Clopidogrel ameliorates high-fat diet-induced hepatic steatosis in mice through activation of the AMPK signaling pathway and beyond

Abstract

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) frequently confers an increased risk of vascular thrombosis; however, the marketed antiplatelet drugs are investigated for the prevention and treatment of MASLD in patients with these coexisting diseases.

Methods: To determine whether clopidogrel could ameliorate high-fat diet (HFD)-induced hepatic steatosis in mice and how it works, mice were fed on normal diet or HFD alone or in combination with or without clopidogrel for 14 weeks, and primary mouse hepatocytes were treated with palmitate/oleate alone or in combination with the compounds examined for 24 h. Body weight, liver weight, insulin resistance, triglyceride and total cholesterol content in serum and liver, histological morphology, transcriptomic analysis of mouse liver, and multiple key MASLD-associated genes and proteins were measured, respectively.

Results and discussion: Clopidogrel mitigated HFD-induced hepatic steatosis (as measured with oil red O staining and triglyceride kit assay) and reduced elevations in serum aminotransferases, liver weight, and the ratio of liver to body weight. Clopidogrel downregulated the expression of multiple critical lipogenic (Acaca/Acacb, Fasn, Scd1, Elovl6, Mogat1, Pparg, Cd36, and Fabp4), profibrotic (Col1a1, Col1a2, Col3a1, Col4a1, Acta2, and Mmp2), and proinflammatory (Ccl2, Cxcl2, Cxcl10, Il1a, Tlr4, and Nlrp3) genes, and enhanced phosphorylation of AMPK and ACC. However, compound C (an AMPK inhibitor) reversed enhanced phosphorylation of AMPK and ACC in clopidogrel-treated primary mouse hepatocytes and alleviated accumulation of intracellular lipids. We concluded that clopidogrel may prevent and/or reverse HFD-induced hepatic steatosis in mice, suggesting that clopidogrel could be repurposed to fight fatty liver in patients.

Keywords: AMPK; MASLD; NAFLD; clopidogrel; fatty liver; hepatic steatosis; steatotic liver.

FIGURE 1

Clopidogrel ameliorates HFD-induced hepatic steatosis in mice. C57BL/6J male mice were fed an ND or an HFD and concomitantly treated with CLP at a low or high dose for 14 weeks (n = 8 each). (A) Changes in body weight over time of feeding. (B) Body weight and liver weight were measured at the end of the study. (C) A ratio of liver weight to body weight was measured at the end of the study. (D) Blood glucose levels of mice were measured for GTT and ITT, and the corresponding AUC values were calculated, respectively. (E) Representative images showing oil red O (upper) and HE (lower) staining of tissue sections of mouse liver (oil red O, scale bar 100 μm; HE 100 ×, scale bar 100 μm; and HE 200 ×, scale bar 50 μm). (F) Oil red O staining-positive areas in the liver sections were analyzed and quantified by ImageJ software. (G) Serum ALT and AST activity levels of mice. (H) Hepatic TG and TC content of mice. (I) Serum TG and TC levels of mice. (J) Serum HDL-c and LDL-c levels of mice. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001, as indicated. AUC, area under the blood glucose concentration-time curve; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CLP, clopidogrel; GTT, glucose tolerance test; HE, hematoxylin and eosin (staining); HFD, high-fat diet; HDL-c, high-density lipoprotein-cholesterol; ITT, insulin tolerance test; LDL-c, low-density lipoprotein-cholesterol; ND, normal (chow) diet; SD, standard deviation; TC, total cholesterol; TG, triglyceride.

