Jörn M. Schattenberg

Jnk1 but not jnk2 promotes the development of steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and varying degrees of necroinflammation. Although chronic oxidative stress, inflammatory cytokines, and insulin resistance have been implicated in the pathogenesis of NAFLD, the mechanisms that underlie the initiation and progression of this disease remain unknown. c‐Jun N‐terminal kinase (JNK) is activated by oxidants and cytokines and regulates hepatocellular injury and insulin resistance, suggesting that this kinase may mediate the development of steatohepatitis. The presence and function of JNK activation were therefore examined in the murine methionine‐ and choline‐deficient (MCD) diet model of steatohepatitis. Activation of hepatic JNK, c‐Jun, and AP‐1 signaling occurred in parallel with the development of steatohepatitis in MCD diet–fed mice. Investigations in jnk1 and jnk2 knockout mice demonstrated that jnk1, but not jnk2, was critical for MCD diet–induced JNK activation. JNK promoted the development of steatohepatitis as MCD diet–fed jnk1 null mice had significantly reduced levels of hepatic triglyceride accumulation, inflammation, lipid peroxidation, liver injury, and apoptosis compared with wild‐type and jnk2 −/− mice. Ablation of jnk1 led to an increase in serum adiponectin but had no effect on serum levels of tumor necrosis factor‐α. In conclusion, JNK1 is responsible for JNK activation that promotes the development of steatohepatitis in the MCD diet model. These findings also provide additional support for the critical mechanistic involvement of JNK1 overactivation in conditions associated with insulin resistance and the metabolic syndrome. (HEPATOLOGY 2006;43:163–172.)

Hepatocyte CYP2E1 Overexpression and Steatohepatitis Lead to Impaired Hepatic Insulin Signaling

Insulin resistance and increased cytochrome P450 2E1 (CYP2E1) expression are both associated with and mechanistically implicated in the development of nonalcoholic fatty liver disease. Although currently viewed as distinct factors, insulin resistance and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signaling. To test this possibility, the effects of in vitro and in vivo CYP2E1 overexpression on hepatocyte insulin signaling were examined. CYP2E1 overexpression in a hepatocyte cell line decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in response to insulin. CYP2E1 overexpression was also associated with increased inhibitory serine 307 and 636/639 IRS-1 phosphorylation. In parallel, the effects of insulin on Akt activation, glycogen synthase kinase 3, and FoxO1a phosphorylation, and glucose secretion were all significantly decreased in CYP2E1 overexpressing cells. This inhibition of insulin signaling by CYP2E1 overexpression was partially c-Jun N-terminal kinase dependent. In the methionine- and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression, insulin-induced IRS-1, IRS-2, and Akt phosphorylation were similarly decreased. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signaling, potentially contributing to the insulin resistance associated with nonalcoholic fatty liver disease.

Non-alcoholic steatohepatitis: pathogenesis and novel therapeutic approaches.

Non-alcoholic fatty liver disease (NAFLD) refers to a disease spectrum, ranging from mere hepatic steatosis to hepatic necroinflammation (NASH, non-alcoholic steatohepatitis). NASH often leads to fibrosis, which can progress to cirrhosis with a high risk of liver failure and hepatocellular carcinoma. The course of NAFLD is highly variable, and only a minority of patients (2-3%) progress to end-stage liver disease. However, due to a dramatic increase of the risk factors for NAFLD, that is obesity and insulin resistance/type 2 diabetes, that affect 15-30% and 7-15% of subjects, in most industrialized countries, respectively, NAFLD has become the most frequent liver disease and is even considered a pace setter of the metabolic syndrome. Sedentary lifestyle, modern Western nutrition, and genetic predispositions have been identified as major causes of NAFLD. These lead to liver injury via insulin resistance and an excess of free fatty acids in hepatocytes, resulting in oxidant stress and lipotoxicity that promote the activation of intracellular stress kinases and apoptosis or necroapoptosis (NASH). The damaged hepatocytes directly trigger inflammation and fibrogenesis, but can also lead to the emergence of fibrogenic progenitor cells. Moreover, NASH is linked to inflammation in peripheral adipose tissues that involves mainly macrophages and humoral factors, such as adipokines and cytokines. The most efficient treatment is by weight loss and exercise, but (adjunctive) pharmacological strategies are urgently needed. Here, we highlight the aspects of NAFLD epidemiology and pathophysiology that are beginning to lead to novel pharmacological approaches to address this growing health-care challenge.

