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Mo1036 Vitamin E and L-Carnitine Prevents Progression of Non-Alcoholic Steatohepatitis With Regulation of Intestinal Inflammasome Activation in NASH Model Mice
Here, we compared the effect of acute obstructive cholestasis on liver injury, inflammation, and homeostasis of bile salts and bilirubin in normal and steatotic rat livers. Methods: Male Wistar rats were fed regular chow or a methionine-choline deficient (MCD) diet for 3 weeks to induce simple steatosis. In the third week of the diet, animals underwent either sham surgery or common bile duct ligation (BDL) to induce obstructive cholestasis for 1 week, followed by euthanasia. Plasma ALT, ALP, GGT, bilirubin, albumin, and coagulation factors were determined enzymatically. Liver tissue was processed for H&E histology and enzymatic measurement of TNF-α, IL-6 and myeloperoxidase (MPO). HPLC was used to determine bile salt spectra in plasma and liver. Hepatic mRNA and protein expression of key transporters and enzymes involved in bile salt and bilirubin homeostasis were quantified using real time PCR and Western blotting. Four groups (sham, MCD, BDL, and MCD+BDL) of 5-6 animals were statistically compared using ANOVA/Tukey's test. Data are provided as mean±SD. Results: Plasma ALT, ALP, and GGT were increased 1.6-fold, 2.0-fold, and 13.0-fold in MCD+BDL over BDL, respectively ( p,0.01). In addition, histological scoring showed that fibrosis, ductular reaction, and neutrophil influx were increased, accompanied by elevated hepatic MPO activity (p,0.01). Notably, the extent of macrovesicular steatosis was .66% of hepatocytes in MCD but ,33% in MCD+BDL (p,0.001). Plasma total bilirubin was 144±53 μM in BDL compared to 270±56 μM in MCD+BDL ( p,0.001). Plasma total bile salt levels were 388±89 μM in BDL compared to 1210±394 μM in MCD+BDL ( p,0.001), accompanied by a shift from tauro- β-muricholate to taurocholate in MCD+BDL compared to BDL. In contrast, hepatic total bile salt levels were 604±98 μM in BDL and 706±260 μM in MCD+BDL (n.s.), with a comparable bile salt composition. Determination of hepatic mRNA levels of Cyp7a1, Bsep, Mrp2, Mrp3, Mrp4, Ntcp, Oatp1a1, Oatp1a4, and Oatp1b2 revealed that expression of the rate-limiting enzyme in bile salt synthesis, Cyp7a1, was induced 1.6-fold in MCD+BDL over BDL ( p,0.05), whereas basolateral conjugate export pump Mrp3 was induced 30-fold over control levels in BDL and 57-fold in MCD+BDL (p,0.01). Conclusions: The present data indicate that experimental steatosis sensitizes rats to liver injury from acute obstructive cholestasis. Plasma bile salt levels were elevated, which may have resulted from increased bile salt synthesis and upregulation of Mrp3.
