Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped.

AIMS/HYPOTHESIS: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1-3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. METHODS: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3-7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1-3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute 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 1-3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. CONCLUSIONS/INTERPRETATION: Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.

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.

Design of Potassium-Selective Mixed Ion/Electron Conducting Polymers.

An approach providing cation-selective poly-(3,4-ethylenedioxythiophene)(PEDOT):polyelectrolyte-mixed conductors is presented in this communication based on the structural modification of this ambivalent (ionic and electronic conductive) polymer complex. First, an 18-crown-6 moiety is integrated into the styrene sulfonate monomer structure as a specific metal cation scavenger particularly targeting K+ versus Na+ detection. This newly functionalized monomer is characterized by 1 H NMR titration to evaluate the ion selectivity. Aqueous PEDOT dispersion inks containing the polymeric ion-selective moieties are designed and their electrical and electrochemical properties analyzed. These biocompatible inks are the first proof-of-concept step towards ion selectivity in view of their interfacing with biological cells and microorgans of interest in the field of biosensors and physiology.

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.

Multilevel control of glucose homeostasis by adenylyl cyclase 8.

AIMS/HYPOTHESIS: Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an important role in glucose-induced signalling and glucose homeostasis. METHODS: We used pharmacological and genetic approaches in beta cells to determine secretion and calcium metabolism. Furthermore, Adcy8 knockout mice were characterised. RESULTS: In clonal beta cells, inhibitors of adenylyl cyclases or their downstream targets reduced the glucose-induced increase in cytosolic calcium and insulin secretion. This was reproduced by knock-down of ADCY8, but not of ADCY1. These agents also inhibited glucose-induced increase in cytosolic calcium and electrical activity in primary beta cells and similar effects were observed after ADCY8 knock-down. Moreover, insulin secretion was diminished in islets from Adcy8 knockout mice. These mice were glucose intolerant after oral or intraperitoneal administration of glucose whereas their levels of glucagon-like peptide-1 remained unaltered. Finally, we knocked down ADCY8 in the ventromedial hypothalamus to evaluate the need for ADCY8 in the central regulation of glucose homeostasis. Whereas mice fed a standard diet had normal glucose levels, high-fat diet exacerbated glucose intolerance and knock-down mice were incapable of raising their plasma insulin levels. Finally we confirmed that ADCY8 is expressed in human islets. CONCLUSIONS/INTERPRETATIONS: Collectively, our findings demonstrate that ADCY8 is required for the physiological activation of glucose-induced signalling pathways in beta cells, for glucose tolerance and for hypothalamic adaptation to a high-fat diet via regulation of islet insulin secretion.

Slow potentials encode intercellular coupling and insulin demand in pancreatic beta cells.

AIMS/HYPOTHESIS: Ion fluxes constitute a major integrative signal in beta cells that leads to insulin secretion and regulation of gene expression. Understanding these electrical signals is important for deciphering the endogenous algorithms used by islets to attain homeostasis and for the design of new sensors for monitoring beta cell function. METHODS: Mouse and human islets were cultured on multielectrode arrays (MEAs) for 3-13 days. Extracellular electrical activities received on each electrode were continuously amplified and recorded for offline characterisation. RESULTS: Differential band-pass filtering of MEA recordings of mouse islets showed two extracellular voltage waveforms: action potentials (lasting 40-60 ms) and very robust slow potentials (SPs, lasting 800-1,500 ms), the latter of which have not been described previously. The frequency of SPs directly correlated with glucose concentration, peaked at 10 mmol/l glucose and was further augmented by picomolar concentrations of glucagon-like peptide-1. SPs required the closure of ATP-dependent potassium channels as they were induced by glucose or glibenclamide but were not elicited by KCl-induced depolarisation. Pharmacological tools and the use of beta cell specific knockout mice showed that SPs reflected cell coupling via connexin 36. Moreover, increasing and decreasing glucose ramps showed hysteresis with reduced glucose sensitivity during the decreasing phase. SPs were also observed in human islets and could be continuously recorded over 24 h. CONCLUSIONS/INTERPRETATION: This novel electrical signature reflects the syncytial function of the islets and is specific to beta cells. Moreover, the observed hysteresis provides evidence for an endogenous algorithm naturally present in islets to protect against hypoglycaemia.

Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells.

