Advertisement

Diabetologia

, Volume 57, Issue 5, pp 970–979 | Cite as

In vivo activation of the PI3K–Akt pathway in mouse beta cells by the EGFR mutation L858R protects against diabetes

  • Elina HakonenEmail author
  • Jarkko Ustinov
  • Décio L. Eizirik
  • Hannu Sariola
  • Päivi J. Miettinen
  • Timo Otonkoski
Article

Abstract

Aims/hypothesis

EGF receptor (EGFR) signalling is required for normal beta cell development and postnatal beta cell proliferation. We tested whether beta cell proliferation can be triggered by EGFR activation at any age and whether this can protect beta cells against apoptosis induced by diabetogenic insults in a mouse model.

Methods

We generated transgenic mice with doxycycline-inducible expression of constitutively active EGFR L858R (CA-EGFR) under the insulin promoter. Mice were given doxycycline at various ages for different time periods, and beta cell proliferation and mass were analysed. Mice were also challenged with streptozotocin and isolated islets exposed to cytokines.

Results

Expression of EGFR L858R led to increased phosphorylation of EGFR and Akt in pancreatic islets. CA-EGFR expression during pancreatic development (embryonic day [E]12.5 to postnatal day [P]1) increased beta cell proliferation and mass in newborn mice. However, CA-EGFR expression in adult mice did not affect beta cell mass. Expression of the transgene improved glycaemia and markedly inhibited beta cell apoptosis after a single high dose, as well as after multiple low doses of streptozotocin. In vitro mechanistic studies showed that CA-EGFR protected isolated islets from cytokine-mediated beta cell death, possibly by repressing the proapoptotic protein BCL2-like 11 (BIM).

Conclusions/interpretation

Our findings show that the expression of CA-EGFR in the developing, but not in the adult pancreas stimulates beta cell replication and leads to increased beta cell mass. Importantly, CA-EGFR protects beta cells against streptozotocin- and cytokine-induced death.

Keywords

Beta cell proliferation Beta cell protection BIM EGFR 

Abbreviations

BIM

BCL2-like 11

CA-EGFR

Constitutively active EGFR L858R

EGFR

EGF receptor

ERK

Extracellular signal-regulated kinase

FOXO

Forkhead box O

INS-CA-EGFR

INS-rtTA::TetOP-EGFR L858R

IPGTT

Intraperitoneal glucose tolerance test

MLDS

Multiple low-dose streptozotocin

OPT

Optical projection tomography

PI3K

Phosphatidylinositol 3-kinase

PUMA

BCL-2-binding component 3

Notes

Acknowledgements

The authors thank H. Varmus (National Cancer Institute, Bethesda, MD, USA) for providing the TetOP-EGFR-L858R mice and Y. Dor (Hebrew University-Hadassah Medical School, Jerusalem, Israel) for the INS-rtTA mice. The authors also thank J. Palgi, V. Hakonen, E. Korhonen and U. Kiiski (all from the University of Helsinki, Helsinki, Finland) for technical assistance. J. Klefström, University of Helsinki, is thanked for providing antibodies.

Funding

These studies were funded by the Diabetes Research Foundation, a research grant from Helsinki University Central Hospital, the Foundation for Pediatric Research, the Sigrid Jusélius Foundation, the Helsinki Biomedical Graduate Program, the Orion-Farmos Research Foundation, the Biomedicum Helsinki Foundation, the Academy of Finland and the European Union (projects Naimit and BetaBat, in the Framework Programme 7 of the European Community to DLE).

Author contributions

EH designed and performed experiments, and wrote the manuscript. JU designed and performed experiments and revised the manuscript. DLE designed experiments and revised the manuscript. HS designed and performed experiments, and revised the manuscript. PJM and TO designed experiments, provided funding and revised the manuscript. All authors approved the final version of the manuscript. EH, PJM and TO are responsible for the integrity of the work as a whole.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Supplementary material

