利用活细胞和分离线粒体的实时核磁共振光谱检测细胞内丙酮酸-乳酸-丙氨酸循环

IF 1.9 3区 化学 Q3 CHEMISTRY, MULTIDISCIPLINARY Magnetic Resonance in Chemistry Pub Date : 2023-12-14 DOI:10.1002/mrc.5419
G. A. Nagana Gowda, John A. Lusk, Vadim Pascua
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引用次数: 0

摘要

丙酮酸是糖酵解的最终产物,是细胞能量的主要燃料。细胞膜丙酮酸的一部分被转运到线粒体,而剩余部分则可逆地转化为乳酸和丙氨酸。有人认为,细胞质乳酸和丙氨酸在线粒体内被运输和代谢。然而,这种机制仍然是一个激烈争论和研究的话题。为了深入了解细胞膜乳酸和丙氨酸的代谢去向,本研究利用稳定同位素标记的三种糖酵解产物(即[3-13C1]丙酮酸、[3-13C1]乳酸和[3-13C1]丙氨酸)作为底物,对小鼠骨骼肌母细胞(C2C12)及其分离的线粒体的代谢进行了探究。利用 1H-13C 二维核磁共振(NMR)光谱分别实时监测了每种底物的摄取和代谢。底物及其代谢产物水平的动态变化随时间的变化而量化。结果表明,所有三种底物都被转运到线粒体中,并且每种底物都被可逆地代谢成另外两种代谢产物。这些结果提供了细胞内丙酮酸-乳酸-丙氨酸循环的直接证据,在这种循环中,细胞内丙酮酸产生的乳酸和丙氨酸被转运到线粒体,并转化回丙酮酸。这种机制表明乳酸和丙氨酸在补充线粒体丙酮酸方面发挥作用,而线粒体丙酮酸是通过氧化磷酸化和电子传递链合成三磷酸腺苷(ATP)的主要来源。这些结果凸显了实时核磁共振光谱在获得细胞和亚细胞功能新见解方面的潜力。
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Intracellular pyruvate–lactate–alanine cycling detected using real-time nuclear magnetic resonance spectroscopy of live cells and isolated mitochondria

Pyruvate, an end product of glycolysis, is a master fuel for cellular energy. A portion of cytosolic pyruvate is transported into mitochondria, while the remaining portion is converted reversibly into lactate and alanine. It is suggested that cytosolic lactate and alanine are transported and metabolized inside mitochondria. However, such a mechanism continues to be a topic of intense debate and investigation. As a part of gaining insight into the metabolic fate of the cytosolic lactate and alanine; in this study, the metabolism of mouse skeletal myoblast cells (C2C12) and their isolated mitochondria was probed utilizing stable isotope-labeled forms of the three glycolysis products, viz. [3-13C1]pyruvate, [3-13C1]lactate, and [3-13C1]alanine, as substrates. The uptake and metabolism of each substrate was monitored, separately, in real-time using 1H-13C 2D nuclear magnetic resonance (NMR) spectroscopy. The dynamic variation of the levels of the substrates and their metabolic products were quantitated as a function of time. The results demonstrate that all three substrates were transported into mitochondria, and each substrate was metabolized to form the other two metabolites, reversibly. These results provide direct evidence for intracellular pyruvate–lactate–alanine cycling, in which lactate and alanine produced by the cytosolic pyruvate are transported into mitochondria and converted back to pyruvate. Such a mechanism suggests a role for lactate and alanine to replenish mitochondrial pyruvate, the primary source for adenosine triphosphate (ATP) synthesis through oxidative phosphorylation and the electron transport chain. The results highlight the potential of real-time NMR spectroscopy for gaining new insights into cellular and subcellular functions.

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来源期刊
CiteScore
4.70
自引率
10.00%
发文量
99
审稿时长
1 months
期刊介绍: MRC is devoted to the rapid publication of papers which are concerned with the development of magnetic resonance techniques, or in which the application of such techniques plays a pivotal part. Contributions from scientists working in all areas of NMR, ESR and NQR are invited, and papers describing applications in all branches of chemistry, structural biology and materials chemistry are published. The journal is of particular interest not only to scientists working in academic research, but also those working in commercial organisations who need to keep up-to-date with the latest practical applications of magnetic resonance techniques.
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