Histochemistry and Cell Biology

, Volume 147, Issue 5, pp 605–623 | Cite as

An immunohistochemical study of nitrotyrosine expression in pancreatic islets of cases with increasing duration of type 1 diabetes and without diabetes

  • Charlton Martin
  • Lars Krogvold
  • Shebani Farik
  • Satya Amirapu
  • Fiona Wu
  • Shiva ReddyEmail author
  • Knut Dahl-Jørgensen
Original Paper


Peroxynitrite-induced nitration of cellular proteins has been shown to associate with various human pathologies. The expression of pancreatic nitrotyrosine and its cellular source relative to insulitis were analysed in cases with increasing duration of type 1 diabetes and compared with non-diabetic autoantibody-negative and -positive cases. Pancreatic tail sections from non-diabetic autoantibody-negative cases (Group 1; n = 7), non-diabetic autoantibody-positive cases (Group 2; n = 6), recently diagnosed cases (Group 3; n = 6), 0.25–5 years of diabetes (Group 4; n = 8) and 7–12 years of diabetes (Group 5; n = 6) were immunostained sequentially for nitrotyrosine, insulin and leucocytes. Nitrotyrosine expression was observed in selective beta cells only. In group 1, the percentage of insulin-positive islets with nitrotyrosine ranged from 7.6 to 58.8%. In group 2, it was minimally expressed in 2 cases and was present in 4.7–19.3% of insulin-positive islets in 3 cases and in all islets in 1 case. In group 3, it was absent in 1 case and in the remaining 5 cases, the values were 17.4–85.7%. In group 4, nitrotyrosine was absent in 6 cases and positive in 1.8 and 22.2% of insulin-positive islets in 2 cases. In group 5, the values were 60% (1 case) and 100% (2 cases), being absent in 3 cases, consistent with insulin-negativity. This case analysis shows that nitrotyrosine immunostaining is independent of the presence and severity of insulitis. Variable nitrotyrosine expression is present in some non‐diabetic cases. Its increased expression in beta cells of recent-onset and long-standing disease requires further studies to determine whether beta cell nitration plays a pathogenic role during T1D.


Beta cells Human type 1 diabetes Nitrosative stress Nitrotyrosine Islets 



Binding immunoglobulin protein


C/EBP homologous protein


Diabetes Virus Detection


Endoplasmic reticulum


Network for Pancreatic Organ Donors with Diabetes



We thank Drs Peter Butler and Tatyana Gurlos from the University of California Medical Centre, Los Angeles, USA, for advice on developing a protocol for nitrotyrosine immunohistochemistry, Dr Sarah Richardson, Exeter Medical School, United Kingdom for recommending antibodies and protocols and Dr. Peter Browett, University of Auckland for ongoing encouragement. This study was supported by a partial grant from the New Zealand Society for the Study of Diabetes. Pancreatic tail sections from newly diagnosed living donors with T1D were from the DiViD Study, funded by South-Eastern Norway Regional Health Authority (Grant to KDJ), The Novo Nordisk Foundation (Grant to KDJ) and through the PEVNET Study Group funded by the European Union’s Seventh Framework Programme (FP7/2007-2013) under Agreement No. 26441 PEVNET. Pancreatic sections from cadaveric pancreas were kindly supplied by nPOD.

Author contribution statement

Satya Amirapu and Fiona Wu provided histological and editing support, respectively. Shiva Reddy led the study and wrote the manuscript.

