| Lipid droplets(LD)in mammary epithelial cells of dairy cows are precursors to the formation of milk fat globules,which are the main form of fat in milk.It is of great significance to study the regulation mechanism of lipid droplets to elucidate the synthesis and secretion of milk fat globules.Previous research has found that phospholipids may affect lipid droplet size,but the mechanism is not clear.The CDP-choline pathway is the main pathway for the synthesis of phosphatidylcholine(PC)in mammals,and phosphatidylcholine cytidine transferase α(CCTα)is an important rate-limiting enzyme in the CDP-choline pathway,which affects the synthesis of PC.In this study,a bovine mammary epithelial cell model with knockout and overexpression of CCTα was constructed.The effects of CCTα on lipid droplet size,phospholipid synthesis,gene and protein expression were analyzed to lay a foundation for elucidating the regulatory mechanism of lipid droplet size in mammary epithelial cells.Effects of CCTα on lipid drops and phospholipid synthesis: Bovine mammary epithelial cells were treated with 100 μmol/L linoleic acid(LA)for 24 h.The localization of CCTαand LD was observed by immunofluorescence staining.It was found that CCTα was mainly localized in the nucleus.After treatment with LA,lipid droplets were gradually formed in the endoplasmic reticulum,and CCTα was transferred from the nucleus to the endoplasmic reticulum and localized on the surface of the lipid droplets.Then,CCT-α knockout(CCT-KO)and overexpressed(CCT-OE)cell lines were constructed.The size and number of lipid droplets were analyzed by oil red O staining.It was found that CCT-α knockout significantly increased the average size of lipid droplets(P<0.05),and the diameter of lipid droplets in normal cells(NC)was 1.53μm.The droplet diameter in CCT-KO group was increased to 1.68 μm,while overexpression decreased the droplet diameter.The mean droplet diameter in CCT-OE group was significantly decreased to 1.18 μm(P<0.001).The results of lipid droplet size distribution ratio showed that compared with the NC group,the ratio of small lipid droplet(size<1 μm)in CCT-OE group increased from 11.39 % to 35.48%,and that of large lipid droplet(size >3 μm)in CCT-KO group increased from 0.67 % to2.88 %.These results indicated that CCTα knockout increased the ratio of large lipid droplet and decreased the ratio of small lipid droplet,and the result of CCTα overexpression was opposite.Lipid droplet fusion was further observed by living cell workstation,and it was found that the number of lipid droplet fusion was larger in CCT-KO group,and the fusion time was shorter,suggesting that the increase of large lipid droplet may be the result of the fusion of small lipid droplet.Phosphatidyl choline and phosphatidyl ethanolamine(PE)were quantitatively analyzed by thin layer chromatography.The results showed that CCT-KO significantly reduced the content of PC and the ratio of PC to PE(P<0.01),and CCT-OE significantly increased the content of PC and the ratio of PC to PE(P<0.05).A total of 1843 lipids were identified by further lipidomics detection.Compared with the NC group,48 lipids were significantly down-regulated in the CCT-KO group and 140 were significantly up-regulated in the CCT-OE group,among which 36 were significantly different in all three groups.Mechanism of CCTα regulation on lipid droplet size: The detection results of triglyceride content in cells and the expression of genes and proteins related to the synthesis and decomposition of lipid droplets showed that CCT-KO significantly increased TG content in cells(P<0.05).Both knockout and overexpression of CCTα significantly increased lipid metabolism-related genes fatty acid synthase(FASN),Peroxisome proliferator activated receptor γ(PPARγ),and SREBP cleavage activating protein(SCAP)(P<0.05),Sterol regulatory element-binding proteins(SREBP1)were significantly increased in the CCT-α knockout group(P<0.05).Lipid droplet protein Xanthine dehydrogenase(XDH)was significantly elevated in the CCTα overexpression group(P<0.05).TG synthetase Diacylglycerol o-Acyltransferase(DGAT)m RNA expression was significantly increased in CCT-KO and significantly decreased in CCT-OE(P<0.05).Adipose triglyceride lipase(ATGL),a key lipolysis enzyme,was significantly decreased in CCT-KO and significantly increased in CCT-OE(P<0.05).Immunofluorescence results showed that the autophagy associated protein microtubule associated protein 1 light chain 3(LC3)was located on the surface of LD.The expression of LC3 B protein was significantly decreased in CCT-KO group(P<0.05),while LC3 B protein expression was significantly increased in CCT-OE group(P<0.05),indicating that CCT-KO inhibited autophagy,while overexpression enhanced autophagy.In conclusion,the knockout of CCTα can reduce the content of PC,increase the average size of lipid droplets,promote the fusion of lipid droplets and increase the content of triglyceride,inhibition of lipid autophagy;overexpression of CCTα can increase the number of lipid droplets,increase the proportion of small lipid droplets and decrease the proportion of large lipid droplets,and increase the content of PC.The expression of lipolysis gene was higher in CCTα overexpression,and it could promote the autophagy of lipid droplets.It is suggested that CCTα plays a regulatory role in lipid droplet synthesis in mammary epithelial cells. |
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