Epigenetic programming of beta-klotho in non-alcoholic fatty liver disease

β-klotho 在非酒精性脂肪肝病中的表观遗传编程

• 批准号: 9918345
• 负责人: Hang Shi
• 金额: $ 34.09万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-25 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918345
• 关键词: Address; Albumins; Biological Process; Cirrhosis; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cues; DNA; DNA Methylation; DNA Methylation Inhibition; DNA Modification Methylases; DNA Sequence; Data; Development; Diet; Disease; Down-Regulation; Environmental Risk Factor; Epigenetic Process; Evaluation; FGF21 gene; Fatty Liver; Fibrosis; Genes; Genetic; Genetic Fingerprintings; Goals; Guide RNA; Hepatic; Hepatocyte; High Fat Diet; High Prevalence; Human; Impairment; Inflammatory; Insulin Resistance; Knock-out; Link; Lipid Mobilization; Lipids; Liver; LoxP-flanked allele; Mediating; Metabolic Diseases; Metabolic Pathway; Methylation; Molecular; Mus; Non-Insulin-Dependent Diabetes Mellitus; Nutritional; Obese Mice; Obesity; Pathogenesis; Pathway interactions; Patients; Prevention; Regulation; Resistance; Role; Saturated Fatty Acids; Signal Transduction; Site; Stimulus; Stress; System; TNF gene; Testing; Triglycerides; Up-Regulation; bisulfite sequencing; cytokine; demethylation; epigenetic regulation; fatty acid oxidation; genome-wide; in vivo; lipid metabolism; methylome; mouse model; new therapeutic target; non-alcoholic fatty liver; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; oxidation; peptide hormone; promoter; receptor; transcriptome sequencing

Although non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic triglyceride (TG) accumulation, its pathogenesis remains poorly understood. The β-Klotho (Klb) gene encodes an obligatory co-receptor necessary for biological functions of FGF21, which promotes fatty acid oxidation in liver, thereby ameliorating hepatic steatosis. However, fatty liver is paradoxically associated with increased FGF21 levels in both obese humans and mice, suggesting a FGF21-resistant state where increased FGF21 fails to block the development of fatty liver. The mechanism underlying FGF21 resistance remains elusive. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors (e.g. diets) and complex diseases (e.g. obesity and NAFLD). Our preliminary data suggested that DNA methylation, regulated by DNA methyltransferases (DNMTs), is an important determinant of hepatic lipid metabolism. Upregulation of DNMTs by saturated fatty acids (SFAs) and the pro-inflammatory cytokines may contribute to deregulated hepatic lipid metabolism and fatty liver in obesity. Moreover, our genome-wide profiling of DNA methylation using reduced representation bisulfite sequencing (RRBS) analysis revealed a significant increase in DNA methylation at the Klb promoter in liver of diet-induced obese (DIO) mice, which is associated with downregulation of Klb expression in RNA-seq analysis. The downregulation of Klb expression may limit the ability of increased FGF21 to promote hepatic lipid oxidation, leading to FGF21 resistance. We therefore hypothesize that epigenetic programming of the Klb promoter by nutritional cues (e.g. SFAs) and pro-inflammatory stimuli (e.g. cytokines), levels of which are commonly elevated in obesity, mediates hepatic lipid accumulation by downregulating the FGF21 pathway, leading to fatty liver in obesity. Aim 1 will determine whether enhanced DNA methylation at the Klb promoter by SFAs and cytokines impairs hepatic FGF21 signaling and lipid metabolism, leading to hepatic TG accumulation. We will first test the hypothesis that DNMT1 mediates the enhanced DNA methylation at the Klb promoter by SFAs and cytokines, leading to hepatic FGF21 resistance and lipid accumulation. We will then determine the direct role of Klb promoter methylation in hepatic FGF21 signaling and lipid metabolism using a novel CRISPR/RNA-guided system to specifically induce methylation/demethylation at the Klb promoter. Aim 2 will determine whether liver-specific inhibition of DNA methylation promotes hepatic FGF21 signaling and ameliorates fatty liver in genetic mouse models fed high fat diet. We have generated liver-specific DNMT1 and 3a knockout (LD1KO and LD3aKO) mice by crossing DNMT1- and 3a-floxed mice with Albumin-Cre mice. We will further determine FGF21 signaling, hepatic lipid metabolism and TG accumulation in LD1KO and LD3aKO mice fed HF diet. We have also established a Cre-dependent CRISPR/RNA-guided approach for targeted DNA methylation/demethylation at the Klb promoter specifically in liver of mice to test the hypothesis that enhanced DNA methylation at the Klb promoter in hepatocytes mediates impaired FGF21signaling and lipid metabolism.

