蛋鸡脂肪代谢相关基因ACACA和FASN表达及调控特性
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摘要
哺乳动物的研究表明,乙酰辅酶A羧化酶A(acetyl-CoA carboxylases alpha, ACACA)与脂肪酸合成酶(fatty acid synthase, FASN)是脂肪酸从头合成的关键限速酶,对脂肪酸的生物合成有着重要的作用,其表达受到多种激素的调控。本研究旨在探讨蛋鸡脂肪代谢相关基因ACACA与FASN基因的表达规律以及雌激素、油脂摄入对其表达量的影响。利用qRT-PCR分析ACACA与FASN基因在蛋鸡不同组织以及不同发育时期肝脏中的表达规律,分别从个体和细胞水平研究不同雌激素和大豆油水平对蛋鸡ACACA与FASN基因表达量的影响。结果表明,ACACA与FASN基因在心、肝、脾、肺、肾、胸肌、十二指肠、腹脂、胰腺、卵巢中均有表达,且在肝脏组织中表达量最高;ACACA与FASN基因在产蛋高峰期鸡肝脏中的表达量极显著高于产蛋前期(P≤0.01);0.5、1.0、2.0 mg/kg的17β-雌二醇处理鸡肝脏组织和25,50,100 nmol/L的17β-雌二醇处理蛋鸡胚肝脏原代细胞后,ACACA与FASN基因的表达量呈现升高趋势,且差异具有显著性(P≤0.05)或极显著性(P≤0.01);饲喂8%大豆油饲粮组蛋鸡肝脏ACACA与FASN基因的表达水平分别显著(P≤0.05)与极显著(P≤0.01)高于对照组,4%大豆油饲粮组与对照组间无明显差异(P>0.05)。综上所述,鸡ACACA与FASN基因参与脂质代谢过程,其表达受雌激素调控,且饲粮中添加8%水平的大豆油可显著提高鸡ACACA与FASN基因的表达量。本研究为进一步解析ACACA与FASN基因对鸡脂质代谢的调控机制提供了科学依据。
Acetyl-CoA carboxylases alpha(ACACA) and fatty acid synthase(FASN), as crucial rate-limiting enzymes, exert significant roles in the biosynthesis of fatty acid and were regulated by multiple hormones in mammals. However, it is still unclear about the expression and regulation characteristics about ACACA and FASN genes in hen(Gallus gallus). The experiment was designed to explore the expression and regulation characteristics of ACACA and FASN genes related to lipid metabolism in hen. A total of 10 tissues includingheart, liver, spleen, lung, kidney, pectorals, duodenum, abdominal fat, pancreas and ovary were obtained from20-and 30-week-old Lushi green eggshell hens. In addition, more liver tissues were obtained from hens at different developmental stages including 5-, 15-, 20-, 30-and 35-week-old Lushi green eggshell hens. These tissues were used to detect the temporal and spatial expression pattern of ACACA and FASN genes via qRTPCR. The hens and chicken embryonic primary hepatocytes(CEPHs) were treated with different concentrations of estrogen, and the regulatory mechanism of ACACA and FASN genes mediated by estrogen in vivo and in vitro were analyzed using qRT-PCR. The hens were fed with the basal diet supplemented with 4%or 8% soybean for 30 days, respectively, to study the effect of different doses of fat feed on ACACA and FASN genes expression. The results demonstrated that ACACA and FASN genes were expressed in all detected tissues including heart, liver, spleen, lung, kidney, pectorals, duodenum, abdominal fat, pancreas and ovary and shared a highest expression in livers. In addition, the expression levels of ACACA and FASN genes were highly significantly higher in the liver of peak-laying hens than that in the liver of pre-laying hens(P≤0.01).Moreover, compared to the control group, ACACA and FASN genes showed significantly or extremely significantly higher expression abundance in livers of hens treated with the 0.5, 1.0, 2.0 mg/kg dose of 17β-estradiol dissolved in olive oil or primary hepatocytes treated with either 25, 50 or 100 nmol/L dose of 17β-estradiol dissolved in ethyl alcohol(P≤0.05 or P≤0.01). Compared to the control group, ACACA and FASN genes were significantly(P≤0.05) and extremely significantly high(P≤0.01) in 8% soybean feed group compared to the control group, but no difference in 4% soybean feed group. Taken together, ACACA and FASN genes were likely to participate in the lipid metabolism in laying hen and their transcriptional expression were regulated via estrogen, and 8% soybean supplement in the basal diet contributes to an significant increase in their expression levels. This study paved the way for the further understanding the regulatory mechanism of ACACA and FASN genes on laying hen lipid metabolism.
