外源性添加磷酸二羟基丙酮对小鼠脂肪细胞三酰甘油及关键代谢酶含量的影响
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摘要
旨在研究磷酸二羟基丙酮对小鼠脂肪细胞三酰甘油及其关键代谢酶的影响。将小鼠3T3-L1前脂细胞诱导成成熟的脂肪细胞,在诱导培养第14天,分别加入含0μmol·L~(-1)(ck组)、20μmol·L~(-1)(试验Ⅰ组)、50μmol·L~(-1)(试验Ⅱ组)、200μmol·L~(-1)(试验Ⅲ组)磷酸二羟基丙酮的完全培养基培养细胞,于0、36、72h收集细胞并进行ELISA及后续检测;利用双抗体夹心法检测三酰甘油(TG)代谢中关键酶含量,如乙酰辅酶A羧化酶(ACC)、脂肪酸合成酶(FAS)、激素敏感脂肪酶(HSL)等。结果表明:1)在36和72h,试验Ⅰ、Ⅱ、Ⅲ组合成的TG量极显著高于对照组(ck组)(P<0.01);试验各组葡萄糖-6-磷酸脱氢酶(G6PD)、二硫辛酰胺转乙酰基酶(DLAT)、二硫辛酸脱氢酶(DLD)和脂肪酸合成酶(FAS)含量均极显著高于对照组(P<0.01);2)在36和72h,试验各组激素敏感脂肪酶(HSL)、肉碱脂酰转移酶1(CPT1)和游离脂肪酸(FFA)含量均极显著低于对照组(P<0.01)。磷酸二羟基丙酮能够促进小鼠脂肪细胞三酰甘油的合成和沉积;磷酸二羟基丙酮还能极显著提高TG合成代谢中G6PD、DLAT、DLD、FAS的含量;能够极显著降低TG分解代谢中HSL、CPT1的含量。
The purpose of this study was to investigate the effect of exogenously added dihydroxyacetone phosphate on the contents of triglyceride and its key metabolic enzymes in mouse adipocytes.The inoculated mouse 3 T3-L1 cell into mature adipocytes were the research object,the cells were cultured in dihydroxyacetone phosphate complete medium containing 0μmol·L~(-1)(ck group),20μmol·L~(-1)(group Ⅰ),50μmol·L~(-1)(group Ⅱ)and 200μmol·L~(-1)(group Ⅲ)on the 14 th day,and the cells were collected at 0,36 and 72 hfor the detection of ELISA and other substances.The contents of key enzymes in triglyceride metabolism were detected by using double antibody sandwich technique,such as acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS),hormone-sensitive lipase(HSL)and other enzymes.The results showed that:1)The contents of triglyceride in the group Ⅰ,Ⅱ andⅢ were significantly higher than that in ck group(P<0.01)at 36 and 72 h;The contents of glucose-6-phosphate dehydrogenase(G6 PD),dihydrolipoyl transacetylase(DLAT),dihydrolipoyl dehydrogenase(DLD)and FAS in the group Ⅰ,Ⅱ and Ⅲwere significantly higher than that in ck group(P<0.01)at 36 and 72 h;2)The contents of HSL,carnitine acyltransferase1(CPT1)and free fatty acid(FFA)in the group Ⅰ,Ⅱ and Ⅲwere significantly lower than that in ck group(P<0.01)at 36 and 72 h.The dihydroxyacetone phosphate can promote the synthesis and deposition of triglyceride in mouse adipocytes;The dihydroxyacetone phosphate can also significantly increase the contents of G6 PD,DLAT,DLD and FAS in triglyceride synthesis,and can significantly reduce the contents of HSL and CPT1 in triglycerides catabolism.
