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

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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.