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Opis: Within the islets of Langerhans, beta cells orchestrate synchronized insulin secretion, a pivotal aspect of metabolic homeostasis. Despite the inherent heterogeneity and multimodal activity of individual cells, intercellular coupling acts as a homogenizing force, enabling coordinated responses through the propagation of intercellular waves. Disruptions in this coordination are implicated in irregular insulin secretion, a hallmark of diabetes. Recently, innovative approaches, such as integrating multicellular calcium imaging with network analysis, have emerged for a quantitative assessment of the cellular activity in islets. However, different groups use distinct experimental preparations, microscopic techniques, apply different methods to process the measured signals and use various methods to derive functional connectivity patterns. This makes comparisons between findings and their integration into a bigger picture difficult and has led to disputes in functional connectivity interpretations. To address these issues, we present here a systematic analysis of how different approaches influence the network representation of islet activity. Our findings show that the choice of methods used to construct networks is not crucial, although care is needed when combining data from different islets. Conversely, the conclusions drawn from network analysis can be heavily affected by the pre-processing of the time series, the type of the oscillatory component in the signals, and by the experimental preparation. Our tutorial-like investigation aims to resolve interpretational issues, reconcile conflicting views, advance functional implications, and encourage researchers to adopt connectivity analysis. As we conclude, we outline challenges for future research, emphasizing the broader applicability of our conclusions to other tissues exhibiting complex multicellular dynamics.
Ključne besede: islets of Langerhans, beta cells, calcium signaling, intercellular communication, functional networks, myosin model
Objavljeno v DKUM: 09.12.2024; Ogledov: 0; Prenosov: 1
Celotno besedilo (4,48 MB)
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Opis: 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.
Ključne besede: beta cells, calcium imaging, glucose-dependence, network analysis
Objavljeno v DKUM: 15.10.2024; Ogledov: 0; Prenosov: 6
Celotno besedilo (4,37 MB)
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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: 193; Prenosov: 14
Celotno besedilo (12,03 MB)
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Opis: V magistrskem delu smo se spoznali s problematiko sladkorne bolezni, ki se odraža z nepravilnim obnašanjem celic beta v trebušni slinavki. Seznanili smo se s postopkom označevanja slik Langerhansovih otočkov, ki vsebujejo celice beta. S ciljem avtomatizacije procesa ročnega označevanja slik smo se odločili za uporabo globoke nevronske mreže za segmentacijo slik. Po analizi podatkovnih množic in preobrazbi slikovnih vrst s postopki agregacije v obliko, primerno za strojno učenje slik, smo s pomočjo nenadzorovanega učenja naučili nevronsko mrežo W-Net in ovrednotili rezultate. Mreža je uspešno identificirala zanimiva območja na slikah, vendar s premalo natančnostjo in prevelikimi območji lažno pozitivnih slikovnih točk.
Ključne besede: segmentacija slik, nenadzorovano učenje, W-Net, nevronska mreža, celice beta
Objavljeno v DKUM: 23.01.2023; Ogledov: 739; Prenosov: 124
Celotno besedilo (10,66 MB)