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

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 (T2DM) 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 to decreased Akt phosphorylation protein levels, suggesting an involvement of the PI3K/Akt pathway in the process. In line with this, PI3K inhibition by 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.

Patient-independent, MHD-robust R-peak detection for retrospective gating in cardiac MRI imaging.

Objective.In cardiovascular magnetic resonance imaging, synchronization of image acquisition with heart motion (calledgating) is performed by detecting R-peaks in electrocardiogram (ECG) signals. Effective gating is challenging with 3T and 7T scanners, due to severe distortion of ECG signals caused by magnetohydrodynamic effects associated with intense magnetic fields. This work proposes an efficient retrospective gating strategy that requires no prior training outside the scanner and investigates the optimal number of leads in the ECG acquisition set.Approach.The proposed method was developed on a data set of 12-lead ECG signals acquired within 3T and 7T scanners. Independent component analysis is employed to effectively separate components related with cardiac activity from those associated to noise. Subsequently, an automatic selection process identifies the components best suited for accurate R-peak detection, based on heart rate estimation metrics and frequency content quality indexes.Main results.The proposed method is robust to different B0 field strengths, as evidenced by R-peak detection errors of 2.4 ± 3.1 ms and 10.6 ± 15.4 ms for data acquired with 3T and 7T scanners, respectively. Its effectiveness was verified with various subject orientations, showcasing applicability in diverse clinical scenarios. The work reveals that ECG leads can be limited in number to three, or at most five for 7T field strengths, without significant degradation in R-peak detection accuracy.Significance.The approach requires no preliminary ECG acquisition for R-peak detector training, reducing overall examination time. The gating process is designed to be adaptable, completely blind and independent of patient characteristics, allowing wide and rapid deployment in clinical practice. The potential to employ a significantly limited set of leads enhances patient comfort.

Mechanisms of action of incretin receptor based dual- and tri-agonists in pancreatic islets.

Simultaneous activation of the incretin G-protein-coupled receptors (GPCRs) via unimolecular dual-receptor agonists (UDRA) has emerged as a new therapeutic approach for type 2 diabetes. Recent studies also advocate triple agonism with molecules also capable of binding the glucagon receptor. In this scoping review, we discuss the cellular mechanisms of action (MOA) underlying the actions of these novel and therapeutically important classes of peptide receptor agonists. Clinical efficacy studies of several UDRAs have demonstrated favorable results both as monotherapies and when combined with approved hypoglycemics. Although the additive insulinotropic effects of dual glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic peptide receptor (GIPR) agonism were anticipated based on the known actions of either glucagon-like peptide-1 (GLP-1) or glucose-dependent insulinotropic peptide (GIP) alone, the additional benefits from GCGR were largely unexpected. Whether additional synergistic or antagonistic interactions among these G-protein receptor signaling pathways arise from simultaneous stimulation is not known. The signaling pathways affected by dual- and tri-agonism require more trenchant investigation before a comprehensive understanding of the cellular MOA. This knowledge will be essential for understanding the chronic efficacy and safety of these treatments.

Cholesterol Redistribution in Pancreatic ß-Cells: A Flexible Path to Regulate Insulin Secretion.

Pancreatic ß-cells, by secreting insulin, play a key role in the control of glucose homeostasis, and their dysfunction is the basis of diabetes development. The metabolic milieu created by high blood glucose and lipids is known to play a role in this process. In the last decades, cholesterol has attracted significant attention, not only because it critically controls ß-cell function but also because it is the target of lipid-lowering therapies proposed for preventing the cardiovascular complications in diabetes. Despite the remarkable progress, understanding the molecular mechanisms responsible for cholesterol-mediated ß-cell function remains an open and attractive area of investigation. Studies indicate that ß-cells not only regulate the total cholesterol level but also its redistribution within organelles, a process mediated by vesicular and non-vesicular transport. The aim of this review is to summarize the most current view of how cholesterol homeostasis is maintained in pancreatic ß-cells and to provide new insights on the mechanisms by which cholesterol is dynamically distributed among organelles to preserve their functionality. While cholesterol may affect virtually any activity of the ß-cell, the intent of this review is to focus on early steps of insulin synthesis and secretion, an area still largely unexplored.

