不同糖及糖水平对松浦镜鲤养殖过程中糖代谢相关基因表达的影响
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摘要
为探讨在养殖不同阶段,不同糖及糖水平对松浦镜鲤肝脏糖酵解酶(GK和HK)、糖异生酶(G6Pase和PEPCK)和糖代谢相关基因(IGF-Ⅰ和GHR)m RNA表达水平的影响,实验采用2×2双因素设计实验,选择淀粉和葡萄糖2种糖源,2个糖水平(25%和50%),共4个实验组,分别为低淀粉组(LS)、高淀粉组(HS)、低葡萄糖组(LG)和高葡萄糖组(HG)。选用初始体质量为(8.30±0.15)g的松浦镜鲤420尾,随机分为4组,每组3个重复,每个重复35尾鱼。实验周期为7周。结果显示,不同养殖阶段,HK基因和IGF-Ⅰ基因在不同实验组中的表达趋势基本一致,HK基因在养殖1周时表达量显著升高,之后开始降低,IGF-Ⅰ基因在养殖3周时表达量显著升高。在不同养殖阶段,GK、PEPCK、G6Pase和GHR基因的表达趋势受糖水平和糖种类的影响。在低糖水平下(LS组和LG组),这些基因表现了相同的变化趋势,GK和GHR基因在1周时表达量最高,之后开始下降;PEPCK基因在3周时表达量最高;G6Pase在1周时表达量升高,之后下降,在7周时表达量又有上升趋势。在高糖水平下,HS组的GK基因的表达量在各个养殖阶段差异不显著,而HG组的GK基因则在1周时表达量显著升高;HS组和HG组的PEPCK基因的表达量在各个养殖阶段差异不显著;HS组的G6Pase基因的表达量在各个养殖阶段差异不显著,而HG组的G6Pase基因则在3周时表达量显著升高;HG组的GHR基因的表达量在各个养殖阶段差异不显著,而HS组的GHR基因则在3周时表达量显著升高。在养殖初期(1周),高糖饲料显著促进了HK基因的表达,并抑制了PEPCK、G6Pase和GHR基因的表达。
This experiment was aimed to study the effect of different carbohydrates and different carbohydrate levels on m RNA expression of glycolytic enzyme(glucose kinase, GK; hexokinase, HK), gluconeogenesis enzyme(Glucose-6-phosphatase, G6 Pase; phosphoenolpyruvate carboxykinase, PEPCK), glucose metabolism related genes(Insulin-like Growth Factor-Ⅰ, IGF-Ⅰ; Growth Hormone Receptor, GHR) in different feeding stages of Songpu mirror carp(Cyprinus carpio). Four experimental diets were formulated to contain two carbohydrates(starch and glucose) diets with two levels(250 g/kg and 500 g/kg)(Low level starch group: LS; High level starch group: HS; Low level glucose group: LG; High level glucose group: HG) two carbohydrates(glucose and starch)and two carbohydrate levels(25% and 50%)(LS/HS/LG/HG) using 2×2 double factor experimental design, 420 Songpu mirror carp(mean weight=[8.30±0.15] g) were randomly divided into 4 dietary treatments with 3 triplicates and 35 fish per triplicate and were reared for 7 weeks. The results showed that in different feeding stages, the m RNA expression levels of HK gene and IGF-Ⅰ gene in different experimental groups were basically in the same trend. The m RNA expression levels of HK gene at feeding 1 W was significantly increased and then decreased. The m RNA expression level of IGF-Ⅰ gene was significantly increased at feeding 3 W. IGF-Ⅰ gene expression increased significantly at feeding 3 W. In different feeding stages, the m RNA expression levels of GK, PEPCK,G6 Pase and GHR genes were affected by carbohydrate levels and carbohydrate types. At low carbohydrate level(LS group and LG group), the m RNA expression levels of these genes showed the same trend. The m RNA expression levels of GK and GHR genes were significantly higher at feeding 1 W and then began to decline. The m RNA expression level of PEPCK gene was the highest at feeding 3 W. The m RNA expression level of G6 Pase genes were significantly higher at feeding 1 W, then began to decline and showed a rising trend at feeding 7 W. At high carbohydrate levels, the m RNA expression level of GK gene in HS group was not significantly different at different feeding stages. But the m RNA expression level of GK gene in HG group was increased significantly at feeding1 W. The m RNA expression level of PEPCK gene in HS group and HG group had no significant difference at different feeding stages. The m RNA expression level of G6 Pase gene in HS group had no significant difference at different feeding stages. But the m RNA expression level of G6 Pase gene in HG group was increased significantly at feeding 3 W. The m RNA expression level of GHR gene in HG group had no significant difference at different feeding stages, while the m RNA expression level of GHR gene in HS group was increased significantly at feeding3 W.In the early feeding stage(1 W), the m RNA expression level of HK gene was significantly promoted by high carbohydrate diet. While, the m RNA expression levels of PEPCK, G6 Pase and GHR genes were suppressed by high carbohydrate diet. In conclusion, this study investigated the effects of different carbohydrates and carbohydrate levels on the expression trend of enzymes and genes involved in glucose metabolism at different feeding stages. It provides a theoretical basis for the application of carbohydrate in feed.
