AbstractMetformin has antihyperglycemic properties and is a commonly prescribed drug for type II diabetes mellitus. Metformin functions in part by activating 5′‐AMP‐activated protein kinase, reducing hepatic gluconeogenesis and blood glucose. Metformin also upregulates peroxisome proliferator‐activated receptor‐gamma coactivator‐1α (PGC‐1α). Several population studies have shown levels of circulating branched‐chain amino acids (BCAA) positively correlate with insulin resistance. Because BCAA catabolic enzyme content is regulated by PGC‐1α, we hypothesized metformin may alter BCAA catabolism. Therefore, the purpose of this work was to investigate the effect of metformin at varying concentrations on myotube metabolism and related gene and protein expression. C2C12 myotubes were treated with metformin at 30 uM (physiological) or 2 mM (supraphysiological) for up to 24 hours. Metabolic gene expression was measured via quantitative real time polymerase chain reaction, protein expression was measured using Western blot, and mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Supraphysiological metformin upregulated PGC‐1α mRNA expression along with related downstream targets, yet the reduced expression of electron transport chain components as well as basal and peak cell metabolism. Supraphysiological metformin also suppressed branched‐chain aminotransferase 2 (BCAT2) and branched‐chain‐alpha‐keto acid dehydrogenase E1a (BCKDHa) mRNA expression as well as BCAT2 protein expression and BCKDHa activity, which was accompanied by decreased Kruppel‐like factor 15 protein expression. Physiological levels of metformin suppressed BCKDHa and cytochrome c oxidase mRNA expression at early time points (4‐12 hours) but had no effect on any other outcomes. Together these data suggest metformin may suppress BCAA catabolic enzyme expression or activity, possibly reducing levels of circulating gluconeogenic substrates.