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Physiological functions of mitochondrial ion transport in insulin-releasing cells

Other Titles
 인슐린 분비세포에서 미토콘드리아 이온운반의 생리적 기능 / 
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Dept. of Medicine/박사
The dominant signaling factor in insulin secretion is the ATP/ADP ratio which determines plasma membrane K+ conductance and voltage-sensitive Ca2+ influx. In pancreatic β-cells, ATP synthesis exclusively depends on oxidative phosphorylation which is regulated by mitochondrial ionic homeostasis. In this study, the functional role of mitochondrial inorganic phosphate (Pi) and Ca2+ transport on metabolism-secretion coupling was investigated in INS-1E rat clonal β-cells. In α-toxin-permeabilised INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and Pi. In the presence of substrates, addition of Pi caused mitochondrial matrix acidification and hyperpolarization which promoted ATP export. The effects of Pi on electrochemical gradient of mitochondria were completely mimicked by inorganic arsenate. Pharmacological inhibition of mitochondrial Pi transport blocked the effects of Pi on electrochemical gradient and ATP export. Knock-down of mitochondrial phosphate carrier (PiC), however, neither prevented Pi-induced mitochondrial activation nor glucose-induced insulin secretion. Dissipation of the mitochondrial pH gradient disabled Pi-induced changes, implying that Pi uptake into mitochondria driven by pH gradient markedly accelerates ATP synthesis and export to cytosol, which provides an efficient signaling mechanism for insulin exocytosis. Mitochondrial Ca2+ influx occurs mainly via mitochondrial Ca2+ uniporter (MCU), encoded by Ccdc109a gene. Knockdown of MCU in INS-1E cells by siRNA treatment attenuated matrix Ca2+ rises in response to extramitochondrial Ca2+ changes. Interestingly, matrix alkalinization by nutrient is markedly reduced in permeabilised cells and also in intact cells with silencing MCU. In addition, knockdown of MCU ablated the acidifying response of matrix pH by extramitochondrial Ca2+ probably caused by Ca2+ efflux. Whereas mitochondrial membrane potential was not altered by silencing MCU, the nigericin-induced hyperpolarization was attenuated due to the reduction in mitochondrial pH gradient. Knockdown of MCU decreased mitochondrial metabolic enzyme activities, expression of respiratory chain complex proteins, and glucose-stimulated oxygen consumption. Consequently, nutrient-stimulated ATP production and insulin secretion were impaired by knockdown of MCU, demonstrating the critical influence of mitochondrial Ca2+ uptake in glucose-stimulated insulin exocytosis. Taken together, mitochondrial Pi and Ca2+ transport plays an important role to increase mitochondrial electrochemical gradient by nutrient, which is critical for ATP generation and metabolism-secretion coupling in insulin-releasing cells.
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