Galactose enhances oxidative stress and alters metabolism of malate-aspartate shuttle in hepatoma cells

  • 張 喬淳

Student thesis: Master's Thesis


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
Date of Award2014 Sept 10
Original languageEnglish
SupervisorShu-Chu Shiesh (Supervisor)

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