The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO₂, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ? l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K⁺, Ca²⁺) across membrane systems and, hence, regulates the net uptake of Ca²⁺ and subsequent release of insulin. The effect of l-lactate upon Ca²⁺ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.     

Calcium and pancreatic β-cell function 7. Evidence for cyclic AMP-induced translocation of intracellular calcium

The effect of cyclic AMP on calcium movements in the pancreatic β-cell was evaluated using an experimental approach based on in situ labelling of intracellular organelles of ob/ob-mouse islets with ⁴⁵Ca. Whereas the glucose-stimulated ⁴⁵Ca incorporation by mitochondria and secretory granules was increased under a condition known to reduce cyclic AMP (starvation), raised levels of this nucleotide (addition of 3-isobutyl-1-methylxanthine or N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic monophosphate) reduced the mitochondrial accumulation of ⁴⁵Ca. Conditions with increased cyclic AMP were associated with a stimulated efflux of ⁴⁵Ca from the secretory granules but not from the mitochondria. The microsomal fraction differed from both the mitochondrial and secretory granule fractions by accumulating more ⁴⁵Ca after the addition of 3-isobutyl-1-methylxanthine. The results suggest that cyclic AMP potentiates glucose-stimulated insulin release by increasing cytoplasmic Ca²⁺ at the expense of the calcium taken up by the organelles of the pancreatic β-cells.     

The stimulus–secretion coupling of amino acid-induced insulin release. Insulinotropic action of l-alanine

Available information on the fate and insulinotropic action of l-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na⁺ co-transport in the insulinotropic action of l-alanine. In the present study conducted in islets prepared from normal adult rats, l-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-¹⁴C]palmitate, (iii) to stimulate ⁴⁵Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or l-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of l-[U-¹⁴C]alanine was unaffected by d-glucose, but inhibited by l-leucine. Inversely, l-alanine decreased the oxidation of d-[U-¹⁴C]glucose, but failed to affect l-[U-¹⁴C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of l-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na⁺ co-transport in the secretory response to l-alanine cannot be ignored.     

Inhibitory effect of clonidine upon adenylate cyclase activity,cyclic AMP production,and insulin release in rat pancreatic islets

Conflicting opinions were recently expressed concerning the possible effect of ₂-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca²⁺), NaF, GTP,, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable , in part at least, to inhibition of adenylate cyclase.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

Glucagon-like peptide-1 (GLP-1): A gut hormone of potential interest in the treatment of diabetes

GLP-1 (glucagon-like peptide-1) is a gut hormone which is released into the blood stream after feeding. Its main action is to stimulate insulin secretion through potentiating the insulinotropic action of glucose. The peptide is encoded in the glucagon gene and expressed mainly in the gut L cells. It exerts its actions through activating specific receptors of the seven transmembraneous domain-G-protein-coupled type with 463 amino acids. Its main signalling mechanism is activation of adenylate cyclase and formation of cyclic AMP. The peptide also increases the cytoplasmic concentration of Ca² which is thought to be executed both through a Na⁺-dependent uptake of extracellular Ca²⁺ and through release of Ca²⁺ from intracellular Ca²⁺ stores. GLP-1 also inhibits glucagon secretion and inhibits gastric emptying and gastric acid and pancreatic exocrine secretion. Its integrated action on carbohydrate metabolism results in reduction of circulating glucose, and GLP-1 has therefore been suggested as a therapeutic alternative in diabetes. Finally, GLP-1 is also expressed in neurons in the hypothalamus, and may be involved in the regulation of feeding behaviour, since it inhibits food intake. BioEssays 20 :642–651, 1998.© 1998 John Wiley & Sons Inc.     

Calcium and pancreatic β-cell function I. Stimulatory effects of pentobarbital on insulin release

Islets microdissected from ob/ob-mice were exposed to 3mM pentobarbital in media which were normal or deficient in Ca²⁺. This treatment resulted in a marked decrease of the islet content of cyclic AMP recorded in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Pentobarbital had a dual effect on insulin release. In addition to being a potent inhibitor of glucose-stimulated insulin release in media containing 2.56 mM Ca²⁺ it increased the amounts of insulin released in high glucose media deficient in Ca²⁺. There was a transient stimulation with ordinary concentrations of Ca²⁺ and 3 mM glucose when the media also contained 3-isobutyl-1-methylxanthine. The stimulatory effect of pentobarbital persisted after replacing part of the Ca²⁺ in the β-cell membrane with lanthanum ions and it could not be mimicked by lowering the oxygen tension of the incubation medium. It is suggested that pentobarbital stimulation of insulin release is the result of a specific action of the drug on the distribution of Ca²⁺ within the pancreatic β-cells.     

