【作者】 白亮；

【导师】 杨公社； Janardan K.Reddy；

【作者基本信息】 西北农林科技大学 ， 动物学， 2011， 博士

【摘要】 非酒精性脂肪肝疾病是一种无过量饮酒史,以肝细胞脂肪变性和脂肪贮积为特征的临床病理综合征。肝脏脂肪代谢长期紊乱,最终会发展为非酒精性肝炎、肝硬化和肝癌。严重影响人类健康。因而,探索调控肝脏脂肪代谢和系统能量平衡的作用机制成为全球科研工作者面临的挑战和关注热点。近年来,研究表明,肝脏脂肪代谢主要受PPARs核受体超家族的调控。PPARα和PPARβ/δ主要参与脂肪酸氧化代谢。而PPARγ主要参与脂肪细胞分化和成熟脂肪细胞脂肪的贮存,掌控着脂肪肝的形成和肝脏脂肪代谢。PPARγ调控的生脂靶基因的转录需要一些辅激活子和辅激活子相关蛋白,如MED1、SRC/p160蛋白家族、PRIP、PIMT、和PRIC285等。研究发现,MED1是Mediator复合物的一个关键成员,在RNA聚合酶II依赖型转录调控中发挥重要作用。周身敲除MED1导致小鼠在胚胎期11.5 d死亡。MED1对PPARα调控的转录活性以及PPARα配体诱导的肝脏肿瘤的形成是必不可少的。而且,MED1在PPARγ诱导的小鼠胚胎成纤维细胞向脂肪细胞分化过程中具有关键作用。然而,目前有关MED1及其他辅激活子在PPARγ功能发挥中的调控作用尚不清楚。本研究中,我们以周身基因敲除鼠和肝脏特异性敲除鼠为实验动物模型,运用腺病毒扩繁及纯化技术、H&E染色、油红O染色、免疫组织化学染色、免疫荧光染色、激光共聚焦技术、甘油三酯和胆固醇酶试剂盒检测、快速蛋白液相色谱分析、原代肝细胞分离和培养、Real-time PCR、Northern Blot、Western Blot、ChIP分析和基因芯片等生物化学、细胞及分子生物学技术,深入探索了转录辅激活子MED1在高脂日粮和PPARγ诱导脂肪肝形成中的调控作用。获得的主要研究结果:1.在体实验表明,MED1是PPARγ介导脂肪肝形成的必需辅激活子。MED1^ΔLiv和MED1^fl/fl鼠经尾静脉注射Ad/PPARγ6 d,MED1^fl/fl鼠在PPARγ诱导下呈现出严重的脂肪肝,而MED1^ΔLiv鼠却能够抵抗PPARγ刺激的脂肪肝生成。基因表达谱分析和Northern Blot检测表明,高表达PPARγ的MED1^ΔLiv鼠肝脏不能有效表达生脂标志基因aP2、adipsin和adiponectin以及脂滴相关蛋白caveolin-1、CideA和S3-12等,而这些基因在PPARγ高表达的MED1^fl/fl鼠脂肪肝中强烈表达。基因芯片分析结果发现,肝脏MED1特异性敲除,PPARγ调控的生脂信号通路,如脂肪生成、贮存、运输和氧化等通路处于失活状态,提示PPARγ信号通路的激活需要完整的MED1。而且,外源表达MED1能够挽救Ad/PPARγ刺激的脂肪肝形成及脂肪相关基因诱导表达。ChIP分析表明,高表达PPARγ的肝脏MED1敲除鼠aP2启动子区无MED1募集,PPARγ的募集显著减少,转录辅激活子PRIP和PIMT的募集能力略微减弱。2.离体实验表明,MED1是PPARγ诱导肝细胞发生脂肪变的必需辅激活子。从MED1^ΔLiv和MED1^fl/fl鼠分离获得的原代肝细胞感染Ad/PPARγ12 h,油红O染色检测发现,MED1阳性肝细胞能够向脂肪细胞转化,而MED1缺失的肝细胞却不能发生脂肪变。Q-PCR检测结果表明,MED1缺失的肝细胞能够抵抗PPARγ诱导的脂肪标志基因aP2以及PPARγ靶基因S3-12和CideA的表达。3.其他转录辅激活子SRC-1、PRIC285、PRIP和PIMT对PPARγ刺激的脂肪肝形成无影响。SRC-1周身敲除(SRC-1^-/-)、PRIC285周身敲除(PRIC285^-/-)、肝脏PRIP特异性敲除(PRIP^ΔLiv)和肝脏PIMT特异性敲除(PIMT^ΔLiv)鼠及其相应的对照鼠经尾静脉注射Ad/PPARγ5 d后,组织学和Northern Blot检测结果发现,SRC-1^-/-、PRIC285^-/-、PRIP^ΔLiv和PIMT^ΔLiv鼠及其相应的对照鼠都形成严重的脂肪肝,并强烈表达脂肪生成标志基因和PPARγ靶基因。因而,转录辅激活子MED1在PPARγ诱导的脂肪肝形成中是必需的。4. MED1是高脂日粮诱导脂肪肝形成的必需调控子。MED1^ΔLiv和MED1^fl/fl鼠饲喂高脂日粮（60% kCal脂肪） 0、1、2、4、8和16 w,组织学检测发现,MED1^ΔLiv鼠能够抵抗高脂日粮诱导的脂肪肝形成,而MED1^fl/fl鼠肝细胞中有大量脂滴积聚,呈明显的脂肪肝状,但并不表达PPARγ的靶基因aP2,提示MED1通过PPARγ非依赖性通路调控肝脏脂肪生成。而且,短期高脂日粮饲喂,MED1^ΔLiv鼠血浆胆固醇含量显著上升。此外,GTT和ITT分析揭示,肝脏MED1特异性敲除鼠葡萄糖耐受性和胰岛素敏感性增加。5.禁食状态下肝脏MED1特异性敲除鼠呈现高脂血症。长期禁食（72 h）,MED1^ΔLiv鼠不发生肝脏脂肪变性。禁食24、48和72 h,与MED1^fl/fl鼠相比,MED1^ΔLiv鼠血浆甘油三酯和胆固醇水平显著升高,呈高脂血症。FPLC检测结果表明,正常饲喂条件下,MED1^ΔLiv鼠和MED1^fl/fl鼠血浆脂蛋白峰相似,而禁食24 h,MED1^ΔLiv鼠血浆携带甘油三酯和胆固醇的VLDL峰严重积聚,提示MED1可能调控VLDL。本文运用肝脏MED1特异性敲除鼠模型,体内和体外研究证实,转录辅激活子MED1在脂肪肝形成过程中发挥关键作用。MED1肝脏特异性敲除鼠能够抵抗高脂日粮和PPARγ诱导的脂肪肝形成和生脂基因表达。而其他转录辅激活子SRC-1、PRIC285、PRIP和PIMT敲除缺对PPARγ调控的肝脏脂肪变性无影响。因而,MED1是脂肪肝形成的正调控基因,是PPARγ功能发挥的必需辅激活子。本研究为理解脂肪肝调控的复杂性开辟了一个崭新的局面,为脂肪肝的预防和治疗提供了新的研究思路和靶点。更多还原

