FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2055857902> ?p ?o ?g. }

• W2055857902 endingPage "4096" @default.
• W2055857902 startingPage "4086" @default.
• W2055857902 abstract "In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca2+ influx, which triggers insulin exocytosis. The mitochondrial Ca2+ uniporter (MCU) mediates Ca2+ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca2+ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca2+. Suppression of the putative Ca2+/H+ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca2+-induced matrix acidification. These results demonstrate that MCU-mediated Ca2+ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells. In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca2+ influx, which triggers insulin exocytosis. The mitochondrial Ca2+ uniporter (MCU) mediates Ca2+ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca2+ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca2+. Suppression of the putative Ca2+/H+ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca2+-induced matrix acidification. These results demonstrate that MCU-mediated Ca2+ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells. Pancreatic β-cells maintain blood glucose homeostasis by adapting insulin secretion to the changes in circulating nutrients. A major signaling molecule in this metabolism-secretion coupling linking nutrient metabolism to insulin secretion is cytosolic ATP most of which is synthesized from oxidative phosphorylation. Mitochondrial ATP synthesis is driven by the electrical (ΔΨmito, membrane potential) and chemical (ΔpHmito) gradients across the mitochondrial inner membrane. These gradients are established as a result of electron transport and the associated export of protons mediated by the respiratory chain. Reducing equivalents in mitochondrial matrix are mainly produced by the tricarboxylic acid (TCA) 2The abbreviations used are: TCAtricarboxylic acidGSISglucose-stimulated insulin secretionLETMleucine zipper-EF hand-containing transmembrane proteinMCUmitochondrial Ca2+ uniporterMTT3-(4,5-dimethylhioazol-2-yl)-2,5-diphenyltetrazolium bromideCOXcytochrome c oxidaseOCRoxygen consumption rate. cycle and mitochondrial metabolite shuttles. Thus, the metabolic status of the β-cell mitochondria critically controls ATP synthesis and insulin secretory activity (1.Wiederkehr A. Wollheim C.B. Mitochondrial signals drive insulin secretion in the pancreatic beta-cell.Mol. Cell Endocrinol. 2012; 353: 128-137Crossref PubMed Scopus (109) Google Scholar). Accumulating evidence suggests that defective mitochondrial function results in impaired glucose-stimulated insulin secretion (GSIS) and may contribute to the development of type 2 diabetes (2.Wollheim C.B. Beta-cell mitochondria in the regulation of insulin secretion: a new culprit in type II diabetes.Diabetologia. 2000; 43: 265-277Crossref PubMed Scopus (166) Google Scholar3.Del Guerra S. Lupi R. Marselli L. Masini M. Bugliani M. Sbrana S. Torri S. Pollera M. Boggi U. Mosca F. Del Prato S. Marchetti P. Functional and molecular defects of pancreatic islets in human type 2 diabetes.Diabetes. 2005; 54: 727-735Crossref PubMed Scopus (391) Google Scholar, 4.Anello M. Lupi R. Spampinato D. Piro S. Masini M. Boggi U. Del Prato S. Rabuazzo A.M. Purrello F. Marchetti P. Functional and morphological alterations of mitochondria in pancreatic beta cells from type 2 diabetic patients.Diabetologia. 2005; 48: 282-289Crossref PubMed Scopus (275) Google Scholar5.Ashcroft F.M. Rorsman P. Diabetes mellitus and the beta cell: the last ten years.Cell. 2012; 148: 1160-1171Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar). tricarboxylic acid glucose-stimulated insulin secretion leucine zipper-EF hand-containing transmembrane protein mitochondrial Ca2+ uniporter 3-(4,5-dimethylhioazol-2-yl)-2,5-diphenyltetrazolium bromide cytochrome c oxidase oxygen consumption rate. The matrix Ca2+ concentration ([Ca2+]mito) is a key activator of mitochondrial metabolic function (1.Wiederkehr A. Wollheim C.B. Mitochondrial signals drive insulin secretion in the pancreatic beta-cell.Mol. Cell Endocrinol. 