Lidija K Bombek

ABSTRACTAlthough mice are a very instrumental model in islet beta cell research, possible phenotypic differences between strains and substrains are largely neglected in the scientific community. In this study, we show important phenotypic differences in beta cell responses to glucose between NMRI, C57BL/6J, and C57BL/6N mice, i.e., the three most commonly used strains. High-resolution multicellular confocal imaging of beta cells in acute pancreas tissue slices was used to measure and quantitatively compare the calcium dynamics in response to a wide range of glucose concentrations. Strain- and substrain-specific features were found in all three phases of beta cell responses to glucose: a shift in the dose-response curve characterizing the delay to activation and deactivation in response to stimulus onset and termination, respectively, and distinct concentration-encoding principles during the plateau phase in terms of frequency, duration, and active time changes with increasing glucos...

Network science has provided new promising tools for studying the structure and function of various complex systems. In the present contribution we demonstrate how network concepts can be used to describe the collective activity of pancreatic β cell populations in islets of Langerhans. In this microorgan, electrically coupled β cells produce and secrete insulin that plays a pivotal role in normal and pathological whole-body nutrient homeostasis. We construct functional networks from correlations between calcium dynamics of individual cells, which is recorded by means of confocal laser-scanning calcium imaging. The extracted connectivity patterns share many similarities with other real-life networks, such as small-worldness, heterogeneity, and modularity. Moreover, by applying the multilayer network formalism, we give particular emphasis to the dynamical evolution of the β cell network after stimulation. By this means, an even deeper insight into the intercellular communication mechanisms in islets is attained.

Background: The selection of embryos with largest implantation potential is an important part in assisted reproduction. Besides the embryo or blastocyst morphology, selection criteria such as position and orientation of pronuclei (PN) in relation to polar body positioning and the number, size and distribution of nucleolar precursor bodies (NPB) have been proposed. In our study, a correlation between PN and NBP morphology with the development of early embryos (day 2 of cultivation) and blastocysts (day 5) was investigated. Methods: 653 zygotes from 113 IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection) patients, younger than 40 years, were assessed 18–20 hours post-insemination. Optimal zygotes (Z1) had thouching centrally located PN with equall numbers of alligned NPB. Other zygote types differred from Z1 in having scattered NPB in both PN (Z2) or alligned NPB in one PN (Z3) or in PN beeing distant from one another (Z4). For each zygote type a percentage of normal early embryos and blastocysts was calculated. Results: Among 653 assessed zygotes 21.8 % were Z1; 29.1 % Z2, 34.6 % Z3 and 14.5 % Z4. The percentage of normal early embryos decreased from Z1 to Z4 zygote type (70.4 % vs. 55.3 % vs. 59.7 % vs.45.3 %; p < 0.05) as well as the percentage of developed blastocysts (63.4 % vs. 55.3 % vs. 58.8 % vs. 43.2 %). However, the percentages of optimal blastocysts in the four groups did not differ (11.3 % vs. 11.1 % vs. 8.4 % vs. 6.3 %). Conclusions: Best grade zygotes result in batter early embryo and blastocyst development suggesting that zygote morphology can be used in combination with embryo and/or blastocyst evaluation as a method for embryo selection prior to embryo transfer.

The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca2+ concentration ([Ca2+]c). To trigger exocytosis, Ca2+ ions enter the cytosol from intracellular Ca2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca2+]c, were extensively described in isolated single cells from various endocrine glands. Notably, less work has been done on endocrine cells in situ to address the heterogeneity of [Ca2+]c events contributing to a collective functional response of a gland. For this, β cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca2+]c dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca2+]c transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca2+ sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca2+]c event identification. Our results demonstrate that under physiological conditions the duration of [Ca2+]c events is variable, and segregated into three reproducible modes, subsecond, second, and tens of seconds time range, and are a result of a progressive temporal summation of the shortest events. Using pharmacological tools we show that activation of intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in β cell collectives, and that a subset of [Ca2+]c events could be triggered even in the absence of Ca2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca2+ receptors in shaping the heterogeneity of [Ca2+]c responses in collectives of endocrine cells in situ.NEW & NOTEWORTHY Physiological glucose or ryanodine stimulation of β cell collectives generates a large number of [Ca2+]c events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca2+]c event identification pipeline. The event durations segregate into three reproducible modes produced by a progressive temporal summation. Using pharmacological tools, we show that activation of ryanodine intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in β cell collectives.

