Synergistic activation of AMPK by AdipoR1/2 agonist and inhibitor of EDPs-EBP interaction recover NAFLD through enhancing mitochondrial function in mice - PubMed (original) (raw)
Synergistic activation of AMPK by AdipoR1/2 agonist and inhibitor of EDPs-EBP interaction recover NAFLD through enhancing mitochondrial function in mice
Nazi Song et al. Acta Pharm Sin B. 2023 Feb.
Abstract
Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), is a common hepatic manifestation of metabolic syndrome. However, there are no effective therapy to treat this devastating disease. Accumulating evidence suggests that the generation of elastin-derived peptides (EDPs) and the inhibition of adiponectin receptors (AdipoR)1/2 plays essential roles in hepatic lipid metabolism and liver fibrosis. We recently reported that the AdipoR1/2 dual agonist JT003 significantly degraded the extracellular matrix (ECM) and ameliorated liver fibrosis. However, the degradation of the ECM lead to the generation of EDPs, which could further alter liver homeostasis negatively. Thus, in this study, we successfully combined AdipoR1/2 agonist JT003 with V14, which acted as an inhibitor of EDPs-EBP interaction to overcome the defect of ECM degradation. We found that combination of JT003 and V14 possessed excellent synergistic benefits on ameliorating NASH and liver fibrosis than either alone since they compensate the shortage of each other. These effects are induced by the enhancement of the mitochondrial antioxidant capacity, mitophagy, and mitochondrial biogenesis via AMPK pathway. Furthermore, specific suppression of AMPK could block the effects of the combination of JT003 and V14 on reduced oxidative stress, increased mitophagy and mitochondrial biogenesis. These positive results suggested that this administration of combination of AdipoR1/2 dual agonist and inhibitor of EDPs-EBP interaction can be recommended alternatively for an effective and promising therapeutic strategy for the treatment of NAFLD and NASH related fibrosis.
Keywords: AMPK; AdipoR1/2 agonist; Combination therapy; EDPs; Liver fibrosis; Mitochondrial biogenesis; Mitochondrial function; Mitophagy; NASH.
© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.
Conflict of interest statement
The authors declare no conflicts of interest.
Figures
Graphical abstract
Figure 1
Histology and serum analysis in human samples along with JT003 + V14 attenuates lipid metabolism, and inflammation in hepatocytes. (A–C) Representative images showing immunohistochemistry (IHC) staining of AdipoR and elastin as well as image analysis of the positive area in formalin-fixed paraffin-embedding (FFPE) sections from non-fibrotic donors and from individuals with liver fibrosis (n = 8). (D, E) The levels of adiponectin and serum elastin-derived peptides (EDPs) were measured in human serum sample from non-fibrotic donors and from individuals with liver fibrosis (n = 14). (F, G) Representative Oil Red O staining in primary hepatocytes treated with palmitic acid (PA) or PA + JT003 + V14 (n = 5). (H) Analysis of triglyceride (TG) contents in the primary hepatocytes (n = 5). (I, J) mRNA levels of lipid metabolism and inflammation in primary hepatocytes treated with PA or PA + JT003 + V14 (n = 5). (K) GSEA pathways were analyzed in primary hepatocytes treated with PA or JT003 + V14 (n = 3). (L) Genes associated to lipid metabolism and inflammation in microarray analysis. For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
Figure 2
JT003 + V14 attenuates methionine-choline deficient (MCD)-induced non-alcoholic steatohepatitis (NASH) features. (A–C) Representative images of hematoxylin–eosin (H&E) staining (n = 6), Oil Red O staining (n = 6) and CD68 (n = 5) in FFPE sections of livers from mice. (D–F) Image analysis of the positive area of the liver. (G, H) Hepatic TG contents and serum Aspartate transaminase (AST) levels and of mice were measured (n = 5). (I–L) mRNA levels of lipogenesis, lipid metabolism and inflammation in the livers from the indicated groups after MCD treatment (n = 6). For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
Figure 3
JT003 + V14 enhance mitochondrial function in vitro and in vivo. (A, B) Representative images of 4-hydroxynonenal (4-HNE) staining and TdT-mediated dUTP nick end labeling (TUNEL) in FFPE sections of livers from MCD-induced NASH mice. (C, D) Image analysis of the positive area of the liver (n = 5). (E) Superoxide dismutase 1 (SOD1) assay to assess hepatic antioxidation in the liver from MCD-induced NASH mice (n = 5). (F) Oxygen consumption rate (OCR) assay was performed in primary hepatocytes. (G) Basal respiration, maximal respiration, ATP production, and spare capacity normalized to bovine albumin (BSA)-treated primary hepatocytes (n = 6). (H, I) mtROS staining and JC-1 staining in primary hepatocytes treated with PA or JT003 + V14 (n = 5). (J) Genes associated to mitochondrial function in microarray analysis from primary hepatocytes (n = 3). For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
Figure 4
