The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reaction were used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

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.

The Selective LAT1 Inhibitor JPH203 Enhances Mitochondrial Metabolism and Content in Insulin-Sensitive and Insulin-Resistant C2C12 Myotubes.

Population data have shown an association between higher circulating branched-chain amino acids (BCAA) and the severity of insulin resistance in people with diabetes. While several studies have assessed BCAA metabolism as a potential target for regulation, less attention has been paid to the role of L-type amino acid transporter 1 (LAT1), the primary transporter of BCAA in skeletal muscle. The aim of this study was to assess the impact of JPH203 (JPH), a LAT1 inhibitor, on myotube metabolism in both insulin-sensitive and insulin-resistant myotubes. C2C12 myotubes were treated with or without 1 µM or 2 µM JPH for 24 h with or without insulin resistance. Western blot and qRT-PCR were used to assess protein content and gene expression, respectively. Mitochondrial and glycolytic metabolism were measured via Seahorse Assay, and fluorescent staining was used to measure mitochondrial content. BCAA media content was quantified using liquid chromatography-mass spectrometry. JPH at 1 µM (but not 2 µM) increased mitochondrial metabolism and content without inducing changes in mRNA expression of transcripts associated with mitochondrial biogenesis or mitochondrial dynamics. Along with increased mitochondrial function, 1µM treatment also reduced extracellular leucine and valine. JPH at 2 µM reduced pAkt signaling and increased extracellular accumulation of isoleucine without inducing changes in BCAA metabolic genes. Collectively, JPH may increase mitochondrial function independent of the mitochondrial biogenic transcription pathway; however, high doses may reduce insulin signaling.