct. Res. 1994; 15: 213-252Crossref Scopus (94) Google Scholar, 8Cypess A.M. Unson C.G. Wu C.R. Sakmar T.P. J. Biol. Chem. 1999; 274: 19455-19464Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The hormone binds to the Gcgr on the cell surface, causing it to interact with stimulatory guanine nucleotide-binding regulatory protein Gs. This liberates the α-subunit of Gs to stimulate adenylate cyclase that catalyzes the conversion of ATP to cAMP, leading to activation of cAMP-dependent protein kinase A (PKA) (6Jelinek L.J. Lok S. Rosenberg G.B. Smith R.A. Grant F.J. Biggs S. Bensch P. Kuijper J.L. Sheppard P.O. Sprecher C.A. O'Hara P.J. Foster D. Walker K.M. Chen L.H.J. McKernan P.A. Kindsvogel W. Science. 1993; 259: 1614-1616Crossref PubMed Scopus (366) Google Scholar, 7Birnbaumer L. Birnbaumer M. J. Recept. Signal. Transduct. Res. 1994; 15: 213-252Crossref Scopus (94) Google Scholar, 8Cypess A.M. Unson C.G. Wu C.R. Sakmar T.P. J. Biol. Chem. 1999; 274: 19455-19464Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Expression of functional Gcgrs in β cell lines, such as MIN6 (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and INS-1 cells (10Kieffer T.J. Heller R.S. Unson C.G. Weir G.C. Habener J.F. Endocrinology. 1996; 137: 5119-5125Crossref PubMed Scopus (60) Google Scholar), and isolated rat pancreatic β cells (10Kieffer T.J. Heller R.S. Unson C.G. Weir G.C. Habener J.F. Endocrinology. 1996; 137: 5119-5125Crossref PubMed Scopus (60) Google Scholar) has been reported, and a role for glucagon in islet hormone secretion during feeding and fasting has been proposed (10Kieffer T.J. Heller R.S. Unson C.G. Weir G.C. Habener J.F. Endocrinology. 1996; 137: 5119-5125Crossref PubMed Scopus (60) Google Scholar, 11Kawai K. Yokota C. Ohashi S. Watanabe Y. Yamashita K. Diabetologia. 1995; 38: 274-276Crossref PubMed Scopus (82) Google Scholar). Glucagon has been shown to potentiate the glucose-stimulated insulin release from β cell lines and from pancreatic islets (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 11Kawai K. Yokota C. Ohashi S. Watanabe Y. Yamashita K. Diabetologia. 1995; 38: 274-276Crossref PubMed Scopus (82) Google Scholar). In β cells models, glucagon increases cAMP, activates PKA which, in turn, phosphorylates the L-type voltage-dependent calcium channel (VDCC), leading to an increased glucose-induced calcium entry elicited through membrane depolarization, thus potentiating insulin secretion (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 11Kawai K. Yokota C. Ohashi S. Watanabe Y. Yamashita K. Diabetologia. 1995; 38: 274-276Crossref PubMed Scopus (82) Google Scholar). This observation is intriguing and paradoxical in that glucagon, which exhibits gluconeogenetic and glycogenolytic effects that are necessary for fighting against hypoglycemia, is also able to release insulin that drives the system in the opposite direction (3Unger R.H. Orci L. de Groot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 1337-1353Google Scholar, 4Cryer P.E. Horm. Res. 1996; 46: 192-194Crossref PubMed Scopus (11) Google Scholar, 9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 11Kawai K. Yokota C. Ohashi S. Watanabe Y. Yamashita K. Diabetologia. 1995; 38: 274-276Crossref PubMed Scopus (82) Google Scholar). We have demonstrated recently (12Dalle S. Fontes G. Lajoix A.D. LeBrigand L. Gross R. Ribes G. Dufour M. Barry L. LeNguyen D. Bataille D. Diabetes. 2002; 51: 406-412Crossref PubMed Scopus (29) Google Scholar) that miniglucagon, the C-terminal glucagon fragment also present in mature secretory granules of the α cells, is released together with glucagon in a low glucose situation (hypoglycemia). Miniglucagon completely blocks any possible insulinotropic action of glucagon, because this peptide is very potent in inhibiting insulin release by closing the β cell VDCC consecutively to plasma membrane repolarization (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 12Dalle S. Fontes G. Lajoix A.D. LeBrigand L. Gross R. Ribes G. Dufour M. Barry L. LeNguyen D. Bataille D. Diabetes. 