The Nuclear Receptor-Aster Pathway in Enterohepatic Metabolism

摘要 脂质是肝肠轴中重要的内分泌信号，通过激活 核激素受体肝脏和肠道脂质代谢是建立系统性 胆固醇和甘油三酯稳态。这些组织中过量的脂质积累与 糖尿病、NASH、癌症和其他疾病。我们的长期目标是揭示基本机制， 其脂质激活的核受体协调细胞和全身脂质稳态。在当前 应用中，我们专注于LXR和FXR的一种新的甾醇转运途径的调节。我们已经发现 在胆固醇运动中起重要作用的哺乳动物蛋白质家族（Aster-A、-B和-C）。的 3 Aster以组织特异性方式表达，并受核受体的差异调节。紫菀- B是由固醇激活的LXR调节的靶点，而Aster-C由胆汁酸激活的FXR调节。 我们假设紫菀在肝肠轴的脂质平衡中起关键作用，包括膳食脂质 吸收、乳糜微粒产生和胆固醇逆向转运。我们进一步假设紫菀是 LXR和FXR激动剂的药理学作用的重要贡献者。我们将解决这些问题 有以下具体目标的假设。具体目标1将定义FXR调节的Aster-C在以下方面的作用： 肝胆固醇转运。我们将结合细胞、生物化学、成像和体内研究 以确定肝细胞中Aster-C依赖性胆固醇转运的途径。我们已经生成了肝脏- 特异性Aster-C基因敲除小鼠，初步分析显示它们的肝脏和血浆脂质发生了改变， 程度.我们将进行代谢分析和体内胆固醇示踪剂研究，以询问系统性 胆固醇流量我们将分析紫菀蛋白的丢失或过度表达如何影响HDL的运动， 基线时和FXR应答时胆固醇进入胆汁和全身胆固醇逆向转运 激动剂具体目标2将阐明紫菀在肠道胆固醇转运中的作用。我们将分析 缺乏Aster-B、Aster-C或两者的小鼠，以测试Aster在膳食胆固醇吸收中的重要性， 乳糜微粒产生。初步分析显示，膳食胆固醇的摄取减少， 肠上皮细胞胆固醇酯含量在没有这两个紫菀。我们将进一步测试阿斯特斯 有助于经肠胆固醇排泄。最后，我们发现批准的药物 依泽替米贝抑制肠道胆固醇摄取，是Aster-C的选择性配体， Aster-C-依折麦布复合物的晶体结构。根据这些发现，我们将确定 Aster途径有助于依折麦布的药理学作用。我们的目标的完成， 提供了对脂质转运途径的基本见解，并可能发现新的机会， 代谢性疾病的干预。

ABSTRACT Lipids are important endocrine signals in the enterohepatic axis that control gene expression by activating nuclear hormone receptors. Hepatic and intestinal lipid metabolism are key factors in establishing systemic cholesterol and triglyceride homeostasis in mammals. Excess lipid accumulation in these tissues is linked to diabetes, NASH, cancer and other diseases. Our long-term objective is to reveal fundamental mechanisms by which lipid-activated nuclear receptors orchestrate cellular and systemic lipid homeostasis. In the current application, we focus on regulation of a novel sterol-transport pathway by LXR and FXR. We have discovered a family of mammalian proteins (Aster-A, -B and -C) that play an important role in cholesterol movement. The 3 Asters are expressed in a tissue-specific manner and are differentially regulated by nuclear receptors. Aster- B is a target for regulation by sterol-activated LXRs, while Aster-C is regulated by the bile acid-activated FXR. We hypothesize the Asters play key roles in lipid homeostasis in the enterohepatic axis, including dietary lipid absorption, chylomicron production and reverse cholesterol transport. We further hypothesize that Asters are important contributors to the pharmacological effects of LXR and FXR agonists. We will address these hypotheses with the following specific aims. Specific Aim 1 will define the role of FXR-regulated Aster-C in hepatic cholesterol transport. We will use a combination of cellular, biochemical, imaging, and in vivo studies to define the pathway for Aster-C-dependent cholesterol transport in hepatocytes. We have generated liver- specific Aster-C knockout mice, and preliminary analysis reveals them to have altered hepatic and plasma lipid levels. We will perform metabolic analyses and in vivo cholesterol tracer studies to interrogate systemic cholesterol flux. We will analyze how loss or overexpression of Aster proteins affects the movement of HDL- cholesterol into bile and for systemic reverse cholesterol transport at baseline and in response to FXR agonists. Specific Aim 2 will elucidate the role of Asters in intestinal cholesterol transport. We will analyze mice lacking Aster-B, Aster-C, or both to test the importance of Asters in dietary cholesterol absorption and chylomicron production. Preliminary analysis has revealed reduced uptake of dietary cholesterol and reduced enterocyte cholesterol ester content in the absence of both Asters. We will further test whether Asters contribute to trans-intestinal cholesterol excretion. Finally, we have discovered that the approved drug ezetimibe, which inhibits intestinal cholesterol uptake, is a selective ligand for Aster-C, and we have solved the crystal structure of the Aster-C–ezetimibe complex. Based on these findings we will determine whether the Aster pathway contributes to the pharmacological effects of ezetimibe. Completion of our aims is expected to provide fundamental insight into pathways governing lipid transport, and may identify new opportunities for intervention in metabolic disease.