Introduction 5/2/2013 IRON RELATED GENES AND OXIDATIVE STRESS IN NON- ALCOHOLIC STEATOHEPATITIS. Iron Physiology. Iron Physiology

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1 // IRON RELATED GENES AND OXIDATIVE STRESS IN NON- ALCOHOLIC STEATOHEPATITIS DIANA MOYA, MD PEDIATRIC GASTROENTEROLOGY FELLOW DIGESTIVE DISEASES & NUTRITION CENTER MAY,. Iron Physiology. /NAFLD. Iron Metabolism in. Our study Iron Physiology Nearly all cells use iron as a cofactor for biochemical processes, such as oxygen transport, energy metabolism and DNA synthesis for cellular growth and proliferation Under physiological conditions, the free form of iron is insoluble and potentially toxic Catalyzes the propagation of reactive oxygen species (ROS) and the generation of highly reactive radicals (hydroxyl radical) through Fenton chemistry Thus, iron is always bound to ligands to make it soluble and nontoxic Iron shuttles between forms: Fe + ferrous (reduced) Fe + ferric (oxidized) Iron Physiology Iron can not freely cross cell membranes and tight junctions, therefore iron enters cells via transporters: Transmembrane proteins and members of the solute carrier (SLC) transporters Carrier protein, transferrin (Tf) in the plasma and its receptors, transferrin receptor (TfR) and transferrin receptor (TfR) Garrick, M. Garrick, L. Cellular Iron Transport. Biochimica et Biophysica Act. 9 Harmful Effects of Iron Related to its capacity to generate ROS by Fenton reaction: Fe + + H O Fe + + OH + OH Quick Review of Iron Physiology Absorption from duodenum:-mg/d Body iron distribution: Hb, erythroid cells and O transport Liver: stores excess iron Macrophages Myoglobin Hydroxyl radicals (OH ): Toxic, leading to damage to DNA, to protein synthesis and to cell proliferation Reacts with unsaturated fatty acids or preformed lipid hydroperoxides to form peroxyl radicals that impair cellular integrity Iron loses: Shedding of mucosal and skin cells Bleeding Not known regulated mechanism for removal of iron (other than blood loss and shedding), thus balance is maintained by dietary- Fe absorption in duodenum cdc.gov/ncbddd/hemochromatosis/training/pathophysiology/ironcycle

2 // Hepatocytes and Iron Iron travels to the liver through portal circulation Hepatocytes serve as a buffer: Stores iron when transferrin is replete after iron uptake Releases iron when transferrin is inadequately loaded Hepatocyte membranes have transferrin receptors for iron uptake via the transferrin (TF) cycle and non-transferrinbound Iron (NTBI) uptake via other receptors 7 Hepatocyte Iron Transport ) TFRC-mediated uptake of diferric Tf Complex TF:TfR is endocytosed Endosome is acidified to dissociate Fe + /TF Fe + is reduced to Fe + by STEAP and transported out of the endosome via DMT Apotransferrin is exocytosed ) Uptake of Non-Tf-bound iron Uptake is concentration dependent Zinc-transporters and DMT ) Transit iron pool Transferred into the cytosolic transit iron pool Transported to the mitochondria or stored as ferritin (H-ferritin and L-ferritin chains) ) Mitochondrial iron Transporter mitoferrin- on inner membrane NTBI Fe + Fe+ DMT ZIP TFR Steap HFE DMT TFR Endocytosis Fe + Fe + Fe + Fe + ph FPN Fe+ 8 Apo-TF Exocytosis Mitoferrin- Mitochondria Transit Iron Pool Ferritin Liver recovers iron from circulating heme and hemoglobin ) Iron release By FPN, oxidized to Fe + by CP and transported by Tf Ceruloplasmin 9 Regulation of Systemic Iron Balance Stimulus that modulate iron homeostasis: Erythroid iron needs Hypoxia Iron deficiency Iron overload Inflammation Control by Hepcidin Hepcidin is a liver-derived hormone Binds to FPN inhibiting Fe + efflux from enterocytes and macrophages Regulation of Systemic Iron Balance: Hepcidin Regulation: C/EBPα (CCAAT/enhancerbinding P α) BMP/SMAD Hemojuvelin (HJV): BMP receptor Increase expression: IL- Decrease expression: Anemia, thalassemia, hypoxia, oxidative stress Wang, J. Regulation of Cellular Iron Metabolism. Biochem. J. Regulation of Cellular Iron Maintain cellular iron homeostasis preventing accumulation of free iron that promotes ROS formation: Ferritin: two chains, L-ferritin (light, liver) H-ferritin (heavy, heart) Iron regulatory proteins (IRPs): Bind to RNA iron regulatory elements (IREs): mrna sequences coding for Tf receptors TfR/TfR and ferritin Iron-responsive element-binding protein (IRE-BP) binds to Tf and ferritin receptors mrna post-transcriptionally In summary: ENTEROCYTES HEPATOCYTES Form of Iron Reduced Fe + Reduced Fe + Iron Uptake Via DMT and heme transporter TfR, TfR NTBI Storage Ferritin Ferritin, mitocondria, labile iron pool Intracellular regulation Iron exported Iron regulatory elements, HIF- Ferroportin (regulated by hepcidin) Ferritin, Iron regulatory elements Ferroportin (regulated by hepcidin) Circulation Transferrin Transferrin