【Abstract】 Nonalcoholic fatty liver disease （NAFLD）, one of metabolic syndrome, is a burgeoning common chronic liver disorder with a morphological spectrum of liver pathology commencing with hepatic steatosis and steatohepatitis which may progress toward the development of cirrhosis and liver cancer, and NAFLD has become a major health concern worldwide. Identification of NAFLD in recent years raises new challenges about its effective precaution and therapy in basic and clinic research. Since the key aspects of lipid metabolism including lipogenesis, fatty acid oxidation, lipoprotein uptake and secretion are regulated by the liver, an understanding of the regulatory mechanisms that influence hepatic lipid homeostasis and systemic energy balance is of paramount importance in gaining insights that might be useful in the management of fatty liver disease. In recent years, increasing attention is being focused on certain transcription factors/nuclear receptors that are known to serve as key regulatory molecules to influence hepatic lipid synthesis, storage and oxidation. In particular, the three members of the peroxisome proliferator-activated receptor （PPAR） subfamily of nuclear receptors, namely PPARα, PPARβ/δ, and PPARγ, govern the regulation of liver lipid metabolism and thus influence the development of hepatic steatosis and fatty liver disease. Of the three members of PPAR subfamily, PPARγis critical for conserving energy as it contributes to adipogenesis, whereas both PPARαand PPARβparticipate in energy expenditure. Overexpression of PPARγin mouse liver leads to adipogenic hepatic steatosis （“hepatic adiposis”） and induces the expression of adipocyte-specific and lipogenesis-related genes. In contrast, liver-specific disruption of PPARγ, exerts an opposite effect in that it dramatically reduces fatty liver. Thus, PPARγplays an important role in liver lipid metabolism and contributes to hepatic steatosis.In the nucleus, PPARs heterodimerize with retinoid X receptorα（RXRα） and bind to peroxisome proliferator response elements （PPREs） in the promoter region of target genes. Transcriptional activity of nuclear receptors and other transcription factors requires certain coactivators and coactivator-associated proteins that include MED1, SRC/p160 family of proteins, CBP/p300, PRIP, PIMT, CARM1, PRIC285, PRIC295, PRIC320, PGC-1αand others. Coactivator MED1 is a key component of Mediator complex, and is required for RNA polymerase II dependent gene transcription. Evidence indicates that MED1 is required for PPARα-mediated transcriptional activity in vivo and PPARαligand induced liver tumor. Invitro experiment displays that MED1 plays an important role in PPARγstimulated adipogenic differentiation. However, the in vivo role of MED1 and other coactivators in liver with regards to PPARγfunction remains unknown.To delineate the in vivo function of coactivator molecules in PPARγ-stimulated