Hyperglycemia induces key genetic and phenotypic changes in human liver epithelial HepG2 cells which parallel the Leprdb/J mouse model of non-alcoholic fatty liver disease (NAFLD)


Autoři: Robin C. Su aff001;  Apurva Lad aff001;  Joshua D. Breidenbach aff001;  Thomas M. Blomquist aff002;  William T. Gunning aff002;  Prabhatchandra Dube aff001;  Andrew L. Kleinhenz aff001;  Deepak Malhotra aff001;  Steven T. Haller aff001;  David J. Kennedy aff001
Působiště autorů: Department of Medicine, The University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, United States of America aff001;  Department of Pathology, The University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, United States of America aff002;  Department of Medical Microbiology and Immunology, The University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0225604

Souhrn

Non-alcoholic fatty liver disease (NAFLD) is a growing global health concern. With a propensity to progress towards non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma, NAFLD is an important link amongst a multitude of comorbidities including obesity, diabetes, and cardiovascular and kidney disease. As several in vivo models of hyperglycemia and NAFLD are employed to investigate the pathophysiology of this disease process, we aimed to characterize an in vitro model of hyperglycemia that was amenable to address molecular mechanisms and therapeutic targets at the cellular level. Utilizing hyperglycemic cell culturing conditions, we induced steatosis within a human hepatocyte cell line (HepG2 cells), as confirmed by electron microscopy. The deposition and accumulation of lipids within hyperglycemic HepG2 cells is significantly greater than in normoglycemic cells, as visualized and quantified by Nile red staining. Alanine aminotransferase (ALT) and alkaline phosphatase (ALP), diagnostic biomarkers for liver damage and disease, were found to be upregulated in hyperglycemic HepG2 cells as compared with normoglycemic cells. Suppression of CEACAM1, GLUT2, and PON1, and elevation of CD36, PCK1, and G6PK were also found to be characteristic in hyperglycemic HepG2 cells compared with normoglycemic cells, suggesting insulin resistance and NAFLD. These in vitro findings mirror the characteristic genetic and phenotypic profile seen in Leprdb/J mice, a well-established in vivo model of NAFLD. In conclusion, we characterize an in vitro model displaying several key genetic and phenotypic characteristics in common with NAFLD that may assist future studies in addressing the molecular mechanisms and therapeutic targets to combat this disease.

Klíčová slova:

Cell staining – Fatty liver – Fluorescence imaging – Gene expression – Lipids – Mouse models – MTT assay


Zdroje

1. Everhart JE, Ruhl CE. Burden of digestive diseases in the United States Part III: Liver, biliary tract, and pancreas. Gastroenterology. 2009;136(4):1134–44. doi: 10.1053/j.gastro.2009.02.038 19245868

2. Grasselli E, Canesi L, Portincasa P, Voci A, Vergani L, Demori I. Models of non-Alcoholic Fatty Liver Disease and Potential Translational Value: the Effects of 3,5-L-diiodothyronine. Ann Hepatol. 2017;16(5):707–19.

3. 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(1):73–84. doi: 10.1002/hep.28431 26707365

4. Kennedy DJ, Khalaf FK, Sheehy B, Weber ME, Agatisa-Boyle B, Conic J, et al. Telocinobufagin, a Novel Cardiotonic Steroid, Promotes Renal Fibrosis via Na(+)/K(+)-ATPase Profibrotic Signaling Pathways. Int J Mol Sci. 2018;19(9).

5. Kaja S, Payne AJ, Naumchuk Y, Koulen P. Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes. Curr Protoc Toxicol. 2017;72:2 26 1–2 10.

6. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63. doi: 10.1016/0022-1759(83)90303-4 6606682

7. Mikolasevic I, Milic S, Turk Wensveen T, Grgic I, Jakopcic I, Stimac D, et al. Nonalcoholic fatty liver disease—A multisystem disease? World J Gastroenterol. 2016;22(43):9488–505. doi: 10.3748/wjg.v22.i43.9488 27920470

8. Amiri Dash Atan N, Koushki M, Motedayen M, Dousti M, Sayehmiri F, Vafaee R, et al. Type 2 diabetes mellitus and non-alcoholic fatty liver disease: a systematic review and meta-analysis. Gastroenterol Hepatol Bed Bench. 2017;10(Suppl1):S1–S7. 29511464

