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691 Mitochondrial adaptations to non alcoholic steatohepatitis
Posters
252 Results: among 1542 subjects, 40 (2.59%) were excluded using security
algorithms, and 1502 included: 50.3% female, median age 49yrs, 957 HP and 545 controls. Among HE 83.4% had cholesterol )200mg]dl, 93.6% LDL-C )100mg/dl, 17.5% Iriglycerides )200mg]dl, 35.9% BMI )27, 33% arterial hypertension, 31.8% insNinernia )10rnUI/ml and 13.7% glucose )6mmol/]. GGT or ALT were elevated ()50IU/1) in 215 HP (22.5%). F2F3F4 were identified by FibroTest in 25/957 (2.6°/6) HE including 13 F2, 8 F3 and 4 F4 but in none (0%) of the 545 controls (P <0.0001). Among 25 HP with fibrosis, 19 had normal ALT, 14 normal GGT, 12 normal ALT and GGT, 4 elevated ALT and GGT and 9 elevated ALT or GGT. Factors associated (p < 0.01) with fibrosis were higher age, BMI, triglycerides, uricemia, and insulinernia. In multivariate logistic regression, including alcohol consumption, insulinemia (P = 0.003) and triglycerides (P = 0.008) were the most significant risk factors. In HP with triglycerides ) 2 0 0 m # d l prevalence of fibrosis was 8.3% and 6.6% with insulinemia ) 10 mUI/ml. Conclusions: Screening strategies for liver fibrosis are feasible with biomarkers in high-risk groups such as HE Without such non-invasive strategies a liver biopsy would have been indicated in up to 22.5% of HP with elevated GGT or ALT and would have probably missed half of HP with fibrosis, who had normal GGT and ALT. References
[1] [2] [3] [41
1•
Comp Hepatol 2004. J Hepatol 2003. Clin Gaslroenterol Hepato12004. EASL 2004.
M O D E R A T E A L C O H O L INTOXICATION I N C R E A S E S TNF-(x A N D C A U S E S HEPATIC A P O P T O S I S IN O B E S E MICE
C. Demeilliers I , M-A. Robin 1, A. Sutton 1, M Paradis 2, S. Dubois 2, R Lettbron I , D. PessayreI , B. Fromenty 1. QNSERM Unit~ 481, Facult~
de MMecine Xavier Bichat, Paris, France," 2Service d'Anatomie et de Cytologic Pathologiques, H@ital Beaujon, Clichy, France Background and Aims: Both obesity and alcohol carl cause oxidative stress, cytokine induction, steatosis and steatohepatitis. In order to determine the consequences of their combination, we compared the hepatic effects of moderate ethanol binges in lean and obese ob/ob mice. Methods: Leptin-deficient C57BL/6J-ob/ob mice and their lean littermates received ethanol (2.5 g/kg daily) or water by gastric intubation for 4 consecutive days, and were killed two hours after the last administration. Results: Serum ethanol concentrations were similar in lean arid obese mice (1.4±0.3 and 1.2±0.6g/1, respectively). There was a 5-fold increase in basal levels o f plasma alanine aminotransferase (ALT) in ob/ob mice compared to lean mice but ethanol intoxication only marginally increased ALT in both groups. In lean mice, ethanol did not significantly increase plasma T N F ~ , hepatic lipid peroxidation nor caspase 3 activity, but triggered some TUNEL-positive hepatocytes. Unintoxicated ob/ob mice exhibited increased hepatic lipid peroxidation, profound mitochondrial (but not cytosolic) GSH depletion and both necrotic and apoptotic hepatocytes, despite adaptive increases in manganese superoxide dismutase (MnSOD), heat shock protein 70 (Hsp70), mitochondrial cytochrome c and mitochondrial DNA (mtDNA). In alcoholized ob/ob mice, plasma T N F ~ was increased, hepatic nuclear p65 NF-KB subunit protein expression and activity were decreased, caspase 3 was significantly activated and hepatocyte death was switched toward more apoptosis. However, despite increased cytochrome P450 2E1 activity and decreased cytosolic glutathion in liver, there was no evidence for overt oxidative stress compared to lean mice. Indeed, hepatic lipid peroxidation as well as plasma antioxidant status were unchanged. In addition, calcium-induced mitochondrial membrane permeabilization remained unchanged and cytochrome c was not detected in the cytosol. Conclusions: Obese mice exhibit hepatic lipid peroxidation, necrosis and apoptosis, despite adaptive increases in MnSOD, Hsp70, mitochondrial
cytochrome c and mtDNA. Moderate ethanol doses markedly increase T N F ~ , decrease nuclear NF-KB activity and activate caspase 3 in obese mice. Oxidative stress is not enhanced, mitochondrial permeabilization is not detected, and hepatocyte death is switched toward more apoptosis and less necrosis. In conclusion, moderate alcohol intoxication causes apoptosis in obese mice, possibly via enhanced generation of T N F ~ by activated cells.
