Functional Characterization of Diacylglycerol Lipases in Mammalian Physiology

描述（由申请人提供）：这项K99/R00职业发展奖提案的总体目标是将创新的化学探针，包括体内活性小分子抑制剂和专门的化学蛋白质组学分析，与成熟的基因敲除和行为小鼠模型结合起来，以获得在肥胖和炎症小鼠模型中破坏2-花生四烯醇甘油酯（2-AG）生物合成的病理生理后果的全局观点。内源性大麻素（内源性大麻素）2-AG是一种脂质递质，可激活G-蛋白偶联受体CB1和CB2，这也是大麻中精神活性成分9-四氢大麻酚的靶点。CB1拮抗剂作为抗肥胖和抗糖尿病药物的临床活性突出了内源性大麻素系统在肥胖相关代谢紊乱中的重要性。临床前模型的补充研究表明，许多有益效果是由于阻断外周部位的CB1受体。这些研究强调需要更深入地了解内源性大麻素系统在调节能量稳态和肥胖相关疾病状态中的中枢和/或外周作用。体内2-AG的生物合成受两种序列相关酶-二酰基甘油脂肪酶- α和β（分别为DAGL α和DAGL β）的差异调节，这为通过DAGL亚型的选择性失活来解耦中枢和外周内源性大麻素信号提供了实验（并最终翻译）机会。该提案将验证DAGL α和DAGL β生物合成参与能量稳态的2-AG的假设，并通过调节cb1依赖性信号在解剖学上不同的部位在能量平衡中发挥互补功能。具体目的是1)测量dagl破坏小鼠体内能量稳态参数和体重组成2)测量dagl破坏小鼠脂肪组织巨噬细胞积累3)确定灭活dagl在代谢综合征发生和进展中的生理影响。我们的初步研究表明，尽管食物摄入量增加，DAGL α仍表现出瘦弱的表型，这为中枢和/或外周2-AG生物合成中断时能量平衡的改变提供了证据。我们的初步研究还表明，在体内使用dagl选择性抑制剂解耦中枢和外周2-AG信号通路是可行的。该候选人的目标是将之前在化学蛋白质组学、合成化学、定量蛋白质组学和代谢组学方面的培训与小鼠代谢和炎症模型的新指导培训结合起来，包括控制能量稳态和体重组成的代谢参数的无创测量，以及使用过继细胞转移和流式细胞术在体内跟踪脂肪组织巨噬细胞积累。

DESCRIPTION (provided by applicant): 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-alpha and beta (DAGL alpha and DAGL beta, 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 DAGL alpha and DAGL beta 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 DAGL alpha 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.