- Email: [email protected]
1293 GENDER DIMORPHISM OF FATTY LIVER DISEASE
POSTERS membranous). 17/24 (70.8%) of cases with stromal SULF2 were GPC3 positive in adjacent tumour cells. In addition, in five cases there was simultaneous SULF2 and GPC3 expression in tumour cells. Conclusions: In combination, these murine and human data implicate a role for SULF2 in the development of steatosis, but also in modulation of signalling between the matrix and hepatocytes in both NAFLD progression and HCC. 1291 COMPLEMENT ALTERNATIVE PATHWAY ACTIVATION IS ASSOCIATED WITH SEVERITY OF NONALCOHOLIC STEATOHEPATITIS F.M. Segers1 , F.J. Verdam1 , C. de Jonge1 , B. Boonen1 , N.D. Bouvy1 , J.W.M. Greve2 , W.A. Buurman1 , S.S. Rensen1 . 1 General Surgery, Maastricht University Medical Center, Maastricht, 2 General Surgery, Atrium Medical Center Parkstad, Heerlen, The Netherlands E-mail: [email protected] Background and Aims: Activation of the innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently, we showed complement activation via classical and lectin pathways in patients with NASH, which was associated with hepatic inflammation, neutrophil infiltration, and increased apoptosis. Here we investigated the role of the complement alternative pathway (AP) in NASH. Methods: Hepatic mRNA and protein expression of various complement components were investigated in liver biopsies of obese subjects with healthy livers (N = 10; NAS score =0) or with severe NASH (N = 12; NAS score >=5). Furthermore, immunohistochemistry for properdin and myeloperoxidase (MPO) was performed, and hepatocyte apoptosis was measured. Results: Although C3 mRNA and protein levels were decreased in subjects with NASH (1.0 vs. 0.5, p = 0.04), increased levels of activated C3 (C3c) were found (2.6-fold, p ≤ 0.01), which positively correlated with lobular inflammation as assessed by Kleiner classification (r2 =0.60, p ≤ 0.01). Hepatic properdin expression was not different between the groups (2.7 vs. 3.7, p = 0.3). However, properdin expression positively correlated with activated C3 levels (r2 =0.69, p = 0.01) and increased with higher lobular inflammation scores (p = 0.04). Immunohistochemical analysis showed colocalization of properdin and infiltrated MPO+ neutrophils surrounding steatotic hepatocytes. Concomitantly, decay accelerating factor mRNA and protein expression were upregulated in patients with NASH, while mRNA expression of factor H was significantly decreased (p = 0.02). Hepatocyte apoptosis, a possible activator of the complement AP was significantly increased in patients with NASH. Conclusions: Collectively, these data suggest a role for AP activation in driving hepatic inflammation during the progression of NASH, and indicate that alterations in host expression of regulatory complement factors might be involved as well. 1292 RESOLVIN D1 PRIMES INFLAMMATION-RESOLUTION PROGRAMMES DURING MODERATE CALORIE RESTRICTION IN OBESITY-INDUCED NASH 1 1 B. Rius1 , E. Titos1,2 , E. Moran-Salvador ´ , C. Lopez-Vicario ´ , 1 1,2 2,3 V. Garc´ıa-Alonso , A. Gonzalez-P ´ eriz ´ , V. Arroyo , J. Claria ` 1,2 . 1 Department of Biochemistry and Molecular Genetics, Hospital Cl´ınic, 2 CIBERehd, 3 Liver Unit, Hospital Cl´ınic, Barcelona, Spain E-mail: [email protected] Background and Aims: Non-alcoholic steatohepatitis (NASH) is a chronic liver disease closely associated with obesity and the metabolic syndrome. Obesity-induced NASH is characterized by aberrant lipid storage in hepatocytes combined with a remarkable unresolved inflammatory component. Both obesity and NASH S522
progression can be effectively attenuated by weight loss following a moderate nutritional intervention aimed to reduce calorie intake. Resolvins of the D-series (RvDs) are a novel family of endogenous anti-inflammatory and pro-resolving lipid mediators that act as agonists of the timely resolution of inflammation and the return to tissue homeostasis. In the current study, we hypothesized that these endogenous autacoids could potentiate and accelerate the inflammation-resolution process in high-fat-fed obese mice with NASH placed on moderate caloric restriction. Methods: Male C57BL/6 mice (n = 27) were fed with a high-fat diet (HFD) for 12 weeks and then randomly assigned into three groups. One group continued on HFD (n = 13) and the rest were switched to chow diet (calorie restriction) and received either RvD1 (300 ng/daily, i.p., n = 7) or placebo (n = 7) for the last 3 weeks. At the end of the intervention