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Physiological levels of adrenaline fail to stop pancreatic beta cell activity at unphysiologically high glucose levels
Nastja Sluga, Lidija Križančić Bombek, Jasmina Kerčmar, Srdjan Sarikas, Sandra Postić, Johannes Pfabe, Maša Skelin, Dean Korošak, Andraž Stožer, Marjan Rupnik, 2022, original scientific article

Abstract: Adrenaline inhibits insulin secretion from pancreatic beta cells to allow an organism to cover immediate energy needs by unlocking internal nutrient reserves. The stimulation of α2-adrenergic receptors on the plasma membrane of beta cells reduces their excitability and insulin secretion mostly through diminished cAMP production and downstream desensitization of late step(s) of exocytotic machinery to cytosolic Ca2+ concentration ([Ca2+]c). In most studies unphysiologically high adrenaline concentrations have been used to evaluate the role of adrenergic stimulation in pancreatic endocrine cells. Here we report the effect of physiological adrenaline levels on [Ca2+]c dynamics in beta cell collectives in mice pancreatic tissue slice preparation. We used confocal microscopy with a high spatial and temporal resolution to evaluate glucose-stimulated [Ca2+]c events and their sensitivity to adrenaline. We investigated glucose concentrations from 8-20 mM to assess the concentration of adrenaline that completely abolishes [Ca2+]c events. We show that 8 mM glucose stimulation of beta cell collectives is readily inhibited by the concentration of adrenaline available under physiological conditions, and that sequent stimulation with 12 mM glucose or forskolin in high nM range overrides this inhibition. Accordingly, 12 mM glucose stimulation required at least an order of magnitude higher adrenaline concentration above the physiological level to inhibit the activity. To conclude, higher glucose concentrations stimulate beta cell activity in a non-linear manner and beyond levels that could be inhibited with physiologically available plasma adrenaline concentration.
Keywords: adrenaline, islets, beta cells, cAMP, concentration dependency, [Ca2+]c oscillations, forskolin
Published in DKUM: 04.07.2024; Views: 73; Downloads: 3
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pH-dependence of glucose- dependent activity of beta cell networks in acute mouse pancreatic tissue slice
Sandra Postić, Marko Gosak, Wen-Hao Tsai, Johannes Pfabe, Srdjan Sarikas, Andraž Stožer, Dean Korošak, Shi-Bing Yang, Marjan Rupnik, 2022, original scientific article

Abstract: Extracellular pH has the potential to affect various aspects of the pancreatic beta cell function. To explain this effect, a number of mechanisms was proposed involving both extracellular and intracellular targets and pathways. Here, we focus on reassessing the influence of extracellular pH on glucose-dependent beta cell activation and collective activity in physiological conditions. To this end we employed mouse pancreatic tissue slices to perform high-temporally resolved functional imaging of cytosolic Ca2+ oscillations. We investigated the effect of either physiological H+ excess or depletion on the activation properties as well as on the collective activity of beta cell in an islet. Our results indicate that lowered pH invokes activation of a subset of beta cells in substimulatory glucose concentrations, enhances the average activity of beta cells, and alters the beta cell network properties in an islet. The enhanced average activity of beta cells was determined indirectly utilizing cytosolic Ca2+ imaging, while direct measuring of insulin secretion confirmed that this enhanced activity is accompanied by a higher insulin release. Furthermore, reduced functional connectivity and higher functional segregation at lower pH, both signs of a reduced intercellular communication, do not necessary result in an impaired insulin release.
Keywords: insulin secretion, membrane excitability, potassium channels, beta cell network, collective activity, calcium waves, pancreatic islets, pH-dependence
Published in DKUM: 01.07.2024; Views: 63; Downloads: 2
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Assessing different temporal scales of calcium dynamics in networks of beta cell populations
Jan Zmazek, Maša Skelin, Rene Markovič, Jurij Dolenšek, Marko Marhl, Andraž Stožer, Marko Gosak, 2021, original scientific article