125_2014_3175_MOESM1_ESM.pdf (1.2 mb)
ESM Fig. 1 Pancreatic tissue sections from MLDS-treated CTRL and CA-EGFR mice. Animals were killed on day 14 and histology performed as described in the methods. To determine the degree of lymphocyte infiltration the islets were categorized into the following groups: no infiltration, minor infiltration, minor peri-insulitis, clear periinsulitis, clear lymphocyte infiltration into the islets (insulitis). Representative images of all the categories from CTRL and CA-EGFR mice. Scale bar 100 μm. (PDF 1199 kb)
125_2014_3175_MOESM2_ESM.pdf (376 kb)
ESM Fig. 2 After one month of doxycycline administration islets were isolated and western blotting was performed using antibodies against phospho-BAD and phosphoglycogen synthase kinase 3 β (GSK-3β). Representative blot showing increased phosphorylation of GSK-3β and BAD upon doxycycline administration (n = 2). (PDF 375 kb)
125_2014_3175_MOESM3_ESM.pdf (371 kb)
ESM Fig. 3 Beta cell mass and proliferation do not change in adult mice during three months expression of EGFR L858R a: Schematic picture of doxycycline administration. be: 3 month-old male mice were given either doxycycline (CA-EGFR) or standard chow (CTRL) for 3 months and weight (b) and fasting blood glucose (c) were measured (n = 6, t-test). d: Beta cell mass (n = 5–6, t-test). e: Beta cell proliferation (n = 5–6, Mann–Whitney U test). Data is shown as means ± SEM. (PDF 371 kb)
125_2014_3175_MOESM4_ESM.pdf (1 mb)
ESM Fig. 4 The expression of CA-EGFR for nine months in insulin-positive cells results in increased body weight, hyperinsulinemia and impaired glucose tolerance but no increase in beta cell mass and no signs of malignancy. a: Schematic picture of doxycycline administration. b: One-month-old mice were treated with doxycycline for nine months to induce EGFR L858R expression (CA-EGFR) or standard chow (CTRL) and beta cell mass was analysed (n = 5–6, NS, t-test). c: Body weight of the doxycycline treated mice increased more than that of the controls (n = 6, t-test). Open circles, controls; black squares, doxycycline (CA-EGFR). de: CA-EGFR mice tended to have higher blood glucose (d, NS, t-test) and serum insulin levels (e, NS, t-test) in intraperitoneal glucose tolerance test at 30 and 60 min time points after nine months of doxycycline treatment. Open circles, controls; black squares, doxycycline (CA-EGFR). f: Relative transgene (hEGFR) mRNA expression from mice on doxycycline (CA-EGFR) from total pancreatic lysate (PANC) and from hypothalamic lysate (HT) (n = 4–5, Mann–Whitney U test). g: No ectopic hEGFR expression protein could be visualized in the hypothalamus of doxycycline-treated mice with mutationspecific anti L858R immunohistochemistry. Pancreas from the same mouse was used as a positive control. Scale bar 100 μm. h: Representative HE-staining from control mouse pancreas (CTRL). i: Representative HE-staining from doxycycline treated mouse pancreas showing normal pancreatic structure without malignant transformation. Scale bar 100 μm. n = 6 in each group. *p < 0.05. (PDF 1070 kb)
125_2014_3175_MOESM5_ESM.pdf (51 kb)
ESM Table 1 (PDF 50 kb)
125_2014_3175_MOESM6_ESM.pdf (85 kb)
ESM Table 2 (PDF 85 kb)
125_2014_3175_MOESM7_ESM.pdf (65 kb)
ESM Table 3 (PDF 64.5 kb)
125_2014_3175_MOESM8_ESM.pdf (75 kb)
ESM Methods (PDF 74 kb)
ESM Video 1

Increased beta cell mass in INS-CA-EGFR mice during pancreatic development. Mice were treated with doxycycline to induce EGFR L858R expression from E12.5 to P30. Representative isosurface reconstructions from OPT-analysed pancreases showing insulin in red and pancreatic tissue in light blue at P30 without (control) and with doxycycline-treatment (CA-EGFR). (MOV 9450 kb)

