PGC-1α调节黄体生成素和醛固酮合成以及葡萄糖激酶表达的分子机制研究

发布时间：2018-05-05 17:07

本文选题：垂体 + 黄体生成素　；参考：《中国协和医科大学》2010年博士论文

【摘要】： 第一部分PGC-1α协同SF-1调节黄体生成素和醛固酮合成的研究过氧化物酶体增殖激活受体γ共激活因子1α(PGC-1α)能共刺激多种转录因子从而调节能量代谢进程。核受体类固醇激素生成因子(SF-1)在垂体、性腺、肾上腺等内分泌组织中高表达,转录调控多种关键基因,同时调节下丘脑-垂体-性腺轴多水平的激素合成。为探索PGC-1α在类固醇激素合成及垂体激素分泌中的作用,本课题以小鼠垂体促性腺αT3-1细胞为研究模型,过表达PGC-1α后,通过real-time PCR实验检测到LHβ和αGSU在水mRNA平的表达量升高,又进一步通过酶联免疫吸附试验(ELISA)证明,PGC-1α刺激αT3-1细胞分泌的黄体生成素增加。分析LHp启动子序列,发现-127 bp～-119 bp位点存在一个SF-1结合元件突变该元件后,双荧光报告实验证明SF-1和PGC-1α对LHβ启动子的刺激作GSE (gonadotrope-specific element),用全部消失。染色质免疫沉淀实验证明PGC-1α与SF-1共定位在LHβ启动子区含GSE元件的区域,并促进LHβ转录。为进一步证明PGC-1α对SF-1的共刺激作用,分析了二者相互作用的分子机制。哺乳动物双杂交实验和免疫共沉淀实验证明PGC-1α与SF-1能在体内形成复合物,进一步实验证明二者能直接相互作用。该作用GST pull-down发生在PGC-1α的N端1～180氨基酸区域(含LXXLL模体,142～146氨基酸)与SF-1 C端187～462氨基酸区之间。构建不同长度的SF-1基因表达片段发现,只有当SF-1的最适激活结构域和AF-2结(proximal activation domain)构域同时存在时,PGC-1α和SF-1才能直接结合。肾上腺皮质是最主要的类固醇激素合成部位,细胞色素P450家族成员在此参与胆固醇代谢、类固醇激素合成的多个催化反应。Cyp11b2基因编码的蛋白质产物负责催化醛固酮合成的最后三步反应,是醛固酮合成的限速酶。在小鼠肾上腺皮质Y-1细胞中,real-time PCR实验证明,PGC-1α能促使Cyp11b2,Cyp11b1和P450scc基因在mRNA水平表达上调,ELISA实验进一步检测到PGC-1α协同SF-1促进细胞醛固酮的分泌。另一方面,通过双荧光报告实验检测到PGC-1α与雌激素受体相关受体家族成员ERRα和ERRγ共同促进SF-1转录,在过表达PGC-1α的αT3-1细胞和Y-1细胞中,SF-1在mRNA水平和蛋白质水平的表达量均显著提高。综上所述,本课题揭示了PGC-1α在类固醇激素和促性腺激素合成中具有重要的调节作用,分析了其刺激激素合成的分子机制,提示PGC-1α可能通过SF-1调节性腺和肾上腺的发育以及性别分化。第二部分PGC-1α协同ERRα调节葡萄糖激酶表达的研究过氧化物酶体增殖激活受体γ共激活因子1α(PGC-1α)是细胞能量代谢的关键调节子。它通过共激活多种核受体和转录因子,调节适应性产热、肝糖异生、脂肪酸氧化及线粒体生物合成等生理进程。葡萄糖激酶是一种存在于哺乳动物肝脏和胰腺中分子量为50 KDa的蛋白质,也被称为Ⅳ型己糖激酶,是糖酵解过程中第一个限速酶。它在调节肝脏葡萄糖利用中发挥重要的作用,并接受胰岛素信号刺激。本课题研究发现,大鼠葡萄糖激酶(glucokinase,Gck)启动子区存在一个保守的雌激素受体相关受体的结合元件ERRE(-52 bp～-39 bp),双荧光报告实验分析了截短和位点突变的Gck启动子,证明PGC-1α和ERRα能利用该元件刺激Gck启动子的活性。EMSA实验进一步证明ERRα能在体外结合ERRE元件,ChIP实验则证实PGC-1α和ERRα共同定位在大鼠肝细胞Gck启动子含该元件的区域,表明PGC-1α和ERRα通过ERRE元件促进大鼠肝脏Gck基因转录。大鼠肝原代细胞感染PGC-1α和ERRα腺病毒后,Gck基因在mRNA和蛋白质水平表达量均明显上调。同时,Gck的催化活性大大增加,这表明PGC-1α和ERRα在调节葡萄糖代谢方面发挥着重要的作用。PGC-1α和ERRα对Gck的促进作用还可以增强胰岛素刺激的效应。最后,引入ERRα特异的siRNA (siERRα)和拮抗剂XCT790抑制ERRα表达后,胰岛素诱导的Gck表达受到影响,充分证明了ERRα介导了胰岛素诱导的Gck表达。综上所述,本研究发现了PGC-1α和ERRα对Gck的促进作用及对肝脏葡萄糖代谢的影响,证明ERRα在胰岛素刺激葡萄糖激酶通路中的作用,并可以促进Ⅱ型糖尿病葡萄糖稳态的形成。
[Abstract]:Part PGC-1 SF-1 synergistic effect of LH on the synthesis of luteinizing hormone and aldosterone
The peroxisome proliferator activated receptor gamma co activating factor 1 alpha (PGC-1 alpha) can stimulate a variety of transcription factors to regulate the process of energy metabolism. The nuclear receptor steroid generating factor (SF-1) is highly expressed in the pituitary, gonadal, adrenal and other endocrine tissues, and regulates multiple key genes in the transcription and regulation of the hypothalamus pituitary gonadal axis. Level of hormone synthesis. In order to explore the role of PGC-1 alpha in the synthesis of steroid hormones and the secretion of pituitary hormones, this subject uses the mouse pituitary gonadotropin alpha T3-1 cells as the research model. After overexpressing PGC-1 alpha, the expression of LH beta and alpha GSU in the water mRNA level is increased by real-time PCR test, and further through the enzyme linked immunosorbent assay (E). LISA) proved that PGC-1 alpha stimulated the increase of luteinizing hormone secreted by alpha T3-1 cells. Analysis of the LHp promoter sequence and the existence of a SF-1 binding element mutation in the -127 BP to -119 BP site, the double fluorescence report showed that SF-1 and PGC-1 alpha were all disappearing. The immunoprecipitation experiment showed that PGC-1 alpha and SF-1 Co located the region of the GSE element in the LH beta promoter region, and promoted the LH beta transcription. In order to further demonstrate the co stimulation of PGC-1 alpha to SF-1, the interaction of the two groups was analyzed.
