Abstract
The endocrine pancreas contains small clusters of 1,000–2,000 neuroendocrine cells termed islets of Langerhans. By secreting insulin, glucagon, or other hormones as circumstances dictate, islets play a central role in the control of glucose homeostasis in mammals. Islets are dispersed throughout the exocrine tissue and comprise only 1–2% of the volume of the whole organ; human pancreas contains about 106 islets whereas rodents have approximately 2 × 103 islets. The isolation of islets from the exocrine tissue usually begins with digestion of the pancreas with collagenase. Collagenase-containing medium is either injected into the pancreatic duct, and the organ left to digest in situ, or added after isolation of the pancreas and its dissection into small pieces ex vivo. Islets can then be separated from the exocrine tissue by gradient density or by handpicking. The islets obtained can either be used intact, for example, to measure insulin or glucagon secretion or be dispersed into single cells with a Ca2+-free medium or with trypsin/dispase. The latter facilitates the introduction of recombinant or trappable probes and microimaging studies of, for example, changes in cytosolic-free Ca2+ concentration or the dynamics of individual organelles or proteins.
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References
Ahren, B. (2000) Autonomic regulation of islet hormone secretion – implications for health and disease. Diabetologia 43, 393–410.
Stagner, J. I., Samols, E., and Bonner-Weir, S. (1988) Beta—alpha—delta pancreatic islet cellular perfusion in dogs. Diabetes 37, 1715–1721.
Leclerc, I., Woltersdorf, W. W., da Sylva Xavier, G., Rowe, R. L., Cross, S. E., Korbutt, G. S., Rajotte, R. V., Smith, R., and Rutter, G. A. (2004) Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. Am. J. Physiol. Endocrinol. Metab. 286, E1023–E1031.
Ravier, M. A., Gilon, P., and Henquin, J. C. (1999) Oscillations of insulin secretion can be triggered by imposed oscillations of cytoplasmic Ca2+ or metabolism in normal mouse islets. Diabetes 48, 2374–2382.
Ravier, M. A., and Rutter, G. A. (2005) Glucose or insulin, but not zinc ions, inhibit glucagon secretion from mouse pancreatic alpha-cells. Diabetes 54, 1789–1797.
Gotoh, M., Maki, T., Kiyoizumi, T., Satomi, S., and Monaco, A. P. (1985) An improved method for isolation of mouse pancreatic islets. Transplantation 40, 437–438.
Gotoh, M., Maki, T., Satomi, S., Porter, J., Bonner-Weir, S., O’Hara, C. J., and Monaco, A. P. (1987) Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase injection. Transplantation 43, 725–730.
Wollheim, C. B., Meda, P., and Halban, P. A. (1990) Isolation of pancreatic islets and primary culture of the intact microorgans or of dispersed islet cells. Methods Enzymol. 192, 188–223.
Ainscow, E. K., and Rutter, G. A. (2001) Mitochondrial priming modifies Ca2+ oscillations and insulin secretion in pancreatic islets. Biochem. J. 353, 175–180.
Kennedy, H. J., Pouli, A. E., Ainscow, E. K., Jouaville, L. S., Rizzuto, R., and Rutter, G. A. (1999) Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria. J. Biol. Chem. 274, 13281–13291.
Rutter, G. A., Theler, J. M., Murgia, M., Wollheim, C. B., Pozzan, T., and Rizzuto, R. (1993) Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion. J. Biol. Chem. 268, 22385–22390.
Pinton, P., Tsuboi, T., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2002) Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells. J. Biol. Chem. 277, 37702–37710.
Pouli, A. E., Emmanouilidou, E., Zhao, C., Wasmeier, C., Hutton, J. C., and Rutter, G. A. (1998) Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera. Biochem. J. 333 (Pt 1), 193–199.
Tsuboi, T., Zhao, C., Terakawa, S., and Rutter, G. A. (2000) Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event. Curr. Biol. 10, 1307–1310.
Tsuboi, T., Ravier, M. A., Parton, L. E., and Rutter, G. A. (2006) Sustained exposure to high glucose concentrations modifies glucose signaling and the mechanics of secretory vesicle fusion in primary rat pancreatic beta-cells. Diabetes 55, 1057–1065.
Riboulet-Chavey, A., Diraison, F., Siew, L. K., Wong, F. S., and Rutter, G. A. (2008) Inhibition of AMP-activated protein kinase protects pancreatic beta-cells from cytokine-mediated apoptosis and CD8+ T-cell-induced cytotoxicity. Diabetes 57, 415–423.
Jonas, J. C., Gilon, P., and Henquin, J. C. (1998) Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells. Diabetes 47, 1266–1273.
Salvalaggio, P. R., Deng, S., Ariyan, C. E., Millet, I., Zawalich, W. S., Basadonna, G. P., and Rothstein, D. M. (2002) Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function. Transplantation 74, 877–879.
Diraison, F., Parton, L., Ferre, P., Foufelle, F., Briscoe, C. P., Leclerc, I., and Rutter, G. A. (2004) Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Biochem. J. 378, 769–778.
Josefsen, K., Stenvang, J. P., Kindmark, H., Berggren, P. O., Horn, T., Kjaer, T., and Buschard, K. (1996) Fluorescence-activated cell sorted rat islet cells and studies of the insulin secretory process. J. Endocrinol. 149, 145–154.
He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998) A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95, 2509–2514.
Liu, Y. J., Vieira, E., and Gylfe, E. (2004) A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic alpha-cell. Cell Calcium 35, 357–365.
Tsien, R. Y. (1992) Intracellular signal transduction in four dimensions: from molecular design to physiology. Am. J. Physiol. 263, C723–C728.
Axelrod, D. (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89, 141–145.
Ravier, M. A., Tsuboi, T., and Rutter, G. A. (2008) Imaging a target of Ca2+ signalling: dense core granule exocytosis viewed by total internal reflection fluorescence microscopy. Methods 46, 233–238.
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Ravier, M.A., Rutter, G.A. (2010). Isolation and Culture of Mouse Pancreatic Islets for Ex Vivo Imaging Studies with Trappable or Recombinant Fluorescent Probes. In: Ward, A., Tosh, D. (eds) Mouse Cell Culture. Methods in Molecular Biology, vol 633. Humana Press. https://doi.org/10.1007/978-1-59745-019-5_12
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DOI: https://doi.org/10.1007/978-1-59745-019-5_12
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