Intestinal Vitamin B2 Absorption: Molecular/Cellular Aspects and Effects of Alcoh

肠道维生素 B2 吸收：分子/细胞方面和酒精的影响

• 批准号: 8803250
• 负责人: HAMID M SAID
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8803250
• 关键词: Actins; Acyl CoA Dehydrogenases; Affect; Alcoholism; Alcohols; Anemia; Animals; Area; Binding; Biological; Biological Assay; Birth Place; Brush Border; C-terminal; Caco-2 Cells; Calories; Carbohydrates; Carcinogens; Cell Differentiation process; Cell physiology; Cells; Cellular biology; Chimeric Proteins; Chronic; Clinical; Computer Analysis; Diabetes Mellitus; Diet; Disease; Down-Regulation; Elderly; Elements; Endoplasmic Reticulum; Enterocytes; Epithelial; Epithelial Cells; Epithelium; Ethanol; Event; Fluorescence Microscopy; Folic Acid; Functional disorder; Funding; Genes; Genetic Transcription; Green Fluorescent Proteins; Growth; Growth and Development function; Health; Heterogeneous Nuclear RNA; High Prevalence; Homeostasis; Human; Human Activities; Image; Impairment; Indium; Individual; Inflammatory Bowel Diseases; Injury; Intestinal Absorption; Intestines; Investigation; Kinetics; Knowledge; Laboratories; Large Intestine; Lead; Life; Lipids; Liquid substance; Mammals; Measures; Mediating; Membrane; Membrane Protein Traffic; Membrane Transport Proteins; Messenger RNA; Metabolic; Metabolism; Microfilaments; Microtubules; Modeling; Molecular; Molecular Biology; Movement; Mutate; N-terminal; Nervous system structure; Nuclear; Nuclear Trans-Acting Factor; Nucleic Acid Regulatory Sequences; Nutrient; Nutritional; Organ Donor; Oxidation-Reduction; Patients; Personal Satisfaction; Physiological; Physiology; Play; Positioning Attribute; Predisposition; Preparation; Process; Proteins; Rattus; Reaction; Recovery; Regulation; Regulatory Element; Retrieval; Riboflavin; Riboflavin Deficiency; Role; Site; Small Intestines; Source; Supplementation; Testing; Therapeutic Agents; Time; Tissues; Transcriptional Regulation; Transport Process; Tyrosine; Up-Regulation; Vertebral column; Vesicle; Villus; Vitamin B6; Vitamins; Western Blotting; Work; absorption; acyl-CoA dehydrogenase; alcohol effect; amino acid metabolism; apical membrane; base; basolateral membrane; brush border membrane; cancer therapy; cell growth regulation; chronic alcohol ingestion; clinically relevant; design; extracellular; feeding; glycosylation; intestinal epithelium; novel; polyclonal antibody; polypeptide; problem drinker; promoter; protein folding; response; secretion process; skin lesion; trafficking; uptake

