PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction
Abstract
:1. Introduction
2. Results
2.1. PNPLA3-I148M Promotes Intracellular LD Accumulation and LX-2 Activation
2.2. PNPLA3-I148M Induces FC Accumulation in LX-2 Cells
2.3. PNPLA3-I148M Disrupts the Function of Mitochondrial
2.4. Impact of PNPLA3-I148M on Mitochondrial Ultrastructure
2.5. Effect of PNPLA3 on the Expression of Mitochondrial Proteins
2.6. PNPLA3-I148M Inhibits Mitochondrial Respiration in LX-2 Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Generating Stable Cell Line
4.3. Lipid Analysis
4.4. BODIPY Staining
4.5. Filipin III Staining
4.6. Co-Localization Analysis
4.7. ATP Measurements
4.8. Analysis of Reactive Oxygen Species
4.9. Measurement of Mitochondrial Transmembrane Potential (ΔΨm)
4.10. Oxygen Consumption Rate Measurements
4.11. Mitochondrial Ultrastructure
4.12. Real-Time PCR
4.13. Western Blot
4.14. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Younossi, Z.; Tacke, F.; Arrese, M.; Chander Sharma, B.; Mostafa, I.; Bugianesi, E.; Wai-Sun Wong, V.; Yilmaz, Y.; George, J.; Fan, J.; et al. Global Perspectives on Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis. Hepatology 2019, 69, 2672–2682. [Google Scholar] [CrossRef] [Green Version]
- Pierantonelli, I.; Svegliati-Baroni, G. Nonalcoholic Fatty Liver Disease: Basic Pathogenetic Mechanisms in the Progression from NAFLD to NASH. Transplantation 2019, 103, e1–e13. [Google Scholar] [CrossRef] [PubMed]
- Cobbina, E.; Akhlaghi, F. Non-alcoholic fatty liver disease (NAFLD)—Pathogenesis, classification, and effect on drug metabolizing enzymes and transporters. Drug Metab. Rev. 2017, 49, 197–211. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bataller, R.; Brenner, D.A. Liver fibrosis. J. Clin. Investig. 2005, 115, 209–218. [Google Scholar] [CrossRef] [PubMed]
- Marrone, G.; Shah, V.H.; Gracia-Sancho, J. Sinusoidal communication in liver fibrosis and regeneration. J. Hepatol. 2016, 65, 608–617. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kamm, D.R.; McCommis, K.S. Hepatic stellate cells in physiology and pathology. J. Physiol. 2022, 600, 1825–1837. [Google Scholar] [CrossRef]
- Senoo, H.; Yoshikawa, K.; Morii, M.; Miura, M.; Imai, K.; Mezaki, Y. Hepatic stellate cell (vitamin A-storing cell) and its relative—Past, present and future. Cell Biol. Int. 2010, 34, 1247–1272. [Google Scholar] [CrossRef]
- Luo, N.; Li, J.; Wei, Y.; Lu, J.; Dong, R. Hepatic Stellate Cell: A Double-Edged Sword in the Liver. Physiol. Res. 2021, 70, 821–829. [Google Scholar] [CrossRef]
- Tsuchida, T.; Friedman, S.L. Mechanisms of hepatic stellate cell activation. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 397–411. [Google Scholar] [CrossRef]
- Dawood, R.M.; El-Meguid, M.A.; Salum, G.M.; El Awady, M.K. Key Players of Hepatic Fibrosis. J. Interferon Cytokine Res. 2020, 40, 472–489. [Google Scholar] [CrossRef]
- Higashi, T.; Friedman, S.L.; Hoshida, Y. Hepatic stellate cells as key target in liver fibrosis. Adv. Drug Deliv. Rev. 2017, 121, 27–42. [Google Scholar] [CrossRef] [PubMed]
- Tomita, K.; Teratani, T.; Suzuki, T.; Shimizu, M.; Sato, H.; Narimatsu, K.; Okada, Y.; Kurihara, C.; Irie, R.; Yokoyama, H.; et al. Free cholesterol accumulation in hepatic stellate cells: Mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice. Hepatology 2014, 59, 154–169. [Google Scholar] [CrossRef] [PubMed]
- Rauchbach, E.; Zeigerman, H.; Abu-Halaka, D.; Tirosh, O. Cholesterol Induces Oxidative Stress, Mitochondrial Damage and Death in Hepatic Stellate Cells to Mitigate Liver Fibrosis in Mice Model of NASH. Antioxidants 2022, 11, 536. [Google Scholar] [CrossRef]
