Endothelial Estrogen Receptor Alpha and Cardiometabolic Disease

项目摘要/摘要 雌激素受体α(ER-α)介导的雌激素作用对血糖稳态和血管的影响 健康。然而，这些行动的分子基础，以及如何有选择地利用它们来 治疗效果仍不得而知。在最近的研究中，我们令人惊讶地发现，ERα缺失完全来自 小鼠的内皮细胞完全否定了雌二醇(E2)的抗糖尿病作用。我们还发现， 我们先前发现的胆固醇代谢产物27-羟基胆固醇(27HC)是一种内源性 ER配体，可引起小鼠的葡萄糖耐量异常。这两种糖尿病表型都是由于骨骼受损所致。 肌肉葡萄糖处理与钝化的肌肉胰岛素输送有关。在先前关于动脉粥样硬化的研究中，我们 虽然E2具有动脉粥样硬化保护作用，但在高胆固醇血症中，27HC的增加被夸大了 老鼠会加重病变的发展。拟议中的小鼠和细胞培养研究的总体目标是 确定雌激素和27HC对内皮ERα的调节如何影响代谢和血管健康。 目的1是确定由E2与27HC连接的内皮ERα是如何影响血糖稳态的。 肌肉胰岛素的输送受胰岛素诱导的微血管募集和灌流以及胰岛素的控制 通过血管内皮细胞单层从循环到肌细胞的细胞穿透。现已透露， E2刺激和27HC钝化培养的内皮细胞的胰岛素转运，我们将阐明一种新的- 鉴定的ERα相互作用蛋白，小GTP酶Septin 11，与ERα合作影响内皮细胞 胰岛素的运输。雌二醇和27HC对肌肉微血管内皮细胞基因表达的影响 将由核糖体相关RNA上的RNA-seq决定。两种内质网配体对微血管的影响 肌肉对胰岛素的反应将使用对比增强超声在活体内进行询问。目标2是 确定由E2与27HC连接的内皮ERα如何影响血管炎症和 动脉硬化。在培养的内皮细胞中，我们先前已经发现，雌激素和雌激素可降低核因子-kB的活性 通过ERα增加了27HC。在发现后一个过程需要Septin 11之后，我们将了解如何 Septin 11介导ERα对核因子-kB的调节。E2和27HC如何改变主动脉中的基因表达 体内的内皮细胞将被核糖体相关RNA上的RNA-seq所询问。在目标3中，认识到 27HC会随着西方饮食摄入量或高胆固醇血症而增加，我们将确定如何降低 内源性27HC影响在这些条件下发生的葡萄糖耐量和动脉粥样硬化， 分别进行了分析。小鼠将接受27HC合成酶Cyp27a1的抑制，或与腺相关的 允许27HC代谢酶CYP7B1过度表达的病毒。将使用带牙线的Cyp27a1小鼠 为了确定巨噬细胞或内皮细胞是否是内源性27HC的关键来源， 精神错乱。拟议的工作将揭示内皮ERα如何作为 内源性ER配体，即雌激素和27HC的作用，影响代谢和血管健康。

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.