The purpose of this study was to investigate the effect of exogenously added dihydroxyacetone phosphate on the contents of triglyceride and its key metabolic enzymes in mouse adipocytes.The inoculated mouse 3 T3-L1 cell into mature adipocytes were the research object,the cells were cultured in dihydroxyacetone phosphate complete medium containing 0μmol·L~(-1)(ck group),20μmol·L~(-1)(group Ⅰ),50μmol·L~(-1)(group Ⅱ)and 200μmol·L~(-1)(group Ⅲ)on the 14 th day,and the cells were collected at 0,36 and 72 hfor the detection of ELISA and other substances.The contents of key enzymes in triglyceride metabolism were detected by using double antibody sandwich technique,such as acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS),hormone-sensitive lipase(HSL)and other enzymes.The results showed that:1)The contents of triglyceride in the group Ⅰ,Ⅱ andⅢ were significantly higher than that in ck group(P<0.01)at 36 and 72 h;The contents of glucose-6-phosphate dehydrogenase(G6 PD),dihydrolipoyl transacetylase(DLAT),dihydrolipoyl dehydrogenase(DLD)and FAS in the group Ⅰ,Ⅱ and Ⅲwere significantly higher than that in ck group(P<0.01)at 36 and 72 h;2)The contents of HSL,carnitine acyltransferase1(CPT1)and free fatty acid(FFA)in the group Ⅰ,Ⅱ and Ⅲwere significantly lower than that in ck group(P<0.01)at 36 and 72 h.The dihydroxyacetone phosphate can promote the synthesis and deposition of triglyceride in mouse adipocytes;The dihydroxyacetone phosphate can also significantly increase the contents of G6 PD,DLAT,DLD and FAS in triglyceride synthesis,and can significantly reduce the contents of HSL and CPT1 in triglycerides catabolism.
引文
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[9]郑雅楠,陈少云,刘文洪,等.基于己糖激酶与葡萄糖-6-磷酸脱氢酶共表达的辅酶NADPH高效再生[J].微生物学通报,2016,43(12):2619-2626.ZHENG Y N,CHEN S Y,LIU W H,et al.Efficient NADPH regeneration based on co-expression of hexokinase and glucose-6-phosphate dehydrogenase[J].Microbiology China,2016,43(12):2619-2626.(in Chinese)
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[19]LORENTE-CEBRIAN S,KULYTE A,HEDEN P.Relationship between site-specific HSL phosphorylation and adipocyte lipolysis in obese women[J].Obes Facts,2011,4(5):365-371.
[20]XU C,HE J H,JIANG H F,et al.Direct effect of glucocorticoids on lipolysis in adipocytes[J].Mol Endocrinol,2009,23(8):1161-1170.
[21]CORNACIU I,BOESZOERMENYI A,LINDERMUTH H,et al.The minimal domain of adipose triglyceride lipase(ATGL)ranges until leucine 254and can be activated and inhibited by cgi-58and G0S2,respectively[J].PLoS One,2011,6(10):e26349.
[22]CHAKRABARTI P,KANDROR K V.FoxO1controls insulin-dependent adipose triglyceride lipase(ATGL)expression and lipolysis in adipocytes[J].J Biol Chem,2009,284(20):13296-13300.
[23]SERR J,SUH Y,LEE K.Regulation of adipose triglyceride lipase by fasting and refeeding in avian species[J].Poult Sci,2009,88(12):2585-2591.