3 //. Iron Physiology NAFLD/ Non-alcoholic Fatty liver disease (NAFLD) Steatosis, steatohepatitis, liver failure, cirrhosis. /NAFLD. Iron Metabolism in Non-alcoholic steatohepatitis () Steatosis, t hepatocyte t ballooning, and inflammation. Our study Cohen, J. Human Fatty Liver Disease: Old Questions and New Insights. Science. Vol, June First hit Obesity Insulin resistance. Iron Physiology LIVER STEATOSIS. /NAFLD Second hit Oxidative Stress. Iron Metabolism in. Research project Inflammation Fibrosis, apoptosis CIRRHOSIS 7 8 Erythropoiesis High-fat, high-caloric diet Obesity TNF-a Hyperinsulinemia, insulin resistance Fe Fe The role of iron in FPN TFRc Impaired insulin signaling Iron overload? Hepcidin FPN Fe Hepatocyte ROS Stimulation Inhibition

4 // 9. Iron Physiology. /NAFLD. Iron Metabolism in Hypothesis: We hypothesized that because of oxidative stress in, the expression of iron related genes is increased. Our study Methods: ) Analysis of serum iron indices in and normal controls (NC) ) Analysis of the microarray data for all iron related genes ) Quantitative Real Time PCR for analysis of gene expression In human samples In cultured cells Methods Inclusion criteria Pediatric patients undergoing percutaneous ultrasound guided liver biopsies between October and November Exclusion criteria Hemochromatosis or other forms of acute or chronic hepatitis Liver biopsy consistent with steastosis only without inflammation or fibrosis Patients with incomplete iron panel Patients who reported alcohol consumption Methods ) Anthropometric and laboratory data: Obtained at the time of the liver biopsy BMI z-score and total body iron were calculated ) Histological evaluation: Liver biopsy were stained with hematoxylin-eosin, Masson s trichrome stain for collagen detection, Pear s Prussian blue for iron detection. Hepatic iron quantification (HIC) and hepatic iron index (HII) were obtained was diagnosed according to Kleiner s criteria: Steatosis (-), lobular inflammation (-) and fibrosis (-) Methods ) Microarray data Used to identify the gene expression of iron related genes and oxidative stress enzymes in patients and NC ) Quantitative Real Time PCR Data (qrt-pcr) Used to analyze the gene expression of iron related genes in and NC Custom primers, forward and reverse were designed for iron related genes Glyceraldehyde -phosphate dehydrogenase(gapdh) was used as the housekeeping gene and the relative gene expression of each gene was normalized with the relative gene expression of GAPDH ) Culture cells and treatment Used to analyzed the gene expression of TF, thioredoxin and catalase HepG cells were treated with H O at the concentrations of,.,. and mm during hours and then harvested for RNA extraction Kleiner DE, Brunt EM, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology, :-.

