Advertisement

Preparation of Islets from Rat Pancreas and Assessment of Islet Function

  • Paul C. Guest
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1916)

Abstract

The islets of Langerhans release vital hormones involved in the regulation of blood sugar and other aspects of metabolism. The islets are housed in diffuse clusters of cells within the exocrine pancreas and, therefore, purification of these cells for research or transplant purposes is a difficult undertaking. Here, a detailed protocol is presented for purification of islets from rat pancreas using limited collagenase digestion and step gradient centrifugation techniques. In addition, a method for assessing islet viability is presented using perifusion under both basal and stimulatory glucose conditions, with measurement of the hormone released using an immunoassay for insulin.

Key words

Islets of Langerhans Collagenase digestion Density gradient centrifugation Insulin Immunoassay 

References

  1. 1.
    Okomoto H (2008) Molecular biology of the islets of Langerhans, 1st edn. Cambridge University Press, Cambridge (13 Oct. 2008). ISBN-10: 0521088003Google Scholar
  2. 2.
    Jo J, Hara M, Ahlgren U, Sorenson R, Periwal V (2012) Mathematical models of pancreatic islet size distributions. Islets 4(1):10–19CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Seino S, Bell GI (eds) (2007) Pancreatic beta cell in health and disease. Springer, Tokyo 2008 edn (18 Dec. 2007). ISBN-10: 4431754512Google Scholar
  4. 4.
    Schatz H, Nierle C, Pfeiffer EF (1975) (Pro-) insulin biosynthesis and release of newly synthesized (pro-) insulin from isolated islets of rat pancreas in the presence of amino acids and sulphonylureas. Eur J Clin Investig 5(6):477–485CrossRefGoogle Scholar
  5. 5.
    Billaudel B, Sutter BC (1982) Effect of corticosterone upon insulin biosynthesis and storage by isolated rat Langerhans islets. Diabete Metab 8(4):283–287PubMedGoogle Scholar
  6. 6.
    Hanson RL, Isaacson CM, Boyajy LD (1985) Stimulation of insulin secretion from isolated rat islets by SaRI 59-801. Diabetes 34(6):548–552CrossRefPubMedGoogle Scholar
  7. 7.
    Grimaldi KA, Siddle K, Hutton JC (1987) Biosynthesis of insulin secretory granule membrane proteins. Control by glucose. Biochem J 245(2):567–573CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Guest PC, Rhodes CJ, Hutton JC (1989) Regulation of the biosynthesis of insulin-secretory-granule proteins. Co-ordinate translational control is exerted on some, but not all, granule matrix constituents. Biochem J 257(2):431–437CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Guest PC, Pipeleers D, Rossier J, Rhodes CJ, Hutton JC (1989) Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans. Biochem J 264(2):503–508CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Guest PC, Bailyes EM, Rutherford NG, Hutton JC (1991) Insulin secretory granule biogenesis. Co-ordinate regulation of the biosynthesis of the majority of constituent proteins. Biochem J 274(Pt 1):73–78CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ortsäter H, Bergsten P (2006) Protein profiling of pancreatic islets. Expert Rev Proteomics 3(6):665–675CrossRefPubMedGoogle Scholar
  12. 12.
    Salvatori AS, Elrick MM, Samson WK, Corbett JA, Yosten GL (2014) Neuronostatin inhibits glucose-stimulated insulin secretion via direct action on the pancreatic α-cell. Am J Physiol Endocrinol Metab 306(11):E1257–E1263CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Guest PC (2017) 2D gel electrophoresis of insulin secretory granule proteins from biosynthetically labelled pancreatic islets. Adv Exp Med Biol 974:167–174CrossRefPubMedGoogle Scholar
  14. 14.
    Lacy PE, Kostianovsky M (1967) Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16(1):35–39CrossRefPubMedGoogle Scholar
  15. 15.
    Hisaoka M, Haratake J, Yamamoto O, Horie A (1990) Three-dimensional observation of the rat endocrine pancreas by a scanning electron microscope. J UOEH 12(3):315–322CrossRefPubMedGoogle Scholar
  16. 16.
    Hanks JH, Wallace RE (1949) Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc Soc Exp Biol Med 71(2):196–200CrossRefPubMedGoogle Scholar
  17. 17.
    Sobey WJ, Beer SF, Carrington CA, Clark PMS, Frank BH, Gray IP et al (1989) Sensitive and specific two site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. Biochem J 260:535–541CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Rabinovitch A, Gutzeit A, Kikuchi M, Cerasi E, Renold AE (1975) Defective early phase insulin release in perifused isolated pancreatic islets of spiny mice (Acomys cahirinus). Diabetologia 11(5):457–465CrossRefPubMedGoogle Scholar
  19. 19.
    Karabatas LM, Arata M, Anaya L, Cresto JC, Pivetta OH, Basabe JC (1988) First phase of insulin secretion stimulated by glucose plus theophylline and inhibitory effect of somatostatin in genetically diabetic mice (C57BL/KsJ-mdb). Diabetologia 31(6):375–378CrossRefPubMedGoogle Scholar
  20. 20.
    Dachicourt N, Serradas P, Giroix MH, Gangnerau MN, Portha B (1996) Decreased glucose-induced cAMP and insulin release in islets of diabetic rats: reversal by IBMX, glucagon, GIP. Am J Phys 271(4 Pt 1):E725–E732Google Scholar

Copyright information

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

Authors and Affiliations

  • Paul C. Guest
    • 1
  1. 1.Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of BiologyUniversity of Campinas (UNICAMP)CampinasBrazil

Personalised recommendations