番鸭产蛋各期肝脂肪酸组成及AMPK信号通路基因表达研究

doi:10.11843/j.issn.0366-6964.2021.09.006

摘要/Abstract

摘要： 旨在探究产蛋各期番鸭肝腺苷酸活化蛋白激酶（AMPK）信号通路基因表达和肝脂肪酸组成，为番鸭肝脂质代谢应答产蛋提供机理研究。本研究选取开产前22周龄、产蛋初期30周龄、产蛋中期40周龄和产蛋末期60周龄母番鸭各15羽，全自动生化仪测定血脂水平，苏木精-伊红（HE）染色和油红O染色观察肝组织学结构，实时荧光定量PCR （qRT-PCR）检测肝AMPK通路基因表达，气相色谱-质谱联用法（GC-MS）检测产蛋各期肝脂肪酸组成。结果表明，总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）、甘油三酯（TG）和极低密度脂蛋白胆固醇（VLDL-C）水平在40周龄显著高于22、30和60周龄（P<0.05）；高密度脂蛋白胆固醇（HDL-C）水平在30和40周龄显著低于22和60周龄（P<0.05）。肝HE和油红O染色切片显示，肝在22周龄呈实质状，至产蛋40和60周龄，肝脂滴沉积明显（P<0.05）。AMPKα1在22、30、40和60周龄呈本底低水平表达，并显著低于产蛋各期肉碱脂酰转移酶1（CPT1）和脂肪酸合成酶（FAS）的表达量（P<0.05），FAS在22、40和60周龄均呈高水平表达（P<0.05）；羟甲基戊二酰辅酶A还原酶（HMGR）、肝细胞核因子4α（HNF4α）、乙酰辅酶A羧化酶1（ACC1）和固醇调控元件结合蛋白-1（SREBP1c）在40周龄表达量显著高于22和30周龄（P<0.05）。肝中饱和脂肪酸（SFA）、单不饱和脂肪酸（MUFA）和多不饱和脂肪酸（PUFA）主要由C16∶0、C18∶0、C18∶1和C20∶2 n-6构成，分别占总脂肪酸含量的32%、16%、30%和9%。C14∶0和C16∶0含量在40周龄显著高于22周龄（P<0.05）；C24∶0、C20∶2 n-6和PUFA含量在60周龄显著高于22和40周龄（P<0.05）。综上，番鸭产蛋期肝通过上调FAS等脂质合成基因表达，合成大量长链脂肪酸，沉积于肝，并增加血脂水平。

关键词: 番鸭, 血脂, 肝, AMPK信号通路, 脂肪酸

Abstract: The aim of this study was to investigate the gene expression in AMP-activated protein kinase (AMPK) signaling pathway and fatty acid composition in the liver of Muscovy duck at different egg-laying stages, so as to illustrate the mechanism of lipid metabolism in response to egg-laying. Fifteen female Muscovy ducks were selected at pre-laying (22 weeks), early laying (30 weeks), laying (40 weeks) and post-laying (60 weeks) stages, respectively. Serum lipid levels were determined by automatic biochemical analyzer. Liver histological structure was observed by hematoxylin-eosin (HE) staining and Oil Red O staining, gene expression in AMPK pathway was determined by quantitative real-time PCR (qRT-PCR), and fatty acid composition was measured by gas chromatography-mass spectrometry (GC-MS). The results showed that the levels of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglyceride (TG) and very low density lipoprotein cholesterol (VLDL-C) were significantly higher at 40 weeks than those at 22, 30 and 60 weeks (P<0.05). The levels of high density lipoprotein cholesterol (HDL-C) at 30 and 40 weeks were significantly lower than those at 22 and 60 weeks (P<0.05). Liver sections stained by HE and Oil Red O showed that the reticular formation was intact at 22 weeks, and lipid deposition significantly increased at 40 and 60 weeks (P<0.05). AMPKα1 was in a low expression level at 22, 30, 40 and 60 weeks, and was significantly lower than the expression of carnitine palmitoyltransferase-1 (CPT1) and fatty acid synthase (FAS) at different egg-laying stages(P<0.05). FAS expressed in a significantly high level at 22, 40 and 60 weeks (P<0.05). The expression levels of hydroxy-methylglutaryl coenzyme A reductase (HMGR), hepatocyte nuclear factor 4α (HNF4α), acetyl-CoA carboxylase 1 (ACC1) and sterol regulatory element binding protein 1 (SREBP1c) at 40 weeks were significantly higher than those at 22 and 30 weeks (P<0.05). Liver saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) were mainly composed of C16:0, C18:0, C18:1 and C20:2 n-6, accounted for 32%, 16%, 30% and 9% of the total fatty acids, respectively. The contents of C14:0 and C16:0 at 40 weeks were significantly higher than those at 22 weeks(P<0.05). The contents of C24:0, C20:2 n-6 and ΣPUFA at 60 weeks were significantly higher than those at 22 and 40 weeks (P<0.05). In conclusion, the liver synthesized large amount of long-chain fatty acids by up-regulating the expression of lipid synthesis genes, including FAS,during egg-laying. The synthesized lipids were deposited in liver and transported into serum thus increased the serum lipid level during laying.

