Intermittent Fasting Primes the Tumor Microenvironment and Improves Nanomedicine Delivery in Hepatocellular Carcinoma.

Fasting has many health benefits, including reduced chemotherapy toxicity and improved efficacy. It is unclear how fasting affects the tumor microenvironment (TME) and tumor-targeted drug delivery. Here the effects of intermittent (IF) and short-term (STF) fasting are investigated on tumor growth, TME composition, and liposome delivery in allogeneic hepatocellular carcinoma (HCC) mouse models. To this end, mice are inoculated either subcutaneously or intrahepatically with Hep-55.1C cells and subjected to IF for 24 d or to STF for 1 d. IF but not STF significantly slows down tumor growth. IF increases tumor vascularization and decreases collagen density, resulting in improved liposome delivery. In vitro, fasting furthermore promotes the tumor cell uptake of liposomes. These results demonstrate that IF shapes the TME in HCC towards enhanced drug delivery. Finally, when combining IF with liposomal doxorubicin treatment, the antitumor efficacy of nanochemotherapy is found to be increased, while systemic side effects are reduced. Altogether, these findings exemplify that the beneficial effects of fasting on anticancer therapy outcomes go beyond modulating metabolism at the molecular level.

Long-term hypercaloric diet exacerbates metabolic liver disease in PNPLA3 I148M animals.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a major health burden associated with the metabolic syndrome leading to liver fibrosis, cirrhosis and ultimately liver cancer. In humans, the PNPLA3 I148M polymorphism of the phospholipase patatin-like phospholipid domain containing protein 3 (PNPLA3) has a well-documented impact on metabolic liver disease. In this study, we used a mouse model mimicking the human PNPLA3 I148M polymorphism in a long-term high fat diet (HFD) experiment to better define its role for NAFLD progression. METHODS: Male mice bearing wild-type Pnpla3 (Pnpla3WT ), or the human polymorphism PNPLA3 I148M (Pnpla3148M/M ) were subjected to HFD feeding for 24 and 52 weeks. Further analysis concerning basic phenotype, inflammation, proliferation and cell death, fibrosis and microbiota were performed in each time point. RESULTS: After 52 weeks HFD Pnpla3148M/M animals had more liver fibrosis, enhanced numbers of inflammatory cells as well as increased Kupffer cell activity. Increased hepatocyte cell turnover and ductular proliferation were evident in HFD Pnpla3148M/M livers. Microbiome diversity was decreased after HFD feeding, changes were influenced by HFD feeding (36%) and the PNPLA3 I148M genotype (12%). Pnpla3148M/M mice had more faecal bile acids. RNA-sequencing of liver tissue defined an HFD-associated signature, and a Pnpla3148M/M specific pattern, which suggests Kupffer cell and monocytes-derived macrophages as significant drivers of liver disease progression in Pnpla3148M/M animals. CONCLUSION: With long-term HFD feeding, mice with the PNPLA3 I148M genotype show exacerbated NAFLD. This finding is linked to PNPLA3 I148M-specific changes in microbiota composition and liver gene expression showing a stronger inflammatory response leading to enhanced liver fibrosis progression.

