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671 Induction of severe insulin resistance and hepatic complications compatible with NASH in mice fed a western-type diet
Category 9: Alcoholic Liver Disease, NAFLD and Drug-Induced Liver Disease ~THE SERUM ENDOTHELIN-1 LEVEL IN STEATOSIS AND NASH, AND ITS RELATION WITH THE SEVERITY OF LIVER FIBROSIS B. Degertekin 1, S. Ozenirler 2, O. Akyol 3. 1Gazi University Faculty
of Medicine Department of Gastroenterology. Ankara, Turkey," 2Gazi University Faculty of Medicine Department of Pathology, Ankara, Turkey," ~Gazi University Faculty of Medicine, Ankara, Turkey Introduction: Nonalcoholic steatohepatitis (NASH) is histologically characterized by steatosis and fibrosis in the liver. The condition is closely related to obesity, diabetes mellitus, insulin resistance, and metabolic syndrome. Endothelin-1 is well known for its role in contraction, proliferation, matrix synthesis and chernotaxis in the fibrotic process led by hepatic stellate cells in the liver. Endothelin-1 is a common mediator in endothelial cell dysfunction, hyperglycernia, oxidative stress and insulin resistance. Aim: The aim of this study was to determine if a mediator with influence on the fibrogenetic processes like endothelin- 1 could be used as an indicator of file degree of fibrosis in patients with NASH. We also investigated the role of non-invasive fibrosis markers like serum ALT level, obesity, diabetes mellitus, hyperlipidernia, AST/ALT ratio and their relation to the severity in patients with NASH. The correlation of these markers with endothelin-1 level was also determined. Material and Methods: The total number of 100 patients were divided into three groups. Group 1: NASH (n=40), Group 2: Biopsy (+) steatosis (n 20) and Group 3: hepatesteatesis on ultrasound with normal liver enzymes (n = 40). Duplicate serum samples from each subject were assayed for ET-1 by a commercially available ELISA kit. Results: Tile mean serum endotelin-1 levels in Group 1, Group 2 and Group 3 were 17.47±4.33, 6.75±2.46 and 5.74±2.34p, mol/ml respectively. Serum endothelin-1 level in Group 1 was found to be significantly higher than the other two groups (p < 0.01). The mean serum endothelin-1 levels in NASH patients with gtade-1, gtade-2, and grade-4 fibrosis were 14.06±0.92, 17.70±2.32 and 20.40±1.40 ~tmol/ml, respectively. Grade-3 fibrosis was not detected in any of the patients. The serum endothelin-1 level in patients with NASH was directly related to tile grade of fibrosis, and tile relationship was statistically significant (p < 0.01). There was a positive correlation between diabetes (p < 0.05) and the serum ALT levels (p <0.01) and fibrosis as well as endothelin-1 levels. Conclusion: The serum endotelin-1 level in NASH patients was higher than patients with steatosis only. The severity of fibrosis in NASH was correlated with the level of serum endothelin-1.