This experiment was aimed to study the effect of different carbohydrates and different carbohydrate levels on m RNA expression of glycolytic enzyme(glucose kinase, GK; hexokinase, HK), gluconeogenesis enzyme(Glucose-6-phosphatase, G6 Pase; phosphoenolpyruvate carboxykinase, PEPCK), glucose metabolism related genes(Insulin-like Growth Factor-Ⅰ, IGF-Ⅰ; Growth Hormone Receptor, GHR) in different feeding stages of Songpu mirror carp(Cyprinus carpio). Four experimental diets were formulated to contain two carbohydrates(starch and glucose) diets with two levels(250 g/kg and 500 g/kg)(Low level starch group: LS; High level starch group: HS; Low level glucose group: LG; High level glucose group: HG) two carbohydrates(glucose and starch)and two carbohydrate levels(25% and 50%)(LS/HS/LG/HG) using 2×2 double factor experimental design, 420 Songpu mirror carp(mean weight=[8.30±0.15] g) were randomly divided into 4 dietary treatments with 3 triplicates and 35 fish per triplicate and were reared for 7 weeks. The results showed that in different feeding stages, the m RNA expression levels of HK gene and IGF-Ⅰ gene in different experimental groups were basically in the same trend. The m RNA expression levels of HK gene at feeding 1 W was significantly increased and then decreased. The m RNA expression level of IGF-Ⅰ gene was significantly increased at feeding 3 W. IGF-Ⅰ gene expression increased significantly at feeding 3 W. In different feeding stages, the m RNA expression levels of GK, PEPCK,G6 Pase and GHR genes were affected by carbohydrate levels and carbohydrate types. At low carbohydrate level(LS group and LG group), the m RNA expression levels of these genes showed the same trend. The m RNA expression levels of GK and GHR genes were significantly higher at feeding 1 W and then began to decline. The m RNA expression level of PEPCK gene was the highest at feeding 3 W. The m RNA expression level of G6 Pase genes were significantly higher at feeding 1 W, then began to decline and showed a rising trend at feeding 7 W. At high carbohydrate levels, the m RNA expression level of GK gene in HS group was not significantly different at different feeding stages. But the m RNA expression level of GK gene in HG group was increased significantly at feeding1 W. The m RNA expression level of PEPCK gene in HS group and HG group had no significant difference at different feeding stages. The m RNA expression level of G6 Pase gene in HS group had no significant difference at different feeding stages. But the m RNA expression level of G6 Pase gene in HG group was increased significantly at feeding 3 W. The m RNA expression level of GHR gene in HG group had no significant difference at different feeding stages, while the m RNA expression level of GHR gene in HS group was increased significantly at feeding3 W.In the early feeding stage(1 W), the m RNA expression level of HK gene was significantly promoted by high carbohydrate diet. While, the m RNA expression levels of PEPCK, G6 Pase and GHR genes were suppressed by high carbohydrate diet. In conclusion, this study investigated the effects of different carbohydrates and carbohydrate levels on the expression trend of enzymes and genes involved in glucose metabolism at different feeding stages. It provides a theoretical basis for the application of carbohydrate in feed.
引文
[1]缪凌鸿,刘波,戈贤平,等.高碳水化合物水平日粮对异育银鲫生长、生理、免疫和肝脏超微结构的影响[J].水产学报,2011,35(2):221-230.Miao L H,Liu B,Ge X P,et al.Effect of high carbohydrate levels in the dietary on growth performance,immunity and transmission electron microscopy(TEM)on hepatic cell of allogynogenetic crucian carp(Carassius auratus gibelio)[J].Journal of Fisheries of China,2011,35(2):221-230(in Chinese).
