| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 102 === Malate-aspartate shuttle (MAS) consists of several carriers and enzymes including oxoglutarate malate carrier, aspartate glutamate carrier, malate dehydrogenase and aspartate aminotransferase. It plays a critical role in regulating cellular redox balance via transporting cytosolic electrons into mitochondria. Citrin deficiency is associated with neonatal intrahepatic cholestasis and adult-onset type II citrullinemia. The outcomes, such as hyperammonemia and citrullinemia, are related to metabolism of MA shuttle. However, the mechanism of disease progression remains unknown. Patients with citrin deficiency usually dislike intake of carbohydrate and suffer from galactosemia. Studies showed that use of carbohydrates exacerbated the symptoms and led to early death in CTLN2 patients. We hypothesized that galactose with low efficiency of glycolysis may cause intracellular stress leading to alters MA shuttle. Thus, in this study, we aimed to investigate the role of galactose on cell metabolism related to MAS in hepatoma cells. HepG2 and Huh-7 cells with or without citrin knockdown (KD) were grown in 25 mM glucose-, 12.5 mM glucose with 12.5 mM galactose-, 10 mM galactose- or 25 mM galactose-supplemented DMEM medium. After 72 hours incubation, the effects of galactose on the oxidative stress and MAS-related metabolites of cells were analyzed. Argininosuccinate synthetase (ASS) protein levels were detected by western blotting, cell viability by ATP levels and culture medium lactate dehydrogenase (LDH) activity and oxidative stress by mitochondrial superoxide. MAS-related metabolites, aspartate, glutamate, malate and succinate were analyzed using liquid chromatography-tandem mass spectrometry. Culture medium ammonium, LDH, lactate and pyruvate were measured by enzymatic methods. HepG2 or Huh-7 cells with or without citrin-KD growing in galactose-supplemented medium had decreased cytosolic ASS protein levels (p〈0.01), lower cell viability (p〈0.01) and increased culture medium LDH activities (p〈0.005). Galactokinase, the first enzyme involved in galactose metabolism, was decreased, indicating low glycolytic activity in galactose-fed cells. Mitochondrial superoxide levels were significantly increased when cells grown in galactose-supplemented medium (p〈0.05). Furthermore, the decreased intracellular lactate (66.0±9.4 vs. 15.6±2.4 mmol/g, p〈0.01) and glutamate (138.8±11.3 vs. 91.76±3.1 μmol/g, p〈0.05) and the increased aspartate (5.1±0.1 vs.9.1±0.1 μmol/g, p〈0.005) and the ratio of aspartate to glutamate (37.5±2.8 vs. 99.4±2.3, p〈0.005) were found in galactose-treated cells. Levels of ammonia in culture medium were increased (p〈0.05), but pyruvate and lactate were decreased (p〈0.005) in cells grown in galactose-supplemented medium. The treatment of pyruvate, NAC, glycine or arginine was effective in increasing cell viability, culture medium lactate and pyruvate. However, only the supplement of pyruvate ameliorated the level of glutamate and aspartate to glutamate ratio to normal. In conclusion, our finding suggested that low glycolytic efficiency of galactose increase oxidative stress and further decrease ASS protein levels and disturbed the metabolism of MAS. Moreover, pyruvate, NAC, glycine or arginine can be beneficial for treating patient with citrin deficiency, and pyruvate may be the most efficient strategy.
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