Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells
Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells
复制标题
DOI：
10.2337/diabetes.47.3.374
复制
发表时间：
1998-03-01
期刊：
影响因子：
7.7
通讯作者：
Wollheim, CB
中科院分区：
文献类型：
--
作者：
Kennedy, ED;Maechler, P;Wollheim, CB
由于线粒体基因组（mtDNA）的改变引起的线粒体功能障碍最近引起了人们的广泛关注，发现线粒体编码的蛋白质中的突变扰乱了细胞功能。几种疾病已经与这种遗传变化联系在一起，包括特定的糖尿病表型。使用溴化乙锭（EtBr）插入mtDNA，我们已经从高度分化的INS-1胰岛素分泌细胞系中有效地消除了mtDNA控制下的功能。我们已经研究了胰岛素分泌，线粒体酶活性，细胞器结构和膜极化的后果，在这样的细胞（INS-1 rho（0）），在这些条件下，线粒体膜电位不能响应于葡萄糖或琥珀酸甲酯而升高。与该发现一致，线粒体的形态在EtBr存在下改变，与线粒体共享相似之处，其中膜电位已被质子载体羰基氰化物对三氟甲氧基苯腙（FCCP）破坏。此外，尽管去极化剂KCl对膜去极化的影响完全保留，但在质膜电位水平上，两种营养促分泌剂均无影响。类似地，葡萄糖和琥珀酸甲酯不能增加胰岛素分泌，而KCl仍然有效。为了进一步测试mtDNA耗竭对胞吐作用的影响，Ne用金黄色葡萄球菌α-毒素透化INS-1细胞，其在质膜上形成小孔。与对照细胞相比，线粒体底物不能刺激mtDNA缺陷细胞中的胰岛素分泌，强调分泌缺陷在于线粒体功能水平而不是胞吐过程。结果表明，线粒体在暴露于高浓度营养促分泌素引起的下游效应中至关重要。
Mitochondrial dysfunction due to alterations in the mitochondrial genome (mtDNA) has recently attracted much attention, with the finding that mutations in the mitochondrially encoded proteins perturb cell function, Several disorders have been kinked to such genetic changes, including a specific diabetic phenotype, Using ethidium bromide (EtBr) that intercalates into mtDNA, we have effectively eliminated functions under the control of mtDNA from the highly differentiated INS-I insulin-secreting cell line. We have investigated the consequences on insulin secretion, mitochondrial enzyme activity, organelle structure, and membrane polarization in such cells (INS-1 rho(0)), Under these conditions, the mitochondrial membrane potential fails to hyperpolarize in response to either glucose or methylsuccinate, In agreement with this finding, the morphology of the mitochondria is altered in the presence of EtBr, sharing similarities with mitochondria in which the membrane potential has been collapsed with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). In addition, there is no effect of either nutrient secretagogue at the level of the plasma membrane potential, although the effect of,he depolarizing agent KCI on membrane depolarization is completely preserved. Similarly glucose and methylsuccinate fail to increase insulin secretion, whereas KCl is still effective, To test further the effects of mtDNA depletion on exocytosis, Ne permeabilized INS-1 cells with Staphylococcus aureus alpha-toxin, which forms small holes in the plasma membrane. In contrast to control cells, mitochondrial substrates were incapable of stimulating insulin secretion in mtDNA-deficient cells, emphasizing that the defect in secretion lies at the level of mitochondrial function rather than in the exocytotic process, The results indicate the paramount importance of the mitochondria in the downstream effects elicited by exposure to elevated concentrations of nutrient secretagogue.