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S1832 Caspase 3 Inactivation Protects Against Diet-Induced Fibrosing Steatohepatitis
Moreover, they outline a novel metabolism-based strategy to overcome the CSC-dependent drug resistance; the potentials of this strategy and its specific effects on the CSC fraction and on tumor growth In Vivo are currently being investigated.
Mitochondrial respiratory chain activity (MRC) was measured by spectrophotometrical analysis, and NFkB activity was measured by ELISA Results: In db/db mice silybin markedly improved liver injury as measured by NAS (P<0.01) and partially restored mitochondria morphology; reduced hepatic triglycerides content (P<0.001) and serum ALT (P<0.01). Silybin administration strongly decreased liver TBARS, 8-OHG and increased GSH (P<0.0001 for all). Strikingly, silybin treatment completely restored hepatic MRC (P<0.0001 for all the five complexes) and inhibited NFkB p65 and p50 subunits binding activity (P<0.0001 for both); consistently, it blocked iNOS expression (P<0.01) and reduced nitrite/nitrates (P<0.05) and 3-nitrotyrosine levels in the liver of db/db mice. Conclusions: In this study, we demonstrated that silybin improves liver injury in the db/db + MCD mouse model of NAFLD. In our opinion, our findings provides a strong rationale for the use of silybin in the clinical management of NAFLD patients. Further experimental studies should clarify additional molecular pathways involved in the therapeutic effects of silybin in NAFLD.
847
AASLD Abstracts
Establishment and Characterization of a Novel Gallbladder Cell Line to Study Gallbladder Cancer and Gallstones Mohammad Z. Abedin, Nandita Suresh, Samuel Jun, Will Sun, Muhammad T. Rahman, Fernando U. Garcia, Mark E. Stearns Background and Aims: Gallbladder cancer (GBC) is the most common malignancy of the biliary tract. The prognosis for patients with GBC is poor as diagnosis is often at late untreatable stages of the disease. Chronic cholelithiasis has been linked to GBC. However, our understanding of the underlying mechanisms for the development of GBC and its association with gallstones (GS) is not clear. The aim of the present study was to establish and characterize a gallbladder cell line to study the molecular mechanisms of pathogenic relationship between gallstone formation and the development of gallbladder cancer. Methods: Primary cultures of gallbladder epithelial cells (GBECs) from prairie dogs were immortalized following previous hTERT method. Sub-cellular organelles were examined under electron microscope(EM) and immunohistochemical analysis was performed to examine the expression of biliary epithelial specific cytokeratines, vimentin and tumor supressor genes. RT-PCR analysis was performed to determine the expression of major Na+ transporter, Na+/H+ exchangers (NHEs) and Megalin. Results: Immortalized GBECs (GBECT) grew twelve passes and became confluent within 48 hrs. EM revealed distinct microvilli and mitochondria. GBECT coexpressed epithelial markers keratins and mesenchymal origin vimentin and showed strong p53 expression. GBECT retained absorbing property of gallbladder by expressing NHE1-3 isoforms. Conclusion: This is a novel gallbladder cell line from the prairie dog that retains gallbladder basic absorbing characteristic while acquires some tumorgenecity and may serve as an important model to study the underlying mechanisms of pathogenic role of GS formation on GBC development.
