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• W2111497148 abstract "We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency. We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency. The essential function of the pancreatic β cell is to secrete insulin in response to increases in circulating glucose. Other nutrients, hormones, and paracrine agents influence pancreatic β cell functions and insulin secretion to optimize glucose homeostasis. Ligands for several G protein-coupled receptors (GPCRs) 3The abbreviations used are:GPCRG protein-coupled receptormAChRmuscarinic acetylcholine receptorERendoplasmic reticulumM3RM3 muscarinic acetylcholine receptor2-APB2-aminoethoxydiphenyl borateSOCEstore-operated calcium entryROCEreceptor-operated calcium entrypERKphospho-ERKRGSregulator of G protein signalingSOCCstore-operated calcium channelKRBHKrebs-Ringer bicarbonate solution. are among the most significant in tuning insulin secretion from β cells, which express several different classes of GPCRs, including muscarinic acetylcholine receptors (mAChRs) (1Gautam D. Ruiz de Azua I. Li J.H. Guettier J.M. Heard T. Cui Y. Lu H. Jou W. Gavrilova O. Zawalich W.S. Wess J. Beneficial metabolic effects caused by persistent activation of β-cell M3 muscarinic acetylcholine receptors in transgenic mice.Endocrinology. 2010; 151: 5185-5194Crossref PubMed Scopus (34) Google Scholar). G protein-coupled receptor muscarinic acetylcholine receptor endoplasmic reticulum M3 muscarinic acetylcholine receptor 2-aminoethoxydiphenyl borate store-operated calcium entry receptor-operated calcium entry phospho-ERK regulator of G protein signaling store-operated calcium channel Krebs-Ringer bicarbonate solution. Five mAChRs subtypes, M1-M5, have been identified (2Wess J. Molecular biology of muscarinic acetylcholine receptors.Crit. Rev. Neurobiol. 1996; 10: 69-99Crossref PubMed Scopus (423) Google Scholar). The M1, M3, and M5 subtypes are Gq-coupled receptors, whereas M2 and M4 are Gi-coupled receptors that are inhibited by pertussis toxin (3Bonner T.I. New subtypes of muscarinic acetylcholine receptors.Trends Pharmacol. Sci. Suppl. 1989; : 11-15PubMed Google Scholar). β cell muscarinic receptors are Gq-coupled because binding of acetylcholine to these receptors results in the well characterized action of Gq to activate phospholipase C β. The resulting hydrolysis of phosphatidylinositol 4,5-bisphosphate generates the second messenger inositol 1,4,5-triphosphate, which binds to its receptor on the ER and induces calcium release from intracellular stores (4Berridge M.J. Bootman M.D. Roderick H.L. Calcium signalling: dynamics, homeostasis and remodelling.Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4189) Google Scholar). Muscarinic agonist-induced mobilization of intracellular calcium (Ca2+i) was absent in mice selectively lacking two members of the β cell Gq protein family, Gαq and Gα11 (5Sassmann A. Gier B. Gröne H.J. Drews G. Offermanns S. Wettschureck N. The Gq/G11-mediated signaling pathway is critical for autocrine potentiation of insulin secretion in mice.J. Clin. Invest. 2010; 120: 2184-2193Crossref PubMed Scopus (59) Google Scholar). The M3 mAChR (M3R) is the predominant receptor subtype expressed in β cells and insulin-secreting cell lines (6Iismaa T.P. Kerr E.A. Wilson J.R. Carpenter L. Sims N. Biden T.J. Quantitative and functional characterization of muscarinic receptor subtypes in insulin-secreting cell lines and rat pancreatic islets.Diabetes. 2000; 49: 392-398Crossref PubMed Scopus (74) Google Scholar, 7Ruiz de Azua I Scarselli M. Rosemond E. Gautam D. Jou W. Gavrilova O. Ebert P.J. Levitt P. Wess J. RGS4 is a negative regulator of insulin release from pancreatic β-cells in vitro and in vivo.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 7999-8004Crossref PubMed Scopus (60) Google Scholar). Parasympathetic nerve endings that innervate the pancreas release acetylcholine during the preabsorptive and absorptive phases of feeding (8Ahrén B. Autonomic regulation of islet hormone secretion: implications for health and disease.Diabetologia. 