Neddylation inhibition ameliorates steatosis in NAFLD by boosting hepatic fatty acid oxidation via the DEPTOR-mTOR axis

Abstract

Objective: Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD.

Methods: Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8.

Results: Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models CONCLUSIONS: Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD.

Keywords: Deptor; Fatty acid oxidation; MLN4924; NAFLD; Neddylation; mTOR.

Graphical abstract

Figure 1

Neddylation is augmented in clinical and pre-clinical mouse models of non-alcoholic fatty liver disease (NAFLD). A. Hepatic neddylation levels assessed by immunohistochemistry and B. Pearson correlation and coefficient between hepatic neddylation and NAS score in NAFLD patients (n = 19) in comparison with a group of healthy controls (n = 7). Hepatic neddylation levels assessed by immunohistochemistry in healthy animals (n = 5) and C. animals maintained during 20 weeks on a high-fat diet (HFD) (n = 11), D. animals maintained during 6 weeks on a choline-deficient high-fat diet (CDHFD) (n = 8) and E. animals maintained during 4 weeks on a 0.1% methionine- and choline-deficient diet (0.1% MCDD) (n = 6). Scale bar corresponds to 100 μm. Data are shown as average ± SEM and Student's t-test was used to compare groups. ∗p < 0.05 and ∗∗∗p < 0.001 are shown.

Figure 2

Neddylation inhibition reduces lipid accumulation in non-alcoholic fatty liver disease (NAFLD) pre-clinical models. A. and B. Bodipy staining in primary mouse hepatocytes isolated from wild-type mice and treated with MLN4924 3 μM (n = 4) or siRNA against Nedd8 (n = 3) in the presence of oleic acid (OA) during 6h. Sudan Red staining and quantification of total hepatic triglycerides in C. animals maintained during 6 weeks on choline-deficient and high-fat diet (CDHFD) (n = 5) and animals on CDHFD and treated with MLN4824 (CDHFD + MLN4924) (n = 5), in D. animals maintained during 4 weeks on 0.1% methionine- and choline-deficient diet (0.1% MCDD) (n = 4) and treated with MLN4924 (0.1%MCDD + MLN4924) (n = 4) and finally, in E. Alfp-Cre/AAV-DIO-shNEDD8 mice were maintained for 6 weeks on CDHFD (n = 5). Transaminase serum levels of ALT (alanine transaminase) and AST (aspartate transaminase) in healthy animals and F. animals maintained during 6 weeks on CDHFD and CDHFD + MLN4924, G. animals maintained during 4 weeks on 0.1% MCDD and 0.1% MCDD + MLN4924, and in H. the hepatic silenced NEDD8 mice model (Alfp-Cre/AAV-DIO-shNEDD8) maintained during 6 weeks on CDHFD. Scale bar corresponds to 100 μm. Data are shown as average ± SEM and One-way ANOVA test was used to compare groups. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 are shown.

Figure 3

Liver proteomics characterization by Liquid Chromatography-Mass Spectrometry (LC-MS) in mouse models of non-alcoholic fatty liver disease (NAFLD) and treated with MLN4924. A. Heatmaps showing the top 50 significantly different most differentially expressed proteins and B. Ingenuity Pathway Analysis (IPA) of top canonical pathways in animals maintained during 6 weeks on choline-deficient and high-fat diet (CDHFD) as well as in animals maintained during 4 weeks on 0.1% methionine- and choline-deficient diet (0.1% MCDD). C. Heatmaps showing the top 50 significantly different most differentially expressed proteins; D. Ingenuity Pathway Analysis (IPA) of Upstream Regulators in animals maintained during 6 weeks on choline-deficient and high-fat diet (CDHFD) and animals on CDHFD and treated with MLN4824 (CDHFD + MLN4924), as well as in animals maintained during 4 weeks on 0.1% methionine- and choline-deficient diet (0.1% MCDD) and animals on 0.1% MCDD and treated with MLN4824 (0.1% MCDD + MLN4924).

Figure 4

DEPTOR accumulation by neddylation inhibition mediates the anti-steatotic effects in pre-clinical models of non-alcoholic fatty liver disease (NAFLD). Western blot analysis of neddylated cullins, DEPTOR, phosphorylated S6 (pS6) and total S6 and beta-actin both in A. animals maintained during 6 weeks on choline-deficient and high-fat diet (CDHFD) (n = 5), and animals on CDHFD and treated with MLN4824 (CDHFD + MLN4924) (n = 5) as well as in B. animals maintained during 4 weeks on 0.1% methionine- and choline-deficient diet (0.1% MCDD) (n = 5) and animals on 0.1%MCDD and treated with MLN4824 (0.1% MCDD + MLN4924) (n = 5). C. Western blot of neddylated cullins, and DEPTOR (n = 3), D. mRNA analysis of DEPTOR levels (n = 3) and E. Bodipy staining of lipids in hepatocytes stimulated with OA and MLN4924 when silencing DEPTOR (n = 3). Data are shown as average ± SEM. One-way ANOVA test and Student's t-test were used to compare groups. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 are shown.

