USP29 alleviates the progression of MASLD by stabilizing ACSL5 through K48 deubiquitination

Abstract

Background/aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic liver disease characterized by hepatic steatosis. Ubiquitin-specific protease 29 (USP29) plays pivotal roles in hepatic ischemiareperfusion injury and hepatocellular carcinoma, but its role in MASLD remains unexplored. Therefore, the aim of this study was to reveal the effects and underlying mechanisms of USP29 in MASLD progression.

Methods: USP29 expression was assessed in liver samples from MASLD patients and mice. The role and molecular mechanism of USP29 in MASLD were assessed in high-fat diet-fed and high-fat/high-cholesterol diet-fed mice and palmitic acid and oleic acid treated hepatocytes.

Results: USP29 protein levels were significantly reduced in mice and humans with MASLD. Hepatic steatosis, inflammation and fibrosis were significantly exacerbated by USP29 deletion and relieved by USP29 overexpression. Mechanistically, USP29 significantly activated the expression of genes related to fatty acid β-oxidation (FAO) under metabolic stimulation, directly interacted with long-chain acyl-CoA synthase 5 (ACSL5) and repressed ACSL5 degradation by increasing ACSL5 K48-linked deubiquitination. Moreover, the effect of USP29 on hepatocyte lipid accumulation and MASLD was dependent on ACSL5.

Conclusion: USP29 functions as a novel negative regulator of MASLD by stabilizing ACSL5 to promote FAO. The activation of the USP29-ACSL5 axis may represent a potential therapeutic strategy for MASLD.

Keywords: ACSL5 protein, human; Lipid metabolism disorders; Metabolic dysfunction-associtaed steatotic liver disease; USP29 protein, human.

Figure 1.

USP29 expression is down-regulated in the pathogenesis of MASLD. (A) The protein level and relative mRNA level of USP29 in liver tissues of mice fed with NC or HFD for 24 weeks (n=4 mice/group); (b, C) The protein level and relative mRNA level of USP29 in liver tissues of mice fed with NC or HFHC for 16 weeks (b) or fed with MCS or MCD for 4 weeks (C) (n=4 mice/group); (D) The protein level and relative mRNA level of USP29 in liver samples from human Normal, MASL and MASH patients (n=4 individual/group); (E) Representative immunohistochemical staining to evaluate USP29 expression in liver samples from human Normal, MASL and MASH patients (n=5 individuals/group). Scale bar, 50 μm; (F) The protein level and relative mRNA level of USP29 in mouse primary hepatocytes induced by bSA or PAOA (0.5/1.0 mM) for 12 h. The protein expression and mRNA expression were normalized to β-actin levels. The data are presented as mean±SD (^*P<0.05, ^**P<0.01, n.s., not significant). NC, normal chow; HFD, high fat; HFHC, high-fat high cholesterol; MCD, methionine and choline deficient; MCS, methionine and choline sufficient; MASL, metabolic dysfunction-associated steatotic liver; MASH, metabolic dysfunction-associated steatohepatitis; PAOA, palmitic acid and oleic acid; bSA, bovine serum albumin.

Figure 2.

USP29 deletion exacerbates insulin resistance and hepatic steatosis induced by a HFD diet. (A) body weight and (b) blood glucose of WT mice and USP29-KO mice after NC chow or HFD diet treatment for indicated times (n=9–10 mice/group). (C) GTTs and (D) ITTs of WT mice and USP29-KO mice were analyzed at the week 22 and 23 fed NC chow or HFD diet, respectively (n=9–10 mice/group). (E) Liver weight and the ratio of liver weight to body weight of WT mice and USP29-KO mice fed NC chow or HFD diet for 24 weeks (n=9–10 mice/group). (F) Hepatic triglyceride (TG) content and (G) serum TG, TC and LDL-C content were detected in WT mice and USP29-KO mice fed NC chow or HFD diet for 24 weeks (n=9–10 mice/group). (H) Representative images and relative quantitative statistical analysis of H&E staining and Oil red O staining of liver tissue from WT and USP29-KO mice fed NC chow or HFD diet for 24 weeks (n=6 mice/group). Scale bar, 50 μm. The data are presented as mean±SD, ^*indicates a statistical analysis between WT-NC group and WT-HFD group (^*P<0.05, ^**P<0.01, n.s., not significant). ^#indicates a statistical analysis between WT-HFD group and USP29-KO-HFD group (^#P<0.05, ^##P<0.01, n.s., not significant). WT, wild type; KO, USP29 knockout; NC, normal chow; HFD, high fat; GTT, glucose tolerance test; ITT, insulin tolerance test; TG, total triglyceride; TC, total cholesterol; LDL-C, low density lipoprotein cholesterol; H&E, hematoxylin and eosin.

