In Vitro Steatosis Model and the Influence of Anti-Lipidemic Drugs on Defatting Steatotic Hepatocytes
2012-11-01

Steatosis as a first step in obesity has become pivotal in surgical interventions and organ transplantation. Postoperatively, this results in decreased liver regeneration, hepatic failure and at worst death of the patient. Therefore, there is need for fast clearance of liver fat from a surgical point of view. Epidemiologic studies suggest natural food compounds with a beneficial influence on lipid metabolism. Our aim was to establish an in vitro steatosis model based on primary human hepatocytes (PHH) in order to investigate effects of natural antilipidemic drugs on hepatic lipid metabolism. PHH were isolated from human liver resectates using a two-step collagenase perfusion technique. Steatosis was then induced by treatment with free fatty acids (FFA). The in vitro hepatic steatosis model showed a significant increase in number and size of lipid droplets compared to control PHH, according to Oil Red O and SRB staining. The XTT assay as well as AST and LDH levels revealed a slightly cytotoxic impact of FFA on PHH whereas urea formation was unchanged. In steatotic PHH we observed a moderate increase of mRNA levels of GLUT2 and insulin receptor. Furthermore, lipid-overloaded hepatocytes showed decreased insulin-induced phosphorylation levels of ERK1/2, AKT, FoXO1 and GSK3α/β measured by western blot analysis. Transcripts of PPAR α, ACSL and CPT1L were increased whereas mRNA of SREBP1c and FASN were decreased. Protein content of PPAR α and SREBP1c was moderately increased in steatotic PHH. Neither application of the antilipidemic drug Fenofibrate (FEN) used as positive control nor Genistein (GEN) a natural anti-lipidemic compound significantly decreased lipid content. An increased phosphorylation of AKT, ERK1/2 and GSK3 α/β in control but not in steatotic PHH was detected. FoXO1 was not detectable in this setting. FEN increased nuclear PPAR α levels in western blot analysis. GEN slightly elevated nuclear PPAR α level and decreased cleaved SREBP1c, both in a concentration-dependent manner in western blot analysis and real time-PCR. Our data show that our human in vitro model of steatosis is a reliable reproduction of in vivo conditions regarding fat accumulation, lipotoxicity and insulin resistance. The latter was observed by impaired phosphorylation levels of insulin-responsive downstream targets suggesting a cellular dysfunction in regulation of gluconeogenesis and glycogen synthesis as well as growth and differentiation. In addition, the detected upregulation of GLUT2 and insulin receptor gene expression is a known factor of a liver in state of insulin resistance. Lipogenesis, characterized by SREBP1c and FASN, was impaired. Beta-oxidation, represented by PPAR α, ACSL and CPT1L, was increased at transcript level whereas nuclear PPAR α protein was increased. Neither FEN nor GEN lowered hepatic lipid content significantly in this setting. However, FEN expectably acted as PPAR α agonist. GEN, by contrast, slightly increased active PPAR α and decreased active SREBP1c, implying a beneficial impact on hepatic lipid metabolism. In summary our in vitro steatosis model is a useful tool for investigations of metabolic and signal pathways of hepatic energy metabolism. Further studies are needed to identify anti-lipidemic drugs and their potential of influencing hepatic lipid metabolism.