References

  1. 1.
    Eizirik DL, Mandrup-Poulsen T (2001) A choice of death—the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia 44:2115–2133PubMedCrossRefGoogle Scholar
  2. 2.
    Thomas HE, McKenzie MD, Angstetra E et al (2009) Beta cell apoptosis in diabetes. Apoptosis 14:1389–1404PubMedCrossRefGoogle Scholar
  3. 3.
    Gurzov EN, Eizirik DL (2011) Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction. Trends Cell Biol 21:424–431PubMedCrossRefGoogle Scholar
  4. 4.
    Herold KC, Vignali DAA, Cooke A, Bluestone JA (2013) Type 1 diabetes: translating mechanistic observations into effective clinical outcomes. Nat Rev Immunol 13:243–256PubMedCrossRefGoogle Scholar
  5. 5.
    Schlessinger J, Ullrich A (1992) Growth factor signaling by receptor tyrosine kinases. Neuron 9:383–391PubMedCrossRefGoogle Scholar
  6. 6.
    Citri A, Yarden Y (2006) EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7:505–516PubMedCrossRefGoogle Scholar
  7. 7.
    Kritzik MR, Krahl T, Good A et al (2000) Expression of ErbB receptors during pancreatic islet development and regrowth. J Endocrinol 165:67–77PubMedCrossRefGoogle Scholar
  8. 8.
    Huotari M-A, Miettinen PJ, Palgi J et al (2002) ErbB signaling regulates lineage determination of developing pancreatic islet cells in embryonic organ culture. Endocrinology 143:4437–4446PubMedCrossRefGoogle Scholar
  9. 9.
    Miettinen PJ, Huotari M, Koivisto T et al (2000) Impaired migration and delayed differentiation of pancreatic islet cells in mice lacking EGF-receptors. Development 127:2617–2627PubMedGoogle Scholar
  10. 10.
    Miettinen PJ, Ustinov J, Ormio P et al (2006) Downregulation of EGF receptor signaling in pancreatic islets causes diabetes due to impaired postnatal beta-cell growth. Diabetes 55:3299–3308PubMedCrossRefGoogle Scholar
  11. 11.
    Cras-Meneur C, Elghazi L, Czernichow P, Scharfmann R (2001) Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation. Diabetes 50:1571–1579PubMedCrossRefGoogle Scholar
  12. 12.
    Miettinen P, Ormio P, Hakonen E et al (2008) EGF receptor in pancreatic beta-cell mass regulation. Biochem Soc Trans 36:280–285PubMedCrossRefGoogle Scholar
  13. 13.
    Elghazi L, Bernal-Mizrachi E (2009) Akt and PTEN: beta-cell mass and pancreas plasticity. Trends Endocrinol Metab 20:243–251PubMedCrossRefGoogle Scholar
  14. 14.
    Choi SH, Mendrola JM, Lemmon MA (2007) EGF-independent activation of cell-surface EGF receptors harboring mutations found in gefitinib-sensitive lung cancer. Oncogene 26:1567–1576PubMedCrossRefGoogle Scholar
  15. 15.
    Shan Y, Eastwood MP, Zhang X et al (2012) Oncogenic mutations counteract intrinsic disorder in the EGFR kinase and promote receptor dimerization. Cell 149:860–870PubMedCrossRefGoogle Scholar
  16. 16.
    Nir T, Melton D, Dor Y (2007) Recovery from diabetes in mice by beta cell regeneration. J Clin Invest 117:2553–2561PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Politi K, Zakowski MF, Fan P-D, Schonfeld EA, Pao W, Varmus HE (2006) Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev 20:1496–1510PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Hakonen E, Ustinov J, Mathijs I et al (2011) Epidermal growth factor (EGF)-receptor signalling is needed for murine beta cell mass expansion in response to high-fat diet and pregnancy but not after pancreatic duct ligation. Diabetologia 54:1735–1743PubMedCrossRefGoogle Scholar
  19. 19.
    Alanentalo T, Asayesh A, Morrison H et al (2007) Tomographic molecular imaging and 3D quantification within adult mouse organs. Nat Methods 4:31–33PubMedCrossRefGoogle Scholar
  20. 20.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  21. 21.
    Liu D, Pavlovic D, Chen MC, Flodström M, Sandler S, Eizirik DL (2000) Cytokines induce apoptosis in beta-cells isolated from mice lacking the inducible isoform of nitric oxide synthase (iNOS−/−). Diabetes 49:1116–1122PubMedCrossRefGoogle Scholar
  22. 22.
    Flodström M, Tyrberg B, Eizirik DL, Sandler S (1999) Reduced sensitivity of inducible nitric oxide synthase-deficient mice to multiple low-dose streptozotocin-induced diabetes. Diabetes 48:706–713PubMedCrossRefGoogle Scholar
  23. 23.
    Wicksteed B, Brissova M, Yan W et al (2010) Conditional gene targeting in mouse pancreatic ß-cells: analysis of ectopic Cre transgene expression in the brain. Diabetes 59:3090–3098PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Irmer D, Funk JO, Blaukat A (2007) EGFR kinase domain mutations—functional impact and relevance for lung cancer therapy. Oncogene 26:5693–5701PubMedCrossRefGoogle Scholar
  25. 25.
    Lenzen S (2008) The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia 51:216–226PubMedCrossRefGoogle Scholar
  26. 26.
    Like AA, Rossini AA (1976) Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus. Science 193:415–417PubMedCrossRefGoogle Scholar
  27. 27.
    Eizirik DL, Colli ML, Ortis F (2009) The role of inflammation in insulitis and beta-cell loss in type 1 diabetes. Nat Rev Endocrinol 5:219–226PubMedCrossRefGoogle Scholar
  28. 28.
    Sandberg JO, Andersson A, Eizirik DL, Sandler S (1994) Interleukin-1 receptor antagonist prevents low dose streptozotocin induced diabetes in mice. Biochem Biophys Res Commun 202:543–548PubMedCrossRefGoogle Scholar
