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1.
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, izvirni znanstveni članek

Opis: 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.
Ključne besede: adrenaline, islets, beta cells, cAMP, concentration dependency, [Ca2+]c oscillations, forskolin
Objavljeno v DKUM: 04.07.2024; Ogledov: 81; Prenosov: 3
.pdf Celotno besedilo (6,21 MB)
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2.
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, izvirni znanstveni članek

Opis: 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.
Ključne besede: insulin secretion, membrane excitability, potassium channels, beta cell network, collective activity, calcium waves, pancreatic islets, pH-dependence
Objavljeno v DKUM: 01.07.2024; Ogledov: 71; Prenosov: 2
.pdf Celotno besedilo (7,45 MB)
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3.
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, izvirni znanstveni članek

Opis: 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.
Ključne besede: pancreas, tissue slices, beta cells, calcium imaging, amplifying pathway, forskolin, Epac2A KO, intercellular network
Objavljeno v DKUM: 27.05.2024; Ogledov: 149; Prenosov: 6
.pdf Celotno besedilo (12,03 MB)
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Vpliv rastnih faktorjev in zaviralcev fibrotičnega tipa celjenja na keratocite v celičnih modelih roženične rane
Tomislav Šarenac, 2018, doktorsko delo/naloga

Opis: 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.
Ključne besede: Roženica, keratocit, miofibroblast, fibroza, celjenje ran, TGF-b, IGF-1, SAHA, halofuginon, Smad, celična kultura, slikovna pretočna citometrija
Objavljeno v DKUM: 14.01.2019; Ogledov: 1813; Prenosov: 233
.pdf Celotno besedilo (69,22 MB)

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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 v DKUM: 23.01.2018; Ogledov: 1665; Prenosov: 383
.pdf Celotno besedilo (3,43 MB)
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8.
Nove metode za ocenjevanje tveganja za razvoj metaboličnega sindroma pri otrocih
Bernarda Vogrin, 2017, doktorsko delo/naloga

Opis: Uvod. Zaradi epidemije otroške debelosti in metaboličnega sindroma lahko v prihodnosti pri otrocih pričakujemo še večje obremenitve srca in žil, čemur smo priča danes. Zgodnje odkrivanje oseb, ki so nagnjene k metaboličnim motnjam in boleznim srca in žil, lahko bistveno pripomore k načrtovanju preventive in zdravljenju bolezni. Hipoteza. Z merjenjem žilne funkcije, telesne sestave in natančno analizo serumskih lipidov lahko odkrijemo nagnjenost k metaboličnemu sindromu in boleznim srca in žil že v otroštvu, še pred pojavom kliničnih znakov. Metode. V študiji je sodelovalo 81 šolarjev starih 11–16 let. Z anketnim vprašalnikom smo pridobili podatke o njihovem socialnem okolju, prehranskih navadah in učnem uspehu. Z analizo športno vzgojnih kartonov smo ocenili njihovo telesno zmogljivost. Opravili smo meritve telesne konstitucije: telesno višino (TV), telesno težo (TT), obseg pasu (OP) in bokov (OB), kožno gubo na nadlahti (KGN) ter izračunali indeks telesne mase (BMI) in standardni odklon glede na spol in starost (SDS BMI). Telesno sestavo (odstotek maščobe (FAT %)) smo ocenjevali z bioimpedanco. Lastnosti arterijskega sistema smo merili z Arteriografom, z določanjem augmentacijskega indeksa (AIx), hitrosti pulznih valov (PWV) in centralnega sistoličnega arterijskega tlaka (SBPao). V vzorcu venozne krvi smo analizirali glukozo (GLU) in serumske lipide vključno s prostimi maščobnimi kislinami (PMK). Rezultati. AIx je značilno koreliral s TV, učnim uspehom, nekaterimi motoričnimi testi in serumskimi lipidi. PWV je značilno koreliral s TT, TV, BMI, SDS BMI, OP, OB, nekaterimi motoričnimi testi in PMK. SBPao je negativno koreliral z učnim uspehom. FAT % je kazal značilne povezave z GLU, PMK in drugimi lipidi. Pri 5 % oziroma 6 % učencev smo ugotovili zelo visoke vrednosti AIx, SBPao in PWV. Zaključki. AIx, PWV, PMK in FAT % so dobri pokazatelji začetnih žilnih in metaboličnih nepravilnosti pri otrocih in mladostnikih. Zvečana AIx in SBPao sta povezana s slabšimi učnimi in motoričnimi sposobnostmi. PWV je pri otrocih in mladostnikih zelo odvisen od telesne konstitucije.
Ključne besede: Metabolični sindrom, augmentacijski indeks, hitrost pulznih valov, otroci, bioimpedanca
Objavljeno v DKUM: 29.11.2017; Ogledov: 1427; Prenosov: 132
.pdf Celotno besedilo (1,91 MB)

