Physiological 4-phenylbutyrate promotes mitochondrial biogenesis and metabolism in C2C12 myotubes

Type 2 diabetes is characterized by elevated circulating blood metabolites such as glucose, insulin, and branched chain amino acids (BCAA), which often coincide with reduced mitochondrial function. 4-Phenylbutyrate (PBA), an ammonia scavenger, has been shown to activate BCAA metabolism, resolve endoplasmic reticulum (ER) stress, and rescue BCAA-mediated insulin resistance. To determine the effect of PBA on the altered metabolic phenotype featured in type 2 diabetes, the present study investigated the effect of PBA on various metabolic parameters including mitochondrial metabolism and mitochondrial biogenesis. C2C12 myotubes were treated with PBA at 0.5 mM (representing physiologically attainable blood concentrations) or 10 mM (representing physiologically unattainable/proof-of-concept levels) for up to 24 h. Mitochondrial and glycolytic metabolism were assessed via oxygen consumption and extracellular acidification rate, respectively. Mitochondrial content, lipid content, and ER stress were measured by fluorescent staining. Metabolic gene expression was measured by qRT-PCR. Both doses of PBA increased expression of indicators of mitochondrial biogenesis, though only PBA at 0.5 mM increased mitochondrial function and content while 10 mM PBA reduced mitochondrial function and content. PBA at 0.5 mM also rescued reduced mitochondrial function during insulin resistance, though PBA also caused a reduced insulin stimulated pAkt expression during insulin resistance. PBA treatment also increased extracellular BCAA accumulation during insulin resistance despite unchanged pBCKDH expression. Taken together, PBA may increase mitochondrial biogenesis, content, and function in a dose-dependent fashion which may have implications for prevention or treatment of metabolic disease such as insulin resistance.