Regulation Of Triacylglycerol And Fatty Acids Synthesis By MTOR Signaling Pathway In BMECs

The content and composition of milk fat is closely related with nutritional quailty of cow’s milk. Triacylglycerol (TG) is the main component of cow’s milk fat. In mammary cells, the fatty acids utilized for TG synthesis is obtained from outside of the cell or synthesised de novo. The synthesis of milk fat in mammary cells is regulated by nutritional factors and various signaling pathways, in which mTOR signaling pathway plays an important role. Mastitis is the most common disease in dairy cows, which affects the content of TG and the composition of fatty acid. S.agalactiae is a common pathogen of mastitis. In the present study, the synthesis and content of TG and fatty acids and regulatory role of mTOR signaling pathway were examed in bovine mammary epithelial cells in the condition of insulin stimulation and S.agalactiae infection.Bovine mammary gland epithelial cells (BMECs) were separated from milk. (1)The expression of key enzymes of TG synthesis were examed by western blot and the content of TQ palmitic acid and DHA are detected by ELISA after inhibition or activation of mTOR singaling pathway in BMECs, which cells treated with mTOR inhibitior rapamycin or transfected with Rheb RNA interference vector and Rheb overexpression vector. (2) The content change of TG was detected by ELISA and 37 kinds of fatty acid were examed by GC-MS after BMECs stimulated with insulin. (3) The content change of TG was detected by ELISA and 37 kinds of fatty acid were examed by GC-MS after BMECs were infected with S.agalactiaeThe results show that:(1) The expression of DGAT1, key enzyme of TG synthesis, was suprressed by mTOR inhibitior rapamycin in BMECs with inhibition of mTOR pathway and cell proliferation at the same time(p<0.01). (2) The content of TG, palmitic acid and DHA were obviously decreased after BMECs were treated with rapamycin (100 nM) for 8 h (p<0.01). (3) The exression of Lipinl and DGAT1, key enzymes of TG synthesis, and the the syntehesis of TG were decreased after posttranscriptional gene silencing of Rheb(p<0.05) in BMECs with suppressed activity of mTOR pathway. (4) The exression of Lipinl and DGAT1, key enzymes of TG synthesis, and the synthesis of TG were increased after BMECs were transfected with Rheb overexpression vector(p<0.01) with enhanced activity of mTOR pathway. (5) The content of TG was obviously increased after 12 h insulin (5 mg/ml) treatment (p<0.05), which process was reversed by rapamycin. (6) Insulin can induce the synthesis of fatty acid in cells. (7) The synthesis and secretion of TG were induced after infection of BMECs with S.agalactiae for a short time (2 h) with the change of component of fatty acid at the same time.In summary, this experiment demonstrated that the mTOR signaling pathway might regulate the expression of key enzymes in the synthesis process to play a positive role in the synthesis of fatty acid and TG in BMECs. Insulin could induce the synthesis of fatty acid in cells. The component of fatty acid were changed due to the BMECs infection with S.agalactiae. However it is not clear yet that whether the mTOR signaling pathway is invovled in this process and the mechanism needs further study.