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

Perinatal Research Center, Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, Colorado Submitted 23 March 2008 ; accepted in final form 14 October 2008 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. translation initiation; mitogen-activated protein kinase pathway; ribosomal protein S6 kinase; protein synthesis Address for reprint requests and other correspondence: L. D. Brown, UCD Perinatal Research Center, F441, 13243 East 23rd Ave., PO Box 6508, Aurora, CO 80045 (e-mail: laura.brown{at}ucdenver.edu )