檸檬素(citrin)是位在粒線體內膜上的運輸蛋白，主要表現在肝臟。檸檬素的功能是將粒線體的天門冬胺酸(aspartate)送到細胞質，並交換麩醯胺酸(glutamate)進入粒線體中。檸檬素也參與蘋果酸－天門冬胺酸運輸器，負責將細胞質的還原態菸草醯胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide reduced form, NADH)相當物質送入粒線體內。瓜胺酸血症(citrullinemia)是一種遺傳性疾病，第I型瓜胺酸血症(CTLN1)是由於ASS1基因異常，導致尿素循環的精胺丁二酸合成酶(argininosuccinate synthetase, ASS)缺失，使得瓜胺酸無法轉換成精胺丁二酸。第II型瓜胺酸血症(CTLN2)是由於SLC25A13基因突變導致檸檬素缺失，並且使肝臟ASS蛋白質變少，是目前台灣最常見的尿素循環代謝疾病。先前的報導指出檸檬素缺乏會有碳水化合物所引起毒性的問題，臨床上以低碳水化合物、高蛋白質的飲食，或是給予丙酮酸(pyruvate)治療病人，以促進NADH轉換成NAD+，減少NADH的堆積。但是檸檬素缺乏致成肝臟ASS蛋白質下降的機制不清楚。我們假設在檸檬素缺乏時，糖類的攝取會改變細胞NAD+/NADH比值，進而影響細胞質ASS的蛋白質表現，以及尿素循環路徑的相關代謝物改變。因此本研究建立了兩個液相層析串聯質譜儀的方法，分別偵測細胞的NAD+和NADH的濃度，以及偵測細胞內尿素循環相關代謝物的濃度；並探討當細胞缺乏檸檬素時，給予不同的葡葡糖濃度之培養基伴隨麩醯胺酸或精胺酸的補充是否會影響其NAD+/NADH比值、ASS蛋白質量，以及尿素循環相關代謝物濃度。近來報導ASS會促使單磷酸腺苷(adenosine monophosphate, AMP)活化蛋白激酶(AMP-activated protein kinase, AMPK)被磷酸化，進而促進β-氧化作用以及抑制脂質的合成代謝。我們想知道給予營養同的培養所造成的ASS蛋白質改變，是否會影響p-AMPK的蛋白質表現。藉由慢病毒為載體的shRNA抑制SLC25A13的表現，建立體外檸檬素缺乏的人類肝癌細胞(HepG2)，檸檬素被抑制達90%，且會使細胞存活率下降。ASS和AMPK的蛋白質量都減少了。細胞質的NAD+與NADH濃度則沒有明顯差異。而精胺酸、瓜胺酸、離胺酸及麩醯胺酸上升趨勢，天門冬胺酸下降趨勢。而在檸檬素缺乏HepG2細胞培養在不同葡萄糖濃度的培養基，細胞存活率則沒有明顯差異，但隨葡萄糖濃度越低而有細胞存活率下降的趨勢。在檸檬素缺乏HepG2細胞ASS的蛋白質量也減少約17%了，而限制葡萄糖可以減緩ASS的蛋白質減少。細胞質的NAD+與NADH濃度則沒有明顯差異。在尿素循環相關代謝物的部分，當細胞培養於低濃度葡萄糖與高濃度葡萄糖相比則沒有明顯差異，但限制醣類的攝取有精胺酸、瓜胺酸與麩醯胺酸下降的趨勢以及天門冬胺酸與麩胺酸上升的趨勢。添加麩醯胺酸或精胺酸到低濃度葡萄糖的培養基，可以緩減低濃度葡萄糖所造成的細胞存活率下降以及ASS、p-AMPK蛋白質量減少。添加精胺酸到低濃度葡萄糖的培養基，可以有效減緩檸檬素缺乏造成ASS下降所導致的天門冬胺酸下降。我們發現細胞限制葡萄糖對ASS蛋白質量和磷酸化AMPK的影響是一致的，顯示ASS蛋白質量改變可能是透過影響AMPK磷酸化。總結來說，我們建立了兩個液相層析串聯式質譜儀的方法，用來偵測細胞質的NAD+與NADH濃度，以及尿素循環相關代謝物的濃度。我們所看到結果與臨床上病人限制碳水化合物的攝取可以減緩檸檬素缺乏所造成的肝臟ASS蛋白質缺乏是一致的。檸檬素缺乏會使ASS及p-AMPK下降，而補充麩醯胺酸或精胺酸可以有效回復檸檬素缺乏所造成的ASS蛋白質量及AMPK的磷酸化下降。

Citrin, expressed mainly in the liver, functions as malate-aspartate shuttle to transport cytosolic NADH-reducing equivalents into mitochondrial. The patients with citrin deficiency demonstrated decrease of liver-specific argininosuccinate synthetase (ASS). Carbohydrate diets may increase cytosolic NADH production and should be avoided in citrin deficiency patients. However, the mechanisms of citrin defect-induced decrease of liver ASS remain unknown. Therefore, we aimed to establish two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to measure cytosolic NAD+ and NADH; and the urea cycle-related metabolites. We also investigated whether citrin-deficiency with low-glucose culture medium would change the NAD+/NADH ratio, metabolites and ASS protein. We examined whether the changes of cytosolic ASS protein levels caused by different nutrition in medium would affect the expression of phosphorylated AMP-activated protein kinase (p-AMPK). Citrin-deficiency HepG2 cells showed the decrease of cell viability, ASS and p-AMPK protein levels and the increase of intracellular lysine and glutamine levels. The results showed that low-glucose treatments decreased the cell viability, cytosolic ASS and p-AMPK protein levels. Addition of glutamine/arginine in low glucose-containing culture medium could reverse the decrease of cell viability, ASS and AMPK. The changes of cytosolic ASS levels cultured in low glucose were in parallel with the decreased AMPK activation. To sum up, two LC-MS/MS methods were established. Our results indicate that restricted glucose intake and replaced glucose with glutamine or arginine could alleviate the decrease of liver-specific ASS caused by citrin deficiency. Citrin deficiency caused low expression of ASS and AMPK, which would be alleviated by glutamine or arginine supplements.

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