ObjectiveUnexplained changes in regulation of branched chain amino acids (BCAA) during diabetes therapy with metformin have been known for years. Here we have investigated mechanisms underlying this effect.MethodsWe used cellular approaches, including single gene/protein measurements, as well as systems-level proteomics. Findings were then cross-validated with electronic health records and other data from human material.ResultsIn cell studies, we observed diminished uptake/incorporation of amino acids following metformin treatment of liver cells and cardiac myocytes. Supplementation of media with amino acids attenuated known effects of the drug, including on glucose production, providing a possible explanation for discrepancies between effective dosesin vivoandin vitroobserved in most studies. Data-Independent Acquisition proteomics identified that SNAT2, which mediates tertiary control of BCAA uptake, was the most strongly suppressed amino acid transporter in liver cells following metformin treatment. Other transporters were affected to a lesser extent. In humans, metformin attenuated increased risk of left ventricular hypertrophy due to the AA allele of KLF15, which is an inducer of BCAA catabolism. In plasma from a double-blind placebo-controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin caused selective accumulation of plasma BCAA and glutamine, consistent with the effects in cells.ConclusionsMetformin restricts tertiary control of BCAA cellular uptake. We conclude that modulation of amino acid homeostasis contributes to therapeutic actions of the drug.