TNF-α对脂联素多聚化修饰及分泌调控的研究

发布时间：2018-09-12 20:22

【摘要】：脂联素是哺乳动物循环系统中含量最丰富的一种脂肪细胞因子。除了调节机体能量平衡外,脂联素还具有增强胰岛素敏感性,抵抗动脉粥样硬化和抵抗炎症的作用。脂联素过表达可降低血糖和改善胰岛素抵抗。相反,脂联素基因敲除可导致胰岛素抵抗和葡萄糖不耐受。肥胖个体血液中脂联素水平呈显著下降。脂联素在循环系统中主要以三种复合体的形式存在:三聚体(LMW),六聚体(MMW),和含有18-36个脂联素单体的高聚体(HMW)形式,HMW在总脂联素中的含量决定胰岛素的增敏能力。脂联素的多聚化依赖于分子间二硫键的形成,而蛋白质二硫键的形成发生在内质网中,内质网分子伴侣ERO1-Lα,Dsb A-L和ERp44在脂联素多聚体尤其是高聚体的组装和分泌过程中发挥重要的作用。作为一种内分泌器官,脂肪组织可分泌大量的细胞因子,如TNF-α,leptin,IL-6,和visfatin等。肥胖个体中巨噬细胞向脂肪组织的浸润可促进脂肪组织炎症环境的形成。TNF-α主要由成熟的脂肪细胞和巨噬细胞分泌,在糖脂代谢中发挥重要作用,是炎症导致的胰岛素抵抗过程中的一个重要的细胞因子。临床研究证实,尽管脂联素和TNF-α作为一种典型的脂肪组织分泌因子,但在肥胖和糖尿病患者的循环系统中,两者的水平则呈显著负相关。这是否因为TNF-α通过旁分泌或自分泌的方式直接调控脂联素的多聚化和分泌,已成为该领域关注的焦点。本文通过离体细胞、高脂饮食和重组TNF-α处理的活体模型,系统的研究了TNF-α对脂联素多聚化和分泌调控的分子机制,旨在回答脂联素和TNF-α同属脂肪组织分泌因子,但在肥胖患者循环系统中为何呈现显著的消涨关系。研究结果如下:1.通过高脂饮食和腹腔注射TNF-α的方法建立了胰岛素抵抗表型的小鼠模型,通过对小鼠模型各生理指标的检测和比较发现:高脂饲喂和TNF-α注射小鼠血糖和血浆胰岛素含量均显著升高;胰岛素抵抗指数分析表明,两组小鼠都表现出明显的胰岛素抵抗;与对照组相比,高脂饮食和腹腔注射TNF-α小鼠循环系统中脂联素的分泌量显著下降,HMW/总脂联素的比值也显著下降。2.TNF-α处理分化的3T3-L1脂肪细胞发现,脂联素分泌显著下降,HMW/总脂联素的比值也显著下降;Real-time和Western blot分析表明,TNF-α处理显著的抑制内质网分子伴侣ERO1-Lα和Dsb A-L的表达。3.PPARγ激动剂、抑制剂处理及PPARγ敲减实验证实,PPARγ可促进ERO1-Lα和Dsb A-L的转录。在克隆ERO1-Lα和Dsb A-L的启动子序列的基础上,生物信息预测及双荧光素酶报告基因实验结果表明,PPARγ可增强ERO1-Lα和Dsb A-L的启动子活性;染色质免疫共沉淀实验证实,PPARγ可特异地与ERO1-Lα和Dsb A-L启动子区-1685/-1533bp和-939/-815bp处的PPRE元件结合,调控ERO1-Lα和Dsb A-L基因的表达,TNF-α处理显著抑制PPARγ与相应PPRE元件结合。4.TNF-α可抑制ERp44的转录,超表达PPARγ不能挽救这种抑制效应;免疫共沉淀实验表明,TNF-α处理能增强ERp44与脂联素分子之间的相互作用。上述结果证明,TNF-α抑制转录因子PPARγ的表达,并通过启动子抑制内质网ERO1-Lα和Dsb A-L的表达,进而降低脂联素的多聚化水平,抑制脂联素的分泌,并通过ERp44对脂联素分子的滞留,从而导致肥胖个体循环系统中脂联素含量及多聚体形成的下调。
[Abstract]:Adiponectin is one of the most abundant adipocytokines in the circulatory system of mammals. In addition to regulating the body's energy balance, adiponectin also enhances insulin sensitivity, resists atherosclerosis and resists inflammation. Overexpression of adiponectin reduces blood sugar and improves insulin resistance. Conversely, adiponectin gene knockout can improve insulin resistance. Adiponectin is present in the circulatory system in the form of three major complexes: trimer (LMW), hexamer (MMW), and high polymer (HMW) containing 18-36 adiponectin monomers. The amount of HMW in total adiponectin determines the increase in insulin. Polymerization of adiponectin depends on the formation of intermolecular disulfide bonds. The formation of protein disulfide bonds occurs in the endoplasmic reticulum. The endoplasmic reticulum molecular chaperones ERO1-Lalpha, Dsb A-L and ERp44 play an important role in the assembly and secretion of adiponectin polymers, especially polymers. As an endocrine organ, adipose tissue is separable. The infiltration of macrophages into adipose tissue can promote the formation of inflammatory environment in adipose tissue. TNF-alpha is mainly secreted by mature adipocytes and macrophages, which play an important role in glucose and lipid metabolism and is one of the processes of insulin resistance induced by inflammation. Clinical studies have shown that although adiponectin and TNF-alpha are