Vitamin E and L-Carnitine Prevents Progression of Non-Alcoholic Steatohepatitis With Regulation of Intestinal Inflammasome Activation in NASH Model Mice Hisashi Ishikawa (Purpose) The inflammasomes trigger the biological maturation of proinflammatory cytokines such as interleukin-1 beta or interleukin 18. They induce inflammation as expected, and also, they mediate host defence against microbial pathogens. Three Nod-like receptors (NLR family; NLRP1, NLRP3, NLRC4) are involved in the assembly of multiprotein complex inflammasomes. Recent advances in understanding pathological organ network such as intestine-to-liver revealed that intestinal inflammasomes have important role in the liver inflammation and fibrosis. It has been reported that intesitinal inflammasome activation plays an important role in the progression of NASH (Nature.2012). The intestinal inflammasome activation induces recovery of NASH and the somatic deletion of inflammasome gene results in progression of NASH. Anti-oxidants, such as vitamin E and pioglitazone have favorable effects in NASH. L-Carnitine plays an important role in mitochondrial beta-oxidation and has been reported to be effective in NASH. In this study, we examined the effects of vitamin E and L-carnitine as antioxidant supplementations on liver pathology and regulation of intestinal inflammasome activation in NASH mouse model.. (Methods) We used streptozotocin(STZ)-treated mouse diabetes model with high fat diet supplementation, known as the NASH hepatocarcinogenesis model (STAM mice). Eight-week-old STAM mice were divided into 3 experimental groups and fed for 4 weeks as follows:(1) high fat diet (S group) (2) high fat diet + L-Carnitine (SC group) (3) high fat diet + vitamin E (SE group). After 4 weeks mice were sacrificed. Following data were compared between these three groups; hepatic histological findings, hepatic 8-OHdG concentration, expression of hepatic inflammatory and hepatic lipogenic genes, and intestinal inflammasome related genes by real time PCR. (Results) In histological analysis, SC group and SE group showed low inflammation and fibrosis compared with S group. The concentration of 8-OHdG was reduced in SC and SE group compared with S group. The hepatic gene expression of inflammatory cytokine TNF-α was down-regulated in SC and SE group. The beta oxidation pathway related genes AOX and MCAD, the peroxisomal proliferator-activated receptor PPAR- α and PPAR-γ were up-regulated in SC and SE group. On the other hand, the intestinal inflammasome related genes were up-regulated in SC and SE group compared with S group. (Conclusion) Vitamin E and L-carnitine may prevent progression of non-alcoholic steatohepatitis with up-regulation of intestinal inflammasome activation.
Mo1039 Honokiol Resolves Hepatic-Steatosis and Inhibits Lipotoxic Effects of Free Fatty Acids in Primary Human Hepatocytes and Huh7 Cells Panjamurthy Kuppusamy, Sandeep Khurana, Dipali Sharma, Neeraj K. Saxena
Mo1037 Dysregulated Methionine Metabolism and Hyperhomocysteinemia in DietInduced Nonalcoholic Fatty Liver Disease Tommy Pacana, Aurora Verdianelli, Haeki Min, Faridoddin Mirshahi, Eoin Quinlivan, Arun J. Sanyal BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is associated with hyperhomocysteinemia, endothelial dysfunction and oxidative stress. Methionine metabolism plays a central role in methylatation reactions, adaptive response to oxidative stress, production of methylarginines which modulate endothelial function and regulating the levels of homocysteine. The mechanisms by which these are affected in NAFLD are not fully understood. AIMS: To perform a metabolomic, molecular and epigenetic analysis of methionine metabolism in a diet-induced model of NAFLD. METHODS: C57BL/S129 mice were fed a chow (n=6) or high-fat high-calorie diet (n=8) for 52 weeks. Serum and liver chemistries were measured. Metabolomic study on methionine and homocysteine metabolism was measured through LC-MS/MS and HPLC. Protein and gene expression of methionine adenosyltransferase I/III (MAT I/III), cystathionine β-synthase (CS), γ-glutamylcysteinesynthetase (GCS), all enzymes involved in methionine and homocysteine metabolism, were also characterized. The methylation status of lipogenic (HMGCoA reductase and fatty acid synthetase), inflammatory (NF κB1, Jun) and apoptosis (BCL-2 and caspase 3) targets were also assessed. RESULTS: HFHCD led to weight gain, marked steatosis and fibrosis. NAFLD was associated with 0.7-fold depletion of methionine (p, 0.01) and 1.3 fold increase homocysteine levels (p , 0.01). While s-adenosylmethionine (SAM) trended upwards, s-adenosylhomocysteine (SAH) was significantly increased (1.7 fold, p , 0.01). There was a trend for increased cystathionine and cysteine whereas glutathione was decreased in NAFLD. There was a trend towards decreased cysteinyl glycine in NAFLD. Among SAMe-dependent methylated products, only monomethylarginine (MMA) and asymmetric dimethylarginines (ADMA) were decreased significantly (p ,0.05), although the decrease in symmetric dimethylarginine (SDMA) almost reached statistical significance (p, 0.07). Other Methylated products measured (sarcosine, betaine, creatine, and choline) showed no siginificant changes between chow and NAFLD group. Although there was decreased gene expression of GCS, MAT I/III or CS, no changes in the protein expression were observed, suggesting postranscriptional modifications. There was an increase in methylation of HMGCR in NAFLD while no changes in methylation status of other targets were noted. CONCLUSION: Under conditions of long-term HFHCD, homocysteine remethylation was blocked resulting in hyperhomocysteinemia and methionine depletion. Accumulation of homocysteine was associated with preserved transulfuration pathway to replete depleted glutathione stores. All methyarginines were depleted suggesting decreased synthesis potentially due to inhibition by SAH. Mo1038 Obstructive Cholestasis in Rats With Simple Steatosis Is Characterized by Exacerbated Liver Injury and Increased Plasma Bile Salts and Bilirubin Daniël A. Lionarons, Michal Heger, Jaap J. Kloek, Dirk R. de Waart, Hendrik A. Marsman, Joanne Verheij, Ulrich Beuers, Coen C. Paulusma, Thomas M. Van Gulik Background: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disorder worldwide. NAFLD comprises a broad and progressive disease spectrum, where simple steatosis is the initial and most common form. Steatosis may sensitize the liver to damage by drugs and toxins. Effects of cholestasis on the steatotic liver are less well studied.
