Zehra Nur Kocas-Kilicarslan

Genetic variants in lipid metabolism influence the risk of developing metabolic dysfunction–associated steatotic liver disease (MASLD), cirrhosis, and end-stage liver disease (ESLD). The mechanisms by which these variants drive disease are poorly understood. Because of the PNPLA3-I148M variant’s strong correlation with all stages of the MASLD spectrum and the lack of tractable therapeutic targets, we sought to understand its impact on cellular function and liver metabolism. Primary human hepatocytes (HAHs) and induced pluripotent stem cell–derived (iPSC-derived) hepatocytes (iHeps) from healthy individuals possessing the PNPLA3-I148M mutation were characterized for changes in lipid metabolism, cellular stress, and survival. Using lipidomics, metabolomics, stable isotope tracing, and flux propensity analysis, we created a comprehensive metabolic profile of the changes associated with the PNPLA3-I148M variant. Functional analysis showed that the presence of the PNPLA3-I148M variant increased endoplasmic reticulum stress, mitochondrial dysfunction, and peroxisomal β-oxidation, ultimately leading to cell death via ferroptosis. Nutritional interventions, ferroptosis-specific inhibitors, and genetic approaches modulating GPX4 activity in PNPLA3-I148M HAHs and iHeps decreased programmed cell death. Our findings indicate that therapies targeting ferroptosis in patients carrying the PNPLA3-I148M variant could affect the development of MASLD and ESLD and highlight the utility of iPSC-based models for the study of genetic contributions to hepatic disorders.

Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals. Multiple genetic variations have been linked to MASLD susceptibility through genome-wide association studies (GWAS), either increasing MASLD risk or protecting against it, such as PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438. As the impact of genetic variants on the levels of drug-metabolizing cytochrome P450 (CYP) enzymes in human hepatocytes has not been thoroughly investigated, this study aims to describe the analysis of metabolic functions for selected phase I and phase II liver enzymes in human hepatocytes. For this purpose, fresh isolated primary hepatocytes were obtained from healthy liver donors (n = 126), and liquid chromatography–mass spectrometry (LC–MS) was performed. For the cohorts, participants were classified into minor homozygotes and nonminor homozygotes (major homozygotes + heterozygotes) for five gene polymorphisms. For phase I liver enzymes, we found a significant difference in the activity of CYP1A2 in human hepatocytes carrying MBOAT7 (p = 0.011) and of CYP2C8 in human hepatocytes carrying PNPLA3 (p = 0.004). It was also observed that the activity of CYP2C9 was significantly lower in human hepatocytes carrying HSD17B13 (p = 0.001) minor homozygous compared to nonminor homozygous. No significant difference in activity of CYP2E1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, ECOD, FMO, MAO, AO, and CES2 and in any of the phase II liver enzymes between human hepatocytes carrying genetic variants for PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438 were observed. These findings offer a preliminary assessment of the influence of genetic variations on drug-metabolizing cytochrome P450 (CYP) enzymes in healthy human hepatocytes, which may be useful for future drug discovery investigations.

Although the underlying cause may vary across countries and demographic groups, liver disease is a major cause of morbidity and mortality globally. Orthotopic liver transplantation is the only definitive treatment for liver failure but is limited by the lack of donor livers. The development of drugs that prevent the progression of liver disease and the generation of alternative liver constructs for transplantation could help alleviate the burden of liver disease. Bioengineered livers containing human induced pluripotent stem cell (iPSC)–derived liver cells are being utilized to study liver disease and to identify and test potential therapeutics. Moreover, bioengineered livers containing pig hepatocytes and endothelial cells have been shown to function and survive after transplantation into pig models of liver failure, providing preclinical evidence toward future clinical applications. Finally, bioengineered livers containing human iPSC-derived liver cells have been shown to function and survive after transplantation in rodents but require considerable optimization and testing prior to clinical use. In conclusion, bioengineered livers have emerged as a suitable tool for modeling liver diseases and as a promising alternative graft for clinical transplantation. The integration of novel technologies and techniques for the assembly and analysis of bioengineered livers will undoubtedly expand future applications in basic research and clinical transplantation.

As diet and lifestyle have changed, fatty liver disease (FLD) has become more and more prevalent. Many genetic risk factors, such as variants of PNPLA3, TM6SF2, GCKR, and MBOAT7, have previously been uncovered via genome wide association studies (GWAS) to be associated with FLD. In 2018, a genetic variant (rs72613567, T > TA) of hydroxysteroid 17-β dehydrogenase family 13 (HSD17B13) was first associated with a lower risk of developing alcoholic liver disease and non-alcoholic fatty liver disease (NAFLD) in minor allele carriers. Other HSD17B13 variants were also later linked with either lower inflammation scores among NAFLD patients or protection against NAFLD (rs6834314, A > G and rs9992651, G > A) respectively. HSD17B13 is a lipid droplet-associated protein, but its function is still ambiguous. Compared to the other genetic variants that increase risk for FLD, HSD17B13 variants serve a protective role, making this gene a potential therapeutic target. However, the mechanism by which these variants reduce the risk of developing FLD is still unclear. Because studies in cell lines and mouse models have produced conflicting results, human liver tissue modeling using induced pluripotent stem cells may be the best way to move forward and solve this mystery.