S1831 Protective Effect of Metformin on the Onset of Fructose-Induced NonAlcoholic Fatty Liver Disease (NAFLD) in Mice Astrid Spruss, Giridhar Kanuri, Valentina Volynets, Stephan C. Bischoff, Ina Bergheim Background and aims: Results of animal and human studies suggest that dietary fructose and bacterial endotoxin both may be critical factors in the development of non-alcoholic fatty liver disease (NAFLD). The antidiabetic drug metformin has been shown to improve NAFLD in patients and ob/ob mice; however, if metformin also protects the liver from fructose-induced NAFLD has not yet been studied. Therefore, the aim of the present study was to investigate the effect of metformin on the onset of fructose-induced NAFLD in a mouse model. Methods: C57/BL-6J mice were either fed 30% fructose solution or plain water for 8 weeks. Some of the animals were concomitantly treated with metformin (300 mg/kg body weight/day) in the drinking solution. Markers of the onset of NAFLD (e.g. plasma ALT and hepatic triglyceride levels) and portal endotoxin concentrations were determined and mRNA expression of tumor necrosis factor (TNF) α, inducible nitric oxide synthase (iNOS), myeloid differentiation factor 88 (MyD88), interferon regulatory factor (IRF) 3 and -7 were measured in liver tissue by realtime RT-PCR. Results: While chronic consumption of 30% fructose solution caused a significant increase in hepatic triglyceride accumulation, concomitant treatment with metformin significantly attenuated this effect of fructose feeding by ~40%. A similar effect was found for plasma ALT levels. Interestingly, the protective effects of the metformin treatment on the onset of fructose-induced NAFLD were associated with a protection against the markedly higher portal endotoxin levels found in mice only fed with fructose (Fructose: ~ 0.72 EU/ml vs. Fructose + Metformin: ~0.09EU/ml, p<0.05). In line with these findings, in fructose fed mice treated with metformin hepatic expression of MyD88, IRF3 and -7 was almost at the level of water controls. Moreover, the induction of hepatic TNFα and iNOS mRNA expression found in fructose fed mice (+ ~2.5-fold and +~1.5-fold in comparison to water controls, p<0.05) was significantly attenuated in livers of fructose fed mice concomitantly treated with metformin. Conclusion: Taken together, these data suggest that metformin not only protects the liver from the onset of NAFLD by mechanisms involving its direct effects on hepatic insulin signaling but rather by mechanisms altering intestinal permeability and subsequent endotoxin-dependent activation of hepatic Kupffer cells. Supported by the German Research Foundation (BE 2376/4-1(IB)).
S1829 Crucial Role of C-Jun N-Terminal Kinase (JNK) in the Pathogenesis of Nonalcoholic Steatohepatitis (NASH) Koji Fujita, Yoshiyasu Shinohara, Kento Imajyo, Hironori Mawatari, Yuichi Nozaki, Masato Yoneda, Hiroyuki Kirikoshi, Satoru Saito, Atsushi Nakajima BACKGROUND/AIMS: We previously demonstrated more marked liver accumulation of FFA in NASH patients than in patients with simple fatty liver (SFL), associated with decreased MTP activity and VLDL synthesis (Fujita K, et al. Hepatology 2009), and also improvement of NASH in an animal model with MAPK signaling inhibited using antiplatelet agents (Fujita K, et al. GUT 2008). Based on a previous report that FFA induce JNK activation (Solinas G, et al. PNAS 2006), we hypothesized that surplus FFAs activate JNK, a member of the MAPK family, to promote insulin resistance and unfolded protein response (UPR)-induced hepatocyte apoptosis, thereby resulting in the development and progression of NASH. To verify our hypothesis, we examined the involvement of JNK signaling in NASH. METHODS: 1) Phosphorylated JNK (p-JNK) protein was measured in human liver biopsy tissues (20 SFL and 32 NASH) to examine the correlation between JNK activity and NAFLD progression. 2) p-JNK protein was measured using murine hepatocyte primary cell cultures, in the presence of several fatty acids, to verify whether FFAs directly activate JNK. 3) Insulin signaling, UPR-associated cell signaling, and UPR-induced hepatocyte apoptosis were measured long-term high fat diet fed mice injected with a JNK inhibitor, to investigate whether inhibition of JNK might inhibit NASH progression. RESULTS: 1) Stronger expression of pJNK protein was significantly observed in NASH than in SFL. 2) Stronger expression of pJNK protein was significantly observed in the FA group than in controls In Vitro. 3) Longterm high fat diet fed mice showed significantly higher liver FFA accumulation and p-JNK protein expression than controls. Injection of a JNK inhibitor resulted in blockade of JNKinduced IRS-1 serine phosphorylation, reversal of the impaired insulin signaling and improved insulin resistance, decrease in expression of activated XBP-1 protein resulting in reversal of the impaired UPR, and finally, improvement of NASH In Vivo animal model. CONCLUSION: Surplus FFA, because of inhibited liver MTP activity to reduce liver VLDL synthesis, activated JNK signaling. Activated JNK signaling led to insulin resistance and unfolded protein response (UPR)-induced hepatocyte apoptosis, resulting in the development and progression of NASH in animal model.
