Riboflavin uptake by the human-derived liver cells Hep G2: Mechanism and regulation

Abstract

The water-soluble vitamin riboflavin (RF) plays a critical role in many metabolic reactions, and thus, is essential for normal cellular functions and growth. The liver plays a central role in normal RF metabolism and is the site of maximal utilization of the vitamin. The mechanism of liver uptake of RF has been studied in animals, but no information is available describing the mechanism of the vitamin uptake in the human situation and its cellular regulation. In this study, we used the human-derived liver cells Hep G2 as an in vitro model system to address these issues. Uptake of RF by Hep G2 cells was found to be temperature- and energy-dependent but Na+-independent in nature. Uptake seemed to involve a carrier-mediated process as indicated by the saturation as a function of substrate concentration (apparent Km 0.41 +/- 0.08 microM), and by the ability of the structural analogs lumiflavin and lumichrome to inhibit the uptake process [inhibition constant (K) of 1.84 and 6.32 microM, respectively]. RF uptake was energy dependent, and was inhibited by the -SH group blocker p-chloromercuriphenylsulfonate (p-CMPS) (Ki of 0.10 mM). Specific modulators of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, and protein tyrosine kinase (PTK)-mediated pathways did not affect RF uptake by Hep G2 cells. On the other hand, specific inhibitors of Ca2+/calmodulin-mediated pathway significantly inhibited the uptake process; this effect seemed to be mediated through a decrease in the Vmax of the substrate uptake process. Maintaining Hep G2 cells in a RF-deficient growth medium was associated with a significant up-regulation in the substrate uptake; this effect was specific for RF and was mediated mainly by means of an increase in the Vmax of the uptake process. These results describe, for the first time, the mechanism and cellular regulation of RF uptake by a human-derived liver cellular preparation, and shows the involvement of a carrier-mediated system in the uptake process. Furthermore, the uptake process seems to be regulated by an intracellular Ca2+/calmodulin-mediated pathway and by extracellular substrate levels.

Transport of High Concentration of Thiamin, Riboflavin and Pyridoxine across Intestinal Epithelial Cells Caco-2

“…The results confirmed the dual mechanism of transport for the studied vitamins and are compatible for the three vitamins and with previous published results ( [23][24][25][26][27][28][29][30][31]; however the experiments for thiamin should be widened to include different concentrations. For low concentrations, normally observed in the human diet, the active mechanism of transport is suggested.…”

“…However, human regulation of intes- tinal absorption is poorly understood. At low vitamin concentration, the results of the experiments showed that riboflavin uptake involves a specialized, carriermediated mechanism of absorption in the alimentary canal (22)(23)(24)(25). In vitro studies using rat everted jejunal sacs have shown that absorption of the vitamin is a saturable and energy-dependent process at physiological concentrations and a simple diffusion takes place at higher concentrations (26).…”

Involvement of riboflavin transporter RFVT2/Slc52a2 in hepatic homeostasis of riboflavin in mice

“…2A). The latter observation was consistent with previous findings of hepatic uptake of riboflavin (Aw et al, 1983;Said et al, 1995Said et al, , 1998. Furthermore, the characteristics of riboflavin uptake observed in this study (Fig.…”

“…Several studies using hepatic plasma membrane vesicles, isolated hepatocytes and human hepatoblastoma cell line Hep G2 have indicated that the riboflavin uptake into the liver is mediated by the Na + -independent transporter(s) (Aw et al, 1983;Said et al, 1995Said et al, , 1998. Moreover, the deprivation of riboflavin leads to the upregulation of hepatic uptake of riboflavin (Aw et al, 1983;Said et al, 1998). Taken together, hepatic riboflavin homeostasis is possibly maintained via the function of specific transporter(s).…”

Involvement of Endocytic Organelles in the Subcellular Trafficking and Localization of Riboflavin

“…Like most water-soluble nutrients, studies have shown that specific transporter systems are involved to facilitate efficient entry of riboflavin across the cell membrane (Said and Arianas, 1991;Said et al, 1998;Huang and Swaan, 2001). Since the molecular identity of the riboflavin-translocating protein(s) remains elusive, most mechanistic studies focused mainly on biochemical characterization of the putative transporter(s) through determinants such as energy or ion dependence.…”