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[2]杨少鹏.由甘油制备1,3-二羟基丙酮的合成研究[D].郑州:郑州大学,2016:3-11.YANG S P.Study on synthesis of 1,3-dihydroxyacetone from Glycerol[D].Zhengzhou:Zhengzhou University,2016:3-11.(in Chinese)
[3]李子杰.利用磷酸二羟基丙酮依赖型醛缩酶在体外和体内进行稀有单糖的合成[D].济南:山东大学,2012:3-19.LI Z J.Enzymatic synthesis of rare sugars with dihydroxyacetone phosphate-dependent aldolases in vitro and in vivo[D].Ji’nan:Shandong University,2012:3-19.(in Chinese)
[4]吴晓茹.利用磷酸二羟基丙酮依赖型醛缩酶和甘油激酶合成稀有糖[D].无锡:江南大学,2016:4-9.WU X R.Enzymatic synthesis of rare sugars with dihydroxyacetone phosphate-dependent aldolases and glycerol kinase[D].Wuxi:Jiangnan University,2016:4-9.(in Chinese)
[5]张文刚,黄家莺,顾欣,等.新型的饲料添加剂——二羟基丙酮[J].上海畜牧兽医通讯,2015(3):64-65.ZHANG W G,HUANG J Y,GU X,et al.The new feed additive-dihydroyacetone[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2015(3):64-65.(in Chinese)
[6]张文刚,吴昊旻,黄家莺,等.二羟基丙酮对肥育猪生长性能、胴体组成和肉质指标的影响[J].饲料研究,2017(4):1-3,40.ZHANG W G,WU H M,HUANG J Y,et al.Effect of dihydroyacetone on growth performance,carcass composition and meat quality of fatten pigs[J].Feed Research,2017(4):1-3,40.(in Chinese)
[7]郭秀玲,徐民岗,张秀丽,等.小鼠3T3-L1前脂肪细胞培养与诱导分化方法的建立[J].中国药物与临床,2013,13(12):1542-1544.GUO X L,XU M G,ZHANG X L,et al.A novel approach for culture and induced differentiation of mouse 3T3-L1preadipocytes[J].Chinese Remedies&Clinics,2013,13(12):1542-1544.(in Chinese)
[8]闫莉,许言午,王晓梁,等.双抗体夹心法检测3-硝基酪氨酸方法的建立及应用[J].中国应用生理学杂志,2009,25(4):569-572.YAN L,XU Y W,WANG X L,et al.Establishment and application of double-antibody sandwich Elisa for determination of 3-nitrotyrosine[J].Chinese Journal of Applied Physiology,2009,25(4):569-572.(in Chinese)
[9]郑雅楠,陈少云,刘文洪,等.基于己糖激酶与葡萄糖-6-磷酸脱氢酶共表达的辅酶NADPH高效再生[J].微生物学通报,2016,43(12):2619-2626.ZHENG Y N,CHEN S Y,LIU W H,et al.Efficient NADPH regeneration based on co-expression of hexokinase and glucose-6-phosphate dehydrogenase[J].Microbiology China,2016,43(12):2619-2626.(in Chinese)
[10]KHU L Y,STOREY K B,RUBTSOV A M,et al.Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus[J].Biochemistry(Mosc),2014,79(7):927-932.
[11]程钰蓉,孙志杰,崔球.丙酮酸脱氢酶竞争性抑制剂调控裂殖壶菌脂肪酸合成的研究[J].食品工业科技,2016,37(16):161-166.CHENG Y R,SUN Z J,CUI Q.Research of a specific competitive inhibitor of pyruvate dehydrogenase complex on Aurantiochytrium synthesizing fatty acids[J].Science and Technology of Food Industry,2016,37(16):161-166.(in Chinese)
[12]李亮,程彦伟.乙酰辅酶A羧化酶在治疗肥胖中的潜在作用[J].生命的化学,2007,27(2):180-182.LI L,CHENG Y W.The potential effects of acetylCoA carboxylase in curing obesity[J].Chemistry of Life,2007,27(2):180-182.(in Chinese)
[13]MAO J Q,DEMAYO F J,LI H G,et al.Liver-specific deletion of acetyl-CoA carboxylase 1reduces hepatic triglyceride accumulation without affecting glucose homeostasis[J].Proc Natl Acad Sci U S A,2006,103(22):8552-8557.
[14]BARBER M C,PRICE N T,TRAVERS M T.Structure and regulation of acetyl-CoA carboxylase genes of metazoa[J].Biochim Biophys Acta,2005,1733(1):1-28.
