不同生物素水平对3T3-L1脂肪细胞脂肪分解及相关基因表达的影响
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摘要
为研究不同生物素水平对3T3-L1脂肪细胞脂肪分解及相关基因表达的影响,本试验利用三联诱导法将3T3-L1细胞经诱导分化为3T3-L1脂肪细胞。当3T3-L1脂肪细胞密集后分别采用0(对照组)、0.2、0.5、1.0μmol/L生物素处理,分别在12、24与48h时检测细胞上清液中甘油释放量、脂肪甘油三酯水解酶(ATGL)、激素敏感性脂肪酶(HSL)、脂滴包被蛋白A(perilipin A)及脂肪分化相关蛋白(ADRP)相对表达量。结果显示,在12h时,1.0μmol/L组甘油释放量、ATGL基因mRNA相对表达量均极显著低于对照组(P<0.01),0.5、1.0μmol/L组HSL基因mRNA相对表达量显著低于对照组(P<0.05),1.0μmol/L组perilipin A、ADRP基因mRNA相对表达量均极显著高于对照组与0.2μmol/L组(P<0.01);在24h时,各试验组甘油释放量、1.0μmol/L组ATGL基因mRNA相对表达量均极显著低于对照组(P<0.01),0.5、1.0μmol/L组perilipin A基因mRNA相对表达量极显著高于对照组与0.2μmol/L组(P<0.01);1.0μmol/L组ADRP基因mRNA相对表达量极显著高于其他组(P<0.01);在48h时,1.0μmol/L组甘油释放量、ATGL基因mRNA相对表达量均极显著或显著低于对照组(P<0.01;P<0.05),1.0μmol/L组perilipin A基因mRNA相对表达量显著高于对照组与0.2μmol/L组(P<0.05),0.5、1.0μmol/L组ADRP基因mRNA相对表达量极显著高于对照组(P<0.01)。综上所述,生物素可通过促进perilipin A与ADRP表达,同时抑制ATGL与HSL表达,达到抑制脂肪分解的效果,且浓度为1.0μmol/L时效果最佳。
In order to explore the effects of different biotin levels on lipolysis and relevant gene expression of 3T3-L1 adipocytes,3T3-L1 cells were differentiate into 3T3-L1 adipocytes by the triplet induction method.3T3-L1 adipocytes were treated with 0(control group),0.2,0.5 and1.0μmol/L biotin upon being concentrated,and relative expression levels of ATGL,HSL,perilipin A and ADRP were measured at 12,24 and 48 h,respectively.The results showed that:At 12 h,the glycerol emission and ATGLgene mRNA relative expression of 1.0μmol/L group were ex-tremely significantly lower than that of control group(P<0.01).The HSLgene mRNA relative expression of 0.5 and 1.0μmol/L groups were significantly lower than that of control group(P<0.05),while the mRNA relative expression of perilipin A and ADRPgenes in 1.0μmol/L group were extremely significantly higher than that of control group and 0.2μmol/L group(P<0.01).At 24 h,the glycerol emission of three test groups and ATGLgene mRNA relative expression of 1.0μmol/L group were extremely significantly lower than that of control group(P<0.01).The perilipin A gene mRNA relative expression of 0.5 and 1.0μmol/L groups were extremely significantly higher than that of control group and 0.2μmol/L group(P<0.01),and the mRNA relative expression of ADRPgene of 1.0μmol/L group were extremely significantly higher than that of other groups(P<0.01).At 48 h,the glycerol emission and ATGLgene mRNA relative expression of 1.0μmol/L group were extremely significantly or significatly lower than that of control group(P<0.01;P<0.05).The perilipin A gene mRNA relative expression of1.0μmol/L group was significantly higher than that of control group and 0.2μmol/L group(P<0.05),and the mRNA relative expression of ADRPgene of 0.5 and 1.0μmol/L groups were extremely significantly higher than that of control group(P<0.01).In conclusion,the biotin could inhibit the lipolysis through promoting the relative expression of perilipin A and ADRP and reducing the relative expression of ATGL and HSL.Moreover,when the concentration was 1.0μmol/L,the effect was optimal.
