Neuronal calcium sensor-1 potentiates glucose-dependent exocytosis in pancreatic β cells through activation of phosphatidylinositol 4-kinase β

Abstract

Cytosolic free Ca 2؉ plays an important role in the molecular mechanisms leading to regulated insulin secretion by the pancreatic ␤ cell. A number of Ca 2؉ -binding proteins have been implicated in this process. Here, we define the role of the Ca 2؉ -binding protein neuronal Ca 2؉ sensor-1 (NCS-1) in insulin secretion. In pancreatic ␤ cells, NCS-1 increases exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in the readily releasable pool. The effect of NCS-1 on exocytosis is mediated through an increase in phosphatidylinositol (PI) 4-kinase ␤ activity and the generation of phosphoinositides, specifically PI 4-phosphate and PI 4,5-bisphosphate. In turn, PI 4,5-bisphosphate controls exocytosis through the Ca 2؉ -dependent activator protein for secretion present in ␤ cells. Our results provide evidence for an essential role of phosphoinositide synthesis in the regulation of glucose-induced insulin secretion by the pancreatic ␤ cell. We also demonstrate that NCS-1 and its downstream target, PI 4-kinase ␤, are critical players in this process by virtue of their capacity to regulate the release competence of the secretory granules.insulin ͉ phosphoinositides ͉ islet ͉ secretion ͉ Ca 2ϩ -dependent activator protein for secretion N euronal Ca 2ϩ sensor-1 (NCS-1) belongs to the family of EF-hand Ca 2ϩ -binding proteins and is mainly expressed in neuronal and neuroendocrine cells, where it enhances neurotransmission and Ca 2ϩ -dependent exocytosis (1-5). NCS-1 interacts with and regulates the activity of phosphatidylinositol 4-kinase ␤ (PI4K␤) (6-9). The members of the PI4K family catalyze the first step in the synthesis of PI 4,5-bisphosphate [PI(4,5)P 2 ], which has recently emerged as an important regulator of Ca 2ϩ -dependent secretion (10-12). Both a PI transfer protein (13) and a PI4P 5-kinase (14) are required for regulated exocytosis of dense core granules. In addition, the presence of synaptic vesicle and dense core granule-associated PI4K activity is essential for exocytosis (15,16). These data imply that generation of PI(4,5)P 2 is an important step in the event of secretion.In pancreatic ␤ cells, glucose dose-dependently increases ATP levels and decreases ADP levels (16). The resulting rise in the ATP at the expense of ADP is an important regulator of the two major signaling pathways involved in glucose-induced insulin secretion. The first of these pathways uses ATP-sensitive K ϩ channels to couple glucose metabolism with electrical activity, Ca 2ϩ influx, and initiation of insulin secretion. The second pathway is exerted at the level of granule priming and regulates the ␤ cell secretory capacity by modulation of the granules' release competence (17). The ␤ cell contains Ϸ10,000 insulincontaining secretory granules (18). Interestingly, as many as Ϸ5% of the granules are docked below the membrane. The readily releasable pool (RRP), defined by functional measurements, represents a subset (50-100 granules) of the docked pool (19). Granules belongin...

The Neuronal Ca2+ Sensor Protein Visinin-like Protein-1 Is Expressed in Pancreatic Islets and Regulates Insulin Secretion

“…This result is consistent with what has been observed with a related NCS protein, NCS-1. Gromada et al (27) demonstrated that NCS-1 is expressed in pancreatic ␤-cells and that overexpression stimulates insulin exocytosis via phosphatidylinositol 4-kinase-␤. Our results in VILIP-1-overexpressing cells employing capacitance recordings suggested that VILIP-1 increased the size and promoted the refilling of the RRP by stimulating the mobilization of granules in the RP, thereby increasing the capacity for exocytosis.…”

“…Recombinant adenovirus particles (ϳ10 10 plaque-forming units/ml) were used to infect isolated mouse islets (multiplicity of infection of 10 4 , assuming 10 3 ␤-cells/islet). Overexpression of VILIP-1 in MIN6 or single islet cells was achieved by transfection of VILIP-1-EGFP using Lipofectamine 2000 (27), and that in intact mouse islets by adenoviral transduction. A pilot study with the control virus Ad-EGFP demonstrated that 70 -80% of the cells in islets were infected as detected by confocal fluorescence microscopy.…”

“…It was shown very recently by cell capacitance measurements that overexpression of NCS-1, a member of NCS family Group II, facilitates glucosedependent exocytosis by increasing RRP via stimulation of phosphatidylinositol 4-kinase-␤ in pancreatic ␤-cells (Fig. 4, E and F) (27). Given these data, we performed cell capacitance measurements on single mouse ␤-cells to verify that VILIP-1 potentiates insulin secretion by directly stimulating exocytosis.…”

Oscillatory control of insulin secretion

“…Phosphatidylinositol-4,5-bisphosphate (PIP 2 ) constitutes only ~1% of the phospholipids in the inner leaflet of the plasma membrane (McLaughlin et al, 2002), but nevertheless plays an important role in secretion by regulating vesicle priming and trafficking (Olsen et al, 2003;Gromada et al, 2005;Waselle et al, 2005) and by serving as precursor for second messengers. One of the best known functions of PIP 2 is as substrate for PLC generation of IP 3 and diacylglycerol (DAG; (Berridge et al, 2000)).…”

Structure of a Ca2+-Myristoyl Switch Protein That Controls Activation of a Phosphatidylinositol 4-Kinase in Fission Yeast

“…When bound to the enzyme, Frq1 occupies residues 121-174 of Pik1, which forms a U-shaped structure that lies upstream of the catalytic domain (residues 792-1066) (26). Mammalian NCS-1 can interact with yeast Pik1 (45) and reportedly regulates PtdIns 4-kinase activity in animal cells (46,47). Ca 2ϩ -dependent activation of PtdIns 4-kinase by NCS-1 may be especially important in neurons because modulation of phosphoinositide synthesis by intracellular Ca 2ϩ controls synaptic vesicle exocytosis (48) and is involved in synaptic plasticity (49).…”