Down-regulation of the monocarboxylate transporter 1 is involved in butyrate deficiency during intestinal inflammation.

BACKGROUND & AIMS: Butyrate oxidation is impaired in intestinal mucosa of patients with inflammatory bowel diseases (IBD). Butyrate uptake by colonocytes involves the monocarboxylate transporter (MCT) 1. We aimed to investigate the role of MCT1 in butyrate oxidation deficiency during colonic inflammation. METHODS: Colonic tissues were collected from patients with IBD or healthy controls and from rats with dextran sulfate sodium (DSS)-induced colitis. The intestinal epithelial cell line HT-29 was treated with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). MCT1 expression was analyzed by real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence. Butyrate uptake and oxidation in HT-29 cells was assessed using [(14)C]-butyrate. The mechanism of MCT1 gene regulation was analyzed by nuclear run-on and reporter gene assays. RESULTS: MCT1 messenger RNA (mRNA) and protein levels were markedly decreased in inflamed colonic mucosa of IBD patients and rats. In HT-29 cells, down-regulation of MCT1 mRNA and protein abundance by IFN-gamma and TNF-alpha correlated with a decrease in butyrate uptake and subsequent oxidation. IFN-gamma and TNF-alpha did not affect MCT1 mRNA stability but rather down-regulated gene transcription. We demonstrate that the cytokine response element is located in the proximal -111/+213 core region of the MCT1 promoter. CONCLUSIONS: The data suggest that butyrate oxidation deficiency in intestinal inflammation is a consequence of reduction in MCT1-mediated butyrate uptake. This reinforces the proposition that butyrate oxidation deficiency in IBD is not a primary defect.

The human colonic monocarboxylate transporter Isoform 1: its potential importance to colonic tissue homeostasis.

BACKGROUND & AIMS: Butyrate serves as the major source of energy for colonic epithelial cells, and has profound effects on their proliferation, differentiation, and apoptosis. Transport of butyrate across the colonocyte luminal membrane is mediated by the monocarboxylate transporter, MCT1; the expression of which is down-regulated dramatically during colon carcinogenesis. We have proposed that the decline in MCT1 expression during colon carcinogenesis may reduce the intracellular availability of butyrate required to regulate expression of genes associated with the processes maintaining tissue homeostasis within the colonic mucosa. METHODS: To test this hypothesis we used the technique of RNA interference to inhibit MCT1 expression specifically, and determined the consequences of this inhibition on the ability of butyrate to exert its recognized effects in vitro using flow cytometry, immunofluorescence, Northern analysis, and Western analysis. RESULTS: We show that inhibition of MCT1 expression, and hence butyrate uptake, has profound inhibitory effects on the ability of butyrate to regulate expression of key target genes: p21waf1/cip1 (p21), intestinal alkaline phosphatase (IAP), and cyclin D1, and their associated processes of proliferation and differentiation. In contrast, inhibition of MCT1 expression had no effect on the ability of butyrate to modulate expression of either bcl-XL or bak, and this was reflected in a corresponding lack of effect on butyrate induction of apoptosis. CONCLUSIONS: Collectively, these results show the importance of MCT1 to the ability of butyrate to induce cell-cycle arrest and differentiation, and suggest fundamental differences in the mechanisms by which butyrate modulates specific aspects of cell function.

The human monocarboxylate transporter, MCT1: genomic organization and promoter analysis.

Uptake of butyrate across the colonocyte luminal membrane is mediated by the monocarboxylate transporter isoform 1 (MCT1). We have demonstrated previously that expression of human colonic MCT1 is responsive to butyrate, and that this involves the dual control of MCT1 gene transcription and stability of the MCT1 transcript. Here we describe the structural organization of the human MCT1 gene, and report the isolation and characterization of the MCT1 gene promoter. The MCT1 gene spans approximately 44 kb, and is organized as 5 exons intervened by 4 introns. The first of these introns is located in the 5'-UTR-encoding DNA, spans >26 kb, and thus accounts for approximately 60% of the entire transcription unit. Analysis of a 1.5 kb fragment of the MCT1 5'-flanking region, shows an absence of the classical TATA-Box motif. However, the region contains potential binding sites for a variety of transcription factors with known association with butyrate's action in the colon. In transient transfections the 5'-flanking region drives high-level expression of a luciferase reporter-gene in cells that endogenously express MCT1. Deletion analyses indicate that the cis-acting elements necessary for basal transcription of MCT1 are contained within the -70/+213 proximal sequence of the promoter.

Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.

Butyrate is the principal source of energy for colonic epithelial cells, and has profound effects on their proliferation, differentiation and apoptosis. Transport of butyrate across the colonocyte luminal membrane is mediated by the monocarboxylate transporter 1 (MCT1). We have examined the regulation of expression of human colonic MCT1 by butyrate, in cultured colonic epithelial cells (AA/C1). Treatment with sodium butyrate (NaBut) resulted in a concentration- and time-dependent upregulation of both MCT1 mRNA and protein. At 2 mM butyrate, the magnitude of induction of mRNA (5.7-fold) entirely accounted for the 5.2-fold increase in protein abundance, and was mediated by both activation of transcription and enhanced mRNA stability. The other monocarboxylates found naturally in the colon, acetate and propionate, had no effect. The properties of butyrate uptake by AA/C1 cells were characteristic of MCT1. Induction of the MCT1 protein resulted in a corresponding increase in the maximal rate of butyrate transport. The V(max) for uptake of [U-(14)C]butyrate was increased 5-fold following pre-incubation with 2 mM NaBut, with no significant change in the apparent K(m). In conclusion, this study is the first to show substrate-induced regulation of human colonic MCT1. The basis of this regulation is a butyrate-induced increase in MCT1 mRNA abundance, resulting from the dual control of MCT1 gene transcription and stability of the MCT1 transcript. We suggest that butyrate-induced increases in the expression and resulting activity of MCT1 serve as a mechanism to maximise intracellular availability of butyrate, to act both as a source of energy and to influence processes maintaining cellular homeostasis in the colonic epithelium.