Abstract
Non-alcoholic fatty liver disease (NAFLD) is already the most common chronic liver disease in western countries. The complex metabolic disorder is strongly associated with insulin resistance. Epidemiological studies suggest beneficial effects of the natural soy isoflavone Genistein (GEN) on hepatic energy metabolism. Our aim was to establish a steatotic liver in vitro model to investigate the impact of GEN on energy metabolism focused on insulin signaling. Primary human hepatocytes (PHH) were isolated from resected liver tissues via a two-step collagenase perfusion technique. Steatosis was induced by adding oleic and palmitic acid for 24h to the culture medium. The lipid content was determined photometrically by Oil Red O and SRB staining. Cytotoxicity was evaluated by assays of XTT, AST/LDH and UREA. Phosphorylation levels of ERK½, AKT, FOXO1 and GSK3α/β were measured by Western blot analysis in PHH with and without insulin as well as after incubation with GEN for 24h. PHH treated with free fatty acids showed a significant increase of fat accumulation and a moderate lipotoxicity. Furthermore, in steatotic PHH we observed decreased insulin induced phosphorylation levels of ERK½, AKT, FOXO1 and GSK3α/β. Additional treatment with GEN showed in control PHH an increase of insulin induced phoshorylation levels of ERK½, AKT and GSK3α/β whereas steatotic PHH revealed decreased levels of ERK½ and AKT in a dose dependent manner. Our data show that the steatotic liver in vitro model is a reliable reproduction of in vivo conditions regarding fat accumulation, insulin resistance and lipotoxicity. Insulin resistance was observed by an impaired phosphorylation of ERK½, AKT, FOXO1 and GSK3α/β. GEN leads only in control PHH to an improvement of insulin signaling by increasing phosphorylation levels. However, in steatotic PHH a beneficial effect was not observed. Therefore we recommend further studies to investigate the impact of GEN on energy metabolism in steatotic liver.
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