蘋果酸(malate)-天門冬胺酸(aspartate)(MA)運輸器由多種載體及酵素組合而成，包含α-酮戊二酸(oxoglutarate)-蘋果酸載體蛋白、天門冬胺酸-榖胺酸(glutamate)、蘋果酸脫氫酶以及天門冬胺酸胺基轉移酶。MA運輸器可將細胞質的電子傳送到粒線體，以維持細胞內氧化還原之平衡。臨床上檸檬素(citrin)缺乏會導致新生兒膽汁淤積症與第二型瓜胺酸血症，而這些疾病的症狀，例如像高血氨症、瓜胺酸血症等，多與MA運輸器之代謝有關。但目前對於新生兒膽汁淤積的患者為何會進一步發展為第二型瓜安酸血症的機制仍不清楚。臨床上發現檸檬素缺乏的病人多半不喜歡醣類食物且有半乳糖血症。近來也有研究指出第二型瓜胺酸血症的病人若食用醣類食物則會使病程加重甚至可能加速病人死亡。我們假設半乳糖的低糖解效率造成氧化壓力並進一步影響了MA運輸器的代謝。因此，本篇研究的目的就是要探討半乳糖對肝腫瘤細胞之MA運輸器相關代謝的影響。我們將一般或檸檬素缺乏的HepG2 及Huh-7 細胞培養在不同的培養液中，包含25 mM高濃度葡萄糖、12.5 mM葡萄糖及半乳糖各半之混合液、10 mM低濃度半乳糖與25 mM高濃度半乳糖四種培養液。經72小時培養後，接著分析細胞內氧化壓力與MA運輸器相關代謝物的變化。以西方點墨法分析半乳糖對精胺丁二酸合成酶(argininosuccinate synthetase, ASS)蛋白表現的影響；細胞中三磷酸腺苷濃度和培養液中乳糖脫氫酶活性反映細胞存活率；並檢測粒線體中超氧化物的濃度以反應氧化壓力。MA運輸器相關代謝物，包括天門冬胺酸、榖胺酸、蘋果酸及琥珀酸(succinate)則利用液相層析質譜儀檢測其變化。另外，以酵素法檢測培養液中氨、乳糖與丙酮酸的濃度。無論檸檬素缺乏與否，在HepG2 及Huh-7 細胞中，半乳糖會導致精胺丁二酸合成酶 (p<0.01) 與細胞存活率明顯降低 (p<0.01)、提升培養液中乳糖脫氫酶活性（p＜0.005）。而調控半乳糖代謝的第一酵素：半乳糖激酶，在給予半乳糖培養的組別中表現量也下降，顯示細胞在半乳糖培養下有較低的糖解活性。細胞粒線體內超氧化物濃度也因為半乳糖的給予而明顯增加(p<0.05)。此外，半乳糖會降低細胞內乳糖(66.0±9.4在葡萄糖培養 vs. 15.6±2.4 mmol/g在半乳糖培養, p<0.01)與榖胺酸濃度(138.8±11.3　vs. 91.76±3.1 μmol/g, p<0.05)，並使天門冬胺酸(5.1±0.1　vs.9.1±0.1 μmol/g, p<0.005)及天門冬胺酸與榖胺酸比值(37.5±2.8 vs. 99.4±2.3, p<0.005)上升。培養液內氨濃度則因半乳糖而堆積(p<0.05)，丙酮酸與乳糖則減少(p<0.005)。給予丙酮酸、精胺酸、N-乙醯基半胱胺酸或甘胺酸可有效提升細胞存活率、培養液乳糖與丙酮酸濃度。其中，只有丙酮酸能有效回復榖胺酸及天門冬胺酸與榖胺酸比值。總結來說，我們的研究顯示半乳糖的低糖解效率會使細胞內氧化壓力增加，並進一步降低精胺丁二酸合成酶的表現，同時干擾MA運輸器相關之代謝；而丙酮酸、N-乙醯基半胱胺酸、苷胺酸或精胺酸可做為檸檬素缺乏患者的新治療策略，其中又以丙酮酸為最有效的治療方式。

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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