ferric-ammonium-citrate and Non-alcoholic-Fatty-Liver-Disease

We have previously demonstrated that iron overload in hepatic reticuloendothelial system cells (RES) is associated with severe nonalcoholic steatohepatitis (NASH) and advanced fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Recruited myeloid-derived macrophages have gained a pivotal position as drivers of NASH progression and fibrosis. In this study, we used bone marrow-derived macrophages (BMDM) from C57Bl6 mice as surrogates for recruited macrophages and examined the effect of iron on macrophage polarization. Treatment with iron (ferric ammonium citrate, FAC) led to increased expression levels of M1 markers: CCL2, CD14, iNOS, IL-1β, IL-6, and TNF-α; it also increased protein levels of CD68, TNF-α, IL-1β, and IL-6 by flow cytometry. This effect could be reversed by desferrioxamine, an iron chelator. Furthermore, iron loading of macrophages in the presence of IL-4 led to the down-regulation of M2 markers: arginase-1, Mgl-1, and M2-specific transcriptional regulator, KLF4. Iron loading of macrophages with IL-4 also resulted in reduced phosphorylation of STAT6, another transcriptional regulator of M2 activation. Dietary iron overload of C57Bl6 mice led to hepatic macrophage M1 activation. Iron overload also stimulated hepatic fibrogenesis. Histologic analysis revealed that iron overload resulted in steatohepatitis. Furthermore, NAFLD patients with hepatic RES iron deposition had increased hepatic gene expression levels of M1 markers, IL-6, IL-1β, and CD40 and reduced gene expression of an M2 marker, TGM2, relative to patients with hepatocellular iron deposition pattern. We conclude that iron disrupts the balance between M1/M2 macrophage polarization and leads to macrophage-driven inflammation and fibrogenesis in NAFLD.

Topics: Adult; Animals; Chemokine CCL2; Deferoxamine; Diet, Fat-Restricted; Female; Femur; Ferric Compounds; Gene Expression Regulation; Humans; Interleukin-1beta; Iron Carbonyl Compounds; Iron Chelating Agents; Iron Overload; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Liver Cirrhosis; Macrophages; Male; Mice; Mice, Inbred C57BL; Middle Aged; Non-alcoholic Fatty Liver Disease; Quaternary Ammonium Compounds; Signal Transduction; STAT6 Transcription Factor; Tibia; Tumor Necrosis Factor-alpha

Iron overload is associated with increased severity of nonalcoholic fatty liver disease (NAFLD) including progression to nonalcoholic steatohepatitis and hepatocellular carcinoma.. To identify potential role(s) of iron in NAFLD, we measured its effects on pathways of oxidative stress and insulin signaling in AML-12 mouse hepatocytes.. Rapid iron overload was induced with 50 μM ferric ammonium citrate and 8-hydroxyquinoline. Insulin response was measured by Western blot of phospho-protein kinase B. Lipid content was determined by staining with Oil Red O. Reactive oxygen species (ROS) were measured by flow cytometry using 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Oxidative stress was measured by Western blots for phospho-jnk and phospho-p38.. Iron increased ROS (p < 0.001) and oxidative stress (p < 0.001) and decreased insulin signaling by 33 % (p < 0.001). Treatment with stearic or oleic acids (200 μM) increased cellular lipid content and differentially modulated effects of iron. Stearic acid potentiated iron-induced ROS levels by two-fold (p < 0.05) and further decreased insulin response 59 % (p < 0.05) versus iron alone. In contrast, cells treated with oleic acid were protected against iron-mediated injury; ROS levels were decreased by half (p < 0.01) versus iron alone while insulin response was restored to control (untreated) levels. The anti-oxidant curcumin reduced effects of iron on insulin signaling, ROS, and oxidative stress (p < 0.01). Curcumin was similarly effective in cells treated with both stearic acid and iron.. An in vitro model of NAFLD progression is described in which iron-induced oxidative stress inhibits insulin signaling. Pathophysiological effects of iron were increased by saturated fat and decreased by curcumin.

Topics: Animals; Biomarkers; Blotting, Western; Cell Line; Fatty Acids, Nonesterified; Fatty Liver; Ferric Compounds; Flow Cytometry; Hepatocytes; Insulin; Insulin Resistance; Iron Overload; Mice; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Oxyquinoline; Quaternary Ammonium Compounds; Reactive Oxygen Species