- Email: [email protected]
720 HEPATIC IRON OVERLOAD IN PATIENTS WITH ALCOHOLIC LIVER DISEASE IS DUE TO INADEQUATE HEPCIDIN INDUCTION
08a: ALCOHOLIC LIVER DISEASE, NAFLD AND DRUG-INDUCED LIVER DISEASE − a) EXPERIMENTAL 718 DIETARY MONOSODIUM GLUTAMATE EXACERBATES TRANS FAT-INDUCED NONALCOHOLIC FATTY LIVER DISEASE K. Collison, Z. Maqbool, S. Saleh, A. Inglis, N. Makhoul, R. Bakheet, M. Al-Johi, R. Al-Rabiah, M. Zaidi, F. Al-Mohanna. Cell Biology & Diabetes Research Unit, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia E-mail: [email protected] The aims of this study were to investigate hepatic and white adipose tissue (WAT) gene expression in diet-induced nonalcoholic fatty liver disease (NAFLD). We examined the effect of dietary Monosodium Glutamate (MSG) on Trans-Fatty Acid (TFA)-induced nonalcoholic fatty liver disease (NAFLD) using Affymetrix microarray analysis of gene expression. In the last 40 years, several changes to the global diet have occurred including [1] a 60% increase in dietary intake of added fats and oils. [2] Widespread use of the flavor enhancer Monosodium Glutamate (MSG), which may cause hepatic steatosis and inflammation. Methods: We used GeneChip Mouse Gene 1.0 ST arrays to examine hepatic and WAT gene expression in 16-week old mice. Four different diet regimens were used in this study: [1] ad lib Standard Chow (Control diet) with ad lib drinking water. [2] Ad lib Standard Chow, with ad lib drinking water containing 91.2±4.6 mg/kg body weight dietary MSG (MSG diet). [3] Ad lib Test Diet Purina 5001 with 20% partially hydrogenated vegetable shortening (Test Diet 5C4M, Purina, USA), and ad lib drinking water (TFA diet). [4] 5C4M and ad lib drinking water containing 91.2±4.6 mg/kg bw MSG (TFA+MSG diet). Results: TFA-fed animals exhibited hepatic macrosteatosis and increased serum leptin, Free Fatty Acid (FFA), HDL-C and total cholesterol (TCHOL) levels. Expression of genes involved in hepatic lipogenesis were elevated, including the transcription factor SREBP1c. Conversely, dietary MSG caused hepatic microsteatosis and the expression of b-oxidative genes. Serum Triglyceride (Tg), FFA and insulin levels were elevated in MSG-treated animals. The abdominal cavities of TFA and MSG diet animals exhibited increased WAT deposition compared to control animals. Microarray analysis of WAT gene expression revealed increased lipid biosynthetic gene expression, together with a 50% decrease in key transcription factor Ppargc1a. A combination of TFA+MSG resulted in the highest levels of serum HDL-C, T-CHOL and Leptin. TFA+MSG-treated livers showed elevated expression of markers of hepatic inflammation, lipid storage, cell damage and cell cycle impairment. TFA+MSG mice also had a high degree of WAT deposition and lipogenic gene expression. Levels of Ppargc1a were further reduced to 25% by TFA+MSG treatment. Conclusion: MSG exacerbates TFA-induced NAFLD. 719 REGULATION OF BILE SALT TRANSPORTERS IN NON-ALCOHOLIC FATTY LIVER DISEASE IN OB/OB-MICE AND HUMAN FATTY LIVERS I.V. Martin1,2 , A. Minkenberg1 , A. Canbay3 , B. Muellhaupt4 , A. Geier1,4,5 . 1 Internal Medicine III, University Hospital Aachen, Aachen, Germany; 2 Division of Gastroenterology and Hepatology, Internal Medicine, University Hospital Zurich, Zurich, Switzerland; 3 Dpt. Internal Medicine, Division of Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany; 4 Swiss HPB-Center and Division of Gastroenterology and Hepatology, Internal Medicine, University Hospital Zurich, 5 Zurich Center for Integrative Human Physiology, Zurich, Switzerland E-mail: [email protected] Introduction: Non-alcoholic fatty liver disease (NAFLD) affects up to 25% of the general population. Bile acids (BA) and several nuclear receptors (NR) have been implied in the progression of NAFLD, e.g. through the regulation of Srebp-1c and FAS. KO-mice-studies assigned Fxr a protective role in fatty liver development whereas the absence of Shp also ameliorates fatty liver, intriguingly. Here we investigated transporter and NR regulation in ob/ob mice and human NAFLD as a prerequisite for further targeted therapeutic strategies.