2012; 353: 128-137Crossref PubMed Scopus (109) Google Scholar, 6.Griffiths E.J. Rutter G.A. Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells.Biochim. Biophys. Acta. 2009; 1787: 1324-1333Crossref PubMed Scopus (254) Google Scholar, 7.Wiederkehr A. Szanda G. Akhmedov D. Mataki C. Heizmann C.W. Schoonjans K. Pozzan T. Spät A. Wollheim C.B. Mitochondrial matrix calcium is an activating signal for hormone secretion.Cell Metab. 2011; 13: 601-611Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The [Ca2+]mito activates several matrix enzymes including α-ketoglutarate dehydrogenase in the TCA cycle (8.Denton R.M. Regulation of mitochondrial dehydrogenases by calcium ions.Biochim. Biophys. Acta. 2009; 1787: 1309-1316Crossref PubMed Scopus (562) Google Scholar). The ATP synthase is also directly activated by a rise in [Ca2+]mito (9.Territo P.R. Mootha V.K. French S.A. Balaban R.S. Ca(2+) activation of heart mitochondrial oxidative phosphorylation: role of the F(0)/F(1)-ATPase.Am. J. Physiol. Cell Physiol. 2000; 278: C423-C435Crossref PubMed Google Scholar). In pancreatic β-cells [Ca2+]mito is strictly required for ATP synthase-dependent respiration stimulated by glucose (10.De Marchi U. Thevenet J. Hermant A. Dioum E. Wiederkehr A. Calcium co-regulates oxidative metabolism and ATP synthase-dependent respiration in pancreatic beta cells.J. Biol. Chem. 2014; 289: 9182-9194Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Given its importance, mitochondrial Ca2+ uptake has been a research focus for decades, starting with the functional characterization in isolated mitochondria. Nevertheless, it took 50 years to elucidate the molecular identity of the mitochondrial Ca2+ uniporter (MCU) (11.Baughman J.M. Perocchi F. Girgis H.S. Plovanich M. Belcher-Timme C.A. Sancak Y. Bao X.R. Strittmatter L. Goldberger O. Bogorad R.L. Koteliansky V. Mootha V.K. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.Nature. 2011; 476: 341-345Crossref PubMed Scopus (1340) Google Scholar, 12.De Stefani D. Raffaello A. Teardo E. Szabò I. Rizzuto R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter.Nature. 2011; 476: 336-340Crossref PubMed Scopus (1357) Google Scholar). Mitochondrial Ca2+ uptake through MCU is regulated by a number of recently discovered proteins, including mitochondrial Ca2+ uptake 1 and 2 (MICU1/2) (13.Csordás G. Golenár T. Seifert E.L. Kamer K.J. Sancak Y. Perocchi F. Moffat C. Weaver D. de la Fuente Perez S. Bogorad R. Koteliansky V. Adijanto J. Mootha V.K. Hajnóczky G. MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca(2)(+) uniporter.Cell Metab. 2013; 17: 976-987Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 14.Plovanich M. Bogorad R.L. Sancak Y. Kamer K.J. Strittmatter L. Li A.A. Girgis H.S. Kuchimanchi S. De Groot J. Speciner L. Taneja N. Oshea J. Koteliansky V. Mootha V.K. MICU2, a paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium handling.PLoS One. 2013; 8: e55785Crossref PubMed Scopus (317) Google Scholar, 15.Mallilankaraman K. Doonan P. Cárdenas C. Chandramoorthy H.C. Müller M. Miller R. Hoffman N.E. Gandhirajan R.K. Molgó J. Birnbaum M.J. Rothberg B.S. Mak D.O. Foskett J.K. Madesh M. MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake that regulates cell survival.Cell. 2012; 151: 630-644Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar), mitochondrial Ca2+ uniporter regulator 1 (MCUR1) (16.Mallilankaraman K. Cárdenas C. Doonan P.J. Chandramoorthy H.C. Irrinki K.M. Golenár T. Csordás G. Madireddi P. Yang J. Müller M. Miller R. Kolesar J.E. Molgó J. Kaufman B. Hajnóczky G. Foskett J.K. Madesh M. MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism.Nat. Cell Biol. 2012; 14: 1336-1343Crossref PubMed Scopus (365) Google Scholar), and essential MCU regulator (EMRE) (17.Sancak Y. Markhard A.L. Kitami T. Kovács-Bogdán E. Kamer K.J. Udeshi N.D. Carr S.A. Chaudhuri D. Clapham D.E. Li A.A. Calvo S.E. Goldberger O. Mootha V.K. EMRE is an essential component of the mitochondrial calcium uniporter complex.Science. 