[2]杨丽萍,郑文佳,秦超彬,等.饲料糖脂比对吉富罗非鱼生长、血液指标和肝脏糖代谢关键酶活性及基因表达的影响[J].水产学报,2016,40(9):1376-1386.Yang L P,Zheng W J,Qin C B,et al.Effects of kind and level of dietary carbohydrate on growth performance and physiological indices of juvenile black carp(Mylopharyngodln piceus Richardson)[J].Journal of Fisheries of China,2016,40(9):1376-1386(in Chinese).
[3]Asaduzzaman M,Wahab M A,Verdegem M C J,et al.Effects of addition of tilapia Oreochromis niloticus and substrates for periphyton developments on pond ecology and production in C/N-controlled freshwater prawn Macrobrachium rosenbergii farming systems[J].Aquaculture,2009,287(3-4):371-380.
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[5]Lee S M,Kim K D.Effects of dietary carbohydrate to lipid ratios on growth and body composition of juvenile and grower rockfish,Sebastes schlegeli[J].Aquaculture Research,2009,40(16):1830-1837.
[6]Hu Y H,Liu Y J,Tian L X,et al.Optimal dietary carbohydrate to lipid ratio for juvenile yellowfin seabream(Sparus latus)[J].Aquaculture Nutrition,2007,13(4):291-297.
[7]Moreira I S,Peres H,Couto A,et al.Temperature and dietary carbohydrate level effects on performance and metabolic utilisation of diets in European sea bass(Dicentrarchus labrax)juveniles[J].Aquaculture,2008,274(1):153-160.
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[9]Jones J I,Clemmons D R.Insulin-like growth factors and their binding protein:Biological actions[J].Endocrine Reviews,1995,16(1):3-34.
[10]Enes P,Sanchez-Gurmaches J,Navarro I,et al.Role of insulin and IGF-Ⅰon the regulation of glucose metabolism in European sea bass(Dicentrarchus labrax)fed with different dietary carbohydrate levels[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2010,157(4):346-353.
[11]李晋南,徐奇友,王常安,等.不同糖及糖水平对松浦镜鲤GH/IGF-Ⅰ基因表达和鱼体组成的影响[J].上海海洋大学学报,2015,24(4):489-495.Li J N,Xu Q Y,Wang C A,et al.Effects of different dietary carbohydrates and carbohydrate levels on GH/IGF-Ⅰm RNA expression and the fish body composition of juvenile mirror carp(Cyprinus carpio)[J].Journal of Shanghai Ocean University,2015,24(4):489-495(in Chinese).
[12]戈贤平,刘波,谢骏,等.饲料中不同碳水化合物水平对翘嘴红鲌生长及血液指标和糖代谢酶的影响[J].南京农业大学学报,2007,30(3):88-93.Ge X P,Liu B,Xie J,et al.Effect of different carbohydrate levels of dietary on growth,plasma biochemical indices and hepaticpancreas carbohydrate metabolic enzymes in topmouth culter(Erythroculter ilishaeformis Bleeker)[J].Journal of Nanjing Agricultural University,2007,30(3):88-93(in Chinese).
[13]Li J N,Xu Q Y,Wang C A,et al.Effects of dietary glucose and starch levels on the growth,haematological indices and hepatic hexokinase and glucokinase m RNAexpression of juvenile mirror carp(Cyprinus carpio)[J].Aquaculture Nutrition,2016,22(3):550-558.
[14]Panserat S,Plagnes-Juan E,Kaushik S.Gluconeogenic enzyme gene expression is decreased by dietary carbohydrates in common carp(Cyprinus carpio)and gilthead seabream(Sparus aurata)[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2002,1579(1):35-42.
[15]谭肖英,罗智,刘永坚.鱼类对饲料中糖的利用研究进展[J].中国饲料,2007(6):19-23.Tan X Y,Luo Z,Liu Y J.Review of carbohydrate utilization in fish feed[J].China Feed,2007(6):19-23(in Chinese).
[16]Furuichi M,Yone Y.Changes in activities of hepatic enzymes related to carbohydrate metabolism of fishes in glucose and insulin-glucose tolerance tests[J].Nippon Suisan Gakkaishi,1982,48(3):463-466.
[17]Panserat S,Médale F,Blin C,et al.Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout,gilthead seabream,and common carp[J].American Journal of Physiology-Regulatory,Integrative and Comparative Physiology,2000,278(5):R1164-R1170.
[18]Tranulis M A,Dregni O,Christophersen B,et al.A glucokinase-like enzyme in the liver of Atlantic salmon(Salmo salar)[J].Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,1996,114(1):35-39.
[19]Caseras A,Metón I,Vives C,et al.Nutritional regulation of glucose-6-phosphatase gene expression in liver of the gilthead sea bream(Sparus aurata)[J].British Journal of Nutrition,2002,88(6):607-614.