Acetyl-CoA carboxylases alpha(ACACA) and fatty acid synthase(FASN), as crucial rate-limiting enzymes, exert significant roles in the biosynthesis of fatty acid and were regulated by multiple hormones in mammals. However, it is still unclear about the expression and regulation characteristics about ACACA and FASN genes in hen(Gallus gallus). The experiment was designed to explore the expression and regulation characteristics of ACACA and FASN genes related to lipid metabolism in hen. A total of 10 tissues includingheart, liver, spleen, lung, kidney, pectorals, duodenum, abdominal fat, pancreas and ovary were obtained from20-and 30-week-old Lushi green eggshell hens. In addition, more liver tissues were obtained from hens at different developmental stages including 5-, 15-, 20-, 30-and 35-week-old Lushi green eggshell hens. These tissues were used to detect the temporal and spatial expression pattern of ACACA and FASN genes via qRTPCR. The hens and chicken embryonic primary hepatocytes(CEPHs) were treated with different concentrations of estrogen, and the regulatory mechanism of ACACA and FASN genes mediated by estrogen in vivo and in vitro were analyzed using qRT-PCR. The hens were fed with the basal diet supplemented with 4%or 8% soybean for 30 days, respectively, to study the effect of different doses of fat feed on ACACA and FASN genes expression. The results demonstrated that ACACA and FASN genes were expressed in all detected tissues including heart, liver, spleen, lung, kidney, pectorals, duodenum, abdominal fat, pancreas and ovary and shared a highest expression in livers. In addition, the expression levels of ACACA and FASN genes were highly significantly higher in the liver of peak-laying hens than that in the liver of pre-laying hens(P≤0.01).Moreover, compared to the control group, ACACA and FASN genes showed significantly or extremely significantly higher expression abundance in livers of hens treated with the 0.5, 1.0, 2.0 mg/kg dose of 17β-estradiol dissolved in olive oil or primary hepatocytes treated with either 25, 50 or 100 nmol/L dose of 17β-estradiol dissolved in ethyl alcohol(P≤0.05 or P≤0.01). Compared to the control group, ACACA and FASN genes were significantly(P≤0.05) and extremely significantly high(P≤0.01) in 8% soybean feed group compared to the control group, but no difference in 4% soybean feed group. Taken together, ACACA and FASN genes were likely to participate in the lipid metabolism in laying hen and their transcriptional expression were regulated via estrogen, and 8% soybean supplement in the basal diet contributes to an significant increase in their expression levels. This study paved the way for the further understanding the regulatory mechanism of ACACA and FASN genes on laying hen lipid metabolism.
引文
李太山.2016.三个地方鸡种部分组织脂肪代谢差异的遗传因素研究[D].硕士学位论文,贵州大学,导师:陈彬pp.35-61.(Li T S.2016.Genetic factors study of three local chicken part of the fat organization of metabolic differences[D].Thesis for M.S.,Guizhou University,Supervisor:Chen B,pp.35-61.)
刘安芳,朱庆,阳泽平.2006.饲料中添加油脂对肉鸭生产性能的影响[J].畜禽业,(10):18-19.(Liu A F,Zhu QYang Z P.2006.Effects of soybean oil on performance of meat ducks[J].Livestock and Poultry Industry,(10)18-19.)
