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.

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.With an estimated 350 million chronically infected people worldwide, hepatitis B virus (HBV) infection represents a major health care problem. Every year nearly 1 million individuals succumb to HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma (9, 18). Although an efficient and safe vaccine is available, HBV is spreading, especially in Asia and Africa (10). Treatment of chronic HBV infection is still unsatisfactory. At present, alpha interferon and lamivudine are the only therapeutic options available in clinical practice. However, alpha interferon yields a sustained suppression of viral replication in only about one-third of patients, and lamivudine resistance with mutations in the viral polymerase gene is a frequent event in lamivudine-treated patients (3, 7). Based on transfection studies of hepatoma cell lines, the mechanisms of HBV replication have been elucidated in great detail (11,13). By contrast, little is known about the early events of the viral life cycle. Unfortunately, permanent cell lines are not permissive to HBV infection, and primary human hepatocytes are not easily available for in vitro infection studies. Furthermore, the quality of liver tissue obtained at surgery for the preparation of primary human hepatocytes is highly variable (5). Therefore, alternative experimental systems for studying HBV infection are urgently needed.Two reports have described transient HBV infection of the Asian tree shrew, Tupaia belangeri, in vivo (17,20). Furthermore, successful infection of tupaias with human herpes simplex virus and hepatitis C virus has been described (2, 19). Tupaias are squirrel-like animals that are closely related to primates and are endemic to subtropical areas of southeast Asia (12). The animals are easily bred in captivity. In addition, cultures of primary tupaia hepatocytes (PTH) can be prepared using well-established liver perfusion protocols.We previously described HBV infection of PTH (17); infection efficiency was too low, h...

Endoplasmic reticulum (ER) stress due to accumulation of hepatoviral or misfolded proteins is increasingly recognized as an important step in the pathogenesis of inflammatory, toxic, and metabolic liver diseases. ER stress results in the activation of several intracellular signaling pathways including Jun N‐terminal kinase (JNK). The AP‐1 (activating protein 1) transcription factor c‐Jun is a prototypic JNK target and important regulator of hepatocyte survival, proliferation, and liver tumorigenesis. Because the functions of c‐Jun during the ER stress response are poorly understood, we addressed this issue in primary hepatocytes and livers of hepatocyte‐specific c‐Jun knockout mice. ER stress was induced pharmacologically in vitro and in vivo and resulted in a rapid and robust induction of c‐Jun protein expression. Interestingly, ER‐stressed hepatocytes lacking c‐Jun displayed massive cytoplasmic vacuolization due to ER distension. This phenotype correlated with exacerbated and sustained activation of canonical ER stress signaling pathways. Moreover, sustained ER stress in hepatocytes lacking c‐Jun resulted in increased cell damage and apoptosis. ER stress is also a strong inducer of macroautophagy, a cell‐protective mechanism of self‐degradation of cytoplasmic components and organelles. Interestingly, autophagosome numbers in response to ER stress were reduced in hepatocytes lacking c‐Jun. To further validate these findings, macroautophagy was inhibited chemically in ER‐stressed wildtype hepatocytes, which resulted in cytoplasmic vacuolization and increased cell damage closely resembling the phenotypes observed in c‐Jun‐deficient cells. Conclusion: Our findings indicate that c‐Jun protects hepatocytes against excessive activation of the ER stress response and subsequent cell death and provide evidence that c‐Jun functionally links ER stress responses and macroautophagy. (HEPATOLOGY 2012)