Compliance with ethical standards

Conflict of interest



  1. Åkerblom HK, Vaarala O, Hyöty H et al (2002) Environmental factors in the etiology of type 1 diabetes. Am J Med Genet 115:18–29. doi: 10.1002/ajmg.10340 CrossRefPubMedGoogle Scholar
  2. Birnboim HC, Lemay A-M, Lam DKY et al (2003) Cutting edge: MHC Class II-restricted peptides containing the inflammation-associated marker 3-nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune response. J Immunol 171:528–532. doi: 10.4049/jimmunol.171.2.528 CrossRefPubMedGoogle Scholar
  3. Buddi R, Lin B, Atilano SR et al (2002) Evidence of oxidative stress in human corneal diseases. J Histochem Cytochem 50:341–351. doi: 10.1177/002215540205000306 CrossRefPubMedGoogle Scholar
  4. Cardozo AK, Ortis F, Storling J, Feng Y-M, Rasschaert J, Tonnesen M, Van Eylen F, Mandrup-Poulsen T, Herchuelz A, Eizirik DL (2005) Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic β-cells. Diabetes 54:452–461CrossRefPubMedGoogle Scholar
  5. Ceriello A, Mercuri F, Quagliaro L et al (2001) Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia 44:834–838. doi: 10.1007/s001250100529 CrossRefPubMedGoogle Scholar
  6. Chung YH, Shin CM, Joo KM et al (2002) Immunohistochemical study on the distribution of nitrotyrosine and neuronal nitric oxide synthase in aged rat cerebellum. Brain Res 951:316–321. doi: 10.1016/S0006-8993(02)03261-4 CrossRefPubMedGoogle Scholar
  7. Cnop M, Welsh N, Jonas JC, Jörns A, Lenzen S, Eizirik DL (2005) Mechanisms of pancreatic beta-cell death in type 1 and 2 diabetes: many differences, few similarities. Diabetes 54:S97–S107CrossRefPubMedGoogle Scholar
  8. Delaney CA, Tyrberg B, Bouwens L et al (1996) Sensitivity of human pancreatic islets to peroxynitrite-induced cell dysfunction and death. FEBS Lett 394:300–306. doi: 10.1016/0014-5793(96)00977-5 CrossRefPubMedGoogle Scholar
  9. Eizirik DL, Colli ML, Ortis F (2009) The role of inflammation in insulitis and beta-cell loss in type 1 diabetes. Nat Rev Endocrinol 5:219–226. doi: 10.1038/nrendo.2009.21 CrossRefPubMedGoogle Scholar
  10. Eizirik DL, Sammeth M, Bouckenooghe T et al (2012) The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines. PLoS Genet 8:e1002552. doi: 10.1371/journal.pgen.1002552 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Fonseca SG, Gromada J, Urano F (2011) Endoplasmic reticulum stress and pancreatic beta cell death. Trends Endocrinol Metab 22:266–274. doi: 10.1016/j.tem.2011.02.008 PubMedPubMedCentralGoogle Scholar
  12. Gerling IC (2009) Oxidative stress, altered-self and autoimmunity. Open Autoimmun J 1:33–36. doi: 10.2174/1876894600901010033 CrossRefGoogle Scholar
  13. Goto T, Haruma K, Kitadai Y et al (1999) Enhanced expression of inducible nitric oxide synthase and nitrotyrosine in gastric mucosa of gastric cancer patients. Clin Cancer Res 5:1411–1415PubMedGoogle Scholar
  14. Haskins K, Bradley B, Powers K et al (2003) Oxidative stress in type 1 diabetes. Ann N Y Acad Sci 1005:43–54. doi: 10.1196/annals.1288.006 CrossRefPubMedGoogle Scholar
  15. Herzog J, Maekawa Y, Cirrito TP et al (2005) Activated antigen-presenting cells select and present chemically modified peptides recognized by unique CD4 T cells. Proc Natl Acad Sci 102:7928–7933. doi: 10.1073/pnas.0502255102 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hober D, Sauter P (2010) Pathogenesis of type 1 diabetes mellitus: interplay between enterovirus and host. Nat Rev Endocrinol 6:279–289. doi: 10.1038/nrendo.2010.27 CrossRefPubMedGoogle Scholar
