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Determining Beta Cell Mass, Apoptosis, Proliferation, and Individual Beta Cell Size in Pancreatic Sections

  • Noèlia TéllezEmail author
  • Eduard Montanya
Protocol
  • 70 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2128)

Abstract

Pancreatic beta cells have a significant remodeling capacity which plays an essential role in the maintenance of glucose homeostasis. Beta cell apoptosis, replication, size, dedifferentiation, and (neo)generation contribute to the beta cell mass regulation. However, the extent of their respective contribution varies significantly depending on the specific condition, and it is the balance among them that determines the eventual change in beta cell mass. Thus, the study of the pancreatic beta cell mass regulation requires the determination of all these factors. In this chapter, we describe the quantification of beta cell replication based on the incorporation of thymidine analogs into replicated DNA strands and on the expression of Ki67 antigen and phosphorylation of histone H3. Beta cell apoptosis is analyzed by the TUNEL technique, and beta cell mass and cross-sectional area of individual beta cells are determined by computerized image processing methods.

Key words

BrdU EdU PHH3 Ki67 antigen Replication TUNEL Apoptosis Beta cell mass Cross-sectional individual beta cell area Pancreatic islets 

Notes

Acknowledgments

This work has been supported by grants from the Catalan Diabetes Association (NT), University of Barcelona (NT), and Carlos III Health Institute (ISCIII) PI16/00462 co-funded by FEDER funds/European Regional Development Fund (ERDF) – “A Way to Build Europe” (EM) – and by CIBERDEM which is a project of ISCIII.

