A new role of p53 in regulating lipid metabolism.

Dear Editor, The p53 tumor suppressor maintains the normal cell growth and genomic stability by launching cell cycle arrest, DNA repair, or apoptosis in response to DNA damage or other forms of cellular stress. Recent studies also suggest that p53 is capable of much broader cellular functions, including the regulation of energy metabolism and autophagy. However, the role of p53 in regulating lipid metabolism is less well understood. Here we report a novel function of p53 in regulating lipid metabolism. Loss of p53 leads to lipid accumulation in both mouse embryonic fibroblast (MEF) cells and mouse liver. Upon high-fat diet (HFD) treatment, p53 knockout mice exhibit marked obesity and hepatic lipid accumulation. Mechanistically, p53 regulates lipid metabolism through transcriptionally regulating aromatase, a key enzyme that converts androgens to estrogens. The importance of aromatase in mediating p53’s function in regulating lipid metabolism is revealed by the observation that transgenic expression of aromatase almost completely reverses the promoting effect of p53 deficiency on lipid accumulation in mouse liver. Our most recent data showed that p53 regulates lipid accumulation by directly binding to G6PD and thus inhibiting pentose phosphate pathway (PPP) (Jiang et al., 2011). To further thoroughly investigate the role of p53 in modulating lipid metabolism, we first compared the lipid content in p53+/+ and p53 MEF cells treated with or without oleic acids, a wellknown stimulator of triglyceride (TG) synthesis. The p53 MEF cells exhibit elevated lipid levels compared with p53+/+ MEF cells as evaluated by Oil Red O (ORO) staining and by BODIPY staining (Supplementary Figure S1A and B), which was consistent with the previous study (Molchadsky et al., 2008). We next evaluated the effect of p53 on the lipid droplet formation in the liver. The liver of p53 mice was shown to have a larger number of lipid droplets compared with that of p53+/+ mice (Supplementary Figure S1C). Consistent with this, primary hepatocytes isolated from p53 mice showed enhanced lipid levels (Supplementary Figure S1D). Also, the knockdown of p53 strongly increased lipid levels in MEF cells (Supplementary Figure S1E). These results demonstrate that the loss of p53 increases lipid accumulation and suggest the physiological function of p53 in regulating lipid metabolism. We next examined whether p53 regulates HFD-induced obesity and hepatic steatosis. When fed with normal diets, p53 and p53+/+ mice showed no obvious difference in either body mass or abdominal fat accumulation. However, HFD treatment resulted in a substantial increase in the body mass of p53 mice compared with p53+/+ mice (Figure 1A and B). Livers from p53 mice were generally larger and paler in color than those from p53+/+ littermates after HFD treatment (Figure 1B). HFD treatment also led to an increased accumulation of epididymal white adipose tissue in p53 mice compared with p53+/+ mice (Figure 1B). Hematoxylin and eosin staining showed that the liver from p53 mice had a greater accumulation of lipid droplets than that from p53 wild-type mice, especially under the HFD treatment condition (Supplementary Figure S2A). Consistently, immunohistochemical staining showed that the lack of p53 resulted in a marked increase in the levels of adipose differentiation related protein (Supplementary Figure S2B), a lipid droplet marker which coats cytoplasmic lipid droplets. Moreover, adipocytes from p53 mice were bigger than those from p53 wild-type mice in response to HFD treatment (Supplementary Figure S2C). Altogether, these results suggest that p53 depletion promotes HFD-induced obesity and liver steatosis in male mice. Interesting enough, after evaluation of the serum levels of testosterone (T) in p53+/+ and p53 mice, we surprisingly found that p53 mice produced dramatically more testosterone than p53+/+ mice upon HFD treatment, while the serum testosterone levels were not significantly different between these two groups of mice under normal diet condition (Supplementary Figure S2D). In the same experiments, the levels of 17b-oestradiol (E2) were comparatively low in p53 and p53+/+ male mice under both normal diet and HFD conditions (Supplementary Figure S2E). As a result, the ratio of T/E2 is significantly higher in p53 mice under HFD treatment conditions (Supplementary Figure S2F). It has been shown that inactivation of aromatase leads to enhanced levels of testosterone and lipid accumulation in the liver (Jones et al., 2000). The observation of elevated levels of testosterone and lipid accumulation in p53 mice upon HFD treatment led us to explore the possibility that the levels of aromatase could be decreased in p53 mice, thus leading to the inhibition of aromatase enzyme activity. After thorough inspection, one putative p53-binding element was found within intron 1 of aromatase gene (Figure 1C). The subsequent chromatin immunoprecipitation (ChIP) assays showed the specific binding of p53 to the chromatin fragments containing the putative p53-binding element (Figure 1C). In addition, pGL3 luciferase reporter plasmid containing the putative p53-binding element, but not the mutant plasmid, showed a p53-responsive transcriptional activity (Supplementary Figure S3A). The lack of p53 resulted in substantially lower levels of both aromatase mRNA and protein expression (Supplementary Figure S3B). Furthermore, real-time RT-PCR analysis showed that various tissues from p53 mice exhibited substantially decreased expression of aromatase compared with those in corresponding tissues from p53+/+ mice doi:10.1093/jmcb/mjs064 Journal of Molecular Cell Biology (2013), 5, 147–150 | 147 Published online December 19, 2012