9.
Tkivni inženiring hrustančnega tkiva na biosintetičnem polimernem polyHIPE nosilcu
Jakob Naranđa, 2017, doktorsko delo/naloga

Opis: Tkivni inženiring hrustančnega tkiva še vedno nudi številne možnosti za izboljšavo, navkljub intenzivnim raziskovalnim naporom v zadnjem času. Razvoj umetnih materialov in 3-D celičnih nosilcev ima pomembno vlogo pri regeneraciji hrustančnega tkiva. Zanimiv pristop pri izdelavi celičnih nosilcev predstavlja izgradnja s pomočjo emulzij. Nastali material, imenovan polyHIPE (PHP), je sintetični visoko porozen polimer, ki ga pripravimo s polimerizacijo visokega deleža notranje faze emulzij (HIPEs – high internal phase emulsions). Glavni cilj te doktorske disertacije je raziskati možnosti za tvorbo hrustančnega tkiva znotraj celičnih nosilcev pripravljenih iz PHP materiala. Proizvodnjo PHP nosilcev smo posebej prilagodili tkivnemu inženiringu hrustanca, tako da smo pripravili porozne (85 %) strukture s primarno velikostjo por v območju 50–170 m. Pokazali smo, da je PHP material biokompatibilen s človeškimi sklepnimi hondrociti, kar smo ovrednotili s pomočjo testa za preživetje celic (Live/Dead kit) in histološko analizo. Opazovali smo hondrocite z okroglimi jedri, ki so bili organizirani v večceličnih plasteh na površini PHP nosilca in so rastli približno 300 m v notranjost nosilca. Kopičenje kolagena tipa 2 smo dokazali s pomočjo imunohistokemije, molekularna analiza je pokazala izražanje hrustančno specifičnih genov z ugodnim razmerjem kolagena tipa 2 in tipa 1. Dodatno so bili PHP vzorci biološko razgradljivi, njihove osnovne mehanske lastnosti pa primerljive z nativnim sklepnim hrustancem. Izsledki raziskave dokazujejo, da je zasnovan PHP celični nosilec primeren za nadaljnjo uporabo v tkivnem inženiringu hrustančnega tkiva.
Ključne besede: humani sklepni hrustanec, tkivni inženiring, biokompatibilni celični nosilec, sintetični polimer, polyHIPE, diferenciacija hondrocitov
Objavljeno v DKUM: 17.10.2017; Ogledov: 2241; Prenosov: 270
.pdf Celotno besedilo (24,38 MB)

10.
SNAP-25b-deficiency increases insulin secretion and changes spatiotemporal profile of $Ca^{2+}$ oscillations in $\beta$ cell networks
Teresa Daraio, Lidija Križančić Bombek, Marko Gosak, Ismael Valladolid-Acebes, Maša Skelin, Essam Refai, Per-Olof Berggren, Kerstin Brismar, Marjan Rupnik, Christina Bark, 2017, izvirni znanstveni članek

Opis: SNAP-25 is a protein of the core SNARE complex mediating stimulus-dependent release of insulin from pancreatic $\beta$ cells. The protein exists as two alternatively spliced isoforms, SNAP-25a and SNAP-25b, differing in 9 out of 206 amino acids, yet their specific roles in pancreatic $\beta$ cells remain unclear. We explored the effect of SNAP-25b-deficiency on glucose-stimulated insulin release in islets and found increased secretion both in vivo and in vitro. However, slow photo-release of caged $Ca^{2+}$ in $\beta$ cells within pancreatic slices showed no significant differences in $Ca^{2+}$-sensitivity, amplitude or rate of exocytosis between SNAP-25b-deficient and wild-type littermates. Therefore, we next investigated if $Ca^{2+}$ handling was affected in glucose-stimulated [beta] cells using intracellular $Ca^{2+}$-imaging and found premature activation and delayed termination of [$Ca^{2+}$] i elevations. These findings were accompanied by less synchronized $Ca^{2+}$-oscillations and hence more segregated functional $\beta$ cell networks in SNAP-25b-deficient mice. Islet gross morphology and architecture were maintained in mutant mice, although sex specific compensatory changes were observed. Thus, our study proposes that SNAP-25b in pancreatic [beta] cells, except for participating in the core SNARE complex, is necessary for accurate regulation of $Ca^{2+}$-dynamics.
Ključne besede: insulin secretion, pre-diabetes
Objavljeno v DKUM: 23.08.2017; Ogledov: 1454; Prenosov: 203
.pdf Celotno besedilo (3,80 MB)
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