, Volume 60, Issue 8, pp 1475–1482 | Cite as

Metabolically inactive insulin analogue does not prevent autoimmune diabetes in NOD mice

  • Juha Grönholm
  • Philippe P. Pagni
  • Minh N. Pham
  • Claire B. Gibson
  • Paul F. Macomber
  • José Luis Vela
  • Matthias von Herrath
  • Michael J. Lenardo



Insulin is widely considered to be a driver antigen in type 1 diabetes in humans and in mouse models of the disease. Therefore, insulin or insulin analogues are candidates for tolerogenic drugs to prevent disease onset in individuals with risk of diabetes. Previous experiments have shown that autoimmune diabetes can be prevented in NOD mice by repeated doses of insulin administered via an oral, nasal or parenteral route, but clinical trials in humans have not succeeded. The hypoglycaemic activity of insulin is dose-limiting in clinical studies attempting tolerance and disease prevention. Here, we aimed to investigate the therapeutic potential of metabolically inactive insulin analogue (MII) in NOD mice.


The tolerogenic potential of MII to prevent autoimmune diabetes was studied by administering multiple i.v. or s.c. injections of MII to non-diabetic 7–12-week-old female NOD mice in three geographical colony locations. The incidence of diabetes was assessed from daily or weekly blood glucose measurements. The effect of MII on insulin autoantibody levels was studied using an electrochemiluminescence-based insulin autoantibody assay. The effect on the number of insulin-reactive CD8+ and CD4+ T lymphocytes in peripheral lymphoid tissue was studied with MHC class I and MHC class II tetramers, respectively.


We found that twice-weekly s.c. administration of MII accelerates rather than prevents diabetes. High-dose i.v. treatment did not prevent disease or affect insulin autoantibody levels, but it increased the amount of insulin-reactive CD4+ T lymphocytes in peripheral lymphoid tissue.


Our data suggest that parenteral MII, even when used in high doses, has little or no therapeutic potential in NOD mice and may exacerbate disease.


Autoimmune diabetes Insulin Non-obese diabetic mouse Tolerance 



Antigen presenting cell




Hen egg lysozyme


Islet-specific glucose-6-phosphatase catalytic subunit-related protein


Metabolically inactive insulin analogue


Novo Nordisk Research Center


Programmed death-ligand 1


Re-stimulation-induced cell death



We thank L. Zheng and K. Shafer-Weaver (both from Laboratory of Immunology, NIAID, NIH, Bethesda, MD, USA) for their scientific advice and B. Chao (Laboratory of Immunology, NIAID, NIH, Bethesda, MD, USA), A. Bel Hani and Y. Manenkova (La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA) for technical assistance. We thank the NIAID Research Technologies Branch for producing the HEL11-25 and insulin B9-23 peptides and the NIH Tetramer Core Facility at Emory University, Atlanta, GA, USA for the tetramers.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


This research was supported by the Division of Intramural Research, NIAID, Clinical Center and National Human Genome Research Institute, NIH. JG was supported by a fellowship grant from the Sigrid Juselius Foundation and by a grant from the Emil Aaltonen Foundation. Wellstat Immunotherapeutics supported this research with Cooperative Research and Development Agreement (CRADA) C-087-2006 (2006-0203) and Wellstat Diagnostics with CRADA C-022-2006 (2006-0369). The study was also funded in part by Novo Nordisk.

Duality of interest

MvH, JLV, PPP, CBG and MNP are employed or affiliated with Novo Nordisk. All other authors declare that there is no duality of interest associated with their contribution to this manuscript.

Contribution statement

JG, MvH, JLV, PPP and MJL designed the experiments. JG, JLV, PPP, MNP, PFM and CBG performed the experiments and analysed the results. JG, PPP, MNP, PFM and JLV drafted the manuscript. All authors performed critical analysis of the manuscript and approved the final version to be published. MJL is a guarantor of this study and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Supplementary material

125_2017_4276_MOESM1_ESM.pdf (577 kb)
ESM 1 (PDF 576 kb)


