The purinergic receptor P2X4R stimulates macro-autophagy with impact on lipid droplet size in fatty liver

Background/aims P2X4R is a lysosomal ATP-gated cation channel receptor, playing roles in liver pathophysiology through poorly understood mechanisms. This study aimed to define the role of P2X4R in macroautophagy and lipid droplet (LD) homeostasis. Methods We used P2X4R-KO, LC3-GFP transgenic and WT mice, a specific P2X4R antagonist (BAY1797), and three experimental models of steatosis (n=5-10 mice/group). Autophagy flux was measured in human embryonic kidney (HEK) reporter cell line, and Atg5-/- mouse embryonic fibroblast (MEF) cells. Oleic acid (OA) cell loading, LC3-II western blots, Oil Red O and LipidTox cell and tissue staining were also performed. Results The autophagic flux in the liver and in primary isolated hepatocytes was decreased in P2X4R-KO as compared with WT mice, as well as in BAY1797 (as compared with vehicle)-treated WT animals and hepatocytes. P2X4R overexpression stimulated the autophagic flux, pointing to an impact of P2X4R on lysosome-autophagosome fusion. BAY1797 treatment in fasted mice as well as in both MCD and HFHSC diet models resulted in small LD, instead of large LD steatosis (vehicle-treated mice) and stimulated cytosolic lipolysis. Similar results were observed in vitro in OA-loaded primary hepatocytes, as well as in MEF in which BAY1797 inhibited lipophagy and stimulated cytosolic lipolysis, resulting in smaller size LDs. Lysosome position analysis upon starvation revealed that P2X4R inhibition interfered with lysosomal trafficking in vitro . In MASLD patients (n=24) a correlation was found between LD size, autophagic flux and P2X4R expression. Conclusion P2X4R inhibition reduced the autophagic flux in hepatocytes, resulting in LD size reduction, during fasting- as well as diet-induced steatosis in mice. P2X4R is proposed as a new therapeutic target to modulate lipophagy in the liver. Impact and implications Our study provides in vitro and in vivo evidence that P2X4R is a regulator of macro-autophagy, and thereby impacts on lipid droplet homeostasis in the liver. Both genetic deletion and pharmacologic specific inhibition of P2X4R results in lipid droplet size reduction in three murine models of fatty liver, in association with inhibition of lipophagy, stimulation of lipolysis and reduction of liver inflammation. Finally in humans, we found in a series of MASLD patients that P2X4R expression was correlated with autophagy flux and lipid droplet size. Altohether, our data support strongly the idea that P2X4R inhibition is hepato-protective in the context of MASLD.