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: 9493612
• 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/9493612
• 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）蓄积， 其发病机制仍知之甚少。 β-Klotho (Klb) 基因编码必需的辅助受体 FGF21 的生物学功能是必需的，可促进肝脏中的脂肪酸氧化，从而改善 肝脂肪变性。然而，脂肪肝与肥胖者和肥胖者中 FGF21 水平升高存在矛盾的相关性。 人类和小鼠，表明存在 FGF21 抵抗状态，其中增加的 FGF21 无法阻止发育 脂肪肝。 FGF21 抗性的潜在机制仍然难以捉摸。表观遗传调控，包括 DNA 甲基化是环境因素（例如饮食）和复杂疾病（例如糖尿病）之间的分子联系。 肥胖和 NAFLD）。我们的初步数据表明，DNA 甲基化受 DNA 调控 甲基转移酶（DNMT）是肝脏脂质代谢的重要决定因素。 DNMT 的上调 饱和脂肪酸（SFA）和促炎细胞因子可能导致肝脂质失调 肥胖中的代谢和脂肪肝。此外，我们使用简化的 DNA 甲基化全基因组分析 代表性亚硫酸氢盐测序 (RRBS) 分析显示 DNA 甲基化显着增加 饮食诱导肥胖 (DIO) 小鼠肝脏中的 Klb 启动子，与 Klb 表达下调相关 在RNA-seq分析中。 Klb表达的下调可能会限制增加的FGF21促进的能力 肝脂质氧化，导致 FGF21 抵抗。因此，我们假设表观遗传编程 Klb 启动子通过营养线索（例如 SFA）和促炎刺激（例如细胞因子）来调节，其水平为 通常在肥胖症中升高，通过下调 FGF21 途径介导肝脏脂质积累， 肥胖导致脂肪肝。目标 1 将确定 Klb 启动子处的 DNA 甲基化是否通过以下方式增强： SFA 和细胞因子会损害肝脏 FGF21 信号传导和脂质代谢，导致肝脏 TG 积累。 我们将首先测试 DNMT1 通过以下方式介导 Klb 启动子处 DNA 甲基化增强的假设： SFA 和细胞因子，导致肝脏 FGF21 抵抗和脂质积累。然后我们将确定 利用一种新的方法研究 Klb 启动子甲基化在肝 FGF21 信号传导和脂质代谢中的直接作用 CRISPR/RNA 引导系统特异性诱导 Klb 启动子甲基化/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 信号传导以及 改善喂食高脂肪饮食的遗传小鼠模型的脂肪肝。我们已经生成了肝脏特异性 DNMT1 和 通过将 DNMT1 和 3a-floxed 小鼠与白蛋白-Cre 小鼠杂交，获得 3a 敲除（LD1KO 和 LD3aKO）小鼠。我们 将进一步确定 LD1KO 和 LD3aKO 中的 FGF21 信号传导、肝脏脂质代谢和 TG 积累 小鼠饲喂 HF 饮食。我们还建立了一种依赖 Cre 的 CRISPR/RNA 引导的靶向 DNA 方法 Klb 启动子处的甲基化/去甲基化特别是在小鼠肝脏中，以检验增强的假设 肝细胞 Klb 启动子处的 DNA 甲基化介导 FGF21 信号传导和脂质代谢受损。