Network representation of multicellular activity in pancreatic islets: Technical considerations for functional connectivity analysis.

Within the islets of Langerhans, beta cells orchestrate synchronized insulin secretion, a pivotal aspect of metabolic homeostasis. Despite the inherent heterogeneity and multimodal activity of individual cells, intercellular coupling acts as a homogenizing force, enabling coordinated responses through the propagation of intercellular waves. Disruptions in this coordination are implicated in irregular insulin secretion, a hallmark of diabetes. Recently, innovative approaches, such as integrating multicellular calcium imaging with network analysis, have emerged for a quantitative assessment of the cellular activity in islets. However, different groups use distinct experimental preparations, microscopic techniques, apply different methods to process the measured signals and use various methods to derive functional connectivity patterns. This makes comparisons between findings and their integration into a bigger picture difficult and has led to disputes in functional connectivity interpretations. To address these issues, we present here a systematic analysis of how different approaches influence the network representation of islet activity. Our findings show that the choice of methods used to construct networks is not crucial, although care is needed when combining data from different islets. Conversely, the conclusions drawn from network analysis can be heavily affected by the pre-processing of the time series, the type of the oscillatory component in the signals, and by the experimental preparation. Our tutorial-like investigation aims to resolve interpretational issues, reconcile conflicting views, advance functional implications, and encourage researchers to adopt connectivity analysis. As we conclude, we outline challenges for future research, emphasizing the broader applicability of our conclusions to other tissues exhibiting complex multicellular dynamics.

Functional characteristics of hub and wave-initiator cells in ß cell networks.

Islets of Langerhans operate as multicellular networks in which several hundred ß cells work in synchrony to produce secretory pulses of insulin, a hormone crucial for controlling metabolic homeostasis. Their collective rhythmic activity is facilitated by gap junctional coupling and affected by their functional heterogeneity, but the details of this robust and coordinated behavior are still not fully understood. Recent advances in multicellular imaging and optogenetic and photopharmacological strategies, as well as in network science, have led to the discovery of specialized ß cell subpopulations that were suggested to critically determine the collective dynamics in the islets. In particular hubs, i.e., ß cells with many functional connections, are believed to significantly enhance communication capacities of the intercellular network and facilitate an efficient spreading of intercellular Ca2+ waves, whereas wave-initiator cells trigger intercellular signals in their cohorts. Here, we determined Ca2+ signaling characteristics of these two ß cell subpopulations and the relationship between them by means of functional multicellular Ca2+ imaging in mouse pancreatic tissue slices in combination with methods of complex network theory. We constructed network layers based on individual Ca2+ waves to identify wave initiators, and functional correlation-based networks to detect hubs. We found that both cell types exhibit a higher-than-average active time under both physiological and supraphysiological glucose concentrations, but also that they differ significantly in many other functional characteristics. Specifically, Ca2+ oscillations in hubs are more regular, and their role appears to be much more stable over time than for initiator cells. Moreover, in contrast to wave initiators, hubs transmit intercellular signals faster than other cells, which implies a stronger intercellular coupling. Our research indicates that hubs and wave-initiator cell subpopulations are both natural features of healthy pancreatic islets, but their functional roles in principle do not overlap and should thus not be considered equal.

High-resolution analysis of the cytosolic Ca2+ events in ß cell collectives in situ.

The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca2+ concentration ([Ca2+]c). To trigger exocytosis, Ca2+ ions enter the cytosol from intracellular Ca2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca2+]c, were extensively described in isolated single cells from various endocrine glands. Notably, less work has been done on endocrine cells in situ to address the heterogeneity of [Ca2+]c events contributing to a collective functional response of a gland. For this, ß cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca2+]c dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca2+]c transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca2+ sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca2+]c event identification. Our results demonstrate that under physiological conditions the duration of [Ca2+]c events is variable, and segregated into three reproducible modes, subsecond, second, and tens of seconds time range, and are a result of a progressive temporal summation of the shortest events. Using pharmacological tools we show that activation of intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in ß cell collectives, and that a subset of [Ca2+]c events could be triggered even in the absence of Ca2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca2+ receptors in shaping the heterogeneity of [Ca2+]c responses in collectives of endocrine cells in situ.NEW & NOTEWORTHY Physiological glucose or ryanodine stimulation of ß cell collectives generates a large number of [Ca2+]c events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca2+]c event identification pipeline. The event durations segregate into three reproducible modes produced by a progressive temporal summation. Using pharmacological tools, we show that activation of ryanodine intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in ß cell collectives.

