Effect of adenosine triphosphate (ATP) on the secretion of insulin induced by tolbutamide in the absence of glucose

Our experiments were carried out on the isolated perfused rat pancreas. The effect of ATP on insulin secretion induced by tolbutamide (a hypoglycemic sulfonylurea) was studied in the absence of glucose. The addition of ATP (0.165 mmol/l) to the perfusion medium did not significantly modify the first phase induced by tolbutamide (0.4 mmol/l) but potentiated the second phase.     

Dual mechanism of the potentiation by glucose of insulin secretion induced by arginine and tolbutamide in mouse islets

Glucose induces insulin secretion (IS) and also potentiates the insulin-releasing action of secretagogues such as arginine and sulfonylureas. This potentiating effect is known to be impaired in type 2 diabetic patients, but its cellular mechanisms are unclear. IS and cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse islets during perifusion with 3-15 mmol/l glucose (G3-G15, respectively) and pulse or stepwise stimulation with 1-10 mmol/l arginine or 5-250 micromol/l tolbutamide. In G3, arginine induced small increases in [Ca(2+)](i) but no IS. G7 alone only slightly increased [Ca(2+)](i) and IS but markedly potentiated arginine effects on [Ca(2+)](i), which resulted in significant IS (already at 1 mmol/l). For each arginine concentration, both responses further increased at G10 and G15, but the relative change was distinctly larger for IS than [Ca(2+)](i). At all glucose concentrations, tolbutamide dose dependently increased [Ca(2+)](i) and IS with thresholds of 25 micromol/l for [Ca(2+)](i) and 100 micromol/l for IS at G3 and of 5 micromol/l for both at G7 and above. Between G7 and G15, the effect of tolbutamide on [Ca(2+)](i) increased only slightly, whereas that on IS was strongly potentiated. The linear relationship between IS and [Ca(2+)](i) at increasing arginine or tolbutamide concentrations became steeper as the glucose concentration was raised. Thus glucose augmented more the effect of each agent on IS than that on [Ca(2+)](i). In conclusion, glucose potentiation of arginine- or tolbutamide-induced IS involves increases in both the rise of [Ca(2+)](i) and the action of Ca(2+) on exocytosis. This dual mechanism must be borne in mind to interpret the alterations of the potentiating action of glucose in type 2 diabetic patients.     

Extracellular bicarbonate ions and insulin secretion

The role of bicarbonate ions in insulin release was studied with incubated and perifused isolated rat islets of Langerhans. In the absence of NaHCO₃, the early phase of glucose-induced secretion was completely abolished and the second phase inhibited by approximately 65%. The insulinotropic effect of the sugar was totally restored after reintroduction of the ion in the medium. The monophasic secretory response to tolbutamide was also markedly diminished by omission of NaHCO₃, whereas the release evoked by a high concentration of K⁺ was very little affected. CO₂ was unable to substitute for HCO₃⁻, but small concentrations of the anion (3 to 5 mM) were sufficient to ensure a normal response to glucose.     

Endogenous ATP potentiates only vasopressin secretion from neurohypophysial terminals

Exogenous ATP induces inward currents and causes the release of arginine-vasopressin (AVP) from isolated neurohypophysial terminals (NHT); both effects are inhibited by the P2X2 and P2X3 antagonists, suramin and PPADS. Here we examined the role of endogenous ATP in the neurohypophysis. Stimulation of NHT caused the release of both AVP and ATP. ATP induced a potentiation in the stimulated release of AVP, but not of oxytocin (OT), which was blocked by the presence of suramin. In loose-patch clamp recordings, from intact neurohypophyses, suramin or PPADS produces an inhibition of action potential currents in a static bath, that can be mimicked by a hyperpolarization of the resting membrane potential (RMP). Correspondingly, in a static versus perfused bath there is a depolarization of the RMP of NHT, which was reduced by either suramin or PPADS. We measured an accumulation of ATP (3.7 +/- 0.7 microM) released from NHT in a static bath. Applications of either suramin or PPADS to a static bath decreased burst-stimulated capacitance increases in NHT. Finally, only vasopressin release from electrically stimulated intact neurohypophyses was reduced in the presence of Suramin or PPADS. These data suggest that there was sufficient accumulation of ATP released from the neurohypophysis during stimulations to depolarize its nerve terminals. This would occur via the opening of P2X2 and P2X3 receptors, inducing an influx of Ca2+. The subsequent elevation in [Ca2+](i) would further increase the stimulated release of only vasopressin from NHT terminals. Such purinergic feedback mechanisms could be physiologically important at most CNS synapses.     

