Creation and characterization of a mitochondrial DNA-depleted pancreatic beta-cell line: impaired insulin secretion induced by glucose, leucine, and sulfonylureas.

Abstract

It has been proposed that mitochondrial oxidative phosphorylation in pancreatic beta-cells plays an important role in insulin secretion. To examine the impact of mitochondrial dysfunction on insulin secretion, we created a MIN6 cell line that depleted mitochondrial DNA (mtDNA) by treatment with ethidium bromide (EtBr), and studied the response of the cell line to various secretagogues. MIN6 cells cultured with 0.5 microg/ml EtBr for over 2 months (termed MIN6 deltamt cells) revealed a marked (>90%) decrease in mtDNA content and a lack of mRNAs encoded by mtDNA. MIN6 deltamt cells showed the defects of cytochrome c oxidase activity, glucose- and leucine-induced increase in cellular ATP content, and respiratory chain-driven ATP synthesis, suggesting that MIN6 deltamt cells lost oxidative phosphorylation activity due to the selective disruption of the subunits of respiratory chain enzymes encoded by mtDNA. MIN6 deltamt cells also showed a decrease in glucose utilization, suggesting the impairment of the glycolytic pathway as well. After stimulation with glucose and leucine, MIN6 deltamt cells showed no response in insulin secretion or intracellular free Ca2+ concentration ([Ca2+]i). On the other hand, arginine stimulated insulin secretion and an increase in [Ca2+]i in MIN6 deltamt cells as in MIN6 cells. Glibenclamide also stimulated insulin secretion and an increase in [Ca2+]i in both types of cells, but the responses of MIN6 deltamt cells were significantly lower than those of MIN6 cells. These results suggest the importance of ATP production in insulin secretion and an increase in [Ca2+]i, both induced by glucose and leucine. Moreover, mitochondrial function turns out to be not essential but important for the activation of sulfonylurea-induced insulin secretion.

pVHL is a regulator of glucose metabolism and insulin secretion in pancreatic β cells

“…That Vhlh −/− ␤ cells have undergone such a shift in glucose metabolism is supported by the presence of enhanced lactate secretion from Vhlh −/− islets as well as the constitutive production of ATP at low glucose. That mitochondrial activity is critical for normal insulin secretion is further supported by previous work demonstrating that depletion of mitochondrial DNA inhibits glucose-stimulated insulin secretion and that manipulating the activities of LDHA or PDH in ␤-cell lines or isolated rat islets likewise affects pyruvate metabolism with consequent effects on insulin secretion (Kennedy et al 1998;Tsuruzoe et al 1998;Ishihara et al 1999;Ainscow et al 2000;Nicholls et al 2002).…”

Metabolic Regulation by Leucine of Translation Initiation Through the mTOR-Signaling Pathway by Pancreatic β-Cells

“…It has also been recently proposed that increases in mitochondrial-derived ATP by ␤-cells may provide localized or privileged elevations of ATP essential for sustained closure of K ATP channels (44). In addition, a ␤-cell line depleted of mitochondrial DNA displayed defects in cytochrome c oxidase activity, glucose, and leucine-induced increases in cellular ATP content and respiratory chain-driven ATP synthesis (45).…”

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

“…Since depletion of mitochondrial DNA abolishes the glucose-induced ATP elevation, mitochondria clearly are a major source of ATP production in pancreatic beta cells [2,3]. Glucose-induced insulin secretion from beta cells is often impaired by exposure to high concentrations of fuels including glucose, NEFAs and ketone bodies, and by administration of diabetogenic pharmacological agents, all of which involve impaired glucose-induced ATP elevation in beta cells [4][5][6][7][8][9][10][11].…”