- Email: [email protected]
Gastric Gene Expression of Inflammatory Cytokines in Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH)
by oil red O stain, reporter gene assay, reverse transcriptase polymerase chain reaction (RT PCR), real time PCR, chromatin immunoprecipitation (ChIP) assay, and immunoblot analysis. Results: Cellular TG content was increased when HepG2 were cultured in the presence of oleic acid, and this increase was prevented by guggulsterone treatment. Guggulsterone prevented LXRα agonist (T0901317) induced activation of SREBP-1c, inhibited its mRNA and protein induction, thereby resulting in the down-regulation of SREBP-1c target genes. This inhibitory effect of guggulsterone was blocked by the protein kinase C (PKC) inhibitor. Moreover, guggulsterone enhanced LXRα phosphorylation at threonine residue and decreased phosphorylation at serine residue, which caused impaired DNA binding activity, leading to inhibition of LXRα transactivation and expression. Conclusion: These results show that guggulsterone prevents LXRα mediated SREBP-1c dependent hepatic steatosis through PKC dependent pathway, and suggests that guggulsterone can therapeutically be used to treat nonalcoholic fatty liver disease.
expressed genes detected in this study may provide novel mechanistic insights into the pathogenesis of NAFLD. Tu1915
Background: Non-alcoholic fatty liver disease (NAFLD) represents a major metabolic health problem worldwide with oxidative stress playing a major role in the harmful progression of liver steatosis towards steatohepatitis. Antioxidants, by counteracting the oxidant effects of reactive oxygen and nitrogen species, might break off the intrahepatic mechanisms leading to a progressive accumulation of neutral lipids within the hepatocytes. Aim: We assessed the antioxidant effect of the silybin-phospholipid-vitamin E complex (SPEC) in different rat models of fatty liver disease. Methods: 5 adult rats in each group were fed nutritionally adequate, calorically equivalent steatogenic or control diets: choline-deprived (CD) for 30 days, high fat (HFD, with 20% fat and 71%, 11% and 18% total calories provided by fat, carbohydrate, and proteins, respectively) for 60 days, and control diet. SPEC (silybin 96 mg, phospholipids 200 mg, vit E 30 mg) or placebo were given by gavage once daily for 30 and 60 days in each group. Circulating and hepatic redox active and nitrogen regulating molecules (thioredoxin, glutathione, and glutathione peroxidase), NO metabolites (nitrosothiols and nitrotyrosine), lipid peroxides (TBARs), and pro-inflammatory keratins (K-18) were measured. Hepatic fatty infiltration was quantitated by automated morphometry. Results: CD and HFD produced remarkable liver steatosis (50% and 25% of liver slices, respectively) without necro-inflammatory changes. In plasma, thioredoxin and nitrosothiol levels were stable but TBARs, nitrotyrosine and K-18 levels increased significantly (1.2-8.0 folds; 0.01
Tu1913 High Fat Diet Increases Susceptibility to Hypoxia-Induced Zone 3-Dominant Hepatocyte Apoptosis in Mice Gentaro Taniguchi, Kazuyoshi Kon, Satoko Hosoya, Kyoko Okumura, Kumiko Arai, Shunhei Yamashina, Kenichi Ikejima, Sumio Watanabe Background: Metabolic-syndrome related non-alcoholic steatohepatitis (NASH) is characterized by acinar zone 3-dominant hepatic steatosis with ballooned hepatocytes and pericellular/ perivenular fibrosis. Although hepatocyte injury in zone 3 is early and prognostically important features of the NASH, it is still not clear how hepatocytes in zone 3 are injured in the pathogenesis of NASH. Obstructive sleep apnea is a common disorder whose prevalence is linked to an epidemic of obesity and causes hypoxia during sleep. Therefore, we evaluated the hypothesis that transient hypoxia causes zone 3-dominant hepatocyte injury in a high fat diet-induced steatotic liver. Methods: Male C57Bl/6 mice 8 weeks after birth fed a high fat diet with 4% fructose water (HFD). Some mice fed a low fat diet with tap water (LFD) as control. After 4 weeks, the animals were moved to the hypoxic chamber and exposed to hypoxia (8.7% O2) for 6 hours, and were sacrificed at 1 and 8 hours after finishing the exposure to hypoxia. Serum ALT levels were measured by the colorimetric method. Hepatocyte apoptosis was evaluated by the expression of caspase-cleaved cytokeratin 18, which was detected by immunohistological staining