I•
M I T O C H O N D R I A L A D A P T A T I O N S TO NON A L C O H O L I C STEATOH EPATITIS
C. Romestaing 1, B. Sibille 1, V Rouleau 2, M. Dautresme2, I. Ollivier2, B. Roy 1, M. Belouze 1, C. Duchamp 1, M.A. Piquet2. 1Physiologic
Intdgrative, UMR 5123 CNRS-UCB, Lyon, France," 2Imagerie Mdtabolique, CHU, Caen, France The causes of tile non alcoholic steatohepatitis (NASH) and its consequences on tile liver mitochondrial functions are still poorly understood. The aim of this study is to evaluate the modifications of the mitochondrial metabolism of choline-methionine deficient diet fed rats, a known model of NASH. Methods: Male Wistar rats fed a standard diet or a choline-methionine deficient diet (CMDD) during 6 weeks. Liver mitochondria were prepared and their respiration and mitochondrial efficiency (P/O) were measured. The reactive oxygen species (ROS) production was estimated from the fluorimetric measure of the mitochondrial H202 formation. The ketone body production and the oxygen consumption of isolated hop atocytes from fasted rats were also measured. Results: Liver triglyceride content (30-fold increase) and histologic analysis confirmed the steatohepatitis in the CMDD group. A modification of tile mitochondrial function in CMDD fed rats was demonslrated by an uncoupling of the oxidative phosphorylation (increase of the respiration with olignmycin: 32.2±2.1 for CMDD fed rats versus 20± 1.3 nanoatomO/mirdmg protein for standard diet fed rats, p < 0.05), and by a decrease of tile mitochondrial efficiency (decrease of tile P/O) and the ROS production (5.6±2.7 versus 19.2±4.2 pmol H202/nmolO2, p < 0.05). Furthermore, an increase of the lipid oxidation was found on CMDD fed rats reflected by a drop of: 1) tile respiration of isolated hepatocytes with dffiydroxyacetone and octanoate as substates (38.90±4.22 versus 27.21±2.60 [tmol O2/unit of citrate synthase) and 2) the ketone body formation (178.23±19.01 versus 139.98±9.11 [tmol/unit of citrate synthase). Conclusion: Choline~methionine deficient diet fed rats shown a mitochondrial dysfimction leading to a liver adaptation to the overload of triglycerides and oxidative stress. Indeed, the mitochondrial uncoupling increased tile lipid oxidation and decreased the ROS production. Such mitochondrial adaptations could limit NASH associated hepatic injuries.