9. Brunt EM, Tiniakos DG. Histopathology of nonalcoholic fatty liver disease. World J Gastroenterol. 2010;16(42):5286–96. doi: 10.3748/wjg.v16.i42.5286 21072891

10. Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr., Ory DS, et al. Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci U S A. 2003;100(6):3077–82. doi: 10.1073/pnas.0630588100 12629214

11. Giannini EG, Testa R, Savarino V. Liver enzyme alteration: a guide for clinicians. CMAJ. 2005;172(3):367–79. doi: 10.1503/cmaj.1040752 15684121

12. Oh RC, Hustead TR, Ali SM, Pantsari MW. Mildly Elevated Liver Transaminase Levels: Causes and Evaluation. Am Fam Physician. 2017;96(11):709–15. 29431403

13. Wilson CG, Tran JL, Erion DM, Vera NB, Febbraio M, Weiss EJ. Hepatocyte-Specific Disruption of CD36 Attenuates Fatty Liver and Improves Insulin Sensitivity in HFD-Fed Mice. Endocrinology. 2016;157(2):570–85. doi: 10.1210/en.2015-1866 26650570

14. Wang B, Yang RN, Zhu YR, Xing JC, Lou XW, He YJ, et al. Involvement of xanthine oxidase and paraoxonase 1 in the process of oxidative stress in nonalcoholic fatty liver disease. Mol Med Rep. 2017;15(1):387–95. doi: 10.3892/mmr.2016.6025 27959408

15. Fedelesova M, Kupcova V, Luha J, Turecky L. Paraoxonase activity in sera of patients with non-alcoholic fatty liver disease. Bratisl Lek Listy. 2017;118(12):719–20. doi: 10.4149/BLL_2017_134a 29322801

16. Garcia-Heredia A, Kensicki E, Mohney RP, Rull A, Triguero I, Marsillach J, et al. Paraoxonase-1 deficiency is associated with severe liver steatosis in mice fed a high-fat high-cholesterol diet: a metabolomic approach. J Proteome Res. 2013;12(4):1946–55. doi: 10.1021/pr400050u 23448543

17. Rao PK, Merath K, Drigalenko E, Jadhav AYL, Komorowski RA, Goldblatt MI, et al. Proteomic characterization of high-density lipoprotein particles in patients with non-alcoholic fatty liver disease. Clin Proteomics. 2018;15:10. doi: 10.1186/s12014-018-9186-0 29527140

18. Hussein O, Zidan J, Abu Jabal K, Shams I, Szvalb S, Grozovski M, et al. Paraoxonase activity and expression is modulated by therapeutics in experimental rat nonalcoholic Fatty liver disease. Int J Hepatol. 2012;2012:265305. doi: 10.1155/2012/265305 22536512

19. Al-Share QY, DeAngelis AM, Lester SG, Bowman TA, Ramakrishnan SK, Abdallah SL, et al. Forced Hepatic Overexpression of CEACAM1 Curtails Diet-Induced Insulin Resistance. Diabetes. 2015;64(8):2780–90. doi: 10.2337/db14-1772 25972571

20. Warrier M, Hinds TD Jr., Ledford KJ, Cash HA, Patel PR, Bowman TA, et al. Susceptibility to diet-induced hepatic steatosis and glucocorticoid resistance in FK506-binding protein 52-deficient mice. Endocrinology. 2010;151(7):3225–36. doi: 10.1210/en.2009-1158 20427484

21. Heinrich G, Ghadieh HE, Ghanem SS, Muturi HT, Rezaei K, Al-Share QY, et al. Loss of Hepatic CEACAM1: A Unifying Mechanism Linking Insulin Resistance to Obesity and Non-Alcoholic Fatty Liver Disease. Front Endocrinol (Lausanne). 2017;8:8.