卢建,王克华,曲亮,等.2013.不同水平大豆油对苏禽青壳蛋鸡产蛋性能、蛋品质和血清脂质指标的影响[C]//,中国畜牧兽医学会家禽学分会(编著),中国畜牧兽医学会家禽学分会第九次代表会议暨第十六次全国家禽学术讨论会论文集.中国家禽,扬州市,pp.292-295.(Lu J,Wang K H,Qu L,et al.2013.Effects of soybean oil on egg performance,egg quality,and serum lipids indexes of laying Hens[C]//,Poultry Science Chapter of Chinese Association of Animal Science and Veterinary Medicine(eds.),Proceedings of 9th representative conference of Poultry Chapter of Chinese Association of Animal Science and Veterinary Medicine and 16th National Poultry Symposium.China Poultry,Yangzhou City,China,pp.292-295.)
欧阳建华,谢亮,刘杰,等.2007.鸡FASN基因2个位点的多样性及其与体重、脂肪沉积性状的相关性[J].畜牧兽医学报,38(1):25-30.(OuYang J H,Xie L,Liu J,et al.2007.Genetic diversity of two sites of FASN gene and its association with growth and fat deposition in chickens[J].Acta Veterinaria et Zootechnica Sinica,38(1):25-30.)
彭音.2009.ChREBP和ACC1在肝细胞糖/脂代谢调控中的作用[D].博士学位论文,华中农业大学,导师:杨在清.pp.7-12.(Peng Y.2009.The role of ChREBP and ACC1 gene in Glyco-and lipometabolism and hepatocyte[D].Thesis for Ph.D.,Huazhong Agricultural University,Supervisor:Yang Z Q,pp.7-12.)
史铭欣,史洪岩,李辉,等.2013.高、低脂系肉鸡肝脏脂肪合成代谢相关基因的表达差异分析[J].农业生物技术学报,21(3):306-312.(Shi M X,Shi H Y,Li H,et al.2013.Expression analysis of lipid synthesis-related genes in the livers of fat and lean broilers[J].Journal of Agricultural Biotechnology,21(3):306-312.)
田方圆,王台安,李艳敏,等.2017.鸡MOGAT和DGAT基因的克隆及表达特性研究[J].畜牧兽医学报,48(2):214-224.(Tian F Y,Wang T A,Li Y M,et al.2017.Cloning and expression characteristics of MOGAT and DGATgenes in chicken(Gallus gallus)[J].Acta Veterinaria et Zootechnica Sinica,48(2):214-224.)
徐乃一,郑航,田卫华,等.2018.鸡SCD基因表达调控特性分析[J].农业生物技术学报,26(1):123-131.(Xu N Y,Zheng H,Tian W H,et al.2013.Characteristics of expression and regulation of SCD gene in chicken(Gallus gallus))[J].Journal of Agricultural Biotechnology,26(1):123-131.)
周淑世.2009.不同脂肪源对朗德鹅产肝性能及FAS mRNA表达的影响[D].硕士学位论文,吉林大学,导师:牛淑玲.pp.23-38.(Zhou S S.2009.Effect of different fatty sources on fatty liver performance and gene expression of FAS mRNA in Landes goose[D].Thesis for M.S.,Jilin University,Supervisor:Niu A L,pp.23-38.)
Baik M,Etchebarne B E,Bong J,et al.2009.Gene expression profiling of liver and mammary tissues of lactating dairy cows[J].Asian Australasian Journal of Animal Sciences,22(6):871-884.
Chow J D Y,Lawrence R T,Healy M E,et al.2014.Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylation[J]Molecular Metabolism,3(4):419-431.
Dorn C,Riener M O,Kirovski G,et al.2010.Expression of fatty acid synthase in nonalcoholic fatty liver disease[J]International Journal of Clinical&Experimental Pathology,3(5):505-514.
Driscoll M D,Sathya G,Muyan M,et al.1998.Sequence requirements for estrogen receptor binding to estrogen response elements[J].Journal of Biological Chemistry273(45):29321-29330.
Fischer P W F,Marks G S.1976.Chick embryo liver cells maintained in serum-free waymouth MD 705/1 medium[J].Tissue Culture Association,2(4):449-452.
Han L Q,Gao T Y,Yang G Y,et al.2018.Overexpression of SREBF chaperone(SCAP)enhances nuclear SREBP1translocation to upregulate fatty acid synthase(FASN)gene expression in bovine mammary epithelial cells[J]Journal of Dairy Science,101(7):6523-6531.
