Effect of palmitate-induced steatosis on paraoxonase-1 and paraoxonase-3 enzymes in human-derived liver (HepG2) cells

Year 2019, Volume: 4 Issue: 3, 118 - 121, 01.12.2019

Gülben Sayılan Özgün Eray Özgün Kıymet Tabakçıoğlu Selma Süer Gökmen Sevgi Eskiocak

https://doi.org/10.25000/acem.623975

Cited By: 1

Abstract

Aim: Palmitate is one of
the most abundant fatty acid in both liver of healthy individuals and in
patients with non-alcoholic fatty liver disease. Palmitate-induced
steatosis in HepG2 cells is an in vitro non-alcoholic fatty
liver disease model to investigate acute harmful effects of fat
overaccumulation in the liver. Non-alcoholic fatty liver disease is strongly
associated with atherosclerosis. Paraoxonase-1 and paraoxonase-3 are
anti-atherosclerotic enzymes which are bound to high density lipoprotein in
circulation and they are primarily synthesized by liver. There is no study that investigated the effect of palmitate-induced
steatosis on paraoxonase-1 and paraoxonase-3 enzymes. The aim of present
study was to investigate the effect of
palmitate-induced steatosis on paraoxonase-1 and paraoxonase-3 enzymes in HepG2 cells.

Methods: To induce steatosis, cells were incubated
with 0.4, 0.7 and 1 mM palmitate for 24 hours. Cell viability was
evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide assay. Cells were stained with oil red O and triglyceride levels
were measured. Paraoxonase-1 and paraoxonase-3 protein levels were
measured by western blotting, their mRNA expression were measured by
quantitative PCR and arylesterase activity was measured spectrophotometrically.

Results: All palmitate
concentrations caused a significant increase on paraoxonase-1 mRNA levels.
Palmitate concentrations did not cause a significant change on paraoxonase-1 and paraoxonase-3 protein levels, paraoxonase-3 mRNA levels and
arylesterase activities.

Conclusion:
Our study showed that palmitate-induced steatosis
up-regulates paraoxonase-1
mRNA, has no effect on paraoxonase-1 and paraoxonase-3
protein levels, paraoxonase-3 mRNA
and arylesterase activity in HepG2 cells.

Keywords

Palmitate, paraoxonase-1, paraoxonase-3, arylesterase, HepG2, non-alcoholic fatty liver disease

Supporting Institution

No funding to declare.

Thanks

None

İnsan kaynaklı karaciğer (HepG2) hücrelerinde palmitat ile oluşturulan yağlanmanın paraoksonaz-1 ve paraoksonaz-3 enzimlerine etkisi

Abstract

Amaç: Palmitat, hem
sağlıklı bireylerin hem de non-alkolik karaciğer yağlanması hastalarının
karaciğerinde en fazla bulunan yağ asitlerinden biridir. HepG2 hücrelerinde
palmitat ile oluşturulan yağlanma, karaciğerdeki yağ birikiminin akut zararlı
etkilerinin araştırılmasında kullanılan in vitro non-alkolik yağlı karaciğer
hastalığı modelidir. Non-alkolik yağlı karaciğer hastalığı ateroskleroz ile
yakından ilişkilidir. Paraoksonaz-1 ve paraoksonaz-3 dolaşımda yüksek dansiteli
lipoproteine bağlı anti-aterosklerotik enzimlerdir ve esas olarak karaciğerde
sentezlenirler. Palmitat ile oluşturulan yağlanmanın paraoksonaz-1 ve
paraoksonaz-3 enzimleri üzerine etkisini araştıran bir çalışma bulunmamaktadır.
Bu çalışmanın amacı HepG2
hücrelerinde palmitat ile oluşturulan yağlanmanın paraoksonaz-1 ve
paraoksonaz-3 enzimlerine etkisini araştırmaktır.

Yöntemler: Yağlanma oluşturmak için hücreler 0.4, 0.7 ve
1 mM palmitat ile 24 saat inkübe edildi. Hücre canlılığı 3-(4,5-Dimetil-2-tiazolil)-2,5-difenil-2H-tetrazolium
bromür testi ile değerlendirildi. Hücreler oil red O ile boyandı ve trigliserit
düzeyleri ölçüldü. Paraoksonaz-1 ve paraoksonaz-3 protein düzeyleri western
blot ile, mRNA’ları ise kantitatif PCR ile ve arilesteraz aktivitesi
spektrofotometrik olarak ölçüldü.