NMDA receptor inhibition increases, synchronizes, and stabilizes the collective pancreatic beta cell activity: Insights through multilayer network analysis.

NMDA receptors promote repolarization in pancreatic beta cells and thereby reduce glucose-stimulated insulin secretion. Therefore, NMDA receptors are a potential therapeutic target for diabetes. While the mechanism of NMDA receptor inhibition in beta cells is rather well understood at the molecular level, its possible effects on the collective cellular activity have not been addressed to date, even though proper insulin secretion patterns result from well-synchronized beta cell behavior. The latter is enabled by strong intercellular connectivity, which governs propagating calcium waves across the islets and makes the heterogeneous beta cell population work in synchrony. Since a disrupted collective activity is an important and possibly early contributor to impaired insulin secretion and glucose intolerance, it is of utmost importance to understand possible effects of NMDA receptor inhibition on beta cell functional connectivity. To address this issue, we combined confocal functional multicellular calcium imaging in mouse tissue slices with network science approaches. Our results revealed that NMDA receptor inhibition increases, synchronizes, and stabilizes beta cell activity without affecting the velocity or size of calcium waves. To explore intercellular interactions more precisely, we made use of the multilayer network formalism by regarding each calcium wave as an individual network layer, with weighted directed connections portraying the intercellular propagation. NMDA receptor inhibition stabilized both the role of wave initiators and the course of waves. The findings obtained with the experimental antagonist of NMDA receptors, MK-801, were additionally validated with dextrorphan, the active metabolite of the approved drug dextromethorphan, as well as with experiments on NMDA receptor KO mice. In sum, our results provide additional and new evidence for a possible role of NMDA receptor inhibition in treatment of type 2 diabetes and introduce the multilayer network paradigm as a general strategy to examine effects of drugs on connectivity in multicellular systems.

Teachers perception of the use on a low-cost pulse rate sensor for biology education.

Information and Communication Technology (ICT) is a commonly used concept in schools, implemented in laboratory work in the form of various digital devices. We evaluated the ICT implementation in cardiovascular physiology in Slovenian primary school education. Surprisingly, we showed a relatively low acceptance rate in biology classes: only 42.8% of involved Slovenian biology teachers used a pulse rate (PR) measuring device. As a part of a Slovenian Project, students designed, developed, and manufactured a device capable of low-cost, automatic, noninvasive, and straightforward PR sampling in real time. The device was named Fingerbeeper, and teachers' perceptions of its efficacy and efficiency were evaluated in the elementary school biology lessons, comparing its ease of use with other commercially available devices: the systems from Vernier, Biopac, and the Gear Sport Samsung smartwatch. The most preferred system was the system from Vernier (36.4%), followed by the Fingerbeeper (29.1%), the system from Biopac (18.2%), and the smartwatch (16.3%). Teachers provided their opinion on the efficiency of the Fingerbeeper in terms of cost compared with the other three measurement devices. Its perception of efficiency was comparable to the other commercially available devices while having the estimated cost of only a few percent of the Biopac or Vernier systems. Considering the general low funding in the public primary schools in Slovenia, the bias toward Fingerbeeper seemed rational, outweighing the superior performance of the commercial systems. Further research and improvement of such low-cost and high-efficiency devices, also in general terms, would lead to broader acceptance and implementation of the ICT in curricula.

Glucose-dependent activation, activity, and deactivation of beta cell networks in acute mouse pancreas tissue slices.

