Glucose-stimulated biphasic insulin secretion involves at least two signaling pathways, the KATP channel-dependent and KATP channel-independent pathways, respectively. In the former, enhanced glucose metabolism increases the cellular adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, closes KATP channels and depolarizes the cell. Activation of voltage-dependent Ca(2+) channels increases Ca(2+) entry and [Ca(2+)]i and stimulates insulin release. The KATP channel-independent pathways augment the response to increased [Ca(2+)]i by mechanisms that are currently unknown. However, they affect different pools of insulin-containing granules in a highly coordinated manner. The beta-cell granule pools can be minimally described as reserve, morphologically docked, readily and immediately releasable. Activation of the KATP channel-dependent pathway results in exocytosis of an immediately releasable pool that is responsible for the first phase of glucose-stimulated insulin release. Following glucose metabolism, the rate-limiting step for the first phase lies in the rate of signal transduction between sensing the rise in [Ca(2+)]i and exocytosis of the immediately releasable granules. The immediately releasable pool of granules can be enlarged by previous exposure to glucose (by time-dependent potentiation, TDP), and by second messengers such as cyclic adenosine monophosphate (cyclic AMP) and diacylglycerol (DAG). The second phase of glucose-stimulated insulin secretion is due mainly to the KATP channel-independent pathways acting in synergy with the KATP channel-dependent pathway. The rate-limiting step here is the conversion of readily releasable granules to the state of immediate releasability, following which, in an activated cell they will undergo exocytosis. In the rat and human beta-cell the KATP channel-independent pathways induce a time-dependent increase in the rate of this step that results in the typical rising second-phase response. In the mouse beta-cell the rate appears not to be changed much by glucose. Potential intermediates involved in controlling the rate-limiting step include increases in cytosolic long-chain acyl-CoA levels, adenosine triphosphate (ATP) and guanosine triphosphate (GTP), DAG binding proteins, including some isoforms of protein kinase (PKC), and protein acyl transferases. Agonists that can change the rate-limiting steps for both phases of insulin release include those like glucagon-like peptide 1 (GLP-1) that raise cyclic AMP levels and those like acetylcholine that act via DAG.

The insulin secretory response by pancreatic ␤-cells to an acute "square wave" stimulation by glucose is characterized by a first phase that occurs promptly after exposure to glucose, followed by a decrease to a nadir, and a prolonged second phase. The first phase of release is due to the ATP-sensitive K ؉ (K ATP ) channel-dependent (triggering) pathway that increases [Ca 2؉ ] i and has been thought to discharge the granules from a "readily releasable pool." It follows that the second phase entails the preparation of granules for release, perhaps including translocation and priming for fusion competency before exocytosis. The pathways responsible for the second phase include the K ATP channel-dependent pathway because of the need for elevated [Ca 2؉ ] i and additional signals from K ATP channel-independent pathways. The mechanisms underlying these additional signals are unknown. Current hypotheses include increased cytosolic long-chain acyl-CoA, the pyruvatemalate shuttle, glutamate export from mitochondria, and an increased ATP/ADP ratio. In mouse islets, the ␤-cell contains some 13,000 granules, of which ϳ100 are in a "readily releasable" pool. Rates of granule release are slow, e.g., one every 3 s, even at the peak of the first phase of glucose-stimulated release. As both phases of glucose-stimulated insulin secretion can be enhanced by agents such as glucagon-like peptide 1, which increases cyclic AMP levels and protein kinase A activity, or acetylcholine, which increases diacylglycerol levels and protein kinase C activity, a single "readily releasable pool" hypothesis is an inadequate explanation for insulin secretion. Multiple pools available for rapid release or rapid conversion of granules to a rapidly releasable state are required. Diabetes 51 (Suppl. 1):S83-S90, 2002

The mechanisms underlying the first phase of glucose-stimulated insulin release, the deterioration of which marks the early stages of both type 1 and type 2 diabetes, are essentially unknown. Among many hypotheses, one holds that the first phase is due to a readily releasable pool of insulin-containing granules. We used current knowledge of the mechanisms of exocytosis and the proteins involved in docking granules at the plasma membrane to test this hypothesis. A docked pool of readily releasable granules was identified by immunoprecipitation of the plasma membrane protein syntaxin with a specific antibody and by co-immunoprecipitation of soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25) and the granule proteins synaptobrevin and synaptotagmin. The four SNARE proteins co-immunoprecipitated each other, thus identifying the core complex associated with docked granules. Using co-immunoprecipitation as a marker for docked granules, we found that the docked pool was rapidly discharged during the first phase of glucose-stimulated insulin release and refilled during the second phase. Other secretagogues also released the pool, whereas the physiological inhibitor norepinephrine blocked its release. Further studies on the nature of this pool of granules should shed light on the causes of its deterioration in the early stages of diabetes and the reasons for deficient insulin release.

