The mechanisms of enterocyte and molecular regulation of biotin uptake are poorly understood. An intestinal cell line possessing the transport characteristics of native intestinal cells is highly desirable to investigate the finer details of the cellular processing and molecular regulation of biotin transport. In the present study, we investigated the uptake of the water-soluble vitamin biotin by a human intestinal cell line Caco-2. Uptake of both low (4 nM) and high (20 μM) concentrations of biotin by confluent monolayers of Caco-2 cells was appreciable and linear for up to 10 min of incubation. Replacement of Na+ in the incubation medium with other monovalent cations - K+, choline, Li+ and NH4+ - caused a significant inhibition of biotin uptake; a relatively lesser inhibition was seen with Li+. Initial rate of uptake of biotin was temperature-dependent and saturable as a function of concentration at 37°C but not at 4°C. The Vmax and apparent Km of the temperature-dependent saturable process were 520 pmol/mg protein per min and 9.5 μM, respectively. The addition of unlabeled biotin and the structural analogue desthiobiotin to the incubation media caused a significant inhibition of the uptake of [3H]biotin. The inhibitory effect of desthiobiotin was competitive in nature with an inhibition constant (Ki) of 41 μM. Biocytin, on the other hand, was a weak inhibitor and biotin methyl ester and diaminobiotin did not have any effect. Pretreatment of Caco-2 cells with the monovalent cation ionophore gramicidin and the Na+,K+-ATPase inhibitor ouabain caused significant inhibition of biotin uptake. Pretreatment with the K+ ionophore valinomycin did not affect biotin uptake. Using the 'Activation Method', the stoichiometric ratio of biotin- to Na+ coupling was found to be 1: 1. Growing confluent Caco-2 cells in a biotin-deficient environment resulted in rapid up-regulation of biotin transport with a marked increase (258%) in the Vmax of biotin uptake. These findings demonstrate that biotin uptake by Caco-2 cells is via a carrier-mediated system. This system is temperature-dependent, driven by Na+-gradient and is regulated by the substrate level. These in-vitro findings are very similar to and further confirm previous findings in human and animal studies and dispute other findings previously reported for Caco-2 cells; the present study also demonstrates the suitability of this system for further characterization of the cellular and molecular regulation of biotin uptake.
All Science Journal Classification (ASJC) codes
- Cell Biology