2002; 51: 406-412Crossref PubMed Scopus (29) Google Scholar). Fully consistent with these findings, endogenously released glucagon from α cells has no effect on the magnitude of glucose-induced insulin secretion (13Moens K. Berger V. Ahn J.M. Van Schravendijk C. Hruby V.J. Pipeleers D. Schuit F. Diabetes. 2002; 51: 669-675Crossref PubMed Scopus (34) Google Scholar), although exogenous glucagon stimulates insulin secretion. On the other hand, glucagon has been shown to exert important long term effects on the pancreatic β cells. Glucagon contributes to the maintenance of the glucose-competent phenotype of native β cells, a key feature commonly impaired in physiopathology of type 2 diabetes (14Huypens P. Ling Z. Pipeleers D. Schuit F. Diabetologia. 2000; 43: 1012-1019Crossref PubMed Scopus (165) Google Scholar), and is required for the differentiation of precursor cells into insulin-secreting β cells in the embryonic pancreas (15Prasadan K. Daume E. Preuett B. Spilde T. Bhatia A. Kobayashi H. Hembree M. Manna P. Gittes G.K. Diabetes. 2002; 51: 3220-3322Crossref PubMed Scopus (61) Google Scholar). Although attention has been focused on the biological role of glucagon in the pancreatic islet physiology, much less is known about the identity and the role of the downstream signaling pathways linked to the Gcgr present in the pancreatic β cells. We first addressed the question of a possible positive coupling of endogenous β cell Gcgr, via cAMP-PKA pathway, to signaling components known to be linked to the tyrosine kinase receptor system. By using the MIN6 cell line and isolated rat pancreatic islets, we report that glucagon treatment of β cells caused a time-dependent activation of ERK1/2 at a low glucose concentration. We used pharmacological approaches to identify the signaling pathway leading from Gcgr activation to ERK1/2 activation. We observed that glucagon-induced MEK-ERK1/2 activities are mediated by the cAMP-PKA pathway but independently from p21Ras, PI 3-kinase, and extracellular calcium influx. An increase in intracellular [Ca2+] through mobilization of intracellular calcium pools was required for a maximal activation of ERK1/2 by PKA. In addition, Gcgr internalization process appears to participate in the ERK1/2 activation. Through nuclear translocation of ERK1/2, we found that glucagon induces phosphorylation of CREB, a major transcription factor allowing, by regulation of gene expression, the maintenance of the glucose-competent phenotype of the β cell, and the disruption of which in the β cell leads to type II diabetes (16Jhala U.S. Canettieri G. Screaton R.A. Kulkarni R.N. Krajewski S. Reed J. Walker J. Lin X. White M. Montminy M. Genes Dev. 2003; 17: 1575-1580Crossref PubMed Scopus (466) Google Scholar, 17Habener J.F. Vallejo M. Hoeffler J.P. Horm. Res. 1989; 32: 61-66Crossref PubMed Scopus (5) Google Scholar). In a second part of our study, we evaluated whether or not miniglucagon, which we have shown to be co-released with glucagon from α cell (12Dalle S. Fontes G. Lajoix A.D. LeBrigand L. Gross R. Ribes G. Dufour M. Barry L. LeNguyen D. Bataille D. Diabetes. 2002; 51: 406-412Crossref PubMed Scopus (29) Google Scholar), exerts a counter-regulation on the signaling network engaged by Gcgr in the β cells. Most interesting, we found that miniglucagon, which blocks the insulinotropic effect of glucagon (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 12Dalle S. Fontes G. Lajoix A.D. LeBrigand L. Gross R. Ribes G. Dufour M. Barry L. LeNguyen D. Bataille D. Diabetes. 2002; 51: 406-412Crossref PubMed Scopus (29) Google Scholar), does not inhibit the glucagon-induced PKA/ERK1/2/CREB signaling pathway leaving untouched the glucagon access to the β cell transcription machinery. Materials—Glucagon, somatostatin, and 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetate/acetoxymethyl ester (BAPTA/AM) were purchased from Calbiochem. Glucagon-like peptide-1-(7-36)-amide (GLP-1) was from Peninsula Laboratories (San Carlos, CA). Miniglucagon was synthesized in our laboratory (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Mouse monoclonal antiphospho-ERK1/2 (p44/42 MAP kinase) antibody, which