In order to explore the effects of different biotin levels on lipolysis and relevant gene expression of 3T3-L1 adipocytes,3T3-L1 cells were differentiate into 3T3-L1 adipocytes by the triplet induction method.3T3-L1 adipocytes were treated with 0(control group),0.2,0.5 and1.0μmol/L biotin upon being concentrated,and relative expression levels of ATGL,HSL,perilipin A and ADRP were measured at 12,24 and 48 h,respectively.The results showed that:At 12 h,the glycerol emission and ATGLgene mRNA relative expression of 1.0μmol/L group were ex-tremely significantly lower than that of control group(P<0.01).The HSLgene mRNA relative expression of 0.5 and 1.0μmol/L groups were significantly lower than that of control group(P<0.05),while the mRNA relative expression of perilipin A and ADRPgenes in 1.0μmol/L group were extremely significantly higher than that of control group and 0.2μmol/L group(P<0.01).At 24 h,the glycerol emission of three test groups and ATGLgene mRNA relative expression of 1.0μmol/L group were extremely significantly lower than that of control group(P<0.01).The perilipin A gene mRNA relative expression of 0.5 and 1.0μmol/L groups were extremely significantly higher than that of control group and 0.2μmol/L group(P<0.01),and the mRNA relative expression of ADRPgene of 1.0μmol/L group were extremely significantly higher than that of other groups(P<0.01).At 48 h,the glycerol emission and ATGLgene mRNA relative expression of 1.0μmol/L group were extremely significantly or significatly lower than that of control group(P<0.01;P<0.05).The perilipin A gene mRNA relative expression of1.0μmol/L group was significantly higher than that of control group and 0.2μmol/L group(P<0.05),and the mRNA relative expression of ADRPgene of 0.5 and 1.0μmol/L groups were extremely significantly higher than that of control group(P<0.01).In conclusion,the biotin could inhibit the lipolysis through promoting the relative expression of perilipin A and ADRP and reducing the relative expression of ATGL and HSL.Moreover,when the concentration was 1.0μmol/L,the effect was optimal.
引文
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[4]ZHAO X,GAO M,HE J,et al.Perilipin1deficiency in whole body or bone marrow-derived cells attenuates lesions in atherosclerosis-prone mice[J].PLoS One,2015,10(4):1537.
[5]WALTHER T C.Lipid droplets and cellular lipid metabolism[J].Annual Review of Biochemistry,2012,81(1):687-714.
[6]郑巍振.生物素高产菌种选育的研究[D].杭州:浙江工业大学,2010.ZHENG W Z.Study on strain screening of high biotin-producing[D].Hangzhou:Zhejiang University of Technology,2010.(in Chinese)
[7]FERNANDEZ-MEJIA C.Biological effects of pharmacological concentrations of biotin[J].Journal of Evidence-Based Complementary&Alternative Medicine,2011,16(1):40-48.
[8]张旭晖,王恬.生物素营养生理作用及其应用研究进展[J].中国饲料,2010,8:5-8.ZHANG X H,WANG T.Biophysical nutrient physiological function and its application research progress[J].China Feed,2010,8:5-8.(in Chinese)
[9]FOUFELLE F,FERRP.New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose:A role for the transcription factor sterol regulatory element binding protein-1c[J].Biochemical Journal,2002,366(2):377-391.
