Skip to main content

Advertisement

SpringerLink
Log in

Human hepatic in vitro models reveal distinct anti-NASH potencies of PPAR agonists

  • Original Article
  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

Non-alcoholic steatohepatitis (NASH) is a highly prevalent, chronic liver disease characterized by hepatic lipid accumulation, inflammation, and concomitant fibrosis. Up to date, no anti-NASH drugs have been approved. In this study, we reproduced key NASH characteristics in vitro by exposing primary human hepatocytes (PHH), human skin stem cell-derived hepatic cells (hSKP-HPC), HepaRG and HepG2 cell lines, as well as LX-2 cells to multiple factors that play a role in the onset of NASH. The obtained in vitro disease models showed intracellular lipid accumulation, secretion of inflammatory chemokines, induced ATP content, apoptosis, and increased pro-fibrotic gene expression. These cell systems were then used to evaluate the anti-NASH properties of eight peroxisome proliferator-activated receptor (PPAR) agonists (bezafibrate, elafibranor, fenofibrate, lanifibranor, pemafibrate, pioglitazone, rosiglitazone, and saroglitazar). PPAR agonists differently attenuated lipid accumulation, inflammatory chemokine secretion, and pro-fibrotic gene expression.

Based on the obtained readouts, a scoring system was developed to grade the anti-NASH potencies. The in vitro scoring system, based on a battery of the most performant models, namely PHH, hSKP-HPC, and LX-2 cultures, showed that elafibranor, followed by saroglitazar and pioglitazone, induced the strongest anti-NASH effects. These data corroborate available clinical data and show the relevance of these in vitro models for the preclinical investigation of anti-NASH compounds.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Bedossa P, Poitou C, Veyrie N, Bouillot JL, Basdevant A, Paradis V, et al. Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients. Hepatology. 2012;56:1751–9.

    PubMed  Google Scholar 

  • Bierhaus A, Schiekofer S, Schwaninger M, Andrassy M, Humpert PM, Chen J, et al. Diabetes-associated sustained activation of the transcription factor nuclear factor-κB. Diabetes. 2001;50:2792–808.

    CAS  PubMed  Google Scholar 

  • Boeckmans J, Natale A, Buyl K, Rogiers V, De Kock J, Vanhaecke T, et al. Human-based systems: mechanistic NASH modelling just around the corner? Pharmacol Res. 2018;134:257–67.

    CAS  PubMed  Google Scholar 

  • Boeckmans J, Buyl K, Natale A, Vandenbempt V, Branson S, De Boe V, et al. Elafibranor restricts lipogenic and inflammatory responses in a human skin stem cell-derived model of NASH. Pharmacol Res. 2019;144:377–89.

    CAS  PubMed  Google Scholar 

  • Boeckmans J, Natale A, Rombaut M, Buyl K, Rogiers V, De Kock J, et al. Anti-NASH drug development hitches a lift on PPAR agonism. Cells. 2020;9:1–20.

    Google Scholar 

  • Brunt EM, Kleiner DE, Wilson LA, Belt P, Neuschwander-Tetri BA. The NAS and the histopathologic diagnosis of NAFLD: distinct clinicopathologic meanings. Hepatology. 2011;53:810–20.

    CAS  PubMed  Google Scholar 

  • Buzzetti E, Pinzani M, Tsochatzis EA. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism. 2016;65:1038–48.

    CAS  PubMed  Google Scholar 

  • Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology. 2012;55:2005–23.

    PubMed  Google Scholar 

  • Chi KR. The NASH drug dash. Nat Rev Drug Discov. 2015;14:447–8.

    CAS  PubMed  Google Scholar 

  • Dash A, Figler RA, Blackman BR, Marukian S, Collado MS, Lawson MJ, et al. Pharmacotoxicology of clinically-relevant concentrations of obeticholic acid in an organotypic human hepatocyte system. Toxicol Vitr. 2017;39:93–103.

    CAS  Google Scholar 

  • Dorn C, Engelmann JC, Saugspier M, Koch A, Hartmann A, Müller M, et al. Increased expression of c-Jun in nonalcoholic fatty liver disease. Lab Investig. 2014;94:394–408.