To evaluate the rate and type of aneuploidies of chromosomes 13, 16, 18, 21 and 22, with respect to the length of in vitro maturation (IVM) period, and to compare the results to previously published studies on aneuploidy rates of unfertilized, uninseminated mature oocytes and first polar bodies. Two hundred and twelve immature germinal vesicle stage oocytes were assigned to two groups. After successful IVM, depending on their maturational period of 24h (Group A) or 36h (Group B), chromosomal analysis was performed by five color fluorescence in situ hybridization (FISH). In Groups A and B the rates of aneuploid oocytes were calculated and compared by chi-square test. Also the rates of hyperhaploidy, hypohaploidy, disomy and nullisomy were determined and compared by chi-square test. The difference was considered statistically significant at p-value of <0.05. The prolonged IVM did not significantly affect the aneuploidy rate compared to the shorter maturation period (48.1% and 45.0%, respectively). Regarding the unbalanced premature chromatid separation, no statistically significant difference was found between hyperhaploidy and hypohaploidy (14.8% versus 8.3%). For chromosome nondisjunction, higher frequency of disomy than nullisomy was observed (30.6% versus 14.8%; p<0.05). The estimated global aneuploidy rate was between 42% and 63%. The aneuploidy rate of IVM GV-oocytes is comparable to the aneuploidy rate of in vivo matured oocytes and first polar bodies, regardless of the length of maturation period. This suggests that the immature oocytes can be used in infertility treatment after they complete maturation.

Knjiga Izbrana poglavja iz fiziologije za študente medicine z navodili za vaje so strukturirana tako, da študent skozi prvi dve poglavji osvoji fiziologijo vzdražnih celic. Sledijo poglavja, ki obravnavajo fiziologijo nekaterih notranjih organov. Več poglavij je namenjenih kardiovaskularni fiziologiji, kjer študent usvoji koncepte arterijskega pritiska, načine merjenja električne aktivnosti srca z elektrokardiografijo ter dejavnike in spremembe minutnega volumna srca. Zadnja tri poglavja postavijo posamezne organe v kontekst celotnega telesa. V njih se študent seznani z delovanjem organov v medsebojni interakciji ter odzivom telesa na različne zunanje stresorje. V vsakem poglavju so podani primeri različnih patoloških stanj, ki so primerni za poglobljen študij obravnavane tematike in predstavljajo uvod v klinične predmete na študiju medicine. Teoretičnemu delu vsakega poglavja sledi navodilo za izvedbo praktične vaje, s pomočjo katerega lahko študent samostojno ali ob pomoči kolegov izvede meritve, jih analizira in tako poglablja svoje znanje.

Extreme or unaccustomed eccentric exercise can cause exercise-induced muscle damage, characterized by structural changes involving sarcomere, cytoskeletal, and membrane damage, with an increased permeability of sarcolemma for proteins. From a functional point of view, disrupted force transmission, altered calcium homeostasis, disruption of excitation-contraction coupling, as well as metabolic changes bring about loss of strength. Importantly, the trauma also invokes an inflammatory response and clinically presents itself by swelling, decreased range of motion, increased passive tension, soreness, and a transient decrease in insulin sensitivity. While being damaging and influencing heavily the ability to perform repeated bouts of exercise, changes produced by exercise-induced muscle damage seem to play a crucial role in myofibrillar adaptation. Additionally, eccentric exercise yields greater hypertrophy than isometric or concentric contractions and requires less in terms of metabolic...

