- Email: [email protected]
Aberrant Neuronal Bile Signaling Alters Brain Cholesterol Homeostasis During Hepatic Encephalopathy Due to Acute Liver Failure
treatment. Activation of nuclear factor-κB (NFκB) mediates TLR4 proinflammatory signaling, leading to proinflammatory cytokine and adhesion molecule formation. Phospho-NFκB p65 increased after LPS, indicating NFκB activation. Tumor necrosis factor-α (TNFα) and intercellular adhesion molecule-1 (ICAM-1) were also increased by LPS. ABC294640 inhibited LPS-induced TLR4, TNFα and ICAM-1 expression and NFκB activation. Myeloperoxidase (marker of neutrophils) and F4/80 (marker of macrophages/monocytes) in liver tissue also increased markedly after LPS treatment, consistent with leukocyte infiltration, which ABC294640 blunted. Conclusion: Inhibition of SK2 decreases LPS-induced inflammatory responses and liver injury. Thus, further studies are warranted to evaluate SK2 inhibition as a potential therapy for systemic inflammation and multiple organ failure caused by sepsis (NIDDK).
HE, increased cholesterol levels in the brain are mediated by bile acid activation of FXR and SHP, which together inhibit Cyp46A1 expression via the inhibition of LXRβ. FXR/SHPmediated signaling may be a possible therapeutic target for the management of HE through its interaction with proteins controlling cholesterol homeostasis.
278 BARIATRIC SURGERY INCREASES THE RISK OF ACUTE LIVER INJURY: RESULTS OF A NATIONWIDE ANALYSIS Praneet Wander, Paul T. Kroner, Violeta Popov, Christopher C. Thompson BACKGROUND: Bariatric surgery provides a durable method of weight loss but is associated with serious adverse events. Some studies report an increase in drug-induced acute liver injury following bariatric surgery. AIM: To assess if bariatric procedures increase the risk of acute liver failure in a large inpatient cohort. Methods: We retrospectively analyzed discharge data on patients who developed acute liver injury (ALI) using the Nationwide Inpatient Sample (NIS) database from 2010-2013. Discharges with an ICD-9 code indicating ALI were included. The primary outcome was ALI in patients with a history of bariatric surgery compared to all other patients with an inpatient diagnosis of ALI. Secondary outcomes were mortality in the two cohorts and independent socio-demographic and medical risk factors for mortality in each cohort. Variables tested include age, gender, race, income, Charlson criteria, hospital factors and medical comorbidities including Malnutrition, HTN, Anemia, CKD, Diabetes, CHF, Coagulopathy, Alcoholism, HBV and HCV. Univariate and multivariate logistic regression analyses were performed to identify independent predictors. Results: During the study period, a total of 437,390 patients were diagnosed with acute liver injury and were included in the study, of which 3,799 had previously undergone bariatric surgery. In the post-bariatric cohort, mean age was 58.7 years and 77% were women . The prevalence of acute liver injury in all inpatient admissions for that time period was higher in patients with history of bariatric surgery (0.85%) than in non-bariatric patients (0.75%), chi-square=21.07, p<0.01. Rate of overall inpatient mortality was higher in nonbariatric cohort (15.9% versus 9.3%). Post-bariatric patients admitted for ALI were more likely to be younger, female, Caucasian and residing in more affluent areas (Table 1) Post-bariatric patients were also more likely to have higher rates of malnutrition, anemia, alcoholism, and significantly lower prevalence of hepatitis B and C, CHF, diabetes and kidney disease. In a multivariate regression model, the presence of CHF and coagulopathy increased mortality risk, and diagnosis of alcoholism was associated with lower mortality risk from ALI in patients with prior history of bariatric surgery (Table 2). Conclusion: Bariatric surgery increases the risk of subsequent acute liver injury. Post-bariatric surgery patients admitted for ALI are more likely to have anemia, malnutrition, and alcoholism, supporting the hypothesis that baseline nutritional status may predispose to drug-induced ALI. Addressing these potentially modifiable risk factors may decrease the significant morbidity and mortality of ALI. Socio-demographic and medical characteristics of patients with a prior bariatric surgery compared to those without history of bariatric surgery admitted for ALI
276
AASLD Abstracts
MIR-155 DEPENDENT DYSREGULATION OF AUTOPHAGY IN HEPATOCYTES AND MACROPHAGES DRIVES THE DEVELOPMENT OF ALD BY EXPLOITING THE EXOSOMAL PATHWAY Istvan Furi, Terence N. Bukong, Shashi Bala, Patrick Lowe, Arvin Iracheta-Vellve, Karen Kodys, Aditya Ambade, Gyongyi Szabo Background: Autophagy is a homeostatic process by which defective cytosolic organelles, proteins, and nucleic acids are sequestered into membrane vesicles, autophagosomes, and delivered for lysosomal degradation. Studies have shown that alcoholic liver disease (ALD) causes significant deregulation of autophagy. However, the mechanism by which autophagy modulates the development of ALD remains elusive. Recently, we showed increased numbers of circulating exosomes in patients with alcoholic hepatitis and in animal models of ALD. We found that these ‘pathogenic' exosomes