NR1H3 Mediates Acetate-induced AGPAT6 Expression In Mammary Gland Of Dairy Cow

Milk fat content influences milk quality to some extent.In ruminants,acetate produced by fermentation of rumen microorganisms in the diet is a precursor substance for milk fat synthesis.The substance acetate has been shown in vitro and in vivo to modulate milk fat synthesis in the epithelial cells of the mammary gland of dairy cows.Acylglycerol-3-phosphate acyltransferase 6(AGPAT6)is a key enzyme in milk lipid synthesis in dairy cows.Previous research has demonstrated that acetate can regulate AGPAT6 expression and promote milk fat production;however,the molecular mechanism by which acetate modulates AGPAT6 expression is unclear.Nuclear receptor subfamily 1H member 3(NR1H3)is a ligand-dependent member of the nuclear hormone receptor superfamily.NR1H3 is highly expressed in tissues with high lipid metabolism.And it is involved in the regulation of cholesterol homeostasis and lipid synthesis in the body.Studies in buffalo and dairy goats have shown that NR1H3 can regulate the expression of genes related to milk lipid synthesis.However,the expression pattern of NR1H3 in the mammary glands of dairy cows and whether NR1H3 is involved in the regulation of AGPAT6 expression induced by acetate remain unclear.To clarify the expression pattern of NR1H3 in mammary tissues of dairy cows and its relationship with milk fat synthesis,this study firstly examined the NR1H3 expressions in mammary tissues of dairy cows at stages of virgin,lactation,and involution by RT-PCR and Western blot.The results showed that NR1H3 m RNA and protein expression levels were significantly higher in mammary tissue during lactation than during virgin or involution(P<0.01),suggesting that NR1H3 expression is associated with mammary lactation function.The expression of NR1H3 and the content of triglyceride in cultured bovine mammary epithelial cells were significantly increased by adding the activator T0901317.And the opposite result was obtained in cells interfered with NR1H3 by RNAi method,indicating that NR1H3 positively regulated the synthesis of milk fat in dairy mammary epithelial cells.To investigate whether acetate can induce NR1H3 expression and regulate AGPAT6 expression and milk fat synthesis through NR1H3,this study first used 12 m M sodium acetate to treat dairy mammary epithelial cells.The result of RT-PCR and Western blot showed that NR1H3 m RNA and protein expression levels were significantly increased in cells with adding sodium acetate(P<0.01),while the expression of PPARγ and AGPAT6 expression were also significantly up-regulated.The induction of AGPAT6 expression and the promotion of triglyceride synthesis by sodium acetate were found to be inhibited by knock-down of NR1H3 expression in mammary epithelial cells of dairy cows treated with sodium acetate.The inhibitory effect of NR1H3 on triglyceride synthesis was restored when NR1H3 was interfered with while overexpressing AGPAT6,indicating that NR1H3 mediates the process of acetate-induced AGPAT6 expression and milk lipid synthesis.PPARγ has been previously found to be involved in the regulation of AGPAT6 expression by acetate in our laboratory.To further clarify whether NR1H3 is involved in the regulation of Acetate-PPARγ-AGPAT6 expression,this study investigated the interaction between NR1H3 and PPARγ by immunoprecipitation assay.The results showed that NR1H3 interacted with PPARγ in the mammary epithelial cells of dairy cows.The interaction of NR1H3 with PPARγ was enhanced in the presence of 12 m M sodium acetate.The same results were observed in bimolecular fluorescence complementation experiments.Previous studies showed that PPARγ can bind to the AGPAT6 promoter region(-110bp～+1bp)to regulate the transcription of AGPAT6.In this study,the transcriptional regulation of AGPAT6 by NR1H3 was investigated by a dual luciferase reporter gene system,and the results showed that the initiation activity of the AGPAT6 promoter(-110bp～+1bp)was significantly decreased after NR1H3 gene interference(P<0.05).The addition of the NR1H3 activator T0901317 significantly increased the initiation activity of the AGPAT6(P<0.05),indicating that NR1H3 and PPARγ can act on the same region of the AGPAT6 promoter.To further investigate whether PPARγ synergistically regulates AGPAT6 transcription with NR1H3 under the action of sodium acetate,we used the PPARγ activator Rosiglitazone to treat the cells while interfering with NR1H3 expression in cells,and the results showed that Rosiglitazone addition increased the transcriptional activity of AGPAT6,but after interfering with NR1H3 Rosiglitazone transcriptional activation of AGPAT6 was inhibited.This result further clarified that under the treatment of acetate,NR1H3 interacts with PPARγ to regulate the expression of AGPAT6,which in turn affects milk lipid synthesis.