杜仲叶对绵羊肝脏糖代谢及其相关基因表达的影响
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摘要
本试验旨在研究杜仲叶(EUL)对绵羊肝脏糖代谢及其相关基因表达的影响。随机选取6～8月龄、体重35～40 kg的绵羊(杜泊羊?×湖羊♀) 12只,平均分为2组,分别为对照组(CTL组)和EUL组(饲粮中添加10%EUL),每组6只。预试期10 d,正试期30 d。采集血液,测定糖代谢相关指标;采集肝脏组织,用于转录组学测序、糖代谢相关酶及核转录因子的mRNA及蛋白表达量分析。结果表明:1)与CTL组相比,EUL组血浆葡萄糖(GLU)、血清胰高血糖素(GC)含量显著降低(P<0.05),血清胰岛素(INS)含量显著升高(P<0.05)。2)转录组学检测结果表明,差异显著基因与糖代谢和能量代谢密切相关,且其在与糖代谢相关的通路中显著富集。3)荧光定量PCR检测结果表明,与CTL组相比,EUL组肝脏胰岛素受体(INSR)及胰岛素受体底物2(IRS2)的mRNA表达量极显著升高(P<0.01),胰高血糖素受体(GCGR)的mRNA表达量极显著降低(P<0.01);糖酵解关键酶磷酸果糖激酶(PFKL)的mRNA表达量显著上调(P <0.05);糖异生相关酶葡萄糖-6-磷酸酶(G 6Pase) mRNA表达量极显著下调(P <0.01),磷酸烯醇式丙酮酸羧激酶1(PEPCK1)的mRNA表达量显著下调(P<0.05);调控糖异生的叉头转录因子1(FoxO1)和环磷酸腺苷应答元件结合蛋白(CREB)的mRNA表达量均极显著下调(P <0.01);肝糖原合成关键酶糖原合酶2(G ys2)的mRNA表达量极显著上调(P <0.01)。4) Western blot结果显示,与CTL组相比,EUL组INSR蛋白表达量无显著变化(P> 0.05); FoxO1、G6Pase和PEPCK1蛋白表达量均极显著下调(P <0.01)。综上所述,EUL显著影响绵羊机体的糖代谢,提高糖酵解关键酶基因的表达,抑制糖异生相关转录因子及酶基因的表达,并促进肝糖原的合成,进而降低血糖。
In order to research the effects of Eucommia ulmoides leaves( EUL) on glucose metabolism and related genes expression in sheep live,12 healthy sheep( dorper sheep ♂×Hu lambs♀) with the similar age( 6 to 8-month-old) and body weight( between 35 to 40 kg) were selected and divided into two groups randomly and equally( 6 sheep in each group),which named control group( CTL group) and EUL group( diet contained 10% of EUL). The adaption period was 7 days,and the experiment period was 30 days. At the end of experiment,blood samples were collected to determine related biochemical indexes and hormones. After the experiment,all sheep were slaughtered and liver tissues were collected to transcriptome sequence,determine genes and protein expression levels for enzymes and factors involved in glucose metabolism. The results showed as follows: 1) compared with CTL group,plasma glucose and serum glucagon contents in EUL group significantly decreased( P<0.05),whereas serum insulin content increased significantly( P < 0.05). 2) The results of transcriptome analysis showed that the differentially significant genes related to glucose metabolism and energy metabolism,and significantly enriched in the glucose metabolic pathways. 3) Fluorescent quantitative PCR results showed that the mRNA expression levels of insulin receptor( INSR) and insulin receptor substrate 2( IRS2) increased significantly( P<0.01),the mRNA expression level of glucagon receptor( GCGR) dropped markedly( P<0.01); the phosphofructokinase( PFKL),the key enzyme in the glycolytic pathway,mRNA expression level up-regulated notably( P<0.05); on the gluconeogenesis pathway,the mRNA expression level of glucose-6-phosphatase( G 6 Pase) decreased significantly( P < 0. 01) and the mRNA expression level of phosphoenolpyruvate carboxykinase 1( PEPCK1) decreased significantly( P < 0.05); the mRNA expression levels of fork head transcription factor 1( FoxO1) and cyclic adenosine monophosphate response element binding protein( CREB) decreased significantly( P < 0. 01); furthermore,glycogen synthase 2( G ys2),which promoted glycogen synthesis,mRNA expression increased significantly( P < 0.01). 4) Western blot results showed that compared with CTL group,the protein expression level of INSR in EUL group had no significant change( P>0.05),but the protein expression levels of FoxO1、G6 Pase and PEPCK1 decreased significantly( P<0.01). In conclusion,EUL greatly affects glucose metabolism of sheep,increases the expression of the key enzyme genes for glycolysis,down-regulates the expression of gluconeogenesis related nuclear transcription factors and enzymes,promotes the synthesis of liver glycogen,and then decreases blood glucose.[Chinese Journal of Animal Nutrition,2019,31( 6) : 2854-2864]
