Effects of FADS2 Interference on Fatty Acid Composition of Mammary Epithelial Cells in Dairy Goats (Capra hircus)
WU Jiao1, HE Qiu-Ya1, LI Zhuang1, LI Cong1, WANG Hui2, SHI Huai-Ping1, LUO Jun1, *
1 Shaanxi Provincial Key Laboratory of Agricultural Molecular Biology / College of Animal Science and Technology, Northwest A & F University, Yangling 712100, China;
2 Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization / Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu 610041, China
Abstract：Fatty acid desaturation 2 (FADS2), as a rate-limiting enzyme in the synthesis of polyunsaturated fatty acids (PUFAs）, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), can dehydrogenate its substrate to form a double bond at the 6~7 position. In this study, FADS2 gene (GenBank No. MK292654), with 1 335 bp length of the CDS and encoding 444 amino acids, was cloned and had high homology with Ovis aries (XM_012146317.2) and Bos taurus (NM_001083444.1). Using siRNA technique in goat mammary epithelial cells (GMECs), the mRNA and protein levels of FADS2 decreased 60%~70% (P<0.01) and 15%~18% (P<0.05), respectively. Interfering FADS2 expression significantly up-regulated the expression of genes related to the synthesis of PUFAs, such as elongase of very long chain fatty acids 2 gene (ELOVL2) (P<0.01), ELOVL6 (P<0.01), fatty acid desaturation 1 gene (FADS1) (P<0.05); It also promoted the expression of sterol regulatory element-binding transcription protein gene (SREBP1a) (P<0.01), fatty acid synthase gene (FASN) (P<0.05), acetyl-CoA carboxylase gene (ACACA) (P<0.05), and stearoyl-CoA desaturase1 gene (SCD1) expression (P<0.05), respectively. In addition, interfering FADS2 expression could significantly decreased the proportion of AA and DHA (P<0.05) and restrained PUFAs synthesis (P<0.05). Furthermore, interfering FADS2 gene expression could significantly inhibit diacylglycerol acyltransferase1 (DGAT1) (P<0.01) and DGAT2 (P<0.05) gene expression to decrease triacylglyceride (TAG) content in GMECs (P<0.05). In conclusion, FADS2 gene plays an important role in regulating PUFAs synthesis and triglyceride metabolism in dairy goats, which provides experimental basis for the study of PUFAs metabolism in goat milk.
邬娇, 赫秋亚, 李壮, 李聪, 王会, 史怀平, 罗军. 干扰FADS2基因对奶山羊乳腺上皮细胞脂肪酸组成的影响[J]. 农业生物技术学报, 2019, 27(11): 1973-1984.
WU Jiao, HE Qiu-Ya, LI Zhuang, LI Cong, WANG Hui, SHI Huai-Ping, LUO Jun. Effects of FADS2 Interference on Fatty Acid Composition of Mammary Epithelial Cells in Dairy Goats (Capra hircus). 农业生物技术学报, 2019, 27(11): 1973-1984.
[1] 曹斌云, 罗军, 姚军虎, 等. 2007. 山羊奶的营养价值与特点[J]. 畜牧兽医杂志, 26(1): 49-50.
(Cao B Y, luo J, Yao J H, et al.2007. Nutritional value and characteristics of goat milk[J]. Journal of Animal Science and Veterinary Medicine, 26(1): 49-50.)
[2] 刘莉, 李明春, 胡国武, 等. 2001. 深黄被孢霉M_(6-22) Δ~6-脂肪酸脱氢酶基因在酿酒酵母中的表达[J]. 微生物学报,41(4): 397-401.
(Liu L, Li M C, Hu G W, et al.2001. Identification of mortieralla isabellna M_(6-22)Δ~6-fatty acid desaturaseby heterologous expression in saccharomyces cerevisiae[J]. Acta Microbiologica Sinica, 41(4): 397-401.)
[3] 王苗, 罗军, 许会芬, 等. 2016. 山羊INSIG1基因超表达对乳腺上皮细胞中脂质合成的影响[J]. 畜牧兽医学报, 47(9): 1806-1816.
(Wang M, Luo J, Xu H F, et al.2016. Effect of INSIG1 overexpression on the lipid synthesis in goat mammary gland epithelial cells[J]. Journal of Animal Science and Veterinary Medicine, 47(9): 1806-1816.)
[4] 许会芬, 2016. SREBP-1基因对山羊乳腺上皮细胞脂肪酸代谢的调控作用研究[D]. 博士学位论文, 西北农林科技大学, 导师: 罗军, pp.19-20.