[10]朱晓波,闫智宏,徐志伟,等.生物素对高糖状态下大鼠胰岛β细胞功能的影响[J].华西药学杂志,2010,25(1):29-31.ZHU X B,YAN Z H,XU Z W,et al.The effect of biotin on the function of rat isletβcell cultured with high glucose[J].West China Journal of Pharmaceutical Sciences,2010,25(1):29-31.(in Chinese)
[11]李萍,岳晶晶,张达,等.小檗碱对3T3-L1脂肪细胞炎症因子、脂肪因子及脂肪酸代谢的影响[J].天津医药,2014,42(6):513-516.LI P,YU J J,ZHANG D,et al.Effects of berberine on inflammatory factors,adipokines and fatty acid metabolism in 3T3-L1 adipocytes[J].Tianjin Medical Journal,2014,42(6):513-516.(in Chinese)
[12]赵艳坤,邵伟,雒诚龙,等.无血清条件下UC-MSCs和BMECs共培养对IGF-Ⅰ表达的影响[J].中国畜牧兽医,2017,44(2):411-416.ZHAO Y K,SHAO W,LUO C L,et al.Effect of umbilical cord mesenchymal stem cells and bovune mammary gland epithelial cells co-culture under the serumfree condition on expression of IGF-Ⅰ[J].China Animal Husbandry&Veterinary Medicine,2017,44(2):411-416.(in Chinese)
[13]赵艳坤.注射脐带间充质干细胞对荷斯坦公犊生长性能及血清生化指标的影响[D].乌鲁木齐:新疆农业大学,2015.ZHAO Y K.Effects on growth performance and biochemical indexes in serum of holstein bull calf after injecting umbilical cord mesenchymal stem cells[D].Urumqi:Xinjiang Agricultural University,2015.(in Chinese)
[14]王镜岩.生物化学[M].北京:高等教育出版社,2002.WANG J Y.Biochemistry[M].Beijing:Higher Education Press,2002.(in Chinese)
[15]郑雪莉,李玉成,鞠大鹏,等.TNF-α对猪脂肪细胞脂肪分解及其相关基因转录表达的影响[J].畜牧兽医学报,2009,40(9):1290-1296.ZHENG X L,LI Y C,JU D P,et al.TNF-αstimulates lipolysis in differentiated porcine adipocytes and down regulated expression of the genes related to lipolysis[J].Chinese Journal of Animal and Veterinary Sciences,2009,40(9):1290-1296.(in Chinese)
[16]李玉成,郑雪莉,杨公社.Leptin对猪原代脂肪细胞脂解及其关键脂酶mRNA表达的影响[J].生物工程学报,2008,24(9):1613-1619.LI Y C,ZHENG X L,YANG G S.Effects of leptin on porcine primary adiocytes lipolysis and mRNA expression of key lipolytic enzymes[J].Chinese Journal of Biotechnology,2008,24(9):1613-1619.(in Chinese)
[17]杜琛,李金泉,陈秀娟.绒山羊不同肌肉组织及肌内脂肪细胞的基因表达分析[J].中国畜牧兽医,2016,43(2):285-295.DU C,LI J Q,CHEN X J.Gene expression analysis on different muscles and intramuscular adipocytes in cashmere goat[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):285-295.(in Chinese)
[18]刘英华.中链脂肪酸对肥胖小鼠脂代谢的调节作用及机制研究[D].北京:中国人民解放军军事医学科学院,2012.LIU Y H.Effects of regulation by medium-chain fatty acids on lipid metabolism and their mechanisms in C57BL/6Jobese mice[D].Beijing:Academy of Military Medical Sciences,2012.(in Chinese)
[19]TANSEY J T,SZTALRYD C,HLAVIN E M,et al.The central role of perilipin a in lipid metabolism and adipocyte lipolysis[J].Iubmb Life,2004,56(7):379-385.
[20]梁静.母猪日粮蛋白水平对仔猪脂肪沉积的影响及leptin介导的脂肪代谢调控研究[D].南京:南京农业大学,2010.LIANG J.Effect of maternal dietary protein level on fat deposition in the piglets at weaning and the research of lipid metabolism regulation mediated by leptin[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese)
[21]赵志武,王君实,马敏,等.下调perilipin 1基因表达对3T3-L1细胞脂解的影响[J].中国生物工程杂志,2016,36(3):17-22.ZHAO Z W,WANG J S,MA M,et al.Effect of down-regulated perilipin1gene expression on lipolysis of 3T3-L1adipocytes[J].China Biotechnology,2016,36(3):17-22.(in Chinese)
[22]杨永青.Perilipin A调控慢性高剂量胰岛素和白细胞介素-6刺激的猪脂肪细胞脂肪分解及其分子机制[D].杨凌:西北农林科技大学,2008.YANG Y Q.Perilipin A regulates lipolysis stimulated by chronic high insulin and IL-6level and the molecular mechanisms[D].Yangling:Northwest A&F University,2008.(in Chinese)
[23]AL D A,PERREAU C,TSIKIS G,et al.Effect of different culture systems on adipocyte differentiation-related protein(ADRP)in bovine embryos[J].Animal Reproduction Science,2014,145(3-4):105-113.
