The short chain fatty acids (SCFA) are the main energy-yielding substrates for the mammalian colonic mucosa and are known to play a pivotal role as regulators of fluid and electrolyte absorption as well as cellular differentiation in the colonic epithelia. Recent studies from the PI's laboratory, utilizing purified membrane vesicles from organ donor colons, have demonstrated the presence of SCFA/HCO3 exchangers in the human colonic apical and basolateral membrane vesicles with kinetically distinct characteristics. To date, however, the molecular nature of these SCFA transporters is not fully defined. Recent studies from the PI's laboratory and others indicated the involvement of monocarboxylate transporter 1 (MCT1) in the SCFA uptake by the human colonic apical membranes as well as the expression of other MCT isoforms in the human colon. The PI hypothesizes that: 1) MCT1 plays a critical role in the SCFA absorption in the human colon, 2) MCT1 regulation involves regulation by kinases, or via alterations in trafficking or targeting to plasma membranes in polarized epithelial cells; and 3) its expression is transcriptionally regulated.
The Specific aims are designed to: i) characterize the expression, distribution and function of the apical membrane MCT transporter(s) of the human intestine; ii) to elucidate the structure-function relationship of the human MCT1 and to delineate the sequences essential for protein trafficking and polarized distribution in epithelial cells; and iii) to characterize genomic clones corresponding to the 5' regulatory region for the human MCT1. For distribution, membrane localization and crypt/surface expression of MCT1 or other MCTs, mRNA and protein in the human intestine, RT-PCR, Rnase protection, Western analysis, in situ hybridization and immunohistochemistry in parallel to an analysis of MCT function will be performed. MCT1 structure/function relationship will be elucidated by utilizing truncation and deletion mutants and expression in a human breast cancer epithelial cell line to determine the relationship of various domains to SCFA transport, inhibition, and possible regulation by kinases. For trafficking studies, green fluorescent protein constructs of hMCT1 and expression in intestinal epithelial cells will be followed by confocal microscopy. MCT1 promoter will be cloned and DNA-protein interactions will be studied by mobility shift and foot printing analyses to yield important information on the cis- and trans-acting elements involved in regulation of MCT1 function and expression. An investigation of the molecular mechanisms of the colonic SCFA transporters, their structure-function analysis and their regulation would be critical for a better understanding of the role of SCFA in basic physiology of the colonocyte as well as in modulation of colonic electrolyte absorption, colitis and colonic malignancies.
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