S-1019
Non-alcoholic fatty liver disease (NAFLD) is characterized by accumulation of fat in the liver; designated as hepatic-steatosis. NAFLD has a histological spectrum which progresses from fatty liver (hepatic-steatosis) through non-alcoholic steatohepatitis (NASH) to cirrhosis, a milieu for hepatocellular carcinoma. In NASH, free fatty acids (FFA) induce endoplasmic reticulum (ER) stress followed by hepatocyte-apoptosis leading to inflammation of liver. Recently, autophagy has been identified as a mechanism for removal of FFA from hepatocytes. Novel therapeutic approaches for NAFLD are desirable since current available therapies are sub-optimal. Natural products have presented an important avenue for development of effective, non-toxic therapies. Honokiol (HNK), a bio-active compound, derived from the bark of Magnolia sp. is used as a dietary supplement in Asian-traditional-medicine. AIM: In the present study, we sought to examine the efficacy and elucidate the mechanism whereby Honokiol resolves hepatic-steatosis. METHODS: Primary human hepatocytes (PHH) and HCC cell line, Huh7 were loaded with FFAs (palmitate or oleate) followed by Honokiol treatment. Fat loading was observed by oil-red-O staining. Lipotoxicity was measured by XTT assay for cell survival and caspase 3-activity for apoptosis. Modulations in expression level of ER stress (PERK, GRP78 and CHOP) and autophagy (Beclin1, LAMP2, Atg7, Atg12 and conversion of LC3-BI to LC3-BII) markers were analyzed by immunoblot assay. Functional importance of autophagy in HNK-mediated fat resolution was examined by ATG7 silencing using siRNA. RESULTS: HNK treatment of FFA loaded PHH and Huh7 cells results in (a) reduced FFA load; (b) suppression of FFA lipotoxicity with reduced cell death and apoptosis (c) reduced ER stress with decreased expression of GRP78 and CHOP (d) increased expression of autophagy markers Beclin1, LAMP2, Atg7, Atg12 and LC3-B. Importantly, ablation of Atg7 in Huh7 cells abrogates protective effects of HNK. CONCLUSIONS: In both PHH and Huh7 cells, Honokiol attenuates lipotoxicity by reducing FFA accumulation, ER stress and cell death via macroautophagy. HNK is a potential novel therapy to resolve hepatic-steatosis and treat NAFLD. Mo1040 Decaffeinated Coffee Reduces Body Weight Gain in Rats Fed With a Hfd by Modulating Intestinal Gpr41 and CCK Expression Giovanna Mazzone, Giuseppe D'Argenio, Filomena Morisco, Vincenzo Lembo, Paola Vitaglione, Rosa Vitiello, Ilaria Loperto, Francesco Auriemma, Vincenzo Fogliano, Nicola Caporaso Background & Aims: A role for coffee beverage on the reduction of weight gain caused by a high-fat diet (HFD) was suggested. Some intestinal chemosensors belonging to the Gprotein-coupled receptor (GPR) family and responding to specific nutrients can play a role to ameliorate metabolic abnormalities caused by HFD. This study aimed to investigate the effect of decaffeinated coffee on body weight and on expressions of nutrient responsive receptors in rats fed with HFD. Methods: Eight rats were fed with a HFD for 5 months. One group (4 rats)drunk simple water while the other group (4 rats) had drinking water added with 1.2 mL decaffeinated coffee per die starting from the 4th month. Contemporarily, 4 rats were fed a standard diet and used as controls. Protein and mRNA expression levels of the intestinal peptides CCK and PYY, and of the fatty acid-responsive receptors GPR40 and GPR41were determined from proximal