In order to research the effects of Eucommia ulmoides leaves( EUL) on glucose metabolism and related genes expression in sheep live,12 healthy sheep( dorper sheep ♂×Hu lambs♀) with the similar age( 6 to 8-month-old) and body weight( between 35 to 40 kg) were selected and divided into two groups randomly and equally( 6 sheep in each group),which named control group( CTL group) and EUL group( diet contained 10% of EUL). The adaption period was 7 days,and the experiment period was 30 days. At the end of experiment,blood samples were collected to determine related biochemical indexes and hormones. After the experiment,all sheep were slaughtered and liver tissues were collected to transcriptome sequence,determine genes and protein expression levels for enzymes and factors involved in glucose metabolism. The results showed as follows: 1) compared with CTL group,plasma glucose and serum glucagon contents in EUL group significantly decreased( P<0.05),whereas serum insulin content increased significantly( P < 0.05). 2) The results of transcriptome analysis showed that the differentially significant genes related to glucose metabolism and energy metabolism,and significantly enriched in the glucose metabolic pathways. 3) Fluorescent quantitative PCR results showed that the mRNA expression levels of insulin receptor( INSR) and insulin receptor substrate 2( IRS2) increased significantly( P<0.01),the mRNA expression level of glucagon receptor( GCGR) dropped markedly( P<0.01); the phosphofructokinase( PFKL),the key enzyme in the glycolytic pathway,mRNA expression level up-regulated notably( P<0.05); on the gluconeogenesis pathway,the mRNA expression level of glucose-6-phosphatase( G 6 Pase) decreased significantly( P < 0. 01) and the mRNA expression level of phosphoenolpyruvate carboxykinase 1( PEPCK1) decreased significantly( P < 0.05); the mRNA expression levels of fork head transcription factor 1( FoxO1) and cyclic adenosine monophosphate response element binding protein( CREB) decreased significantly( P < 0. 01); furthermore,glycogen synthase 2( G ys2),which promoted glycogen synthesis,mRNA expression increased significantly( P < 0.01). 4) Western blot results showed that compared with CTL group,the protein expression level of INSR in EUL group had no significant change( P>0.05),but the protein expression levels of FoxO1、G6 Pase and PEPCK1 decreased significantly( P<0.01). In conclusion,EUL greatly affects glucose metabolism of sheep,increases the expression of the key enzyme genes for glycolysis,down-regulates the expression of gluconeogenesis related nuclear transcription factors and enzymes,promotes the synthesis of liver glycogen,and then decreases blood glucose.[Chinese Journal of Animal Nutrition,2019,31( 6) : 2854-2864]
引文
[1]马山,卢少海,田景振.杜仲药效成分和药理学的研究概况[J].食品与药品,2013,15(6):449-451.
