Intracellular pyruvate-lactate-alanine cycling detected using real-time nuclear magnetic resonance spectroscopy of live cells and isolated mitochondria.
Intracellular pyruvate-lactate-alanine cycling detected using real-time nuclear magnetic resonance spectroscopy of live cells and isolated mitochondria.
复制标题
使用活细胞和分离线粒体的实时核磁共振波谱检测细胞内丙酮酸-乳酸-丙氨酸循环。
DOI：
10.1002/mrc.5419
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Pascua,Vadim
中科院分区：
文献类型：
--
作者：
NaganaGowda,GA;Lusk,JohnA;Pascua,Vadim
丙酮酸，糖酵解的最终产物，是细胞能量的主要燃料。一部分胞质丙酮酸被转运到线粒体中，而其余部分可逆地转化为乳酸和丙氨酸。这表明，胞浆乳酸和丙氨酸的运输和代谢内线粒体。然而，这一机制仍然是一个激烈辩论和调查的主题。作为深入了解胞质乳酸和丙氨酸代谢命运的一部分，在这项研究中，小鼠骨骼肌成肌细胞（C2 C12）及其分离的线粒体的代谢利用稳定的同位素标记形式的三种糖酵解产物，即[3- 13 C1]丙酮酸，[3 - 13 C1]乳酸和[3- 13 C1]丙氨酸，作为底物进行了探测。使用1H-13 C 2D NMR光谱法分别实时监测每种底物的吸收和代谢。底物及其代谢产物水平的动态变化被定量为时间的函数。结果表明，所有三种底物都被转运到线粒体中，并且每种底物被可逆地代谢形成另外两种代谢物。这些结果为细胞内丙酮酸-乳酸-丙氨酸循环提供了直接证据，其中由胞质丙酮酸产生的乳酸和丙氨酸被转运到线粒体中并转化回丙酮酸。这种机制表明乳酸和丙氨酸补充线粒体丙酮酸的作用，线粒体丙酮酸是通过氧化磷酸化和电子传递链合成ATP的主要来源。这些结果突出了实时NMR光谱学对细胞和亚细胞功能的新见解的潜力。
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 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 ATP synthesis through oxidation 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.