The glutamate receptor GluK2 contributes to the regulation of glucose homeostasis and its deterioration during aging

OBJECTIVE: Islets secrete neurotransmitters including glutamate which participate in fine regulation of islet function. The excitatory ionotropic glutamate receptor GluK2 of the kainate receptor family is widely expressed in brain and also found in islets, mainly in alpha and gamma cells. alpha cells co-release glucagon and glutamate and the latter increases glucagon release via ionotropic glutamate receptors. However, neither the precise nature of the ionotropic glutamate receptor involved nor its role in glucose homeostasis is known. As isoform specific pharmacology is not available, we investigated this question in constitutive GluK2 knock-out mice (GluK2-/-) using adult and middle-aged animals to also gain insight in a potential role during aging. METHODS: We compared wild-type GluK2+/+ and knock-out GluK2-/- mice using adult (14-20 weeks) and middle-aged animals (40-52 weeks). Glucose (oral OGTT and intraperitoneal IPGTT) and insulin tolerance as well as pyruvate challenge tests were performed according to standard procedures. Parasympathetic activity, which stimulates hormones secretion, was measured by electrophysiology invivo. Isolated islets were used invitro to determine islet beta-cell electrical activity on multi-electrode arrays and dynamic secretion of insulin as well as glucagon was determined by ELISA. RESULTS: Adult GluK2-/- mice exhibit an improved glucose tolerance (OGTT and IPGTT), and this was also apparent in middle-aged mice, whereas the outcome of pyruvate challenge was slightly improved only in middle-aged GluK2-/- mice. Similarly, insulin sensitivity was markedly enhanced in middle-aged GluK2-/- animals. Basal and glucose-induced insulin secretion invivo was slightly lower in GluK2-/- mice, whereas fasting glucagonemia was strongly reduced. Invivo recordings of parasympathetic activity showed an increase in basal activity in GluK2-/- mice which represents most likely an adaptive mechanism to counteract hypoglucagonemia rather than altered neuronal mechanism. Invitro recording demonstrated an improvement of glucose-induced electrical activity of beta-cells in islets obtained from GluK2-/- mice at both ages. Finally, glucose-induced insulin secretion invitro was increased in GluK2-/- islets, whereas glucagon secretion at 2mmol/l of glucose was considerably reduced. CONCLUSIONS: These observations indicate a general role for kainate receptors in glucose homeostasis and specifically suggest a negative effect of GluK2 on glucose homeostasis and preservation of islet function during aging. Our observations raise the possibility that blockade of GluK2 may provide benefits in glucose homeostasis especially during aging.Transistors multimodaux sensibles aux ions à polymères ambivalents pour biocapteurs hybridesIdentification de biomarqueurs du stress et de l'inflammation des cellules B pancréatiques en explorant les communications inter-organes dans des modèles précliniques d'obésité et de diabète de type

Communications intercellulaires dynamiques au sein des îlots pancréatiques analysées par multi-electrode arrays : rôles physiologiques et applications biotechnologiques en diabétologie