Glucose progressively stimulates insulin release over a wide range of concentrations. However, the nutrient coding underlying activation, activity, and deactivation of beta cells affecting insulin release remains only partially described. Experimental data indicate that nutrient sensing in coupled beta cells in islets is predominantly a collective trait, overriding to a large extent functional differences between cells. However, some degree of heterogeneity between coupled beta cells may play important roles. To further elucidate glucose-dependent modalities in coupled beta cells, the degree of functional heterogeneity, and uncover the emergent collective operations, we combined acute mouse pancreas tissue slices with functional multicellular calcium imaging. We recorded beta cell calcium responses from threshold (7 mM) to supraphysiological (16 mM) glucose concentrations with high spatial and temporal resolution. This enabled the analysis of both classical physiological parameters ...

The accumulation of oxidative damage to DNA and other biomolecules plays an important role in the etiology of aging and age-related diseases such as type 2 diabetes mellitus (T2D), atherosclerosis, and neurodegenerative disorders. Mitochondrial DNA (mtDNA) is especially sensitive to oxidative stress. Mitochondrial dysfunction resulting from the accumulation of mtDNA damage impairs normal cellular function and leads to a bioenergetic crisis that accelerates aging and associated diseases. Age-related mitochondrial dysfunction decreases ATP production, which directly affects insulin secretion by pancreatic beta cells and triggers the gradual development of the chronic metabolic dysfunction that characterizes T2D. At the same time, decreased glucose oxidation in skeletal muscle due to mitochondrial damage leads to prolonged postprandial blood glucose rise, which further worsens glucose homeostasis. ROS are not only highly reactive by-products of mitochondrial respiration capable of oxid...

Obesity and accompanying type 2 diabetes are among major and increasing worldwide problems that occur fundamentally due to excessive energy intake during its expenditure. Endotherms continuously consume a certain amount of energy to maintain core body temperature via thermogenic processes, mainly in brown adipose tissue and skeletal muscle. Skeletal muscle glucose utilization and heat production are significant and directly linked to body glucose homeostasis at rest, and especially during physical activity. However, this glucose balance is impaired in diabetic and obese states in humans and mice, and manifests as glucose resistance and altered muscle cell metabolism. Uncoupling proteins have a significant role in converting electrochemical energy into thermal energy without ATP generation. Different homologs of uncoupling proteins were identified, and their roles were linked to antioxidative activity and boosting glucose and lipid metabolism. From this perspective, uncoupling protei...

To evaluate the rate and type of aneuploidies of chromosomes 13, 16, 18, 21 and 22, with respect to the length of in vitro maturation (IVM) period, and to compare the results to previously published studies on aneuploidy rates of unfertilized, uninseminated mature oocytes and first polar bodies. Two hundred and twelve immature germinal vesicle stage oocytes were assigned to two groups. After successful IVM, depending on their maturational period of 24h (Group A) or 36h (Group B), chromosomal analysis was performed by five color fluorescence in situ hybridization (FISH). In Groups A and B the rates of aneuploid oocytes were calculated and compared by chi-square test. Also the rates of hyperhaploidy, hypohaploidy, disomy and nullisomy were determined and compared by chi-square test. The difference was considered statistically significant at p-value of <0.05. The prolonged IVM did not significantly affect the aneuploidy rate compared to the shorter maturation period (48.1% and 45.0%, respectively). Regarding the unbalanced premature chromatid separation, no statistically significant difference was found between hyperhaploidy and hypohaploidy (14.8% versus 8.3%). For chromosome nondisjunction, higher frequency of disomy than nullisomy was observed (30.6% versus 14.8%; p<0.05). The estimated global aneuploidy rate was between 42% and 63%. The aneuploidy rate of IVM GV-oocytes is comparable to the aneuploidy rate of in vivo matured oocytes and first polar bodies, regardless of the length of maturation period. This suggests that the immature oocytes can be used in infertility treatment after they complete maturation.

