Metformin 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.

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By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.