period, hepatic steatosis, inflammation, macrophage accumulation, insulin resistance and serum and tissue adipokine levels were determined. RvD1 (10 nM) actions on liver cells were tested in precision cut liver slices (PCLS). RvD1 gene targets and microRNA networks were screened by real-time PCR and miRNA arrays. Results: Moderate caloric restriction induced anti-obesogenic and anti-steatotic effects, reduced serum leptin and resistin levels and ameliorated insulin resistance, reflected by decreased JNK phosphorylation. On top of these actions, RvD1 reduced hepatic inflammatory infiltrate and MCP-1, IL-1b, IL-6, TNFa and CCR7 expression, while up-regulating adiponectin and arginase 1. The anti-inflammatory and pro-resolving actions of RvD1 on liver cells were confirmed in organotypic cultures of PCLS, where this lipid mediator attenuated hypoxia-induced COX-2, IL-1b and IL-6 upregulation. Ingenuity® analysis revealed that miRNAs differentially regulated by RvD1 were intimately related to cytokine signaling pathways. Conclusion: RvD1 primes the resolution process initiated by moderate caloric restriction through reducing the inflammatory component of obesity-induced NASH. 1293 GENDER DIMORPHISM OF FATTY LIVER DISEASE M. Schiffrin, F. Gilardi, B. Desvergne. Center for Integrative Genomics, Unil Lausanne, Lausanne, Switzerland E-mail: [email protected] Objective: NAFLD comprises a spectrum of clinical-histological disturbances, ranging from simple lipid accumulation in the cytoplasm of hepatocytes to steatosis combined with inflammation and liver injury (NASH). The disease can potentially progress to more severe forms, culminating in liver fibrosis and cirrhosis. Among patients with NAFLD men are more exposed to NASH and liver fibrosis than women. However, this gender dimorphism of NAFLD evolution was poorly studied and the molecular mechanism is still unclear. The aims of this project are to characterize the gender-specific evolution of liver steatosis and to establish the molecular basis of this gender specificity in mouse. Methods: Mouse carrying a null mutation in both PPARg alleles was fed with normal chow diet. PPARg null mice have no adipose tissue and develop a massive accumulation of fat in the liver. Characterization of the gender-specific evolution of liver steatosis was assessed and microarray analysis was performed to establish the molecular basis of the gender specificity. Results: While in females we observed macro and micro-vescicular steatosis at all time points, in males lipid droplets almost disappeared starting from 20 weeks. Furthermore, at 20 weeks the hepatic content of triglycerides is significantly higher in KO females than in KO males, whereas no differences are detected at 7 weeks. Further supporting the different response of the two sexes to lipid loading, the expression of a panel of genes involved in lipid synthesis and sequestration in lipid droplets was increased
Journal of Hepatology 2013 vol. 58 | S409–S566
POSTERS only in KO females. Interestingly, several evidences suggest that TG accumulation is less toxic for the liver than gathering abnormal quantities of other lipids, such as ceramide, cholesterol and diacylglycerol (DAG). Thus, it seems reasonable to hypothesize that KO female livers could be less damaged by steatosis. Microarrays analyses highlighted pathways differently activated during the progression of the pathology in the two genders. Conclusions: PPARg null mice show a clear gender dimorphism of hepatosteatosis. The comprehension of sex-related difference molecular basis could be important to identify the mechanisms that protect females from a worst progression of the pathology. 1294 THE ROLE OF CD36 IN THE PATHOGENESIS OF NAFLD F. Sheedfar1 , M. Aparicio-Vergara1 , B. Moesker1 , A. de Bruin2 , M. Febbraio3 , M.H. Hofker1 , D.P.Y. Koonen1 , Department of Molecular Genetics. 