β-Hydroxy-β-methylbutyrate (HMB) is a breakdown product of leucine, which promotes muscle growth. Although some studies indicate that HMB activates AKT and mTOR, others show activation of the downstream effectors, P70S6K and S6, independent of mTOR. Our aim was to study the metabolic effect of HMB around the circadian clock in order to determine more accurately the signaling pathway involved. C2C12 myotubes were treated with HMB and clock, metabolic and myogenic markers were measured around the clock. HMB-treated C2C12 myotubes showed no activation of AKT and mTOR, but did show activation of P70S6K and S6. Activation of P70S6K and S6 was also found when myotubes were treated with HMB combined with metformin, an indirect mTOR inhibitor, or rapamycin, a direct mTOR inhibitor. The activation of the P70S6K and S6 independent of AKT and mTOR, was accompanied by increased activation of phospholipase D2 (PLD). In addition, HMB led to high amplitude and advanced circadian rhythms. In conclusion, HMB induces myogenesis in C2C12 by activating P70S6K and S6 via PLD2, rather than AKT and mTOR, leading to high amplitude advanced rhythms.

中文翻译：

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β-羟基-β-甲基丁酸酯 (HMB) 导致磷脂酶 D2 (PLD2) 激活并改变肌管的昼夜节律

β-羟基-β-甲基丁酸 (HMB) 是亮氨酸的分解产物，可促进肌肉生长。尽管一些研究表明 HMB 激活 AKT 和 mTOR，但其他研究表明下游效应器 P70S6K 和 S6 的激活与 mTOR 无关。我们的目的是研究 HMB 在生物钟周围的代谢作用，以便更准确地确定所涉及的信号通路。 C2C12 肌管用 HMB 处理，并全天候测量时钟、代谢和生肌标记物。 HMB 处理的 C2C12 肌管未显示 AKT 和 mTOR 激活，但显示 P70S6K 和 S6 激活。当用 HMB 联合二甲双胍（一种间接 mTOR 抑制剂）或雷帕霉素（一种直接 mTOR 抑制剂）处理肌管时，也发现了 P70S6K 和 S6 的激活。 P70S6K 和 S6 的激活独立于 AKT 和 mTOR，伴随着磷脂酶 D2 (PLD) 激活的增加。此外，HMB 还会导致高振幅和提前的昼夜节律。总之，HMB 通过PLD2（而不是 AKT 和 mTOR）激活 P70S6K 和 S6 来诱导 C2C12 中的肌生成，从而导致高振幅提前节律。