Biology subjects

Kravets, V.

Publications and source records attributed to Kravets, V..

12 recordsLinked to original sources

bioRxiv2026

Computational modelling of natural cell-to-cell heterogeneity reveals key parameters that control the diversity of human pancreatic islet β-cell excitability in response to glucose

Insulin-producing {beta}-cells demonstrate remarkable heterogeneity in their individual responsiveness to glucose, and that cellular heterogeneity contributes to coordinating islet activity and glucose homeostasis. Our current understanding of how variation in cell-intrinsic factors control cellular excitability and insulin secretion is informed by foundational experiments conducted on dispersed single {beta}-cells. Such studies are limited in their ability to link multiple electrical or metabolic properties within a single cell and preclude the ability to relate, post hoc, each parameters contribution to glucose responsiveness. Computational modelling represents a unique and underutilized tool to integrate and investigate the role of natural {beta}-cell heterogeneity in physiologic glucose responses. Herein, we utilize a high-volume single-cell electrophysiology "patch-seq" dataset to define the physiologically relevant sources of variability in human {beta}-cell electrophysiology and model their influence on single-cell glucose responses. Three thousand in silico human {beta}-cells were fitted to physiologically relevant variations in glucokinase activity, K+ current, Na+ current, Ca2+ current, and exocytotic function. Four dominant electrical phenotypes arose at low (2 mM) and high (20 mM) glucose: silent, bursting, spiking, and depolarized. Approximately 50% of uncoupled {beta}-cells remained electrically silent at high glucose. Furthermore, Na+ channel half-inactivation voltage was a major predictor of the silent and spiking phenotypes at each glucose concentration, and of cells that transition from silent to spiking when glucose increased. Indeed, experimentally observed variation in Na+ channel voltage dependence was second only to variation in ATP-sensitive potassium channel conductance in determining {beta}-cell excitability. Our data-driven computational modelling highlights the functional importance of electrical heterogeneity in human {beta}-cell glucose responses, and provides a useful tool for generating testable hypotheses.

physiology↗

bioRxiv2025

Differentiating the Roles of Metabolic Similarity and Ionic Coupling in Determining the Beta Hub Cell Phenotype

Pancreatic beta cells regulate circulating glucose levels by releasing insulin. Beta cells transduce elevated blood glucose into electrical activity through an intrinsic cascade of metabolic and electrophysiologic responses. These responses are synchronized across the electrically connected network of beta cells, such that insulin release is also relatively synchronous. Despite their coordinated behavior, individual beta cells exhibit significant functional heterogeneity. This heterogeneity is thought to provide cells with specific functional phenotypes the ability to control the activity of the broader network. Hub cells, identified by their synchronized [Ca2+] activity, are one such functional subpopulation, and are believed to orchestrate the second, oscillatory phase of insulin release. However, it remains unclear whether the cell-autonomous characteristics of hub cells, such as their metabolic activity and electrophysiologic properties, are more important than their network characteristics (i.e. gap junctional coupling) for their ability to influence broader network activity. In this study, we investigate the roles of intrinsic metabolic and electrophysiologic properties and ionic coupling in determining the beta hub cell phenotype. Using a computational islet model of 1,000 beta cells, our analysis revealed that both intrinsic metabolic properties and structural coupling via gap junctions are crucial for determining the hub cell phenotype. After investigating the intrinsic coupling conductance of neighboring cells and the number of structural (direct electrical) links as independent contributors to the hub cells local electrical coupling, we find that the number of cells to which a beta cell is directly coupled may be a key determinant of its propensity to serve as a hub cell in the model. As predicted for this subpopulation, we also demonstrate that decoupling hub cells impairs the functional connectivity of the entire network. Our findings indicate the importance of both autonomous cellular dynamics and non-autonomous structural coupling for the hub cell phenotype. These insights help build a fundamental understanding of hub cells, which in turn may contribute to identifying potential approaches to preserve or improve beta cell function and thereby manage the progression of diabetes.

bioengineering↗

bioRxiv2025

Exploring the intrinsic and extrinsic determinants of heterogeneity in a β-cell network

