de Andrade P B M, Rubi B, Frigerio F, van den Ouweland J M W, Maassen J A, Maechler P
Department of Cell Physiology and Metabolism, University Medical Center, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland.
Diabetologia. 2006 Aug;49(8):1816-26. doi: 10.1007/s00125-006-0301-9. Epub 2006 May 31.
AIMS/HYPOTHESIS: Mitochondrial DNA (mtDNA) mutations cause several diseases, including mitochondrial inherited diabetes and deafness (MIDD), typically associated with the mtDNA A3243G point mutation on tRNALeu gene. The common hypothesis to explain the link between the genotype and the phenotype is that the mutation might impair mitochondrial metabolism expressly required for beta cell functions. However, this assumption has not yet been tested.
We used clonal osteosarcoma cytosolic hybrid cells (namely cybrids) harbouring mitochondria derived from MIDD patients and containing either exclusively wild-type or mutated (A3243G) mtDNA. According to the importance of mitochondrial metabolism in beta cells, we studied the impact of the mutation on key parameters by comparing stimulation of these cybrids by the main insulin secretagogue glucose and the mitochondrial substrate pyruvate.
Compared with control mtDNA from the same patient, the A3243G mutation markedly modified metabolic pathways leading to a high glycolytic rate (2.8-fold increase), increased lactate production (2.5-fold), and reduced glucose oxidation (-83%). We also observed impaired NADH responses (-56%), negligible mitochondrial membrane potential, and reduced, only transient ATP generation. Moreover, cybrid cells carrying patient-derived mutant mtDNA exhibited deranged cell calcium handling with increased cytosolic loads (1.4-fold higher), and elevated reactive oxygen species (2.6-fold increase) under glucose deprivation.
CONCLUSIONS/INTERPRETATION: The present study demonstrates that the mtDNA A3243G mutation impairs crucial metabolic events required for proper cell functions, such as coupling of glucose recognition to insulin secretion.
目的/假说：线粒体DNA（mtDNA）突变会引发多种疾病，包括线粒体遗传性糖尿病和耳聋（MIDD），通常与tRNALeu基因上的mtDNA A3243G点突变相关。解释基因型与表型之间联系的常见假说是，该突变可能会损害β细胞功能明确所需的线粒体代谢。然而，这一假设尚未得到验证。
我们使用了克隆性骨肉瘤胞质杂种细胞（即细胞杂交体），其线粒体来源于MIDD患者，且仅含有野生型或突变型（A3243G）mtDNA。鉴于线粒体代谢在β细胞中的重要性，我们通过比较主要胰岛素促分泌剂葡萄糖和线粒体底物丙酮酸对这些细胞杂交体的刺激作用，研究了该突变对关键参数的影响。
与同一患者的对照mtDNA相比，A3243G突变显著改变了代谢途径，导致糖酵解速率大幅提高（增加2.8倍）、乳酸生成增加（2.5倍）以及葡萄糖氧化减少（-83%）。我们还观察到NADH反应受损（-56%）、线粒体膜电位可忽略不计以及ATP生成仅短暂减少。此外，携带患者来源突变mtDNA的细胞杂交体在葡萄糖剥夺条件下表现出细胞钙处理紊乱，胞质钙负荷增加（高1.4倍），活性氧水平升高（增加2.6倍）。
结论/解读：本研究表明，mtDNA A3243G突变会损害细胞正常功能所需的关键代谢事件，如葡萄糖识别与胰岛素分泌的偶联。
