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Impaired mitophagy triggers NLRP3 inflammasome activation during the progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis

Laboratory Investigation (2019) | Download Citation


Activation of inflammation is an important mechanism in the development of nonalcoholic steatohepatitis (NASH). This study aims to delineate how mitophagy affects NLRP3 inflammasome activation in hepatic lipotoxicity. Mice were fed a high fat/calorie diet (HFCD) for 24 weeks. Primary rat hepatocytes were treated with palmitic acid (PA) for various periods of time. Mitophagy was measured by protein levels of LC3II and P62. NLRP3, caspase-1, interleukin (IL)-18, and IL-1β at mRNA and protein levels were used as indicators of inflammasome activation. Along with steatotic progression in HFCD-fed mice, ratio of LC3II/β-actin was decreased concurrently with increased levels of liver P62, NLRP3, caspase-1, IL-1β, IL-18, and serum IL-1β levels in late-stage NASH. PA treatment resulted in mitochondrial oxidative stress and initiated mitophagy in primary hepatocytes. The addition of cyclosporine A did not change LC3II/Τοmm20 ratios; but P62 levels were increased after an extended duration of PA exposure, indicating a defect in autophagic activity. Along with impaired mitophagy, mRNA and protein levels of NLRP3, caspase-1, IL-18 and IL-1β were upregulated by PA treatment. Pretreatment with MCC950, N-acetyl cysteine or acetyl-l-carnitine reversed inflammasome activation and a pyroptotic cascade. Additionally, mitophagic flux was partially recovered as indicated by increases in LC3II/Tomm20 ratio, parkin, and PINK1 expression, and decreased P62 expression. The findings suggest that impaired mitophagy triggers hepatic NLRP3 inflammasome activation in a murine NASH model and primary hepatocytes. The new insights into inflammasome activation through mitophagy advance our understanding of how fatty acids elicit lipotoxicity through oxidant stress and autophagy in mitochondria.

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  1. 1.

    Diehl AM, Day C. Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis. N Engl J Med. 2017;377:2063–72.

  2. 2.

    Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018;67:328–57.

  3. 3.

    Brown GT, Kleiner DE. Histopathology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Metabolism. 2016;65:1080–6.

  4. 4.

    Mota M, Banini BA, Cazanave SC, Sanyal AJ. Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease. Metabolism 2016;65:1049–61.

  5. 5.

    Sfunny NE, Bril F, Cusi K. Mitochondrial adaptation in nonalcoholic fatty liver disease: novel mechanisms and treatment strategies. Trends Endocrinol Metab. 2017;28:250–60.

  6. 6.

    Simoes ICM, Fontes A, Pinton P, Zischka H, Wieckowski MR. Mitochondria in non-alcoholic fatty liver disease. Int J Biochem Cell Biol 2018;95:93–9.

  7. 7.

    Kojima H, Sakurai S, Uemura M, Fukui H, Morimoto H, Tamagawa Y. Mitochondrial abnormality and oxidative stress in nonalcoholic steatohepatitis. Alcohol Clin Exp Res 2007;31:S61–6.

  8. 8.

    Marra F, Svegliati-Baroni G. Lipotoxicity and the gut-liver axis in NASH pathogenesis. J Hepatol. 2018;68:280–95.

  9. 9.

    Zhan SS, Jiang JX, Wu J, Halsted C, Friedman SL, Zern MA, et al. Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo. Hepatology 2006;43:435–43.

  10. 10.

    Wang L, Liu X, Nie J, Zhang J, Kimball SR, Zhang H, et al. ALCAT1 controls mitochondrial etiology of fatty liver diseases, linking defective mitophagy to steatosis. Hepatology 2015;61:486–96.

  11. 11.

    Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 2016;12:1–222.

  12. 12.

    Szabo G, Csak T. Inflammasomes in liver diseases. J Hepatol. 2012;57:642–54.

  13. 13.

    Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay A, et al. NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology 2014;59:898–910.

  14. 14.

