Iron overload causes oxidative stress and impaired insulin signaling in AML-12 hepatocytes

Background: Iron overload is associated with increased severity of nonalcoholic fatty liver disease (NAFLD) including progression to nonalcoholic steatohepatitis and hepatocellular carcinoma. Aims: 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. Methods: 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. Results: 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. Conclusions: 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.