PNPLA3-I148M genetic variant rewires lipid metabolism to drive programmed cell death in human hepatocytes
Rodrigo M. Florentino, Olamide Animasahun, Nils Haep, Minal Nenwani, Kehinde Omoloja, Leyla Nurcihan Altay, Abhinav Achreja, Kazutoyo Morita, Takashi Motomura, Ricardo Diaz-Aragon, Lanuza A.P. Faccioli, Yiyue Sun, Zhenghao Liu, Zhiping Hu, Bo Yang, Fulei Wuchu, Ajay Shankaran, Miya Paserba, Annalisa M. Baratta, Shohrat Arazov, Zehra N. Kocas-Kilicarslan, Noah Meurs, Jaideep Behari, Edgar N. Tafaleng, Jonathan Franks, Alina Ostrowska, Takahiro Tomiyama, Kyohei Yugawa, Akinari Morinaga, Zi Wang, Kazuki Takeishi, Dillon C. Gavlock, Mark Miedel, D. Lansing Taylor, Ira J. Fox, Tomoharu Yoshizumi, Deepak Nagrath, Alejandro Soto-Gutierrez
Rodrigo M. Florentino, Olamide Animasahun, Nils Haep, Minal Nenwani, Kehinde Omoloja, Leyla Nurcihan Altay, Abhinav Achreja, Kazutoyo Morita, Takashi Motomura, Ricardo Diaz-Aragon, Lanuza A.P. Faccioli, Yiyue Sun, Zhenghao Liu, Zhiping Hu, Bo Yang, Fulei Wuchu, Ajay Shankaran, Miya Paserba, Annalisa M. Baratta, Shohrat Arazov, Zehra N. Kocas-Kilicarslan, Noah Meurs, Jaideep Behari, Edgar N. Tafaleng, Jonathan Franks, Alina Ostrowska, Takahiro Tomiyama, Kyohei Yugawa, Akinari Morinaga, Zi Wang, Kazuki Takeishi, Dillon C. Gavlock, Mark Miedel, D. Lansing Taylor, Ira J. Fox, Tomoharu Yoshizumi, Deepak Nagrath, Alejandro Soto-Gutierrez
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Research Article Gastroenterology Hepatology

PNPLA3-I148M genetic variant rewires lipid metabolism to drive programmed cell death in human hepatocytes

Abstract

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.

Authors

Rodrigo M. Florentino, Olamide Animasahun, Nils Haep, Minal Nenwani, Kehinde Omoloja, Leyla Nurcihan Altay, Abhinav Achreja, Kazutoyo Morita, Takashi Motomura, Ricardo Diaz-Aragon, Lanuza A.P. Faccioli, Yiyue Sun, Zhenghao Liu, Zhiping Hu, Bo Yang, Fulei Wuchu, Ajay Shankaran, Miya Paserba, Annalisa M. Baratta, Shohrat Arazov, Zehra N. Kocas-Kilicarslan, Noah Meurs, Jaideep Behari, Edgar N. Tafaleng, Jonathan Franks, Alina Ostrowska, Takahiro Tomiyama, Kyohei Yugawa, Akinari Morinaga, Zi Wang, Kazuki Takeishi, Dillon C. Gavlock, Mark Miedel, D. Lansing Taylor, Ira J. Fox, Tomoharu Yoshizumi, Deepak Nagrath, Alejandro Soto-Gutierrez

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Figure 4

Peroxisomal β-oxidation is increased in human PNPLA3-I148M hepatocytes.

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Peroxisomal β-oxidation is increased in human PNPLA3-I148M hepatocytes. ...
(A) Western blot analysis of ACOX1 intensity in Hep-PNPLA3-I148M and Hep-PNPLA3-WT. Intensity ratio of the peroxisomal 50 kDA ACOX1 and precursor 70 kDa ACOX1 (*P = 0.05, Welch’s t test, WT: n = 3, I148M: n = 3). PMP70 intensity (#P = 0.8128, Welch’s t test, WT: n = 3, I148M: n = 3). (B) ACAA1 immunohistochemistry signal quantification (P = 0.001, Mann-Whitney U test, WT: n = 3 normal livers/5 different fields each, I418M: n = 3 normal livers/5 fields each). (C) Schematic of the generation of M+1 Acetyl-CoA from C1-labeled palmitic acid via mitochondrial β-oxidation. Inserted is the scatterplot indicating the enrichment of M+1 Acetyl-CoA in Hep-PNPLA3-I481M extracts (n = 9) compared with Hep-PNPLA3-WT (n = 8). Horizontal bar indicates the mean and data are analyzed using 2-tailed Mann-Whitney U test. (D) Schematic of the generation of M+2 Acetyl-CoA from docosanoic-1,2,3,4- 13C4 acid via peroxisomal β-oxidation. Inserted is the scatterplot indicating the enrichment of M+2 Acetyl-CoA in Hep-PNPLA3-I481M extracts (n = 9) compared with Hep-PNPLA3-WT (n = 8). Horizontal bar indicates the mean and data are analyzed using 2-tailed Mann-Whitney U test.