1 Pathology and Medical Biology, UMCG, Groningen, 2 Pathobiology, UU, Utrecht, The Netherlands; 3 Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH, USA E-mail: [email protected] Non-alcoholic fatty liver disease (NAFLD) is the main cause of chronic liver injury worldwide. Hepatic steatosis, characterized by excess triglyceride accumulation in the cytoplasm of hepatocytes, is the hallmark of NAFLD. While steatosis itself is generally considered an innocent incident, the presence of inflammation can lead to further progression of non-alcoholic steatohepatitis (NASH), resulting in more advanced stages of liver disease. Since CD36 mediates long-chain fatty acid uptake in the liver, we hypothesize that absence of CD36 in the liver will protect against NAFLD/NASH. To investigate this, CD36 knockout (CD36 KO mice) and wild type (WT) mice were fed a low fat (LF; 10% fat from lard) and a high fat (HF; 60% fat from lard) for 12 weeks. Moreover mice were fed a high fat cholesterol diet (HFC; 0.2% cholesterol, 21% milk butter) to induce hepatic inflammation triggered by Kupffer cell activation. Livers were dissected for gene expression (RT-PCR) and pathological assessment of NAFLD. In contrast to our hypothesis we show that CD36 ablation does not protect against NAFLD in mice fed a HF diet. However, CD36 KO mice are prone to develop NASH when fed with a HFC diet, consistent with enhanced Kupffer cell function. As CD36 is expressed in Kupffer cells and upregulated by cholesterol supplementation to the diet, our data suggest an inhibitory role of CD36 in Kupffer cells to dampen inflammation in the liver. Therefore, we propose that CD36 in Kupffer cells may serve as a protective mechanism, dampening liver inflammation and thereby attenuating the progression towards NASH. This research was supported by the Center for Translational Molecular Medicine (CTMM) and the Netherlands Heart Foundation, Dutch Diabetes Research Foundation and Dutch Kidney Foundation (PREDICCt). 1295 THE INSULIN-LIKE GROWTH FACTOR 2 (IGF2) mRNA BINDING PROTEIN p62/IMP2–2 ACCELERATES STEATOSIS, INFLAMMATION AND FIBROSIS IN A DIETARY MODEL OF NON-ALCOHOLIC STEATOHEPATITIS (NASH) Y. Simon1 , S.M. Kessler1,2 , N. Van Roijen3 , J. Haybaeck2 , A.K. Kiemer1 . 1 Department of Pharmaceutical Biology, Saarland University, Saarbruecken, Germany; 2 Institute of Pathology, Medical University Graz, Graz, Austria; 3 Department of Molecular Cell Biology, Vrije University Medical Center, Amsterdam, The Netherlands E-mail: [email protected] Background: Liver-specific overexpression of p62 induces steatosis. Increased cytoplasmic levels of the NF-úB subunit p65 suggested an increased susceptibility towards an inflammatory stimulus.
Aim of this study was to investigate the effect of p62 on inflammation and fibrosis in a dietary model of NASH. Methods: p62 transgenic mice (tg) and wild type (wt) littermates were fed a methionine-choline deficient diet (MCD) or a methionine-choline supplemented control diet for 2, 4, or 12 weeks (n = 10 animals, each). Liver tissue specimens were investigated by histology and immunohistochemistry, real-time RT-PCR, and a fluorimetric TBARS assay. Kupffer cells (KCs) were depleted by clodronate liposomes. Results: Serum transaminase levels were comparable between wt and tg mice at all time points. MCD-induced steatosis was more pronounced in tg compared to wt animals only at 2 weeks (p = 0.02). Most notably, a pronounced ductular reaction was observed as early as 2 and 4 weeks in tg on MCD (p = 0.04 and 0.01, respectively). Wt mice showed ductular reactions only after MCD feeding for 12 weeks. Determination of lipid peroxidation revealed increased TBARS levels in tg compared to wt animals upon MCD diet for 4 weeks (p = 0.03). This was accompanied by pronounced lobular lymphocytic and neutrophilic inflammation in tg at all time points. We also observed a significantly increased mRNA expression of the chemokine MCP-1 (p = 0.03) and the cytokines TNFa (p = 0.02) and IL-1b (p = 0.02). Activation of the proinflammatory transcription factor NF-úB was increased in hepatocytes of tg mice as assessed by nuclear translocation of p65. Fibrosis development was accelerated in tg animals with increased collagen 1a1 mRNA expression already after feeding for 2 or 4 weeks (p = 0.006 and 0.026, respectively), while no expression was observed in wt mice. Histological analysis revealed detectable fibrosis in tg animals at 4 weeks MCD feeding (p = 0.03). These data indicated that the proinflammatory effects of p62 in the MCD model are derived from injured hepatocytes rather than from the tissue resident macrophages, the KCs. In fact, KC depletion neither altered steatosis development nor expression of inflammatory cytokines and chemokines in MCD-fed animals. Conclusion: p62 promotes inflammation and fibrogenesis in the described murine NASH model. 1296 SAFETY AND EFFICACY OF THE PAN-CASPASE INHIBITOR IDN-6556 IN THE TREATMENT OF NONALCOHOLIC FATTY LIVER AND INSULIN RESISTANCE W. Lu1 , A. Eguchi1 , D. Sirbu1 , C. Jonhson1 , P. Contreras2 , A. Wree1 , M. Lazic1 , D. Povero1 , A. Feldstein1 . 