The oral-gut axis: Salivary and fecal microbiome dysbiosis in patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders that fall into two main categories: Crohn's disease (CD) and ulcerative colitis (UC). The gastrointestinal tract extends from the mouth to the anus and harbors diverse bacterial communities. Several sequencing-based studies have identified an intestinal enrichment of oral-associated bacteria and demonstrated their ability to induce intestinal inflammation in mice, suggesting that intestinal pathobionts originate from the oral cavity, particularly members of the genus Streptococcus. This study aimed to investigate the composition of the salivary and fecal microbiome of IBD patients (n = 14) compared to healthy controls (n = 12) and to determine the abundance of common bacterial taxa in both niches. Metagenomic DNA was extracted from saliva and fecal samples, and the 16S rRNA gene was targeted for sequencing. Our results revealed that the overall microbial composition of saliva was significantly altered in the IBD patients compared to the control subjects (p = 0.038). At the genus level, Veillonella and Prevotella were highly abundant in IBD (median: 25.4% and 22.2%, respectively) compared to the control group (17.9% and 13.4%, respectively). In contrast, Neisseria, Streptococcus, Haemophilus, and Fusobacterium were associated with a healthy gut state. Regarding the fecal microbiome, the IBD group had a significantly higher abundance of Clostridium sensu stricto 1 and Escherichia-Shigella (both comprising pathogenic bacteria) compared with the control group. Members of both bacterial groups have previously been shown to positively correlate with intestinal inflammation and high expression of pro-inflammatory cytokines that disrupt intestinal barrier integrity. In addition, we demonstrate that the increased abundance of Clostridium sensu stricto 1 and Escherichia-Shigella has also been associated with significant upregulation of certain metabolic pathways in the feces of the IBD group, including bacterial invasion of epithelial cells. Streptococcus was the only common genus detected in both the salivary and fecal microbiome and represented the oral-gut axis in our study. Using culture-based methods, we isolated 57 and 91 Streptococcus strains from saliva as well as 40 and 31 strains from fecal samples of the controls and IBD patients, respectively. The phylogenetic tree of streptococci based on sodA sequences revealed several patient-specific clusters comprising salivary and fecal streptococcal isolates from the same patient and belonging to the same species, suggesting that the oral cavity is an endogenous reservoir for intestinal strains.

Gut microbiota depletion exacerbates cholestatic liver injury via loss of FXR signalling.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology for which there are no approved therapeutic options. Patients with PSC display changes in gut microbiota and in bile acid (BA) composition; however, the contribution of these alterations to disease pathogenesis remains controversial. Here we identify a role for microbiota-dependent changes in BA synthesis that modulates PSC pathophysiology. In a genetic mouse model of PSC, we show that loss of microbiota-mediated negative feedback control of BA synthesis results in increased hepatic BA concentrations, disruption of bile duct barrier function and, consequently, fatal liver injury. We further show that these changes are dependent on decreased BA signalling to the farnesoid X receptor, which modulates the activity of the rate-limiting enzyme in BA synthesis, CYP7A1. Moreover, patients with advanced stages of PSC show suppressed BA synthesis as measured by serum C4 levels, which is associated with poor disease prognosis. Our preclinical data highlight the microbiota-dependent dynamics of BA metabolism in cholestatic liver disease, which could be important for future therapies targeting BA and gut microbiome interactions, and identify C4 as a potential biomarker to functionally stratify patients with PSC and predict disease outcomes.

Obesity/Type 2 Diabetes-Associated Liver Tumors Are Sensitive to Cyclin D1 Deficiency.

Type 2 diabetes, which is mainly linked to obesity, is associated with increased incidence of liver cancer. We have previously found that in various models of obesity/diabetes, hyperinsulinemia maintains heightened hepatic expression of cyclin D1, suggesting a plausible mechanism linking diabetes and liver cancer progression. Here we show that cyclin D1 is greatly elevated in human livers with diabetes and is among the most significantly upregulated genes in obese/diabetic liver tumors. Liver-specific cyclin D1 deficiency protected obese/diabetic mice against hepatic tumorigenesis, whereas lean/nondiabetic mice developed tumors irrespective of cyclin D1 status. Cyclin D1 dependency positively correlated with liver cancer sensitivity to palbociclib, an FDA-approved CDK4 inhibitor, which was effective in treating orthotopic liver tumors under obese/diabetic conditions. The antidiabetic drug metformin suppressed insulin-induced hepatic cyclin D1 expression and protected against obese/diabetic hepatocarcinogenesis. These results indicate that the cyclin D1-CDK4 complex represents a potential selective therapeutic vulnerability for liver tumors in obese/diabetic patients. SIGNIFICANCE: Obesity/diabetes-associated liver tumors are specifically vulnerable to cyclin D1 deficiency and CDK4 inhibition, suggesting that the obese/diabetic environment confers cancer-selective dependencies that can be therapeutically exploited.