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Methods: 8 week-old ob/ob-mice (n = 5) and WT-controls were fed standard chow ad libitum for 6 weeks. Liver biopsies from morbidly obese patients (n = 7) were compared to controls. Liver mRNA, microsomes & nuclear protein was prepared and expression was analyzed by RT-PCR and Western Blotting. Binding activity of FXR to the IR-1 element was examined by EMSA. Results: Oatp1- & Oatp2-expression decreased dramatically in ob/ob-mice (mRNA: 2% and 55%, protein: ~10%), while Oatp4-mRNA is elevated 2-fold and Ntcp remains unchanged. Mrp3-mRNA increases to 330% and Mrp4 is strongly induced 5−10-fold (protein and mRNA). Bsep- and Mrp2mRNA is upregulated to ~200% while the proteins are decreased to ~60%. PPARg and Srebp-1c mRNA are drastically induced 7- and 60-fold. Pxr-, Lxr- and Fxr-expression is increased between 1.3- and 3.3-fold in ob/obmice. Despite Fxr-induction expression of Shp remained unchanged. DNA binding activity of Fxr is increased ~2-fold in obese mice. In human NAFLD NTCP expression is even increased to 170% while OATP-C, BSEP and FXR expression are largely unchanged. In contrast to ob/obmice human MRP4 is strongly decreased by −90%. SREBP-1c induction (2-fold) is less pronounced in human NAFLD. Conclusions: Increased Fxr-expression and binding activity represents a central event in murine FLD. Shp-independent effects of Fxr might be involved in a cross-talk between BA-signalling and the induction of their adipogenic target genes. The physiological SHP-induction with subsequent Ntcp suppression is absent in mice. Moreover, adaptive suppression of NTCP and induction of MRP4 are even inversely regulated in humans. Bile acid transporter maladaptation could play a role in the progression of fatty liver disease. 720 HEPATIC IRON OVERLOAD IN PATIENTS WITH ALCOHOLIC LIVER DISEASE IS DUE TO INADEQUATE HEPCIDIN INDUCTION G. Millonig1 , G.N. Waite1 , M.U. Muckenthaler2 , H.K. Seitz1 , S. Mueller1 . 1 Salem Medical Center, University of Heidelberg, 2 Dept. of Pediatrics, Univ. of Heidelberg, Heidelberg, Germany E-mail: [email protected] Background: Alcoholic liver disease (ALD) leads to secondary iron overload in more than 50% patients that is considered a major factor in the progression of fibrosis and the development of hepatocellular carcinoma. Hepcidin is the major systemic iron sensor in mammals and typically induced under conditions of iron overload. Hepcidin blocks duodenal iron absorption and the release of iron by inflammatory cells. Suppressed hepcidin levels have been recently reported from animals chronically exposed to alcohol. Methods and Results: Using cDNA microarray platform containing over 250 iron relevant genes (IRON CHIP), we first screened liver tissue of a control group and patients with ALD with or without histological deposition of iron for significantly regulated iron-associated gene expression (each group n = 4). However, none of the classical iron genes (hepcidin, transferrin receptor, transferrin, ferroportin, hemojuvelin) were significantly regulated when ALD patients with and without iron overload were compared. These results were validated by qRT-PCR in an extended group of patients with ALD (n = 30) with or without iron overload (serum ferritin as serological marker and Prussian Blue staining in liver biopsies). Patients were also matched for age, alcohol consumption and fibrosis stage. Thus, ALD patients with iron accumulation showed an inadequate induction of hepcidin. Serum analysis of hepcidin also showed no difference in circulating hepcidin in both groups. In vitro exposure of human hepatoma cells (Hep3B) to a continuous flux of non-toxic H2 O2 a central reactive oxygen species, significantly suppresses hepcidin levels. Conclusion: ALD patients with increased hepatic iron deposits show an inadequate secretion of hepcidin. Ethanol-mediated oxidative stress mechanisms could be pivotal in the suppression of hepcidin despite iron overload.