5 // Methods ) Statistical analysis Student t-test with a two-tailed distribution was used to analyze the difference of gene expression on NC and in microarray and qrt-pcr data Spearman s rank correlation analysis was used to examine the correlation of gene expression in human samples Pearson s product-moment correlation analysis was used for cultured cells A p value less than. was considered to be statistically significant Results Average NC Age (years). ±.. ±. Body mass index (%). ± 7.7 *. ± Body Mass index (z score). ±.8.9 ±. ALT (IU/L) 7. ± 9. *. ± 7. AST (IU/L) 9. ± 8. *. ±.7 Hemoglobin (g/dl).7 ±..9 ±. Sedimentation rate (mm/hr) 7. ±. * 9. ±. Serum Ferritin (ng/ml) 8.8 ± 7.8 * 7. ±. Serum Iron (mcg/dl) 8. ±. 8.8 ±.8 Total body Iron (mg/kg). ±. 8. ±.8 Total iron binding capacity (mcg/dl) 7. ±. ±. Transferrin saturation (%) 8.7 ± 9. ± 8. Soluble transferrin receptor (mg/l). ±.7. ±. * Significant p value <. 7 8 Gene Expression: Microarray Data Results: RT-PCR analysis. Genes Average NC Average /NC p Value TF 9. ±..7 ±...9 TFR. ±.. ±.8.. Genes involved in systemic iron transport (Tf) and hepatocyte uptake (TFR, TFR) TFR p =.7 CAT.9 ±.7.8 ± p =.. p =. Transferrin TFR.. Results: RT-PCR analysis 9 Results: RT-PCR analysis 7 Genes involved in iron export (FPN) and iron transport into the mitochondria (Mitoferrin-) Ferroportin p =. Oxidative stress related enzymes Catalase p =.. Mitoferrin- 8 8 p =.7 Hemopexin 8 8 p =. Thioredoxin p =.

6 // Correlation Analysis between TF and CAT per microarray sion TF Gene Express R² =.88 CAT Gene Expression Results ) Gene Expression in H O treated HepG cells To determine if there is an induction in the gene expression of Tf, CAT and TXN by H O in HepG cells at different concentrations RT-PCR: Significant correlation of TF and TXN Significant correlation of TF and CAT Gene Expression CAT, TF and TRX. Correlation Analysis between CAT and TF in H O treated HepG cells per qrt-pcr data Correlation Analysis between TXN and TF in H O treated HepG cells per qrt-pcr data. Gene Ex pression. mm. mm. mm Catalase 8 R² =.87 n Thioredoxi...8. R² =.89. TF CAT TXN... Transferrin..... Transferrin Discussion. Iron Physiology. /NAFLD. Iron Metabolism in. Our study Studies in patients in regards to hepatic iron accumulation have yielded conflicting results No significant iron accumulation in patients with Younossi, Z.M., et al., Hepatic iron and nonalcoholic fatty liver disease. Hepatology, 999. (): p Angulo, P., et al., Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology, 999. (): p. -. Increase iron stores may be a major determinant of fibrosis and hepatic damage in patients, especially with HFE mutation George, D.K., et al., Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis. Gastroenterology, 998. (): p. -8. Bonkovsky, H.L., et al., Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis. J Hepatol, 999. (): p. -9.

7 // Discussion Hyperferritinemia can be induced by inflammation and hepatic iron overload Karam et al, postulated that the increase in ferritin correlates mainly with the inflammation present in, as opposed to an increase in hepatic iron content Karam, L.B., et al., Liver biopsy results in patients with sickle cell disease on chronic transfusions: poor correlation with ferritin levels. Pediatr Blood Cancer, 8. (): p Discussion: Our Findings This is the first study conducted in pediatric patients in iron related genes in livers and their correlation with oxidative stress is considered an inflammatory condition related to increase oxidative stress. The significant increase of serum ferritin in patients when compared to controls correlates with this observation 8 Discussion: Our Findings There is an increased expression of iron related genes involved in iron transport and hepatocyte uptake (TF, TFR, TFR), mitochondrial uptake (mitoferrin-) and iron export (FPN) in human livers when compared to controls There is an increased expression of oxidative stress related enzymes (CAT, TXN and HPX) in livers when compared to controls 9 Discussion: Our Findings There is a positive relationship between iron related genes and the gene expression of oxidative stress related enzymes In vitro study with cultured cells showed that H O increases the expression of TF, TXN and CAT. This in vitro approach also confirmed a correlation of the gene expression between iron related genes and oxidative stress enzymes Conclusion Although total body iron is not elevated in, there is an increase in gene expression of iron related genes in livers in response to the oxidative stress present in these patients THANK YOU 7

8 // Acknowledgment Dr. Susan Baker Dr. Robert Baker Dr. Lixin Zhu Dr. Wensheng Liu Dr. Michael Garrick Dr. Rafel Kozielski Dr. Abdur Kahn 8

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