[15]刘作华,杨飞云,孔路军,等.日粮能量水平对生长育肥猪肌内脂肪含量以及脂肪酸合成酶和激素敏感脂酶mRNA表达的影响[J].畜牧兽医学报,2007,38(9):934-941.LIU Z H,YANG F Y,KONG L J,et al.Effects of dietary energy level on the content of intramuscular fat and mRNA expression for fatty acid synthase and hormone-sensitive lipase in growing-finishing pigs[J].Acta Veterinaria et Zootechnica Sinica,2007,38(9):934-941.(in Chinese)
[16]DENTIN R,BENHAMED F,PEGORIER J P,et al.Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation[J].J Clin Invest,2005,115(10):2843-2854.
[17]NESTEL P J.Fish oil and cardiovascular disease:Lipids and arterial function[J].Am J Clin Nutr,2000,71(S1):228S-231S.
[18]张晓图,杜晨红,丁小娟,等.多不饱和脂肪酸的生物学功能及其在动物生产中的应用[J].动物营养学报,2017,29(9):3059-3067.ZHANG X T,DU C H,DING X J,et al.Biological functions of polyunsaturated fatty acids and its application in animal production[J].Chinese Journal of Animal Nutrition,2017,29(9):3059-3067.(in Chinese)
[19]LORENTE-CEBRIAN S,KULYTE A,HEDEN P.Relationship between site-specific HSL phosphorylation and adipocyte lipolysis in obese women[J].Obes Facts,2011,4(5):365-371.
[20]XU C,HE J H,JIANG H F,et al.Direct effect of glucocorticoids on lipolysis in adipocytes[J].Mol Endocrinol,2009,23(8):1161-1170.
[21]CORNACIU I,BOESZOERMENYI A,LINDERMUTH H,et al.The minimal domain of adipose triglyceride lipase(ATGL)ranges until leucine 254and can be activated and inhibited by cgi-58and G0S2,respectively[J].PLoS One,2011,6(10):e26349.
[22]CHAKRABARTI P,KANDROR K V.FoxO1controls insulin-dependent adipose triglyceride lipase(ATGL)expression and lipolysis in adipocytes[J].J Biol Chem,2009,284(20):13296-13300.
[23]SERR J,SUH Y,LEE K.Regulation of adipose triglyceride lipase by fasting and refeeding in avian species[J].Poult Sci,2009,88(12):2585-2591.
[24]何微.辛伐他汀和非诺贝特对NAFLD大鼠ATGL酶和TNF-α表达调控的实验研究[D].广州:南方医科大学,2013:3-19.HE W.An experimental study on the regulation of ATGL and TNF-a for NAFLD rats by Fenofibrate and Simvastatin[D].Guangzhou:Southern Medical University,2013:3-19.(in Chinese)
[25]袁禹惠.饲料中脂肪及花生四烯酸水平对半滑舌鳎(Cynoglossus semilaevis)稚鱼生长、脂肪酸组成及代谢相关基因表达的影响[D].青岛:中国海洋大学,2015:2-9.YUAN Y H.Effects of dietary lipid and arachidonic acid level on growth performance,fatty acid composition and genes expression of metabolism related enzymes of larval half-smooth tongue sole,Cynoglossus semilaevis[D].Qingdao:Ocean University of China,2015:2-9.(in Chinese)
[26]胡伟.草鱼脂代谢相关酶的分离纯化、分子克隆、酶动力学性质及对铜和锌的响应研究[D].武汉:华中农业大学,2015:4-11.HU W.Isolation,purification,cloning,kinetic properties of enzymes related to lipid metabolism and response to copper and zinc in Ctenopharyngodon idella[D].Wuhan:Huazhong Agricultural University,2015:4-11.(in Chinese)
[27]董婧.肉碱对鲤鱼幼鱼脂肪酸代谢调控作用的研究[D].长春:吉林农业大学,2014:2-6.DONG J.Studies on regulatory mechanism of supplementing L-carnitine on fatty acids metabolism in juvenile common carp(Cyprinus carpio)[D].Changchun:Jilin Agricultural University,2014:2-6.(in Chinese)
[28]ABU-ELHEIGA L,MATZUK M M,ABO-HASHEMA K A H,et al.Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2[J].Science,2001,291(5513):2613-2616.
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