Regulation of fatty acid oxidation by liraglutide via SIRT1 in hepatocytes: A mechanistic study

Technical Summary: Regulation of fatty acid oxidation by liraglutide via SIRT1 in hepatocytes

Problem Statement
Metabolic-associated fatty liver disease (MAFLD) is a prevalent chronic liver condition characterized by hepatic lipid accumulation and impaired fatty acid metabolism. While the pathogenesis involves complex interactions between genetic, environmental, and lifestyle factors leading to insulin resistance and oxidative stress, a specific therapeutic target remains elusive. A critical mechanism underlying MAFLD progression is the disruption of fatty acid β-oxidation, particularly within the mitochondrial and peroxisomal pathways. The SIRT1/PGC-1α/PPARα signaling pathway is recognized as a central regulator of this process; its dysfunction leads to reduced expression of rate-limiting enzymes such as carnitine palmitoyltransferase 1a (CPT1A) and acyl-CoA oxidase 1 (ACOX1), resulting in lipid deposition. Although liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has shown clinical efficacy in improving histological outcomes in nonalcoholic steatohepatitis (NASH), its specific molecular mechanism regarding the regulation of fatty acid oxidation via the SIRT1 pathway in obesity-related MAFLD remains incompletely understood.

Methodology
This study employed a dual approach combining bioinformatics analysis with in vitro experimental validation:

1. Bioinformatics Screening:

   • Data Source: The GSE37031 dataset from the Gene Expression Omnibus (GEO) was utilized, comprising samples from healthy controls and MAFLD patients.
   • Differential Analysis: Differentially expressed genes (DEGs) were screened using criteria of |log2FC| > 1.5 and P < 0.05.
   • Network Analysis: Weighted Gene Co-expression Network Analysis (WGCNA) identified gene modules correlated with MAFLD phenotypes. A PPI network was constructed via the STRING database, and core genes were selected using Cytoscape (CytoHubba plugin).
   • Target Prediction: Potential targets of liraglutide were predicted using PubChem and SEA databases. The intersection of these drug targets with the identified core MAFLD genes was analyzed.
   • Validation: Molecular docking (AutoDock Vina) was performed to assess the binding affinity between liraglutide and the identified key target.

2. In Vitro Experimental Validation:

   • Cell Model: HepG2 hepatocytes were treated with free fatty acids (FFA, 1 mmol/L) to induce a lipid accumulation model.
   • Intervention: Cells were treated with varying concentrations of liraglutide (25–200 nmol/L) to determine optimal dosage via CCK-8 assays. The optimal concentration (100 nmol/L) was used for subsequent experiments.
   • Inhibition: To verify the mechanism, the SIRT1 inhibitor EX527 was applied to the liraglutide-treated group.
   • Assays:
     • Lipid Deposition: Visualized via Oil Red O staining.
     • Biochemical Markers: Intracellular levels of triglycerides (TG), total cholesterol (TC), LDL-C, HDL-C, AST, and ALT were quantified.
     • Gene and Protein Expression: RT-qPCR and Western blotting were used to measure the expression of SIRT1, PGC-1α, PPARα, CPT1A, and ACOX1.

Key Results

• Bioinformatics Findings: The analysis identified 724 DEGs, primarily enriched in lipid metabolism pathways. WGCNA and PPI analyses narrowed these down to 10 core genes. SIRT1 emerged as a key intersection between the core MAFLD genes and the predicted targets of liraglutide. Molecular docking confirmed a stable interaction between liraglutide and SIRT1 with a binding energy of −5.5 kcal/mol.
• Cell Viability and Lipid Reduction: FFA treatment significantly reduced HepG2 cell viability and increased lipid droplet deposition. Liraglutide treatment (100 nmol/L) significantly restored cell viability and reduced lipid droplet accumulation (P < 0.05).
• Biochemical Improvements: Liraglutide intervention significantly decreased intracellular TG, LDL-C, and liver injury markers (AST, ALT) compared to the FFA model group.
• Pathway Activation:
  • Gene/Protein Upregulation: Liraglutide treatment significantly upregulated the mRNA and protein expression of SIRT1, PGC-1α, and PPARα, as well as the fatty acid oxidation enzymes CPT1A and ACOX1.
  • Reversal by Inhibitor: The addition of the SIRT1 inhibitor EX527 significantly reversed the upregulation of SIRT1, PGC-1α, PPARα, CPT1A, and ACOX1 induced by liraglutide. This confirmed that the therapeutic effect of liraglutide is dependent on SIRT1 activation.

Significance and Claims
The authors claim that this study provides a mechanistic basis for the therapeutic effect of liraglutide in MAFLD. Specifically, the paper concludes that liraglutide alleviates hepatic lipid deposition by activating the SIRT1/PGC-1α/PPARα signaling pathway, which subsequently enhances the expression of CPT1A and ACOX1 to promote fatty acid β-oxidation. The study identifies SIRT1 as a critical molecular target through which liraglutide exerts its protective effects on hepatocytes.

The authors maintain a modest tone regarding the scope of their findings. They explicitly acknowledge that the study was conducted using a HepG2 cell model, which simulates lipid deposition but cannot fully replicate the complex metabolic environment and multi-factor interactions of in vivo MAFLD. Consequently, they state that further verification using animal models is necessary to fully validate these conclusions. The paper does not propose new clinical applications beyond the theoretical basis for treating MAFLD but emphasizes the identification of SIRT1 as an important therapeutic target.