45 results on '"Dolenšek J"'
Search Results
2. Triple arterial blood supply to the liver and double cystic arteries
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Dolenšek J
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Male ,medicine.medical_specialty ,Arterial blood supply ,Histology ,Left hepatic artery ,Left gastric artery ,03 medical and health sciences ,Hepatic Artery ,0302 clinical medicine ,Cadaver ,Internal medicine ,medicine.artery ,medicine ,Humans ,Superior mesenteric artery ,Upper abdomen ,Right hepatic artery ,business.industry ,Gallbladder ,medicine.anatomical_structure ,Liver ,030220 oncology & carcinogenesis ,Cardiology ,030211 gastroenterology & hepatology ,Anatomy ,business - Abstract
A rare combination of variations in the arterial supply of the liver and gallbladder was encountered in a male cadaver. The simultaneous occurrence of an accessory left hepatic artery and an accessory right hepatic artery from which double cystic arteries arose (one of which was low-lying). This combination has not yet been reported. The accessory left hepatic artery originated from the left gastric artery. The accessory right hepatic artery originated from the superior mesenteric artery. Such arterial variations are caused by differences in embryological development. This, however, may lead to complications related to diagnostic and therapeutic procedures in the upper abdomen.
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- 2017
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3. Simulation of pulmonary ventilation and its control by negative feedback
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Dolenšek, J., Runovc, F., and Kordaš, M.
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- 2005
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4. New approach for the investigation of pancreatic duct in mice
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Gál, E., Dolensek, J., Stozer, A., Pohorec, V., Ébert, A., and Venglovecz, V.
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- 2020
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5. Calcium Dependencies of Regulated Exocytosis in Different Endocrine Cells
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DOLENŠEK, J., primary, SKELIN, M., additional, and RUPNIK, M. S., additional
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- 2011
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6. Calcium oscillations and waves in beta cells from acute mouse pancreas tissue slices.
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Stozer, A., Dolenšek, J., Klemen, M. S., and Rupnik, M. S.
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LANGERHANS cells , *GLUCOSE , *CALCIUM ions - Abstract
In beta cells from Langerhans islets, cell stimulation is coupled to insulin secretion. Upon stimulation with glucose, the concentration of intracellular calcium ions ([Ca2+]i) oscillates in phase with accompanying oscillatory changes in membrane potential and insulin secretion. The calcium oscillations in different cells within a single islet of Langerhans are well synchronized, with phase differences between oscillations in individual cells of up to a few seconds (1,2). Furthermore, some studies have reported the presence of calcium waves in islets of Langerhans, with a speed of 20-200 µm/s (3). However, many different types of calcium oscillations have been reported in isolated islets which are the most widely used experimental model. It has been suggested that the duration and conditions of islet culture influence the types of responses and that calcium waves occur only in cultured islets (4-6). Additionally, due to limited diffusion of fluorescent calcium indicators, calcium imaging experiments performed on isolated islets enable insight only into the most peripheral parts of islets, where beta cells are least abundant in mice and where other types of cells are present (7). To enable access to all cellular layers of an islet and clarify the existing controversy regarding types of oscillations present in islets and the existence of calcium waves, we combined the recently introduced acute pancreas tissue slice method (8) with confocal laser scanning calcium imaging, using the Oregon green® 488 BAPTA-1 fluorescent calcium indicator. The study was conducted in accordance with all national and European recommendations pertaining to work with isolated tissue and our protocol was approved by the Veterinary Administration of the Republic of Slovenia. The tissue slices were prepared from agarose-injected pancreata of 10-20 week old NMRI mice of either sex, upon sacrifice by cervical dislocation as described in detail previously (8). Taking advantage of previous reports that islet cell types can be identified by their characteristic responses to glucose (7), this enabled simultaneous recording of [Ca2+]i in a large number of cells from all layers of an islet. We showed that fast [Ca2+]i oscillations (46 /min) superimposed on a sustained increase in [Ca2+]i are the predominant type of response to 12 mM glucose in beta cells in mice. Additionally, we were able to detect other types of responses, characteristic of alpha and delta cells, predominantly at the periphery of islets. The changes in [Ca2+]i were well synchronized exclusively between coupled beta cells (Figure 1). Finally, employing high speed calcium imaging (20 frames/s) we detected calcium waves spreading in an orderly manner across the beta cell syncitium at a velocity of 80-90 µm/s as the mechanistic substrate of synchronicity between beta cells in uncultured acute pancreas tissue slices (Figure 2). A detailed quantification of activation and deactivation phases before and after the sustained plateau with superimposed oscillations revealed that large amplitude changes in [Ca2+]i are insufficient to ensure intercellular synchronization during stimulation with glucose and that metabolic activation is necessary to align the activity of a heterogeneous population of beta cells. [ABSTRACT FROM AUTHOR]
- Published
- 2013
7. Calcium Imaging and Analysis in Beta Cells in Acute Mouse Pancreas Tissue Slices.
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Paradiž Leitgeb E, Pohorec V, Križančić Bombek L, Skelin Klemen M, Duh M, Gosak M, Dolenšek J, and Stožer A
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- Animals, Mice, Pancreas metabolism, Pancreas cytology, Glucose metabolism, Insulin-Secreting Cells metabolism, Calcium metabolism, Microscopy, Confocal methods, Calcium Signaling
- Abstract
Ca
2+ ions play a central role in the stimulus-secretion coupling cascade of pancreatic beta cells. The use of confocal microscopy in conjunction with the acute pancreas tissue slice technique offers valuable insights into changes in the intracellular calcium concentration following stimulation by secretagogues. This allows the study of beta cells on a single cell level, as well as their behavior on a multicellular scale within an intact environment. With the use of advanced analytical tools, this approach offers insight into how single cells contribute to the functional unit of islets of Langerhans and processes underlying insulin secretion. Here we describe a comprehensive protocol for the preparation and utilization of acute pancreas tissue slices in mice, the use of high-resolution confocal microscopy for observation of glucose-stimulated calcium dynamics in beta cells, and the computational analysis for objective evaluation of calcium signals., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)- Published
- 2025
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8. Network representation of multicellular activity in pancreatic islets: Technical considerations for functional connectivity analysis.
