Monitoring live mitochondrial metabolism in real‐time using NMR spectroscopy

Abstract

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. [3‐13C1]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 [2‐13C1]acetyl coenzyme A, which is produced from [3‐13C1]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.

Intracellular pyruvate–lactate–alanine cycling detected using real‐time nuclear magnetic resonance spectroscopy of live cells and isolated mitochondria

“…Mitochondrial metabolism was investigated using live mitochondria isolated from the C2C12 cells. The protocol used for isolation of mitochondria was evaluated by (a) testing the purity of mitochondria using Western blot analysis to ensure that there was no contamination from the cytoplasm and (b) testing the viability based on the visualization of optical images of mitochondria treated with a fluorescent dye under a confocal microscope as described earlier 27 …”

“…Briefly, commercially available kits were used to isolate mitochondria from ~0.5 to 1 × 10 8 cells following the protocol provided by the manufacturer. The purity and viability of mitochondria isolated using this kit were tested based on Western blot analysis and measurement of fluorescence signal from the membrane potential indicator dye as described in our recent study 27 . The isolated mitochondrial pellet was washed with PBS and suspended in 100 μl respiratory buffer that contained 120 mM KCl, 5 mM KH 2 PO 4 , 1 mM EGTA, and 3 mM HEPES, 28 and incubated for 30 min at 37°C.…”

“…Metabolites were identified based on the combination of literature 25,29,30 as well as publicly available databases, including the Human Metabolome Database (HMDB), 31 Biological Magnetic Resonance Bank (BMRB), 32 our earlier study on isolated mitochondria, 27 and in combination with the comparison of blank spectra obtained without adding cells or mitochondria. Peaks from isotope‐labeled pyruvate, lactate, and alanine were further confirmed by comparing 2D NMR spectra obtained before and after spiking with authentic compounds.…”

“…Protocols for isolation of mitochondria, and purity and viability assay were used as described in our recent study 27 . Briefly, commercially available kits were used to isolate mitochondria from ~0.5 to 1 × 10 8 cells following the protocol provided by the manufacturer.…”

“…Peaks from isotope‐labeled pyruvate, lactate, and alanine were further confirmed by comparing 2D NMR spectra obtained before and after spiking with authentic compounds. Processed 2D spectra were imported using the software program developed for automated integration of 2D NMR peaks from multiple spectra in a single step, as described previously 27 . Metabolites were quantified by integrating 2D NMR peaks and comparing them with the internal standard (TSP).…”