Hansmannel F,Mordier S,Iynedjian P.2006.Insulin induction of glucokinase and fatty acid synthase in hepatocytes:Analysis of the roles of sterol-regulatory-elementbinding protein-1c and liver X receptor[J].Biochemica Journal,399(2):275-283.
Hillgartner F B,Charron T,Chesnut K A.1997.Triiodothyronine stimulates and glucagon inhibits transcription of the acetyl-CoA carboxylase gene in chick embryo hepatocytes:Glucose and insulin amplify the effect of triiodothyronine[J].Archives of Biochemistry&Biophysics337(2):159-168.
Kim Y J,Yang J L,Kim K Y H.2003.Regulation of acetylCoA carboxylase gene expression by hormones and nutrients[J].Preventive Nutrition&Food Science,8(1)61-65.
Knobloch M,Braun S M,Zurkirchen L,et al.2013.Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis[J].Nature,493(7431):226-230.
Kumar V,Chambon P.1988.The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer[J].Cell,55(1):145-156.
Li H,Wang T,Xu C,et al.2015.Transcriptome profile of liver at different physiological stages reveals potential mode for lipid metabolism in laying hens[J].BMC Genomics,16(1):763-775.
Li J,Leghari I H,He B,et al.2014.Estrogen stimulates expression of chicken hepatic vitellogeninⅡand very low-density apolipoproteinⅡthrough ER-α[J].Theriogenology,82(3):517-524.
Mildner A M,Clarke S D.1991.Porcine fatty acid synthase:Cloning of a complementary DNA,tissue distribution of its mRNA and suppression of expression by somatotropin and dietary protein[J].Journal of Nutrition,121(6):900-907.
Palmisano B T,Zhu L,Stafford J M.2017.Role of estrogens in the regulation of liver lipid metabolism[J].Advances in Experimental Medicine&Biology,1043:227-256.
Peckett A J,Wright D C,Riddell M C.2011.The effects of glucocorticoids on adipose tissue lipid metabolism[J].Metabolism-clinical&Experimental,60(11):1500-1510.
Pellegrini M,Pallottini V,Marin R,et al.2014.Role of the sex hormone estrogen in the prevention of lipid disorder[J].Current Medicinal Chemistry,21(24):2734-2742.
Phil G W D.1996.Nutritional and hormonal regulation of fatty acid synthase[J].Nutrition Reviews,54(4):122-123.
Saha A K,Ruderman N B.2003.Malonyl-CoA and AMP-activated protein kinase:An expanding partnership[J].Molecular&Cellular Biochemistry,253(2):65-70.
Sha R,Gui L,Khan R,et al.2017.Association between FASNgene polymorphisms ultrasound carcass traits and intramuscular fat in Qinchuan cattle[J].Gene,645:55-59.
Smith S,Witkowski A,Joshi A K.2003.Structural and functional organization of the animal fatty acid synthase[J].Progress in Lipid Research,42(4):289-317.
Tian J,Wang S,Wang G,et al.2009.A single nucleotide polymorphism of chicken acetyl-CoA carboxylase A gene associated with fatness traits[J].Animal Biotechnology,21(1):42-50.
Tong L.2005.Acetyl-coenzyme A carboxylase:Crucial metabolic enzyme and attractive target for drug discovery[J].Cellular&Molecular Life Sciences,62(16):1784-1803.
toran-allerand c d.1999.Novel mechanisms of estrogen action in the developing brain[J].Frontiers in Neuroendocrinology,20(2):97-121.
Williams J,Harvey S.1986.Plasma concentrations of luteinizing hormone growth hormone,oestradiol,testosterone and androstenedione in the domestic hen from 6 weeks of age to sexual maturity[J].Reproduction Nutrition Developpement,26(2A):515-522.
刘安芳,朱庆,阳泽平.2006.饲料中添加油脂对肉鸭生产性能的影响[J].畜禽业,(10):18-19.(Liu A F,Zhu QYang Z P.2006.Effects of soybean oil on performance of meat ducks[J].Livestock and Poultry Industry,(10)18-19.)
