Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways.

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus.

The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.

Synthesis and applicability of a photolabile 7,7-azi analogue of 3-sulfated taurine-conjugated bile salts.

For the identification of proteins involved in hepatobiliary transport, the photolabile derivative 7,7-ASLCT ((7,7-azi-3 alpha-sulfato-5 beta-cholan-24-oyl)-2'-aminoethanesulfonate) was synthesized. 7,7-ASLCT is taken up into liver and excreted into bile completely unmetabolized at a rate between the excretion rate of SLCT ((3 alpha-sulfato-5 beta-cholan-24-oyl)-2'- aminoethanesulfonate) and SCCT ((7 alpha-hydroxy-3 alpha-sulfato-5 beta- cholan-24-oyl)-2'-aminoethanesulfonate). The dependency of flux rate of uptake into freshly isolated hepatocytes on the concentration of 7,7-ASLCT in presence of Na+ (143 mM) and with Na+ depletion (1 mM) is best described by the assumption of two simple transport systems, the kinetic parameters of which are similar to those of SLCT. As studied in the presence of Na+, 7,7-ASLCT and SLCT exhibit a clearly competitive cross-inhibition of uptake with inhibition constants that are similar to the corresponding half-saturation constants. Photoaffinity labeling of isolated hepatocytes using 7,7-ASLCT (400 microM) resulted in the irreversible inhibition of the uptake of 7,7-ASLCT and SLCT to similar extents, confirming the kinetic data that 7,7-ASLCT is a true competing substrate for the uptake of SLCT. Because in intact rat liver 7,7-ASLCT and SLCT mutually inhibit their biliary excretion, the photolabile derivative shares with SLCT the same pathways in uptake and in excretion. Thus, 7,7-ASLCT should be appropriate for the study of hepatobiliary transport of sulfated and taurine-conjugated bile salts by photoaffinity labeling.

Zonal changes of hepatobiliary taurocholate transport in intrahepatic cholestasis induced by 17 alpha-ethinyl estradiol: a histoautoradiographic study in rats.

The liver has a great reserve capacity for hepatobiliary bile salt transport. This study was performed to elucidate the significance of this capacity in ethinyl estradiol-induced cholestasis by direct visualization of the zonal involvement in taurocholate transport. The acinar distribution of [3H]taurocholate was determined by histoautoradiographical study of cryopreserved liver slices in normal rats and rats treated with ethinyl estradiol for 5 days. Silver grain densities over the different acinar zones were estimated on digitized image analysis. In control animals, histoautoradiographical study performed 4 min after the start of perfusion showed restriction of taurocholate to acinar zone 1. In contrast, in ethinyl estradiol-treated animals, taurocholate was also found in zone 2 and, in smaller concentrations, in zone 3. In control animals, the relative blackenings by silver grains of acinar zones 1, 2 and 3 were 66% +/- 1.2%, 25% +/- 1.6% and 5% +/- 0.6%, respectively. After 5 days of ethinyl estradiol treatment, blackenings were 58% +/- 1.5%, 36% +/- 2.1% and 12% +/- 0.8%, respectively. As early as 15 sec after injection of [3H]taurocholate, the bile canalicular areas of the cell plates and the bile ductules of ethinyl estradiol-treated animals were labeled as intensely as those of control animals. Our results demonstrate ethinyl estradiol-induced recruitment of the acinar zones 2 and 3 for hepatobiliary taurocholate transport. This recruitment may largely compensate for reduction of transport capacity of periportal hepatocytes in early cholestasis.

The acinar location of the sodium-independent and the sodium-dependent component of taurocholate uptake. A histoautoradiographic study of rat liver.

The acinar location of tritium-labelled taurocholate taken up from sodium-containing and sodium-free perfusion media in isolated perfused rat liver was made visible by means of histoautoradiography on cryoslices at low, medium and high bile salt concentrations. In antegrade perfusion studies, in the presence of sodium, with taurocholate concentrations of 1 and 20 microM, respectively, the silver grain label was mainly restricted to acinar zones 1. At an 80 microM concentration, zones 2 and 3 were also labelled but with decreasing intensities towards the terminal hepatic venules. At 120 microM taurocholate, all acinar zones were nearly equally labelled. In the absence of sodium, antegrade liver perfusions with 1, 20 and 120 microM taurocholate resulted in an almost homogenous labelling of all acinar zones and retrograde perfusions in a silver grain density slightly decreasing towards the terminal portal venules. The results indicate that hepatocytes of all acinar zones are capable of taking up taurocholate by both a sodium-dependent and a sodium-independent pathway. The contribution of the sodium-independent uptake to the overall uptake of taurocholate increases with increasing zonal recruitment at higher concentrations. The sodium-dependent uptake, however, is always dominant.

The histoautoradiographic localization of taurocholate in rat liver after bile duct ligation. Evidence for ongoing secretion and reabsorption processes.

To determine whether in complete obstructive cholestasis taurocholate is taken up by hepatocytes and if so whether it is secreted into bile, tritium-labelled taurocholate was localized by histoautoradiography on cryoslices from normal rat livers and from those after bile duct ligation. In non-cholestatic livers the hepatocytes of acinar zones 1 as well as the lumina and the epithelia of bile ductules and ducts became intensely labelled directly after injection of [3H]taurocholate into a mesenterial vein. Four hours and 4 days after bile duct ligation, hepatocytes of all three acinar zones became labelled, but in contrast to the normal state, pericanalicular concentration of silver grains was not observed, not even within 5 min. Fifteen days after bile duct obstruction, cryoslices taken 2 min after injection of [3H]taurocholate exhibited an intense silver grain labelling of all acinar zones, with the highest density at bile canalicular areas of the liver cell plates as well as the proliferated bile ductules and bile ducts. The biliary epithelium of small bile ductules and ducts of non-cholestatic and of bile duct-obstructed livers were also covered with silver grains; the epithelium of larger ducts exhibited significant labelling predominantly at the lateral sites of the cells. The biliary epithelium of the common bile duct was not significantly labelled. The results indicate that in complete obstructive cholestasis (a) taurocholate continues to be taken up from blood by hepatocytes and secreted into bile, but in terms of varying duration of obstruction, (b) all acinar zones are involved in bile salt transport, (c) in the initial phase (4 h and 4 days respectively after bile duct obstruction) hepatocytes fail to concentrate taurocholate at the canalicular site, (d) in a consecutive phase, in which bile ductules and ducts proliferate (demonstrated for a 15-day cholestasis), the taurocholate concentration at the canalicular site of hepatocytes is re-established and biliary secretion seems to be enhanced, (e) the biliary epithelium of bile ductules and ducts may play a significant role in the reabsorption and/or regurgitation of bile salts from bile to blood. Reabsorption/regurgitation of biliary constituents may also be operative in the non-cholestatic state but may become significantly enhanced with bile ductular proliferation.