Ethidium Bromide-induced Inhibition of Mitochondrial Gene Transcription Suppresses Glucose-stimulated Insulin Release in the Mouse Pancreatic β-Cell Line βHC9

Hayakawa, Tadao; Noda, Mitsuhiko; Yasuda, Kazuki; Yorifuji, Hiroshi; Taniguchi, Shigeki; Miwa, Ichitomo; Sakura, Hiroshi; Terauchi, Yasuo; Hayashi, Jun‐Ichi; Sharp, Geoffrey W. G.; Kanazawa, Yasunori; Akanuma, Yasuo; Yazaki, Yoshio; Kadowaki, Takashi

doi:10.1074/jbc.273.32.20300

Cited by 107 publications

(73 citation statements)

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“…After dehydration with a graded series of ethanol, they were washed by propylene oxide and embedded in Spurr's low viscosity resin. Silver to gold sections were cut and examined using a transmission electron microscope (CM 10; Philips, Eindhoven, the Netherlands) at a 60 kV accelerating voltage [32]. Measurements were made on ten individual adipocytes treated with or without LA and/or ALC.…”

Section: Methodsmentioning

confidence: 99%

R-α-Lipoic acid and acetyl-l-carnitine complementarily promote mitochondrial biogenesis in murine 3T3-L1 adipocytes

et al. 2007

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Aims/hypothesis The aim of the study was to address the importance of mitochondrial function in insulin resistance and type 2 diabetes, and also to identify effective agents for ameliorating insulin resistance in type 2 diabetes. We examined the effect of two mitochondrial nutrients, R-α-lipoic acid (LA) and acetyl-L-carnitine (ALC), as well as their combined effect, on mitochondrial biogenesis in 3T3-L1 adipocytes. Methods Mitochondrial mass and oxygen consumption were determined in 3T3-L1 adipocytes cultured in the presence of LA and/or ALC for 24 h. Mitochondrial DNA and mRNA from peroxisome proliferator-activated receptor gamma and alpha (Pparg and Ppara) and carnitine palmitoyl transferase 1a (Cpt1a), as well as several transcription factors involved in mitochondrial biogenesis, were evaluated by real-time PCR or electrophoretic mobility shift (EMSA) assay. Mitochondrial complexes proteins were measured by western blot and fatty acid oxidation was measured by quantifying CO 2 production from [1-14 C] palmitate. Results Treatments with the combination of LA and ALC at concentrations of 0.1, 1 and 10 μmol/l for 24 h significantly increased mitochondrial mass, expression of mitochondrial DNA, mitochondrial complexes, oxygen consumption and fatty acid oxidation in 3T3L1 adipocytes. These changes were accompanied by an increase in expression of Pparg, Ppara and Cpt1a mRNA, as well as increased expression of peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha (Ppargc1a), mitochondrial transcription factor A (Tfam) and nuclear respiratory factors 1 and 2 (Nrf1 and Nrf2). However, the treatments with LA or ALC alone at the same concentrations showed little effect on mitochondrial function and biogenesis. Conclusions/interpretation We conclude that the combination of LA and ALC may act as PPARG/A dual ligands to complementarily promote mitochondrial synthesis and adipocyte metabolism.

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Section: Methodsmentioning

confidence: 99%

R-α-Lipoic acid and acetyl-l-carnitine complementarily promote mitochondrial biogenesis in murine 3T3-L1 adipocytes

et al. 2007

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“…Mitochondrial metabolism may set the maximal limit of fuel-stimulated insulin secretion in ␤-cells by controlling the rate of ATP production (10). Inhibition of mitochondrial gene transcription, and presumably oxidation of glucose-derived pyruvate in the mitochondria, is found to suppress glucose-induced insulin secretion in ␤HC9 cells (15,16). Thus, glucokinase may not be the only rate-limiting step in glucose-induced insulin secretion of pancreatic ␤-cells.…”

