CHARACTERIZATION OF INTESTINAL ABSORPTION OF VITAMIN B1

该提案涉及在细胞和分子水平上表征人体肠道中维生素B1（硫胺素）的吸收过程。 硫胺素是一种水溶性维生素，在不同的细胞代谢反应中起着重要作用。 硫胺素对正常人类健康和福祉的重要性表现在硫胺素缺乏症中发生的严重临床异常，包括心血管和神经系统疾病。 硫胺素缺乏症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、乳糜泻患者、肾病患者、老年人、先天性硫胺素代谢缺陷和长期使用利尿剂者中。 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源来源获得维生素。 因此，肠道在决定和调节硫胺素正常体内平衡中起着关键作用。 硫胺素通过两个来源进入人体肠道：饮食和大肠中正常微生物菌群的细菌合成产物。 关于人体小肠中膳食硫胺素的吸收机制知之甚少，也没有关于大肠中细菌合成硫胺素吸收机制的信息。硫胺素跨功能极化的小肠和结肠上皮细胞的转运代表维生素跨两个结构和功能不同的膜结构域（即，腔和基底外侧膜区域）。 我们建议使用纯化的管腔和基底外侧膜囊泡制剂来表征硫胺素跨单个膜转运的机制。近年来研究表明，多种营养物质在肠道的转运过程受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，对人体肠道中硫胺素吸收过程的细胞调节知之甚少。 我们建议在本申请中解决这个问题。肠道硫胺素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S. cerevisiae NKC 6，一种硫胺素转运活性缺陷的酵母突变体。 我们还建议克隆和鉴定人肠道硫胺素转运蛋白基因的5 '调控区。在这些研究中将使用各种生理和分子生物学技术。 有价值的信息，预计将出现从这些调查有关的细胞/分子机制和调节硫胺素吸收在人体肠道。 这将最终帮助我们设计有效的策略，以优化硫胺素体内稳态，并帮助我们了解在某些病理生理条件下硫胺素营养发生畸变的原因。

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.