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Šterk M, Zhang Y, Pohorec V, Leitgeb EP, Dolenšek J, Benninger RKP, Stožer A, Kravets V, and Gosak M
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- Animals, Computational Biology methods, Mice, Insulin metabolism, Humans, Insulin-Secreting Cells physiology, Insulin-Secreting Cells metabolism, Insulin-Secreting Cells cytology, Insulin Secretion physiology, Models, Biological, Calcium metabolism, Calcium Signaling physiology, Islets of Langerhans physiology, Islets of Langerhans metabolism, Islets of Langerhans cytology
- Abstract
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.)
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- 2024
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9. Western diet-induced ultrastructural changes in mouse pancreatic acinar cells.
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Lipovšek S, Dolenšek J, Dariš B, Valladolid-Acebes I, Vajs T, Leitinger G, Stožer A, and Skelin Klemen M
- Abstract
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.)
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- 2024
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10. The prevalence of the anconeus epitrochlearis muscle in a Central European population.
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Dolenšek J
- Abstract
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.
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- 2024
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11. Exendin-4 affects calcium signalling predominantly during activation and activity of beta cell networks in acute mouse pancreas tissue slices.
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Paradiž Leitgeb E, Kerčmar J, Križančić Bombek L, Pohorec V, Skelin Klemen M, Slak Rupnik M, Gosak M, Dolenšek J, and Stožer A
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- Mice, Animals, Exenatide pharmacology, Calcium, Glucose pharmacology, Calcium, Dietary, Incretins pharmacology, Insulin-Secreting Cells
- Abstract
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 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., 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.)- Published
- 2024
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12. Human Beta Cell Functional Adaptation and Dysfunction in Insulin Resistance and Its Reversibility.
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Skelin Klemen M, Kopecky J, Dolenšek J, and Stožer A
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- Humans, Insulin metabolism, Glucose metabolism, Insulin Resistance physiology, Diabetes Mellitus, Type 2, Renal Insufficiency, Chronic
- Abstract
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.)
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- 2024
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13. The effect of forskolin and the role of Epac2A during activation, activity, and deactivation of beta cell networks.
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Skelin Klemen M, Dolenšek J, Križančić Bombek L, Pohorec V, Gosak M, Slak Rupnik M, and Stožer A
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- Animals, Mice, Colforsin pharmacology, Cyclic AMP, Glucose pharmacology, Mice, Knockout, Calcium, Calcium, Dietary
- Abstract
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.)
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- 2023
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14. Measurement of extraction forces of non-absorbable suture and different anchoring systems used for pelvic organ prolapse surgery using soft-embalmed cadavers: A feasibility study.
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Serdinšek T, Rakuša M, Kocbek Šaherl L, Pejković B, Dolenšek J, and But I
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- Humans, Female, Feasibility Studies, Cadaver, Sutures, Pelvis, Pelvic Organ Prolapse surgery
- Abstract
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.)
- Published
- 2023
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15. Functional characteristics of hub and wave-initiator cells in β cell networks.
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Šterk M, Dolenšek J, Skelin Klemen M, Križančić Bombek L, Paradiž Leitgeb E, Kerčmar J, Perc M, Slak Rupnik M, Stožer A, and Gosak M
- Subjects
- Mice, Animals, Calcium Signaling physiology, Insulin metabolism, Insulin Secretion, Calcium metabolism, Glucose metabolism, Insulin-Secreting Cells metabolism, Islets of Langerhans metabolism
- Abstract
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 Ca2+ signaling characteristics of these two β 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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16. High-resolution analysis of the cytosolic Ca 2+ events in β cell collectives in situ.
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Postić S, Sarikas S, Pfabe J, Pohorec V, Križančić Bombek L, Sluga N, Skelin Klemen M, Dolenšek J, Korošak D, Stožer A, Evans-Molina C, Johnson JD, and Slak Rupnik M
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- Cytosol metabolism, Ryanodine metabolism, Ryanodine pharmacology, Glucose metabolism, Calcium metabolism, Calcium Signaling, Insulin-Secreting Cells metabolism, Islets of Langerhans metabolism
- Abstract
The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca
2+ 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, β 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 β 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 β 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 β cell collectives.- Published
- 2023
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17. From Isles of Königsberg to Islets of Langerhans: Examining the Function of the Endocrine Pancreas Through Network Science.
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Stožer A, Šterk M, Paradiž Leitgeb E, Markovič R, Skelin Klemen M, Ellis CE, Križančić Bombek L, Dolenšek J, MacDonald PE, and Gosak M
- Subjects
- Animals, Cell Communication, Insulin, Mice, Pancreas, Insulin-Secreting Cells, Islets of Langerhans
- Abstract
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.)
