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THE EFFECT OF STEATOSIS ON IRON HOMEOSTASIS IN HuH-7 HEPATOMA CELLS
MD Beaton, SJ Wilkins, DM Frazer, JP Whitehead, JH Moffitt, GA Macdonald, LW Powell, GJ Anderson
Iron Metabolism Laboratory, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Queensland
AIMS: Hepatic iron is often found in patients with non-alcoholic fatty liver disease (NAFLD). Why this occurs is unclear. We sought to investigate the effects of steatosis on cellular iron metabolism using a well defined cell culture model.
METHODS: All studies used the human hepatoma cell line HuH-7. Cells were maintained in RPMI1640 medium with 10% foetal bovine serum and were used at 90% confluence. Lipid loading was achieved by overnight (16h) incubation with 1mM oleic acid. Cells were iron loaded with ferric ammonium citrate (200µg/mL) (with or without 1mM oleic acid) or depleted of iron with desferrioxamine (DFO, 100µM). Lipid loading was confirmed by Oil Red O stain and immunofluorescence. Hepatocyte viability was assessed by Trypan Blue exclusion. Western blotting was used to evaluate the levels of ferritin, transferrin receptor 1 (TfR1), divalent metal-ion transporter 1 (DMT1) and ferroportin (FPN), all key proteins in iron transport and storage. Expression of the iron regulatory peptide hepcidin was studied at the mRNA level using Real Time PCR.
RESULTS: Cells treated with oleic acid demonstrated significant lipid loading on both Oil Red O staining and immunofluorescent staining for adipophilin. Treated cells remained 85-90% viable. Expression of ferritin, the diferric transferrin binding protein TfR1, and FPN was readily detected in HuH-7 cells, but their levels were unaffected by oleic acid treatment. No expression of the membrane ferrous iron transport protein DMT1 could be detected. Treatment of cells with both oleic acid and ferric ammonium citrate stimulated ferritin synthesis, but there was no enhanced effect over that seen with the iron treatment alone. Treatment with DFO reduced ferritin levels confirming that this protein was being appropriately regulated by iron in HuH-7 cells. The expression of the liver-derived iron regulatory peptide hepcidin was examined at the mRNA level but was unaltered by oleic acid treatment
CONCLUSIONS: Although hepatic steatosis has been suggested to alter the iron status of cells, in this study we found no evidence that excess lipid exerts a direct effect on key molecules involved in cellular iron homeostasis.