Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non‑alcoholic steatohepatitis (NASH), there is a lack of in vitro models of non‑alcoholic fatty liver disease (NAFLD) with similar findings. Despite being the most commonly used immortalized cell line for in vitro models of NAFLD, HepG2 cells exposed to free fatty acids (FFAs) exhibit a decreased mitochondrial respiration. On the other hand, the use of HepaRG cells to study mitochondrial respiratory changes following exposure to FFAs has not yet been fully explored. Therefore, the present study aimed to assess cellular energy metabolism, particularly mitochondrial respiration, and lipotoxicity in FFA‑treated HepaRG and HepG2 cells. HepaRG and HepG2 cells were exposed to FFAs, followed by comparative analyses that examained cellular metabolism, mitochondrial respiratory enzyme activities, mitochondrial morphology, lipotoxicity, the mRNA expression of selected genes and triacylglycerol (TAG) accumulation. FFAs stimulated mitochondrial respiration and glycolysis in HepaRG cells, but not in HepG2 cells. Stimulated complex I, II‑driven respiration and β‑oxidation were linked to increased complex I and II activities in FFA‑treated HepaRG cells, but not in FFA‑treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA‑treated HepaRG cells than in FFA‑treated HepG2 cells. TAG accumulation was less prominent in HepaRG cells than in HepG2 cells. On the whole, the present study demonstrates that stimulated mitochondrial respiration is associated with lipotoxicity in FFA‑treated HepaRG cells, but not in FFA‑treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed in vitro model of early‑stage NASH.

Department of Histology and Embryology Charles University Faculty of Medicine in Hradec Kralove 500 03 Hradec Kralove Czech Republic

Department of Medical Biophysics Charles University Faculty of Medicine in Hradec Kralove 500 03 Hradec Kralove Czech Republic

Department of Pharmacology and Toxicology Charles University Faculty of Pharmacy in Hradec Kralove 500 05 Hradec Kralove Czech Republic

Department of Physiology Charles University Faculty of Medicine in Hradec Kralove 500 03 Hradec Kralove Czech Republic

Zobrazit více v PubMed

Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64:73–84. PubMed

Anderson EL, Howe LD, Jones HE, Higgins JPT, Lawlor DA, Fraser A. The prevalence of non-alcoholic fatty liver disease in children and adolescents: A systematic review and meta-analysis. PLoS One. 2015;10:e0140908. PubMed PMC

Maurice J, Manousou P. Non-alcoholic fatty liver disease. Clin Med (Lond) 2018;18:245–250. PubMed PMC

Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui JM, Fung C, Karim R, Lin R, Samarasinghe D, Liddle C, et al. NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology. 2002;35:373–379. PubMed

Simões ICM, Fontes A, Pinton P, Zischka H, Wieckowski MR. Mitochondria in non-alcoholic fatty liver disease. Int J Biochem Cell Biol. 2018;95:93–99. PubMed

Nassir F, Ibdah JA. Role of mitochondria in nonalcoholic fatty liver disease. Int J Mol Sci. 2014;15:8713–8742. PubMed PMC

Lu Q, Tian X, Wu H, Huang J, Li M, Mei Z, Zhou L, Xie H, Zheng S. Metabolic changes of hepatocytes in NAFLD. Front Physiol. 2021;12:710420. PubMed PMC

Sunny NE, Bril F, Cusi K. Mitochondrial adaptation in nonalcoholic fatty liver disease: Novel mechanisms and treatment strategies. Trends Endocrinol Metabolism. 2017;28:250–260. PubMed

Koliaki C, Szendroedi J, Kaul K, Jelenik T, Nowotny P, Jankowiak F, Herder C, Carstensen M, Krausch M, Knoefel WT, et al. Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab. 2015;21:739–746. PubMed

Satapati S, Sunny NE, Kucejova B, Fu X, He TT, Méndez-Lucas A, Shelton JM, Perales JC, Browning JD, Burgess SC. Elevated TCA cycle function in the pathology of diet-induced hepatic insulin resistance and fatty liver. J Lipid Res. 2012;53:1080–1092. PubMed PMC

Begriche K, Massart J, Robin MA, Bonnet F, Fromenty B. Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease. Hepatology. 2013;58:1497–1507. PubMed

Green CJ, Parry SA, Gunn PJ, Ceresa CDL, Rosqvist F, Piche ME, Hodson L. Studying non-alcoholic fatty liver disease: The ins and outs of in vivo, ex vivo and in vitro human models. Horm Mol Biol Clin Investig. 2018;41 doi: 10.1515/hmbci-2018-0038. PubMed DOI