- Published
- 2022
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18. Calcium imaging in intact mouse acinar cells in acute pancreas tissue slices.
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Marolt U, Paradiž Leitgeb E, Pohorec V, Lipovšek S, Venglovecz V, Gál E, Ébert A, Menyhárt I, Potrč S, Gosak M, Dolenšek J, and Stožer A
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- Acetylcholine pharmacology, Animals, Calcium, Dietary, Ceruletide, Mice, Microscopy, Confocal, Pancreas, Acinar Cells, Calcium
- Abstract
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.
- Published
- 2022
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19. Teachers perception of the use on a low-cost pulse rate sensor for biology education.
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Dolenšek J, Kos T, Stožer A, and Špernjak A
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- Biology, Heart Rate, Humans, Perception, School Teachers, Schools, Students
- Abstract
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.
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- 2022
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20. 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.
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Pohorec V, Križančić Bombek L, Skelin Klemen M, Dolenšek J, and Stožer A
- Subjects
- Animals, Calcium, Male, Mice, Mice, Inbred C57BL, Reproducibility of Results, Glucose pharmacology, Insulin-Secreting Cells
- Abstract
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.)
- Published
- 2022
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21. Peripherally active dextromethorphan derivatives lower blood glucose levels by targeting pancreatic islets.
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Scholz O, Otter S, Welters A, Wörmeyer L, Dolenšek J, Klemen MS, Pohorec V, Eberhard D, Mrugala J, Hamacher A, Koch A, Sanz M, Hoffmann T, Hogeback J, Herebian D, Klöcker N, Piechot A, Mayatepek E, Meissner T, Stožer A, and Lammert E
- Subjects
- Animals, Apoptosis drug effects, Blood-Brain Barrier drug effects, Blood-Brain Barrier metabolism, Calcium metabolism, Dextromethorphan analogs & derivatives, Dextromethorphan metabolism, Dextromethorphan therapeutic use, Diabetes Mellitus, Experimental chemically induced, Diabetes Mellitus, Experimental drug therapy, Diabetes Mellitus, Type 2 pathology, Drug Design, Glucagon-Like Peptide-1 Receptor agonists, Glucagon-Like Peptide-1 Receptor metabolism, Humans, Hypoglycemic Agents chemistry, Hypoglycemic Agents metabolism, Hypoglycemic Agents therapeutic use, Insulin blood, Insulin metabolism, Islets of Langerhans cytology, Islets of Langerhans metabolism, Male, Membrane Potentials drug effects, Mice, Inbred C57BL, Mice, Blood Glucose analysis, Dextromethorphan pharmacology, Hypoglycemic Agents pharmacology, Islets of Langerhans drug effects
- Abstract
Dextromethorphan (DXM) acts as cough suppressant via its central action. Cell-protective effects of this drug have been reported in peripheral tissues, making DXM potentially useful for treatment of several common human diseases, such as type 2 diabetes mellitus (T2DM). Pancreatic islets are among the peripheral tissues that positively respond to DXM, and anti-diabetic effects of DXM were observed in two placebo-controlled, randomized clinical trials in humans with T2DM. Since these effects were associated with central side effects, we here developed chemical derivatives of DXM that pass the blood-brain barrier to a significantly lower extent than the original drug. We show that basic nitrogen-containing residues block central adverse events of DXM without reducing its anti-diabetic effects, including the protection of human pancreatic islets from cell death. These results show how to chemically modify DXM, and possibly other morphinans, as to exclude central side effects, while targeting peripheral tissues, such as pancreatic islets., Competing Interests: Declaration of interests S.O., A.W., D.H., T. M., E.M., and E. L. declare the following competing financial interests: these authors are inventors of the US patent 10,464,904 entitled “Dextrorphan-derivatives with suppressed central nervous activity”; and T.M. and E.L. are inventors of the US patent 9,370,511 entitled “Morphinan-derivatives for treating diabetes and related disorders.”, (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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22. Autopoietic Influence Hierarchies in Pancreatic β Cells.
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Korošak D, Jusup M, Podobnik B, Stožer A, Dolenšek J, Holme P, and Rupnik MS
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- Animals, Humans, Cell Communication physiology, Insulin-Secreting Cells cytology, Models, Biological
- Abstract
β 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 β 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).
- Published
- 2021
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23. Mechanisms of Post-Pancreatitis Diabetes Mellitus and Cystic Fibrosis-Related Diabetes: A Review of Preclinical Studies.
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Gál E, Dolenšek J, Stožer A, Czakó L, Ébert A, and Venglovecz V
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- Animals, Diabetes Mellitus etiology, Humans, Cystic Fibrosis complications, Diabetes Mellitus pathology, Pancreatitis complications
- Abstract
Anatomical proximity and functional correlations between the exocrine and endocrine pancreas warrant reciprocal effects between the two parts. Inflammatory diseases of the exocrine pancreas, such as acute or chronic pancreatitis, or the presence of cystic fibrosis disrupt endocrine function, resulting in diabetes of the exocrine pancreas. Although novel mechanisms are being increasingly identified, the intra- and intercellular pathways regulating exocrine-endocrine interactions are still not fully understood, making the development of new and more effective therapies difficult. Therefore, this review sought to accumulate current knowledge regarding the pathogenesis of diabetes in acute and chronic pancreatitis, as well as cystic fibrosis., 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 © 2021 Gál, Dolenšek, Stožer, Czakó, Ébert and Venglovecz.)
