Effect Of T10, C12 Conjugated Linoleic Acid On The Regulation Mechanism Of SREBP1 In Bovine Mammary Epithelial Cells

The t10, c12 CLA may reduce the milk fat to 50% percentage through decreasing the SREBP1. This study was conducted to explore the regulation mechanism of t10, c12 CLA on the gene and translation expression of SREBP1 in MAC-T cells, using the mammary epithelial cell lines as model, and supply some implication to release the MFD causing by high concentrated ratio and improve the milk quality.Trial 1: Effects of t10, c12 CLA on fatty acid synthesis network.MAC-T was used as a model, extracting the third of forth generation total RNA for PCR after recovery. The gene expression was detected by 2% agarose gel electrophoresis, and lipid drops secretion was observed by oil red O staining. The MAC-T was treated by 0, 75, 150 and 300 nM t10, c12 CLA, the best inhibiting concentration of t10, c12 CLA on nSREBP1 was determined by western blot. And MAC-T was treated by the best inhibiting concentration t10, c12 CLA, mRNA and protein abundance were detected by RT-qPCR and western blot in the fatty acid synthesis network respectively. The results showed that 1) MAC-T can express 22 genes in the fatty acid synthesis and secret lipid drops; 2) 150 nM t10, c12 CLA had the inhibiting effect on nSREBP1 and reduced mRNA abundance of ACACA, FASN, SCD1 and protein abundance of SCD1. The results indicated that MAC-T was a good model for this study and t10, c12 CLA inhibits the de novo lipid synthesis in mammary epithelial tissues through reducing nSREBP1 level and down-regulating mRNA expression of ACACA, FASN, and SCD1.Trial 2: The regulation mechanism of t10, c12 CLA on the transcription and post-translational processing of nSREBP1In order to reveal the regulation mechanism of t10, c12 CLA on nSREBP1, this study detected the different expression levels of nSREBP1 when MAC-T was treated by 150 nM t10, c12 CLA. The results showed that the protein abundance of SCAP and INSIG1 reduced significantly, so t10, c12 CLA may impede pSREBP1 transportation from endoplasmic reticulum to Golgi and reduce nSREBP1 through down-regulating protein expression of SCAP and INSIG1.Trial 3: Effects of t10, c12 CLA on nSREBP1 degradation.On the basis of restricting 26 S proteasome activity, adding the 150 nM t10, c12 CLA significantly down-regulated the SREBP1 mRNA abundance, pSREBP1 and nSREBP1 protein abundance, but up-regulated SCAP mRNA abundance, which accounted for no significant difference between SCAP protein abundance and control group. The results demonstrated t10, c12 CLA down-regulated SREBP1 mRNA expression through some mechanism; even if SCAP protein had no significant variation, pSREBP1 and nSREBP1 level also reduced significantly, after the 26 S proteasome activity was inhibited.In conclusion, MFD phenomenon may be caused by t10, c12 CLA down-regulating protein expression of SCAP and INSIG1, which affected the pSREBP1 transportation from endoplasmic reticulum to Golgi, and then reducing pSREBP1 in Golgi.