- Zhou, Y.; Long, D.; Zhao, Y.; Li, S.; Liang, Y.; Wan, L.; Zhang, J.; Xue, F.; Feng, L. Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation. Cell Death Dis. 2022, 13, 689. [Google Scholar] [CrossRef]
- Romeo, S.; Kozlitina, J.; Xing, C.; Pertsemlidis, A.; Cox, D.; Pennacchio, L.A.; Boerwinkle, E.; Cohen, J.C.; Hobbs, H.H. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat. Genet. 2008, 40, 1461–1465. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Cherubini, A.; Casirati, E.; Tomasi, M.; Valenti, L. PNPLA3 as a therapeutic target for fatty liver disease: The evidence to date. Expert Opin. Ther. Targets 2021, 25, 1033–1043. [Google Scholar] [CrossRef] [PubMed]
- Min, H.K.; Sookoian, S.; Pirola, C.J.; Cheng, J.; Mirshahi, F.; Sanyal, A.J. Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells. Am. J. Physiol.-Gastrointest. Liver Physiol. 2014, 307, G66–G76. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dong, X.C. PNPLA3-A Potential Therapeutic Target for Personalized Treatment of Chronic Liver Disease. Front. Med. 2019, 6, 304. [Google Scholar] [CrossRef]
- Basu Ray, S.; Smagris, E.; Cohen, J.C.; Hobbs, H.H. The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation. Hepatology 2017, 66, 1111–1124. [Google Scholar] [CrossRef] [Green Version]
- Pirazzi, C.; Valenti, L.; Motta, B.M.; Pingitore, P.; Hedfalk, K.; Mancina, R.M.; Burza, M.A.; Indiveri, C.; Ferro, Y.; Montalcini, T.; et al. PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells. Hum. Mol. Genet. 2014, 23, 4077–4085. [Google Scholar] [CrossRef] [Green Version]
- Pingitore, P.; Dongiovanni, P.; Motta, B.M.; Meroni, M.; Lepore, S.M.; Mancina, R.M.; Pelusi, S.; Russo, C.; Caddeo, A.; Rossi, G.; et al. PNPLA3 overexpression results in reduction of proteins predisposing to fibrosis. Hum. Mol. Genet. 2016, 25, 5212–5222. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bruschi, F.V.; Claudel, T.; Tardelli, M.; Caligiuri, A.; Stulnig, T.M.; Marra, F.; Trauner, M. The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells. Hepatology 2017, 65, 1875–1890. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bruschi, F.V.; Claudel, T.; Tardelli, M.; Starlinger, P.; Marra, F.; Trauner, M. PNPLA3 I148M Variant Impairs Liver X Receptor Signaling and Cholesterol Homeostasis in Human Hepatic Stellate Cells. Hepatol. Commun. 2019, 3, 1191–1204. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liu, Z.; Zhang, Y.; Graham, S.; Wang, X.; Cai, D.; Huang, M.; Pique-Regi, R.; Dong, X.C.; Chen, Y.E.; Willer, C.; et al. Causal relationships between NAFLD, T2D and obesity have implications for disease subphenotyping. J. Hepatol. 2020, 73, 263–276. [Google Scholar] [CrossRef]
- Dhar, D.; Loomba, R. Emerging Metabolic and Transcriptomic Signature of PNPLA3-Associated NASH. Hepatology 2021, 73, 1248–1250. [Google Scholar] [CrossRef] [PubMed]
- Kim, H.S.; Xiao, X.; Byun, J.; Jun, G.; DeSantis, S.M.; Chen, H.; Thrift, A.P.; El-Serag, H.B.; Kanwal, F.; Amos, C.I. Synergistic Associations of PNPLA3 I148M Variant, Alcohol Intake, and Obesity with Risk of Cirrhosis, Hepatocellular Carcinoma, and Mortality. JAMA Netw. Open 2022, 5, e2234221. [Google Scholar] [CrossRef]
- Salameh, H.; Raff, E.; Erwin, A.; Seth, D.; Nischalke, H.D.; Falleti, E.; Burza, M.A.; Leathert, J.; Romeo, S.; Molinaro, A.; et al. PNPLA3 Gene Polymorphism Is Associated with Predisposition to and Severity of Alcoholic Liver Disease. Am. J. Gastroenterol. 2015, 110, 846–856. [Google Scholar] [CrossRef] [Green Version]