Animal Models of Non-Alcoholic Steatohepatitis: Of Mice and Man

The epidemic occurrence of obesity has led to a rapid increase in the incidence of non-alcoholic fatty liver disease (NAFLD) in industrial countries. The disease spectrum includes hepatic steatosis, lobular inflammation with steatohepatitis (NASH) and varying degrees of liver fibrosis, which can progress to cirrhosis. Hepatocellular carcinoma can develop in patients with NASH, even in the absence of cirrhosis. The majority of patients with primary NASH exhibit risk factors that define the metabolic syndrome including insulin resistance and visceral obesity. However, only a minority of patients with NAFLD progress to end-stage liver disease and, so far, predictors to identify these patients are not available. The course of disease progression appears to be slow and develops progressively over years, modulated by genetic susceptibility, nutritional misbehavior and environmental factors. Although risk factors have been identified in epidemiological studies, little is known about disease initiation and progression. This review summarizes the existing animal models of NAFLD, focusing on genetic and dietary models, and discusses their applicability in studying signaling events involved in steatohepatitis. Despite the shortcomings inherent to all experimental models, research in this field has helped to identify potential therapeutic targets and, thus, contributed significantly to our understanding of this disease. The validation and search for new in vivo and in vitro models will propagate the understanding of NASH and help clinicians to develop new treatment modalities.

Apoptosis in liver disease.

Abstract: The description of the morphological hallmarks of programmed cell death, apoptosis, in 1972 by Kerr, Wyllie and Currie started a field of research that revolutionized our understanding of cellular proliferation, tissue homeostasis and pathophysiology of many diseases. In the following years, a series of proteins involved in signaling and intracellular death pathways were identified and 30 years later the Noble Prize for physiology and medicine was awarded to S. Brenner, H. R. Horvitz and J. E. Sulston for their discoveries related to describing the mechanisms of cell death (apoptosis). The delineation of the signaling pathways that mediate apoptosis changed the paradigms of understanding in many liver diseases. The most detailed analyzed mode of apoptosis involves a cell surface‐based receptor–ligand system. Death receptors are typically members of the tumor necrosis factor‐receptor superfamily and comprise an intracellular death domain. Following ligand binding to the receptor, intracellular adapter molecules are recruited to the receptor and subsequently transmit the apoptotic signal. Intracellular organelle‐dependent signaling occurs, and effector molecules then augment the receptor‐initiated apoptosis process. Cell death and degradation follows the activation of a highly regulated set of cytosolic and nuclear proteases and DNAses. Receptor‐independent activation of the apoptotic process can occur as part of the cytotoxicity related to UV radiation, chemotherapeuticals or other DNA‐damaging agents through activation of intracellular sensors of cellular integrity, e.g. the tumor suppressor gene p53. In contrast to necrosis, apoptosis is not commonly accompanied by an inflammatory response that causes collateral cell damage. The apoptotic program is highly effective in eliciting cell death and thus must be tightly controlled. This is achieved through continuous integration of pro‐ and antiapoptotic signals at the individual cell level. Dysregulation of the apoptotic process, resulting in too much or too little cell death, has potentially devastating effects and has been implicated in many forms of liver disease like acute liver failure or hepatocellular carcinoma. This review will focus initially on recent progress in signaling events of hepatocellular apoptosis and subsequently discuss the consequences for the hepatic pathophysiology that involves disarrangement of hepatocellular apoptosis.

