Tsuruzoe K, Araki E, Furukawa N, Shirotani T, Matsumoto K, Kaneko K, Motoshima H, Yoshizato K, Shirakami A, Kishikawa H, Miyazaki J, Shichiri M
Department of Metabolic Medicine, Kumamoto University School of Medicine, Honjo, Japan.
Diabetes. 1998 Apr;47(4):621-31. doi: 10.2337/diabetes.47.4.621.
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.
有人提出，胰腺β细胞中的线粒体氧化磷酸化在胰岛素分泌中起重要作用。为了研究线粒体功能障碍对胰岛素分泌的影响，我们创建了一个MIN6细胞系，通过用溴化乙锭（EtBr）处理来耗尽线粒体DNA（mtDNA），并研究该细胞系对各种促分泌剂的反应。用0.5μg/ml EtBr培养超过2个月的MIN6细胞（称为MIN6 deltamt细胞）显示mtDNA含量显著降低（>90%），且缺乏mtDNA编码的mRNA。MIN6 deltamt细胞表现出细胞色素c氧化酶活性缺陷、葡萄糖和亮氨酸诱导的细胞ATP含量增加以及呼吸链驱动的ATP合成缺陷，这表明MIN6 deltamt细胞由于mtDNA编码的呼吸链酶亚基的选择性破坏而失去了氧化磷酸化活性。MIN6 deltamt细胞还显示葡萄糖利用率降低，这也表明糖酵解途径受损。用葡萄糖和亮氨酸刺激后，MIN6 deltamt细胞在胰岛素分泌或细胞内游离Ca2+浓度（[Ca2+]i）方面无反应。另一方面，精氨酸像在MIN6细胞中一样刺激MIN6 deltamt细胞的胰岛素分泌和[Ca2+]i增加。格列本脲也刺激两种类型细胞的胰岛素分泌和[Ca2+]i增加，但MIN6 deltamt细胞的反应明显低于MIN6细胞。这些结果表明由葡萄糖和亮氨酸诱导的ATP产生在胰岛素分泌和[Ca2+]i增加中的重要性。此外，线粒体功能对于磺酰脲诱导的胰岛素分泌激活并非必不可少，但很重要。