jejunum of all rats. Results are expressed as fold changes compared to controls. Results: A mean 47% higher weight gain in HFD+ water vs HFD + coffee rats was recorded at the end of study ( p ,0.01). CCK gene expression was significantly reduced in rats fed with HFD vs control (0.46+0.4 vs 1.03+0.05, p ,0.03). GPR41 expression was up regulated in HFD + water vs control rats (2.33+0.55 vs 1.03+0.06, p,0.05) and down regulated in HFD + coffee vs HFD + water (1.35+0.28 vs 2.33+0.55, p,0.05). No significant difference were observed for PYY and GPR40 among groups. Conclusions: It was demonstrated that decaffeinated coffee beverage reduces body weight gain in
AASLD Abstracts
AASLD Abstracts
Mo1036
Vitamin E and L-Carnitine Prevents Progression of Non-Alcoholic Steatohepatitis With Regulation of Intestinal Inflammasome Activation in NASH Model Mice Hisashi Ishikawa (Purpose) The inflammasomes trigger the biological maturation of proinflammatory cytokines such as interleukin-1 beta or interleukin 18. They induce inflammation as expected, and also, they mediate host defence against microbial pathogens. Three Nod-like receptors (NLR family; NLRP1, NLRP3, NLRC4) are involved in the assembly of multiprotein complex inflammasomes. Recent advances in understanding pathological organ network such as intestine-to-liver revealed that intestinal inflammasomes have important role in the liver inflammation and fibrosis. It has been reported that intesitinal inflammasome activation plays an important role in the progression of NASH (Nature.2012). The intestinal inflammasome activation induces recovery of NASH and the somatic deletion of inflammasome gene results in progression of NASH. Anti-oxidants, such as vitamin E and pioglitazone have favorable effects in NASH. L-Carnitine plays an important role in mitochondrial beta-oxidation and has been reported to be effective in NASH. In this study, we examined the effects of vitamin E and L-carnitine as antioxidant supplementations on liver pathology and regulation of intestinal inflammasome activation in NASH mouse model.. (Methods) We used streptozotocin(STZ)-treated mouse diabetes model with high fat diet supplementation, known as the NASH hepatocarcinogenesis model (STAM mice). Eight-week-old STAM mice were divided into 3 experimental groups and fed for 4 weeks as follows:(1) high fat diet (S group) (2) high fat diet + L-Carnitine (SC group) (3) high fat diet + vitamin E (SE group). After 4 weeks mice were sacrificed. Following data were compared between these three groups; hepatic histological findings, hepatic 8-OHdG concentration, expression of hepatic inflammatory and hepatic lipogenic genes, and intestinal inflammasome related genes by real time PCR. (Results) In histological analysis, SC group and SE group showed low inflammation and fibrosis compared with S group. The concentration of 8-OHdG was reduced in SC and SE group compared with S group. The hepatic gene expression of inflammatory cytokine TNF-α was down-regulated in SC and SE group. The beta oxidation pathway related genes AOX and MCAD, the peroxisomal proliferator-activated receptor PPAR- α and PPAR-γ were up-regulated in SC and SE group. On the other hand, the intestinal inflammasome related genes were up-regulated in SC and SE group compared with S group. (Conclusion) Vitamin E and L-carnitine may prevent progression of non-alcoholic steatohepatitis with up-regulation of intestinal inflammasome activation.
