Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells

Abstract

The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were 2.1- or 3.1-fold higher, respectively, than in their bovine L-1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase (S6K1) on Thr(389) but not on Ser(411) was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E-BP1 or S6K1 phosphorylation than deprivation of all AA. In AA-deprived CID-9 cells, Leu specifically restored BLG synthesis from pre-existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E-BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu- and AA-stimulated BLG translation initiation in a dose-dependent manner. A further reduction was observed for Leu-treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells.

Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway

“…According to previous reports, Leu was involved in cell growth and differentiation through the mTOR pathway (Proud, 2007;Stipanuk, 2007). Moreover, Leu produced a dosedependent regulation of mTOR and served as a key intracellular regulatory factor of the mTORC1 pathway in CMECs (Moshel et al, 2006;Prizant and Barash, 2008;Appuhamy et al, 2012;Durán and Hall, 2012). Kim et al (2001) found that His was the first limiting factor for milk protein secretion.…”

“…When activated by AAs, mTORC1 in turn catalyzed S6K1 and 4EBP1 phosphorylation, followed by phosphorylating the ribosomal protein S6 (RPS6) (Wang and Proud, 2006). S6K1 was a target of mTOR activated in Thr 389 whose phosphorylation status was a main indicator of the AA-induced mTORC1-activated signaling pathway (Moshel et al, 2006;Prizant and Barash, 2008;Appuhamy et al, 2012;Arriola Apelo et al, 2014c). In addition, phosphorylation of 4EBP1 by mTOR increased the initial reaction rates (Beugnet et al, 2003).…”

“…Although the effects of EAAs on mTOR cell signaling and milk protein synthesis have been extensively explored (Moshel et al, 2006;Burgos and Cant, 2010;Toerien et al, 2010) The αs1-casein antibody (SAB1401093) and κ-casein antibody (SAB1401094) were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd. China, the αs2-casein antibody (bs-10034R) was purchased from Bioss (Beijing Biosynthesis Biotechnology Co., Ltd., China) and the β-casein antibody (orb18512) was purchased from Biorbyt (Cambridge, UK). β-Actin (ab8226), used as a loading control, was purchased from Abcam Trading (Shanghai) Company Ltd. (China).…”

Milk Protein Synthesis in the Lactating Mammary Gland: Insights from Transcriptomics Analyses

“…Bovine mammary acini cultured in lactogenic hormones and in a nutrient-rich medium (i.e., high glucose and 4-fold the concentration of amino acids found in plasma of well-fed cows) had an increase of S6K1 and 4EBP1 phosphorylation and dissociation between 4EBP1 and eIF2E [29]. Mixtures of all AA or Leu alone in medium strongly affects synthesis of beta-lactoglobulin in mammary epithelial cells of the mouse and the cow, with all AA being more potent than Leu alone [162]. The latter study demonstrated that synthesis of beta-lactoglobulin in bovine cells was associated with phosphorylation of 4EBP1 and S6K1 and that the major effect on protein synthesis of all AA or Leu was through mTOR.…”

Amino acid and mineral composition of milk from local Ukrainian cows and their crossbreedings with Brown Swiss and Montbeliarde breeds

“…One of the most important components of milk proteins is the functional group of immuneactive AAs, which, in addition form the immune active proteins of the body, accelerate the production of T-lymphocytes, specific antibodies, and also have their own immunological activity and are perspective as immune modulators (Niven et al,1998;Moshel et al, 2006;Mazhitova et al, 2015). The highest amount of immune-active AAs was in the milk protein of crossbred cows at the crosses of URS with Montbeliarde breeds cows, this indicator was 501.6 mg/g, which is by 9.6 242 J.Indonesian Trop.Anim.Agric.…”