S1832 Caspase 3 Inactivation Protects Against Diet-Induced Fibrosing Steatohepatitis Samjhana Thapaliya, Michael P. Berk, Laura Dixon, Ariel E. Feldstein Hepatocyte cell death is a key feature of nonalcoholic steohepatitis (NASH). Emerging data suggest that inhibition of caspases, intracellular proteases that are central for initiation and execution of the cell death program, may be an attractive therapeutic approach for patients with NASH. However, the contribution of specific caspases to liver injury and fibrosis remains unclear. Our aims were to ascertain if inactivation of caspase 3 attenuates liver injury and fibrogenesis in NASH using murine models including specific knock out mice. Methods: C57BL/6 wild-type (WT), and caspase 3 knock out (Casp3-/-) mice were placed on either a methionine- and choline-deficient (MCD) diet, or control (CTL) diet (n= 4-6 in each group). After 6 wks plasma and liver tissue were collected. Hepatic steatosis, hepatocellular damage, apoptosis, inflammatory activity, and fibrosis were assessed by triglyceride (TG) quantification, Oil Red O staining, serum ALT, active caspase 3 staining and its activity, cytokeratin-18 fragment staining, liver histopathology with determination of NAFLD activity score (NAS), and Sirius Red staining coupled to morphometric quantitation. Markers of inflammation including TNF-α, IL-6, F4/80 and makers of stellate cell activation (HSC) including alpha-smooth muscle actin (αSMA), collagen 1-alpha (COL1A1), and transforming growth factor-Beta (TGF-β) were determined by real time PCR. Results: WT mice on the MCD diet showed significant increased in caspase 3 activity, and apoptosis, in conjunction with marked steatohepatitis with increased hepatic triglyceride levels, hepatocyte ballooning, inflammation and fibrosis. Casp3-/- on the MCD diet showed similar ALT levels and NAS compared to WT mice on the MCD diet. However Casp3-/- mice on the MCD diet showed a marked reduction in mRNA expression of genes involved in fibrogenesis (αSMA was 10.8fold greater in WT vs. Casp3-/- MCD-fed mice; COL1A1 was 1.5-fold greater in WT vs. Casp3-/- MCD-fed mice; and TGF-β was 2.7-fold greater in WT vs. Casp3-/- MCD-fed mice). Furthermore, Sirius red staining for hepatic collagen deposition was significantly reduced in Casp3-/- MCD-fed mice compared to WT MCD-fed animals. These changes were associated with the decreased levels of apoptosis, and a significant reduction in the expression of cytokines involved in inflammatory signaling Conclusion: These findings support a prominent role for caspase 3 activation in NASH related apoptosis, fibrogenesis and fibrosis. This concept has important implications for the development of novel treatment strategies for patients with this condition.
S1830 Silybin Enhances Mitochondrial Function and Inhibits NFkB Activation in Experimental NAFLD Federico Salamone, Giovanni Li Volti Background and aims: NonAlcoholic Fatty Liver Disease (NAFLD) is a chronic metabolic disorder with significant impact on liver and cardiovascular morbidity and mortality. Silybin, a flavonolignan extracted from milk thistle, exerts a marked liver protecting action in a variety of experimental liver injuries, mostly because of free radicals scavenging properties. Because oxidative stress plays a pivot role in NAFLD pathophysiology and contributes to liver damage directly and via mitochondrial dysfunction and NFkB activation, we aimed to clarify the putative therapeutic role of silybin in a murine model of NAFLD. Methods: We explored the effects of a 4-week daily (20 mg/kg i.p.) administration of silybin in 6-weekold db/db mice feeding a methionine-choline deficient (MCD) diet . Liver histology was evaluated following the NAFLD activity score (NAS); mitochondria morphology was evaluated by electron microscopy. Liver triglycerides were determined using a colorimetric kit; liver 8-hydroxyguanosine (8-OHG) was determined by ELISA; thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) were measured by a TBARS and a GSH assay kit. Hepatic nitrite/nitrate was measured colorimetrically using Griess reaction. Expression of iNOS and 3-nitrotyrosine (3-NT) was evaluated by western blot and immunofluorescence.