    Liu XJ, Duan NN, Liu C, Niu C, Liu XP, Wu J. Characterization of a murine nonalcoholic steatohepatitis model induced by high fat high calorie diet plus fructose and glucose in drinking water. Lab Invest 2018;98:1184–99.

  15. 15.

    Kamari Y, Shaish A, Vax E, Shemesh S, Kandel-Kfir M, Arbel Y, et al. Lack of interleukin-1alpha or interleukin-1beta inhibits transformation of steatosis to steatohepatitis and liver fibrosis in hypercholesterolemic mice. J Hepatol 2011;55:1086–94.

  16. 16.

    Csak T, Ganz M, Pespisa J, Kodys K, Dolganiuc A, Szabo G. Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells. Hepatology 2011;54:133–44.

  17. 17.

    Vandanmagsar B, Youm YH, Ravussin A, Galgani JE, Stadler K, Mynatt RL, et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat Med 2011;17:179–88.

  18. 18.

    Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature 2011;469:221–5.

  19. 19.

    Wu J, Li X, Zhu G, Zhang Y, He M, Zhang J. The role of Resveratrol-induced mitophagy/autophagy in peritoneal mesothelial cells inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS. Exp Cell Res 2016;341:42–53.

  20. 20.

    Roberts RF, Tang MY, Fon EA, Durcan TM. Defending the mitochondria: The pathways of mitophagy and mitochondrial-derived vesicles. Int J Biochem Cell Biol 2016;79:427–36.

  21. 21.

    Kim MJ, Yoon JH, Ryu JH. Mitophagy: a balance regulator of NLRP3 inflammasome activation. BMB Rep. 2016;49:529–35.

  22. 22.

    Wu J, Soderbergh H, Karlsson K, Danielsson A. Protective effect of S-adenosyl-L-methionine on bromobenzene- and D-galactosamine-induced toxicity to isolated rat hepatocytes. Hepatology 1996;23:359–65.

  23. 23.

    Ni HM, Bockus A, Boggess N, Jaeschke H, Ding WX. Activation of autophagy protects against acetaminophen-induced hepatotoxicity. Hepatology 2012;55:222–32.

  24. 24.

    Pesce V, Nicassio L, Fracasso F, Musicco C, Cantatore P, Gadaleta MN. Acetyl-L-carnitine activates the peroxisome proliferator-activated receptor-gamma coactivators PGC-1alpha/PGC-1beta-dependent signaling cascade of mitochondrial biogenesis and decreases the oxidized peroxiredoxins content in old rat liver. Rejuvenation Res 2012;15:136–9.

  25. 25.

    Wu J, Karlsson K, Danielsson A. Effects of vitamins E, C and catalase on bromobenzene- and hydrogen peroxide-induced intracellular oxidation and DNA single-strand breakage in Hep G2 cells. J Hepatol. 1997;26:669–77.

  26. 26.

    Zhang Y, Venugopal SK, He S, Liu P, Wu J, Zern MA. Ethanol induces apoptosis in hepatocytes by a pathway involving novel protein kinase C isoforms. Cell Signal 2007;19:2339–50.

  27. 27.

    Yan HM, Ramachandran A, Bajt ML, Lemasters JJ, Jaeschke H. The oxygen tension modulates acetaminophen-induced mitochondrial oxidant stress and cell injury in cultured hepatocytes. Toxicol Sci 2010;117:515–23.

  28. 28.

    Marnef A, Jady BE, Kiss T. Human polypyrimidine tract-binding protein interacts with mitochondrial tRNA(Thr) in the cytosol. Nucleic Acids Res. 2016;44:1342–53.

  29. 29.

    Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 2011;12:222–30.

  30. 30.

    Wong A, Cortopassi G. Reproducible quantitative PCR of mitochondrial and nuclear DNA copy number using the LightCycler. Methods Mol Biol. 2002;197:129–37.

  31. 31.

    Wu J, Lizarzaburu ME, Kurth MJ, Liu L, Wege H, Zern MA, et al. Cationic lipid polymerization as a novel approach for constructing new DNA delivery agents. Bioconjug Chem 2001;12:251–7.

  32. 32.

