A Novel, Non-Radioactive Method for Measuring Fatty Acid Oxidation and Cellular Metabolism in C2C12 Myocytes

Assays to identify metabolic pathways and quantify cellular bioenergetics are few and not well suited for drug screening. The protocols that exist require the use of radioactivity or expensive low-throughput technologies, such as Mass Spectrometry. Seahorse Bioscience has developed a novel microplate based technology which measures the Extracellular Flux (XF) of molecular oxygen and protons simultaneously. By measuring the rate of oxygen consumption (OCR), a measure of mitochondrial respiration, and extracellular acidification (ECAR), a measure of glycolysis, the XF24 Analyzer provides a means for both quantifying cellular bioenergetics and differentiating metabolic pathways for drug discovery. We have previously shown that OCR and ECAR are valid measurements for examining cellular metabolism in a variety of cell types including C2C12 myoblasts and cancer lines. To validate these parameters for use in a fatty acid oxidation (FAO) assay we simultaneously measured OCR and ECAR in C2C12 myocytes over a 40 minute time period. Knowing the analytes that should be consumed and excreted during FAO we anticipated that the OCR values should increase and the ECAR values decrease (due to the decreased amount of protons released per ATP in the oxidative state). Using the ratio of OCR to ECAR to represent the relative amount of FAO we found that upon stimulation with palmitate there was a significant increase (>100%) over baseline. To confirm that the increase in the ratio was specific for oxidation of palmitate and not glucose we added the CPT-1 inhibitor Etomoxir which is known to block FAO by preventing the translocation of palmitate into the mitochondria. Etomoxir significantly reduced the OCR:ECAR ratio by greater than 50% compared to palmitate alone. In parallel, we performed these studies using a radiometric assay which measured 3 H-water accumulation and found equivalent results. Data is presented in which we challenged C2C12 myocytes with varying doses of the AMP Kinase agonist AICAR (5-Aminoimidazole-4-carboxamide-1-β-riboside Z-riboside). Upon palmitate addition AICAR increased oxygen consumption over control indicating an increase in FAO at the lower doses compared to the highest dose which appeared to inhibit the response. These findings suggest that this approach will have utility in identifying modulators and elucidating mechanisms of metabolic pathways.