PPDPF alleviates hepatic steatosis through inhibition of mTOR signaling

Abstract

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease in the world, however, no drug treatment has been approved for this disease. Thus, it is urgent to find effective therapeutic targets for clinical intervention. In this study, we find that liver-specific knockout of PPDPF (PPDPF-LKO) leads to spontaneous fatty liver formation in a mouse model at 32 weeks of age on chow diets, which is enhanced by HFD. Mechanistic study reveals that PPDPF negatively regulates mTORC1-S6K-SREBP1 signaling. PPDPF interferes with the interaction between Raptor and CUL4B-DDB1, an E3 ligase complex, which prevents ubiquitination and activation of Raptor. Accordingly, liver-specific PPDPF overexpression effectively inhibits HFD-induced mTOR signaling activation and hepatic steatosis in mice. These results suggest that PPDPF is a regulator of mTORC1 signaling in lipid metabolism, and may be a potential therapeutic candidate for NAFLD.

Fig. 1. Liver- specific PPDPF deficiency leads to the development of fatty liver disease.

a The body weight of PPDPF-LKO (n = 6) and WT (n = 6) mice at 8 months. Mean ± SEM, ***p = 0.0005 by two-tailed unpaired Student’s t test. b The ratio of liver-to-body weight (LW/BW) of PPDPF-LKO (n = 6) and WT (n = 6) mice at 8 months. Mean ± SEM, *p = 0.003 by two-tailed unpaired Student’s t test. c Liver images of WT and LKO mice at 8 months of age. d Images of H&E and Oil Red O staining of liver tissues from the WT and LKO mice at 8 months. Scale bars, 100 μm. e, f The triglyceride (TG) and nonestesterified fatty acid (NEFA) in the livers of WT and LKO mice at 8 months of age (n = 6 mice for each group). Mean ± SEM, e **p = 0.002, f **p = 0.001 by two tailed unpaired Student’s t test. g The relative mRNA levels of genes involved in lipid metabolism, including lipogenesis genes, oxidation-related genes, lipid uptake, and lipid secretion in WT and LKO mice at 8 months of age (n = 6 for each group). The mRNA expression levels of the genes are normalized to that of 18 s. Mean ± SEM, SREBP1: *p = 0.025, FASN: *p = 0.035, ACLY: *p = 0.045, ME: *p = 0.012, PPARG: *p = 0.011 by two-tailed unpaired Student’s t test. h Representative images of immunohistochemical staining of PPDPF in liver sections from controls and NAFLD patients. Scale bars,100 μm. All experiments were repeated three times independently.

Fig. 2. PPDPF deficiency exacerbates HFD-induced hepatic steatosis.

Body weight (a) and liver weight (b) of WT(n = 6) and LKO (n = 6) mice fed a chow diet or HFD for 16 weeks. Mean ± SEM, *p = 0.01 by two tailed unpaired Student’s t test. The triglyceride (TG) (c) and nonesterified fatty acid (NEFA) (d) levels in the livers of WT and LKO mice at the end of 16 weeks of HFD feeding (n = 6 mice for each group). Mean ± SEM, (c) *p = 0.032 (NC16W), *p = 0.019 (HFD16W), (d) **p = 0.001 (HFD16W) by two tailed unpaired Student’s t test. e GTTs and ITTs are performed in WT and LKO mice after 20 weeks of NC or 21 weeks of HFD feeding (n = 6 in each group). Mean ± SEM. See Supplementary Data 1 for statistics. f Images of H&E and Oil Red O staining of liver tissues from WT and LKO mice fed HFD for 16 weeks. Scale bars, 100 μm. g The mRNA levels of lipogenic genes in the livers of the WT and LKO mice (n = 6 in each group). Mean ± SEM, SREBP1: **p = 0.006, FASN: *p = 0.019, ACLY: **p = 0.004, PPARG: ****p < 0.0001, ME: *p = 0.026 by two tailed unpaired Student’s t test. h PPDPF expression is measured by qPCR in primary hepatocytes. Mean±SEM, ****p < 0.0001 by two-tailed unpaired Student’s t test. i Oil Red O staining of the primary hepatocytes from WT and LKO mice exposed to 0.4 mM plamitic acid (PA). Scale bars, 100um. j Triglyceride quantification of the primary hepatocytes from WT (n = 3) and LKO (n = 3) mice exposed to 0.4 mM PA. Mean ± SEM, *p = 0.01 (WT-BSA Vs LKO-BSA), *p = 0.04 (WT-BSA Vs WT-PA), ***p = 0.002 (LKO-BSA Vs LKO-PA) by two tailed unpaired Student’s t test. k The relative mRNA levels of the indicated molecules in cells from WT (n = 6) and LKO (n = 6) mice. Mean ± SEM, SREBP1: **p < 0.005, FASN: *p = 0.015, ACLY: **p = 0.003, PPARG: ***p = 0.0002, ME: **p = 0.0049 by two-tailed unpaired Student’s t test. All experiments were repeated three times independently.