Cell death and hepatocarcinogenesis: Dysregulation of apoptosis signaling pathways

Hepatocellular carcinoma (HCC) remains a disease with a poor prognosis despite recent advances in the pathophysiology and treatment. Although the disease is biologically heterogeneous, dysregulation of cellular proliferation and apoptosis both occur frequently and contribute to the malignant phenotype. Chronic liver disease is associated with intrahepatic inflammation which promotes dysregulation of cellular signaling pathways; this triggers proliferation and thus lays the ground for expansion of premalignant cells. Cancer emerges when immunological control fails and transformed cells develop resistance against cell death signaling pathways. The same mechanisms underlie the poor responsiveness of HCC towards chemotherapy. Only recently advances in understanding the signaling pathways involved has led to the development of an effective pharmacological therapy for advanced disease. The current review will discuss apoptosis signaling pathways and focus on apoptosis resistance of HCC involving derangements in cell death receptors (e.g. tumor necrosis factor‐alpha [TNF], CD95/Apo‐1, TNF‐related apoptosis‐inducing ligand [TRAIL]) and associated adapter molecules (e.g. FADD and FLIP) of apoptotic signaling pathways. In addition, the role of the transcription factor nuclear factor‐kappaB (NFκB) and members of the B cell leukemia‐2 (Bcl‐2) family that contribute to the regulation of apoptosis in hepatocytes are discussed. Eventually, the delineation of cell death signaling pathways could contribute to the implementation of new therapeutic strategies to treat HCC.

Nonalcoholic steatohepatitis: the therapeutic challenge of a global epidemic.

Purpose of review Nonalcoholic fatty liver (NAFL) and especially its inflammatory variant nonalcoholic steatohepatitis (NASH) have become a major challenge to healthcare systems worldwide because of the increasing prevalence of its major risk factors obesity and type 2 diabetes, which are closely linked to overeating, physical inactivity, and the metabolic syndrome. Recent findings Between 10 and 20% of patients with NAFL develop NASH, which can progress to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. The overall mortality in these patients is significantly increased because of both cardiovascular and liver-related complications. Sustained weight loss by diet and exercise, which is the most effective therapeutic measure, is only achieved by a minority of patients, having led to a great yet unmet need for medical therapies of NASH. Summary Pharmacological therapies should target the underlying pathophysiology that involves insulin resistance, enhanced peripheral lipolysis and release of free fatty acids, oxidative stress, accumulation of toxic lipids, adipose tissue inflammation, sensitization of hepatocytes toward apoptotic cell death, and fibrogenesis. However, pharmacological therapy that is well tolerated, cost-effective, and poses an acceptable risk-to-benefit ratio has still to be identified. This review summarizes the current and promising treatment options and their implications for future research and clinical practice.

Hepatocyte transplantation activates hepatic stellate cells with beneficial modulation of cell engraftment in the rat

We investigated whether transplanted hepatocytes interact with hepatic stellate cells, as cell–cell interactions could modulate their engraftment in the liver. We transplanted Fischer 344 rat hepatocytes into syngeneic dipeptidyl peptidase IV–deficient rats. Activation of hepatic stellate cells was analyzed by changes in gene expression, including desmin and α‐smooth muscle actin, matrix proteases and their inhibitors, growth factors, and other stellate cell‐associated genes with histological methods or polymerase chain reaction. Furthermore, the potential role of hepatic ischemia, Kupffer cells, and cytokine release in hepatic stellate cell activation was investigated. Hepatocyte transplantation activated desmin‐positive hepatic stellate cells, as well as Kupffer cells, including in proximity with transplanted cells. Inhibition of Kupffer cells by gadolinium chloride, blockade of tumor necrosis factor alpha (TNF‐α) activity with etanercept or attenuation of liver ischemia with nitroglycerin did not decrease this hepatic stellate cell perturbation. After cell transplantation, soluble signals capable of activating hepatic stellate cells were rapidly induced, along with early upregulated expression of matrix metalloproteinases‐2, ‐3, ‐9, ‐13, ‐14, and their inhibitors. Moreover, prior depletion of activated hepatic stellate cells with gliotoxin decreased transplanted cell engraftment. In conclusion, cell transplantation activated hepatic stellate cells, which, in turn, contributed to transplanted cell engraftment in the liver. Manipulation of hepatic stellate cells might provide new strategies to improve liver repopulation after enhanced transplanted cell engraftment. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:1072–1081.)

