The long-term objectives of this application are to characterize the molecular mechanisms involved in the regulation of the intestinal absorption of vitamin B2 (riboflavin, RF), and those involved in intracellular trafficking and membrane targeting of the involved membrane transporter. We also aim at examining the effect of chronic alcohol use on intestinal RF absorption and determining the cellular and molecular mechanisms involved. RF is involved in critical cellular metabolic reactions, and thus, is essential for normal human health and well-being. Its deficiency, which occurs in a variety of conditions like chronic alcoholism, leads to serious clinical abnormalities that include degenerative changes in the nervous system, anemia, growth retardation, and skin lesions. Humans (and other mammals) cannot synthesize RF, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Thus, the human intestine plays a central role in maintaining and regulating normal RF body homeostasis. For these reasons understanding the physiology/pathophysiology as well as cell/molecular biology of the intestinal RF absorption process is of significant importance and is the aim of this proposal. We have been investigating the physiology of intestinal RF transport for over two decades, but more is needed at the molecular level to fully understand the process. With the recent molecular identification of mammalian RF transporters, we are now in an excellent position to carry out such investigations. Thus, in new preliminary studies we have shown that the up-regulation in intestinal RF uptake in RF deficiency shown by us previously is mediated (at least in part) via a transcriptional regulatory mechanism(s) affecting hRFT-2. We also showed that the intestinal RF uptake process undergoes differentiation - dependent regulation and that this regulation again appears to be mediated (at least in part) via a transcriptional mechanism(s). Using confocal imaging of living human intestinal epithelia cells and Western blot analysis of native human intestinal brush border and basolateral membrane preparations, our preliminary studies further showed exclusive expression of the human RF transporter -2 (hRFT-2;the most relevant intestinal RF transporter) at the apical membrane domain of polarized human intestinal epithelial cells. Furthermore, distinct trafficking vesicles appear to be involved in intracellular movement of hRFT2 in human intestinal epithelial cells. Finally, we showed for the first time that chronic alcohol feeding significantly inhibit RF transport across the jejunal BBM which is associated with a significant decrease in the level of expression of RFT-2. Our Specific Objectives in this proposal are: 1) To continue our investigations into the molecular mechanisms involved in the adaptive up-regulation in intestinal RF uptake in RF deficiency and during cell differentiation;2) To study the mechanism(s) involved in the targeting of the hRFT-2 protein to the apical membrane domain of polarized human intestinal epithelial cells, and to determine the factor(s) involved in its intracellular trafficking;and 3) to extend our basic physiological/nutritional investigations on the intestinal RF uptake process into a clinically-relevant area and will examine the effect of chronic alcohol consumption on cell and molecular parameters of intestinal RF absorption process. Collectively, results of these studies should provide novel and valuable information regarding the physiology/pathophysiology and cell/molecular biology of intestinal RF uptake and of the factors that interfere with the process. This should ultimately assist us in the designing of effective strategies to optimize RF body homeostasis, especially in conditions of RF deficiency and sub- optimal levels. 1

Public Health Relevance

Riboflavin (RF) is essential for normal human health and its deficiency leads to a variety of clinical abnormalities. Humans cannot synthesize RF and must obtain the vitamin from exogenous sources via intestinal absorption. Thus, the gut plays a central role in regulating normal RF body level. The objectives of this application are to characterize the molecular mechanisms involved in the regulation of the intestinal RF absorption and those involved in intracellular trafficking and membrane targeting of the involved transporter, as well as investigate the effect of chronic alcohol consumption on this process. Results of these studies are expected to provide important information on how the intestine regulates the absorption of vitamin B2, and how chronic alcohol use interferes with this process. Such knowledge should help in the designing of effective strategies to optimize RF homeostasis, especially in conditions associated with RF deficiency and sub-optimal level. 1

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
1I01BX001142-01
Application #
8139616
Study Section
Gastroenterology (GAST)
Project Start
2011-04-01
Project End
2016-06-30
Budget Start
2011-04-01
Budget End
2013-06-30
Support Year
1
Fiscal Year
2011
Total Cost
Indirect Cost
Name
Veterans Health Administration
Department
Type
DUNS #
625399951
City
Long Beach
State
CA
Country
United States
Zip Code
90822
Subramanian, Veedamali S; Sabui, Subrata; Moradi, Hamid et al. (2018) Inhibition of intestinal ascorbic acid uptake by lipopolysaccharide is mediated via transcriptional mechanisms. Biochim Biophys Acta Biomembr 1860:556-565
Elahi, Asif; Sabui, Subrata; Narasappa, Nell N et al. (2018) Biotin Deficiency Induces Th1- and Th17-Mediated Proinflammatory Responses in Human CD4+ T Lymphocytes via Activation of the mTOR Signaling Pathway. J Immunol 200:2563-2570
Lakhan, Ram; Subramanian, Veedamali S; Said, Hamid M (2017) Role of MicroRNA-423-5p in posttranscriptional regulation of the intestinal riboflavin transporter-3. Am J Physiol Gastrointest Liver Physiol 313:G589-G598
Subramanian, Veedamali S; Srinivasan, Padmanabhan; Wildman, Alexis J et al. (2017) Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract. Am J Physiol Gastrointest Liver Physiol 312:G340-G347
Sabui, Subrata; Subramanian, Veedamali S; Kapadia, Rubina et al. (2017) Adaptive regulation of pancreatic acinar mitochondrial thiamin pyrophosphate uptake process: possible involvement of epigenetic mechanism(s). Am J Physiol Gastrointest Liver Physiol 313:G448-G455
Lakhan, Ram; Said, Hamid M (2017) Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway. Am J Physiol Cell Physiol 312:C376-C384
Subramanian, Veedamali S; Constantinescu, Alexandru R; Benke, Paul J et al. (2017) Mutations in SLC5A6 associated with brain, immune, bone, and intestinal dysfunction in a young child. Hum Genet 136:253-261
Anandam, Kasin Yadunandam; Srinivasan, Padmanabhan; Subramanian, Veedamali S et al. (2017) Molecular mechanisms involved in the adaptive regulation of the colonic thiamin pyrophosphate uptake process. Am J Physiol Cell Physiol 313:C655-C663
Subramanian, Veedamali S; Sabui, Subrata; Teafatiller, Trevor et al. (2017) Structure/functional aspects of the human riboflavin transporter-3 (SLC52A3): role of the predicted glycosylation and substrate-interacting sites. Am J Physiol Cell Physiol 313:C228-C238
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

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