尽管非酒精性脂肪性肝病(NAFLD)的特点是肝脏甘油三酯(TG)积聚， 其发病机制尚不清楚。β-Klotho(Klb)基因编码一种必需的辅助受体 FGF21的生物学功能所必需的，它促进肝脏的脂肪酸氧化，从而改善 肝脏脂肪变性。然而，在这两种肥胖者中，脂肪肝与FGF21水平升高之间存在矛盾的联系 人类和小鼠，提示FGF21抵抗状态，其中增加的FGF21未能阻止发育 脂肪肝的症状。FGF21耐药的机制仍不清楚。表观遗传调控，包括 DNA甲基化是环境因素(如饮食)和复杂疾病(如饮食)之间的分子联系。 肥胖和非酒精性脂肪肝)。我们的初步数据表明，受DNA调控的DNA甲基化 甲基转移酶(DNMT)是肝脂代谢的重要决定因素。通过以下方式上调DNMT 饱和脂肪酸(SFAs)和促炎细胞因子可能导致肝脂代谢紊乱 肥胖症的代谢与脂肪肝。此外，我们的全基因组DNA甲基化图谱使用Reduced 代表性亚硫酸氢盐测序(RRBS)分析显示，在 饮食诱导肥胖(DIO)小鼠肝脏中KLb启动子的表达下调 在RNA-seq分析中。KLb表达下调可能限制FGF21促进 肝脏脂质氧化，导致FGF21抵抗。因此，我们假设表观遗传编程 KLB启动子受营养信号(如SFA)和促炎刺激(如细胞因子)的影响，其水平为 通常在肥胖中升高，通过下调FGF21途径来调节肝脏脂肪堆积， 肥胖会导致脂肪肝。AIM 1将确定KLb启动子上的DNA甲基化增强是否通过 SFas和细胞因子损害肝脏FGF21信号转导和脂质代谢，导致肝脏TG蓄积。 我们将首先验证DNMT1通过以下方式介导KLb启动子上增强的DNA甲基化的假设 SFas和细胞因子，导致肝脏FGF21抵抗和脂质堆积。然后我们将确定 利用一种新的基因研究KLb启动子甲基化在肝脏FGF21信号和脂质代谢中的直接作用 CRISPR/RNA引导的系统，特异性地诱导KLB启动子的甲基化/去甲基化。目标2将 确定肝脏特异性DNA甲基化抑制是否促进肝脏FGF21信号转导和 改善饲喂高脂饮食的遗传性小鼠脂肪肝。我们已经产生了肝脏特异的DNMT1和 3A基因敲除(LD1KO和LD3aKO)小鼠与白蛋白-Cre小鼠杂交。我们 将进一步确定LD1KO和LD3aKO中的FGF21信号、肝脂代谢和甘油三酯积累 饲喂HF饲料的小鼠。我们还建立了一种依赖CRE的CRISPR/RNA引导的靶向DNA方法 小鼠肝脏特异性KLb启动子的甲基化/去甲基化验证增强的假说 肝细胞KLb启动子DNA甲基化介导FGF21信号和脂代谢受损。