Relation of cytosolic reducing power to argininosuccinate synthetase activity in hepatocyte

  • 李 冠瑩

Student thesis: Master's Thesis


Argininosuccinate synthetase (ASS) is a ubiquitous enzyme with most abundant levels in liver and kidney ASS plays a key role in pathways of urea cycle polyamine synthesis creatinine synthesis and arginine-citrulline cycle for NO production Decreased liver-specific ASS activity was found in adult-onset type II citrullinemia (CTLN2) patients which is due to the deficiency of citrin an aspartate-glutamate carrier on the inner membrane of mitochondria to maintain cellular redox states However ASS activity was at variable levels in neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) Metabolic disorders such as galactosemia are shown in NICCD patients Some NICCD patients progress to CTLN2 decades later with unknown mechanism Lactose-restricted formula improved clinical symptoms in NICCD patients Moreover increased cytosolic reducing power was found in cells grown in galactose-containing culture medium Therefore this study aimed to establish two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the measurements of ASS activity and arginine and its related metabolites and to investigate the impact of galactose fructose and low glucose (9 mM glucose) on reducing power ASS activity and cellular stress in primary hepatocytes with or without citrin-knockdown In addition we examined the feasibility of ASS activity measurement in peripheral lymphocytes and the comparison with liver ASS activity Mice hepatocytes AML12 were transfected with Slc25a13-shRNA to knockdown citrin and cultured in medium with or without galactose fructose or low glucose Decreased ASS specific activity was found in hepatocytes cultured in galactose- (154 7 ± 8 0 mU/g) fructose- (140 0 ± 9 2 mU/g) or low glucose-containing medium (159 8 ± 11 4 mU/g) compared with 18 mM glucose medium (203 1 ± 3 0 mU/g) Increased lactate/pyruvate ratio in culture media was also found in 9 mM glucose + 9 mM galactose or fructose or 9 mM glucose only (64 5 63 7 and 101 1 v s 53 3) as well as cytosolic NADH concentrations (44 6 40 8 and 52 0 v s 22 5 nmol/g) Citrin knockdown did not alter ASS protein levels measured by western bloting However glucose insufficient culture medium caused impaired mitochondrial membrane potential (MMP) in citrin-KD cells as well as the decreased ASS activity increased lactate/pyruvate ratio and increased NADH concentration The above changes were similar with AML12 cells without citrin-KD Moreover hepatocytes cultured in glucose insufficient medium displayed increased degree of vulnerability to H2O2 as indicated by decreased MMP and cell viability The lactate/pyruvate ratio and ASS activity showed a negative correlation (r=-0 647 p
Date of Award2014 Sep 10
Original languageEnglish
SupervisorShu-Chu Shiesh (Supervisor)

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