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. 2023 Jul;55(7):1520-1530.
doi: 10.1038/s12276-023-01036-7. Epub 2023 Jul 3.

Ablation of the deubiquitinase USP15 ameliorates nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

Jung-Hwan Baek #  1 , Myung Sup Kim #  1 , Hye Ryeon Jung  2 , Min-Seon Hwang  1 , Chan-Ho Lee  1 , Dai Hoon Han  3 , Yong-Ho Lee  4 , Eugene C Yi  2 , Seung-Soon Im  5 , Ilseon Hwang  6 , Kyungeun Kim  7 , Joon-Yong Chung  8 , Kyung-Hee Chun  9
Affiliations

Ablation of the deubiquitinase USP15 ameliorates nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

Jung-Hwan Baek et al. Exp Mol Med. 2023 Jul.

Erratum in

Abstract

Nonalcoholic fatty liver disease (NAFLD) occurs due to the accumulation of fat in the liver, leading to fatal liver diseases such as nonalcoholic steatohepatitis (NASH) and cirrhosis. Elucidation of the molecular mechanisms underlying NAFLD is critical for its prevention and therapy. Here, we observed that deubiquitinase USP15 expression was upregulated in the livers of mice fed a high-fat diet (HFD) and liver biopsies of patients with NAFLD or NASH. USP15 interacts with lipid-accumulating proteins such as FABPs and perilipins to reduce ubiquitination and increase their protein stability. Furthermore, the severity of NAFLD induced by an HFD and NASH induced by a fructose/palmitate/cholesterol/trans-fat (FPC) diet was significantly ameliorated in hepatocyte-specific USP15 knockout mice. Thus, our findings reveal an unrecognized function of USP15 in the lipid accumulation of livers, which exacerbates NAFLD to NASH by overriding nutrients and inducing inflammation. Therefore, targeting USP15 can be used in the prevention and treatment of NAFLD and NASH.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. USP15 is upregulated in fatty livers.
a, b The mRNA and protein levels of USP15 in the liver from mice fed a normal-fat diet (NFD) or a high-fat diet (HFD) for 12 weeks (n = 4/group). c Representative images of mouse normal and NAFLD liver tissues with low and high immunohistochemical staining for USP15. Hepatic steatosis was visualized with hematoxylin and eosin (H&E) staining. Scale bar indicates 100 μm. d The level of USP15 mRNA in the livers of individuals without NAFLD (normal) or with NAFLD (n = 5/group). e Representative immunohistochemical staining for USP15 in formalin-fixed paraffin-embedded liver tissues. H&E staining of formalin-fixed, paraffin-embedded human liver samples diagnosed as normal, NAFLD, and NASH is shown at the top of the image. Scale bar indicates 100 μm. f USP15 levels were significantly increased in NAFLD (p = 0.041) and NASH (p = 0.001) specimens compared to those in normal controls. g Positive correlation between USP15 levels and serum AST concentrations and serum ALT concentrations and BMI. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the NFD or normal group.
Fig. 2
Fig. 2. USP15 interacts with FABP1 and FABP4 and Perilipin1 and Perilipin2.
a USP15 interactome analysis. USP15 binding proteins were immunoprecipitated using a FLAG antibody and identified via mass spectrometry. b Interaction between endogenous USP15 and FABP1, FABP4, Perilipin1, and Perilipin2. The AML12 cells were immunoprecipitated using IgG and USP15 antibodies. c The interaction between exogenous USP15 and FABP4 or Perilipin1. HEK293 cells were transfected with FLAG-USP15, HA-FABP4, or HA-Perilipin1 plasmids and immunoprecipitated using a FLAG antibody. d GST pulldown assay showing a direct interaction between USP15 and FABP4 or Perilipin1. e Representative immunohistochemical staining for USP15 and Perilipin1 in patients with nonalcoholic fatty liver disease. The scale bar indicates 100 µm. f Overexpression of Perilipin1 was observed in NAFLD (p < 0.001) and NASH (p < 0.001). g There was a statistically significant correlation between USP15 and Perilipin1 in nonalcoholic fatty liver specimens (Pearson’s X2 test = 7.483, p = 0.006). ***p < 0.001 compared to normal.
Fig. 3
Fig. 3. USP15 increases the protein stability of FABP1/4 and Perilipin1/2 and lipid accumulation in hepatocytes.
a Western blot analysis showing the dose dependency of HA-FABP4 or HA-PLIN1 in HEK293 cells with increasing FLAG-USP15. b Effects of USP15 knockdown on the protein stability of FABP1/4 and Perilipin1/2. AML12 cells were transfected with siRNA against USP15, and cells were treated with or without 20 μM MG132 for 8 h. c Western blots of AML12 cells treated with cycloheximide as indicated. d Normalized protein levels of FABP1/4 and Perilipin1/2 from (c). e Representative image of Oil Red O staining of AML12 cells treated with oleic acid as indicated with or without siRNA against USP15. f Normalization of Oil Red O staining from (e). Data were represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to scRNA. g Western blots of AML12 cells transfected with either USP15 WT or C298A followed by oleic acid treatments as indicated. Data were represented as the mean ± SEM. *p < 0.05 and **p < 0.01 compared to USP15 WT. h Representative image of Oil Red O staining of AML12 cells transfected with either USP15 WT or C298A mutant. Cells were treated with oleic acid as indicated. i Normalization of Oil Red O staining from (h). Data were represented as the mean ± SEM. *p < 0.05 and **p < 0.01 compared to empty vector.
Fig. 4
Fig. 4. USP15 regulates the protein stability of FABP1/4 and Perilipin1/2 by deubiquitination.