Figure 3.

USP29 knockout accelerates hepatic steatosis, inflammation and fibrosis induced by a HFHC diet. (A) blood glucose of WT and USP29-KO mice after NC or HFHC diet treatment for 16weeks (n=8–10 mice/group). (b) GTTs of WT mice and USP29-KO mice were analyzed at the week 15 fed NC chow or HFHC diet (n=8–10 mice/group). (C) The ratio of liver weight to body weight of WT mice and USP29-KO mice fed NC chow or HFD diet for 16 weeks (n=8–10 mice/group). (D) Hepatic TG content and (E) serum TG, TC and LDL-C content were detected in WT mice and USP29-KO mice fed NC or HFHC diet for 16 weeks (n=8–10 mice/group). (F) Representative images and relative quantitative statistical analysis of H&E, Oil red O staining of liver tissue from WT and USP29-KO mice fed HFHC diet for 16 weeks (n=6 mice/group). Scale bar, 50 μm. (G) Representative images and relative quantitative statistical analysis of CD11b, F4/80, PSR and a-SMA staining of liver tissue from WT and USP29-KO mice fed HFHC diet for 16 weeks (n=4–6 mice/group). Scale bar, 50 μm. The data are presented as mean±SD, ^*indicates a statistical analysis between WT-NC group and WT-HFHC group (^**P<0.01, n.s., not significant). ^#indicates a statistical analysis between WT-HFHC group and USP29-KO-HFHC group (^#P<0.05, ^##P<0.01, n.s., not significant). WT, wild type; KO, USP29 knockout; NC, normal chow; HFHC, high-fat high cholesterol; GTT, glucose tolerance test; TG, total triglyceride; TC, total cholesterol; LDL-C, low density lipoprotein cholesterol; H&E, hematoxylin and eosin; PSR, picro Sirius Red.

Figure 4.

USP29 alleviates hepatocyte lipid deposition and inflammation and promotes the fatty acid degradation pathway. (A) Representative images of Nile red staining and (b) cellular TG contents in primary hepatocytes isolated from WT and USP29-KO mice and induced by PAOA or bSA for 12 h (n=3 independent experiments). Scale bar, 25 μm. (C) Representative images of Nile red staining and (D) cellular TG content in primary hepatocytes infected with AdUSP29 and AdGFP and induced by PAOA or bSA for 12 h (n=3 independent experiments). Scale bar, 25 μm. (E) The Venn diagram shows the 16 pathways presenting with a histogram according to Robust rank aggregation down-regulated by USP29-KO but up-regulated by USP29-overexpressing hepatocytes under PAOA treatment, and then the GSEA show the pathway of fatty acid degradation regulated by USP29-KO and USP29 overexpression. (F) The Venn diagram shows the 12 pathways presenting with a histogram according to Robust rank aggregation up-regulated by USP29-KO but down-regulated by USP29-overexpressing hepatocytes under PAOA treatment, and then the GSEA show the pathway of cytokine - cytokine receptor interaction regulated by USP29-KO and USP29 overexpression. The data are presented as mean±SD (^**P<0.01, n.s., not significant). bSA, bovine serum albumin; PAOA, palmitate acid and oleic acid; WT, wild type; KO, USP29 knock out; OE, over-expression; TG, total triglyceride.

Figure 5.

USP29 interacts with ACSL5 and upregulates the ACSL5 expression. (A) Flow diagram of IP-MS in AdUSP29 and AdGFP group. (b) Endogenous IP assays were performed to evaluate the interaction between USP29 and ACSL5, ALDH2, ACOX1 in AdUSP29 hepatocytes treated with PAOA. (C) The protein level of ACSL5, ALDH2 and ACOX1 in AdUSP29 hepatocytes induced by PAOA. (D–F) Western blotting analysis to assay ACSL5 protein level in indicated mouse primary hepatocytes under PAOA treatment and liver tissue of WT and USP29-KO mice fed for 16 weeks with HFHC (n=3 mice/group). (G, H) The protein level and representative immunohistochemical image of ACSL5 and in liver samples from normal, MASL and MASH patients (n=3–4 individuals/group). (I) The protein level of ACSL5 in primary hepatocytes induced with bSA or PAOA. The protein expression and mRNA expression were normalized to β-actin levels. The data are presented as mean±SD (^**P<0.01, n.s., not significant). bSA, bovine serum albumin; PAOA, palmitate acid and oleic acid; WT, wild type; KO, USP29 knock out; HFHC, high-fat high cholesterol; MASL, metabolic dysfunction-associated steatotic liver; MASH, metabolic dysfunction-associated steatohepatitis.