Pancreatic islets are micro-organs composed of multiple endocrine cell types. {beta}-cells are the most common of these and are highly heterogeneous in both their intrinsic properties, such as ion channel conductances and metabolic activity, and extrinsic properties, including gap junction coupling and paracrine signaling. Capturing these diverse sources of heterogeneity is essential for computational models that aim to reproduce islet function. We evaluate two established multicellular models, the coupled Cha-Noma model of the mouse {beta}-cell network, and the Riz human model. Prior work with these two models suggest that the Cha-Noma model involves minimal intrinsic heterogeneity and therefore bursts with highly synchronized activity, whereas heterogeneity in the Riz model prescribed from in vitro patch-clamp results in highly unsynchronized behavior of the coupled network. We hypothesize that adjusting the number of bursting cells in both model formulations may invoke more physiologically realistic network coordination. We applied a categorical sensitivity analysis to establish which parameters are most important for determining bursting in single cells of the Riz model. We also introduced new heterogeneity (based on single-cell gene expression) in parameters that had previously been treated as invariant among {beta}-cells. We hypothesized that introducing heterogeneity in the small conductance Ca2+ K+ channel in particular could promote a higher proportion of bursting cells. Lastly, both models fail to incorporate the influences of non-{beta}-cells. We introduced paracrine signaling between and {beta}-cells into the coupled Cha-Noma model and showed it plays a role in accelerating the response time of {beta}-cells to acute glucose stimulation (i.e. promotes a 1st responder phenotype).

bioengineering↗

bioRxiv2025

Role of GLP1-receptor-mediated α-β-cell communication in functional β-cell heterogeneity.

While islet {beta}-cells were first viewed as a singular functional entity, since the 1970s findings reveal that individual {beta}-cells differ in their insulin secretion. More recently distinct functional subpopulations based on differential calcium dynamics have been demonstrated to drive islet function. Here, we investigate how paracrine signaling, specifically glucagon-like peptide receptor (GLP-1R)-mediated -{beta}-cell communication shapes functional {beta}-cell heterogeneity. To address this, we utilized confocal imaging of calcium responses in isolated islets from GCaMP6s mice and in islets from pancreatic slices of C57BL/6 mice, both before and after a GLP-1R antagonist (exendin-9) treatment. Inhibiting -{beta}-cell communication prolonged response time, increased 1st phase heterogeneity, and decreased the 1st phase response peak. Additionally, it reduced 2nd phase oscillation frequency and heterogeneity, thereby enhancing 2nd phase coordination across {beta}-cells. These changes were more pronounced in -neighboring {beta}-cells. Moreover, addition of exendin-9 disrupted the temporal consistency and -cell proximity of hub-cells and (to a lesser degree) 1st responder {beta}-cells. Together, these findings underscore the importance of engineering islets containing both - and {beta}-cells for stem cellderived islet replacement therapies for Type-1diabetes. Article Highlights{whitebullet} Role of GLP-1R mediated -{beta} cell communication in functional {beta}-cell heterogeneity was unclear. {whitebullet}Does GLP-1R inhibition affect all {beta}-cells uniformly, or will -neighboring cells be affected more? Is existence of 1st responder and hub cell subpopulations shaped by GLP-1R signaling? {whitebullet}GLP-1R inhibition decreases multiple metrics or {beta}-cell responsiveness - especially in -neighboring {beta}-cells. It diminishes spatiotemporal consistency of hub {beta}-cells and (to a lesser degree) 1st responders. {whitebullet}Islet-local GLP-1R communication in absence of exogenous GLP-1 is sufficient for significant control of {beta}-cell function. Incorporating -cells into the engineered islets can improve islet replacement outcomes.

bioengineering↗

bioRxiv2024

Diminished gap junction coupling under diabetogenic conditions does not drive loss of functional β-cell sub-populations