Mo1039 Honokiol Resolves Hepatic-Steatosis and Inhibits Lipotoxic Effects of Free Fatty Acids in Primary Human Hepatocytes and Huh7 Cells Panjamurthy Kuppusamy, Sandeep Khurana, Dipali Sharma, Neeraj K. Saxena
Mo1037 Dysregulated Methionine Metabolism and Hyperhomocysteinemia in DietInduced Nonalcoholic Fatty Liver Disease Tommy Pacana, Aurora Verdianelli, Haeki Min, Faridoddin Mirshahi, Eoin Quinlivan, Arun J. Sanyal BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is associated with hyperhomocysteinemia, endothelial dysfunction and oxidative stress. Methionine metabolism plays a central role in methylatation reactions, adaptive response to oxidative stress, production of methylarginines which modulate endothelial function and regulating the levels of homocysteine. The mechanisms by which these are affected in NAFLD are not fully understood. AIMS: To perform a metabolomic, molecular and epigenetic analysis of methionine metabolism in a diet-induced model of NAFLD. METHODS: C57BL/S129 mice were fed a chow (n=6) or high-fat high-calorie diet (n=8) for 52 weeks. Serum and liver chemistries were measured. Metabolomic study on methionine and homocysteine metabolism was measured through LC-MS/MS and HPLC. Protein and gene expression of methionine adenosyltransferase I/III (MAT I/III), cystathionine β-synthase (CS), γ-glutamylcysteinesynthetase (GCS), all enzymes involved in methionine and homocysteine metabolism, were also characterized. The methylation status of lipogenic (HMGCoA reductase and fatty acid synthetase), inflammatory (NF κB1, Jun) and apoptosis (BCL-2 and caspase 3) targets were also assessed. RESULTS: HFHCD led to weight gain, marked steatosis and fibrosis. NAFLD was associated with 0.7-fold depletion of methionine (p, 0.01) and 1.3 fold increase homocysteine levels (p , 0.01). While s-adenosylmethionine (SAM) trended upwards, s-adenosylhomocysteine (SAH) was significantly increased (1.7 fold, p , 0.01). There was a trend for increased cystathionine and cysteine whereas glutathione was decreased in NAFLD. There was a trend towards decreased cysteinyl glycine in NAFLD. Among SAMe-dependent methylated products, only monomethylarginine (MMA) and asymmetric dimethylarginines (ADMA) were decreased significantly (p ,0.05), although the decrease in symmetric dimethylarginine (SDMA) almost reached statistical significance (p, 0.07). Other Methylated products measured (sarcosine, betaine, creatine, and choline) showed no siginificant changes between chow and NAFLD group. Although there was decreased gene expression of GCS, MAT I/III or CS, no changes in the protein expression were observed, suggesting postranscriptional modifications. There was an increase in methylation of HMGCR in NAFLD while no changes in methylation status of other targets were noted. CONCLUSION: Under conditions of long-term HFHCD, homocysteine remethylation was blocked resulting in hyperhomocysteinemia and methionine depletion. Accumulation of homocysteine was associated with preserved transulfuration pathway to replete depleted glutathione stores. All methyarginines were depleted suggesting decreased synthesis potentially due to inhibition by SAH. Mo1038 Obstructive Cholestasis in Rats With Simple Steatosis Is Characterized by Exacerbated Liver Injury and Increased Plasma Bile Salts and Bilirubin Daniël A. Lionarons, Michal Heger, Jaap J. Kloek, Dirk R. de Waart, Hendrik A. Marsman, Joanne Verheij, Ulrich Beuers, Coen C. Paulusma, Thomas M. Van Gulik Background: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disorder worldwide. NAFLD comprises a broad and progressive disease spectrum, where simple steatosis is the initial and most common form. Steatosis may sensitize the liver to damage by drugs and toxins. Effects of cholestasis on the steatotic liver are less well studied.