Loss of c-Jun N-terminal Kinase 1 and 2 Function in Liver Epithelial Cells Triggers Biliary Hyperproliferation Resembling Cholangiocarcinoma.

Targeted inhibition of the c-Jun N-terminal kinases (JNKs) has shown therapeutic potential in intrahepatic cholangiocarcinoma (CCA)-related tumorigenesis. However, the cell-type-specific role and mechanisms triggered by JNK in liver parenchymal cells during CCA remain largely unknown. Here, we aimed to investigate the relevance of JNK1 and JNK2 function in hepatocytes in two different models of experimental carcinogenesis, the dethylnitrosamine (DEN) model and in nuclear factor kappa B essential modulator (NEMO)hepatocyte-specific knockout (Δhepa) mice, focusing on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development. Moreover, regulation of essential genes was assessed by reverse transcription polymerase chain reaction, immunoblottings, and immunostainings. Additionally, specific Jnk2 inhibition in hepatocytes of NEMOΔhepa/JNK1Δhepa mice was performed using small interfering (si) RNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors. Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocellular carcinoma (HCC) in both the DEN model and in NEMOΔhepa mice but in contrast caused massive proliferation of the biliary ducts. Indeed, Jnk1/2 deficiency in hepatocytes of NEMOΔhepa (NEMOΔhepa/JNKΔhepa) animals caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression but reduced HCC. Furthermore, siJnk2 treatment in NEMOΔhepa/JNK1Δhepa mice recapitulated the phenotype of NEMOΔhepa/JNKΔhepa mice. Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NEMOΔhepa mice. We found that NEMOΔhepa/JNKΔhepa livers exhibited overexpression of the interleukin-6/signal transducer and activator of transcription 3 pathway in addition to epidermal growth factor receptor (EGFR)-rapidly accelerated fibrosarcoma (Raf)-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade. The functional relevance was tested by administering lapatinib, which is a dual tyrosine kinase inhibitor of erythroblastic oncogene B-2 (ErbB2) and EGFR signaling, to NEMOΔhepa/JNKΔhepa mice. Lapatinib effectively inhibited cystogenesis, improved transaminases, and effectively blocked EGFR-Raf-MEK-ERK signaling. Conclusion: We define a novel function of JNK1/2 in cholangiocyte hyperproliferation. This opens new therapeutic avenues devised to inhibit pathways of cholangiocarcinogenesis.

Influence of Liver Fibrosis on Lobular Zonation.

Little is known about how liver fibrosis influences lobular zonation. To address this question, we used three mouse models of liver fibrosis, repeated CCl4 administration for 2, 6 and 12 months to induce pericentral damage, as well as bile duct ligation (21 days) and mdr2-/- mice to study periportal fibrosis. Analyses were performed by RNA-sequencing, immunostaining of zonated proteins and image analysis. RNA-sequencing demonstrated a significant enrichment of pericentral genes among genes downregulated by CCl4; vice versa, periportal genes were enriched among the upregulated genes. Immunostaining showed an almost complete loss of pericentral proteins, such as cytochrome P450 enzymes and glutamine synthetase, while periportal proteins, such as arginase 1 and CPS1 became expressed also in pericentral hepatocytes. This pattern of fibrosis-associated 'periportalization' was consistently observed in all three mouse models and led to complete resistance to hepatotoxic doses of acetaminophen (200 mg/kg). Characterization of the expression response identified the inflammatory pathways TGFß, NFκB, TNFα, and transcription factors NFKb1, Stat1, Hif1a, Trp53, and Atf1 among those activated, while estrogen-associated pathways, Hnf4a and Hnf1a, were decreased. In conclusion, liver fibrosis leads to strong alterations of lobular zonation, where the pericentral region adopts periportal features. Beside adverse consequences, periportalization supports adaptation to repeated doses of hepatotoxic compounds.