Metabolic and secretory effects of methylamine in pancreatic islets

The metabolic and secretory effects of methylamine in rat pancreatic islets were investigated. Methylamine accumulated in islet cells, was incorporated into endogenous islet proteins, and inhibited the incorporation of [2,5-³H] histamine into either N,N-dimethylcasein or endogenous islet proteins. Methylamine (2 mM ) did not affect the oxidation of glucose or endogenous nutrients or the intracellular pH in islet cells. Glucose did not affect the activity of transglutaminase in islet homogenates, the uptake of ¹⁴C-methylamine by intact islets or its incorporation into endogenous islet proteins. Methylamine inhibited insulin release evoked by glucose, other nutrient secretagogues, and non-nutrient insulinotropic agents such as L -arginine or gliclazide. The inhibitory effect of methylamine upon insulin release was diminished in the presence of cytochalasin B or at low extracellular pH. Methylamine retarded the conversion of proinsulin to insulin. Trimethylamine (0.7 mM ) was more efficiently taken up by islet cells than methylamine (2.0 mM ), and yet caused only a modest inhibition of insulin release. These findings suggest that methylamine interferes with a late step in the secretory sequence, possibly by inhibiting the access of secretory granules to their exocytotic site.     

Somatostatin promotes glucose generation of Ca2+oscillations in pancreatic islets both in the absence and presence of tolbutamide

Many cellular processes, including pulsatile release of insulin, are triggered by increase of cytoplasmic Ca²⁺. This study examines how somatostatin affects glucose generation of cytoplasmic Ca²⁺ oscillations in mouse islets in absence and presence of tolbutamide blockade of the K_ATP channels. Ca²⁺ was measured with dual wavelength microflurometry in isolated islets loaded with the indicator Fura-2. Rise of glucose from 3 to 20 mM evoked introductory lowering of Ca²⁺ prolonged by activation of somatostatin receptors. During continued superfusion exposure to somatostatin triggered oscillations mediated by periodic increase from the basal level (absence of tolbutamide) or by periodic interruption of an elevated level (presence of tolbutamide). In the latter situation the oscillations were transformed into sustained elevation by activation of muscarinic receptors (acetylcholine) or increase of cyclic AMP (IBMX, 8-bromo-cyclic AMP, forskolin). The observed effect of cyclic AMP raises the question whether high proportions of the glucagon-producing α-cells promote steady-state elevation of Ca²⁺. In support for this idea somatostatin was found to trigger glucose-induced Ca²⁺ oscillations essentially in small islets that contain very few α-cells. The results indicate that somatostatin promotes glucose generation of Ca²⁺oscillations with similar characteristics both in the absence and presence of functional K_ATP channels.     

Ba++ stimulates accumulation of cyclic AMP in rat pancreatic islets.

In pancreatic islets prelabelled with (³H) adenine, Ba⁺⁺ augmented (³H) cyclic AMP in 1–10 min incubations. 3-isobutyl-l-methylxanthine markedly enhanced and prolonged the Ba⁺⁺-induced nucleotide as well as the insulin response. In the presence of the methyl xanthine 1.6 mM Ba⁺⁺ was a maximally and 0.4 mM a submaximally effective concentration both for the stimulation of (³H) cyclic AMP and insulin. A 5-fold excess of Ca⁺⁺ partly inhibited the Ba⁺⁺-induced nucleotide and — more profoundly — the insulin response. Increasing Mg⁺⁺ from 2 to 10 mM was also inhibitory. Stimulation by Ba⁺⁺ was observed in the absence as well as in the presence of D-glucose. It is concluded that the insulinotropic action of Ba⁺⁺ is at least partly mediated by cyclic AMP.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.     

An incretin-based tri-agonist promotes superior insulin secretion from murine pancreatic islets via PLC activation

