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.
Showing the most recent 10 out of 101 publications