2000; 43: 393-410Crossref PubMed Scopus (694) Google Scholar) to activate this receptor. Studies utilizing M3R knockout mice have implicated M3R as the receptor subtype responsible for cholinergic potentiation of glucose-stimulated insulin secretion (9Duttaroy A. Zimliki C.L. Gautam D. Cui Y. Mears D. Wess J. Muscarinic stimulation of pancreatic insulin and glucagon release is abolished in m3 muscarinic acetylcholine receptor-deficient mice.Diabetes. 2004; 53: 1714-1720Crossref PubMed Scopus (160) Google Scholar, 10Zawalich W.S. Zawalich K.C. Tesz G.J. Taketo M.M. Sterpka J. Philbrick W. Matsui M. Effects of muscarinic receptor type 3 knockout on mouse islet secretory responses.Biochem. Biophys. Res. Commun. 2004; 315: 872-876Crossref PubMed Scopus (33) Google Scholar). Furthermore, mice selectively deficient in β cell M3Rs demonstrated impaired glucose tolerance and reduced insulin release, whereas mice overexpressing M3Rs in β cells exhibited a significant increase in glucose tolerance and insulin release (11Gautam D. Han S.J. Hamdan F.F. Jeon J. Li B. Li J.H. Cui Y. Mears D. Lu H. Deng C. Heard T. Wess J. A critical role for β cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.Cell Metab. 2006; 3: 449-461Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Similar observations were made in mice overexpressing constitutively active β cell M3Rs (1Gautam D. Ruiz de Azua I. Li J.H. Guettier J.M. Heard T. Cui Y. Lu H. Jou W. Gavrilova O. Zawalich W.S. Wess J. Beneficial metabolic effects caused by persistent activation of β-cell M3 muscarinic acetylcholine receptors in transgenic mice.Endocrinology. 2010; 151: 5185-5194Crossref PubMed Scopus (34) Google Scholar). Signaling by the M3R also activates ERK1/2 in β cells, most likely downstream of elevated intracellular calcium (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 13Jain S. Ruiz de Azua I Lu H. White M.F. Guettier J.M. Wess J. Chronic activation of a designer G(q)-coupled receptor improves β cell function.J. Clin. Invest. 2013; 123: 1750-1762Crossref PubMed Scopus (101) Google Scholar, 14Selway J.L. Moore C.E. Mistry R. John Challiss R.A. Herbert T.P. Molecular mechanisms of muscarinic acetylcholine receptor-stimulated increase in cytosolic free Ca2+ concentration and ERK1/2 activation in the MIN6 pancreatic β-cell line.Acta Diabetol. 2012; 49: 277-289Crossref PubMed Scopus (11) Google Scholar). ERK1/2 activation enhances insulin gene transcription following nutrient-induced insulin secretion (15Benes C. Poitout V. Marie J.C. Martin-Perez J. Roisin M.P. Fagard R. Mode of regulation of the extracellular signal-regulated kinases in the pancreatic β-cell line MIN6 and their implication in the regulation of insulin gene transcription.Biochem. J. 1999; 340: 219-225Crossref PubMed Scopus (51) Google Scholar16Khoo S. Griffen S.C. Xia Y. Baer R.J. German M.S. Cobb M.H. Regulation of insulin gene transcription by extracellular-signal regulated protein kinases (ERK) 1 and 2 in pancreatic β cells.J. Biol. Chem. 2003; 278: 32969-32977Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 17Lawrence M.C. McGlynn K. Park B.H. Cobb M.H. ERK1/2-dependent activation of transcription factors required for acute and chronic effects of glucose on the insulin gene promoter.J. Biol. Chem. 2005; 280: 26751-26759Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18Lawrence M.C. McGlynn K. Shao C. Duan L. Naziruddin B. Levy M.F. Cobb M.H. Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in β-cells.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13315-13320Crossref PubMed Scopus (55) Google Scholar). We have reported previously that the GPCR complex T1R1/T1R3 is an early amino acid sensor in the MIN6 pancreatic β cell line and in other cell types (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Similar to M3R, T1R1/T1R3 activation leads to a rise in Ca2+i and ERK1/2 phosphorylation that is partially dependent upon phospholipase C β activation. Reduced expression of T1R3 in MIN6 cells resulted in a decrease of amino acid-induced ERK1/2 and mammalian target of rapamycin complex 1 activation. Signaling defects in cells in which the receptor had been depleted included a reduction in the ability of amino acids to induce changes in Ca2+i (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Despite the impaired ability of amino acids to stimulate ERK1/2 in T1R3-depleted MIN6 cells, carbachol, a muscarinic receptor agonist, activated ERK1/2 better in T1R3-depleted cells than in control cells (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We explored the underlying mechanisms for the enhanced carbachol response in MIN6 cells to determine whether similar mechanisms were enlisted to compensate for amino acid deficiency. Fura-2/AM was purchased from Molecular Probes. Nifedipine was purchased from Calbiochem. 