a Western blots of deubiquitination assays of FABP1/4 and Perilipin1/2. AML12 cells were transfected with or without siRNA against USP15 followed by 20 μM MG132 for 8 h. b Western blots of Ni2+ NTA pulldown show the deubiquitination of overexpressed FABP4 or Perilipin1 with 20 μM MG132 for 8 h. c Western blots of deubiquitination assays in HEK293 cells after transfection with either USP15 WT or C298A mutant followed by 20 μM MG132 for 8 h. d Effects of USP15 WT or C298A mutant on the protein stability of FABP4 or Perilipin1 in a USP15 dose-dependent manner. e Normalization of Western blots of protein levels in (d). Data were represented as the mean ± SEM. *p < 0.05, **p < 0.01 compared to USP15 WT.
Fig. 5
Fig. 5. Liver-specific USP15 knockout mice exhibit improved hepatic steatosis in PPARγ-overexpressing livers.
a Schematic representation of adenovirus injection (n = 4/group). b Macroscopic view of the liver from adenovirus-injected mice. c Slide sections of the liver from adenovirus-injected mice. Liver sections were stained with H&E, FABP4, Perilipin1, and F4/80. d Liver weight and the ratio of liver weight to body weight of adenovirus-injected mice (n = 4/group). e Expression of genes involved in fatty acid uptake and lipogenesis (n = 4/group). Data in (d, e) are represented as the mean ± SEM. ***p < 0.001 compared to WT AV-PPARγ.
Fig. 6
Fig. 6. Adenovirus-mediated USP15 overexpression aggravates hepatic steatosis.
a Schematic representation of adenovirus injection (n = 4/group). b Macroscopic view of the liver from adenovirus-injected mice. c Slide sections of the liver from adenovirus-injected mice. The liver sections were stained with H&E. d The liver weight and the ratio of liver weight to body weight of adenovirus-injected mice (n = 4/group). Data were represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to AV-Cont or AV-PPARγ + USP15. e Contents of triglycerides and free fatty acids in the liver from adenovirus-injected mice (n = 4/group). Data were represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to AV-Cont or AV-PPARγ + USP15. f The in vivo effects of USP15 WT or C298A mutant on FABP1/4 and Perilipin1/2 protein stability from the mouse liver (n = 4/group). g Normalization of protein levels from (d). Data are represented as the mean ± SEM. *p < 0.05 and **p < 0.01 compared to AV-USP15.
Fig. 7
Fig. 7. Liver-specific USP15 knockout mice show high-fat diet-induced fatty liver improvement.
Both wild-type and USP15LKO mice were fed a high-fat diet (HFD) for 12 weeks. a Macroscopic view of livers from the wild-type and USP15LKO mice fed an HFD. b Body weight, liver weight, and the ratio of liver weight to body weight of the wild-type and USP15LKO mice fed an HFD (WT: n = 10, USP15LKO: n = 9). c Triglyceride and free fatty acid contents in the livers of the wild-type and USP15LKO mice fed an HFD (n = 5/group). d Slide sections of livers from the wild-type and USP15LKO mice fed an HFD. Liver sections were stained with H&E or Oil Red O. Scale bar indicates 100 μm. e Concentrations of ALT and AST in the serum from the wild-type and USP15LKO mice fed an HFD (n = 4/group). f The expression of genes involved in gluconeogenesis, fatty acid uptake, lipogenesis, and fatty acid oxidation in the livers of the wild-type and USP15LKO mice fed an HFD (n = 4/group). g Protein expression of FABP1, FABP4, Perilipin1, and Perilipin2 in the liver from the wild-type and USP15LKO mice fed an HFD. h Slide sections of livers from the wild-type and USP15LKO mice fed an HFD. Liver sections were immunohistochemically stained using antibodies against FABP4 and PLIN1. The scale bar indicates 100 μm. i The expression of factors involved in inflammation in the liver from the wild-type and USP15LKO mice fed an HFD (n = 4/group). Slide sections of the liver were stained using an antibody against F4/80. The scale bar indicates 100 μm. The mRNA expression was analyzed with quantitative RT‒PCR. Data were represented as the mean ± SEM. *p < 0.05 and **p < 0.01 compared to WT.
Fig. 8
Fig. 8. Hepatocyte deletion of USP15 reduces liver steatosis, inflammation, and fibrosis in FPC-fed mice.
Both wild-type and USP15LKO mice were fed a fructose-palmitate-cholesterol (FPC) diet for 16 weeks. a Macroscopic view of the liver from the wild-type and USP15LKO mice fed an FPC diet. b Body weight, liver weight, and the ratio of liver weight to body weight of the wild-type and USP15LKO mice fed an FPC diet (WT: n = 8, USP15LKO: n = 8). c Triglyceride and free fatty acid contents in the livers of the wild-type and USP15LKO mice fed an FPC diet (n = 5/group). d Concentrations of ALT and AST in the serum from the wild-type and USP15LKO mice fed an FPC diet (n = 5/group). e Slide sections of the liver from the wild-type and USP15LKO mice fed an FPC diet. Liver sections were stained with H&E, Oil Red O, and Sirius red. Liver sections were immunohistochemically stained using antibodies against α-SMA, F4/80, and p-SMAD3. The left panels represent 200x magnifications (x200), and the right panels represent 400x magnifications (x 400). The scale bar indicates 100 μm. f The expression of factors involved in inflammation in the liver from wild-type and USP15LKO mice fed an FPC diet (n = 4/group). The mRNA expression was analyzed with quantitative RT‒PCR. g The expression of liver fibrosis markers in the livers from the wild-type and USP15LKO mice fed an FPC diet (n = 4/group). The mRNA expression was analyzed with quantitative RT‒PCR. h Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of liver sections from the wild-type and USP15LKO mice fed an FPC diet. The scale bar indicates 100 μm. Data were represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to WT.
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