Lysine/methionine Pattern And Glucose Level Regulate The Expression Of Genes Related To Casein Synthesis

The synthesis of milk protein requires the availability of amino acids and a large supply of energy. Lysine, methionine and glucose regulate milk protein production of lactating dairy cows in vivo. Moreover, the content and ratio of lysine to methionine can regulate milk protein synthesis via cellular signaling pathways involving JAK2-STAT5 and mammalian target of rapamycin(m TOR). This study was conducted to investigate the effects of different Lysine/Methionine pattern and Gluose level on milk protein synthesis in vitro, mainly focused on genes related to JAK2-STAT5 and m TOR. Primary bovine mammary epithelial cells(BMEC) were obtained from Holstein dairy cows and cultured in Dulbecco’s modified Eagle’s medium-F12 medium containing 10% fetal bovine serum. BMEC were subjected to four treatments arranged in a 2 x 2 factorial design with Lysine/Methionine ratio(3: 1 vs 2.3: 1, namely balanced vs unbalanced) and glucose level(17.5 mmol/L vs 2.5 mmol/L, namely high vs low) as two factors. The experiment was repeat 3 times. The study included three parts:1. Effects of different lysine/methionine patterns and Glucose levels on mammary epithelial cells proliferation were evaluated by MTT colorimetric assay. The results showed that OD values of groups with high level of Glucose was significantly increased(P<0.05) compared with groups with low level of glucose. That is, the effects of groups with high level of glucose on mammary epithelial cells proliferation was significantly higher than groups with low level of glucose. No significant differences(P>0.05) was observed between HB and HU, the same as LB and LU(P>0.05). Effects of lysine/methionine patterns and glucose levels on mammary epithelial cells proliferation have no interaction.2. Effects of different lysine/methionine patterns and glucose levels on total casein content in mammary epithelial cells were evaluated by ELISA. The results show that effects of different lysine/methionine patterns and glucose levels on casein content in mammary epithelial cells were significant difference(P<0.01). Casein content was highest in HB group, HU, LB, LU were in descending order. Compared with low level of glucose groups, casein content increased in groups with high level of glucose(P<0.01). Casein content was also higher in Lysine/Methionine balanced groups than in unbalanced groups(P<0.01).3. Effects of different lysine/methionine patterns and glucose levels on expression of genes related to casein synthesis in mammary epithelial cells were evaluated by Real Time-PCR. The results show that effects of different lysine/methionine patterns and glucose levels on expression of genes related to casein synthesis in mammary epithelial cells were significant difference(P<0.05). Expression of CSN1S2, CSN2, LALBA, STAT5, ELF5, m TOR(P<0.01) and CSN1S1(P<0.05) were up regulated in groups with high level of glucose compared with low level groups. The up regulation of CSN1S2, CSN2, LALBA, JAK2, STAT5, ELF5, m TOR(P<0.01) and CSN1S1, CSN3(P<0.05) were also observed in Lysine/Methionine balanced groups compared with unbalanced groups, while EIF4EBP1 was down regulated(P<0.05). Effects of lysine/methionine patterns and glucose levels on expression of genes related to casein synthesis in mammary epithelial cells have interaction. Lysine/methionine patterns were more sensitive to regulate expression of genes related to casein synthesis than glucose level.In conclusion, proper ratio of Lysine to methionine and high level of glucose may directly accelerate the BMEC proliferation and regulate the expression of genes related to milk protein transcription, which can increase milk protein synthesis.