Biomarker Department, Daiichi Sankyo Co., Ltd, Tokyo, Japan.
Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.
Sci Rep. 2020 Aug 3;10(1):13065. doi: 10.1038/s41598-020-70000-6.
Fumarate hydratase (FH) is an enzyme in the tricarboxylic acid (TCA) cycle, biallelic loss-of-function mutations of which are associated with hereditary leiomyomatosis and renal cell cancer. However, how FH defect modulates intracellular metabolic fluxes in human cells has remained unclear. This study aimed to reveal metabolic flux alterations induced by reduced FH activity. We applied C metabolic flux analysis (C-MFA) to an established cell line with diminished FH activity (FH) and parental HEK293 cells. FH cells showed reduced pyruvate import flux into mitochondria and subsequent TCA cycle fluxes. Interestingly, the diminished FH activity decreased FH flux only by about 20%, suggesting a very low need for FH to maintain the oxidative TCA cycle. Cellular ATP production from the TCA cycle was dominantly suppressed compared with that from glycolysis in FH cells. Consistently, FH cells exhibited higher glucose dependence for ATP production and higher resistance to an ATP synthase inhibitor. In summary, using FH cells we demonstrated that FH defect led to suppressed pyruvate import into mitochondria, followed by downregulated TCA cycle activity and altered ATP production pathway balance from the TCA cycle to glycolysis. We confirmed that C-MFA can provide direct and quantitative information on metabolic alterations induced by FH defect.
延胡索酸水合酶（FH）是三羧酸（TCA）循环中的一种酶，其双等位基因失活突变与遗传性平滑肌瘤病和肾细胞癌有关。然而，FH 缺陷如何调节人类细胞内的细胞内代谢通量仍不清楚。本研究旨在揭示 FH 活性降低引起的代谢通量变化。我们应用 C 代谢通量分析（C-MFA）对 FH 活性降低的已建立细胞系（FH）和亲本 HEK293 细胞进行分析。FH 细胞表现出进入线粒体的丙酮酸摄取通量减少，随后 TCA 循环通量减少。有趣的是，降低的 FH 活性仅降低 FH 通量约 20%，表明 FH 维持氧化 TCA 循环的需求非常低。与糖酵解相比，FH 细胞中来自 TCA 循环的细胞 ATP 产生受到明显抑制。一致地，FH 细胞表现出更高的葡萄糖依赖性 ATP 产生和对 ATP 合酶抑制剂的更高抗性。总之，使用 FH 细胞，我们证明 FH 缺陷导致进入线粒体的丙酮酸摄取减少，随后 TCA 循环活性降低，以及从 TCA 循环到糖酵解的 ATP 产生途径平衡改变。我们证实 C-MFA 可以提供 FH 缺陷引起的代谢变化的直接和定量信息。