AASLD Abstracts
S-798
Mitochondrial respiratory chain activity (MRC) was measured by spectrophotometrical analysis, and NFkB activity was measured by ELISA Results: In db/db mice silybin markedly improved liver injury as measured by NAS (P<0.01) and partially restored mitochondria morphology; reduced hepatic triglycerides content (P<0.001) and serum ALT (P<0.01). Silybin administration strongly decreased liver TBARS, 8-OHG and increased GSH (P<0.0001 for all). Strikingly, silybin treatment completely restored hepatic MRC (P<0.0001 for all the five complexes) and inhibited NFkB p65 and p50 subunits binding activity (P<0.0001 for both); consistently, it blocked iNOS expression (P<0.01) and reduced nitrite/nitrates (P<0.05) and 3-nitrotyrosine levels in the liver of db/db mice. Conclusions: In this study, we demonstrated that silybin improves liver injury in the db/db + MCD mouse model of NAFLD. In our opinion, our findings provides a strong rationale for the use of silybin in the clinical management of NAFLD patients. Further experimental studies should clarify additional molecular pathways involved in the therapeutic effects of silybin in NAFLD.
847
AASLD Abstracts
Establishment and Characterization of a Novel Gallbladder Cell Line to Study Gallbladder Cancer and Gallstones Mohammad Z. Abedin, Nandita Suresh, Samuel Jun, Will Sun, Muhammad T. Rahman, Fernando U. Garcia, Mark E. Stearns Background and Aims: Gallbladder cancer (GBC) is the most common malignancy of the biliary tract. The prognosis for patients with GBC is poor as diagnosis is often at late untreatable stages of the disease. Chronic cholelithiasis has been linked to GBC. However, our understanding of the underlying mechanisms for the development of GBC and its association with gallstones (GS) is not clear. The aim of the present study was to establish and characterize a gallbladder cell line to study the molecular mechanisms of pathogenic relationship between gallstone formation and the development of gallbladder cancer. Methods: Primary cultures of gallbladder epithelial cells (GBECs) from prairie dogs were immortalized following previous hTERT method. Sub-cellular organelles were examined under electron microscope(EM) and immunohistochemical analysis was performed to examine the expression of biliary epithelial specific cytokeratines, vimentin and tumor supressor genes. RT-PCR analysis was performed to determine the expression of major Na+ transporter, Na+/H+ exchangers (NHEs) and Megalin. Results: Immortalized GBECs (GBECT) grew twelve passes and became confluent within 48 hrs. EM revealed distinct microvilli and mitochondria. GBECT coexpressed epithelial markers keratins and mesenchymal origin vimentin and showed strong p53 expression. GBECT retained absorbing property of gallbladder by expressing NHE1-3 isoforms. Conclusion: This is a novel gallbladder cell line from the prairie dog that retains gallbladder basic absorbing characteristic while acquires some tumorgenecity and may serve as an important model to study the underlying mechanisms of pathogenic role of GS formation on GBC development.