and oxidative stress was measured by the expression of 4-HNE, which also was detected by immunohistological staining. Results: After 4 weeks of consuming HFD, mice had an increase in body weight consisting of 126±2% compared with 118±1% in LFD-fed mice (p<0.01) and developed liver steatosis. Serum ALT levels of HFD-fed mice were significantly elevated from 1 hour (10.7±0.7 IU/L) to 8 hours (15.3±1.2 IU/L, p<0.01) after finishing of hypoxia, which is significantly more than LFD after 8 hours (10.0±1.0 IU/L, p<0.01). The frequency of apoptotic hepatocytes in zone 3 in the liver of HFD-fed mice was significantly increased after 8 hours reaching to 18.6±2.5% in comparison with the liver of LFD-fed mice (11.1±2.1%, p<0.05). The ratio of the 4HNE expression in hepatocytes in the acinar zone 3 to that in zone 1 was markedly elevated to 2.5±0.6 in HFD-fed mice, as compared with 1.4±0.4 in LHD fed mice (p<0.05). Conclusions: Hypoxia for 6 hours caused zone-3 dominant oxidative stress and apoptosis of hepatocyte in the liver of HFD-fed mice. These findings demonstrates that reactive oxygen species are easily increased even by transient hypoxia in acinar zone 3 of steatotic liver. Therefore, it is indicated that hepatic steatosis is susceptible to hypoxia-induced oxidative stress and obstructive sleep apnea has a potential to be a trigger of development of NASH.
Tu1916 Non-Alcoholic Fatty Liver Disease as a Harbinger of Subclinical Coronary Artery Disease Donghee Kim, W. Ray Kim, Min Jung Park, Yoon Jun Kim, Jung-Hwan Yoon, Hyo-Suk Lee Background/Aims: Non-alcoholic fatty liver disease (NAFLD) and coronary artery disease (CAD) often coexist because of overlapping risk factors that point to the underlying metabolic syndrome. For example, serum concentrations of alanine aminotransferase (ALT) and gammaglutamyl transpeptidase (gGT) have been associated CAD and overall mortality. However, it is unclear whether the presence of NAFLD represents an additional risk of CAD over and above the established risk factors of the metabolic syndrome such as diabetes, hypertension and hyperlipidemia. The aim of this study is to investigate whether NAFLD was predictive of CAD in asymptomatic individuals after adjustment for these known factors. Methods: In a health screen clinic, 4,023 subjects were recruited for this study between 2004 and 2008. The inclusion criteria consisted of absence of known liver disease such as hepatitis B or C or alcoholic liver disease and absence of symptoms suggestive of CAD. Eligible subjects underwent comprehensive screening examinations including laboratory tests and abdominal ultrasonography as well as filling out a health behavior questionnaire. In addition, multidetector computerized tomography was performed to determine coronary artery calcification (CAC), which was quantified using the Agaston method: A CAC score of 0 indicates absence of calcification, whereas scores up to 10 represent mild CAD, those up to 100 moderate CAD and those above 100 severe CAD. Ultrasonography was used to diagnose NAFLD by the characteristic increase in echogenicity of the liver parenchyma in subjects who reported daily alcohol consumption of <20g. Results: The mean age of the study subjects was 56.9 ± 9.4 years and men accounted for 60.7%. The prevalence of CAD (CAC score>0) was 32% (n= 1,286), which was mostly of mild degree (26%, n=1,035). The mean ALT was 28.2 ± 10.2 U/L with 20% of men and 8% of women having abnormal values. The mean gGT was 34.7 ± 18.9 U/L with 15% of men and 11% of women with abnormal values. In univariate analyses, ALT, gGT and NAFLD were associated with CAC as well as age, sex, body mass index, total cholesterol, triglycerides, HDL cholesterol, diabetes, and hypertension. The Table summarizes multivariable ordinal logistic regression analyses in which NAFLD was independently associated with CAC (p<0.01) after adjustment for other established risk factors. In these models, ALT was no longer significant (p=0.41), whereas GGT remained significant (p=0.046). Conclusion: These data demonstrate that NAFLD is an independent risk factor for asymptomatic CAD even after adjustment for classical cardiovascular risk factors of atherosclerosis. Although imperfect, ultrsonographic diagnosis of NAFLD is a powerful indicator of CAD. In the presence of these more direct predictors, elevated gGT but not ALT carries additional diagnostic information.