- Valenti, L.; Dongiovanni, P.; Ginanni Corradini, S.; Burza, M.A.; Romeo, S. PNPLA3 I148M variant and hepatocellular carcinoma: A common genetic variant for a rare disease. Dig. Liver Dis. 2013, 45, 619–624. [Google Scholar] [CrossRef] [Green Version]
- Wang, Y.; Kory, N.; BasuRay, S.; Cohen, J.C.; Hobbs, H.H. PNPLA3, CGI-58, and Inhibition of Hepatic Triglyceride Hydrolysis in Mice. Hepatology 2019, 69, 2427–2441. [Google Scholar] [CrossRef] [Green Version]
- Ioannou, G.N. The Role of Cholesterol in the Pathogenesis of NASH. Trends Endocrinol. Metab. 2016, 27, 84–95. [Google Scholar] [CrossRef]
- Horn, C.L.; Morales, A.L.; Savard, C.; Farrell, G.C.; Ioannou, G.N. Role of Cholesterol-Associated Steatohepatitis in the Development of NASH. Hepatol. Commun. 2022, 6, 12–35. [Google Scholar] [CrossRef] [PubMed]
- Teratani, T.; Tomita, K.; Suzuki, T.; Oshikawa, T.; Yokoyama, H.; Shimamura, K.; Tominaga, S.; Hiroi, S.; Irie, R.; Okada, Y.; et al. A high-cholesterol diet exacerbates liver fibrosis in mice via accumulation of free cholesterol in hepatic stellate cells. Gastroenterology 2012, 142, 152–164.e110. [Google Scholar] [CrossRef] [PubMed]
- Furuhashi, H.; Tomita, K.; Teratani, T.; Shimizu, M.; Nishikawa, M.; Higashiyama, M.; Takajo, T.; Shirakabe, K.; Maruta, K.; Okada, Y.; et al. Vitamin A-coupled liposome system targeting free cholesterol accumulation in hepatic stellate cells offers a beneficial therapeutic strategy for liver fibrosis. Hepatol. Res. 2018, 48, 397–407. [Google Scholar] [CrossRef] [PubMed]
- Tomita, K.; Teratani, T.; Suzuki, T.; Shimizu, M.; Sato, H.; Narimatsu, K.; Usui, S.; Furuhashi, H.; Kimura, A.; Nishiyama, K.; et al. Acyl-CoA:cholesterol acyltransferase 1 mediates liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells. J. Hepatol. 2014, 61, 98–106. [Google Scholar] [CrossRef]
- Twu, Y.C.; Lee, T.S.; Lin, Y.L.; Hsu, S.M.; Wang, Y.H.; Liao, C.Y.; Wang, C.K.; Liang, Y.C.; Liao, Y.J. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation. Int. J. Mol. Sci. 2016, 17, 1122. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chen, L.; Zhao, Z.W.; Zeng, P.H.; Zhou, Y.J.; Yin, W.J. Molecular mechanisms for ABCA1-mediated cholesterol efflux. Cell Cycle 2022, 21, 1121–1139. [Google Scholar] [CrossRef] [PubMed]
- Yu, X.H.; Tang, C.K. ABCA1, ABCG1, and Cholesterol Homeostasis. Adv. Exp. Med. Biol. 2022, 1377, 95–107. [Google Scholar] [CrossRef]
- Garcia-Ruiz, C.; Mari, M.; Colell, A.; Morales, A.; Caballero, F.; Montero, J.; Terrones, O.; Basanez, G.; Fernandez-Checa, J.C. Mitochondrial cholesterol in health and disease. Histol. Histopathol. 2009, 24, 117–132. [Google Scholar] [CrossRef]
- Martin, L.A.; Kennedy, B.E.; Karten, B. Mitochondrial cholesterol: Mechanisms of import and effects on mitochondrial function. J. Bioenerg. Biomembr. 2016, 48, 137–151. [Google Scholar] [CrossRef]
- Elustondo, P.; Martin, L.A.; Karten, B. Mitochondrial cholesterol import. Biochim. Biophys. Acta (BBA)-Mol. Cell Biol. Lipids 2017, 1862, 90–101. [Google Scholar] [CrossRef]
- Zorova, L.D.; Popkov, V.A.; Plotnikov, E.Y.; Silachev, D.N.; Pevzner, I.B.; Jankauskas, S.S.; Babenko, V.A.; Zorov, S.D.; Balakireva, A.V.; Juhaszova, M.; et al. Mitochondrial membrane potential. Anal. Biochem. 2018, 552, 50–59. [Google Scholar] [CrossRef] [PubMed]
- Li, X.; Zhang, W.; Cao, Q.; Wang, Z.; Zhao, M.; Xu, L.; Zhuang, Q. Mitochondrial dysfunction in fibrotic diseases. Cell Death Discov. 2020, 6, 80. [Google Scholar] [CrossRef] [PubMed]