Pancreatic beta cells secrete insulin in response to stimulation with glucose and other nutrients, and impaired insulin secretion plays a central role in development of diabetes mellitus. Pharmacological management of diabetes includes various antidiabetic drugs, including incretins. The incretin hormones, glucagon-like peptide-1 and gastric inhibitory polypeptide, potentiate glucose-stimulated insulin secretion by binding to G protein-coupled receptors, resulting in stimulation of adenylate cyclase and production of the secondary messenger cAMP, which exerts its intracellular effects through activation of protein kinase A or the guanine nucleotide exchange protein 2A. The molecular mechanisms behind these two downstream signaling arms are still not fully elucidated and involve many steps in the stimulus–secretion coupling cascade, ranging from the proximal regulation of ion channel activity to the central Ca2+ signal and the most distal exocytosis. In addition to modifying intracel...

Beta cells couple stimulation by glucose with insulin secretion and impairments in this coupling play a central role in diabetes mellitus. To clarify the effect of cAMP and the role of Epac2A in intracellular calcium signals and intercellular coupling, we performed functional multicellular calcium imaging in beta cells in mouse pancreas tissue slices after stimulation with glucose and forskolin in wild-type and Epac2A knock-out mice. Increased cAMP evoked calcium signals in otherwise sub-stimulatory 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 increased cAMP increased the active time via a large increase in oscillation frequency and small decrease in oscillation duration in both Epac2A knock-out and wild-type mice. Functional network properties during stimulation with glucose did no...

Progesterone in sublethal concentrations temporarily inhibits growth of Hortaea werneckii. This study investigates some of the compensatory mechanisms which are activated in the presence of progesterone and are most probably contributing to escape from growth inhibition. ...

SNAP-25 is a protein of the core SNARE complex mediating stimulus-dependent release of insulin from pancreatic β 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 β 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 β 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 β 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 β 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 β cells, except for participating in the core SNARE complex, is necessary for accurate regulation of Ca 2+-dynamics. A controlled insulin secretion from β cells in the islets of Langerhans is essential to preserve healthy levels of blood glucose during basal and stimulated conditions 1. Glucose-driven insulin secretion is mediated by SNARE proteins (Soluble NSF-attachment Protein (SNAP) REceptors; NSF, N-ethylmaleimide-sensitive fusion protein) and requires an increase in cytosolic Ca 2+2, 3. When β cells sense, import and metabolize glucose from the blood, molecular processes increase cytosolic levels of Ca 2+ , which activate the SNARE machinery mediating insulin granule fusion with the plasma membrane. Glucose stimulation of β cells normally induces a biphasic response of insulin secretion, consisting of an initial fast first phase lasting a few minutes, followed by a sustained long-lasting second phase characterized by pulsatile insulin release 1, 4, 5. The excitable β cell expresses essentially the same repertoire of SNARE proteins as neuroendo-crine and neuronal cells 2, 3, 6. The SNARE complex includes the plasma membrane proteins syntaxin and synaptosomal-associated protein of 25 kD (SNAP-25), and the vesicular protein VAMP/synaptobrevin. SNAP-25 is essential for stimulus-dependent exocytosis and exists as two isoforms, SNAP-25a and SNAP-25b. Alternative splicing between two exons 5 in the Snap25 gene results in two proteins, differing in only 9 out of 206 amino acids 7-9. Messenger RNAs for both SNAP-25 isoforms are expressed in primary pancreatic β cells and both variants support insulin secretion 10, 11. The functional difference between the two isoforms is not fully understood, however, in mouse brain SNAP-25b forms more stable SNARE complexes than SNAP-25a 12. Furthermore, in embryonic SNAP-25-deficient chromaffin cells, introduction of exogenous SNAP-25b induces a larger pool