AIMS/HYPOTHESIS: Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca(2+) and cAMP: exocytosis is triggered by Ca(2+) and mediated by the cAMP/protein kinase A (PKA) signalling pathway. However, the causal link between these two processes in primary beta cells remains undefined. METHODS: Time-resolved confocal imaging of fluorescence resonance energy transfer signals was performed to visualise PKA activity, and combined membrane capacitance recordings were used to monitor insulin secretion from patch-clamped rat beta cells. RESULTS: Membrane depolarisation-induced Ca(2+) influx caused an increase in cytosolic PKA activity via activating a Ca(2+)-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca(2+) oscillations and PKA activation. ADCY8 knockdown significantly reduced the level of depolarisation-evoked PKA activation and impaired replenishment of the readily releasable vesicle pool. Pharmacological inhibition of PKA by two inhibitors reduced depolarisation-induced PKA activation to a similar extent and reduced the capacity for sustained vesicle exocytosis and insulin release. CONCLUSIONS/INTERPRETATION: Our findings suggest that depolarisation-induced Ca(2+) influx plays dual roles in regulating exocytosis in rat pancreatic beta cells by triggering vesicle fusion and replenishing the vesicle pool to support sustained insulin release. Therefore, Ca(2+) influx may be important for glucose-stimulated insulin secretion.

Extracellular electrophysiology on clonal human ß-cell spheroids.

Background: Pancreatic islets are important in nutrient homeostasis and improved cellular models of clonal origin may very useful especially in view of relatively scarce primary material. Close 3D contact and coupling between ß-cells are a hallmark of physiological function improving signal/noise ratios. Extracellular electrophysiology using micro-electrode arrays (MEA) is technically far more accessible than single cell patch clamp, enables dynamic monitoring of electrical activity in 3D organoids and recorded multicellular slow potentials (SP) provide unbiased insight in cell-cell coupling. Objective: We have therefore asked whether 3D spheroids enhance clonal ß-cell function such as electrical activity and hormone secretion using human EndoC-ßH1, EndoC-ßH5 and rodent INS-1 832/13 cells. Methods: Spheroids were formed either by hanging drop or proprietary devices. Extracellular electrophysiology was conducted using multi-electrode arrays with appropriate signal extraction and hormone secretion measured by ELISA. Results: EndoC-ßH1 spheroids exhibited increased signals in terms of SP frequency and especially amplitude as compared to monolayers and even single cell action potentials (AP) were quantifiable. Enhanced electrical signature in spheroids was accompanied by an increase in the glucose stimulated insulin secretion index. EndoC-ßH5 monolayers and spheroids gave electrophysiological profiles similar to EndoC-ßH1, except for a higher electrical activity at 3 mM glucose, and exhibited moreover a biphasic profile. Again, physiological concentrations of GLP-1 increased AP frequency. Spheroids also exhibited a higher secretion index. INS-1 cells did not form stable spheroids, but overexpression of connexin 36, required for cell-cell coupling, increased glucose responsiveness, dampened basal activity and consequently augmented the stimulation index. Conclusion: In conclusion, spheroid formation enhances physiological function of the human clonal ß-cell lines and these models may provide surrogates for primary islets in extracellular electrophysiology.

Electrophysiological characterisation of iPSC-derived human ß-like cells and an SLC30A8 disease model.

iPSC-derived human ß-like cells (BLC) hold promise for both therapy and disease modelling, but their generation remains challenging and their functional analyses beyond transcriptomic and morphological assessments remain limited. Here, we validate an approach using multicellular and single cell electrophysiological tools to evaluate BLCs functions. The Multi-Electrode Arrays (MEAs) measuring the extracellular electrical activity revealed that BLCs are electrically coupled, produce slow potential (SP) signals like primary ß-cells that are closely linked to insulin secretion. We also used high-resolution single-cell patch-clamp measurements to capture the exocytotic properties, and characterize voltage-gated sodium and calcium currents. These were comparable to those in primary ß and EndoC-ßH1 cells. The KATP channel conductance is greater than in human primary ß cells which may account for the limited glucose responsiveness observed with MEA. We used MEAs to study the impact of the type 2 diabetes protective SLC30A8 allele (p.Lys34Serfs*50) and found that BLCs with this allele have stronger electrical coupling. Our data suggest that with an adapted approach BLCs from pioneer protocol can be used to evaluate the functional impact of genetic variants on ß-cell function and coupling.

Non-invasive long-term and real-time analysis of endocrine cells on micro-electrode arrays.