S-1019
Non-alcoholic fatty liver disease (NAFLD) is characterized by accumulation of fat in the liver; designated as hepatic-steatosis. NAFLD has a histological spectrum which progresses from fatty liver (hepatic-steatosis) through non-alcoholic steatohepatitis (NASH) to cirrhosis, a milieu for hepatocellular carcinoma. In NASH, free fatty acids (FFA) induce endoplasmic reticulum (ER) stress followed by hepatocyte-apoptosis leading to inflammation of liver. Recently, autophagy has been identified as a mechanism for removal of FFA from hepatocytes. Novel therapeutic approaches for NAFLD are desirable since current available therapies are sub-optimal. Natural products have presented an important avenue for development of effective, non-toxic therapies. Honokiol (HNK), a bio-active compound, derived from the bark of Magnolia sp. is used as a dietary supplement in Asian-traditional-medicine. AIM: In the present study, we sought to examine the efficacy and elucidate the mechanism whereby Honokiol resolves hepatic-steatosis. METHODS: Primary human hepatocytes (PHH) and HCC cell line, Huh7 were loaded with FFAs (palmitate or oleate) followed by Honokiol treatment. Fat loading was observed by oil-red-O staining. Lipotoxicity was measured by XTT assay for cell survival and caspase 3-activity for apoptosis. Modulations in expression level of ER stress (PERK, GRP78 and CHOP) and autophagy (Beclin1, LAMP2, Atg7, Atg12 and conversion of LC3-BI to LC3-BII) markers were analyzed by immunoblot assay. Functional importance of autophagy in HNK-mediated fat resolution was examined by ATG7 silencing using siRNA. RESULTS: HNK treatment of FFA loaded PHH and Huh7 cells results in (a) reduced FFA load; (b) suppression of FFA lipotoxicity with reduced cell death and apoptosis (c) reduced ER stress with decreased expression of GRP78 and CHOP (d) increased expression of autophagy markers Beclin1, LAMP2, Atg7, Atg12 and LC3-B. Importantly, ablation of Atg7 in Huh7 cells abrogates protective effects of HNK. CONCLUSIONS: In both PHH and Huh7 cells, Honokiol attenuates lipotoxicity by reducing FFA accumulation, ER stress and cell death via macroautophagy. HNK is a potential novel therapy to resolve hepatic-steatosis and treat NAFLD. Mo1040 Decaffeinated Coffee Reduces Body Weight Gain in Rats Fed With a Hfd by Modulating Intestinal Gpr41 and CCK Expression Giovanna Mazzone, Giuseppe D'Argenio, Filomena Morisco, Vincenzo Lembo, Paola Vitaglione, Rosa Vitiello, Ilaria Loperto, Francesco Auriemma, Vincenzo Fogliano, Nicola Caporaso Background & Aims: A role for coffee beverage on the reduction of weight gain caused by a high-fat diet (HFD) was suggested. Some intestinal chemosensors belonging to the Gprotein-coupled receptor (GPR) family and responding to specific nutrients can play a role to ameliorate metabolic abnormalities caused by HFD. This study aimed to investigate the effect of decaffeinated coffee on body weight and on expressions of nutrient responsive receptors in rats fed with HFD. Methods: Eight rats were fed with a HFD for 5 months. One group (4 rats)drunk simple water while the other group (4 rats) had drinking water added with 1.2 mL decaffeinated coffee per die starting from the 4th month. Contemporarily, 4 rats were fed a standard diet and used as controls. Protein and mRNA expression levels of the intestinal peptides CCK and PYY, and of the fatty acid-responsive receptors GPR40 and GPR41were determined from proximal jejunum of all rats. Results are expressed as fold changes compared to controls. Results: A mean 47% higher weight gain in HFD+ water vs HFD + coffee rats was recorded at the end of study ( p ,0.01). CCK gene expression was significantly reduced in rats fed with HFD vs control (0.46+0.4 vs 1.03+0.05, p ,0.03). GPR41 expression was up regulated in HFD + water vs control rats (2.33+0.55 vs 1.03+0.06, p,0.05) and down regulated in HFD + coffee vs HFD + water (1.35+0.28 vs 2.33+0.55, p,0.05). No significant difference were observed for PYY and GPR40 among groups. Conclusions: It was demonstrated that decaffeinated coffee beverage reduces body weight gain in
AASLD Abstracts
AASLD Abstracts
Mo1036