Many details of glucose-stimulated intracellular calcium changes in ß cells during activation, activity, and deactivation, as well as their concentration-dependence, remain to be analyzed. Classical physiological experiments indicated that in islets, functional differences between individual cells are largely attenuated, but recent findings suggest considerable intercellular heterogeneity, with some cells possibly coordinating the collective responses. To address the above with an emphasis on heterogeneity and describing the relations between classical physiological and functional network properties, we performed functional multicellular calcium imaging in mouse pancreas tissue slices over a wide range of glucose concentrations. During activation, delays to activation of cells and any-cell-to-first-responder delays are shortened, and the sizes of simultaneously responding clusters increased with increasing glucose concentrations. Exactly the opposite characterized deactivation. The frequency of fast calcium oscillations during activity increased with increasing glucose up to 12 mM glucose concentration, beyond which oscillation duration became longer, resulting in a homogenous increase in active time. In terms of functional connectivity, islets progressed from a very segregated network to a single large functional unit with increasing glucose concentration. A comparison between classical physiological and network parameters revealed that the first-responders during activation had longer active times during plateau and the most active cells during the plateau tended to deactivate later. Cells with the most functional connections tended to activate sooner, have longer active times, and deactivate later. Our findings provide a common ground for recent differing views on ß cell heterogeneity and an important baseline for future studies of stimulus-secretion and intercellular coupling.NEW & NOTEWORTHY We assessed concentration-dependence in coupled ß cells, degree of functional heterogeneity, and uncovered possible specialized subpopulations during the different phases of the response to glucose at the level of many individual cells. To this aim, we combined acute mouse pancreas tissue slices with functional multicellular calcium imaging over a wide range from threshold (7 mM) and physiological (8 and 9 mM) to supraphysiological (12 and 16 mM) glucose concentrations, classical physiological, and advanced network analyses.

Autopoietic Influence Hierarchies in Pancreatic ß Cells.

ß cells are biologically essential for humans and other vertebrates. Because their functionality arises from cell-cell interactions, they are also a model system for collective organization among cells. There are currently two contradictory pictures of this organization: the hub-cell idea pointing at leaders who coordinate the others, and the electrophysiological theory describing all cells as equal. We use new data and computational modeling to reconcile these pictures. We find via a network representation of interacting ß cells that leaders emerge naturally (confirming the hub-cell idea), yet all cells can take the hub role following a perturbation (in line with electrophysiology).

ß Cells Operate Collectively to Help Maintain Glucose Homeostasis.

Residing in the islets of Langerhans in the pancreas, ß cells contribute to glucose homeostasis by managing the body's insulin supply. Although it has been acknowledged that healthy ß cells engage in heavy cell-to-cell communication to perform their homeostatic function, the exact role and effects of such communication remain partly understood. We offer a novel, to our knowledge, perspective on the subject in the form of 1) a dynamical network model that faithfully mimics fast calcium oscillations in response to above-threshold glucose stimulation and 2) empirical data analysis that reveals a qualitative shift in the cross-correlation structure of measured signals below and above the threshold glucose concentration. Combined together, these results point to a glucose-induced transition in ß-cell activity thanks to increasing coordination through gap-junctional signaling and paracrine interactions. Our data and the model further suggest how the conservation of entire cell-cell conductance, observed in coupled but not uncoupled ß cells, emerges as a collective phenomenon. An overall implication is that improving the ability to monitor ß-cell signaling should offer means to better understand the pathogenesis of diabetes mellitus.

Simultaneous Pure Laparoscopic Resection of Primary Colorectal Cancer and Synchronous Liver Metastases: A Single Institution Experience with Propensity Score Matching Analysis.

BACKGROUND: The aim of the study was to compare the outcome of pure laparoscopic and open simultaneous resection of both the primary colorectal cancer and synchronous colorectal liver metastases (SCLM). PATIENTS AND METHODS: From 2000 to 2016 all patients treated by simultaneous resection were assessed for entry in this single center, clinically nonrandomized trial. A propensity score matching was used to compare the laparoscopic group (LAP) to open surgery group (OPEN). Primary endpoints were perioperative and oncologic outcomes. Secondary endpoints were overall survival (OS) and disease-free survival (DFS). RESULTS: Of the 82 patients identified who underwent simultaneous liver resection for SCLM, 10 patients underwent LAP. All these consecutive patients from LAP were matched to 10 comparable OPEN. LAP reduced the length of hospital stay (P = 0.044) and solid food oral intake was faster (P = 0.006) in this group. No patient undergoing the laparoscopic procedure experienced conversion to the open technique. No difference was observed in operative time, blood loss, transfusion rate, narcotics requirement, clinical risk score, resection margin, R0 resections rate, morbidity, mortality and incisional hernias rate. The two groups did not differ significantly in terms of the 3-year OS rate (90 vs. 75%; P = 0.842) and DFS rate (60 vs. 57%; P = 0.724). CONCLUSIONS: LAP reduced the length of hospital stay and offers faster solid food oral intake. Comparable oncologic and survival outcomes can be achieved. LAP is beneficial for well selected patients in high volume centers with appropriate expertise.

