Autoantibodies against the glial glutamate transporter GLT1/EAAT2 in Type 1 diabetes mellitus-Clues to novel immunological and non-immunological therapies.

Islet cell surface autoantibodies were previously found in subjects with type 1 diabetes mellitus (T1DM), but their target antigens and pathogenic mechanisms remain elusive. The glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2) is expressed on the membrane of pancreatic ß-cells and physiologically controls extracellular glutamate concentrations thus preventing glutamate-induced ß-cell death. We hypothesized that GLT1 could be an immunological target in T1DM and that autoantibodies against GLT1 could be pathogenic. Immunoprecipitation and ELISA experiments showed that sera from T1DM subjects recognized GLT1 expressed in brain, pancreatic islets, and GLT1-transfected COS7-cell extracts. We validated these findings in two cohorts of T1DM patients by quantitative immunofluorescence assays. Analysis of the combined data sets indicated the presence of autoantibodies against GLT1 in 32 of the 87 (37%) T1DM subjects and in none of healthy controls (n = 64) (p < 0.0001). Exposure of pancreatic ßTC3 cells and human islets to purified IgGs from anti-GLT1 positive sera supplemented with complement resulted in plasma membrane ruffling, cell lysis and death. The cytotoxic effect was prevented when sera were depleted from IgGs. Furthermore, in the absence of complement, 6 out of 16 (37%) anti-GLT1 positive sera markedly reduced GLT1 transport activity in ßTC3 cells by inducing GLT1 internalization, also resulting in ß-cell death. In conclusion, we provide evidence that GLT1 is a novel T1DM autoantigen and that anti-GLT1 autoantibodies cause ß-cell death through complement-dependent and independent mechanisms. GLT1 seems an attractive novel therapeutic target for the prevention of ß-cell death in individuals with diabetes and prediabetes.

Exenatide regulates pancreatic islet integrity and insulin sensitivity in the nonhuman primate baboon Papio hamadryas.

The glucagon-like peptide-1 receptor agonist exenatide improves glycemic control by several and not completely understood mechanisms. Herein, we examined the effects of chronic intravenous exenatide infusion on insulin sensitivity, ß cell and α cell function and relative volumes, and islet cell apoptosis and replication in nondiabetic nonhuman primates (baboons). At baseline, baboons received a 2-step hyperglycemic clamp followed by an l-arginine bolus (HC/A). After HC/A, baboons underwent a partial pancreatectomy (tail removal) and received a continuous exenatide (n = 12) or saline (n = 12) infusion for 13 weeks. At the end of treatment, HC/A was repeated, and the remnant pancreas (head-body) was harvested. Insulin sensitivity increased dramatically after exenatide treatment and was accompanied by a decrease in insulin and C-peptide secretion, while the insulin secretion/insulin resistance (disposition) index increased by about 2-fold. ß, α, and δ cell relative volumes in exenatide-treated baboons were significantly increased compared with saline-treated controls, primarily as the result of increased islet cell replication. Features of cellular stress and secretory dysfunction were present in islets of saline-treated baboons and absent in islets of exenatide-treated baboons. In conclusion, chronic administration of exenatide exerts proliferative and cytoprotective effects on ß, α, and δ cells and produces a robust increase in insulin sensitivity in nonhuman primates.

Neurotransmitters and Neuropeptides: New Players in the Control of Islet of Langerhans' Cell Mass and Function.

Islets of Langerhans control whole body glucose homeostasis, as they respond, releasing hormones, to changes in nutrient concentrations in the blood stream. The regulation of hormone secretion has been the focus of attention for a long time because it is related to many metabolic disorders, including diabetes mellitus. Endocrine cells of the islet use a sophisticate system of endocrine, paracrine and autocrine signals to synchronize their activities. These signals provide a fast and accurate control not only for hormone release but also for cell differentiation and survival, key aspects in islet physiology and pathology. Among the different categories of paracrine/autocrine signals, this review highlights the role of neurotransmitters and neuropeptides. In a manner similar to neurons, endocrine cells synthesize, accumulate, release neurotransmitters in the islet milieu, and possess receptors able to decode these signals. In this review, we provide a comprehensive description of neurotransmitter/neuropetide signaling pathways present within the islet. Then, we focus on evidence supporting the concept that neurotransmitters/neuropeptides and their receptors are interesting new targets to preserve ß-cell function and mass. A greater understanding of how this network of signals works in physiological and pathological conditions would advance our knowledge of islet biology and physiology and uncover potentially new areas of pharmacological intervention. J. Cell. Physiol. 231: 756-767, 2016. © 2015 Wiley Periodicals, Inc.