Within the islets of Langerhans, gap junction coupling is important for synchronizing oscillatory free-calcium activity ([Ca2+]) and regulating pulsatile insulin release. In islets from multiple models of diabetes, gap junction coupling is disrupted, and [Ca2+] synchronization and pulsatile insulin is lost. Functional sub-populations have been identified within the islet that are linked to driving synchronized [Ca2+] and insulin release. These sub-populations can be disrupted under conditions associated with diabetes such as glucolipotoxicity and inflammatory environments, and their loss may drive islet dysfunction. Here we investigated how loss of gap junction coupling influences functional subpopulations under diabetogenic environments. We treated islets with a cocktail of pro-inflammatory cytokines, and protected gap junction coupling via co-treatment with a Cx36 peptide S293 that was previously shown to specifically prevent a decline in gap junction permeability and synchronized [Ca2+] dynamics. We performed calcium imaging and ChR2 stimulation, analyzed islet [Ca2+] dynamics and the presence of functional sub-populations including hubs and first-responders. 1h or 24h cytokine-treatment disrupted gap junction coupling, which was fully prevented by S293 peptide co-treatment. Treatment with pro-inflammatory cytokines decreased the recruitment of [Ca2+] upon ChR2 stimulation, increased the time between first and last responding cells upon glucose stimulation, and reduced the number and consistency of hub cells. When preserving gap junction coupling by S293 during cytokine treatment, the presence and consistency of these sub-populations was only marginally improved. We therefore concluded that while gap junction coupling is important for functional sub-populations to exert their influence on islet function, restoration of gap junctions alone is not sufficient to recover functional sub-populations upon diabetogenic conditions. Thus, preventing a disruption to intrinsic {beta}-cell properties that define functional subpopulations is likely important for preserving these sub-populations during diabetes.

biophysics↗

bioRxiv2024

Network approach reveals preferential T-cell and macrophage association with α-linked β-cells in early stage of insulitis in NOD mice.

One of the challenges in studying islet inflammation - insulitis - is that it is a transient phenomenon. Traditional reporting of the insulitis progression is based on cumulative, donor-averaged values of leucocyte density in the vicinity of pancreatic islets, that hinders intra- and inter-islet heterogeneity of disease progression. Here, we aimed to understand why insulitis is non-uniform, often with peri-insulitis lesions formed on one side of an islet. To achieve this, we demonstrated applicability of network theory in detangling intra-islet multi-cellular interactions during insulitis. Specifically, we asked the question "what is unique about regions of the islet which interact with immune cells first". This study utilized the non-obese diabetic mouse model of type one diabetes and examined the interplay among -, {beta}-, T-cells, myeloid cells, and macrophages in pancreatic islets during the progression of insulitis. Disease evolution was tracked based on T/{beta} cell ratio in individual islets. In the early stage, we found that immune cells are preferentially interacting with -cell-rich regions of an islet. At the islet periphery -linked {beta}-cells were found to be targeted significantly more compared to those without -cell neighbors. Additionally, network analysis revealed increased T-myeloid, and T-macrophage interactions with all {beta}-cells.

bioengineering↗

bioRxiv2022

Engineered vasculature induces functional maturation of pluripotent stem cell-derived islet organoids

Blood vessels play a critical role in pancreatic islet health and function, yet current culture methods to generate islet organoids from human pluripotent stem cells (SC-islets) lack a vascular component. Here, we engineered 3D vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs) and fibroblasts both in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-{beta}-cells, a hallmark of {beta}-cell function that is blunted in non-vascularized SC-islets. We show that an islet-like basement membrane is formed by vasculature and contributes to the functional improvement of SC-{beta}-cells. Furthermore, cell-cell communication networks based on scRNA-seq data predicted BMP2/4-BMPR2 signaling from ECs to SC-{beta}-cells. Correspondingly, BMP4 augmented the SC-{beta}-cell Ca2+ response and insulin secretion. These vascularized SC-islet models will enable further studies of crosstalk between {beta}-cells and ECs and can serve as in vivo-mimicking platforms for disease modeling and therapeutic testing.

cell biology↗

bioRxiv2022

What do Functional Synchronization Networks Indicate About Underlying Structure and System Dynamics? A network theory study in the islet

Diabetes is caused by dysfunction of electrically coupled heterogeneous {beta}-cells within the pancreatic islet. Functional networks have been used to represent cellular synchronization and study {beta}-cells subpopulations, which play an important role in driving dynamics. The mechanism by which highly synchronized {beta}-cell subpopulations drive islet function is not clear. We used experimental and computational techniques to investigate the relationship between functional networks, structural (gap-junction) networks, and underlying {beta}-cell dynamics. Highly synchronized subpopulations in the functional network were differentiated by metabolic dynamics rather than structural coupling. Consistent with this, metabolic similarities were more predictive of edges in the islet functional network. Finally, removal of gap junctions, as occurs in diabetes, caused decreases in the efficiency and clustering of the functional network. These results indicate that metabolism rather than structure drives connections in the function network, deepening our interpretation of functional networks and the formation of functional sub-populations in dynamic tissues such as the islet.

