Endothelial Estrogen Receptor Alpha and Cardiometabolic Disease

项目总结/摘要 雌激素受体α（ERα）介导的雌激素作用影响葡萄糖稳态和血管 健康然而，这些行动的分子基础，以及如何选择性地利用它们， 治疗增益仍然未知。在最近的研究中，我们惊奇地发现ERα缺失只发生在 在小鼠中的内皮完全否定雌二醇（E2）的抗糖尿病作用。我们还发现， 胆固醇代谢物27-羟基胆固醇（27 HC），我们以前发现是一种内源性的胆固醇代谢物。 ER配体，导致小鼠葡萄糖耐受不良。这两种糖尿病表型都是由于骨骼受损所致。 肌肉葡萄糖处置与肌肉胰岛素输送钝化有关。在先前的动脉粥样硬化研究中，我们 确定，尽管E2具有动脉粥样硬化保护作用，但高胆固醇血症患者27 HC的过度增加， 小鼠的病变发展。小鼠和细胞培养拟议研究的总体目标是 确定雌激素和27 HC对内皮ERα的调节如何影响代谢和血管健康。 目的1是确定E2与27 HC如何配体内皮ERα影响葡萄糖稳态。 肌肉胰岛素输送受胰岛素诱导的微血管募集和灌注控制， 从循环到肌细胞的转胞吞作用穿过内皮单层。现在已经揭示了， E2刺激和27 HC通过培养的内皮细胞钝化胰岛素转胞吞作用，我们将阐明新的- 一种已鉴定的ERα相互作用蛋白，即小GT β Septin 11，与ERα结合，影响内皮细胞的生长， 胰岛素转运E2与27 HC如何影响体内肌肉微血管内皮细胞的基因表达 将通过核糖体相关RNA上的RNA-seq确定。两种雌激素受体配体对微血管的影响 将使用对比增强超声在体内询问肌肉中对胰岛素的反应。目标二是 确定E2与27 HC的内皮ERα配体如何影响血管炎症， 动脉粥样硬化在培养的内皮细胞中，我们先前表明E2和E2均能降低NF-κ B的活化， 通过ERα增加27 HC。在发现后一个过程需要Septin 11之后， Septin 11介导ERα对NF-κ B的调节。E2和27 HC如何改变主动脉中的基因表达 将通过核糖体相关RNA上的RNA-seq来询问体内内皮。目标3认识到， 无论是西方饮食摄入或高胆固醇血症增加27 HC，我们将确定如何降低 内源性27 HC影响在这些条件下发生的葡萄糖耐受不良和动脉粥样硬化， 分别小鼠将接受27 HC合成酶Cyp 27 a1的抑制剂，或腺相关抗体。 允许27 HC代谢酶Cyp 7 b1过表达的病毒。将使用Floxed Cyp 27 a1小鼠 确定巨噬细胞或内皮细胞是否是导致这些的内源性27 HC的关键来源， 紊乱这项工作将揭示内皮细胞ERα是如何作为一个临界点的收敛， 内源性ER配体，即雌激素和27 HC，影响代谢和血管健康的作用。

Project Summary/ Abstract Actions of estrogens mediated by estrogen receptor alpha (ERα) influence glucose homeostasis and vascular health. However, the molecular underpinnings of these actions, and how they can be selectively leveraged for therapeutic gain remain unknown. In recent studies we surprisingly found that ERα deletion exclusively from endothelium in mice fully negates the antidiabetogenic actions of estradiol (E2). We also found that elevating the cholesterol metabolite 27-hydroxycholesterol (27HC), which we previously discovered is an endogenous ER ligand, causes glucose intolerance in mice. Both these diabetic phenotypes were due to impaired skeletal muscle glucose disposal related to blunted muscle insulin delivery. In prior studies of atherosclerosis, we determined that whereas E2 is atheroprotective, an exaggerated increase in 27HC in hypercholesterolemic mice worsens lesion development. The Overall Goal of the proposed research in mice and cell culture is to determine HOW endothelial ERα modulation by estrogens and 27HC influences metabolic and vascular health. Aim 1 is to determine how endothelial ERα liganding by E2 versus 27HC influences glucose homeostasis. Muscle insulin delivery is governed by insulin-induced microvascular recruitment and perfusion, and insulin transcytosis from the circulation to the myocytes across the endothelial monolayer. Having now revealed that E2 stimulates and 27HC blunts insulin transcytosis by cultured endothelium, we will elucidate how a newly- identified ERα interacting protein, the small GTPase septin 11, partners with ERα to influence endothelial insulin transport. How E2 versus 27HC impacts gene expression in muscle microvascular endothelium in vivo will be determined by RNA-seq on ribosome-associated RNA. Effects of the two ER ligands on microvascular responses to insulin in muscle will be interrogated in vivo using contrast-enhanced ultrasound. Aim 2 is to determine how endothelial ERα liganding by E2 versus 27HC influences vascular inflammation and atherosclerosis. In cultured endothelium we previously showed that NF-kB activation is decreased by E2 and increased by 27HC via ERα. Having found that the latter process requires septin 11, we will discern how septin 11 mediates ERα regulation of NF-kB. How E2 and 27HC alter gene expression in the aortic endothelium in vivo will be interrogated by RNA-seq on ribosome-associated RNA. In Aim 3, recognizing that 27HC increases with either western diet intake or hypercholesterolemia, we will determine how lowering endogenous 27HC impacts the glucose intolerance and atherosclerosis that occur under these conditions, respectively. Mice will receive an inhibitor of the 27HC synthesizing enzyme Cyp27a1, or an adeno-associated virus allowing overexpression of the 27HC-metabolizing enzyme Cyp7b1. Floxed Cyp27a1 mice will be used to determine if macrophages or endothelial cells are key sources of endogenous 27HC contributing to these disorders. The proposed work will reveal how endothelial ERα serves as a critical point of convergence of the actions of endogenous ER ligands, namely estrogens and 27HC, to influence metabolic and vascular health.