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Grant Abstract

Grant Number: 1R21AA021948-01
PI Name: Zastre
Project Title: Adaptive Regulation of Vitamin B1 Transport

Abstract: DESCRIPTION (provided by applicant): Vitamin B1 (thiamine) is a critical enzyme cofactor within the glycolytic metabolism network that is fundamentally required to sustain the bioenergetic and anabolic needs of all cells. When deficiencies arise, an adaptive up-regulation in thiamine transporter expression has been described that attempts to restore thiamine homeostasis. However, thiamine deficiencies during chronic alcohol consumption are associated with a down- regulation of thiamine transporters. This antagonism between alcohol and thiamine availability on gene regulation of thiamine transporters is not clearly understood because there is a lack of fundamental knowledge on how thiamine transporters are adaptively regulated. Hence, this application endeavors to understand the cis- and trans-regulatory elements that are responsible for mediating an adaptive up-regulation during thiamine deficiency. Until this mechanistic insight into adaptive regulation is known, understanding how alcohol attenuates the gene expression of thiamine transporters during associated thiamine deficiency will not be realized. The overall objective we have for this application is to determine the transcriptional pathways involved in the adaptive regulation of thiamine transporters during thiamine deficiency. It is our central hypothesis that hypoxia inducible factor-1 (HIF-1 is a centrl transcription factor modulating thiamine availability by trans- activating expression of the thiamine transporter SLC19A3. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratory. The rationale for the proposed research is that once the transcriptional pathways for adaptive thiamine transporter expression are known, new insights and approaches into the underlying effects of alcohol consumption on thiamine transport capacity can be achieved. Guided by our strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Establishing the connection between thiamine deficiency and pseudo-hypoxia on adaptive thiamine transport expression. It is our contention that the metabolic consequences of thiamine deficiency can lead to an accumulation of pyruvate which in turn activates HIF-1 mediated gene regulation. Thus we will use a combination of metabolic profiling and assessment of HIF-1 target gene expression and in particular SLC19A3 during thiamine deficiency. We will also determine if the consequences of alcohol metabolism attenuates the level of pyruvate to counteract this adaptive regulation. 2) Determining HIF-1 mediated trans- activation of SLC19A3 gene expression. Our working hypothesis is that adaptive up-regulation of the thiamine transporter SLC19A3 is conferred through hypoxia response elements in the proximal promoter region. Using a combination of promoter-luciferase assays and ChIP we will establish HIF-1 as an essential trans-activator for SLC19A3 expression. Overall, the proposed research is significant because it is expected to lead to a mechanistic understanding of thiamine transporter regulation within thiamine deficiency related disorders. PUBLIC HEALTH RELEVANCE: Vitamin B1 is an essential enzyme cofactor that cannot be synthesized de novo by humans and as such requires adaptively regulated transport processes to maintain availability during nutritional and pathological stress. This application proposes to better understand the pathways by which gene expression of vitamin B1 transport processes are controlled within deficiencies associated with chronic alcohol use. The proposed research is relevant to public health and the NIH's mission, because it is expected to increase our understanding into the effects of nutrition on gene regulation and the impact of disease pathologies on vitamin B1 homeostasis.

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