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Methods: 8 week-old ob/ob-mice (n = 5) and WT-controls were fed standard chow ad libitum for 6 weeks. Liver biopsies from morbidly obese patients (n = 7) were compared to controls. Liver mRNA, microsomes & nuclear protein was prepared and expression was analyzed by RT-PCR and Western Blotting. Binding activity of FXR to the IR-1 element was examined by EMSA. Results: Oatp1- & Oatp2-expression decreased dramatically in ob/ob-mice (mRNA: 2% and 55%, protein: ~10%), while Oatp4-mRNA is elevated 2-fold and Ntcp remains unchanged. Mrp3-mRNA increases to 330% and Mrp4 is strongly induced 5−10-fold (protein and mRNA). Bsep- and Mrp2mRNA is upregulated to ~200% while the proteins are decreased to ~60%. PPARg and Srebp-1c mRNA are drastically induced 7- and 60-fold. Pxr-, Lxr- and Fxr-expression is increased between 1.3- and 3.3-fold in ob/obmice. Despite Fxr-induction expression of Shp remained unchanged. DNA binding activity of Fxr is increased ~2-fold in obese mice. In human NAFLD NTCP expression is even increased to 170% while OATP-C, BSEP and FXR expression are largely unchanged. In contrast to ob/obmice human MRP4 is strongly decreased by −90%. SREBP-1c induction (2-fold) is less pronounced in human NAFLD. Conclusions: Increased Fxr-expression and binding activity represents a central event in murine FLD. Shp-independent effects of Fxr might be involved in a cross-talk between BA-signalling and the induction of their adipogenic target genes. The physiological SHP-induction with subsequent Ntcp suppression is absent in mice. Moreover, adaptive suppression of NTCP and induction of MRP4 are even inversely regulated in humans. Bile acid transporter maladaptation could play a role in the progression of fatty liver disease. 720 HEPATIC IRON OVERLOAD IN PATIENTS WITH ALCOHOLIC LIVER DISEASE IS DUE TO INADEQUATE HEPCIDIN INDUCTION G. Millonig1 , G.N. Waite1 , M.U. Muckenthaler2 , H.K. Seitz1 , S. Mueller1 . 1 Salem Medical Center, University of Heidelberg, 2 Dept. of Pediatrics, Univ. of Heidelberg, Heidelberg, Germany E-mail: [email protected] Background: Alcoholic liver disease (ALD) leads to secondary iron overload in more than 50% patients that is considered a major factor in the progression of fibrosis and the development of hepatocellular carcinoma. Hepcidin is the major systemic iron sensor in mammals and typically induced under conditions of iron overload. Hepcidin blocks duodenal iron absorption and the release of iron by inflammatory cells. Suppressed hepcidin levels have been recently reported from animals chronically exposed to alcohol. Methods and Results: Using cDNA microarray platform containing over 250 iron relevant genes (IRON CHIP), we first screened liver tissue of a control group and patients with ALD with or without histological deposition of iron for significantly regulated iron-associated gene expression (each group n = 4). However, none of the classical iron genes (hepcidin, transferrin receptor, transferrin, ferroportin, hemojuvelin) were significantly regulated when ALD patients with and without iron overload were compared. These results were validated by qRT-PCR in an extended group of patients with ALD (n = 30) with or without iron overload (serum ferritin as serological marker and Prussian Blue staining in liver biopsies). Patients were also matched for age, alcohol consumption and fibrosis stage. Thus, ALD patients with iron accumulation showed an inadequate induction of hepcidin. Serum analysis of hepcidin also showed no difference in circulating hepcidin in both groups. In vitro exposure of human hepatoma cells (Hep3B) to a continuous flux of non-toxic H2 O2 a central reactive oxygen species, significantly suppresses hepcidin levels. Conclusion: ALD patients with increased hepatic iron deposits show an inadequate secretion of hepcidin. Ethanol-mediated oxidative stress mechanisms could be pivotal in the suppression of hepcidin despite iron overload.