Effects of nutrient stress on malate-aspartate shuttle-silencing cells

  • 王 貝慈

Student thesis: Master's Thesis


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
Date of Award2014 Feb 6
Original languageEnglish
SupervisorShu-Chu Shiesh (Supervisor)

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