Functional Characterization of Diacylglycerol Lipases in Mammalian Physiology

项目摘要/摘要 这项K99/R00职业发展奖提案的总体目标是整合创新的化学探针， 包括体内活性小分子抑制剂和专门的化学蛋白质组分析，具有良好的 建立了基因敲除和行为小鼠模型，以获得病理生理学的全局视图 干扰2-花生四烯酸甘油(2-AG)生物合成对肥胖和肥胖小鼠的影响 发炎。内源性大麻素(内源性大麻素)2-AG是一种脂类递质，激活G- 蛋白质偶联受体CB1和CB2也是大麻中精神活性成分的靶标， ∆9-四氢大麻酚。内源性大麻素系统在肥胖相关代谢中的重要性 CB1拮抗剂作为抗肥胖和抗糖尿病药物的临床活性突出了疾病。 临床前模型的补充研究表明，许多有益的影响是由于 阻断外周部位的CB1受体。这些研究突显了需要更深入地了解 内源性大麻素系统在调节能量稳态中的中枢和/或外周作用 以及与肥胖相关的疾病状态。2-AG在体内的生物合成受两个序列的差异调控- 相关酶，二酰甘油脂肪酶-α和β(分别为DAGLα和DAGLβ)，提供了一种实验性的 (并最终翻译)解开中枢和外周内源性大麻素信号的机会 通过选择性地灭活DAGL亚型。这一提议将检验这样的假设，即DAGLα和 DAGLβ生物合成2-AG参与能量动态平衡并在能量上发挥互补功能 通过在解剖上不同的部位调节CB1依赖的信号来实现平衡。具体目标是 1)测定DAGL损伤小鼠体内能量稳态参数和体质量组成2) 测量DAGL破坏的小鼠脂肪组织中巨噬细胞的聚集和3)确定 DAGLS失活在代谢综合征发生发展中的生理作用我们的 初步研究表明，尽管摄食量增加，但DAGLα表现出瘦肉型，提供了 中枢和/或外周2-AG生物合成中断时能量平衡改变的证据。我们的 初步研究还表明，使用DAGL选择性抑制剂来解偶联中枢和 体内外周2-AG信号通路。 这位候选人的目标是将之前在化学蛋白质组学、合成化学和定量方面的培训结合起来 蛋白质组学和代谢组学在小鼠代谢和炎症模型中的新指导训练 包括控制能量稳态和体重的代谢参数的非侵入性测量 过继细胞转移法测定脂肪组织巨噬细胞聚集的组成及体内示踪 和流式细胞术。

Project Summary/Abstract The overall goal of this K99/R00 career development award proposal is to integrate innovate chemical probes, including in vivo-active small-molecule inhibitors and specialized chemoproteomic assays, with well- established genetic knockout and behavioral mouse models to obtain a global view of the pathophysiological consequence of disrupting 2-arachidonoylglycerol (2-AG) biosynthesis in mouse models of obesity and inflammation. The endogenous cannabinoid (endocannabinoid) 2-AG is a lipid transmitter that activates the G- protein-coupled receptors CB1 and CB2, which are also targets for the psychoactive ingredient in marijuana, ∆9-tetrahydrocannabinol. The importance of the endocannabinoid system in obesity-associated metabolic disorders is highlighted by the clinical activity of CB1 antagonists as anti-obesity and anti-diabetic drugs. Complementary studies in preclinical models have since shown that many of the beneficial effects are due to blocking CB1 receptors at peripheral sites. These studies highlight the need for a deeper understanding of the central and/or peripheral contributions of the endocannabinoid system in regulating energy homeostasis as well as obesity-related disease states. 2-AG biosynthesis in vivo is differentially regulated by two sequence- related enzymes, diacylglycerol lipase-α and β (DAGLα and DAGLβ, respectively), providing an experimental (and, eventually translational) opportunity to uncouple central and peripheral endocannabinoid signaling through selective inactivation of DAGL isoforms. This proposal will test the hypothesis that DAGLα and DAGLβ biosynthesize 2-AG involved in energy homeostasis and perform complementary functions in energy balance through regulation of CB1-dependent signaling at anatomically-distinct sites. The specific aims are to 1) measure energy homeostasis parameters and body mass composition in vivo in DAGL-disrupted mice 2) measure adipose tissue macrophage accumulation in DAGL-disrupted mice and 3) Determine the physiological effects of inactivating DAGLs in the development and progression of metabolic syndrome. Our preliminary studies show that DAGLα display a lean phenotype despite increased food intake, providing evidence of altered energy balance upon central and/or peripheral disruption of 2-AG biosynthesis. Our preliminary studies also show the feasibility of using DAGL-selective inhibitors to uncouple central and peripheral 2-AG signaling pathways in vivo. The candidate aims to combine previous training in chemoproteomics, synthetic chemistry, and quantitative proteomics and metabolomics with new mentored training in mouse models of metabolism and inflammation including non-invasive measurements of metabolic parameters governing energy homeostasis and body mass composition as well as in vivo tracking of adipose tissue macrophage accumulation using adoptive cell transfer and flow cytometry.