Intestinal dysbiosis augments liver disease progression via NLRP3 in a murine model of primary sclerosing cholangitis.

OBJECTIVE: There is a striking association between human cholestatic liver disease (CLD) and inflammatory bowel disease. However, the functional implications for intestinal microbiota and inflammasome-mediated innate immune response in CLD remain elusive. Here we investigated the functional role of gut-liver crosstalk for CLD in the murine Mdr2 knockout (Mdr2-/-) model resembling human primary sclerosing cholangitis (PSC). DESIGN: Male Mdr2-/-, Mdr2-/- crossed with hepatocyte-specific deletion of caspase-8 (Mdr2-/- /Casp8∆hepa) and wild-type (WT) control mice were housed for 8 or 52 weeks, respectively, to characterise the impact of Mdr2 deletion on liver and gut including bile acid and microbiota profiling. To block caspase activation, a pan-caspase inhibitor (IDN-7314) was administered. Finally, the functional role of Mdr2-/- -associated intestinal dysbiosis was studied by microbiota transfer experiments. RESULTS: Mdr2-/- mice displayed an unfavourable intestinal microbiota signature and pronounced NLRP3 inflammasome activation within the gut-liver axis. Intestinal dysbiosis in Mdr2-/- mice prompted intestinal barrier dysfunction and increased bacterial translocation amplifying the hepatic NLRP3-mediated innate immune response. Transfer of Mdr2-/- microbiota into healthy WT control mice induced significant liver injury in recipient mice, highlighting the causal role of intestinal dysbiosis for disease progression. Strikingly, IDN-7314 dampened inflammasome activation, ameliorated liver injury, reversed serum bile acid profile and cholestasis-associated microbiota signature. CONCLUSIONS: MDR2-associated cholestasis triggers intestinal dysbiosis. In turn, translocation of endotoxin into the portal vein and subsequent NLRP3 inflammasome activation contribute to higher liver injury. This process does not essentially depend on caspase-8 in hepatocytes, but can be blocked by IDN-7314.

Disruption of the FasL/Fas axis protects against inflammation-derived tumorigenesis in chronic liver disease.

Fas Ligand (FasL) and Fas (APO-1/CD95) are members of the TNFR superfamily and may trigger apoptosis. Here, we aimed to elucidate the functional role of Fas signaling in an experimental model of chronic liver disease, the hepatocyte-specific NEMO knockout (NEMOΔhepa) mice. We generated NEMOΔhepa /Faslpr mice, while NEMOΔhepa, NEMOf/f as well as Faslpranimals were used as controls, and characterized their phenotype during liver disease progression. Liver damage was evaluated by serum transaminases, histological, immunofluorescence procedures, and biochemical and molecular biology techniques. Proteins were detected by western Blot, expression of mRNA by RT-PCR, and infiltration of inflammatory cells was determined by FACs analysis, respectively. Faslpr mutation in NEMOΔhepa mice resulted in overall decreased liver injury, enhanced hepatocyte survival, and reduced proliferation at 8 weeks of age compared with NEMOΔhepa mice. Moreover, NEMOΔhepa/Faslpr animals elicited significantly decreased parameters of liver fibrosis, such as Collagen IA1, MMP2, and TIMP1, and reduced proinflammatory macrophages and cytokine expression. At 52 weeks of age, NEMOΔhepa/Faslpr exhibited less malignant growth as evidenced by reduced HCC burden associated with a significantly decreased number of nodules and LW/BW ratio and decreased myeloid populations. Deletion of TNFR1 further reduced tumor load of 52-weeks-old NEMOΔhepa/Faslpr mice. The functionality of FasL/Fas might affect inflammation-driven tumorigenesis in an experimental model of chronic liver disease. These results help to develop alternative therapeutic approaches and extend the limitations of tumor therapy against HCC.

Inactivation of caspase 8 in liver parenchymal cells confers protection against murine obstructive cholestasis.