    CAS  PubMed  Google Scholar 

  • Ekstedt M, Hagström H, Nasr P, Fredrikson M, Stål P, Kechagias S, et al. Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up. Hepatology. 2015;61:1547–54.

    CAS  PubMed  Google Scholar 

  • Estes C, Razavi H, Loomba R, Younossi Z, Sanyal AJ. Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease. Hepatology. 2018;67:123–33.

    CAS  PubMed  Google Scholar 

  • Feaver RE, Cole BK, Lawson MJ, Hoang SA, Marukian S, Blackman BR, et al. Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis. J Clin Invest. 2016;1:e90954.

    Google Scholar 

  • Fernández-Miranda C, Pérez-Carreras M, Colina F, López-Alonso G, Vargas C, Solís-Herruzo JA. A pilot trial of fenofibrate for the treatment of non-alcoholic fatty liver disease. Dig Liver Dis. 2008;40:200–5.

    PubMed  Google Scholar 

  • Frades I, Andreasson E, Mato JM, Alexandersson E, Matthiesen R, Martínez-Chantar ML. Integrative genomic signatures of hepatocellular carcinoma derived from nonalcoholic fatty liver disease. PLoS One. 2015;10:e0124544.

    PubMed  PubMed Central  Google Scholar 

  • Francque S, Verrijken A, Caron S, Prawitt J, Paumelle R, Derudas B, et al. PPAR-α gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis. J Hepatol. 2015;63:164–73.

    CAS  PubMed  Google Scholar 

  • Franko A, Neschen S, Rozman J, Rathkolb B, Aichler M, Feuchtinger A, et al. Bezafibrate ameliorates diabetes via reduced steatosis and improved hepatic insulin sensitivity in diabetic TallyHo mice. Mol Metab. 2017;6:256–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat Med. 2018;24:908–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Garbacz WG, Lu P, Miller TM, Poloyac SM, Eyre NS, Mayrhofer G, et al. Hepatic overexpression of CD36 improves glycogen homeostasis and attenuates high-fat diet-induced hepatic steatosis and insulin resistance. Mol Cell Biol. 2016;36:2715–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gross B, Pawlak M, Lefebvre P, Staels B. PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nat Rev Endocrinol. 2017;13:36–49.

    CAS  PubMed  Google Scholar 

  • Haas JT, Vonghia L, Mogilenko DA, Verrijken A, Molendi-Coste O, Fleury S, et al. Transcriptional network analysis implicates altered hepatic immune function in NASH development and resolution. Nat Metab. 2019;1:604–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Han J, Hajjar DP, Tauras JM, Feng J, Gotto AM, Nicholson AC. Transforming growth factor-β1 (TGF-β1) and TGF-β2 decrease expression of CD36, the type B scavenger receptor, through mitogen-activated protein kinase phosphorylation of peroxisome proliferator-activated receptor-γ. J Biol Chem. 2000;275:1241–6.

    CAS  PubMed  Google Scholar 

  • Heikkinen S, Auwerx J, Argmann CA. PPARγ in human and mouse physiology. Biochim Biophys Acta—Mol Cell Biol Lipids. 2007;1771:999–1013.

    CAS  Google Scholar 

  • Holden PR, Tugwood JD. Peroxisome proliferator-activated receptor alpha: role in rodent liver cancer and species differences. J Mol Endocrinol. 1999;22:1–8.

    CAS  PubMed  Google Scholar 

  • Honda Y, Kessoku T, Ogawa Y, Tomeno W, Imajo K, Fujita K, et al. Pemafibrate, a novel selective peroxisome proliferator-activated receptor alpha modulator, improves the pathogenesis in a rodent model of nonalcoholic steatohepatitis. Sci Rep. 2017;7:1–11.

