Induction and siRNA-mediated downregulation of the lipid droplet-associated PAT-proteins adipophilin and TIP47 in cell culture models of hepatocyte steatosis

Hepatocellular lipid droplet (LD) accumulation is the most frequent liver pathology in western countries, and is caused by a broad range of disorders such as alcohol abuse and metabolic syndrome. The LD-surface is governed by amphiphilic proteins of the PAT-family (perilipin, adipophilin, TIP47;=PERILIPIN 1–3). Downregulation of adipophilin has been shown to inhibit diet-induced fatty liver in mice. For human liver, we demonstrated that other PAT-proteins including perilipin are enrolled as well and are differentially expressed. In this study, we characterized the expression pattern of LD-associated proteins in human steatotic liver and in cell culture models of hepatocyte steatosis. Additionally, the effects of LD-accumulation with oleate and of siRNA-mediated downregulation of PAT-proteins in cell culture were evaluated with immunofluorescence microscopy, biochemical and functional assays. Perilipin, adipophilin and TIP47 localized to different size LDs in hepatocytes of human steatotic liver in situ. In hepatoma-derived cells of the lines PLC, HuH7, Hep3B, and HepG2, adipophilin and TIP47 surrounded small LDs. Whilst oleate treatment of HuH7 cells caused a strong increase in adipophilin expression, the protein amount of TIP47 remained unchanged. So far, perilipin was not detected in normal or induced cultured cells. SiRNA-mediated downregulation of adipophilin and TIP47 reduced the number of LDs despite oleate treatment and an increase of larger LDs was observed. However, inhibition of adipophilin did not significantly change TIP47-expression levels and vice versa.In conclusion, LDs in common hepatoma-derived cell culture models contain the PAT-proteins adipophilin, TIP47, but not perilipin, which is found in human steatotic hepatocytes in situ. Downregulation of adipophilin and TIP47 reduce hepatic steatosis in vitro. Thereby adipophilin and TIP47 may represent future targets for therapy of LD-associated diseases such as hepatocyte steatosis.

PAT-protein - lipid droplet - steatosis