I•
INSULIN RESISTANCE P R O M O T E S FIBROSIS P R O G R E S S I O N A N D PREDICTS N E C R O - I N F L A M M A T O R Y ACTIVITY IN PATIENTS WITH N O N - A L C O H O L I C FATTY LIVER DISEASE
D. S{mchez-MnfiozI E. Sn{trez 1, M.V Galen t, L. Grande t, G. Mufioz2,
M. Romero-Gdmez I . ; Hepatology Unit, Hospital Universitario de Valme, Sevilla, Spain," 2Pathology Unit, Hospital Universitario de Valme, Sevilla, Spain Aims: To assess the presence of metabolic syndrome X (MSx) and insulin
resistance (IR), andits relationship with histologic damage, in patients with non-alcoholic fatty liver disease (NAFLD). Patients and Methods: Thirty-five patients, 25 male and 10 female, with an average age of 45.7±12.7 [24-76] years, diagnosed by NAFLD, were included. Liver biopsy was carried out after persistence for, at least, six months of altered liver enzymes with appropriate diet and physical activity
252 Results: among 1542 subjects, 40 (2.59%) were excluded using security
algorithms, and 1502 included: 50.3% female, median age 49yrs, 957 HP and 545 controls. Among HE 83.4% had cholesterol )200mg]dl, 93.6% LDL-C )100mg/dl, 17.5% Iriglycerides )200mg]dl, 35.9% BMI )27, 33% arterial hypertension, 31.8% insNinernia )10rnUI/ml and 13.7% glucose )6mmol/]. GGT or ALT were elevated ()50IU/1) in 215 HP (22.5%). F2F3F4 were identified by FibroTest in 25/957 (2.6°/6) HE including 13 F2, 8 F3 and 4 F4 but in none (0%) of the 545 controls (P <0.0001). Among 25 HP with fibrosis, 19 had normal ALT, 14 normal GGT, 12 normal ALT and GGT, 4 elevated ALT and GGT and 9 elevated ALT or GGT. Factors associated (p < 0.01) with fibrosis were higher age, BMI, triglycerides, uricemia, and insulinernia. In multivariate logistic regression, including alcohol consumption, insulinemia (P = 0.003) and triglycerides (P = 0.008) were the most significant risk factors. In HP with triglycerides ) 2 0 0 m # d l prevalence of fibrosis was 8.3% and 6.6% with insulinemia ) 10 mUI/ml. Conclusions: Screening strategies for liver fibrosis are feasible with biomarkers in high-risk groups such as HE Without such non-invasive strategies a liver biopsy would have been indicated in up to 22.5% of HP with elevated GGT or ALT and would have probably missed half of HP with fibrosis, who had normal GGT and ALT. References
[1] [2] [3] [41
1•
Comp Hepatol 2004. J Hepatol 2003. Clin Gaslroenterol Hepato12004. EASL 2004.
M O D E R A T E A L C O H O L INTOXICATION I N C R E A S E S TNF-(x A N D C A U S E S HEPATIC A P O P T O S I S IN O B E S E MICE
C. Demeilliers I , M-A. Robin 1, A. Sutton 1, M Paradis 2, S. Dubois 2, R Lettbron I , D. PessayreI , B. Fromenty 1. QNSERM Unit~ 481, Facult~
de MMecine Xavier Bichat, Paris, France," 2Service d'Anatomie et de Cytologic Pathologiques, H@ital Beaujon, Clichy, France Background and Aims: Both obesity and alcohol carl cause oxidative stress, cytokine induction, steatosis and steatohepatitis. In order to determine the consequences of their combination, we compared the hepatic effects of moderate ethanol binges in lean and obese ob/ob mice. Methods: Leptin-deficient C57BL/6J-ob/ob mice and their lean littermates received ethanol (2.5 g/kg daily) or water by gastric intubation for 4 consecutive days, and were killed two hours after the last administration. Results: Serum ethanol concentrations were similar in lean arid obese mice (1.4±0.3 and 1.2±0.6g/1, respectively). There was a 5-fold increase in basal levels o f plasma alanine aminotransferase (ALT) in ob/ob mice compared to lean mice but ethanol intoxication only marginally increased ALT in both groups. In lean mice, ethanol did not significantly increase plasma T N F ~ , hepatic lipid peroxidation nor caspase 3 activity, but triggered some TUNEL-positive hepatocytes. Unintoxicated ob/ob mice exhibited increased hepatic lipid peroxidation, profound mitochondrial (but not cytosolic) GSH depletion and both necrotic and apoptotic hepatocytes, despite adaptive increases in manganese superoxide dismutase (MnSOD), heat shock protein 70 (Hsp70), mitochondrial cytochrome c and mitochondrial DNA (mtDNA). In alcoholized ob/ob mice, plasma T N F ~ was increased, hepatic nuclear p65 NF-KB subunit protein expression and activity were decreased, caspase 3 was significantly activated and hepatocyte death was switched toward more apoptosis. However, despite increased cytochrome P450 2E1 activity and decreased cytosolic glutathion in liver, there was no evidence for overt oxidative stress compared to lean mice. Indeed, hepatic lipid peroxidation as well as plasma antioxidant status were unchanged. In addition, calcium-induced mitochondrial membrane permeabilization remained unchanged and cytochrome c was not detected in the cytosol. Conclusions: Obese mice exhibit hepatic lipid peroxidation, necrosis and apoptosis, despite adaptive increases in MnSOD, Hsp70, mitochondrial
cytochrome c and mtDNA. Moderate ethanol doses markedly increase T N F ~ , decrease nuclear NF-KB activity and activate caspase 3 in obese mice. Oxidative stress is not enhanced, mitochondrial permeabilization is not detected, and hepatocyte death is switched toward more apoptosis and less necrosis. In conclusion, moderate alcohol intoxication causes apoptosis in obese mice, possibly via enhanced generation of T N F ~ by activated cells.
