Valsartan Rescues Suppressed Mitochondrial Metabolism during Insulin Resistance in C2C12 Myotubes.

Elevated circulating branched-chain amino acids (BCAA) have been linked with the severity of insulin resistance across numerous populations, implicating heightened BCAA metabolism as a potential therapy for insulin resistance. Recently, the angiotensin II type 1 receptor (AT1R) inhibitor Valsartan (VAL) was identified as a potent inhibitor of branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), a negative regulator of BCAA metabolism. This work investigated the effect of VAL on myotube metabolism and insulin sensitivity under both insulin sensitive and insulin resistant conditions. C2C12 myotubes were treated with or without VAL at 8 µM for 24 h, both with and without hyperinsulinemic-induced insulin resistance. Oxygen consumption and extracellular acidification were used to measure mitochondrial and glycolytic metabolism, respectively. Gene expression was assessed via qRT-PCR, and insulin sensitivity was assessed via Western blot. Insulin resistance significantly reduced both basal and peak mitochondrial function which were rescued to control levels by concurrent VAL. Changes in mitochondrial function occurred without substantial changes in mitochondrial content or related gene expression. Insulin sensitivity and glycolytic metabolism were unaffected by VAL, as was lipogenic signaling and lipid content. Additionally, both VAL and insulin resistance depressed Bckdha expression. Interestingly, an interaction effect was observed for extracellular isoleucine, valine, and total BCAA (but not leucine), suggesting VAL may alter BCAA utilization in an insulin sensitivity-dependent manner. Insulin resistance appears to suppress mitochondrial function in a myotube model which can be rescued by VAL. Further research will be required to explore the implications of these findings in more complex models.

Effect of mTORC Agonism via MHY1485 with and without Rapamycin on C2C12 Myotube Metabolism.

The mechanistic target of rapamycin complex (mTORC) regulates protein synthesis and can be activated by branched-chain amino acids (BCAAs). mTORC has also been implicated in the regulation of mitochondrial metabolism and BCAA catabolism. Some speculate that mTORC overactivation by BCAAs may contribute to insulin resistance. The present experiments assessed the effect of mTORC activation on myotube metabolism and insulin sensitivity using the mTORC agonist MHY1485, which does not share structural similarities with BCAAs. METHODS: C2C12 myotubes were treated with MHY1485 or DMSO control both with and without rapamycin. Gene expression was assessed using qRT-PCR and insulin sensitivity and protein expression by western blot. Glycolytic and mitochondrial metabolism were measured by extracellular acidification rate and oxygen consumption. Mitochondrial and lipid content were analyzed by fluorescent staining. Liquid chromatography-mass spectrometry was used to assess extracellular BCAAs. RESULTS: Rapamycin reduced p-mTORC expression, mitochondrial content, and mitochondrial function. Surprisingly, MHY1485 did not alter p-mTORC expression or cell metabolism. Neither treatment altered indicators of BCAA metabolism or extracellular BCAA content. CONCLUSION: Collectively, inhibition of mTORC via rapamycin reduces myotube metabolism and mitochondrial content but not BCAA metabolism. The lack of p-mTORC activation by MHY1485 is a limitation of these experiments and warrants additional investigation.

Fructose Reduces Mitochondrial Metabolism and Increases Extracellular BCAA during Insulin Resistance in C2C12 Myotubes.

Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been correlated with the severity of insulin resistance. Most recently, the effect of fructose consumption on the downregulation of BCAA catabolic enzymes was observed. Thus, this mechanistic study investigated the effects of physiologically attainable levels of fructose, both with and without concurrent insulin resistance, in a myotube model of skeletal muscle. METHODS: C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. RESULTS: Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. DISCUSSION: Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.