Abstract
Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.
Highlights
Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting TC3 cell
Syt III/VII are located in insulincontaining secretory granules, and we suggest that Syt III/VII may be the Ca2؉ sensor or one of the Ca2؉ sensors for insulin exocytosis of the -cell
Identification of Synaptotagmin Isoforms in Islets and Insulin-secreting -Cells—The expression of synaptotagmin mRNA in pancreatic islet cells and insulinsecreting cell lines TC3 and RINm5F was examined by RTPCR with specific primers (Fig. 1). mRNAs of Syt isoform III, IV, V, and VII were found in pancreatic islet cells, whereas isoform I, II, III, IV, V, VII, and VIII were found in TC3 cells
Summary
Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting TC3 cell. Syt III mRNA was present in MIN6, RINm5F, HIT-T15, and TC6-f7 [29] cells and pancreatic islets [28], and the protein expression of Syt III in MIN6 cell and pancreatic islets was confirmed by one group [28] but not by another [29]. The aims of the current study were to examine the expression of various Syt isoforms in pancreatic islets as well as insulinsecreting -cell lines, the subcellular localization of Syt, and the functional role of Syt in insulin exocytosis using a -cell line overexpressing Syt. Insulin exocytosis from the -cell of the islets of Langerhans is stimulated by various physiological secretagogues that include glucose, amino acids, and receptor-mediated agonists such as acetylcholine, cholecystokinin, and glucagon like-peptide 1 [1,2,3,4,5,6,7]. The mechanisms by which Ca2ϩ induces insulin granule fusion with the plasma membrane of -cell remain unclear [1, 16, 18, 20, 21]
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