title: Iron overload causes oxidative stress and impaired insulin signaling in AML-12 hepatocytes.
abstract: BACKGROUND
  Iron overload is associated with increased severity of nonalcoholic fatty liver disease (NAFLD) including progression to nonalcoholic steatohepatitis and hepatocellular carcinoma.
  
  
  OBJECTIVE
  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.
authors.affiliation: Bastyr University, 14500 Juanita Drive NE, Kenmore, WA 98028-4966, USA. dmessner@bastyr.edu
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Messner
authors.forename: Byung Han
authors.initials: BH
authors.lastname: Rhieu
authors.forename: Kris V
authors.initials: KV
authors.lastname: Kowdley
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005234:Fatty Liver / Q000209:etiology* / Q000378:metabolism; D005290:Ferric Compounds; D005434:Flow Cytometry; D022781:Hepatocytes / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance / Q000502:physiology*; D019190:Iron Overload / Q000139:chemically induced / Q000150:complications* / Q000378:metabolism; D051379:Mice; D065626:Non-alcoholic Fatty Liver Disease; D018384:Oxidative Stress; D015125:Oxyquinoline; D000644:Quaternary Ammonium Compounds; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D015415:Biomarkers; D005230:Fatty Acids, Nonesterified; D005290:Ferric Compounds; D007328:Insulin; D000644:Quaternary Ammonium Compounds; D017382:Reactive Oxygen Species; D015125:Oxyquinoline; C013531:ferric ammonium citrate
references.citation: Crit Rev Clin Lab Sci. 2008;45(1):1-23
references.pmid: 18293179
references.citation: J Hepatol. 2004 Dec;41(6):926-34
references.pmid: 15582125
references.citation: N Engl J Med. 1964 Dec 31;271:1391-5
references.pmid: 14214656
references.citation: Hepatology. 1999 Dec;30(6):1356-62
references.pmid: 10573511
references.citation: Clin Sci (Lond). 2008 Sep;115(5):141-50
references.pmid: 18662168
references.citation: Trends Mol Med. 2008 Feb;14(2):72-81
references.pmid: 18218340
references.citation: J Biol Chem. 2006 Apr 28;281(17):12093-101
references.pmid: 16505490
references.citation: Histochemistry. 1992 Jul;97(6):493-7
references.pmid: 1385366
references.citation: Nat Clin Pract Gastroenterol Hepatol. 2005 Jan;2(1):46-53
references.pmid: 16265100
references.citation: Gastroenterology. 1998 Feb;114(2):311-8
references.pmid: 9453491
references.citation: Antioxid Redox Signal. 2005 Jul-Aug;7(7-8):1040-52
references.pmid: 15998259
references.citation: Hepatol Res. 2009 Mar;39(3):213-22
references.pmid: 19261002
references.citation: Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):674-8
references.pmid: 7904757
references.citation: Diabetes Care. 1998 Dec;21(12):2190
references.pmid: 9839115
references.citation: J Clin Invest. 2008 Sep;118(9):2992-3002
references.pmid: 18769626
references.citation: Endocrinology. 2008 Jul;149(7):3549-58
references.pmid: 18403477
references.citation: J Hepatol. 2009 Feb;50(2):351-7
references.pmid: 19070395
references.citation: Hepatology. 2010 Aug;52(2):774-88
references.pmid: 20683968
references.citation: Hepatology. 2004 Jan;39(1):179-87
references.pmid: 14752836
references.citation: Hepatology. 2007 Sep;46(3):723-9
references.pmid: 17680648
references.citation: Nutr Res Rev. 2005 Dec;18(2):212-21
references.pmid: 19079906
references.citation: Gastroenterology. 1994 Oct;107(4):1103-9
references.pmid: 7523217
references.citation: Braz J Med Biol Res. 2003 Jun;36(6):739-45
references.pmid: 12792703
references.citation: J Clin Invest. 2004 Jul;114(2):147-52
references.pmid: 15254578
references.citation: Carcinogenesis. 2001 Aug;22(8):1163-72
references.pmid: 11470744
references.citation: Free Radic Biol Med. 2004 Dec 15;37(12):1921-42
references.pmid: 15544913
references.citation: J Hepatol. 1999 Sep;31(3):421-9
references.pmid: 10488699
references.citation: Arq Gastroenterol. 2006 Jul-Sep;43(3):224-8
references.pmid: 17160239
references.citation: J Hepatol. 2011 Oct;55(4):920-32
references.pmid: 21718726
references.citation: Hepatology. 2012 Nov;56(5):1730-40
references.pmid: 22611049
references.citation: Curr Opin Clin Nutr Metab Care. 2010 Nov;13(6):709-14
references.pmid: 20842026
references.citation: J Gastroenterol Hepatol. 2009 May;24(5):830-40
references.pmid: 19207680
references.citation: J Biol Chem. 1989 Mar 15;264(8):4417-22
references.pmid: 2466835
references.citation: Ann N Y Acad Sci. 1975 Dec 30;264:373-86
references.pmid: 1062961
references.citation: Toxicology. 2000 Aug 14;149(1):43-50
references.pmid: 10963860
references.citation: Nature. 2006 Apr 13;440(7086):944-8
references.pmid: 16612386
references.citation: J Mol Med (Berl). 2010 May;88(5):477-86
references.pmid: 20169331
references.citation: Annu Rev Physiol. 2007;69:69-85
references.pmid: 17014365
references.citation: Diabetes. 2006 Dec;55 Suppl 2:S9-S15
references.pmid: 17130651
references.citation: Am J Pathol. 2008 Mar;172(3):738-47
references.pmid: 18245813
references.citation: Lancet. 2010 Jun 26;375(9733):2267-77
references.pmid: 20609972
references.citation: Liver Int. 2003 Aug;23(4):232-42
references.pmid: 12895262
references.citation: Free Radic Biol Med. 2002 May 1;32(9):833-40
references.pmid: 11978485
references.citation: Trends Mol Med. 2006 Dec;12(12):555-8
references.pmid: 17049925
references.citation: Hepatology. 2011 Feb;53(2):448-57
references.pmid: 21274866
references.citation: Free Radic Biol Med. 2007 Oct 1;43(7):995-1022
references.pmid: 17761297
references.citation: Endocrinology. 2001 Jan;142(1):229-40
references.pmid: 11145586
references.citation: Liver Int. 2009 Jan;29(1):63-72
references.pmid: 18492020