莱菔硫烷对脂肪细胞的代谢调控作用及分子机制研究
发布时间：2019-04-13 16:58
【摘要】：目的:以3T3-L1成熟脂肪细胞为模型,研究莱菔硫烷(sulforaphane,SFN)对3T3-L1成熟脂肪细胞糖、脂代谢的影响及其潜在的分子机制。方法:经典激素鸡尾酒(methylisobutylxanthine,dexamethasone and insulin,MDI)法诱导3T3-L1前脂肪细胞分化成为成熟脂肪细胞。油红O染色法直接观察3T3-L1前脂肪细胞在诱导分化过程中的脂质积聚情况,2-N[7-硝基苯-2-乙二酸,34羟氨基]-2-脱氧葡萄糖(2-NBDG)染色法检测诱导分化过程中脂肪细胞的葡萄糖摄取变化,以探索SFN干预的最佳时点。低浓度(0-10.0μmol/L)SFN干预3T3-L1成熟脂肪细胞48h,经2-NBDG染色后,分别用共聚焦和多功能酶标仪定性、定量检测SFN处理的成熟脂肪细胞内的葡萄糖摄取变化,蛋白印迹法和实时荧光定量PCR法分别检测SFN干预48h对葡萄糖摄取和代谢相关蛋白GLUT-4、GCK、CS及基因ACC、FAS表达的影响;甘油GPO-POD酶法检测成熟脂肪细胞经SFN处理后培养基中的甘油含量,间接评价SFN对白色脂肪细胞脂质水解的影响,蛋白印迹法检测SFN干预48h对白色脂肪细胞脂质分解代谢相关蛋白ATGL、HSL、p-HSL~(Ser563)、p-HSLSer565、p-HSL~(Ser660)、Perilipin、CPT1A、CS、DGAT-1表达的影响,综合评价SFN处理对3T3-L1成熟脂肪细胞葡萄糖和脂质代谢的影响,探讨SFN调控白色脂肪细胞的糖、脂代谢作用及分子机制。结果:诱导分化过程中的3T3-L1前脂肪细胞葡萄糖摄取和脂质积累逐渐增加,到分化第10天逐渐趋于稳定,故本课题选择诱导分化第10天的3T3-L1脂肪细胞作为本研究对象。2-NBDG染色显示,SFN可显著增强3T3-L1成熟脂肪细胞的葡萄糖摄取。甘油GPO-POD酶法检测提示,SFN干预可明显增加脂肪细胞的脂质水解作用。蛋白印迹法和实时荧光定量PCR法检测表明,SFN可上调葡萄糖摄取相关蛋白GLUT-4,葡萄糖有氧氧化相关蛋白GCK、CS,脂质水解相关蛋白HSL、p-HSL~(Ser563)、p-HSL~(Ser660)和脂肪酸β氧化相关蛋白CPT1A的表达,同时下调脂肪酸合成相关基因ACC、FAS,脂质水解相关蛋白p-HSLSer565、Perilipin,甘油三酯合成相关蛋白DGAT-1的表达。结论:低浓度(0-10μmol/L)SFN干预可通过上调葡萄糖摄取和代谢相关蛋白GLUT-4、GCK、CS的表达,增强3T3-L1成熟脂肪细胞的葡萄糖摄取和有氧氧化;上调脂质水解和脂肪酸β氧化相关蛋白HSL及磷酸化p-HSL~(Ser563)、p-HSL~(Ser660)和CPT1A水平,增强成熟脂肪细胞的脂质代谢,下调ACC、FAS基因表达和DGAT-1蛋白水平,抑制葡萄糖合成脂肪酸以及甘油三酯的再合成。上述结果表明,低浓度SFN干预可改善白色脂肪细胞糖、脂代谢作用,为以白色脂肪细胞为靶点,探索肥胖的防治策略提供科学依据。
[Abstract]:Aim: to study the effects of sulforaphane (sulforaphane,SFN) on glucose and lipid metabolism in 3T3-L1 mature adipocytes and its potential molecular mechanism using 3T3-L1 mature adipocytes as a model. Methods: 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes by classical hormone cocktail (methylisobutylxanthine,dexamethasone and insulin,MDI). Oil red O staining was used to observe the lipid accumulation of 3T3-L1 preadipocytes in the process of induced differentiation, 2N [7-nitrobenzene-2-oxalic acid], 34 hydroxyamino]-2-deoxyglucose (2-NBDG) staining was used to detect the changes of glucose uptake in adipocytes during induction and differentiation in order to explore the optimal time point of SFN intervention. 3T3-L1 mature adipocytes were treated with low concentration (0-10.0 渭 mol / L) SFN) for 48 h. After 2-NBDG staining, the glucose uptake in mature adipocytes treated with SFN was quantitatively detected by confocal and multi-functional enzyme labeling apparatus. Protein blot and real-time fluorescence quantitative PCR were used to detect the effects of SFN intervention for 48 h on glucose uptake and metabolism-related protein GLUT-4,GCK,CS and gene ACC,FAS expression. Glycerol GPO-POD method was used to detect the glycerol content in the medium of mature adipocytes treated with SFN, and the effect of SFN on lipid hydrolysis of white adipocytes was evaluated indirectly. Western blot assay was used to detect the effects of SFN on the expression of lipid catabolism-related proteins ATGL,HSL,p-HSL~ (Ser563), pJHSLSer565, pHSLSer660 and Perilipin,CPT1A,CS,DGAT-1 in white adipocytes after 48 h intervention. To evaluate the effects of SFN treatment on glucose and lipid metabolism in mature adipocytes of 3T3-L1, and to explore the effects and molecular mechanisms of SFN on glucose and lipid metabolism in white adipocytes. Results: glucose uptake and lipid accumulation of 3T3-L1 preadipocytes increased gradually during differentiation, and became stable on the 10th day of differentiation. In this study, 3T3-L1 adipocytes on the 10th day of differentiation were selected as the object of this study. 