The long-term objective of this renewal application is to continue our investigations into the cellular and molecular mechanisms involved in the intestinal absorption process of the water- soluble vitamin B1 (thiamin) and their regulation. We also aim at examining the effect of infection with enteropathogenic E. coli (EPEC), a common intestinal pathogen, on the intestinal thiamin absorption process. Thiamin is essential for normal cellular functions and its deficiency (which represents a significant nutritional problem) leads to a variety of clinical abnormalities including cardiovascular and neurological disorders. Humans (and other mammals) cannot synthesize thiamin, and thus, must obtain the vitamin via intestinal absorption. Studies during the current funding period have characterized many aspects of the intestinal thiamin uptake process. These include characterization of the 5'-regulatory regions of the genes of the human thiamin transporters 1 &2 (hTHTR-1 &hTHTR-2) both in vitro and in vivo, demonstration that the intestinal thiamin uptake process is adaptively up-regulated in thiamin deficiency via transcriptionally-mediated mechanism(s) and that the process also undergoes differentiation- and developmental- dependent regulation. We have also characterized the mechanisms involved in membrane targeting and intracellular trafficking of the thiamin transporters in epithelial cells. Using gene specific siRNA approaches, recent studies from our laboratory have shown that both the hTHTR 1 &2 are involved in thiamin uptake by a human intestinal epithelial Caco- 2 cell line in vitro. Nothing is known about the role of these thiamin transporters in thiamin uptake in the native intestine. Thus, in new preliminary studies we have developed an Slc19a3-/- knockout mouse model and showed significant impairment in intestinal thiamin uptake compared to wild-type mice;we have also established a colony of Slc19a2-/- knockout mice in our laboratory. In addition, we have used the bacterial two-hybrid system to screen a human intestinal cDNA library and have identified two putative proteins that can interact with hTHTR-1 (tetraspanin and E-cadherin-1). Furthermore, we have examined the effect of the enteropathogenic Escherichia coli (EPEC) and found significant inhibition in thiamin uptake. Based on our previous and new preliminary findings, our aims in this proposal are: 1) To further characterize the intestinal thiamin absorption process using Slc19a2 -/-and Slc19a3 -/- knockout mouse models and to determine the role of THTR-1 and THTR-2 in trans-epithelial and trans- membrane transport events in the native intestine, 2) To identify the cis-regulatory elements and trans-acting nuclear factors involved in the adaptive up-regulation of thiamin uptake in thiamin deficiency, 3) To identify proteins that interact with hTHTR-1 and hTHTR-2 in human intestinal epithelial cells and to understand their biological/physiological roles, and 4) to determine the cellular and molecular mechanisms involved in EPEC inhibition of the intestinal thiamin uptake process. Results of these studies should continue to provide novel and valuable information regarding the cellular and molecular mechanisms involved in the intestinal thiamin uptake process and their regulation as well as of the factors that negatively impact the process. This should ultimately assist us in the designing of effective strategies to optimize thiamin body homeostasis in conditions associated with thiamin deficiency and sub-optimal levels, and in minimizing the effect of exogenous factors that may negatively impact this nutritional parameter.

Public Health Relevance

Humans and other mammals cannot synthesize vitamin B1 (an essential micronutrient for normal health), and thus, must obtain it from exogenous sources via intestinal absorption. The aims of this proposal since its inception ten years ago were (and continue to be) the delineation of the mechanisms involved in intestinal thiamin absorption, how the process is regulated, and what factors affect its function. Results of these investigations should help in the designing of effective strategies to optimize thiamin body levels, especially in conditions associated with thiamin deficiency and sub-optimal levels.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (ZRG1-DIG-D (02))
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Maruvada, Padma
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University of California Irvine
Internal Medicine/Medicine
Schools of Medicine
United States
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Srinivasan, Padmanabhan; Subramanian, Veedamali S; Said, Hamid M (2014) Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake. Am J Physiol Gastrointest Liver Physiol 306:G631-9
Nabokina, Svetlana M; Inoue, Katsuhisa; Subramanian, Veedamali S et al. (2014) Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter. J Biol Chem 289:4405-16
Subramanian, Veedamali S; Nabokina, Svetlana M; Said, Hamid M (2014) Association of TM4SF4 with the human thiamine transporter-2 in intestinal epithelial cells. Dig Dis Sci 59:583-90
Nabokina, Svetlana M; Subramanian, Veedamali S; Valle, Judith E et al. (2013) Adaptive regulation of human intestinal thiamine uptake by extracellular substrate level: a role for THTR-2 transcriptional regulation. Am J Physiol Gastrointest Liver Physiol 305:G593-9
Subramanian, Veedamali S; Subramanya, Sandeep B; Ghosal, Abhisek et al. (2013) Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: studies utilizing Caco-2 cells and Rab8a knockout mice. Dig Dis Sci 58:641-9
Biswas, Arundhati; Elmatari, Daniel; Rothman, Jason et al. (2013) Identification and functional characterization of the Caenorhabditis elegans riboflavin transporters rft-1 and rft-2. PLoS One 8:e58190
Subramanian, Veedamali S; Ghosal, Abhisek; Subramanya, Sandeep B et al. (2013) Differentiation-dependent regulation of intestinal vitamin B(2) uptake: studies utilizing human-derived intestinal epithelial Caco-2 cells and native rat intestine. Am J Physiol Gastrointest Liver Physiol 304:G741-8
Nabokina, Svetlana M; Valle, Judith E; Said, Hamid M (2013) Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: a role for NF-Y in regulating basal transcription. Gene 528:248-55
Subramanian, Veedamali S; Subramanya, Sandeep B; Ghosal, Abhisek et al. (2013) Chronic alcohol feeding inhibits physiological and molecular parameters of intestinal and renal riboflavin transport. Am J Physiol Cell Physiol 305:C539-46
Kumar, Jeyan S; Subramanian, Veedamali S; Kapadia, Rubina et al. (2013) Mammalian colonocytes possess a carrier-mediated mechanism for uptake of vitamin B3 (niacin): studies utilizing human and mouse colonic preparations. Am J Physiol Gastrointest Liver Physiol 305:G207-13

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