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Focal secretion of insulin within intact islets of langerhans

J. Low, J. Mitchell, J. Bax and P. Thorn, School of Biomedical Sciences, The University of Queensland, St Lucia, QLD 4072, Australia.

The biochemical pathways that link glucose sensing in pancreatic β-cells to insulin secretion are well understood. However, in a more physiological context it is not clear how glucose controls/coordinates insulin secretion from the many hundreds of β-cells within the islets of Langerhans. Here we have set out to determine the basis of glucose sensing from β-cells within intact mouse pancreatic islets. With two-photon imaging of whole islets we identify each individual exocytic event within individual cells, induced by a range of different glucose concentrations.

We used 2-photon microscopy to measure insulin granule fusion from intact living islets isolated from CD-1 mice. The mice were humanely killed according to local ethics guidelines. The islets were isolated by a collagenase digestion procedure and cultured for 2-3 days prior to use.   Insulin granule fusion was recorded in response to a range of glucose concentrations. We observed a glucose dose-dependence in the numbers of fusion events that is very similar to our measured dose-dependence of insulin secretion. A Chi-squared test proves (P<0.01) that the exocytic responses are not evenly distributed; a few cells show many fusion events and ∼54% of cells (for example at 15 mM glucose) apparently have none. To explore this result we captured responses at 1 plane, then moved focus 5.5 μm and captured responses at a 2nd plane. We reasoned that if cells were non-responsive, then doubling the sampling volume should not disclose a response. Our data however show that now 44% of cells have no responses suggesting that cells are responding and that exocytosis must be unevenly distributed across single β cells.

To test this we calculated the ratio of numbers of fusion events in one plane compared to the other. An even distribution of exocytosis around a cell would give a ratio of 1:1. In contrast, we get a ratio of 1:6.6 which provides evidence for strong polarization of exocytosis in the β cells. This conclusion means our measurements of maximal exocytic activity must be in regions of hot spots of secretion within that cell. Here the measured fusion density is 1 granule per 2.29 μm2. We then measured insulin secretion and based on estimates of granule insulin content and numbers of β cells within islets we calculate that each β cell secretes 132 granules (in 20 minutes of 15 mM glucose). If all this exocytosis occurs within a focal hot-spot then 132 granules would fuse over an area of 302 μm2. The total surface area of a β cell is 784 μm2.

  We conclude that insulin granule exocytosis is focused within a small area, 38%, of β cells within intact islets.