The relationship between node degree and dissipation rate in networks of diffusively coupled oscillators and its significance for pancreatic beta cells.

Self-sustained oscillatory dynamics is a motion along a stable limit cycle in the phase space, and it arises in a wide variety of mechanical, electrical, and biological systems. Typically, oscillations are due to a balance between energy dissipation and generation. Their stability depends on the properties of the attractor, in particular, its dissipative characteristics, which in turn determine the flexibility of a given dynamical system. In a network of oscillators, the coupling additionally contributes to the dissipation, and hence affects the robustness of the oscillatory solution. Here, we therefore investigate how a heterogeneous network structure affects the dissipation rate of individual oscillators. First, we show that in a network of diffusively coupled oscillators, the dissipation is a linearly decreasing function of the node degree, and we demonstrate this numerically by calculating the average divergence of coupled Hopf oscillators. Subsequently, we use recordings of intracellular calcium dynamics in pancreatic beta cells in mouse acute tissue slices and the corresponding functional connectivity networks for an experimental verification of the presented theory. We use methods of nonlinear time series analysis to reconstruct the phase space and calculate the sum of Lyapunov exponents. Our analysis reveals a clear tendency of cells with a higher degree, that is, more interconnected cells, having more negative values of divergence, thus confirming our theoretical predictions. We discuss these findings in the context of energetic aspects of signaling in beta cells and potential risks for pathological changes in the tissue.

Difference Between Bladder and Esophageal Temperatures in Mild Induced Hypothermia.

BACKGROUND: Mild induced hypothermia is an established treatment strategy for comatose survivors of cardiac arrest. The goal of the induction phase of mild induced hypothermia is to cool the patient's core body temperature to 32°-34°C. OBJECTIVE: The main goal of this study was to compare temperature changes measured in the esophagus and urinary bladder in survivors of cardiac arrest undergoing mild induced hypothermia using cold saline infusion. METHODS: We performed a prospective study in a 12-bed adult medical intensive care unit at a tertiary level hospital in comatose adult survivors of nontraumatic cardiac arrest admitted from January to April 2012. Paired temperature readings from bladder and esophageal probes were recorded every 5 min for 95 min (20 readings). Cold fluid infusion was terminated when the measured temperature from either of the probes reached 33.9°C. Factorial repeated-measures analysis of variance was used to determine the effect of time and site of measurement on temperature readings. RESULTS: Measurements were performed in 8 patients. Target temperature was achieved in 33 ± 15 min in the esophagus and in 63 ± 15 min in the bladder (p = 0.006). We discovered a significant interaction effect (p < 0.001) between time and site of measurement, indicating that temperature changes differently depending on the site of measurement, with esophageal temperatures decreasing faster than temperatures measured in urinary bladder. CONCLUSIONS: Our results indicate that esophageal temperature measurements show a faster response rate compared to temperature measured in the bladder when cold saline infusion is used to induce mild hypothermia.

Membrane Potential and Calcium Dynamics in Beta Cells from Mouse Pancreas Tissue Slices: Theory, Experimentation, and Analysis.

