Cultured equine satellite cells as a model system to assess leucine stimulated protein synthesis in horse muscle.

Abstract

Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was to evaluate the effect of leucine on the mTOR signaling pathway in cultured equine satellite. Protein synthesis was evaluated by measuring the incorporation of [3H] Phenylalanine (3HPhe) in equine satellite cell myotube cultures treated with a leucine titration ranging from 0 to 408 µM. Our results show a 1.8-fold increase (P < 0.02) in protein synthesis at levels slightly greater than those found in the general circulation, 204 and 408 µM when compared to a no leucine control (0 µM). Puromycin incorporation, a nonradioactive surface sensing of translation (SUnSET) methodology, was also measured in cells treated with leucine (LEU; 408 µM), a no-leucine control (CON), and a puromycin-negative vehicle (PURO-). These results demonstrated a 180% increase (P = 0.0056) in puromycin incorporation in LEU compared to CON cultures. To evaluate the mTOR signaling pathway, equine satellite cell myotube cultures were treated with leucine (LEU; 408 µM) or a no-leucine control (CON) in the presence or absence of rapamycin (LR and CR, respectively), an inhibitor of mTOR. The mTOR inhibitor, rapamycin, suppressed phosphorylation of mTOR (P < 0.01) and rS6 (P < 0.01) with an increase in phosphorylation of rS6 in leucine-treated cultures observed when compared to control cultures (P < 0.05). Similarly, there was a 27% increase (P < 0.005) in the hyperphosphorylated γ-form of 4E-BP1 compared to total 4E-BP1 in LEU compared to CON cultures with leucine-induced phosphorylation of 4E-BP1 completely blocked by rapamycin with a smaller decrease observed in CR compared to CON cultures. The major finding of this study was that leucine activated the mTOR translation initiation pathway and increased transcription of global proteins in cultured equine satellite cells. Use of the cell culture system with primary equine muscle cell lines provides the opportunity to distinguish the impact of leucine on muscle and protein synthesis, independent of systemic interactions.