Key words: Muscovy duck, serum lipid, liver, AMPK signaling pathway, fatty acid

中图分类号:

S834.2

朱文俊, 陈兴勇, 刘乐, 刘政权, 赵羽彤, 耿照玉. 番鸭产蛋各期肝脂肪酸组成及AMPK信号通路基因表达研究[J]. 畜牧兽医学报, 2021, 52(9): 2429-2438.

ZHU Wenjun, CHEN Xingyong, LIU Le, LIU Zhengquan, ZHAO Yutong, GENG Zhaoyu. Fatty Acid Composition and Gene Expression of AMPK Signaling Pathway in Liver of Muscovy Duck at Different Egg-laying Stages[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2429-2438.

参考文献 33

[1]	YE P F, LI M, LIAO W, et al.Hypothalamic transcriptome analysis reveals the neuroendocrine mechanisms in controlling broodiness of Muscovy duck (Cairina moschata)[J].PLoS One, 2019, 14(5):e0207050.
[2]	YE P F, GE K, LI M, et al.Egg-laying and brooding stage-specific hormonal response and transcriptional regulation in pituitary of Muscovy duck (Cairina moschata)[J].Poult Sci, 2019, 98(11):5287-5296.
[3]	朱文俊, 杨万里, 韦聪聪, 等.番鸭产蛋前期和产蛋期肝脏组织非靶向代谢组学比较分析[J].中国农业大学学报, 2020, 25(12):76-85.ZHU W J, YANG W L, WEI C C, et al.Comparative analysis of nontargeted metabolomics in liver tissue of Muscovy duck in pre-laying and laying stages[J].Journal of China Agricultural University, 2020, 25(12):76-85.(in Chinese)
[4]	WALZEM R L, HANSEN R J, WILLIAMS D L, et al.Estrogen induction of VLDLy assembly in egg-laying hens[J].J Nutr, 1999, 129(2S Suppl):467S-472S.
[5]	SCHMIDINGER B, WEIJLER A M, SCHNEIDER W J, et al.Hepatosteatosis and estrogen increase apolipoprotein O production in the chicken[J].Biochimie, 2016, 127:37-43.
[6]	RATNA W N, BHATT V D, CHAUDHARY K, et al.Estrogen-responsive genes encoding egg yolk proteins vitellogenin and apolipoprotein II in chicken are differentially regulated by selective estrogen receptor modulators[J].Theriogenology, 2016, 85(3):376-383.
[7]	LI H, WANG T A, XU C L, et al.Transcriptome profile of liver at different physiological stages reveals potential mode for lipid metabolism in laying hens[J].BMC Genomic, 2015, 16:763.
[8]	陈兴勇, 耿照玉, 廖望, 等.一种基于脂联素基因ADP鉴定鸭繁殖性状的分子标记及其应用:中国, 201711459759.6[P].2020-05-08.CHEN X Y, GENG Z Y, LIAO W, et al.Molecular marker for identifying duck reproduction trait based on adiponectin (ADP) gene and application of molecular marker:CN, 201711459759.6[P].2020-05-08.(in Chinese)
[9]	ALVES-BEZERRA M, COHEN D E.Triglyceride metabolism in the liver[J].Compr Physiol, 2017, 8(1):1-8.
[10]	TUMOVÁ E, TEIMOURI A.Fat deposition in the broiler chicken:a review[J].Sci Agric Bohemica, 2010, 41(2):121-128.
[11]	KNIGHT B L, HEBBACHI A, HAUTON D, et al.A role for PPARalpha in the control of SREBP activity and lipid synthesis in the liver[J].Biochem J, 2005, 389(Pt 2):413-421.
[12]	SKOP V, CAHOVÁ M, PAPÁČKOVÁ Z, et al.Autophagy-lysosomal pathway is involved in lipid degradation in rat liver[J]. Physiol Res, 2012, 61(3):287-297.
[13]	MA Z, LI H, ZHENG H, et al.MicroRNA-101-2-5p targets the ApoB gene in the liver of chicken (Gallus Gallus)[J].Genome, 2017, 60(8):673-678.
[14]	ALVARENGA R R, ZANGERONIMO M G, PEREIRA L J, et al.Lipoprotein metabolism in poultry[J].World's Poult Sci J, 2011, 67(3):431-440.
[15]	DE MORENTIN P B M, GONZÁLEZ C R, SAHA A K, et al.Hypothalamic AMP-activated protein kinase as a mediator of whole body energy balance[J].Rev Endocr Metab Disord, 2011, 12(3):127-140.
[16]	周华金, 胡希怡, 杨家昶, 等.热应激对肉仔鸡肝AMPKα1及脂肪代谢相关分子基因表达的影响[J].畜牧兽医学报, 2018, 49(1):102-110.ZHOU H J, HU X Y, YANG J C, et al.Effects of heat stress on gene expression of AMPKα1 and lipid metabolism related molecules in the liver of broiler chickens[J].Acta Veterinaria et Zootechnica Sinica, 2018, 49(1):102-110.(in Chinese)
