Leucine and a-ketoisocaproic acid, but not norleucine, stimulate skeletal muscle protein synthesis in neonatal pigs
Journal Title (Medline/Pubmed accepted abbreviation):  J Nutr
Year:  2010
Volume: 140
Number: 8
Page numbers: 1418-1424
doi (if applicable): 

Summary of Background and Research Design

Hypothesis/Research Question:Leucine is a branched-chain amino acid that can specifically stimulate protein synthesis. However, it is not clear if leucine itself or one of its downstream metabolites is responsible for stimulating protein synthesis. The authors explored the roles of leucine, its keto-acid a-ketoisocaproate (KIC), or a norleucine (a structural analog of leucine with a non-branched side chain) in the promotion of protein synthesis in a piglet model.

Subjects:Groups of 6-8 healthy piglets at about 6 days of age

Experimental design:Independent groups, random assignment to groups

Treatments and protocol:The piglets were cannulated into the left jugular vein and left carotid artery and returned to the sow for recovery from surgery. After recovery, piglets were randomized to receive one of 4 different IV infusions: 1) leucine; 2) KIC; 3) norleucine; or 4) saline (control). The initial infusion was 148 mmol/kg, followed by continuous infusion of 400 mg/kg for the first 3 treatments. This dose level was shown in previous studies to provide a plasma leucine concentration that equates to postprandial levels. The piglet group receiving saline received an equal volume of saline as the pigs receiving leucine. The infusion periods were 60 min long (10 min for initial infusion). An infusion of L[4-3H-]phenylalanine was also given to evaluate protein synthesis (via incorporate of labeled phenylalanine into muscle tissue). Piglets were killed at the end of the infusion and muscle tissue (longissimus dorsi) and blood were examined for protein synthesis, amino acid levels, transcription factors, and enzyme activities.

Summary of research findings:
The leucine and KIC treatments raised plasma leucine levels, while the saline did not, compared with baseline. Norleucine lowered plasma leucine levels vs. baseline. Both leucine and KIC increased fractional protein synthesis to a comparable degree (from ~13.5% with saline to ~ 20-21%). The authors also measured the phosphorylation (inactivation) of a protein (4E-BP1). This protein, when phosphorylated, has reduced binding with eukaryotic initiation factor 4E (eIF4E) and decreases its ability to stimulate translation. Both leucine and KIC stimulated 4E-BP1 phosphorylation, while norleucine and saline had no significant effects.Correspondingly, the authors also measured lower levels of the complex of eIF4E with 4E-BP1.
At the same time, leucine and KIC stimulated the formation of a complex of eurkaryotic initiation factor 4G (eIF4G) with eIF4E, a combination that tends to promote the start of translation. Leucine and KIC apparently operated by phosphorylation of a serine residue (Ser1108) on eIF4G. Saline and norleucine had no significant effects.

Interpretation of findings/Key practice applications:

This research clearly shows, in a neonatal piglet model, that leucine and/or its metabolism to KIC is/are needed for stimulation of protein synthesis. Norleucine, as a structural isomer of leucine, but with no branching of the side chain, did not have significant effects. The ability of leucine and KIC to stimulate protein synthesis is in agreement with other findings in the literature, although it should be noted that previous studies in cultured adipocytes or rats have shown that norleucine can stimulate protein synthesis in these models. It is not clear why there were differences between animal models or how these results might be representative of humans, but it does highlight the importance of the branching of leucine’s side chain in the ultimate “turning on” of protein translation in the muscle cell.
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