Skip to main content

Advertisement

SpringerLink
Log in

An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

DMEM/F12 nutrient mixture, a recommended media for ARPE-19 culture, contains glucose concentration of 17.5 mM. But, several recent studies employed normal glucose media (5.5 mM) that was shown to affect the growth and function of ARPE-19 cells. Here, we set a protocol to study the effect of hyperglycemia on intracellular oxidative stress and redox status in ARPE-19 using DMEM/F12 as control. The WST-1 assay was performed to analyze the viability of ARPE-19 upon glucose treatment. The intracellular oxidative stress was measured by a dichlorofluorescein assay. The mitochondrial membrane potential (MMP) was monitored by using a JC-10 MMP assay kit. The expression of antioxidant marker proteins was analyzed by western blotting. Exogenous addition of glucose (7.5 and 12.5 mM) for 24 and 48 h did not change the viability and morphology of ARPE-19 cells. Hyperglycemia increased intracellular ROS level and decreased MMP in a dose-dependent manner. High-glucose treatment for 24 h down-regulated the protein expression of redox-specific transcription factors Nrf-2, XBP-1 and NF-κB, and subsequently decreased the expression of HO-1, catalase, and SOD-2. This study offers baseline information for the subsequent use of DMEM/F12 nutrient mixture to study glucose-mediated changes in intracellular oxidative stress and redox status of ARPE-19 without affecting its basic functions.

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

Similar content being viewed by others

Abbreviations

ARE:

Antioxidant response element

ARPE-19:

Adult retinal pigment epithelial cell line-19

ATCC:

American type culture collection

CCCP:

Carbonyl cyanide m-chlorophenyl hydrazine

CRALBP:

Cellular retinaldehyde-binding protein

DCF:

Dichlorofluorescein

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

DMEM/F12:

Dulbecco’s modified eagle medium nutrient mixture F-12

ER:

Endoplasmic reticulum

FBS:

Fetal bovine serum

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HO-1:

Heme oxygenase 1

H2O2 :

Hydrogen peroxide

MMP:

Mitochondrial membrane potential

NFκB:

Nuclear factor kappa B

Nrf2:

Nuclear factor erythroid-2 related factor 2

POS:

Photoreceptor outer segment

ROS:

Reactive oxygen species

RPE:

Retinal pigmental epithelium

RPE-65:

Retinal pigment epithelium-specific 65 kDa protein

SOD-2/MnSOD:

Superoxide dismutase 2/manganese-dependent superoxide dismutase

WST-1:

Water-soluble tetrazolium salt-1

XBP-1:

X-box binding protein

ZO-1:

Zonula occludens-1

References

  1. Hamann S (2002) Molecular mechanisms of water transport in the eye. Int Rev Cytol 215:395–431

    Article  CAS  PubMed  Google Scholar 

  2. Simo R, Villarroel M, Corraliza L, Hernandez C, Garcia-Ramirez M (2010) The retinal pigment epithelium: Something more than a constituent of the blood-retinal barrier-implications for the pathogenesis of diabetic retinopathy. J Biomed Biotechnol 2010:190724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Davidson AE, Millar ID, Urquhart JE, Burgess-Mullan R, Shweikh Y, Parry N, O’Sullivan J, Maher GJ, McKibbin M, Downes SM, Lotery AJ (2009) Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa. Am J Hum Genet 85:581–592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bianchi E, Scarinci F, Ripandelli G, Feher J, Pacella E, Magliulo G, Gabrieli CB, Plateroti R, Plateroti P, Mignini F, Artico M (2013) Retinal pigment epithelium, age-related macular degeneration and neurotrophic keratouveitis. Int J Mol Med 31:232–242

    Article  PubMed  Google Scholar 

  5. Dunn KC, Aotaki-keen AE, Putkey FR, Hjelmeland LM (1996) ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62:155–170