Beta cells in the pancreatic islets of Langerhans are precise biological sensors for glucose and play a central role in balancing the organism between catabolic and anabolic needs. A hallmark of the beta cell response to glucose are oscillatory changes of membrane potential that are tightly coupled with oscillatory changes in intracellular calcium concentration which, in turn, elicit oscillations of insulin secretion. Both membrane potential and calcium changes spread from one beta cell to the other in a wave-like manner. In order to assess the properties of the abovementioned responses to physiological and pathological stimuli, the main challenge remains how to effectively measure membrane potential and calcium changes at the same time with high spatial and temporal resolution, and also in as many cells as possible. To date, the most wide-spread approach has employed the electrophysiological patch-clamp method to monitor membrane potential changes. Inherently, this technique has many advantages, such as a direct contact with the cell and a high temporal resolution. However, it allows one to assess information from a single cell only. In some instances, this technique has been used in conjunction with CCD camera-based imaging, offering the opportunity to simultaneously monitor membrane potential and calcium changes, but not in the same cells and not with a reliable cellular or subcellular spatial resolution. Recently, a novel family of highly-sensitive membrane potential reporter dyes in combination with high temporal and spatial confocal calcium imaging allows for simultaneously detecting membrane potential and calcium changes in many cells at a time. Since the signals yielded from both types of reporter dyes are inherently noisy, we have developed complex methods of data denoising that permit for visualization and pixel-wise analysis of signals. Combining the experimental approach of high-resolution imaging with the advanced analysis of noisy data enables novel physiological insights and reassessment of current concepts in unprecedented detail.

Functional connectivity in islets of Langerhans from mouse pancreas tissue slices.

We propose a network representation of electrically coupled beta cells in islets of Langerhans. Beta cells are functionally connected on the basis of correlations between calcium dynamics of individual cells, obtained by means of confocal laser-scanning calcium imaging in islets from acute mouse pancreas tissue slices. Obtained functional networks are analyzed in the light of known structural and physiological properties of islets. Focusing on the temporal evolution of the network under stimulation with glucose, we show that the dynamics are more correlated under stimulation than under non-stimulated conditions and that the highest overall correlation, largely independent of Euclidean distances between cells, is observed in the activation and deactivation phases when cells are driven by the external stimulus. Moreover, we find that the range of interactions in networks during activity shows a clear dependence on the Euclidean distance, lending support to previous observations that beta cells are synchronized via calcium waves spreading throughout islets. Most interestingly, the functional connectivity patterns between beta cells exhibit small-world properties, suggesting that beta cells do not form a homogeneous geometric network but are connected in a functionally more efficient way. Presented results provide support for the existing knowledge of beta cell physiology from a network perspective and shed important new light on the functional organization of beta cell syncitia whose structural topology is probably not as trivial as believed so far.

Human Beta Cell Functional Adaptation and Dysfunction in Insulin Resistance and Its Reversibility.

BACKGROUND: Beta cells play a key role in the pathophysiology of diabetes since their functional adaptation is able to maintain euglycemia in the face of insulin resistance, and beta cell decompensation or dysfunction is a necessary condition for full-blown type 2 diabetes (T2D). The mechanisms behind compensation and decompensation are incompletely understood, especially for human beta cells, and even less is known about influences of chronic kidney disease (CKD) or immunosupressive therapy after transplantation on these processes and the development of posttransplant diabetes. SUMMARY: During compensation, beta cell sensitivity to glucose becomes left-shifted, i.e., their sensitivity to stimulation increases, and this is accompanied by enhanced signals along the stimulus-secretion coupling cascade from membrane depolarization to intracellular calcium and the most distal insulin secretion dynamics. There is currently no clear evidence regarding changes in intercellular coupling during this stage of disease progression. During decompensation, intracellular stimulus-secretion coupling remains enhanced to some extent at low or basal glucose concentrations but seems to become unable to generate effective signals to stimulate insulin secretion at high or otherwise stimulatory glucose concentrations. Additionally, intercellular coupling becomes disrupted, lowering the number of cells that contribute to secretion. During progression of CKD, beta cells also seem to drift from a compensatory left-shift to failure, and immunosupressants can further impair beta cell function following kidney transplantation. KEY MESSAGES: Beta cell stimulus-secretion coupling is enhanced in compensated insulin resistance. With worsening insulin resistance, both intra- and intercellular coupling become disrupted. CKD can progressively disrupt beta cell function, but further studies are needed, especially regarding changes in intercellular coupling.

Western diet-induced ultrastructural changes in mouse pancreatic acinar cells.

