Distinct anabolic signalling responses to amino acids in C2C12 skeletal muscle cells
Distinct anabolic signalling responses to amino acids in C2C12 skeletal muscle cells
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DOI：
10.1007/s00726-009-0377-x
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发表时间：
2010-05-01
期刊：
影响因子：
3.5
通讯作者：
Rennie, Michael J.
中科院分区：
文献类型：
--
作者：
Atherton, Philip J.;Smith, Ken;Rennie, Michael J.
必需氨基酸（EAA）通过机制激活合成代谢信号传导，这些机制的细节尚不清楚，但包括通过雷帕霉素复合物1（mTORC 1）的哺乳动物靶点增加信号传导。在所有的EAA中，支链氨基酸（BCAA）亮氨酸被认为是刺激蛋白质合成的最有效的，尽管还没有研究调查每种EAA对合成代谢信号通路的影响。因此，我们进行了每种EAA对C2 C12肌管中mTORC 1信号传导的影响的系统分析，其中在用2 mM的每种氨基酸刺激之前，使细胞血清（4小时）和氨基酸（1小时）饥饿。免疫印迹法用于检测蛋白激酶B（Akt）/mTORC 1信号传导酶的磷酸化形式。Akt的磷酸化通过与EAA孵育而未改变。mTOR和4 E结合蛋白-1（4 EBP 1）的磷酸化分别响应于亮氨酸刺激而增加1.67 +/-A0.1倍和2.5 +/-A0.1倍，但不响应于任何其他EAA。核糖体S6激酶（p70 S6 K1）的磷酸化增加的刺激与所有的EAA的异亮氨酸和缬氨酸除外。然而，与非BCAA EAA的1.6-2.0倍相比，亮氨酸的增加显著更大，为5.9 +/- A 0.3倍。这种激活模式在核糖体蛋白s6（RPS 6）中是相同的，其中亮氨酸的额外作用是3.8 +/- A 0.3倍对1.5-2.0倍。真核起始/延伸因子eIF 2 α和eEF 2的磷酸化不受EAA的影响。我们的结论是，亮氨酸是独特的氨基酸之间的能力，刺激mTOR和4 EBP 1磷酸化和增强p70 S6 K1信号。
The essential amino acids (EAA) activate anabolic signalling through mechanisms, which are unclear in detail but include increased signalling through the mammalian target of rapamycin complex 1 (mTORC1). Of all the EAA, the branched chain amino acid (BCAA) leucine has been suggested as the most potent in stimulating protein synthesis, although there have been no studies investigating the effects of each EAA on anabolic signalling pathways. We therefore undertook a systematic analysis of the effect of each EAA on mTORC1 signalling in C2C12 myotubes whereby cells were serum (4 h) and amino acid (1 h) starved before stimulation with 2 mM of each amino acid. Immunoblotting was used to detect phosphorylated forms of protein kinase B (Akt)/mTORC1 signalling enzymes. The phosphorylation of Akt was unchanged by incubation with EAA. Phosphorylation of mTOR and 4E binding protein-1 (4EBP1) were increased 1.67 +/- A 0.1-fold and 2.5 +/- A 0.1-fold, respectively, in response to leucine stimulation but not in response to any other EAA. The phosphorylation of ribosomal s6 kinase (p70S6K1) was increased by stimulation with all EAA with the exceptions of isoleucine and valine. However, the increase with leucine was significantly greater, 5.9 +/- A 0.3-fold compared to 1.6-2.0-fold for the non-BCAA EAA. This pattern of activation was identical in ribosomal protein s6 (RPS6) with the additional effect of leucine being 3.8 +/- A 0.3-fold versus 1.5-2.0-fold. Phosphorylation of eukaryotic initiation/elongation factors eIF2 alpha and eEF2 were unaffected by EAA. We conclude that leucine is unique amongst the amino acids in its capacity to stimulate both mTOR and 4EBP1 phosphorylation and to enhance p70S6K1 signalling.