Sub mechanism. Both mammalian two hybrid experiments and immunoprecipitation experiments show that PGC-1 alpha and SF-1 can form complex in the body. Further experiments show that the two can interact directly. The action GST pull-down occurs between the 1~180 amino acid regions of the N end of PGC-1 alpha (including LXXLL module, 142~146 amino acid) and the 187~462 amino acid region of SF-1 C terminal. The construction of SF-1 gene expression fragments of different lengths showed that only when the optimal active domain of SF-1 and the AF-2 junction (proximal activation domain) domain existed simultaneously, PGC-1 alpha and SF-1 were directly combined. The adrenal cortex is the main steroid hormone synthesis site, and the cytochrome P450 family members are
This is involved in cholesterol metabolism, the multiple catalytic reaction of the steroid hormone synthesis, the protein product encoded by the.Cyp11b2 gene, is responsible for the final three step reaction of aldosterone synthesis, which is a speed limiting enzyme in the aldosterone synthesis. In the Y-1 cells of the adrenal cortex of mice, the real-time PCR experiment proved that PGC-1 alpha could induce the Cyp11b2, Cyp11b1 and P450scc genes in M. The level of RNA was upregulated. ELISA test further detected that PGC-1 alpha synergy SF-1 promoted aldosterone secretion.
On the other hand, PGC-1 alpha and estrogen receptor related receptor family members ERR alpha and ERR gamma were detected by double fluorescence report experiments to promote SF-1 transcription. The expression of SF-1 at mRNA level and protein level was significantly increased in PGC-1 alpha alpha T3-1 cells and Y-1 cells. To sum up, the subject revealed that PGC-1 a is steroid in steroid. The regulation of hormone and gonadotropin synthesis is important, and the molecular mechanism of its stimulatory hormone synthesis is analyzed. It is suggested that PGC-1 alpha may regulate the development of gonadal and adrenal glands and sex differentiation through SF-1.
The second part is to study the regulation of glucokinase expression by PGC-1 alpha and ERR alpha.
Peroxisome proliferator activated receptor gamma CO activation factor 1 alpha (PGC-1 alpha) is a key regulator of cell energy metabolism. It regulates physiological processes such as adaptive heat production, liver sugar isogenesis, fatty acid oxidation and mitochondrial biosynthesis by activating a variety of nuclear receptors and transcription factors. The protein of 50 KDa in the pancreas, also known as type IV hexokinase, is the first speed limiting enzyme in glycolysis. It plays an important role in regulating glucose utilization in the liver and is stimulated by insulin signal.
The study found that there is a conserved estrogen receptor related receptor binding element ERRE (-52 BP to -39 BP) in the promoter region of the glucokinase (Gck) promoter of the rat. The double fluorescence report experiment analyses the Gck promoter of the truncated and loci mutation, proving that PGC-1 alpha and ERR alpha can use this element to stimulate the active.EMSA solid of the Gck promoter. The test further demonstrated that ERR alpha could be combined with ERRE elements in vitro, and the ChIP experiment confirmed that PGC-1 A and ERR alpha were Co located in the Gck promoter region of rat hepatocytes, indicating that PGC-1 A and ERR a promoted the transcription of Gck genes in rat liver through ERRE components. The level of white matter expression is obviously up. Meanwhile, the catalytic activity of Gck is greatly increased, which indicates that PGC-1 alpha and ERR alpha play an important role in regulating glucose metabolism..PGC-1 A and ERR a can enhance the effect of Gck stimulation. Finally, the specific siRNA (siERR alpha) and antagonist XCT790 inhibition ERR are introduced. After the expression of alpha, the expression of insulin induced Gck was affected, which fully demonstrated that ERR a mediated the expression of Gck induced by insulin. To sum up, this study found that PGC-1 A and ERR alpha have a promoting effect on Gck and the effect on glucose metabolism in the liver. It is proved that ERR a plays a role in insulin stimulation of glucose kinase pathway and can promote type II. The formation of glucose homeostasis in diabetes.

【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R341

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