Effect of Pathophysiological Conditions on Intestinal Absorption of Free Thiamin

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PROJECT SUMMARY/ABSTRACT Research in our laboratory focuses on studying the molecular physiology, pathophysiology and cell biology of the transport processes of water-soluble vitamins in organs of the digestive system. In this proposal, we aim to continue our investigations into the physiology, pathophysiology and cell biology of the absorption processes of both dietary and microbiota-generated forms of vitamin B1 [i. e., free thiamin and that of thiamin pyrophosphate (TPP)] in the small and large intestine. Vitamin B1 (in its biologically active form, i. e., TPP) is essential for normal physiology and health of all cells due to the critical roles it plays in oxidative energy metabolism, ATP production, and reduction of cellular oxidative stress. Deficiency of vitamin B1 occurs in a variety of conditions including inflammatory bowel diseases, sepsis and chronic alcoholism. Humans/mammals cannot synthesize vitamin B1 endogenously, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Two sources of the vitamin are available to the host: dietary and microbiota-generated. We have previously characterized different aspects of the uptake process of free thiamin along the intestinal tract, and showed involvement of two transport systems: thiamin transporter-1 & -2 (THTR- 1 & -2; products of the SLC19A2 & SLC19A3 genes, respectively). As to the microbiota-generated vitamin B1, this source provides thiamin in both free and phosphorylated (TPP) forms. Studies from our laboratory have shown that both of these forms are absorbable in the colon; absorption of free thiamin occurs as in the small intestine via a carrier-mediated process that involves THTR-1 & -2, while that of TPP occurs via a distinct, high- affinity and specific (i.e., does not transport free thiamin) carrier-mediated process. Subsequent studies from our laboratory have cloned a specific TPP transporter from the colon (the cTPPT; product of the SLC44A4 gene) and found its expression along the intestinal tract to be restricted to the large intestine only, and occurs exclusively at the apical membrane domain of the lining epithelia. Our objectives in this proposal are: 1) To determine the contribution of cTPPT toward total carrier-mediated uptake of the microbiota-generated TPP in the native colon, its role in normal colon physiology/health, and to study aspects of its cell biology; and 2) To examine the effect of conditions/factors that the intestinal tract is exposed to under certain disease states [namely: hypoxia, pro-inflammatory cytokines, and bacterial lipopolysaccharides (LPS) and flagellin] on colonic/small intestinal TPP and free thiamin uptake. Thus, in new preliminary studies we have generated a Slc44a4 knockout (KO) mouse model, which upon initial characterization showed clear phenotype (with impaired colonic TPP uptake) compared to wild-type littermates. We also identified putative interactors with cTPPT in colonocytes, and showed an essential role for its cytoplasmic tail in apical targeting. Furthermore, we obtained evidence to show that exposure of colonic/small intestinal epithelia to hypoxia, pro-inflammatory cytokines, LPS and flagellin to lead to inhibition in uptake of vitamin B1 forms. Based on these new (and previous) findings, our working hypotheses in this proposal are that the cTPPT (SLC44A4) is the predominant system involved in carrier- mediated uptake of the microbiota-generated TPP in native colon, that this transporter is important for colon physiology and health, that cTPPT has interacting partner(s), and that apical targeting of cTPPT is dictated by specific structural motif(s) in its c-terminal domain. We also hypothesize that exposure of colonic/small intestinal epithelia to hypoxia, pro-inflammatory cytokines, and to bacterial products to lead to inhibition in TPP and free thiamin uptake. We will test these hypotheses by accomplishing two specific aims and will utilize state-of the-art physiological/cellular/molecular approaches. Results of these studies should provide new information regarding vitamin B1 absorption along the intestinal tract in health and disease. This should assist in the designing effective strategies to optimize global (body) and local (gut mucosal) vitamin B1 nutrition, and thus, improve health.