Function Of SREBP1 In The Milk Fat Synthesis Of Dairy Cow Mammary Gland Epithelial Cells

Sterol regulatory element-binding proteins (SREBPs) belong to a family of nuclear transcription factors. SREBPs play an important role in regulating lipid homeostasis and the transcription of lipogenic genes that can control the expression of lipogenic and cholesterogenic genes. The question of which is the most important positive regulator in milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) between SREBPs or other nuclear transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ), remains a controversial one. Recent studies have found that mTORC1 (the mammalian target of rapamycin C1) regulates SREBP1 to promote fat synthesis. Thus far, however, the effect of SREBP1 on mTOR(the mammalian target of rapamycin)pathways in the regulation of milk fat synthesis remains poorly understood. This study aimed to identify the function of SREBP1 in milk fat synthesis and to characterize the effect of SREBP1 on the expression of mTOR in DCMECs.By studying the influence of SREBP1 overexpression and gene silencing on milk fat synthesis identified the function of SREBP1 in milk fat synthesis. By adding stearic acid and serum, we detected the expression of SREBP1 and the effect on the upregulation of milk fat synthesis and determined whether stearic acid and serum regalute the milk fat sysnthesis through SREBP1 further illustrating the effect of SREBP1 on milk fat synthesis. In this study, the mRNA levels of ACC, FAS, SCD, m-TOR, FABP3 and PPARy were detected by real-time quantitative PCR; the protein levels of SREBP1,p-SREBP1,m-TOR and p-mTOR were detected by western blotting, the expression and localization of SREBP1 and p-mTOR were detected by immunostaining; the lipid droplet contents were detected by lipid droplet staining; the triglyceride contents were detected by detection kits of triglyceride.The results showed that (1) SREBP1 over-expression resulted in an significantly increase in the mRNA levels of ACC, FAS, SCD, m-TOR,and FABP3 (p<0.01), PPARγ mRNA had no significant change (p>0.05); the protein levels of SREBP1 and p-SREBP1 were significantly increased (p<0.01), whereas the expression of m-TOR and p-mTOR had a slight inccrease (p<0.05); the expression of SREBP1 were significantly increase (p<0.01) by immunofluorescence and p-mTOR had a slight increase (p<0.05); the contents of lipid drops and triglyceride were significantly increased (p<0.01). (2) While SREBP1 gene silencing, the mRNA levels of ACC, FAS, SCD and FABP3 were significantly decreased (p<0.01), m-TOR and PPARγ mRNA had no significant change (p>0.05); the protein levels of SREBP1, p-SREBP1 and m-TOR were significantly decreased (p<0.01), whereas the expression of p-mTOR had a slight decrease (p<0.05); the expression of SREBP1 and p-mTOR were significantly decrease (p<0.01) by immunofluorescence; the contents of lipid drops and triglyceride were significantly dencreased O<0.01). (3) By the addition of stearic acid and serum, ACC, FAS, SCD, PPARy, m-TOR and FABP3 mRNA levels were significantly increased (p<0.01); the protein levels of SREBP1 and p-SREBP1 were increased in D+S+B,D+B and D+S groups, significantly increased in D+S+B (p<0.01); by immunofluorescencethe, expression of SREBP1 and p-mTOR were significantly increased in treatment groups (p<0.01); the contents of lipid drops and triglyceride were significantly increased (p<0.01).The results of this study showed that SREBP1 was a key regulator of milk fat synthesis and could be positively regulated milk fat synthesis. Stearic acid and serum as nutrients could promote SREBP1 gene expression and enhanced the ability of dairy cow mammary epithelial cells milk fat synthesis. This study also provided the basis for the establish of lactation model of dairy cow mammary epithelial cells.