selectively recognizes the doubly phosphorylated, active forms of these kinases, mouse monoclonal anti-phosphoserine 133-CREB antibody, which detects endogenous levels of CREB specifically when phosphorylated at Ser133 (and also the phosphorylated form of the CREB-related protein ATF-1), and rabbit polyclonal anti-CREB antibody were obtained from Cell Signaling Technology (New England Biolabs, Beverly, MA). Anti-ERK1/2 and anti-Shc proteins antibodies were from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-linked anti-rabbit and anti-mouse antibodies, antiphosphotyrosine (PY20) antibody, and protein A/G-Plus-agarose were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dulbecco's modified Eagle's medium and fetal calf serum were purchased from Invitrogen. Nitrocellulose transfer membranes (Protran) were purchased from Schleicher & Schuell. 45CaCl2 was obtained from Amersham Biosciences. All other reagents were purchased from Sigma. Pancreatic Islet Preparation and Cell Culture—Pancreatic islet were isolated from fed male Wistar rats (Iffa-Credo, France) weighing 280-320 g the day of sacrifice. Islets were isolated by collagenase digestion followed by Ficoll gradient separation, as described previously (18Lacy P.E. Kostianovsky M. Diabetes. 1967; 16: 35-39Crossref PubMed Scopus (2480) Google Scholar). For Western blotting experiments, pancreatic islets were stabilized for 2 h at 37 °C in HEPES-balanced Krebs-Ringer bicarbonate buffer (119 mm NaCl; 4 mm KCl; 1.2 mm KH2PO4; 1.2 mm MgSO4; 2.5 mm CaCl2; 20 mm HEPES, pH 7.2) containing 0.1% bovine serum albumin (BSA) (KRB buffer) and 2.8 mm glucose, either in the absence or the presence of inhibitor. Islets were then washed and further incubated by groups of 100 islets for 10 min at 37 °C in KRB buffer supplemented with stimulants and inhibitor as described in the figure legends. Following the 10-min incubation, islets were rapidly centrifuged, and 1 μl/islet of cold lysis buffer (50 mm HEPES, 1% Nonidet P-40, 1 mm Na3VO4, 100 mm NaF, 10 mm pyrophosphate, 4 mm EDTA, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml leupeptin, 1 μg/ml aprotinin) was added. After 30 min of incubation in lysis buffer, islets were sonicated (10 s) and stored at -20 °C until use for subsequent protein determination by Bradford assay and Western blotting experiments. MIN6 cells were cultured as described previously (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 16Jhala U.S. Canettieri G. Screaton R.A. Kulkarni R.N. Krajewski S. Reed J. Walker J. Lin X. White M. Montminy M. Genes Dev. 2003; 17: 1575-1580Crossref PubMed Scopus (466) Google Scholar, 19Gomez E. Pritchard C. Herbert T.P. J. Biol. Chem. 2002; 277: 48146-48151Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) in Dulbecco's modified Eagle's medium containing 25 mm glucose supplemented with 15% fetal calf serum, 100 μg/ml streptomycin, 100 units/ml penicillin sulfate, and 50 μm β-mercaptoethanol. Cultures were never allowed to become completely confluent. Each batch of MIN6 cells used in the experiments was tested for the dose-dependent insulin response to glucose that always was in the physiological range (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Measurement of cAMP Production—MIN6 cells were grown in 24-well plates and incubated in Dulbecco's modified Eagle's medium without fetal calf serum containing 1% BSA, 1 mm isobutylmethylxanthine as an inhibitor of cAMP phosphodiesterase, and the test substances. After a 10-min incubation at room temperature, cells were extracted using 60% perchloric acid. Samples were neutralized with 9 n KOH and succinylated to increase the sensitivity of the assay, and cAMP was quantified by radioimmunoassay (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Measurement of Ca2+ Influx—MIN6 cells were preincubated for 30 min at 37 °C in KRB containing 0.1% BSA and 1 mm glucose in a 5% CO2 environment. The preincubation solution was then replaced by KRB containing 8 μCi/ml 45CaCl2 (5-50 mCi/mg Ca+) and the test agents. The reaction, performed at 37 °C, was stopped by aspiration of the medium. Cells were then