S1831 Protective Effect of Metformin on the Onset of Fructose-Induced NonAlcoholic Fatty Liver Disease (NAFLD) in Mice Astrid Spruss, Giridhar Kanuri, Valentina Volynets, Stephan C. Bischoff, Ina Bergheim Background and aims: Results of animal and human studies suggest that dietary fructose and bacterial endotoxin both may be critical factors in the development of non-alcoholic fatty liver disease (NAFLD). The antidiabetic drug metformin has been shown to improve NAFLD in patients and ob/ob mice; however, if metformin also protects the liver from fructose-induced NAFLD has not yet been studied. Therefore, the aim of the present study was to investigate the effect of metformin on the onset of fructose-induced NAFLD in a mouse model. Methods: C57/BL-6J mice were either fed 30% fructose solution or plain water for 8 weeks. Some of the animals were concomitantly treated with metformin (300 mg/kg body weight/day) in the drinking solution. Markers of the onset of NAFLD (e.g. plasma ALT and hepatic triglyceride levels) and portal endotoxin concentrations were determined and mRNA expression of tumor necrosis factor (TNF) α, inducible nitric oxide synthase (iNOS), myeloid differentiation factor 88 (MyD88), interferon regulatory factor (IRF) 3 and -7 were measured in liver tissue by realtime RT-PCR. Results: While chronic consumption of 30% fructose solution caused a significant increase in hepatic triglyceride accumulation, concomitant treatment with metformin significantly attenuated this effect of fructose feeding by ~40%. A similar effect was found for plasma ALT levels. Interestingly, the protective effects of the metformin treatment on the onset of fructose-induced NAFLD were associated with a protection against the markedly higher portal endotoxin levels found in mice only fed with fructose (Fructose: ~ 0.72 EU/ml vs. Fructose + Metformin: ~0.09EU/ml, p<0.05). In line with these findings, in fructose fed mice treated with metformin hepatic expression of MyD88, IRF3 and -7 was almost at the level of water controls. Moreover, the induction of hepatic TNFα and iNOS mRNA expression found in fructose fed mice (+ ~2.5-fold and +~1.5-fold in comparison to water controls, p<0.05) was significantly attenuated in livers of fructose fed mice concomitantly treated with metformin. Conclusion: Taken together, these data suggest that metformin not only protects the liver from the onset of NAFLD by mechanisms involving its direct effects on hepatic insulin signaling but rather by mechanisms altering intestinal permeability and subsequent endotoxin-dependent activation of hepatic Kupffer cells. Supported by the German Research Foundation (BE 2376/4-1(IB)).
S1829 Crucial Role of C-Jun N-Terminal Kinase (JNK) in the Pathogenesis of Nonalcoholic Steatohepatitis (NASH) Koji Fujita, Yoshiyasu Shinohara, Kento Imajyo, Hironori Mawatari, Yuichi Nozaki, Masato Yoneda, Hiroyuki Kirikoshi, Satoru Saito, Atsushi Nakajima BACKGROUND/AIMS: We previously demonstrated more marked liver accumulation of FFA in NASH patients than in patients with simple fatty liver (SFL), associated with decreased MTP activity and VLDL synthesis (Fujita K, et al. Hepatology 2009), and also improvement of NASH in an animal model with MAPK signaling inhibited using antiplatelet agents (Fujita K, et al. GUT 2008). Based on a previous report that FFA induce JNK activation (Solinas G, et al. PNAS 2006), we hypothesized that surplus FFAs activate JNK, a member of the MAPK family, to promote insulin resistance and unfolded protein response (UPR)-induced hepatocyte apoptosis, thereby resulting in the development and progression of NASH. To verify our hypothesis, we examined the involvement of JNK signaling in NASH. METHODS: 1) Phosphorylated JNK (p-JNK) protein was measured in human liver biopsy tissues (20 SFL and 32 NASH) to examine the correlation between JNK activity and NAFLD progression. 2) p-JNK protein was measured using murine hepatocyte primary cell cultures, in the presence of several fatty acids, to verify whether FFAs directly activate JNK. 3) Insulin signaling, UPR-associated cell signaling, and UPR-induced hepatocyte apoptosis were measured long-term high fat diet fed mice injected with a JNK inhibitor, to investigate whether inhibition of JNK might inhibit NASH progression. RESULTS: 1) Stronger expression of pJNK protein was significantly observed in NASH than in SFL. 2) Stronger expression of pJNK protein was significantly observed in the FA group than in controls In Vitro. 3) Longterm high fat diet fed mice showed significantly higher liver FFA accumulation and p-JNK protein expression than controls. Injection of a JNK inhibitor resulted in blockade of JNKinduced IRS-1 serine phosphorylation, reversal of the impaired insulin signaling and improved insulin resistance, decrease in expression of activated XBP-1 protein resulting in reversal of the impaired UPR, and finally, improvement of NASH In Vivo animal model. CONCLUSION: Surplus FFA, because of inhibited liver MTP activity to reduce liver VLDL synthesis, activated JNK signaling. Activated JNK signaling led to insulin resistance and unfolded protein response (UPR)-induced hepatocyte apoptosis, resulting in the development and progression of NASH in animal model.
