Cyclic AMP (cAMP) and Ca(2+) are key regulators of exocytosis in many cells, including insulin-secreting beta cells. Glucose-stimulated insulin secretion from beta cells is pulsatile and involves oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse beta cells. These oscillations were preceded and enhanced by elevations of [Ca(2+)](i). However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca(2+)](i) rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.

Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.

Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro.

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...

SummaryPhosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5)P 2 concentration, little is known about its precursor, PtdIns(4)P, in particular whether there are stimulusinduced alterations independent of those of PtdIns(4,5)P 2 . We investigated plasma-membrane PtdIns(4)P and PtdIns(4,5)P 2 dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5)P 2 induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4)P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4)P elevation required voltage-gated Ca 2+ entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca 2+ independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca 2+ -and PKC-dependent changes in the plasmamembrane PtdIns(4)P concentration that are independent of the effects on PtdIns(4,5)P 2 . These findings indicate that enhanced formation of PtdIns(4)P, apart from ensuring efficient replenishment of the PtdIns(4,5)P 2 pool, might serve an independent signalling function by regulating the association of PtdIns(4)P-binding proteins with the plasma membrane.