Effect Of PLIN3 On Milk Fat Synthesis In Mammary Epithelial Cells Of Dairy Cows

The main component of milk fat was Triglyceride(TAG),which was the nutrient closest to breast milk and had the advantages of easy gastrointestinal absorption and high utilization rate.Milk fat synthesis is regulated by multiple gene expressions in lactating dairy cows.The PLIN gene family is a class of highly conserved proteins that related to lipid metabolism,which participate in the regulation of lipid metabolism through specific binding to lipid droplets.In order to learn the relationship between PLIN family and milk production,we first detected the expression pattern of mammary tissue of five PLIN family members in lactation and dry milk period by q PCR method,and the results showed that the expression of PLIN3 m RNA in mammary tissue of lactation was significantly higher than that in dry milk period.PLIN2 m RNA was expressed in milk tissues of dairy cows,but the m RNA levels of PLIN1,PLIN4 and PLIN5 m RNA were not detected in milk tissues of dairy cows during lactation compared with dry milk,suggesting that the expression of PLIN3 in various PLIN family members may be related to milk lactation.Western blot was used to detect the expression of PLIN3 protein in mammary tissues of dairy cows at different physiological stages.The results also showed that the expression of PLIN3 protein in mammary tissues of lactating cows was significantly higher than that in dry milk stage.Immunofluorescence method was used to detect the expression site of PLIN3 in mammary gland tissues of dairy cows.The results showed that there were abundant milk fat droplets in mammary epithelial cells during lactation,and PLIN3 was mainly detected in acinar epithelial cells enriched with milk fat droplets,suggesting that PLIN3 may be involved in the regulation of milk fat synthesis of lactating dairy cows.To further verify the relationship between PLIN3 expression and milk fat synthesis in dairy cows,we used gene overexpression and gene silencing methods to change the expression level of PLIN3 in mammary epithelial cells of dairy cows.TAG detection results showed that PLIN3 overexpression significantly increased the TAG content and lipid drop number in mammary epithelial cells of dairy cows.After PLIN3 silencing,the intracellular TAG content decreased.These results indicate that PLIN3 can positively regulate milk fat synthesis.Oleic acid is the main octadecarbonate unsaturated fatty acid in milk.Studies have shown that dietary supplementation with oleic acid can change fatty acid composition in milk fat and improve milk fat yield of dairy cows.To investigate the effects of oleic acid on milk fat synthesis in mammary epithelial cells of dairy cows and whether PLIN3 mediates this process,we first stimulated mammary epithelial cells with 100 μm/L oleic acid in fat-free and serumfree medium.The results showed that oleic acid supplementation significantly increased the TAG level in mammary epithelial cells of dairy cows.RT-PCR results showed that oleic acid could induce up-regulation of the m RNA expression of PLIN2 and PLIN3,but the m RNA expression of PLIN1,PLIN4 and PLIN5 was not detected.Western blot results also showed that oleic acid treatment could increase PLIN3 protein expression in bovine mammary epithelial cells.In addition to oleic acid,PLIN3 gene was silenced and oleic acid induced milk fat synthesis was inhibited.The lipid droplets in bovine mammary epithelial cells were labeled by Bodipy,and the localization of PLIN3 and intracellular lipid droplets was observed by laser confocal microscope at 0.5h,1h,2h,4h,6h and 24 h after oleic acid treatment.The results showed that lipid droplets began to form after oleic acid addition,and the number and volume of lipid droplets increased with the increase of treatment time.PLIN3 was always localized on the surface of small lipid droplets,and no expression of PLIN3 was observed on the surface of large lipid droplets.These results suggest that PLIN3 is involved in the initial formation of lipid droplets in mammary epithelial cells of dairy cows.The mTOR signaling pathway can sense the stimulation of nutrient signals in cells.Studies have shown that short-chain fatty acids can activate the mTOR signaling pathway to regulate milk fat synthesis.In order to further study whether mTOR signaling pathways can be oleic acid activation,mediated oleic acid of PLIN3 expression and regulation of milk fat synthesis,we in the cow mammary gland epithelial cells induced by oleic acid medium,add the mTOR inhibitors rapamycin,results showed that rapamycin suppresses the activation of mTOR and the signaling pathways downstream of molecular and PLIN3 expression,Oleic acid induced TAG synthesis was also inhibited.However,milk fat synthesis could be restored by overexpression of PLIN3 in bovine mammary epithelial cells under rapamycin inhibition.These results suggest that oleic acid can regulate PLIN3 protein expression by activating the mTOR signaling pathway,and thus regulate milk fat synthesis.In conclusion,the expression of PLIN3 is related to the lactation process of dairy cows.PLIN3 can promote the synthesis of milk fat in dairy cow mammary epithelial cells.In dairy cow mammary epithelial cells,oleic acid regulates the expression of PLIN3 through the mTOR signaling pathway,and then regulates the synthesis of milk fat.This study clarified the role of PLIN3 in milk fat synthesis of lactating dairy cows,which can provide a theoretical basis for improving the regulatory network of milk fat synthesis genes in dairy cows.