  17. Krogvold L, Edwin B, Buanes T et al (2014a) Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes. Diabetes. doi: 10.2337/db14-1370 PubMedGoogle Scholar
  18. Krogvold L, Edwin B, Buanes T et al (2014b) Pancreatic biopsy by minimal tail resection in live adult patients at the onset of type 1 diabetes: experiences from the DiViD study. Diabetologia 57:841–843. doi: 10.1007/s00125-013-3155-y CrossRefPubMedGoogle Scholar
  19. Lakey JR, Suarez-Pinzon WL, Strynadka K et al (2001) Peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta-cells. Lab Investig 81:1683–1692. doi: 10.1038/labinvest.3780381 CrossRefPubMedGoogle Scholar
  20. Lenzen S (2008) Oxidative stress: the vulnerable beta-cell. Biochem Soc Trans 36:343–347. doi: 10.1042/BST0360343 CrossRefPubMedGoogle Scholar
  21. Lenzen S, Drinkgern J, Tiedge M (1996) Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radic Biol Med 20:463–466. doi: 10.1016/0891-5849(96)02051-5 CrossRefPubMedGoogle Scholar
  22. Loeser RF, Carlson CS, Del Carlo M, Cole A (2002) Detection of nitrotyrosine in aging and osteoarthritic cartilage: correlation of oxidative damage with the presence of interleukin-1β and with chondrocyte resistance to insulin-like growth factor 1. Arthritis Rheum 46:2349–2357. doi: 10.1002/art.10496 CrossRefPubMedGoogle Scholar
  23. Maganti A, Evans-Molina C, Mirmira RG (2014) From immunobiology to beta-cell biology: the changing perspective on type 1 diabetes. Islets 6:e28778. doi: 10.4161/isl.28778 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Marhfour I, Lopez XM, Lefkaditis D et al (2012) Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes. Diabetologia 55:2417–2420. doi: 10.1007/s00125-012-2604-3 CrossRefPubMedGoogle Scholar
  25. Meier JJ, Bhushan A, Butler AE et al (2005) Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration? Diabetologia 48:2221–2228. doi: 10.1007/s00125-005-1949-2 CrossRefPubMedGoogle Scholar
  26. O’Sullivan-Murphy B, Urano F (2012) ER stress as a trigger for beta-cell dysfunction and autoimmunity in type 1 diabetes. Diabetes 61:780–781. doi: 10.2337/db12-0091 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Oleszak EL, Zaczynska E, Bhattacharjee M et al (1998) Inducible nitric oxide synthase and nitrotyrosine are found in monocytes/macrophages and/or astrocytes in acute, but not in chronic, multiple sclerosis. Clin Diagn Lab Immunol 5:438–445PubMedPubMedCentralGoogle Scholar
  28. Padalko E, Ohnishi T, Matsushita K et al (2004) Peroxynitrite inhibition of Coxsackievirus infection by prevention of viral RNA entry. Proc Natl Acad Sci USA 101:11731–11736. doi: 10.1073/pnas.0400518101 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Pirot P, Cardozo AK, Eizirik DL (2008) Mediators and mechanisms of pancreatic beta-cell death in type 1 diabetes. Arq Bras Endocrinol Metabol 52:156–165. doi: 10.1590/S0004-27302008000200003 CrossRefPubMedGoogle Scholar
  30. Pugliese A, Yang M, Kusmarteva I et al (2014) The Juvenile Diabetes Research Foundation Network for Pancreatic Organ Donors with Diabetes (nPOD) Program: goals, operational model and emerging findings. Pediatr Diabetes 15:1–9. doi: 10.1111/pedi.12097 CrossRefPubMedGoogle Scholar
  31. Reddy S, Bradley J (2004) Immunohistochemical demonstration of nitrotyrosine, a biomarker of oxidative stress, in islet cells of the NOD mouse. Ann N Y Acad Sci 1037:199–202. doi: 10.1196/annals.1337.032 CrossRefPubMedGoogle Scholar
  32. Reddy S, Zeng N, Al-Diery H et al (2015) Analysis of peri-islet CD45-positive leucocytic infiltrates in long-standing type 1 diabetic patients. Diabetologia 58:1024–1035. doi: 10.1007/s00125-015-3519-6 CrossRefPubMedGoogle Scholar