Tu1914 Gastric Gene Expression of Inflammatory Cytokines in Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) Rohini Mehta, Aybike Birerdinc, Lei Wang, Arian Afendy, Maria Stepanova, Hazem A. Elariny, Zachary Goodman, Vikas Chandhoke, Ancha Baranova, Zobair M. Younossi Obesity results from a failure in normal homeostatic mechanisms regulating food intake, fat storage, and energy utilization. Obesity is strongly associated with NAFLD and NASH. In NASH, steatosis is associated with varying degrees hepatocyte ballooning, lobular inflammation and fibrosis. In obesity, there is interplay of substances produced by various organs (visceral adipose, liver, muscle & gastric tissues). Soluble molecules produced by gastric tissue and their receptors may play a role in the pathogenesis of NAFLD. Aim: To assess the role of inflammatory molecules/receptors related genes expressed in gastric tissue of obese patients with NAFLD. Methods: The study used gastric tissue specimens from obese patients undergoing bariatric surgery. Liver biopsies were performed at the time of surgery and read by a single hepatopathologist using a pathologic protocol for NAFLD. Gene expression profiling of inflammatory cytokines and receptors in the gastric tissue was carried out using a panel which includes 84 chemokine-cytokine encoding genes involved in inflammatory response as well as their receptors (SABiosciences, Frederick, MD). Spearman's correlations of all the 84 genes were calculated for age, BMI, AST/ALT ratio, presence of hepatic inflammation (lobular or portal) and fibrosis (portal or pericellular). For group comparisons, p-values were calculated using Mann-Whitney tests. Regression models were built by stepwise bidirectional selection of predictors including clinical and laboratory parameters as well as gene expression data. Result: 20 NAFLD patients (65% NASH, age=43.4 ± 10.6, BMI=48.7 ± 8.9, AST=24.2 ± 7.1, ALT=31.3 ± 13, AST/ALT ratio=0.8 ± 0.3) were included. Expression levels of 9 genes (CCL21, CCL3, CCL4, CCR5, FNA2, IL19, IL1F8, IL8, IL8RB) significantly (p<0.05)correlated with hepatic inflammation. Additionally, 9 genes (CCL1, CCR3, CCR9, CXCL12, IL1RN, IL5, IL8RA, IL8RB,I L9) significantly (p<0.05) correlated with histologic NASH. The multivariate model predicting high grade hepatic inflammation (≥3) included male gender, age and gene expression levels of CCL4, CCR5, IFNA2, IL1F8 , IL8 and CCL21 [P=0.0009, Multiple R2 =0.858]. The model showed reduced expression of CCL4/MIP-1β and IL1F8 in NAFLD patients with high grade hepatic inflammation, while CCR5, CCL21, IL8, IFNA2, IL1F8 expressions were increased in the same patients. According to previous studies, CCL21 & IL8 are involved in granulocyte adhesion and diapedesis while CCL21, IL8 along with CCL4 participate in MAPK, TGF-β, ERK signaling pathways. Conclusions: This data indicates that gastric tissue may play a previously unexplored role in the pathogenesis of NAFLD/NASH through inflammatory pathways. With further validation, the differentially
S-985
AASLD Abstracts
AASLD Abstracts
The Silybin-Phospholipids-Vitamin E Complex Protects Against Fatty Degeneration, Nitrosative and Oxidative Stress in Rat Models of Liver Steatosis Ignazio Grattagliano, Catia V. Diogo, Domenico Ferri, David Q. Wang, Piero Portincasa
expressed genes detected in this study may provide novel mechanistic insights into the pathogenesis of NAFLD. Tu1915
