title: Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells.
abstract: Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.
authors.affiliation: Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061. Electronic address: jaappuhamy@ucdavis.edu.
authors.forename: J A D R N
authors.initials: JA
authors.lastname: Appuhamy
authors.affiliation: Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: W A
authors.initials: WA
authors.lastname: Nayananjalie
authors.affiliation: Department of Animal and Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: E M
authors.initials: EM
authors.lastname: England
authors.affiliation: Department of Animal and Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: D E
authors.initials: DE
authors.lastname: Gerrard
authors.affiliation: Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: R M
authors.initials: RM
authors.lastname: Akers
authors.affiliation: Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: M D
authors.initials: MD
authors.lastname: Hanigan
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000601:Amino Acids, Essential / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005947:Glucose / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000166:cytology; D008894:Milk Proteins / Q000096:biosynthesis*; D010316:Particle Size; D010766:Phosphorylation; D014176:Protein Biosynthesis; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000601:Amino Acids, Essential; D008894:Milk Proteins; D058570:TOR Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D005947:Glucose
keywords: AMP-activated protein kinase (AMPK); casein synthesis; energy substrate; essential amino acid