    Article  CAS  PubMed  Google Scholar 

  6. Kannan R, Zhang N, Sreekumar PG, Spee CK, Rodriguez A, Barron E, Hinton DR (2006) Stimulation of apical and basolateral VEGF-A and VEGF-C secretion by oxidative stress in polarized retinal pigment epithelial cells. Mol Vis 12:1649–1659

    CAS  PubMed  Google Scholar 

  7. Maminishkis A, Chen S, Jalickee S, Banzon T, Shi G, Wang FE, Ehalt T, Hammer JA, Miller SS (2006) Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci 47:3612–3624

    Article  PubMed  PubMed Central  Google Scholar 

  8. Mao Y, Finnemann SC (2012) Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture. Humana Press, Totowa, pp 285–295

    Google Scholar 

  9. Azad MB, Chen Y, Gibson SB (2009) Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid Redox Signal 11:777–790

    Article  CAS  PubMed  Google Scholar 

  10. Fragoso MA, Patel AK, Nakamura REI, Yi H, Surapaneni K, Hackam AS (2012) The Wnt/β-catenin pathway cross-talks with STAT3 signaling to regulate survival of retinal pigment epithelium cells. PLoS ONE 7:e46892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhong Y, Li J, Wang JJ, Chen C, Tran JTA, Saadi A, Yu Q, Le YZ, Mandal MNA, Anderson RE, Zhang SX (2012) X-Box binding protein 1 is essential for the anti-oxidant defense and cell survival in the retinal pigment epithelium. PLoS ONE 7:e38616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sheu SJ, Liu NC, Ou CC, Bee YS, Chen SC, Lin HC, Chan JY (2013) Resveratrol stimulates mitochondrial bioenergetics to protect retinal pigment epithelial cells from oxidative damage. Invest Ophthalmol Vis Sci 54:6426–6438

    Article  CAS  PubMed  Google Scholar 

  13. Kowluru RA, Mishra M (2015) Oxidative stress, mitochondrial damage and diabetic retinopathy. Biochim Biophys Acta 1852:2474–2483

    Article  CAS  PubMed  Google Scholar 

  14. Kensler TW, Wakabayashi N, Biswal S (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 47:89–116

    Article  CAS  PubMed  Google Scholar 

  15. Lewis KN, Mele J, Hayes JD, Buffenstein R (2010) Nrf2, a guardian of healthspan and gatekeeper of species longevity. Integr Comp Biol 50:829–843

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bai Y, Cui W, Xin Y, Miao X, Barati MT, Zhang C, Chen Q, Tan Y, Cui T, Zheng Y, Cai L (2013) Prevention by sulforaphane of diabetic cardiomyopathy is associated with up-regulation of Nrf2 expression and transcription activation. J Mol Cell Cardiol 57:82–95

    Article  CAS  PubMed  Google Scholar 

  17. Siewert S, González I, Santillán L, Lucero R, Ojeda MS, Gimenez MS (2013) Downregulation of Nrf2 and HO-1 expression contributes to oxidative stress in type 2 diabetes mellitus: a study in Juana Koslay City, San Luis, Argentina. J Diabetes Mellit 3:71–78

    Article  CAS  Google Scholar 

  18. Luo Y, Zhuo Y, Fukuhara M, Rizzolo LJ (2006) Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line. Invest Ophthalmol Vis Sci 47:3644–3655

    Article  PubMed  Google Scholar 

  19. Heimsath EG, Unda R, Vidro E, Muniz A, Villazana-Espinoza ET, Tsin A (2006) ARPE-19 cell growth and cell functions in euglycemic culture media. Curr Eye Res 31:1073–1080

    Article  CAS  PubMed  Google Scholar 

  20. Nicholls DG (2004) Mitochondrial membrane potential and aging. Aging Cell 3:35–40

    Article  CAS  PubMed  Google Scholar 

  21. Obuobi S, Karatayev S, Chai CLL, Ee PLR, Ma´tyus P (2016) The role of modulation of antioxidant enzyme systems in the treatment of neurodegenerative diseases. J Enzyme Inhib Med Chem 31:194–204