BACKGROUND & AIMS: Caspase 8 (CASP8) is the apical initiator caspase in death receptor-mediated apoptosis. Strong evidence for a link between death receptor signaling pathways and cholestasis has recently emerged. Herein, we investigated the role of CASP8-dependent and independent pathways during experimental cholestasis. METHODS: Liver injury was characterized in a cohort of human sera (n = 28) and biopsies from patients with stage IV primary biliary cholangitis. In parallel, mice with either specific deletion of Casp8 in liver parenchymal cells (Casp8Δhepa) or hepatocytes (Casp8Δhep), and mice with constitutive Ripk3 (Ripk3-/-) deletion, were subjected to surgical ligation of the common bile duct (BDL) from 2 to 28 days. Floxed (Casp8fl/fl) and Ripk3+/+ mice were used as controls. Moreover, the pan-caspase inhibitor IDN-7314 was used, and cell death mechanisms were studied in primary isolated hepatocytes. RESULTS: Overexpression of activated caspase 3, CASP8 and RIPK3 was characteristic of liver explants from patients with primary biliary cholangitis. Twenty-eight days after BDL, Casp8Δhepamice showed decreased necrotic foci, serum aminotransferase levels and apoptosis along with diminished compensatory proliferation and ductular reaction. These results correlated with a decreased inflammatory profile and ameliorated liver fibrogenesis. A similar phenotype was observed in Ripk3-/- mice. IDN-7314 treatment decreased CASP8 levels but failed to prevent BDL-induced cholestasis, independently of CASP8 in hepatocytes. CONCLUSION: These findings show that intervention against CASP8 in liver parenchymal cells - specifically in cholangiocytes - might be a beneficial option for treating obstructive cholestasis, while broad pan-caspase inhibition might trigger undesirable side effects. LAY SUMMARY: Loss of caspase 8 - a protein involved in programmed cell death - in liver parenchymal cells protects against experimental cholestasis. Therefore, specific pharmacological intervention against caspase 8 might be a valid alternative for the treatment of obstructive cholestasis in the clinic, whereas broad pan-caspase inhibiting drugs might trigger undesirable side effects.

Insulin regulation of gluconeogenesis.

The coordinated regulation between cellular glucose uptake and endogenous glucose production is indispensable for the maintenance of constant blood glucose concentrations. The liver contributes significantly to this process by altering the levels of hepatic glucose release, through controlling the processes of de novo glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis). Various nutritional and hormonal stimuli signal to alter hepatic gluconeogenic flux, and suppression of this metabolic pathway during the postprandial state can, to a significant extent, be attributed to insulin. Here, we review some of the molecular mechanisms through which insulin modulates hepatic gluconeogenesis, thus controlling glucose production by the liver to ultimately maintain normoglycemia. Various signaling pathways governed by insulin converge at the level of transcriptional regulation of the key hepatic gluconeogenic genes PCK1 and G6PC, highlighting this as one of the focal mechanisms through which gluconeogenesis is modulated. In individuals with compromised insulin signaling, such as insulin resistance in type 2 diabetes, insulin fails to suppress hepatic gluconeogenesis, even in the fed state; hence, an insight into these insulin-moderated pathways is critical for therapeutic purposes.

ERRα Maintains Mitochondrial Oxidative Metabolism and Constitutes an Actionable Target in PGC1α-Elevated Melanomas.

The uncontrolled growth of tumors provides metabolic dependencies that can be harnessed for therapeutic benefit. Although tumor cells exhibit these increased metabolic demands due to their rapid proliferation, these metabolic processes are general to all cells, and furthermore, targeted therapeutic intervention can provoke compensatory adaptation that alters tumors' characteristics. As an example, a subset of melanomas depends on the transcriptional coactivator PGC1α function to sustain their mitochondrial energy-dependent survival. However, selective outgrowth of resistant PGC1α-independent tumor cells becomes endowed with an augmented metastatic phenotype. To find PGC1α-dependent components that would not affect metastasis in melanomas, an unbiased proteomic analyses was performed and uncovered the orphan nuclear receptor ERRα, which supports PGC1α's control of mitochondrial energetic metabolism, but does not affect the antioxidant nor antimetastatic regulatory roles. Specifically, genetic or pharmacologic inhibition of ERRα reduces the inherent bioenergetic capacity and decreases melanoma cell growth, but without altering the invasive characteristics. Thus, within this particularly aggressive subset of melanomas, which is characterized by heighted expression of PGC1α, ERRα specifically mediates prosurvival functions and represents a tangible therapeutic target.Implications: ERRα, a druggable protein, mediates the bioenergetic effects in melanomas defined by high PGC1α expression, suggesting a rational means for therapeutic targeting of this particularly aggressive melanoma subtype. Mol Cancer Res; 15(10); 1366-75. ©2017 AACR.

