The abundance and activation of mTORC1 regulators in skeletal muscle of neonatal pigs are modulated by insulin, amino acids, and age
Journal Title (Medline/Pubmed accepted abbreviation):  J. Appl. Physiol.
Year: 2010
Volume: 109
Page numbers: 1448-1454
doi (if applicable):  10.1152/japplphysiol.00428.2010

Summary of Background and Research Design

Background:The mTOR pathway regulates muscle protein synthesis. There is a high level of protein synthesis, specifically in neonatal pigs so these animals are a convenient model to test the regulation of the mTOR pathway. After meals, there are changes in mRNA translation (i.e., enzyme synthesis) caused by an increase in glycemia, insulinemia, and blood amino acids as well as the activity of existing enzymes through phosphorylation and dephosphorylation. The mTOR pathway, specifically, is regulated by physiological phenomena such as amino acids, insulin, cellular energy levels (AMP:ATP), and stress. It is unknown what specific enzymes are involved in the effects of amino acids on the upregulation of mTORC1 (one of the major protein complexes in the mTOR pathway) and muscle protein synthesis.

Hypothesis/Research Question:What is the role of the postprandial rise in insulin and amino acids in the regulation of the mTORC1 pathway in an in vivo model? How do these roles change with development?

Subjects:Piglets from four litters at 6 days old and 26 days old (both ages are considered “neonatal”).

Experimental design:randomized into three treatment groups, 4-6 piglets/age/group:
1) euinsulinemic-euglycemic-euaminoacidemic (control)
2) euinsulinemic-euglycemic-hyperaminoacidemic (AA)
3) hyperinsulinemic-euglycemic-euaminoacidemic (INS)

Treatments and protocol:Baseline blood samples were acquired. A constant infusion of insulin was administered to achieve a blood insulin concentration of about 3 µU/mL (fasted, euinsulinemic) or about 30 µU/mL (postprandial, hyperinsulinemic). Glucose was infused to achieve blood glucose concentrations within ±10% of fasting glycemia (euglycemia). Euaminoacidemia was achieved by adjusting branched chain amino acid (BCAA) infusion rates to equal ±10% of fasting levels. Hyperaminoacidemia was achieved by administering BCAAs to equal twice the fasting level. Rates of protein synthesis were monitored by injecting tritium-labeled phenylalanine (3H) 30 min before the end of the infusion and then assessing the percent of labeled phenylalanine retained by muscle tissue. The pigs were euthanized 120 min after the infusion and the longissimus dorsi muscles (back muscle along the spine) were collected for protein analysis.
                Protein immunoblots were used to quantify various signaling compounds in the mTORC1 complex including: mTOR, PRAS40, Rheb, Raptor, PLD1, Vps34, and Rag B. Protein-protein interactions were examined by the same technique for Rheb-FKBP38, Rheb-PLD1, Vps34-mTOR, and Rag A/B-Raptor.

Summary of research findings:
  • PRAS40 displayed a significant increase in phosphorylation at Thr-246 with insulin but not amino acids and the effect was more pronounced in 6-day old piglets compared with 26-day old piglets. (Phosphorylation of PRAS40 causes it to dissociate from the mTOR complex and increase protein synthesis.)
  • PLD1 quantities were higher in 6-day old piglets compared to 26-day old piglets and phosphorylation of PLD1 decreased with development.
  •   Levels and phosphorylation states of FKBP38 and Vps34 were not affected by postprandial conditions or age of the piglet.
  • RagB abundance and RagB-Raptor associations were higher in younger piglets.

Interpretation of findings/Key practice applications:

The authors did not find an effect of the amino acids on any of the signaling compounds studied, although it was noted that insulin did increase phosphorylation of PRAS40. The reasons for the lack of effects of amino acids are not clear. Possible explanations include insufficient dose of amino acids, timing of collection of muscle samples relative to amino acid administration, or that growth was already so highly stimulated due to developmental state that the amino acids could not provide additional effects.
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