卢建,王克华,曲亮,等.2013.不同水平大豆油对苏禽青壳蛋鸡产蛋性能、蛋品质和血清脂质指标的影响[C]//,中国畜牧兽医学会家禽学分会(编著),中国畜牧兽医学会家禽学分会第九次代表会议暨第十六次全国家禽学术讨论会论文集.中国家禽,扬州市,pp.292-295.(Lu J,Wang K H,Qu L,et al.2013.Effects of soybean oil on egg performance,egg quality,and serum lipids indexes of laying Hens[C]//,Poultry Science Chapter of Chinese Association of Animal Science and Veterinary Medicine(eds.),Proceedings of 9th representative conference of Poultry Chapter of Chinese Association of Animal Science and Veterinary Medicine and 16th National Poultry Symposium.China Poultry,Yangzhou City,China,pp.292-295.)
欧阳建华,谢亮,刘杰,等.2007.鸡FASN基因2个位点的多样性及其与体重、脂肪沉积性状的相关性[J].畜牧兽医学报,38(1):25-30.(OuYang J H,Xie L,Liu J,et al.2007.Genetic diversity of two sites of FASN gene and its association with growth and fat deposition in chickens[J].Acta Veterinaria et Zootechnica Sinica,38(1):25-30.)
彭音.2009.ChREBP和ACC1在肝细胞糖/脂代谢调控中的作用[D].博士学位论文,华中农业大学,导师:杨在清.pp.7-12.(Peng Y.2009.The role of ChREBP and ACC1 gene in Glyco-and lipometabolism and hepatocyte[D].Thesis for Ph.D.,Huazhong Agricultural University,Supervisor:Yang Z Q,pp.7-12.)
史铭欣,史洪岩,李辉,等.2013.高、低脂系肉鸡肝脏脂肪合成代谢相关基因的表达差异分析[J].农业生物技术学报,21(3):306-312.(Shi M X,Shi H Y,Li H,et al.2013.Expression analysis of lipid synthesis-related genes in the livers of fat and lean broilers[J].Journal of Agricultural Biotechnology,21(3):306-312.)
田方圆,王台安,李艳敏,等.2017.鸡MOGAT和DGAT基因的克隆及表达特性研究[J].畜牧兽医学报,48(2):214-224.(Tian F Y,Wang T A,Li Y M,et al.2017.Cloning and expression characteristics of MOGAT and DGATgenes in chicken(Gallus gallus)[J].Acta Veterinaria et Zootechnica Sinica,48(2):214-224.)
徐乃一,郑航,田卫华,等.2018.鸡SCD基因表达调控特性分析[J].农业生物技术学报,26(1):123-131.(Xu N Y,Zheng H,Tian W H,et al.2013.Characteristics of expression and regulation of SCD gene in chicken(Gallus gallus))[J].Journal of Agricultural Biotechnology,26(1):123-131.)
周淑世.2009.不同脂肪源对朗德鹅产肝性能及FAS mRNA表达的影响[D].硕士学位论文,吉林大学,导师:牛淑玲.pp.23-38.(Zhou S S.2009.Effect of different fatty sources on fatty liver performance and gene expression of FAS mRNA in Landes goose[D].Thesis for M.S.,Jilin University,Supervisor:Niu A L,pp.23-38.)
Baik M,Etchebarne B E,Bong J,et al.2009.Gene expression profiling of liver and mammary tissues of lactating dairy cows[J].Asian Australasian Journal of Animal Sciences,22(6):871-884.
Chow J D Y,Lawrence R T,Healy M E,et al.2014.Genetic inhibition of hepatic acetyl-CoA carboxylase activity increases liver fat and alters global protein acetylation[J]Molecular Metabolism,3(4):419-431.
Dorn C,Riener M O,Kirovski G,et al.2010.Expression of fatty acid synthase in nonalcoholic fatty liver disease[J]International Journal of Clinical&Experimental Pathology,3(5):505-514.
Driscoll M D,Sathya G,Muyan M,et al.1998.Sequence requirements for estrogen receptor binding to estrogen response elements[J].Journal of Biological Chemistry273(45):29321-29330.
Fischer P W F,Marks G S.1976.Chick embryo liver cells maintained in serum-free waymouth MD 705/1 medium[J].Tissue Culture Association,2(4):449-452.