TIRFM and pH-sensitive GFP-probes to evaluate neurotransmitter vesicle dynamics in SH-SY5Y neuroblastoma cells: cell imaging and data analysis.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease. Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (<150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events. In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented. To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling. With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

Chronic continuous exenatide infusion does not cause pancreatic inflammation and ductal hyperplasia in non-human primates.

In this study, we aimed to evaluate the effects of exenatide (EXE) treatment on exocrine pancreas of nonhuman primates. To this end, 52 baboons (Papio hamadryas) underwent partial pancreatectomy, followed by continuous infusion of EXE or saline (SAL) for 14 weeks. Histological analysis, immunohistochemistry, Computer Assisted Stereology Toolbox morphometry, and immunofluorescence staining were performed at baseline and after treatment. The EXE treatment did not induce pancreatitis, parenchymal or periductal inflammatory cell accumulation, ductal hyperplasia, or dysplastic lesions/pancreatic intraepithelial neoplasia. At study end, Ki-67-positive (proliferating) acinar cell number did not change, compared with baseline, in either group. Ki-67-positive ductal cells increased after EXE treatment (P = 0.04). However, the change in Ki-67-positive ductal cell number did not differ significantly between the EXE and SAL groups (P = 0.13). M-30-positive (apoptotic) acinar and ductal cell number did not change after SAL or EXE treatment. No changes in ductal density and volume were observed after EXE or SAL. Interestingly, by triple-immunofluorescence staining, we detected c-kit (a marker of cell transdifferentiation) positive ductal cells co-expressing insulin in ducts only in the EXE group at study end, suggesting that EXE may promote the differentiation of ductal cells toward a ß-cell phenotype. In conclusion, 14 weeks of EXE treatment did not exert any negative effect on exocrine pancreas, by inducing either pancreatic inflammation or hyperplasia/dysplasia in nonhuman primates.

Neurosteroid allopregnanolone regulates EAAC1-mediated glutamate uptake and triggers actin changes in Schwann cells.

Recent evidence shows that neurotransmitters (e.g., GABA, Ach, adenosine, glutamate) are active on Schwann cells, which form myelin sheaths in the peripheral nervous system under different pathophysiologic conditions. Glutamate, the most important excitatory neurotransmitter, has been recently involved in peripheral neuropathies, thus prevention of its toxic effect is desirable to preserve the integrity of peripheral nervous system and Schwann cells physiology. Removal of glutamate from the extracellular space is accomplished by the high affinity glutamate transporters, so we address our studies to analyze their functional presence in Schwann cells. We first demonstrate that Schwann cells express the EAAC1 transporter in the plasma membrane and in intracellular vesicular compartments of the endocytic recycling pathways. Uptake experiments confirm its presence and functional activity in Schwann cells. Secondly, we demonstrate that the EAAC1 activity can be modulated by exposure to the neurosteroid allopregnanolone 10 nM (a progesterone metabolite proved to support Schwann cells). Transporter up-regulation by allopregnanolone is rapid, does not involve protein neo-synthesis and is prevented by actin depolymerization. Allopregnanolone modulation involves GABA-A receptor and PKC activation, promotes the exocytosis of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips, and modifies the morphology of cell processes. Finally, we provide evidence that glutamate transporters control the allopregnanolone-mediated effects on cell proliferation. Our findings are the first to demonstrate the presence of a functional glutamate uptake system, which can be dynamically modulated by allopregnanolone in Schwann cells. Glutamate transporters may represent a potential therapeutic target to control Schwann cell physiology.

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death.

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal ß-cell lines and human islet ß-cells but not in α-cells. In human islets, glutamate-induced ß-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of ß-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced ß-cell death, whereas GLT1 up-regulation promoted ß-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that ß-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.