Automatic Optimization of Multichannel Electrode Configurations for Robust Fetal Heart Rate Detection by Blind Source Separation.

OBJECTIVE: Fetal heart rate (fHR) evaluation is fundamental to guarantee timely medical intervention in case of pregnancy complications. Due to the limitations of traditional cardiotocography, multichannel electrophysiological recording was proposed as a viable alternative, which requires Blind Source Separation (BSS) techniques. Yet effective and reliable separation of the fetal ECG remains challenging due to multiple noise sources and the effects of varying fetal position. In this work, we demonstrate that the adopted electrode configuration plays a key role in the effectiveness of BSS and propose guidelines for optimal electrode positioning. Moreover, a model is proposed to automatically predict the most suited configuration for accurate BSS-based fHR estimation with a minimal number of leads, to facilitate practical implementation. METHODS: We compared fHR estimation accuracy with different electrode configurations on in-silico data, identifying the optimal configuration for a recent BSS method. Based on features extracted from raw signals, we proposed a support vector regression model to automatically identify the best electrode configuration in terms of fHR estimation accuracy and to dynamically adjust it to varying fetal presentation. Evaluation was performed on real and synthetic data. RESULTS: Guidelines for the optimal electrode configuration are proposed by using 4 leads. Prediction of configuration quality shows 80.9% accuracy; the optimal configurat- ion is recognized in 92.2% of the subjects. CONCLUSION: The proposed method successfully predicts the quality of the configurations, demonstrating the impact of the electrode configuration on the BSS performance. SIGNIFICANCE: The method holds potential for long-term fetal monitoring, by dynamically choosing the optimal configuration.

Pancreatic PCSK9 controls the organization of the ß-cell secretory pathway via LDLR-cholesterol axis.

BACKGROUND: Cholesterol is central to pancreatic ß-cell physiology and alterations of its homeostasis contribute to ß-cell dysfunction and diabetes. Proper intracellular cholesterol levels are maintained by different mechanisms including uptake via the low-density lipoprotein receptor (LDLR). In the liver, the proprotein convertase subtilisin/kexin type 9 (PCSK9) routes the LDLR to lysosomes for degradation, thus limiting its recycling to the membrane. PCSK9 is also expressed in the pancreas and loss of function mutations of PCSK9 result in higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Aim of this study was to investigate whether PCSK9 also impacts ß-cells function. METHODS: Pancreas-specific Pcsk9 null mice (Pdx1Cre/Pcsk9 fl/fl) were generated and characterized for glucose tolerance, insulin release and islet morphology. Isolated Pcsk9-deficient islets and clonal ß-cells (INS1E) were employed to characterize the molecular mechanisms of PCSK9 action. RESULTS: Pdx1Cre/Pcsk9 fl/fl mice exhibited normal blood PCSK9 and cholesterol levels but were glucose intolerant and had defective insulin secretion in vivo. Analysis of PCSK9-deficient islets revealed comparable ß-cell mass and insulin content but impaired stimulated secretion. Increased proinsulin/insulin ratio, modifications of SNARE proteins expression and decreased stimulated­calcium dynamics were detected in PCSK9-deficient ß-cells. Mechanistically, pancreatic PCSK9 silencing impacts ß-cell LDLR expression and cholesterol content, both in vivo and in vitro. The key role of LDLR is confirmed by the demonstration that LDLR downregulation rescued the phenotype. CONCLUSIONS: These findings establish pancreatic PCSK9 as a novel critical regulator of the functional maturation of the ß-cell secretory pathway, via modulation of cholesterol homeostasis.

Surgical rarities: case report of appendicular diverticulitis and literature review.

Appendicular diverticulosis is a rare condition observed in about 0.004-2% of all appendectomy specimens. Risk of perforation/bleeding is high and a relevant association with mucinous neoplasms is known. Appendectomy is indicated even in case of occasional finding. We present the case of a 22-year-old man who entered the Emergency Room for pain in right iliac fossa. Blood tests showed only a slight increase in C-reactive protein. Abdominal ultrasound (US) evidenced an appendix with thickened walls and a rounded fluid-filled hypoechoic lesion at its distal tip. Laparoscopic appendectomy confirmed the presence of mucocele. Postoperative course was uneventful and the patients discharged on postoperative day 3. Histological examination indicated diverticulitis/peridiverticulitis of the appendix and acute suppurative appendicitis. No perforation of the diverticula was detected. No neoplastic epithelium/mucous material was observed. In our case, preoperative US proved to be a useful alternative to computed tomography for the diagnosis.