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INDUCTION OF SEVERE INSULIN RESISTANCE AND HEPATIC COMPLICATIONS COMPATIBLE WITH NASH IN MICE FED A WESTERN-TYPE DIET
C. Dewever. Y. Horsmans, I. Leclercq. Gastroenterology Unit, Universitd
Catholique de Louvain, Brussels, Belgium Insulin resistance is a major factor implicated in the pathogenesis o f nonalcoholic steatohepatitis (NASH). Our aim was to evaluate the effects of high-glucose and/or high-satutated fat/cholesterol diet on emergence of insulin resistance and liver pathology. Methods: Male C57BL6/J mice were fed ad libitum for 5 weeks a standard chow (CT group, n = 4 ) , a standard chow with high-glucose in drin!dng water (glucose group, n = 6 ) or a diet enriched in saturated fat and cholesterol together with high-glucose in drinldng water (SFA group, n = 6). Intraperitoneal glucose tolerance and insulin resistance tests were performed in vivo. At the end of the experimental period, blood and tissue samples were obtained for analyses. Results: Compared with CT, mice from glucose and SFA groups exhibited hyperglycemia (145±5 and 177±8 versus 124±2mg/dL, both p < 0.001) and hyperinsulinemia (25.8-t-4.7 and 30.3±1.6 versus 2.8±0.1ngjml, both p<0.001). As assessed in vivo, mice of glucose group and, to a
245
greater extent, mice of SFA group had glucose intolerance (p = 0.004 and p < 0.001, respectively) and insulin resistance (p =0.007 and p ~<0.001 respectively), which was confirmed by a significant (p < 0.001) rise in the HOMA-IR index (0.3±0.1, 3.3±0.6 and 4.7±0.3 in CT, glucose and SFA, respectively). Liver histology appeared normal in mice from CT and glucose groups, but mice in SFA group developed significant macrovesicnlar steatosis, hepatocyte ballooning and apoptosis and variable panlobular inflammation. Consistently, inwahepatic lipids were sig'nificanfly increased in S FA versus CT (9.0±4.7 versus 1.8±0.5 rng lipid/100 m g liver, p = 0.02). Importantly, steatohepatitis in SFA mice was associated with a significant up-regnlation of collagen Ial mRNA expression (1.7±0.4 versus 1.0±0.2 in CT, p = 0.01). Conclusions: A diet enriched in glucose, saturated fatty acids and cholesterol induced a severe and prolonged insulin resistance in mice. This was associated with hepatic damage, including activation of fibrogenesis, compatible with NASH. We believe that this original mouse model will be useful to analyse the role of insulin resistance and nutritional factors in the pathogenesis of NASH and for evaluation of treatment.
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ALTERED DRUG DISPOSITION OF BOSENTAN, AN ENDOTHELIN RECEPTOR ANTAGONIST, IN MRP2 DEFICIENT RATS L. Fouassier I , A. Trieber2, S. Delahaie 2, C. Rey I , C. HoussetI . llnserm,
UPMC, Paris, France," 2Actelion Pharmaceuticals Ltd, Allschwil, Switzerland MRP2, a canalicular transporter of organic anions, which is deficient in Dubin Johnson syndrome, has an important role in deternfinlng drug disposition. Bosentan, a non-peptidic endothelin receptor antagonist, is extensively metabolized in the liver and may cause cholestatic changes in treated patients. We previously showed that in normal tats, bosentan causes maj or changes in canalicular bile formation including a stimulation of bilesalt independent bile flow and of bilirubin secretion, and an uenupling of phospholipid from bile salt secretion. Because these changes were abolished in MRP2-ttansport-deficient ( T R ) tats, tile aim of the present study was to determine whether MRP2 affects tile hepatobiliary disposition of bosentan. Methods: A single dose of bosentan (15mg/kg body) was injected in pentobarbital-anesthetized normal Wistar and T R tats. Plasma and bile samples were collected at different time points, and were analyzed for bosentan and/or its metabolites, Ro 48-5033, Ro 47-8634 and Ro 64 1056. Liver microsomes were prepared from normal and T R rats and were tested for their bosentan metabolizing activities. Results: Normal tats showed bile excretions of Ro 48-5033 > bosentan > Ro 47-8634 >> Ro 64-1056. Pharmacokinetic analyses showed that the plasma clearance of bosentan was lower in T R than in normal vats (3.8±0.6ml/min/kg vs 18±3 ml/rnin/kg). Bosentan major metabolite, Ro 48-5033, was produced to a lesser extent by liver mirosomes from T R rats as compared with normal (Vma,,: 177.5±1.6 vs 423±5pmol[min&ng protein) and excreted in lower amounts in their bile. Ro 47-8634 was produced to a lesser extent by liver mirosomes from T R - tats (Vmax 69±2 vs 259±6 pmol/min/mg protein) but was excreted in higher amounts in their bile. The biliary excretions of bosentan itself and o f Ro 64-1056 were also higher in T R - than in normal tats. Conclusions: These results indicate that bosentan and its metabolites are eliminated in bile and transported at the canalicular membrane of hepatocytes by other transporters than mrp2. In mrp2 deficient rats, the hepatobiliary disposition of bosentan is profoundly affected, which may be attributed to an up-regulation of such transporter(s) and to a decrease in cytochrome P450 activities, previously shown to occur in response to rnrp2 defect.