- Published
- 2021
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24. Glucose-dependent activation, activity, and deactivation of beta cell networks in acute mouse pancreas tissue slices.
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Stožer A, Skelin Klemen M, Gosak M, Križančić Bombek L, Pohorec V, Slak Rupnik M, and Dolenšek J
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- Animals, Calcium Signaling, Female, Male, Mice, Glucose metabolism, Insulin-Secreting Cells metabolism
- Abstract
Many details of glucose-stimulated intracellular calcium changes in β 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 β cell heterogeneity and an important baseline for future studies of stimulus-secretion and intercellular coupling. NEW & NOTEWORTHY We assessed concentration-dependence in coupled β 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.
- Published
- 2021
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25. The Role of cAMP in Beta Cell Stimulus-Secretion and Intercellular Coupling.
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Stožer A, Paradiž Leitgeb E, Pohorec V, Dolenšek J, Križančić Bombek L, Gosak M, and Skelin Klemen M
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- Animals, Glucose metabolism, Humans, Insulin Resistance, Models, Biological, Cyclic AMP metabolism, Insulin-Secreting Cells metabolism, Intracellular Space metabolism
- Abstract
Pancreatic beta cells secrete insulin in response to stimulation with glucose and other nutrients, and impaired insulin secretion plays a central role in development of diabetes mellitus. Pharmacological management of diabetes includes various antidiabetic drugs, including incretins. The incretin hormones, glucagon-like peptide-1 and gastric inhibitory polypeptide, potentiate glucose-stimulated insulin secretion by binding to G protein-coupled receptors, resulting in stimulation of adenylate cyclase and production of the secondary messenger cAMP, which exerts its intracellular effects through activation of protein kinase A or the guanine nucleotide exchange protein 2A. The molecular mechanisms behind these two downstream signaling arms are still not fully elucidated and involve many steps in the stimulus-secretion coupling cascade, ranging from the proximal regulation of ion channel activity to the central Ca
2+ signal and the most distal exocytosis. In addition to modifying intracellular coupling, the effect of cAMP on insulin secretion could also be at least partly explained by the impact on intercellular coupling. In this review, we systematically describe the possible roles of cAMP at these intra- and inter-cellular signaling nodes, keeping in mind the relevance for the whole organism and translation to humans.- Published
- 2021
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26. Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices.
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Stožer A, Dolenšek J, Križančić Bombek L, Pohorec V, Slak Rupnik M, and Klemen MS
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- Animals, Mice, Pancreas cytology, Calcium metabolism, Calcium Signaling, Image Processing, Computer-Assisted methods, Microscopy, Confocal methods, Pancreas metabolism
- Abstract
The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.
- Published
- 2021
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27. Assessing Different Temporal Scales of Calcium Dynamics in Networks of Beta Cell Populations.
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Zmazek J, Klemen MS, Markovič R, Dolenšek J, Marhl M, Stožer A, and Gosak M
- Abstract
Beta cells within the pancreatic islets of Langerhans respond to stimulation with coherent oscillations of membrane potential and intracellular calcium concentration that presumably drive the pulsatile exocytosis of insulin. Their rhythmic activity is multimodal, resulting from networked feedback interactions of various oscillatory subsystems, such as the glycolytic, mitochondrial, and electrical/calcium components. How these oscillatory modules interact and affect the collective cellular activity, which is a prerequisite for proper hormone release, is incompletely understood. In the present work, we combined advanced confocal Ca
2+ imaging in fresh mouse pancreas tissue slices with time series analysis and network science approaches to unveil the glucose-dependent characteristics of different oscillatory components on both the intra- and inter-cellular level. Our results reveal an interrelationship between the metabolically driven low-frequency component and the electrically driven high-frequency component, with the latter exhibiting the highest bursting rates around the peaks of the slow component and the lowest around the nadirs. Moreover, the activity, as well as the average synchronicity of the fast component, considerably increased with increasing stimulatory glucose concentration, whereas the stimulation level did not affect any of these parameters in the slow component domain. Remarkably, in both dynamical components, the average correlation decreased similarly with intercellular distance, which implies that intercellular communication affects the synchronicity of both types of oscillations. To explore the intra-islet synchronization patterns in more detail, we constructed functional connectivity maps. The subsequent comparison of network characteristics of different oscillatory components showed more locally clustered and segregated networks of fast oscillatory activity, while the slow oscillations were more global, resulting in several long-range connections and a more cohesive structure. Besides the structural differences, we found a relatively weak relationship between the fast and slow network layer, which suggests that different synchronization mechanisms shape the collective cellular activity in islets, a finding which has to be kept in mind in future studies employing different oscillations for constructing networks., 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 © 2021 Zmazek, Klemen, Markovič, Dolenšek, Marhl, Stožer and Gosak.)- Published
- 2021
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28. β Cells Operate Collectively to Help Maintain Glucose Homeostasis.
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Podobnik B, Korošak D, Skelin Klemen M, Stožer A, Dolenšek J, Slak Rupnik M, Ivanov PC, Holme P, and Jusup M
- Subjects
- Glucose, Homeostasis, Insulin, Insulin-Secreting Cells, Islets of Langerhans
- Abstract
Residing in the islets of Langerhans in the pancreas, β cells contribute to glucose homeostasis by managing the body's insulin supply. Although it has been acknowledged that healthy β 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 β-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 β cells, emerges as a collective phenomenon. An overall implication is that improving the ability to monitor β-cell signaling should offer means to better understand the pathogenesis of diabetes mellitus., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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29. A Novel in situ Approach to Studying Detrusor Smooth Muscle Cells in Mice.
