Exocytosis In Pancreatic Beta-cells Research Articles

Reduced Incretin Receptor Trafficking upon Activation Enhances Glycemic Control and Reverses Obesity in Diet-Induced Obese Mice.

Activation of incretin receptors by their cognate agonists augments sustained cAMP generation both from the plasma membrane as well as from the endosome. To address the functional outcome of this spatiotemporal signaling, we developed a non-acylated glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor dual agonist I-M-150847 that reduced receptor internalization following activation of the incretin receptors. The incretin receptor dual agonist I-M-150847 was developed by replacing the tryptophan cage of exendin-4 tyrosine substituted at the amino terminus with the C-terminal undecapeptide sequence of oxyntomodulin that placed lysine 30 of I-M-150847 in frame with the corresponding lysine residue of GIP. The peptide I-M-150847 is a partial agonist of GLP-1R and GIPR; however, the receptors, upon activation by I-M-150847, undergo reduced internalization that promotes agonist-mediated iterative cAMP signaling and augments glucose-stimulated insulin exocytosis in pancreatic beta cells. Chronic administration of I-M-150847 improved glycemic control, enhanced insulin sensitivity, and provided profound weight loss in diet-induced obese (DIO) mice. Our results demonstrated that despite being a partial agonist, I-M-150847, by reducing the receptor internalization upon activation, enhanced the incretin effect and reversed obesity.

Coupling of mDia1‐dependent fast endocytosis to exocytosis in pancreatic beta cells helps to maintain normal glucose tolerance in mice

Previously we have identified a novel Ca2+‐triggered clathrin‐independent but actin‐dependent fast endocytosis (CIE) in pancreatic beta cells. In contrast to well‐defined clathrin‐dependent endocytosis, neither the molecular mechanism nor physiological function of this clathrin‐independent endocytosis is clear. By combining patch clamp capacitance recording, super‐resolution imaging and specific molecular interventions, we have identified that an actin polymerization factor, mDia1, mediates this fast CIE in pancreatic beta‐cells. Using shRNA of mDia1 and knockout mice model, we have shown that removal of CIE leads to impaired exocytosis evoked by sustained stimulation, and disturbed glucose tolerance in mice, which cause ER stress and cellular function degeneration. Interestingly, during the progression of type II diabetes in KKAy mice, we have found out that down‐regulation of mDia1 expression and defects in fast CIE precede the dysregulation of insulin secretion in beta cells, highlighting defects of CIE may cause the beta cell failure. Overall, we conclude that exocytosis‐coupled fast CIE in pancreatic beta cells is essential to sustain insulin secretion during glucose elevation in chronic experiments, and is important for the maintaining of beta cell function and glucose tolerance in a long run.Support or Funding InformationThis study was supported by grants from the National Science and Technology Major 224 Project Program (2016YFA0500400), the Major State Basic Research Program of China 225 (2013CB531200), and the National Natural Science Foundation of China (31327901, 226 31521062, 31570839, 31428004, 61375018, and 61672253).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Mechanisms of V-SNARE Function in Secretory Granule Exocytosis

Insulin is released by regulated exocytosis and docking of insulin granules at the plasma membrane, followed by the assembly of the secretory machinery is required for insulin release. Newly formed granules require a complex maturation process to become release-ready and the formation of these fusion competent mature secretory granules involves a series of discrete and unique events comprised of granule sorting, maturation, docking, priming, and finally Ca2+-dependent fusion. Soluble NSF attachment receptor (SNARE) proteins are the conserved core machinery required for all intracellular membrane fusion events. While SNAREs specifically control fusion, vesicular SNAREs (v-SNAREs) perform additional roles that are still elusive. In secretory cell types, v-SNAREs are pivotal for targeting vesicles to different locations, mediating both sorting and membrane protein distribution and reductions in v-SNAREs result in defective regulated exocytosis. Using total internal reflection fluorescence (TIRF) microscopy, we studied the role of VAMP8 in secretory granule exocytosis in pancreatic beta cells. We found that VAMP8 associates with early, late and recycling endosome compartment, rather than insulin containing granules. However, VAMP8 traffics and fuses at the plasma membrane specifically with the recycling endosomal compartment. VAMP8 expression compromised secretory granule fusion, whereas VAMP2 expression facilitated granule fusion, delineating a VAMP8-dependent fusion step between recycling endosomes and the plasma membrane. Truncation of the VAMP8 C-terminal region resulted in attenuated exocytosis and a change in secretion kinetics. Moreover, secreting insulin measurements are negatively correlated with increased VAMP8 copy number in human islets. We conclude that VAMP8 vesicles play an important role in granule genesis and sorting of essential membrane proteins regulating insulin secretion. Additionally, the TMD of VAMP8 may be necessary for initiating fusion upon stimulation and appears to have some direct interaction on insulin secretion. This work should fill critical gaps in our understanding of regulated secretion and the treatment of type-2 diabetes.