Recently, a unimolecular tri-agonist with activity at glucagon-like peptide 1 receptor (GLP-1R), glucose dependent insulinotropic receptor, and the glucagon receptor was reported to improve glycemic control in mice. Here, we defined the underlying molecular mechanisms of enhanced insulin secretion in murine pancreatic islets for a specific tri-agonist. The tri-agonist induced an increase in insulin secretion from murine islets compared to the respective mono-agonists. GLP-1R mainly signals via activation of the Gα_s pathway, but inhibition of protein kinase A (H89) and exchange protein activation by cAMP (EPAC) (ESI-09) could not completely block insulin release induced by tri-agonist. Electrophysiological observations identified a strong increase of intracellular Ca²⁺ concentration and whole-cell currents induced by tri-agonist via transient receptor potential channels (TRPs). Although, EPAC activation mobilizes intracellular Ca²⁺ via TRPs, the TRPs blockers (La³⁺ and Ruthenium Red) had a larger inhibitory impact than ESI-09 on tri-agonist stimulatory effects. To test for other potential mechanisms, we blocked PLC activity (U73122) which reduced the superior effect of tri-agonist to induce insulin secretion, and partially inhibited the induced Ca²⁺ influx. This result suggests that the relative effect of tri-agonist on insulin secretion caused by GLP-1R agonism is mediated mainly via Gα_s signaling and partially by activation of PLC. Therefore, the large portion of the increased intracellular Ca²⁺ concentration and the enhanced whole-cell currents induced by tri-agonist might be attributable to TRP channel activation resulting from signaling through multiple G-proteins. Here, we suggest that broadened intracellular signaling may account for the superior in vivo effects observed with tri-agonism.     

cAMP-enhancing agents “permit” stimulus-secretion coupling in canine pancreatic islet β-cells

Isolated canine islets of Langerhans differ from isolated islets of other species (including rodents and man) in that elevated glucose concentrations are unable to stimulate insulin secretion. Here we demonstrate that addition to the perifusate of isobutylmethylxanthine (IBMX), forskolin or 8-CPT-cAMP, all of which enhance cytosolic cAMP, permits insulin secretion in response to glucose, leucine or tolbutamide. These cAMP enhancers increase secretogogue-induced electrical activity in β-cells and restore depolarization-induced, Ca²⁺-dependent granule exocytosis measured as stepwise increases in membrane capacitance. We propose that the primary permissive action of cAMP is to tightly link Ca²⁺ entry to insulin granule release, while a secondary action is to tighten the link between glucose metabolism and cell depolarization.     

Activation,but not inhibition,by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell

The effect of glucose on the Ca²⁺-activated K⁺ permeability in pancreatic islet cells was investigated by measuring the rate of ⁸⁶Rb efflux, ⁴⁵Ca efflux and insulin release from perifused rat pancreatic islets exposed to step-wise increased in glucose concentration. When the glucose concentration was raised from intermediate (8.3 or 11.1 mM) to higher values, a rapid and sustained increase in ⁸⁶Rb outflow, ⁴⁵Ca outflow and insulin release was observed. Likewise, in the presence of 8.3 or 16.7 mM glucose, tolbutamide increased ⁸⁶Rb and ⁴⁵Ca efflux, as well as insulin release. In the two series of experiments, a tight correlation was found between the magnitude of the changes in ⁸⁶Rb and ⁴⁵Ca outflow, respectively. It is concluded that, at variance with current ideas, glucose does not inhibit the response to cytosolic Ca²⁺ of the Ca²⁺-sensitive modality of K⁺ extrusion. On the contrary, as a result of its effect upon Ca²⁺ handling, glucose stimulates the Ca²⁺-activated K⁺ permeability.     

Accumulation of cadmium in pancreatic β cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd²⁺ and the effects of this ion on secretory activity and metabolism were investigated in β cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 μmol/kg dry wt. After 60 min of incubation in a Ca²⁺-deficient medium containing 2.5 μM Cd²⁺ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca²⁺. The incorporation of Cd²⁺ was stimulated either by raising the concentration of glucose to 20 mM or K⁺ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K⁺. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd²⁺-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd²⁺ efflux into a Ca²⁺-deficient medium. Concentrations of Cd²⁺ up to 2.5 μM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd²⁺ concentration was above 10 μM. Basal insulin release was stimulated by 5 μM Cd²⁺. At a concentration of 160 μM, Cd²⁺ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the β cell uptake of Cd²⁺ is facilitated by the activation of voltage-dependent Ca²⁺ channels. Apparently, the accumulation of Cd²⁺ mimics that of Ca²⁺ also involving a component of intracellular sequestration promoted by glucose.     

Cold-induced insulin release in vitro: Evidence for exocytosis

Summary Exposure of isolated pancreatic islets (mouse or rat) to low temperature (2° C) evoked a threefold increase in insulin release irrespective of the glucose concentration in the incubation medium. Cold-induced release was transient and rewarming to 37° C restored the sensitivity of B-cells to glucose stimulation. In islets cooled to 2° C, exocytotic profiles could easily be detected both by thin-section and freeze-fracture electron microscopy. As revealed by the freeze-fracture technique, the number of exocytotic profiles per membrane area was increased three-to fourfold as compared to islet cells incubated at 20° C. This was paralleled by intracellular fusion of secretory vesicles. Cold-induced insulin release was not affected by theophylline, cytochalasin B, omission of extracellular Ca⁺⁺ or D600. Replacement of extracellular Na⁺ with choline or sucrose suppressed the increase in insulin release and in frequency of exocytotic profiles recorded after exposure to 2° C. It is suggested that a redistribution of Ca⁺⁺ from intracellular stores, possibly mediated by an increase in intracellular Na⁺, triggers exocytosis of insulin granules upon exposure to cold.     

