Slow-release Urea, Rumen-protected Methionine, and Histidine: Effects on Expression and Activation of the mTOR Signaling Pathway in Skeletal Muscle of Dairy Cows Receiving a Diet Deficient in Metabolizable Protein
The mammalian target of rapamycin (mTOR) signaling pathway is mediated by two functionally distinct multi-protein complexes, mTORC1 and mTORC2. The mTORC1 is a nutrient sensor, in particular of amino acids, activating protein synthesis by phosphorylation of ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E-binding protein (4E-BP1). The mTORC2 responds to growth factors but is largely nutrient insensitive and phosphorylates protein kinase B (Akt) on Ser473, resulting in activation of cell growth, and survival. We hypothesized that supplementation of diets deficient in metabolizable protein (MP) with slow-release urea or rumen-protected (RP) Met and His will affect the gene expression of key factors of the mTOR pathway, in particular of mTORC1, and will alter their activation by phosphorylation in skeletal muscle of dairy cows in support of protein synthesis. Sixty Holstein cows were blocked based on DIM and milk yield and within block randomly assigned to 1 of 5 diets in a 10-wk experiment (including the first 2 wk as covariate period): MP-adequate diet (AMP); MP-deficient diet [DMP; 5% below MP requirements (NRC, 2001)]; DMP supplemented with slow-release urea as Optigen (Alltech Inc.; DMPO); DMPO supplemented with RPMet (Mepron; Evonik Industries AG; DMPOM); and DMPOM supplemented with RPHis (Balchem Corp.; DMPOMH). Muscle biopsies were collected from Longissimus dorsi during the last wk of the experiment. The mRNA abundance of the following target genes was quantified by qPCR: mTOR, S6K1, and 4E-BP1. Western blotting was used to assess total (t)- and phosphorylated (p)-S6K1 (Thr389), t-Akt and p-Akt (Ser473), p-mTOR (Ser2481), and p-S6 (Ser240/244). Data were analyzed by the MIXED procedure of SAS. The mRNA abundance of the target genes was not affected by the treatments; treatment effects were limited to p-mTOR and p-S6: p-mTOR values in DMPO were decreased when compared against DMP (P = 0.03) and also tended to be lower (P = 0.07) in DMPOMH than in DMPOM. The p-S6 values in DMPOM tended to be greater (P = 0.07) than in DMPO. There was also a trend (P= 0.09) for decreased p-S6 in DMPO versus DMPOM and DMPOMH. In conclusion, supplementation of the DMP diet with slow-release urea, or RPMet and RPHis, respectively, altered the phosphorylation status of mTOR-associated signaling proteins in muscle. No such effects were observed when comparing the AMP and DMP diets, thus indicating specific effects of the supplements.
Keywords: rumen-protected amino acid, mTOR, dairy cow