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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK056061-15
Application #
8519984
Study Section
Special Emphasis Panel (ZRG1-DIG-D (02))
Program Officer
Maruvada, Padma
Project Start
1999-08-01
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
15
Fiscal Year
2013
Total Cost
$314,489
Indirect Cost
$102,458
Name
University of California Irvine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Nabokina, Svetlana M; Ramos, Mel Brendan; Said, Hamid M (2016) Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter. PLoS One 11:e0149255
Nabokina, Svetlana M; Subramanian, Veedamali S; Said, Hamid M (2016) The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function. Biochim Biophys Acta 1858:866-71
Sabui, Subrata; Subramanian, Veedamali S; Kapadia, Rubina et al. (2016) Structure-function characterization of the human mitochondrial thiamin pyrophosphate transporter (hMTPPT; SLC25A19): Important roles for Ile(33), Ser(34), Asp(37), His(137) and Lys(291). Biochim Biophys Acta 1858:1883-90
Srinivasan, Padmanabhan; Thrower, Edwin C; Gorelick, Fred S et al. (2016) Inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Am J Physiol Gastrointest Liver Physiol 310:G874-83
Subramanian, Veedamali S; Lambrecht, Nils; Lytle, Christian et al. (2016) Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption. Am J Physiol Gastrointest Liver Physiol 310:G285-93
Srinivasan, Padmanabhan; Nabokina, Svetlana; Said, Hamid M (2015) Chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse 266-6 cell line and primary cells. Am J Physiol Gastrointest Liver Physiol 309:G750-8
Said, Hamid M (2015) Water-soluble vitamins. World Rev Nutr Diet 111:30-7
Nabokina, Svetlana M; Ramos, Mel Brendan; Valle, Judith E et al. (2015) Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells. Am J Physiol Cell Physiol 308:C750-7
Subramanian, Veedamali S; Ghosal, Abhisek; Kapadia, Rubina et al. (2015) Molecular Mechanisms Mediating the Adaptive Regulation of Intestinal Riboflavin Uptake Process. PLoS One 10:e0131698
Ghosal, Abhisek; Sabui, Subrata; Said, Hamid M (2015) Identification and characterization of the minimal 5'-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells. Am J Physiol Cell Physiol 308:C189-96

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