2-Aminoethoxydiphenyl borate (2-APB) was purchased from Sigma. Thapsigargin was purchased from Santa Cruz Biotechnology. MIN6 cells were cultured, and stable cell lines with T1R3 expression reduced following expression of a short hairpin were created and maintained as described previously (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Cells were plated at 80% confluency in white-walled, 96-well plates (Costar 3903). After 48 h, the cells were washed twice with PBS (0.2 ml/well) and incubated with 5 μm Fura-2/AM diluted in Krebs-Ringer bicarbonate solution (KRBH) containing 115 mm NaCl, 5 mm KCl, 24 mm NaHCO3, 1 mm MgCl2, 2.5 mm CaCl2, 25 mm HEPES (pH 7.4), 0.1% BSA, and 4.5 mm glucose for 1 h (0.1 ml/well). Cells were then washed twice with KRBH (0.2 ml/well) and equilibrated in the same buffer for 30 min (0.1 ml/well). Agents were applied (0.1 ml/well) to triplicate wells at 2× concentrations using injectors at a rate of 225 μl/s. Changes in Ca2+i were assessed every 0.74 s by dual excitation of Fura-2 at 340/11 and 380/20 nm (center/bandpass) and emission at 508/20 nm using the SynergyTM 2 multimode microplate reader (BioTek) with Gen5TM software. Cells were pretreated with the indicated inhibitors for 30 min prior to stimulation. For experiments performed in the absence of calcium, cells were loaded, washed, and equilibrated with calcium-free KRBH in which MgCl2 was substituted for 2.5 mm CaCl2. To assess store-operated calcium entry (SOCE), intracellular stores were depleted using 10 μm thapsigargin. Calcium was then replenished with a second injection of KRBH containing 12.5 mm CaCl2 (5× concentration, 50 μl/well). To assess receptor-operated calcium entry (ROCE), after calcium repletion, a third injection was required to apply 0.6 mm carbachol (6× concentration, 50 μl/well). Final concentrations of all agents were 1×. For experiments involving nifedipine or 2-APB, cells were pretreated with inhibitors for 30 min prior to stimulation. All steps in each assay were performed at room temperature. MIN6 cells were plated as above for calcium assays or in 12-well plates for RNA or protein isolation. To examine the effects of reduced amino acids, cells nearing confluency were washed twice with PBS and incubated with KRBH supplemented with 10% dialyzed serum, 4.5 mm glucose, and either 1.0× amino acids (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) or 0.1× amino acids for 16 h at 37 °C and 10% CO2 prior to stimulation with carbachol or cell lysis. Calcium was measured as above with reduced amino acids throughout. To examine the effects of reduced glucose, cells were incubated as above in KRBH containing 10% dialyzed serum, 1× amino acids, and either 25 or 2 mm glucose. Human islets were provided by the Integrated Islet Distribution Program. Islets were washed twice in KRBH and then once in KRBH containing 10% dialyzed serum, 4.5 mm glucose, and either 0.1× or 1× amino acids prior to treatment overnight. Cells were lysed in 50 mm HEPES (pH 7.5), 150 mm NaCl, 1% Triton X-100, 10 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 0.2 mg/ml PMSF, 100 mm NaF, and 2 mm Na3VO4. For lysates from stable cell lines, 40 μg of protein, as determined by BCA assay (Pierce), was resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature. Membranes were incubated with primary antibodies overnight at 4 °C. Antibodies were diluted in 5% milk/TBST as follows: M3R muscarinic receptor (1:250, Millipore, catalog no. AB9018, rabbit, polyclonal) and ERK1/2 (1:2000, Abcam, catalog no. ab54230, mouse, monoclonal). For the carbachol time course, pERK1/2/ERK1/2 blots used 20 μg of protein. Antibodies were diluted in 5% milk/TBST as follows: pERK1/2 (1:1000, Sigma, catalog no. M8159, mouse, monoclonal) and ERK1/2 (1:1000, 691 rabbit (19Boulton T.G. Cobb M.H. Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies.Cell Regul. 