| Sterol regulatory element binding protein-1(SREBP-1) is one of the most important nuclear transcriptional factors in mammals, it mainly participate in the regulation of endogenous cholesterol, fatty acid, phospholipid and triacylglycerol(TAG) biosynthesis metabolism. In the mammary gland of ruminants, SREBP-1 is the master regulator of milk fat synthesis, it can promote the synthesis of de novo fatty acid(C≤16) goat milk by activating the expression of key genes and enzymes of milk fat synthesis and secretion, further regulate the composition and content of beneficial fatty acids. Goat milk possess very high nutritional value and health care function for the high content of short and medium chain fatty acids(C≤16). Thus, investigating the function and regulatory effect of SREBP-1 on fatty acid metabolism related genes are of great importance for clarifying the function of SREBP-1 in milk fat metabolism and promoting the nutritional value of goat milk. However, there is rare report about SREBP-1 regulating goat mammary gland fatty acid synthesis metabolism.The present study investigate the function of SREBP-1 on fatty acid synthesis by using adenovirus-mediated overexpression and si RNA-mediated interference, and further validate its function by using a specific agonist(T0901317) of LXRα-SREBP1. Cloning and mutation analysis of goat SREBP-1c and ACSS2 promoter provide evidences for revealing their transcriptional regulation mechanism. Knockdown of two key enzymes(ACSS2 and ACLY) of de novo fatty acid synthesis result in the decrease in TAG content in goat mammary gland epithelial cells. The main results are as follows:1. Overexpression of goat mature SREBP-1(n SREBP-1)By using adenovirus-mediated overexpression technology, we constructed the mature form of SREBP-1 gene into p Adtrack-CMV vector, and then recombined it with backbone vector p Ad Easy-1, transfected it into HEK 293 for packaging and amplifying high titer adenovirus Ad-n SREBP1. After 48 h infection with Ad-n SREBP1, the m RNA and protein expression of SREBP-1 increased significantly, and the expression of fatty acid synthesis and metabolism related genes: ACSL1, FABP3, ELOVL6, SCD1, ACSS2, ACLY, ACACA, FASN, IDH1, INSIG1,NR1H3, PPARG, all upregulated significantly(P<0.05),while the chaperone protein: SCAP,decreased significantly(P<0.05). SREBP-1 overexpression resulted in a significant upregulation of TAG synthesis related genes(LPIN1 and DGAT1) and total cellular TAGcontent(P<0.01), C16:0 and C18:1 fatty acid(P<0.05) were increased by SREBP-1overexpression, while the content of C18:0 and C18:2 decreased significantly(P<0.05).However, the content of long chain polyunsaturated fatty acid(LCPUFA, C20:4 and C22:6)did not show any obvious changes(P>0.05).2. RNA interference of goat SREBP-1 geneSi RNA targeted specifically the N-terminal active fragment of SREBP-1 was transfected into GMEC for 48 h, expression of SREBF1 and SREBP-1a were decreased significantly for more than 60%(P<0.01), mature SREBP1 protein were decreased. Interference of SREBP1 gene resulted in a remarkable decrease in the expression of cellular fatty acid synthesis,transport and lipid metabolism regulator(SCD1, ELOVL6, ACLY, ACSS2, IDH1, FABP3,ACSL1, SLC27A6, INSIG1, SCAP, PPARG and CPT1A)(P<0.05), expression of PLIN3, XDH,PPARA and ACOX increased(P<0.05), ACACA, FASN, CD36, GPAM, DGAT1, PLIN2,NR1H3, ATGL and HSL did not show any obvious changes(P>0.05). Cellular TAG(P<0.05)and cholesterol(P>0.05) content were decreased by SREBP-1 knockdown.3. Effects of LXRα-SREBP1 pathway activation on fatty acid metabolismAfter 48 h treatment with different dose of T0901317(LXRα agonist), the m RNA and protein expression of SREBP-1 all increased in a dose-dependent manner, accompanying with the accumulation of mature SREBP-1 in cell