Cullin-associated and neddylation-dissociated protein 1 (CAND1) alleviates NAFLD by reducing ubiquitinated degradation of ACAA2

Figures

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  Fig. 1. CAND1 expression is decreased in the liver during hepatic steatosis. a Western blotting of CAND1 in liver samples from normal donors (n = 6) and NAFLD patients (n = 7). b CAND1 mRNA level in liver samples from normal donors (n = 6) and NAFLD patients (n = 7). c Western blotting of CAND1 in the livers from HFD-fed and NC-fed mice (n = 6). NC, Normal chow. d The mRNA levels of CAND1 in the livers from HFD-fed and NC-fed mice (n = 7). e Western blotting of CAND1 in AML12 cells treated with PA (n = 4). f CAND1 mRNA level in AML12 cells treated with PA (n = 7). g Western blotting of CAND1 in THLE-2 cells treated with PA (n = 4). h CAND1 mRNA level in THLE-2 cells treated with PA (n = 7). p values obtained via two-tailed unpaired Student’s t tests, one-way ANOVA with Tukey’s multiple comparisons test. The data were shown as means ± SD of independent biological replicates. Source data are provided as a Source data file.

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  Fig. 2. Hepatocyte-specific CAND1 deficiency exacerbates HFD-induced liver injury in male mice. a Food intake of CAND1 cKO and WT male mice after 16 weeks of NC or HFD feeding (n = 7). b Body weight in indicated groups (WT, n = 10; cKO, n = 9; WT-HFD, n = 10; cKO-HFD, n = 9). c Epididymal fat weight (n = 8). d LW/BW ratio (WT, n = 10; cKO, n = 9; WT-HFD, n = 10; cKO-HFD, n = 9). e, f TG and TC content in indicated groups (n = 6). g H&E staining of liver sections (n = 8). Scale bar=50 μm. Magnification 200×. h Oil red O staining of liver sections (n = 8). Scale bar = 50 μm. Magnification 200×. i Fasting blood glucose (n = 10). j Fasting insulin levels (WT, n = 7; cKO, n = 7; WT-HFD, n = 9; cKO-HFD, n = 8). k HOMA-IR index (WT, n = 7; cKO, n = 7; WT-HFD, n = 9; cKO-HFD, n = 8). l GTTs (WT, n = 8; cKO, n = 8; WT-HFD, n = 9; cKO-HFD, n = 9). m Areas under the curve (AUC) of GTT (WT, n = 8; cKO, n = 8; WT-HFD, n = 9; cKO-HFD, n = 9). n ITTs (WT, n = 8; cKO, n = 8; WT-HFD, n = 9; cKO-HFD, n = 9). o AUC of ITT (WT, n = 8; cKO, n = 8; WT-HFD, n = 9; cKO-HFD, n = 9). p mRNA levels of inflammatory cytokines in the livers (WT, n = 5; cKO, n = 6; WT-HFD, n = 7; cKO-HFD, n = 6). p values obtained via…

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  Fig. 3. Hepatocyte-specific CAND1 overexpression attenuates HFD-induced liver injury in male mice. a Food intake of hepatocyte-specific conditional knockin (cKI) and WT male mice after 16 weeks of NC or HFD feeding (n = 7). b Body weight in indicated groups (WT, n = 9; cKI, n = 9; WT-HFD, n = 10; cKI-HFD, n = 10). c Epididymal fat weight (n = 8). d LW/BW ratio (WT, n = 9; cKI, n = 9; WT-HFD, n = 10; cKI-HFD, n = 10). e, f TG and TC content in the indicated groups (WT, n = 9; cKI, n = 8; WT-HFD, n = 10; cKI-HFD, n = 8). g H&E staining of liver sections (n = 8). Scale bar = 50 μm. Magnification 200×. h Oil red O staining of liver sections (n = 8). Scale bar = 50 μm. Magnification 200×. i Fasting blood glucose (WT, n = 9; cKI, n = 9; WT-HFD, n = 10; cKI-HFD, n = 10). j Fasting insulin levels (n = 8). k HOMA-IR index (n = 8). l GTTs (n = 9). m AUC of GTT (n = 9). n ITTs (n = 9). o AUC of ITT (n = 9). p mRNA levels of inflammatory cytokines in the livers (WT, n = 6; cKI, n = 7; WT-HFD, n = 6; cKI-HFD, n = 6). p values obtained via one-way ANOVA with Tukey’s multiple comparisons test. The data were shown as means ± SD of independent biological replicates. Source data are provided as a Source…