Fig. 3. AAV8-mediated reintroduction of PPDPF rescues the phenotype of PPDPF-null in PPDPF-LKO mice fed a chow diet for 8 months.

The body weight (a), liver weight (b), TG test (c) and NEFA test (d) of WT + AAV8-con (n = 8), LKO + AAV8-con (n = 7), and LKO + AAV8-PPDPF (n = 7) mice at 8 months on chow diets. Mean ± SEM, (a) **p = 0.0017 (WT + AAV8-con Vs LKO + AAV8-con), ****p < 0.0001 (LKO + AAV8-con Vs LKO + AAV8-PPDPF), (b) **p = 0.0055 (WT + AAV8-con Vs LKO + AAV8-con), **p = 0.01 (LKO + AAV8-con Vs LKO + AAV8-PPDPF), (c) ****p < 0.0001 (WT + AAV8-con Vs LKO + AAV8-con), ****p < 0.0001 (LKO + AAV8-con Vs LKO + AAV8-PPDPF), (d) ****p < 0.0001 (WT + AAV8-con Vs LKO + AAV8-con), ****p < 0.0001 (LKO + AAV8-con Vs LKO + AAV8-PPDPF) by two-tailed unpaired Student’s t test. e Representative images of H&E and Oil Red O staining of liver sections from the mice injected with indicated adenovirus at 8 months. Scale bars, 100 um. f The mRNA expression levels of lipogenesis-related genes in WT + AAV8-con (n = 6), LKO + AAV8-con (n = 6) and LKO + AAV8-PPDPF (n = 6) mice at 8months. Mean ± SEM, SREBP1: **p = 0.0015, *p = 0.0463; FASN: **p = 0.0050, *p = 0.0029; ACLY: *p = 0.0322, *p = 0.0123; PPARG: *p = 0.0134, *p = 0.0281; ME: *p = 0.459, *p = 0.011 by two-tailed unpaired Student’s t test. All experiments were repeated three times independently.

Fig. 4. PPDPF represses mTOR signaling pathway during the development of hepatic steatosis.

Western blot analysis of phosphorylated S6K, total S6K, cleaved-SREBP1, and FASN in the livers from NC (a) (n = 4 per group) or HFD (b) (n = 5 per group) fed mice in PPDPF-WT and PPDPF-LKO groups. Western blot analysis of phosphorylated S6K, total S6K, cleaved-SREBP1, and FASN in primary hepatocytes (c) and HepG2 (d) cells upon PA treatment. e Expression of p-S6K, S6K, SREBP1 and FASN in liver samples of WT + AAV8-con, LKO + AAV8-con and LKO + AAV8-PPDPF mice in rescue experiment (n = 4 per group). f Expression of p-S6K, S6K, SREBP1, and FASN in liver samples of WT + AAV8-con, LKO + AAV8-con and LKO + AAV8-PPDPF mice in rescue experiment after 3-month HFD feeding (n = 4 per group). All experiments were repeated three times independently.

Fig. 5. PPDPF interacts with Raptor and inhibits the ubiquitination of Raptor.

a, b Co-immunoprecipitation assay in 293T cells co-transfected with Flag-Raptor and HA-PPDPF. c, d Examination of the interaction between endogenous PPDPF and Raptor by co-immunoprecipitation. e PPDPF interacts with Raptor in vitro, purified GST was used as a control. f GST-pull down assay using different truncation mutants of PPDPF. g, h 293 T cells were transfected with control vector and PPDPF WT, PPDPF mut, HA-Raptor plasmid, respectively. After 48 h, co-immunoprecipitation assay was performed. i GST-pull down assay examining the interaction between GST-PPDPF, GST-PPDPF mut with Raptor. j The ubiquitination of Raptor in 293T cells was examined using immunoprecipitation with Raptor antibody. All experiments were repeated 3 times independently.

