Biology subjects

Benninger, R. K.

Publications and source records attributed to Benninger, R. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Laminin-511 protects pancreatic β-cells from cytokine-induced death through integrin-mediated pro-survival signaling and modulation of protein kinase C δ

During the progression of type 1 diabetes (T1D), the extracellular matrix (ECM) surrounding pancreatic islets is degraded concurrent with infiltration of autoreactive immune cells and {beta}-cell death. Among the lost ECM proteins, laminin-511 is known to be essential for islet survival under healthy and T1D associated conditions, including high levels of pro-inflammatory cytokines. However, the key {beta}-cell signaling pathways regulated by laminin and the contributions to T1D pathogenesis when these cues are lost are poorly understood. This study utilizes a biomimetic reverse thermal gel (RTG) with laminin-511 to determine if laminin protects {beta}-cells against cytokine-induced death and elucidate the signaling pathways involved. MIN6 cells, C57Bl/6 mouse islets and human islets were encapsulated in RTG scaffolds with laminin-511 and treated with a cytokine cocktail for 24 hours. Islet viability and the activities of several pro- and anti-apoptotic proteins were studied. Laminin-511 was shown to protect islets against cytokine-induced death by interacting with {beta}1 integrins and activating pro-survival Akt signaling. Pro-survival signaling was mediated by reduced activity of protein kinase C{delta} (PKC{delta}), a key mediator of cytokine-induced {beta}-cell death, at the cell membrane in the presence of laminin via reduced levels of diacylglycerol (DAG), a canonical activator of PKC{delta}. Taken together, these results demonstrate that laminin-511 is an essential factor in protecting {beta}-cells against cytokine-induced death by downregulation of membrane DAG, inhibiting activation of pro-apoptotic PKC{delta}. Our results suggest that loss of ECM in T1D may make {beta}-cells more susceptible to cytokine-induced death by increasing activation of PKC{delta}. HighlightsO_LILaminin-511 improves islet survival under cytokine treatment C_LIO_LIPro-survival Akt is upregulated by laminin-511 C_LIO_LIPro-apoptotic PKC{delta} activity is downregulated at the cell membrane by laminin-511 C_LIO_LILaminin-511 decreases membrane DAG levels leading to reduced PKC{delta} activation C_LIO_LILaminin-511 is an essential ECM component for islet survival during type 1 diabetes C_LI

cell biology↗

bioRxiv2025

The Chromatin Remodeler Protein CHD4 Cooperates With NKX2.2 to Regulate Pancreatic Beta Cell Integrity

NKX2.2 is a transcription factor that regulates pancreatic islet beta ({beta}) cell identity and function; however, cofactor proteins that modulate the functional activity of NKX2.2 in {beta} cells are relatively unexplored. An unbiased proteomics screen identified chromodomain helicase DNA-binding protein 4 (CHD4) as an NKX2.2 interacting partner. CHD4 is a nucleosome remodeler that directs the appropriate differentiation, maturation and function of many cell types. To characterize the roles of CHD4 in {beta} cells, we generated Chd4 {beta}KO mice. Deletion of Chd4 substantially impaired the function of {beta} cells. The Chd4 {beta}KO mice became diabetic due to the disruption of islet integrity, calcium signaling and downregulation of essential {beta} cell regulatory genes. We also discovered CHD4 is required to bind at and repress non-beta cell genes, including Kcnj5, the gene that encodes the GIRK4 potassium channel in {beta} cells. Aberrant upregulation of GIRK4 causes impaired glucose-stimulated insulin secretion. These studies demonstrate that CHD4 is an essential transcriptional cofactor of NKX2.2 that is required for the proper maturation and function of pancreatic {beta} cells. Article HighlightsO_LINKX2.2 interacts with the Nucleosome Remodeling and Deacetylase (NuRD) complex through its interaction with CHD4. C_LIO_LIDeletion of CHD4 from developing pancreatic {beta} cells in mice causes diabetes due to a loss of islet integrity, disrupted calcium signaling and impaired insulin secretion. C_LIO_LIBeta cells lacking CHD4 inappropriately upregulate the GIRK4 potassium channel; inhibition of GIRK4 rescues the insulin secretion defect. C_LI

developmental biology↗

bioRxiv2025

Physics-Informed Digital Twin Can Predict Cerebral Blood Flow and Cerebral Vascular Regulation Mechanisms in Neurocritical Care Patients

1Cerebral blood flow is vital for brain function and is acutely controlled through a set of physiological mechanisms known as cerebral vascular regulation (CVR)[1-3]. It remains challenging to directly measure the dynamics and function of individual CVR mechanisms[4], limiting our ability to understand and optimize brain perfusion, particularly for neurologically injured patients. Digital twins offer an ideal tool for overcoming this gap because they enable estimation, tracking, and forecasting of unmeasured physiological states[5, 6]. Here, we introduce CereBRLSIM (Cerebral Blood Regulation Latent State Inference and Modeling), a digital twin that integrates physiological knowledge and patient data to infer CVR function and predict cerebral dynamics. Using both in vivo experiments and simulated data, CereBRLSIM predicted cerebral hemodynamics with high accuracy and estimated the dynamics of myogenic, endothelial, and metabolic mechanisms underlying CVR. When personalized to neurocritical care patient data, CereBRLSIM differentiated cerebral hemodynamic phenotypes, predicted patient outcomes, and forecasted blood flow with significantly higher accuracy than machine learning models. This work provides a novel, interpretable, and clinically compatible approach for quantifying CVR function and forecasting cerebral blood flow, enabling new opportunities in precision diagnostics and foundational understanding of cerebral hemodynamics.

