Huh-7細胞剔除粒線體NADH運輸器-檸檬酸之研究 : 甘胺酸之效用

Abstract

Malate-aspartate shuttle (MAS) plays an important role in equilibrating the cellular bioenergetic states by transferring cytosolic NADH equivalent across the inner mitochondrial membrane into the mitochondria for oxidation. Citrin, encoded by SLC25A13, is a liver-type mitochondrial aspartate-glutamate carrier. It plays an important role in malate-aspartate nicotinamide adenine dinucleotide (NADH) shuttle, and the synthesis of urea, protein and nucleotide. Citrin deficiency results in type II citrullinemia (CTLN2). Patients with CTLN2 are characterized by liver-specific argininosuccinate synthetase (ASS) deficiency, a decreased cytosolic nicotinamide adenine dinucleotide (NAD) to NADH ratio and increased oxidative stress. This study aimed to establish a method for simultaneous determination of NAD+ and NADH concentration by LC-tandem mass. Furthermore, we investigated the effects of genetic silencing of citrin by SLC25A13 shRNA or chemically shut-down of MA shuttle by aminooxyacetate (AOA) on Huh-7 cells; whether glycine, arginine or pyruvate treatment would reverse the changes. The mechanism of citrin deficiency–induced ASS expression would be further evaluated. Cells were examined for mitochondrial membrane potential (MMP) using the fluorescent dye Rhodamine 123, and cell apoptosis level by staining of Annexin V and propidium iodide (PI) by flow cytometry and detecting of caspase-3 activity. Oxidative stress was examined by malondialdehyde (MDA), a lipid peroxidation indicator, by high performance liquid chromatography, and intracellular hydrogen peroxide using H2O2-sensitive fluorescent probe, 2',7'- dichlorofluorescin diacetate (DCFH-DA), respectively. Cytosolic ASS and mitochondrial citrin protein were measured by western blotting. The imprecision (CV%) were <6%. The average recovery of NAD+ was 101.7%, NADH was 118.3%. Compared to control cells, citrin-KD cells showed increased MDA (0.18±0.02 μmol/g vs. 0.25±0.02 μmol/g, p<0.05), caspase-3 activity and cell apoptosis level. Furthermore, decreased MMP, cytosolic NAD+/NADH ratio (2.44±0.06 vs. 1.832±0.1, p<0.005) and cytosolic ASS expression were also seen in citrin-KD cells. The increase of apoptosis and decrease of MMP, cytosolic ASS expression, cytosolic NAD+/NADH ratio were restored with glycine, arginine or pyruvate treatments in citrin-KD cells. Furthermore, glycine treatment reduced the citrin-KD induced MDA increase. AOA-treated cells also showed increased MDA (0.16±0.01 μmol/g vs. 0.31±0.02 μmol/g, p<0.0005), caspase-3 activity and cell apoptosis, decreased MMP and cytosolic NAD+/NADH ratio (2.72±0.30 vs. 2.00±0.15, p<0.05). The decreased MMP and increased caspase-3 activity and apoptosis level induced by AOA treatment were recovered by glycine, arginine or pyruvate treatment. Taken together, glycine has the potential in decreasing the oxidative stress and apoptosis, maintaining the mitochondrial membrane potential and increasing ASS expression. Glycine treatment may be a new therapeutic strategy for patients with CTLN2.