2013; 342: 1379-1382Crossref PubMed Scopus (444) Google Scholar). Especially MICU1/2 negatively regulate MCU activity under resting cytosolic Ca2+ ([Ca2+]i). At stimulating [Ca2+]i (>10 μm), however, MICU1 activates MCU activity, implying that the regulatory subunits of the MCU complex modulate mitochondrial Ca2+ loads of ΔΨmito-driven Ca2+ uptake without perturbing the important signal propagation from ER to mitochondria (13.Csordás G. Golenár T. Seifert E.L. Kamer K.J. Sancak Y. Perocchi F. Moffat C. Weaver D. de la Fuente Perez S. Bogorad R. Koteliansky V. Adijanto J. Mootha V.K. Hajnóczky G. MICU1 controls both the threshold and cooperative activation of the mitochondrial Ca(2)(+) uniporter.Cell Metab. 2013; 17: 976-987Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 18.Patron M. Checchetto V. Raffaello A. Teardo E. Vecellio Reane D. Mantoan M. Granatiero V. Szabò I. De Stefani D. Rizzuto R. MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity.Mol. Cell. 2014; 53: 726-737Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 19.de la Fuente S. Matesanz-Isabel J. Fonteriz R.I. Montero M. Alvarez J. Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells.Biochem. J. 2014; 458: 33-40Crossref PubMed Scopus (33) Google Scholar). Mitochondrial Ca2+ homeostasis is maintained by balanced Ca2+ influx and efflux. Mitochondrial Ca2+ export is mediated by antiporters exchanging Ca2+ for H+ or Na+ (20.Nicholls D.G. Crompton M. Mitochondrial calcium transport.FEBS Lett. 1980; 111: 261-268Crossref PubMed Scopus (205) Google Scholar). Two mitochondrial antiporters promoting Ca2+ efflux have been identified: The leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) and the mitochondrial sodium calcium exchanger (NCLX). LETM1, which is defective in Wolf-Hirschhorn syndrome (WHS), works as a K+/H+ exchanger in yeast mitochondria (21.Froschauer E. Nowikovsky K. Schweyen R.J. Electroneutral K+/H+ exchange in mitochondrial membrane vesicles involves Yol027/Letm1 proteins.Biochim. Biophys. Acta. 2005; 1711: 41-48Crossref PubMed Scopus (63) Google Scholar) or mammalian ER (22.Kuum M. Veksler V. Liiv J. Ventura-Clapier R. Kaasik A. Endoplasmic reticulum potassium-hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes.J. Cell Sci. 2012; 125: 625-633Crossref PubMed Scopus (45) Google Scholar). LETM1 was also shown to mediate Ca2+/H+ exchange in mitochondria with a [Ca2+]mito-dependent biphasic mode (23.Jiang D. Zhao L. Clapham D.E. Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.Science. 2009; 326: 144-147Crossref PubMed Scopus (398) Google Scholar). NCLX was confirmed as an electrogenic Na+/Ca2+ antiporter (exchanging 3 or 4 Na+ per Ca2+) (24.Palty R. Silverman W.F. Hershfinkel M. Caporale T. Sensi S.L. Parnis J. Nolte C. Fishman D. Shoshan-Barmatz V. Herrmann S. Khananshvili D. Sekler I. NCLX is an essential component of mitochondrial Na+/Ca2+ exchange.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 436-441Crossref PubMed Scopus (556) Google Scholar). Inhibition of NCLX in pancreatic β-cells increases [Ca2+]mito, accelerates mitochondrial oxidative metabolism and GSIS (25.Lee B. Miles P.D. Vargas L. Luan P. Glasco S. Kushnareva Y. Kornbrust E.S. Grako K.A. Wollheim C.B. Maechler P. Olefsky J.M. Anderson C.M. Inhibition of mitochondrial Na+-Ca2+ exchanger increases mitochondrial metabolism and potentiates glucose-stimulated insulin secretion in rat pancreatic islets.Diabetes. 2003; 52: 965-973Crossref PubMed Scopus (67) Google Scholar26.Tarasov A.I. Semplici F. Ravier M.A. Bellomo E.A. Pullen T.J. Gilon P. Sekler I. Rizzuto R. Rutter G.A. The mitochondrial Ca2+ uniporter MCU is essential for glucose-induced ATP increases in pancreatic beta-cells.PLoS One. 2012; 7: e39722Crossref PubMed Scopus (131) Google Scholar, 27.Gauthier B.R. Wiederkehr A. Baquié M. Dai C. Powers A.C. Kerr-Conte J. Pattou F. MacDonald R.J. Ferrer J. Wollheim C.B. PDX1 deficiency causes mitochondrial dysfunction and defective insulin secretion through TFAM suppression.Cell Metab. 