Han L Q,Gao T Y,Yang G Y,et al.2018.Overexpression of SREBF chaperone(SCAP)enhances nuclear SREBP1translocation to upregulate fatty acid synthase(FASN)gene expression in bovine mammary epithelial cells[J]Journal of Dairy Science,101(7):6523-6531.
Hansmannel F,Mordier S,Iynedjian P.2006.Insulin induction of glucokinase and fatty acid synthase in hepatocytes:Analysis of the roles of sterol-regulatory-elementbinding protein-1c and liver X receptor[J].Biochemica Journal,399(2):275-283.
Hillgartner F B,Charron T,Chesnut K A.1997.Triiodothyronine stimulates and glucagon inhibits transcription of the acetyl-CoA carboxylase gene in chick embryo hepatocytes:Glucose and insulin amplify the effect of triiodothyronine[J].Archives of Biochemistry&Biophysics337(2):159-168.
Kim Y J,Yang J L,Kim K Y H.2003.Regulation of acetylCoA carboxylase gene expression by hormones and nutrients[J].Preventive Nutrition&Food Science,8(1)61-65.
Knobloch M,Braun S M,Zurkirchen L,et al.2013.Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis[J].Nature,493(7431):226-230.
Kumar V,Chambon P.1988.The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer[J].Cell,55(1):145-156.
Li H,Wang T,Xu C,et al.2015.Transcriptome profile of liver at different physiological stages reveals potential mode for lipid metabolism in laying hens[J].BMC Genomics,16(1):763-775.
Li J,Leghari I H,He B,et al.2014.Estrogen stimulates expression of chicken hepatic vitellogeninⅡand very low-density apolipoproteinⅡthrough ER-α[J].Theriogenology,82(3):517-524.
Mildner A M,Clarke S D.1991.Porcine fatty acid synthase:Cloning of a complementary DNA,tissue distribution of its mRNA and suppression of expression by somatotropin and dietary protein[J].Journal of Nutrition,121(6):900-907.
Palmisano B T,Zhu L,Stafford J M.2017.Role of estrogens in the regulation of liver lipid metabolism[J].Advances in Experimental Medicine&Biology,1043:227-256.
Peckett A J,Wright D C,Riddell M C.2011.The effects of glucocorticoids on adipose tissue lipid metabolism[J].Metabolism-clinical&Experimental,60(11):1500-1510.
Pellegrini M,Pallottini V,Marin R,et al.2014.Role of the sex hormone estrogen in the prevention of lipid disorder[J].Current Medicinal Chemistry,21(24):2734-2742.
Phil G W D.1996.Nutritional and hormonal regulation of fatty acid synthase[J].Nutrition Reviews,54(4):122-123.
Saha A K,Ruderman N B.2003.Malonyl-CoA and AMP-activated protein kinase:An expanding partnership[J].Molecular&Cellular Biochemistry,253(2):65-70.
Sha R,Gui L,Khan R,et al.2017.Association between FASNgene polymorphisms ultrasound carcass traits and intramuscular fat in Qinchuan cattle[J].Gene,645:55-59.
Smith S,Witkowski A,Joshi A K.2003.Structural and functional organization of the animal fatty acid synthase[J].Progress in Lipid Research,42(4):289-317.
Tian J,Wang S,Wang G,et al.2009.A single nucleotide polymorphism of chicken acetyl-CoA carboxylase A gene associated with fatness traits[J].Animal Biotechnology,21(1):42-50.
Tong L.2005.Acetyl-coenzyme A carboxylase:Crucial metabolic enzyme and attractive target for drug discovery[J].Cellular&Molecular Life Sciences,62(16):1784-1803.
toran-allerand c d.1999.Novel mechanisms of estrogen action in the developing brain[J].Frontiers in Neuroendocrinology,20(2):97-121.
Williams J,Harvey S.1986.Plasma concentrations of luteinizing hormone growth hormone,oestradiol,testosterone and androstenedione in the domestic hen from 6 weeks of age to sexual maturity[J].Reproduction Nutrition Developpement,26(2A):515-522.