1 Pediatrics, University of California, San Diego, La Jolla, 2 Conatus Pharmaceuticals, San Diego, CA, USA E-mail: [email protected] Hepatocellular and extrahepatic cell death are key features of obesity-associated fatty liver disease (NAFLD) and metabolic syndrome. Emerging data suggest that inhibition of caspases may be an attractive therapeutic approach for patients with these conditions. Our aim was to test the hypothesis that the pan-caspase inhibitor IDN-6556 will improve hepatic steatosis and metabolic abnormalities commonly observed with NAFLD, such as insulin resistance and dyslipidemia. Methods: C57BL/6 mice, aged 6 to 8 weeks at the beginning of the study, were fed either a high fat (HFAT) “western” diet or a low fat control diet for 20 weeks. During the last 5 weeks mice fed the HFAT died were treated with 3 mg/kg/day of IDN-6556 (n = 12) or with a placebo (n = 12), via gavage. Mice were then sacrificed and their livers, adipose tissue (epididymal) and blood were collected. Liver and adipose tissue inflammation and cell death was assessed by histopathology, TUNEL assay, immunoblotting, F4/80 immunohistochemistry, and real time qPCR for pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IL-6). Liver injury was further determined by serum ALT levels and alpha-smooth muscle actin (alpha-SMA), collagen 1-alpha (COL1A1), and transforming growth factor-Beta (TGF-beta) by real time qPCR.
Journal of Hepatology 2013 vol. 58 | S409–S566
S523
progression can be effectively attenuated by weight loss following a moderate nutritional intervention aimed to reduce calorie intake. Resolvins of the D-series (RvDs) are a novel family of endogenous anti-inflammatory and pro-resolving lipid mediators that act as agonists of the timely resolution of inflammation and the return to tissue homeostasis. In the current study, we hypothesized that these endogenous autacoids could potentiate and accelerate the inflammation-resolution process in high-fat-fed obese mice with NASH placed on moderate caloric restriction. Methods: Male C57BL/6 mice (n = 27) were fed with a high-fat diet (HFD) for 12 weeks and then randomly assigned into three groups. One group continued on HFD (n = 13) and the rest were switched to chow diet (calorie restriction) and received either RvD1 (300 ng/daily, i.p., n = 7) or placebo (n = 7) for the last 3 weeks. At the end of the intervention period, hepatic steatosis, inflammation, macrophage accumulation, insulin resistance and serum and tissue adipokine levels were determined. RvD1 (10 nM) actions on liver cells were tested in precision cut liver slices (PCLS). RvD1 gene targets and microRNA networks were screened by real-time PCR and miRNA arrays. Results: Moderate caloric restriction induced anti-obesogenic and anti-steatotic effects, reduced serum leptin and resistin levels and ameliorated insulin resistance, reflected by decreased JNK phosphorylation. On top of these actions, RvD1 reduced hepatic inflammatory infiltrate and MCP-1, IL-1b, IL-6, TNFa and CCR7 expression, while up-regulating adiponectin and arginase 1. The anti-inflammatory and pro-resolving actions of RvD1 on liver cells were confirmed in organotypic cultures of PCLS, where this lipid mediator attenuated hypoxia-induced COX-2, IL-1b and IL-6 upregulation. Ingenuity® analysis revealed that miRNAs differentially regulated by RvD1 were intimately related to cytokine signaling pathways. Conclusion: RvD1 primes the resolution process initiated by moderate caloric restriction through reducing the inflammatory component of obesity-induced NASH. 1293 GENDER DIMORPHISM OF FATTY LIVER DISEASE M. Schiffrin, F. Gilardi, B. Desvergne. Center for Integrative Genomics, Unil Lausanne, Lausanne, Switzerland E-mail: [email protected] Objective: NAFLD comprises a spectrum of clinical-histological disturbances, ranging from simple lipid accumulation in the cytoplasm of hepatocytes to steatosis combined with inflammation and liver injury (NASH). The disease can potentially progress to more severe forms, culminating in liver fibrosis and cirrhosis. Among patients with NAFLD men are more exposed to NASH and liver fibrosis than women. However, this gender dimorphism of NAFLD evolution was poorly studied and the molecular mechanism is still unclear. The aims of this project are to characterize the gender-specific evolution of liver steatosis and to establish the molecular basis of this gender specificity in mouse. Methods: Mouse carrying a null mutation in both