THE INTEGRATED ROLES OF IPLA2G IN OBESITY, INFLAMMATION AND HEPATIC DYSFUNCTION

描述(申请人提供)：工业化社会中死亡和发病的主要原因是高脂饮食诱导的肥胖(HFDIO)的多重下游后遗症。在此之前，我们通过构建和鉴定一只全球iPLA2γKO小鼠，证明了非钙依赖性磷脂酶A2γ(iPLA2γ)作为一种整合细胞信号和生物体生物能量学的中心酶介质的重要性。值得注意的是，iPLA2γ基因敲除小鼠对HFDIO和胰岛素抵抗的发展是完全抵抗的。然而，由于在整个动物iPLA2γ基因敲除过程中涉及多个器官系统，iPLA2γ在每个组织中的作用机制尚不清楚，从而导致该小鼠对hFDIO的完全抵抗。这项拟议研究的首要目标是从机制上确定iPLA2γ在促进炎症、细胞信号适应不良和导致胰岛素抵抗和HFDIO病理性末端器官后遗症的生物能量学功能障碍中的组织和细胞器特异性作用。因此，我们产生了含有iPLA2γ活性部位的花状结构的小鼠，该小鼠已与组织特异性Cre小鼠杂交，从而产生了肝细胞特异性和骨骼肌特异性iPLA2γ基因敲除小鼠。这项拟议的研究将协同使用这些使能遗传模型，以及我们开发的集成脂质组学和代谢组学平台，以确定iPLA2γ在高强度脂肪抑制期间参与肥胖、炎症和胰岛素抵抗的机制。第一个特定的目的是确定肝细胞特异性的iPLA2γ在不受iPLA2γ在种系敲除中存在的其他细胞类型的功能丧失的影响的情况下，在HFDIO期间在脂类第二信使的产生、脂代谢、肝骨病和生物能量学中的调节作用。在特定的目标2中，我们将检测骨骼肌细胞特异性地去除iPLA2γ对骨骼肌代谢、胰岛素抵抗和线粒体功能障碍的影响，这些在高脂肪喂养的WT小鼠中存在，但在生殖系基因敲除小鼠中得到挽救。最后，在特定的目标3中，我们将通过将线粒体特异性或过氧化物体特异性的iPLA2γ亚型转基因重新导入肝细胞特异性的iPLA2γ基因敲除小鼠中，来确定细胞器特异性的iPLA2γ亚型的相互作用机制。此外，由iPLA2γ在肝细胞、骨骼肌细胞和脂肪细胞中产生的脂质第二信使介导这些代谢交织的组织之间的器官间通讯的机制将通过我们开发/改进的协同高穿透技术来确定。通过使用新的遗传试剂、高质量精度的质谱学技术和综合的化学生理学方法的多学科方法，可以确定减轻HFDIO后遗症的新的药理靶点。

DESCRIPTION (provided by applicant): The major causes of mortality and morbidity in industrialized societies result from the multiple downstream sequelae of high fat diet-induced obesity (HFDIO). Previously, we demonstrated the importance of calciumindependent phospholipase A2γ(iPLA2γ) as a central enzymic mediator integrating cellular signaling and organismal bioenergetics through the generation and characterization of a global iPLA2γ KO mouse. Remarkably, the iPLA2γ knockout mouse was completely resistant to HFDIO and the development of insulin resistance. However, due to the multiple organ systems affected in the whole animal iPLA2γ knockout, the mechanistic roles of iPLA2γ in each tissue contributing to the complete resistance of this mouse to HFDIO are unknown. The overarching goal of the proposed research is the mechanistic determination of the tissue- and organelle-specific roles of iPLA2γ in promoting inflammation, maladaptive cellular signaling and dysfunctional bioenergetics that result in insulin resistance and the pathologic end-organ sequelae of HFDIO. Accordingly, we generated mice containing a floxed construct of the iPLA2γ active site which has been crossed with tissue specific Cre mice resulting in the generation of hepatocyte-specific and skeletal muscle myocyte-specific iPLA2γ knockout mice. The proposed research will synergistically use these enabling genetic models in conjunction with the integrated lipidomics and metabolomics platforms we developed to identify the mechanisms through which iPLA2γ participates in the development of obesity, inflammation, and insulin resistance during HFDIO. The first specific aim will identify the roles of hepatocyte-specific iPLA2γ in mediating alterations in the generation of lipid 2nd messengers, lipid metabolism, hepatosteatosis and bioenergetics during HFDIO unencumbered by the effects of iPLA2γ loss of function in other cell types that are present in the germline knockout. In Specific Aim 2, we will examine the effects of skeletal muscle myocyte-specific ablation of iPLA2γ on skeletal muscle metabolism, insulin resistance and mitochondrial dysfunction that are present during high fat feeding in WT mice, but are rescued in the germline knockout mouse. Finally, in Specific Aim 3, we will determine the interactive mechanistic roles of the organelle-specific isoforms of iPLA2γ through the transgenic reintroduction of either the mitochondrial-specific or peroxisomal-specific isoforms of iPLA2γ into the hepatocyte-specific iPLA2γ knockout mouse. Moreover, the mechanisms through which lipid 2nd messengers generated by iPLA2γ in hepatocytes, skeletal muscle myocytes and adipocytes mediate inter-organ communication between these metabolically interwoven tissues will be identified through synergistic highly penetrating technologies we have developed/refined. Through this multidisciplinary approach employing novel genetic reagents, high mass accuracy mass spectrometry technologies and integrated chemophysiologic approaches, novel pharmacologic targets to attenuate the sequelae of HFDIO can be identified.