Jason W. Frank

Leucine Supplementation of a Low-Protein Meal Increases Skeletal Muscle and Visceral Tissue Protein Synthesis in Neonatal Pigs by Stimulating mTOR-Dependent Translation Initiation

Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with amino acids. Leucine appears to be the most effective single amino acid to trigger these effects. To examine the response to enteral leucine supplementation, overnight food-deprived 5-d-old pigs were gavage fed at 0 and 60 min a: 1) low-protein diet (LP); 2) LP supplemented with leucine (LP+L) to equal leucine in the high-protein diet (HP); or 3) HP diet. Diets were isocaloric and equal in lactose. Fractional protein synthesis rates and translation initiation control mechanisms were examined in skeletal muscles and visceral tissues 90 min after feeding. Protein synthesis rates in longissimus dorsi, gastrocnemius, and masseter muscles, heart, jejunum, kidney, and pancreas, but not liver, were greater in the LP+L group compared with the LP group and did not differ from the HP group. Feeding LP+L and HP diets compared with the LP diet increased phosphorylation of mammalian target of rapamycin (mTOR), 4E-binding protein 1, ribosomal protein S6 kinase-1, and eIF4G and formation of the active eIF4E·eIF4G complex in longissimus dorsi muscle. In all tissues except liver, activation of mTOR effectors increased in pigs fed LP+L and HP vs. LP diets. Our results suggest that leucine supplementation of a low-protein meal stimulates protein synthesis in muscle and most visceral tissues to a rate similar to that achieved by feeding a high-protein meal and this stimulation involves activation of mTOR downstream effectors.

Leucine and α-Ketoisocaproic Acid, but Not Norleucine, Stimulate Skeletal Muscle Protein Synthesis in Neonatal Pigs

The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, alpha-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 micromol kg(-1) h(-1) for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1.eIF4E complex, and greater (P < 0.05) active eIF4G.eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.

Expression of the TGF-|[beta]| Family of Ligands Is Developmentally Regulated in Skeletal Muscle of Neonatal Rats

To dissect the possible role of the transforming growth factor-β (TGF-β) family in the regulation of skeletal muscle growth during the early postnatal period, the protein abundances of the TGF-β family and their correlation with protein synthesis were determined in skeletal muscle of neonatal rats. To obtain direct evidence of the role of these growth factors in the regulation of protein synthesis, the TGF-β inhibitor, follistatin, was infused into 10-d-old rats for 11 d and protein synthesis and phosphorylation of S6 kinase 1 (S6K1) and ribosomal protein (rpS6) were measured. TGF-β2 abundance and protein synthesis in muscle decreased with development and were positively correlated. The abundances of bone morphogenetic protein 2 (BMP-2), BMP-7, and myostatin increased with development and were negatively correlated with protein synthesis. The abundances of BMP-2 and BMP-7 were positively correlated with BMP receptor IA (BMP-RIA) abundance. Activin A abundance was positively correlated with follistatin abundance and activin receptor IIB (Act-RIIB) abundance. Infusion of follistatin increased muscle protein synthesis and S6K1 and rpS6 phosphorylation. The results provide indirect and direct evidence of TGF-β family involvement in the regulation of muscle protein synthesis during the neonatal period.

Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin

Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.