Novel roles of the intestine to regulate whole body lipid homeostasis using pathways of nutritional physiology and molecular regulation.

该计划的工作假设是，肠道是脂质和类固醇前体的主要调节器，用于全身脂质代谢，并通过包括炎症在内的新型机制途径网络采用代偿机制。通过转变这种新的整合生物学，我们将揭示和调和平衡和失调营养状态的新途径。主要目标利用高阶动物模型（猪）评估肠源性（淋巴）HDL对全身胆固醇稳态的营养和生理意义。短期和长期目标包括比较肠道和肝脏合成的HDL及其主要蛋白（载脂蛋白AI）；了解其对胆固醇运输和排泄的作用和动力学；绘制与淋巴中高密度脂蛋白部分相关的miR的图谱，并发现它们对调节代谢的贡献。我们将利用猪的留置淋巴插管（我们小组开发的）来评估淋巴减少对HDL/胆固醇代谢的影响；采用示踪剂测定来自肝脏和肠系膜淋巴管的apoAI动力学；继续开发猪淋巴样本的miR阵列，并表征其在不同营养状态下的特征。2. 描述miR-150在肠系膜淋巴中的新作用及其对脂质和高密度脂蛋白代谢的影响。我们将测定在喂食和禁食条件下的免疫反应；研究miR-150在脂质代谢受损期间的关系，以评估其在这一状态下的促炎反应作用。我们将使用啮齿动物和猪模型来生成不同营养状态下的淋巴样本。炎症状态将通过体外细胞增殖技术（含或不含miR-150）、细胞分选（FACS）、细胞表面标记物和门控方法进行评估。我们还建议开发新的功能方法学方法来验证miR-150的相关性，例如使用不同浓度的安塔戈米尔-150或安塔戈米尔-scr预孵育的分离淋巴细胞。3. 揭示由我们的初步数据确定的肠细胞内调节TICE的分子机制；(i)细胞内转录，（ii）类固醇激素受体的HSP90伴侣周期，（iii）肠细胞运输和代谢以及（iv）细胞间通讯。我们将蛋白质组学和基因阵列技术应用于分离的整个肠细胞和特定的刷状边界膜（BBM），并使用系统定位来确定涉及TICE的典型途径。我们已经开发了营养和药理学制剂，可以损害或刺激体内的TICE。我们将把这些综合的生物学方法结合起来。令人兴奋的初步数据表明，来自淋巴的HDL可能是TICE（离体使用腔法）的有效供体，代表了该领域的开创性观点。

Hypothesis and Significance The working hypothesis of this program is that the intestine is a primary regulator of lipid and steroid precursors for whole body lipid metabolism and employs compensatory mechanisms through a network of novel mechanistic pathways, including inflammation. By transforming this new integrated biology, we will reveal and reconcile novel pathways for homeostatic and perturbed nutritional states. Primary Objectives 1. Assess the nutritional and physiological significance of intestinal-derived (lymph) HDL to whole body cholesterol homoeostasis using a higher order animal model (swine). Short- and long-term objectives include comparing HDL and its primary protein (apolipoprotein AI) synthesized by both the intestine and the liver; understand its contrition and kinetics to cholesterol transport and excretion; map the profile of miR's associated within the HDL fraction from lymph and discover their contributions to regulate metabolism. We will utilize the indwelling lymph-cannulation in swine (developed by our group) to assess the impact of lymphopenia on HDL/cholesterol metabolism; employ tracers to determine apoAI kinetics derived from hepatic and mesenteric lymphatics; continue to develop miR arrays for lymphatic samples from swine and characterize their profile under different nutritional states. 2. Delineate the novel role of miR-150 in mesenteric lymph and impact to lipid and HDL metabolism. We will determine the immune response under fed and fasted conditions; investigate the relationship of miR-150 during impaired lipid metabolism to assess its role pro-inflammatory responses during this state. We will use both rodent and swine models to generate lymph samples under different nutritional states. Inflammatory status will be assessed using ex vivo cell proliferation techniques (with and without miR-150), as well as cell sorting (FACS), cell-surface markers and gating approaches. We also propose to develop new functional methodological approaches to validate the relevance of miR-150 such as using isolated lymphocytes pre-incubated with antagomir-150 or antagomir-scr at varying concentrations. 3. Reveal the molecular mechanisms that regulate TICE within the enterocyte identified by our preliminary data; (i) intracellular transcription, (ii) HSP90 chaperone cycle for steroid hormone receptors, (iii) enterocytic transport and metabolism and (iv) cell-cell communication. We have applied proteomic and gene array technologies to isolates of whole enterocytes as well as specific brush boarder membrane (BBM) and used system mapping to identify the canonical pathways involved in TICE. We have developed nutritional and pharmacological agents that can impair or stimulate TICE in vivo. We will combine these integrated biological approaches. Exciting preliminary data suggests that HDL derived from the lymphatics maybe an effective donor for TICE (ex vivo Ussing Chamber methods) and represents a pioneering perspective for the field.