Non-invasive high-throughput and long-term monitoring of endocrine cells is important for drug research, phenotyping, tissue engineering and pre-transplantation quality control. Here we report a novel approach to obtain simultaneous long-term electrical recordings of different islet cell types using multi-electrode arrays. We implemented wavelet transforms to resolve the low signal/noise ratio inherent to these measurements and extracted on-line a signature specific of cell activity. The architecture employed allows multiplexing a large number of electrodes for high-throughput screening. This method should be of considerable advantage in endocrine research and may be extended to other excitable cells previously not accessible to the technique.

Vertical Organic Electrochemical Transistors and Electronics for Low Amplitude Micro-Organ Signals.

Electrical signals are fundamental to key biological events such as brain activity, heartbeat, or vital hormone secretion. Their capture and analysis provide insight into cell or organ physiology and a number of bioelectronic medical devices aim to improve signal acquisition. Organic electrochemical transistors (OECT) have proven their capacity to capture neuronal and cardiac signals with high fidelity and amplification. Vertical PEDOT:PSS-based OECTs (vOECTs) further enhance signal amplification and device density but have not been characterized in biological applications. An electronic board with individually tuneable transistor biases overcomes fabrication induced heterogeneity in device metrics and allows quantitative biological experiments. Careful exploration of vOECT electric parameters defines voltage biases compatible with reliable transistor function in biological experiments and provides useful maximal transconductance values without influencing cellular signal generation or propagation. This permits successful application in monitoring micro-organs of prime importance in diabetes, the endocrine pancreatic islets, which are known for their far smaller signal amplitudes as compared to neurons or heart cells. Moreover, vOECTs capture their single-cell action potentials and multicellular slow potentials reflecting micro-organ organizations as well as their modulation by the physiological stimulator glucose. This opens the possibility to use OECTs in new biomedical fields well beyond their classical applications.

Integrating an Islet-Based Biosensor in the Artificial Pancreas: In Silico Proof-of-Concept.

OBJECTIVE: Current treatment of type 1 diabetes by closed-loop therapy depends on continuous glucose monitoring. However, glucose readings alone are insufficient for an artificial pancreas to truthfully restore nutrient homeostasis where additional physiological regulators of insulin secretion play a considerable role. Previously, we have developed an electrophysiological biosensor of pancreatic islet activity, which integrates these additional regulators through electrical measurements. This work aims at investigating the performance of the biosensor in a blood glucose control loop as potential in silico proof-of-concept. METHODS: Two islet algorithm models were identified on experimental data recorded with the biosensor. First, we validated electrical measurement as a means to exploit the inborn regulation capabilities of islets for intravenous glucose measurement and insulin infusion. Subsequently, an artificial pancreas integrating the islet-based biosensor was compared to standard treatment approaches using subcutaneous routes. The closed-loop simulations were performed in the UVA/Padova T1DM Simulator where a series of realistic meal scenarios were applied to virtual diabetic patients. RESULTS: With intravenous routes, the endogenous islet algorithms successfully restored glucose homeostasis for all patient categories (mean time in range exceeds 90%) while mitigating the risk of adverse glycaemic events (mean BGI < 2). Using subcutaneous routes, the biosensor-based artificial pancreas was as efficient as standard treatments, and outperformed them under challenging conditions. CONCLUSION: This work validates the concept of using inborn pancreatic islets algorithms in an artificial pancreas in silico. SIGNIFICANCE: Pancreatic islet endogenous algorithms obtained via an electrophysiological biosensor successfully regulate blood glucose levels of virtual type 1 diabetic patients.

Towards the Integration of an Islet-Based Biosensor in Closed-Loop Therapies for Patients With Type 1 Diabetes.

In diabetes mellitus (DM) treatment, Continuous Glucose Monitoring (CGM) linked with insulin delivery becomes the main strategy to improve therapeutic outcomes and quality of patients' lives. However, Blood Glucose (BG) regulation with CGM is still hampered by limitations of algorithms and glucose sensors. Regarding sensor technology, current electrochemical glucose sensors do not capture the full spectrum of other physiological signals, i.e., lipids, amino acids or hormones, relaying the general body status. Regarding algorithms, variability between and within patients remains the main challenge for optimal BG regulation in closed-loop therapies. This work highlights the simulation benefits to test new sensing and control paradigms which address the previous shortcomings for Type 1 Diabetes (T1D) closed-loop therapies. The UVA/Padova T1DM Simulator is the core element here, which is a computer model of the human metabolic system based on glucose-insulin dynamics in T1D patients. That simulator is approved by the US Food and Drug Administration (FDA) as an alternative for pre-clinical testing of new devices and closed-loop algorithms. To overcome the limitation of standard glucose sensors, the concept of an islet-based biosensor, which could integrate multiple physiological signals through electrical activity measurement, is assessed here in a closed-loop insulin therapy. This investigation has been addressed by an interdisciplinary consortium, from endocrinology to biology, electrophysiology, bio-electronics and control theory. In parallel to the development of an islet-based closed-loop, it also investigates the benefits of robust control theory against the natural variability within a patient population. Using 4 meal scenarios, numerous simulation campaigns were conducted. The analysis of their results then introduces a discussion on the potential benefits of an Artificial Pancreas (AP) system associating the islet-based biosensor with robust algorithms.