Background:The affinity of soluble adenylyl cyclase (sAC) for its substrate ATP suggested that it might be sensitive to fluctuations in ATP. Results: In sAC-overexpressing and glucose-responsive cells, sAC-generated cAMP reflects intracellular ATP levels. Conclusion: sAC can be an ATP sensor inside cells. Significance: sAC serves as a metabolic sensor via regulation by three cellular metabolites: ATP, bicarbonate, and calcium.

Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling.

The molecular mechanisms responsible for the 'distal' effect by which noradrenaline (NA) blocks exocytosis in the β-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 β-cells. NA inhibited Ca Abbreviations BoNT/A, botulinum toxin A; Im, imaginary part of the patch admittance; mSIRK, myristoylated βγ-binding/activating peptide; NA, noradrenaline; PTX, pertussis toxin; Re, real part of the patch admittance; RRP, readily releasable pool; SNAP-25, synaptosomal-associated protein 25; SNARE, SNAP (soluble NSF attachment protein) receptors.

The biphasic secretory response of pancreatic beta-cells to abrupt and sustained exposure to glucose is well documented. Some of the ATP-sensitive K(+) (K(ATP)) channel-dependent mechanisms underlying the first phase of insulin release are known; the mechanisms underlying the second phase are less well known. The hypothesis we propose is that one rate-limiting step, controlling the conversion of granules in a readily releasable (RR) docked granule pool to an immediately releasable (IR) pool, is responsible for the magnitude of both phases of release. Furthermore, we propose that the K(ATP) channel-independent signaling pathway regulates this rate-limiting step. The size of the IR pool of granules that constitutes the first phase is determined under resting conditions by the forward and reverse rates of conversion of granules in the RR and IR pools. The resulting equilibrium position determines the maximum number of beta-cell granules available for release during the first phase upon exposure to glucose. At the nadir between the two phases, the IR pool has been depleted so that the rate of granule release is equal to the low forward rate for the conversion of RR to IR granules. After the nadir, the forward rate is accelerated during the rising portion of the second phase until it reaches a maximum rate at the plateau.

Electron microscopy and quantitative stereological techniques were used to study the dynamics of the docked granule pool in the rat pancreatic ␤-cell. The mean number of granules per ␤-cell was 11,136. After equilibration in RPMI containing 5.6 mmol/l glucose, 6.4% of the granules (ϳ700) were docked at the plasma membrane (also measured as [means ؎ SE] 4.3 ؎ 0.6 docked granules per 10 m of plasma membrane at the perimeter of the cell sections). After a 40-min exposure to 16.7 mmol/l glucose, 10.2% of the granules (ϳ1,060) were docked (6.4 ؎ 0.8 granules per 10 m of plasma membrane). Thus, the docked pool increased by 50% during stimulation with glucose. Islets were also exposed to 16.7 mmol/l glucose in the absence or presence of 10 mol/l nitrendipine. In the absence and presence of nitrendipine, there were 6.1 ؎ 0.7 and 6.3 ؎ 0.6 granules per 10 m of membrane, respectively. Thus, glucose increased granule docking independently of increased [Ca 2؉ ]i and exocytosis. The data suggest a limit to the number of docking sites. As the rate of docking exceeded the rate of exocytosis, docking is not rate limiting for insulin release. Only with extremely high release rates, glucose stimulation after a 4-h incubation with a high concentration of fatty acid-free BSA, was the docked granule pool reduced in size. Diabetes 53:3179 -3183, 2004 I n glucose-stimulated biphasic insulin secretion, the first phase is due to the ATP-sensitive K ϩ channeldependent pathway of glucose signaling, depolarization of the cell, increased Ca 2ϩ influx, and exocytosis of an "immediately releasable" pool of docked granules (1-4). After the first phase, in rat and human, the second phase is characterized by an increasing rate of secretion to a plateau. It has been suggested that the second phase is due to time-dependent potentiating signals (5-7) generated by the ATP-sensitive K ϩ channelindependent or -amplifying pathway (7). The mechanisms underlying this pathway and the second phase of release are unknown (1,7-9), although it has been suggested that its action is at the level of the docked granules to increase the rate at which they enter the immediately releasable pool or state (7). Second-phase secretion over a lengthy period requires that the ␤-cell replace the released docked granules. For this to happen, a flow of granules from the large reserve pool to the plasma membrane followed by docking and subsequent priming and preparation for release has to occur. Little is known about this movement or about the dynamics of the docked granules. The aim of this research was to determine whether the increased rate of insulin secretion during the second phase was associated with any change in the number of granules docked at the ␤-cell plasma membrane. RESEARCH DESIGN AND METHODSMale Sprague-Dawley rats weighing 250 -350 g were used for the study and fed ad libitum. They were killed by CO 2 asphyxiation and the pancreata removed. Pancreatic islets were isolated by collagenase digestion (10), as previously described using a Krebs-Ringer bicarbonate...