NAFLD: Mechanisms and Treatments

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）是一种常见的肝病，影响世界三分之一的人口。NAFLD的第一阶段是肝脂肪变性，其特征在于肝细胞的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的年龄相关途径。我们发现C/EBP家族的两个成员C/EBPα和C/EBP β以及染色质重塑蛋白p300在与衰老相关的NAFLD中起关键作用。我们的研究结果表明，细胞周期蛋白依赖性激酶4（cdk 4）磷酸化C/EBPα在Ser 193，触发形成的三重C/EBPα/p300复合物。使用四种C/EBPα/p300复合物水平升高或降低的动物模型，我们发现C/EBPα/p300复合物激活五种关键甘油三酯（TG）合成酶的表达。甘油三酯是NAFLD患者肝细胞中积累的脂肪滴的主要成分。这导致了我们的主要假设，即NAFLD的发生是由cdk 4-C/EBPα-p300- TG通路的上调介导的，并且cdk 4激酶介导的C/EBPα Ser 193磷酸化的减少将抑制NAFLD的发生。我们建议使用NAFLD动物模型和NAFLD患者的肝活检来验证这一假设。具体目标1将检查携带C/EBPα-S193 A（一种不能被cdk 4磷酸化的C/EBPα构建体）的基因敲入小鼠是否对NAFLD的发生具有抗性。我们最近产生了这些C/EBPα-S193 A小鼠。我们将检查在C/EBPα-S193 A小鼠中，我们通常随年龄增长和长期高脂饮食条件下观察到的NAFLD的发展是否会减少。具体目标2将测试小分子药物对cdk 4活性的抑制是否会抑制NAFLD。野生型小鼠将维持高脂肪或低脂肪饮食，并用cdk 4抑制剂如PD 0332991治疗。将在未处理和处理的小鼠之间比较cdk 4-C/EBPα-p300-TG途径的活性和NAFLD的发展。具体目标3将确定cdk 4-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C/EBPα水平升高。因此，我们将：1）确定cdk 4及其活化伴侣细胞周期蛋白D3的表达在NAFLD患者的肝脏中是否升高，2）确定C/EBPα-p300复合物的水平在NAFLD患者的肝脏样品中是否升高，和3）检查TG合成酶水平在NAFLD患者中是否升高。这些拟议的研究将为开发基于cdk 4的NAFLD新疗法提供基础。由于cdk 4/6抑制剂PD 033991已经用于其他疾病的临床试验，因此拟议的研究可能会迅速转化为NAFLD患者的治疗。

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.