Studies of Metabolic Profile Changes in Human Cells Harboring High Levels of A8344G mtDNA Mutation
| Autor: | Yu-Jen Lai, 賴郁仁 |
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| Rok vydání: | 2008 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 In mammalian cells, more than 90% of the ATP is synthesized by mitochondrial respiration and oxidative phosphorylation. Under normoxia, many metabolites can be further metabolized to CO2 and H2O by mitochondria in aerobic metabolism. With limited oxygen, a large proportion of pyruvate made from glycolysis is converted to lactate by lactate dehydrogenase-A (LDH-A) in anaerobic metabolism. Many previous studies have demonstrated that lactic acidosis is one of the features of many mitochondrial diseases. We have hypothesized that metabolic shift is a general phenomenon in patients with mitochondrial dysfunction, and that is a result of gene expression pattern change from aerobic metabolism to glycolysis. In this study, I used human cells harboring high levels of A8344G mitochondrial DNA (mtDNA) mutation as an experimental model to investigate the effect of mitochondrial dysfunction on cellular metabolism and the underlying mechanisms. We found that the respiratory functions were defective in mutant cells, which had lower oxygen consumption rate (30-40%) and lower level of endogenous superoxide anions (22%). Moreover, mutant cells had higher rate of lactic acid production. We then investigated the alterations in the expression of genes involved in aerobic metabolism and glycolysis. The results showed that the mutant cells exhibited decreased expression of pyruvate dehydrogenase (PDH) and increased expression of glucose transporter 1 (GLUT1), lactate dehydrogenase-A (LDH-A) and PDH kinase (PDK) that inhibits the PDH activity. In addition, the protein expression level of hypoxia-inducible facor-1�� (HIF-1��) in mutant cells was higher than that in normal cells. After treatment of mutant cells with the HIF-1 inhibitor echinomycin, the expression profile of metabolic enzymes and lactic acid production rate resumed to the normal level. Furthermore, mutant cells were found to be more sensitive than normal cells to the treatment of echinomycin. After treatment of cells with cycloheximide and MG132, respectively, we found no significant difference in the protein stability of HIF-1���nbetween the mutant and wild-type cells. However, the rate of HIF-1���nsynthesis was higher in mutant cells. This indicates that in mutant cells the HIF-1 activity is increased by an increase of HIF-1���nexpression to regulate the metabolic shift, which is very important for mutant cells to survive. Taken together, we suggest that mitochondrial dysfunction-elicited metabolic shift is one of the major contributory factors for lactic acidosis frequently manifested in patients with the mitochondrial diseases. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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