ADK Regulation of Fat Metabolism and Insulin Sensitivity

脂肪在肝细胞中过度沉积会引起炎症反应，损害胰岛素信号，进而促进全身性胰岛素抵抗和代谢失调的发展。作为腺苷磷酸化的酶，腺苷激酶(ADK)在肝脏中的表达水平最高，并决定着细胞内和细胞外的腺苷水平。然而，ADK如何调节肝脏和全身脂肪代谢与全身胰岛素敏感性的确切关系尚不清楚。在本项目中，肝细胞中的ADK已被证实能促进小鼠肝脏脂肪沉积和增加全身脂肪质量。其他初步结果还表明：1)小鼠肝细胞特异性ADK过表达可增加肝脏炎症反应，引起全身性胰岛素抵抗，而肝细胞特异性ADK干扰可减轻高脂饮食(HFD)诱导的胰岛素抵抗的严重程度；2)ADK干扰可增加肝脏和巨噬细胞腺苷2A受体(A2AR)的表达，从而增加肝脏ADK数量，加剧饮食诱导的肝脏脂肪堆积和胰岛素抵抗，损害肝细胞胰岛素信号转导，并刺激巨噬细胞促炎(M1)激活；以及3)在肝细胞特异性ADK缺陷小鼠的肝脏和髓系细胞特异性ADK缺陷小鼠的巨噬细胞中，DNA甲基化减少。基于这些发现，本项目将验证这一中心假设，即过量的ADK通过失调肝细胞-巨噬细胞串扰促进肝脏脂肪沉积，损害肝脏胰岛素信号，增加肝脏炎症，从而导致肝脏和全身胰岛素抵抗。从机制上讲，ADK的作用包括A2AR信号受损和肝细胞脂肪酸氧化和巨噬细胞抗炎反应基因内DNA甲基化的增加。因此，将追求三个具体目标。目的1：明确肝细胞ADK在调节脂肪代谢和胰岛素敏感性中的作用。体内实验将检验肝细胞特异性ADK过度表达或中断在多大程度上改变HFD诱导的肝脏脂肪沉积、炎症和胰岛素抵抗的严重程度。体外实验将会 用于研究ADK驱动的肝细胞因子如何改变巨噬细胞/枯否细胞的炎症状态。目的2：明确巨噬细胞ADK在调节脂肪代谢和胰岛素敏感性中的作用。体内实验将检查髓系细胞特异性ADK过度表达或中断在多大程度上改变HFD诱导的肝脏脂肪沉积、炎症和胰岛素抵抗的严重程度。体外实验将检验ADK驱动的巨噬细胞因子是否以及如何促进肝细胞脂肪沉积和炎症反应。目的3：确定ADK通过减少A2AR信号和/或增加DNA甲基化，损害肝细胞脂肪代谢和胰岛素信号转导，促进巨噬细胞M1活化，从而发挥作用的程度。该项目的成功完成将加速开发新的基于ADK抑制的方法来管理肥胖相关的慢性疾病。

Excessive deposition of fats in hepatocytes causes inflammation and impairs insulin signaling, which in turn promotes the development of systemic insulin resistance and metabolic dysregulation. As the enzyme that phosphorylates adenosine, adenosine kinase (ADK) is expressed at the highest levels in the liver and critically determines the levels of both intracellular and extracellular adenosine. However, precisely how ADK regulates hepatic and systemic fat metabolism in relation to whole body insulin sensitivity is unknown. For this project, the ADK in hepatocytes has been validated to promote hepatic fat deposition and increase whole body fat mass in mice. Additional preliminary results also indicate: 1) hepatocyte-specific ADK overexpression in mice increases liver inflammation and causes systemic insulin resistance whereas hepatocyte-specific ADK disruption in mice decreases the severity of high-fat diet (HFD)-induced insulin resistance; 2) ADK disruption increases hepatic and macrophage expression of adenosine 2A receptor (A2AR), whose disruption increases hepatic ADK amount, exacerbates diet-induced hepatic fat accumulation and insulin resistance, impairs hepatocyte insulin signaling, and stimulates macrophage proinflammatory (M1) activation; and 3) DNA methylation was decreased in livers of hepatocyte-specific ADK-deficient mice and in macrophages from myeloid cell-specific ADK-deficient mice. Based on these findings, this project will test the central hypothesis that excessive ADK acts through dysregulating hepatocyte-macrophage crosstalk to promote hepatic fat deposition, impair liver insulin signaling, and increase liver inflammation, thereby bringing about hepatic and systemic insulin resistance. Mechanistically, ADK actions involve impaired A2AR signaling and increased DNA methylation within genes for hepatocyte fatty acid oxidation and macrophage anti-inflammatory responses. Accordingly, three Specific Aims will be pursued. Aim 1: Define the role of hepatocyte ADK in regulating fat metabolism and insulin sensitivity. In vivo experiments will be performed to examine the extent to which hepatocyte-specific ADK overexpression or disruption alters the severity of HFD-induced hepatic fat deposition, inflammation, and insulin resistance. In vitro experiments will be used to examine how ADK-driven hepatocyte factors alter the inflammatory status of macrophages/Kupffer cells. Aim 2: Define the role of macrophage ADK in regulating fat metabolism and insulin sensitivity. In vivo xperiments will be performed to examine the extent to which myeloid cell-specific ADK overexpression or disruption alters the severity of HFD-induced hepatic fat deposition, inflammation, and insulin resistance. In vitro experiments will be performed to examine whether and how ADK-driven macrophage factors promote hepatocyte fat deposition and inflammatory responses. Aim 3: Determine the extent to which ADK acts through decreasing A2AR signaling and/or increasing DNA methylation to impair hepatocyte fat metabolism and insulin signaling and enhance macrophage M1 activation. The successful completion of this project will accelerate the development of novel ADK inhibition-based approaches for managing obesity-associated chronic diseases.