adipogenic hepatic steatosis, we used in this study genetically altered mouse lineages, and cellular and molecular biotechnology, including amplification and purification of adenovirus, H&E staining, Oil Red O staining, immunohistochemistry, immunofluorescence, confocal microscopy, biochemical assays of triglyceride and cholesterol, fast protein liquid chromatography （FPLC）, primary hepatocyte isolation and culture, Real-time PCR, Northern Blot, Western Blot, Chromatin immunoprecipitation （ChIP） assay and microarray analysis. The reulsts demonstrate that deletion of MED1 in mouse liver impairs high fat diet and PPARγ-stimulated adipogenic steatosis, whereas deficiency of coactivators such as SRC-1, PRIC285, PRIP, and PIMT had no effect. The specific results are as follows:1. MED1 is required for PPARγ-stimulated hepatic steatosis and the expression of adipogenic genes in vivo. MED1Δliv mice injected with adenovirus-PPARγ（Ad/PPARγ） by tail vein for 6 days did not develop fatty liver, whereas MED1^fl/fl mice injected with Ad/PPARγdeveloped severe hepatic steatosis. Gene expression profiling and Northern blot analyses of Ad/PPARγinjected mouse livers showed impaired induction in MED1Δliv mouse liver of adipogenic markers, such as aP2, adipsin, adiponectin and lipid droplet-associated genes, including caveolin-1, CideA, S3-12 and others. These adipocyte-specific and lipogenesis-related genes are strongly induced in MED1^fl/fl mouse liver in response to Ad/PPARγ. cDNA microarray analysis showed that upregulation of lipogenesis related gene networks by PPARγrequires intact MED1 gene, and in the absence of MED1 in liver the levels of expression of these genes were markedly subdued, which clearly establish that MED1 plays a key role in facilitating the transcriptional regulation of PPARγtarget genes. Furthermore, re-expression of MED1 using Ad/MED1 in MED1^ΔLiv mouse liver restored PPARγ-stimulated hepatic adipogenic response. In addition, ChIP reveals no recruitment of MED1 and slightly reduced association in MED1Δliv mouse liver of PRIP and PIMT with aP2 gene promoter, suggesting that MED1 is required for the transcriptional activation of target genes of PPARγby its ability to stabilize Mediator complex necessary for RNA polymerase II dependent transcription.2. MED1 is required for PPARγ-induced transdifferentiation of hepatocytes toward adipocytes and the expression of adipogenic genes in vitro. Primary hepatocytes isolated from MED1^ΔLiv mouse were infected with Ad/PPARγfor 12 hours. Histology and Real time PCR showed that hepatocytes from MED1^ΔLiv mouse are failed to PPARγ-stimulated hepatic adiposis and expression of adipocyte mark genes aP2 and PPARγtarget genes S3-12 and CideA.3. Other transcription