Studies on proteins involved in the molecular regulation of insulin exocytosis

Abstract: Type 2 diabetes mellitus is a disease characterized by impaired insulin secretion and insulin resistance. Better understanding of molecular mechanisms of insulin secretion may help to optimize type 2 diabetes management. The present work has thus investigated the expression of exocytotic proteins in insulin-producing beta-cells and their regulatory roles in insulin exocytosis. Western blotting and immunofluorescence histochemistry revealed that the syntaxin-interacting protein, mammalian homologue of C. elegans unc-18 gene (Munc-18), was expressed in insulin-secreting beta-cells. Munc-18 was present in both cytosol- and membrane-enriched fractions and was associated with syntaxin 1 in HIT-T15 cells. Administration of Munc-18 peptide or Munc-18 antiserum to permeabilized HIT-T15 cells increased insulin release, but had no effect on voltage-dependent Ca2+ channel activity. The data indicate that Munc-18 is a regulator of the insulin secretory machinery via a mechanism not involving syntaxin-associated Ca2+ channels. Tomosyn, another syntaxin-interacting protein, was also identified in pancreatic beta-cells. RT-PCR revealed that mRNAs for all three so far identified tomosyn isoforms were expressed in RINm5F cells and rat pancreatic islets. Overexpression of tomosyn in mouse beta-cells significantly decreased exocytosis as measured by changes in membrane capacitance using the patch-clamp technique, indicating an essential role of tomosyn in insulin secretion. The Munc-18-interacting protein Mint1 was demonstrated in beta-cells by immunohistochemistry and Western blotting. RT-PCR showed the expression of amplified PCR products corresponding to Mint1, Mint2 and Mint3 in RINm5F cells and rat islets. Immunohistochemistry with double-labeling revealed that Mint1 immunoreactivity was colocalized with Munc-18 in rat pancreatic islet cells. Mint1 immunoreactivity was preferentially distributed in the periphery of the cells, whereas Munc-18 immunoreactivity was mainly punctate in the cytosol. The presence of Mint in insulin-secreting beta-cells and the colocalization of Mint1 with Munc-18 suggest a functional role for Mint in the regulation of insulin secretion. Exocytosis of insulin-containing secretory granules depends on phosphatidylinositol (PI) 4-kinase activity, since inhibition of this enzyme suppressed glucose-stimulated insulin secretion. Intracellular application of PI 4-phosphate (PI(4)P) and PI 4,5-biphosphates (PI(4,5)P2) stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules in a readily releasable pool. Reducing cytoplasmic ADP concentration, which mimicks the effects of glucose stimulation, activated PI 4-kinase and increased exocytosis, whereas changes of the ATP-concentration in the physiological range had little effect. The PI(4,5)P2-binding protein, Ca2+-dependent activator protein for secretion (CAPS), was present in beta-cells and neutralization of the protein abolished both Ca2+- and PI(4,5)P2-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P2 represents a metabolic sensor in the beta-cell by virtue of its capacity to regulate the release competence of the secretory granules. In type 2 diabetic Goto-Kakizaki (GK) rats, the pancreatic islets frequently showed an irregular structure with cells separated from each other. The immunoreactivities for vesicle-associated membrane protein-2 (VAMP-2), synaptotagmin III, cysteine string protein (CSP), Munc-18, tomosyn, N-ethylmaleimide-sensitive factor (NSF), alpha-soluble NSF attachment protein (alpha-SNAP), synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin in pancreatic islets of GK rats were weaker. Insulin immunoreactivity was also decreased in GK rat beta-cells, whereas no detectable alteration in the actin immunoreactivity was shown. The data suggest that reduced expression of exocytotic proteins and decreased insulin content may contribute to the diabetic syndrome of GK rats. Taken together, we have demonstrated the presence of Munc-18, tomosyn, Mint and CAPS in beta-cells. Munc-18 and tomosyn are associated with syntaxin and are involved in the regulation of insulin secretion from beta-cells. CAPS is essential for insulin secretion and functions downstream of PI(4,5)P2. PI 4-kinase serves as a metabolic sensor of beta-cells and regulates priming of secretory granules via generation of PI(4,5)P2. In pancreatic islets of type 2 diabetic GK rats, the immunoreactivities for exocytotic proteins and insulin are decreased, which may be associated with impaired insulin secretion and the diabetic syndrome present in GK rats.