番鸭产蛋各期肝脂肪酸组成及AMPK信号通路基因表达研究

Fatty Acid Composition and Gene Expression of AMPK Signaling Pathway in Liver of Muscovy Duck at Different Egg-laying Stages

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。

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PUFA 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.