【作者】 韩春春；

【导师】 王继文； 李学伟； 龙乔明；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 肝内脂质沉积受到很多因素的影响,高能食物、葡萄糖、胰岛素、胆固醇和肝脏X受体(LXR)激活剂等都可以诱导哺乳动物肝脂肪变性,水禽的肝脂肪变性与哺乳动物有不同的特性,关于水禽肝脂肪变性的机制目前还不清楚。在组织水平,通过对朗德鹅和四川白鹅进行填饲高能碳水化合物检测了填饲对血浆葡萄糖、胰岛素和胆固醇浓度、肝内脂质含量及肝内三酰甘油(TG)含量、与脂肪酸和TG合成、及VLDL-TG(极低密度脂蛋白)组装与分泌相关基因的mRNA表达水平影响在两个品种间的差异；在细胞水平通过添加不同浓度的葡萄糖、胰岛素、胆固醇和LXR激活剂T0901317培养鹅原代肝细胞,检测生酯酶脂肪酸合成酶(FAS)和乙酰辅酶A羧化酶(ACC)酶活性、细胞内外TG含量及总TG含量、细胞外VLDL浓度、相关基因表达水平；并用western blot和凝胶电泳迁移实验(EMSA)方法检测了葡萄糖和胰岛素、胆固醇和LXR激活剂T0901317处理细胞后转录因子固醇调节元件结合蛋白-1 (SREBP-1)的蛋白变化及SREBP-1的核蛋白与靶基因ACCa的结合情况。主要结果如下：(1)填饲引起脂质大量沉积在鹅肝脏内,朗德鹅肝内脂质沉积强度要高于四川白鹅；填饲引起的血浆胰岛素和胆固醇水平的增加程度也存在品种差异,朗德鹅的变化大于四川白鹅(P<0.05)；而填饲后血浆葡萄糖在朗德鹅中上升,在四川白鹅中下降(P<0.05)。填饲后与脂肪酸合成相关的基因ACCa、FAS, SREBP-1和碳水化合物反应元件结合蛋白(ChREBP)在肝组织中的表达水平降低(P<0.05)；脂蛋白酯酶(LPL夕和LXRa mRNA表达量显著升高(P<0.05); VLDL-TG组装与分泌相关基因微粒体甘油三酯转运蛋白(MTTP)基因表达显著降低(P<0.05)；填饲诱导的基因表达水平的变化在两个品种中显著不同,SREBP-1, ChREBP和MTTP基因表达水平在朗德鹅中的降低程度大于四川白鹅(P＜O.05), LPL基因表达的升高程度在朗德鹅中大于四川白鹅(P<0.05)。(2)葡萄糖和胰岛素分别单独与肝细胞培养后显著增加生酯酶FAS和ACC的活性、脂肪酸合成相关基因FAS, ACCa、SREBP-1、ChREBP、LXRa和硬脂酰辅酶A去饱和酶1 (SCD1)的基因表达水平及细胞内TG含量,并且葡萄糖和胰岛素共同培养存在显著协同效应P<0.05)。另外,5 mmol/L葡萄糖和50 nmol/L胰岛素分别单独培养对SREBP-1的核蛋白浓度及SREBP-1核蛋白与靶基因ACCa探针的结合没有显著影响,但当5 mmol/L葡萄糖和50 nmol／L胰岛素共同培养时显著增加了他们的结合P<0.05)。葡萄糖和胰岛素单独培养鹅原代肝细胞都能显著提高细胞外TG和VLDL的浓度(P<0.05),同时对VLDL-TG合成与分泌相关基因的表达具有明显诱导作用。葡萄糖和胰岛素共同培养肝细胞,发现对甘油二酯酰基转移酶1(DFAT1)、DGAT2.叉头蛋白1(FoxO1)和MTTP的基因表达有显著协同增加作用(P<0.05),高胰岛素浓度和葡萄糖的诱导作用高于低胰岛素浓度和葡萄糖的作用(P<0.05)；葡萄糖和胰岛素共处理细胞对载脂蛋白B(apoB)的基因表达也有显著协同作用(P<0.05),但低浓度胰岛素和葡萄糖的诱导作用要高于高浓度胰岛素和葡萄糖的作用(P<0.05)；葡萄糖和胰岛素共处理对细胞外TG和VLDL的浓度的诱导作用也要高于其单独处理作用(P<0.05)。(3)胆固醇对细胞内TG含量、细胞外TG含量、总TG含量、细胞外胆固醇浓度和VLDL浓度的调控模式与其对脂肪合成相关基因(FAS.ACCa.SREBP.1.ChREBP. LXRa和SCD1)及VLDL-TG组装与分泌相关基因(DGAT2、MTTP和FoxO1)mRNA表达水平的调控模式类似。其中,10μg/mL胆固醇对以上各指标具有促进作用；20μg/mL的胆固醇的诱导作用最明显(P<0.05)；与20 gg／mL的胆固醇作用相比,30μg/mL的胆固醇表现出了对以上各检测指标的抑制作用(P<0.05)。胆固醇培养细胞能显著增加生酯酶FAS活性、SREBP-1的核蛋白浓度及SREBP-1核蛋白与靶基因ACCα探针的结合；胆固醇对DGAT1和apoB基因表达水平的调控与对其他基因的调控不同,对DGAT1基因表达水平的调控呈现剂量依赖模式,对apoB的mRNA水平没有显著影响(P>0.05)。(4)LXR激活剂(T0901317)以剂量依赖方式上调细胞内TG含量和总TG含量、上调生酯酶FAS和ACC活性和与脂肪酸合成有关基因(FAS.ACCa.SREBP-1. ChREBP、LXPa和SCD1)的mRNA表达,也同样上调LPL和DGAT1基因表达水平。另外,LXR激活剂(T0901317)可以增加SREBP-1的核蛋白浓度及SREBP-1核蛋白与靶基因ACCa探针的结合；LXR激活剂(T0901317)对细胞外TG和VLDL浓度的调控模式与对MTTP.DGAT2.FOxxO1和apoB基因水平的调控类似,随着T0901317浓度的增加,诱导作用增加,当T0901317浓度达到10μmol／L时,对基因水平的诱导效果降低,要低于1μmol/L T0901317处理组的水平。结论：填饲、葡萄糖、胰岛素、胆固醇和LXR激活剂(T0901317)都可以通过影响鹅肝细胞内脂肪酸和TG合成、VLDL-TG的组装和分泌诱导肝细胞内的脂质沉积,进而导致肝细胞脂肪变性；在葡萄糖、胰岛素、胆固醇和LXR激活剂(T0901317)诱导的肝脂肪酸合成中,可以通过SREBP-1激活靶基因ACCa的转录实现间接调控作用。更多还原

