Hyperglycemia impairs EAAT2 glutamate transporter trafficking and glutamate clearance in islets of Langerhans: implications for type 2 diabetes pathogenesis and treatment.

Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and ß-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to ß-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high-glucose concentrations led to the transporter internalization in vesicular compartments and reduced [H3]-d-glutamate uptake (65 ± 5% inhibition), phenocopying the findings in T2DM pancreatic sections. The transporter relocalization was associated with decreased Akt phosphorylation protein levels, suggesting an involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the process. In line with this, PI3K inhibition by a 100-µM LY294002 treatment in human and clonal ß-cells caused the transporter relocalization in intracellular compartments and significantly reduced the glutamate uptake compared to control conditions, suggesting that hyperglycemia changes the trafficking of the transporter to the plasma membrane. Upregulation of the glutamate transporter upon treatment with the antibiotic ceftriaxone rescued hyperglycemia-induced ß-cells dysfunction and death. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve ß-cell survival and function in diabetes.NEW & NOTEWORTHY The glutamate transporter SLC1A2/excitatory amino acid transporter 2 (EAAT2) is expressed on the plasma membrane of pancreatic ß-cells and controls islet glutamate clearance and ß-cells survival. We found that the EAAT2 membrane expression is lost in the islets of Langerhans from type 2 diabetes mellitus (T2DM) patients due to hyperglycemia-induced downregulation of the phosphoinositide 3-kinase/Akt pathway and modification of its intracellular trafficking. Pharmacological rescue of EAAT2 expression prevents ß-cell dysfunction and death, suggesting EAAT2 as a new potential target of intervention in T2DM.

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.

Nodding and shaking of the head as simulated approach and avoidance responses.

Our recent study within the embodiment perspective showed that the evaluation of true and false information activates the simulation of vertical and horizontal head movements involved in nodding and shaking of the head (Moretti & Greco, 2018). This result was found in an explicit evaluation task where motion detection software was deployed to enable participants to assess a series of objectively true or false statements by moving them with the head vertically and horizontally on a computer screen, under conditions of compatibility and incompatibility between simulated and performed action. This study replicated that experiment, but with subjective statements about liked and disliked food, in both explicit and implicit evaluation tasks. Two experiments, plus one control experiment, were devised to test the presence of a motor-affective compatibility effect (vertical-liked; horizontal-disliked) and whether the motor-semantic compatibility found with objective statements (vertical-true; horizontal-false) could be a sub-effect of a more general and automatic association (vertical-accepted; horizontal-refused). As expected, response times were shorter when statements about liked foods and disliked foods were moved vertically and horizontally respectively by making head movements, even when participants were not explicitly required to evaluate them. In contrast, the truth compatibility effect only occurred in the explicit evaluation task. Overall results support the idea that head-nodding and shaking are simulated approach-avoidance responses. Different aspects of the meaning of these gestures and the practical implications of the study for cognitive and social research are discussed.

Author Correction: Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

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Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans.

Ex vivo expansion and differentiation of human pancreatic ß-cell are enabling steps of paramount importance for accelerating the development of therapies for diabetes. The success of regenerative strategies depends on their ability to reproduce the chemical and biophysical properties of the microenvironment in which ß-cells develop, proliferate and function. In this paper we focus on the biophysical properties of the extracellular environment and exploit the cluster-assembled zirconia substrates with tailored roughness to mimic the nanotopography of the extracellular matrix. We demonstrate that ß-cells can perceive nanoscale features of the substrate and can convert these stimuli into mechanotransductive processes which promote long-term in vitro human islet culture, thus preserving ß-cell differentiation and function. Proteomic and quantitative immunofluorescence analyses demonstrate that the process is driven by nanoscale topography, via remodelling of the actin cytoskeleton and nuclear architecture. These modifications activate a transcriptional program which stimulates an adaptive metabolic glucose response. Engineered cluster-assembled substrates coupled with proteomic approaches may provide a useful strategy for identifying novel molecular targets for treating diabetes mellitus and for enhancing tissue engineering in order to improve the efficacy of islet cell transplantation therapies.

Truth is in the head. A nod and shake compatibility effect.