Pancreatic islets are the main sensor of glycaemia and they integrate all the metabolic and hormonal inputs to adapt in real time the secretion of hormones such as insulin by β cells and glucagon by α cells. In type 1 diabetes (T1D) β cells are destroyed by immune attack, and in T2D, β cell mass, function and the intra-islet network are altered. The islet micro-organs are highly reactive due to their electrical properties encoding rapid information and due to intercellular communications between β cells and β/non-β cells. Nevertheless, non-invasive, high resolution and long-term approaches for analysis are still lacking. Extracellular electrophysiology with multi-electrode arrays (MEAs) allows this analysis of islets by measuring both cellular as well as multicellular signals (SPs) due to β cell coupling. During my PhD, I used MEAs (i) to explore islet physiology/pathophysiology and (ii) for biotechnological applications in diabetology. I have shown that biphasic kinetics of insulin secretion are encoded by SPs through dynamic changes in β cell coupling. An important intestinal hormone (GLP-1) increases the 2nd phase of β-cell activity while diabetic conditions (glucotoxicity) reduce the 1st phase. Islet responses to nutrients also require α/β cell cooperation since α cell ablation in the inducible GluDTR mice model reduced both the basal and 2nd phase of β cell activity generated by glucose and a physiological mix of amino acids. I have also performed the electrophysiological characterization of human β cells derived from induced pluripotent stem cells (iPSC), determined their coupling, established their quality control and shown the functional impact of a mutation of interest (ZnT8) edited by CRISPR/Cas9. A functional quality control of human islets prior to transplantation in T1D patients was also performed for correlations with clinical data. Finally, my SP recordings analyzed in real time by microelectronics has contributed to validate an in silico model of biosensor in a FDA-approved simulator of T1D patients. In conclusion, my work demonstrates (i) the role of intra-islet communications in the dynamic physiological adaptation of these micro-organs, (ii) and that detailed characterization of SPs opens new applications from artificial pancreas to personalized cell therapy.Les îlots pancréatiques sont le principal capteur de glycémie et intègrent toutes les informations métaboliques et hormonales pour adapter en temps réel la sécrétion des hormones, telles que l'insuline par les cellules β majoritaires et le glucagon par les cellules α. Dans le diabète de type 1 (DT1) les cellules β sont détruites par réaction auto-immune et dans le DT2 la masse de cellules β, la fonction et le réseau intra-îlot sont altérés. La réactivité de ces micro-organes est due à leurs propriétés électriques, encodant rapidement l’information, et aux communications entre cellules β/β et β/non-β. Cependant des outils non-invasifs à haute résolution et long terme pour les analyser manquent. L’électrophysiologie extracellulaire par multi-electrode arrays (MEAs) le permet en mesurant des signaux cellulaires mais aussi multicellulaires (SPs) dus aux couplages entre cellules β. J’ai donc utilisé les MEAs (i) pour explorer la physiologie/physiopathologie des îlots et (ii) pour des applications en diabétologie. J’ai montré que la cinétique biphasique de la sécrétion d’insuline était encodée par les SPs avec des changements dynamiques de couplages entre cellules β. Une hormone intestinale importante (GLP-1) augmente la 2de phase alors que des conditions diabétiques (glucotoxicité) réduisent la 1re. La réponse aux nutriments requiert de plus la coopération avec les cellules α, car leur suppression (modèle inductible GluDTR) réduit l’activité basale et la 2de phase des cellules β en présence d’un mélange physiologique d’acides aminés. J’ai également caractérisé électrophysiologiquement les cellules β humaines dérivées de cellules souches pluripotentes induites (iPSC), déterminé leurs couplages, établi leur contrôle qualité et démontré l’impact fonctionnel d’une mutation d’intérêt (ZnT8) éditée par CRISPR/Cas9. Un contrôle qualité fonctionnel des îlots humains avant greffe chez des patients DT1 a également été réalisé et comparé aux résultats cliniques. Enfin, mes enregistrements de SPs analysées par microélectronique en temps réel ont permis de valider un modèle in silico de biocapteur dans un simulateur de référence de patients DT1. En conclusion, mes travaux montrent (i) l’importance des communications intra-îlots dans leur adaptation physiologique dynamique, (ii) et que l’exploitation des SPs ouvre des applications allant du pancréas artificiel à la thérapie cellulaire personnalisée

Dynamic intercellular communications within pancreatic islets analyzed by multi-electrode arrays : physiological roles and biotechnological applications in diabetology