CYP2E1 overexpression alters hepatocyte death from menadione and fatty acids by activation of ERK1/2 signaling

Chronic oxidative stress induced by overexpression of the cytochrome P450 isoform 2E1 (CYP2E1) has been implicated in hepatocyte injury and death. However, the mechanism by which CYP2E1 overexpression may promote cell death is unknown. Acute oxidative stress activates mitogen‐activated protein kinases (MAPK), suggesting that chronic oxidant generation by CYP2E1 may regulate cellular responses through these signaling pathways. The effect of CYP2E1 overexpression on MAPK activation and their function in altering death responses of CYP2E1‐overexpressing hepatocytes were investigated. Chronic CYP2E1 overexpression led to increased extracellular signal‐regulated kinase 1/2 (ERK1/2) activation constitutively and in response to oxidant stress from the superoxide generator menadione. CYP2E1‐overexpressing cells were resistant to menadione toxicity through an ERK1/2‐dependent mechanism. Similar to menadione, the polyunsaturated fatty acid (PUFA) arachidonic acid (AA) induced an increased activation of ERK1/2 in hepatocytes that overexpressed CYP2E1. However, CYP2E1‐overexpressing cells were sensitized to necrotic death from AA and the PUFA γ‐linolenic acid, but not from saturated or monounsaturated fatty acids. Death from PUFA resulted from oxidative stress and was blocked by inhibition of ERK1/2, but not p38 MAPK or activator protein‐1 signaling. CYP2E1 expression induced ERK1/2 activation through increased epidermal growth factor receptor (EGFR)/c‐Raf signaling. Inhibition of EGFR signaling reversed CYP2E1‐induced resistance to menadione and sensitization to AA toxicity. In conclusion, chronic CYP2E1 overexpression leads to sustained ERK1/2 activation mediated by EGFR/c‐Raf signaling. This adaptive response in hepatocytes exposed to chronic oxidative stress confers differential effects on cellular survival, protecting against menadione‐induced apoptosis, but sensitizing to necrotic death from PUFA. (HEPATOLOGY 2004;39;444–445.)

Ablation of c-FLIP in hepatocytes enhances death-receptor mediated apoptosis and toxic liver injury in vivo.

BACKGROUND & AIMS Apoptosis is crucially involved in acute and chronic liver injury, including viral, cholestatic, toxic, and metabolic liver disease. Additionally, dysregulation of apoptosis signaling pathways has been implicated in hepatocarcinogenesis. The most prominent members of the apoptosis-mediating tumor necrosis factor receptor superfamily are the TNF-R1 (CD120a) and the CD95 (Apo-1/Fas) receptor. Although extensively studied, the intracellular signaling events in hepatocytes are only incompletely understood. METHODS To examine the role of the caspase-8 homolog cellular FLICE-inhibitory protein (c-FLIP) in liver injury, we generated mice with hepatocyte specific deletion of c-FLIP. Three models of acute liver injury were employed: the agonistic anti-CD95 antibody Jo2, d-galactosamine and LPS (GalN/LPS), and concanavalin A. RESULTS Conditional ablation of c-FLIP in hepatocytes augmented liver injury and cell death in all three models of liver injury. CD95- and GalN/LPS-induced liver injury was ameliorated by a pancaspase inhibitor, while ConA-induced injury was unaffected by caspase inhibition. Augmented activation of the MAPK JNK was observed in parallel to liver injury in c-FLIP knockout mice in all injury models; however, inhibition of JNK only affected TNF- and ConA-mediated injury. CONCLUSIONS In summary, c-FLIP is a central regulator of cell death in hepatocytes, involving increased activation of caspases and the MAPK JNK.