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Serdinšek T, Lipovšek S, Leitinger G, But I, Stožer A, and Dolenšek J
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- Animals, Calcium metabolism, Cells, Cultured, Mice, Microscopy, Confocal, Muscle Contraction physiology, Myocytes, Smooth Muscle ultrastructure, Urinary Bladder ultrastructure
- Abstract
The aim of our study was to develop a novel approach to investigating mouse detrusor smooth muscle cell (SMC) physiological activity, utilizing an acute tissue dissection technique and confocal calcium imaging. The bladder of a sacrificed adult female NMRI mouse was dissected. We used light and transmission electron microscopy to assess morphology of SMCs within the tissue. Calcium imaging in individual SMCs was performed using confocal microscopy during stimulation with increasing concentrations of carbamylcholine (CCh). SMCs were identified according to their morphology and calcium activity. We determined several parameters describing the SMC responses: delays to response, recruitment, relative activity, and contraction of the tissue. CCh stimulation revealed three different SMC phenotypes: spontaneously active SMCs with and without CCh-enhanced activity and SMCs with CCh-induced activity only. SMCs were recruited into an active state in response to CCh-stimulation within a narrow range (1-25 µM); causing activation of virtually all SMCs. Maximum calcium activity of SMCs was at about 25 µM, which coincided with a visible tissue contraction. Finally, we observed shorter time lags before response onsets with higher CCh concentrations. In conclusion, our novel in situ approach proved to be a robust and reproducible method to study detrusor SMC morphology and physiology.
- Published
- 2020
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30. Modelling of dysregulated glucagon secretion in type 2 diabetes by considering mitochondrial alterations in pancreatic α-cells.
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Grubelnik V, Markovič R, Lipovšek S, Leitinger G, Gosak M, Dolenšek J, Valladolid-Acebes I, Berggren PO, Stožer A, Perc M, and Marhl M
- Abstract
Type 2 diabetes mellitus (T2DM) has been associated with insulin resistance and the failure of β-cells to produce and secrete enough insulin as the disease progresses. However, clinical treatments based solely on insulin secretion and action have had limited success. The focus is therefore shifting towards α-cells, in particular to the dysregulated secretion of glucagon. Our qualitative electron-microscopy-based observations gave an indication that mitochondria in α-cells are altered in Western-diet-induced T2DM. In particular, α-cells extracted from mouse pancreatic tissue showed a lower density of mitochondria, a less expressed matrix and a lower number of cristae. These deformities in mitochondrial ultrastructure imply a decreased efficiency in mitochondrial ATP production, which prompted us to theoretically explore and clarify one of the most challenging problems associated with T2DM, namely the lack of glucagon secretion in hypoglycaemia and its oversecretion at high blood glucose concentrations. To this purpose, we constructed a novel computational model that links α-cell metabolism with their electrical activity and glucagon secretion. Our results show that defective mitochondrial metabolism in α-cells can account for dysregulated glucagon secretion in T2DM, thus improving our understanding of T2DM pathophysiology and indicating possibilities for new clinical treatments., Competing Interests: M.P. was a member of the Royal Society Open Science Editorial Board at the time of submission and publication of this paper, but was not involved in the assessment of the manuscript., (© 2020 The Authors.)
- Published
- 2020
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31. A Novel in situ Approach to Studying Pancreatic Ducts in Mice.
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Gál E, Dolenšek J, Stožer A, Pohorec V, Ébert A, and Venglovecz V
- Abstract
Introduction: The tissue slice technique offers several benefits compared to isolated cells and cell clusters that help us understand the (patho)physiology of several organs in situ . The most prominent features are preserved architecture and function, with intact homotypic and heterotypic interactions between cells in slices. In the pancreas, this technique has been utilized successfully to study acinar and endocrine islet cells. However, it has never been used to investigate ductal function. Since pancreatic ductal epithelial cells (PDECs) play an essential role in the physiology of the pancreas, our aim was to use this technique to study PDEC structure and function in situ . Materials and methods: Eight- to sixteen weeks old C57BL/6 mice were used for preparation of pancreas tissue slices. Low melting point agarose was injected into the common bile duct and the whole organ was extracted. For morphological studies, pieces of tissue were embedded in agarose and cryosectioned to obtain 15 μm thick slices. In order to visualize pancreatic ducts, (i) the Giemsa dye was added to the agarose and visualized using light microscopy or (ii) immunostaining for the cystic fibrosis transmembrane conductance regulator (CFTR) was performed. For functional characterization, agarose-embedded tissue was immediately cut to 140 μm thick tissue slices that were loaded with the cell permeant form of the Oregon Green 488 BAPTA-1 dye and used for confocal calcium imaging. Results: Giemsa staining has shown that the injected agarose reaches the head and body of the pancreas to a greater extent than the tail, without disrupting the tissue architecture. Strong CFTR expression was detected at the apical membranes of PDECs and acinar cells, whereas islet cells were completely negative for CFTR. Stimulation with chenodeoxycholic acid (CDCA, 1 mM) resulted in a robust transient increase in intracellular calcium concentration that was readily visible in >40 ductal cells per slice. Conclusion: Our results confirm that the acutely-isolated pancreas tissue slice technique is suitable for structural and functional investigation of PDECs and their relationship with other cell types, such as acini and endocrine cells in situ . In combination with different genetic, pharmacological or dietary approaches it could become a method of choice in the foreseeable future.
