| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 100 === Myoclonic epilepsy and ragged-red fibers (MERRF) syndrome is a maternally inherited mitochondrial encephalomyopathy characterized by various clinical presentations involving both muscular and nervous systems. It has been documented that 80-90% of the patients with MERRF syndrome are caused by the A8344G mutation in the tRNALys gene of mitochondrial DNA (mtDNA) that leads to severe defects in mitochondrial protein synthesis. Mitochondrial dysfunction caused by mtDNA mutation results in not only inefficient generation of ATP but also increased production of reactive oxygen species (ROS). Previously, primary cultures of skin fibroblasts from several patients with MERRF syndrome were established in our laboratory. In the present study, I first investigated the energy metabolism shifts from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis as a bioenergetic adaptation in skin fibroblasts from MERRF patients as compared to those from age-matched normal subjects. I contended that the bioenergetic metabolism in skin fibroblasts would be perturbed by oxidative stress. In order to unravel the molecular mechanism involved in the alteration of energy metabolism in response to oxidative stress, I treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H2O2 to study the response of bioenergetic adaptation. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase A (LDHA) and glucose transporter 1 (GLUT1) were up-regulated in H2O2-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H2O2 in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, I found that the AMPK-mediated increase of glycolytic flux was accompanied by an increase of intracellular NADPH content in response to H2O2–induced oxidative stress via the action of glucose-6-phosphate dehydrogenase (G6PD). By treatment of the CCD-966SK cells with glycolytic inhibitors, an AMPK inhibitor, and genetic knockdown of AMPK, respectively, I found that the H2O2-induced increase of NADPH was abrogated and resulted in the overproduction of intracellular ROS and cell death. Significantly, I showed that the phosphorylation level of AMPK and glycolytic flux were also increased to confer an advantage of survival for MERRF skin fibroblasts. Taken together, I suggest that the increased production of NADPH by AMPK- mediated increase of the glycolytic flux contributes to the oxidative stress adaptation of MERRF skin fibroblasts and H2O2-treated normal skin fibroblasts, respectively. The findings of this study have provided new information for us to better understand the bioenergetic response to oxidative stress of human skin fibroblasts and shed new light in the unraveling of the molecular basis of the pathophysiology of mitochondrial diseases such as MERRF syndrome.
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