References

  1. 1.
    Montanya E (2014) Insulin resistance compensation: not just a matter of β-Cells? Diabetes 63(3):832–834.  https://doi.org/10.2337/db13-1843CrossRefPubMedGoogle Scholar
  2. 2.
    Tellez N, Joanny G, Escoriza J, Vilaseca M, Montanya E (2011) Gastrin treatment stimulates {beta}-cell regeneration and improves glucose tolerance in 95% pancreatectomized rats. Endocrinology 152(7):2580–2588CrossRefGoogle Scholar
  3. 3.
    Montanya E, Nacher V, Biarnes M, Soler J (2000) Linear correlation between beta-cell mass and body weight throughout the lifespan in Lewis rats: role of beta-cell hyperplasia and hypertrophy. Diabetes 49(8):1341–1346CrossRefGoogle Scholar
  4. 4.
    Knip M (2017) Diabetes: loss of β-cell mass - an acute event before T1DM presentation? Nat Rev Endocrinol 13(5):253–254.  https://doi.org/10.1038/nrendo.2017.33CrossRefPubMedGoogle Scholar
  5. 5.
    Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC (2003) Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52(1):102–110CrossRefGoogle Scholar
  6. 6.
    Eriksson O, Laughlin M, Brom M, Nuutila P, Roden M, Hwa A, Bonadonna R, Gotthardt M (2016) In vivo imaging of beta cells with radiotracers: state of the art, prospects and recommendations for development and use. Diabetologia 59(7):1340–1349.  https://doi.org/10.1007/s00125-016-3959-7CrossRefPubMedGoogle Scholar
  7. 7.
    Brom M, Woliner-van der Weg W, Joosten L, Frielink C, Bouckenooghe T, Rijken P, Andralojc K, Göke BJ, de Jong M, Eizirik DL, Béhé M, Lahoutte T, Oyen WJ, Tack CJ, Janssen M, Boerman OC, Gotthardt M (2014) Non-invasive quantification of the beta cell mass by SPECT with 111In-labelled exendin. Diabetologia 57(5):950–959.  https://doi.org/10.1007/s00125-014-3166-3CrossRefPubMedGoogle Scholar
  8. 8.
    Tellez N, Montanya E (2014) Gastrin induces ductal cell dedifferentiation and beta-cell neogenesis after 90% pancreatectomy. J Endocrinol 223(1):67–78.  https://doi.org/10.1530/JOE-14-0222CrossRefPubMedGoogle Scholar
  9. 9.
    Spijker HS, Ravelli RB, Mommaas-Kienhuis AM, van Apeldoorn AA, Engelse MA, Zaldumbide A, Bonner-Weir S, Rabelink TJ, Hoeben RC, Clevers H, Mummery CL, Carlotti F, de Koning EJ (2013) Conversion of mature human β-cells into glucagon-producing α-cells. Diabetes 62(7):2471–2480.  https://doi.org/10.2337/db12-1001CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Téllez N, Vilaseca M, Martí Y, Pla A, Montanya E (2016) β-Cell dedifferentiation, reduced duct cell plasticity, and impaired β-cell mass regeneration in middle-aged rats. Am J Physiol Endocrinol Metab 311(3):E554–E563.  https://doi.org/10.1152/ajpendo.00502.2015CrossRefPubMedGoogle Scholar
  11. 11.
    Talchai C, Xuan S, Lin HV, Sussel L, Accili D (2012) Pancreatic β cell dedifferentiation as a mechanism of diabetic β cell failure. Cell 150(6):1223–1234.  https://doi.org/10.1016/j.cell.2012.07.029CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Inada A, Nienaber C, Katsuta H, Fujitani Y, Levine J, Morita R, Sharma A, Bonner-Weir S (2008) Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth. Proc Natl Acad Sci U S A 105(50):19915–19919CrossRefGoogle Scholar
  13. 13.
    Thorel F, Nepote V, Avril I, Kohno K, Desgraz R, Chera S, Herrera PL (2010) Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss. Nature 464(7292):1149–1154CrossRefGoogle Scholar
  14. 14.
    Russ HA, Bar Y, Ravassard P, Efrat S (2008) In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing. Diabetes 57(6):1575–1583.  https://doi.org/10.2337/db07-1283CrossRefPubMedGoogle Scholar
  15. 15.
    Téllez N, Montolio M, Estil-les E, Escoriza J, Soler J, Montanya E (2007) Adenoviral overproduction of interleukin-1 receptor antagonist increases beta cell replication and mass in syngeneically transplanted islets, and improves metabolic outcome. Diabetologia 50(3):602–611.  https://doi.org/10.1007/s00125-006-0548-1CrossRefPubMedGoogle Scholar
  16. 16.
    Estil les E, Téllez N, Escoriza J, Montanya E (2012) Increased β-cell replication and β-cell mass regeneration in syngeneically transplanted rat islets overexpressing insulin-like growth factor II. Cell Transplant 21(10):2119–2129.  https://doi.org/10.3727/096368912X638955CrossRefPubMedGoogle Scholar
  17. 17.
    Estil les E, Téllez N, Soler J, Montanya E (2009) High sensitivity of beta-cell replication to the inhibitory effects of interleukin-1beta: modulation by adenoviral overexpression of IGF2 in rat islets. J Endocrinol 203(1):55–63.  https://doi.org/10.1677/JOE-09-0047CrossRefPubMedGoogle Scholar
  18. 18.
    Téllez N, Montolio M, Biarnés M, Castaño E, Soler J, Montanya E (2005) Adenoviral overexpression of interleukin-1 receptor antagonist protein increases beta-cell replication in rat pancreatic islets. Gene Ther 12(2):120–128.  https://doi.org/10.1038/sj.gt.3302351CrossRefPubMedGoogle Scholar
  19. 19.
    Hija A, Salpeter S, Klochendler A, Grimsby J, Brandeis M, Glaser B, Dor Y (2014) G0-G1 transition and the restriction point in pancreatic β-cells in vivo. Diabetes 63(2):578–584.  https://doi.org/10.2337/db12-1035CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Diermeier-Daucher S, Clarke ST, Hill D, Vollmann-Zwerenz A, Bradford JA, Brockhoff G (2009) Cell type specific applicability of 5-ethynyl-2′-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A 75(6):535–546.  https://doi.org/10.1002/cyto.a.20712CrossRefPubMedGoogle Scholar
  21. 21.
    Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105(7):2415–2420.  https://doi.org/10.1073/pnas.0712168105CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Carballar R, Canyelles ML, Fernández C, Martí Y, Bonnin S, Castaño E, Montanya E, Téllez N (2017) Purification of replicating pancreatic β-cells for gene expression studies. Sci Rep 7(1):17515.  https://doi.org/10.1038/s41598-017-17776-2CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.CIBER of Diabetes and Metabolic Diseases, CIBERDEMBarcelonaSpain
  2. 2.Bellvitge Biomedical Research Institute, IDIBELLBarcelonaSpain
  3. 3.University of BarcelonaBarcelonaSpain
  4. 4.Endocrine UnitHospital Universitari de BellvitgeBarcelonaSpain

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