Les îlots pancréatiques sont le principal capteur de glycémie et intègrent toutes les informations métaboliques et hormonales pour adapter en temps réel la sécrétion des hormones, telles que l'insuline par les cellules β majoritaires et le glucagon par les cellules α. Dans le diabète de type 1 (DT1) les cellules β sont détruites par réaction auto-immune et dans le DT2 la masse de cellules β, la fonction et le réseau intra-îlot sont altérés. La réactivité de ces micro-organes est due à leurs propriétés électriques, encodant rapidement l’information, et aux communications entre cellules β/β et β/non-β. Cependant des outils non-invasifs à haute résolution et long terme pour les analyser manquent. L’électrophysiologie extracellulaire par multi-electrode arrays (MEAs) le permet en mesurant des signaux cellulaires mais aussi multicellulaires (SPs) dus aux couplages entre cellules β. J’ai donc utilisé les MEAs (i) pour explorer la physiologie/physiopathologie des îlots et (ii) pour des applications en diabétologie. J’ai montré que la cinétique biphasique de la sécrétion d’insuline était encodée par les SPs avec des changements dynamiques de couplages entre cellules β. Une hormone intestinale importante (GLP-1) augmente la 2de phase alors que des conditions diabétiques (glucotoxicité) réduisent la 1re. La réponse aux nutriments requiert de plus la coopération avec les cellules α, car leur suppression (modèle inductible GluDTR) réduit l’activité basale et la 2de phase des cellules β en présence d’un mélange physiologique d’acides aminés. J’ai également caractérisé électrophysiologiquement les cellules β humaines dérivées de cellules souches pluripotentes induites (iPSC), déterminé leurs couplages, établi leur contrôle qualité et démontré l’impact fonctionnel d’une mutation d’intérêt (ZnT8) éditée par CRISPR/Cas9. Un contrôle qualité fonctionnel des îlots humains avant greffe chez des patients DT1 a également été réalisé et comparé aux résultats cliniques. Enfin, mes enregistrements de SPs analysées par microélectronique en temps réel ont permis de valider un modèle in silico de biocapteur dans un simulateur de référence de patients DT1. En conclusion, mes travaux montrent (i) l’importance des communications intra-îlots dans leur adaptation physiologique dynamique, (ii) et que l’exploitation des SPs ouvre des applications allant du pancréas artificiel à la thérapie cellulaire personnalisée.Pancreatic islets are the main sensor of glycaemia and they integrate all the metabolic and hormonal inputs to adapt in real time the secretion of hormones such as insulin by β cells and glucagon by α cells. In type 1 diabetes (T1D) β cells are destroyed by immune attack, and in T2D, β cell mass, function and the intra-islet network are altered. The islet micro-organs are highly reactive due to their electrical properties encoding rapid information and due to intercellular communications between β cells and β/non-β cells. Nevertheless, non-invasive, high resolution and long-term approaches for analysis are still lacking. Extracellular electrophysiology with multi-electrode arrays (MEAs) allows this analysis of islets by measuring both cellular as well as multicellular signals (SPs) due to β cell coupling. During my PhD, I used MEAs (i) to explore islet physiology/pathophysiology and (ii) for biotechnological applications in diabetology. I have shown that biphasic kinetics of insulin secretion are encoded by SPs through dynamic changes in β cell coupling. An important intestinal hormone (GLP-1) increases the 2nd phase of β-cell activity while diabetic conditions (glucotoxicity) reduce the 1st phase. Islet responses to nutrients also require α/β cell cooperation since α cell ablation in the inducible GluDTR mice model reduced both the basal and 2nd phase of β cell activity generated by glucose and a physiological mix of amino acids. I have also performed the electrophysiological characterization of human β cells derived from induced pluripotent stem cells (iPSC), determined their coupling, established their quality control and shown the functional impact of a mutation of interest (ZnT8) edited by CRISPR/Cas9. A functional quality control of human islets prior to transplantation in T1D patients was also performed for correlations with clinical data. Finally, my SP recordings analyzed in real time by microelectronics has contributed to validate an in silico model of biosensor in a FDA-approved simulator of T1D patients. In conclusion, my work demonstrates (i) the role of intra-islet communications in the dynamic physiological adaptation of these micro-organs, (ii) and that detailed characterization of SPs opens new applications from artificial pancreas to personalized cell therapy

Cellules α et β du pancréas

Les diabètes sucrés sont des maladies métaboliques graves en constante augmentation. Ils sont dus à des déficits de sécrétion et d’action de l’insuline, la seule hormone qui diminue efficacement la glycémie. L’insuline est sécrétée par les cellules β des îlots pancréatiques. Les cellules α, également présentes dans les îlots, libèrent du glucagon et ont des effets opposés à ceux des cellules β sur la glycémie. Longtemps considérée comme néfaste dans le diabète, la cellule α apparaît désormais comme un modulateur des cellules β, ce qui nécessite de prendre désormais en compte cette cellule sur le plan thérapeutique. Cette revue présente le fonctionnement des cellules β et des cellules α. L’implication des interactions dynamiques entre ces deux types cellulaires dans l’homéostasie du glucose, mais aussi celle des autres nutriments, est également décrite

