攝取檸檬酸對果蠅生理的影響與誘發長壽的機制

Abstract

三羧酸循環是生物在粒線體進行醣類、脂類及胺基酸代謝與合成，以及製造提供能量的腺苷三磷酸的途徑，而檸檬酸是其中重要的中間代謝物。我們讓 w1118 果蠅攝取加入 0.1% 檸檬酸的高卡路里食物，發現添加檸檬酸具有延長果蠅壽命的效果，同時並不影響果蠅的體重、食量與活動力，因此本研究探討攝取檸檬酸對果蠅生理的影響，以及檸檬酸誘發的長壽機制與原因。利用轉基因果蠅，我們將可能相關的分子之基因表達減少，並測量其壽命是否會因攝取檸檬酸而延長。由於檸檬酸的運輸需要透過細胞膜上的檸檬酸運輸蛋白，我們先測試 Indy 突變果蠅，發現檸檬酸誘發的長壽機制需要 Indy 的參與。過去的研究指出 Indy 突變果蠅的生理表現類似飲食節制，因此我們接著分析飲食節制相關的分子機制AMPK 、 TOR ，以及 PGC-1 ，將這些基因表達缺失的果蠅培養在含有檸檬酸的食物，失去了檸檬酸誘發的長壽效果，說明攝取檸檬酸誘發長壽的機制與飲食節制有關。於是我們接著驗證飲食節制的下游，酮體代謝的相關分子，包含酮生成的 Hmgs 與 Hmgcl ，以及酮分解的 SCOT ，發現降低這些基因表達的果蠅攝取檸檬酸，也沒有長壽的結果。先前的研究指出飲食節制會提升酮體代謝，而透過測量果蠅體內酮體β-羥基丁酸的濃度，發現攝取檸檬酸的 w1118 果蠅有較高的的酮體濃度。綜合以上結果，我們推測檸檬酸誘發的長壽機制與飲食節制一致，需要 Indy，營養和能量感應途徑，以及酮體代謝的參與。

Citrate plays an essential role in the metabolic pathway. As a critical intermediate in the tricarboxylic acid cycle, citrate mediates energy production and contributes to fatty acid and sterol synthesis. In this study, we aim to investigate the effect of citrate supplementation on Drosophila physiology. Our results showed that 0.1% citrate intake extended the lifespan of Drosophila on a higher yeast diet (15%), without affecting their body weight, food intake, and locomotor activity. Citrate-induced lifespan extension requires Indy encoding citrate transporters, and genetic disruptions of dietary restriction (DR) pathway-related molecules, including AMP-activated protein kinase (AMPK), target of rapamycin (dTOR) and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1/ spargel) in flies diminished citrate-induced lifespan extension. To further elucidate, we analyzed downstream pathways of DR which includes ketogenesis. We observed knockdown in ketogenesis pathway molecules, such as hydroxymethylglutaryl-CoA synthase (Hmgs) and HMG-CoA lyase (Hmgcl), as well as ketolysis molecules succinyl-CoA:3-oxaloacid CoA transferase (SCOT) failed to prolong lifespan with citrate supplementation. Moreover, administration of 0.1% citrate to w1118 flies significantly increased ketone body levels. These data establish the role of INDY transporters, nutrient and energy-sensing pathways, as well as ketone body metabolism under citrate-induction conditions which provides a mechanistic link between citrate supplementation and DR pathways.