An Overview of the Sensors for Heart Rate Monitoring Used in Extramural Applications.

This work presents an overview of the main strategies that have been proposed for non-invasive monitoring of heart rate (HR) in extramural and home settings. We discuss three categories of sensing according to what physiological effect is used to measure the pulsatile activity of the heart, and we focus on an illustrative sensing modality for each of them. Therefore, electrocardiography, photoplethysmography, and mechanocardiography are presented as illustrative modalities to sense electrical activity, mechanical activity, and the peripheral effect of heart activity. In this paper, we describe the physical principles underlying the three categories and the characteristics of the different types of sensors that belong to each class, and we touch upon the most used software strategies that are currently adopted to effectively and reliably extract HR. In addition, we investigate the strengths and weaknesses of each category linked to the different applications in order to provide the reader with guidelines for selecting the most suitable solution according to the requirements and constraints of the application.

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.

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure-Function Relationships in Mammalian SLC6 Transporters.

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional-structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts.

Iron Metabolism in Pancreatic Beta-Cell Function and Dysfunction.

Iron is an essential element involved in a variety of physiological functions. In the pancreatic beta-cells, being part of Fe-S cluster proteins, it is necessary for the correct insulin synthesis and processing. In the mitochondria, as a component of the respiratory chain, it allows the production of ATP and reactive oxygen species (ROS) that trigger beta-cell depolarization and potentiate the calcium-dependent insulin release. Iron cellular content must be finely tuned to ensure the normal supply but also to prevent overloading. Indeed, due to the high reactivity with oxygen and the formation of free radicals, iron excess may cause oxidative damage of cells that are extremely vulnerable to this condition because the normal elevated ROS production and the paucity in antioxidant enzyme activities. The aim of the present review is to provide insights into the mechanisms responsible for iron homeostasis in beta-cells, describing how alteration of these processes has been related to beta-cell damage and failure. Defects in iron-storing or -chaperoning proteins have been detected in diabetic conditions; therefore, the control of iron metabolism in these cells deserves further investigation as a promising target for the development of new disease treatments.

Dedicated Algorithm for Unobtrusive Fetal Heart Rate Monitoring Using Multiple Dry Electrodes.

Multi-channel measurements from the maternal abdomen acquired by means of dry electrodes can be employed to promote long-term monitoring of fetal heart rate (fHR). The signals acquired with this type of electrode have a lower signal-to-noise ratio and different artifacts compared to signals acquired with conventional wet electrodes. Therefore, starting from the benchmark algorithm with the best performance for fHR estimation proposed by Varanini et al., we propose a new method specifically designed to remove artifacts typical of dry-electrode recordings. To test the algorithm, experimental textile electrodes were employed that produce artifacts typical of dry and capacitive electrodes. The proposed solution is based on a hybrid (hardware and software) pre-processing step designed specifically to remove the disturbing component typical of signals acquired with these electrodes (triboelectricity artifacts and amplitude modulations). The following main processing steps consist of the removal of the maternal ECG by blind source separation, the enhancement of the fetal ECG and identification of the fetal QRS complexes. Main processing is designed to be robust to the high-amplitude motion artifacts that corrupt the acquisition. The obtained denoising system was compared with the benchmark algorithm both on semi-simulated and on real data. The performance, quantified by means of sensitivity, F1-score and root-mean-square error metrics, outperforms the performance obtained with the original method available in the literature. This result proves that the design of a dedicated processing system based on the signal characteristics is necessary for reliable and accurate estimation of the fHR using dry, textile electrodes.

Accurate hemodynamic response estimation by removal of stimulus-evoked superficial response in fNIRS signals.