FIGURE 2

RNA-seq analysis was used to systematically demonstrate the effects of clopidogrel on the expression profiling of genes involved in MASLD. RNA-seq data were acquired from liver tissues of mice, with ND-vehicle, HFD-vehicle, or HFD-CLP categorized (n = 4 each). (A) Clustering tree illustrating global sample distribution profiles by hierarchical clustering analysis. (B) A correlation heatmap showing a correlation between samples by Pearson’s correlation coefficients. (C) Volcano plot exhibiting the fold change and p values of all genes, using different colors to highlight DEGs of different expression patterns, and using pie charts showing the number of DEGs regulated by HFD feeding and CLP intervention, respectively. (D) Dot plot representing pairwise comparisons of GSEA enrichment in KEGG pathways involved in lipid metabolism, inflammation, and fibrosis, all of which were upregulated by HFD feeding but downregulated by CLP intervention. Heat maps displaying different expression patterns of representative DEGs involved in lipid metabolism (E), inflammation and fibrosis (F) that were upregulated by HFD feeding but reversed by CLP. DEG, differentially expressed gene; FDR, false discovery rate; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score. Other abbreviations, see those shown in Figure 1.

FIGURE 3

Clopidogrel activates the AMPK pathway in mice. (A) KEGG pathway enrichment analysis showing the pathways involved in lipid metabolism, inflammation, and fibrosis, all of which were regulated by clopidogrel in HFD-fed mice (n = 4 each). (B) A heatmap illustrating the expression profiles of genes responsible for lipid metabolism, inflammation, and fibrosis present in the liver of mice on either HFD-vehicle or HFD-CLP, which was associated with differentially phosphorylated AMPK signaling molecules based on STRING and PubMed database (n = 4 each). qRT-PCR analysis demonstrating relative mRNA expression levels of (C) lipid metabolism-related genes (Acaca, Acacb, Cd36, Elovl6, Fabp4, Fasn, Mogat1, Pparg, and Scd1), (D) proinflammatory genes (Ccl2, Cxcl2, Cxcl10, Il1a, Nlrp3, and Tlr4), and (E) profibrotic genes (Acta2, Col1a1, Col1a2, Col3a1, Col4a1, and Mmp2) in the liver of mice on either HFD-vehicle or HFD-CLP (n = 6 each). (F) Western blot assay of total and phosphorylated AMPKα and ACC in the liver of mice on HFD-vehicle or HFD-CLP (n = 4 each). β-Actin was used as a loading control for all cell lysates. Each target protein was quantified by ImageJ software. Other abbreviations, see those shown in Figures 1, 2.

FIGURE 4

Clopidogrel reduces lipid accumulation by enhanced phosphorylation of the AMPK pathway in mouse primary hepatocytes. (A–C) Mouse primary hepatocytes were treated with BSA or a combination of palmitic acid (PA) and oleic acid (OA) (PA/OA, 0.3 mM), and then treated with DMSO or CLP (50 μM) for 24 h. (A) Representative images showing oil red O staining of mouse primary hepatocytes. Scale bar, 100 μm. (B) qRT-PCR analysis showing relative mRNA expression levels of genes responsible for lipid metabolism (Acaca, Fasn, and Scd1) in mouse primary hepatocytes as indicated, whose mRNA expression was normalized to that of Actb. (C) Western blot assay of total and phosphorylated AMPKα and ACC in primary mouse hepatocytes. β-Actin was used as a loading control for all cell lysates. (D, E) Primary mouse hepatocytes were challenged by PA/OA (0.3 mM) and treated with CLP (50 μM) and/or Compound C (CC, 5 μM) for 24 h. (D) Representative images showing oil red O staining of primary mouse hepatocytes. Scale bar, 100 μm. (E) Western blot assay of total and phosphorylated AMPKα and ACC in primary mouse hepatocytes. β-Actin was used as a loading control for all cell lysates. Results shown are representative of at least three independent experiments. ACC, acetyl-CoA carboxylase; Actb; gene encoding β-actin; AMPK, AMP-activated protein kinase; BSA, bovine serum albumin; CC, compound C; DMSO, dimethyl sulfoxide; PA/OA, palmitic acid and oleic acid. Other abbreviations, see those shown in Figures 1–3.