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)(+) ratio.

Simultaneous Imaging of Vesicle Trafficking and Calcium-Mediated Exocytosis in Pancreatic Beta-Cells

It has long been known that only a small fraction (<10%) of the insulin in pancreatic beta-cells can be released. Clinical treatments of type 2 diabetes focus on increasing insulin release, so an understanding of insulin vesicle trafficking and release may lead to novel therapeutic strategies. Towards this understanding, we have utilized a novel quantitative imaging assay, based on a double fusion to the integral vesicle protein phogrin. In this construct, a pH-dependent ecliptic pHluorinFP is inside the lumen of the granule and a mOrangeFP is outside the granule in the cytoplasm. The pHluorin fluorescence is quenched by the low pH inside the granule until exocytosis when the lumen of the granule mixes with the extracellular media. This increases the local pH and the green fluorescence can then be measured as a readout of exocytosis. Experiments were done on the multicolor TIRF system with a photoactivation module. We photoconverted the mOrange-phogrin to a deep red protein in the perinuclear region, and then watched where that population of granules went as we stimulated the cells with glucose. Having only a small subset of vesicles labeled greatly facilitates tracking. We used this approach with TIRF microscopy to measure secreted granules and determine if they came from previously docked granules or from regions beyond the TIRF imaging field. In beta-cells, we find that secreted vesicles do not come from a docked pool, with secretion happening within about 1 second of the vesicle's arrival at the membrane. This time is decreased even further with the addition of glucose. These data suggest that the releasable insulin pool in beta-cells may not be docked at the membrane as are synaptic vesicles, but rather are maintained deeper with in the cell.

Microtubule-Driven Migration of Clathrin-Coated Pits Towards Vesicle Fusion Sites for Rapid Recycling in Pancreatic Beta Cells

Due to the long time required for de novo formation of deeply invaginated clathrin-coated pits that mostly appear at sites different from exocytosis before fission, clathrin-dependent vesicle recycling in secretory cells is usually regarded as a slow process. Here we show that regulated exocytosis in pancreatic beta cells is associated with glucose-dependent recruitment of clathrin and dynamin-1 puncta at the release sites shortly or long after fusion pore opens. The subsequent disassembly of these clathrin puncta mediates internalization of synaptotagmin VII clusters in the plasma membrane. The fast clathrin-dependent endocytosis contributes significantly to the total vesicle recycling process, and involves microtubule-dependent linear movement of preformed clathrin-coated pits to vesicle release sites prior to fission. These results have revealed an unexpected close spatio-temporal coupling of clathrin-dependent endocytosis to vesicle fusion in pancreatic beta cells, and highlighted a novel pathway to replenish the “readily retrieval pool” of clathrin that sustains fast clathrin-dependent endocytosis under intense stimulation.

AMPA receptor-mediated signaling acts on KATP channel and exocytosis in pancreatic beta-cells

AMPA receptor-mediated signaling acts on KATP channel and exocytosis in pancreatic beta-cells

Glutaredoxin-1 Mediates NADPH-Dependent Stimulation of Calcium-Dependent Insulin Secretion

Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca(2+)-induced exocytosis in pancreatic beta-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated beta-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat beta-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca(2+)-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca(2+) currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca(2+) currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic beta-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates beta-cell exocytosis and, in turn, insulin secretion.

CAPS1 and CAPS2 Regulate Stability and Recruitment of Insulin Granules in Mouse Pancreatic β Cells

CAPS1 and CAPS2 regulate dense-core vesicle release of transmitters and hormones in neuroendocrine cells, but their precise roles in the secretory process remain enigmatic. Here we show that CAPS2(-/-) and CAPS1(+/-);CAPS2(-/-) mice, despite having increased insulin sensitivity, are glucose intolerant and that this effect is attributable to a marked reduction of glucose-induced insulin secretion. This correlates with diminished Ca(2+)-dependent exocytosis, a reduction in the size of the morphologically docked pool, a decrease in the readily releasable pool of secretory vesicles, slowed granule priming, and suppression of second-phase (but not first-phase) insulin secretion. In beta cells of CAPS1(+/-);CAPS2(-/-) mice, the lowered insulin content and granule numbers were associated with an increase in lysosome numbers and lysosomal enzyme activity. We conclude that although CAPS proteins are not required for Ca(2+)-dependent exocytosis to proceed, they exert a modulatory effect on insulin granule priming, exocytosis, and stability.

