Leucine stimulates mTOR signalling in C2C12 myoblast cells through inhibition of AMP‐activated protein kinase (AMPK)

Abstract

Leucine stimulates mammalian target of rapamycin (mTOR) signalling and protein synthesis. However, mechanisms are poorly understood. The objective of this study is to show that leucine stimulates mTOR signalling partially through inhibition of AMPK activity. C2C12 myoblast cells and primary myoblast cells which over-expressed dominant negative AMPKα were used in this study. Cells were subjected to 2 mM leucine and/or 2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribonucleoside (AICAr) treatments, and collected 30 min following treatments. Leucine treatment enhanced the phosphorylation of mTOR and ribosomal protein S6 kinase, and increased the amount of eIF4G.eIF4E complex. Leucine treatment decreased the AMP/ATP ratio by 36.4 ± 9.1 % (p≤0.05) in myoblast cells which was associated with a reduction in the phosphorylation of AMPKα at Thr172 (28.6 ± 4.9 % reduction, p≤0.05) and inhibition of AMPK activity (43.6 ± 3.5% reduction, p≤0.05). On the other hand, the phosphorylation of mTOR at Ser2448 was increased for 63.5 ± 10.0% (p≤0.05) and protein synthesis for 30.6 ± 6.1% (p≤0.05). Maximal stimulation of AMPK activity by applying AICAr, an activator of AMPK, partially abolished the stimulation of leucine on mTOR signalling. In myoblast cells over-expressing a dominant negative AMPKα subunit which results in very low AMPK activity in these cells, the stimulation of mTOR phosphorylation by leucine was also attenuated. In both these two cases, leucine did not induce a significant change in AMPK activity, which reveals that the change of AMPK activity during leucine stimulation contributes to the activation of mTOR signalling. In conclusion, besides direct stimulation of mTOR signalling by leucine, leucine also stimulates mTOR signalling by improving the energy status in muscle cells, via inhibition of AMPK.