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1.
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: 101; Downloads: 2
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Vpliv rastnih faktorjev in zaviralcev fibrotičnega tipa celjenja na keratocite v celičnih modelih roženične rane
Tomislav Šarenac, 2018, doctoral dissertation

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: 1770; Downloads: 233
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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, original scientific article

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.
Keywords: beta cells, islets of Langerhans, self-organized criticality, intercellular dynamics, calcium waves, glucose oscillations, computational model, confocal calcium imaging
Published in DKUM: 23.01.2018; Views: 1637; Downloads: 380
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6.
Nove metode za ocenjevanje tveganja za razvoj metaboličnega sindroma pri otrocih
Bernarda Vogrin, 2017, doctoral dissertation

Abstract: 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.
Keywords: Metabolični sindrom, augmentacijski indeks, hitrost pulznih valov, otroci, bioimpedanca
Published in DKUM: 29.11.2017; Views: 1405; Downloads: 128
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7.
Tkivni inženiring hrustančnega tkiva na biosintetičnem polimernem polyHIPE nosilcu
Jakob Naranđa, 2017, doctoral dissertation

Abstract: 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.
Keywords: humani sklepni hrustanec, tkivni inženiring, biokompatibilni celični nosilec, sintetični polimer, polyHIPE, diferenciacija hondrocitov
Published in DKUM: 17.10.2017; Views: 2200; Downloads: 270
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8.
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, original scientific article

Abstract: 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.
Keywords: insulin secretion, pre-diabetes
Published in DKUM: 23.08.2017; Views: 1426; Downloads: 200
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9.
Increased augmentation index and central systolic arterial pressure are associated with lower school and motor performance in young adolescents
Bernarda Vogrin, Marjan Rupnik, Dušanka Mičetić-Turk, 2017, original scientific article

Abstract: Objective: In adults, improper arterial function has been linked to cognitive impairment. The pulse wave velocity (PWV), augmentation index (AIx) and other vascular parameters are useful indicators of arterial health. In our study, we monitored arterial properties, body constitution, school success, and motor skills in young adolescents. We hypothesize that reduced cognitive and motor abilities have a vascular origin in children. Methods: We analysed 81 healthy school children aged 11-16 years. Anthropometry central systolic arterial pressure, body mass index (BMI), standard deviation scores (SDS) BMI, general school performance grade, and eight motor tests were assessed. PWV, AIx, and central systolic arterial pressure (SBPao) were measured. Results: AIx and SBPao correlated negatively with school performance grades. Extremely high AIx, PWVand SBPao values were observed in 5% of children and these children had average to low school performance. PWV correlated significantly with weight, height, and waist and hip circumference. AIx, PWV, school success, and BMI correlated strongly with certain motor functions. Conclusions: Increased AIx and SBPao are associated with lower school and motor performance in children. PWV is influenced by the body's constitution.
Keywords: augmentation index, pulse wave velocity, school success, motor functions, young adolescents, central systolic arterial pressure, body mass index
Published in DKUM: 13.07.2017; Views: 1211; Downloads: 339
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10.
Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-ß/Smad pathway of fibrosis in human keratocytes in vitro
Tomislav Šarenac, Martin Trapečar, Lidija Gradišnik, Marjan Rupnik, Dušica Pahor, 2016, original scientific article

Abstract: Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.
Keywords: cornea, wounds, treatment, antifibrotic treatment, keratocytes
Published in DKUM: 23.06.2017; Views: 1371; Downloads: 172
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