Enhanced supply of methionine or arginine alters mechanistic target of rapamycin signaling proteins, messenger RNA, and microRNA abundance in heat-stressed bovine mammary epithelial cells in vitro

摘要

Heat stress (HS) causes reductions in milk production,but it is unclear whether this effect is due to reducednumber or functional capacity (or both) of mammarycells. Methionine supplementation improves milkprotein, whereas Arg is taken up in excess by mammarycells to produce energy and nonessential AA that canbe incorporated into milk protein. To evaluate molecularmechanisms by which mammary functional capacityis affected by HS and Met or Arg, mammary alveolar(MAC-T) cells were incubated at thermal-neutral(37°C) or HS (42°C) temperatures. Treatments wereoptimal AA profiles (control; Lys: Met = 2.9:1.0; Lys:Arg = 2.1:1.0), control plus Met (Lys: Met = 2.5:1.0), orcontrol plus Arg (Lys: Arg = 1.0:1.0). After incubationfor 6 h, cells were harvested and RNA and protein wereextracted for quantitative real-time PCR and Westernblotting. Protein abundance of mechanistic target ofrapamycin (MTOR), eukaryotic initiation factor 2a,serine-threonine protein kinase (AKT), 4E binding protein1 (EIF4EBP1), and phosphorylated EIF4EBP1 waslower during HS. The lower phosphorylated EIF4EBP1with HS would diminish translation initiation and reduceprotein synthesis. Both Met and Arg had no effecton MTOR proteins, but the phosphorylated EIF4EBP1decreased by AA, especially Arg. Additionally, Met butnot Arg decreased the abundance of phosphorylatedeukaryotic elongation factor 2, which could be positivefor protein synthesis. Although HS upregulated theheat shock protein HSPA1A, the apoptotic gene BAX,and the translation inhibitor EIF4EBP1, the mRNAabundance of PPARG, FASN, ACACA (lipogenesis),and BCL2L1 (antiapoptotic) decreased. Greater supplyof Met or Arg reversed most of the effects of HS occurringat the mRNA level and upregulated the abundanceof HSPA1A. In addition, compared with the control,supply of Met or Arg upregulated genes related to transcriptionand translation (MAPK1, MTOR, SREBF1,RPS6KB1, JAK2), insulin signaling (AKT2, IRS1),AA transport (SLC1A5, SLC7A1), and cell proliferation(MKI67). Upregulation of microRNA related tocell growth arrest and apoptosis (miR-34a, miR-92a,miR-99, and miR-184) and oxidative stress (miR-141and miR-200a) coupled with downregulation of fatsynthesis-related microRNA (miR-27ab and miR-221)were detected with HS. Results suggest that HS has adirect negative effect on synthesis of protein and fat,mediated in part by coordinated changes in mRNA,microRNA, and protein abundance of key networks.The positive responses with Met and Arg raise the possibilitythat supplementation with these AA during HSmight have a positive effect on mammary metabolism.