    Google Scholar 

  • Ishibashi S, Arai H, Yokote K, Araki E, Suganami H, Yamashita S. Efficacy and safety of pemafibrate (K-877), a selective peroxisome proliferator-activated receptor α modulator, in patients with dyslipidemia: results from a 24-week, randomized, double blind, active-controlled, phase 3 trial. J Clin Lipidol. 2018;12:173–84.

    PubMed  Google Scholar 

  • Jain MR, Giri SR, Bhoi B, Trivedi C, Rath A, Rathod R, et al. Dual PPARα/γ agonist saroglitazar improves liver histopathology and biochemistry in experimental NASH models. Liver Int. 2018;38:1084–94.

    CAS  PubMed  Google Scholar 

  • Kanmani P, Kim H. Protective effects of lactic acid bacteria against TLR4 induced inflammatory response in hepatoma HepG2 cells through modulation of toll-like receptor negative regulators of mitogen-activated protein kinase and NF-κB signaling. Front Immunol. 2018;9:1537.

    PubMed  PubMed Central  Google Scholar 

  • Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–21.

    PubMed  Google Scholar 

  • Kücükoglu Ö, Labenz C, Sydor S, Schlattjan M, Best J, Gerken G, et al. Free fatty acids enhance CD36 knockdown in primary human hepatocytes and abrogate PTEN expression—recapitulation of NASH-associated HCC. J Hepatol. 2017;66:S464.

    Google Scholar 

  • LeBleu VS, O’Connell JT, Gonzalez Herrera KN, Wikman H, Pantel K, Haigis MC, et al. PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation to promote metastasis. Nat Cell Biol. 2014;16:992–1003.

    CAS  PubMed  PubMed Central  Google Scholar 

  • López-Riera M, Conde I, Tolosa L, Zaragoza Á, Castell JV, Gómez-Lechón MJ, et al. New microRNA biomarkers for drug-induced steatosis and their potential to predict the contribution of drugs to non-alcoholic fatty liver disease. Front Pharmacol. 2017;8:1–12.

    Google Scholar 

  • Mandard S, Müller M, Kersten S. Peroxisome proliferator-activated receptor α target genes. Cell Mol Life Sci. 2004;61:393–416.

    CAS  PubMed  Google Scholar 

  • Mannaerts I, Leite SB, Verhulst S, Claerhout S, Eysackers N, Thoen LFR, et al. The hippo pathway effector YAP controls mouse hepatic stellate cell activation. J Hepatol. 2015;63:679–88.

    CAS  PubMed  Google Scholar 

  • Marchesini G, Day CP, Dufour JF, Canbay A, Nobili V, Ratziu V, et al. EASL-EASD-EASO clinical practice guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016;64:1388–402.

    Google Scholar 

  • Maréchal L, Laviolette M, Rodrigue-Way A, Sow B, Brochu M, Caron V, et al. The CD36-PPARγ pathway in metabolic disorders. Int J Mol Sci. 2018;19:1–16.

    Google Scholar 

  • Marra F, Tacke F. Roles for chemokines in liver disease. Gastroenterology. 2014;147:577–94.

    CAS  PubMed  Google Scholar 

  • Miquilena-Colina ME, Lima-Cabello E, Sánchez-Campos S, García-Mediavilla MV, Fernández-Bermejo M, Lozano-Rodríguez T, et al. Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C. Gut. 2011;60:1394–402.

    CAS  PubMed  Google Scholar 

  • Moylan CA, Pang H, Dellinger A, Suzuki A, Garrett ME, Guy CD, et al. Hepatic gene expression profiles differentiate pre-symptomatic patients with mild versus severe nonalcoholic fatty liver disease. Hepatology. 2014;59:471–82.

    CAS  PubMed  Google Scholar 

  • Ohashi T, Tanabe J, Ishikawa T, Okumura A, Sato K, Ayada M, et al. Inflammatory cytokines modulate chemokine production patterns of HepG2 cells toward initially inclined direction. Hepatol Res. 2009;39:510–9.

    CAS  PubMed  Google Scholar 

  • Ratziu V, Giral P, Jacqueminet S, Charlotte F, Hartemann-Heurtier A, Serfaty L, et al. Rosiglitazone for nonalcoholic steatohepatitis: one-year results of the randomized placebo-controlled fatty liver improvement with rosiglitazone therapy (FLIRT) trial. Gastroenterology. 2008;135:100–10.

