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Assessment of β-Cell Replication in Isolated Rat Islets of Langerhans

  • Louise T. DalgaardEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2029)

Abstract

Pancreatic β-cells in the islets of Langerhans secrete insulin in response to the rise in glucose levels following food intake. The hypoglycemic action of insulin applies a strong evolutionary brake on β-cell division. However, under some conditions β-cells can be stimulated to enter cell cycle progression and divide, for example following exposure to increased glucose levels or during pregnancy. Here, a protocol is described for the isolation of rat adult islets of Langerhans, followed by culture of intact islets in Matrigel and measurement of β-cell replication by the incorporation of ethynyldeoxyuridine (EdU). EdU positive cells are revealed by a click reaction, nuclei are visualized using a DNA-binding fluorophore (Hoechst 33342), and β-cells are identified using immunofluorescence detection.

Key words

Islet of Langerhans β-Cell Proliferation Mitosis Ethynyldeoxyuridine (EdU) Glucose Insulin Hoechst 33342 

References

  1. 1.
    Meier JJ, Butler AE, Saisho Y, Monchamp T, Galasso R, Bhushan A, Rizza RA, Butler PC (2008) Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans. Diabetes 57(6):1584–1594CrossRefGoogle Scholar
  2. 2.
    Butler PC, Meier JJ, Butler AE, Bhushan A (2007) The replication of beta cells in normal physiology, in disease and for therapy. Nat Clin Pract Endocrinol Metab 3(11):758–768CrossRefGoogle Scholar
  3. 3.
    Van Assche FA, Aerts L, de PF (1978) A morphological study of the endocrine pancreas in human pregnancy. Br J Obstet Gynaecol 85(11):818–820CrossRefGoogle Scholar
  4. 4.
    Butler AE, Cao-Minh L, Galasso R, Rizza RA, Corradin A, Cobelli C, Butler PC (2010) Adaptive changes in pancreatic beta cell fractional area and beta cell turnover in human pregnancy. Diabetologia 53(10):2167–2176.  https://doi.org/10.1007/s00125-010-1809-6CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Saisho Y, Butler AE, Meier JJ, Monchamp T, Allen-Auerbach M, Rizza RA, Butler PC (2007) Pancreas volumes in humans from birth to age one hundred taking into account sex, obesity, and presence of type-2 diabetes. Clin Anat 20(8):933–942.  https://doi.org/10.1002/ca.20543CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Rhodes CJ (2005) Type 2 diabetes-a matter of beta-cell life and death? Science 307(5708):380–384CrossRefGoogle Scholar
  7. 7.
    Ackermann AM, Gannon M (2007) Molecular regulation of pancreatic beta-cell mass development, maintenance, and expansion. J Mol Endocrinol 38(1-2):193–206CrossRefGoogle Scholar
  8. 8.
    Dor Y, Brown J, Martinez OI, Melton DA (2004) Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429(6987):41–46CrossRefGoogle Scholar
  9. 9.
    Georgia S, Bhushan A (2004) Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass. J Clin Invest 114(7):963–968.  https://doi.org/10.1172/JCI22098CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Schmidt SF, Madsen JG, Frafjord KO, Poulsen L, Salo S, Boergesen M, Loft A, Larsen BD, Madsen MS, Holst JJ, Maechler P, Dalgaard LT, Mandrup S (2016) Integrative genomics outlines a biphasic glucose response and a ChREBP-RORgamma axis regulating proliferation in beta cells. Cell Rep 16(9):2359–2372.  https://doi.org/10.1016/j.celrep.2016.07.063CrossRefPubMedGoogle Scholar
  11. 11.
    Donath MY, Halban PA (2004) Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications. Diabetologia 47(3):581–589CrossRefGoogle Scholar
  12. 12.
    Yesil P, Michel M, Chwalek K, Pedack S, Jany C, Ludwig B, Bornstein SR, Lammert E (2009) A new collagenase blend increases the number of islets isolated from mouse pancreas. Islets 1(3):185–190.  https://doi.org/10.4161/isl.1.3.9556CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Science and EnvironmentRoskilde UniversityRoskildeDenmark

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