Abstract
BackgroundPepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte.ResultsWe found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by ∼2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles.ConclusionsCollectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.
Highlights
Butyrate is a short-chain fatty acid (SCFA) produced in the colonic lumen by bacterial fermentation of carbohydrates and dietary fibers [1]
To test the response of the human PepT1 (hPepT1) promoter to SCFAs, human intestinal epithelial Caco2-BBE cells were transfected with a reporter construct encoding the putative proximal promoter region of hPepT1 gene (722 bp) previously cloned in our laboratory ([27]; GeneBankTM accession number DQ370174)
Our results revealed that of the six examined SCFAs, butyrate was the most potent enhancer of hPepT1 promoter activity (Figure 1A)
Summary
Butyrate is a short-chain fatty acid (SCFA) produced in the colonic lumen by bacterial fermentation of carbohydrates and dietary fibers [1]. The colonic lumen contains 100–150 mM total SCFAs [2,3]. 95–99% of SCFAs produced in the colonic lumen are absorbed [4,5]. SCFAs uptake by epithelial cells occurs by simple diffusion of the unionized forms across cell membranes, whereas uptake of the ionized form is mediated by apical membrane monocarboxylic transporter (MCT)-1 [6]. Of the three major SCFAs, butyrate is the main intestinal fuel even in the presence of competing substrates such as glucose and glutamine [8]. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte
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