Figure 5

Neddylation inhibition boosts fatty acid oxidation coupled with oxidative phosphorylation in non-alcoholic fatty liver disease (NAFLD) pre-clinical models. A. Fatty acid oxidation (FAO) fluxes (n = 3); and B. Oxygen consumption rate (OCR) as analyzed using a Seahorse analyzer in primary mouse hepatocytes stimulated with oleic acid (OA) during 6 h in the presence and absence of MLN4924. C. Immunofluorescence staining of Bodipy with or without stimulation with etomoxir, an inhibitor of FAO in primary mouse hepatocytes isolated from wild type mice and treated with MLN4924 in the presence of OA during 6h (n = 3–4). D. and E. Fatty acid oxidation fluxes in animal models maintained during 6 weeks on choline-deficient and high-fat diet (CDHFD), and animals on CDHFD and treated with MLN4824 (CDHFD + MLN4924) as well as in animals maintained during 4 weeks on 0.1% methionine- and choline-deficient diet (0.1% MCDD) and animals on 0.1%MCDD and treated with MLN4824 (0.1% MCDD + MLN4924) (n = 5 per group). F. OCR as analyzed using a Seahorse analyzer in isolated liver mitochondria from mice maintained during 6 weeks on CDHFD or CDHFD + MLN4924. Scale bar corresponds to 100 μm. One-way ANOVA test and Student's t-test, respectively, were used to compare groups. # p = 0.09, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 are shown.

Figure 6

Neddylation inhibition reduces lipid peroxidation and inflammation in non-alcoholic fatty liver disease (NAFLD) pre-clinical mouse models. A. and B. dihydroethidium (DHE), a fluorescent marker of reactive oxygen species (ROS), 4-hydroxynonenal (4-HNE) and F4/80 immunostaining and respective quantification (n = 4–5); C. and D. mRNA levels of genes involved in the inflammatory and fibrotic process (Tnf-tumor necrosis factor, Ccl2- C–C motif chemokine ligand 2, Il-6- Interleukin-6, Il-1β- Interleukin-1 β, Timp2, tissue inhibitor of metalloproteinases 2) both in animals maintained during 6 weeks on choline-deficient and high-fat diet (CDHFD), and animals on CDHFD and treated with MLN4824 (CDHFD + MLN4924), and compared to controls on standard chow diet, as well as in animals maintained during 4 weeks on 0.1% methionine- and choline-deficient diet (0.1% MCDD) and animals on 0.1%MCDD and treated with MLN4824 (0.1% MCDD + MLN4924), and compared to controls on standard chow diet (n = 3–5 per group). Scale bar corresponds to 100 μm. Data are shown as average ± SEM and One-way ANOVA tests were used to compare groups. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 are shown.

Figure 7

Serum NEDD8 levels correlate with NAFLD severity and response to treatment. A. Serum NEDD8 levels in NAFLD patients (n = 46) in comparison with a group of healthy controls (n = 8). B. Pearson correlation and coefficient between serum NEDD8 levels and NAS score. C. Comparison of serum Nedd8 levels between high-fat diet for 20 weeks (HFD), choline-deficient high-fat diet for 6 weeks (CDHFD), and 0.1% methionine- and choline-deficient diet for 4 weeks (0.1% MCDD) and healthy animals on a standard chow diet (control), as well as animals on CDHFD and treated with MLN4824 (CDHFD + MLN4924), and 0.1%MCDD and treated with MLN4824 (0.1% MCDD + MLN4924) (n = 4–5 per group). D. Hematoxylin and Eosin (H&E) staining; E. Serum transaminases (alanine aminotransferase- ALT, and aspartate aminotransferase- AST); and F. Serum Nedd8 levels in healthy animals (control), in a group of animals maintained during 3 weeks on a 0.1% methionine, choline-deficient, high-fat diet (3 wk CDAAHFD) and in another group where this 3-week diet period was followed by one week of standard chow diet (SCD) (3 wk CDAAHFD + 1wk SCD). Data are shown as average ± SEM. One-way ANOVA test and Student's t-test, respectively, were used to compare groups. ∗∗p < 0.01 and ∗∗∗p < 0.001 are shown.