I•
M I T O C H O N D R I A L A D A P T A T I O N S TO NON A L C O H O L I C STEATOH EPATITIS
C. Romestaing 1, B. Sibille 1, V Rouleau 2, M. Dautresme2, I. Ollivier2, B. Roy 1, M. Belouze 1, C. Duchamp 1, M.A. Piquet2. 1Physiologic
Intdgrative, UMR 5123 CNRS-UCB, Lyon, France," 2Imagerie Mdtabolique, CHU, Caen, France The causes of tile non alcoholic steatohepatitis (NASH) and its consequences on tile liver mitochondrial functions are still poorly understood. The aim of this study is to evaluate the modifications of the mitochondrial metabolism of choline-methionine deficient diet fed rats, a known model of NASH. Methods: Male Wistar rats fed a standard diet or a choline-methionine deficient diet (CMDD) during 6 weeks. Liver mitochondria were prepared and their respiration and mitochondrial efficiency (P/O) were measured. The reactive oxygen species (ROS) production was estimated from the fluorimetric measure of the mitochondrial H202 formation. The ketone body production and the oxygen consumption of isolated hop atocytes from fasted rats were also measured. Results: Liver triglyceride content (30-fold increase) and histologic analysis confirmed the steatohepatitis in the CMDD group. A modification of tile mitochondrial function in CMDD fed rats was demonslrated by an uncoupling of the oxidative phosphorylation (increase of the respiration with olignmycin: 32.2±2.1 for CMDD fed rats versus 20± 1.3 nanoatomO/mirdmg protein for standard diet fed rats, p < 0.05), and by a decrease of tile mitochondrial efficiency (decrease of tile P/O) and the ROS production (5.6±2.7 versus 19.2±4.2 pmol H202/nmolO2, p < 0.05). Furthermore, an increase of the lipid oxidation was found on CMDD fed rats reflected by a drop of: 1) tile respiration of isolated hepatocytes with dffiydroxyacetone and octanoate as substates (38.90±4.22 versus 27.21±2.60 [tmol O2/unit of citrate synthase) and 2) the ketone body formation (178.23±19.01 versus 139.98±9.11 [tmol/unit of citrate synthase). Conclusion: Choline~methionine deficient diet fed rats shown a mitochondrial dysfimction leading to a liver adaptation to the overload of triglycerides and oxidative stress. Indeed, the mitochondrial uncoupling increased tile lipid oxidation and decreased the ROS production. Such mitochondrial adaptations could limit NASH associated hepatic injuries.
I•
INSULIN RESISTANCE P R O M O T E S FIBROSIS P R O G R E S S I O N A N D PREDICTS N E C R O - I N F L A M M A T O R Y ACTIVITY IN PATIENTS WITH N O N - A L C O H O L I C FATTY LIVER DISEASE
D. S{mchez-MnfiozI E. Sn{trez 1, M.V Galen t, L. Grande t, G. Mufioz2,
M. Romero-Gdmez I . ; Hepatology Unit, Hospital Universitario de Valme, Sevilla, Spain," 2Pathology Unit, Hospital Universitario de Valme, Sevilla, Spain Aims: To assess the presence of metabolic syndrome X (MSx) and insulin
resistance (IR), andits relationship with histologic damage, in patients with non-alcoholic fatty liver disease (NAFLD). Patients and Methods: Thirty-five patients, 25 male and 10 female, with an average age of 45.7±12.7 [24-76] years, diagnosed by NAFLD, were included. Liver biopsy was carried out after persistence for, at least, six months of altered liver enzymes with appropriate diet and physical activity