Adipose Tissue CLK2 Promotes Energy Expenditure during High-Fat Diet Intermittent Fasting.

A promising approach to treating obesity is to increase diet-induced thermogenesis in brown adipose tissue (BAT), but the regulation of this process remains unclear. Here we find that CDC-like kinase 2 (CLK2) is expressed in BAT and upregulated upon refeeding. Mice lacking CLK2 in adipose tissue exhibit exacerbated obesity and decreased energy expenditure during high-fat diet intermittent fasting. Additionally, tissue oxygen consumption and protein levels of UCP1 are reduced in CLK2-deficient BAT. Phosphorylation of CREB, a transcriptional activator of UCP1, is markedly decreased in BAT cells lacking CLK2 due to enhanced CREB dephosphorylation. Mechanistically, CREB dephosphorylation is rescued by the inhibition of PP2A, a phosphatase that targets CREB. Our results suggest that CLK2 is a regulatory component of diet-induced thermogenesis in BAT through increased CREB-dependent expression of UCP1.

Brown Adipose YY1 Deficiency Activates Expression of Secreted Proteins Linked to Energy Expenditure and Prevents Diet-Induced Obesity.

Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight.

Haematopoietic cell-derived Jnk1 is crucial for chronic inflammation and carcinogenesis in an experimental model of liver injury.

BACKGROUND & AIMS: Chronic liver injury triggers complications such as liver fibrosis and hepatocellular carcinoma (HCC), which are associated with alterations in distinct signalling pathways. Of particular interest is the interaction between mechanisms controlled by IKKγ/NEMO, the regulatory IKK subunit, and Jnk activation for directing cell death and survival. In the present study, we aimed to define the relevance of Jnk in hepatocyte-specific NEMO knockout mice (NEMO(Δhepa)), a genetic model of chronic inflammatory liver injury. METHODS: We generated Jnk1(-/-)/NEMO(Δhepa) and Jnk2(-/-)/NEMO(Δhepa) mice by crossing NEMO(Δhepa) mice with Jnk1 and Jnk2 global deficient animals, respectively, and examined the progression of chronic liver disease. Moreover, we investigated the expression of Jnk during acute liver injury, evaluated the role of Jnk1 in bone marrow-derived cells, and analysed the expression of NEMO and p-JNK in human diseased-livers. RESULTS: Deletion of Jnk1 significantly aggravated the progression of liver disease, exacerbating apoptosis, compensatory proliferation and carcinogenesis in NEMO(Δhepa) mice. Conversely, Jnk2(-/-)/NEMO(Δhepa) displayed hepatic inflammation. By using bone marrow transfer, we observed that Jnk1 in haematopoietic cells had an impact on the progression of chronic liver disease in NEMO(Δhepa) livers. These findings are of clinical relevance since NEMO expression was downregulated in hepatocytes of patients with HCC whereas NEMO and p-JNK were expressed in a large amount of infiltrating cells. CONCLUSIONS: A synergistic function of Jnk1 in haematopoietic cells and hepatocytes might be relevant for the development of chronic liver injury. These results elucidate the complex function of Jnk in chronic inflammatory liver disease.

Hepatocyte specific deletion of c-Met leads to the development of severe non-alcoholic steatohepatitis in mice.

