Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.
Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle.
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DOI：
10.1016/j.cmet.2015.08.021
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发表时间：
2015-11-03
期刊：
影响因子：
29
通讯作者：
Kibbey RG
中科院分区：
文献类型：
--
作者：
Alves TC;Pongratz RL;Zhao X;Yarborough O;Sereda S;Shirihai O;Cline GW;Mason G;Kibbey RG
质量同位素异构体多纵标光谱分析（MIMOSA）是一种逐步通量分析平台，用于测量离散的糖酵解和线粒体代谢速率。重要的是，通过对[U-13 C6]-D-葡萄糖标记产生的质谱进行去卷积，获得直接柠檬酸盐合成速率，用于线粒体乙酰辅酶A、草酰乙酸和柠檬酸盐的位置特异性富集。根据13 C从连续前体到其产物的位置特异性转移，计算关键代谢率（丙酮酸脱氢酶、β-氧化、丙酮酸羧化酶、异柠檬酸脱氢酶和PEP/丙酮酸循环）的全面稳态和动态分析。确定了以前技术的重要局限性。在INS-1细胞中，柠檬酸合酶速率与胰岛素分泌和氧消耗相关。丙酮酸羧化酶率大大低于以前报道的，但显示出最高的倍数变化响应于葡萄糖刺激。总之，MIMOSA测量代谢物之间13 C标记的前体/产物位置特异性转移的关键代谢率，并对任何葡萄糖氧化细胞具有广泛的适用性。
Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-13C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, β-oxidation, pyruvate carboxylase, isocitrate dehydrogenase and PEP/pyruvate cycling) were calculated from the position-specific transfer of 13C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of 13C label between metabolites and has broad applicability to any glucose-oxidizing cell.