Varying the ratio of Lys:Met while maintaining the ratios of Thr:Phe, Lys:Thr, Lys:His, and Lys:Val alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription

Abstract

Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys:Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys:Met 2.9:1 (ideal AA ratio; IPAA), Lys:Met 2.5:1 (LM2.5), and Lys:Met 2.0:1 (LM2.0). The ratios of Thr:Phe (1.05:1), Lys:Thr (1.8:1), Lys:His (2.38:1), and Lys:Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography-mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys:Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of β-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells.

Supply of methionine and arginine alters phosphorylation of mechanistic target of rapamycin (mTOR), circadian clock proteins, and α-s1-casein abundance in bovine mammary epithelial cells

“…Thus, the levels of Met and Arg used in the present study were in accordance with our previous work. 16,17 Within those in vitro studies, although there was an overall positive effect on mammary cell metabolism, the negative effects on phosphorylation of 4E binding protein 1 (EIF4EBP1), eukaryotic elongation factor 2 (EEF2), 16 mTOR, 17 and mRNA abundance of β-casein and AA transporters 17 were partly consistent with the present results. Similarly, despite using EAA profiles similar to those in IPAA, previous in vivo studies also reported results contrary to ours, for instance phosphorylation ratio of ribosomal protein S6 (RPS6) was lower at a Lys : Met = 2.9 : 1 41 and milk protein synthesis or the efficiency of N utilization was not modified at an Lys : Arg = 2 : 1.…”

“…The modified lactogenic media was prepared according to . 17,33 The 10 EAA (L-isomer, Sigma-Aldrich, St Louis, MO) were individually added into the custom highglucose DMEM (devoid of these 10 EAA, custom made from Gibco, Carlsbad, CA). Treatment media were prepared by increasing only Met, Arg or both while keeping the concentrations of 7 other EAA (histidine, isoleucine, leucine, phenylalanine, threonine, tryptophan, valine) the same across all media (Table 1).…”

“…Recent in vitro studies reported a positive effect of greater Arg supply (Lys : Arg at 1 : 1) on mRNA and protein abundance of various components of the milk protein synthesis network, 16 but a negative impact of supplying Met beyond the 3 : 1 ratio of Lys : Met. 17 Although most published studies have focused on the optimal supply of Met and Arg with respect to the role of mTORC1 downstream factors on milk protein synthesis, recent research has identified important roles of factors upstream of mTORC1, 18,19 AA transporters 20 and AA sensors 21,22 in the overall regulation of protein synthesis. Thus, detailed mechanisms encompassing not only mTORC1 in response to changes in AA supply merit further research.…”

“…In this study, we hypothesized that a greater supply of Met or Arg based on an ideal AA profile which has been used in cell culture studies from our laboratory 17,30,31 stimulates casein protein abundance in bovine mammary epithelial cells (BMEC). We used mRNA and protein abundance analyses to explore the potential molecular mechanisms involved in the coordination of upstream components of mTORC1 signaling and the circadian clock network.…”

Advances on Research in Protein-Enriched Cow’s Milk: A Review from Omics Perspective

“…The Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 18 October 2018 doi:10.20944/preprints201810.0411.v1 effect of increasing supplemental Met according to the ideal 3:1 Lys to Met ratio related to protein synthesis and mTOR pathway-related proteins phosphorylation status has also been determined recently [112]. Using gas chromatography-mass spectrometry metabolomics, that study revealeddistinct clusters of metabolites that were differentially concentrated in response to different Met-Lys ratios [112].…”