Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

Abstract

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.

Fig. 1.

Current concepts of the amino acid signaling pathway leading to protein synthesis. IRS, insulin receptor substrate; PKB, protein kinase B; AMPK, AMP-activated protein kinase; TSC, tuberous sclerosis complex; GβL, G protein β-subunit-like protein; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; eIF, eukaryotic initiation factor; 4E-BP1, eIF4E-binding protein-1; S6K1, S6 kinase 1; eEF, eukaryotic elongation factor.

Fig. 2.

Fractional rates of protein synthesis in skeletal muscle of 7-day-old pigs after 60 min of infusion of saline (Sal), saline with rapamycin (Sal+Rap), 400 μmol·kg⁻¹·h⁻¹ leucine without rapamycin (Leu), and 400 μmol·kg⁻¹·h⁻¹ leucine with rapamycin (Leu+Rap). Values are means ± pooled SE; n = 5–7 per treatment. Values with different superscripts differ significantly (P < 0.05).

Fig. 3.

Phosphorylation of PKB at Ser473 (A), AMPK at Thr172 (B), and TSC2 at Thr1462 (C) in skeletal muscle of 7-day-old pigs after 60 min of infusion of saline, saline with rapamycin, 400 μmol·kg⁻¹·h⁻¹ leucine without rapamycin, and 400 μmol·kg⁻¹·h⁻¹ leucine with rapamycin. Values for phosphorylation (P) of PKB, AMPK, and TSC2 were corrected by total PKB, AMPK, and TSC2, respectively. Values are means ± pooled SE; n = 5–7 per treatment. AU, arbitrary unit.

Fig. 4.

Association between mTOR and raptor (A), mTOR and GβL (B), and mTOR and rictor (C) in skeletal muscle of 7-day-old pigs after 60 min of infusion of saline, saline with rapamycin, 400 μmol·kg⁻¹·h⁻¹ leucine without rapamycin, and 400 μmol·kg⁻¹·h⁻¹ leucine with rapamycin. Association between mTOR and its partners was corrected by total mTOR in the immunoprecipitant. Values are means ± pooled SE; n = 5–7 per treatment. Values with different superscripts differ significantly (P < 0.05).

Fig. 5.

Phosphorylation of mTOR at Ser2481 (A) or at Ser2448 (B) in skeletal muscle of 7-day-old pigs after 60 min of infusion of saline, saline with rapamycin, 400 μmol·kg⁻¹·h⁻¹ leucine without rapamycin, and 400 μmol·kg⁻¹·h⁻¹ leucine with rapamycin. Values for phosphorylation of mTOR were corrected by total mTOR. Values are means ± pooled SE; n = 5–7 per treatment. Values with different superscripts differ significantly (P < 0.05).

Fig. 6.

Phosphorylation of the 70-kDa ribosomal protein S6K1 at Thr389 (A) and 4E-BP1 at Thr70 (B) in skeletal muscle of 7-day-old pigs after 60 min of infusion of saline, saline with rapamycin, 400 μmol·kg⁻¹·h⁻¹ leucine without rapamycin, and 400 μmol·kg⁻¹·h⁻¹ leucine with rapamycin. Values for phosphorylation of S6K1 and 4E-BP1 were corrected by total S6K1 and 4E-BP1, respectively. Values are means ± pooled SE; n = 5–7 per treatment. Values with different superscripts differ significantly (P < 0.05).

Fig. 7.

Association of eIF4G with eIF4E (A) and 4E-BP1 with eIF4E (B) in skeletal muscle of 7-day-old pigs after 60 min of infusion of saline, saline with rapamycin, 400 μmol·kg⁻¹·h⁻¹ leucine without rapamycin, and 400 μmol·kg⁻¹·h⁻¹ leucine with rapamycin. Formation of eIF4G·eIF4E and 4E-BP1·eIF4E complexes was corrected by total eIF4E in the immunoprecipitant. Values are means ± pooled SE; n = 5–7 per treatment. Values with different superscripts differ significantly (P < 0.05).

Fig. 8.

Phosphorylation of eEF2 at Thr56 in skeletal muscle of 7-day-old pigs after 60 min of infusion of saline, saline with rapamycin, 400 μmol·kg⁻¹·h⁻¹ leucine without rapamycin, and 400 μmol·kg⁻¹·h⁻¹ leucine with rapamycin. Phosphorylation of eEF2 was corrected by total eEF2. Values are means ± pooled SE; n = 5–7 per treatment.