The Ratio Between Lysine And Methionine On Casein Synthesis In Bovine Mammary Epithelial Cells

This study was to use the bovine mammary epithelial cells as a model to determine the best ratio between lysine and methionine, and focused on the effects of additional lysine and methionine on the regulation of genes related to milk protein synthesis. Study included three parts:1. Effects of lysine and methionine on bovine mammary epithelial cells proliferation were evaluated by MTT colorimetric assay. The lysine was added to the DMEM/F12 culture medium at concentrations of 0, 0.05, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 mmol/L and the methionine was added to the DMEM/F12 culture medium at concentrations of 0, 0.025, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8 mmol/L, respectively. Experiment with 24, 48, 72 h. All experiments repeated 3 times. The results showed that 0.8 to 1.6 mmol/L lysine and 0.4 to 0.8 mmol/L methionine at 48 h significantly improved the proliferation of bovine mammary epithelial cells (P<0.01).2. The cells were subsequently treated with lysine (0,0.4,0.8,1.2,1.6,2.0,2.4 mmol/L) and methionine (0,0.2,0.4,0.6,0.8,1.0 mmol/L) for 48 h. All experiments repeated 3 times. I) the content of casein was increased with the increasing levels of lysine and peaked at 0.8 m mol/L. From the fitted curve of the content of casein (y) and Lysine levels (x, mmol/L): y= -0.336X~2+0.792X+1.983 (R~2=0.965), the highest content of casein expression occurred at 1.2 mmol/L of Lysine. Expression of several genes regulating milk protein syntheses were measured at the 1.2 mmol/L Lysine concentration. The expression of genes CSN1S1, CSN1S2, LALBA, CSN2 and CSN3 were different (P<0.01). The amino acid transporter genes CAT-1 was significantly increased (P<0.01), the expression of genes related to JAK2-STAT5 pathway were up-regulated, JAK2 and STAT5were significantly increased (P<0.01). mTOR and S6K related to mTOR signaling pathway were increased, but S6K was not significantly (P>0.05). EIF4E-BP1 expression was significantly down-regulated (P<0.01). II) The content of casein was increased with the increasing levels of Methionine and peaked at 0.6 m mol/L. From the fitted curve the content of casein (y) and Methionine levels (x, mmol/L): y= -2.736X~2+2.851X+1.984 (R~2=0.95), it was estimated that the highest content of casein occurred at 0.5 mmol/L of Methionine. Expression of several genes regulating milk protein syntheses were measured at the 0.5 mmol/L Methionine concentration. CSN1S1, LALBA, CSN3 and CSN1S2 were significantly increased by the addition of methionine (P<0.01), however there was no change in CSN2 expression. The amino acid transporter genes ASCT-2 was significantly increased (P<0.01).The genes of JAK2-STAT5 pathway expression were increased, but ELF5 was not significantly, JAK2 and STAT5 were significantly increased (P<0.01).The genes mTOR and S6K of mTOR signaling pathway expression were increased, but they were not significantly. EIF4E-BP1 expression was significantly down-regulated (P<0.01).3. The experiment was conducted with a 5×3 factorial design: with lysine (1.0, 1.2, 1.4, 1.5, 1.6 mmol/L in DMEM/F12 medium) and methionine (0.4, 0.5, 0.6 mmol/L in DMEM/F12 medium). The expression of important genes related to milk protein synthesis process included the genes of the JAK2-STAT5 pathway and the mTOR signaling pathway. All experiments repeated 3 times. The expression of genes and caseins content were detected by RT-qPCR method and Casein ELISA Kit, respectively. Data were analyzed with the PROC GLM and PROC ANOVA. The results showed that when the concentration of lysine was 1.2 m mol/L and the concentration of methionine was 0.4 mmol/L, the content of casein in medium increased significantly (P<0.01) and peaked at 2.95ppm. We measured the genes expression at this ratio (3:1). CSN1S1 and LALBA were the most highly expressed genes encoding caseins and increased more than 2-fold (P<0.01),followed by CSN2, CSN3 and CSN2S2, and these genes expression were also up-regulated (P<0.01). The expression of genes related to JAK2-STAT5 pathway were up-regulated, and JAK2 and ELF5 expression increased significantly (P<0.01). The expression of genes related to mTOR signaling pathway was increased, but S6K was not significantly (P>0.05). EIF4E-BP1 expression was significantly down-regulated (P<0.01). The expression of amino acid transporter CAT-1 and ASCT-2 had no change.These observations together with the fact that genes expression changed in the cells with the ratio between lysine and methionine revealed that the lysine, methionine and the ratio between lysine and methionine may directly regulate the expression of genes related to milk protein transcription and translation, leading to improved milk protein synthesis.