JT003 + V14 induces mitochondrial mitophagy and biogenesis in vitro and in vivo. (A) Images of electron microscopy analyses in primary hepatocytes treated with PA or JT003 + V14 (n = 5). (B, C) The ratio of microtubule-associated protein 1 light chain 3 (LC3BII/I) and sequestosome 1 (P62) protein expression levels were measured using Western blot (WB) in the PA-induced primary hepatocytes (n = 5). (D) Confocal microscopy in primary hepatocytes treated with PA or JT003 + V14, LC3 protein was detected by GFP, and mitochondria were detected by MitoTracker® Red FM (n = 5). (E) Confocal microscopy in primary hepatocytes, lysosome was detected by Lyso-Tracker Red FM and mitochondria were detected by MitoTracker® Green FM (n = 5). (F, G) Unc-51-like kinase 1 (ULK) phosphorylation and the ratio of LC3BII/I levels in the mitochondrion were measured using WB in the MCD-induced NASH models (n = 5). (H) Genes associated to mitophagy in microarray analysis from primary hepatocytes treated with PA or JT003 + V14 (n = 3). (I, J) The ratio of LC3BII/I were measured using WB in the PA-induced primary hepatocytes with chloroquine (CQ) (n = 5). (K) mtDNA copy number as evaluated by qPCR decreased by JT003 + V14 treatment in MCD-induced NASH model (n = 5). For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
Figure 5
JT003 + V14 attenuates high fat diet (HFD)-induced NASH features. (A–C) Representative images of H&E staining, Oil Red O staining, and IHC of CD68 in FFPE sections of livers in HFD-induced NASH. (D–F) Image analysis of the positive area of the liver from HFD-induced NASH mice (n = 5). (G) Serum levels of AST of mice were measured in NASH mice that fed an HFD (n = 5). (H, I) mRNA levels of lipid metabolism and inflammation in the livers from the indicated groups after HFD treatment (n = 5). For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
Figure 6
JT003 + V14 enhance mitochondrial function and turnover in HFD mice. (A, B) ROS staining and TUNEL staining of liver sections in HFD-induced NASH mice. (C, D) Image analysis of the positive area of the liver from HFD-induced NASH mice (n = 5). (E) Genes associated to oxidation, reduction, and apoptosis in microarray analysis from the liver of NASH mice that fed an HFD (n = 3). (F) Representative images of electron microscopy analyses in HFD mice (n = 3). (G, H) mRNA levels of mitophagy (Bnip3, mTor) in the livers from the HFD-induced NASH mice (n = 5). (I, J) ULK phosphorylation and the ratio of LC3BII/I levels in the mitochondrion were measured using WB in HFD mice (n = 5). (K) Genes associated to mitophagy and mitochondrial biogenesis in microarray analysis from the liver of NASH mice that fed an HFD (n = 3). (L, M) WB analysis of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1_α_ in MCD-induced NASH models (n = 5). (N) mtDNA copy number as evaluated by qPCR decreased by JT003 + V14 treatment (n = 5). For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
Figure 7
JT003 + V14 inhibit the progress of carbon tetrachloride (CCl4)-induced liver fibrosis. (A–C) H&E, Sirius Red staining and IHC of CD68 in FFPE sections of the livers form the CCl4-induced mice. (D, E) ROS staining and TUNEL staining of liver sections. (F–I) Image analysis of the positive area of Sirius Red staining, CD68 staining, 4-HNE staining and TUNEL staining (n = 5). (J, K) qPCR of gene linked to extracellular matrix (ECM) metabolism and inflammation in liver tissue (n = 5). (L–O) AST, ALT, glutathione (GSH) and SOD levels in serum were analyzed in CCl4-induced NASH mice (n = 5). For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
Figure 8
Decreased mitophagy and mitochondrial biogenesis by compound C in PA-induced primary hepatocytes. (A) Representative WB analysis for adenosine monophosphate-activated protein kinase (AMPK) expression in the livers of MCD, HFD, and CCl4-induced mice (n = 5). (B) Representative images of electron microscopy analyses in primary hepatocytes with compound C (n = 5), lipid drops were marked by the white arrow and autophagosome as well as isolation membranes were marked by yellow arrow. (C) OCR was analyzed in primary hepatocytes treated with compound C (n = 5). (D–F) Basal respiration, maximal respiration, and ATP production normalized to BSA-treated primary hepatocytes (n = 5). (G) Representative images of mtROS staining treated with compound C in primary hepatocytes treated with compound C (n = 5). (H) Confocal microscopy of mitophagy in primary hepatocytes treated with compound C. LC3 protein was detected by GFP, and mitochondria were detected by MitoTracker® Red FM (n = 5). (I) Confocal microscopy in primary hepatocytes, lysosome was detected by Lyso-Tracker Red FM and mitochondria were detected by MitoTracker® Green FM (n = 5). (J) Scheme of JT003 + V14 in recovering NAFLD. For graphs, all data are represented as mean ± SEM using Dunnett's test as the post hoc test following the one-way ANOVA or Tukey's test as the post hoc test following the two-way ANOVA. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns represents no significance.
References
- Wree A., Broderick L., Canbay A., Hoffman H.M., Feldstein A.E. From NAFLD to NASH to cirrhosis—new insights into disease mechanisms. Nat Rev Gastroenterol Hepatol. 2013;10:627–636. - PubMed
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