washed with ice-cold buffer (135 mm NaCl, 5 mm KCl, 2.5 mm CaCl2, 1 mm LaCl2, 10 mm HEPES) and solubilized in KRB containing 0.1% Triton for 1 h at room temperature. An aliquot of the solution (100 μl) was then assayed for 45Ca2+ content in a β counter after addition of a liquid scintillation medium (Complete Phase Combining system, Amersham Biosciences). Western Blotting and Immunoprecipitation—After a 2-h stabilization period at 37 °C in KRB buffer (16Jhala U.S. Canettieri G. Screaton R.A. Kulkarni R.N. Krajewski S. Reed J. Walker J. Lin X. White M. Montminy M. Genes Dev. 2003; 17: 1575-1580Crossref PubMed Scopus (466) Google Scholar, 19Gomez E. Pritchard C. Herbert T.P. J. Biol. Chem. 2002; 277: 48146-48151Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), either in the absence or the presence of inhibitors, 6-well plates of MIN6 cells (60-80% confluency) were incubated in KRB containing different glucose concentrations and other test agents for various times as indicated in figure legends. MIN6 cells were then lysed in a cold lysis buffer containing 50 mm HEPES, 1 mm EDTA, 1% Triton X-100, 0.1% SDS, 1 mm Na3VO4, 1 mm PMSF, 10 mm pyrophosphate, 100 mm NaF, and 1 mg/ml bacitracin. After a 30-min incubation in lysis buffer, cell lysates were centrifuged at 14,000 rpm for 30 min to remove insoluble materials. Protein content was determined by Bradford assay. Cell lysates were denatured by boiling for 3 min in Laemmli sample buffer containing 100 mm dithiothreitol. Equal amounts of lysate proteins (25-35 μg of protein per lane) were resolved by SDS-PAGE. For immunoprecipitation, the supernatants (400-800 μg of total protein) were incubated with primary antibody as indicated for 4 h at 4 °C. Immunocomplexes were precipitated from the supernatant with protein A/G-Plus agarose, washed three times with ice-cold cell lysis buffer, and boiled for 3 min in Laemmli sample buffer and resolved by SDS-PAGE. Nitrocellulose membranes were blocked, probed with the specific antibodies, and incubated with horseradish peroxidase-linked secondary antibody followed by enhanced chemiluminescence detection. Visualization and quantification of the bands were obtained using a Kodak Image Station 2000 system (Eastman Kodak Co.). Immunostaining for Phospho-p44/42 MAP Kinase—MIN6 cells were grown on glass coverslips for 3-5 days. Cells were fixed with 3.7% formaldehyde for 30 min at 4 °C. Cells were permeabilized by incubation with 0.1 m Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Triton X-100 (TBST buffer) for 10 min. After incubation in blocking buffer (5% normal horse serum in TBST buffer) for 1 h at room temperature, coverslips were probed with the anti-phospho-ERK1/2 primary antibody in 5% BSA TBST buffer overnight at 4 °C (1:400 dilution). After washing, phospho-ERK1/2 antibody was detected by incubation with fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:100 in 3% BSA TBST, for 1 h at room temperature. Coverslips were mounted using a polyvinyl alcohol medium at least 1 h before observation using a Zeiss dual photon confocal microscope (Oberkocher, Germany). Subcellular Fractionation—After 2 h of starvation in KRB, 10-cm dishes plated MIN6 cells were stimulated with 10 nm glucagon for 5 min. Cells are washed twice with ice-cold phosphate-buffered saline and scraped in 500 μl of hypotonic buffer (10 mm HEPES; 10 mm NaCl; 1 mm KH2PO4; 5 mm NaHCO3; 1 mm CaCl2; 0.5 mm MgCl2; 5 mm EDTA, pH 8; 1 mm PMSF; 10 μg/ml aprotinin; 10 μg/ml leupeptin; 1 μg/ml pepstatin). After 15 min of incubation, cells were disrupted (50 times) using a Dounce homogenizer (pestle B) at 4 °C and centrifuged for 5 min at 7500 rpm at 4 °C. Supernatant containing cytosol and membranes was collected and preserved for protein content determination and Western blot analysis, and the pellet containing nuclei was disrupted (30 times) using a Dounce homogenizer (pestle B) in 10 mm Tris, pH 7.5, 300 mm sucrose, 1 mm EDTA, pH 8, 0.1% Nonidet P-40, 1 mm PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin (TSE buffer) and centrifuged for 5 min at 5000 rpm at 4 °C. Final pellet containing