Protective role of adenosine 2A receptor in NAFLD

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）是终末期肝病（包括肝硬化和肝细胞癌）的最常见原因之一。越来越多的证据表明炎症在NAFLD的发病机制中至关重要。例如，营养过载引发炎症，其可以通过刺激脂肪生成来增加肝脂肪变性。后者反过来会加剧肝脏炎症并进展为非酒精性脂肪性肝炎（NASH）。然而，肝脂肪变性和肝脏炎症之间相互作用的确切机制仍有待阐明。因此，拟议研究的长期目标是剖析NAFLD的代谢和炎症机制，以便开发新的循证方法来预防和/或治疗NASH。腺苷2A受体（adenosine 2A receptor，A2 AR）是一种G蛋白偶联受体，在免疫细胞中大量表达，具有很强的抗炎作用。A2 AR也在肝细胞中高度表达，其中A2 AR功能在很大程度上是未知的。对于该项目，中心假设是肝细胞和巨噬细胞中的A2 AR以细胞类型依赖性方式保护NAFLD的不同方面的发展。这一假说基于以下新发现：1）肝细胞中A2 AR缺陷比骨髓细胞中A2 AR缺陷发挥更重要的作用（巨噬细胞）在加剧高脂饮食（HFD）诱导的肝脂肪变性中的作用，这与脂肪生成酶的肝表达增加有关; 2）A2 AR缺乏加剧HFD诱导的肝脏炎症，这可能归因于增加的巨噬细胞/枯否细胞促炎活化;和3）通过特异性激动剂激活A2 AR保护小鼠免受HFD诱导的NAFLD。因此，该项目的目标是确定A2 AR在NAFLD中的新保护作用。为此目的，产生肝细胞和/或骨髓细胞中缺乏A2 AR的小鼠。对于特定目标1，将进行体内实验以检查肝细胞中A2 AR通过抑制脂肪生成来保护免于NAFLD的程度。此外，将进行细胞实验以阐明SREBP 1c和ChREBP参与A2 AR抑制脂肪生成基因表达。对于特定目标2，将进行体内实验以检查巨噬细胞或肝细胞中的A2 AR通过抑制肝脏炎症反应而保护免受NAFLD的程度。对于特定目标3，将进行体内实验以确定NAFLD中肝细胞-巨噬细胞串扰的A2 AR协调。此外，将进行体外共培养实验以检查由A2 AR缺陷型巨噬细胞产生的因子，即，肿瘤坏死因子和IL-6刺激肝细胞脂肪生成，以及由A2 AR缺陷肝细胞产生的因子，即，棕榈酸，刺激巨噬细胞促炎激活。总之，拟议的研究将阐明NAFLD的新范式，并通过A2 AR激活为预防和/或治疗NASH提供实验基础。

DESCRIPTION (provided by applicant): Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of terminal liver diseases including liver cirrhosis and hepatocellular carcinoma. Growing evidence demonstrates the critical importance of inflammation in the pathogenesis of NAFLD. For instance, nutrient overload triggers inflammation, which can act through stimulating lipogenesis to increase hepatic steatosis. The latter, in turn, can exacerbate liver inflammation and progress to non-alcoholic steatohepatitis (NASH). However, the precise mechanisms underlying the interaction between hepatic steatosis and liver inflammation remain to be elucidated. Thus, the long-term goal of the proposed research is to dissect the metabolic and inflammatory mechanisms underlying NAFLD in order that novel evidence-based approaches can be developed for preventing and/or treating NASH. As a G-protein-coupled receptor, adenosine 2A receptor (A2AR) is abundantly expressed in immune cells and exhibits powerful anti-inflammatory properties. A2AR is also highly expressed in hepatocytes, in which A2AR functions are largely unknown. For this project, the central hypothesis is that the A2AR in hepatocytes and macrophages protects against the development of different aspects of NAFLD in a cell-type-dependent manner. This hypothesis is based on the following novel findings: 1) A2AR deficiency in hepatocytes plays a more important role than A2AR deficiency in myeloid cells (macrophages) in exacerbating high-fat diet (HFD)- induced hepatic steatosis, which is associated with increased hepatic expression of lipogenic enzymes; 2) A2AR deficiency exacerbates HFD-induced liver inflammation, which is likely attributed to increased macrophage/Kupffer cell proinflammatory activation; and 3) A2AR activation by a specific agonist protects mice from HFD-induced NAFLD. Thus, the goal of this project is to define a novel protective role for A2AR in NAFLD. For this purpose, mice that lack A2AR in hepatocytes and/or myeloid cells are generated. For Specific Aim 1, in vivo experiments will be performed to examine the extent to which the A2AR in hepatocytes acts through inhibiting lipogenesis to protect against NAFLD. Moreover, cellular experiments will be performed to elucidate the involvement of SREBP1c and ChREBP in A2AR inhibition of lipogenic gene expression. For Specific Aim 2, in vivo experiments will be performed to examine the extent to which the A2AR in macrophages or hepatocytes protects against NAFLD by suppressing liver inflammatory response. For Specific Aim 3, in vivo experiments will be performed to define A2AR coordination of hepatocyte-macrophage crosstalk in NAFLD. Moreover, in vitro co-culture experiments will be performed to examine the extent to which factors generated by A2AR-deficient macrophages, i.e., TNF? and IL-6, stimulate hepatocyte lipogenesis, and the extent to which factors generated by A2AR-deficient hepatocytes, i.e., palmitate, stimulate macrophage proinflammatory activation. Together, the proposed research will illustrate a new paradigm on NAFLD, and provide the experimental basis for prevention and/or treatment of NASH by means of A2AR activation.