ATP signalling is crucial for the response of human keratinocytes to mechanical stimulation by hypo-osmotic shock.

Touch is detected through receptors located in the skin and the activation of channels in sensory nerve fibres. Epidermal keratinocytes themselves, however, may sense mechanical stimulus and contribute to skin sensation. Here, we showed that the mechanical stimulation of human keratinocytes by hypo-osmotic shock releases adenosine triphosphate (ATP) and increases intracellular calcium. We demonstrated that the release of ATP was found to be calcium independent because emptying the intracellular calcium stores did not cause ATP release; ATP release was still observed in the absence of external calcium and it persisted on chelating cytosolic calcium. On the other hand, the released ATP activated purinergic receptors and mobilized intracellular calcium stores. The resulting depletion of stored calcium led to the activation of capacitative calcium entry. Increase in cytosolic calcium concentration was blocked by the purinergic receptor blocker suramin, phospholipase C inhibitor and apyrase, which hydrolyses ATP. Collectively, our data demonstrate that human keratinocytes are mechanically activated by hypo-osmotic shock, leading first to the release of ATP, which in turn stimulates purinergic receptors, resulting in the mobilization of intracellular calcium and capacitative calcium entry. These results emphasize the crucial role of ATP signalling in the transduction of mechanical stimuli in human keratinocytes.

[Pancreatic α and ß cells: Best enemies or partners for life?] / Cellules α et ß du pancréas - Meilleures ennemies ou partenaires pour la vie ?

Diabetes are major metabolic diseases constantly increasing in the population, caused by reduced secretion and action of insulin, the only hormone lowering efficiently the glycaemia. Insulin is secreted by ß cells within the pancreatic islets of Langerhans. The islet micro-organs also contain 15 to 35% of α cells, well-known for their opposite effects on glycaemia. Considered until now as potentially harmful in diabetes, α cells are emerging as potent enhancers of ß cell activity when studied in physiological nutritional setting and should therefore be reconsidered in a therapeutic point of view. This review summarizes the latest concepts regarding ß cell function in physiological states and the involvement of dynamic functional interactions between α and ß cells for the regulation of nutrient homeostasis.

TITLE: Cellules α et ß du pancréas - Meilleures ennemies ou partenaires pour la vie ? ABSTRACT: 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 ß des îlots pancréatiques. Les cellules α, également présentes dans les îlots, libèrent du glucagon et ont des effets opposés à ceux des cellules ß sur la glycémie. Longtemps considérée comme néfaste dans le diabète, la cellule α apparaît désormais comme un modulateur des cellules ß, ce qui nécessite de prendre désormais en compte cette cellule sur le plan thérapeutique. Cette revue présente le fonctionnement des cellules ß 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.

Dynamic Uni- and Multicellular Patterns Encode Biphasic Activity in Pancreatic Islets.

Biphasic secretion is an autonomous feature of many endocrine micro-organs to fulfill physiological demands. The biphasic activity of islet ß-cells maintains glucose homeostasis and is altered in type 2 diabetes. Nevertheless, underlying cellular or multicellular functional organizations are only partially understood. High-resolution noninvasive multielectrode array recordings permit simultaneous analysis of recruitment, of single-cell, and of coupling activity within entire islets in long-time experiments. Using this unbiased approach, we addressed the organizational modes of both first and second phase in mouse and human islets under physiological and pathophysiological conditions. Our data provide a new uni- and multicellular model of islet ß-cell activation: during the first phase, small but highly active ß-cell clusters are dominant, whereas during the second phase, electrical coupling generates large functional clusters via multicellular slow potentials to favor an economic sustained activity. Postprandial levels of glucagon-like peptide 1 favor coupling only in the second phase, whereas aging and glucotoxicity alter coupled activity in both phases. In summary, biphasic activity is encoded upstream of vesicle pools at the micro-organ level by multicellular electrical signals and their dynamic synchronization between ß-cells. The profound alteration of the electrical organization of islets in pathophysiological conditions may contribute to functional deficits in type 2 diabetes.