coactivators SRC-1, PRIP, PIMT, and PRIC285 are dispensable for PPARγ-stimulated fatty liver development while MED1 is necessary for PPARγdependent transcription of downstream target genes and the development of hepatic steatosis. Other PPARs coactivators germ-line knockout SRC-1 (SRC-1^-/-) and PRIC285 (PRIC285^-/-) mice and liver conditional null (PRIP^ΔLiv) and (PIMT^ΔLiv) mice and their corresponding control mice were injected with Ad/PPARγand killed 5 days later. Fatty liver developed in mice lacking SRC-1, PRIC285, PRIP, and PIMT and their corresponding intact floxed controls after Ad/PPARγadministration. Northern Blot analysis revealed similar levels of increases in hepatic mRNA levels of adipogenesis genes in knockout and control mice following PPARγoverexpression.4. MED1 is required for high fat diet induced fatty liver. MED1^ΔLiv and MED1^fl/fl mice were fed high fat diet （60% kcal fat） for 0, 1, 2, 3, 4, 8 and 16 weeks. Histolocial test revealed that MED1Δliv mice when fed a high fat diet for up to 16 weeks failed to develop fatty liver, whereas MED1^fl/fl fed a high-fat diet developed severe hepatic steatosis, which was not associated with induction of PPARγtarget gene aP2. These results suggest that MED1 has significant PPARγ-independent effects on hepatic steatosis. On the other hand, PPARγ-stimulated hepatic steatosis is dependent upon MED1. Glucose and insulin tolerance tests revealed that MED1^ΔLiv mice fed a high fat diet for 4 or 16 weeks displayed lower glucose levels and exhibited greater insulin sensitivity than MED1^fl/fl mice. These results suggest that MED1 deficiency increases glucose tolerance and insulin sensitivity. In addition, MED1^ΔLiv mice showed significant elevated plasma cholesterol under short term high fat diet.5. Hepatic MED1 deficient mice showed hyperlipidemia in response to fasting. There was no fat accumulation in livers of MED1^ΔLiv mice compared to MED1^fl/fl and PPARα^-/- control mice after 72 hours of fasting. Compared with MED1^fl/fl mice, plasma triglycerides and cholesterol in MED1^ΔLiv mice were significantly increased after 24, 48 and 72 hours of fasting. FPLC showed that lipoprotein profiles were similar in fed MED1^fl/fl and MED1^ΔLiv mice. However, VLDL was significantly increased in MED1^ΔLiv mice after 24 hours of fasting, which suggests MED1 may regulate VLDL and plays a pivotal role in triglyceride and cholesterol metabolism.We conclude that transcription coactivator MED1 is required for high-fat diet-induced and PPARγ-stimulated fatty liver development in vivo, which points to a new layer of regulatory complexity in the development of hepatic steatosis and suggests that MED1 may be considered a potential therapeutic target for hepatic steatosis.更多还原