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  Fig. 4. CAND1 controls the ubiquitination and degradation of fatty acid β-oxidation gene ACAA2. a Volcano plot of Cullin1-binding protein in L02 induced by PA with NC or siCAND1. b Heatmap of deregulated binding proteins in L02 cells induced by PA after knockdown of CAND1. c Molecular docking of Cullin1, FBXO42 and ACAA2. d Immunoprecipitated with anti-Cullin1 antibody of NC or SI CAND1 groups after PA treatment in AML12, and then immunoblotted with antibodies specific for FBXO42 and ACAA2 (n = 3). e Immunoprecipitated with anti-Cullin1 antibody of NC or SI CAND1 groups after PA treatment in THLE-2, and then immunoblotted with antibodies specific for FBXO42 and ACAA2 (n = 3). f Western blotting of ACAA2 in the livers of WT and cKO mice after 16 weeks of NC or HFD feeding (n = 4). g Ubiquitination of ACAA2 in the livers of WT and cKO mice after 16 weeks of NC or HFD feeding (n = 3). h Western blotting of CAND1 in the livers of WT and cKI mice after 16 weeks of NC or HFD feeding (n = 4). i Ubiquitination of ACAA2 in the livers of WT and cKI mice after 16 weeks of NC or HFD feeding (n = 3). p values obtained via two-tailed unpaired Student’s t tests, one-way ANOVA with Tukey’s multiple…

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  Fig. 5. CAND1 restrains the formation of Cullin1, FBXO42 and ACAA2 complexes. a Livers of WT and cKO mice after 16 weeks of NC or HFD-feeding immunoprecipitated with anti-ACAA2/Cullin1/FBXO42 antibodies and then immunoblotted with antibodies specific for ACAA2/FBXO42/Cullin1, and quantification of the relative ACAA2/FBXO42/Cullin1 level (n = 3). b Livers of WT and cKI mice after 16 weeks of NC or HFD-feeding immunoprecipitated with anti-ACAA2/Cullin1/FBXO42 antibodies and then immunoblotted with antibodies specific for ACAA2/FBXO42/Cullin1, and quantification of the relative ACAA2/FBXO42/Cullin1 level (n = 3). p values obtained via one-way ANOVA with Tukey’s multiple comparisons test. The data were shown as means ± SD of independent biological replicates. Source data are provided as a Source data file.

