NAFLD: Mechanisms and Treatments

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）是一种常见的肝脏疾病，影响着世界三分之一的人口。NAFLD的第一阶段是肝脂肪变性，其特征是肝细胞细胞质中脂肪滴的积累。肝脂肪变性进一步发展为非酒精性脂肪性肝炎（NASH）、肝硬化和肝细胞癌（HCC）。年龄和饮食/脂肪摄入是NAFLD的危险因素。我的实验室研究NAFLD与年龄相关的途径。我们发现C/EBP家族的两个成员，C/EBPα和C/EBPß，以及染色质重塑蛋白p300在与衰老相关的NAFLD中发挥关键作用。我们的研究结果表明，细胞周期蛋白依赖性激酶4 （cdk4）使C/EBPα在Ser193位点磷酸化，引发三方C/EBPα/ß-p300复合物的形成。通过四种C/EBPα/ß-p300复合物水平升高或降低的动物模型，我们发现C/EBPα/ß-p300复合物激活了五种关键甘油三酯（TG）合成酶的表达。甘油三酯是NAFLD患者肝细胞内积聚的脂肪滴的主要成分。这导致我们的主要假设，NAFLD的发展是通过上调cdk4-C/EBPα-p300- TG途径介导的，cdk4抑制剂介导的C/EBPα Ser193磷酸化的降低将抑制NAFLD的发展。我们建议使用NAFLD动物模型和NAFLD患者的肝脏活检来验证这一假设。特异性目的1将检测携带C/EBPα- S193A（一种不能被cdk4磷酸化的C/EBPα构建体）的敲入小鼠是否对NAFLD的发展具有抗性。我们最近生成了这些C/EBPα-S193A小鼠。我们将研究C/EBPα-S193A小鼠NAFLD的发展是否会随着年龄的增长和长期高脂肪饮食条件下的减少。特异性Aim 2将测试小分子药物抑制cdk4活性是否会抑制NAFLD。野生型小鼠将维持高脂或低脂饮食，并使用cdk4抑制剂（如PD0332991）进行治疗。将比较未治疗和治疗小鼠的cdk4-C/EBPα-p300-TG通路的活性和NAFLD的发展。特异性Aim 3将确定cdk4-C/EBPα-p300-TG合成途径在NAFLD患者中是否升高。我们最近的数据显示，在NAFLD小鼠模型和NAFLD患者中，s193磷酸化的C/EBPα水平升高。因此，我们将：1)确定NAFLD患者肝脏中cdk4及其激活伴侣细胞周期素D3的表达是否升高，2)确定NAFLD患者肝脏样本中C/EBPα-p300复合物的水平是否升高，3)检查NAFLD患者中TG合成酶水平是否升高。这些拟议的研究将为开发一种新的基于cdk4的NAFLD治疗方法提供基础。由于cdk4/6抑制剂PD033991已经用于其他疾病的临床试验，因此拟议的研究可能很快转化为治疗人类NAFLD患者。

DESCRIPTION (provided by applicant): Nonalcoholic fatty liver disease (NAFLD) is a common liver disease that affects one-third of the world's population. The first stage of NAFLD is hepatic steatosis, which is characterized by accumulation of fat droplets in the cytoplasm of hepatocytes. Hepatic steatosis further develops into nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Age and diet/fat intake are risk factors for NAFLD. My laboratory investigates age-associated pathways of NAFLD. We found that two members of the C/EBP family, C/EBPα and C/EBPß, as well as the chromatin remodeling protein p300 play a critical role in NAFLD associated with aging. Our findings show that cyclin-dependent kinase 4 (cdk4) phosphorylates C/EBPα at Ser193, triggering formation of the tripartite C/EBPα/ß-p300 complex. Using four animal models with increased or reduced levels of C/EBPα/ß-p300 complexes, we found that the C/EBPα/ß-p300 complex activates expression of five key triglyceride (TG) synthesis enzymes. Triglycerides are the main components of the fat droplets that are accumulated in the hepatocytes of NAFLD patients. This led to our main hypothesis that the development of NAFLD is mediated by up-regulation of the cdk4-C/EBPα-p300- TG pathway and that cdk4 inhibitor-mediated reduction in phosphorylation of C/EBPα at Ser193 will inhibit development of NAFLD. We propose to test this hypothesis using animal models of NAFLD and liver biopsies from patients with NAFLD. Specific Aim 1 will examine if knock-in mice carrying C/EBPα- S193A, a C/EBPα construct that cannot be phosphorylated by cdk4, are resistant to development of NAFLD. We recently generated these C/EBPα-S193A mice. We will examine if the development of NAFLD that we normally observe with age and under long-term high fat diet conditions will be reduced in C/EBPα-S193A mice. Specific Aim 2 will test if the inhibition of cdk4 activity by small molecule drugs will inhibit NAFLD. Wild-type mice will be maintained on either high-fat or low-fat diets and treated with inhibitors of cdk4, such as PD0332991. Activity of the cdk4-C/EBPα-p300-TG pathway and development of NAFLD will be compared between un-treated and treated mice. Specific Aim 3 will determine if the cdk4-C/EBPα-p300-TG synthesis pathway is elevated in patients with NAFLD. Our recent data showed that levels of S193-phosphorylated C/EBPα are increased in mouse models of NAFLD and in NAFLD patients. We will therefore: 1) determine if the expression of cdk4 and its activating partner cyclin D3 are elevated in the livers of patients with NAFLD, 2) determine if levels of C/EBPα-p300 complexes are elevated in NAFLD patient liver samples, and 3) examine if TG synthesis enzyme levels are increased in patients with NAFLD. These proposed studies will provide a basis for the development of a novel cdk4-based therapy for NAFLD. Because the cdk4/6 inhibitor PD033991 is already used in the clinical trials for other diseases, the proposed studies might be quickly translated to treatment of human patients with NAFLD.