Molecular Mechanism Of Methionine Regulating Fat Synthesis,Autophagy And Proliferation Of Bovine Mammary Epithelial Cells

Amino acids,as small organic molecules,are involved in various biological processes,including synthesizing proteins and lipids,regulating cell metabolism,cell growth,cell differentiation,cell proliferation and autophagy.Amino acids as exogenous signals regulates cellular metabolism via activating phosphatidylinositol 3-kinase(PI3K)and the mammalian target of rapamycin(m TOR).The sodium-coupled neutral amino acid transporter 2(SNAT2),which is highly expressed in BMECs,is a solute carrier transporter that transports neutral amino acids such as methionine(Met)and leucine(Leu)into cells,and promotes the milk synthesis in BMECs,but the mechanism needs further study.Previous studies in the laboratory found that SNAT2 can mediate Met is involved in the regulation of milk synthesis in BMECs,as well as BMECs proliferation,apoptosis and autophagy during lactation,but the mechanism needs further study.The present research studied the roles and molecular mechanism of Met on milk synthesis in,proliferation of,and autophagy in BMECs,providing a theoretical and experimental foundation to the usage of Met in milk productive process.In this study,BMECs were cultured by tissue block method.Immunofluorescence combined with Western blotting were used to identify the expression of cytokeratin 18(CK18)and ?-casein in BMECs,which proved that purified BMECs were obtained.Different concentrations of Met(0,0.3,0.6,0.9 and 1.2 m M)were administrated to BMECs.BODIPY staining and triglyceride(TG)detection showed that,with the increase of Met concentration,lipid droplets and TG increased gradually in BMECs,peaked at a Met concentration of 0.6 m M and then decreased.These results indicated that Met promoted milk fat synthesis in a dose-dependent manner.CASY cell counting and flow cytometry were used to detect cell number and cell cycle.As Met concentration increased,cell population and cell proportions in S and G2/M phases were gradually increased,also peaked at 0.6 m M and then decreased.These results indicated that Met dose-dependently affected the proliferation and cell cycle of BMECs.The results of Western blotting showed that the protein levels of p-m TOR,SREBP-1c and Cyclin D1 were significantly increased in a dose-dependent manner(peaked at 0.6 m M and then gradually decreased),indicating that Met could activate the m TOR,SREBP-1c and Cyclin D1 signaling pathways.The optimal concentration of Met was 0.6 m M,and Met might be cytotoxic when over 0.6 m M.To investigate the effect of Met on autophagy in BMECs,Western blotting was used to detect the effects of SNAT2 on the expression of autophagic biomarker LC3-II,and immunofluorescence was used to detect the change of autophagic spots(LC3-II).The results showed that the expression level of p-m TOR was decreased,whereas LC3-II levels and the autophagy spots were increased when SNAT2 was inhibited.Trehalose(Tre)(the autophagy enhancer)and Met were both added to the culture medium of BMECs,and the results showed that,compared with Tre and Met administration group,the expression of p-m TOR was higher but the expression of LC3-II.The autophagy spots were lower in cells treated with Met alone.Tre and Met were added together with SNAT2 si RNA transfection,and the results showed that the expression level of p-m TOR was decreased,whereas LC3-II levels and the autophagy spots were increased after this treatment,compared with the control group(Tre and Met administration group).These above results demonstrate that Met inhibits autophagy in a SNAT2 dependent manner.The effects of overexpression and knockdown of SNAT2 gene showed that SNAT2 positively regulated the secretion of triglyceride and the formation of lipid droplets in BMECs.Western blotting detected that SNAT2 overexpression up-regulated the protein levels of p-m TOR,SREBP-1c and Cyclin D1,whereas SNAT2 knockdown had the opposite effects.The results reveal that SNAT2 is an upstream signal molecule of m TOR,SREBP-1c and Cyclin D1,and positively regulates milk fat synthesis and cell proliferation.To investigate the molecular mechanism through which Met regulates SNAT2 expression,PI3 K inhibitor Wortmannin(Wm)was administrated into the culture medium of BMECs overexpressing SNAT2.Western blotting detected that PI3 K inhibition abolished the stimulation of protein levels of p-m TOR,SREBP-1c and Cyclin D1 caused by SNAT2 overexpression,indicating that SNAT2 activated m TOR,and SREBP-1c and Cyclin D1 signaling pathways via PI3 K activation.Met(0.6 m M)was administrated into the culture medium of BMECs transfected with SNAT2 si RNA,and the results showed that SNAT2 knockdown significantly inhibited the up-regulation of Met on the protein levels of p-PI3 K,p-m TOR,SREBP-1c and Cyclin D1.Western blotting combined with q RTPCR showed that 0.6 m M Met treatment significantly increased the protein and m RNA levels of SNAT2 in BMECs.Immunofluorescence assay showed that 0.6 m M Met treatment significantly increased the cytoplasmic localization of SNAT2.These data reveal that Met regulates the m TOR,SREBP-1c and Cyclin D1 signaling pathways via the SNAT2-PI3 K signaling.In summary,Met regulates the expression of m TOR,SREBP-1c and Cyclin D1 in a dosedependent manner,and over-dosage Met is cytotoxic.Met positively regulates milk fat synthesis,cell proliferation and autophagy in BMECs by regulating the m TOR,SREBP-1c and Cyclin D1 signaling pathways via the SNAT2-PI3K signaling.