were enriched in miR-155, a central regulator of inflammation and miR-155 knockout mice were protected from ALD. Here we hypothesized that molecular cross-talks between miRNA-155, autophagy and release of ‘pathogenic' exosomes contribute to ALD. Methods: C57BL/6 mice and miR-155 deficient (KO) mice received Lieber De Carli 5% alcohol or control diet for 6 weeks. In vitro studies made use of primary mouse hepatocytes, Kupffer cells (KC) as well as RAW267.4 and Hepa1-6 cells. Translational studies were performed on liver samples from control donors and patients with ALD. Experimental analyses of samples was done by RT-qPCR, western blotting and NanoSight analysis. Results: We found that ALD in mice was associated with incomplete autophagy indicated by significantly higher levels of hepatic LC3II and p62 in alcohol-fed compared to control mice. Strikingly, miR-155 KO alcohol-fed mice maintained functional autophagy demonstrated by lower LC3II and decreased p62 levels. Further, miR-155-KO alcohol-fed mice had significantly lower numbers of circulating exosomes compared to WT alcohol-fed mice. Functionally, we found that increased miR-155 targets Rheb and mTOR leading to decreased cellular homoeostasis inducing autophagy. Mechanistically, in vitro studies showed that alcohol treatment (50mM, 24h) of KCs increased cellular release of exosomes with high levels of miR-155. Co-culture of miR-155 enriched exosomes and or transfection of miR-155 mimic into Hepa1-6 hepatocytes or RAW267.4 macrophages induced a significant increase in exosome release. Functionally, in both cell types, increase in miR155 significantly reduced mTOR and Rheb protein expression. Simultaneously, cellular expression of LC3II significantly increased and p62 decreased demonstrating a direct role of miR-155 in autophagy induction. Furthermore, analysis of ALD liver samples revealed decreased Rheb and mTOR but increased LC3II and p62 suggesting defective autolysosomal degradation in human ALD. Conclusions: Our observations suggest a cross-talk between miR-155 and cellular autophagy in the production of ‘pathogenic' exosomes in ALD. These results define a mechanistic role for alcohol-related dysregulation of autophagy both in hepatocytes and macrophages and highlight miR-155 as a potential therapeutic target in ALD.
277 ABERRANT NEURONAL BILE SIGNALING ALTERS BRAIN CHOLESTEROL HOMEOSTASIS DURING HEPATIC ENCEPHALOPATHY DUE TO ACUTE LIVER FAILURE Shamyal H. Khan, Matthew McMillin, Stephanie Grant, Gabriel Frampton, Sharon DeMorrow During acute liver failure (ALF), we have demonstrated that increased serum bile acids induce permeability of the blood-brain barrier and contribute to the development of hepatic encephalopathy (HE) via activation of farnesoid X receptor (FXR) in neurons. The events downstream of FXR activation contributing to HE are unknown. FXR activation is known to decrease the expression of liver X receptor beta (LXRβ). Highly expressed in neurons, LXRβ has a role in regulation of Cyp46A1, an enzyme responsible for converting cholesterol to 24(S)-hydroxycholesterol (24S-HC), the main mechanism of cholesterol clearance in the brain. We hypothesize that increased levels of cholesterol in the brain during ALF and HE result from bile acid-mediated activation of FXR and LXRβ suppression leading to a dysregulation of cholesterol clearance. Methods: C57Bl/6 mice or Cyp7A1 knockout (Cyp7A1-/-) mice were injected with azoxymethane (AOM) to induce ALF and HE. In parallel, C57Bl/6 mice were given an intracerebroventricular infusion of FXR Vivo-Morpholino sequences to reduce FXR protein expression, or methyl-β-cyclodextrin (Mβ-CD) to reduce brain cholesterol prior to AOM injection. Cognitive impairment was monitored by assessment of reflex responses and ataxia. Primary neurons were treated with taurocholic acid (TCA) and/or guggulsterone (an FXR inhibitor). Liver damage was assessed by hematoxylin and eosin staining and serum liver enzyme measurement. Cyp46A1, SHP, and LXRβ expression were assessed by immunoblotting and qPCR. Brain and serum levels of cholesterol and 24SHC were measured using commercially available kits. Results: Cortical LXRb and Cyp46A1 expression were decreased in AOM-treated mice with a concomitant increase in total cortical cholesterol and decrease in 24S-HC compared to controls. These alterations were not observed in Cyp7A1-/- mice that have a reduced bile acid pool, nor in mice treated with FXR VivoMorpholino sequences to block cortical FXR expression. Treatment of primary neurons with DCA reduced Cyp46A1 protein and mRNA expression, which was inhibited when cotreated with guggulsterone. Pre-treatment with Mβ-CD decreased levels of AST, ALT, and bilirubin, reduced liver damage, lowered serum and brain cholesterol levels, and reduced cognitive impairment compared to AOM-treated mice. Conclusion: During AOM-induced ALF and
AASLD Abstracts
S-1046
HE, increased cholesterol levels in the brain are mediated by bile acid activation of FXR and SHP, which together inhibit Cyp46A1 expression via the inhibition of LXRβ. FXR/SHPmediated signaling may be a possible therapeutic target for the management of HE through its interaction with proteins controlling cholesterol homeostasis.
