Uptake of biotin by human hepatoma cell line, Hep G2: a carrier-mediated process similar to that of normal liver.
Little is known about the cellular and molecular regulation of the uptake 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 allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human-derived liver cells, Hep G2. Uptake of biotin by Hep G2 cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na+ gradient-dependent as indicated by studies of Na+ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of 3H-biotin uptake at 37 degrees C but not 4 degrees C. Initial rate of biotin uptake was saturable as a function of concentration at 37 degrees C but was lower and linear at 4 degrees C. Pretreatment of Hep G2 cells with sulfhydryl group inhibitors (e.g., p-chloromercuribenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G2 cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na+ uptake in Hep G2 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 dependent on Na(+)-gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G2 cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver.
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Medical Research Service, VA Medical Center, Long Beach, California 90822.
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Kamanna
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D001692:Biological Transport / Q000187:drug effects; D001710:Biotin / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins / Q000378:metabolism; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D012964:Sodium / Q000502:physiology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014634:Valinomycin / Q000494:pharmacology
D002352:Carrier Proteins; D013439:Sulfhydryl Reagents; D006096:Gramicidin; D014634:Valinomycin; D010042:Ouabain; D001710:Biotin; D012964:Sodium