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，肠道是全身脂肪代谢的脂质和类固醇前体的主要调节器，并通过包括炎症在内的一系列新的机制网络使用代偿机制。通过改变这种新的综合生物学，我们将揭示和调和动态平衡和扰动营养状态的新途径。主要目的1.利用高阶动物模型(猪)评价肠源性(淋巴)高密度脂蛋白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高密度脂蛋白及其主要蛋白质(载脂蛋白AI)由肠道和肝脏合成；了解其对胆固醇运输和排泄的悔悟和动力学；绘制淋巴中高密度脂蛋白组分中与miR相关的图谱，并发现它们对调节新陈代谢的贡献。我们将利用猪的留置淋巴管(由我们团队开发)来评估淋巴细胞减少对高密度脂蛋白/胆固醇代谢的影响；使用示踪剂来确定源自肝脏和肠系膜淋巴管的载脂蛋白AI动力学；继续开发猪淋巴样本的miR阵列，并表征它们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高密度脂蛋白代谢的影响。我们将测定进食和禁食条件下的免疫反应；研究miR-150在脂代谢受损过程中的关系，以评估其在这一状态下的促炎反应中的作用。我们将使用啮齿动物和猪的模型来产生不同营养状态下的淋巴样本。炎症状态将使用体外细胞增殖技术(使用和不使用miR-150)以及细胞分选(FAC)、细胞表面标志物和门控方法进行评估。我们还建议开发新的功能方法来验证miR-150的相关性，例如使用分离的淋巴细胞预先与不同浓度的antagomir-150或antagomir-scr孵育。3.揭示了我们的初步数据确定的在肠细胞内调节Tice的分子机制：(I)细胞内转录，(Ii)类固醇激素受体的HSP90伴侣循环，(Iii)肠道细胞的运输和代谢，以及(Iv)细胞间的通讯。我们将蛋白质组学和基因芯片技术应用于整个肠上皮细胞的分离以及特定的刷状边界膜(BBM)，并使用系统图谱来鉴定TICE中涉及的典型途径。我们已经开发出可以在体内损害或刺激Tice的营养和药理制剂。我们将结合这些综合的生物学方法。令人兴奋的初步数据表明，来自淋巴管的高密度脂蛋白可能是TICE(体外使用小室方法)的有效供体，代表了该领域的一个开创性的前景。

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.