Bulgular: Tüm palmitat konsantrasyonları
paraoksonaz-1 mRNA düzeylerinde anlamlı bir artışa yol açtı. Palmitat
konsantrasyonları paraoksonaz-1 ve paraoksonaz-3 protein düzeylerinde,
paraoksonaz-3 mRNA düzeylerinde ve arilesteraz aktivitesinde anlamlı bir
değişime yol açmadı.

Sonuç: Çalışmamız,
HepG2 hücrelerinde palmitat ile oluşturulan yağlanmanın paraoksonaz-1 mRNA
düzeyini arttırdığını, paraoksonaz-1 ve paraoksonaz-3
protein düzeylerine, paraoksonaz-3 mRNA düzeylerine ve arilesteraz aktivitesine
etkisi olmadığını gösterdi.

Keywords

Palmitat, paraoksonaz-1, paraoksonaz-3, arilesteraz, HepG2, non-alkolik yağlı karaciğer hastalığı

References

• 1. 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.
• 2. 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.
• 3. DeFilippis AP, Blaha MJ, Martin SS, Reed RM, Jones SR, Nasir K, et al. Nonalcoholic fatty liver disease and serum lipoproteins: the Multi-Ethnic Study of Atherosclerosis. Atherosclerosis. 2013;227:429-36.
• 4. Byrne CD, Targher G. NAFLD: a multisystem disease. J Hepatol. 2015; 62: 47-64.
• 5. Sookoian S, Pirola CJ. Non-alcoholic fatty liver disease is strongly associated with carotid atherosclerosis: a systematic review. J Hepatol. 2008;49:600-07.
• 6. Araya J, Rodrigo R, Videla LA, Thielemann L, Orellana M, Pettinelli, P et al. Increase in long-chain polyunsaturated fatty acid n - 6/n - 3 ratio in relation to hepatic steatosis in patients with non-alcoholic fatty liver disease. Clin Sci (Lond). 2004;106:635-43.
• 7. Bouma ME, Rogier E, Verthier N, Labarre C, Feldmann G. Further cellular investigation of the human hepatoblastoma-derived cell line HepG2: morphology and immunocytochemical studies of hepatic-secreted proteins. In Vitro Cell Dev Biol. 1989;25:267-75.
• 8. Gómez-Lechón MJ, Donato MT, Martínez-Romero A, Jiménez N, Castell JV, O'Connor JE. A human hepatocellular in vitro model to investigate steatosis. Chem Biol Interact. 2007;165:106-16.
• 9. She ZG, Chen HZ, Yan Y, Li H, Liu DP. The human paraoxonase gene cluster as a target in the treatment of atherosclerosis. Antioxid Redox Signal. 2012;16:597-632.
• 10. Podrez EA. Anti-oxidant properties of high-density lipoprotein and atherosclerosis. Clin Exp Pharmacol Physiol. 2010;37:719-25.
• 11. Précourt LP, Amre D, Denis MC, Lavoie JC, Delvin E, Seidman E, et al. The three-gene paraoxonase family: physiologic roles, actions and regulation. Atherosclerosis. 2011;214:20-36.
• 12. 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.
• 13. Pereira RR, de Abreu IC, Guerra, JF, Lage NN, Lopes JM, Silva M, et al. Açai (Euterpe oleracea Mart.) Upregulates Paraoxonase 1 Gene Expression and Activity with Concomitant Reduction of Hepatic Steatosis in High-Fat Diet-Fed Rats. Oxid Med Cell Longev. 2016;2016:8379105.
• 14. Desai S, Bake SS, Liu W, Moya DA, Browne RW, Mastrandrea L, et al. Paraoxonase 1 and oxidative stress in paediatric non-alcoholic steatohepatitis. Liver Int. 2014;34:110-17.
• 15. 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;15:387-95.
• 16. Yang X, Chan C. Repression of PKR mediates palmitate-induced apoptosis in HepG2 cells through regulation of Bcl-2. Cell Res. 2009;19:469-86.
• 17. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55-63.