278 BARIATRIC SURGERY INCREASES THE RISK OF ACUTE LIVER INJURY: RESULTS OF A NATIONWIDE ANALYSIS Praneet Wander, Paul T. Kroner, Violeta Popov, Christopher C. Thompson BACKGROUND: Bariatric surgery provides a durable method of weight loss but is associated with serious adverse events. Some studies report an increase in drug-induced acute liver injury following bariatric surgery. AIM: To assess if bariatric procedures increase the risk of acute liver failure in a large inpatient cohort. Methods: We retrospectively analyzed discharge data on patients who developed acute liver injury (ALI) using the Nationwide Inpatient Sample (NIS) database from 2010-2013. Discharges with an ICD-9 code indicating ALI were included. The primary outcome was ALI in patients with a history of bariatric surgery compared to all other patients with an inpatient diagnosis of ALI. Secondary outcomes were mortality in the two cohorts and independent socio-demographic and medical risk factors for mortality in each cohort. Variables tested include age, gender, race, income, Charlson criteria, hospital factors and medical comorbidities including Malnutrition, HTN, Anemia, CKD, Diabetes, CHF, Coagulopathy, Alcoholism, HBV and HCV. Univariate and multivariate logistic regression analyses were performed to identify independent predictors. Results: During the study period, a total of 437,390 patients were diagnosed with acute liver injury and were included in the study, of which 3,799 had previously undergone bariatric surgery. In the post-bariatric cohort, mean age was 58.7 years and 77% were women . The prevalence of acute liver injury in all inpatient admissions for that time period was higher in patients with history of bariatric surgery (0.85%) than in non-bariatric patients (0.75%), chi-square=21.07, p<0.01. Rate of overall inpatient mortality was higher in nonbariatric cohort (15.9% versus 9.3%). Post-bariatric patients admitted for ALI were more likely to be younger, female, Caucasian and residing in more affluent areas (Table 1) Post-bariatric patients were also more likely to have higher rates of malnutrition, anemia, alcoholism, and significantly lower prevalence of hepatitis B and C, CHF, diabetes and kidney disease. In a multivariate regression model, the presence of CHF and coagulopathy increased mortality risk, and diagnosis of alcoholism was associated with lower mortality risk from ALI in patients with prior history of bariatric surgery (Table 2). Conclusion: Bariatric surgery increases the risk of subsequent acute liver injury. Post-bariatric surgery patients admitted for ALI are more likely to have anemia, malnutrition, and alcoholism, supporting the hypothesis that baseline nutritional status may predispose to drug-induced ALI. Addressing these potentially modifiable risk factors may decrease the significant morbidity and mortality of ALI. Socio-demographic and medical characteristics of patients with a prior bariatric surgery compared to those without history of bariatric surgery admitted for ALI
276
AASLD Abstracts
MIR-155 DEPENDENT DYSREGULATION OF AUTOPHAGY IN HEPATOCYTES AND MACROPHAGES DRIVES THE DEVELOPMENT OF ALD BY EXPLOITING THE EXOSOMAL PATHWAY Istvan Furi, Terence N. Bukong, Shashi Bala, Patrick Lowe, Arvin Iracheta-Vellve, Karen Kodys, Aditya Ambade, Gyongyi Szabo Background: Autophagy is a homeostatic process by which defective cytosolic organelles, proteins, and nucleic acids are sequestered into membrane vesicles, autophagosomes, and delivered for lysosomal degradation. Studies have shown that alcoholic liver disease (ALD) causes significant deregulation of autophagy. However, the mechanism by which autophagy modulates the development of ALD remains elusive. Recently, we showed increased numbers of circulating exosomes in patients with alcoholic hepatitis and in animal models of ALD. We found that these ‘pathogenic' exosomes were enriched in miR-155, a central regulator of inflammation and miR-155 knockout mice were protected from ALD. Here we hypothesized that molecular cross-talks between miRNA-155, autophagy and release of ‘pathogenic' exosomes contribute to ALD. Methods: C57BL/6 mice and miR-155 deficient (KO) mice received Lieber De Carli 5% alcohol or control diet for 6 weeks. In vitro studies made use of primary mouse hepatocytes, Kupffer cells (KC) as well as RAW267.4 and Hepa1-6 cells. Translational studies were