CHARACTERIZATION OF INTESTINAL ABSORPTION OF VITAMIN B1

维生素 B1 肠道吸收的表征

• 批准号: 6612826
• 负责人: HAMID M SAID
• 金额: $ 18.47万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612826
• 关键词: Saccharomyces cerevisiae; gastrointestinal nutrient absorption; genetic library; human tissue; membrane transport proteins; microorganism culture; molecular cloning; mutant; nucleic acid sequence; nutrition related tag; protein kinase; radiotracer; thiamine; tissue /cell culture; vesicle /vacuole

This proposal deals with characterization of the absorption process of vitamin B1(thiamine) in the human intestine at the cellular and molecular levels. Thiamine, a water-soluble vitamin, plays an essential role in different cellular metabolic reactions. The importance of thiamine to normal human health and well being is manifested by the serious clinical abnormalities that occur in thiamine deficiency which include cardiovascular and neurological disorders. Thiamine deficiency represents a significant nutritional problem in developed countries and occur in alcoholics, diabetics, coeliac disease patients, renal disease patients, the elderly, in inborn errors of thiamine metabolism, and in long-term users of diuretics. Humans and other mammals can not synthesize thiamine, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Therefore, the intestine plays a critical role in determining and regulating thiamine normal body homeostasis. Thiamine is presented to the human intestine from two sources: the diet, and as a product of bacterial synthesis by the normal microflora in the large intestine. Very little is known about the absorption mechanism of dietary thiamine in the human small intestine, and no information is available regarding the mechanism of absorption of the bacterially synthesized thiamine in the large intestine. Transport of thiamine across the functionally polarized small intestinal and colonic epithelial cells represents transport of the vitamin across two structurally and functionally different membrane domains (i.e., the luminal and basolateral membrane domains). We propose to characterize the mechanism(s) of thiamine transport across the individual membrane using purified luminal and basolateral membrane vesicle preparations. Intestinal transport processes of variety of nutrients have been shown in recent years to be regulated by specific intracellular protein kinase-mediated pathways, and by extracellular substrate levels. Very little, however, is known about the cellular regulation of the thiamine absorption process in the human intestine. We propose to address this issue in this application. The molecular characteristics of the intestinal thiamine transport process is not known. We propose to clone and characterize the human intestinal thiamine transporters(s) using a method that is based on the strategy of complementation with a human cDNA library of the yeast S. cerevisiae NKC6, a yeast mutant defective in thiamine transport activity. We also propose to clone and characterize the 5'-regulatory region of the human intestinal thiamine transporter(s) gene. Variety of physiological and molecular biology techniques will be used in these studies. Valuable information are expected to emerge from these investigations regarding the cellular/molecular mechanisms and regulation of thiamine absorption in the human intestine. This should ultimately assist us in designing effective strategies to optimize thiamine body homeostasis, and help us understand the causes of aberrations that occur in thiamine nutrition under certain pathophysiological conditions.

这项建议涉及在细胞和分子水平上表征维生素B1(硫胺素)在人体肠道中的吸收过程。硫胺素是一种水溶性维生素，在细胞的不同代谢反应中起着至关重要的作用。硫胺素缺乏症引起的严重临床异常，包括心血管和神经疾病，证明了硫胺素对正常人类健康和福祉的重要性。硫胺素缺乏在发达国家是一个严重的营养问题，发生在酗酒者、糖尿病患者、腹部疾病患者、肾脏疾病患者、老年人、硫胺素代谢先天缺陷以及长期使用利尿剂的人中。人类和其他哺乳动物不能合成硫胺素，因此必须通过肠道吸收从外部来源获得维生素。因此，肠道在决定和调节硫胺素正常体内稳态方面起着至关重要的作用。硫胺素通过两种途径进入人体肠道：一种是饮食，另一种是由大肠中的正常微生物群合成的产物。人们对食物中硫胺素在人体小肠中的吸收机制知之甚少，也没有关于细菌合成的硫胺素在大肠中吸收机制的信息。硫胺素跨功能极化的小肠和结肠上皮细胞的转运代表着维生素跨越两个结构和功能不同的膜域(即腔膜域和基侧膜域)的转运。我们建议使用纯化的管腔和基侧膜泡制剂来表征硫胺素跨膜转运的机制(S)。近年来，各种营养素的肠道转运过程被证明受到特定的细胞内蛋白激酶介导的途径和细胞外底物水平的调节。然而，人们对人体肠道硫胺素吸收过程的细胞调控知之甚少。我们建议在此应用程序中解决此问题。硫胺素在肠道的转运过程的分子特征尚不清楚。我们提出了一种基于与酵母突变株酿酒酵母NKC6互补的方法来克隆和鉴定人肠道硫胺转运蛋白(S)。我们还打算克隆和鉴定人肠道硫胺转运蛋白(S)基因的5‘-调控区。在这些研究中将使用各种生理学和分子生物学技术。有价值的信息有望从这些研究中出现，关于人体肠道中硫胺素吸收的细胞/分子机制和调节。这最终将有助于我们设计有效的策略来优化硫胺素体内的动态平衡，并帮助我们了解在某些病理生理条件下硫胺素营养异常的原因。