PPARg alleles was fed with normal chow diet. PPARg null mice have no adipose tissue and develop a massive accumulation of fat in the liver. Characterization of the gender-specific evolution of liver steatosis was assessed and microarray analysis was performed to establish the molecular basis of the gender specificity. Results: While in females we observed macro and micro-vescicular steatosis at all time points, in males lipid droplets almost disappeared starting from 20 weeks. Furthermore, at 20 weeks the hepatic content of triglycerides is significantly higher in KO females than in KO males, whereas no differences are detected at 7 weeks. Further supporting the different response of the two sexes to lipid loading, the expression of a panel of genes involved in lipid synthesis and sequestration in lipid droplets was increased
Journal of Hepatology 2013 vol. 58 | S409–S566
POSTERS only in KO females. Interestingly, several evidences suggest that TG accumulation is less toxic for the liver than gathering abnormal quantities of other lipids, such as ceramide, cholesterol and diacylglycerol (DAG). Thus, it seems reasonable to hypothesize that KO female livers could be less damaged by steatosis. Microarrays analyses highlighted pathways differently activated during the progression of the pathology in the two genders. Conclusions: PPARg null mice show a clear gender dimorphism of hepatosteatosis. The comprehension of sex-related difference molecular basis could be important to identify the mechanisms that protect females from a worst progression of the pathology. 1294 THE ROLE OF CD36 IN THE PATHOGENESIS OF NAFLD F. Sheedfar1 , M. Aparicio-Vergara1 , B. Moesker1 , A. de Bruin2 , M. Febbraio3 , M.H. Hofker1 , D.P.Y. Koonen1 , Department of Molecular Genetics. 1 Pathology and Medical Biology, UMCG, Groningen, 2 Pathobiology, UU, Utrecht, The Netherlands; 3 Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH, USA E-mail: [email protected] Non-alcoholic fatty liver disease (NAFLD) is the main cause of chronic liver injury worldwide. Hepatic steatosis, characterized by excess triglyceride accumulation in the cytoplasm of hepatocytes, is the hallmark of NAFLD. While steatosis itself is generally considered an innocent incident, the presence of inflammation can lead to further progression of non-alcoholic steatohepatitis (NASH), resulting in more advanced stages of liver disease. Since CD36 mediates long-chain fatty acid uptake in the liver, we hypothesize that absence of CD36 in the liver will protect against NAFLD/NASH. To investigate this, CD36 knockout (CD36 KO mice) and wild type (WT) mice were fed a low fat (LF; 10% fat from lard) and a high fat (HF; 60% fat from lard) for 12 weeks. Moreover mice were fed a high fat cholesterol diet (HFC; 0.2% cholesterol, 21% milk butter) to induce hepatic inflammation triggered by Kupffer cell activation. Livers were dissected for gene expression (RT-PCR) and pathological assessment of NAFLD. In contrast to our hypothesis we show that CD36 ablation does not protect against NAFLD in mice fed a HF diet. However, CD36 KO mice are prone to develop NASH when fed with a HFC diet, consistent with enhanced Kupffer cell function. As CD36 is expressed in Kupffer cells and upregulated by cholesterol supplementation to the diet, our data suggest an inhibitory role of CD36 in Kupffer cells to dampen inflammation in the liver. Therefore, we propose that CD36 in Kupffer cells may serve as a protective mechanism, dampening liver inflammation and thereby attenuating the progression towards NASH. This research was supported by the Center for Translational Molecular Medicine (CTMM) and the Netherlands Heart Foundation, Dutch Diabetes Research Foundation and Dutch Kidney Foundation (PREDICCt). 1295 THE INSULIN-LIKE GROWTH FACTOR 2 (IGF2) mRNA BINDING PROTEIN p62/IMP2–2 ACCELERATES STEATOSIS, INFLAMMATION AND FIBROSIS IN A DIETARY MODEL OF NON-ALCOHOLIC STEATOHEPATITIS (NASH) Y. Simon1 , S.M. Kessler1,2 , N. Van Roijen3 , J. Haybaeck2 , A.K. Kiemer1 . 1 Department of Pharmaceutical Biology, Saarland University, Saarbruecken, Germany; 2 Institute of Pathology, Medical University Graz, Graz, Austria; 3 Department of Molecular Cell Biology, Vrije University Medical Center, Amsterdam, The Netherlands E-mail: [email protected] Background: Liver-specific overexpression of p62 induces steatosis. Increased cytoplasmic levels of the NF-úB subunit p65 suggested an increased susceptibility towards an inflammatory stimulus.