    CAS  PubMed  Google Scholar 

  • Ratziu V, Charlotte F, Bernhardt C, Giral P, Halbron M, Lenaour G, et al. Long-term efficacy of rosiglitazone in nonalcoholic steatohepatitis: results of the fatty liver improvement by rosiglitazone therapy (FLIRT 2) extension trial. Hepatology. 2010;51:445–53.

    CAS  PubMed  Google Scholar 

  • Ratziu V, Harrison SA, Francque S, Bedossa P, Lehert P, Serfaty L, et al. Elafibranor, an agonist of the peroxisome proliferator-activated receptor-α and -δ, induces resolution of nonalcoholic steatohepatitis without fibrosis worsening. Gastroenterology. 2016;150:1147–59.

    CAS  PubMed  Google Scholar 

  • Rodrigues RM, De Kock J, Branson S, Vinken M, Meganathan K, Chaudhari U, et al. Human skin-derived stem cells as a novel cell source for in vitro hepatotoxicity screening of pharmaceuticals. Stem Cells Dev. 2014;23:44–55.

    CAS  PubMed  Google Scholar 

  • Rodrigues RM, Branson S, De Boe V, Sachinidis A, Rogiers V, De Kock J, et al. In vitro assessment of drug-induced liver steatosis based on human dermal stem cell-derived hepatic cells. Arch Toxicol. 2016a;90:677–89.

    CAS  PubMed  Google Scholar 

  • Rodrigues RM, Heymans A, De Boe V, Sachinidis A, Chaudhari U, Govaere O, et al. Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems. Toxicol Lett. 2016b;240:50–9.

    CAS  PubMed  Google Scholar 

  • Rogue A, Lambert C, Jossé R, Antherieu S, Spire C, Claude N, et al. Comparative gene expression profiles induced by PPARγ and PPARα/γ agonists in human hepatocytes. PLoS One. 2011;6:e18816.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rogue A, Anthérieu S, Vluggens A, Umbdenstock T, Claude N, De la Moureyre-Spire C, et al. PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells. Toxicol Appl Pharmacol. 2014;276:73–81.

    CAS  PubMed  Google Scholar 

  • Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med. 2010;362:1675–85.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sato O, Kuriki C, Fukui Y, Motojima K. Dual promoter structure of mouse and human fatty acid translocase/CD36 genes and unique transcriptional activation by peroxisome proliferator-activated receptor α and γ ligands. J Biol Chem. 2002;277:15703–11.

    CAS  PubMed  Google Scholar 

  • Scarpulla RC. Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network. Biochim Biophys Acta. 2011;1813:1269–78.

    CAS  PubMed  Google Scholar 

  • Sumida Y, Yoneda M. Current and future pharmacological therapies for NAFLD/NASH. J Gastroenterol. 2018;53:362–76.

    CAS  PubMed  Google Scholar 

  • Tacke F, Zimmermann HW, Trautwein C, Schnabl B. CXCL5 plasma levels are decreased in patients with chronic liver disease. J Gastroenterol Hepatol. 2011;26:523–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tarek I, Tamini A-R, Elgouhari HM, Alkhouri N, Yerian LM, Berk MP, et al. An apoptosis panel for nonalcoholic steatohepatitis diagnosis. J Hepatol. 2011;54:1224–9.

    Google Scholar 

  • Ten RM, Paya CV, Israel N, Le Bail O, Mattei MG, Virelizier JL, et al. The characterization of the promoter of the gene encoding the p50 subunit of NF-κB indicates that it participates in its own regulation. EMBO J. 1992;11:195–203.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Thomas E, Gonzalez VD, Li Q, Modi AA, Chen W, Noureddin M, et al. HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons. Gastroenterology. 2012;142:978–88.