Mouse models of diet-induced type 2 diabetes mellitus provide powerful tools for studying the structural and physiological changes that are related to the disease progression. In this study, diabetic-like glucose dysregulation was induced in mice by feeding them a western diet, and light and transmission electron microscopy were used to study the ultrastructural changes in the pancreatic acinar cells. Acinar necrosis and vacuolization of the cytoplasm were the most prominent features. Furthermore, we observed intracellular and extracellular accumulation of lipid compounds in the form of lipid droplets, structural enlargement of the cisternae of the rough endoplasmic reticulum (RER), and altered mitochondrial morphology, with mitochondria lacking the typical organization of the inner membrane. Last, autophagic structures, i.e., autophagosomes, autolysosomes, and residual bodies, were abundant within the acinar cells of western diet-fed mice, and the autolysosomes contained lipids and material of varying electron density. While diets inducing obesity and type 2 diabetes are clearly associated with structural changes and dysfunction of the endocrine pancreas, we here demonstrate the strong effect of dietary intervention on the structure of acinar cells in the exocrine part of the organ before detectable changes in plasma amylase activity, which may help us better understand the development of non-alcoholic fatty pancreas disease and its association with endo- and exocrine dysfunction.

Both electrical and metabolic coupling shape the collective multimodal activity and functional connectivity patterns in beta cell collectives: A computational model perspective.

Pancreatic beta cells are coupled excitable oscillators that synchronize their activity via different communication pathways. Their oscillatory activity manifests itself on multiple timescales and consists of bursting electrical activity, subsequent oscillations in the intracellular Ca^{2+}, as well as oscillations in metabolism and exocytosis. The coordination of the intricate activity on the multicellular level plays a key role in the regulation of physiological pulsatile insulin secretion and is incompletely understood. In this paper, we investigate theoretically the principles that give rise to the synchronized activity of beta cell populations by building up a phenomenological multicellular model that incorporates the basic features of beta cell dynamics. Specifically, the model is composed of coupled slow and fast oscillatory units that reflect metabolic processes and electrical activity, respectively. Using a realistic description of the intercellular interactions, we study how the combination of electrical and metabolic coupling generates collective rhythmicity and shapes functional beta cell networks. It turns out that while electrical coupling solely can synchronize the responses, the addition of metabolic interactions further enhances coordination, the spatial range of interactions increases the number of connections in the functional beta cell networks, and ensures a better consistency with experimental findings. Moreover, our computational results provide additional insights into the relationship between beta cell heterogeneity, their activity profiles, and functional connectivity, supplementing thereby recent experimental results on endocrine networks.

The effect of forskolin and the role of Epac2A during activation, activity, and deactivation of beta cell networks.

Beta cells couple stimulation by glucose with insulin secretion and impairments in this coupling play a central role in diabetes mellitus. Cyclic adenosine monophosphate (cAMP) amplifies stimulus-secretion coupling via protein kinase A and guanine nucleotide exchange protein 2 (Epac2A). With the present research, we aimed to clarify the influence of cAMP-elevating diterpene forskolin on cytoplasmic calcium dynamics and intercellular network activity, which are two of the crucial elements of normal beta cell stimulus-secretion coupling, and the role of Epac2A under normal and stimulated conditions. To this end, we performed functional multicellular calcium imaging of beta cells in mouse pancreas tissue slices after stimulation with glucose and forskolin in wild-type and Epac2A knock-out mice. Forskolin evoked calcium signals in otherwise substimulatory glucose and beta cells from Epac2A knock-out mice displayed a faster activation. During the plateau phase, beta cells from Epac2A knock-out mice displayed a slightly higher active time in response to glucose compared with wild-type littermates, and stimulation with forskolin increased the active time via an increase in oscillation frequency and a decrease in oscillation duration in both Epac2A knock-out and wild-type mice. Functional network properties during stimulation with glucose did not differ in Epac2A knock-out mice, but the presence of Epac2A was crucial for the protective effect of stimulation with forskolin in preventing a decline in beta cell functional connectivity with time. Finally, stimulation with forskolin prolonged beta cell activity during deactivation, especially in Epac2A knock-out mice.

Exendin-4 affects calcium signalling predominantly during activation and activity of beta cell networks in acute mouse pancreas tissue slices.

