Optimal ratios of essential amino acids stimulate β-casein synthesis via activation of the mammalian target of rapamycin signaling pathway in MAC-T cells and bovine mammary tissue explants

Abstract

Amino acids are the building blocks of proteins and serve as key molecular components upstream of the signaling pathways that regulate protein synthesis. The objective of this study was to systematically investigate the effect of essential AA ratios on milk protein synthesis in vitro and to elucidate some of the underlying mechanisms. Triplicate cultures of MAC-T cells and bovine mammary tissue explants (MTE) were incubated with the optimal AA ratio (OPAA; Lys:Met, 2.9:1; Thr:Phe, 1.05:1; Lys:Thr, 1.8:1; Lys:His, 2.38:1; and Lys:Val, 1.23:1) in the presence of rapamycin (control), OPAA, a Lys:Thr ratio of 2.1:1, a Lys:Thr ratio of 1.3:1, a Lys:His ratio of 3.05:1, or a Lys:Val ratio of 1.62:1 for 12 h; the other AA concentrations were equal to OPAA. In some experiments, the cells were cultured with OPAA with or without rapamycin (100 ng/mL) or with mammalian target of rapamycin (mTOR) small interference RNA, and the MTE were exposed to OPAA with rapamycin for β-casein expression. Among the treatments, the expression of β-casein was greatest in the MTE cultured with OPAA. In MAC-T cells, the OPAA upregulated the mRNA expression of SLC1A5 and SLC7A5 but downregulated the expression of IRS1, AKT3, EEF1A1, and EEF2 compared with the control. The OPAA had no effect on the mTOR phosphorylation status but increased the phosphorylation of S6K1 and RPS6. When the MTE were treated with rapamycin in the presence of OPAA, the expression of β-casein was markedly decreased. The phosphorylation of RPS6 and 4EBP1 also was reduced in MAC-T cells. A similar negative effect on the expression of RPS6KB1 and EIF4EBP1 was detected when the cells were cultured with either rapamycin or mTOR small interference RNA. The optimal AA ratio stimulated β-casein expression partly by enhancing the transport of AA into the cells, cross-talk with insulin signaling and a subsequent enhancement of mTOR signaling, or translation elongation in both MAC-T cells and bovine MTE.

Seryl-tRNA synthetase-mediated essential amino acids regulate β-casein synthesis via cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells

“…The increased phosphorylation of mTOR and its downstream factors (S6K1 and 4EBP1) stimulated by EAA confirmed the regulatory effects of EAA on promotion of milk protein synthesis by motivating the mTOR signaling pathway in BMEC. Additionally, these changes in mTOR-related protein abundance in response to EAA are in line with previous studies conducted in MAC-T cells (Appuhamy et al, 2011), bovine mammary gland tissue explants (Li et al, 2017), and BMEC (Gao et al, 2015). It appears that SARS inhibition blocks the stimulating effects of EAA on the mTOR system and then prevents casein synthesis.…”

“…The activation of the mTORC1 signaling pathway in response to EAA is critical for the regulation of protein synthesis in mammals (Jewell and Guan, 2013;Kim et al, 2013). Furthermore, mTORC1 is critical for milk protein synthesis in dairy cows (Appuhamy et al, 2011;Li et al, 2017). Additionally, phosphorylation of S6K1 and 4EBP1 was demonstrated previously as a marker of mTOR activation (Hara et al, 1998).…”

“…Several factors, including hormones (e.g., prolactin and insulin; Bequette et al, 2001;Burgos et al, 2010), nutrients (e.g., amino acids and glucose; Appuhamy et al, 2011Appuhamy et al, , 2014, and environmental stressors (e.g., heat stress and cold stress; Collier et al, 2006;Hu et al, 2016) are known to affect milk protein synthesis. Of those dietary nutrients understood to influence milk protein synthesis, the EAA are among the most important because they provide the substrates for protein synthesis and act as regulators of casein synthesis in the bovine mammary gland tissues (Appuhamy et al, 2011;Li et al, 2017). Importantly, several studies have demonstrated that EAA stimulate cell proliferation of bovine mammary epithelial cells (BMEC; Theodorou et al, 2011;Luo et al, 2018).…”

“…Therefore, it appears that EAA-stimulated expansion of epithelial cell populations in the mammary gland especially during lactation may further promote milk yield (Ellis and Capuco, 2002;Arendt and Kuperwasser, 2015). An additional regulator of milk protein syn-Seryl-tRNA synthetase-mediated essential amino acids regulate β-casein synthesis via cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells 10457 thesis is the mammalian target of rapamycin (mTOR) signaling pathway, one of the main mechanisms by which EAA regulate casein synthesis (Appuhamy et al, 2011;Guo et al, 2017;Li et al, 2017). Once activated, mammalian target of rapamycin complex 1 (mTORC1) is phosphorylated and subsequently stimulates the downstream proteins-ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E binding protein 1 (4EBP1)-which promote the release of 4EBP1 from the key initiation factor (eIF4E) and strengthen the translation initiation process (Appuhamy et al, 2011;Shimobayashi and Hall, 2014).…”

Methionine and valine activate the mammalian target of rapamycin complex 1 pathway through heterodimeric amino acid taste receptor (TAS1R1/TAS1R3) and intracellular Ca2+ in bovine mammary epithelial cells

“…For mammary epithelial cells, instantly sensing alterations in extracellular AA availability is likely crucial for priming downstream protein synthesis-regulating pathways (Loor et al, 2013). Although an AA transporter-mediated regulatory role in milk protein synthesis has recently been identified (Suryawan and Davis, 2011), less is understood about upstream pathways consisting of AA-sensing components (Averous et al, 2016;Li et al, 2016Li et al, , 2017.…”