| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 102 === Malate-aspartate (MA) shuttle, locating on the inner membrane of mitochondria, is essential for maintaining cellular bioenergetic states via the redox and transaminase reactions. The defect of citrin, a component of MA shuttle, is associated with neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). NICCD patients present metabolic abnormalities including galactosemia. However, it’s unknown why symptoms of NICCD patients resolve within first year of life and some of patients develop CTLN2 decades later. Treating NICCD patients with lactose (galactose)-restricted formula has been shown to improve clinical symptoms. Therefore, we aimed to investigate the effects of high glucose-, low glucose- or galactose on MA shuttle-silencing HepG2 cells. The silencing was achieved genetically with SLC25A13 shRNA. Furthermore, we examined whether supplement of specific amino acids would alleviate these effects. Mitochondrial membrane potential (MMP) using Rhodamine 123 or JC-1 dye and cell viability by ATP levels were measured. Oxidative stress was assessed by cytosolic NADH/NAD+ ratio and intracellular reactive oxygen species (ROS) levels using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and fluorescence spectrophotometer, respectively. The concentrations of intracellular metabolites were measured by LC-MS/MS. In citrin knockdown-(KD) cells, galactose-supplemented medium significantly caused reduced levels of cytosolic argininosuccinate synthetase (ASS) (p〈0.01), and increased oxidative stress (p〈0.01) and intracellular aspartate/glutamate ratio (0.06 in galactose supplement vs. 0.04 in low glucose supplement, p〈0.05), compared to low glucose supplement. The addition of H2O2 induced the decrease of MMP (p〈0.05) in galactose-supplemented citrin-KD cells. Furthermore, citrin-KD cells grown in galactose medium showed higher degree of vulnerability to H2O2, indicated by increased cytosolic NADH/NAD+ (p〈0.05), ROS (p〈0.005) and intracellular aspartate/glutamate (p〈0.005), and decreased cell viability (p〈0.005), compared to low glucose medium. The pretreatment of pyruvate, glycine or NAC was effective in increasing the cell viability and MMP, and decreasing the accumulation of cytosolic NADH, intracellular ROS and intracellular aspartate/glutamate; thus protecting citrin-KD cells against nutrient and oxidative stress. The supplement of pyruvate ameliorated the reduction of cytosolic ASS in galactose-treated citrin-KD cells (p〈0.05). Taken together, our results suggest that patients with citrin defect should avoid galactose-containing food and oxidative stress, and pyruvate, glycine or NAC may be beneficial for patients with citrin deficiency.
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