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.