Fig. 6. AAV8-mediated reintroduction of PPDPF and PPDPF-mut in PPDPF-LKO mice.

The liver TG test (a) and NEFA test (b) of WT + con, LKO + con, LKO + PPDPF, and LKO + PPDPF mut mice (n = 5 per group) at 8 months on chow diets. Mean ± SEM, n.s (not significant), ****p < 0.0001 by two-tailed unpaired Student’s t test. c Representative images of H&E and Oil Red O staining of liver sections from the mice injected with indicated adenovirus at 8 months. Scale bars, 100 um. d Expression of p-S6K, S6K, SREBP1 and FASN in the liver tissues of WT + con, LKO + con, LKO + PPDPF and LKO + PPDPF mut mice (n = 3 per group) fed HFD for 3 months. The liver TG test (e) and NEFA test (f) of WT + con, LKO + con, LKO + PPDPF, and LKO + PPDPF mut mice (n = 5 per group) fed HFD for 3 months. Mean ± SEM, n.s (not significant), ****p < 0.0001 by two-tailed unpaired Student’s t-test. g Representative images of H&E and Oil Red O staining of liver sections from the mice injected with indicated adenovirus fed HFD for 3 months. Scale bars, 100 um. h Expression of p-S6K, S6K, SREBP1, and FASN in each group in HFD mouse model. All experiments were repeated three times independently.

Fig. 7. PPDPF disrupts the interaction between Raptor and DDB1.

a, b The interaction between PPDPF and DDB1 is detected by Co-immunoprecipitation assay in 293T cells co-transfected with Flag-DDB1 and HA-PPDPF. c GST pull-down is performed to confirm the interaction between PPDPF and DDB1, purified GST as the control. d PPDPF inhibits DDB1-mediated increase in Raptor ubiquitination. e, f PPDPF inhibites Raptor-DDB1 and Raptor-mTOR interaction in 293T and HepG2 cells by co-immunoprecipitation with Raptor antibody. g The interaction between Raptor with DDB1, mTOR is examined by co-immunoprecipitation upon PA stimulation in control and PPDPF-overexpressing HepG2 cells. h Raptor-DDB1 and Raptor-mTOR interaction in primary hepatocytes isolated from PPDPF-WT and PPDPF-LKO mice upon PA stimulation. i AAV-mediated reintroduction of PPDPF in the liver of LKO mice inhibits the Raptor-DDB1 and Raptor-mTOR interaction. All experiments were repeated three times independently.

Fig. 8. PPDPF overexpression reduces HFD-induced hepatic steatosis.

Body weight (a) and liver weight (b) of AAV8-con and AAV8-PPDPF mice fed a chow diet of HFD for 12 weeks (n = 4 for each group). Mean ± SEM, n.s (not significant), *p = 0.014 (a); n.s (not significant), **p = 0.01 (b) by two-tailed unpaired Student’s t test. The triglyceride (TG) (c) and nonesterified fatty acid (NEFA) (d) levels in the livers of AAV8-con and AAV8-PPDPF mice at the end of 12-week HFD feeding (n = 4 mice for each group). Mean±SEM, n.s (not significant), **p = 0.004 (c); n.s (not significant), ***p = 0.0003 (d) by two-tailed unpaired Student’s t test. e Representative H&E and Oil Red O staining of liver sections from AAV8-con and AAV8-PPDPF mice after 12-week HFD feeding. Scale bars, 100 um. GTTs (f) and ITTs (g) were performed in AAV8-con (n = 3) and AAV8-PPDPF (n = 3) mice fed a chow diet or AAV8-con (n = 3) and AAV8-PPDPF (n = 4) mice on HFD diet for 10 weeks and 11weeks. Mean ± SEM. See Supplementary Data 2 for statistics. h The mRNA levels of lipogenic genes in the livers of AAV8-con (n = 4) and AAV8-PPDPF (n = 4) mice on HFD diet. Mean ± SEM, SREBP1: *p = 0.018, FASN: *p = 0.045, ACLY: *p = 0.026, PPARG: **p = 0.009, ME: *p = 0.038 by two-tailed unpaired Student’s t test. i Expression of p-S6K, S6K, SREBP1, and FASN in the liver samples of AAV8-con and AAV8-PPDPF micefed HFD for 3 months (n = 4 per group). All experiments were repeated three times indenpendently.