BACKGROUND & AIMS: Non-alcoholic-fatty-liver disease (NAFLD) is part of the metabolic syndrome. The spectrum of NAFLD includes NASH (non-alcoholic steatohepatitis), which is characterised by progressive inflammation associated with oxidative stress and apoptosis, finally triggering liver cirrhosis and hepatocellular carcinoma. HGF (hepatocyte growth factor)/mesenchymal-epithelial transition factor (c-Met) receptor signalling is known to activate distinct intracellular pathways mediating among others anti-apoptotic properties to hepatocytes. Therefore, the aim was to characterise the role of c-Met during NASH development. METHODS: Hepatocyte specific c-Met knockout mice (c-MetΔ(hepa)) using the cre-loxP system and wild type controls (c-Met(loxP/loxP)) were fed a methionine-choline deficient (MCD) diet. RESULTS: MCD feeding triggered massive steatosis, decreased survival and higher transaminases in c-MetΔ(hepa) livers compared to c-Met(loxP/loxP). Gene array analysis demonstrated that genes involved in fatty acid metabolism were strongly upregulated in c-MetΔ(hepa) livers correlating with higher amounts of hepatic free fatty acids. Consequently, c-MetΔ(hepa) mice showed significantly more TUNEL positive cells and more superoxide anion production than c-Met(loxPloxP) animals. Additionally, c-MetΔ(hepa) livers showed significantly larger fractions of infiltrating neutrophils, macrophages, and cytotoxic T cells. These changes correlated with an enhanced progression of liver fibrosis as evidenced by higher collagen deposition in c-MetΔ(hepa) livers. As increased apoptosis was a prominent feature in c-MetΔ(hepa) livers, we generated c-Met/Casp8Δ(hepa) double knockout mice. In these animals compared to c-MetΔ(hepa) animals the increase in apoptosis could be reverted. CONCLUSIONS: c-Met deletion in hepatocytes triggers NASH progression. A prominent mechanism is higher fatty acid accumulation and increased apoptosis, which in part can be reverted by blocking caspase 8.

Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis.

OBJECTIVE: The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. DESIGN: Jnk1(f/f) wildtype (WT), Jnk1(-/-) and Jnk1(Δhepa) (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. RESULTS: Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1(-/-) compared with WT and Jnk1(Δhepa) mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and α-smooth muscle actin expression were downregulated in Jnk1(-/-) compared with WT and Jnk1(Δhepa) livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow-derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1(-/-) livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. CONCLUSIONS: Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.

Hepatocyte caspase-8 is an essential modulator of steatohepatitis in rodents.

UNLABELLED: In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death-receptor-mediated apoptosis activity and therefore its modulation might be critical for the pathogenesis of NASH. The aim was to dissect the role of hepatocyte Casp8 in a murine model of steatohepatitis. We generated hepatocyte-specific Casp8 knockout (Casp8(Δhep) ) mice. Animals were fed with a methionine-choline-deficient (MCD) diet. Liver injury was assessed by histopathological analysis, apoptotic death, serum alanine aminotransferase (ALT), fluorescent-activated cell sorter (FACS), analysis of liver infiltration and inflammation, reactive oxygen species (ROS), and liver fibrosis. MCD feeding triggered steatosis, hepatic lipid storage, and accumulation of free fatty acid (FFA) in wildtype (WT) livers, which were significantly reduced in Casp8(Δhep) animals. Additionally, lack of Casp8 expression in hepatocytes reduced the MCD-dependent increase in apoptosis and decreased expression of proinflammatory cytokines as well as hepatic infiltration. As a consequence, ROS production was lower, leading to a reduction in the progression of liver fibrosis in Casp8(Δhep) livers. CONCLUSION: Selective ablation of Casp8 in hepatocytes ameliorates development of NASH by modulating liver injury. Casp8-directed therapy might be a plausible treatment for patients with steatohepatitis. (HEPATOLOGY 2013;57:2189-2201).

Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury.

BACKGROUND & AIMS: Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury. METHODS: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.