22. Gomez-Valades AG, Mendez-Lucas A, Vidal-Alabro A, Blasco FX, Chillon M, Bartrons R, et al. Pck1 gene silencing in the liver improves glycemia control, insulin sensitivity, and dyslipidemia in db/db mice. Diabetes. 2008;57(8):2199–210. doi: 10.2337/db07-1087 18443203

23. Marzban L, Rahimian R, Brownsey RW, McNeill JH. Mechanisms by which bis(maltolato)oxovanadium(IV) normalizes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression in streptozotocin-diabetic rats in vivo. Endocrinology. 2002;143(12):4636–45. doi: 10.1210/en.2002-220732 12446591

24. Zhang F, Xu X, Zhang Y, Zhou B, He Z, Zhai Q. Gene expression profile analysis of type 2 diabetic mouse liver. PLoS One. 2013;8(3):e57766. doi: 10.1371/journal.pone.0057766 23469233

25. Li X, Li J, Lu X, Ma H, Shi H, Li H, et al. Treatment with PPARdelta agonist alleviates non-alcoholic fatty liver disease by modulating glucose and fatty acid metabolic enzymes in a rat model. Int J Mol Med. 2015;36(3):767–75. doi: 10.3892/ijmm.2015.2270 26133486

26. Lau JK, Zhang X, Yu J. Animal models of non-alcoholic fatty liver disease: current perspectives and recent advances. J Pathol. 2017;241(1):36–44. doi: 10.1002/path.4829 27757953

27. Liu XL, Ming YN, Zhang JY, Chen XY, Zeng MD, Mao YM. Gene-metabolite network analysis in different nonalcoholic fatty liver disease phenotypes. Exp Mol Med. 2017;49(1):e283. doi: 10.1038/emm.2016.123 28082742

28. Chiappini F, Coilly A, Kadar H, Gual P, Tran A, Desterke C, et al. Metabolism dysregulation induces a specific lipid signature of nonalcoholic steatohepatitis in patients. Sci Rep. 2017;7:46658. doi: 10.1038/srep46658 28436449

29. Kohjima M, Higuchi N, Kato M, Kotoh K, Yoshimoto T, Fujino T, et al. SREBP-1c, regulated by the insulin and AMPK signaling pathways, plays a role in nonalcoholic fatty liver disease. Int J Mol Med. 2008;21(4):507–11. 18360697

30. Wu J, Wang C, Li S, Li S, Wang W, Li J, et al. Thyroid hormone-responsive SPOT 14 homolog promotes hepatic lipogenesis, and its expression is regulated by liver X receptor alpha through a sterol regulatory element-binding protein 1c-dependent mechanism in mice. Hepatology. 2013;58(2):617–28. doi: 10.1002/hep.26272 23348573

31. Fisher CD, Lickteig AJ, Augustine LM, Ranger-Moore J, Jackson JP, Ferguson SS, et al. Hepatic cytochrome P450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease. Drug Metab Dispos. 2009;37(10):2087–94. doi: 10.1124/dmd.109.027466 19651758

32. Sookoian S, Castano G, Gianotti TF, Gemma C, Pirola CJ. Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease. J Nutr Biochem. 2009;20(10):765–70. doi: 10.1016/j.jnutbio.2008.07.005 18926681

33. Liu JF, Ma Y, Wang Y, Du ZY, Shen JK, Peng HL. Reduction of lipid accumulation in HepG2 cells by luteolin is associated with activation of AMPK and mitigation of oxidative stress. Phytother Res. 2011;25(4):588–96. doi: 10.1002/ptr.3305 20925133

34. Ashraf U. Nissar LS, Tasdug Sheikh A. Palmitic acid induced lipotoxicity is associated with altered lipid metabolism, enhanced CYP450 2E1 and intracellular calcium mediated ER stress in human hepatoma cells Toxicology Research. 2015(5).

35. Huang Y, Liu J, Xu Y, Dai Z, Alves MH. Reduction of insulin resistance in HepG2 cells by knockdown of LITAF expression in human THP-1 macrophages. J Huazhong Univ Sci Technolog Med Sci. 2012;32(1):53–8. doi: 10.1007/s11596-012-0009-7 22282245

36. Gremlich S, Roduit R, Thorens B. Dexamethasone induces posttranslational degradation of GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with the effects of fatty acids. J Biol Chem. 1997;272(6):3216–22. doi: 10.1074/jbc.272.6.3216 9013557

37. Westerink WM, Schoonen WG. Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol In Vitro. 2007;21(8):1581–91. doi: 10.1016/j.tiv.2007.05.014 17637504


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2019 Číslo 12