CIDEC Regulates FASN Expression In Mammary Epithelial Cells Of Dairy Cows

Milk fat is a kind of high quality natural fat,which is the main nutritional component of milk.In our country,lower milk fat percentage limits dairy industry development.Recently,the milk yield increases,however,the milk fat percentage does not change.The mil k fat percentage is 3%~4%.Generally,the milk yield restricts the increase of milk fat percentage.Then how to increase milk fat percentage become a hot research area.Studies have reported that dietary nutrients regulate milk fat synthesis by regulating gene expression.Acetate and ?-hydroxybutyrate(BHBA)are precursors of de novo FA synthesis in mammary gland.In cultured bovine mammary epithelial cells(BMECs),acetate and BHBA treatment significantly increased intracellular triacylglycerol(TAG)content.However,little is known about the molecular mechanisms of acetate and BHBA regulating milk fat synthesis.Cell death-inducing DNA fragmentation factor-?-like effector C(CIDEC)is a lipid droplet surface protein.CIDEC promotes fat storage and lipid accumulation,which in turn regulates fat metabolism and body energy balance.Previous studies have shown that CIDEC could regulate milk fat synthesis in BMECs.However,the specific regulatory pathway was not clear.Fatty acid synthetase(FASN)is one of lipogenic enzymes required for mammary gland de novo FA synthesis.In order to investigate the relationship between CIDEC and FASN expression and milk fat synthesis,purified BMECs were used.Western blot results showed that 8 mmol/L acetate and 1 mmol/L BHBA treatment increased the expression of p-4EBP1,p-P70S6 K in BMECs,but decreased the expression of p AMPK?,suggesting that milk fat synthesis precursors can induce CIDEC expression by activating m TOR signaling pathway and AMPK signaling pathway.Then we investigated the effects of CIDEC on FASN expression and milk lipid synthesis in BMESs treated with acetae and BHBA.The results showed that the overexpression of CIDEC markedly increased the m RNA and protein levels of FASN,as well as intracellular TAG content and lipid droplet number.The knockdown of CIDEC had the adverse consequences.There results revealed that CIDEC can positively regulate FASN expression,thereby inducing milk fat synthesis in BMECs.In order to investigate the regulation of CIDEC on FASN transcription,we predicted the binding sites of transcription factors in the FASN promoter and found potential C/EBP? binding sites in the promoter region of FASN.Then gene overexpression and interference were used to investigate the effects of C/EBP? on FASN expression and mik fat synthesis.The results displayed that C/EBP? overexpression in BMECs increased the m RNA and protein levels of FASN.The intracellular TAG content was also increase in C/EBP? overexpressed cells.The knockdown of C/EBP? had the opposite effects.These results further demonstrated that C/EBP? positively regulates FASN transcription,thus affects milk fat synthesis.Then we validated the binding site of C/EBP? in FASN promoter by dual-luciferase reporter assay and point mutation.The results showed that C/EBP? binding site was between-110 bp and-50 bp.Finally,to study whether CIDEC regulates FASN transcription through C/EBP?,BMECs were transfected with pc DNA3.1-CIDEC and C/EBP? si RNA.TAG assay showed that intracellular TAG content was decreased in cells transfected with pc DNA3.1-CIDEC and C/EBP? si RNA compared with cells transfected with pc DNA3.1-CIDEC.q PCR results also showed that FASN expresseion was downregulated in cells transfected with pc DNA3.1-CIDEC and C/EBP? si RNA compared with cells transfected with pc DNA3.1-CIDEC.Taken together,these results revealed that CIDEC regulates FASN transcription via C/EBP? in BMECs,thus affecting the synthesis of milk fat.The results could enrich the molecular mechanism of milk fat synthesis in mammary glands of lactating dairy cows,which provides valuable experimental and theoretical basis for improving the economic benefits of dairy cow breeding.