(The) study for pathophysiological roles of NR1D1 in fatty liver disease
지방간질환에서 핵수용체 NR1D1의 병태생리학적 역할에 대한 연구

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 약학과, 2017. 2. 이미옥.-
dc.description.abstractThe recent studies of nonalcoholic fatty liver diseases (NAFLD) caused by multiple steps actively involve studies of nuclear receptors which are useful as drug targets. Nuclear receptor subfamily 1, group D, member 1 (Nr1d1) is abundantly expressed in the metabolic tissues and organs such as adipose tissues, skeletal muscle, and liver, and specifically expressed in non- parenchymal cells such as hepatic stellate cells, macrophages, and T cells. Expressions of the Nr1d1 in various tissues and cells reflect its roles and functions, and Nr1d1 has been reported to contribute the adipocyte differentiation, muscle development, gluconeogenesis, inflammation and immune response. However, the role and function of NR1D1 in NAFLD was not clearly addressed. To clarify the role of NR1D1 in hepatic lipid metabolism and to understand the molecular pathogenesis of NAFLD, the Nr1d1 Δex3/4 mice, in which deletion of Nr1d1 exons 3 and 4 for a functional disruption was introduced, were generated by crossing Nr1d1 floxed mice with transgenic Zp3-Cre deleter mice. To induce NAFLD stage, the Nr1d1 Δex3/4 mice were challenged by a high fat diet (HFD), and the metabolic phenotypes that responded to these stimuli were observed. The Nr1d1 Δex3/4 mice displayed severe hepatic steatosis compared to the WT mice, and it resulted from the interaction effect between diet and genotype. In addition, to understand the potential molecular mechanism, the differential gene expression was profiled using microarray, and upstream factors and gene coexpression networks were predicted. Interestingly, neutrophils homeostasis and cAMP signaling pathways were affected by the interaction effect, and they were thought to elevate severity of hepatic steatosis in response of HFD-fed functional disruption of Nr1d1. In addition, CCAAT/enhancer-binding protein alpha and hepatocyte nuclear factor 4 alpha were predicted to tether the function of HFD-responsive Nr1d1 Δex3/4, and thyroid hormone-responsive (Thsrp) was a potential target gene of HFD-responsive Nr1d1 Δex3/4. Taken together, loss of transcriptional function of Nr1d1 was associated with deterioration in hepatic steatosis. The interaction between the Nr1d1 ex3/4 genotype with an HFD might mediate these phenotypic changes, probably through a nonclassical transcriptional function of Nr1d1.-
dc.description.tableofcontents1. Introduction 1
1.1. Background 2
1.1.1. Definition of nonalcoholic fatty liver diseases (NAFLD) 2
1.1.2. Association between NAFLD and chronic metabolic syndromes 4
1.1.3. Nuclear receptors as therapeutic targets for NAFLD. 4
1.2. Nuclear receptor subfamily 1, group D, member 1 (NR1D1) 8
1.2.1. Expression and structure of NR1D1 8
1.2.2. Roles and functions of Nr1d1 in pathophysiological processes 10
1.2.3. In vitro and in vivo models of Nr1d1 in NAFLD 17
1.3. Outline of this thesis 19

2. Materials and methods 20
2.1. Animal studies 21
2.1.1. The generation of Nr1d1 exon3/4 mice 21
2.1.2. The treatment of HFD in Nr1d1 exon3/4 mice 21
2.1.3. Histology and serum/liver biochemistry analysis 22
2.2. Cells and cell culture 23
2.2.1. NIH3T3 cells culture 23
2.2.2. MEF preparation and culture 23
2.3. Transient transfection, and reporter gene analysis 24
2.3.1. Transient transfection 24
2.3.2. Reporter gene analysis 24
2.4. Quantitative reverse transcription PCR (RT-qPCR) 24
2.5. Western blotting, fluorescence microscopy, and chromatin immunoprecipitation (ChIP) assay 25
2.5.1. Western blotting 25
2.5.2. Fluorescence microscopy 25
2.5.3. Chromatin immunoprecipitation (ChIP) assay 27
2.6. Microarray experiments and data analysis 27
2.6.1. Microarray experiments 27
2.6.2. Data analysis 28
2.7. Statistical analysis 29

3. Results 30
3.1. Generation of whole-body Nr1d1 Δex3/4 mice 31
3.1.1. The strategy of floxed Nr1d1 and generation of whole-body Nr1d1 Δex3/4 mice 31
3.1.2. The Nr1d1 Δex3/4 mutant protein is located in both the nuclei and cytoplasm of the cells 36
3.1.3. The Nr1d1 Δex3/4 protein does not have transcriptional activity 41
3.2. Enhanced hepatic steatosis in the Nr1d1 Δex3/4 mice after HFD feeding 46
3.2.1. The Nr1d1 Δex3/4 mice are more prone to HFD-induced obesity 46
3.2.2. The severe liver injuries in the Nr1d1 Δex3/4 mice after HFD feeding 50
3.2.3. The enhanced HFD-induced hepatic steatosis in the Nr1d1 Δex3/4 mice 56
3.3. Expression of hepatic genes in the Nr1d1 Δex3/4 mice after HFD feeding 61
3.3.1. Expression of metabolic genes is disrupted in the Nr1d1 Δex3/4 mice 61
3.3.2. Expression of inflammatory genes is disrupted in the Nr1d1 Δex3/4 mice 67
3.4. Gene expression profiling in the Nr1d1 Δex3/4 mice after HFD feeding 69
3.4.1. Differential hepatic gene expression analysis 69
3.4.2. Prediction of upstream regulators for altered hepatic gene expression in the Nr1d1 Δex3/4 mice after HFD feeding 78
3.4.3. A putative target gene associated with function of HFD-responsive Nr1d1 82
3.5. The hepatic steatosis in liver-specific Nr1d1 Δex3/4 mice 85
3.5.1. The generation of liver-specific Nr1d1 Δex3/4 mice 85
3.5.2. The little difference of HFD-induced hepatic steatosis in the Nr1d1 LΔex3/4 mice 87
3.5.3. The biochemical assessment of liver damage in the Nr1d1 LΔex3/4 mice after HFD feeding 94

4. Discussion 98
4.1. The importances of nuclear receptors in NAFLD 99
4.2. The strategic validity of the Nr1d1 Δex3/4 mice 100
4.3. Experimental models of NAFLD in the Nr1d1 Δex3/4 mice 102
4.4. Understanding the pathophysiological meaning of gene set affected by interaction effect 104
4.5. Nonclassical functions of the Nr1d1 in hepatic lipid metabolism 105
4.6. The discrepancies of hepatic steatosis phenotypes between the whole-body and liver-specific Nr1d1 Δex3/4 mice 107

5. Conclusion 109
Bibliography 111
초록 123
dc.format.extent2345791 bytes-
dc.publisher서울대학교 대학원-
dc.subjectnuclear recptor-
dc.title(The) study for pathophysiological roles of NR1D1 in fatty liver disease-
dc.title.alternative지방간질환에서 핵수용체 NR1D1의 병태생리학적 역할에 대한 연구-
dc.contributor.affiliation약학대학 약학과-
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Ph.D. / Sc.D._약학과)
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