Pannexin 1 attenuates hepatic steatosis and insulin resistance via AMPK-autophagy axis activation.

Abstract

Background: Metabolic dysfunction-associated fatty liver disease (MASLD) affects over 25% of the global population, yet effective therapies remain limited. While Pannexin 1 (Panx1) has been implicated in metabolic regulation, its role in hepatic lipid metabolism remains unexplored. We hypothesized that Panx1 regulates the AMPK-autophagy axis to control hepatic lipid accumulation and insulin sensitivity.

Methods: We employed a comprehensive experimental strategy combining genetic manipulation in mice and cultured hepatocytes with multiple complementary analytical approaches. In vivo studies utilized both Panx1 knockout (KO) mice and wild-type littermates subjected to high-fat diet (HFD) feeding to induce MASLD. In vitro experiments employed Hepa1-6 hepatocytes treated with palmitic acid (PA) and oleic acid (OA) to simulate lipid overload, with Panx1 expression modulated through siRNA-mediated knockdown or plasmid-mediated overexpression. We integrated histological analyses (Oil Red O staining, PAS staining), molecular techniques (Western blotting, quantitative RT-PCR), flow cytometry for glucose uptake assessment, transmission electron microscopy for autophagosome visualization, and dual-fluorescence LC3 assays for autophagic flux monitoring. To establish causality, we performed rescue experiments using the autophagy inhibitors chloroquine (CQ) and bafilomycin A1 (BafA1), as well as the autophagy activator rapamycin (Rapa).

Results: Our investigations revealed that Panx1 expression is significantly downregulated in both human MASLD patients and experimental MASLD models, establishing its clinical relevance. Through loss-of-function studies, we demonstrated that Panx1 knockout dramatically exacerbated HFD-induced metabolic dysfunction, manifesting as markedly increased body weight gain, severe glucose intolerance, pronounced insulin resistance, elevated serum triglycerides and cholesterol, and massive hepatic lipid accumulation accompanied by upregulation of key lipogenic genes (SREBP1c, FASN, ACC1, SCD1, DGAT1). Conversely, gain-of-function experiments using Panx1 overexpression yielded striking protective effects: significantly reduced hepatic steatosis, improved glucose tolerance and insulin sensitivity, decreased serum lipid profiles, and suppressed expression of lipogenic genes in HFD-fed mice. Mechanistically, we uncovered that Panx1 functions as a critical activator of the AMPK-autophagy signaling cascade. Panx1 overexpression robustly activated AMPK phosphorylation, enhanced LC3-II accumulation, accelerated P62 degradation, and increased autophagosome formation as visualized by transmission electron microscopy and dual-fluorescence LC3 assays. These molecular changes translated into enhanced autophagic flux and lipophagy, effectively clearing accumulated lipids. Importantly, our rescue experiments definitively established autophagy as the essential mediator of Panx1's metabolic effects: pharmacological autophagy inhibition with CQ or BafA1 completely abolished the beneficial effects of Panx1 overexpression on lipid accumulation and insulin signaling, while rapamycin-induced autophagy activation successfully rescued the metabolic defects caused by Panx1 deficiency. Furthermore, we demonstrated that Panx1's effects on insulin sensitivity are autophagy-dependent, as evidenced by restored GLUT4 expression, IRS phosphorylation, and AKT activation following autophagy modulation.

Conclusion: This study identifies Panx1 as a novel regulator of hepatic lipid metabolism and insulin sensitivity via the AMPK-autophagy pathway. Given its downregulation in MASLD patients and the lack of FDA-approved treatments, Panx1 represents a promising therapeutic target for this prevalent metabolic disorder.