胺基酸對Aminooxyacetate所引發之早發性細胞老化的抑制作用探討

Abstract

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 態時，必須進行以粒線體角色轉換為主軸的代謝重整(metabolic reprogramming)： 粒線體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 為cataplerosis 作用。而細胞生長所需的大量ATP 則轉由glycolysis 提供，並提高 LDH-A活性，將pyruvate快速轉換為lactate排出，同時維持細胞質NAD+/NADH ratio，確保glycolysis 的進行。同時，細胞必須補充流失的TCA cycle 中間產物， 此即為補充效應(anaplerosis)，它給予細胞使用TCA cycle的中間產物作為生合成 所需前驅物的能力，是細胞生長的重要特徵。其中，胺基酸glutamine 的代謝為 一anaplerosis 的重要來源，可被glutaminase 轉成glutamate，再經由GDH 轉成 aKG，而進入TCA cycle進行補充。 在人類纖維母細胞WI38 中，處理粒線體malate-aspartate shuttle 的抑制物 AOA，會造成細胞停止增生、細胞老化(senescence)，以及mTORC1 活性下降， 而mTORC2 活性上升的現象。此些AOA 處理所引發的效應，會在與aKG 或 NEAA 共同處理之下而受到阻礙。我們推論aKG 是藉由anaplerosis 補充TCA cycle，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， 逐一測試其中成分之後，發現唯有aspartate與aspargine有阻礙AOA 的效果，以 aspartate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， 或是透過提升asparagine synthetase 活性而產生作用。此外，leucine 是促進 mTORC1 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 係，但實驗發現leucine沒有反轉AOA的效果。 本研究透過在aKG 或aspartate 添加之下可阻斷AOA 所誘發的細胞停止增 生及老化的效應顯示，粒線體malate-aspartate shuttle活性為細胞增長所必須，可 能在anaplerosis 中扮演重要角色，突顯出粒線體功能轉換之代謝重整(metabolic reprogramming)在細胞生長過程中的重要性。

Mitochondria play a crucial role in cell growth. The switching role of mitochondria TCA cycle from producing maximal amount of ATP to exporting much of the intermediates for lipid, protein, and nucleic acid synthesis is the main part of “metabolic reprogramming” process that necessary for cells to proliferate. This results in a continuous efflux of intermediates, which is so-called cataplerosis. In response to cataplerosis, cells switch the generation of ATP from TCA cycle to glycolysis and enhance LDH-A activity to convert pyruvate into lactate and also maintain the NAD+/NADH ratio, ensuring high glycolytic flux in proliferating cells. In order to sustain TCA cycle function under cataplerosis, cells must re-supply TCA cycle intermediates, which is so-called anaplerosis. Anaplerosis is critical for cell growth because it enables cells to use TCA cycle as a supply of biosynthetic precursors. Glutamine metabolism is one of the anaplerotic sources. Proliferating cells metabolize glutamine into glutamate and then aKG, which is convenient for cells to use as carbon source for TCA cycle, providing anaplerosis sources in growing cells. In WI38 cells system, the malate-aspartate shuttle inhibitor, AOA, induces cell cycle arrest and senescence with the reduction of mTORC1 and promotion of mTORC2 activity. These effects are blocked by co-treating aKG or NEAA. The reversal effect of aKG on AOA treatment is believed as replenishing TCA cycle intermediates through anaplerosis pathway. On the other hand, after testing individual amino acid in NEAA, we discovered that only aspartate and asparagine can rescue the cell cycle arrest and senescence ratio caused by AOA and the effect of aspartate is better than asparagine. Aspartate may function through enhancing asparagine synthetase activity or as a competitor of AOA thus attenuating its effect. We also tried leucine because of its greatest effect on activating mTORC1. However, addition of leucine has no effect to reverse AOA-induced cell cycle arrest and senescence. iv In this study, we found that the function of malate-aspartate shuttle is important for cell growth, implying that malate-aspartate shuttle is essential in anaplerosis. This study also points out the importance of mitochondria switching role in metabolic reprogramming in cell growth.