Pharmacological Activation of AMP-activated Protein Kinase Ameliorates Liver Fibrosis in a Metabolic Dysfunction-Associated Steatohepatitis Mouse Model

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) is a significant contributor to hepatocellular carcinoma (HCC). To validate AMPK activation as a therapeutic strategy for MASH-associated liver fibrosis, we investigated the effects of a 4-chloro-benzenesulfonamide derivative named KN21, a novel AMPK activator, on the liver fibrogenic process in a MASH model. In mice fed a choline-deficient, L-amino acid-defined, high fat diet (CDAHFD), KN21 reduced hepatic steatosis, lipid accumulation, and liver fibrosis. In hepatocyte cells treated with palmitic acid and oleic acid (PO), KN21 attenuated lipid accumulation and the release of reactive oxygen species (ROS) and fibrotic mediators. Hepatic stellate cells stimulated with hepatocyte-derived conditioned medium (CM) exhibited increased expression of fibrosis markers, whereas hepatic stellate cells exposed to CM from KN21-treated hepatocytes showed a decrease of fibrosis marker expression. Additionally, KN21 inhibited the activation of human hepatic stellate cells and demonstrated potent antifibrotic activity. These findings underscore the therapeutic potential of pharmacological AMPK activation for the treatment of MASH-associated liver fibrosis.

Keywords: 4-chloro-benzenesulfonamide derivative; AMP-activated protein kinase (AMPK); lipid accumulation; liver fibrosis; metabolic dysfunction-associated steatohepatitis (MASH); synthetic AMPK activator.

Figure 1

KN21 stimulates AMPK activation by directly binding to the AMP binding site located on the AMPKγ subunit. (A) Chemical structure of KN21, a 4-chloro-benzenesulfonamide derivative. (B, C) Concentration-dependent effects of KN21 on AMPKα phosphorylation in HepG2 and LX-2 cells, analyzed via immunoblotting. (D) Assessment of the interaction between KN21 and AMPKγ protein in LX-2 cells through cell thermal shift analysis (CETSA). (E) Docking studies to identify the interaction residues between AMPKγ and AMP as well as between AMPKγ and KN21. (F) CETSA assessment of the interaction between KN21 and both wild-type and mutant AMPKγ protein in HEK293 cells.

Figure 2

KN21 mitigates hepatic steatosis, liver damage, and lipid accumulation in CDAHFD-fed mouse model. (A) Experimental timeline showing CDAHFD feeding and KN21 (15 mg/kg) administration. (B, C) Body weight and food intake of mice across treatment groups during the experimental period (n=5). (D) Representative liver images from each group (n=5). (E, F) Liver weights and liver-to-body weight ratio for each group (n=5). (G, H) Serum levels of ALT and AST, indicators of liver function (n=5). (I) Representative images of H&E and immunohistochemical staining for FASN in liver sections (n=5). (J) Immunoblotting analysis of proteins involved in lipid metabolism (SREBP-1, FASN and PPARγ) (n=5, combined with results in Figure S6A). (K) Quantitative real-time PCR analysis of lipid metabolism-related genes, including SREBF1, FASN, and CD36 (n=5). ***P < 0.001, **P < 0.01, *P < 0.05 vs. the ND group; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 vs. the CDAHFD group (one-way ANOVA); “n.s.” indicates a nonsignificant difference.

Figure 3

KN21 improves liver fibrosis induced by CDAHFD in mice as an activator of AMPK. (A) Immunoblotting analysis showing total and phosphorylated AMPKα proteins in CDAHFD-fed mice treated with 15 mg/kg of KN21 (n=5, combined with results in Figure S6B). (B) Representative images of Sirius red staining and immunohistochemical staining for α-SMA in liver sections (n=5). (C) Immunoblotting analysis of proteins involved in fibrosis (COL1A1 and α-SMA) (n=5, combined with results in Figure S6C). (D) Quantitative real-time PCR analysis of fibrosis-related genes (TIMP1, COL1A1, COL3A1, PDFGB, PDGFA, ACTA2) (n=5). ***P < 0.001, *P < 0.05 vs. the ND group; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 vs. the CDAHFD group (one-way ANOVA).