[2]ZHOU J F,ZHANG T M,CHEN W A,et al.Comparative analysis of chemical components betw een barks and leaves of Eucommia ulmoides Oliver[J].Journal of Central South University,2009,16(3):371-379.
[3]DO M H,HUR J,CHOI J,et al.Eucommia ulmoides ameliorates glucotoxicity by suppressing advanced glycation end-products in diabetic mice kidney[J].Nutrients,2018,10(3):265.
[4]杨改青,王林枫,廉红霞,等.杜仲叶对绵羊营养物质消化利用、生长性能及屠宰性能的影响[J].动物营养学报,2017,29(4):1383-1391.
[5]孙燕荣,董俊兴,吕秋军,等.杜仲对脂肪细胞糖代谢的影响[J].中医药学刊,2004,22(8):1552-1553.
[6]ZHANG Y Y,ZHANG H X,WANG F,et al.The ethanol extract of Eucommia ulmoides Oliv.leaves inhibits disaccharidase and glucose transport in Caco-2 cells[J].Journal of Ethnopharmacology,2015,163:99-105.
[7]JIN X,AMITANI K,ZAMAMI Y,et al.Ameliorative effect of Eucommia ulmoides Oliv.leaves extract(ELE)on insulin resistance and abnormal perivascular innervation in fructose-drinking rats[J].Journal of Ethnopharmacology,2010,128(3):672-678.
[8]周芳,刘韶,邓梦如,等.GC-MS/SIM法测定不同产地杜仲叶中的氨基酸[J].中南药学,2012,10(4):257-260.
[9]LEE M K,KIM M J,CHO S Y,et al.Hypoglycemic effect of Duzhong(Eucommia ulmoides Oliv.)leaves in streptozotocin-induced diabetic rats[J].Diabetes Research and Clinical Practice,2005,67(1):22-28.
[10]PARK S A,CHOI M S,KIM M J,et al.Hypoglycemic and hypolipidemic action of Duzhong(Eucommia ulmoides Oliver)leaves w ater extract in C57BL/KsJ-db/db mice[J].Journal of Ethnopharmacology,2006,107(3):412-417.
[11]田吉,岳永花,秦大莲.杜仲叶降血糖作用的实验研究[J].现代医药卫生,2011,27(7):961-962.
[12]CAI Y,WANG Y,ZHI F,et al.The effect of sanggua drink extract on insulin resistance through the PI3K/AKT signaling pathw ay[J].Evidence-Based Complementary and Alternative M edicine,2018,2018:9407945.
[13]CHENG Z Y,TSENG Y,WHITE M.Insulin signaling meets mitochondria in metabolism[J].Trends in Endocrinology&M etabolism,2010,21(10):589-598.
[14]XIE W,YE Y L,FENG Y,et al.Linderane suppresses hepatic gluconeogenesis by inhibiting the cAM P/PKA/CREB pathw ay through indirect activation of PDE 3 via ERK/STAT3[J].Frontiers in Pharmacology,2018,9:476.
[15]杨杰.磷酸果糖激酶相关微小RNA影响肿瘤细胞糖代谢的机制研究[D].硕士学位论文.宁波:宁波大学,2015.
[16]WANG T,JIANG H M,CAO S J,et al.Baicalin and its metabolites suppresses gluconeogenesis through activation of AM PK or AKT in insulin resistant HepG-2cells[J].European Journal of M edicinal Chemistry,2017,141:92-100.
[17]XIONG X P,SONG Q,HAN C C,et al.Insulin promotes the expression of the gluconeogenic rate-limiting enzymes phosphoenolpyruvate carboxykinase(Pepck)and glucose 6-phosphatase(G6pase)through PI3k/Akt/mTOR signaling pathw ay in goose hepatocytes[J].Revista Brasileira de Ciência Avícola,2016,18(3):395-400.
[18]NAKAE J,KITAMURA T,SILVER D L,et al.The forkhead transcription factor Foxo1(Fkhr)confers insulin sensitivity onto glucose-6-phosphatase expression[J].Journal of Clinical Investigation,2001,108(9):1359-1367.