Aim of this study was to investigate the effect of p62 on inflammation and fibrosis in a dietary model of NASH. Methods: p62 transgenic mice (tg) and wild type (wt) littermates were fed a methionine-choline deficient diet (MCD) or a methionine-choline supplemented control diet for 2, 4, or 12 weeks (n = 10 animals, each). Liver tissue specimens were investigated by histology and immunohistochemistry, real-time RT-PCR, and a fluorimetric TBARS assay. Kupffer cells (KCs) were depleted by clodronate liposomes. Results: Serum transaminase levels were comparable between wt and tg mice at all time points. MCD-induced steatosis was more pronounced in tg compared to wt animals only at 2 weeks (p = 0.02). Most notably, a pronounced ductular reaction was observed as early as 2 and 4 weeks in tg on MCD (p = 0.04 and 0.01, respectively). Wt mice showed ductular reactions only after MCD feeding for 12 weeks. Determination of lipid peroxidation revealed increased TBARS levels in tg compared to wt animals upon MCD diet for 4 weeks (p = 0.03). This was accompanied by pronounced lobular lymphocytic and neutrophilic inflammation in tg at all time points. We also observed a significantly increased mRNA expression of the chemokine MCP-1 (p = 0.03) and the cytokines TNFa (p = 0.02) and IL-1b (p = 0.02). Activation of the proinflammatory transcription factor NF-úB was increased in hepatocytes of tg mice as assessed by nuclear translocation of p65. Fibrosis development was accelerated in tg animals with increased collagen 1a1 mRNA expression already after feeding for 2 or 4 weeks (p = 0.006 and 0.026, respectively), while no expression was observed in wt mice. Histological analysis revealed detectable fibrosis in tg animals at 4 weeks MCD feeding (p = 0.03). These data indicated that the proinflammatory effects of p62 in the MCD model are derived from injured hepatocytes rather than from the tissue resident macrophages, the KCs. In fact, KC depletion neither altered steatosis development nor expression of inflammatory cytokines and chemokines in MCD-fed animals. Conclusion: p62 promotes inflammation and fibrogenesis in the described murine NASH model. 1296 SAFETY AND EFFICACY OF THE PAN-CASPASE INHIBITOR IDN-6556 IN THE TREATMENT OF NONALCOHOLIC FATTY LIVER AND INSULIN RESISTANCE W. Lu1 , A. Eguchi1 , D. Sirbu1 , C. Jonhson1 , P. Contreras2 , A. Wree1 , M. Lazic1 , D. Povero1 , A. Feldstein1 . 1 Pediatrics, University of California, San Diego, La Jolla, 2 Conatus Pharmaceuticals, San Diego, CA, USA E-mail: [email protected] Hepatocellular and extrahepatic cell death are key features of obesity-associated fatty liver disease (NAFLD) and metabolic syndrome. Emerging data suggest that inhibition of caspases may be an attractive therapeutic approach for patients with these conditions. Our aim was to test the hypothesis that the pan-caspase inhibitor IDN-6556 will improve hepatic steatosis and metabolic abnormalities commonly observed with NAFLD, such as insulin resistance and dyslipidemia. Methods: C57BL/6 mice, aged 6 to 8 weeks at the beginning of the study, were fed either a high fat (HFAT) “western” diet or a low fat control diet for 20 weeks. During the last 5 weeks mice fed the HFAT died were treated with 3 mg/kg/day of IDN-6556 (n = 12) or with a placebo (n = 12), via gavage. Mice were then sacrificed and their livers, adipose tissue (epididymal) and blood were collected. Liver and adipose tissue inflammation and cell death was assessed by histopathology, TUNEL assay, immunoblotting, F4/80 immunohistochemistry, and real time qPCR for pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IL-6). Liver injury was further determined by serum ALT levels and alpha-smooth muscle actin (alpha-SMA), collagen 1-alpha (COL1A1), and transforming growth factor-Beta (TGF-beta) by real time qPCR.
Journal of Hepatology 2013 vol. 58 | S409–S566
S523