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  Fig. 6. ACAA2 overexpression ameliorates CAND1 deficiency-potentiated hepatic steatosis, insulin resistance and inflammation in male mice. a LW/BW ratio in the indicated groups after 16 weeks of HFD feeding (WT-HFD, n = 10; cKO-HFD, n = 9; WT-HFD + ACAA2, n = 10; cKO-HFD + ACAA2, n = 10). b, c TG and TC content in the indicated group after 16 weeks of HFD feeding (n = 6). d H&E staining of liver sections in the indicated groups (n = 8). Scale bar = 50 μm. Magnification 200×. e Oil red O of liver sections staining (n = 8). Scale bar=50 μm. Magnification 200×. f Fasting blood glucose (WT-HFD, n = 10; cKO-HFD, n = 9; WT-HFD + ACAA2, n = 10; cKO-HFD + ACAA2, n = 10). g Fasting insulin levels (WT-HFD, n = 9; cKO-HFD, n = 8; WT-HFD + ACAA2, n = 8; cKO-HFD + ACAA2, n = 8). h HOMA-IR index (WT-HFD, n = 9; cKO-HFD, n = 8; WT-HFD + ACAA2, n = 8; cKO-HFD + ACAA2, n = 8). i GTTs (WT-HFD, n = 10; cKO-HFD, n = 9; WT-HFD + ACAA2, n = 10; cKO-HFD + ACAA2, n = 10). j AUC of GTT (WT-HFD, n = 10; cKO-HFD, n = 9; WT-HFD + ACAA2, n = 10; cKO-HFD + ACAA2, n = 10). k ITTs (WT-HFD, n = 10; cKO-HFD, n = 9; WT-HFD + ACAA2, n = 10; cKO-HFD + ACAA2, n = 10). l AUC of ITT (WT-HFD, n = 10; cKO-HFD, n = 9; WT-HFD +…

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  Fig. 7. Hepatocyte-specific CAND1 knockout promotes starvation-induced fatty liver in male mice. a Gross images of liver and H&E staining from WT and cKO male mice that were fasted for 24 h (n = 5). Scale bar=2 mm. Magnification 2×. b Oli red O and H&E staining from WT and cKO male mice that were fasted for 24 h (n = 5). Scale bar=50 μm. Oil red O magnification 200×, H&E magnification 400×. c, d TG and TC content from WT and cKO male mice that were fasted for 24 h (n = 4). e Western blotting of ACAA2 (n = 6). p values obtained via unpaired two-sided Student’s t-test. The data were shown as means ± SD of independent biological replicates. Source data are provided as a Source data file. TG, Triglyceride. TC, Total cholesterol.

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  Fig. 8. Androgen/Androgen receptor (AR) signaling regulates CAND1 transcription. a AR was predicted as a transcription factor of CAND1 by JASPAR. b Plasma testosterone levels of normal chow and HFD mice (Normal chow, n = 5; HFD, n = 6). c mRNA level of AR in the livers of normal chow and HFD mice (Normal chow, n = 9; HFD, n = 8). d mRNA level of AR in PA-treatment AML12 cells (NC, n = 6; PA, n = 7). e Serially truncated CAND1 promoter constructs and AR plasmids were cloned and transfected into cells. The relative luciferase activities were determined after testosterone treatment (n = 4). f mRNA level of CAND1 in AML12 cells treated with indicated concentrations of testosterone (n = 8). g Western blotting of CAND1 in AML12 cells treated with indicated concentrations (10 nM) of testosterone (n = 6). h Oil red O staining of SI-CAND1, SI-AR, SI-CAND1 + SI-AR and SI-AR + OE-CAND1 groups after PA treatment (n = 6). Scale bar =50 μm. Magnification 200×. i, j TG and TC contents in the indicated group after PA treatment (n = 6). k CAND1 protein level in WT mice treated with AR antagonist, enzalutamide by western blot (n = 6). l CAND1 mRNA level in WT mice treated with AR antagonist, enzalutamide…

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  Fig. 9. Schematic diagram of the role of CAND1 in regulating ubiquitination and degradation of ACAA2 and NAFLD. Under normal condition, AR regulates the transcription of CAND1 by binding to its promoter region. CAND1 finetunes the ubiquitination and degradation of ACAA2 via the Cullin1/Fbxo42/ACAA2 complex to maintain fatty acid metabolism homeostasis. In NAFLD, decreased AR expression reduced CAND1 transcription. Reduction of CAND1 promotes the assembly of Cullin1, FBXO42, and ACAA2 complexes, and accelerates ubiquitinated degradation of ACAA2. which causes the insufficient β-oxidative capacity of fatty acids, accumulation of fatty acids, increased lipotoxicity in hepatocytes, and eventually NAFLD. The diagram was created using BioRender.