BMP4-BMPR1A Signaling in β Cells Is Required for and Augments Glucose-Stimulated Insulin Secretion

Impaired glucose-stimulated insulin secretion (GSIS) and perturbed proinsulin processing are hallmarks of beta cell dysfunction in type 2 diabetes. Signals that can preserve and/or enhance beta cell function are therefore of great therapeutic interest. Here we show that bone morphogenetic protein 4 (Bmp4) and its high-affinity receptor, Bmpr1a, are expressed in beta cells. Mice with attenuated BMPR1A signaling in beta cells show decreased expression of key genes involved in insulin gene expression, proinsulin processing, glucose sensing, secretion stimulus coupling, incretin signaling, and insulin exocytosis and develop diabetes due to impaired insulin secretion. We also show that transgenic expression of Bmp4 in beta cells enhances GSIS and glucose clearance and that systemic administration of BMP4 protein to adult mice significantly stimulates GSIS and ameliorates glucose tolerance in a mouse model of glucose intolerance. Thus, BMP4-BMPR1A signaling in beta cells plays a key role in GSIS.

Munc13-1 is required for the sustained release of insulin from pancreatic β cells

Munc13-1 is a presynaptic protein that is essential for synaptic vesicle priming. Deletion of Munc13-1/unc13 causes total arrest of synaptic transmission due to a complete loss of fusion-competent synaptic vesicles. The requirement of Munc13-1 for large dense-core vesicles (LDCVs), however, has not been established. In the present study, we use Munc13-1 knockout (KO) and diacylglycerol (DAG) binding-deficient Munc13-1(H567K) mutant knockin (KI) mice to determine the role of Munc13-1 in the secretion of insulin-containing LDCVs from primary cultured pancreatic beta cells. We show that Munc13-1 is required for the sustained insulin release upon prolonged stimulation. The sustained release involves signaling of DAG second messenger, since it is also reduced in KI mice. Insulin secretion in response to glucose stimulation is characterized by a biphasic time course. Our data show that Munc13-1 plays an essential role in the development of the second phase of insulin secretion by priming insulin-containing LDCVs.

Evidence that an isoform of calpain-10 is a regulator of exocytosis in pancreatic beta-cells.

Calpain-10 (CAPN10) is the first type 2 diabetes susceptibility gene to be identified through a genome scan, with polymorphisms being associated with altered CAPN10 expression. Functional data have been hitherto elusive, but we report here a corresponding increase between CAPN10 expression level and regulated insulin secretion. Pancreatic beta-cell secretory granule exocytosis is mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor protein complex of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin 1, and vesicle-associated membrane protein 2. We report, for the first time, direct binding of a calpain-10 isoform with members of this complex. Furthermore, SNAP-25 undergoes a Ca2+-dependent partial proteolysis during exocytosis, with calpain protease inhibitor similarly suppressing both insulin secretion and SNAP-25 proteolysis. Based upon these findings, we postulate that an isoform of calpain-10 is a Ca2+-sensor that functions to trigger exocytosis in pancreatic beta-cells.

Interaction of ATP Sensor, cAMP Sensor, Ca2+ Sensor, and Voltage-dependent Ca2+ Channel in Insulin Granule Exocytosis

ATP, cAMP, and Ca(2+) are the major signals in the regulation of insulin granule exocytosis in pancreatic beta cells. The sensors and regulators of these signals have been characterized individually. The ATP-sensitive K(+) channel, acting as the ATP sensor, couples cell metabolism to membrane potential. cAMP-GEFII, acting as a cAMP sensor, mediates cAMP-dependent, protein kinase A-independent exocytosis, which requires interaction with both Piccolo as a Ca(2+) sensor and Rim2 as a Rab3 effector. l-type voltage-dependent Ca(2+) channels (VDCCs) regulate Ca(2+) influx. In the present study, we demonstrate interactions of these molecules. Sulfonylurea receptor 1, a subunit of ATP-sensitive K(+) channels, interacts specifically with cAMP-GEFII through nucleotide-binding fold 1, and the interaction is decreased by a high concentration of cAMP. Localization of cAMP-GEFII overlaps with that of Rim2 in plasma membrane of insulin-secreting MIN6 cells. Localization of Rab3 co-incides with that of Rim2. Rim2 mutant lacking the Rab3 binding region, when overexpressed in MIN6 cells, is localized exclusively in cytoplasm, and impairs cAMP-dependent exocytosis in MIN6 cells. In addition, Rim2 and Piccolo bind directly to the alpha(1)1.2-subunit of VDCC. These results indicate that ATP sensor, cAMP sensor, Ca(2+) sensor, and VDCC interact with each other, which further suggests that ATP, cAMP, and Ca(2+) signals in insulin granule exocytosis are integrated in a specialized domain of pancreatic beta cells to facilitate stimulus-secretion coupling.

