Objective

IGF-I and branched-chain amino acids have been reported to promote muscle hypertrophy via the stimulation of protein synthesis. Sestrin2, the function of which is regulated by leucine, has been reported to attenuate the activity of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) that stimulates protein synthesis. The objective of this study was to examine whether IGF-I modulates Sestrin2 abundance and to clarify the involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1.

Design

C2C12 and L6 myocytes were stimulated by leucine (1 mM) with or without pretreatment with IGF-I (100 ng/mL). Phosphorylation of p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), both of which are targets of the mTORC1, was examined by western blotting. Effects of Sestrin2 small interfering RNA (siRNA) on the actions of leucine and IGF-I were examined. Sestrin2 mRNA and protein levels were also determined after Sestrin2 siRNA.

Results

Leucine increased the phosphorylation of S6K and 4E-BP1 in a dose-dependent manner. Pretreatment with IGF-I for 5 h further increased the stimulatory effect of leucine on the phosphorylation of S6K and 4E-BP1 in C2C12 cells. IGF-I increased Sestrin2 protein and messenger RNA levels. Sestrin2 siRNA increased or tended to increase basal phosphorylation of 4E-BP1 and decreased the leucine-induced phosphorylation in C2C12 and L6 cells, in particular after IGF-I treatment, suggesting the involvement of Sestrin2 in the action of leucine and IGF-I. The net increase in leucine-induced 4E-BP1 phosphorylation appeared to be attenuated by Sestrin2 siRNA. Likewise, Sestrin2 siRNA attenuated leucine-induced S6K phosphorylation in L6 cells. However, Sestrin2 siRNA did not influence leucine-induced S6K phosphorylation in C2C12 cells.

Conclusions

IGF-I and leucine cooperatively increased mTORC1 activity in C2C12 cells. IGF-I increased Sestrin2. Sestrin2 siRNA experiments showed that Sestrin2 was involved in the effect of leucine and IGF-I on mTORC1 activity in C2C12 and L6 cells, and suggested that increased Sestrin2 by IGF-I pretreatment might play a role in enhancing the effect of leucine on mTORC1.

中文翻译：

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Sestrin2 参与 IGF-I 和亮氨酸对 C2C12 和 L6 肌细胞中 mTROC1 活性的影响

客观的

据报道，IGF-I 和支链氨基酸通过刺激蛋白质合成来促进肌肉肥大。据报道，Sestrin2 的功能受亮氨酸调节，可减弱刺激蛋白质合成的雷帕霉素 (mTOR) 复合物 1 (mTORC1) 的机械靶标的活性。本研究的目的是检查 IGF-I 是否调节 Sestrin2 的丰度，并阐明 Sestrin2 参与 IGF-I 和亮氨酸对 mTROC1 的影响。

设计

C2C12 和 L6 肌细胞被亮氨酸 (1 mM) 刺激，有或没有用 IGF-I (100 ng/mL) 预处理。通过蛋白质印迹检查 p70 S6 激酶 (S6K) 和 4E 结合蛋白 1 (4E-BP1) 的磷酸化，这两者都是 mTORC1 的靶标。检测了 Sestrin2 小干扰 RNA (siRNA) 对亮氨酸和 IGF-I 作用的影响。在 Sestrin2 siRNA 后也测定了 Sestrin2 mRNA 和蛋白质水平。

结果

亮氨酸以剂量依赖性方式增加 S6K 和 4E-BP1 的磷酸化。用 IGF-I 预处理 5 小时进一步增加了亮氨酸对 C2C12 细胞中 S6K 和 4E-BP1 磷酸化的刺激作用。IGF-I 增加了 Sestrin2 蛋白和信使 RNA 水平。Sestrin2 siRNA一世增加或倾向于增加 4E-BP1 的基础磷酸化并降低 C2C12 和 L6 细胞中亮氨酸诱导的磷酸化，特别是在 IGF-I 处理后，表明 Sestrin2 参与亮氨酸和 IGF-I 的作用。Sestrin2 siRNA 似乎减弱了亮氨酸诱导的 4E-BP1 磷酸化的净增加。同样，Sestrin2 siRNA 减弱了 L6 细胞中亮氨酸诱导的 S6K 磷酸化。然而，Sestrin2 siRNA 不影响 C2C12 细胞中亮氨酸诱导的 S6K 磷酸化。

结论

IGF-I 和亮氨酸协同增加 C2C12 细胞中的 mTORC1 活性。IGF-I 增加了 Sestrin2。Sestrin2 siRNA实验表明，Sestrin2参与了亮氨酸和IGF-I对C2C12和L6细胞mTORC1活性的影响，提示IGF-I预处理增加的Sestrin2可能在增强亮氨酸对mTORC1的作用中发挥作用。