Alberto Fachado

Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans

Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets 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. Cholinergic signaling within islets represents a potential therapeutic target in diabetes, highlighting the relevance of this advance to future drug development.

Glutamate is a positive autocrine signal for glucagon release.

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.

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic β cell

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 cells sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca2+ concentration, [Ca2+]i, and hormone release in vitro, we show that human β cells express ionotropic ATP receptors of the P2X3 type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X3 receptors in the β-cell plasma membrane, resulting in increased [Ca2+]i and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the β cells 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.

The Thyroid Hormone-Inactivating Type III Deiodinase Is Expressed in Mouse and Human β-Cells and Its Targeted Inactivation Impairs Insulin Secretion

Deiodinases are selenoproteins that activate or inactivate thyroid hormone. During vertebrate development, these pathways control thyroid hormone action in a cell-specific fashion explaining how systemic thyroid hormone can affect local control of tissue embryogenesis. Here we investigated the role of the thyroid hormone-inactivating deiodinase (D3) in pancreatic islet function and glucose homeostasis. D3 expression was determined by real-time PCR, immunofluorescence, and enzyme activity. Embryonic and adult wild-type mice and Mice with targeted disruption of Dio3 gene (D3KO) as well as human fetal pancreas and adult islets were studied. Insulin secretion was evaluated in adult mouse isolated islets. We found Dio3 gene expression and protein highly expressed in embryonic and adult pancreatic islets, predominantly in β-cells in both humans and mice. However, mRNA levels were barely detectable for both the thyroid hormone-activating deiodinases types 1 and 2. D3KO animals were found to be glucose intolerant due to in vitro and in vivo impaired glucose-stimulated insulin secretion, without changes in peripheral sensitivity to insulin. D3KO neonatal (postnatal day 0) and adult pancreas exhibited reduced total islet area due to reduced β-cell mass, insulin content, and impaired expression of key β-cells genes. D3 expression in perinatal pancreatic β-cells prevents untimely exposure to thyroid hormone, the absence of which leads to impaired β-cell function and subsequently insulin secretion and glucose homeostasis. An analogous role is likely in humans, given the similar D3 expression pattern.

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.

Control Of Insulin Secretion By Cholinergic Signaling In The Human Pancreatic Islet

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.

Automated, High-Throughput Assays for Evaluation of Human Pancreatic Islet Function

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.

Long-Term Protective Immune Response Elicited by Vaccination with an Expression Genomic Library of Toxoplasma gondii

ABSTRACT Immunization of BALB/c mice with an expression genomic library of Toxoplasma gondii induces a Th1-type immune response, with recognition of several T. gondii proteins (21 to 117 kDa) and long-term protective immunity against a lethal challenge. These results support further investigations to achieve a multicomponent anti-T. gondii DNA vaccine.

Maternal Inheritance of an Inactive Type III Deiodinase Gene Allele Affects Mouse Pancreatic β-Cells and Disrupts Glucose Homeostasis

Dio3 is the most distal gene of the imprinted Dlk1-Dio3 gene locus and is expressed according to parental origin. Dio3 encodes the type 3 deiodinase (D3), a thioredoxin-fold like containing selenoenzyme that inactivates thyroid hormone and dampens thyroid hormone signaling. Here we used heterozygous animals with disruption of the Dio3 gene to study the allelic expression pattern of Dio3 in pancreatic β-cells and the metabolic phenotype resulting from its inactivation. Adult heterozygous mice with disruption of the Dio3 gene with maternal inheritance of the inactive Dio3 allele exhibited a total loss of D3 activity in isolated pancreatic islets, approximately 30% reduction in total pancreatic islet area, a marked decrease in insulin2 mRNA and in vivo glucose intolerance. In contrast, inheritance of the inactive Dio3 allele from the father did not affect D3 activity in isolated pancreatic islets and did not result in a pancreatic phenotype. Furthermore, exposure of pancreatic explants, D3-expressing MIN6-C3 cells or isolated pancreatic islets to 100 nM T3 for 24 hours reduced insulin2 mRNA by approximately 50% and the peak of glucose-induced insulin secretion. An unbiased analysis of T3-treated pancreatic islets revealed the down-regulation of 21 gene sets (false discovery rate q value < 25%) involved in nucleolar function and transcription of rRNA, ribonucleotide binding, mRNA translation, and membrane organization. We conclude that the Dio3 gene is preferentially expressed from the maternal allele in pancreatic islets and that the inactivation of this allele is sufficient to disrupt glucose homeostasis by reducing the pancreatic islet area, insulin2 gene expression, and glucose-stimulated insulin secretion.

Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor

Nephrin belongs to a family of highly conserved proteins with a well characterized function as modulators of cell adhesion and guidance, and nephrin may have a role in metabolic pathways linked to podocyte and pancreatic β-cell survival. However, this role is incompletely characterized. In this study, we developed floxed nephrin mice for pancreatic β-cell-specific deletion of nephrin, which had no effect on islet size and glycemia. Nephrin deficiency, however, resulted in glucose intolerance in vivo and impaired glucose-stimulated insulin release ex vivo Glucose intolerance was also observed in eight patients with nephrin mutations compared with three patients with other genetic forms of nephrotic syndrome or nine healthy controls.In vitro experiments were conducted to investigate if nephrin affects autocrine signaling through insulin receptor A (IRA) and B (IRB), which are both expressed in human podocytes and pancreatic islets. Coimmunoprecipitation of nephrin and IRB but not IRA was observed and required IR phosphorylation. Nephrin per se was sufficient to induce phosphorylation of p70S6K in an phosphatidylinositol 3-kinase-dependent but IR/Src-independent manner, which was not augmented by exogenous insulin. These results suggest a role for nephrin as an independent modulator of podocyte and pancreatic β-cell nutrient sensing in the fasting state and the potential of nephrin as a drug target in diabetes.