Abstract 569: Pnpla2 Influences Secretion of Triglyceride-rich Lipoproteins but Does Not Affect Lipid-droplet Metabolism of Human Hepatoma Cells

Background: Increased hepatic secretion of triglyceride (TG)-rich lipoproteins and cellular TG-accumulation are associated with enhanced risk for cardiovascular disease and diabetes mellitus but the factors regulating these processes are largely unknown. KO studies demonstrated that patatin-domain containing protein 2 (PNPLA2), also known as ATGL, is largely responsible for hepatic TG-hydrolysis in mice but the physiological role of PNPLA2 in human liver metabolism has thus far not been evaluated. Aim: To investigate the role of PNPLA2 in the secretion of TG-rich lipoproteins and TG-accumulation in human hepatoma Huh7 and HepG2 cell-lines. Results: Gene-specific siRNA silencing of PNPLA2 generated mRNA reductions of 88±9% and 82±5% in Huh7 and HepG2 cells, respectively. PNPLA2-silencing was associated with 48±5% and 45±12% decreases in cellular TG-hydrolysis in both hepatoma cell-lines as measured by an in vivo assay. Secretion of TG-rich lipoproteins was analyzed using an ELISA for apolipoprotein B (APOB) and quantification of 14 C -TG following incubation with 14 C-TG labelled glycerol. It was found that PNPLA2-silencing reduced the secretion of APOB and TG by 35±3% and 37±10% in Huh7 cells and by 23±13% and 29±6% in HepG2 cells, respectively. Surprisingly, no changes in cellular TG-accumulation were observed following PNPLA2-inhibition in Huh7 and HepG2 cells. Confocal microscopy studies confirmed that PNPLA2 inhibition did not change the lipid-droplet content of the hepatoma cells. Subcellular localization studies with a monoclonal PNPLA2 antibody found no evidence for the colocalization of PNPLA2 with lipid droplets. In contrast, considerable overlap was observed between PNPLA2 and the endoplasmic reticulum (ER) marker protein disulphide isomerase (PDI). The colocalization was quantified using Pearson correlation analysis (Rcoloc). The Rcoloc value for PNPLA2 and PDI was 0.78 ±0.06, compatible with predominant localization of PNPLA2 in the ER in human hepatoma cells. Conclusion: These studies demonstrate that PNPLA2 acts as a TG-hydrolase in human hepatoma cells and it is involved in the secretion of TG-rich lipoproteins. However, PNPLA2 does not influence hepatic lipid-droplet homeostasis and is not co-localized with large lipid-droplets.