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confidence: 99%

Leucine Regulation of Glucokinase and ATP Synthase Sensitizes Glucose-Induced Insulin Secretion in Pancreatic β-Cells

et al. 2006

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We have recently shown that leucine culture upregulates ATP synthase ␤-subunit (ATPS␤) and increases ATP level, cytosolic Ca 2؉ , and glucose-induced insulin secretion in rat islets. The aim is to test whether glucokinase expression is also affected in rat islets and its role in glucose sensitization during leucine culture. Leucine culture increased glucose-induced NAD(P)H level at 1 and 2 days but not at 1 week. The half-maximal effective concentration of the glucose response curve for NAD(P)H was left-shifted from 5-7 to 2-3 mmol/l. The effect was dose dependent and rapamycin insensitive. Leucine culture did not affect glyceraldehyde effects on NAD(P)H. Leucine pretreatment for 30 min had no effects on NAD(P)H levels. Leucine culture for 2 days also increased glucose-induced cytosolic Ca 2؉ elevation, ATP level, and insulin secretion. Leucine increase of glucokinase mRNA levels occurred as early as day 1 and lasted through 1 week. That of ATPS␤ did not occur until day 2 and lasted through 1 week. Leucine effects on both mRNAs were dose dependent. The upregulation of both genes was confirmed by Western blotting. Leucine culture also increased glucose-induced insulin secretion, ATP level, glucokinase, and ATPS␤ levels of type 2 diabetic human islets. In conclusion, leucine culture upregulates glucokinase, which increases NAD(P)H level, and ATPS␤, which increases oxidation of NADH and production of ATP. The combined upregulation of both genes increases glucoseinduced cytosolic Ca 2؉ and insulin secretion. ] (6 -8). As the rate-limiting enzyme of glycolysis, it is believed that glucokinase sets a strict control on glucose metabolism in pancreatic ␤-cells. However, overexpression of glucokinase or hexokinase I fails to increase the maximal insulin output induced by glucose, although it decreases the threshold for glucose-induced insulin secretion in pancreatic ␤-cells (9 -12). In pancreatic ␤-cells, most of the intracellular ATP comes from the oxidation of glucosederived pyruvate and oxidation of NADH in the mitochondria via the electron transport chain. Alternations in ATP synthesis results in changes of ␤-cell function and glucosestimulated insulin secretion (13,14). Mitochondrial metabolism may set the maximal limit of fuel-stimulated insulin secretion in ␤-cells by controlling the rate of ATP production (10). Inhibition of mitochondrial gene transcription, and presumably oxidation of glucose-derived pyruvate in the mitochondria, is found to suppress glucose-induced insulin secretion in ␤HC9 cells (15,16). Thus, glucokinase may not be the only rate-limiting step in glucose-induced insulin secretion of pancreatic ␤-cells. Other factor(s) may also be rate-limiting and contribute to the tight control of glucose-induced insulin secretion. As the key enzyme catalyzing the conversion of ADP and inorganic phosphate (Pi) to ATP by proton gradient in the electron transport chain, ATP synthase (complex V) may play an important role in ATP synthesis and hence glucose-induced insulin secretion. It had been established that ...

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“…The critical regulatory role of oxidative ATP production in glucose-stimulated insulin secretion (GSIS) is underscored by the observation that disrupting mitochondrial oxidative metabolism blocks nutrient-mediated insulin secretion. For example, inhibition of oxidative phosphorylation (7,8), blockade of NADH transport into mitochondria (9,10), or elimination of mitochondrial DNA from ␤-cells in vitro (11)(12)(13)(14)(15) or in vivo (16) all prevent GSIS. We tested the hypothesis that enhancing oxidative ATP production in response to glucose can increase insulin secretion.…”

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confidence: 99%

Inhibition of Mitochondrial Na+-Ca2+ Exchanger Increases Mitochondrial Metabolism and Potentiates Glucose-Stimulated Insulin Secretion in Rat Pancreatic Islets

