Leucine inhibits proteolysis by the mTOR kinase signaling pathway in skeletal muscle

Abstract

Introduction. The branched-chain amino acids, particularly leucine, appear to activate protein synthesis in skeletal muscle by a signal transduction pathway involving the protein kinase mTOR (mammalian target of rapamycin). While leucine also inhibits proteolysis in skeletal muscle, the mechanism for this action is poorly understood. Recently it has been found that another important anabolic factor for muscle, insulin, inhibits proteolysis in this tissue by sequential activation of a “nutrient-sensing” pathway requiring PI3K (phosphatidylinositol 3-kinase), Akt kinase, and mTOR. We studied the role of these kinases in mediating the effects of leucine on protein breakdown, and whether leucine or its metabolites are the critical signal for inhibition of proteolysis. Methods. L6 myotubes (a rat skeletal muscle cell line) were incubated with leucine (5 mM) in the presence or absence of wortmannin or rapamycin (100 nM each), inhibitors of PI3K and mTOR, respectively. Rates of protein degradation were measured by the release of acid-soluble 3H-tyrosine from pre-labeled muscle protein. Statistical analysis was performed using one-way ANOVA. Results. Leucine caused a significant reduction in proteolysis compared to controls (−8 to 12%, P < 0.001). This effect of leucine was not influenced by wortmannin. Conversely, rapamycin completely blocked the ability of leucine to inhibit proteolysis. Furthermore (data not shown), the transamination product of leucine, α-KIC (ketoisocaproic acid), was equally if not more effective in inhibiting proteolysis in the myotubes than leucine itself (up to 20% inhibition). Conclusions. Leucine or its metabolites appear to inhibit skeletal muscle proteolysis by activation of mTOR signaling, but unlike the known effects of insulin on this process, the upstream kinase PI3K is not required. TABLE—ABSTRACT P16 Effect of Leucine on Proteolysis in Myotubes (%/24hrs) Control + Leucine +Leucine +Wortmannin +Leucine +Rapamycin 29.6 ± 0.5 27.2 ± 0.4 26.7 ± 0.4 ∗ 31.2 ± 0.3 Note. Shown are means ± SEM; n = 6; ∗ P < 0.001 versus Control.