Background: Non-alcoholic fatty liver disease (NAFLD) represents a major metabolic health problem worldwide with oxidative stress playing a major role in the harmful progression of liver steatosis towards steatohepatitis. Antioxidants, by counteracting the oxidant effects of reactive oxygen and nitrogen species, might break off the intrahepatic mechanisms leading to a progressive accumulation of neutral lipids within the hepatocytes. Aim: We assessed the antioxidant effect of the silybin-phospholipid-vitamin E complex (SPEC) in different rat models of fatty liver disease. Methods: 5 adult rats in each group were fed nutritionally adequate, calorically equivalent steatogenic or control diets: choline-deprived (CD) for 30 days, high fat (HFD, with 20% fat and 71%, 11% and 18% total calories provided by fat, carbohydrate, and proteins, respectively) for 60 days, and control diet. SPEC (silybin 96 mg, phospholipids 200 mg, vit E 30 mg) or placebo were given by gavage once daily for 30 and 60 days in each group. Circulating and hepatic redox active and nitrogen regulating molecules (thioredoxin, glutathione, and glutathione peroxidase), NO metabolites (nitrosothiols and nitrotyrosine), lipid peroxides (TBARs), and pro-inflammatory keratins (K-18) were measured. Hepatic fatty infiltration was quantitated by automated morphometry. Results: CD and HFD produced remarkable liver steatosis (50% and 25% of liver slices, respectively) without necro-inflammatory changes. In plasma, thioredoxin and nitrosothiol levels were stable but TBARs, nitrotyrosine and K-18 levels increased significantly (1.2-8.0 folds; 0.01
Tu1913 High Fat Diet Increases Susceptibility to Hypoxia-Induced Zone 3-Dominant Hepatocyte Apoptosis in Mice Gentaro Taniguchi, Kazuyoshi Kon, Satoko Hosoya, Kyoko Okumura, Kumiko Arai, Shunhei Yamashina, Kenichi Ikejima, Sumio Watanabe Background: Metabolic-syndrome related non-alcoholic steatohepatitis (NASH) is characterized by acinar zone 3-dominant hepatic steatosis with ballooned hepatocytes and pericellular/ perivenular fibrosis. Although hepatocyte injury in zone 3 is early and prognostically important features of the NASH, it is still not clear how hepatocytes in zone 3 are injured in the pathogenesis of NASH. Obstructive sleep apnea is a common disorder whose prevalence is linked to an epidemic of obesity and causes hypoxia during sleep. Therefore, we evaluated the hypothesis that transient hypoxia causes zone 3-dominant hepatocyte injury in a high fat diet-induced steatotic liver. Methods: Male C57Bl/6 mice 8 weeks after birth fed a high fat diet with 4% fructose water (HFD). Some mice fed a low fat diet with tap water (LFD) as control. After 4 weeks, the animals were moved to the hypoxic chamber and exposed to hypoxia (8.7% O2) for 6 hours, and were sacrificed at 1 and 8 hours after finishing the exposure to hypoxia. Serum ALT levels were measured by the colorimetric method. Hepatocyte apoptosis was evaluated by the expression of caspase-cleaved cytokeratin 18, which was detected by immunohistological staining and oxidative stress was measured by the expression of 4-HNE, which also was detected by immunohistological staining. Results: After 4 weeks of consuming HFD, mice had an increase in body weight consisting of 126±2% compared with 118±1% in LFD-fed mice (p<0.01) and developed liver steatosis. Serum ALT levels of HFD-fed mice were significantly elevated from 1 hour (10.7±0.7 IU/L) to 8 hours (15.3±1.2 IU/L, p<0.01) after finishing of hypoxia, which is significantly more than LFD after 8 hours (10.0±1.0 IU/L, p<0.01). The frequency of apoptotic hepatocytes in zone 3 in the liver of HFD-fed mice was significantly increased after 8 hours reaching to 18.6±2.5% in comparison with the liver of LFD-fed mice (11.1±2.1%, p<0.05). The ratio of the 4HNE expression in hepatocytes in the