Abstract
Synaptotagmin (Syt) is involved in Ca2+ -regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+ -dependent secretory process. Previous studies have yielded conflicting results as to which Syt isoform is present on the secretory granules in the native beta-cell. Here we show by western blotting and RT-PCR analysis, the presence of both Syt V and Syt IX in rat pancreatic islets and in the clonal beta-cell line INS-1E. The subcellular distribution of the two Syt isoforms was assessed by confocal microscopy and by sedimentation in a continuous sucrose density gradient in INS-1E cells. These experiments show that both proteins colocalize with insulin-containing secretory granules but are absent from synaptic-like microvesicles. Further immunofluorescence studies performed in primary pancreatic endocrine cells revealed that Syt V is present in glucagon-secreting alpha-cells, whereas Syt IX is associated with insulin granules in beta-cells. Transient overexpression of Syt V and Syt IX did not alter exocytosis in INS-1E cells. Finally, reduction of the expression of both Syt isoforms by RNA interference did not change basal secretion. Remarkably, hormone release in response to glucose was selectively and strongly reduced, indicating that Syt V and Syt IX are directly involved in the Ca2+ -dependent stimulation of exocytosis.
Highlights
Insulin exocytosis from the β-cells of the islets of Langerhans is stimulated by various physiological secretagogues that include glucose, amino acids and receptor agonists such as acetylcholine, cholecystokinin and glucagon like-peptide 1
We examine the functional impact of Syt V and Syt IX on insulin exocytosis in INS-1E cells, comparing their overexpression as well as suppression using RNA interference (RNAi)
We investigated the presence of Syt IX mRNA in the insulin-secreting cell line INS-1E, rat pancreatic islets and rat brain
Summary
Insulin exocytosis from the β-cells of the islets of Langerhans is stimulated by various physiological secretagogues that include glucose, amino acids and receptor agonists such as acetylcholine, cholecystokinin and glucagon like-peptide 1. The mechanism by which Ca2+ induces insulin-granule fusion with the plasma membrane in the β-cell is poorly understood (Wollheim and Maechler, 2002; Rorsman and Renstrom, 2003). Vertebrates express at least 15 isoforms, which share a common structure composed of an intravesicular Nterminal domain and a single membrane-spanning region, followed by the two C-terminal C2A and C2B domains (Südhof and Rizo, 1996; Rickman et al, 2004). These domains mediate the Ca2+-dependent phospholipid binding that is essential for membrane interactions
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