184 Supplementation with Leucine and the Leucine Metabolite, Beta-hydroxy-beta-methylbutyrate, Stimulates Muscle Protein Synthesis in Neonatal Pigs via Different Signaling Mechanisms

Background: Early weaning of pigs frequently results in growth faltering. Supplementation with leucine or its metabolite, beta-hydroxy-beta-methylbutyrate (HMB), enhances protein synthesis in skeletal muscle of neonatal pigs by stimulating mTORC1-dependent translation initiation. Recent studies have identified signaling components that may be involved in leucine-induced mTORC1 activation, however, the mechanism by which HMB activates mTORC1 is unclear. Objective: We investigated potential mechanisms of HMB-induced mTORC1 activation through amino acid-sensing components in neonatal muscle. In study 1, overnight-fasted neonatal pigs were fed one of five diets for 24 h: low protein (LP), high protein (HP), or LP supplemented with 4 (LP+HMB4), 40 (LP+HMB40), or 80 (LP+HMB80) μmol HMB/(kg body wt·day). In study 2, fasted neonatal pigs were fed for 24 h: LP, LP supplemented with leucine (LP+Leu), or HP diets. Upstream signaling components relevant to mTORC1 activation were analyzed. Results: Phosphorylation of mTOR (Ser2448 and Ser2481) was greater in LP+HMB40, LP+HMB80, and LP+Leu than LP, and was greater in HP than all HMB. Rheb-mTOR formation was higher in HP, but not HMB and LP+Leu, than LP. RagA-mTOR and RagC-mTOR complexes were higher in LP+Leu and HP than LP and HMB. Sestrin2-GATOR2 formation was lower in LP+Leu and HP than LP, but was unaffected by HMB. Phosphorylation of Erk1/2 and TSC2, but not AMPK, was greater in HP than LP and unaffected by HMB or leucine. There were no treatment effects on RagB-SH3BP4, Vps34-LRS, and RagD-LRS complex formation. Conclusions: Our results demonstrate that supplementation with either leucine or HMB stimulates mTORC1 activation in muscle of neonatal pigs. This leucine-stimulated process involves dissociation of the Sestrin2-GATOR2 complex and increased Rag A/C binding to mTOR. HMB’s action on mTORC1 is independent of these leucine-sensing components. Support: USDA NIFA 2013-67015-20438, NIH HD085573, NIH HD072891, Abbott Nutrition, USDA CRIS 6250-51000-055.