2-NBDG staining showed that SFN significantly enhanced glucose uptake of 3T3-L1 mature adipocytes. Glycerol GPO-POD enzyme assay suggested that SFN intervention could significantly increase the lipid hydrolysis of adipocytes. Protein blot and real-time fluorescence quantitative PCR assay showed that SFN could up-regulate glucose uptake-related protein GLUT-4, glucose oxidation-related protein GCK,CS, lipid hydrolysis-associated protein HSL,p-HSL~ (Ser563). The expression of CPT1A, a fatty acid 尾-oxidation-related protein, and the expression of lipid hydrolysis-related protein (ACC,FAS,) related to fatty acid synthesis genes ACC,FAS, perilipin and DGAT-1 were also down-regulated. The expression of Ser660 and 尾-oxidation-related protein DGAT-1 of fatty acid synthesis was also down-regulated. Conclusion: low concentration (0-10 渭 mol / L) SFN) can enhance glucose uptake and aerobic oxidation of 3T3-L1 mature adipocytes by up-regulating glucose uptake and GLUT-4,GCK,CS expression. By up-regulating lipid hydrolysis and fatty acid 尾-oxidation-associated protein HSL and phospho-hsl ~ (Ser563), phospho-HSL ~ (Ser660) and CPT1A levels, enhancing lipid metabolism of mature adipocytes and down-regulating ACC,FAS gene expression and DGAT-1 protein level, the expression of ACC,FAS gene and DGAT-1 protein were up-regulated in mature adipocytes. Inhibition of glucose synthesis of fatty acids and triglyceride re-synthesis. These results suggest that low concentration of SFN can improve glucose and lipid metabolism of white adipocytes and provide scientific basis for the prevention and treatment of obesity by targeting white adipocytes.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589.2
[Abstract]:Aim: to study the effects of sulforaphane (sulforaphane,SFN) on glucose and lipid metabolism in 3T3-L1 mature adipocytes and its potential molecular mechanism using 3T3-L1 mature adipocytes as a model. Methods: 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes by classical hormone cocktail (methylisobutylxanthine,dexamethasone and insulin,MDI). Oil red O staining was used to observe the lipid accumulation of 3T3-L1 preadipocytes in the process of induced differentiation, 2N [7-nitrobenzene-2-oxalic acid], 34 hydroxyamino]-2-deoxyglucose (2-NBDG) staining was used to detect the changes of glucose uptake in adipocytes during induction and differentiation in order to explore the optimal time point of SFN intervention. 