[20]Panserat S,Blin C,Médale F,et al.Molecular cloning,tissue distribution and sequence analysis of complete glucokinase c DNAs from gilthead seabream(Sparus aurata),rainbow trout(Oncorhynchus mykiss)and common carp(Cyprinus carpio)[J].Biochimica et Biophysica Acta(BBA)-General Subjects,2000,1474(1):61-69.
[21]Gao W,Liu Y J,Tian L X,et al.Effect of dietary carbohydrate-to-lipid ratios on growth performance,body composition,nutrient utilization and hepatic enzymes activities of herbivorous grass carp(Ctenopharyngodon idella)[J].Aquaculture Nutrition,2010,16(3):327-333.
[22]Li X F,Wang Y,Liu W B,et al.Effects of dietary carbohydrate/lipid ratios on growth performance,body composition and glucose metabolism of fingerling blunt snout bream Megalobrama amblycephala[J].Aquaculture Nutrition,2013,19(5):701-708.
[23]Hanson R W,Reshef L.Regulation of phosphoenolpyruvate carboxykinase(GTP)gene expression[J].Annual Review of Biochemistry,1997,66:581-611.
[24]Panserat S,Plagnes-Juan E,Brèque J,et al.Hepatic phosphoenolpyruvate carboxykinase gene expression is not repressed by dietary carbohydrates in rainbow trout(Oncorhynchus mykiss)[J].Journal of Experimental Biology,2001,204(2):359-365.
[25]Forsyth I A,Wallis M.Growth hormone and prolactinmolecular and functional evolution[J].Journal of Mammary Gland Biology and Neoplasia,2002,7(3):291-312.
[26]刘丽丽.胰岛素样生长因子I的研究进展[J].中国草食动物科学,2013,33(6):57-59.Liu L L.Pregress in insulin like growth factor I[J].China Herbivore Science,2013,33(6):57-59(in Chinese).
[2]杨丽萍,郑文佳,秦超彬,等.饲料糖脂比对吉富罗非鱼生长、血液指标和肝脏糖代谢关键酶活性及基因表达的影响[J].水产学报,2016,40(9):1376-1386.Yang L P,Zheng W J,Qin C B,et al.Effects of kind and level of dietary carbohydrate on growth performance and physiological indices of juvenile black carp(Mylopharyngodln piceus Richardson)[J].Journal of Fisheries of China,2016,40(9):1376-1386(in Chinese).
[3]Asaduzzaman M,Wahab M A,Verdegem M C J,et al.Effects of addition of tilapia Oreochromis niloticus and substrates for periphyton developments on pond ecology and production in C/N-controlled freshwater prawn Macrobrachium rosenbergii farming systems[J].Aquaculture,2009,287(3-4):371-380.
[4]Asaduzzaman M,Wahab M A,Verdegem M C J,et al.Effects of carbohydrate source for maintaining a high C∶N ratio and fish driven re-suspension on pond ecology and production in periphyton-based freshwater prawn culture systems[J].Aquaculture,2010,301(1-4):37-46.
[5]Lee S M,Kim K D.Effects of dietary carbohydrate to lipid ratios on growth and body composition of juvenile and grower rockfish,Sebastes schlegeli[J].Aquaculture Research,2009,40(16):1830-1837.
[6]Hu Y H,Liu Y J,Tian L X,et al.Optimal dietary carbohydrate to lipid ratio for juvenile yellowfin seabream(Sparus latus)[J].Aquaculture Nutrition,2007,13(4):291-297.
[7]Moreira I S,Peres H,Couto A,et al.Temperature and dietary carbohydrate level effects on performance and metabolic utilisation of diets in European sea bass(Dicentrarchus labrax)juveniles[J].Aquaculture,2008,274(1):153-160.
[8]Wilson R P.Utilization of dietary carbohydrate by fish[J].Aquaculture,1994,124(1-4):67-80.
[9]Jones J I,Clemmons D R.Insulin-like growth factors and their binding protein:Biological actions[J].Endocrine Reviews,1995,16(1):3-34.
[10]Enes P,Sanchez-Gurmaches J,Navarro I,et al.Role of insulin and IGF-Ⅰon the regulation of glucose metabolism in European sea bass(Dicentrarchus labrax)fed with different dietary carbohydrate levels[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2010,157(4):346-353.