DESCRIPTION (provided by applicant): The long-term objectives of this application are to characterize the molecular mechanisms involved in the regulation of the intestinal absorption of vitamin B2 (riboflavin, RF), and those involved in intracellular trafficking and membrane targeting of the involved membrane transporter. We also aim at examining the effect of chronic alcohol use on intestinal RF absorption and determining the cellular and molecular mechanisms involved. RF is involved in critical cellular metabolic reactions, and thus, is essential for normal human health and well-being. Its deficiency, which occurs in a variety of conditions like chronic alcoholism, leads to serious clinical abnormalities that include degenerative changes in the nervous system, anemia, growth retardation, and skin lesions. Humans (and other mammals) cannot synthesize RF, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Thus, the human intestine plays a central role in maintaining and regulating normal RF body homeostasis. For these reasons understanding the physiology/pathophysiology as well as cell/molecular biology of the intestinal RF absorption process is of significant importance and is the aim of this proposal. We have been investigating the physiology of intestinal RF transport for over two decades, but more is needed at the molecular level to fully understand the process. With the recent molecular identification of mammalian RF transporters, we are now in an excellent position to carry out such investigations. Thus, in new preliminary studies we have shown that the up-regulation in intestinal RF uptake in RF deficiency shown by us previously is mediated (at least in part) via a transcriptional regulatory mechanism(s) affecting hRFT-2. We also showed that the intestinal RF uptake process undergoes differentiation - dependent regulation and that this regulation again appears to be mediated (at least in part) via a transcriptional mechanism(s). Using confocal imaging of living human intestinal epithelia cells and Western blot analysis of native human intestinal brush border and basolateral membrane preparations, our preliminary studies further showed exclusive expression of the human RF transporter -2 (hRFT-2; the most relevant intestinal RF transporter) at the apical membrane domain of polarized human intestinal epithelial cells. Furthermore, distinct trafficking vesicles appear to be involved in intracellular movement of hRFT2 in human intestinal epithelial cells. Finally, we showed for the first time that chronic alcohol feeding significantly inhibit RF transport across the jejunal BBM which is associated with a significant decrease in the level of expression of RFT-2. Our Specific Objectives in this proposal are: 1) To continue our investigations into the molecular mechanisms involved in the adaptive up-regulation in intestinal RF uptake in RF deficiency and during cell differentiation; 2) To study the mechanism(s) involved in the targeting of the hRFT-2 protein to the apical membrane domain of polarized human intestinal epithelial cells, and to determine the factor(s) involved in its intracellular trafficking; and 3) to extend our basic physiological/nutritional investigations on the intestinal RF uptake process into a clinically-relevant area and will examine the effect of chronic alcohol consumption on cell and molecular parameters of intestinal RF absorption process. Collectively, results of these studies should provide novel and valuable information regarding the physiology/pathophysiology and cell/molecular biology of intestinal RF uptake and of the factors that interfere with the process. This should ultimately assist us in the designing of effective strategies to optimize RF body homeostasis, especially in conditions of RF deficiency and sub- optimal levels. 1

描述(由申请人提供)： 这一应用的长期目标是表征参与调节维生素B2(核黄素，RF)肠道吸收的分子机制，以及参与相关膜转运体的细胞内转运和膜靶向的分子机制。我们还旨在研究长期饮酒对肠道RF吸收的影响，并确定相关的细胞和分子机制。RF参与关键的细胞代谢反应，因此对正常的人类健康和福祉是必不可少的。它的缺乏会在各种情况下发生，如慢性酒精中毒，会导致严重的临床异常，包括神经系统的退行性变化、贫血、生长迟缓和皮肤损害。人类(和其他哺乳动物)不能合成RF，因此必须通过肠道吸收从外部来源获得维生素。因此，人体肠道在维持和调节正常的RF体内稳态方面起着核心作用。由于这些原因，了解肠道射频吸收过程的生理学/病理生理学以及细胞/分子生物学是非常重要的，也是本提案的目的。二十多年来，我们一直在研究肠道射频传输的生理学，但还需要在分子水平上更多地研究才能完全了解这一过程。随着最近对哺乳动物射频转运蛋白的分子鉴定，我们现在处于开展此类研究的有利地位。因此，在新的初步研究中，我们已经表明，我们先前显示的RF缺乏时肠道RF摄取的上调是通过影响hRFT-2的转录调控机制(S)介导的(至少部分)。我们还表明，肠道RF摄取过程经历了依赖于分化的调节，并且这种调节似乎再次(至少部分地)通过转录机制介导(S)。利用活体人肠上皮细胞的共聚焦成像和天然人肠刷状缘和基侧膜制备的Western印迹分析，我们的初步研究进一步表明，人RF转运蛋白-2(hRFT-2；最相关的肠道RF转运蛋白)在极化的人肠上皮细胞的顶膜区是唯一表达的。此外，不同的运输小泡似乎参与了hRFT2在人肠上皮细胞内的运动。最后，我们首次发现长期饮酒显著抑制了RF通过空肠BBM的运输，这与RFT-2的表达水平显著降低有关。我们在这项建议中的具体目标是：1)继续研究在RF缺乏和细胞分化过程中，hRFT-2蛋白适应性上调的分子机制；2)研究hRFT-2蛋白靶向极化的人肠上皮细胞顶膜区的机制(S)，并确定其细胞内转运的相关因素(S)；以及3)将我们对肠道RF吸收过程的基本生理/营养调查扩展到临床相关领域，并研究长期饮酒对肠道RF吸收过程中细胞和分子参数的影响。总之，这些研究的结果应该提供有关肠道RF摄取的生理学/病理生理学和细胞/分子生物学以及干扰这一过程的因素的新的和有价值的信息。这最终将有助于我们设计有效的策略，以优化射频身体稳态，特别是在射频不足和次优水平的条件下。1