Functional Characterization of Diacylglycerol Lipases in Mammalian Physiology

项目总结/摘要 这个K99/R 00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学测定， 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， 在肥胖小鼠模型中破坏2-花生四烯酸甘油（2-AG）生物合成的结果， 炎症内源性大麻素（endocannabinoid）2-AG是一种脂质递质，可激活G- 蛋白偶联受体CB 1和CB 2也是大麻中精神活性成分的靶点， - 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 CB 1拮抗剂作为抗肥胖症和抗糖尿病药物的临床活性突出了疾病的风险。 在临床前模型中的补充研究已经表明，许多有益的效果是由于 阻断外周部位的CB 1受体。这些研究突出表明，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。体内2-AG生物合成受两种序列的差异调节- 相关酶，二酰基甘油脂肪酶-α和β（分别为DAGLα和DAGLβ），提供了一个实验性的 (and最终翻译）的机会来解偶联中枢和外周内源性大麻素信号传导 通过DAGL同种型的选择性失活。该提议将检验DAGLα和 DAGLβ生物合成2-AG参与能量稳态并在能量中发挥补充功能 通过在解剖学上不同的位点调节CB 1依赖的信号传导来平衡。具体目标是 1)在DAGL破坏的小鼠中测量体内能量稳态参数和体重组成2） 测量DAGL-破坏的小鼠中脂肪组织巨噬细胞的积累，和3）测定DAGL-破坏的小鼠中脂肪组织巨噬细胞的积累。 失活DAGL在代谢综合征的发展和进展中的生理作用。我们 初步研究表明，尽管食物摄入量增加，DAGLα仍显示出瘦型， 在2-AG生物合成的中枢和/或外周破坏后改变的能量平衡的证据。我们 初步研究还显示了使用DAGL选择性抑制剂解偶联中枢和中枢神经系统的可行性。 体内外周2-AG信号通路。 候选人的目标是联合收割机在化学蛋白质组学，合成化学和定量 蛋白质组学和代谢组学，在代谢和炎症小鼠模型中进行新的指导培训 包括控制能量稳态和体重的代谢参数的非侵入性测量 组合物以及使用过继细胞转移体内追踪脂肪组织巨噬细胞积聚 和流式细胞仪

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.