Monitoring live mitochondrial metabolism in real-time using NMR spectroscopy

Investigation of mitochondrial metabolism is gaining increased interest owing to the growing recognition of the role of mitochondria in health and numerous diseases. Studies of isolated mitochondria promise novel insights into the metabolism devoid of confounding effects from other cellular organelles such as cytoplasm. This study describes the isolation of mitochondria from mouse skeletal myoblast cells (C2C12) and the investigation of live mitochondrial metabolism in real-time using isotope tracer-based NMR spectroscopy. [3-¹³ C₁ ]pyruvate was used as the substrate to monitor the dynamic changes of the downstream metabolites in mitochondria. The results demonstrate an intriguing phenomenon, in which lactate is produced from pyruvate inside the mitochondria and the results were confirmed by treating mitochondria with an inhibitor of mitochondrial pyruvate carrier (UK5099). Lactate is associated with health and numerous diseases including cancer and, to date, it is known to occur only in the cytoplasm. The insight that lactate is also produced inside mitochondria opens avenues for exploring new pathways of lactate metabolism. Further, experiments performed using inhibitors of the mitochondrial respiratory chain, FCCP and rotenone, show that [2-¹³ C₁ ]acetyl coenzyme A, which is produced from [3-¹³ C₁ ]pyruvate and acts as a primary substrate for the tricarboxylic acid cycle in mitochondria, exhibits a remarkable sensitivity to the inhibitors. These results offer a direct approach to visualize mitochondrial respiration through altered levels of the associated metabolites.