acinar zone 3 to that in zone 1 was markedly elevated to 2.5±0.6 in HFD-fed mice, as compared with 1.4±0.4 in LHD fed mice (p<0.05). Conclusions: Hypoxia for 6 hours caused zone-3 dominant oxidative stress and apoptosis of hepatocyte in the liver of HFD-fed mice. These findings demonstrates that reactive oxygen species are easily increased even by transient hypoxia in acinar zone 3 of steatotic liver. Therefore, it is indicated that hepatic steatosis is susceptible to hypoxia-induced oxidative stress and obstructive sleep apnea has a potential to be a trigger of development of NASH.
Tu1916 Non-Alcoholic Fatty Liver Disease as a Harbinger of Subclinical Coronary Artery Disease Donghee Kim, W. Ray Kim, Min Jung Park, Yoon Jun Kim, Jung-Hwan Yoon, Hyo-Suk Lee Background/Aims: Non-alcoholic fatty liver disease (NAFLD) and coronary artery disease (CAD) often coexist because of overlapping risk factors that point to the underlying metabolic syndrome. For example, serum concentrations of alanine aminotransferase (ALT) and gammaglutamyl transpeptidase (gGT) have been associated CAD and overall mortality. However, it is unclear whether the presence of NAFLD represents an additional risk of CAD over and above the established risk factors of the metabolic syndrome such as diabetes, hypertension and hyperlipidemia. The aim of this study is to investigate whether NAFLD was predictive of CAD in asymptomatic individuals after adjustment for these known factors. Methods: In a health screen clinic, 4,023 subjects were recruited for this study between 2004 and 2008. The inclusion criteria consisted of absence of known liver disease such as hepatitis B or C or alcoholic liver disease and absence of symptoms suggestive of CAD. Eligible subjects underwent comprehensive screening examinations including laboratory tests and abdominal ultrasonography as well as filling out a health behavior questionnaire. In addition, multidetector computerized tomography was performed to determine coronary artery calcification (CAC), which was quantified using the Agaston method: A CAC score of 0 indicates absence of calcification, whereas scores up to 10 represent mild CAD, those up to 100 moderate CAD and those above 100 severe CAD. Ultrasonography was used to diagnose NAFLD by the characteristic increase in echogenicity of the liver parenchyma in subjects who reported daily alcohol consumption of <20g. Results: The mean age of the study subjects was 56.9 ± 9.4 years and men accounted for 60.7%. The prevalence of CAD (CAC score>0) was 32% (n= 1,286), which was mostly of mild degree (26%, n=1,035). The mean ALT was 28.2 ± 10.2 U/L with 20% of men and 8% of women having abnormal values. The mean gGT was 34.7 ± 18.9 U/L with 15% of men and 11% of women with abnormal values. In univariate analyses, ALT, gGT and NAFLD were associated with CAC as well as age, sex, body mass index, total cholesterol, triglycerides, HDL cholesterol, diabetes, and hypertension. The Table summarizes multivariable ordinal logistic regression analyses in which NAFLD was independently associated with CAC (p<0.01) after adjustment for other established risk factors. In these models, ALT was no longer significant (p=0.41), whereas GGT remained significant (p=0.046). Conclusion: These data demonstrate that NAFLD is an independent risk factor for asymptomatic CAD even after adjustment for classical cardiovascular risk factors of atherosclerosis. Although imperfect, ultrsonographic diagnosis of NAFLD is a powerful indicator of CAD. In the presence of these more direct predictors, elevated gGT but not ALT carries additional diagnostic information.
