To establish an accurate molecular model of human branched-chain amino acid (BCAA) metabolism, the distribution, activity, and expression of the first 2 enzymes in the catabolic pathway--branched-chain-amino-acid aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) complex--were determined in human tissues. The same enzyme activities were measured in rat and African green monkey tissues. Overall, the activities of BCAT and BCKD were higher in rat than in human and monkey tissues; nevertheless, the ratio of the 2 activities was similar in most tissues in the 3 species. Total oxidative capacity was concentrated in skeletal muscle and liver (> 70%) with muscle having a higher proportion of the total in humans and monkeys. In humans, brain (10-20%) and kidney (8-13%) may contribute significantly to whole-body BCAA metabolism. Furthermore, in primates the high ratio of transaminase to oxidative capacity in the entire gastrointestinal tract serves to prevent loss of essential BCAA carbon and raises the possibility that the gastrointestinal tract contributes to the plasma branched-chain alpha-keto acid pool. Quantitative polymerase chain reaction was used to examine expression of human branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), the key enzyme that regulates the activity state of the human BCKD complex and human BCAT isoenzymes. To design the primers for the polymerase chain reaction, human BCKDK was cloned. BCKDK message was found in all human tissues tested, with the highest amount in human muscle. As in rats, there was ubiquitous expression of mitochondrial BCAT, whereas mRNA for the cytosolic enzyme was at or below the limit of detection outside the brain. Finally, the role of BCAA in body nitrogen metabolism is discussed.

Protein synthesis in skeletal muscle of adult rats increases in response to oral gavage of supraphysiological doses of leucine. However, the effect on protein synthesis of a physiological rise in plasma leucine has not been investigated in neonates, an anabolic population highly sensitive to amino acids and insulin. Therefore, in the current study, fasted pigs were infused intra-arterially with leucine (0, 200, or 400 micromol.kg(-1).h(-1)), and protein synthesis was measured after 60 or 120 min. Protein synthesis was increased in muscle, but not in liver, at 60 min. At 120 min, however, protein synthesis returned to baseline levels in muscle but was reduced below baseline values in liver. The increase in protein synthesis in muscle was associated with increased plasma leucine of 1.5- to 3-fold and no change in plasma insulin. Leucine infusion for 120 min reduced plasma essential amino acid levels. Phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein-1 (4E-BP1), ribosomal protein (rp) S6 kinase, and rpS6 was increased, and the amount of eIF4E associated with its repressor 4E-BP1 was reduced after 60 and 120 min of leucine infusion. No change in these biomarkers of mRNA translation was observed in liver. Thus a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle of neonatal pigs in association with increased eIF4E availability for eIF4F assembly. This response appears to be insulin independent, substrate dependent, and tissue specific. The results suggest that the branched-chain amino acid leucine can act as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonates.

Infusion of physiological levels of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates. To determine whether insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonates, insulin secretion was blocked with somatostatin in fasted 7-day-old pigs (n = 8-12/group) while glucose and glucagon were maintained at fasting levels and insulin was infused to simulate either less than fasting, fasting, intermediate, or fed insulin levels. At each dose of insulin, amino acids were clamped at either the fasting or fed level; at the highest insulin dose, amino acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of l-[4-(3)H]phenylalanine. Hyperinsulinemia increased protein synthesis in skeletal muscle during hypoaminoacidemia and euaminoacidemia. Hyperaminoacidemia increased muscle protein synthesis during hypoinsulinemia and euinsulinemia. There was a dose-response effect of both insulin and amino acids on muscle protein synthesis. At each insulin dose, hyperaminoacidemia increased muscle protein synthesis. The effects of insulin and amino acids on muscle protein synthesis were largely additive until maximal rates of protein synthesis were achieved. Amino acids enhanced basal protein synthesis rates but did not enhance the sensitivity or responsiveness of muscle protein synthesis to insulin. The results suggest that insulin and amino acids independently stimulate protein synthesis in skeletal muscle of the neonate.