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 α and γ cells. α 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 in vivo. Isolated islets were used in vitro to determine islet ß-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 in vivo was slightly lower in GluK2-/- mice, whereas fasting glucagonemia was strongly reduced. In vivo 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. In vitro recording demonstrated an improvement of glucose-induced electrical activity of ß-cells in islets obtained from GluK2-/- mice at both ages. Finally, glucose-induced insulin secretion in vitro was increased in GluK2-/- islets, whereas glucagon secretion at 2 mmol/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.

Mechanosensor Channels in Mammalian Somatosensory Neurons.

Mechanoreceptive sensory neurons innervating the skin, skeletal muscles andviscera signal both innocuous and noxious information necessary for proprioception, touchand pain. These neurons are responsible for the transduction of mechanical stimuli intoaction potentials that propagate to the central nervous system. The ability of these cells todetect mechanical stimuli impinging on them relies on the presence of mechanosensitivechannels that transduce the external mechanical forces into electrical and chemical signals.Although a great deal of information regarding the molecular and biophysical properties ofmechanosensitive channels in prokaryotes has been accumulated over the past two decades,less is known about the mechanosensitive channels necessary for proprioception and thesenses of touch and pain. This review summarizes the most pertinent data onmechanosensitive channels of mammalian somatosensory neurons, focusing on theirproperties, pharmacology and putative identity.

Simultaneous monitoring of single cell and of micro-organ activity by PEDOT:PSS covered multi-electrode arrays.

Continuous and long-term monitoring of cellular and micro-organ activity is required for new insights into physiology and novel technologies such as Organs-on-Chip. Moreover, recent advances in stem cell technology and especially in the field of diabetes call for non-invasive approaches in quality testing of the large quantities of surrogate pancreatic islets to be generated. Electrical activity of such a micro-organ results in single cell action potentials (APs) of high frequency and in low frequency changes in local field potentials (slow potentials or SPs), reflecting coupled cell activity and overall organ physiology. Each of them is indicative of different physiological stages in islet activation. Action potentials in islets are of small amplitude and very difficult to detect. The use of PEDOT:PSS to coat metal electrodes is expected to reduce noise and results in a frequency-dependent change in impedance, as shown here. Whereas detection of high-frequency APs improves, low frequency SPs are less well detected which is, however, an acceptable trade off in view of the strong amplitude of SPs. Using a dedicated software, recorded APs and SPs can be automatically diagnosed and analyzed. Concomitant capture of the two signals will considerably increase the diagnostic power of monitoring islets and islet surrogates in fundamental research as well as drug screening or the use of islets as biosensors.

Guiding pancreatic beta cells to target electrodes in a whole-cell biosensor for diabetes.

We are developing a cell-based bioelectronic glucose sensor that exploits the multi-parametric sensing ability of pancreatic islet cells for the treatment of diabetes. These cells sense changes in the concentration of glucose and physiological hormones and immediately react by generating electrical signals. In our sensor, signals from multiple cells are recorded as field potentials by a micro-electrode array (MEA). Thus, cell response to various factors can be assessed rapidly and with high throughput. However, signal quality and consequently overall sensor performance rely critically on close cell-electrode proximity. Therefore, we present here a non-invasive method of further exploiting the electrical properties of these cells to guide them towards multiple micro-electrodes via electrophoresis. Parameters were optimized by measuring the cell's zeta potential and modeling the electric field distribution. Clonal and primary mouse or human ß-cells migrated directly to target electrodes during the application of a 1 V potential between MEA electrodes for 3 minutes. The morphology, insulin secretion, and electrophysiological characteristics were not altered compared to controls. Thus, cell manipulation on standard MEAs was achieved without introducing any external components and while maintaining the performance of the biosensor. Since the analysis of the cells' electrical activity was performed in real time via on-chip recording and processing, this work demonstrates that our biosensor is operational from the first step of electrically guiding cells to the final step of automatic recognition. Our favorable results with pancreatic islets, which are highly sensitive and fragile cells, are encouraging for the extension of this technique to other cell types and microarray devices.