Objective.We address the problem of hemodynamic response (HR) estimation when task-evoked extra-cerebral components are present in functional near-infrared spectroscopy (fNIRS) signals. These components might bias the HR estimation; therefore, careful and accurate denoising of data is needed.Approach.We propose a dictionary-based algorithm to process each single event-related segment of the acquired signal for both long separation (LS) and short separation (SS) channels. Stimulus-evoked components and physiological noise are modeled by means of two distinct waveform dictionaries. For each segment, after removal of the physiological noise component in each channel, a template is employed to estimate stimulus-evoked responses in both channels. Then, the estimate from the SS channel is employed to correct the evoked superficial response and refine the HR estimate from the LS channel.Main results.Analysis of simulated, semi-simulated and real data shows that, by averaging single-segment estimates over multiple trials in an experiment, reliable results and improved accuracy compared to other methods can be obtained. The average estimation error of the proposed method for the semi-simulated data set is 34% for oxy-hemoglobin (HbO) and 78% for deoxy-hemoglobin (HbR), considering 40 trials. The proposed method outperforms the results of the methods proposed in the literature. While still far from the possibility of single-trial HR estimation, a significant reduction in the number of averaged trials can also be obtained.Significance.This work proves that dedicated dictionaries can be successfully employed to model all different components of fNIRS signals. We demonstrate the effectiveness of a specifically designed algorithm structure in dealing with a complex denoising problem, enhancing the possibilities of fNIRS-based HR analysis.

Verbascoside Protects Pancreatic ß-Cells against ER-Stress.

Substantial epidemiological evidence indicates that a diet rich in polyphenols protects against developing type 2 diabetes. The phenylethanoid glycoside verbascoside/acteoside, a widespread polyphenolic plant compound, has several biological properties including strong antioxidant, anti-inflammatory and neuroprotective activities. The aim of this research was to test the possible effects of verbascoside on pancreatic ß-cells, a target never tested before. Mouse and human ß-cells were incubated with verbascoside (0.8-16 µM) for up to five days and a combination of biochemical and imaging techniques were used to assess the ß-cell survival and function under normal or endoplasmic reticulum (ER)-stress inducing conditions. We found a dose-dependent protective effect of verbascoside against oxidative stress in clonal and human ß-cells. Mechanistic studies revealed that the polyphenol protects ß-cells against ER-stress mediated dysfunctions, modulating the activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response and promoting mitochondrial dynamics. As a result, increased viability, mitochondrial function and insulin content were detected in these cells. These studies provide the evidence that verbascoside boosts the ability of ß-cells to cope with ER-stress, an important contributor of ß-cell dysfunction and failure in diabetic conditions and support the therapeutic potential of verbascoside in diabetes.

Proteomic Analysis Reveals a Mitochondrial Remodeling of ßTC3 Cells in Response to Nanotopography.

Recently, using cluster-assembled zirconia substrates with tailored roughness produced by supersonic cluster beam deposition, we demonstrated that ß cells can sense nanoscale features of the substrate and can translate these stimuli into a mechanotransductive pathway capable of preserveing ß-cell differentiation and function in vitro in long-term cultures of human islets. Using the same proteomic approach, we now focused on the mitochondrial fraction of ßTC3 cells grown on the same zirconia substrates and characterized the morphological and proteomic modifications induced by the nanostructure. The results suggest that, in ßTC3 cells, mitochondria are perturbed by the nanotopography and activate a program involving metabolism modification and modulation of their interplay with other organelles. Data were confirmed in INS1E, a different ß-cell model. The change induced by the nanostructure can be pro-survival and prime mitochondria for a metabolic switch to match the new cell needs.

Shaping Pancreatic ß-Cell Differentiation and Functioning: The Influence of Mechanotransduction.

Embryonic and pluripotent stem cells hold great promise in generating ß-cells for both replacing medicine and novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these ß-like cells still fail to fully mirror the adult ß-cell physiology. For their proper growth and functioning, ß-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals. We here review the current status of knowledge of biophysical cues provided by the niche where ß-cells normally live and differentiate, and we underline the possible machinery designated for mechanotransduction in ß-cells. Although the regulatory mechanisms remain poorly understood, the analysis reveals that ß-cells are equipped with all mechanosensors and signaling proteins actively involved in mechanotransduction in other cell types, and they respond to mechanical cues by changing their behavior. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the ß-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of ß-cell differentiation capacity in vitro.