3T3-L1 mature adipocytes were treated with low concentration (0-10.0 渭 mol / L) SFN) for 48 h. After 2-NBDG staining, the glucose uptake in mature adipocytes treated with SFN was quantitatively detected by confocal and multi-functional enzyme labeling apparatus. Protein blot and real-time fluorescence quantitative PCR were used to detect the effects of SFN intervention for 48 h on glucose uptake and metabolism-related protein GLUT-4,GCK,CS and gene ACC,FAS expression. Glycerol GPO-POD method was used to detect the glycerol content in the medium of mature adipocytes treated with SFN, and the effect of SFN on lipid hydrolysis of white adipocytes was evaluated indirectly. Western blot assay was used to detect the effects of SFN on the expression of lipid catabolism-related proteins ATGL,HSL,p-HSL~ (Ser563), pJHSLSer565, pHSLSer660 and Perilipin,CPT1A,CS,DGAT-1 in white adipocytes after 48 h intervention. To evaluate the effects of SFN treatment on glucose and lipid metabolism in mature adipocytes of 3T3-L1, and to explore the effects and molecular mechanisms of SFN on glucose and lipid metabolism in white adipocytes. Results: glucose uptake and lipid accumulation of 3T3-L1 preadipocytes increased gradually during differentiation, and became stable on the 10th day of differentiation. In this study, 3T3-L1 adipocytes on the 10th day of differentiation were selected as the object of this study. 2-NBDG staining showed that SFN significantly enhanced glucose uptake of 3T3-L1 mature adipocytes. Glycerol GPO-POD enzyme assay suggested that SFN intervention could significantly increase the lipid hydrolysis of adipocytes. Protein blot and real-time fluorescence quantitative PCR assay showed that SFN could up-regulate glucose uptake-related protein GLUT-4, glucose oxidation-related protein GCK,CS, lipid hydrolysis-associated protein HSL,p-HSL~ (Ser563). The expression of CPT1A, a fatty acid 尾-oxidation-related protein, and the expression of lipid hydrolysis-related protein (ACC,FAS,) related to fatty acid synthesis genes ACC,FAS, perilipin and DGAT-1 were also down-regulated. The expression of Ser660 and 尾-oxidation-related protein DGAT-1 of fatty acid synthesis was also down-regulated. Conclusion: low concentration (0-10 渭 mol / L) SFN) can enhance glucose uptake and aerobic oxidation of 3T3-L1 mature adipocytes by up-regulating glucose uptake and GLUT-4,GCK,CS expression. By up-regulating lipid hydrolysis and fatty acid 尾-oxidation-associated protein HSL and phospho-hsl ~ (Ser563), phospho-HSL ~ (Ser660) and CPT1A levels, enhancing lipid metabolism of mature adipocytes and down-regulating ACC,FAS gene expression and DGAT-1 protein level, the expression of ACC,FAS gene and DGAT-1 protein were up-regulated in mature adipocytes. Inhibition of glucose synthesis of fatty acids and triglyceride re-synthesis. These results suggest that low concentration of SFN can improve glucose and lipid metabolism of white adipocytes and provide scientific basis for the prevention and treatment of obesity by targeting white adipocytes.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R589.2
【参考文献】
相关期刊论文 前5条
1 Surapon Tangvarasittichai;;Oxidative stress,insulin resistance,dyslipidemia and type 2 diabetes mellitus[J];World Journal of Diabetes;2015年03期
2 谢述琼;何s，
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