Differential beta cell coupling patterns drive biphasic activity

Background and aims: After food intake, pancreatic islets secrete insulin with a biphasic pattern, which is impaired in type 2 diabetic patients. The mechanisms underlying this pattern have not been fully elucidated and the presence of distinct vesicle pools has been proposed as explanation. Electrical activity of islets consists of individual β cell activity (action potentials, APs) and the multicellular electrical response due to coupling between β cells (slow potentials, SPs). We addressed here the contribution of these two distinct activities to the 1 st and the 2 nd phase of β cell activity, and their modulation by physiological concentrations of GLP-1. Materials and methods: Electrical activity (SPs and APs) of entire mice (C57Bl6/J, age 10-14 weeks) or human islets have been recorded on polymer-coated microelectrode arrays (MEA). These new electrodes allow simultaneous detection of APs (of very low amplitude) and SPs at a high time resolution (10'000 points/s x60 electrodes) for a prolonged period mimicking physiological digestion (2 h). Specific filters differentially detect SPs and APs and 3 parameters were analyzed at the same time: SP frequencies, SP amplitudes and AP frequencies. To investigate synchrony of SPs between different regions of the same islet, we used high density MEAs with an inter-electrode distance of 30 instead of 200 µm followed by analysis via Matlab. Results: Islets were stimulated with glucose concentrations in the physiological range (5.5-8.2 mM). Electrical responses were biphasic for both SPs and APs. APs were mainly present during the 1 st phase while the transition between the 1 st and the 2 nd phase is driven by SPs. In 2 nd phase, the SP amplitude and synchronisation increased significantly (1 st phase: 18.1±2.3 µV; 2 nd phase: 47.4±5.5 µV, p<0.0001), reflecting further electrical coupling and synchronisation of β cells. The intra-islet synchronisation was also further correlate using high density MEAs. The incretin GLP-1, at a physiological postprandial concentration (50 pM), did not change the individual activity of cells (APs) but increased specifically coupling (SPs) and only in the 2nd phase (37.7±3.0 µV vs 47.0±4.2 µV with GLP-1, p<0.0001). Furthermore, when GLP-1 was applied in the presence of a subthreshold glucose concentration (5.5 mM), the hormone triggered only a 2 nd phase. The biphasic electric profile was confirmed in human islets. Their exposure to a glucotoxic medium (20 mM glucose, 65 h) considerably increased basal activity and abolished the biphasic response as well as the discrimination between glucose concentrations. These glucotoxic effects were partially reversible. Conclusion: Our data show that (i) electrical activity pattern shape the biphasic secretion and (ii) the transition period between the 1 st and the 2 nd phase results from increasing electrical synchronisation. Thus biphasic secretion is primarily dictated by changes in electrical activity rather than vesicle pools. The effects of GLP-1 on only coupling SP signals and only during the 2 nd phase explain its clinical effects

Multimed: An Integrated, Multi-Application Platform for the Real-Time Recording and Sub-Millisecond Processing of Biosignals

Enhanced understanding and control of electrophysiology mechanisms are increasingly being hailed as key knowledge in the fields of modern biology and medicine. As more and more excitable cell mechanics are being investigated and exploited, the need for flexible electrophysiology setups becomes apparent. With that aim, we designed Multimed, which is a versatile hardware platform for the real-time recording and processing of biosignals. Digital processing in Multimed is an arrangement of generic processing units from a custom library. These can freely be rearranged to match the needs of the application. Embedded onto a Field Programmable Gate Array (FPGA), these modules utilize full-hardware signal processing to lower processing latency. It achieves constant latency, and sub-millisecond processing and decision-making on 64 channels. The FPGA core processing unit makes Multimed suitable as either a reconfigurable electrophysiology system or a prototyping platform for VLSI implantable medical devices. It is specifically designed for open- and closed-loop experiments and provides consistent feedback rules, well within biological microseconds timeframes. This paper presents the specifications and architecture of the Multimed system, then details the biosignal processing algorithms and their digital implementation. Finally, three applications utilizing Multimed in neuroscience and diabetes research are described. They demonstrate the system’s configurability, its multi-channel, real-time processing, and its feedback control capabilities.Senseur Hybride Bioélectronique du Pancréas endocrine (Criblage et Thérapie du Diabète)ISLET CHIP: Contrôle de Qualité d’Îlots pour la GreffeHYbridation de REseaux de Neurones pour l'exploration de fonctions de réhabilitatio

