Elevated Porcupine Disrupts Lipid Metabolism and Promotes Inflammatory Response in MASLD
Yalin Zhang
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorFengyu Ju
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorLi Yan
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorXin Shen
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorShiqing Guo
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorMuchen Yu
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorCorresponding Author
Yujia Cao
Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
Correspondence:
Yujia Cao ([email protected])
Wenhui Wang ([email protected])
Search for more papers by this authorCorresponding Author
Wenhui Wang
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Correspondence:
Yujia Cao ([email protected])
Wenhui Wang ([email protected])
Search for more papers by this authorYalin Zhang
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorFengyu Ju
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorLi Yan
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorXin Shen
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorShiqing Guo
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorMuchen Yu
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Search for more papers by this authorCorresponding Author
Yujia Cao
Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
Correspondence:
Yujia Cao ([email protected])
Wenhui Wang ([email protected])
Search for more papers by this authorCorresponding Author
Wenhui Wang
Department of Pharmacology and School of Basic Medicine Clinical Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China
Correspondence:
Yujia Cao ([email protected])
Wenhui Wang ([email protected])
Search for more papers by this authorFunding: This study is supported by National Natural Science Foundation of China (81902480), the Fundamental Research Funds for the Central Universities (2632019PY04), the Jiangsu Provincial Double-Innovation Doctor Program and Nanjing Science and Technology Innovation Project to Wenhui Wang.
Yalin Zhang and Fengyu Ju contributed equally to this study.
Wenhui Wang: Lead contact.
Handling Editor: Dr. Luca Valenti
ABSTRACT
Background and Aims
Metabolic dysfunction-associated steatotic liver disease (MASLD) presents a high incidence globally and is a major cause of cirrhosis and hepatocellular carcinoma, lacking of efficient interventions. Patients with MASLD exhibit exceeded serum levels of palmitic acid (PA). However, the association between PA and MASLD remains obscure.
Methods
Gene expression omnibus dataset analysis, western blotting, mRNA-sequencing, RT-qPCR, a click chemistry-immunoprecipitation-immunofluorescence system, ELISA, lipid extraction and UHPLC–MS/MS analysis, CyTOF mass cytometry, gene knockdown via lentivirus-mediated shRNA, and high-fat methionine and choline-deficient diet-fed WT and db/db mice models were used to reveal the expression and functions of Porcupine in MASLD development both in vitro and in vivo.
Results
Our findings show that PA, as a crucial substrate for protein palmitoylation, induced the expression of palmitoyltransferase Porcupine in a time-dependent manner. This induction was closely associated with dysregulated lipid metabolism and stimulated inflammatory response observed in vitro. Porcupine protein levels were significantly increased in liver tissues from both MASLD mice models, which was predominantly localised in lipid droplet-rich hepatocytes. Pharmacological inhibition of Porcupine by Wnt974 markedly ameliorated the aberrant lipid accumulation and inflammatory response in mouse livers. Furthermore, increased Porcupine positively correlated with CD36 at protein levels, and its inhibition or knockdown decreased CD36 protein levels via mechanisms irrelevant to transcriptional regulation, but primarily dependent on protein palmitoylation.
Conclusions
The current study reveals that PA-induced Porcupine disrupts lipid metabolism and promotes inflammatory response during MASLD development, which can be ameliorated by the Porcupine inhibitor Wnt974. Therefore, Porcupine may be a potential pharmacological target for the treatment of MASLD.
Conflicts of Interest
The authors declare no conflicts of interest.
Open Research
Data Availability Statement
The data associated with this paper are available upon request to the corresponding author.