nucleus. Addition of T0901317 leaded to the activation of long chain fatty acid activation, transport, desaturation, de novo fatty acid and TAG synthesis, so as to the lipid metabolism regulator: INSIG1, SCAP, PPARA and PPARG.Activation of LXRα-SREBP1 pathway resulted in the increase of cellular lipid droplet accumulation and TAG content. Content of C16:0 and C18:2(P>0.05), C18:0(P<0.05) and LCPUFA(C20:3, C20:4, C20:5, C22:4, C22:5 and C22:6, P<0.01) decreased, while monounsaturated fatty acid(MUFA, C16:1,C18:1,C20:1 and C22:1) increased significantly(P<0.05).4. Transcriptional regulation of goat SREBP-1cWe cloned and sequenced a 2 186 bp sequence of goat SREBP-1c 5’ flanking region, 2 012 bp fragment upstream of transcriptional stat site(TSS). Bioinformatics analysis revealed that there are two SRE and two LXRE elements in goat SREBP-1c promoter, NF-Y and Sp1 elements are found nearby SRE elements. Luciferase activity analysis indicated that T0901317 activate the promoter activity of SREBP-1c significantly(P<0.05), while SREBP1 interference decreased SREBP-1c promoter activity(P<0.05). Results of deleted mutation indicated that the core promoter region of SREBP-1c was located in-395 bp to +1 bp, and the deletion from-395 bp to-86 bp resulted in the disappearance of T0901317-induced activation of SREBP-1c promoter. Site-directed mutation of SRE element, not LXRE element, decreasedthe basal transcriptional activity of SREBP-1c significantly(P<0.01). Mutation of either SRE or LXRE element weaken the activation effect of T0901317 on SREBP-1c promoter, while double mutation of two LXRE elements totally abolished the activation effect of T0901317 on SREBP-1c promoter. These results indicated that goat SREBP-1c gene is transcriptionally regulated by LXRα and n SREBP1.5. Transcriptional regulation of goat ACSS2 gene by SREBP-1We cloned and sequenced a 2 359 bp fragment of goat ACSS2 gene 5’ flanking region,containing 1 976 bp upstream of transcriptional stat site(TSS). Online promoter analysis results revealed that there are binding sites for SREBP-1, LXR, NF-Y, Sp1 and STAT5 in ACSS2 gene promoter, si RNA of SREBP1 and T0901317 can significantly decreased and increased the promoter activity of ACSS2 gene(P<0.05). Results of deleted mutation indicated that the core promoter region of SREBP-1c was located in-515 bp to +1 bp, and one conserved SRE element were identified in this region. Site-directed mutation of this SRE element resulted in the decrease of ACSS2 promoter activity. Chromatin immunoprecipitation(Ch IP) results also showed that SREBP1 protein can bind with the SRE element in ACSS2 promoter, indicated the direct regulation of ACSS2 gene by SREBP1.6. Effects of ACSS2 and ACLY interference on fatty acid and TAG synthesisSi RNAs were designed to knockdown ACSS2 and ACLY gene, after 48 h transfection of these two si RNAs into GMEC, q RT-PCR results showed that the m RNA expression of both ACSS2 and ACLY were decreased by 70%, and the expression of lipid metabolism related genes: ACACA, FASN, SCD1, ELOVL6, DGAT1, DGAT2, AGPAT6, FABP3, CD36, PPARA,ACOX, CPT1 A, PLIN2 and PLIN3, were all decreased significantly(P<0.05). Cellular lipid droplet accumulation and TAG content were decreased.Our results indicated that, SREBP1 gene can extensively regulate the expression of genes related to fatty acid biosynthesis, transport, and TAG synthesis, increase the content of MUFA,and decreased the content of LCPUFA. LXRα gene can regulate goat fatty acid metabolism by directly activating SREBP-1 gene expression, and goat SREBP-1c gene expression is transcriptionally regulated by both LXRα and n SREBP1. Goat ACSS2 gene is directly regulated by SREBP-1 on transcription level, and further regulate fatty acid and TAG synthesis. |
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