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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Šterk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lipovšek, Dolenšek, Dariš, Valladolid-Acebes, Vajs, Leitinger, Stožer and Skelin Klemen.)

Background: An anconeus epitrochlearis muscle (AE) is a common anatomical variant in the upper extremity, located at the medial aspect of the elbow. Its anatomical position contributes to the roof of the cubital tunnel. While it plays a role in protecting the ulnar nerve, it may also pose a risk for ulnar nerve compression. This study aimed to determine the true prevalence of AE in a Central European population., Materials and Methods: The presence of AE was evaluated in 115 cadaveric upper extremities from an undetermined number of subjects. The limbs for dissection were assumed to be healthy, and AE identification involved anatomical description and measurements. Data analysis aimed to determine the true prevalence, considering 95% confidence intervals., Results: AE was present in 5 of the 115 cadaveric limbs (4.3%). Specimens lacking AE were observed, depicting normal cubital tunnel roof anatomy. When present, AE replaced the proximal part of the cubital tunnel roof, superficially coursing to the ulnar nerve. Morphological variations were noted., Conclusions: The true prevalence of AE was 4.3% (95% CI = 0.2%-8.4%), consistent with recent studies. Historical reports indicate varying true prevalence up to 26%, possibly linked to manual labor changes. Contrary to prior assumptions, our study did not find a significantly higher true prevalence in the European population. AE's association with cubital tunnel syndrome is complex, with both protective and potentially compressive roles.

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 [Ca 2+ ] 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 Ca 2+ 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 [Ca 2+ ] 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 [Ca 2+ ] IC during beta cell activation and activity, indicating that the effect of incretins may, to an important extent, be explained by enhanced [Ca 2+ ] IC signals. During deactivation, previous incretin stimulation does not critically prolong cellular activity, which corroborates their low risk of hypoglycemia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Paradiž Leitgeb, Kerčmar, Križančić Bombek, Pohorec, Skelin Klemen, Slak Rupnik, Gosak, Dolenšek and Stožer.)

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., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Skelin Klemen, Dolenšek, Križančić Bombek, Pohorec, Gosak, Slak Rupnik and Stožer.)

Objective: Success of pelvic organ prolapse (POP) mesh procedures also depends on reliable anchoring systems (AS). Our primary aim was to assess the use of soft-embalmed cadavers in testing of different AS and our secondary aim was to compare extraction forces (EF) of different AS and non-absorbable suture (NAS)., Study Design: IRB approval was obtained. NAS (Ti-cron®) and different AS were attached to force-measuring instrument (Dynamometer SS25LA) and anchored to anterior longitudinal (ALL) and pectineal ligament (PL) (Protack®, Uplift®, NAS), and sacrospinous ligament (SSL) (Surelift®, Elevate PC®, NAS) of Thiel soft-embalmed cadavers. EF were measured 2-4 times in each cadaver. Data were compared using non-parametric tests. Statistical significance was set at p < 0.05., Results: Three female cadavers (age 59, 77 and 87) were used. NAS EF were significantly higher than AS EF for ALL and SSL, but not PL. Thiel soft-embalmed cadavers proved to be useful in testing of different AS., Conclusions: Use of soft-embalmed cadavers in testing of different AS is feasible. According to our results, the NAS provides most reliable intra-corporeal fixation. However, significant inter- and intra-subject variability indicates that results may also be dependent on the tissue properties and anchoring procedure. Further testing using soft-embalmed cadavers could help optimise mesh procedures and establish a threshold EF necessary for reliable fixation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Islets of Langerhans operate as multicellular networks in which several hundred β 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 β cell subpopulations that were suggested to critically determine the collective dynamics in the islets. In particular hubs, i.e., β cells with many functional connections, are believed to significantly enhance communication capacities of the intercellular network and facilitate an efficient spreading of intercellular Ca 2+ waves, whereas wave-initiator cells trigger intercellular signals in their cohorts. Here, we determined Ca 2+ signaling characteristics of these two β cell subpopulations and the relationship between them by means of functional multicellular Ca 2+ imaging in mouse pancreatic tissue slices in combination with methods of complex network theory. We constructed network layers based on individual Ca 2+ 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, Ca 2+ 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca 2+ concentration ([Ca 2+ ] c ). To trigger exocytosis, Ca 2+ ions enter the cytosol from intracellular Ca 2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca 2+ ] 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 [Ca 2+ ] c events contributing to a collective functional response of a gland. For this, β cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca 2+ ] c dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca 2+ ] c transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca 2+ sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca 2+ ] c event identification. Our results demonstrate that under physiological conditions the duration of [Ca 2+ ] 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 Ca 2+ receptors is both sufficient and necessary for glucose-dependent [Ca 2+ ] c oscillations in β cell collectives, and that a subset of [Ca 2+ ] c events could be triggered even in the absence of Ca 2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca 2+ receptors in shaping the heterogeneity of [Ca 2+ ] c responses in collectives of endocrine cells in situ. NEW & NOTEWORTHY Physiological glucose or ryanodine stimulation of β cell collectives generates a large number of [Ca 2+ ] c events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca 2+ ] 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 Ca 2+ receptors is both sufficient and necessary for glucose-dependent [Ca 2+ ] c oscillations in β cell collectives.