- Published
- 2019
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32. Heterogeneity and Delayed Activation as Hallmarks of Self-Organization and Criticality in Excitable Tissue.
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Stožer A, Markovič R, Dolenšek J, Perc M, Marhl M, Slak Rupnik M, and Gosak M
- Abstract
Self-organized critical dynamics is assumed to be an attractive mode of functioning for several real-life systems and entails an emergent activity in which the extent of observables follows a power-law distribution. The hallmarks of criticality have recently been observed in a plethora of biological systems, including beta cell populations within pancreatic islets of Langerhans. In the present study, we systematically explored the mechanisms that drive the critical and supercritical behavior in networks of coupled beta cells under different circumstances by means of experimental and computational approaches. Experimentally, we employed high-speed functional multicellular calcium imaging of fluorescently labeled acute mouse pancreas tissue slices to record calcium signals in a large number of beta cells simultaneously, and with a high spatiotemporal resolution. Our experimental results revealed that the cellular responses to stimulation with glucose are biphasic and glucose-dependent. Under physiological as well as under supraphysiological levels of stimulation, an initial activation phase was followed by a supercritical plateau phase with a high number of global intercellular calcium waves. However, the activation phase displayed fingerprints of critical behavior under lower stimulation levels, with a progressive recruitment of cells and a power-law distribution of calcium wave sizes. On the other hand, the activation phase provoked by pathophysiologically high glucose concentrations, differed considerably and was more rapid, less continuous, and supercritical. To gain a deeper insight into the experimentally observed complex dynamical patterns, we built up a phenomenological model of coupled excitable cells and explored empirically the model's necessities that ensured a good overlap between computational and experimental results. It turned out that such a good agreement between experimental and computational findings was attained when both heterogeneous and stimulus-dependent time lags, variability in excitability levels, as well as a heterogeneous cell-cell coupling were included into the model. Most importantly, since our phenomenological approach involved only a few parameters, it naturally lends itself not only for determining key mechanisms of self-organized criticality at the tissue level, but also points out various features for comprehensive and realistic modeling of different excitable systems in nature.
- Published
- 2019
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33. Beta Cell Functional Adaptation and Dysfunction in Insulin Resistance and the Role of Chronic Kidney Disease.
- Author
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Stožer A, Hojs R, and Dolenšek J
- Subjects
- Animals, Calcium metabolism, Humans, Renal Insufficiency, Chronic physiopathology, Adaptation, Physiological physiology, Insulin Resistance, Insulin-Secreting Cells physiology, Renal Insufficiency, Chronic etiology
- Abstract
Background: Beta cells are central in the pathophysiology of diabetes, since their functional adaptation maintains euglycemia in insulin-resistant individuals and beta cell dysfunction is required for the clinical picture of frank diabetes. The pathophysiological mechanisms driving compensation and decompensation are incompletely understood and little is known about the influence of chronic kidney disease (CKD) on beta cell function., Summary: In compensated insulin resistance, beta cells enhance their function at all stages in the stimulus-secretion coupling cascade, from the most proximal membrane depolarization to the intermediate increase in intracellular calcium concentration and the most distal granule fusion. Intercellular coupling is not disrupted at this early stage during disease progression. Later during progression, when hyperglycemia becomes more apparent owing to insufficient beta cell compensation, intracellular stimulus-secretion coupling becomes enhanced to an even larger degree, but intercellular coupling becomes disrupted, indicating that ineffective cell-to-cell signal transmission may be the earliest event in progression to frank diabetes. CKD can negatively affect beta cell function through increased levels of urea that reduce beta cell glucose utilization and impair insulin secretion, and possibly also through factors other than urea. It remains to be investigated whether urea and other factors of CKD can also affect intercellular coupling. Key messages: Beta cells enhance intracellular stimulus-secretion coupling in early insulin resistance. With worsening insulin resistance, intracellular coupling enhances even more, but intercellular coupling becomes disrupted. CKD negatively impacts beta cell function, but its influence on intercellular coupling awaits further studies., (© 2019 S. Karger AG, Basel.)
- Published
- 2019
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34. Loosening the shackles of scientific disciplines with network science: Reply to comments on "Network science of biological systems at different scales: A review".
- Author
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Gosak M, Markovič R, Dolenšek J, Rupnik MS, Marhl M, Stožer A, and Perc M
- Subjects
- Biological Science Disciplines, Signal Transduction
- Published
- 2018
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35. Network science of biological systems at different scales: A review.
- Author
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Gosak M, Markovič R, Dolenšek J, Slak Rupnik M, Marhl M, Stožer A, and Perc M
- Subjects
- Brain cytology, Brain physiology, Humans, Islets of Langerhans cytology, Islets of Langerhans physiology, Nerve Net cytology, Nerve Net physiology, Models, Biological
- Abstract
Network science is today established as a backbone for description of structure and function of various physical, chemical, biological, technological, and social systems. Here we review recent advances in the study of complex biological systems that were inspired and enabled by methods of network science. First, we present research highlights ranging from determination of the molecular interaction network within a cell to studies of architectural and functional properties of brain networks and biological transportation networks. Second, we focus on synergies between network science and data analysis, which enable us to determine functional connectivity patterns in multicellular systems. Until now, this intermediate scale of biological organization received the least attention from the network perspective. As an example, we review the methodology for the extraction of functional beta cell networks in pancreatic islets of Langerhans by means of advanced imaging techniques. Third, we concentrate on the emerging field of multilayer networks and review the first endeavors and novel perspectives offered by this framework in exploring biological complexity. We conclude by outlining challenges and directions for future research that encompass utilization of the multilayer network formalism in exploring intercellular communication patterns in tissues, and we advocate for network science being one of the key pillars for assessing physiological function of complex biological systems-from organelles to organs-in health and disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2018
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36. Critical and Supercritical Spatiotemporal Calcium Dynamics in Beta Cells.