  1. 1.
    Atkinson MA, Eisenbarth GS, Michels AW (2014) Type 1 diabetes. Lancet 383:69–82CrossRefPubMedGoogle Scholar
  2. 2.
    Anderson MS, Bluestone JA (2005) The NOD mouse: a model of immune dysregulation. Annu Rev Immunol 23:447–485CrossRefPubMedGoogle Scholar
  3. 3.
    Unanue ER (2014) Antigen presentation in the autoimmune diabetes of the NOD mouse. Annu Rev Immunol 32:579–608CrossRefPubMedGoogle Scholar
  4. 4.
    Reed JC, Herold KC (2015) Thinking bedside at the bench: the NOD mouse model of T1DM. Nat Rev Endocrinol 11:308–314CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Nakayama M, Abiru N, Moriyama H et al (2005) Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature 435:220–223CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lieberman SM, Evans AM, Han B et al (2003) Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. Proc Natl Acad Sci U S A 100:8384–8388CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Grönholm J, Lenardo MJ (2015) Novel diagnostic and therapeutic approaches for autoimmune diabetes—a prime time to treat insulitis as a disease. Clin Immunol 156:109–118CrossRefPubMedGoogle Scholar
  8. 8.
    Yu L, Robles DT, Abiru N et al (2000) Early expression of antiinsulin autoantibodies of humans and the NOD mouse: evidence for early determination of subsequent diabetes. Proc Natl Acad Sci U S A 97:1701–1706CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Atkinson MA, Maclaren NK, Luchetta R (1990) Insulitis and diabetes in NOD mice reduced by prophylactic insulin therapy. Diabetes 39:933–937CrossRefPubMedGoogle Scholar
  10. 10.
    Muir A, Peck A, Clare-Salzler M et al (1995) Insulin immunization of nonobese diabetic mice induces a protective insulitis characterized by diminished intraislet interferon-gamma transcription. J Clin Invest 95:628–634CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Karounos DG, Bryson JS, Cohen DA (1997) Metabolically inactive insulin analog prevents type I diabetes in prediabetic NOD mice. J Clin Invest 100:1344–1348CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Daniel C, Weigmann B, Bronson R, von Boehmer H (2011) Prevention of type 1 diabetes in mice by tolerogenic vaccination with a strong agonist insulin mimetope. J Exp Med 208:1501–1510CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhang JZ, Davidson L, Eisenbarth G et al (1994) Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. J Endocrinol Investig 17:573–580CrossRefGoogle Scholar
  14. 14.
    Harrison LC, Dempsey-Collier M, Kramer DR, Takahashi K (1996) Aerosol insulin induces regulatory CD8 gamma delta T cells that prevent murine insulin-dependent diabetes. J Exp Med 184:2167–2174CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Pham MN, Gibson C, Rydén AKE et al (2016) Oral insulin (human, murine, or porcine) does not prevent diabetes in the non-obese diabetic mouse. Clin Immunol 164:28–33CrossRefPubMedGoogle Scholar
  16. 16.
    Snow AL, Pnadiyan P, Zheng L et al (2010) The power and the promise of restimulation-induced cell death in human immune diseases. Immunol Rev 236:68–82CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Critchfield JM, Racke MK, Zuniga-Pflucker JC et al (1994) T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis. Science 263:1139–1143CrossRefPubMedGoogle Scholar
  18. 18.
    Mcfarland HI, Lobito AA, Johnson MM et al (2001) Effective antigen-specific immunotherapy in the marmoset model of multiple sclerosis. J Immunol 166:2116–2121CrossRefPubMedGoogle Scholar
  19. 19.
    Chaillous L, Lefèvre H, Thivolet C et al (2000) Oral insulin administration and residual beta-cell function in recent-onset type 1 diabetes: a multicentre randomised controlled trial. Lancet 356:545–549CrossRefPubMedGoogle Scholar
  20. 20.
    Diabetes Prevention Trial—Type 1 Diabetes Study Group (2002) Effects of insulin in relatives of patients with type 1 diabetes mellitus. N Engl J Med 346:1685–1691CrossRefGoogle Scholar
  21. 21.
    Vølund A, Brange J, Drejer K et al (1991) In vitro and in vivo potency of insulin analogues designed for clinical use. Diabet Med 8:839–847CrossRefPubMedGoogle Scholar
  22. 22.
    Homann D, Dyrberg T, Petersen J et al (1999) Insulin in oral immune “tolerance”: a one-amino acid change in the B chain makes the difference. J Immunol 163:1833–1838PubMedGoogle Scholar
  23. 23.
    Ansari MJI, Salama AD, Chitnis T et al (2003) The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 198:63–69CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Levisetti MG, Suri A, Petzold SJ, Unanue ER (2007) The insulin-specific T cells of nonobese diabetic mice recognize a weak MHC-binding segment in more than one form. J Immunol 178:6051–6057CrossRefPubMedGoogle Scholar
  25. 25.
    Lo B, Swafford ADE, Shafer-Weaver KA et al (2011) Antibodies against insulin measured by electrochemiluminescence predicts insulitis severity and disease onset in non-obese diabetic mice and can distinguish human type 1 diabetes status. J Transl Med 9:203CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Gill RG, Pagni PP, Kufper T et al (2015) A preclinical consortium approach for assessing the efficacy of combined anti-CD3 plus IL-1 blockade in reversing new-onset autoimmune diabetes in NOD mice. Diabetes 65:1310–1316CrossRefPubMedGoogle Scholar
  27. 27.
    Mohan JF, Levisetti MG, Calderon B et al (2010) Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes. Nat Immunol 11:350–354CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Shoda LD, Young DL, Ramanujan S et al (2005) A comprehensive review of interventions in the NOD mouse and implications for translation. Immunity 2:115–126CrossRefGoogle Scholar
  29. 29.
    Liddi R, Beales PE, Rosignoli G, Pozzilli P (2000) Incomplete Freund’s adjuvant reduces diabetes in the non-obese diabetic mouse. Horm Metab Res 6:201–206CrossRefGoogle Scholar

Copyright information

© Springer-Verlag (outside the USA) 2017

Authors and Affiliations

  • Juha Grönholm
    • 1
  • Philippe P. Pagni
    • 2
    • 3
  • Minh N. Pham
    • 2
  • Claire B. Gibson
    • 2
  • Paul F. Macomber
    • 4
  • José Luis Vela
    • 2
  • Matthias von Herrath
    • 2
  • Michael J. Lenardo
    • 1
  1. 1.Molecular Development of the Immune System Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases (NIAID) and Clinical Genomics Program, NIAIDNational Institutes of HealthBethesdaUSA
  2. 2.Novo Nordisk Type 1 Diabetes CenterNovo Nordisk Research CenterSeattleUSA
  3. 3.La Jolla Institute for Allergy and ImmunologyLa JollaUSA
  4. 4.Wellstat DiagnosticsGaithersburgUSA

Personalised recommendations