P16 promotes aging-induced lipid droplet accumulation via up-regulation of PCSK9 in hepatocytes

Abstract

Abstract Background: The accumulation of senescent cells promotes hepatic fat accumulation. P16, a proto-typical marker of senescent cells, is closely correlated to hepatic lipid accumulation. PCSK9 (proprotein convertase subtilisin/kexin type 9) plays a critical role in lipid metabolism via PCSK9/LDLR (low-density lipoprotein receptor) axis. This study aimed to explore the mechanism of p16 modulating PCSK9 expression to enhance hepatic lipid accumulation. Methods: All aging mice (12 months old) were randomly assigned two groups: control group with HF (high fat) diet for 6 months, and medicine group with ABT263 (senolytic drug) treatment for 6 months in the presence of HF diet. To induce the senescent cells, cells were treated with bleomycin or adenovirus overexpressing p16 (ad-p16). Cells were treated with cell culture medium containing oleic acid (OA) and palmitic acid (PA) to mimic hepatic steatosis in vivo. The senescent cells were evaluated by SA-β-gal staining. For lipid droplets visualization, Oil red O and Nile red staining were performed. Eventually, the effect of p16 on PCSK9/LDLR axis was determined by Western blot and real-time quantitative polymerase chain reaction (RT-qPCR). Results: We found ABT263 treatment markedly reduced lipid droplets, accompanied with dramatically decreased expression of p16 and PCSK9 in the liver. P16 silencing in senescent HL7702 inhibited lipid droplet accumulation, while p16 overexpression in AML12 remarkably increased lipid droplets, cellular content of total cholesterol and low-density lipoprotein cholesterol. Moreover, total PCSK9 protein level enhanced in p16-overexpressed hepatocytes, while LDLR significantly decreased in membrane and increased in cytoplasm in these cells. Mechanically, we found p16 overexpression inhibited K48-linked polyubiquitination of PCSK9. Conclusions: These results indicate a novel role of p16 in lipid droplet accumulation through aberrant regulation of PCSK9/LDLR axis with PCSK9 ubiquitination in hepatocytes. Lowering p16 expression may be a novel strategy to reduce aberrant lipid metabolism in aging-related diseases.