[19]张兰涛,常翠青,刘阳,等.氯原酸对db/db小鼠糖脂代谢紊乱的影响及其作用机制[J].中国医学科学院学报,2011,33(3):281-286.
[20]SHI Y N,QIAO J Y,MU B,et al.3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione(K145)ameliorated dexamethasone induced hepatic gluconeogenesis through activation of Akt/FoxO1 pathw ay[J].Biochemical and Biophysical Research Communications,2017,493(1):286-290.
[21]WANG Q,WANG N,DONG M,et al.GdCl3reduces hyperglycaemia through Akt/FoxO1-induced suppression of hepatic gluconeogenesis in Type 2 diabetic mice[J].Clinical Science,2014,127(2):91-100.
[22]SAMUEL V T,CHOI C S,PHILLIPS T G,et al.Targeting FoxO1 in mice using antisense oligonucleotide improves hepatic and peripheral insulin action[J].Diabetes,2006,55(7):2042-2050.
[23]SAMUEL V T,SHULMAN G I.The pathogenesis of insulin resistance:integrating signaling pathw ays and substrate flux[J].Journal of Clinical Investigation,2016,126(1):12-22.
[24]LEE J,KIM M S.The role of GSK3 in glucose homeostasis and the development of insulin resistance[J].Diabetes Research and Clinical Practice,2007,77(Suppl.3):S49-S57.
[25]XU H,ZHIOU Y,LIU Y X,et al.Metformin improves hepatic IRS2/PI3K/Akt signaling in insulin-resistant rats of NASH and cirrhosis[J].Journal of Endocrinology,2016,229(2):133-144.
[26]LIU T Y,SHI C X,GAO R,et al.Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K/Akt pathw ay in type 2 diabetic mice and hepatocytes[J].Clinical Science,2015,129(10):839-850.
[2]ZHOU J F,ZHANG T M,CHEN W A,et al.Comparative analysis of chemical components betw een barks and leaves of Eucommia ulmoides Oliver[J].Journal of Central South University,2009,16(3):371-379.
[3]DO M H,HUR J,CHOI J,et al.Eucommia ulmoides ameliorates glucotoxicity by suppressing advanced glycation end-products in diabetic mice kidney[J].Nutrients,2018,10(3):265.
[4]杨改青,王林枫,廉红霞,等.杜仲叶对绵羊营养物质消化利用、生长性能及屠宰性能的影响[J].动物营养学报,2017,29(4):1383-1391.
[5]孙燕荣,董俊兴,吕秋军,等.杜仲对脂肪细胞糖代谢的影响[J].中医药学刊,2004,22(8):1552-1553.
[6]ZHANG Y Y,ZHANG H X,WANG F,et al.The ethanol extract of Eucommia ulmoides Oliv.leaves inhibits disaccharidase and glucose transport in Caco-2 cells[J].Journal of Ethnopharmacology,2015,163:99-105.
[7]JIN X,AMITANI K,ZAMAMI Y,et al.Ameliorative effect of Eucommia ulmoides Oliv.leaves extract(ELE)on insulin resistance and abnormal perivascular innervation in fructose-drinking rats[J].Journal of Ethnopharmacology,2010,128(3):672-678.
[8]周芳,刘韶,邓梦如,等.GC-MS/SIM法测定不同产地杜仲叶中的氨基酸[J].中南药学,2012,10(4):257-260.
[9]LEE M K,KIM M J,CHO S Y,et al.Hypoglycemic effect of Duzhong(Eucommia ulmoides Oliv.)leaves in streptozotocin-induced diabetic rats[J].Diabetes Research and Clinical Practice,2005,67(1):22-28.
[10]PARK S A,CHOI M S,KIM M J,et al.Hypoglycemic and hypolipidemic action of Duzhong(Eucommia ulmoides Oliver)leaves w ater extract in C57BL/KsJ-db/db mice[J].Journal of Ethnopharmacology,2006,107(3):412-417.
