Purpose To determine if amino acids (AA), independent of insulin, modulate key signal transduction proteins that regulate translation initiation in skeletal muscle in the late gestation ovine fetus
Methods Three groups of late gestation fetal lambs were studied. The first group received an infusion of AA (Trophamine TM), which provokes a concomitant physiologic increase in fetal insulin (AA + I group). The second group received an infusion of AA plus a continuous somatostatin infusion to suppress amino acid-stimulated fetal insulin secretion (AA-only group). Responses were compared to a control group, which received a saline infusion. The AA infusion was adjusted to increase fetal AA concentrations by 2-fold greater than baseline. Glucose, insulin and amino acid concentrations were measured at baseline and at infusion steady-state. After a 2 hour infusion period, fetal skeletal muscle was harvested for quantification of mTOR, 4EBP-1, eIF4E and p70S6k by Western blot analysis.
Results AA infusion stimulated a 3-fold increase in fetal plasma insulin concentration from baseline in the AA + I group, and somatostatin prevented this increase in the AA-only group. Branched chain amino acid concentrations were unchanged in the control group, and were increased 2-fold with AA infusion. Fetal glucose concentration was stable in all groups. Compared to control, skeletal muscle concentrations of total mTOR were increased 5-fold in the AA + I and AA-only groups (p≤0.05). Phosphorylated, activated mTOR was increased 3-fold in the AA + I group (p≤0.05). Total 4E-BP1 was increased 5-fold in AA + I group and 2-fold in the AA-only group (p≤0.05). Phosphorylated, activated 4E-BP1 was increased 3-fold in the AA + I group (p≤0.05). Total eIF4E was increased 4-fold in the AA + I group (p≤0.05) and trended upward in the AA-only group. The phosphorylation of eIF4E was unchanged in both groups. Total p70S6k trended upward in the AA + I group and was increased 3-fold in the AA-only group (p≤0.05).
Conclusions AA infusion, independent of insulin, increases skeletal muscle concentration of mTOR, p70S6k, 4E-BP1, and eIF4E. Increases in phosphorylated, activated mTOR and 4E-BP1 were only demonstrated when insulin concentrations were also increased. These results suggest that increased supply of AA maximizes muscle protein synthesis not just by mass effect of increased substrate, but by increasing the total amount of critical regulatory proteins, thereby augmenting the effect of insulin to activate these proteins by phosphorylation.