typical adipose tissue secreting factors, their levels are negatively correlated in the circulatory system of obese and diabetic patients. Is this because TNF-alpha directly regulates adiponectin polymerization and secretion by paracrine or autocrine means? In this paper, we systematically studied the molecular mechanism of TNF-alpha on adiponectin accumulation and secretion through in vitro cell, high-fat diet and recombinant TNF-alpha treatment in vivo. The aim is to answer why adiponectin and TNF-alpha are both adipose tissue secretory factors, but they are significant in the circulatory system of obese patients. The results are as follows: 1. Insulin resistance phenotype mice were established by high-fat diet and intraperitoneal injection of TNF-alpha. By testing and comparing the physiological indexes of the mice model, it was found that the blood glucose and plasma insulin content of the mice fed with high-fat diet and injected with TNF-alpha increased significantly. Compared with the control group, the secretion of adiponectin and the ratio of HMW to total adiponectin in the circulatory system of high fat diet and intraperitoneal injection of TNF-alpha decreased significantly. 2. TNF-alpha treated differentiated 3T3-L1 adipocytes showed a significant decrease in adiponectin secretion and HMW/total adiponectin ratio. Real-time and Western blot analysis showed that TNF-a treatment significantly inhibited the expression of ERO1-Lalpha and Dsb A-L. 3. PPAR gamma agonist, inhibitor treatment and PPAR gamma knockdown assay confirmed that PPAR gamma could promote the transcription of ERO1-Lalpha and Dsb A-L. Bioinformatics prediction and double luciferase reporter gene assay showed that PPAR gamma could enhance the promoter activity of ERO1-Lalpha and Dsb A-L. Chromatin immunoprecipitation assay confirmed that PPAR gamma could specifically bind to the PPRE elements in the promoter regions of ERO1-Lalpha and Dsb A-L, and regulate the expression of ERO1-Lalpha and Dsb A-L genes. TNF-alpha treatment significantly inhibited the binding of PPAR-gamma to the corresponding PPRE elements. 4. TNF-alpha could inhibit the transcription of ERP-44, but the over-expression of PPAR-gamma could not rescue the inhibitory effect. Immunocoprecipitation assay showed that TNF-alpha treatment could enhance the interaction between ERP-44 and adiponectin molecules. These results demonstrated that TNF-alpha inhibited the expression of PPAR-gamma and initiated it. Motors inhibit the expression of ERO1-Lalpha and Dsb A-L in the endoplasmic reticulum, thereby reducing the level of adiponectin polymerization and inhibiting the secretion of adiponectin. The retention of adiponectin molecules through ERp44 results in the down-regulation of adiponectin content and the formation of polymers in the circulatory system of obese individuals.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q78;R363

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