Studies from the embodiment perspective on language processing have shown facilitation or interference effects depending on the compatibility between verbal contents, concrete or abstract, and the motion of various parts of the body. The aim of the present study was to test whether such compatibility effects can be found when a higher cognitive process like truth evaluation is accomplished with head movements. Since nodding is a vertical head gesture typically performed with positive and affirmative responses, and shaking is a horizontal head gesture associated with negative and dissenting contents, faster response times can be expected when true information is evaluated by making a vertical head movement and false information by making a horizontal head movement. Three experiments were designed in order to test this motor compatibility effect. In the first experiment a series of very simple sentences were asked to be evaluated as true or false by dragging them vertically and horizontally with the head. It resulted that truth-value was assessed faster when it was compatible with the direction of the head movement, compared to when it was incompatible. In the second experiment participants were asked to evaluate the same sentences as the first experiment but by moving them with the mouse. In the third experiment, a non-evaluative classification task was given, where sentences concerning animals or objects were to be dragged by vertical and horizontal head movements. In the second and third experiment no compatibility effect was observed. Overall results support the hypothesis of an embodiment effect between the abstract processing of truth evaluation and the direction of the two head movements of nodding and shaking. Cultural aspects, cognitive implications, and the limits of these findings are discussed.

Use of evidence in a categorization task: analytic and holistic processing modes.

Category learning performance can be influenced by many contextual factors, but the effects of these factors are not the same for all learners. The present study suggests that these differences can be due to the different ways evidence is used, according to two main basic modalities of processing information, analytically or holistically. In order to test the impact of the information provided, an inductive rule-based task was designed, in which feature salience and comparison informativeness between examples of two categories were manipulated during the learning phases, by introducing and progressively reducing some perceptual biases. To gather data on processing modalities, we devised the Active Feature Composition task, a production task that does not require classifying new items but reproducing them by combining features. At the end, an explicit rating task was performed, which entailed assessing the accuracy of a set of possible categorization rules. A combined analysis of the data collected with these two different tests enabled profiling participants in regard to the kind of processing modality, the structure of representations and the quality of categorial judgments. Results showed that despite the fact that the information provided was the same for all participants, those who adopted analytic processing better exploited evidence and performed more accurately, whereas with holistic processing categorization is perfectly possible but inaccurate. Finally, the cognitive implications of the proposed procedure, with regard to involved processes and representations, are discussed.

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.

Delta cell death in the islet of Langerhans and the progression from normal glucose tolerance to type 2 diabetes in non-human primates (baboon, Papio hamadryas).

AIMS/HYPOTHESIS: The cellular composition of the islet of Langerhans is essential to ensure its physiological function. Morphophysiological islet abnormalities are present in type 2 diabetes but the relationship between fasting plasma glucose (FPG) and islet cell composition, particularly the role of delta cells, is unknown. We explored these questions in pancreases from baboons (Papio hamadryas) with FPG ranging from normal to type 2 diabetic values. METHODS: We measured the volumes of alpha, beta and delta cells and amyloid in pancreatic islets of 40 baboons (Group 1 [G1]: FPG < 4.44 mmol/l [n = 10]; G2: FPG = 4.44-5.26 mmol/l [n = 9]; G3: FPG = 5.27-6.94 mmol/l [n = 9]; G4: FPG > 6.94 mmol/l [n = 12]) and correlated islet composition with metabolic and hormonal variables. We also performed confocal microscopy including TUNEL, caspase-3, and anti-caspase cleavage product of cytokeratin 18 (M30) immunostaining, electron microscopy, and immuno-electron microscopy with anti-somatostatin antibodies in baboon pancreases. RESULTS: Amyloidosis preceded the decrease in beta cell volume. Alpha cell volume increased ∼ 50% in G3 and G4 (p < 0.05), while delta cell volume decreased in these groups by 31% and 39%, respectively (p < 0.05). In G4, glucagon levels were higher, while insulin and HOMA index of beta cell function were lower than in the other groups. Immunostaining of G4 pancreatic sections with TUNEL, caspase-3 and M30 showed apoptosis of beta and delta cells, which was also confirmed by immuno-electron microscopy with anti-somatostatin antibodies. CONCLUSIONS/INTERPRETATION: In diabetic baboons, changes in islet composition correlate with amyloid deposition, with increased alpha cell and decreased beta and delta cell volume and number due to apoptosis. These data argue for an important role of delta cells in type 2 diabetes.

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.