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Background/aims: Efficient uptake at the basolateral plasma membrane of hepatocytes is required for maintenance of the enterohepatic circulation of bile salts. Uptake occurs mainly via a Na+-dependent process mediated by ntcp, a recently cloned and characterized 51 kDa glycoprotein. The aim of this study was to evaluate the role of variations in hepatic bile salt flux through the liver in the regulation of ntcp activity and expression under non-cholestatic conditions., Methods: We determined the kinetics of Na+-dependent taurocholate transport in isolated basolateral plasma membrane vesicles as well as hepatic ntcp protein and ntcp mRNA levels in long-term (8 days) bile-diverted rats, with a transhepatic bile salt flux of 0, and in streptozotocin-induced diabetic rats with a 2.5-fold increased bile salt flux., Results: We found no changes in the kinetics of taurocholate transport in the absence of transhepatic bile salt flux due to bile diversion. Ntcp protein and ntcp mRNA levels were also unaffected in bile-diverted rats. Likewise, no changes in taurocholate transport kinetics, ntcp protein or ntcp mRNA levels were detected in streptozotocin-diabetic rats when compared to non-diabetic controls. Thus, variation in hepatic bile salt flux from 0 to 250% of normal values had no effect on hepatic ntcp expression or taurocholate transport activity in basolateral plasma membrane vesicles in rats. In contrast, 4 days of bile duct ligation resulted in a strong decrease in ntcp mRNA and protein levels, as recently also reported by others., Conclusions: Our data indicate that ntcp is not regulated by the transhepatic flux of bile acids under non-cholestatic conditions.

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months. Albumin- and fibrinogen-secreting cells were selected and cloned by limiting dilution to obtain homologous cell populations. The established IHH (immortalized human hepatocyte) cell lines were evaluated for their usefulness in studying the regulation of cell growth and of certain differentiated hepatocyte functions. IHH cells retain several differentiated features of normal hepatocytes. They display albumin secretion at a level comparable to cultured primary human hepatocytes (30 micrograms albumin/ml per day). A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous organic anions accumulate. The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles. The polarized features allowed the use of IHH cells for the study of localization of the newly characterized multidrug resistance protein MRP1. The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates. In differentiated hepatocytes, MRP1 expression is extremely low. In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes. A highly differentiated feature of short-term cultured primary hepatocytes which is not detectable in IHH cells is active uptake of the bile salt taurocholate. Furthermore, IHH cells secrete triglyceride (TG)-rich lipoproteins, apolipoprotein B (0.6 microgram/ml per day), and apolipoprotein A-I (1 microgram/ml per day). However, they secrete apoB-containing TG-rich lipoproteins mainly in the LDL density range, while short-term cultured primary hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range. In conclusion, functions that are rapidly lost in short-term hepatocyte cultures are, in general, not displayed by IHH cells. Immortalized human hepatocytes provide a valuable tool for studying the regulation of hepatocyte proliferation-related phenomena.

Background & Aims: The multidrug resistance protein (MRP) functions as an organic anion efflux carrier. Recent studies suggest that hepatocytes contain two mrp homologues, named mrp1 and mrp2, localized on the lateral and canalicular membrane, respectively. The aim of this study was to evaluate the role of MRP1. Protein levels and localization of MRP1 in human hepatocytes, HepG2 hepatoma cells, and SV40 large T antigen-immortalized human hepatocytes were studied., Methods: Using specific antibodies, MRP1 protein levels and cellular localization were examined by Western blotting and fluorescence confocal microscopy, respectively. In addition, a fluorescent substrate, glutathione-methylfluorescein, was used to measure plasma membrane transport activity and to observe intracellular transport activity., Results: The level of MRP1 in normal hepatocytes is very low. In contrast, MRP1 is highly increased in both HepG2 and immortalized hepatocytes. In these cells MRP1 is localized in lateral membranes of adjacent cells. Plasma membrane staining is absent in separate cells. Glutathione-methylfluorescein is transported in the medium and intracellular vesicles., Conclusions: MRP1 protein level is greatly increased in the lateral membrane of proliferating hepatocyte-derived cells. The presence of a lateral domain seems necessary for plasma membrane localization. These results suggest that MRP1-mediated organic anion transport is important in proliferating hepatocytes, but not in quiescent cells.

