PNPLA3 P.I148M VARIANT INCREASES LIPID DROPLETS NUMBER AND SIZE IN PATIENT-DERIVED LIVER ORGANOIDS

PNPLA3 rs738409 C>G p.I148M variant is the main genetic determinant of metabolic associated steatotic liver disease (MASLD). To this day, this association still lacks a molecular characterization, due to the lack of comprehensive human models. To overcome this gap in knowledge, we generated patient-derived human liver organoids (HLOs) and studied lipid accumulation across different PNPLA3 genotypes. HLOs were differentiated toward a hepatocyte-like phenotype (Hep-HLO) and exposed to 300 µM Palmitic and Oleic fatty acids (FAs) for 72h. Lipid droplets (LDs) average size and number (normalized for nuclei) were quantified by NileRed staining and compared between each PNPLA3 genotype. Regardless of FA treatment, M/M Hep-HLOs show increased LDs size when compared to I/M and I/I. Conversely, LDs number and total area showed direct correlation with the number of mutated alleles, indicating that the p.I148M substitution promotes the formation of new LDs, which was exacerbated by exposure to FAs. Overall, these data confirm on novel 3D cellular models the seminal role of rs738409 in the development of MASLD and suggest that LDs size and number are regulated by independent mechanisms. Preliminary lipidomic analyses highlighted an enrichment in sphyngomyelins, lysophosphatidylcholines and ceramides in PNPLA3 M/M Hep-HLO, which was exacerbated by FAs administration. These lipid species are associated with cellular damage, inflammation, Insulin resistance, oxidative stress and MASH development. The opposite trend was observed for phosphatidylcholines, which are essential for LDL loading and triacylglycerols export from the liver and whose levels drop during MASLD development; highlighting an unbalance towards lipid species that are proinflammatory and pro-apoptotic in Hep-HLO homozygous for PNPLA3 rs738409. Overall, these data corroborate the robustness of Hep-HLO as a preclinical in vitro model for genetic predisposition for MASLD. We are now developing siRNAs specific for silencing PNPLA3 rs738409 transcripts to examine the impact on LD phenotypes.