A HIGH FAT DIET AND PARENTERAL IRON CAUSES INCREASED INFLAMMATION AND OXIDATIVE STRESS BUT LESS STEATOSIS IN AN OBESE, DIABETIC MOUSE MODEL OF NAFLD James E. Nelson 1 , Bryan Maliken 1 , Barjinder Dhillon 1 , Heather Klintworth 1 , Matthew M. Yeh 2 , Kris V. Kowdley 1,2 1 Center for Liver Disease, Digestive Disease Institute, and Benaroya Research Institute, Virginia Mason Medical Center Seattle WA. 2 Dept of Pathology, University of Washington Medical Center, Seattle WA METHODS Lepr db/db mice were fed either a 71% high-fat diet (61% unsaturated fat) or normal chow for 8 weeks. A subset of mice on both diets were administered a single dose of 1.25 mg/g wt Fe-dextran by IP injection at the start of the study. Histological features of NASH and iron deposition were scored using NASH CRN scoring criteria. Malondialdehyde (MDA), iron and hydroxyproline were assessed in liver tissue. Liver and adipose tissue were collected for expression of proinflammatory, lipid and oxidative stress genes, determined by RT- PCR, normalized to GAPDH mRNA 0 1 2 3 Steatosis (0-3) Mean Score # 0 1 2 3 4 Lobular Inflammation (0-3) Mean Score †# †# 0 0.4 Hepatocellular Ballooning (0-2) Mean Score 0 2 4 NAFLD Activity Score (0-8) †# † p<0.05 vs Normal Chow; # p<0.05 vs HFD High Fat Diet (20x) High Fat Diet + Iron (20x) 0 1 2 3 4 Hepatic IL6 expression Relative IL6/GAPDH mRNA †# 0 1 2 3 4 Hepatic TNFα expression Relative TNFα/GAPDH mRNA †# † p<0.05 vs Normal Chow; # p<0.05 vs HFD 0 1.5 3 Hepatic Srebp1c Relative Srebp1c/GAPDH mRNA †# # 0 1000 2000 3000 4000 5000 6000 7000 8000 Hepatic Iron Concentration HIC µg iron/g liver tissue †# †# Normal Chow HFD Parenteral Iron HFD +Parenteral Iron 0 3 6 9 Hepatic HAMP expression Relative HAMP/GAPDH mRNA †# † BACKGROUND Iron is thought to contribute to NAFLD progression by increasing oxidative stress as a catalyst for the production of reactive oxygen species. Iron may also potentiate progression of NAFLD by altering insulin signaling and lipid metabolism. The aim of this study was to examine the effects of parenteral iron loading in an obese, diabetic murine model of NAFLD. FIGURE 1: Histology Scores IGURE 2: Liver Histology and Iron Staining H&E stain Iron stain IGURE 3: Relationship of Iron and Hepcidin Expression 0 1 2 3 4 Hepatic Heme Oxygenase-1 Expression Relative HO-1/GAPDH mRNA †# †# 0 10 20 30 40 50 Hepatic Malondialdehyde MDA (umoles/g tissue) †# †# 0 2 4 6 8 10 Hepatic Hydroxyproline Assay ug HP/gram tissue †# † † p<0.05 vs Normal Chow; # p<0.05 vs HFD FIGURE 5: Relationship of Iron, Oxidative Stress and Collagen Production FIGURE 4: Relationship of Iron and Inflammatory and Lip Gene Expression B CONCLUSIONS PI administration causes a strong inflammatory response PI administration leads to KC activation and phagocytosis of iron PI caused decreased steatosis PI induces oxidative stress and initiates fibrogenesis Combination of HFD and PI led to more inflammation, oxidative stress and collagen production than either iron or HFD alone