UPTAKE OF BIOTIN BY HUMAN HEPATOMA-CELL LINE, HEP G(2) - A CARRIER-MEDIATED PROCESS SIMILAR TO THAT

Little is known about the cellular and molecular regulation of the upt ake process of the water-soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allow s examination oi prolonged exposure to an agent(s) or a particular con dition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the speci alized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) oi biotin uptake by the cultured human-deriv ed liver cells, Hep G(2). Uptake of biotin by Hep G(2) cells was appre ciable and linear for up to 10 min of incubation. The uptake process w as Na+ gradient-dependent as indicated by studies of Na+ replacement a nd pretreatment of cells with gramicidin and ouabain. Biotin uptake wa s also dependent on both incubation temperature and intracellular ener gy. Unlabeled biotin and the structural analogs with free carboxyl gro ups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused sign ificant inhibition of H-3-biotin uptake at 37 degrees C but not 4 degr ees C. Initial rate of biotin uptake was saturable as a function of co ncentration at 37 degrees C but was lower and linear at 4 degrees C. P retreatment of Hep G(2) cells with sulfhydryl group inhibitors (e.g., p-chloromercuribenzene sulfonate) led to a significant inhibition in b iotin uptake; this inhibition was effectively reversed by reducing age nts (e.g., dithiothreitol). Biotin uptake was also inhibited by the me mbrane transport inhibitors probenecid (noncompetitively), DIDS and fu rosemide but not by amiloride. Pretreatment of Hep G(2) cells with val inomycin did not alter biotin uptake. The stoichiometric ratio of biot in to Na+ uptake in Hep G(2) cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is d ependent on Na+-gradient, temperature, and energy and transports the v itamin by an electroneutral process. These findings are similar to tho se seen with native liver tissue preparations and demonstrate the suit ability of Hep G(2) cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver. (C) 1994 Wiley -Liss, inc.