Ramos MJ, Bandiera L, Menolascina F, Fallowfield JA. In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications. iScience. 2022;25:103549. PubMed PMC

Green CJ, Johnson D, Amin HD, Sivathondan P, Silva MA, Wang LM, Stevanato L, McNeil CA, Miljan EA, Sinden JD, et al. Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line. Am J Physiol Endocrinol Metab. 2015;309:E511–E522. PubMed PMC

Amorim R, Simões ICM, Veloso C, Carvalho A, Simões RF, Pereira FB, Thiel T, Normann A, Morais C, Jurado AS, et al. Exploratory data analysis of cell and mitochondrial high-fat, high-sugar toxicity on human HepG2 cells. Nutrients. 2021;13:1723. PubMed PMC

Garcia-Ruiz I, Solis-Munoz P, Fernandez-Moreira D, Munoz-Yague T, Solis-Herruzo JA. In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis. Dis Model Mech. 2015;8:183–191. PubMed PMC

Pérez-Carreras M, Del Hoyo P, Martín MA, Rubio JC, Martín A, Castellano G, Colina F, Arenas J, Solis-Herruzo JA. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology. 2003;38:999–1007. PubMed

Donato MT, Tolosa L, Gómez-Lechón MJ. Culture and functional characterization of human hepatoma HepG2 cells. Methods Mol Biol. 2015;1250:77–93. PubMed

Gibbons GF, Khurana R, Odwell A, Seelaender MC. Lipid balance in HepG2 cells: Active synthesis and impaired mobilization. J Lipid Res. 1994;35:1801–1808. PubMed

Tascher G, Burban A, Camus S, Plumel M, Chanon S, Le Guevel R, Shevchenko V, Van Dorsselaer A, Lefai E, Guguen-Guillouzo C, Bertile F. In-depth proteome analysis highlights HepaRG cells as a versatile cell system surrogate for primary human hepatocytes. Cells. 2019;8:192. PubMed PMC

Berlanga A, Guiu-Jurado E, Porras JA, Auguet T. Molecular pathways in non-alcoholic fatty liver disease. Clin Exp Gastroenterol. 2014;7:221–239. PubMed PMC

Zhou Y, Orešič M, Leivonen M, Gopalacharyulu P, Hyysalo J, Arola J, Verrijken A, Francque S, Van Gaal L, Hyötyläinen T, Yki-Järvinen H. Noninvasive detection of nonalcoholic steatohepatitis using clinical markers and circulating levels of lipids and metabolites. Clin Gastroenterol Hepatol. 2016;14:1463–1472.e6. PubMed

Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005;115:1343–1351. PubMed PMC

Ipsen DH, Lykkesfeldt J, Tveden-Nyborg P. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cell Mol Life Sci. 2018;75:3313–3327. PubMed PMC

Geng Y, Faber KN, de Meijer VE, Blokzijl H, Moshage H. How does hepatic lipid accumulation lead to lipotoxicity in non-alcoholic fatty liver disease? Hepatol Int. 2021;15:21–35. PubMed PMC

Tsilingiris D, Tzeravini E, Koliaki C, Dalamaga M, Kokkinos A. The role of mitochondrial adaptation and metabolic flexibility in the pathophysiology of obesity and insulin resistance: An updated overview. Curr Obes Rep. 2021;10:191–213. PubMed

Stefela A, Kaspar M, Drastik M, Holas O, Hroch M, Smutny T, Skoda J, Hutníková M, Pandey AV, Micuda S, et al. 3β-Isoobeticholic acid efficiently activates the farnesoid X receptor (FXR) due to its epimerization to 3α-epimer by hepatic metabolism. J Steroid Biochem Mol Biol. 2020;202:105702. PubMed

Geng Y, Villanueva AH, Oun A, Buist-Homan M, Blokzijl H, Faber KN, Dolga A, Moshage H. Protective effect of metformin against palmitate-induced hepatic cell death. Biochim Biophys Acta. 2020;1866:165621. PubMed

Elkalaf M, Vaněčková K, Staňková P, Červinková Z, Polák J, Kučera O. Measuring mitochondrial substrate flux in recombinant Perfringolysin O-Permeabilized cells. J Vis Exp. 2021;13 doi: 10.3791/62902. PubMed DOI

Iuso A, Repp B, Biagosch C, Terrile C, Prokisch H. Assessing mitochondrial bioenergetics in isolated mitochondria from various mouse tissues using seahorse XF96 analyzer. Methods Mol Biol. 2017;1567:217–230. PubMed

Spinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc. 2012;7:1235–1246. PubMed

Elkalaf M, Tůma P, Weiszenstein M, Polák J, Trnka J. Mitochondrial probe methyltriphenylphosphonium (TPMP) inhibits the krebs cycle Enzyme 2-Oxoglutarate Dehydrogenase. PLoS One. 2016;11:e0161413. PubMed PMC

Cechakova L, Ondrej M, Pavlik V, Jost P, Cizkova D, Bezrouk A, Pejchal J, Amaravadi RK, Winkler JD, Tichy A. A potent autophagy inhibitor (Lys05) enhances the impact of ionizing radiation on human lung cancer cells H1299. Int J Mol Sci. 2019;20:5881. PubMed PMC

Kucera O, Endlicher R, Rousar T, Lotkova H, Garnol T, Drahota Z, Cervinková Z. The effect of tert-butyl hydroperoxide-induced oxidative stress on lean and steatotic rat hepatocytes in vitro. Oxid Med Cell Longev. 2014;2014:752506. PubMed PMC

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–408. PubMed

Nagarajan SR, Paul-Heng M, Krycer JR, Fazakerley DJ, Sharland AF, Hoy AJ. Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: A comprehensive resource for in vitro studies of hepatic metabolism. Am J Physiol Endocrinol Metab. 2019;316:E578–E589. PubMed

Judge A, Dodd MS. Metabolism. Essays Biochem. 2020;64:607–647. PubMed PMC

Staňková P, Kučera O, Peterová E, Lotková H, Maseko TE, Nožičková K, Červinková Z. Adaptation of mitochondrial substrate flux in a mouse model of nonalcoholic fatty liver disease. Int J Mol Sci. 2020;21:1101. PubMed PMC

Staňková P, Kučera O, Peterová E, Elkalaf M, Rychtrmoc D, Melek J, Podhola M, Zubáňová V, Červinková Z. Western diet decreases the liver mitochondrial oxidative flux of succinate: Insight from a Murine NAFLD model. Int J Mol Sci. 2021;22:6908. PubMed PMC

Pfleger J. Measurements of mitochondrial respiration in intact cells, permeabilized cells, and isolated tissue mitochondria using the seahorse XF analyzer. Methods Mol Biol. 2022;2497:185–206. PubMed

Larsen S, Nielsen J, Hansen CN, Nielsen LB, Wibrand F, Stride N, Schroder HD, Boushel R, Helge JW, Dela F, Hey-Mogensen M. Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J Physiol. 2012;590:3349–3360. PubMed PMC

Urra FA, Muñoz F, Lovy A, Cárdenas C. The mitochondrial complex(I)ty of cancer. Front Oncol. 2017;7:118. PubMed PMC

Peyta L, Jarnouen K, Pinault M, Guimaraes C, Pais de Barros JP, Chevalier S, Dumas JF, Maillot F, Hatch GM, Loyer P, Servais S. Reduced cardiolipin content decreases respiratory chain capacities and increases ATP synthesis yield in the human HepaRG cells. Biochim Biophys Acta. 2016;1857:443–453. PubMed

de Sousa IF, Migliaccio V, Lepretti M, Paolella G, Di Gregorio I, Caputo I, Ribeiro EB, Lionetti L. Dose- and time-dependent effects of oleate on mitochondrial Fusion/Fission proteins and cell viability in HepG2 cells: Comparison with palmitate effects. Int J Mol Sci. 2021;22:9812. PubMed PMC

Sasi US, Sindhu G, Raghu KG. Fructose-palmitate based high calorie induce steatosis in HepG2 cells via mitochondrial dysfunction: An in vitro approach. Toxicol In Vitro. 2020;68:104952. PubMed

Grasselli E, Baldini F, Vecchione G, Oliveira PJ, Sardão VA, Voci A, Portincasa P, Vergani L. Excess fructose and fatty acids trigger a model of non-alcoholic fatty liver disease progression in vitro: Protective effect of the flavonoid silybin. Int J Mol Med. 2019;44:705–712. PubMed

Feaver RE, Cole BK, Lawson MJ, Hoang SA, Marukian S, Blackman BR, Figler RA, Sanyal AJ, Wamhoff BR, Dash A. Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis. JCI Insight. 2016;1:e90954. PubMed PMC

Longhitano L, Distefano A, Amorini AM, Orlando L, Giallongo S, Tibullo D, Lazzarino G, Nicolosi A, Alanazi AM, Saoca C, et al. (+)-lipoic acid reduces lipotoxicity and regulates mitochondrial homeostasis and energy balance in an in vitro model of liver steatosis. Int J Mol Sci. 2023;24:14491. PubMed PMC