- Author
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Gosak M, Stožer A, Markovič R, Dolenšek J, Perc M, Rupnik MS, and Marhl M
- Abstract
A coordinated functioning of beta cells within pancreatic islets is mediated by oscillatory membrane depolarization and subsequent changes in cytoplasmic calcium concentration. While gap junctions allow for intraislet information exchange, beta cells within islets form complex syncytia that are intrinsically nonlinear and highly heterogeneous. To study spatiotemporal calcium dynamics within these syncytia, we make use of computational modeling and confocal high-speed functional multicellular imaging. We show that model predictions are in good agreement with experimental data, especially if a high degree of heterogeneity in the intercellular coupling term is assumed. In particular, during the first few minutes after stimulation, the probability distribution of calcium wave sizes is characterized by a power law, thus indicating critical behavior. After this period, the dynamics changes qualitatively such that the number of global intercellular calcium events increases to the point where the behavior becomes supercritical. To better mimic normal in vivo conditions, we compare the described behavior during supraphysiological non-oscillatory stimulation with the behavior during exposure to a slightly lower and oscillatory glucose challenge. In the case of this protocol, we observe only critical behavior in both experiment and model. Our results indicate that the loss of oscillatory changes, along with the rise in plasma glucose observed in diabetes, could be associated with a switch to supercritical calcium dynamics and loss of beta cell functionality.
- Published
- 2017
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37. The triggering pathway to insulin secretion: Functional similarities and differences between the human and the mouse β cells and their translational relevance.
- Author
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Skelin Klemen M, Dolenšek J, Slak Rupnik M, and Stožer A
- Subjects
- Animals, Diabetes Mellitus, Type 2 drug therapy, Diabetes Mellitus, Type 2 metabolism, Diabetes Mellitus, Type 2 physiopathology, Humans, Hypoglycemic Agents pharmacology, Hypoglycemic Agents therapeutic use, Insulin Secretion, Insulin-Secreting Cells drug effects, Mice, Secretory Vesicles drug effects, Species Specificity, Translational Research, Biomedical methods, Calcium Signaling drug effects, Insulin metabolism, Insulin-Secreting Cells metabolism, Models, Biological, Secretory Pathway drug effects, Secretory Vesicles physiology
- Abstract
In β cells, stimulation by metabolic, hormonal, neuronal, and pharmacological factors is coupled to secretion of insulin through different intracellular signaling pathways. Our knowledge about the molecular machinery supporting these pathways and the patterns of signals it generates comes mostly from rodent models, especially the laboratory mouse. The increased availability of human islets for research during the last few decades has yielded new insights into the specifics in signaling pathways leading to insulin secretion in humans. In this review, we follow the most central triggering pathway to insulin secretion from its very beginning when glucose enters the β cell to the calcium oscillations it produces to trigger fusion of insulin containing granules with the plasma membrane. Along the way, we describe the crucial building blocks that contribute to the flow of information and focus on their functional role in mice and humans and on their translational implications.
- Published
- 2017
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38. Complete occipitalization of the atlas with bilateral external auditory canal atresia.
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Dolenšek J, Cvetko E, Snoj Ž, and Meznaric M
- Subjects
- Abnormalities, Multiple, Cadaver, Female, Humans, Middle Aged, Cervical Atlas abnormalities, Congenital Abnormalities, Ear abnormalities, Occipital Bone abnormalities
- Abstract
Fusion of the atlas with the occipital bone is a rare congenital dysplasia known as occipitalization of the atlas, occipitocervical synostosis, assimilation of the atlas, or atlanto-occipital fusion. It is a component of the paraxial mesodermal maldevelopment and commonly associated with other dysplasias of the craniovertebral junction. External auditory canal atresia or external aural atresia is a rare congenital absence of the external auditory canal. It occurs as the consequence of the maldevelopment of the first pharyngeal cleft due to defects of cranial neural crest cells migration and/or differentiation. It is commonly associated with the dysplasias of the structures derived from the first and second pharyngeal arches including microtia. We present the coexistence of the occipitalization of the atlas and congenital aural atresia, an uncommon combination of the paraxial mesodermal maldevelopment, and defects of cranial neural crest cells. The association is most probably syndromic as minimal diagnostic criteria for the oculoariculovertebral spectrum are fulfilled. From the clinical point of view, it is important to be aware that patients with microtia must obtain also appropriate diagnostic imaging studies of the craniovetebral junction due to eventual concomitant occipitalization of the atlas and frequently associated C1-C2 instability.
- Published
- 2017
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39. Membrane Potential and Calcium Dynamics in Beta Cells from Mouse Pancreas Tissue Slices: Theory, Experimentation, and Analysis.
- Author
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Dolenšek J, Špelič D, Klemen MS, Žalik B, Gosak M, Rupnik MS, and Stožer A
- Subjects
- Animals, Islets of Langerhans physiology, Mice, Models, Biological, Optical Imaging, Calcium metabolism, Islets of Langerhans cytology, Membrane Potentials physiology
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
40. The relationship between node degree and dissipation rate in networks of diffusively coupled oscillators and its significance for pancreatic beta cells.
