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Subramanya SB, Subramanian VS, Kumar JS, Hoiness R, Said HM. Inhibition of intestinal biotin absorption by chronic alcohol feeding: cellular and molecular mechanisms. Am J Physiol Gastrointest Liver Physiol 300: G494–G501, 2011. First published December 9, 2010; doi:10.1152/ajpgi.00465.2010.—The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels, and animal studies have shown inhibition in intestinal biotin absorption by chronic alcohol feeding. Little, however, is known about the cellular and molecular mechanisms involved in the inhibition in intestinal biotin transport by chronic alcohol use. These mechanisms were investigated in this study by using rats and transgenic mice carrying the human full-length SLC5A6 5=-regulatory region chronically fed alcohol liquid diets; human intestinal epithelial Caco-2 cells chronically exposed to alcohol were also used as models. The results showed chronic alcohol feeding of rats to lead to a significant inhibition in carrier-mediated biotin transport events across jejunal brush border and basolateral membrane domains. This inhibition was associated with a significant reduction in level of expression of the SMVT protein, mRNA, and heterogenous nuclear RNA. Chronic alcohol feeding also inhibited carrier-mediated biotin uptake in rat colon. Studies with transgenic mice confirmed the above findings and further showed chronic alcohol feeding significantly inhibited the activity of SLC5A6 5=-regulatory region. Finally, chronic exposure of Caco-2 cells to alcohol led to a significant decrease in the activity of both promoters P1 and P2 of the human SLC5A6 gene. These studies identify for the first time the cellular and molecular parameters of the intestinal biotin absorptive processes that are affected by chronic alcohol feeding.

uptake by YAMC cells was: 1) energyand temperature(but not Na-) dependent and occurred without metabolic alteration of the transported substrate; 2) saturable as a function of concentration with an apparent Km and Vmax of 2.1 ± 0.5 μM and 53.4 ± 4.3 pmol/ mg protein/3min, respectively; 3) inhibited by pyridoxine structural analogs but not by unrelated compounds; 4) trans-stimulated by unlabeled pyridoxine; 5) inhibited by amiloride in a concentration-dependent manner; 6) adaptively up-regulated by extracellular substrate level via what appears to be a transcriptional/translationalmediated mechanism(s); and, 7) affected by modulators of intracellular Ca2+-calmodulin-mediated pathway. Evidence was also obtained to show that pyridoxine uptake by native human colonic AMV was carriermediated. Conclusions: These studies demonstrate, for the first time, the existence of a specific carrier-mediated mechanism for pyridoxine uptake by mammalian colonocytes and show that the process is regulated by extracellular pyridoxine level and by an intracellular Ca2+-calmodulin-mediated pathway. [Supported by DVA and NIH grants].

Said ZM, Subramanian VS, Vaziri ND, Said HM. Pyridoxine uptake by colonocytes: a specific and regulated carrier-mediated process. Am J Physiol Cell Physiol 294: C1192–C1197, 2008. First published March 19, 2008; doi:10.1152/ajpcell.00015.2008.—The water-soluble vitamin B6 (pyridoxine) is important for normal cellular functions, growth, and development. The vitamin is obtained from two exogenous sources: a dietary source, which is absorbed in the small intestine, and a bacterial source, where the vitamin is synthesized in significant quantities by the normal microflora of the large intestine. Evidence exists to suggest the bioavailability of the latter source of the vitamin, but nothing is known about the mechanism involved and its regulation. In this study, we addressed these issues using young adult mouse colonic epithelial (YAMC) cells and human colonic apical membrane vesicles (AMV) as models and using [H]pyridoxine as the uptake substrate. The results showed the initial rate of [H]pyridoxine uptake by YAMC cells to be 1) energyand temperature(but not Na-) dependent and to occur without metabolic alteration in the transported substrate; 2) saturable as a function of concentration with an apparent Km and Vmax of 2.1 0.5 M and 53.4 4.3 pmol mg protein 1 3 min , respectively; 3) cis-inhibited by unlabeled pyridoxine and its structural analogs, but not by the unrelated compounds theophylline, penicillamine, and isoniazid; 4) trans-stimulated by unlabeled pyridoxine; 5) amiloride sensitive; and 6) regulated by extracellular and intracellular factors. Uptake of pyridoxine by native human colonic AMV was also found to involve a carrier-mediated process. These studies demonstrate, for the first time, the functional existence of a specific and regulatable carriermediated process for pyridoxine uptake by mammalian colonocytes.