Figure 4

KN21 inhibits lipid accumulation in PO-induced hepatocytes. (A, B) Oil red O staining in primary hepatocytes and HepG2 cells stimulated with BSA or PO (0.5 mM PA and 1.0 mM OA), and subsequently treated with DMSO (vehicle), 10 μM KN21, or 50 μM KN21 for 12 h. (C, D) Immunofluorescence staining of SREBP-1 in PO-stimulated primary hepatocytes and HepG2 cells treated with 10 μM KN21 for 12 h. (E, F) Immunoblotting analysis of precursor and mature forms of SREBP-1 in PO-stimulated primary hepatocytes and HepG2 cells treated with 10 μM KN21 for 12 h. (G, H) Immunoblotting analysis of FASN and PPARγ in primary hepatocytes and HepG2 cells stimulated with PO and treated with 10 μM KN21 for 12 h. (I, J) Quantitative real-time PCR analysis of lipid metabolism-related genes (FASN, PPARγ, and SCD1) in PO-stimulated primary hepatocytes and HepG2 cells treated with 10 μM KN21 for 12 h. (K, L) Immunoblotting analysis showing total and phosphorylated AMPKα proteins in PO-stimulated primary hepatocytes and HepG2 cells treated with 10 μM KN21 for 12 h. **P < 0.01, *P < 0.05 vs. the control group; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 vs. the PO-stimulated group (one-way ANOVA)..

Figure 5

KN21 reduces lipid accumulation in hepatocytes under metabolic stress via AMPK activation. (A, B) Immunoblotting analysis showing total and phosphorylated AMPKα proteins in PO-stimulated primary hepatocytes and HepG2 cells treated with KN21, Compound C, or their combination for 12 h. (C, D) Oil red O staining of PO-stimulated primary hepatocytes and HepG2 cells treated with KN21, Compound C, or their combination for 12 h, indicating lipid accumulation. (E, F) Quantitative real-time PCR analysis of lipid metabolism-related gene expression (FASN, PPARγ, and SCD1) in PO-stimulated primary hepatocytes and HepG2 cells treated with KN21, Compound C, or their combination for 12 h. **P < 0.01, *P < 0.05 vs. the PO-stimulated group; ^##P < 0.01, ^#P < 0.05 vs. the PO + KN21-treated group (one-way ANOVA).

Figure 6

KN21 suppresses ROS production and the release of profibrotic mediators from hepatocytes. (A) Schematic representation measuring ROS production and the release of profibrotic mediators in hepatocytes. (B) Measurement of intracellular ROS production in HepG2 cells. (C, D) Quantification of the profibrotic mediators PDGFβ and TGF-β1 in the culture media from HepG2 cells. (E) Immunoblotting analysis of fibrosis-related proteins (Fibronectin, COL1A1 and α-SMA) in LX-2 cells treated with CM derived from HepG2 cells. (F) Quantitative real-time PCR analysis of profibrotic gene expression (CTGF, ACTA2, COL1A1, FN) in LX-2 cells treated with CM derived from HepG2 cells. (G) Immunofluorescence staining of LX-2 cells treated with CM derived from HepG2 cells, showing α-SMA (green) in confocal images. (H) Transwell migration assay of LX-2 cells treated with CM derived from HepG2 cells. (I) Morphological images of primary HSCs treated with CM derived from primary hepatocytes. (J) Immunoblotting analysis of α-SMA protein expression in primary HSCs treated with CM derived from primary hepatocytes. ***P < 0.001, **P < 0.01, *P < 0.05 vs. the control group; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 vs. the PO-treated group (one-way ANOVA).

Figure 7

KN21 reduces TGF-β1-induced activation of hepatic stellate cells. (A) Immunoblotting analysis showing total and phosphorylated AMPKα proteins in LX-2 cells treated with 10 μM A769662 or 5 μM KN21 for 12 h. (B) Immunoblotting analysis of protein expression in LX-2 cells treated with 2 ng/mL TGF-β1 for 12 h, followed by incubation with 10 μM A769662 or 5 μM KN21 for 12 h. (C) Quantitative real-time PCR analysis of fibrosis-related gene expression (CTGF. ACTA2, COL1A1, FN) in LX-2 cells treated with 2 ng/mL TGF-β1 for 12 h, followed by incubation with 10 μM A769662 or 5 μM KN21 for 12 h. (D) Morphological images of primary HSCs cultured for a specific period. (E) Immunoblotting analysis of protein expression in primary HSCs treated with 5 μM KN21. ***P < 0.001 vs. the control group; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 vs. the TGF-β1-treated group (one-way ANOVA).