The Regulatory Role Of FASN Gene In Fatty Acid Metabolism Of Goat Mammary Gland

Fatty acid synthase(FASN) is the crucial enzyme of fatty acid de novo synthesis. Despite of the important role in fatty acid synthesis, the exact mechanism of how FASN regulating milk fat formation remains unknown.In the present study, on the basis of transcriptome sequencing, digital gene expression profiles(DGE) of goat mammary gland tissues from lactation period, dry-off period and non-lactating period were sequenced, to screen the key genes in regulating milk fat metabolism during goat lactating; Analysing the correlationship among the expression of genes related to fatty acid metabolism and milk fatty acid composition,to find out the crucial genes in regulating goat milk fatty acid composition; Using orlistat, C75 and sh RNA processing goat mammary epithelial cells, to research the role of FASN in regulating fatty acid metabolism in goat mammary epithelial cells(GMECs); cloning goat MAT sequences, and constructing adenoviral overexpression vector, to verify their role in regulating the expression of genes related to fatty acid metabolism in GMECs. The main results as follows:1. The results of digital gene expression profiles(DGE) showed that FABP3, FASN and SCD et al. gained high expression level in various lactation period, their expression in lactating goat mammary gland were signifigantly higher than in dry-off. The results of crucial gene screen,performed by pearson method, showed that CPT1, ACSL1, AMPT and AKT et al. may play important role during goat lactating.2. The results of correlationship analyses between goat milk fatty acid composition and the expression level of genes related to fatty acid metabolism in mammary gland showed that close correlationship was observed between short and medium chain fatty acids(C6:0、C8:0、C10:0 and C12:0)(P<0.01). Although there was not significant correlationship with other genes related to fatty acid metabolism, the expression of FASN in mammary gland gained significant correlation with n3-UFA in goat milk.3. Role of FASN in regualting medium chain fatty acids formation. The results of olistat treatment(0, 5, 15, 25 and 35 μM) showed that there was a significant suppression of FASN activity by orlistat at concentrations higher than 15 μM(P<0.05). The effect appeared to be concentration dependent with the maximal concentration, 35 μM orlistat, having the greatest inhibition effect in GMEC. 35 μM olistat inhibited the activity of FASN by 40%, and also suppressed the expression of genes related to medium chain fatty acid formation(including HFABP, ACC and LPL). The silencing of FASN reduced the relative content of C12:0 and C10:0, and increased the content of C14:0 in GMECs.4. Role of FASN in regulating milk fat formation and secretion. Different concentrations of C75(0, 1, 5, 7, and 10 ng/μL) were used for GMEC incubation. The results of activity determination revealed that 10 ng/μL of C75 was most effective in inhibiting FASN activity. 10 ng of C75/μL was used for GMEC treatment, the results showed that C75 significantly reduced the relative content of C16:1n9 and C18:1n9 in GMECs. Both C75 treatment and FASN silencing significantly suppressed the expression of genes related to triglyceride synthesis(including GAPM、DGAT2 and AGPAT6), enhanced the expression of lipolysis(including HSL and ATGL), inhibited the expression of genes related to lipid droplets formation and secretion(including TIP47 、 BTN1A1 and XDH), and reduced cellular triglyceride content and lipid driplets accumulation.5. The mechanism underlying FASN regulating the expression of genes related to triglyceride synthesis. The silencing of FASN increased the content of malonyl-Co A significantly in GMECs. Malonyl-Co A treatment suppressed the expression of genes related to triglyceride synthesis(GPAM, AGPAT6 and DGAT2). Supplementary of malonyl-Co A enhanced the inhibition effect induced by FASN silencing on the expression of genes mentioned above. These results showed an important role of malonyl-Co A in mediating the function of FASN silencing.6. We cloned the sequences of goat MAT, and constructed the expression adenovirus. Overexpression of bovine MAT significantly increased the expression of PPARγ and LPL, both of which were not affected by overexpression of goat MAT and TE. Overexpression of goat MAT increased the expression of SCD1 and suppressed that of TIP47 significantly. Both MATs from goat and bovine suppressed the expression of THRSP and XDH significantly, with a slighter reduction by goat TE adenovirus treatment.In conclusion, FASN plays an important role in regulating fatty acid metabolism in goat mammary gland. Silencing of FASN can suppress the accumulation of GMECs lipid by suppressing the synthesis of triglyceride and stimulating the hydrolysis of triglyceride. FASN can regulate the expression of genes related to triglyceride synthesis via the control of malonyl-Co A concentration.