Lee¹,

Miles²,

Vargas³

et al. 2003

Diabetes

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؉ . We show that inhibition of the mNCE enhances mitochondrial oxidative metabolism and increases glucose-stimulated insulin secretion in rat islets and INS-1 cells. The benzothiazepine CGP37157 inhibited mNCE activity in INS-1 cells (50% inhibition at IC 50 ‫؍‬ 1.5 mol/l) and increased the glucose-induced rise in mitochondrial Ca 2؉ ([Ca 2؉ ] m ) 2.1 times. Cellular ATP content was increased by 13% in INS-1 cells and by 49% in rat islets by CGP37157 (1 mol/l). Krebs cycle flux was also stimulated by CGP37157 when glucose was present. Insulin secretion was increased in a glucosedependent manner by CGP37157 in both INS-1 cells and islets. In islets, CGP37157 increased insulin secretion dose dependently (half-maximal efficacy at EC 50 ‫؍‬ 0.06 mol/l) at 8 mmol/l glucose and shifted the glucose dose response curve to the left. In perifused islets, mNCE inhibition had no effect on insulin secretion at 2.8 mmol/l glucose but increased insulin secretion by 46% at 11 mmol/l glucose. The effects of CGP37157 could not be attributed to interactions with the plasma membrane sodium calcium exchanger, L-type calcium channels, ATP-sensitive K ؉ channels, or [Ca 2؉ ] m uniporter. In hyperglycemic clamp studies of Wistar rats, CGP37157 increased plasma insulin and C-peptide levels only during the hyperglycemic phase of the study. These results illustrate the potential utility of agents that affect mitochondrial metabolism as novel insulin secretagogues. Diabetes 52:965-973, 2003 M itochondrial oxidative metabolism plays an important role in the insulin secretory process in pancreatic ␤-cells. Stimulus-secretion coupling in the ␤-cell depends on the metabolism of glucose and the subsequent mitochondrial oxidative phosphorylation that generates ATP. ATP closes ATPsensitive K ϩ (K ATP ) channels, causing depolarization of the ␤-cell membrane, opening of voltage-dependent calcium channels, and Ca 2ϩ influx (1,2). Although two main processes, glycolysis and oxidative phosphorylation, are responsible for ATP synthesis during glucose metabolism, oxidative phosphorylation is the predominant pathway in the pancreatic ␤-cell (3,4). Insulin secretion induced by other secretagogues, such as leucine and glyceraldehydes, is also mediated by the production of ATP (5,6). The critical regulatory role of oxidative ATP production in glucose-stimulated insulin secretion (GSIS) is underscored by the observation that disrupting mitochondrial oxidative metabolism blocks nutrient-mediated insulin secretion. For example, inhibition of oxidative phosphorylation (7,8), blockade of NADH transport into mitochondria (9,10), or elimination of mitochondrial DNA from ␤-cells in vitro (11-15) or in vivo (16) all prevent GSIS. We tested the hypothesis that enhancing oxidative ATP production in response to glucose can increase insulin secretion. The normal feed-forward regulatory role of mitochondrial Ca 2ϩ ([Ca 2ϩ ] m ) in the ␤-cell was exploited to enhance oxidative metabolism.The influx of calcium into the cytoplasm of the ␤-cell in response to a nutrient l...

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Ethidium Bromide-induced Inhibition of Mitochondrial Gene Transcription Suppresses Glucose-stimulated Insulin Release in the Mouse Pancreatic β-Cell Line βHC9

Cited by 107 publications

References 53 publications

R-α-Lipoic acid and acetyl-l-carnitine complementarily promote mitochondrial biogenesis in murine 3T3-L1 adipocytes

R-α-Lipoic acid and acetyl-l-carnitine complementarily promote mitochondrial biogenesis in murine 3T3-L1 adipocytes

Leucine Regulation of Glucokinase and ATP Synthase Sensitizes Glucose-Induced Insulin Secretion in Pancreatic β-Cells

Inhibition of Mitochondrial Na+-Ca2+ Exchanger Increases Mitochondrial Metabolism and Potentiates Glucose-Stimulated Insulin Secretion in Rat Pancreatic Islets

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