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 Ca2+ imaging in fresh mouse pancreas tissue slices with time series analysis and network science approaches to unveil the glucosedependent 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.
Keywords: islets of Langerhans, beta cell network, calcium oscillations, multimodal activity analysis, confocal imaging, functional connectivity, multiplex network
Published in DKUM: 06.06.2024; Views: 104; Downloads: 4
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The effect of forskolin and the role of Epac2A during activation, activity, and deactivation of beta cell networks
Maša Skelin, Jurij Dolenšek, Lidija Križančić Bombek, Viljem Pohorec, Marko Gosak, Marjan Rupnik, Andraž Stožer, 2023, original scientific article

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.
Keywords: pancreas, tissue slices, beta cells, calcium imaging, amplifying pathway, forskolin, Epac2A KO, intercellular network
Published in DKUM: 27.05.2024; Views: 148; Downloads: 6
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Uporaba večceličnih modelov in mrežnih analiz za preučevanje kolektivne celične dinamike v Langerhansovih otočkih : doktorska disertacija po skandinavskem modelu
Marko Šterk, 2024, doctoral dissertation

Abstract: V doktorski disertaciji smo se osredotočili na pereča vprašanja s področja fiziologije Langerhansovih otočkov, ki igrajo ključno vlogo pri uravnavanju glukozne homeostaze v telesu. Z uporabo inovativnih računalniških tehnik smo analizirali vpliv 3D citoarhitekture otočkov na večcelično dinamiko v opazovani 2D optični rezini, vpliv inhibicije NMDA receptorjev na kolektivno dinamiko celic beta, različne subpopulacije celic beta in njihov vpliv na kolektivno dinamiko ter vpliv metabolne sklopitve celic na kompleksnost funkcionalnih omrežij. Ugotovili smo, da je pri analizi medceličnih kalcijevih valov treba upoštevati 3D citoarhitekturo Langerhansovih otočkov, saj velik delež medceličnih kalcijevih valov izvira izven opazovane optične rezine, kar vpliva na opazovano hitrost širjenja valovne fronte. Nadalje je naša analiza pokazala, da inhibicija NMDA receptorjev blagodejno vpliva na delovanje posameznih celic beta kakor tudi na njihovo kolektivno dinamiko, kar se odraža v povečani stabilnosti poteka medceličnih kalcijevih valov. Podrobna analiza subpopulacij celic beta je razkrila, da centralne celice in celice, ki sprožajo valove, igrajo ključno vlogo pri širjenju medceličnih valov in sinhronosti celic, kar je izjemnega pomena za zagotavljanje normalne fiziološke funkcije. Naši izsledki kažejo, da ti dve vrsti celic predstavljata različni subpopulaciji, ki se med seboj ne prekrivata. Poleg tega s teoretičnim raziskovanjem mehanizmov sinhronizacije celic beta podrobneje prikazujemo, da je za koordinirano aktivnost celic beta dovolj samo električna sklopitev, vendar dodatna metabolna sklopitev še dodatno okrepi sinhrono delovanje in s tem vpliva na lastnosti pridobljenih funkcionalnih omrežij, zaradi česar so ta bolj podobna tistim, ki jih opazimo v eksperimentih. Z izvedenimi raziskavami smo prispevali k razumevanju, kako je vzpostavljeno koordinirano delovanje v kolektivih celic beta, kar odpira nove možnosti za razvoj zdravil za sladkorno bolezen tipa 2.
Keywords: celice beta, kompleksna omrežja, diabetes tipa 2, večcelični modeli, celična dinamika
Published in DKUM: 25.04.2024; Views: 223; Downloads: 20
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Human beta cell functional adaptation and dysfunction in insulin resistance and its reversibility
Maša Skelin, Jan Kopecky, Jurij Dolenšek, Andraž Stožer, 2023, other scientific articles

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.
Keywords: human beta cell, functional adaptation, dysfunction, insulin resistance
Published in DKUM: 15.04.2024; Views: 142; Downloads: 10
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Vpliv inkretinov in njihovih analogov na dinamiko kalcija v celicah beta v svežih rezinah trebušne slinavke
Eva Paradiž Leitgeb, 2024, doctoral dissertation

