Rapid Turnover of Phosphatidylinositol-4,5-Bisphosphate in Insulin-Secreting Cells Mediated by Ca2+ and the ATP-to-ADP Ratio

Abstract

P hosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a minor membrane component of eukaryotic cells constituting ϳ1% of the phospholipids in the inner leaflet of the plasma membrane. Nevertheless, the phospholipid plays important roles in the regulation of a variety of cell functions. Apart from serving as a precursor for the messenger molecules inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol generated on activation of phospholipase C (PLC) (1), and for phosphoinositide 3-kinase-generated phosphatidylinositol-3,4,5-trisphosphate (2), PIP 2 is known to regulate ion channel activity (3), proteins involved in organization of the cytoskeleton (4), and the trafficking of vesicles in endo-and exocytosis (5). PIP 2 is mainly synthesized in two steps by phosphorylation of phosphatidylinositol to phosphatidylinositol-4-phosphate (PIP) by phosphatidylinositol 4-kinases (PI 4-kinases), followed by PIP phosphorylation to PIP 2 by type I PIP 5-kinases (6). Some PIP 2 is also formed via phosphorylation of phosphatidylinositol-5-phosphate by type II PIP 4-kinases (6).Pancreatic ␤-cells secrete insulin after elevation of the ambient glucose concentration. The rapid uptake and metabolism of the glucose lead to an increase of the intracellular ATP-to-ADP ratio, closure of ATP-sensitive K ϩ channels (K ATP channels) in the plasma membrane, depolarization, and opening of voltage-dependent Ca 2ϩ channels. The resulting increase of the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ) triggers exocytosis of insulin secretory granules (7). A large amount of data indicates that PIP 2 plays an important role in the insulin secretory process. It was recognized early that the rate of phosphoinositide metabolism is increased in glucose-stimulated islets (8,9). This effect is due to PLC-mediated hydrolysis of PIP 2 (10). However, there are different opinions about the mechanisms underlying glucose-induced PLC activation. Whereas several studies indicate that the glucoseinduced phosphoinositide hydrolysis depends on the presence of extracellular Ca 2ϩ (9,11,12), other reports indicate that the process is at least in part Ca 2ϩ independent (13-15). Recent observations in single insulinoma cells (16) and intact mouse islets (17) have demonstrated that elevation of [Ca 2ϩ ] i is sufficient to trigger PLC activity and that [Ca 2ϩ ] i oscillations are associated with periodic activation of PLC. However, the kinetics of the early changes in PIP 2 concentration and how it is related to [Ca 2ϩ ] i after glucose stimulation are unknown. PIP 2 has been found to be important for secretion independent of its role as substrate for PLC. A role for PIP 2 in exocytosis was first suggested by the observation that phosphatidylinositol transfer protein and a type I PIP 5-kinase are essential components of the ATP-dependent priming of exocytotic vesicles in permeabilized chromaffin cells (18,19). The importance of PIP 2 was later confirmed by experiments showing that exocytosis is negatively affected by inhibition of PIP 2 synthesis in various type...

Distinct plasma-membrane PtdIns(4)P and PtdIns(4,5)P2 dynamics in secretagogue-stimulated β-cells

“…Inhibition of PI4K activity not only affected PtdIns(4)P levels, but also abolished the synthesis of PtdIns(4,5)P 2 , in accordance with previous observations with phenylarsine oxide (Thore et al, 2007). When 200 mM LY294002 was added during the ATPinduced increase in the concentration of the phosphoinositides, the PtdIns(4)P levels immediately decreased, whereas those of PtdIns(4,5)P 2 started to decline after a delay of 25±4 seconds (Fig.…”

“…5E). Glucose has previously been found to induce oscillations of the plasma-membrane PtdIns(4,5)P 2 concentration (Tamarina et al, 2005;Thore et al, 2007). Parallel recordings with OSBP PH -CFP and PLCd1 PH -YFP demonstrated that the periodic peaks in the plasma-membrane concentration of PtdIns(4)P coincided with pronounced PtdIns(4,5)P 2 nadirs (Fig.…”

“…We next investigated PtdIns(4)P turnover using an approach with cells permeabilized by a-toxin from Staphylococcus aureus that was previously used in studies of PtdIns(4,5)P 2 (Thore et al, 2007). In contrast to digitonin, which causes release of large intracellular proteins, a-toxin creates small pores that only allow passage of molecules smaller than ~3 kDa.…”

Oscillatory control of insulin secretion

“…Real-time imaging studies in isolated insulinoma cells and intact islets (Thore et al, 2004;Tamarina et al, 2005;Thore et al, 2007) have recently confirmed that glucose stimulates PLC activity secondary to the elevation of [Ca 2+ ] i and that membrane depolarization alone (Gromada et al, 1996;Liu et al, 1996) or increase of [Ca 2+ ] i (Mathias et al, 1985;Best et al, 1987;Biden et al, 1987) In addition to contributing to the generation and shaping of Ca 2+ signals, PLC activation can be anticipated to regulate insulin secretion kinetics via generation of DAG and activation of PKC. Several conventional, novel and atypical PKC isoforms are expressed in islets and insulin-secreting cells (Jones and Persaud, 1998;Warwar et al, 2006).…”

Imaging Phosphoinositide Dynamics in Living Cells

“…1C). Studies in the authors' laboratory have employed TIRF microscopy for measurements of the plasma membrane PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 concentrations using the PH domains from PLCδ1 and protein kinase B/Akt (16)(17)(18)(19). TIRF microscopy provides better resolution as the optical section is several-fold thinner than in confocal microscopy.…”