Fusion pore dynamics of insulin granules in intact islets of db/db mice — ASN Events
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Fusion pore dynamics of insulin granules in intact islets of db/db mice (#266)

Oanh H. Do 1 , Jiun T. Low 1 , Justin Mitchell 1 , Peter Thorn 1
  1. The University of Queensland, St Lucia, QLD, Australia

Type-2 diabetes manifests as a disorder not only in insulin action but also as a reduction in insulin secretion from the islets of Langerhans. It has been suggested that the latter is due to differences in the mechanism of fusion on insulin-containing granules (Rorsman and Renström, 2003). In this study, we measured single granule fusion events to evaluate how a stimulated beta-cell secretes insulin in intact islets of db/db mice; a progressive model of type-2 diabetes.

Db/db mice (13–20 weeks old) were diagnosed with diabetes by glucose tolerance test before sacrificing. Isolated islets were cultured for 2-3 days in RPMI-1640 and preincubated for 30 minutes in an extracellular buffer containing 3mM glucose before stimulating with different glucose concentrations. Two-photon imaging was recorded in 20 minutes at 35oC with exocytic events identified as the appearance of small fluorescent spots (diameter∼400nm), reflecting the entry of extracellular sulforhodamine-B (Takahashi and Kasai, 2007). We have evidence that these fusion events are due to the exocytosis of insulin granules. Islets from db/+ and +/+ were used as control.

In the two-photon focal plane, we observed a cross-section through 23.6 ±0.26 cells (mean ±SEM), approximately 2048 µm3 of the islet volume (N=95 islets). Within this volume of islet, the number of exocytic events in the wild-type was glucose-dependent: 6.8±1.4(3mM),10.9 ±3.2(6mM), 31.5±3.7(15mM) and 31.4±3.8(20mM) (N=39 islets, 4 mice). The heterozygotes showed a similar pattern in terms of glucose induced insulin secretion (N=34 islets). However, there was a significant decrease of exocytic events in the homozygotes compared to their lean littermates 2±0.8 (3mM), 6±2.4 (6mM), 5.2±2.4 (15mM) and 6.8±1.6 (20mM) (N=22 islets).

These results indicated that the number of exocytic events in an intact islet was glucose dependent. Db/db islets, however, demonstrated a reduction in granule recruitment. We are looking further into the individual event to explore the defect in insulin granule fusion of the db/db mice.