Involvement of Protein Kinase C-ϵ in Inositol Hexakisphosphate-induced Exocytosis in Mouse Pancreatic β-Cells

Inositolhexakisphosphate (InsP6) plays a pivotal role in the pancreatic beta-cell stimulus-secretion coupling. We have used capacitance measurements to study the effects of InsP6 on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. In the presence of inhibitors of the protein phosphatase calcineurin to block endocytosis, intracellular application of InsP6 produced a dose-dependent stimulation of exocytosis, and half-maximal effect was observed at 22 microM. The stimulatory effect of InsP6 was dependent on protein kinase C (PKC) activity. Antisense oligonucleotides directed against specific PKC isoforms (alpha, beta II, delta, epsilon, xi) revealed the involvement of PKC-epsilon in InsP6-induced exocytosis. Furthermore, expression of dominant negative PKC-epsilon abolished InsP6-evoked exocytosis, whereas expression of wild-type PKC-epsilon led to a significant stimulation of InsP6-induced exocytosis. These data demonstrate that PKC-epsilon is involved in InsP6-induced exocytosis in pancreatic beta-cells.

Piccolo, a Ca2+ Sensor in Pancreatic β-Cells: INVOLVEMENT OF cAMP-GEFII·Rim2·PICCOLO COMPLEX IN cAMP-DEPENDENT EXOCYTOSIS

We have previously shown that cAMP-binding protein cAMP-guanidine nucleotide exchange factor II (GEFII) (or Epac2) interacting with Rim2 is involved in cAMP-dependent, protein kinase A-independent exocytosis in pancreatic beta-cells. The action of the cAMP-GEFII.Rim2 complex requires both intracellular cAMP and Ca(2+). Although Rim2 has C(2) domains, its role as a Ca(2+) sensor has remained unclear. In the present investigation, we have discovered that Piccolo, a CAZ (cytoskeletal matrix associated with the active zone) protein in neurons that is structurally related to Rim2, also binds to cAMP-GEFII and that it forms both homodimer and heterodimer with Rim2 in a Ca(2+)-dependent manner, whereas Rim2 alone does not form the homodimer. The association of Piccolo.Rim2 heterodimerization is stronger than Piccolo. Piccolo homodimerization. Treatment of pancreatic islets with antisense oligodeoxynucleotides against Piccolo inhibits insulin secretion induced by cAMP analog 8-bromo-cyclic AMP plus high glucose stimulation. These results suggest that Piccolo serves as a Ca(2+) sensor in exocytosis in pancreatic beta-cells and that the formation of a cAMP-GEFII.Rim2. Piccolo complex is important in cAMP-induced insulin secretion. In addition, this study suggests that CAZ proteins similar to those in neurons are also function in pancreatic beta-cells.

Mitochondria as the conductor of metabolic signals for insulin exocytosis in pancreatic beta-cells.

Mitochondrial metabolism is crucial for the coupling of glucose recognition to the exocytosis of the insulin granules. This is illustrated by in vitro and in vivo observations discussed in the present review. Mitochondria generate ATP, which is the main coupling messenger in insulin secretion. However, the subsequent Ca2+ signal in the cytosol is necessary but not sufficient for full development of sustained insulin secretion. Hence, mitochondria generate ATP and other coupling factors serving as fuel sensors for the control of the exocytotic process. Numerous studies have sought to identify the factors that mediate the amplifying pathway over the Ca2+ signal in glucose-stimulated insulin secretion. Predominantly, these factors are nucleotides (GTP, ATP, cAMP, NADPH), although metabolites have also been proposed, such as long-chain acyl-CoA derivatives and glutamate. Hence, the classical neurotransmitter glutamate receives a novel role, that of an intracellular messenger or co-factor in insulin secretion. This scenario further highlights the importance of glutamate dehydrogenase, a mitochondrial enzyme well recognized to play a key role in the control of insulin secretion. Therefore, additional putative messengers of mitochondrial origin are likely to participate in insulin secretion.

Fast and cAMP-sensitive mode of Ca(2+)-dependent exocytosis in pancreatic beta-cells.