Tight control of beta cell stimulus-secretion coupling is crucial for maintaining homeostasis of energy-rich nutrients. While glucose serves as a primary regulator of this process, incretins augment beta cell function, partly by enhancing cytosolic [Ca2+] dynamics. However, the details of how precisely they affect beta cell recruitment during activation, their active time, and functional connectivity during plateau activity, and how they influence beta cell deactivation remain to be described. Performing functional multicellular Ca2+ imaging in acute mouse pancreas tissue slices enabled us to systematically assess the effects of the GLP-1 receptor agonist exendin-4 (Ex-4) simultaneously in many coupled beta cells with high resolution. In otherwise substimulatory glucose, Ex-4 was able to recruit approximately a quarter of beta cells into an active state. Costimulation with Ex-4 and stimulatory glucose shortened the activation delays and accelerated beta cell activation dynamics. More specifically, active time increased faster, and the time required to reach half-maximal activation was effectively halved in the presence of Ex-4. Moreover, the active time and regularity of [Ca2+]IC oscillations increased, especially during the first part of beta cell response. In contrast, subsequent addition of Ex-4 to already active cells did not significantly enhance beta cell activity. Network analyses further confirmed increased connectivity during activation and activity in the presence of Ex-4, with hub cell roles remaining rather stable in both control experiments and experiments with Ex-4. Interestingly, Ex-4 demonstrated a biphasic effect on deactivation, slightly prolonging beta cell activity at physiological concentrations and shortening deactivation delays at supraphysiological concentrations. In sum, costimulation by Ex-4 and glucose increases [Ca2+]IC during beta cell activation and activity, indicating that the effect of incretins may, to an important extent, be explained by enhanced [Ca2+]IC signals. During deactivation, previous incretin stimulation does not critically prolong cellular activity, which corroborates their low risk of hypoglycemia.

A Promising Method for the Determination of Cell Viability: The Membrane Potential Cell Viability Assay.

Determining the viability of cells is fraught with many uncertainties. It is often difficult to determine whether a cell is still alive, approaching the point of no return, or dead. Today, there are many methods for determining cell viability. Most rely on an indirect determination of cell death (metabolism, molecular transport, and leakage, to name a few). In contrast, we have developed a promising novel method for a "direct" determination of cell viability. The potential method assesses cell membrane integrity (which is essential for all viable cells) by measuring the electrical potential of the cell membrane. To test the assay, we chose two different cell types, blood macrophages (TLT) and breast cancer epithelial cells (MCF 7). We exposed them to seven different toxic scenarios (arsenic (V), UV light, hydrogen peroxide, nutrient starvation, Tetrabromobisphenol A, fatty acids, and 5-fluorouracil) to induce different cell death pathways. Under controlled test conditions, the assay showed good accuracy when comparing the toxicity assessment with well-established methods. Moreover, the method showed compatibility with live cell imaging. Although we know that further studies are needed to confirm the performance of the assay in other situations, the results obtained are promising for their wider application in the future.

Glucose-Stimulated Calcium Dynamics in Beta Cells From Male C57BL/6J, C57BL/6N, and NMRI Mice: A Comparison of Activation, Activity, and Deactivation Properties in Tissue Slices.

Although mice are a very instrumental model in islet beta cell research, possible phenotypic differences between strains and substrains are largely neglected in the scientific community. In this study, we show important phenotypic differences in beta cell responses to glucose between C57BL/6J, C57BL/6N, and NMRI mice, i.e., the three most commonly used strains. High-resolution multicellular confocal imaging of beta cells in acute pancreas tissue slices was used to measure and quantitatively compare the calcium dynamics in response to a wide range of glucose concentrations. Strain- and substrain-specific features were found in all three phases of beta cell responses to glucose: a shift in the dose-response curve characterizing the delay to activation and deactivation in response to stimulus onset and termination, respectively, and distinct concentration-encoding principles during the plateau phase in terms of frequency, duration, and active time changes with increasing glucose concentrations. Our results underline the significance of carefully choosing and reporting the strain to enable comparison and increase reproducibility, emphasize the importance of analyzing a number of different beta cell physiological parameters characterizing the response to glucose, and provide a valuable standard for future studies on beta cell calcium dynamics in health and disease in tissue slices.