【Abstract】 The lipids deposition in liver can be affected by many factors. High energy food, glucose, insulin, cholesterol and LXRa agnonist all can induce hepatic steatosis in mammals. The feature of hepatic steatosis in waterfowl is different from that in mammals, and the mechanism of hepatic steatosis in waterfowls remains unclear yet. In this study, after overfeeding carbohydrate to Landes goose and Sichuan Landes goose, we measured the plasma level of glucose, insulin and cholesterol; lipid content in liver, hepatic TG content and mRNA expression of genes involved in the lipids metabolism in vivo; also measured the effect of glucose, insulin, cholesterol and LXRa agnonist on activities of lipogeneic enzymes FAS and ACC, total TG accumulation, intracellular and extracellular TG content and extracellular VLDL content, mRNA level of related genes in vitro; at last, we measured the SREBP-1 nuclear protein content using western blot andand the binding of SREBP-1 nuclear protein and SRE probe of ACCa gene using EMSA. There rsults are as followed:(1) Overfeeding induced the lipids deposition in goose liver, and the lipid deposition intensity of Lands goose was higher than that of Sichuan white goose; the increase of the plasma level of insulin and cholesterol induced by overfeeding showed a difference between the two breeds, and the increase of Lands goose was bigger than that of Sichuan white goose (P<0.05); the plasma glucose concentration increased in Landes goose after overfeeding, but decreased in Sichuan white goose (P<0.05). After overfeeding, the mRNA level of genes (ACCa, FAS, SREBP-1 and ChREBP)involved in the lipogenesis decreased in the two breeds, and the gene expression of LPL and LXRa increased (P＜0.05); the mRNA level of genes (MTTP) related with assembly and secretion of VLDL-TG decreased (P＜0.05); the change of mRNA level induced by overfeeding is different in the two breeds, and the decease of mRNA level of SREBP-1, ChREBP and MTTP in Lands goose is bigger than that in Sichuan white goose; the increase of mRNA level of LPL gene in Lands goose is bigger than that in Sichuan white goose.(2) Glucose and insulin cultured with hepatocytes separately evidently increase the activity of lipogenic enzyme FAS and ACC, mRNA level of lipogenic genes (FAS, ACCa, SREBP-1, ChREBP, LXRa and SCD1), and intracellular TG content (P<0.05). There was evident synergetic effect when glucose and insulin cultured together (P<0.05). In addition,5 mmol/L glucose and 50 nmol/L insulin cultured alone had no significant effect on the SREBP-1 nuclear protein levels and the binding of SREBP-1 nuclear protein with a target gene ACCa probe, but 5 mmol/L glucose and 50 nmol/L insulin cultured together significantly increased the binding (P<0.05). Glucose could increase the extracellular concentration of TG and VLDL (P<0.05), and evidently increase the mRNA level of genes related with VLDL-TG synthesis and secretion. Glucose and insulin cultured