Supporting Information
| Filename | Description |
|---|---|
| liv16130-sup-0001-FigureS1.tifTIFF image, 6.7 MB |
FIGURE S1. Relative hepatic mRNA levels of Porcupine in MASLD patients. The GEO data set GSE48452 (HC, healthy control, n = 14; HO, healthy obese, n = 27; MASL, n = 14; MASH, n = 18) is analysed to compare hepatic PORCN mRNA levels among MASLD patients. |
| liv16130-sup-0002-FigureS2.tifTIFF image, 36.3 MB |
FIGURE S2. The expression levels of Porcupine in liver cell lines treated with PA. Oil red O staining of lipid droplets and relative red oil intensity in HepG2 (A) and Hepa1-6 (C) treated with PA at different concentrations for 18 h or with 0.4 mM PA for 6, 12, 18 and 24 h, respectively (400×). Protein (B, D, E, G, H) and mRNA (F, I) levels of Porcupine in HepG2, Hepa1-6 and Raw264.7 cells treated with PA at different concentrations for 18 h. Data are depicted as mean ± SEM, n = 3–5. Comparisons among groups were carried out using one-way ANOVA with Dunnett’s multiple comparisons test.*p < 0.05. |
| liv16130-sup-0003-FigureS3.tifTIFF image, 8.1 MB |
FIGURE S3. PA treatment caused aberrant lipid metabolism in vitro. (A–D) mRNA levels of lipid metabolism related Fasn, Srebp1c, Pparα and Atgl genes in Hepa1-6 cells treated with indicated-concentration PA for 18 h (n = 5–6). Data in histograms are depicted as mean ± SEM. Comparisons among groups were carried out using one-way ANOVA with Dunnett’s multiple comparisons test.*p < 0.05, **p < 0.01, ****p < 0.0001. |
| liv16130-sup-0004-FigureS4.tifTIFF image, 11.1 MB |
FIGURE S4. Pharmacological inhibition of Porcupine ameliorates MASLD in vivo. The quantification of hepatic caspase3 and PCNA (A and B, n = 3) and serum ALT (C) from WT mice fed with CD (normal, n = 9) or HFMCD (treated with DMSO as control or Wnt974, n = 11–13). Body weight curves (D, Control, n = 9; Wnt974, n = 16) and the quantification of hepatic caspase3 (E) from db/db mice fed with HFMCD (treated with DMSO as control or Wnt974, n = 3). Comparisons among groups were carried out using one-way ANOVA with Dunnett’s multiple comparisons test (for ≥ three groups) and two-way ANOVA (for body weight; ##p = 0.0069, vs. control group). Data in histograms are depicted as mean ± SEM. *p < 0.05, ****p < 0.0001. |
| liv16130-sup-0005-FigureS5.tifTIFF image, 13.3 MB |
FIGURE S5. Pharmacological inhibition of Porcupine ameliorates dysregulated lipid metabolism in vivo. (A) Hepatic and serum LDL in db/db control and Wnt974 groups (n = 3–5). These data are depicted as mean ± SEM. Comparisons between groups were carried out using unpaired-t test. **p < 0.01. (B) Hepatic and serum TG and TCHO in WT mice fed with CD (normal group) or HFMCD (control and Wnt974 groups), n = 7–13. These data are depicted as mean ± SEM. Comparisons among groups were carried out using one-way ANOVA with Tukey’s multiple comparisons test (for ≥ three groups). *p < 0.05, **p < 0.01, ***p < 0.001. (C) Log2 fod change of lipogenic and lipolytic genes analysed using RNAseq data from HFMCD feeding WT mice (control and Wnt974 groups). Data are depicted as log2 fold change± standard error, n = 3. Differences considered significant at a padj < 0.05. |
| liv16130-sup-0006-FigureS6.tifTIFF image, 5 MB |
FIGURE S6. Pharmacological inhibition of Porcupine dampens down inflammatory response in vivo. (A) Inflammation score of liver sections form control (n = 9) and Wnt974 (n = 16) groups of db/db mice. Hepatic CCL2 (B) and CXCL2 (C) in control and Wnt974 groups of WT mice feeding with HFMCD (n = 7–8). These data are depicted as mean ± SEM. Comparisons between groups were carried out using unpaired-t test. *p < 0.05, **p < 0.01. |
| liv16130-sup-0007-FigureS7.tifTIFF image, 18.7 MB |
FIGURE S7. Porcupine is important to maintain CD36 expression. (A, B) The protein levels of CD36 in HepG2 cells treated with 0.4 mM PA for 6, 12, 18 and 24 h, respectively (n = 7). Comparisons between groups were carried out using one-way ANOVA with Dunnett’s multiple comparisons test. (C) Porcupine inhibition by Wnt974 decreases CD36 levels in HepG2 cells. (D) Representative CD36 staining (IHC, 200×) and its expression scores in the liver tissues from db/db mice fed with HFMCD (Control group and Wnt974 group, n = 3). Comparisons between groups were carried out using unpaired-t test. Data are depicted as mean ± SEM. NC, normal control. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. |
| liv16130-sup-0008-TableS1.docxWord 2007 document , 29.3 KB |
TABLE S1. Sequences of primers used for RT-qPCR. |
| liv16130-sup-0009-DataS1.docxWord 2007 document , 34.1 KB |
DATA S1. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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