biophysics↗

bioRxiv2020

Functional architecture of the pancreatic islets: First responder cells drive the first-phase response

Insulin-secreting {beta}-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine first responder cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified "hub" and "leader" cells. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of {beta}-cells in the islet was spatially organized, dependent on the cells distance to the first responder cell, and consistent over time up to [~]24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder {beta}-cell state, critical for the islet first-phase response to glucose.

biophysics↗

bioRxiv2020

RESTORING CONNEXIN-36 FUNCTION IN DIABETOGENIC ENVIRONMENTS PRECLUDES MOUSE AND HUMAN ISLET DYSFUNCTION

The secretion of insulin from {beta}-cells in the islet of Langerhans is governed by a series of metabolic and electrical events, which can fail during the progression of type2 diabetes (T2D). {beta}-cells are electrically coupled via Cx36 gap junction channels, which coordinates the pulsatile dynamics of [Ca2+] and insulin release across the islet. Factors such as pro-inflammatory cytokines and free fatty acids disrupt gap junction coupling under invitro conditions. Here we test whether gap junction coupling and coordinated [Ca2+] dynamics are disrupted in T2D, and whether recovery of gap junction coupling can recover islet function. We examine islets from donors with T2D, from db/db mice, and islets treated with proinflammatory cytokines (TNF-, IL-1{beta}, IFN-{gamma}) or free fatty acids (palmitate). We modulate gap junction coupling using Cx36 over-expression or pharmacological activation via modafinil. We also develop a peptide mimetic (S293) of the c-terminal regulatory site of Cx36 designed to compete against its phosphorylation. Cx36 gap junction permeability and [Ca2+] dynamics were disrupted in islets from both human donors with T2D and db/db mice, and in islets treated with proinflammatory cytokines or palmitate. Cx36 over-expression, modafinil treatment and S293 peptide all enhanced Cx36 gap junction coupling and protected against declines in coordinated [Ca2+] dynamics. Cx36 over-expression and S293 peptide also reduced apoptosis induced by proinflammatory cytokines. Critically S293 peptide rescued gap junction coupling and [Ca2+] dynamics in islets from both db/db mice and a sub-set of T2D donors. Thus, recovering or enhancing Cx36 gap junction coupling can improve islet function in diabetes. KEY POINTS- Cx36 gap junction permeability and associated coordination of [Ca2+] dynamics is diminished in human type 2 diabetes (T2D) and mouse models of T2D. - Enhancing Cx36 gap junction permeability protects against disruptions to the coordination of [Ca2+] dynamics. - A novel peptide mimetic of the Cx36 c-terminal regulatory region protects against declines in Cx36 gap junction permeability. - Pharmacological elevation in Cx36 or Cx36 peptide mimetic recovers [Ca2+] dynamics and GSIS in human T2D and mouse models of T2D.

biophysics↗

bioRxiv2020

Caloric Restriction recovers impaired β-cell-β-cell coupling, calcium oscillation coordination and insulin secretion in prediabetic mice

Caloric restriction has been shown to decrease the incidence of metabolic diseases such as obesity and type 2 diabetes mellitus (T2DM). The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis and are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine if caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high fat diet (HFD), showing indications of prediabetes after 2 months, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to one month of 40% caloric restriction (2g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+ oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+ dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

physiology↗

bioRxiv2019

Optogenetic Stimulation of Pancreatic Function via Vagal Cholinergic Axons

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion; concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools enable high specificity in neural stimulation using cell-type specific targeting of opsins and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting vagal parasympathetic axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to both ultrasound image-guided optical stimulation of axon terminals in the pancreas and optical stimulation of axons of the cervical vagus nerve, together with ultrasound-based measures of pancreas blood flow. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and vagal stimulation, accompanying rapid reductions in glycemic levels. Additionally, a significant increase in pancreatic blood flow was measured following optical stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest therapeutic potential for the control of insulin secretion and glucose homeostasis.

bioengineering↗