2009; 10: 110-118Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar28.Nita II Hershfinkel M. Fishman D. Ozeri E. Rutter G.A. Sensi S.L. Khananshvili D. Lewis E.C. Sekler I. The mitochondrial Na+/Ca2+ exchanger upregulates glucose dependent Ca2+ signalling linked to insulin secretion.PLoS One. 2012; 7: e46649Crossref PubMed Scopus (55) Google Scholar). In addition to [Ca2+]mito, the matrix pH has been identified as a regulator of mitochondrial energy metabolism in β-cells. In contrast to other cell types, pancreatic β-cells have acidic pHmito under resting conditions. Nutrient stimulation causes matrix alkalinization without any marked cytosolic pH change (29.Wiederkehr A. Park K.S. Dupont O. Demaurex N. Pozzan T. Cline G.W. Wollheim C.B. Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic beta-cell activation.EMBO J. 2009; 28: 417-428Crossref PubMed Scopus (59) Google Scholar). Preventing the resulting nutrient-induced increase of the ΔpHmitochanges using ionophores abrogated proton-coupled mitochondrial ion/metabolite transport, ATP synthesis, and GSIS regardless of elevated ΔΨmito (29.Wiederkehr A. Park K.S. Dupont O. Demaurex N. Pozzan T. Cline G.W. Wollheim C.B. Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic beta-cell activation.EMBO J. 2009; 28: 417-428Crossref PubMed Scopus (59) Google Scholar, 30.Akhmedov D. Braun M. Mataki C. Park K.S. Pozzan T. Schoonjans K. Rorsman P. Wollheim C.B. Wiederkehr A. Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal beta cells.FASEB J. 2010; 24: 4613-4626Crossref PubMed Scopus (49) Google Scholar, 31.Quan X. Das R. Xu S. Cline G.W. Wiederkehr A. Wollheim C.B. Park K.S. Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells.Mol. Cell Endocrinol. 2013; 381: 198-209Crossref PubMed Scopus (9) Google Scholar). Therefore, pathogenic conditions causing a reduction of ΔpHmito may seriously deteriorate ATP generation and insulin secretion in pancreatic β-cells. Several recent reports demonstrate the functional role of MCU in pancreatic β-cells (26.Tarasov A.I. Semplici F. Ravier M.A. Bellomo E.A. Pullen T.J. Gilon P. Sekler I. Rizzuto R. Rutter G.A. The mitochondrial Ca2+ uniporter MCU is essential for glucose-induced ATP increases in pancreatic beta-cells.PLoS One. 2012; 7: e39722Crossref PubMed Scopus (131) Google Scholar, 32.Alam M.R. Groschner L.N. Parichatikanond W. Kuo L. Bondarenko A.I. Rost R. Waldeck-Weiermair M. Malli R. Graier W.F. Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial Ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic beta-cells.J. Biol. Chem. 2012; 287: 34445-34454Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). MCU mediates glucose-stimulated [Ca2+]mito rise and second phase ATP/ADP increase (26.Tarasov A.I. Semplici F. Ravier M.A. Bellomo E.A. Pullen T.J. Gilon P. Sekler I. Rizzuto R. Rutter G.A. The mitochondrial Ca2+ uniporter MCU is essential for glucose-induced ATP increases in pancreatic beta-cells.PLoS One. 2012; 7: e39722Crossref PubMed Scopus (131) Google Scholar). Knockdown of either MCU or MICU1 diminishes insulin secretion associated with defects in mitochondrial Ca2+ uptake (32.Alam M.R. Groschner L.N. Parichatikanond W. Kuo L. Bondarenko A.I. Rost R. Waldeck-Weiermair M. Malli R. Graier W.F. Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial Ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic beta-cells.J. Biol. Chem. 2012; 287: 34445-34454Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Mice lacking MCU show a significant reduction of [Ca2+]mito and Ca2+-stimulated oxygen consumption in muscle mitochondria, without changes in the basal respiration in embryonic fibroblasts (33.Pan X. Liu J. Nguyen T. Liu C. Sun J. Teng Y. Fergusson M.M. Rovira II Allen M. Springer D.A. Aponte A.M. Gucek M. Balaban R.S. Murphy E. Finkel T. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter.Nat. Cell Biol. 2013; 15: 1464-1472Crossref PubMed Scopus (465) Google Scholar). It remains unclear, however, how reduced MCU activity attenuates mitochondrial signal generation in pancreatic β-cell metabolism-secretion coupling. In this study, we observed that reduced mitochondrial Ca2+ uptake following silencing of MCU significantly attenuated respiratory chain activity and ΔpHmito increase in permeabilized as well as in intact insulin-secreting cells. These defects lead to impaired ATP synthesis and insulin secretion, demonstrating the crucial role of mitochondrial Ca2+ uptake for the establishment of the ΔpHmito in metabolism-secretion coupling. We also provide evidence for a novel role of the putative Ca2+/H+ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) as a Ca2+ efflux route in insulin secreting cells, the role of which is altered in the absence of MCU. Rat insulinoma INS-1E cells were cultured in a humidified atmosphere (37 °C) containing 5% CO2 in a complete medium composed of RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen), 1 mm sodium pyruvate, 50 μm 2-mercaptoethanol, 2 mm glutamine, 10 mm HEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin (HyClone, Thermo Fisher Scientific Inc., Lafayette, CO). Experiments were performed with cells of passage number 80–120. Most chemicals were purchased from Sigma except JC-1 from Molecular Probes (Eugene, OR). Pancreatic islets were isolated from 200–300-g male Sprague-Dawley rats (Orient Bio, Seongnam, Korea) by collagenase (Sigma) digestion (29.Wiederkehr A. Park K.S. Dupont O. Demaurex N. Pozzan T. Cline G.W. Wollheim C.B. Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic beta-cell activation.EMBO J. 2009; 28: 417-428Crossref PubMed Scopus (59) Google Scholar) and dispersed by a brief incubation with trypsin (Invitrogen). Islet cells were seeded on multi-well-plates coated with 804G matrix and cultured in RPMI 1640 medium supplemented with 10% FBS, 10 mm HEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin (7.Wiederkehr A. Szanda G. Akhmedov D. Mataki C. Heizmann C.W. Schoonjans K. Pozzan T. Spät A. Wollheim C.B. Mitochondrial matrix calcium is an activating signal for hormone secretion.Cell Metab. 2011; 13: 601-611Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). INS-1E cells were seeded and cultured onto well-plates or coverslips coated with 804G matrix. Cells were washed with Ca2+-free Krebs-Ringer bicarbonate (KRB) solution (mm; 140 NaCl, 3.6 KCl, 0.5 NaH2PO4, 0.5 MgSO4, 1.5 CaCl2, 10 HEPES, 2 NaHCO3, 5.5 glucose, pH 7.4 titrated with NaOH) and then incubated for 10 min at 37 °C with 1 μg/ml of Staphylococcus aureus α-hemolysin toxin (Sigma) in an intracellular buffer (mm; 140 KCl, 5 NaCl, 7 MgSO4, 1 KH2PO4, 20 HEPES, 10.2 EGTA, 1.65 CaCl2, 0.1 ATP, pH 7.0 with KOH), which has about 120 nm of free Ca2+ concentration. After α-toxin permeabilization, cells were washed once with 0.5% bovine serum albumin (BSA) containing intracellular buffer and used for experiments (29.Wiederkehr A. Park K.S. Dupont O. Demaurex N. Pozzan T. Cline G.W. Wollheim C.B. Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic beta-cell activation.EMBO J. 2009; 28: 417-428Crossref PubMed Scopus (59) Google Scholar). Cells were transfected with non-targeting or target-specific small interfering RNA (siRNA) using DharmaFECT1 (Dharmacon, Thermo Fisher Scientific Inc.). The target-specific siRNAs for rat MCU and LETM1 were purchased from Dharmacon, which is composed of siRNAs for four different targets of each gene (SMARTpool, Dharmacon). Total RNA was isolated from cells 72 h after siRNA transfection using the RNeasy kit (Cat. 74134, Qiagen GmbH, Hilden, Germany). First strand cDNA was synthesized from 1 μg of total RNA with a reverse transcription kit (Applied Bioscience, Foster City, CA) using oligo-dT. Quantitative PCR was performed using sequence-specific primers for rat MCU (forward: 5′-GAAGTAGGTGACCGGTTCCA-3′, reverse: 5′-AGGAAAGCGGAGAAGAGGAC-3′), and LETM1 (forward: 5′-GGCTGGACTTGCACCTGTAT-3′, reverse: 5′-CAAGGTGGACTTCAGCTGGT-3′). Rat β-actin was used as the reference control. For the analysis of each gene expression, experiments were conducted in a triplicate in the real-time PCR system (7900HT, Applied Bioscience) using SYBR Green PCR Master Mix (Cat. 204143, Qiagen GmbH). Data were analyzed following ΔΔCT method (34.Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔC(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123424) Google Scholar). The protein level of MCU or LETM1 was determined in a cell extract using Western blotting as described previously (31.Quan X. Das R. Xu S. Cline G.W. Wiederkehr A. Wollheim C.B. Park K.S. Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells.Mol. Cell Endocrinol. 2013; 381: 198-209Crossref PubMed Scopus (9) Google Scholar). Primary antibodies for MCU (1:1000, Cat. HPA016480, Sigma), LETM1 (1:500, Cat. sc134672, Santa Cruz Biotechnology, Dallas, TX), complex I NDUFA9 (1:2000 dilution, Cat. 459100, Invitrogen, Carlsbad, CA), complex III UQCRC2 (1:2500, Cat. MS304, Mitosciences, Abcam), complex IV subunit I (1:1000, Cat. 459600, Invitrogen), complex V (1:5000, Cat. MS604-300, Mitosciences, Abcam), TOM20 (1:1000, Cat. sc11415, Santa Cruz Biotechnology), and β-actin (1:5000, Cat. ab6276, Abcam, Cambridge, UK) were used. Horseradish peroxidase (HRP)-conjugated secondary antibody against either mouse or rabbit IgG (Cat. 31450 and 31460, Thermo Scientific) was incubated for 1 h at room temperature. The bands were visualized with a UVP Biospectrum-600 imaging system using enhanced chemiluminescence (ECL) solution (Luminata Forte, Millipore). To measure mitochondrial matrix Ca2+ level, we used a mitochondria-targeted ratio-pericam (RPmit) plasmid, generously provided by Prof. Roger Tsien (UC San Diego). Cells were transfected with siRNA, and 24 h after, transfected with RPmit using X-tremeGENE (Roche Diagnostics GmbH, Mannheim, Germany). Fluorescence imaging of Ca2+ was performed by using an inverted microscope (IX-81, Olympus, Tokyo, Japan) with an array laser confocal spinning disk (CSU10, Yokogawa Electric Corporation, Tokyo, Japan) and a cooled charge-coupled device (CCD) camera (Cascade 512B, Photometrics, Tucson, AZ). Intact or permeabilized cells on the confocal microscope were perfused with KRB solution or intracellular buffer, respectively, and fluorescence images (435 nm excitation and 535 nm emission) were acquired every 10 s and analyzed using Metafluor 6.3 software (Universal Imaging, Molecular Devices). Cells plated onto 48 well-plates (2 × 105 cells/well) were permeabilized with α-toxin and incubated for 5 or 15 min in an intracellular buffer containing ADP (10 μm) with or without succinate (3 mm). To determine mitochondrial ATP release, the supernatant was harvested after incubation, and ATP level was measured by using the microplate reader (SynergyTM2, BioTek Instruments Inc., Winooski, VT) with a bioluminescence assay kit (HS II, Roche Diagnostics, Mannheim, Germany). For static insulin secretion measurement, cells in a 804G-coated 24 well-plate (1.5 × 105 cells/well) were transfected with siRNA and grown for 72 h. After deprivation of glucose for 1 h, cells were preincubated for 30 min with a KRB solution containing 2.8 mm glucose and 0.1% BSA. Then, cells were washed and incubated for 30 min with 2.8 mm or 16.7 mm glucose-containing KRB solution. Supernatant was collected for estimation of insulin release. Cellular insulin contents were determined in acid-ethanol extracts. Insulin levels were measured by using an insulin ELISA kit (Shibayagi Co., Gunma, Japan). For cytochrome c oxidase (COX) activity measurement, INS-1E cells were permeabilized by freeze-thaw cycle three times and mixed with