• 18. Ahmadian S, Barar J, Saei AA, Fakhree, MA Omidi Y. Cellular toxicity of nanogenomedicine in MCF-7 cell line: MTT assay. J Vis Exp. 2009;26:1191.
• 19. Jang E, Shin MH, Kim KS, Kim Y, Na YC, Woo HJ, et al. Anti-lipoapoptotic effect of Artemisia capillaris extract on free fatty acids-induced HepG2 cells. BMC Complement Altern Med. 2014;14:253.
• 20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-75.
• 21. 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;25:402-8.
• 22. Beltowski J, Jamroz-Wiśniewska A, Borkowska E, Wójcicka G. Differential effect of antioxidant treatment on plasma and tissue paraoxonase activity in hyperleptinemic rats. Pharmacol Res. 2005;51:523-32.
• 23. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671-75.
• 24. Ozgun E, Sayilan Ozgun G, Tabakcioglu K, Suer Gokmen S, Sut N, Eskiocak S. Effect of lipoic acid on paraoxonase-1 and paraoxonase-3 protein levels, mRNA expression and arylesterase activity in liver hepatoma cells. Gen Physiol Biophys. 2017;36:465-70.
• 25. Gan KN, Smolen A, Eckerson HW, La Du BN. Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab Dispos. 1991;19:100-6.
• 26. Wang GL, Fu YC, Xu WC, Feng YQ, Fang SR, Zhou XH. Resveratrol inhibits the expression of SREBP1 in cell model of steatosis via Sirt1-FOXO1 signaling pathway. Biochem Biophys Res Commun. 2009;380:644-49.
• 27. Gorgani-Firuzjaee S, Adeli K, Meshkani R. Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells. Biochem Biophys Res Commun. 2015;464:441-46.
• 28. 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;25:588-96.
• 29. Ma S, Yang D, Li D, Tan Y, Tang B, Yang Y. Inhibition of uncoupling protein 2 with genipin exacerbates palmitate-induced hepatic steatosis. Lipids Health Dis. 2012;11:154.
• 30. Choi YJ, Choi SE, Ha ES, Kang Y, Han SJ, Kim DJ, et al. Involvement of visfatin in palmitate-induced upregulation of inflammatory cytokines in hepatocytes. Metabolism. 2011;60:1781-89.
• 31. Joshi-Barve S, Barve SS, Amancherla K, Gobejishvili L, Hill D, Cave M, et al. Palmitic acid induces production of proinflammatory cytokine interleukin-8 from hepatocytes. Hepatology. 2007;46:823-30.
• 32. Kudchodkar BJ, Lacko AG, Dory L, Fungwe TV. Dietary fat modulates serum paraoxonase 1 activity in rats. J Nutr. 2000;130:2427-33.
• 33. Boshtam M, Razavi AE, Pourfarzam M, Ani M, Naderi GA, Basati G, et al. Serum paraoxonase 1 activity is associated with fatty acid composition of high density lipoprotein. Dis Markers. 2013;35:273-80.
• 34. Perry BD, Rahnert JA, Xie Y, Zheng B, Woodworth-Hobbs ME, Price SR. Palmitate induced ER stress and inhibition of protein synthesis in cultured myotubes does not require Toll-like receptor 4. PLoS One. 2018;13:e0191313.
• 35. Reddy ST, Wadleigh DJ, Grijalva V, Ng C, Hama S, Gangopadhyay A. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids. Arterioscler Thromb Vasc Biol. 2001;21:542-47.
• 36. Sayılan Özgün G, Özgün E, Tabakçıoğlu K, Süer Gökmen S, Eskiocak S, Çakır E. Caffeine Increases Apolipoprotein A-1 and Paraoxonase-1 but not Paraoxonase-3 Protein Levels in Human-Derived Liver (HepG2) Cells. Balkan Med J. 2017;34:534-39.
• 37. Draganov DI, Stetson PL, Watson CE, Billecke SS, La Du BN. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation. J Biol Chem. 2000;275:33435-42.