[24]刘梅芳,徐国恒.PAT家族蛋白在细胞内脂滴代谢过程中的作用[J].生理科学进展,2006,37(2):103-107.LIU M F,XU G H.Function of PAT family proteins in the lipid metabolism[J].Progress in Physiological Sciences,2006,37(2):103-107.(in Chinese)
[25]IMAMURA M,INOGUCHI T,IKUYAMA S,et al.ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts[J].American Journal of Physiology-Endocrinology and Metabolism,2002,283(4):775-783.
[26]LISTENBERGER L L,OSTERMEYERFAY A G,GOLDBERG E B,et al.Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover[J].Journal of Lipid Research,2007,48(12):2751-2755.
[2]MACPHERSON R E K,RAMOS S V,VANDENBOOM R,et al.Skeletal muscle PLIN proteins,ATGL and CGI-58,interactions at rest and following stimulated contraction[J].American Journal of Physiology Regulatory Integrative&Comparative Physiology,2013,304(8):644-650.
[3]余康.奶山羊ADRP基因对GMEC脂滴的影响及其分子机制的初步研究[D].杨凌:西北农林科技大学,2014.YU K.Effects of ADRP gene of dairy goat to lipid droplets in GMEC(goat mammary gland epithelial cells)and its preliminary molecular mechanism exploration[D].Yangling:Northwest A&F University,2014.(in Chinese)
[4]ZHAO X,GAO M,HE J,et al.Perilipin1deficiency in whole body or bone marrow-derived cells attenuates lesions in atherosclerosis-prone mice[J].PLoS One,2015,10(4):1537.
[5]WALTHER T C.Lipid droplets and cellular lipid metabolism[J].Annual Review of Biochemistry,2012,81(1):687-714.
[6]郑巍振.生物素高产菌种选育的研究[D].杭州:浙江工业大学,2010.ZHENG W Z.Study on strain screening of high biotin-producing[D].Hangzhou:Zhejiang University of Technology,2010.(in Chinese)
[7]FERNANDEZ-MEJIA C.Biological effects of pharmacological concentrations of biotin[J].Journal of Evidence-Based Complementary&Alternative Medicine,2011,16(1):40-48.
[8]张旭晖,王恬.生物素营养生理作用及其应用研究进展[J].中国饲料,2010,8:5-8.ZHANG X H,WANG T.Biophysical nutrient physiological function and its application research progress[J].China Feed,2010,8:5-8.(in Chinese)
[9]FOUFELLE F,FERRP.New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose:A role for the transcription factor sterol regulatory element binding protein-1c[J].Biochemical Journal,2002,366(2):377-391.
[10]朱晓波,闫智宏,徐志伟,等.生物素对高糖状态下大鼠胰岛β细胞功能的影响[J].华西药学杂志,2010,25(1):29-31.ZHU X B,YAN Z H,XU Z W,et al.The effect of biotin on the function of rat isletβcell cultured with high glucose[J].West China Journal of Pharmaceutical Sciences,2010,25(1):29-31.(in Chinese)
[11]李萍,岳晶晶,张达,等.小檗碱对3T3-L1脂肪细胞炎症因子、脂肪因子及脂肪酸代谢的影响[J].天津医药,2014,42(6):513-516.LI P,YU J J,ZHANG D,et al.Effects of berberine on inflammatory factors,adipokines and fatty acid metabolism in 3T3-L1 adipocytes[J].Tianjin Medical Journal,2014,42(6):513-516.(in Chinese)
[12]赵艳坤,邵伟,雒诚龙,等.无血清条件下UC-MSCs和BMECs共培养对IGF-Ⅰ表达的影响[J].中国畜牧兽医,2017,44(2):411-416.ZHAO Y K,SHAO W,LUO C L,et al.Effect of umbilical cord mesenchymal stem cells and bovune mammary gland epithelial cells co-culture under the serumfree condition on expression of IGF-Ⅰ[J].China Animal Husbandry&Veterinary Medicine,2017,44(2):411-416.(in Chinese)