isotonic solution (10 mm KH2PO4, 250 mm sucrose, 0.1% BSA, pH 6.5) with detergent (laurylmaltoside, 2.5 mm). Traces were started with the addition of reduced cytochrome c (25 nm; with a tiny amount of sodium hydrosulfite) and the enzymatic activity of COX was estimated by measuring absorbance at 550 nm continuously with a spectrophotometer (Amersham Biosciences, GE Healthcare Biosciences, Pittsburgh, PA). COX activity was expressed as moles of oxidized cytochrome c per min. Citrate synthase activity was measured with citrate synthase assay kit (Sigma) based on the manufacturer's instructions. For the MTT assay, siRNA-transfected cells were incubated with 3-(4,5-dimethylhioazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (50 μg/well) for 2 h and then treated with dimethyl sulfoxide (100 μl/well). The absorbance (A570 − A630) of each well was measured by using a microplate reader (Molecular Devices, Sunnyvale, CA). Cellular oxygen consumption rate (OCR) was determined by Extracellular Flux Analyzer (XF-24, Seahorse Bioscience, North Billerica, MA). Cells (2 × 104 cells/well) seeded on 24-well plates (Seahorse Bioscience) were transfected with siRNA and cultured for 72 h. On the experiment day, cells were incubated for 1 h at 37 °C with KRB solution containing 2.8 mm glucose prior to 20 min of basal OCR measurement. Then, glucose (16.7 mm), oligomycin (3 μg/ml), FCCP (3 μm), and antimycin A (3 μm) were added consecutively and the changes in OCR analyzed. Mitochondrial matrix pH (pHmito) was measured using adenovirus expressing mtAlpHi (Ad-tetON-mAlpHi) as described previously (29.Wiederkehr A. Park K.S. Dupont O. Demaurex N. Pozzan T. Cline G.W. Wollheim C.B. Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic beta-cell activation.EMBO J. 2009; 28: 417-428Crossref PubMed Scopus (59) Google Scholar). Intact or permeabilized cells on the confocal microscope were perfused with KRB solution (pH 7.4) or intracellular buffer (pH 7.0), respectively, and the fluorescence signals (488 nm excitation and 535 nm emission) were recorded. Titration of the mitochondrial pH was performed by clamping the matrix pH with high K+ buffer (mm) (125 KCl, 5 NaCl, 1 NaH2PO4, 1 MgSO4, 10 HEPES) of defined pH containing the ionophores nigericin (5 μm) and monensin (5 μm) (31.Quan X. Das R. Xu S. Cline G.W. Wiederkehr A. Wollheim C.B. Park K.S. Mitochondrial phosphate transport during nutrient stimulation of INS-1E insulinoma cells.Mol. Cell Endocrinol. 2013; 381: 198-209Crossref PubMed Scopus (9) Google Scholar). To measure the mitochondrial membrane potential (ΔΨmito), cells seeded onto black-walled 96-well plates (5 × 104 cells/well) were loaded with JC-1 (500 nm, Invitrogen) for 30 min and then permeabilized with α-toxin. The ratio of red (540 nm excitation and 590 nm emission) over green (490 nm excitation and 540 nm emission) fluorescence intensity was monitored from permeabilized cells in the presence of intracellular buffer containing JC-1 (500 nm) using a multi-well fluorescence reader (FlexStation, Molecular Devices) (35.Park K.S. Wiederkehr A. Kirkpatrick C. Mattenberger Y. Martinou J.C. Marchetti P. Demaurex N. Wollheim C.B. Selective actions of mitochondrial fissi" @default.
• W2055857902 created "2016-06-24" @default.
• W2055857902 creator A5005873228 @default.
• W2055857902 creator A5006036350 @default.
• W2055857902 creator A5006602429 @default.
• W2055857902 creator A5010264333 @default.
• W2055857902 creator A5019529769 @default.
• W2055857902 creator A5026303073 @default.
• W2055857902 creator A5040844514 @default.
• W2055857902 creator A5059335787 @default.
• W2055857902 creator A5062719341 @default.
• W2055857902 creator A5072228093 @default.
• W2055857902 creator A5080944317 @default.
• W2055857902 date "2015-02-01" @default.
• W2055857902 modified "2023-10-16" @default.
• W2055857902 title "Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells" @default.
• W2055857902 cites W1966852147 @default.