  33. Regnéll SE, Lernmark Å (2013) The environment and the origins of islet autoimmunity and Type 1 diabetes. Diabet Med 30:155–160. doi: 10.1111/dme.12099 CrossRefPubMedCentralGoogle Scholar
  34. Rivera JF, Costes S, Gurlo T et al (2014) Autophagy defends pancreatic beta-cells from human islet amyloid polypeptide-induced toxicity. J Clin Invest 124:3489–3500. doi: 10.1172/JCI71981 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Robertson RP (2004) Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem 279:42351–42354. doi: 10.1074/jbc.R400019200 CrossRefPubMedGoogle Scholar
  36. Rodriguez-Calvo T, Ekwall O, Amirian N et al (2014) Increased immune cell infiltration of the exocrine pancreas: a possible contribution to the pathogenesis of type 1 diabetes. Diabetes 63:3880–3890. doi: 10.2337/db14-0549 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Roep BO, Atkinson M (2004) Animal models have little to teach us about type 1 diabetes: 1. In support of this proposal. Diabetologia 10:1650–1656CrossRefGoogle Scholar
  38. Roep BO, Atkinson M, von Herrath M (2004) Satisfaction (not) guaranteed: re-evaluating the use of animal models of type 1 diabetes. Nat Rev Immunol 12:989–997CrossRefGoogle Scholar
  39. Sandhu JK, Robertson S, Birnboim HC, Goldstein R (2003) Distribution of protein nitrotyrosine in synovial tissues of patients with rheumatoid arthritis and osteoarthritis. J Rheumatol 30:1173–1181PubMedGoogle Scholar
  40. Shin CM, Chung YH, Kim MJ et al (2002) Age-related changes in the distribution of nitrotyrosine in the cerebral cortex and hippocampus of rats. Brain Res 931:194–199. doi: 10.1016/S0006-8993(01)03391-1 CrossRefPubMedGoogle Scholar
  41. Suarez-Pinzon WL, Szabó C, Rabinovitch A (1997) Development of autoimmune diabetes in NOD mice is associated with the formation of peroxynitrite in pancreatic islet beta-cells. Diabetes 46:907–911. doi: 10.2337/diab.46.5.907 CrossRefPubMedGoogle Scholar
  42. Szabó C, Ischiropoulos H, Radi R (2007) Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6:662–680. doi: 10.1038/nrd2222 CrossRefPubMedGoogle Scholar
  43. Tersey SA, Nishiki Y, Templin AT et al (2012) Islet beta-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model. Diabetes 61:818–827. doi: 10.2337/db11-1293 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Trigwell SM, Radford PM, Page SR et al (2001) Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus. Clin Exp Immunol 126:242–249. doi: 10.1046/j.1365-2249.2001.01653.x CrossRefPubMedPubMedCentralGoogle Scholar
  45. Wang X, Misawa R, Zielinski MC et al (2013) Regional differences in islet distribution in the human pancreas–preferential beta-cell loss in the head region in patients with type 2 diabetes. PLoS ONE 8:e67454. doi: 10.1371/journal.pone.0067454 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Charlton Martin
    • 1
  • Lars Krogvold
    • 4
    • 5
  • Shebani Farik
    • 1
  • Satya Amirapu
    • 2
  • Fiona Wu
    • 3
  • Shiva Reddy
    • 1
    Email author
  • Knut Dahl-Jørgensen
    • 4
    • 5
  1. 1.Department of Molecular Medicine and Pathology, Faculty of Medical and Health SciencesUniversity of AucklandAucklandNew Zealand
  2. 2.Department of Anatomy with Radiology, Faculty of Medical and Health SciencesUniversity of AucklandAucklandNew Zealand
  3. 3.Diabetes UnitAuckland District Health BoardAucklandNew Zealand
  4. 4.Division of Paediatric and Adolescent MedicineOslo University HospitalOsloNorway
  5. 5.Faculty of MedicineUniversity of OsloOsloNorway

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