[11]李晋南,徐奇友,王常安,等.不同糖及糖水平对松浦镜鲤GH/IGF-Ⅰ基因表达和鱼体组成的影响[J].上海海洋大学学报,2015,24(4):489-495.Li J N,Xu Q Y,Wang C A,et al.Effects of different dietary carbohydrates and carbohydrate levels on GH/IGF-Ⅰm RNA expression and the fish body composition of juvenile mirror carp(Cyprinus carpio)[J].Journal of Shanghai Ocean University,2015,24(4):489-495(in Chinese).
[12]戈贤平,刘波,谢骏,等.饲料中不同碳水化合物水平对翘嘴红鲌生长及血液指标和糖代谢酶的影响[J].南京农业大学学报,2007,30(3):88-93.Ge X P,Liu B,Xie J,et al.Effect of different carbohydrate levels of dietary on growth,plasma biochemical indices and hepaticpancreas carbohydrate metabolic enzymes in topmouth culter(Erythroculter ilishaeformis Bleeker)[J].Journal of Nanjing Agricultural University,2007,30(3):88-93(in Chinese).
[13]Li J N,Xu Q Y,Wang C A,et al.Effects of dietary glucose and starch levels on the growth,haematological indices and hepatic hexokinase and glucokinase m RNAexpression of juvenile mirror carp(Cyprinus carpio)[J].Aquaculture Nutrition,2016,22(3):550-558.
[14]Panserat S,Plagnes-Juan E,Kaushik S.Gluconeogenic enzyme gene expression is decreased by dietary carbohydrates in common carp(Cyprinus carpio)and gilthead seabream(Sparus aurata)[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2002,1579(1):35-42.
[15]谭肖英,罗智,刘永坚.鱼类对饲料中糖的利用研究进展[J].中国饲料,2007(6):19-23.Tan X Y,Luo Z,Liu Y J.Review of carbohydrate utilization in fish feed[J].China Feed,2007(6):19-23(in Chinese).
[16]Furuichi M,Yone Y.Changes in activities of hepatic enzymes related to carbohydrate metabolism of fishes in glucose and insulin-glucose tolerance tests[J].Nippon Suisan Gakkaishi,1982,48(3):463-466.
[17]Panserat S,Médale F,Blin C,et al.Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout,gilthead seabream,and common carp[J].American Journal of Physiology-Regulatory,Integrative and Comparative Physiology,2000,278(5):R1164-R1170.
[18]Tranulis M A,Dregni O,Christophersen B,et al.A glucokinase-like enzyme in the liver of Atlantic salmon(Salmo salar)[J].Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,1996,114(1):35-39.
[19]Caseras A,Metón I,Vives C,et al.Nutritional regulation of glucose-6-phosphatase gene expression in liver of the gilthead sea bream(Sparus aurata)[J].British Journal of Nutrition,2002,88(6):607-614.
[20]Panserat S,Blin C,Médale F,et al.Molecular cloning,tissue distribution and sequence analysis of complete glucokinase c DNAs from gilthead seabream(Sparus aurata),rainbow trout(Oncorhynchus mykiss)and common carp(Cyprinus carpio)[J].Biochimica et Biophysica Acta(BBA)-General Subjects,2000,1474(1):61-69.
[21]Gao W,Liu Y J,Tian L X,et al.Effect of dietary carbohydrate-to-lipid ratios on growth performance,body composition,nutrient utilization and hepatic enzymes activities of herbivorous grass carp(Ctenopharyngodon idella)[J].Aquaculture Nutrition,2010,16(3):327-333.
[22]Li X F,Wang Y,Liu W B,et al.Effects of dietary carbohydrate/lipid ratios on growth performance,body composition and glucose metabolism of fingerling blunt snout bream Megalobrama amblycephala[J].Aquaculture Nutrition,2013,19(5):701-708.
[23]Hanson R W,Reshef L.Regulation of phosphoenolpyruvate carboxykinase(GTP)gene expression[J].Annual Review of Biochemistry,1997,66:581-611.
[24]Panserat S,Plagnes-Juan E,Brèque J,et al.Hepatic phosphoenolpyruvate carboxykinase gene expression is not repressed by dietary carbohydrates in rainbow trout(Oncorhynchus mykiss)[J].Journal of Experimental Biology,2001,204(2):359-365.
[25]Forsyth I A,Wallis M.Growth hormone and prolactinmolecular and functional evolution[J].Journal of Mammary Gland Biology and Neoplasia,2002,7(3):291-312.
[26]刘丽丽.胰岛素样生长因子I的研究进展[J].中国草食动物科学,2013,33(6):57-59.Liu L L.Pregress in insulin like growth factor I[J].China Herbivore Science,2013,33(6):57-59(in Chinese).