The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin‐sensitive and insulin‐resistant C2C12 myotubes

Abstract Elevated circulating branched‐chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2 (3,6‐dichlorobenzo[ b ]thiophene‐2‐carboxylic acid), an inhibitor of branched‐chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real‐time polymerase chain reaction were used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography‐mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24‐h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose‐dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small‐molecule uncoupler. Additionally, BT2 treatment consistently and dose‐dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small‐molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.