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

假设和意义该计划的工作假设是，肠道是全身脂质代谢的脂质和类固醇前体的主要调节剂，并通过包括炎症在内的新机制途径网络采用代偿机制。通过改变这种新的综合生物学，我们将揭示和协调稳态和扰动营养状态的新途径。主要目标1。使用高阶动物模型（猪）评估脑源性（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HDL及其由肠和肝脏合成的主要蛋白质（载脂蛋白AI）;了解其与胆固醇转运和排泄的相关性和动力学;绘制来自淋巴的HDL组分内相关的miR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管插管（由我们的小组开发）来评估淋巴细胞减少对HDL/胆固醇代谢的影响;使用示踪剂来确定来自肝脏和肠系膜动脉的apoAI动力学;继续开发猪淋巴样品的miR阵列并表征其在不同营养状态下的特征。2.阐明miR-150在肠系膜淋巴中的新作用及其对脂质和HDL代谢的影响。 我们将确定进食和禁食条件下的免疫应答;研究miR-150在脂质代谢受损期间的关系，以评估其在这种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品。将使用离体细胞增殖技术（有和没有miR-150）以及细胞分选（FACS）、细胞表面标志物和门控方法来评估炎症状态。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Eschomir-150或Eschomir-scr预孵育的分离淋巴细胞。3.揭示了我们的初步数据确定的肠上皮细胞内的TICE调节的分子机制;（一）细胞内转录，（二）热休克蛋白90伴侣蛋白的类固醇激素受体的周期，（三）肠细胞运输和代谢和（四）细胞-细胞通信。我们已经应用蛋白质组学和基因阵列技术分离的整个肠上皮细胞以及特定的刷状缘膜（BBM），并使用系统映射，以确定参与TICE的典型途径。我们已经开发了可以在体内损害或刺激TICE的营养和药理学试剂。我们将联合收割机结合这些综合生物学方法。令人兴奋的初步数据表明，HDL来源于胆固醇可能是一个有效的供体TICE（离体Ussing室方法），并代表了该领域的开拓性观点。

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.