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Glucose-stimulated calcium dynamics in Islets of Langerhans in acute mouse pancreas tissue slices
Andraž Stožer, Jurij Dolenšek, Marjan Rupnik, 2013, izvirni znanstveni članek

Opis: 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 ofcalcium 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 evidencethat even a large amplitude calcium signal was not sufficient and thatmetabolic 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.
Ključne besede: glucose, pancreas, islets of Langerhans, mice
Objavljeno: 10.07.2015; Ogledov: 619; Prenosov: 238
.pdf Celotno besedilo (2,83 MB)
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Intracellular serotonin modulates insulin secretion from pancreatic ß-cells by protein serotonylation
Nils Paulmann, Maik Grohmann, Jörg-Peter Voigt, Bettina Bert, Jakob Vowinckel, Michael Bader, Maša Skelin, Marko Jevšek, Heidrun Fink, Marjan Rupnik, Diego Walther, 2009, izvirni znanstveni članek

Opis: While serotonin (5-HT) co-localization with insulin in granules of pancreatic ß-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is stili unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1-/-) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1-/- ß-cells, which clearly showed that the secretory defect is downstream of Ca2+ -signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in ß-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic ß-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.
Ključne besede: insulin secretion, serotonin, insulin, glucose, diabetes mellitus, guanosine triphosphatase, exocytosis, pancreas
Objavljeno: 16.06.2017; Ogledov: 582; Prenosov: 58
.pdf Celotno besedilo (774,75 KB)
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The relationship between membrane potential and calcium dynamics in glucose-stimulated beta cell syncytium in acute mouse pancreas tissue slices
Jurij Dolenšek, Andraž Stožer, Maša Skelin, Evan Miller, Marjan Rupnik, 2013, izvirni znanstveni članek

Opis: 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 ([Ca2+]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 [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]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 [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]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 [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.
Ključne besede: glucose, pancreas, mice
Objavljeno: 19.06.2017; Ogledov: 491; Prenosov: 211
.pdf Celotno besedilo (2,57 MB)
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Magnetic field effects on redox potential of reduction and oxidation agents
Mojca Božič, Lucija Črepinšek-Lipuš, Vanja Kokol, 2008, izvirni znanstveni članek

Opis: Redox potentials of two reducing (sodium dithionite and glucose) and two oxidizing (hydrogen peroxide and sodium hypochlorite) agents were monitored at various concentrations and at different temperatures for 30-75 minutes after the exposure of their water solutions (glucose and hypochlorite solutions once; sodium dithionite and hydrogen peroxide solutions one, two and/or three-times) to the static magnetic field of flux density of 0.9 V s M-2 . The aim of the investigation was to suggest improvements, i.e., intensification and stability, of the reduction-oxidation ability of selected agents applicable in textile fibre processing, primarily bleaching and vat dyeing. Results of the experiments show that magnetic treatment (of solutions) raises both the reducing ability of glucose and the oxidation ability of hydrogen peroxide and sodium hypochlorite, promising some technological and economical benefits for the textile industry as well as forother fields of chemistry.
Ključne besede: magnetic water treatment, sodium dithionite, glucose, hydrogen peroxide, sodium hypochlorite, redox potential, textile vat processing
Objavljeno: 05.07.2017; Ogledov: 352; Prenosov: 54
.pdf Celotno besedilo (206,88 KB)
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Critical and supercritical spatiotemporal calcium dynamics in beta cells
Marko Gosak, Andraž Stožer, Rene Markovič, Jurij Dolenšek, Matjaž Perc, Marjan Rupnik, Marko Marhl, 2017, izvirni znanstveni članek

Opis: 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.
Ključne besede: beta cells, islets of Langerhans, self-organized criticality, intercellular dynamics, calcium waves, glucose oscillations, computational model, confocal calcium imaging
Objavljeno: 23.01.2018; Ogledov: 537; Prenosov: 233
.pdf Celotno besedilo (3,43 MB)
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