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Imaging of Insulin Exocytosis from Pancreatic Beta Cells

  • Mica Ohara-Imaizumi
  • Kyota Aoyagi
  • Shinya Nagamatsu
Protocol
Part of the Neuromethods book series (NM, volume 83)

Abstract

The pancreatic beta cells are highly sensitive to the glucose concentration, and the minute to minute regulation of insulin secretion by glucose occurs at the level of exocytosis of insulin, transcriptional rate of the insulin gene, translation of the mRNA, and processing of the proinsulin to mature insulin (Mahato et al., Biochem J 174:517–526, 1978; Hedeskov, Physiol Rev 60:442–509, 1980; Welsh et al., J Biol Chem 260:13590–13594, 1985; Welsh et al., Biochem J 235:459–467, 1986; Newgard and McGarry, Annu Rev Biochem 64:689–719,1995). Thus, glucose is the most important physiological regulator of insulin gene transcription, biosynthesis, and secretion. The insulin biosynthetic rate is not always proportional to the secretion rate. Insulin secretion occurs by the fusion of insulin granules and plasma membrane (Nagamatsu, Pancreatic beta cells in health and disease 177–194, 2008). Insulin granules exist as large–dense core structures that are discernible by electron microscopy as an electron-dense interior surrounded by a clear region in a 300–350-nm intracellular membrane-delineated compartment (Greider et al., J Cell Biol 41:162–166, 1969; Lange, J Ultrastruct Res 46:301–307, 1974).

It is generally accepted that SNARE proteins play a crucial role in insulin exocytotic process (Wollheim et al, Diabetes Rev 4:276–297, 1996). Several reports revealed that t-SNARE protein syntaxin 1(syt1) and SNAP-25 are plasma membrane localized, whereas the v-SNARE protein VAMP2 is associated with insulin secretory granules (Regazzi et al., EMBO J 14:2723–2730, 1995; Sadoul et al., J Cell Biol 128:1019–1028, 1995; Boyd et al., J Biol Chem 270:18216–18218, 1995; Nagamatsu et al., J Biol Chem 271:1160–1165, 1996). Furthermore, we previously reported that alteration of SNARE proteins expression is closely associated with type 2 diabetes (Nagamatsu et al., Diabetes 48:2367–2373, 1999; Ohara-Imaizumi et al., Diabetologia 47:2200–2207, 2004). In order to explore the mechanism of insulin exocytosis, methods for gene transfer and imaging system are powerful tools. Here, we describe about the methods for studying the insulin exocytosis from the point of view of imaging techniques and gene transfer system.

Key words

Exocytosis Insulin Electroporation Fluorescence TIRF 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Mica Ohara-Imaizumi
    • 1
  • Kyota Aoyagi
    • 1
  • Shinya Nagamatsu
    • 1
  1. 1.Department of BiochemistryKyorin University School of MedicineTokyoJapan

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