Essential Amino Acids Regulate Both Initiation and Elongation of mRNA Translation Independent of Insulin in MAC-T Cells and Bovine Mammary Tissue Slices

Abstract

Current nutrient requirement models assume fixed efficiencies of absorbed amino acid (AA) conversion to milk protein. Regulation of mammary protein synthesis (PS) potentially violates this assumption by changing the relationship between AA supply and milk protein output. The objective of this study was to investigate the effects of essential AA (EAA) and insulin on cellular signaling and PS rates in bovine mammary cells. MAC-T cells were subjected to 0 or 100% of normal EAA concentrations in DMEM/F12 and 0 or 100 μg insulin/L in a 2 × 2 factorial arrangement of treatments. Lactogenic bovine mammary tissue slices (MTS) were subjected to the same treatments, except low-EAA was 5% of normal DMEM/F12 concentrations. In MAC-T cells, EAA increased phosphorylation of mammalian target of rapamycin (mTOR; Ser2448), ribosomal protein S6 kinase 1 (S6K1; Thr389), eIF4E binding protein 1 (4EBP1; Thr37/46), and insulin receptor substrate 1 (IRS1; Ser1101), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2; Thr56) and eukaryotic initiation factor (eIF) 2-α (Ser51). In the presence of insulin, phosphorylation of Akt (Ser473), mTOR, S6K1, 4EBP1, and IRS1 increased in MAC-T cells. In MTS, EAA had similar effects on phosphorylation of signaling proteins and increased mammary PS rates. Insulin did not affect MTS signaling, perhaps due to inadequate levels. Effects of EAA and insulin were independent and additive for mTOR signaling in MAC-T cells. EAA did not inhibit insulin stimulation of Akt phosphorylation. PS rates were strongly associated with phosphorylation of 4EBP1 and eEF2 in MTS. EAA availability affected translation initiation and elongation control points to more strongly regulate PS than insulin.

Association of a polymorphism in the 3' untranslated region of the <i>OLR1</i> gene with milk fat and protein in dairy cows

“…On the other hand, there is increasing evidence that Ox-LDL induces beta cell dysfunction and reduction of insulin production (Maziere et al 2004, Abderrahmani et al 2007, Favre et al 2011. Considering the role of insulin in stimulating protein synthesis in mammary gland (Mackle et al 2000, Molento et al 2002, Menzies et al 2009, Appuhamy et al 2011, it can be hypothesized that Ox-LDL may decrease protein synthesis by reducing insulin production in beta cells and its signalling in mammary cells. OLR1, as a major receptor for Ox-LDL, is able to remove Ox-LDL from circulating blood and as a result reduces its deleterious effect on insulin production and signalling.…”

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

“…mTORC1 consists of mTOR, regulatory associated protein of TOR (Raptor), and a G protein β subunit-like protein (GβL) (Kim, 2009). A great deal of evidence indicated that P-mTOR at Ser 2448 was an indicator of mTOR pathway activity in immortalized bovine mammary epithelial cells (MAC-T) (Appuhamy et al, 2011b;. Prizant and Barash (2008) showed that P-mTOR in Ser 2448 was completely inhibited by His supplementation, but phosphorylation of the ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein (4EBP1) could still be detected.…”

“…When 4EBP1 was unphosphorylated, cap-dependent translation was inhibited by competitively blocking the binding of eukaryotic translation initiation factor 4G (eIF4G) to eIF4E (Gingras et al, 1999;Harris and Lawrence, 2003). The elongation step of mRNA translation was enhanced by eukaryotic elongation factor 2 (eEF2) (Wang and Proud, 2006) and was inhibited by its phosphorylation on Thr 56 , which suggested that eEF2 may be a limiting factor in milk protein synthesis (Christophersen et al, 2002;Appuhamy et al, 2011b).…”

WISP3 (CCN6) Regulates Milk Protein Synthesis and Cell Growth Through mTOR Signaling in Dairy Cow Mammary Epithelial Cells

“…In addition to this well-known function, in recent years, studies have found that Lys is also an important regulator of various signaling pathways, cell growth, and protein synthesis (Appuhamy et al, 2011a;Lu et al, 2013). In this study, after treatment with 1.2 mM Lys for 24 h, the expression of CSN2 in DCMECs and cell viability were assessed.…”

“…Currently, the transcriptional process of milk protein synthesis is known to be regulated by prolactin through the JAK/STAT5 signaling pathway just as its translational process is regulated by amino acids through the mTOR/S6K1 signaling pathway (Yang et al, 2000b;Galbaugh et al, 2006;Jankiewicz et al, 2006;Wang and Proud, 2006;Prizant and Barash, 2008;Toerien et al, 2010). The activity of mTOR also appears to be modulated by branched-chain amino acids (AA) (Kimball and Jefferson, 2006;Moshel et al, 2006), and the availability of amino acids is the main limiting factor for milk protein synthesis (Reynolds et al, 1994;Appuhamy et al, 2011a), and according to previous reports, lysine and methionine were the most limiting in bovine mammary glands (Bequette et al, 1998;Hanigan et al, 2002). In addition, many previous reports have shown that lysine, as the most limiting, could modulate mammary protein synthesis (Vyas and Erdman, 2009;Appuhamy et al, 2011b;Lee et al, 2012;Wang et al, 2012;Nan et al, 2014;Paz and Kononoff, 2014).…”