of Medicine Department of Gastroenterology. Ankara, Turkey," 2Gazi University Faculty of Medicine Department of Pathology, Ankara, Turkey," ~Gazi University Faculty of Medicine, Ankara, Turkey Introduction: Nonalcoholic steatohepatitis (NASH) is histologically characterized by steatosis and fibrosis in the liver. The condition is closely related to obesity, diabetes mellitus, insulin resistance, and metabolic syndrome. Endothelin-1 is well known for its role in contraction, proliferation, matrix synthesis and chernotaxis in the fibrotic process led by hepatic stellate cells in the liver. Endothelin-1 is a common mediator in endothelial cell dysfunction, hyperglycernia, oxidative stress and insulin resistance. Aim: The aim of this study was to determine if a mediator with influence on the fibrogenetic processes like endothelin- 1 could be used as an indicator of file degree of fibrosis in patients with NASH. We also investigated the role of non-invasive fibrosis markers like serum ALT level, obesity, diabetes mellitus, hyperlipidernia, AST/ALT ratio and their relation to the severity in patients with NASH. The correlation of these markers with endothelin-1 level was also determined. Material and Methods: The total number of 100 patients were divided into three groups. Group 1: NASH (n=40), Group 2: Biopsy (+) steatosis (n 20) and Group 3: hepatesteatesis on ultrasound with normal liver enzymes (n = 40). Duplicate serum samples from each subject were assayed for ET-1 by a commercially available ELISA kit. Results: Tile mean serum endotelin-1 levels in Group 1, Group 2 and Group 3 were 17.47±4.33, 6.75±2.46 and 5.74±2.34p, mol/ml respectively. Serum endothelin-1 level in Group 1 was found to be significantly higher than the other two groups (p < 0.01). The mean serum endothelin-1 levels in NASH patients with gtade-1, gtade-2, and grade-4 fibrosis were 14.06±0.92, 17.70±2.32 and 20.40±1.40 ~tmol/ml, respectively. Grade-3 fibrosis was not detected in any of the patients. The serum endothelin-1 level in patients with NASH was directly related to tile grade of fibrosis, and tile relationship was statistically significant (p < 0.01). There was a positive correlation between diabetes (p < 0.05) and the serum ALT levels (p <0.01) and fibrosis as well as endothelin-1 levels. Conclusion: The serum endotelin-1 level in NASH patients was higher than patients with steatosis only. The severity of fibrosis in NASH was correlated with the level of serum endothelin-1.
[6•
INDUCTION OF SEVERE INSULIN RESISTANCE AND HEPATIC COMPLICATIONS COMPATIBLE WITH NASH IN MICE FED A WESTERN-TYPE DIET
C. Dewever. Y. Horsmans, I. Leclercq. Gastroenterology Unit, Universitd
Catholique de Louvain, Brussels, Belgium Insulin resistance is a major factor implicated in the pathogenesis o f nonalcoholic steatohepatitis (NASH). Our aim was to evaluate the effects of high-glucose and/or high-satutated fat/cholesterol diet on emergence of insulin resistance and liver pathology. Methods: Male C57BL6/J mice were fed ad libitum for 5 weeks a standard chow (CT group, n = 4 ) , a standard chow with high-glucose in drin!dng water (glucose group, n = 6 ) or a diet enriched in saturated fat and cholesterol together with high-glucose in drinldng water (SFA group, n = 6). Intraperitoneal glucose tolerance and insulin resistance tests were performed in vivo. At the end of the experimental period, blood and tissue samples were obtained for analyses. Results: Compared with CT, mice from glucose and SFA groups exhibited hyperglycemia (145±5 and 177±8 versus 124±2mg/dL, both p < 0.001) and hyperinsulinemia (25.8-t-4.7 and 30.3±1.6 versus 2.8±0.1ngjml, both p<0.001). As assessed in vivo, mice of glucose group and, to a
245
