MPEP combines mGluR5 inhibition and AMPK activation to reduce hepatic steatosis

Abstract

Introduction and Aim

Recent studies highlight the role of metabotropic glutamate receptor type 5 (mGluR5) in hepatic steatosis, where its hyperactivation in hepatic stellate cells (HSCs) drives fat accumulation in hepatocytes (Choi et al., 2019). Previous research demonstrated that 2-methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of mGluR5, reduces lipid deposition in HepG2 cells exposed to an oleate/palmitate-induced steatosis model (Ferrigno et al., 2020). This study aimed to evaluate mGluR5’s role in lipid metabolism using negative allosteric modulators (MPEP, Fenobam), an orthosteric antagonist (carboxyphenylglycine, CPG), and an orthosteric agonist ((S)-3,5-dihydroxyphenylglycine, DHPG). Additionally, we explored whether MPEP's effects involve activation of AMP-activated protein kinase (AMPK), a critical regulator of lipid metabolism linked to ATP depletion (Ferrigno et al., 2018).

Materials and Methods

Lipid accumulation was induced in HepG2 cells using 2 mM oleate/palmitate (O/P) for 24 hours. Cells were treated with MPEP (0.3-3-30 μM), Fenobam (1-25-50 μM), and CPG (100-150-200 μM), alone or with the AMPK inhibitor (Compound C 0.1-1-10 μM). Non-steatotic cells received DHPG (100-200-300 μM) alone or with 30 μM MPEP. Lipid content was visualized with Nile Red dye and quantified via ImageJ; cell viability was assessed via MTT assay. ATP levels were measured using a luciferin-luciferase assay, and the p-AMPK/AMPK ratio was analyzed by Western blot.

Results

In steatotic HepG2 cells, MPEP, Fenobam, and CPG significantly reduced lipid accumulation dose-dependently. DHPG increased lipid content in non-steatotic cells, but co-treatment with MPEP reversed this effect. MPEP, uniquely among the compounds, depleted ATP levels and activated AMPK. Compound C abolished MPEP's lipid-lowering effects, confirming AMPK's role. Western blot showed increased p-AMPK/AMPK ratios in MPEP-treated steatotic cells.

Conclusion

mGluR5 inhibition reduces lipid accumulation in an in vitro steatosis model. MPEP engages an AMPK-mediated pathway, providing a dual mechanism against hepatic steatosis and highlighting its therapeutic potential.