Identification of a human-specific alteration of beta cell function after prolonged culture of pancreatic islets under glucotoxic conditions

Background and aims: The rapid reversibility of recent type 2 diabetes (T2D) by very low-calorie diet in some patients correlates with a marked improvement of glucose-stimulated insulin secretion (GSIS), emphasizing the role of β-cell dysfunction in the early stages of the disease. In search of human-specific mechanisms of β-cell dysfunction, we extensively characterized the glucotoxic alterations of insulin secretion and upstream coupling events in human pancreatic islets cultured for 1 to 3 weeks at ~5, 8, 10 or 20 mmol/l glucose and acutely stimulated by a stepwise increase in glucose concentration. Materials and methods: Islets from 46 non-diabetic (ND) and 6 type 2 diabetic (T2D) donors were obtained from 5 isolation centers over the last 10 years. The islets were precultured 3-7 days in RPMI containing 5 mmol/l glucose and 10% FBS. They were then cultured for 1-3 weeks in the same medium containing 5.5, 8.5, 10.5 or 20.5 mmol/l glucose before measurements of insulin secretion during culture, islet insulin/DNA content ratio, β-cell apoptosis, cytosolic and mitochondrial thiol redox state, and assessment of dynamic insulin secretion and upstream coupling events during stepwise stimulation with glucose (NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)). Results: Culture of ND-islets for 1 to 3 weeks at ~8, 10 or 20 vs 5 mmol/l glucose did not increase β-cell apoptosis or oxidative stress but concentration-dependently decreased insulin content and increased the β-cell sensitivity to subsequent stimulation with glucose. The islet glucose responsiveness (max amplitude of GSIS per islet) was larger after culture at 8 or 10 vs 5 mmol/l glucose but was markedly reduced after culture at 20 vs 5 mmol/l glucose. In the latter islets, [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were bell-shaped, with a maximal stimulation at 5 to 10 mmol/l glucose followed by a rapid sustained inhibition above that concentration. This glucotoxic alteration was a robust characteristic of human islets. It resulted from long-term stimulation of glucose metabolism and was fully reversible after culture at 5 mmol/l glucose. Finally, acute activation/inhibition of glucokinase during perifusion of islets cultured at 20 mmol/l glucose indicated that the acute reduction of [Ca2+]c and insulin secretion above 10 mmol/l glucose was due to overstimulation rather than inhibition of glucose metabolism. Similar results were obtained in islets from T2D-donors. Conclusion: Long-term exposure of human islets to mildly elevated glucose concentrations markedly increases their glucose sensitivity and reveals a bell-shaped glucose response curve for changes in [Ca2+]c and insulin secretion. This human-specific glucotoxic alteration may contribute to β-cell dysfunction in T2D independently from a detectable increase in β-cell apoptosis and oxidative stress

Islets-on-chip : a tool for real-time assessment of islet function prior to transplantation

Islet transplantation improves metabolic control in patients with unstable type 1 diabetes. Clinical outcomes have been improving over the last decade, and the widely used beta-score allows the evaluation of transplantation results. However, predictive pre-transplantation criteria of islet quality for clinical outcomes are lacking. In this proof-of-concept study, we examined whether characterization of the electrical activity of donor islets could provide a criterion. Aliquots of 8 human donor islets from the STABILOT study, sampled from islet preparations before transplantation, were characterized for purity and split for glucose-induced insulin secretion and electrical activity using multi-electrode-arrays. The latter tests glucose concentration dependencies, biphasic activity, hormones, and drug effects (adrenalin, GLP-1, glibenclamide) and provides a ranking of CHIP-scores from 1 to 6 (best) based on electrical islet activity. The analysis was performed online in real time using a dedicated board or offline. Grouping of beta-scores and CHIP-scores with high, intermediate, and low values was observed. Further analysis indicated correlation between CHIP-score and beta-score, although significance was not attained (R = 0.51,p= 0.1). This novel approach is easily implantable in islet isolation units and might provide means for the prediction of clinical outcomes. We acknowledge the small cohort size as the limitation of this pilot study

Décodage des algorithmes endogènes des îlots en temps réel et leur utilisation dans les simulateurs in-silico du sujet sain ou atteint de diabète de type 1

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