[11]田吉,岳永花,秦大莲.杜仲叶降血糖作用的实验研究[J].现代医药卫生,2011,27(7):961-962.
[12]CAI Y,WANG Y,ZHI F,et al.The effect of sanggua drink extract on insulin resistance through the PI3K/AKT signaling pathw ay[J].Evidence-Based Complementary and Alternative M edicine,2018,2018:9407945.
[13]CHENG Z Y,TSENG Y,WHITE M.Insulin signaling meets mitochondria in metabolism[J].Trends in Endocrinology&M etabolism,2010,21(10):589-598.
[14]XIE W,YE Y L,FENG Y,et al.Linderane suppresses hepatic gluconeogenesis by inhibiting the cAM P/PKA/CREB pathw ay through indirect activation of PDE 3 via ERK/STAT3[J].Frontiers in Pharmacology,2018,9:476.
[15]杨杰.磷酸果糖激酶相关微小RNA影响肿瘤细胞糖代谢的机制研究[D].硕士学位论文.宁波:宁波大学,2015.
[16]WANG T,JIANG H M,CAO S J,et al.Baicalin and its metabolites suppresses gluconeogenesis through activation of AM PK or AKT in insulin resistant HepG-2cells[J].European Journal of M edicinal Chemistry,2017,141:92-100.
[17]XIONG X P,SONG Q,HAN C C,et al.Insulin promotes the expression of the gluconeogenic rate-limiting enzymes phosphoenolpyruvate carboxykinase(Pepck)and glucose 6-phosphatase(G6pase)through PI3k/Akt/mTOR signaling pathw ay in goose hepatocytes[J].Revista Brasileira de Ciência Avícola,2016,18(3):395-400.
[18]NAKAE J,KITAMURA T,SILVER D L,et al.The forkhead transcription factor Foxo1(Fkhr)confers insulin sensitivity onto glucose-6-phosphatase expression[J].Journal of Clinical Investigation,2001,108(9):1359-1367.
[19]张兰涛,常翠青,刘阳,等.氯原酸对db/db小鼠糖脂代谢紊乱的影响及其作用机制[J].中国医学科学院学报,2011,33(3):281-286.
[20]SHI Y N,QIAO J Y,MU B,et al.3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione(K145)ameliorated dexamethasone induced hepatic gluconeogenesis through activation of Akt/FoxO1 pathw ay[J].Biochemical and Biophysical Research Communications,2017,493(1):286-290.
[21]WANG Q,WANG N,DONG M,et al.GdCl3reduces hyperglycaemia through Akt/FoxO1-induced suppression of hepatic gluconeogenesis in Type 2 diabetic mice[J].Clinical Science,2014,127(2):91-100.
[22]SAMUEL V T,CHOI C S,PHILLIPS T G,et al.Targeting FoxO1 in mice using antisense oligonucleotide improves hepatic and peripheral insulin action[J].Diabetes,2006,55(7):2042-2050.
[23]SAMUEL V T,SHULMAN G I.The pathogenesis of insulin resistance:integrating signaling pathw ays and substrate flux[J].Journal of Clinical Investigation,2016,126(1):12-22.
[24]LEE J,KIM M S.The role of GSK3 in glucose homeostasis and the development of insulin resistance[J].Diabetes Research and Clinical Practice,2007,77(Suppl.3):S49-S57.
[25]XU H,ZHIOU Y,LIU Y X,et al.Metformin improves hepatic IRS2/PI3K/Akt signaling in insulin-resistant rats of NASH and cirrhosis[J].Journal of Endocrinology,2016,229(2):133-144.
[26]LIU T Y,SHI C X,GAO R,et al.Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K/Akt pathw ay in type 2 diabetic mice and hepatocytes[J].Clinical Science,2015,129(10):839-850.