The fast component (mode 1) of Ca(2+)-dependent exocytosis in pancreatic beta-cells, unlike that in adrenal chromaffin cells, is regulated by cytosolic ATP in a concentration-dependent manner. This action of ATP is apparent within 3 min and does not require ATP hydrolysis; rather, it requires the production of cAMP by adenylate cyclase. Moreover, the effect of cAMP is ATP dependent, as revealed by the observation that the fast component of exocytosis is facilitated by ATP, even in the presence of a saturating concentration of cAMP (200 micromol/l). Thus, the amplitude of mode-1 exocytosis depends quadratically on the cytosolic ATP concentration and is facilitated by ATP, even in the absence of an increase in the concentration of cAMP. Given that high glucose concentrations increase the cytosolic ATP concentration, glucose-induced insulin secretion likely involves this action of ATP on mode-1 exocytosis, together with its effect on ATP-dependent K(+) channels. In contrast to the fast component of exocytosis, the slow component (mode 2) of this process is independent of cAMP and ATP and can account for the slow component of insulin secretion, which does not require these nucleotides.

Localized exocytosis detected by spatially resolved amperometry in single pancreatic beta-cells.

Spatially resolved measurements of exocytosis in pancreatic beta-cells were made using amperometry with 1-microm radius electrodes. These measurements revealed that certain portions of a cell actively undergo exocytosis following stimulation with depolarizing agents, but other regions are inactive. The amperometric measurements were compared to measurements made with the membrane indicator dye, FM1-43, which showed uneven increases in fluorescence around the surface of the cell, with amperometric secretion being detected only at the brightest regions. In some instances, a large number of exocytotic events were detected from one electrode position. The number of events was larger than what would be expected based on the number of vesicles that could fit under an electrode of the dimensions used. These results suggest a mechanism of vesicle traffic that allows multiple fusions at a small membrane area.

Direct effects of tolbutamide on mitochondrial function, intracellular Ca2+ and exocytosis in pancreatic beta-cells.

Using the whole-cell voltage-clamp method to measure ATP-sensitive K+(KATP) currents, changes in cell capacitance to measure secretion and microfluorimetry to monitor intracellular Ca2+ and mitochondrial function, we have investigated the direct effect of sulphonylureas on exocytosis in pancreatic beta-cells. Tolbutamide (100 microM) and 100 nM 4-beta-12-phorbolmyristate-13-acetate (PMA), which activates the protein kinase C (PKC) isoforms found in beta-cells, potentiated exocytosis in a non-additive manner. These effects were blocked by down-regulation of PKC. Our data support the idea that tolbutamide can potentiate secretion from beta-cells via a PKC-dependent pathway. Because PKC and sulphonylureas can modulate the activity of KATP channels, we explored whether the above effects are caused by inhibition of this channel. PMA increased whole-cell KATP currents but did not affect their sensitivity to tolbutamide. Down-regulation of PKC affected neither the magnitude nor the tolbutamide sensitivity of the KATP current. Both tolbutamide and the mitochondrial uncoupler FCCP (1 microM) mobilized intracellular Ca2+ and prolonged Ca2+ transients elicited by cholinergic mobilization of intracellular Ca2+ stores. Tolbutamide (0.1-0.5 mM), like FCCP, depolarized the mitochondrial membrane potential and activated KATP currents. We suggest that sulphonylureas can directly potentiate exocytosis by impairing mitochondrial function and Ca2+ handling, which ultimately leads to activation of Ca2+-dependent enzymes such as PKC.

Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells.

The role of cytosolic ATP in exocytosis was investigated by using amperometric measurement of insulin exocytosis in pancreatic beta cells, which were stimulated with photolysis of caged Ca2+ compounds. Insulin exocytosis occurred with two rates. We found that ATP hastened and augmented the exocytosis via selective enhancement of the exocytosis with the faster rate. A nonhydrolysable analog of ATP, adenosine 5'-O-(3-thiotriphosphate), which blocks ATPase, was even more effective than ATP, indicating that the phosphorylation event occurred downstream of ATP-dependent vesicle transportation and priming. The action of ATP was eliminated by a competitive antagonist of cAMP, and by an inhibitor of adenylate cyclase. These data characterize an ATP sensing mechanism for the Ca2+-dependent exocytosis involving adenylate-cyclase, cAMP-dependent protein kinase, and, possibly, the fusion machinery itself. Thus, the fast exocytotic machinery requires both phosphorylation and Ca2+ for the final triggering and likely constitutes a distal ATP sensor for insulin exocytosis that acts in concert with ATP-sensitive K+ channels.