  29. 29.
    Moore F, Naamane N, Colli ML et al (2011) STAT1 is a master regulator of pancreatic β-cell apoptosis and islet inflammation. J Biol Chem 286:929–941PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Wang H, Gambosova K, Cooper ZA et al (2010) EGF regulates survivin stability through the Raf-1/ERK pathway in insulin-secreting pancreatic β-cells. BMC Mol Biol 11:66PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Teta M, Long S, Wartschow L, Rankin M, Kushner J (2005) Very slow turnover of beta-cells in aged adult mice. Diabetes 54:2557–2567PubMedCrossRefGoogle Scholar
  32. 32.
    Meier JJ, Butler AE, Saisho Y et al (2008) Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans. Diabetes 57:1584–1594PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Zeng N, Yang K-T, Bayan J-A et al (2013) PTEN controls β-cell regeneration in aged mice by regulating cell cycle inhibitor p16(ink4a.). Aging Cell 12:1000–1011PubMedCrossRefGoogle Scholar
  34. 34.
    Bernal-Mizrachi E, Wen W, Stahlhut S, Welling CM, Permutt MA (2001) Islet beta cell expression of constitutively active Akt1/PKB alpha induces striking hypertrophy, hyperplasia, and hyperinsulinemia. J Clin Invest 108:1631–1638PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Tuttle RL, Gill NS, Pugh W et al (2001) Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha. Nat Med 7:1133–1137PubMedCrossRefGoogle Scholar
  36. 36.
    Elghazi L, Weiss AJ, Barker DJ et al (2009) Regulation of pancreas plasticity and malignant transformation by Akt signaling. Gastroenterology 136:1091–1103PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Ji H, Li D, Chen L et al (2006) The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies. Cancer Cell 9:485–495PubMedCrossRefGoogle Scholar
  38. 38.
    Sordella R, Bell DW, Haber DA, Settleman J (2004) Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305:1163–1167PubMedCrossRefGoogle Scholar
  39. 39.
    Oh YS, Shin S, Lee Y-J, Kim EH, Jun H-S (2011) Betacellulin-induced beta cell proliferation and regeneration is mediated by activation of ErbB-1 and ErbB-2 receptors. PLoS ONE 6:e23894PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Yamamoto H, Uchigata Y, Okamoto H (1981) Streptozotocin and alloxan induce DNA strand breaks and poly(ADP-ribose) synthetase in pancreatic islets. Nature 294:284–286PubMedCrossRefGoogle Scholar
  41. 41.
    Kantwerk-Funke G, Burkart V, Kolb H (1991) Low dose streptozotocin causes stimulation of the immune system and of anti-islet cytotoxicity in mice. Clin Exp Immunol 86:266–270PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Cnop M, Welsh N, Jonas J-C, Jörns A, Lenzen S, Eizirik DL (2005) Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 54(Suppl 2):S97–S107PubMedCrossRefGoogle Scholar
  43. 43.
    Barthson J, Germano CM, Moore F et al (2011) Cytokines tumor necrosis factor-α and interferon-γ induce pancreatic β-cell apoptosis through STAT1-mediated Bim protein activation. J Biol Chem 286:39632–39643PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Santin I, Moore F, Colli ML et al (2011) PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic β-cell apoptosis via regulation of the BH3-only protein Bim. Diabetes 60:3279–3288PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Nogueira TC, Paula FM, Villate O et al (2013) GLIS3, a susceptibility gene for type 1 and type 2 diabetes, modulates pancreatic beta cell apoptosis via regulation of a splice variant of the BH3-only protein Bim. PLoS Genet 9:e1003532PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Ley R, Ewings KE, Hadfield K, Cook SJ (2005) Regulatory phosphorylation of Bim: sorting out the ERK from the JNK. Cell Death Differ 12:1008–1014PubMedCrossRefGoogle Scholar
  47. 47.
    Qi X-J, Wildey GM, Howe PH (2006) Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function. J Biol Chem 281:813–823PubMedCrossRefGoogle Scholar
  48. 48.
    Dijkers PF, Medema RH, Lammers JW, Koenderman L, Coffer PJ (2000) Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 10:1201–1204PubMedCrossRefGoogle Scholar
  49. 49.
    Gong Y, Somwar R, Politi K et al (2007) Induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors in mutant EGFR-dependent lung adenocarcinomas. PLoS Med 4:e294PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Costa DB, Halmos B, Kumar A et al (2007) BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations. PLoS Med 4:1669–1679PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Elina Hakonen
    • 1
    Email author
  • Jarkko Ustinov
    • 1
  • Décio L. Eizirik
    • 2
  • Hannu Sariola
    • 3
  • Päivi J. Miettinen
    • 1
    • 4
  • Timo Otonkoski
    • 1
    • 4
  1. 1.Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell CenterUniversity of HelsinkiHelsinkiFinland
  2. 2.Laboratory of Experimental Medicine, ULB Center for Diabetes ResearchUniversite Libre de BruxellesBrusselsBelgium
  3. 3.Institute of Biomedicine, Biochemistry and Developmental BiologyUniversity of HelsinkiHelsinkiFinland
  4. 4.Children’s HospitalUniversity of Helsinki and Helsinki University Central HospitalHelsinkiFinland

Personalised recommendations