In this study the effects of bile acids and other organic anions on the secretion of very-low-density lipoproteins (VLDLs) were evaluated in rat hepatocytes in primary culture. Incubation of cells with portal blood concentrations (10-200 microM) of bile acids resulted in dose-dependent suppression of secretion of VLDL-associated [3H]triglyceride (TG) formed from [3H]glycerol, and also of TG mass. The degree of the inhibition was highly correlated with intracellular bile acid concentration. Prolonged incubation with 100 microM extracellular taurocholic acid (TC) decreased the secretion of [3H]TG and TG mass to 35% and 50% of the controls respectively. Cellular content of mass and of [3H]TG during prolonged incubation with TC were about 20% and 60% higher than the controls respectively. The inhibitory effect remained for at least 24 h in the presence of TC without altering VLDL-lipid and VLDL-apolipoprotein compositions or the size distribution of the particles. Secretion of apoB-100 and of apoB-48 was inhibited to a similar extent. Cells largely lost their capacity to accumulate bile acids intracellularly after 48 h in culture. In these cells TC was unable to exert its suppressive effects. Taurine and glycine conjugates of all common bile acids were capable of suppressing [3H]TG secretion. Trihydroxylated (cholic acid) and various dihydroxylated (deoxycholic, chenodeoxycholic and ursodeoxycholic acids) bile acids had similar capacities in this respect, suggesting that their common sterol-3 alpha-OH structure is required for the suppressive effect. Neither non-bile acid organic anions, e.g. bilirubin ditaurate and dibromosulphthalein, nor dianionic bile acid metabolites, e.g. sulphated taurolithocholic acid and lithocholate-3-O-glucuronide, showed any effect on [3H]TG secretion. These results indicate that bile acids might play a physiological role in regulating VLDL production by the liver, especially in the postprandial state when their enterohepatic circulation is stimulated.

The chicken very low density apolipoprotein II (apoVLDLII) gene is estrogen-inducible and specifically expressed in liver. We examined the possible involvement of the retinoid X receptor (RXR) and its ligand 9-cis-retinoic acid (9-cis-RA) in the activation of the apoVLDLII promoter. We first concentrated on a potential RXR recognition site, which deviates at only one position from a perfect direct A/GGGTCA repeat spaced by one nucleotide (DR-1) and was earlier identified as a common HNF-4/COUP-TF recognition site. However, band shift analysis revealed that this imperfect DR-1 motif does not interact with RXR alpha-homodimers. In accordance with this observation we found that this regulatory element does not mediate transactivation through RXR alpha in the presence of 9-cis-RA. However, our experiments revealed another, unexpected, effect of 9-cis-RA. Instead of stimulating, 9-cis-RA attenuated estrogen-induced expression of transfected estrogen-responsive VLDL-CAT reporter plasmids. This repression appeared to take place through the main estrogen response element (ERE) of the gene. Importantly, 9-cis-RA also strongly repressed the estrogen-induced expression of the endogenous apoVLDLII gene in cultured chicken hepatoma cells.

The gene coding for chicken very low density apolipoprotein II (apoVLDLII) is expressed exclusively in liver in response to estrogen. Previous work in our laboratory identified several protein binding sites, identified by the letters A to F, and their cognate factors within the first 300 bp flanking the gene. Here we present an extensive functional analysis of the apoVLDLII promoter by gene transfer experiments using a chicken hepatoma cell line and cultured non-hepatic cells. Deletion analysis revealed that the -301 to -163-bp promoter region, comprising elements E1, E2 and F, is sufficient for strong estrogen-dependent expression. Mutation analysis demonstrated that efficient transcription requires the interplay of the major estrogen response element E1 with several other cis-acting elements. Analysis of individual protein binding sites showed that element E1 is sufficient by itself to confer weak estrogen-induced transcription from the apoVLDLII promoter, and that additional promoter elements are required for full estrogen-responsiveness. Elements F and B1 were capable of strongly potentiating the activity of element E1. In general, the activity of certain cis-acting elements appeared to be strongly promoter-context dependent. Cultured non-liver cells expressed transfected VLDL-CAT reporter plasmids in the presence of cotransfected estrogen receptor expression vector in a hormone-dependent way, indicating that for the control of tissue specificity the 5'-proximal promoter region is not sufficient.

The chicken Very-Low-Density Apolipoprotein II (apoVLDL II) gene is specifically expressed in liver in response to estrogen. In this study, we performed a functional analysis of the 300-base pair region immediately 5' to the gene by gene transfer of chloramphenicol acetyl transferase (CAT) constructs into chicken embryonic hepatocytes (CEH). Two estrogen response elements (EREs) could be distinguished which together form a potent estrogen response unit. Stimulation of transient expression by co-transfection with a plasmid expressing rat C/EBP confirmed that a similar protein in chicken liver may be involved in apoVLDL II transcription. In vitro DNaseI footprinting and band-shift analysis with liver, oviduct and spleen nuclear extract revealed the tissue distribution of the proteins binding to the promoter region. A liver-specific protein bound to multiple sites of which some resembled the recognition sequence of the CCAAT/Enhancer binding protein, C/EBP. Of the other proteins binding to the apoVLDL II promoter, one was identified as the liver-specific LF-A1 by mobility shift analysis, using purified bovine LF-A1, and another as the general COUP-transcription factor, using an antiserum against the human COUP-TF.