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1 VA Medical Center, Long Beach, CA, USA; University of California College of Medicine, Irvine, CA, USA
2 University of Minnesota Medical School, Minneapolis, MN, USA
* To whom correspondence should be addressed. E-mail: hmsaid{at}uci.edu.
The aim of this study was to investigate the expression and relative contribution of the human thiamin transporter hTHTR-2 toward overall carrier- mediated thiamin uptake by human intestinal epithelial cells. Northern blot analysis showed the message of the hTHTR-2 to be expressed along the native human gastrointestinal tract with the highest expression being in the proximal part of the small intestine. The hTHTR-2 protein was found, by Western blotting, to be expressed at the brush border membrane, but not at the basolateral membrane, of native human enterocytes. This pattern of expression was confirmed in studies using a fusion protein of hTHTR-2 with the enhanced green fluorescent protein (hTHTR2-EGFP) expressed in living Caco-2 cells grown on filter. Pre-treating Caco-2 cells (which also express the hTHTR-2 at the RNA and protein levels) with hTHTR-2 gene specific small interfering RNA (siRNA) led to a significant (p < 0.01) and specific inhibition (48%) in carrier-mediated thiamin uptake. Similarly, pre-treating Caco-2 cells with siRNA that specifically target the human thiamin transporter hTHTR-1 (which is also expressed in Caco-2 cells) also significantly (p < 0.01) and specifically inhibited (by 56%) the carrier-mediated thiamin uptake. When Caco-2 cells were pre-treated with siRNAs against both of the hTHTR-2 and hTHTR-1 genes, an almost comp lete inhibition in carrier-mediated thiamin uptake was observed. These results show that the message of the hTHTR-2 is expressed along the human gastrointestinal tract, and that expression of its protein in intestinal epithelia is mainly localized to the apical brush border membrane domain. In addition, the results show that this transporter plays a significant role in carrier-mediated thiamin uptake in the human intestine.
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