柯逢春臺灣大學:分子與細胞生物學研究所蔡孟真Tsai, Meng-JenMeng-JenTsai2010-06-022018-07-062010-06-022018-07-062009U0001-0508200914341800http://ntur.lib.ntu.edu.tw//handle/246246/184720粒線體在細胞生長(cell growth)上扮演重要角色，當細胞進入生長、增生狀時，必須進行以粒線體角色轉換為主軸的代謝重整(metabolic reprogramming)：線體TCA cycle將其中間產物輸出，提供生長所需之巨分子合成的材料，此即cataplerosis 作用。而細胞生長所需的大量ATP 則轉由glycolysis 提供，並提高DH-A活性，將pyruvate快速轉換為lactate排出，同時維持細胞質NAD+/NADHatio，確保glycolysis 的進行。同時，細胞必須補充流失的TCA cycle 中間產物，即為補充效應(anaplerosis)，它給予細胞使用TCA cycle的中間產物作為生合成需前驅物的能力，是細胞生長的重要特徵。其中，胺基酸glutamine 的代謝為anaplerosis 的重要來源，可被glutaminase 轉成glutamate，再經由GDH 轉成KG，而進入TCA cycle進行補充。人類纖維母細胞WI38 中，處理粒線體malate-aspartate shuttle 的抑制物OA，會造成細胞停止增生、細胞老化(senescence)，以及mTORC1 活性下降，mTORC2 活性上升的現象。此些AOA 處理所引發的效應，會在與aKG 或EAA 共同處理之下而受到阻礙。我們推論aKG 是藉由anaplerosis 補充TCAycle，而反轉AOA 所造成的效應。而NEAA 是七種非必需氨基酸的混合物，一測試其中成分之後，發現唯有aspartate與aspargine有阻礙AOA 的效果，以spartate效果最好。Aspartate的作用可能是透過競爭性抑制而弱化AOA的效果，是透過提升asparagine synthetase 活性而產生作用。此外，leucine 是促進TORC1 活性效果最明顯的胺基酸，又mTORC1 與粒線體活性間互有影響關，但實驗發現leucine沒有反轉AOA的效果。研究透過在aKG 或aspartate 添加之下可阻斷AOA 所誘發的細胞停止增及老化的效應顯示，粒線體malate-aspartate shuttle活性為細胞增長所必須，可在anaplerosis 中扮演重要角色，突顯出粒線體功能轉換之代謝重整(metaboliceprogramming)在細胞生長過程中的重要性。Mitochondria play a crucial role in cell growth. The switching role ofitochondria TCA cycle from producing maximal amount of ATP to exporting muchf the intermediates for lipid, protein, and nucleic acid synthesis is the main part ofmetabolic reprogramming” process that necessary for cells to proliferate. This resultsn a continuous efflux of intermediates, which is so-called cataplerosis. In response toataplerosis, cells switch the generation of ATP from TCA cycle to glycolysis andnhance LDH-A activity to convert pyruvate into lactate and also maintain theAD+/NADH ratio, ensuring high glycolytic flux in proliferating cells. In order toustain TCA cycle function under cataplerosis, cells must re-supply TCA cyclentermediates, which is so-called anaplerosis. Anaplerosis is critical for cell growthecause it enables cells to use TCA cycle as a supply of biosynthetic precursors.lutamine metabolism is one of the anaplerotic sources. Proliferating cells metabolizelutamine into glutamate and then aKG, which is convenient for cells to use as carbonource for TCA cycle, providing anaplerosis sources in growing cells.n WI38 cells system, the malate-aspartate shuttle inhibitor, AOA, induces cellycle arrest and senescence with the reduction of mTORC1 and promotion ofTORC2 activity. These effects are blocked by co-treating aKG or NEAA. Theeversal effect of aKG on AOA treatment is believed as replenishing TCA cyclentermediates through anaplerosis pathway. On the other hand, after testing individualmino acid in NEAA, we discovered that only aspartate and asparagine can rescue theell cycle arrest and senescence ratio caused by AOA and the effect of aspartate isetter than asparagine. Aspartate may function through enhancing asparagineynthetase activity or as a competitor of AOA thus attenuating its effect. We also triedeucine because of its greatest effect on activating mTORC1. However, addition ofeucine has no effect to reverse AOA-induced cell cycle arrest and senescence.vn this study, we found that the function of malate-aspartate shuttle is importantor cell growth, implying that malate-aspartate shuttle is essential in anaplerosis. Thistudy also points out the importance of mitochondria switching role in metaboliceprogramming in cell growth.誌謝 i要 iibstract iii錄 v目錄 vii索表 viii言 1線體(mitochondria)在細胞生長(cell growth)所扮演的角色 1胞生長(cell growth)的中心調節者: mTOR 3基酸(amino acid)調控mTORC1的活性 6驗目的 12料與方法 14料 14胞培養 14長曲線 15化比例測定(Senescence Associated β-galatosidase stain) 15果 17EAA可以阻礙AOA造成的cell cycle arrest以及senescence 17EAA若去除aspartate和asparagine則失去反轉AOA所引發之cell cycle arrest和senescence的能力 18spartate以及asparagine有效阻礙AOA所引發的cell cycle arrest和senescence 19spartate透過其transporter運送至細胞內 19eucine不能反轉由AOA引起的cell cycle arrest和senescence 21論 22考文獻 37-46application/pdf1015647 bytesapplication/pdfen-US粒線體代謝重整AOAmalate-aspartate shuttle細胞老化mitochondriacell growthmetabolic reprogramminghttp://ntur.lib.ntu.edu.tw/bitstream/246246/184720/1/ntu-98-R95b43021-1.pdf