S1832 Caspase 3 Inactivation Protects Against Diet-Induced Fibrosing Steatohepatitis Samjhana Thapaliya, Michael P. Berk, Laura Dixon, Ariel E. Feldstein Hepatocyte cell death is a key feature of nonalcoholic steohepatitis (NASH). Emerging data suggest that inhibition of caspases, intracellular proteases that are central for initiation and execution of the cell death program, may be an attractive therapeutic approach for patients with NASH. However, the contribution of specific caspases to liver injury and fibrosis remains unclear. Our aims were to ascertain if inactivation of caspase 3 attenuates liver injury and fibrogenesis in NASH using murine models including specific knock out mice. Methods: C57BL/6 wild-type (WT), and caspase 3 knock out (Casp3-/-) mice were placed on either a methionine- and choline-deficient (MCD) diet, or control (CTL) diet (n= 4-6 in each group). After 6 wks plasma and liver tissue were collected. Hepatic steatosis, hepatocellular damage, apoptosis, inflammatory activity, and fibrosis were assessed by triglyceride (TG) quantification, Oil Red O staining, serum ALT, active caspase 3 staining and its activity, cytokeratin-18 fragment staining, liver histopathology with determination of NAFLD activity score (NAS), and Sirius Red staining coupled to morphometric quantitation. Markers of inflammation including TNF-α, IL-6, F4/80 and makers of stellate cell activation (HSC) including alpha-smooth muscle actin (αSMA), collagen 1-alpha (COL1A1), and transforming growth factor-Beta (TGF-β) were determined by real time PCR. Results: WT mice on the MCD diet showed significant increased in caspase 3 activity, and apoptosis, in conjunction with marked steatohepatitis with increased hepatic triglyceride levels, hepatocyte ballooning, inflammation and fibrosis. Casp3-/- on the MCD diet showed similar ALT levels and NAS compared to WT mice on the MCD diet. However Casp3-/- mice on the MCD diet showed a marked reduction in mRNA expression of genes involved in fibrogenesis (αSMA was 10.8fold greater in WT vs. Casp3-/- MCD-fed mice; COL1A1 was 1.5-fold greater in WT vs. Casp3-/- MCD-fed mice; and TGF-β was 2.7-fold greater in WT vs. Casp3-/- MCD-fed mice). Furthermore, Sirius red staining for hepatic collagen deposition was significantly reduced in Casp3-/- MCD-fed mice compared to WT MCD-fed animals. These changes were associated with the decreased levels of apoptosis, and a significant reduction in the expression of cytokines involved in inflammatory signaling Conclusion: These findings support a prominent role for caspase 3 activation in NASH related apoptosis, fibrogenesis and fibrosis. This concept has important implications for the development of novel treatment strategies for patients with this condition.
S1830 Silybin Enhances Mitochondrial Function and Inhibits NFkB Activation in Experimental NAFLD Federico Salamone, Giovanni Li Volti Background and aims: NonAlcoholic Fatty Liver Disease (NAFLD) is a chronic metabolic disorder with significant impact on liver and cardiovascular morbidity and mortality. Silybin, a flavonolignan extracted from milk thistle, exerts a marked liver protecting action in a variety of experimental liver injuries, mostly because of free radicals scavenging properties. Because oxidative stress plays a pivot role in NAFLD pathophysiology and contributes to liver damage directly and via mitochondrial dysfunction and NFkB activation, we aimed to clarify the putative therapeutic role of silybin in a murine model of NAFLD. Methods: We explored the effects of a 4-week daily (20 mg/kg i.p.) administration of silybin in 6-weekold db/db mice feeding a methionine-choline deficient (MCD) diet . Liver histology was evaluated following the NAFLD activity score (NAS); mitochondria morphology was evaluated by electron microscopy. Liver triglycerides were determined using a colorimetric kit; liver 8-hydroxyguanosine (8-OHG) was determined by ELISA; thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) were measured by a TBARS and a GSH assay kit. Hepatic nitrite/nitrate was measured colorimetrically using Griess reaction. Expression of iNOS and 3-nitrotyrosine (3-NT) was evaluated by western blot and immunofluorescence.
AASLD Abstracts
S-798