Neural Control of Emotional Actions in Response to Affective Vocalizations.

Social-emotional cues, such as affective vocalizations and emotional faces, automatically elicit emotional action tendencies. Adaptive social-emotional behavior depends on the ability to control these automatic action tendencies. It remains unknown whether neural control over automatic action tendencies is supramodal or relies on parallel modality-specific neural circuits. Here, we address this largely unexplored issue in humans. We consider neural circuits supporting emotional action control in response to affective vocalizations, using an approach-avoidance task known to reliably index control over emotional action tendencies elicited by emotional faces. We isolate supramodal neural contributions to emotional action control through a conjunction analysis of control-related neural activity evoked by auditory and visual affective stimuli, the latter from a previously published data set obtained in an independent sample. We show that the anterior pFC (aPFC) supports control of automatic action tendencies in a supramodal manner, that is, triggered by either emotional faces or affective vocalizations. When affective vocalizations are heard and emotional control is required, the aPFC supports control through negative functional connectivity with the posterior insula. When emotional faces are seen and emotional control is required, control relies on the same aPFC territory downregulating the amygdala. The findings provide evidence for a novel mechanism of emotional action control with a hybrid hierarchical architecture, relying on a supramodal node (aPFC) implementing an abstract goal by modulating modality-specific nodes (posterior insula, amygdala) involved in signaling motivational significance of either affective vocalizations or faces.

Cholesterol metabolism, pancreatic ß-cell function and diabetes.

Cholesterol plays an essential role in determining cell membrane physico-chemical characteristics and functions. A proper membrane structure is critical in pancreatic ß-cells for glucose-mediated insulin secretion, and alterations in cellular cholesterol content may negatively affect this process, leading to ß-cell dysfunction. The low density lipoprotein receptor (LDL-R) appears to play a relevant role in ß-cell dysfunction due to cholesterol accumulation. This observation raised the question of whether hypocholesterolemic drugs which increase LDL-R expression might bear diabetogenic properties, thus increasing the risk of new-onset diabetes or worsen glycaemic parameters in diabetic patients. Being at higher cardiovascular risk, diabetic patients are usually treated with hypolipidemic drugs to correct the atherogenic dyslipidemia characteristic of this pathological condition. Statin therapy has been associated with an increased incidence of new-onset diabetes (NOD), being the diabetogenic effect depending on the type and dose of statin. However, it is worth noting that the benefits on cardiovascular mortality largely exceed the increased risk associated with the development of diabetes. Although genetic variants associated with lower levels of LDL-C are also associated with an increased NOD risk, clinical trials with lipid-lowering drugs other than statins, namely ezetimibe or monoclonal antibodies against PCSK9, did not observe an increase of developing diabetes. In summary, molecular evidence clearly points to a key role for cholesterol homeostasis in pancreatic ß-cell function which, in humans, is negatively affected by statins. Available data exclude that this could be the case for other hypocholesterolemic approaches, but long-term studies are warranted to explore this critical aspect.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Pancreatic islet of Langerhans' cytoarchitecture and ultrastructure in normal glucose tolerance and in type 2 diabetes mellitus.

While a number of structural and cellular abnormalities occur in the islet of Langerhans in diabetes, and in particular in type 2 diabetes, the focus has been mostly on the insulin producing ß-cells and only more recently on glucagon producing α- and δ-cells. There is ample evidence that in type 2 diabetes mellitus (T2DM), in addition to a progressive decline in ß-cell function and associated insulin resistance in a number of insulin-sensitive tissues, alterations in glucagon secretion are also present and may play an important role in the pathogenesis of hyperglycemia both in the fasting and in the postprandial state. Recently, a number of studies have showed that there are also functional and structural alterations in glucagon-producing α-cells and somatostatin-producing δ-cells. Thus, it is becoming increasingly clear that multiple cellular alterations of multiple cell types occur, which adds even more complexity to our understanding of the pathophysiology of this common and severe disease. We believe that persistent efforts to increase the understanding of the pathophysiology of hormone secretion in the islets of Langerhans will also improve our capability to better prevent and treat diabetes mellitus.