Spatial transcriptomics maps the metabolic zonation of hepatocytes in MASLD I148M-PNPLA3 carriers

Abstract

Introduction

The I148M PNPLA3 polymorphism is the main genetic predictor of MASLD. Few data showed that I148M overexpression in hepatocytes correlated with metabolic switching and mitochondrial (mt)-dysfunction.

Aims

To deepen the impact of PNPLA3 variation on mt-function by overexpressing I148M protein in HepG2 and Hep3B cells, to force or introduce the mutation, respectively; to dissect hepatocytes metabolic zonation in liver biopsies of MASLD patients, wild-type (WT) and homozygous for PNPLA3-I148M with similar disease severity (NAS=4), through spatial transcriptomic.

Materials and Methods Results

PNPLA3-I148M protein was upregulated in hepatoma cells by lentiviral transfection. Spatial transcriptomics was performed by Visium CytAssist (10X-Genomics). I148M-protein upregulation in hepatoma cells fostered lipid accumulation, thus increasing PGC1a to clear fat. Both I148M-overexpressed models showed lower OXPHOS capacity and ATP production alongside higher release of ROS and mtDNA fragments, corroborating the I148M-mediated mt-dysfunction. To explore the role of PNPLA3 I148M in hepatocytes zonation, we spatially mapped pericentral (PC) and periportal (PP) hepatocytes of WT and I148M MASLD-biopsies by using CYP3A4/CYP2E1 and HAL/SDS genes as zonation markers, respectively. Among DEGs, we found increased lipogenesis (SREPF1, DGAT2, ACACA, FABP1) and mitobiogenesis (PGC1a), the latter as response to fat accumulation, in both WT-I148M-PP areas. However, WT-PP zones showed high expression of DEGs related to physiologic fusion-mitophagy (Mfn1, Mfn2, Opa1, BnipL, Bnip3), ðœ·-oxidation (PPARa) and mt-respiration (SDHA, ATP5MF), that conversely were decreased in I148M-PP areas suggesting a mt-impairment. As concern pathways related to glycolysis, fatty acids metabolism, autophagy/mitophagy and PPAR/Wnt signaling, we observed an opposite trend between WT and I148M as they were enhanced in PC zones in the former and PP areas in the latter.

Conclusion

The in vitro I148M overexpression dampens mitochondrial respiration triggering oxidative stress. In MASLD patients, the I148M mutation leads to unbalanced PC-PP metabolic zonation providing new insights into its impact in the disease progression.