A role for polyamines in stimulus-secretion coupling in the pancreatic β-cell

Measurement of the content of polyamines in pancreatic islets indicated that no significant change in their concentration took place during glucose-stimulated insulin release. The finding, together with the absence of any effect of -difluoromethylornithine on glucosestimulated insulin release suggested that rapid synthesis of polyamines is not involved in stimulus-secretion coupling in the -cell. The concentration of polyamines found in islets were high enough for them to act as substrates for the Ca²⁺-dependent islet transglutaminase during insulin release. This was further demonstrated by the ability of islet transglutaminase to incorporate [¹⁴C]putrescine into proteins from islet homogenates and by the demonstration of an increase in the covalent incorporation of [¹⁴C]putrescine into the proteins of intact islets following their challenge with glucose. Unlike monoamine substrates of transglutaminase, putrescine failed to effectively inhibit insulin release when its intracellular concentration was increased. A role for polyamines in the secretory process through their incorporation into islet proteins is suggested.     

Modifying actions of calcium ionophores on insulin release.

Beta-Cell-rich pancreatic islets were microdissected from noninbred ob/obmice and exposed to the calcium ionophores X-537A and A-23187. X-537A differed from A-23187 in being a potent insulin secretagogue at non-stimulating glucose concentrations. Both ionophores inhibited the stimulation of insulin release obtained after adding 20 mM glucose to the incubation medium. The latter observation is consistent with the idea of a reduced beta-cell function when the Ca-2+ in the functionally important intracellular pool (s) exceeds a certain concentration. The ionophore inhibition of the glucose-stimulated insulin release may at least in part result from decreased formation of cyclic AMP, since X-537A proved to be as effective as L-epinephrine in reducing the islet content of this nucleotide in the presence of a phosphodiesterase inhibitor. The secretagogic action of X-537A at a low glucose concentration persisted when different ions were omitted from the incubation medium and was actually considerably enhanced in the absence of extracellular Ca-2+. The insulin-releasing action of X-537A was neither influenced by 3-O-methyglucose nor by drugs blocking the alpha or beta-adrenergic receptor sites. Exposure of the pancreatic beta-cells to metabolic inhibitors in concentrations which significantly reduced the secretory response to glucose, potentiated stimulation of insulin release by X-537A, suggesting that this effect may in part be accounted for by intracellular dissolution of secretory granules.     

Glucose-stimulated Single Pancreatic Islets Sustain Increased Cytosolic ATP Levels during Initial Ca2+ Influx and Subsequent Ca2+ Oscillations

In pancreatic islets, insulin secretion occurs via synchronous elevation of Ca²⁺ levels throughout the islets during high glucose conditions. This Ca²⁺ elevation has two phases: a quick increase, observed after the glucose stimulus, followed by prolonged oscillations. In these processes, the elevation of intracellular ATP levels generated from glucose is assumed to inhibit ATP-sensitive K⁺ channels, leading to the depolarization of membranes, which in turn induces Ca²⁺ elevation in the islets. However, little is known about the dynamics of intracellular ATP levels and their correlation with Ca²⁺ levels in the islets in response to changing glucose levels. In this study, a genetically encoded fluorescent biosensor for ATP and a fluorescent Ca²⁺ dye were employed to simultaneously monitor the dynamics of intracellular ATP and Ca²⁺ levels, respectively, inside single isolated islets. We observed rapid increases in cytosolic and mitochondrial ATP levels after stimulation with glucose, as well as with methyl pyruvate or leucine/glutamine. High ATP levels were sustained as long as high glucose levels persisted. Inhibition of ATP production suppressed the initial Ca²⁺ increase, suggesting that enhanced energy metabolism triggers the initial phase of Ca²⁺ influx. On the other hand, cytosolic ATP levels did not fluctuate significantly with the Ca²⁺ level in the subsequent oscillation phases. Importantly, Ca²⁺ oscillations stopped immediately before ATP levels decreased significantly. These results might explain how food or glucose intake evokes insulin secretion and how the resulting decrease in plasma glucose levels leads to cessation of secretion.