Glucose-dependent docking and SNARE protein-mediated exocytosis in mouse pancreatic alpha-cell

Abstract

The function of alpha-cells in patients with type 2 diabetes (T2D) is often disturbed; glucagon secretion is increased at hyperglycaemia, yet fails to respond to hypoglycaemia. A crucial mechanism behind the fine-tuned release of glucagon relies in the exocytotic machinery including SNARE proteins. Here we aimed to investigate the temporal role of syntaxin 1A and SNAP-25 in mouse alpha-cell exocytosis. First, we used confocal imaging to investigate glucosedependency in the localisation of SNAP-25 and syntaxin 1A. SNAP-25 was mainly distributed in the plasma membrane at 2.8 mM glucose whereas the syntaxin 1A distribution in the plasma membrane, as compared to the cytosolic fraction, was highest at 8.3 mM glucose. Further, following inclusion of an antibody against SNAP-25 or syntaxin 1A, exocytosis evoked by a train of ten depolarisations and measured as an increase in membrane capacitance was reduced by ~50%. Closer inspection revealed a reduction in the refilling of granules from the reserve pool (RP), but also showed a decreased size of the readily releasable pool (RRP) by ~45%. Disparate from the situation in pancreatic beta-cells, the voltage-dependent Ca 2+ -current was not reduced, but the Ca 2+ -sensitivity of exocytosis decreased by the antibody against syntaxin 1A. Finally, ultrastructural analysis revealed that the number of docked granules was >2-fold higher at 16.7 mM than at 1 mM glucose. We conclude that syntaxin 1A and SNAP-25 are necessary for alphacell exocytosis and regulate fusion of granules belonging to both the RRP and RP without affecting the Ca 2+ -current.3

Calcium Current Inactivation Rather than Pool Depletion Explains Reduced Exocytotic Rate with Prolonged Stimulation in Insulin-Secreting INS-1 832/13 Cells

“…Our data showed a near-linear ΔC m to Q relation (Fig. 1C), as previously observed in mouse beta- [8], [10], [20] and alpha-cells [30], [36]. In contrast, human beta-cells in situ show a nonlinear, concave ΔC m to Q relation [21].…”

“…Responses to the various depolarizations applied to the same cell are likely correlated, while responses in different cells are not, but reflect genuine biological heterogeneity. In order to handle this scenario appropriately, we applied statistical mixed-effects modeling, as done previously in our studies of exocytosis in mouse beta- and alpha-cells [20], [30].…”

“…As in our previous studies [20], [30] a significant between-cell variation was present, and we proceeded by fitting linear mixed-effects models [23], which are more appropriate for representing clustered data, such as in our case where several observations are done on each cell.…”

CFTR and Anoctamin 1 (ANO1) contribute to cAMP amplified exocytosis and insulin secretion in human and murine pancreatic beta-cells

“…Islets were collected after insulin secretion assay and prepared for electron microscopy, examined and analyzed as previously described [26]. The granule volume density (N v ) and surface density (N s ) were calculated using in-house software programmed in MatLab.…”

Functional identification of islet cell types by electrophysiological fingerprinting

“…The first is that β-cells are larger than non-β-cells [3,34–44]. The second is that α-, β- and δ-cells possess distinct ionic channels or similar channels that exhibit different properties.…”