Figure 6.

The effect of USP29 on hepatocyte lipid accumulation and MASLD is dependent on ACSL5. (A) The protein expression of USP29 and ACSL5 in WT or USP29-KO primary hepatocytes infected with ACSL5 overexpression and control adenovirus after PAOA stimulation. (b) Representative images of Nile red staining and (C) cellular TG content in indicated primary hepatocytes induced by PAOA (n=3 independent experiments). Scale bar, 25 μm. (D) The mRNA levels of Cpt1a, Ehhadh, Cxcl2 and Ccl5 in indicated primary hepatocytes induced by PAOA (n=4 mice/group). (E) The liver weight and the ratio of liver weight to body weight in USP29-KO and WT mice injected with AAV8-ACSL5 and controls fed a HFHC diet for 16 weeks (n=9 mice/group). (F) The serum lipid contents including TG and TC were detected in USP29-KO and WT mice injected with AAV8-ACSL5 and controls fed HFHC diet for 16 weeks (n=9 mice/group). (G) Representative images and relative quantitative statistical analysis of H&E, Oil red O, CD11b and PSR staining in liver tissue from USP29-KO and WT mice injected with AAV8-ACSL5 and controls fed HFHC diet for 16 weeks (n=4–6 mice/group). Scale bar, 50 μm. The data are presented as mean±SD (^*P<0.05, ^**P<0.01, n.s., not significant). WT, wild type; KO, knock out; AAV, adeno-associated virus; HFHC, high-fat high cholesterol; TG, total triglyceride; TC, total cholesterol; H&E, hematoxylin and eosin; PSR, picro Sirius Red.

Figure 7.

USP29 directly interacts with ACSL5. (A, b) Co-IP analysis of interaction between USP29 and ACSL5 in HEK293T cells cotransfected Flag-USP29 and HA-ACSL5. (C, D) GST pull down assays showing direct binding of USP29 and ACSL5. Purified GST was used as a control. (E, F) The interaction between USP29 and ACSL5 domains was investigated by IP analysis through transfection of HEK293T cells with both USP29 or ACSL5 full-length and truncated expression plasmids. IP, immunoprecipitation.

Figure 8.

USP29 stabilizes ACSL5 through the suppression of K48-linked ubiquitination. (A) The protein expression of ACSL5 in primary hepatocytes treated with PAOA and protein synthesis inhibitor cycloheximide (CHX; 25 µg/mL). (b, C) Ubiquitination assays determining the ubiquitination of endogenous ACSL5 in AdUSP29 primary hepatocytes (b) and USP29-KO primary hepatocytes (C) with PAOA treatment. (D) Ubiquitination assays screening the potential lysine ubiquitin type of HA-ACSL5 in response to USP29 overexpression in HEK293T cells transfected with wild type (WT) or different mutant Myc-Ub plasmid. (E) Ubiquitination assays determining the k48 linked ubiquitination of endogenous ACSL5 in primary hepatocytes from WT and USP29-KO mice after PAOA treatment. (F) Ubiquitination assays determining the k48 linked ubiquitination of endogenous ACSL5 in primary hepatocytes infected with adenovirus AdUSP29 and AdUSP29-mutant (AdUSP29-m) under PAOA treatment. (G) The protein expression of ACSL5 in primary hepatocytes infected with adenovirus AdUSP29 and AdUSP29-mutant (AdUSP29-m) under PAOA treatment. (H) Representative images of Nile red staining and (I) cellular TG content in indicated primary hepatocytes induced by PAOA for 12 h (n=3 independent experiments). Scale bar, 25 μm. The protein expression and mRNA expression were normalized to β-actin levels. The data are presented as mean±SD (^**P<0.01, n.s., not significant). PAOA, palmitate acid and oleic acid; CHX, cycloheximide; IP, immunoprecipitation; WT, wild type; KO, USP29 knock out; TG, total triglyceride.