- Author
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Gosak M, Stožer A, Markovič R, Dolenšek J, Marhl M, Rupnik MS, and Perc M
- Subjects
- Animals, Cells, Cultured, Computer Simulation, Diffusion, Energy Transfer physiology, Mice, Biological Clocks physiology, Calcium Signaling physiology, Cell Communication physiology, Insulin-Secreting Cells physiology, Models, Biological, Nonlinear Dynamics
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
41. Progressive glucose stimulation of islet beta cells reveals a transition from segregated to integrated modular functional connectivity patterns.
- Author
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Markovič R, Stožer A, Gosak M, Dolenšek J, Marhl M, and Rupnik MS
- Subjects
- Algorithms, Animals, Calcium metabolism, Female, Insulin-Secreting Cells cytology, Insulin-Secreting Cells metabolism, Male, Metabolic Networks and Pathways, Mice, Microscopy, Confocal, Glucose pharmacology, Insulin-Secreting Cells drug effects
- Abstract
Collective beta cell activity in islets of Langerhans is critical for the supply of insulin within an organism. Even though individual beta cells are intrinsically heterogeneous, the presence of intercellular coupling mechanisms ensures coordinated activity and a well-regulated exocytosis of insulin. In order to get a detailed insight into the functional organization of the syncytium, we applied advanced analytical tools from the realm of complex network theory to uncover the functional connectivity pattern among cells composing the intact islet. The procedure is based on the determination of correlations between long temporal traces obtained from confocal functional multicellular calcium imaging of beta cells stimulated in a stepwise manner with a range of physiological glucose concentrations. Our results revealed that the extracted connectivity networks are sparse for low glucose concentrations, whereas for higher stimulatory levels they become more densely connected. Most importantly, for all ranges of glucose concentration beta cells within the islets form locally clustered functional sub-compartments, thereby indicating that their collective activity profiles exhibit a modular nature. Moreover, we show that the observed non-linear functional relationship between different network metrics and glucose concentration represents a well-balanced setup that parallels physiological insulin release.
- Published
- 2015
- Full Text
- View/download PDF
42. Structural similarities and differences between the human and the mouse pancreas.
- Author
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Dolenšek J, Rupnik MS, and Stožer A
- Subjects
- Animals, Disease Models, Animal, Humans, Insulin-Secreting Cells metabolism, Islets of Langerhans anatomy & histology, Islets of Langerhans metabolism, Mice, Pancreas blood supply, Pancreas innervation, Pancreas anatomy & histology, Pancreas metabolism
- Abstract
Mice remain the most studied animal model in pancreas research. Since the findings of this research are typically extrapolated to humans, it is important to understand both similarities and differences between the 2 species. Beside the apparent difference in size and macroscopic organization of the organ in the 2 species, there are a number of less evident and only recently described differences in organization of the acinar and ductal exocrine tissue, as well as in the distribution, composition, and architecture of the endocrine islets of Langerhans. Furthermore, the differences in arterial, venous, and lymphatic vessels, as well as innervation are potentially important. In this article, the structure of the human and the mouse pancreas, together with the similarities and differences between them are reviewed in detail in the light of conceivable repercussions for basic research and clinical application.
- Published
- 2015
- Full Text
- View/download PDF
43. The relationship between membrane potential and calcium dynamics in glucose-stimulated beta cell syncytium in acute mouse pancreas tissue slices.
- Author
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Dolenšek J, Stožer A, Skelin Klemen M, Miller EW, and Slak Rupnik M
- Subjects
- Animals, Giant Cells metabolism, Glucose pharmacology, Insulin-Secreting Cells drug effects, Islets of Langerhans physiology, Mice, Microscopy, Confocal, Patch-Clamp Techniques, Tetraethylammonium pharmacology, Calcium metabolism, Calcium Signaling drug effects, Glucose metabolism, Insulin-Secreting Cells metabolism, Membrane Potentials physiology
- Abstract
Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca(2+)]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca(2+)]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca(2+)]i oscillations. Here, we aimed to correlate the plateau [Ca(2+)]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca(2+)]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca(2+)]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca(2+)]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca(2+)]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.
- Published
- 2013
- Full Text
- View/download PDF
44. Glucose-stimulated calcium dynamics in islets of Langerhans in acute mouse pancreas tissue slices.
- Author
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Stožer A, Dolenšek J, and Rupnik MS
- Subjects
- Animals, Female, In Vitro Techniques, Islets of Langerhans metabolism, Kinetics, Male, Mice, Calcium metabolism, Glucose pharmacology, Islets of Langerhans drug effects
- Abstract
In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous in vitro studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings in vivo. The main controversies regard the types of calcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the in situ acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidence that even a large amplitude calcium signal was not sufficient and that metabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future.
- Published
- 2013
- Full Text
- View/download PDF
45. Functional connectivity in islets of Langerhans from mouse pancreas tissue slices.
- Author
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Stožer A, Gosak M, Dolenšek J, Perc M, Marhl M, Rupnik MS, and Korošak D
- Subjects
- Animals, Cluster Analysis, Histocytochemistry, Mice, Microscopy, Confocal, Models, Biological, Systems Theory, Calcium analysis, Calcium metabolism, Calcium Signaling physiology, Computational Biology methods, Islets of Langerhans cytology, Islets of Langerhans metabolism
- Abstract
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.
- Published
- 2013
- Full Text
- View/download PDF
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