Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development.

An important feature of glucose homeostasis is the effective release of glucagon from the pancreatic alpha cell. The molecular mechanisms regulating glucagon secretion are still poorly understood. We now demonstrate that human alpha cells express ionotropic glutamate receptors (iGluRs) that are essential for glucagon release. A lowering in glucose concentration results in the release of glutamate from the alpha cell. Glutamate then acts on iGluRs of the AMPA/kainate type, resulting in membrane depolarization, opening of voltage-gated Ca(2+) channels, increase in cytoplasmic free Ca(2+) concentration, and enhanced glucagon release. In vivo blockade of iGluRs reduces glucagon secretion and exacerbates insulin-induced hypoglycemia in mice. Hence, the glutamate autocrine feedback loop endows the alpha cell with the ability to effectively potentiate its own secretory activity. This is a prerequisite to guarantee adequate glucagon release despite relatively modest changes in blood glucose concentration under physiological conditions.

Sodium selenite (Na 2 SeO 3 ) is the selenium form used in the composition of dietary supplements, and diphenyl diselenide (PhSe) 2 is an important intermediate in organic synthesis, which increases the risk of human exposure to this chemical in the workplace. These compounds have been reported to inhibit the cerebral and hepatic aminolevulinic acid dehydratase (ALA-D) in vitro, and now we show that ascorbic acid can reverse some alterations caused by in vivo selenium exposure, but not ALA-D inhibition. The effect of Na 2 SeO 3 or (PhSe) 2 and ascorbic acid on selenium distribution, total non-protein thiol, ascorbic acid content (liver and brain) and haemoglobin was also examined. Mice were exposed to 250 mmol/kg (PhSe) 2 , or 18.75 mmol/kg Na 2 SeO 3 subcutaneously, and to ascorbic acid, twice a day, 1 mmol/kg intraperitonially, for 10 days. Hepatic ALA-D of mice treated with (PhSe) 2 was inhibited about 58% and similar results were observed in the animals that received ascorbic acid supplementation (PϽ0.01, for (PhSe) 2 -treated and (PhSe) 2 πascorbic acid-treated mice). The haemoglobin content decreased after treatment with (PhSe) 2 (PϽ0.01). However, the haemoglobin content of the (PhSe) 2 πascorbic acid group was significantly higher than in the (PhSe) 2 -treated mice (PϽ0.05), and similar to control (PϾ0.10). Ascorbic acid treatment decreased significantly the hepatic and cerebral deposition of Se in (PhSe) 2 -exposed mice (PϽ0.01). Hepatic non-protein thiol content was not changed by treatment with (PhSe) 2 , ascorbic acid or (PhSe) 2 πascorbic acid. Hepatic content of ascorbic acid was twice that in mice that received (PhSe) 2 , independent of ascorbic acid treatment (PϽ0.001). The results of this study suggest that vitamin C may have a protective role in organodiselenide intoxication.

Acetylcholine regulates hormone secretion from the pancreatic islet and is thus crucial for glucose homeostasis. Little is known, however, about acetylcholine (cholinergic) signaling in the human islet. We recently reported that in the human islet, acetylcholine is primarily a paracrine signal released from α-cells rather than primarily a neural signal as in rodent islets. In this study, we demonstrate that the effects acetylcholine produces in the human islet are different and more complex than expected from studies conducted on cell lines and rodent islets. We found that endogenous acetylcholine not only stimulates the insulin-secreting β-cell via the muscarinic acetylcholine receptors M3 and M5, but also the somatostatin-secreting δ-cell via M1 receptors. Because somatostatin is a strong inhibitor of insulin secretion, we hypothesized that cholinergic input to the δ-cell indirectly regulates β-cell function. Indeed, when all muscarinic signaling was blocked, somatostatin secretion decreased and insulin secretion unexpectedly increased, suggesting a reduced inhibitory input to β-cells. Endogenous cholinergic signaling therefore provides direct stimulatory and indirect inhibitory input to β-cells to regulate insulin secretion from the human islet.

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human β cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca 2+ concentration, [Ca 2+ ] i , and hormone release in vitro, we show that human β cells express ionotropic ATP receptors of the P2X 3 type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X 3 receptors in the β-cell plasma membrane, resulting in increased [Ca 2+ ] i and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the β cell's secretory machinery. This may explain how the human pancreatic β cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.extracellular ATP | human pancreatic β cell | insulin secretion | P2X receptor | positive autocrine feedback G lucose homeostasis is tightly controlled by hormone secretion from the endocrine pancreas, the islets of Langerhans. Even small physiological deviations (e.g., 10%) in plasma glucose are effectively counteracted by sharp (e.g., 3-fold) increases in the secretion of the islet hormones insulin and glucagon (1). Intraislet autocrine and paracrine signaling are pivotal mechanisms for proper function of the islet, making islet cells extremely sensitive and responsive to plasma glucose fluctuations. The roles of different compounds such as GABA, glutamate, Zn 2+ , insulin, and ATP as autocrine and paracrine regulators of islet hormone release have been examined extensively (2-8). Extracellular ATP seems important because it is present in insulin-containing secretory granules and is released during glucose stimulation in sufficient amounts to stimulate ATP receptors (9-12).Extracellular ATP is an important neurotransmitter signal in the brain as well as in vascular, immune, and endocrine cells (13-15). The purinergic system comprises receptors for extracellular ATP and adenosine, the P2 and P1 receptors, respectively. P2 purinergic receptors can be divided into metabotropic P2Y receptors (G protein coupled) and ionotropic P2X receptors (ligand-gated ion channels) (16). The ionotropic P2X family comprises seven subtypes named P2X 1 -P2X 7 that regulate cell function by opening cation channels permeable to Na + , K + , and Ca 2+ (15,17). Activation of these channels regulates the release of neurotransmitters and hormones, either through direct Ca 2+ influx or by promoting membrane depolarization and thereby inducing action potentials (18-21).The role of ATP signaling in the physiology of pancreatic islets has been studied in rodent models, but the results in the literature are conflicting (22-28). In rat islets, purinergic agonists...

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1 Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X 7 subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2 P2Y and P2X receptor agonists ATP, uridine 5 0 -triphosphate (UTP) and 2 0 ,3 0 -O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration-and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X 7 receptor. Activation was maximal at 5-10 min and was sustained for 60 min; the EC 50 for BzATP was approximately 50 mM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3 The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X 7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5 0 -phosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4 Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5 In conclusion, our data indicate that stimulation of astrocytic P2X 7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

An important challenge in pancreatic islet transplantation in association with type 1 diabetes is to define automatic high-throughput assays for evaluation of human islet function. The physiological techniques presently used are amenable to small-scale experimental samples and produce descriptive results. The postgenomic era provides an opportunity to analyze biological processes on a larger scale, but the transition to high-throughput technologies is still a challenge. As a first step to implement high-throughput assays for the study of human islet function, we have developed two methodologies: multiple automated perifusion to determine islet hormone secretion and high-throughput kinetic imaging to examine islet cellular responses. Both technologies use fully automated devices that allow performing simultaneous experiments on multiple islet preparations. Our results illustrate that these technologies can be applied to study the functional status and explore the pharmacological profiles of islet cells. These methodologies will enable functional characterization of human islet preparations before transplantation and thereby provide the basis for the establishment of predictive tests for β-cell potency.