The objectives of this proposal continue to focus on investigating the physiology/pathophysiology and cell biology of the absorption processes of vitamin B1 in the intestinal tract. Vitamin B1 (in its biologically active form, TPP) is essential for normal physiology and health of all cells due to its critical roles 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 (SLC19A2 and SLC19A3, respectively). As to the microbiota-generated vitamin B1, a considerable amount of this source is provided in the form of TPP, and we have identified a high-affinity and specific carrier-mediated process for its uptake by colonocytes. We have also cloned a specific TPP transporter from the colon (the cTPPT; product SLC44A4 gene), showed its expression along the GI tract to be restricted to the large intestine, and demonstrated its exclusive localization at the apical membrane domain of epithelia. Our objectives in this proposal are to determine the contribution of cTPPT toward total carrier-mediated uptake of TPP in native colonic tissue, its role in colon physiology/health, whether it has interactor(s) that influence its physiology/cell biology, and molecular determinant(s) that dictates its apical targeting. We also aim to investigate the effects of conditions/factors that the intestinal tract is exposed to under certain disease states [namely, hypoxia, pro-inflammatory cytokines, and the bacterial lipopolysaccharides (LPS) and flagellin] on TPP uptake. In new preliminary studies, we have generated homozygous Slc44a4 loxP+/+ mice [a critical step towards generating a large intestine-specific Slc44a4 knockout (KO) mice], identified putative interactors with cTPPT, and found a critical role for its cytoplasmic tail in apical targeting. We also found that exposure of colonic epithelia to hypoxia, pro-inflammatory cytokines, LPS and flagellin to lead to inhibition in vitamin B1 uptake. 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 TPP uptake in native colon, it is important for colon physiology/health, it has interacting partner(s), and that its apical targeting is dictated by specific structural motif(s) in its structure. We also hypothesize that exposure of colonic epithelia to hypoxia, pro- inflammatory cytokines, and to bacterial products to lead to inhibition in TPP uptake. We will test these hypotheses by accomplishing two specific aims and will utilize state-of the-art physiological/molecular approaches. Results of these studies should provide new information regarding intestinal vitamin B1 absorption in health and disease, which should assist in the designing effective strategies to optimize its homeostasis/nutrition, and thus, improve health.