Differential regulation of mTORC1 activation by leucine and β-hydroxy-β-methylbutyrate in skeletal muscle of neonatal pigs

Leucine (Leu) and its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent protein synthesis in the skeletal muscle of neonatal pigs. This study aimed to determine whether HMB and Leu utilize common nutrient-sensing mechanisms to activate mTORC1. In study 1, 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^-1·day^-1. In study 2, neonatal pigs were fed for 24 h: LP, LP supplemented with Leu (LP+Leu), or HP diets delivering 9, 18, and 18 mmol Leu·kg body wt^-1·day^-1, respectively. The upstream signaling molecules that regulate mTORC1 activity were analyzed. mTOR phosphorylation on Ser2448 and Ser2481 was greater in LP+HMB40, LP+HMB80, and LP+Leu than in LP and greater in HP than in HMB-supplemented groups (P < 0.05), whereas HP and LP+Leu were similar. Rheb-mTOR complex formation was lower in LP than in HP (P < 0.05), with no enhancement by HMB or Leu supplementation. The Sestrin2-GATOR2 complex was more abundant in LP than in HP and was reduced by Leu (P < 0.05) but not HMB supplementation. RagA-mTOR and RagC-mTOR complexes were higher in LP+Leu and HP than in LP and HMB groups (P < 0.05). There were no treatment differences in RagB-SH3BP4, Vps34-LRS, and RagD-LRS complex abundances. Phosphorylation of Erk1/2 and TSC2, but not AMPK, was lower in LP than HP (P < 0.05) and unaffected by HMB or Leu supplementation. Our results demonstrate that HMB stimulates mTORC1 activation in neonatal muscle independent of the leucine-sensing pathway mediated by Sestrin2 and the Rag proteins.NEW & NOTEWORTHY Dietary supplementation with either leucine or its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulates protein synthesis in skeletal muscle of the neonatal pig. Our results demonstrate that both leucine and HMB stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) phosphorylation in neonatal muscle. This leucine-stimulated process involves dissociation of the Sestrin2-GATOR2 complex and increased binding of Rag A/C to mTOR. However, HMB's activation of mTORC1 is independent of this leucine-sensing pathway.