1991; 2: 357-371Crossref PubMed Scopus (281) Google Scholar)). The membranes were then washed with TBST and incubated for 1 h at room temperature with secondary antibodies: donkey anti-rabbit IRDye 680RD or donkey anti-mouse IRDye 800CW (1:10,000, Li-Cor Biosciences). The membranes were washed with TBST and then imaged using the LI-Cor Odyssey infrared imaging system. Blots were quantified using Li-Cor Odyssey application software (version 3.0). Cells or human islets were harvested in TRI reagent® solution, and RNA was extracted according to the instructions of the manufacturer (Applied Biosystems). cDNA was generated using a high-capacity cDNA reverse transcription kit (Applied Biosystems). SYBR Green Supermix with ROX was purchased from Bio-Rad. GAPDH was used as an internal expression control. The primers were as follows: GAPDH, 5′-CTGGAGAAACCTGCCAAGTA-3′ (forward) and 5′-TGTTGCTGTAGCC GTATTCA-3′ (reverse); actin, 5′-AGGTCATCACTATTGCAACGA-3′ (forward) and 5′-CACTTCATGATGGAATTGAATGTAGTT-3′ (reverse); M3R (human) muscarinic receptor, 5′-ATTAAGCACTTGTGTTCTGATTAGT-3′ (forward) and 5′-CACGCCACAGCAAAACCTTA-3′ (reverse); M1R muscarinic receptor, 5′-CCCTGGCAGGTGGCCTTC ATC-3′ (forward) and 5′ AGCACAGGCCAGGCTCAGCAG-3′ (reverse); mouse M3R muscarinic receptor, 5′-ACAGCCACCTGGAG CACGGC-3′ (forward) and 5′-AACGCAGCACTTCAAGAGGAGAGTC-3′ (reverse); M5R muscarinic receptor, 5′-GGCCAAGAAGAGGGGAGGCCA-3′ (forward) and 5′-CCGGGGTGCCGTTGACAGTG-3′ (reverse); Gαq, 5′-GCCGACCCTTCCTATCTGC-3′ (forward) and 5′-CCCCCTACATCGAC CATTCTGA-3′ (reverse); RGS4, 5′-TCTGCCGGCTTCCTGCCTGA-3′ (forward) and 5′-TCTTGGCTTACCCTCTGGCAAGTT-3′ (reverse); and TRPC6, 5′-GGAGACGACGGCTACCCG CA-3′ (forward) and 5′-AATCGTCTGCCGCCGGTGAG-3′ (reverse). Validated T1R3 primers were purchased from Bio-Rad (unique assay no. qmmuCED0004159). Results were expressed as means ± S.E. determined from three independent experiments. Statistical significance was calculated using Student's t test. To determine the basis for the increased carbachol-induced ERK1/2 phosphorylation in MIN6 cells with reduced T1R3 expression (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), we first examined the effect of carbachol on Ca2+i after loading cells with the ratiometric calcium indicator Fura-2. Stimulation of β cells with carbachol produced a biphasic rise in Ca2+i, composed of a rapid and transient peak followed by a sustained plateau phase (20Gylfe E. Carbachol induces sustained glucose-dependent oscillations of cytoplasmic Ca2+ in hyperpolarized pancreatic β cells.Pflugers Arch. 1991; 419: 639-643Crossref PubMed Scopus (40) Google Scholar, 21Liu Y.J. Gylfe E. Store-operated Ca2+ entry in insulin-releasing pancreatic β-cells.Cell Calcium. 1997; 22: 277-286Crossref PubMed Scopus (58) Google Scholar). Compared with the control, we observed a larger rise in peak Ca2+i in MIN6 cells with depleted T1R3 as well as a faster decline during the second phase of the carbachol response (Fig. 1, A and B). One possible reason for enhanced carbachol-induced changes in Ca2+i in MIN6 T1R3 knockdown cells could be increased expression of mAChRs. Therefore, we performed real -time quantitative PCR to compare changes in expression of the Gq-coupled mAChRs, M1, M3, and M5, in MIN6 control and T1R3 knockdown cells. There was 2.35 + 0.06-fold increase in M3R mRNA in the T1R3knockdown cells compared with the control, whereas the expression of M1 and M5 receptors did not change significantly (Fig. 1C). The increase in M3R mRNA in T1R3 knockdown cells was mirrored by an increase in M3R protein expression that was found by immunoblotting lysates from control and T1R3 knockdown cells (Fig. 1D). Quantitation of the immunoblot analyses indicated a 3.8 + 0.9-fold increase in M3R protein in MIN6 cells with suppressed T1R3 expression (Fig. 1D). In addition to mAChRs, we also examined the expression of the regulator of G protein signaling 4 (RGS4) and transient receptor potential channel 6 (TRPC6). RGS proteins are GTPase-activating proteins that enhance Gα-GTP hydrolysis, thereby decreasing the lifetime of active states of G protein subunits, and RGS4 is expressed in β cells (22Ross E.M. Wilkie T.M. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins.Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (926) Google Scholar). TRPC6 has been reported to be a receptor-operated cation channel that is activated upon GPCR stimulation and subsequent phospholipase C β activation (23Estacion M. Li S. Sinkins W.G. Gosling M. Bahra P. Poll C. Westwick J. Schilling W.P. Activation of human TRPC6 channels by receptor stimulation.J. Biol. Chem. 2004; 279: 22047-22056Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Zhang L. Guo F. Kim J.Y. Saffen D. Muscarinic acetylcholine receptors activate TRPC6 channels in PC12D cells via Ca2+ store-independent mechanisms.J. Biochem. 2006; 139: 459-470Crossref PubMed Scopus (16) Google Scholar). Calcium entry occurring through plasma membrane channels as a result of GPCR activation independent of the state of Ca2+i stores is referred to as ROCE (25Hofmann T. Schaefer M. Schultz G. Gudermann T. Transient receptor potential channels as molecular substrates of receptor-mediated cation entry.J. Mol. Med. 2000; 78: 14-25Crossref PubMed Scopus (0) Google Scholar). It has been demonstrated that carbachol is capable of inducing ROCE by activating and promoting cell surface expression of TRPC6 downstream of muscarinic receptor binding (26Boulay G. Ca2+-calmodulin regulates receptor-operated Ca2+ entry activity of TRPC6 in HEK-293 cells.Cell Calcium. 2002; 32: 201-207Crossref PubMed Scopus (75) Google Scholar, 27Cayouette S. Lussier M.P. Mathieu E.L. Bousquet S.M. Boulay G. Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq protein-coupled receptor activation.J. Biol. Chem. 2004; 279: 7241-7246Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Expression of neither RGS4 nor TRPC6 was significantly different in T1R3 knockdown cells compared with the control, suggesting that the enhanced carbachol response was not a result of decreased RGS4 or increased TRPC6 expression (Fig. 1C). The initial rapid rise in calcium observed upon carbachol stimulation in β cells has been shown to be due to the inositol 1,4,5-triphosphate-mediated release of calcium from ER stores, whereas the second phase is maintained by SOCE (21Liu Y.J. Gylfe E. Store-operated Ca2+ entry in insulin-releasing pancreatic β-cells.Cell Calcium. 1997; 22: 277-286Crossref PubMed Scopus (58) Google Scholar). SOCE refers to calcium influx that occurs through store-operated calcium channels (SOCCs) as a result of Ca2+i store depletion (28Parekh A.B. Penner R. Store depletion and calcium influx.Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1289) Google Scholar). We investigated whether the contributions of Ca2+i stores or SOCE to carbachol-induced changes in Ca2+i differed in MIN6 cells with suppressed T1R3 expression compared with control cells. Cells were stimulated with carbachol in the absence of extracellular calcium to determine whether activation of M3R in the T1R3 knockdown cells induced a larger rise in Ca2+i as a result of the release of calcium from intracellular stores. We found that carbachol stimulated a larger rise in Ca2+i in the absence of extracellular calcium in the T1R3 knockdown cells compared with control cells (Fig. 2, A and B). This finding suggests that, in addition to elevated M3R, a larger release of calcium from intracellular stores may contribute to the enhanced first phase of the carbachol response in the MIN6 T1R3 knockdown cells. We next determined whether the larger carbachol-induced release from intracellular stores was a consequence of more calcium stored in the ER of cells with reduced T1R3 by treating cells with thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase, to deplete ER stores of calcium (29Lytton J. Westlin M. Hanley M.R. