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.
CONCLUSIONS: 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.
方法:C2C12肌管用MHY1485或DMSO对照在有和没有雷帕霉素的情况下处理。使用qRT-PCR评估基因表达,通过蛋白质印迹评估胰岛素敏感性和蛋白质表达。通过细胞外酸化速率和耗氧量来测量糖酵解和线粒体代谢。通过荧光染色分析线粒体和脂质含量。使用液相色谱-质谱来评估细胞外BCAA。
结果:雷帕霉素降低p-mTORC表达,线粒体含量,和线粒体功能。令人惊讶的是,MHY1485不改变p-mTORC表达或细胞代谢。两种治疗均未改变BCAA代谢或细胞外BCAA含量的指标。
结论:总的来说,通过雷帕霉素抑制mTORC会降低肌管代谢和线粒体含量,但不会降低BCAA代谢。MHY1485缺乏p-mTORC激活是这些实验的限制,并需要进一步的研究。