greater extent, mice of SFA group had glucose intolerance (p = 0.004 and p < 0.001, respectively) and insulin resistance (p =0.007 and p ~<0.001 respectively), which was confirmed by a significant (p < 0.001) rise in the HOMA-IR index (0.3±0.1, 3.3±0.6 and 4.7±0.3 in CT, glucose and SFA, respectively). Liver histology appeared normal in mice from CT and glucose groups, but mice in SFA group developed significant macrovesicnlar steatosis, hepatocyte ballooning and apoptosis and variable panlobular inflammation. Consistently, inwahepatic lipids were sig'nificanfly increased in S FA versus CT (9.0±4.7 versus 1.8±0.5 rng lipid/100 m g liver, p = 0.02). Importantly, steatohepatitis in SFA mice was associated with a significant up-regnlation of collagen Ial mRNA expression (1.7±0.4 versus 1.0±0.2 in CT, p = 0.01). Conclusions: A diet enriched in glucose, saturated fatty acids and cholesterol induced a severe and prolonged insulin resistance in mice. This was associated with hepatic damage, including activation of fibrogenesis, compatible with NASH. We believe that this original mouse model will be useful to analyse the role of insulin resistance and nutritional factors in the pathogenesis of NASH and for evaluation of treatment.
•
ALTERED DRUG DISPOSITION OF BOSENTAN, AN ENDOTHELIN RECEPTOR ANTAGONIST, IN MRP2 DEFICIENT RATS L. Fouassier I , A. Trieber2, S. Delahaie 2, C. Rey I , C. HoussetI . llnserm,
UPMC, Paris, France," 2Actelion Pharmaceuticals Ltd, Allschwil, Switzerland MRP2, a canalicular transporter of organic anions, which is deficient in Dubin Johnson syndrome, has an important role in deternfinlng drug disposition. Bosentan, a non-peptidic endothelin receptor antagonist, is extensively metabolized in the liver and may cause cholestatic changes in treated patients. We previously showed that in normal tats, bosentan causes maj or changes in canalicular bile formation including a stimulation of bilesalt independent bile flow and of bilirubin secretion, and an uenupling of phospholipid from bile salt secretion. Because these changes were abolished in MRP2-ttansport-deficient ( T R ) tats, tile aim of the present study was to determine whether MRP2 affects tile hepatobiliary disposition of bosentan. Methods: A single dose of bosentan (15mg/kg body) was injected in pentobarbital-anesthetized normal Wistar and T R tats. Plasma and bile samples were collected at different time points, and were analyzed for bosentan and/or its metabolites, Ro 48-5033, Ro 47-8634 and Ro 64 1056. Liver microsomes were prepared from normal and T R rats and were tested for their bosentan metabolizing activities. Results: Normal tats showed bile excretions of Ro 48-5033 > bosentan > Ro 47-8634 >> Ro 64-1056. Pharmacokinetic analyses showed that the plasma clearance of bosentan was lower in T R than in normal vats (3.8±0.6ml/min/kg vs 18±3 ml/rnin/kg). Bosentan major metabolite, Ro 48-5033, was produced to a lesser extent by liver mirosomes from T R rats as compared with normal (Vma,,: 177.5±1.6 vs 423±5pmol[min&ng protein) and excreted in lower amounts in their bile. Ro 47-8634 was produced to a lesser extent by liver mirosomes from T R - tats (Vmax 69±2 vs 259±6 pmol/min/mg protein) but was excreted in higher amounts in their bile. The biliary excretions of bosentan itself and o f Ro 64-1056 were also higher in T R - than in normal tats. Conclusions: These results indicate that bosentan and its metabolites are eliminated in bile and transported at the canalicular membrane of hepatocytes by other transporters than mrp2. In mrp2 deficient rats, the hepatobiliary disposition of bosentan is profoundly affected, which may be attributed to an up-regulation of such transporter(s) and to a decrease in cytochrome P450 activities, previously shown to occur in response to rnrp2 defect.