    Ye J, Li TS, Xu G, Zhao YM, Zhang NP, Fan J, et al. JCAD Promotes Progression of Nonalcoholic Steatohepatitis to Liver Cancer by Inhibiting LATS2 Kinase Activity. Cancer Res 2017;77:5287–300.

  33. 33.

    Xu G, Ye J, Liu XJ, Zhang NP, Zhao YM, Fan J, et al. Activation of pluripotent genes in hepatic progenitor cells in the transition of nonalcoholic steatohepatitis to pre-malignant lesions. Lab Invest 2017;97:1201–17.

  34. 34.

    Karasawa T, Kawashima A, Usui-Kawanishi F, Watanabe S, Kimura H, Kamata R, et al. Saturated Fatty Acids Undergo Intracellular Crystallization and Activate the NLRP3 Inflammasome in Macrophages. Arterioscler Thromb Vasc Biol 2018;38:744–56.

  35. 35.

    Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011;147:728–41.

  36. 36.

    Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, et al. Autophagy regulates lipid metabolism. Nature 2009;458:1131–5.

  37. 37.

    Yang L, Li P, Fu S, Calay ES, Hotamisligil GS. Defective hepatic autophagy in obesity promotes ER stress and causes insulin resistance. Cell Metab 2010;11:467–78.

  38. 38.

    Flores-Toro JA, Go KL, Leeuwenburgh C, Kim JS. Autophagy in the liver: cell's cannibalism and beyond. Arch Pharm Res 2016;39:1050–61.

  39. 39.

    Petrosillo G, Portincasa P, Grattagliano I, Casanova G, Matera M, Ruggiero FM, et al. Mitochondrial dysfunction in rat with nonalcoholic fatty liver Involvement of complex I, reactive oxygen species and cardiolipin. Biochim Biophys Acta 2007;1767:1260–7.

  40. 40.

    Cassel SL, Joly S, Sutterwala FS. The NLRP3 inflammasome: a sensor of immune danger signals. Semin Immunol. 2009;21:194–8.

  41. 41.

    Franchi L, Munoz-Planillo R, Nunez G. Sensing and reacting to microbes through the inflammasomes. Nat Immunol. 2012;13:325–32.

  42. 42.

    Lazarou M. Keeping the immune system in check: a role for mitophagy. Immunol Cell Biol. 2015;93:3–10.

  43. 43.

    Kiriyama Y, Nochi H. Intra- and intercellular quality control mechanisms of mitochondria. Cells 2017;7:1.

  44. 44.

    Pickles S, Vigie P, Youle RJ. Mitophagy and quality control mechanisms in mitochondrial maintenance. Curr Biol. 2018;28:R170–85.

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The authors are grateful to Mrs. Ke Qiao in the Department of Medical Microbiology and Parasitology, Fudan University School of Basic Medical Sciences for her technical support in the use of confocal microscope and Operetta High Content Imaging System. Part of this work was presented at the Annual Meeting of the American Association for the Study of Liver Disease (AASLD), Nov. 11–15, 2016, Boston, MA, USA, and published as an abstract in Hepatology 2016;64(Suppl):777A.


This work is supported by the National Natural Science Foundation of China (NSFC #81272436, 81572356, 81871997 to JW), (#81472673 and 81672720 to X-ZS), Shanghai Commission of Sciences and Technologies (#16140903700), and the Ministry of Science & Technology of China (#2016YFE0107400) to JW. Young Investigator of Zhongshan Hospital, Fudan University (#2016ZSQN03) to N-PZ.

Author information


  1. Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, 200032, Shanghai, China

    • Ning-Ping Zhang
    •  & Xi-Zhong Shen
  2. Shanghai Institute of Liver Diseases, Shanghai, China

    • Ning-Ping Zhang
    • , Xi-Zhong Shen
    •  & Jian Wu
  3. Department of Medical Microbiology, School of Basic Medical Sciences of Fudan University, 200032, Shanghai, China

    • Xue-Jing Liu
    • , Li Xie
    •  & Jian Wu


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The authors declare that they have no conflict of interest.

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Correspondence to Xi-Zhong Shen or Jian Wu.

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