The involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1 activity in C2C12 and L6 myocytes

IF 1.6 4区 医学 Q4 CELL BIOLOGY Growth Hormone & Igf Research Pub Date : 2021-08-01 DOI:10.1016/j.ghir.2021.101406
Ran Sawa , Ikumi Wake , Yu Yamamoto, Yasuhiko Okimura
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引用次数: 4

Abstract

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.

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Sestrin2参与IGF-I和亮氨酸对C2C12和L6肌细胞mTROC1活性的影响
目的已报道igf - 1和支链氨基酸通过刺激蛋白质合成促进肌肉肥大。据报道,功能受亮氨酸调节的Sestrin2可以减弱刺激蛋白质合成的雷帕霉素(mTOR)复合物1 (mTORC1)的机制靶点的活性。本研究的目的是研究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肌细胞分别用亮氨酸(1 mM)和IGF-I (100 ng/mL)进行刺激。western blotting检测了p70 S6激酶(S6K)和4e结合蛋白1 (4E-BP1)的磷酸化,两者都是mTORC1的靶点。研究了Sestrin2小干扰RNA (siRNA)对亮氨酸和igf - 1作用的影响。同时检测Sestrin2 siRNA后的Sestrin2 mRNA和蛋白水平。结果亮氨酸增加了S6K和4E-BP1的磷酸化,并呈剂量依赖性。IGF-I预处理5 h进一步增强亮氨酸对C2C12细胞中S6K和4E-BP1磷酸化的刺激作用。igf - 1增加了Sestrin2蛋白和信使RNA水平。在C2C12和L6细胞中,特别是在IGF-I处理后,Sestrin2 siRNA增加或倾向于增加4E-BP1的基础磷酸化,并降低亮氨酸诱导的磷酸化,提示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,在L6细胞中,Sestrin2 siRNA减弱亮氨酸诱导的S6K磷酸化。然而,在C2C12细胞中,Sestrin2 siRNA不影响亮氨酸诱导的S6K磷酸化。结论sigf - i和亮氨酸共同提高C2C12细胞mTORC1活性。igf - 1增加了Sestrin2。Sestrin2 siRNA实验显示,Sestrin2参与了亮氨酸和IGF-I对C2C12和L6细胞mTORC1活性的影响,提示通过IGF-I预处理增加Sestrin2可能起到增强亮氨酸对mTORC1作用的作用。
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来源期刊
Growth Hormone & Igf Research
Growth Hormone & Igf Research 医学-内分泌学与代谢
CiteScore
3.30
自引率
0.00%
发文量
38
审稿时长
57 days
期刊介绍: Growth Hormone & IGF Research is a forum for research on the regulation of growth and metabolism in humans, animals, tissues and cells. It publishes articles on all aspects of growth-promoting and growth-inhibiting hormones and factors, with particular emphasis on insulin-like growth factors (IGFs) and growth hormone. This reflects the increasing importance of growth hormone and IGFs in clinical medicine and in the treatment of diseases.
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