Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs
 
 
Journal Title (Medline/Pubmed accepted abbreviation):  Amino Acids
Year: 2011
Issue: 40
Page numbers: 157-165
doi (if applicable): 10.1007/s00726-010-0629-9

Summary of Background and Research Design

Background: Leucine, a branched-chain amino acid, has been shown to stimulate transient skeletal protein synthesis via the mTOR pathway post-ingestion. Its effect has shown to be longer-lasting with the administration of other amino acids in order to prevent a decline of amino acid concentrations in the blood. Continuous administration of leucine in conjunction with other amino acids has shown an increase in prolonged skeletal muscle synthesis in fast-twitch glycolytic fibers.
 
Mechanism of leucine-activated protein synthesis:
Leucine activates the mammalian target of rapamycin complex (mTORC1). This mTORC1 activation, in turn, increases the activity of eukaryotic initiation factor 4E (eIF-4E). The mTORC1 phosphorylates a binding protein (4E-BP) for eIF-4E, preventing it from binding to eIF-4E. Ultimately, this free eIF-4E allows for the formation of complexes that allow the transcription process to be turned on. Also, mTORC1 activates S6K1, a signaling factor within the ribosome that also helps protein synthesis

Hypothesis:Amino acid infusion will stimulate prolonged skeletal muscle synthesis in many types of muscle types in addition to fast-twitch glycolytic fibers.

Subjects:Five-day old piglets, n=6 per treatment group

Experimental design:randomized into 1 of 3 treatment groups: 1) saline solution (control), 2) leucine infusion, 3) leucine + balanced amino acid infusion. The leucine infusion rate was chosen to simulate postprandial blood concentrations of leucine. The balanced amino acid infusion rate was chosen to maintain fasting amino acid concentrations. (It is common for blood amino acid concentration to drop below fasting levels with increased protein synthesis.)

Treatments and protocol: Infusions lasted for 24 hrs. Isotopically labeled phenylalanine was administered 30 min before the end of the infusion to measure the rate of protein synthesis. The piglets were sacrificed at the end of the 24 hr period and muscle samples were collected from the gastrocnemius (calf) muscle (mixed slow- and fast-twitched oxidative fibers), masseter (jaw) muscle (slow-twitched muscle fibers), left and right chamber of the heart, liver, kidney, pancreas, and small intestine. Tissues were analyzed for protein content, rate and capacity of protein synthesis, specific enzyme activity, and amount of mRNA.

Summary of research findings:
  • Infusion of leucine caused a decrease in essential amino acids (EAA, [EAA] at 24 hrs = 502 ± 38.6 nmol/mL while at t=0 [EAA] = 1,059 ± 35.5 nmol/mL). An infusion of leucine plus other amino acids allowed retention of baseline EAA quantities ([EAA] at t=24 hrs = 1,167 ± 71.5 nmol/mL).
  • The piglets who underwent the infusion of leucine plus amino acids showed a higher rate of protein synthesis in both the gastrocnemius (P=0.057) and the masseter muscle (P=0.009) compared to both other sets of piglets. Protein synthesis in cardiac muscle appeared to not be affected.
  • The rate of translational efficiency was estimated as the total protein synthesized in a day per total RNA in the muscle. Translation showed increased efficiency in the gastrocnemius, masseter, and liver, but not in the heart, pancreas, kidney, or jejunum.
  • Phosphorylation (ie activation) of 4E-BP1 and S6K1, two downstream targets of mTORC1, was exhibited most markedly in the two skeletal muscles after infusion of leucine with or without other amino acids (P<0.03). Phosphorylation of these proteins also occurred in the liver with leucine with or without other amino acids.

Interpretation of findings/Key practice applications:

It is important to maintain high leucine blood levels to keep the mTOR pathway activated and therefore maintain a high rate of protein synthesis in skeletal muscle. It is also important to maintain blood levels of other amino acids to feed the production of new proteins.

Limitations:

 These subjects were neonatal pigs whose genetic expression is set to increase the mass of the entire body of the piglet, not just skeletal muscle. Gene expression and/or regulation may be slightly different in adult mammals who have finished developmental maturation.
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