Perry RJ, Kim T, Zhang XM, Lee HY, Pesta D, Popov VB, Zhang D, Rahimi Y, Jurczak MJ, Cline GW, et al. Reversal of hypertriglyceridemia, fatty liver disease, and insulin resistance by a liver-targeted mitochondrial uncoupler. Cell Metab. 2013;18:740–748. PubMed PMC

Serviddio G, Bellanti F, Tamborra R, Rollo T, Romano AD, Giudetti AM, Capitanio N, Petrella A, Vendemiale G, Altomare E. Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model. Eur J Clin Invest. 2008;38:245–252. PubMed

Jastroch M, Divakaruni AS, Mookerjee S, Treberg JR, Brand MD. Mitochondrial proton and electron leaks. Essays Biochem. 2010;47:53–67. PubMed PMC

Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev. 2014;94:909–950. PubMed PMC

Zhao RZ, Jiang S, Zhang L, Yu ZB. Mitochondrial electron transport chain, ROS generation and uncoupling (Review) Int J Mol Med. 2019;44:3–15. PubMed PMC

Nassir F, Arndt JJ, Johnson SA, Ibdah JA. Regulation of mitochondrial trifunctional protein modulates nonalcoholic fatty liver disease in mice. J Lipid Res. 2018;59:967–973. PubMed PMC

Kamalian L, Douglas O, Jolly CE, Snoeys J, Simic D, Monshouwer M, Williams DP, Park BK, Chadwick AE. The utility of HepaRG cells for bioenergetic investigation and detection of drug-induced mitochondrial toxicity. Toxicol In Vitro. 2018;53:136–147. PubMed

Porceddu M, Buron N, Rustin P, Fromenty B, Borgne-Sanchez A. In vitro assessment of mitochondrial toxicity to predict drug-induced liver injury. Methods Pharmacol Toxicol. 2018;21:283–300.

Calabrese C, Iommarini L, Kurelac I, Calvaruso MA, Capristo M, Lollini PL, Nanni P, Bergamini C, Nicoletti G, Giovanni CD, et al. Respiratory complex I is essential to induce a Warburg profile in mitochondria-defective tumor cells. Cancer Metab. 2013;1:11. PubMed PMC

Ye JH, Chao J, Chang ML, Peng WH, Cheng HY, Liao JW, Pao LH. Pentoxifylline ameliorates non-alcoholic fatty liver disease in hyperglycaemic and dyslipidaemic mice by upregulating fatty acid β-oxidation. Sci Rep. 2016;6:33102. PubMed PMC

Liemburg-Apers DC, Willems PH, Koopman WJ, Grefte S. Interactions between mitochondrial reactive oxygen species and cellular glucose metabolism. Arch Toxicol. 2015;89:1209–1226. PubMed PMC

Zheng Y, Wang S, Wu J, Wang Y. Mitochondrial metabolic dysfunction and non-alcoholic fatty liver disease: New insights from pathogenic mechanisms to clinically targeted therapy. J Transl Med. 2023;21:510. PubMed PMC

Amorim R, Simões ICM, Teixeira J, Cagide F, Potes Y, Soares P, Carvalho A, Tavares LC, Benfeito S, Pereira SP, et al. Mitochondria-targeted anti-oxidant AntiOxCIN(4) improved liver steatosis in Western diet-fed mice by preventing lipid accumulation due to upregulation of fatty acid oxidation, quality control mechanism and antioxidant defense systems. Redox Biol. 2022;55:102400. PubMed PMC

Chen W, Zhao H, Li Y. Mitochondrial dynamics in health and disease: Mechanisms and potential targets. Signal Transduct Target Ther. 2023;8:333. PubMed PMC

Doczi J, Karnok N, Bui D, Azarov V, Pallag G, Nazarian S, Czumbel B, Seyfried TN, Chinopoulos C. Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics. Sci Rep. 2023;13:10822. PubMed PMC

Engin AB. What is lipotoxicity? Adv Exp Med Biol. 2017;960:197–220. PubMed

Zhang D, Liu ZX, Choi CS, Tian L, Kibbey R, Dong J, Cline GW, Wood PA, Shulman GI. Mitochondrial dysfunction due to long-chain Acyl-CoA dehydrogenase deficiency causes hepatic steatosis and hepatic insulin resistance. Proc Natl Acad Sci USA. 2007;104:17075–17080. PubMed PMC

Collagen I Increases Palmitate-Induced Lipotoxicity in HepG2 Cells via Integrin-Mediated Death