pure nuclei was dissolved in TSE buffer for protein determination before denaturation in Laemmli buffer and analysis by SDS-PAGE. Statistical Analysis—Results are presented as means ± S.E. Differences between results were analyzed by Student's t test for unpaired data. ERK1/2 Phosphorylation by Glucagon in MIN6 Cells—We have demonstrated previously (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) that activated Gcgrs induce cAMP production through adenylate cyclase activation in MIN6 cells. Because the cAMP/PKA pathway has been shown to activate the ERK1/2 signaling cascade in β cells (19Gomez E. Pritchard C. Herbert T.P. J. Biol. Chem. 2002; 277: 48146-48151Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 20Frödin M. Sekine N. Roche E. Filloux C. Prentki M. Wollheim C.B. Van Obberghen E. J. Biol. Chem. 1995; 270: 7882-7889Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 21Arnette D. Gibson T.B. Lawrence M.C. January B. Khoo S. McGlynn K. Vanderbilt C.A. Cobb M.H. J. Biol. Chem. 2003; 278: 32517-32525Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), we evaluated whether glucagon activates ERK1/2 under noninsulinotropic (low) glucose concentration (2.8 mm), which corresponds to physiological conditions for glucagon release from pancreatic α cells in vivo, when glucagon is present in extracellular spaces inside the islet at the vicinity of the insulin-secreting β cells (3Unger R.H. Orci L. de Groot L.J. Endocrinology. 3rd Ed. W. B. Saunders Co., Philadelphia1995: 1337-1353Google Scholar, 4Cryer P.E. Horm. Res. 1996; 46: 192-194Crossref PubMed Scopus (11) Google Scholar). In this glucose situation, glucagon has no effect on insulin secretion (9Dalle S. Smith P. Blache P. Le-Nguyen D. Le Brigand L. Bergeron F. Ashcroft F.M. Bataille D. J. Biol. Chem. 1999; 274: 10869-10876Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 12Dalle S. Fontes G. Lajoix A.D. LeBrigand L. Gross R. Ribes G. Dufour M. Barry L. LeNguyen D. Bataille D. Diabetes. 2002; 51: 406-412Crossref PubMed Scopus (29) Google Scholar, 13Moens K. Berger V. Ahn J.M. Van Schravendijk C. Hruby V.J. Pipeleers D. Schuit F. Diabetes. 2002; 51: 669-675Crossref PubMed Scopus (34) Google Scholar). As shown in Fig. 1 (A and B), in the presence of a non-insulinotropic glucose concentration (2.8 mm), MIN6 cells exhibited a small but significant transient phosphorylation of ERK1/2 (Fig. 1A). The amounts of phosphorylated ERK2 (p42 MAP kinase) peaked at 5 min and returned to the basal level by 10-20 min (Fig. 1B). Addition of 10 nm glucagon significantly enhanced glucose-induced activation of ERK1/2 at all time points (Fig. 1, A and B). We also tested the hypothesis whether activated Gcgrs transduce ERK1/2 cascade in MIN6 cells in the absence of glucose. Most interesting, we observed that, even under these extra-physiological conditions, glucagon alone induced a rapid and transient phosphorylation of ERK1/2 with a maximal effect observed at 5 min which returned to basal by 30 min (Fig. 1, C and D). Mechanisms of Glucagon-induced ERK1/2 Activation—We used pharmacological approaches to identify the mechanisms of glucagon-induced ERK1/2 activation in the absence of glucose during the glucagon stimulation. We tested the hypothesis that, under non-physiological conditions in which there is no calcium influx, a mechanism linked to the cAMP/PKA pathway exists in the β cells, activating the ERK1/2 cascade independently from calcium entry. In line with the fact that glucagon alone had no significant effect on basal calcium uptake (Fig. 7A), we observed that glucagon-mediated ERK1/2 activation in the absence of glucose (10 min) (Fig. 2, A and B), or in the presence of 2.8 mm glucose (data not shown), was not significantly inhibited by the VDCC blocker nifedipine. The role of PKA in the ERK1/2 activation by glucagon was addressed in this context. The complete suppression of the glucagon-induced ERK1/2 activation by a non-cytotoxic treatment with a low concentration of PKA-specific inhibitor (H89) clearly indicated that the glucagon-stimul

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