, 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



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.


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.


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).


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.


Beta cell proliferation Beta cell protection BIM EGFR 



BCL2-like 11


Constitutively active EGFR L858R


EGF receptor


Extracellular signal-regulated kinase


Forkhead box O




Intraperitoneal glucose tolerance test


Multiple low-dose streptozotocin


Optical projection tomography


Phosphatidylinositol 3-kinase


BCL-2-binding component 3



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.


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)


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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

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