Extracellular ATP and zinc are co-secreted with insulin and activate multiple P2X purinergic receptor channels expressed by islet beta-cells to potentiate insulin secretion

It is well established that ATP is co-secreted with insulin and zinc from pancreatic beta-cells (β-cells) in response to elevations in extracellular glucose concentration. Despite this knowledge, the physiological roles of extracellular secreted ATP and zinc are ill-defined. We hypothesized that secreted ATP and zinc are autocrine purinergic signaling molecules that activate P2X purinergic receptor (P2XR) channels expressed by β-cells to enhance glucose-stimulated insulin secretion (GSIS). To test this postulate, we performed ELISA assays for secreted insulin at fixed time points within a “real-time” assay and confirmed that the physiological insulin secretagogue glucose stimulates secretion of ATP and zinc into the extracellular milieu along with insulin from primary rat islets. Exogenous ATP and zinc alone or together also induced insulin secretion in this model system. Most importantly, the presence of an extracellular ATP scavenger, a zinc chelator, and P2 receptor antagonists attenuated GSIS. Furthermore, mRNA and protein were expressed in immortalized β-cells and primary islets for a unique subset of P2XR channel subtypes, P2X₂, P2X₃, P2X₄, and P2X₆, which are each gated by extracellular ATP and modulated positively by extracellular zinc. On the basis of these results, we propose that, within endocrine pancreatic islets, secreted ATP and zinc have profound autocrine regulatory influence on insulin secretion via ATP-gated and zinc-modulated P2XR channels.     

Diffusion induced oscillatory insulin secretion

Oscillatory secretion of insulin has been observed in many different experimental preparations. Here we examine a mathematical model for in vitro insulin secretion from pancreatic beta cells in a flow-through reactor. The analysis shows that oscillations result because of an important interplay between flow rate of the reactor and insulin diffusion. In particular, if the ratio of flow rate to volume of the reaction bed is too large, oscillations are eliminated, in contradiction to the conclusions of Maki and Keizer (L. W. Maki and Keizer J. Mathematical analysis of a proposed mechanism for oscillatory insulin secretion in perifused HIT-15 cells. Bull. Math. Biol., 57 (1995), 569–591). Furthermore, with reasonable numbers for the experimental parameters and the diffusion of insulin, the model equations do not exhibit oscillations.     

K(ATP) channels and insulin secretion disorders

ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP. Nutrient oxidation in beta-cells leads to a rise in [ATP]-to-[ADP] ratios, which in turn leads to reduced K(ATP) channel activity, depolarization, voltage-dependent Ca(2+) channel activation, Ca(2+) entry, and exocytosis. Persistent hyperinsulinemic hypoglycemia of infancy (HI) is a genetic disorder characterized by dysregulated insulin secretion and, although rare, causes severe mental retardation and epilepsy if left untreated. The last five or six years have seen rapid advance in understanding the molecular basis of K(ATP) channel activity and the molecular genetics of HI. In the majority of cases for which a genotype has been uncovered, causal HI mutations are found in one or the other of the two genes, SUR1 and Kir6.2, that encode the K(ATP) channel. This article will review studies that have defined the link between channel activity and defective insulin release and will consider implications for future understanding of the mechanisms of control of insulin secretion in normal and diseased states.     

Leptin fully suppresses acetylcholine-induced insulin secretion and is reversed by tolbutamide in isolated perfused chicken pancreas.

So far, there has been no evidence for any direct pancreatic effect of leptin in the chicken. The present study was aimed at detecting chicken leptin receptor (cOb-R) expression in isolated chicken islets of Langerhans and to examine the direct effect of leptin on insulin secretion after stimulation by acetylcholine (1 micro M) + glucose (14 mM) from isolated perfused chicken pancreas. We will show that i) full length cOb-R mRNA was expressed in isolated pancreatic islets of chickens, ii) recombinant chicken leptin (10 nM) or diazoxide (100 micro M) rapidly (within 2 min) and significantly suppressed insulin secretion induced by acetylcholine stimulation without any change in volume outflow rate, iii) tolbutamide (100 micro M) introduced 10 min after leptin and perfused for 10 min fully reversed the suppressive effect of leptin on pre-established acetylcholine-induced insulin release. In conclusion, we found that leptin has a profound inhibitory influence upon insulin secretion in perfused chicken pancreas. The results suggest that leptin inhibits insulin secretion by acting before or at the level of K ATP channels in chicken pancreatic beta-cells. Further studies are warranted to clarify the specific inhibitory mechanism.     

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.     

AICAR induced AMPK activation potentiates neuronal insulin signaling and glucose uptake

Insulin signaling is extensively studied in peripheral tissues while comparatively understudied in neuronal cells. AMPK is considered to be a fuel gauge of our body and activation of the same has been reported to increase insulin sensitivity in skeletal muscles thereby increasing glucose transport. However its role in neuronal insulin signaling is not established yet. Here we report positive regulation of insulin signaling as well as glucose uptake by AICAR, a pharmacological activator of AMPK, in cultured Neuro-2a cells in vitro. Compound C, a specific AMPK inhibitor, completely blocked the potentiating effects of AICAR on insulin signaling and glucose uptake, thus suggesting that AMPK mediates effects of AICAR on insulin signaling. Our study provides valuable insight in understanding the role of AMPK in neuronal insulin signal transduction.