Macrophage AMPK, Inflammation, and Atherosclerosis

描述(申请人提供)：AMPK是一种进化保守的细胞能量感受器，调节脂肪、胆固醇和葡萄糖代谢的代谢途径。我们已经报道了巨噬细胞AMPK在体外对脂质诱导的炎症和胰岛素抵抗的保护作用。此外，在载脂蛋白E-/-小鼠中，用5-aminoimidazole-4-carboxamide-1-2-4-ribofuranoside(AICAR)激活AMPK可以减轻动脉粥样硬化的负担。然而，AMPK的多效性功能使得很难明确巨噬细胞AMPK在炎症相关代谢紊乱的发病机制中的作用，包括动脉粥样硬化和胰岛素抵抗。在这项计划中，将使用巨噬细胞特异性过度表达显性负或固有活性11AMPK的转基因小鼠(分别为DN11和CA11)和巨噬细胞特异性11MAPK基因敲除(MAKO)小鼠来研究巨噬细胞AMPK在这些代谢紊乱中的作用。我们发现，CA11小鼠的巨噬细胞显著降低了脂质和胆固醇诱导的内质网应激、炎症和细胞凋亡，这与参与脂肪酸氧化和胆固醇外流的基因上调有关。我们假设巨噬细胞AMPK通过促进脂肪氧化和胆固醇外流来调节巨噬细胞的脂质和胆固醇动态平衡，从而防止脂质和胆固醇诱导的内质网应激、炎症和细胞凋亡。这可能与巨噬细胞AMPK对动脉粥样硬化和胰岛素抵抗的保护作用有关。具体目标1将利用CA11、DN11和Mako小鼠的巨噬细胞确定巨噬细胞AMPK防止脂质和胆固醇诱导的内质网应激、炎症和凋亡的机制。我们将确定：1)AMPK是否通过促进胆固醇外流和脂肪酸氧化来调节巨噬细胞的脂质和胆固醇动态平衡，以及2)这些途径是否在介导AMPK抑制脂质和胆固醇诱导的内质网应激、炎症和细胞凋亡方面是必要的。目的2利用CA11、DN11和Mako小鼠与低密度脂蛋白受体基因敲除小鼠杂交，确定巨噬细胞AMPK对动脉粥样硬化的保护作用。我们将确定：1)血浆脂蛋白表型，2)体内巨噬细胞胆固醇的反向转运，3)动脉粥样硬化程度，4)动脉粥样硬化病变中巨噬细胞的数量、脂质含量、内质网应激、炎症和细胞凋亡，以及5)巨噬细胞积聚、内质网应激、炎症和细胞凋亡发展的动力学。通过CA11、DN11和Mako小鼠实验，确定巨噬细胞AMPK对胰岛素抵抗的保护作用。高脂饮食小鼠的胰岛素敏感性、脂肪组织巨噬细胞浸润、内质网应激和炎症反应将被检测。完成这些研究将极大地提高我们对巨噬细胞AMPK在预防炎症相关代谢紊乱(包括动脉粥样硬化和胰岛素抵抗)中的作用的了解。

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) is an evolutionally conserved cellular energy sensor that regulates metabolic pathways in lipid, cholesterol and glucose metabolism. We have reported that macrophage AMPK protects against lipid-induced inflammation and insulin resistance in vitro. In addition, activating AMPK by 5- aminoimidazole-4-carboxamide-1-2-4-ribofuranoside (AICAR) in ApoE-/- mice alleviates atherosclerosis burden. However, the pleiotropic functions of AMPK make it difficult to determine specifically the role of macrophage AMPK in the pathogenesis of inflammation-associated metabolic disorders, including atherosclerosis and insulin resistance. In this proposal, transgenic mice with macrophage-specific over- expression of dominant negative or constitutively active 11AMPK (DN11 and CA11, respectively), and macrophage-specific 11MAPK knockout (MAKO) mice will be used to study the role of macrophage AMPK in these metabolic disorders. We find that macrophages from CA11 mice have significantly lower lipid- and cholesterol-induced ER stress, inflammation and apoptosis, which is associated with up-regulation of genes involved in fatty acid oxidation and cholesterol efflux. We hypothesize that macrophage AMPK regulates macrophage lipid and cholesterol homeostasis by promoting fatty oxidation and cholesterol efflux, thereby preventing lipid- and cholesterol-induced ER stress, inflammation and apoptosis. This may contribute to macrophage AMPK's protective effects against atherosclerosis and insulin resistance. Specific aim 1 will determine mechanisms by which macrophage AMPK prevents lipid- and cholesterol-induced ER stress, inflammation and apoptosis using macrophages from CA11, DN11 and MAKO mice. We will determine: 1) whether AMPK regulates macrophage lipid and cholesterol homeostasis by promoting cholesterol efflux and fatty acid oxidation, and 2) whether these pathways are necessary in mediating AMPK's suppression of lipid- and cholesterol-induced ER stress, inflammation and apoptosis. Specific aim 2 will determine the protective effects of macrophage AMPK against atherosclerosis using CA11, DN11 and MAKO mice crossed with low density lipoprotein receptor knockout mice. We will determine: 1) plasma lipoprotein phenotype, 2) in vivo reverse cholesterol transport from macrophages, 3) atherosclerosis extent, 4) macrophage number, lipid content, ER stress, inflammation and apoptosis in atherosclerotic lesions, and 5) kinetics of macrophage accumulation, ER stress, inflammation and apoptosis development in early and late atherosclerotic lesions in these mice fed an atherogenic diet. Specific aim 3 will determine the protective effects of macrophage AMPK against insulin resistance using CA11, DN11 and MAKO mice. The insulin sensitivity and adipose tissue macrophage infiltration, ER stress and inflammation will be determined in these mice on a high fat diet. Completing these studies will greatly improve our knowledge on the role of macrophage AMPK in the protection against inflammation-associated metabolic disorders, including atherosclerosis and insulin resistance.