(Xu H F.2016. The Regulation function Of SREBP-1 gene on fatty acid metabolism in goat mammary gland epithelial cells[D]. Thesis for Ph.D., Northwest A & F University, Supervisor: Luo J, pp .19-20.)
[5] 朱江江, 2011. 奶山羊脂肪酸合酶基因乙酰/丙二酸单酰转移酶区域的过表达研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 罗军. pp. 33-34.
(Zhu J J, 2011. Overexpression of MAT domain of fatty acid synthesis (FASN) gene in dairy goat[D]. Thesis for M.S., Northwest A & F University, Supervisor: Luo J, pp. 33-34.)
[6] Aki T, Shimada Y, Inagaki K, et al.1999. Molecular cloning and functional characterization of rat delta-6 fatty acid desaturase[J]. Biochemical & Biophysical Research Communications, 255(3): 575-579.)
[7] Alex S, Lange K, Amolo T, et al.2013. Short-chain fatty acids stimulate angiopoietin-like 4 synthesis in human colon adenocarcinoma cells by activating peroxisome proliferator-activated receptor γ[J]. Molecular & Cellular Biology, 33: 1303-1316.
[8] Ali M, Heyob K, Rogers L K.2016. DHA-mediated regulation of lung cancer cell migration is not directly associated with gelsolin or vimentin expression[J]. Life Sciences, 155: 1-9.
[9] Castro L F, Tocher D R, Monroig O.2016. Long-chain polyunsaturated fatty acid biosynthesis in chordates: Insights into the evolution of Fads and Elovl gene repertoire[J]. Progress in Lipid Research, 62: 25-40.
[10] Cho H P, Nakamura M T, Clarke S D.1999. Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase[J]. Journal of Biological Chemistry, 274(1): 471-477.
[11] Cunnane S C.2003. Problems with essential fatty acids: Time for a new paradigm?[J]. Progress in Lipid Research, 42(6): 544.
[12] Das U N.2010. Essential fatty acids: Biochemistry, physiology and pathology[J]. Biotechnology Journal, 1(4): 420-439.
[13] de Toro-Martín J, Guénard F, Rudkowska I, et al.2018. A common variant in ARHGEF10 alters delta-6 desaturase activity and influence susceptibility to hypertriglyceridemia[J]. Journal of Clinical Lipidology, 12(2): 311-320.
[14] Deng X, Dong Q, Bridges D, et al.2015. Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase[J]. BBA-Molecular and Cell Biology of Lipids, 1851: 1521-1529.
[15] Georgiadi A, Kersten S.2012. Mechanisms of gene regulation by fatty acids[J]. Advances in Nutrition, 3(2): 127-134.
[16] Graziela R R, Maria M B, Tharcisio C T, et al.2015. Docosahexaenoic acid modulates a HER2-associated lipogenic phenotype, induces apoptosis, and increases trastuzumab action in HER2-overexpressing breast carcinoma cells[J]. Biomed Research International, 2015: 1-13.
[17] Guillou H, Zadravec D, Martin P G, et al.2010. The key roles of elongases and desaturases in mammalian fatty acid metabolism: Insights from transgenic mice[J]. Progress in Lipid Research, 49(2): 186-199.
[18] Hammond S M, Boettcher S, Caudy A A, et al .2001. Argonaute2, a link between genetic and biochemical analyses of RNAi[J]. Science, 293: 1146-1150.
[19] Hayashi Y, Shimamura A, Ishikawa T, et al.2018. FADS2 inhibition in essential fatty acid deficiency induces hepatic lipid accumulation via impairment of very low-density lipoprotein (VLDL) secretion[J]. Biochemical & Biophysical Research Communications, 496(2): 549-555.
[20] Heijden R, Sheedfar F, Morrison M C, et al.2015. High-fat diet induced obesity primes inflammation in adipose tissue prior to liver in C57BL/6j mice[J]. Aging, 7(4): 256-267.
[21] Hervé G, Sabine D, Vincent R, et al.2004. Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat delta6-desaturase activity[J]. Journal of Lipid Research, 45: 32-40.
[22] Hwang J K, Yu H N, Noh E M, et al.2017. DHA blocks TPA-induced cell invasion by inhibiting MMP-9 expression via suppression of the PPAR-γ/NF-κB pathway in MCF-7 cells[J]. Oncology Letters, 13: 243-249.
[23] Hyungjae L, Woo J P.2014. Unsaturated fatty acids, desaturases, and human health[J]. Journal of Medicinal Food, 17(2): 189-197.