    Article  CAS  PubMed  Google Scholar 

  22. Jiménez-Osorio AS, Picazo A, González-Reyes S, Barrera-Oviedo D, Rodríguez-Arellano ME, Pedraza-Chaverri J (2014) Nrf2 and redox status in prediabetic and diabetic patients. Int J Mol Sci 15:20290–20305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhou LZH, Johnson AP, Rando TA (2001) NFκB and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells. Free Radic Biol Med 31:1405–1416

    Article  CAS  PubMed  Google Scholar 

  24. Liu Y, Adachi M, Zhao S, Hareyama M, Koong AC, Luo D, Rando PA, Imai K, Shinomura Y (2009) Preventing oxidative stress: a new role for XBP1. Cell Death Differ 16:847–857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gutiérrez-Uzquiza Á, Arechederra M, Bragado P, Aguirre-Ghiso JA, Porras A (2012) p38α mediates cell survival in response to oxidative stress via induction of antioxidant genes: effect on the p70S6K pathway. J Biol Chem 287:2632–2642

    Article  CAS  PubMed  Google Scholar 

  26. Beatty S, Koh H-H, Phil M, Henson D, Boulton M (2000) The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol 45:115–134

    Article  CAS  PubMed  Google Scholar 

  27. Shen J, Yang X, Dong A, Petters RM, Peng YW, Wong F, Campochiaro PA (2005) Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa. J Cell Physiol 203:457–464

    Article  CAS  PubMed  Google Scholar 

  28. Kowluru RA, Chan P-S (2007) Oxidative stress and diabetic retinopathy. Exp Diabetes Res 2007:1–12

    Google Scholar 

  29. Ye ZW, Zhang J, Townsend DM, Tew KD (2015) Oxidative stress, redox regulation and diseases of cellular differentiation. Biochim Biophys Acta 1850:1607–1621

    Article  CAS  PubMed  Google Scholar 

  30. Samuels IS, Cutler AH, Tarchick MJ, Anand-Apte B (2016) The contribution of RPE-specific insulin signaling to the development of outer retina dysfunction associated with diabetes. Invest Ophthalmol Vis Sci 57:5422

    Google Scholar 

  31. Ahmado A, Carr AJ, Vugler AA, Semo M, Gias C, Lawrence JM, Chen LL, Chen FK, Turowski P, da Cruz L, Coffey PJ (2011) Induction of differentiation by pyruvate and DMEM in the human retinal pigment epithelium cell line ARPE-19. Invest Ophthalmol Vis Sci 52:7148–7159

    Article  CAS  PubMed  Google Scholar 

  32. Fronk AH, Vargis E (2016) Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations. J Tissue Eng 7:1–23

    Article  CAS  Google Scholar 

  33. Samuel W, Jaworski C, Postnikova OA, Kutty RK, Duncan T, Tan LX, Poliakov E, Lakkaraju A, Redmond TM (2017) Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells. Mol Vis 23:60–89

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Xie P, Fujii I, Zhao J, Shinohara M, Matsukura M (2012) A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro. Biol Pharm Bull 35:1447–1453

    Article  CAS  PubMed  Google Scholar 

  35. Arnal E, Johnsen-Soriano S, Lopez-Malo D, Perez-Pastor G, Vidal-Gil L, Morillas N, Sancho-Pelluz J, Romero FJ, Barcia JM (2016) Docosahexaenoic acid protects against high glucose-induced oxidative stress in human retinal pigment epithelial cells. ROS 2:298–307

    Google Scholar 

  36. Fu D, Tian X (2017) Effect of astaxanthin on retinal pigment epithelial cells in high glucose: an in-vitro study. Biomed Res 28:6839–6843

    CAS  Google Scholar 

  37. Yang H, Jin X, Kei Lam CW, Yan S-K (2011) Oxidative stress and diabetes mellitus. Clin Chem Lab Med 49:1773–1782

    CAS  PubMed  Google Scholar 

  38. Wu Y, Tang L, Chen B (2014) Oxidative stress: implications for the development of diabetic retinopathy and antioxidant therapeutic perspectives. Oxid Med Cell Longev 2014:1–12

