Analysis of endocrine pancreas using two-photon excitation imaging

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Regulated insulin secretion is a key step in glucose metabolism, and its dysfunction is one of the major factors of pathophysiology in type2 diabetes mellitus. From the studies of patients with type 2 diabetes or animal models, some defects were reported in the process of exocytosis. We constructed various kinds of cDNA expression vector carrying exocytic protein or granule protein labeled with photoactivatable GFPs. We transfected these cDNAs into the insulin-secreting cells, and analyzed the fluorescent properties. We also studied the mechanism of exocytosis, using FRET probe of SNAP25, one of the SNARE proteins expressed in plasma membrane, by fusing two kinds of GFP variants. We measured the conformational change of SNAP25 inside the intact pancreatic islets, and analyzed the correlation with quantal insulin exocytic events.