[13]赵艳坤.注射脐带间充质干细胞对荷斯坦公犊生长性能及血清生化指标的影响[D].乌鲁木齐:新疆农业大学,2015.ZHAO Y K.Effects on growth performance and biochemical indexes in serum of holstein bull calf after injecting umbilical cord mesenchymal stem cells[D].Urumqi:Xinjiang Agricultural University,2015.(in Chinese)
[14]王镜岩.生物化学[M].北京:高等教育出版社,2002.WANG J Y.Biochemistry[M].Beijing:Higher Education Press,2002.(in Chinese)
[15]郑雪莉,李玉成,鞠大鹏,等.TNF-α对猪脂肪细胞脂肪分解及其相关基因转录表达的影响[J].畜牧兽医学报,2009,40(9):1290-1296.ZHENG X L,LI Y C,JU D P,et al.TNF-αstimulates lipolysis in differentiated porcine adipocytes and down regulated expression of the genes related to lipolysis[J].Chinese Journal of Animal and Veterinary Sciences,2009,40(9):1290-1296.(in Chinese)
[16]李玉成,郑雪莉,杨公社.Leptin对猪原代脂肪细胞脂解及其关键脂酶mRNA表达的影响[J].生物工程学报,2008,24(9):1613-1619.LI Y C,ZHENG X L,YANG G S.Effects of leptin on porcine primary adiocytes lipolysis and mRNA expression of key lipolytic enzymes[J].Chinese Journal of Biotechnology,2008,24(9):1613-1619.(in Chinese)
[17]杜琛,李金泉,陈秀娟.绒山羊不同肌肉组织及肌内脂肪细胞的基因表达分析[J].中国畜牧兽医,2016,43(2):285-295.DU C,LI J Q,CHEN X J.Gene expression analysis on different muscles and intramuscular adipocytes in cashmere goat[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):285-295.(in Chinese)
[18]刘英华.中链脂肪酸对肥胖小鼠脂代谢的调节作用及机制研究[D].北京:中国人民解放军军事医学科学院,2012.LIU Y H.Effects of regulation by medium-chain fatty acids on lipid metabolism and their mechanisms in C57BL/6Jobese mice[D].Beijing:Academy of Military Medical Sciences,2012.(in Chinese)
[19]TANSEY J T,SZTALRYD C,HLAVIN E M,et al.The central role of perilipin a in lipid metabolism and adipocyte lipolysis[J].Iubmb Life,2004,56(7):379-385.
[20]梁静.母猪日粮蛋白水平对仔猪脂肪沉积的影响及leptin介导的脂肪代谢调控研究[D].南京:南京农业大学,2010.LIANG J.Effect of maternal dietary protein level on fat deposition in the piglets at weaning and the research of lipid metabolism regulation mediated by leptin[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese)
[21]赵志武,王君实,马敏,等.下调perilipin 1基因表达对3T3-L1细胞脂解的影响[J].中国生物工程杂志,2016,36(3):17-22.ZHAO Z W,WANG J S,MA M,et al.Effect of down-regulated perilipin1gene expression on lipolysis of 3T3-L1adipocytes[J].China Biotechnology,2016,36(3):17-22.(in Chinese)
[22]杨永青.Perilipin A调控慢性高剂量胰岛素和白细胞介素-6刺激的猪脂肪细胞脂肪分解及其分子机制[D].杨凌:西北农林科技大学,2008.YANG Y Q.Perilipin A regulates lipolysis stimulated by chronic high insulin and IL-6level and the molecular mechanisms[D].Yangling:Northwest A&F University,2008.(in Chinese)
[23]AL D A,PERREAU C,TSIKIS G,et al.Effect of different culture systems on adipocyte differentiation-related protein(ADRP)in bovine embryos[J].Animal Reproduction Science,2014,145(3-4):105-113.
[24]刘梅芳,徐国恒.PAT家族蛋白在细胞内脂滴代谢过程中的作用[J].生理科学进展,2006,37(2):103-107.LIU M F,XU G H.Function of PAT family proteins in the lipid metabolism[J].Progress in Physiological Sciences,2006,37(2):103-107.(in Chinese)
[25]IMAMURA M,INOGUCHI T,IKUYAMA S,et al.ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts[J].American Journal of Physiology-Endocrinology and Metabolism,2002,283(4):775-783.
[26]LISTENBERGER L L,OSTERMEYERFAY A G,GOLDBERG E B,et al.Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover[J].Journal of Lipid Research,2007,48(12):2751-2755.