    CAS  PubMed  Google Scholar 

  • Tong L, Wang L, Yao S, Jin L, Yang J, Zhang Y, et al. PPAR δ attenuates hepatic steatosis through autophagy-mediated fatty acid oxidation. Cell Death Dis. 2019;10:1–14.

    Google Scholar 

  • Vaidyanathan S, Maboudian M, Warren V, Yeh C, Dieterich HA, Howard D, et al. A study of the pharmacokinetic interactions of the direct renin inhibitor aliskiren with metformin, pioglitazone and fenofibrate in healthy subjects. Curr Med Res Opin. 2008;24:2313–26.

    CAS  PubMed  Google Scholar 

  • Varga ZV, Ferdinandy P, Liaudet L, Pacher P. Drug-induced mitochondrial dysfunction and cardiotoxicity. Am J Physiol—Heart Circ Physiol. 2015;309:1453–67.

    Google Scholar 

  • Wettstein G, Luccarini J-M, Poekes L, Faye P, Kupkowski F, Adarbes V, et al. The new-generation pan-peroxisome proliferator-activated receptor agonist IVA337 protects the liver from metabolic disorders and fibrosis. Hepatol Commun. 2017;1:524–37.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xu L, Hui AY, Albanis E, Arthur MJ, Blaner WS, Mukherjee P, et al. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut. 2005;54:142–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yan F, Wang Q, Xu C, Cao M, Zhou X, Wang T, et al. Peroxisome proliferator-activated receptor α activation induces hepatic steatosis, suggesting an adverse effect. PLoS One. 2014;9:e99245.

    PubMed  PubMed Central  Google Scholar 

  • Yang L, Roh YS, Song J, Zhang B, Liu C, Loomba R, et al. TGF-β signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism. Hepatology. 2014;59:483–95.

    CAS  PubMed  Google Scholar 

  • Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2018;15:11–20.

    PubMed  Google Scholar 

Download references

Funding

This work was funded by grants of Research Foundation Flanders (1S10518N, 12H2216N, 1S73019N, and G042019N), Onderzoeksraad Vrije Universiteit Brussel, and the Research Chair Mireille Aerens for Alternatives to Animal Testing.

Author information

Author notes

  1. Tamara Vanhaecke and Robim M Rodrigues are equally contributing senior authors.

Authors and Affiliations

  1. Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium

    Joost Boeckmans, Alessandra Natale, Matthias Rombaut, Karolien Buyl, Brent Cami, Anja Heymans, Vera Rogiers, Joery De Kock, Tamara Vanhaecke & Robim M Rodrigues

  2. Department of Urology, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090, Brussels, Belgium

    Veerle De Boe

Authors
  1. Joost Boeckmans
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alessandra Natale
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias Rombaut
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karolien Buyl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Brent Cami
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Veerle De Boe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Anja Heymans
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vera Rogiers
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Joery De Kock
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tamara Vanhaecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Robim M Rodrigues
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conception and design: J.B. and R.M.R; data analysis and interpretation: J.B., J.D.K., and R.M.R.; collection and assembly of data: J.B., A.N., M.R., K.B., B.C., and A.H.; financial support: V.R., T.V., and R.M.R.; manuscript writing: J.B., A.N., M.R., K.B., B.C., A.H., V.R., J.D.K., T.V., and R.M.R.; administrative support: V.R. and T.V.; provision of study material: V.D.B.; project supervision: V.R., T.V., and R.M.R.; final approval of manuscript: J.B., T.V., and R.M.R.

Corresponding author

Correspondence to Robim M Rodrigues.

Ethics declarations

Conflict of interest

The authors have no declarations of interest.

Ethics approval

Ethical approval was obtained from the medical ethical committee of the UZ Brussel (Belgium).

Consent to participate

Informed consent was obtained where applicable.

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 3441 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Boeckmans, J., Natale, A., Rombaut, M. et al. Human hepatic in vitro models reveal distinct anti-NASH potencies of PPAR agonists. Cell Biol Toxicol 37, 293–311 (2021). https://doi.org/10.1007/s10565-020-09544-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10565-020-09544-2

Keywords

Search

Navigation