Insulin is required for amino acid stimulation of dual pathways for translational control in skeletal muscle in the late-gestation ovine fetus

Abstract

During late gestation, amino acids and insulin promote skeletal muscle protein synthesis. However, the independent effects of amino acids and insulin on the regulation of mRNA translation initiation in the fetus are relatively unknown. The purpose of this study was to determine whether acute amino acid infusion in the late-gestation ovine fetus, with and without a simultaneous increase in fetal insulin concentration, activates translation initiation pathway(s) in skeletal muscle. Fetuses received saline (C), mixed amino acid infusion plus somatostatin infusion to suppress amino acid-stimulated fetal insulin secretion (AA+S), mixed amino acid infusion with concomitant physiological increase in fetal insulin (AA), or high-dose insulin infusion with euglycemia and euaminoacidemia (HI). After a 2-h infusion period, fetal skeletal muscle was harvested under in vivo steady-state conditions and frozen for quantification of proteins both upstream and downstream of mammalian target of rapamycin (mTOR). In the AA group, we found a threefold increase in ribosomal protein S6 kinase (p70(S6k)) and Erk1/2 phosphorylation; however, blocking the physiological rise in insulin with somatostatin in the AA+S group prevented this increase. In the HI group, Akt, Erk1/2, p70(S6k), and ribosomal protein S6 were highly phosphorylated and 4E-binding protein 1 (4E-BP1) associated with eukaryotic initiation factor (eIF)4E decreased by 30%. These data show that insulin is a significant regulator of intermediates involved in translation initiation in ovine fetal skeletal muscle. Furthermore, the effect of amino acids is dependent on a concomitant increase in fetal insulin concentrations, because amino acid infusion upregulates p70(S6k) and Erk only when amino acid-stimulated increase in insulin occurs.

Fig. 1.

Schematic of experimental study design. euAA, euaminoacidemia; euG, euglycemia.

Fig. 2.

Fetal arterial plasma amino acid concentrations in four treatment groups. Open bars, values at baseline; gray bars, values at experimental infusion steady state. A: saline control (C). B: high-dose insulin infusion (HI). C: amino acid infusion (AA). D: amino acid + somatostatin infusion (AA+S). Minor changes were observed in some amino acids in the C and HI groups. Significant increases in all essential and most nonessential amino acids as a result of mixed amino acid infusion were demonstrated in the AA and AA+S groups. *P ≤ 0.05.

Fig. 3.

Representative Western blots with data graphed to show phosphorylation differences in skeletal muscle from animals treated with saline (C), amino acids and somatostatin reflecting independent AA effects (AA+S), amino acids with permissive physiological rise in fetal plasma insulin (AA), and pharmacological hyperinsulinemia (HI). All data are expressed as a ratio of the phosphorylated protein to respective total protein content. A: Akt. B: Erk. C: ribosomal S6 kinase (p70^S6k). D: ribosomal protein (rp) S6. E: 4E-binding protein 1 (4E-BP1). β-Actin was used as an internal control. Means with different superscripts differ at P ≤ 0.01. *Experimental group differs from C group by Student's t-test at P ≤ 0.01.

Fig. 4.

Association of eukaryotic initiation factor (eIF)4E with 4E-BP1 in C, AA+S, AA, and HI treatment groups. eIF4E and its bound proteins were immunoprecipitated (IP) with m⁷GTP-Sepharose 4B. Immunoblot (IB) analysis was performed on the resin-bound fraction with anti-4E-BP1 antibody and normalized to total eIF4E (representative blots shown at top). Means with different superscripts differ at P ≤ 0.05.