title: Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices.
abstract: Current nutrient requirement models assume fixed efficiencies of absorbed amino acid (AA) conversion to milk protein. Regulation of mammary protein synthesis (PS) potentially violates this assumption by changing the relationship between AA supply and milk protein output. The objective of this study was to investigate the effects of essential AA (EAA) and insulin on cellular signaling and PS rates in bovine mammary cells. MAC-T cells were subjected to 0 or 100% of normal EAA concentrations in DMEM/F12 and 0 or 100 μg insulin/L in a 2 × 2 factorial arrangement of treatments. Lactogenic bovine mammary tissue slices (MTS) were subjected to the same treatments, except low-EAA was 5% of normal DMEM/F12 concentrations. In MAC-T cells, EAA increased phosphorylation of mammalian target of rapamycin (mTOR; Ser2448), ribosomal protein S6 kinase 1 (S6K1; Thr389), eIF4E binding protein 1 (4EBP1; Thr37/46), and insulin receptor substrate 1 (IRS1; Ser1101), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2; Thr56) and eukaryotic initiation factor (eIF) 2-α (Ser51). In the presence of insulin, phosphorylation of Akt (Ser473), mTOR, S6K1, 4EBP1, and IRS1 increased in MAC-T cells. In MTS, EAA had similar effects on phosphorylation of signaling proteins and increased mammary PS rates. Insulin did not affect MTS signaling, perhaps due to inadequate levels. Effects of EAA and insulin were independent and additive for mTOR signaling in MAC-T cells. EAA did not inhibit insulin stimulation of Akt phosphorylation. PS rates were strongly associated with phosphorylation of 4EBP1 and eEF2 in MTS. EAA availability affected translation initiation and elongation control points to more strongly regulate PS than insulin.
authors.affiliation: Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
authors.forename: J A D Ranga Niroshan
authors.initials: JA
authors.lastname: Appuhamy
authors.forename: Ashley L
authors.initials: AL
authors.lastname: Bell
authors.forename: W A Deepthi
authors.initials: WA
authors.lastname: Nayananjalie
authors.forename: Jeffery
authors.initials: J
authors.lastname: Escobar
authors.forename: Mark D
authors.initials: MD
authors.lastname: Hanigan
mesh_terms: D000601:Amino Acids, Essential / Q000008:administration & dosage / Q000494:pharmacology*; D000824:Animal Nutritional Physiological Phenomena; D000818:Animals; D002417:Cattle; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D007328:Insulin / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008894:Milk Proteins / Q000096:biosynthesis / Q000235:genetics; D009751:Nutritional Requirements; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D000601:Amino Acids, Essential; D007328:Insulin; D008894:Milk Proteins; D012333:RNA, Messenger; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases