Iron Overload Causes Oxidative Stress and Impaired Insulin Signaling in AML-12 Hepatocytes

Abstract

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.