- Pessayre, D.; Fromenty, B. NASH: A mitochondrial disease. J. Hepatol. 2005, 42, 928–940. [Google Scholar] [CrossRef] [PubMed]
- Cichoz-Lach, H.; Michalak, A. Oxidative stress as a crucial factor in liver diseases. World J. Gastroenterol. 2014, 20, 8082–8091. [Google Scholar] [CrossRef]
- Mansouri, A.; Gattolliat, C.H.; Asselah, T. Mitochondrial Dysfunction and Signaling in Chronic Liver Diseases. Gastroenterology 2018, 155, 629–647. [Google Scholar] [CrossRef] [Green Version]
- Solsona-Vilarrasa, E.; Fucho, R.; Torres, S.; Nunez, S.; Nuno-Lambarri, N.; Enrich, C.; Garcia-Ruiz, C.; Fernandez-Checa, J.C. Cholesterol enrichment in liver mitochondria impairs oxidative phosphorylation and disrupts the assembly of respiratory supercomplexes. Redox Biol. 2019, 24, 101214. [Google Scholar] [CrossRef]
- Torres, S.; Garcia-Ruiz, C.M.; Fernandez-Checa, J.C. Mitochondrial Cholesterol in Alzheimer’s Disease and Niemann-Pick Type C Disease. Front. Neurol. 2019, 10, 1168. [Google Scholar] [CrossRef] [Green Version]
- Cogliati, S.; Enriquez, J.A.; Scorrano, L. Mitochondrial Cristae: Where Beauty Meets Functionality. Trends Biochem. Sci. 2016, 41, 261–273. [Google Scholar] [CrossRef] [Green Version]
- Norambuena, A.; Sun, X.; Wallrabe, H.; Cao, R.; Sun, N.; Pardo, E.; Shivange, N.; Wang, D.B.; Post, L.A.; Ferris, H.A.; et al. SOD1 mediates lysosome-to-mitochondria communication and its dysregulation by amyloid-beta oligomers. Neurobiol. Dis. 2022, 169, 105737. [Google Scholar] [CrossRef]
- Horspool, A.M.; Chang, H.C. Superoxide dismutase SOD-1 modulates C. elegans pathogen avoidance behavior. Sci. Rep. 2017, 7, 45128. [Google Scholar] [CrossRef] [Green Version]
- Sakiyama, H.; Fujiwara, N.; Yoneoka, Y.; Yoshihara, D.; Eguchi, H.; Suzuki, K. Cu, Zn-SOD deficiency induces the accumulation of hepatic collagen. Free Radic. Res. 2016, 50, 666–677. [Google Scholar] [CrossRef] [PubMed]
- Hernandez-Alvarez, M.I.; Sebastian, D.; Vives, S.; Ivanova, S.; Bartoccioni, P.; Kakimoto, P.; Plana, N.; Veiga, S.R.; Hernandez, V.; Vasconcelos, N.; et al. Deficient Endoplasmic Reticulum-Mitochondrial Phosphatidylserine Transfer Causes Liver Disease. Cell 2019, 177, 881–895.e817. [Google Scholar] [CrossRef]
- Zhu, H.; Shan, Y.; Ge, K.; Lu, J.; Kong, W.; Jia, C. Specific Overexpression of Mitofusin-2 in Hepatic Stellate Cells Ameliorates Liver Fibrosis in Mice Model. Hum. Gene Ther. 2020, 31, 103–109. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Xu, L.; Hui, A.Y.; Albanis, E.; Arthur, M.J.; O’Byrne, S.M.; Blaner, W.S.; Mukherjee, P.; Friedman, S.L.; Eng, F.J. Human hepatic stellate cell lines, LX-1 and LX-2: New tools for analysis of hepatic fibrosis. Gut 2005, 54, 142–151. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Gou, Y.; Wang, L.; Zhao, J.; Xu, X.; Xu, H.; Xie, F.; Wang, Y.; Feng, Y.; Zhang, J.; Zhang, Y. PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction. Int. J. Mol. Sci. 2023, 24, 9681. https://doi.org/10.3390/ijms24119681
Gou Y, Wang L, Zhao J, Xu X, Xu H, Xie F, Wang Y, Feng Y, Zhang J, Zhang Y. PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction. International Journal of Molecular Sciences. 2023; 24(11):9681. https://doi.org/10.3390/ijms24119681
Chicago/Turabian StyleGou, Yusong, Lifei Wang, Jinhan Zhao, Xiaoyi Xu, Hangfei Xu, Fang Xie, Yanjun Wang, Yingmei Feng, Jing Zhang, and Yang Zhang. 2023. "PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction" International Journal of Molecular Sciences 24, no. 11: 9681. https://doi.org/10.3390/ijms24119681