• W2055857902 cites W1968264041 @default.
• W2055857902 cites W1981295521 @default.
• W2055857902 cites W1984675604 @default.
• W2055857902 cites W1985276381 @default.
• W2055857902 cites W1986213783 @default.
• W2055857902 cites W1998577671 @default.
• W2055857902 cites W2002475003 @default.
• W2055857902 cites W2002791298 @default.
• W2055857902 cites W2005146560 @default.
• W2055857902 cites W2005641666 @default.
• W2055857902 cites W2008682063 @default.
• W2055857902 cites W2008925266 @default.
• W2055857902 cites W2010572143 @default.
• W2055857902 cites W2016783751 @default.
• W2055857902 cites W2025447674 @default.
• W2055857902 cites W2033810389 @default.
• W2055857902 cites W2037657568 @default.
• W2055857902 cites W2040651543 @default.
• W2055857902 cites W2041874682 @default.
• W2055857902 cites W2048377098 @default.
• W2055857902 cites W2055791664 @default.
• W2055857902 cites W2057315336 @default.
• W2055857902 cites W2061113221 @default.
• W2055857902 cites W2065569764 @default.
• W2055857902 cites W2067609194 @default.
• W2055857902 cites W2067841051 @default.
• W2055857902 cites W2070869619 @default.
• W2055857902 cites W2072240385 @default.
• W2055857902 cites W2078179312 @default.
• W2055857902 cites W2084824261 @default.
• W2055857902 cites W2092340487 @default.
• W2055857902 cites W2101840868 @default.
• W2055857902 cites W2103329317 @default.
• W2055857902 cites W2107277218 @default.
• W2055857902 cites W2112384117 @default.
• W2055857902 cites W2115010141 @default.
• W2055857902 cites W2136416442 @default.
• W2055857902 cites W2139625864 @default.
• W2055857902 cites W2142903428 @default.
• W2055857902 cites W2147607863 @default.
• W2055857902 cites W2156007429 @default.
• W2055857902 cites W2159570963 @default.
• W2055857902 cites W2165573061 @default.
• W2055857902 cites W4252911401 @default.
• W2055857902 doi "https://doi.org/10.1074/jbc.m114.632547" @default.
• W2055857902 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4326818" @default.
• W2055857902 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25548283" @default.
• W2055857902 hasPublicationYear "2015" @default.
• W2055857902 type Work @default.
• W2055857902 sameAs 2055857902 @default.
• W2055857902 citedByCount "61" @default.
• W2055857902 countsByYear W20558579022015 @default.
• W2055857902 countsByYear W20558579022016 @default.
• W2055857902 countsByYear W20558579022017 @default.
• W2055857902 countsByYear W20558579022018 @default.
• W2055857902 countsByYear W20558579022019 @default.
• W2055857902 countsByYear W20558579022020 @default.
• W2055857902 countsByYear W20558579022021 @default.
• W2055857902 countsByYear W20558579022022 @default.
• W2055857902 countsByYear W20558579022023 @default.
• W2055857902 crossrefType "journal-article" @default.
• W2055857902 hasAuthorship W2055857902A5005873228 @default.
• W2055857902 hasAuthorship W2055857902A5006036350 @default.
• W2055857902 hasAuthorship W2055857902A5006602429 @default.
• W2055857902 hasAuthorship W2055857902A5010264333 @default.
• W2055857902 hasAuthorship W2055857902A5019529769 @default.
• W2055857902 hasAuthorship W2055857902A5026303073 @default.
• W2055857902 hasAuthorship W2055857902A5040844514 @default.
• W2055857902 hasAuthorship W2055857902A5059335787 @default.
• W2055857902 hasAuthorship W2055857902A5062719341 @default.
• W2055857902 hasAuthorship W2055857902A5072228093 @default.
• W2055857902 hasAuthorship W2055857902A5080944317 @default.
• W2055857902 hasBestOaLocation W20558579021 @default.
• W2055857902 hasConcept C12554922 @default.
• W2055857902 hasConcept C131584629 @default.
• W2055857902 hasConcept C134018914 @default.
• W2055857902 hasConcept C181199279 @default.
• W2055857902 hasConcept C185592680 @default.
• W2055857902 hasConcept C191897082 @default.
• W2055857902 hasConcept C192562407 @default.
• W2055857902 hasConcept C2779306644 @default.

• next