It is well established that changes in insulin secretion play an important role in the pathogenesis of type 2 diabetes. However, the underlying mechanisms of secretory changes occurring at the pancreatic beta-cell level are not fully elucidated. Therefore, this thesis focuses on the modulation of beta-cell function, particularly insulin granule exocytosis, during the progression of type 2 diabetes using the BKS.Cg-Dock7m+/+Leprdb/J (Leprdb mice) as a model of disease.
I applied a variety of in vivo and in vitro methods to this project and further refined a live-cell two-photon assay which I used to measure the numbers and kinetics of granule fusion events within intact islets.
Using this latter technique, in the first part of the project I aimed to test a prevalent hypothesis that granule fusion behaviour changes in disease. Three indices of the post-fusion granule behaviour were compared between islets from animals with overt diabetes (db/db) and wild type. There was no difference in two indices of the fusion-granule behaviour; lifetime of granule fusion and post-fusion fluorescence intensity. The only change was a small increase in the percentage of recaptured granules (or “kiss-and-run” exocytosis) in db/db islets. In contrast to this minor change in granule fusion kinetics, further work showed that the main secretory deficit of frank diabetes, in db/db islets, is due to the loss of responding beta-cells and a reduction in the numbers of exocytic fusing granules in the remaining responsive cells.
In the second part of the project I studied disease progression in the Leprdb model. Based on the area under the curve of the glucose tolerance tests, each db/db mouse was classified into one of four stages of disease, ranging from pre-diabetes (stage 1) to severe diabetes (stage 4). The results indicated that changes in islet size and insulin content occurred across all stages of disease severity. At stage 1, there was an increase in islet insulin content due to both an increased number of beta-cells and increased insulin content in each cell, whereas at stages 2-4, there was a progressive loss of islet insulin content that is a reflection of loss of content from individual beta-cells.
Finally, in terms of beta-cell function, the data revealed that modulation of function characterised all stages of disease severity in db/db mice. In stage 1 there was an upregulation in the number of fusion of insulin-containing granules as well as the prevalence of compound exocytosis. At later disease stages there was a loss of glucose-induced insulin-granule fusion which my data indicated was due to impairment in glucose sensing. This work demonstrates that beta-cell function is intimately associated with pre-diabetes and progression to diabetes and adds weight to clinical strategies for therapeutic intervention as early as possible.
In summary, the results show that the major factors in the disease phenotype are changes in the numbers of responding beta-cells and the numbers of fusing granules rather than the kinetics of the fusion granules. Moreover, my work provides further evidence for the importance of modulation in beta-cell function at all stages of disease.