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar). In the absence of extracellular calcium, thapsigargin induced a similar rise in Ca2+i in T1R3 knockdown and control cells (Fig. 2, C and D). When extracellular calcium was replenished, allowing SOCE to occur, calcium entry through SOCCs was similar in both T1R3 knockdown and control cells. We also explored ROCE, after calcium restoration and SOCE had occurred, by stimulating cells with carbachol. Despite little or no change in TRPC6 expression in MIN6 cells depleted of T1R3, ROCE was absent (Fig. 2, C and D), suggesting that another, as yet unidentified channel may contribute to ROCE in MIN6 cells. Consistent with the larger release of calcium from intracellular stores induced by carbachol, a greater portion of the peak carbachol response was sensitive to inhibition by thapsigargin in the T1R3 knockdown cells (Fig. 3A). Thapsigargin blocked a greater portion of carbachol-induced changes in Ca2+i at 2 min in the control cell line. It is possible that SOCE triggered by carbachol generates a smaller influx of Ca2+ through SOCCs in the T1R3 knockdown cells compared with control cells. This might explain why the second phase of the calcium response declined at a faster rate and was less affected by thapsigargin in T1R3 knockdown cells. 2-APB, an inositol 1,4,5-triphosphate receptor antagonist, inhibited peak and 2 mincarbachol-induced rises in Ca2+i to a similar degree in both control and T1R3 knockdown cells (Fig. 3, A and B). It is conceivable that the inhibitory effect of 2-APB on carbachol-stimulated changes in Ca2+i was due to its actions on SOCCs rather than through inhibition of inositol 1,4,5-triphosphate receptors (30Missiaen L. Callewaert G. De Smedt H. Parys J.B. 2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2+ pump and the non-specific Ca2+ leak from the non-mitochondrial Ca2+ stores in permeabilized A7r5 cells.Cell Calcium. 2001; 29: 111-116Crossref PubMed Scopus (153) Google Scholar). Using an ER-localized FRET sensor, it has been reported that, in MIN6 cells, carbachol-induced reductions in ER calcium were not inhibited by pretreatment with 2-APB despite lower overall Ca2+i (14Selway J.L. Moore C.E. Mistry R. John Challiss R.A. Herbert T.P. Molecular mechanisms of muscarinic acetylcholine receptor-stimulated increase in cytosolic free Ca2+ concentration and ERK1/2 activation in the MIN6 pancreatic β-cell line.Acta Diabetol. 2012; 49: 277-289Crossref PubMed Scopus (11) Google Scholar). To determine whether or not 2-APB had an effect on SOCE, calcium was depleted and then added back to MIN6 cells, as shown in Fig. 2C, and pretreated with 2-APB for 30 min. In 2-APB-treated cells, the ability of thapsigargin to deplete stores and the subsequent SOCE was reduced significantly, whereas ROCE was not altered (Fig. 3, C and D). It has been shown previously that nifedipine, an L-type voltage-gated calcium channel blocker, inhibits carbachol-induced changes in Ca2+i in MIN6 cells, suggesting that carbachol causes membrane depolarization (12Wauson E.M. Zaganjor E. Lee A.Y. Guerra M.L. Ghosh A.B. Bookout A.L. Chambers C.P. Jivan A. McGlynn K. Hutchison M.R. Deberardinis R.J. Cobb M.H. The G protein-coupled receptor T1R1/T1R3 regulates mTORC1 and autophagy.Mol. Cell. 2012; 47: 851-862Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). It has been proposed that phosphatidylinositol 4,5-bisphosphate hydrolysis, resulting from carbachol-induced phospholipase C activation, leads to decreased KATP channel activity. Decreased KATP channel activity results in membrane depolarization and activation of voltage-gated calcium channels. Nifedipine had a greater effect on the carbachol response in the control cells compared with those with reduced T1R3, consistent with the observation that calcium released from intracellular stores contributes a greater portion of the carbachol-induced rise in Ca2+i in the T1R3 knockdown cells than does extracellular calcium (Fig. 3, A and B). To determine whether nifedipine was having an effect on SOCE or ROCE, MIN6 cells were pretreated with nifedipine for 30 min prior to stimulation. Unlike what was observed with 2-APB, nifedipine only had an effect on ROCE (Fig. 3, C and D). This is consistent with the data in Fig. 3, A and B, which demonstrated that nifedipine had a smaller effect on carbachol-induced changes in Ca2+i in the T1R3 knockdown cells in which ROCE was absent. Because we observed these changes in carbachol signaling in MIN6 cells in which T1R3 expression was stably suppressed and because we have shown previously that the T1R1/T1R3 complex is an early sensor of amino acids, we hypothesized that depriving" @default.
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• W2111497148 title "Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling" @default.
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