Abstract: Natančno uravnavanje sklopitve stimulacije in izločanja v celicah beta je ključnega pomena za vzdrževanje normoglikemije. Čeprav glukoza v tem procesu predstavlja primarni dražljaj, inkretini ključno prispevajo k povečanemu izločanju inzulina, delno tudi preko ojačitve znotrajcelične dinamike [Ca2+] ([Ca2+]IC). Podrobnosti o tem, kako inkretini vplivajo na rekrutacijo celic beta v fazi aktivacije, na aktivni čas in funkcionalno povezanost v fazi platoja in kako na njihovo deaktivacijo, še niso pojasnjene. S pomočjo konfokalne mikroskopije smo v svežih tkivnih rezinah trebušne slinavke z visoko resolucijo istočasno zajemali [Ca2+]IC signale v številnih celicah in tako sistematično proučili vpliv agonistov receptorjev GLP-1 (GLP-1RA), eksendina-4 (Ex-4) in GLP-1, na aktivnost sklopljenih celic beta. V nestimulativni koncentraciji glukoze (6 mM) je Ex-4 aktiviral približno četrtino celic beta v otočku. Kostimulacija z Ex-4 in glukozo (10 mM) je skrajšala zamik do aktivacije celic beta in pospešila dinamiko njihove aktivacije. Tako se je čas, ki je bil potreben, da so celice dosegle polovično vrednost maksimalnega aktivnega časa, v prisotnosti Ex-4 prepolovil. Aktivni čas in regularnost oscilacij [Ca2+]IC sta se povečala, zlasti v začetnem delu odziva celic beta. Kasnejše dodajanje Ex-4, ko so bile celice že aktivne, ni privedel do tako izrazitega porasta aktivnosti. Mrežne analize so potrdile povečano povezanost celic med fazo aktivacije in fazo platoja, pri čemer je vloga centralnih celic ostala stabilna tako v kontrolnih poskusih kot ob stimulaciji z Ex-4. Zanimivo je, da smo pri Ex-4 opazili dvojen učinek na deaktivacijo celic beta, tako da se je celična aktivnost pri nizkih koncentracijah podaljšala, pri visokih pa skrajšala. Naši poskusi so dosledno pokazali, da je GLP-1 šibkejši agonist. Kostimulacija z GLP-1RA in glukozo tako povzroči porast [Ca2+]IC med aktivacijo in aktivnostjo celic beta, kar kaže, da je učinek inkretinov v veliki meri mogoče pojasniti z ojačano dinamiko [Ca2+]IC. Predhodna stimulacija z inkretini ne privede do kritičnega podaljšanja aktivnosti celic beta, kar potrjuje njihovo nizko tveganje za razvoj hipoglikemije.
Keywords: celice beta, tkivna rezina, agonisti receptorjev GLP-1, konfokalna mikroskopija, kalcijeve oscilacije
Published in DKUM: 26.03.2024; Views: 166; Downloads: 21
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Novel in vitro model for toxicological analysis of drugs
Eneko Jose Madorran Esteiro, 2024, doctoral dissertation

Abstract: Despite there being a substantial range of available drugs on the market, many patients still lack effective and safe medications for their specific conditions, showing a constant need to develop new ones. Toxicological models enable us to assess their safety to various degrees, but all such models have certain limitations. Considering the advantages and drawbacks of these models, we have developed a novel viability method, Membrane Potential Cell Viability Assay (MPCVA), and a novel cell-based in vitro liver model to overcome some of the limitations of these models. The MPCVA evaluates the changes in the membrane potential, so we tested the membrane potential and cell viability relation with a cell-by-cell analysis. The MPCVA was further tested in case of cell exposure to various toxic agents that induce different cell death pathways. Finally, we used MPCVA to determine the viability of our novel cell-based in vitro liver model, which we built by combining four different types of liver cells (hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells). Along with the MPCVA, we used clinical instrumentation to test the toxicity of three hepatotoxic drugs (5-fluorouracil, ibuprofen, and rifampicin). We observed that, under the given conditions, the MPCVA had a similar toxicity assessment as standard viability methods. We also observed that the toxicity assessment of hepatotoxic drugs is similar to the in vivo toxicity evaluation (clinical data). Therefore, the suggested approach can potentially improve existing toxicological models. However, we understand its translation to clinical practice warrants further experimentation in exposures to other drugs/toxins and by also using other cell types.
Keywords: Keywords: Toxicological model, translational analysis, liver in vitro model, cell viability.
Published in DKUM: 23.02.2024; Views: 294; Downloads: 12
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