performed on liver samples from control donors and patients with ALD. Experimental analyses of samples was done by RT-qPCR, western blotting and NanoSight analysis. Results: We found that ALD in mice was associated with incomplete autophagy indicated by significantly higher levels of hepatic LC3II and p62 in alcohol-fed compared to control mice. Strikingly, miR-155 KO alcohol-fed mice maintained functional autophagy demonstrated by lower LC3II and decreased p62 levels. Further, miR-155-KO alcohol-fed mice had significantly lower numbers of circulating exosomes compared to WT alcohol-fed mice. Functionally, we found that increased miR-155 targets Rheb and mTOR leading to decreased cellular homoeostasis inducing autophagy. Mechanistically, in vitro studies showed that alcohol treatment (50mM, 24h) of KCs increased cellular release of exosomes with high levels of miR-155. Co-culture of miR-155 enriched exosomes and or transfection of miR-155 mimic into Hepa1-6 hepatocytes or RAW267.4 macrophages induced a significant increase in exosome release. Functionally, in both cell types, increase in miR155 significantly reduced mTOR and Rheb protein expression. Simultaneously, cellular expression of LC3II significantly increased and p62 decreased demonstrating a direct role of miR-155 in autophagy induction. Furthermore, analysis of ALD liver samples revealed decreased Rheb and mTOR but increased LC3II and p62 suggesting defective autolysosomal degradation in human ALD. Conclusions: Our observations suggest a cross-talk between miR-155 and cellular autophagy in the production of ‘pathogenic' exosomes in ALD. These results define a mechanistic role for alcohol-related dysregulation of autophagy both in hepatocytes and macrophages and highlight miR-155 as a potential therapeutic target in ALD.
277 ABERRANT NEURONAL BILE SIGNALING ALTERS BRAIN CHOLESTEROL HOMEOSTASIS DURING HEPATIC ENCEPHALOPATHY DUE TO ACUTE LIVER FAILURE Shamyal H. Khan, Matthew McMillin, Stephanie Grant, Gabriel Frampton, Sharon DeMorrow During acute liver failure (ALF), we have demonstrated that increased serum bile acids induce permeability of the blood-brain barrier and contribute to the development of hepatic encephalopathy (HE) via activation of farnesoid X receptor (FXR) in neurons. The events downstream of FXR activation contributing to HE are unknown. FXR activation is known to decrease the expression of liver X receptor beta (LXRβ). Highly expressed in neurons, LXRβ has a role in regulation of Cyp46A1, an enzyme responsible for converting cholesterol to 24(S)-hydroxycholesterol (24S-HC), the main mechanism of cholesterol clearance in the brain. We hypothesize that increased levels of cholesterol in the brain during ALF and HE result from bile acid-mediated activation of FXR and LXRβ suppression leading to a dysregulation of cholesterol clearance. Methods: C57Bl/6 mice or Cyp7A1 knockout (Cyp7A1-/-) mice were injected with azoxymethane (AOM) to induce ALF and HE. In parallel, C57Bl/6 mice were given an intracerebroventricular infusion of FXR Vivo-Morpholino sequences to reduce FXR protein expression, or methyl-β-cyclodextrin (Mβ-CD) to reduce brain cholesterol prior to AOM injection. Cognitive impairment was monitored by assessment of reflex responses and ataxia. Primary neurons were treated with taurocholic acid (TCA) and/or guggulsterone (an FXR inhibitor). Liver damage was assessed by hematoxylin and eosin staining and serum liver enzyme measurement. Cyp46A1, SHP, and LXRβ expression were assessed by immunoblotting and qPCR. Brain and serum levels of cholesterol and 24SHC were measured using commercially available kits. Results: Cortical LXRb and Cyp46A1 expression were decreased in AOM-treated mice with a concomitant increase in total cortical cholesterol and decrease in 24S-HC compared to controls. These alterations were not observed in Cyp7A1-/- mice that have a reduced bile acid pool, nor in mice treated with FXR VivoMorpholino sequences to block cortical FXR expression. Treatment of primary neurons with DCA reduced Cyp46A1 protein and mRNA expression, which was inhibited when cotreated with guggulsterone. Pre-treatment with Mβ-CD decreased levels of AST, ALT, and bilirubin, reduced liver damage, lowered serum and brain cholesterol levels, and reduced cognitive impairment compared to AOM-treated mice. Conclusion: During AOM-induced ALF and
AASLD Abstracts
S-1046