Tu1914 Gastric Gene Expression of Inflammatory Cytokines in Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) Rohini Mehta, Aybike Birerdinc, Lei Wang, Arian Afendy, Maria Stepanova, Hazem A. Elariny, Zachary Goodman, Vikas Chandhoke, Ancha Baranova, Zobair M. Younossi Obesity results from a failure in normal homeostatic mechanisms regulating food intake, fat storage, and energy utilization. Obesity is strongly associated with NAFLD and NASH. In NASH, steatosis is associated with varying degrees hepatocyte ballooning, lobular inflammation and fibrosis. In obesity, there is interplay of substances produced by various organs (visceral adipose, liver, muscle & gastric tissues). Soluble molecules produced by gastric tissue and their receptors may play a role in the pathogenesis of NAFLD. Aim: To assess the role of inflammatory molecules/receptors related genes expressed in gastric tissue of obese patients with NAFLD. Methods: The study used gastric tissue specimens from obese patients undergoing bariatric surgery. Liver biopsies were performed at the time of surgery and read by a single hepatopathologist using a pathologic protocol for NAFLD. Gene expression profiling of inflammatory cytokines and receptors in the gastric tissue was carried out using a panel which includes 84 chemokine-cytokine encoding genes involved in inflammatory response as well as their receptors (SABiosciences, Frederick, MD). Spearman's correlations of all the 84 genes were calculated for age, BMI, AST/ALT ratio, presence of hepatic inflammation (lobular or portal) and fibrosis (portal or pericellular). For group comparisons, p-values were calculated using Mann-Whitney tests. Regression models were built by stepwise bidirectional selection of predictors including clinical and laboratory parameters as well as gene expression data. Result: 20 NAFLD patients (65% NASH, age=43.4 ± 10.6, BMI=48.7 ± 8.9, AST=24.2 ± 7.1, ALT=31.3 ± 13, AST/ALT ratio=0.8 ± 0.3) were included. Expression levels of 9 genes (CCL21, CCL3, CCL4, CCR5, FNA2, IL19, IL1F8, IL8, IL8RB) significantly (p<0.05)correlated with hepatic inflammation. Additionally, 9 genes (CCL1, CCR3, CCR9, CXCL12, IL1RN, IL5, IL8RA, IL8RB,I L9) significantly (p<0.05) correlated with histologic NASH. The multivariate model predicting high grade hepatic inflammation (≥3) included male gender, age and gene expression levels of CCL4, CCR5, IFNA2, IL1F8 , IL8 and CCL21 [P=0.0009, Multiple R2 =0.858]. The model showed reduced expression of CCL4/MIP-1β and IL1F8 in NAFLD patients with high grade hepatic inflammation, while CCR5, CCL21, IL8, IFNA2, IL1F8 expressions were increased in the same patients. According to previous studies, CCL21 & IL8 are involved in granulocyte adhesion and diapedesis while CCL21, IL8 along with CCL4 participate in MAPK, TGF-β, ERK signaling pathways. Conclusions: This data indicates that gastric tissue may play a previously unexplored role in the pathogenesis of NAFLD/NASH through inflammatory pathways. With further validation, the differentially
S-985
AASLD Abstracts
AASLD Abstracts
The Silybin-Phospholipids-Vitamin E Complex Protects Against Fatty Degeneration, Nitrosative and Oxidative Stress in Rat Models of Liver Steatosis Ignazio Grattagliano, Catia V. Diogo, Domenico Ferri, David Q. Wang, Piero Portincasa