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Abstract: Calcium signals in pancreatic � cell collectives show a sharp transition from uncorrelated to correlated state resembling a phase transition as the slowly increasing glucose concentration crosses the tipping point. However, the exact nature or the order of this phase transition is not well understood. Using confocal microscopy to record the collective calcium activation of � cells in an intact islet under changing glucose concentration in an increasing and then decreasing way, we first show that in, addition to the sharp transition, the coordinated calcium response exhibits a hysteresis indicating a critical, first-order transition. A network model of � cells combining link selection and coordination mechanisms capture the observed hysteresis loop and the critical nature of the transition. Our results point towards an understanding of the role of islets as tipping elements in the pancreas that, interconnected by perfusion, diffusion, and innervation, cause the tipping dynamics and abrupt insulin release.
Keywords: cellular organization, physiology & dynamics, phase transitions in biological systems, complex networks, endocrine system, optical microscopy
Published in DKUM: 19.03.2025; Views: 0; Downloads: 2
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Abstract: Many details of glucose-stimulated intracellular calcium changes in [beta] 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 [beta] cell heterogeneity and an important baseline for future studies of stimulus-secretion and intercellular coupling. NEW & NOTEWORTHY: We assessed concentration-dependence in coupled [beta] 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.
Keywords: beta cells, calcium imaging, glucose-dependence, network analysis
Published in DKUM: 15.10.2024; Views: 0; Downloads: 18
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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: 155; Downloads: 14
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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: 193; Downloads: 14
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Abstract: Celjenje roženičnih ran je pogosto omejeno s fibrozo in tvorjenjem brazgotin, ki jih lahko povzroča transformni rastni faktor β (angl. transforming growth factor – TGF). Nadzirana fibroza, ki jo lahko usmerimo z inzulinu podobnim rastnim faktorjem – 1 (angl. insulin-like growth factor – IGF) in protifibrotičnimi učinkovinami, bi lahko prispevala k ohranjanju prozornosti roženice med celjenjem. S pomočjo stimulacije primarnih človeških keratocitov s TGF-β v brezserumskem gojitvenem mediju smo ustvarili celični model roženične stromalne rane. S slikovno pretočno citometrijo smo analizirali posamezne celice iz celičnih kultur in določali stopnjo nuklearizacije Smad3 in znotrajcelično fluorescenčno intenziteto obarvanega Smad7 in roženičnega Kristalina – aldehidne dehidrogenaze 3A1. Pri preučevanju izločanja proteoglikanov Biglikana in Keratokana v zunajcelični matriks smo uporabili teste ELISA. Skupaj s stimulacijo s TGF-β smo celice obravnavali samo z IGF-1, s suberoilanilidehidroksiamično kislino (SAHA) ali halouginonom; ločenim populacijam smo poleg protifibrotikov dodali še IGF-1. Pri samostojni obravnavi z IGF-1 smo ugotovili zmanjšano translokacijo Smad3 in zvišano količino Aldehidne dehidrogenaze 3A1 znotraj celic. Poleg tega je bilo izločanje proteoglikanov prav tako ugodno za ustvarjanje pogojev prozornosti. SAHA je povzročila zvišanje Smad7 v celicah in inhibirala translokacijo Smad3 v jedra – tudi v kombinaciji z IGF-1. Imunofluorescenčna mikroskopija je pokazala, da je dodatek IGF-1 in v kombinaciji s protifibrotičnimi učinkovinami zavrl transdiferenciacijo v miofibroblaste in spodbudil nastanek fibroblastov. TGF-β/ Smad signalna pot fibroze in zamotnjenosti roženice je bila inhibirana s strani IGF-1; še posebej ob dodatku SAHA kakor tudi s halofuginonom. Zaključujemo, da bi lahko IGF-1 uspešno dodali k zdravljenju s protifibrotičnimi učinkovinami, kar bi omogočilo boljše celjenje roženične rane in tvorbo bolj prozornega tkiva.
Keywords: Roženica, keratocit, miofibroblast, fibroza, celjenje ran, TGF-b, IGF-1, SAHA, halofuginon, Smad, celična kultura, slikovna pretočna citometrija
Published in DKUM: 14.01.2019; Views: 1884; Downloads: 246
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