Abstract
Apoptosis of pancreatic beta cells is a feature of type 1 and type 2 diabetes, although by different effector mechanisms. In type 1 diabetes, beta cells are the targets of cytotoxic CD8+ T cells that kill by releasing the contents of their cytotoxic granules into the immunological synapse with the target beta cell. In type 2 diabetes, the mechanisms of beta cell apoptosis are less clear, but believed to be due to cellular stresses including endoplasmic reticulum stress and oxidative stress induced by chronic exposure to high concentrations of glucose, lipids, inflammatory cytokines, or islet amyloid polypeptide. Measuring apoptosis in primary islets can be more difficult than in a beta cell line because islets exist as a cluster of cells and it is often difficult to obtain sufficient cells for any particular type of assay. Here, we describe two different methods for measuring islet cell apoptosis. The first method is the measurement of DNA fragmentation, a hallmark of apoptosis, of islets that have been cultured with reagents that induce stress. The second method is the measurement of islet lysis by activated cytotoxic T cells. We describe methods using mouse islets, but these can easily be adapted for human islets.
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References
Thomas HE, McKenzie MD, Angstetra E, Campbell PD, Kay TW (2009) Beta cell apoptosis in diabetes. Apoptosis 14:1389–1404
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 52:102–110
Rahier J, Guiot Y, Goebbels RM, Sempoux C, Henquin JC (2008) Pancreatic beta-cell mass in European subjects with type 2 diabetes. Diabetes Obes Metab 10(Suppl 4):32–42
Yoon KH, Ko SH, Cho JH, Lee JM, Ahn YB, Song KH, Yoo SJ, Kang MI, Cha BY, Lee KW, Son HY, Kang SK, Kim HS, Lee IK, Bonner-Weir S (2003) Selective beta-cell loss and alpha-cell expansion in patients with type 2 diabetes mellitus in Korea. J Clin Endocrinol Metab 88:2300–2308
Weir GC, Bonner-Weir S (2013) Islet beta cell mass in diabetes and how it relates to function, birth, and death. Ann N Y Acad Sci 1281:92–105
Szot GL, Koudria P, Bluestone JA (2007) Murine pancreatic islet isolation. J Vis Exp 7:255
Hotchkiss RS, Strasser A, McDunn JE, Swanson PE (2009) Cell death. N Engl J Med 361:1570–1583
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279
Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461
McKenzie MD, Carrington EM, Kaufmann T, Strasser A, Huang DC, Kay TW, Allison J, Thomas HE (2008) Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells. Diabetes 57:1284–1292
McKenzie MD, Jamieson E, Jansen ES, Scott CL, Huang DC, Bouillet P, Allison J, Kay TW, Strasser A, Thomas HE (2010) Glucose induces pancreatic islet cell apoptosis that requires the BH3-only proteins Bim and Puma and multi-BH domain protein Bax. Diabetes 59:644–652
Brunner KT, Mauel J, Cerottini JC, Chapuis B (1968) Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14:181–196
Campbell PD, Estella E, Dudek NL, Jhala G, Thomas HE, Kay TW, Mannering SI (2008) Cytotoxic T-lymphocyte-mediated killing of human pancreatic islet cells in vitro. Hum Immunol 69:543–551
Dudek NL, Thomas HE, Mariana L, Sutherland RM, Allison J, Estella E, Angstetra E, Trapani JA, Santamaria P, Lew AM, Kay TW (2006) Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways. Diabetes 55:2412–2418
Verdaguer J, Schmidt D, Amrani A, Anderson B, Averill N, Santamaria P (1997) Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice. J Exp Med 186:1663–1676
Sutton VR, Estella E, Li C, Chen M, Thomas HE, Kay TW, Trapani JA (2006) A critical role for granzyme B, in addition to perforin and TNFalpha, in alloreactive CTL-induced mouse pancreatic beta cell death. Transplantation 81:146–154
Acknowledgements
The authors would like to thank Dr Nadine L Dudek for originally optimizing the protocol for 51Cr release assays with islets. This work was supported by a project (APP1032610) and a program (APP1037321) grant from the National Health and Medical Research Council of Australia (NHMRC), and a NHMRC/Juvenile Diabetes Research Foundation (JDRF) joint special program grant in type 1 diabetes (APP466658). H.E.T. is supported by a fellowship from the NHMRC (APP1042735) and J.A.W. is supported by a University of Melbourne Viola Edith Reid Bequest Scholarship. St Vincent’s Institute is supported in part by the Victorian Government’s Operational Infrastructure Support Program.
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Wali, J.A., Trivedi, P., Kay, T.W., Thomas, H.E. (2015). Measuring Death of Pancreatic Beta Cells in Response to Stress and Cytotoxic T Cells. In: Oslowski, C. (eds) Stress Responses. Methods in Molecular Biology, vol 1292. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2522-3_12
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DOI: https://doi.org/10.1007/978-1-4939-2522-3_12
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2521-6
Online ISBN: 978-1-4939-2522-3
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