A novel human hepatocyte cell line to study PNPLA3-associated steatotic liver disease

Abstract

Patatin-like phospholipase domain-containing protein 3 (PNPLA3) p.I148M is a well-established variant associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). Conflicting in vitro and in vivo data about the impact of the variant suggest that the PNPLA3 p.I148M variant could be gain- or loss-of-function, or neomorphic. Most in vitro models used to study MASLD are cancer-derived hepatoma cell lines such as HepG2 and Huh7, which already endogenously express the homozygous PNPLA3 p.I148M variant. This highlights the need to develop models that better reflect disease and allow comparisons with wild-type cells. Clustered regularly interspaced short palindromic repeats (CRISPR) prime editing was used to introduce the PNPLA3 p.I148M gene variant into a healthy-derived immortalized human hepatocyte (IHH) cell line to generate a new in vitro model of MASLD that would better reflect PNPLA3-associated MASLD/MASH. Heterozygous and homozygous PNPLA3 p.I148M IHH cell lines were generated and validated with Sanger sequencing. Mutant cell lines exhibited lipid accumulation, increased cluster of differentiation 36 (CD36) gene expression and a decline in carnitine palmitoyltransferase 1 alpha (CPT1A) gene expression compared with the wild-type control, basally or in the presence of free fatty acid (FFA)-induced steatosis. The homozygous PNPLA3 p.I148M IHH cell line also demonstrated reduced PNPLA3 gene and protein expression compared with the wild-type control. We have developed a new human hepatocyte cell line and in vitro model to help understand PNPLA3-associated steatotic liver disease and provide a new resource for developing potential therapeutics.NEW & NOTEWORTHY We have developed a novel in vitro model for studying the PNPLA3 p.I148M variant in steatotic liver disease using a normal, healthy-derived hepatocyte cell line, which does not endogenously express the variant. We show that carrying the homozygous PNPLA3 p.I148M variant results in reduced PNPLA3 gene and protein expression, more lipid accumulation, increased lipid uptake, and reduced mitochondrial lipid oxidation-associated gene expressions and altered expression of genes associated with lipid synthesis and transport.

Keywords: clustered regularly interspaced short palindromic repeats; metabolic dysfunction-associated steatotic liver disease; patatin-like phospholipase domain-containing protein 3; prime editing; steatosis.