[17]	National Research Council.Nutrient requirements of poultry[M].9th ed.Washington, DC:National Academy Press, 1994.
[18]	国家卫生和计划生育委员会, 国家食品药品监督管理总局.GB 5009.168-2016食品安全国家标准食品中脂肪酸的测定[S].北京:中国标准出版社, 2017.National Health and Family Planning Commission of the People's Republic of China, China Food and Drug Administration.GB 5009.168-2016 National food safety Stan-dard:determination of fatty acid in foods[S].Beijing:China Standard Press, 2017.(in Chinese)
[19]	陈兴勇, 赵宁, 张燕, 等.皖西白鹅育肥期肌肉脂肪酸组成及肝PPARα、FADS2和ME1基因表达规律的研究[J].畜牧兽医学报, 2017, 48(10):1912-1919.CHEN X Y, ZHAO N, ZHANG Y, et al.The fatty acid profile in muscles and expression of PPARα, FADS2 and ME1 genes in liver of Chinese Wanxi white geese in fattening period[J].Acta Veterinaria et Zootechnica Sinica, 2017, 48(10):1912-1919.(in Chinese)
[20]	VON ECKARDSTEIN A, NOFER J R, ASSMANN G.High density lipoproteins and arteriosclerosis:role of cholesterol efflux and reverse cholesterol transport[J].Arterioscler, Thromb, Vasc Biol, 2001, 21(1):13-27.
[21]	KISS R S, SNIDERMAN A.Shunts, channels and lipoprotein endosomal traffic:a new model of cholesterol homeostasis in the hepatocyte[J].J Biomed Res, 2017, 31(2):95-107.
[22]	OLZMANN J A, CARVALHO P.Dynamics and functions of lipid droplets[J].Nat Rev Mol Cell Biol, 2019, 20(3):137-155.
[23]	WANG Q, LIU S D, ZHAI A H, et al.AMPK-mediated regulation of lipid metabolism by phosphorylation[J].Biol Pharm Bull, 2018, 41(7):985-993.
[24]	NADA M A, ABDEL-ALEEM S, SCHULZ H.On the rate-limiting step in the β-oxidation of polyunsaturated fatty acids in the heart[J].Biochim Biophys Acta, 1995, 1255(3):244-250.
[25]	JOHNSON A L.Ovarian follicle selection and granulosa cell differentiation[J].Poult Sci, 2015, 94(4):781-785.
[26]	EBERLÉ D, HEGARTY B, BOSSARD P, et al.SREBP transcription factors:master regulators of lipid homeostasis[J].Biochimie, 2004, 86(11):839-848.
[27]	邢智洋, 张梦璐, 张菡, 等.沉默SCAP基因对奶牛乳腺上皮细胞脂滴的影响[J].畜牧兽医学报, 2019, 50(3):507-516.XING Z Y, ZHANG M L, ZHANG H, et al.Effect of silencing SCAP gene on lipid droplets in dairy mammary epithelial cells[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(3):507-516.(in Chinese)
[28]	KIM C W, ADDY C, KUSUNOKI J, et al.Acetyl CoA carboxylase inhibition reduces hepatic steatosis but elevates plasma triglycerides in mice and humans:a bedside to bench investigation[J].Cell Metab, 2017, 26(2):394-406.
[29]	HAMPTON R Y.Proteolysis and sterol regulation[J].Annu Rev Cell Dev Biol, 2002, 18:345-378.
[30]	YU D S, CHEN G, PAN M L, et al.High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice[J].J Cell Physiol, 2018, 233(6):4770-4782.
[31]	TVRZICKA E, KREMMYDA L S, STANKOVA B, et al.Fatty acids as biocompounds:their role in human metabolism, health and disease-a review.Part 1:classification, dietary sources and biological functions[J].Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2011, 155(2):117-130.
[32]	TVRZICKÁ E, VECKA M, STAŇKOVÁ B, et al.Analysis of fatty acids in plasma lipoproteins by gas chromatography-flame ionization detection:quantitative aspects[J].Anal Chim Acta, 2002, 465(1-2):337-350.
[33]	ROSQVIST F, MCNEIL C A, PRAMFALK C, et al.Fasting hepatic de novo lipogenesis is not reliably assessed using circulating fatty acid markers[J].Am J Clin Nutr, 2019, 109(2):260-268.