together with goose primary hepatocytes increased the mRNA level of DGAT1, DGAT2 and MTTP genes (P＜0.05), and the induction of high concentration of insulin and glucose was higher than the effect of low concentration of insulin and glucose (P<0.05); Glucose and insulin cultured together had synergetic effect on gene level of apoB, and the effect of low concentration of insulin and glucose was higher than the effect of high concentration of insulin and glucose(P<0.05); the effect of glucose and insulin cultured together on extracellelμlar TG and VLDL concentration was also higher than the effect when cultured with glucose and insulin seperatly (P<0.05).(3) The regulation mode of intracellular and extrcellular TG level, total TG level and extrcellular cholesterol level by cholesterol was similar with the regulation of mRNA level of lipogenic genes (FAS, ACCa, SREBP-1, ChREBP, LXRa and SCD1) and the genes involved in the assembly and secretion of VLDL-TG (DGAT2, MTTP and FoxO1).10μg/mL cholesterol up-regulated the level of the detected indexes; the induction by 20μg/mL cholesterol is most evident, and 30μg/mL cholesterol showed an inhibiting role (P＜0.05). The regulation of gene expression of DGAT1 and apoB by cholesterol is different from the regulation of other genes. Cholesterol could increase the mRNA level of DGAT1 gene in a dose-dependent manner, and had no evident effect on mRNA level of apoB gene (P>0.05).(4) LXR agonist (T0901317) increased intracellular TG content and total TG levels, activity of FAS and ACC, mRNA level of genes involved in lipogensis(FAS, ACCa, SREBP-1, ChREBP, LXRa and SCD1), and also increased the gene expression of LPL and DGAT1 genes in a dose-dependent manner. In addition, T0901317 could increase the concentration of SREBP-1 nuclear protein and the binding of SREBP-1 nuclear protein and its target gene ACCa probe. The regulation of extracellular concentration of TG and VLDL by T0901317 was similar with that of gene expression of MTTP, DGAT2, FoxO1 and apoB. With the increase of T0901317, the induction increased. But when the concentration of T0901317 reached 10μmol/L, the induction to gene expression decreased, and the gene level after the treatment of 10μmol/L T0901317 was lower than that in 1μmol/L T0901317.Conclusion:Overfeeding, culture with glucose, insulin, cholesterol and LXR agonist (T0901317) could induce the lipids deposition in goose hepatocytes by affecting the fatty acid and TG synthesis and VLDL-TG assembly and secretion, then could lead to hepatic steatosis; The induction of fatty acid synthesis by glucose, insulin and LXR agonist (T0901317) can be indirectly achieved by SREBP-1 activting transcription of its target gene ACCa.更多还原