Actions of chronic physiological 3-hydroxyisobuterate treatment on mitochondrial metabolism and insulin signaling in myotubes

Abstract

Branched-chain amino acids (BCAAs) are essential in the diet and may provide benefit for those who partake in regular physical activity and resistance training, yet circulating BCAAs have been repeatedly shown to correlate with severity of insulin resistance in obese/diseased populations. Recently, the valine catabolite 3-hydroxyisobuterate (3HIB) was shown to promote insulin resistance in skeletal muscle by increasing lipid content in vivo. The purpose of this study was to investigate the mechanistic effects of 3HIB on skeletal muscle insulin signaling, metabolism, and related gene expression in vitro. Given these previous observations, we hypothesized that 3HIB would depress skeletal muscle metabolism and insulin sensitivity. C2C12 myotubes were treated with 3HIB for up to 48 hours using both physiological (25-100 μmol/L) and supraphysiological (5 mmol/L) concentrations. Metabolic gene expression was measured via quantitative real-time polymerase chain reaction, mitochondrial metabolism was measured via O2 consumption, and glycolytic metabolism was quantified using extracellular acidification rate. Western blot was used to assess insulin sensitivity following insulin stimulation (indicated by phospho-AKT expression). 3HIB did not alter expressional indicators of mitochondrial biogenesis, glycolysis, BCAA catabolism, or lipogenesis. Chronic physiological 3HIB treatment significantly increased peak oxygen consumption, whereas supraphysiological 3HIB treatment suppressed basal and peak mitochondrial and glycolytic metabolism. Both physiological and supraphysiological 3HIB reduced pAkt expression during insulin stimulation. These findings suggest that 3HIB may reduce muscle insulin sensitivity in cultured myotubes, supporting a potentially causal role of 3HIB in the development of insulin resistance in highly metabolic cell types.