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

虽然非酒精性脂肪性肝病（NAFLD）的特征在于肝脏甘油三酯（TG）积累， 其发病机制仍然知之甚少。β-Klotho（Klb）基因编码一种专性共受体 FGF 21的生物学功能所必需的，其促进肝脏中的脂肪酸氧化，从而改善 肝脂肪变性然而，脂肪肝是矛盾的与增加FGF 21水平在两个肥胖 人和小鼠，提示FGF 21耐药状态，其中增加的FGF 21不能阻止发展。 脂肪肝FGF 21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（如饮食）和复杂疾病（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，由DNA调节， 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脏脂质失调 脂肪肝的发病机制此外，我们使用还原的DNA甲基化的全基因组分析， 代表性亚硫酸氢盐测序（RRBS）分析显示，在DNA甲基化的显着增加， 饮食诱导肥胖（DIO）小鼠肝脏中的Klb启动子，其与Klb表达下调相关 RNA测序分析。Klb表达的下调可能限制了增加的FGF 21促进细胞凋亡的能力。 肝脏脂质氧化，导致FGF 21抗性。因此，我们假设， Klb启动子通过营养提示物（例如SFA）和促炎刺激物（例如细胞因子），其水平是 通常在肥胖症中升高，通过下调FGF 21途径介导肝脏脂质蓄积， 导致肥胖症中的脂肪肝。目的1将确定是否增强DNA甲基化在Klb启动子， SFA和细胞因子损害肝脏FGF 21信号传导和脂质代谢，导致肝脏TG蓄积。 我们将首先检验DNMT 1介导Klb启动子DNA甲基化增强的假设， SFA和细胞因子，导致肝脏FGF 21抵抗和脂质积聚。然后我们将确定 Klb基因启动子甲基化在肝脏FGF 21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启动子的甲基化/去甲基化。目标2将 确定DNA甲基化的肝特异性抑制是否促进肝FGF 21信号传导， 改善高脂饮食遗传小鼠模型的脂肪肝。我们已经产生了肝脏特异性DNMT 1， 通过将DNMT 1-和3a-floxed小鼠与白蛋白-Cre小鼠杂交，获得3a敲除（LD 1 KO和LD 3aKO）小鼠。我们 将进一步确定LD 1 KO和LD 3aKO中的FGF 21信号传导、肝脏脂质代谢和TG蓄积 喂食HF饮食的小鼠。我们还建立了一种Cre依赖的CRISPR/RNA引导的靶向DNA方法， 在小鼠肝脏中特异性地在Klb启动子处的甲基化/去甲基化，以测试增强Klb启动子甲基化/去甲基化的假设。 肝细胞中Klb启动子的DNA甲基化介导FGF 21信号转导和脂质代谢受损

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.