Islets of Langerhans are multicellular microorgans located in the pancreas that play a central role in whole-body energy homeostasis. Through secretion of insulin and other hormones they regulate postprandial storage and interprandial usage of energy-rich nutrients. In these clusters of hormone-secreting endocrine cells, intricate cell-cell communication is essential for proper function. Electrical coupling between the insulin-secreting beta cells through gap junctions composed of connexin36 is particularly important, as it provides the required, most important, basis for coordinated responses of the beta cell population. The increasing evidence that gap-junctional communication and its modulation are vital to well-regulated secretion of insulin has stimulated immense interest in how subpopulations of heterogeneous beta cells are functionally arranged throughout the islets and how they mediate intercellular signals. In the last decade, several novel techniques have been proposed to assess cooperation between cells in islets, including the prosperous combination of multicellular imaging and network science. In the present contribution, we review recent advances related to the application of complex network approaches to uncover the functional connectivity patterns among cells within the islets. We first provide an accessible introduction to the basic principles of network theory, enumerating the measures characterizing the intercellular interactions and quantifying the functional integration and segregation of a multicellular system. Then we describe methodological approaches to construct functional beta cell networks, point out possible pitfalls, and specify the functional implications of beta cell network examinations. We continue by highlighting the recent findings obtained through advanced multicellular imaging techniques supported by network-based analyses, giving special emphasis to the current developments in both mouse and human islets, as well as outlining challenges offered by the multilayer network formalism in exploring the collective activity of islet cell populations. Finally, we emphasize that the combination of these imaging techniques and network-based analyses does not only represent an innovative concept that can be used to describe and interpret the physiology of islets, but also provides fertile ground for delineating normal from pathological function and for quantifying the changes in islet communication networks associated with the development of diabetes mellitus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stožer, Šterk, Paradiž Leitgeb, Markovič, Skelin Klemen, Ellis, Križančić Bombek, Dolenšek, MacDonald and Gosak.)

The physiology and pathophysiology of the exocrine pancreas are in close connection to changes in intra-cellular Ca2+ concentration. Most of our knowledge is based on in vitro experiments on acinar cells or acini enzymatically isolated from their surroundings, which can alter their structure, physiology, and limit our understanding. Due to these limitations, the acute pancreas tissue slice technique was introduced almost two decades ago as a complementary approach to assess the morphology and physiology of both the endocrine and exocrine pancreas in a more conserved in situ setting. In this study, we extend previous work to functional multicellular calcium imaging on acinar cells in tissue slices. The viability and morphological characteristics of acinar cells within the tissue slice were assessed using the LIVE/DEAD assay, transmission electron microscopy, and immunofluorescence imaging. The main aim of our study was to characterize the responses of acinar cells to stimulation with acetylcholine and compare them with responses to cerulein in pancreatic tissue slices, with special emphasis on inter-cellular and inter-acinar heterogeneity and coupling. To this end, calcium imaging was performed employing confocal microscopy during stimulation with a wide range of acetylcholine concentrations and selected concentrations of cerulein. We show that various calcium oscillation parameters depend monotonically on the stimulus concentration and that the activity is rather well synchronized within acini, but not between acini. The acute pancreas tissue slice represents a viable and reliable experimental approach for the evaluation of both intra- and inter-cellular signaling characteristics of acinar cell calcium dynamics. It can be utilized to assess many cells simultaneously with a high spatiotemporal resolution, thus providing an efficient and high-yield platform for future studies of normal acinar cell biology, pathophysiology, and screening pharmacological substances., Competing Interests: The authors have declared that no competing interests exist.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pohorec, Križančić Bombek, Skelin Klemen, Dolenšek and Stožer.)