    Article  CAS  Google Scholar 

  39. Chen JY, Chou HC, Chen YH, Chan HL (2013) High glucose-induced proteome alterations in hepatocytes and its possible relevance to diabetic liver disease. J Nutr Biochem 24:1889–1910

    Article  CAS  PubMed  Google Scholar 

  40. Zhang W, Song J, Zhang Y, Ma Y, Yang J, He G, Chen S (2018) Intermittent high glucose-induced oxidative stress modulates retinal pigmented epithelial cell autophagy and promotes cell survival via increased HMGB. BMC Opthalmol 18:192

    Article  CAS  Google Scholar 

  41. Fardoonian M, Halbach C, Slinger C, Pattnaik BR, Sorenson CM, Sheibani N (2016) High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression. Am J Physiol Cell Physiol 311:C418–C436

    Article  Google Scholar 

  42. Schieber M, Chandel NS (2014) ROS function in redox signaling and oxidative stress. Current Biol 24:R453–R462

    Article  CAS  Google Scholar 

  43. Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S, Yamamoto M (2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 275:16023–16029

    Article  CAS  PubMed  Google Scholar 

  44. Kowluru RA, Kowluru V, Xiong Y, Ho YS (2006) Overexpression of mitochondrial superoxide dismutase in mice protects the retina from diabetes-induced oxidative stress. Free Radic Biol Med 41:1191–2006

    Article  CAS  PubMed  Google Scholar 

  45. Góth L (2008) Catalase deficiency and type 2 diabetes. Diabetes Care 31:e93

    Article  CAS  PubMed  Google Scholar 

  46. Farnoodian M, Halbach C, Slinger C, Pattnaik BR, Sorenson CM, Sheibani N (2016) High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression. Am J Physiol Cell Physiol 311:418–436

    Article  Google Scholar 

  47. Wang J, Fields J, Zhao C, Langer J, Thimmulappa RK, Kensler TW, Yamamoto M, Biswal S, Dore S (2007) Role of Nrf2 in protection against intracerebral hemorrhage injury in mice. Free Radic Biol Med 43:408–414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Stefanson AL, Bakovic M (2014) Dietary regulation of Keap1/Nrf2/ARE pathway: focus on plant-derived compounds and trace minerals. Nutrients 6:3777–3801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang SH, Lee WC, Chou HC (2015) Retinal proteins associated with redox regulation and protein folding play central roles in response to high glucose conditions. Electrophoresis 36:902–909

    Article  CAS  PubMed  Google Scholar 

  50. Yao J, Tao ZF, Li CP, Li XM, Cao GF, Jiang Q, Yan B (2014) Regulation of autophagy by high glucose in human retinal pigment epithelium. Cell Physiol Biochem 33:107–116

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The author, Arpitha H S acknowledges the University Grants Commission (UGC), New Delhi, India for granting Research Fellowship. The authors thank the Academy of Scientific & Innovative Research (AcSIR) and the Director, CSIR-CFTRI for the constant support to carry out this work.

Funding

This study was funded by the 12th Five Year Plan Project (BSC-0404) of CSIR, New Delhi.

Author information

Authors and Affiliations

  1. Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute (CFTRI), Mysore, Karnataka, 570 020, India

    Arpitha Haranahalli Shivarudrappa, Sowmya Shree Gopal & Ganesan Ponesakki

Authors
  1. Arpitha Haranahalli Shivarudrappa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sowmya Shree Gopal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ganesan Ponesakki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ganesan Ponesakki.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Publisher’s Note

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Haranahalli Shivarudrappa, A., Gopal, S.S. & Ponesakki, G. An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells. Mol Biol Rep 46, 1263–1274 (2019). https://doi.org/10.1007/s11033-019-04597-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-019-04597-x

Keywords

Search

Navigation