[24] Ikuyo I, Nozomu K, Yuka A, et al.2014. Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency[J]. Biochim Biophys Acta, 1841: 204-213.
[25] Jin A, Shi X C, Liu Y, Sun J, et al.2018. Docosahexaenoic acid induces PPARγ-dependent preadipocytes apoptosis in grass carp Ctenopharyngodon idella[J]. General & Comparative Endocrinology, 266: 211-219.
[26] Julia A, Ramón R, Videla L A, et al.2004. Increase in long-chain polyunsaturated fatty acid n - 6/n - 3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease[J]. Clinical Science, 106(6): 635.
[27] Kazutoshi Y, Eishiro M, Hajime S, et al.2015. Characteristics of hepatic fatty acid compositions in patients with nonalcoholic steatohepatitis[J]. Liver International, 35(2): 582-590.
[28] Manuel R R, Stroud C K, Haschek W M, et al.2010. Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice[J]. The Journal of Lipid Research, 51(2): 360-367.
[29] Markiewicz-Kęszycka M, Czyżak-Runowska G, Lipińska P, et al.2013. Fatty acid profile of milk-a review[J]. Bulletin-Veterinary Institute in Pulawy, 57(2): 135-139.
[30] Marsman H A, Graaf W D, Heger M, et al.2013. Hepatic regeneration and functional recovery following partial liver resection in an experimental model of hepatic steatosis treated with omega-3 fatty acids[J]. British Journal of Surgery, 100(5): 674-683.
[31] Meegalla R L, Billheimer J T, Cheng D.2002. Concerted elevation of acyl-coenzyme A: Diacylglycerol acyltransferase (DGAT) activity through independent stimulation of mRNA expression of DGAT1 and DGAT2 by carbohydrate and insulin[J]. Biochemical and Biophysical Research Communications, 298(3): 317-323.
[32] Alferez M J M, Barrionuevo M, Aliaga I L, et al.2001. Digestive utilization of goat and cow milk fat in malabsorption syndrome[J]. Journal of Dairy Research, 68(03): 451-461.
[33] Poulsen L L, Siersbæk M, Mandrup S.2012. PPARs: Fatty acid sensors controlling metabolism[J]. Seminars in Cell & Developmental Biology, 23(6): 631-639.
[34] Qi B, Fraser T, Mugford S, Dobson G, et al.2004. Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants[J]. Nature Biotechnology, 22(6): 739-745.
[35] Reddy A S, Nuccio M L, Gross L M, et al.1993. Isolation of a delta 6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp. strain PCC 7120[J]. Plant Molecular Biology, 22(2): 293-300.
[36] Stoffel W, Hammels I, Jenke B, et al.2014. Obesity resistance and deregulation of lipogenesis in Δ6-fatty acid desaturase (FADS2) deficiency[J]. Embo Reports, 15(1): 110-120.
[37] Stoffel W, Holz B, Jenke B, et al.2008. Δ6-desaturase (FADS2) deficiency unveils the role of ω3- and ω6-polyunsaturated fatty acids[J]. The EMBO Journal, 27(17):2281-2292.
[38] Stroud C K, Nara T Y, Manuel R R, et al.2009. Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration[J]. The Journal of Lipid Research, 50(9): 1870.
[39] Tsai C H, Shen Y C, Chen H W, et al.2017. Docosahexaenoic acid increases the expression of oxidative stress-induced growth inhibitor 1 through the PI3K/Akt/Nrf2 signaling pathway in breast cancer cells[J]. Food & Chemical Toxicology, 108: 276-288.
[40] Tuschl T.2001. RNA interference and small interfering RNAs[J]. Chembiochem, 2(4): 239-245.
[41] Vaittinen M, Walle P, Kuosmanen E, et al.2015. FADS2 genotype regulates delta-6 desaturase activity and inflammation in human adipose tissue[J]. Journal of Lipid Research, 57(1): 56-65.
[42] Walle P, Takkunen M, Mã Nnistã V,et al.2016. Fatty acid metabolism is altered in non-alcoholic steatohepatitis independent of obesity[J]. Metabolism-clinical & Experimental, 65(5): 655-666.
[43] Wallis J G, Watts J L, Browse J.2002. Polyunsaturated fatty acid synthesis: What will they think of next?[J]. Trends in Biochemical Sciences, 27(9): 467-473.
[44] Xu H F, Luo J, Zhao W S, et al.2016. Overexpression of SREBP1 (sterol regulatory element binding protein 1) promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells[J]. Journal of Dairy Science, 99(1): 783-795.