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

CRISP: Correlation-Refined Image Segmentation Process

Calcium imaging enables real-time recording of cellular activity across various biological contexts. To assess the activity of individual cells, scientists typically manually outline the cells based on visual inspection. This manual cell masking introduces potential user error. To ameliorate this error, we developed the Correlation-Refined Image Segmentation Process (CRISP), a two-part automated algorithm designed to both enhance the accuracy of user-drawn cell masks and to automatically identify cell masks. We developed and tested CRISP on calcium images of densely packed {beta}-cells within the islet of Langerhans. Because these {beta}-cells are densely packed within the islet, traditional clustering-based image segmentation methods struggle to identify individual cell outlines. Using {beta}-cells isolated from two different mouse phenotypes and imaged on two different confocal microscopes, we show that CRISP is generalizable and accurate. To test the benefit of using CRISP in functional biological analyses, we show that CRISP improves accuracy of functional network analysis and utilize CRISP to characterize the distribution of {beta}-cell size.

bioengineering↗

bioRxiv2024

Glucokinase activity controls subpopulations of lower case Greek beta-cells that alternately lead islet Ca2+ oscillations

Oscillations in insulin secretion, driven by islet Ca2+ waves, are crucial for glycemic control. Prior studies, performed with single-plane imaging, suggest that subpopulations of electrically coupled {beta}-cells have privileged roles in leading and coordinating the propagation of Ca2+ waves. Here, we used 3D light- sheet imaging to analyze the location and Ca2+ activity of single {beta}-cells within the entire islet at >2 Hz. In contrast with single-plane studies, 3D network analysis indicates that the most highly synchronized {beta}-cells are located at the islet center, and remain regionally but not cellularly stable between oscillations. This subpopulation, which includes hub cells, is insensitive to changes in fuel metabolism induced by glucokinase and pyruvate kinase activation. {beta}-cells that initiate the Ca2+ wave ( leaders) are located at the islet periphery, and strikingly, change their identity over time via rotations in the wave axis. Glucokinase activation, which increased oscillation period, reinforced leader cells and stabilized the wave axis. Pyruvate kinase activation, despite increasing oscillation frequency, had no effect on leader cells, indicating the wave origin is patterned by fuel input. These findings emphasize the stochastic nature of the {beta}-cell subpopulations that control Ca2+ oscillations and identify a role for glucokinase in spatially patterning leader {beta}-cells. Highlights* Studies of islet Ca2+ oscillations by 3D light-sheet imaging provide a more complete picture of {beta}- cell subpopulations than prior 2D studies. * Highly synchronized {beta}-cells (including hub cells) are a regionally-stable subpopulation located at the islet center that is insensitive to metabolic perturbation. * Glucokinase activation patterns the Ca2+ wave axis, which originates from stochastic {beta}-cell subpopulations on the islet periphery that change between oscillations. * The stochasticity of leader {beta}-cells, and the stability of hubs, is geographically consistent with the peripheral location of /{delta}-cells in mouse islets.

biophysics↗

bioRxiv2023

Resolving spatiotemporal electrical signaling within the islet via CMOS microelectrode arrays

Glucose-stimulated beta-cells synchronize calcium waves across the islet to recruit more beta-cells for insulin secretion. Compared to calcium dynamics, the formation and cell-to-cell propagation of electrical signals within the islet are poorly characterized. To determine factors that influence the propagation of electrical activity across the islet underlying calcium oscillations and beta-cell synchronization, we used high-resolution CMOS multielectrode arrays (MEA) to measure voltage changes associated with the membrane potential of individual cells within intact mouse islets. We measured both fast (milliseconds, spikes) and slow (seconds, waves) voltage changes and analyzed the spatiotemporal voltage dynamics. Treatment of islets from C57BL6 mice with increasing glucose concentrations revealed that single spike activity and wave signal velocity were both glucose-dependent. A repeated glucose stimulus involved a highly active subset of cells in terms of spike activity. When islets were pretreated for 72 hours with glucolipotoxic medium, the wave velocity was significantly reduced. Network analysis confirmed that the synchrony of islet cells was affected due to slower propagating electrical waves and not due to altered spike activity. In summary, this approach provided novel insight regarding the propagation of electrical activity and opens a wide field for further studies on signal transduction in the islet cell network. Article HighlightsThis study presents a new method for characterizing islet spatiotemporal electrical dynamics and subpopulations of beta-cells. We asked whether a high-resolution CMOS-MEA is suited to detect electrical signals on a level close to single cells, and whether we can track the propagation of electrical activity through the islet on a cellular scale. A highly active subpopulation of islet cells was identified by action potential-like spike activity, whereas slower waves were a measure for synchronized electrical activity. Further, propagating waves were slowed by glucolipotoxicity. The technique is a useful tool for exploring the pancreatic islet network in health and disease.

physiology↗

bioRxiv2022

Cleavage of Protein Kinase C δ by Caspase-3 Mediates Pro-inflammatory Cytokine-Induced Apoptosis in the Pancreatic Islet

In type 1 diabetes (T1D), autoreactive immune cells infiltrate the pancreas and secrete pro-inflammatory cytokines that initiate cell death in insulin producing islet {beta}-cells. Protein kinase C {delta} (PKC{delta}) plays a role in mediating cytokine-induced {beta}-cell death; however, the exact mechanisms are not well understood. To address this, we utilized an inducible {beta}-cell specific PKC{delta} KO mouse as well as a small peptide specific inhibitor of PKC{delta}. We identified a role for PKC{delta} in mediating cytokine-induced {beta}-cell death and have shown that inhibiting PKC{delta} protects pancreatic {beta}-cells from cytokine-induced apoptosis in both mouse and human islets. We determined that cytokines induced nuclear translocation and activity of PKC{delta} and that caspase-3 cleavage of PKC{delta} may be required for cytokine-mediated islet apoptosis. Further, cytokine-activated PKC{delta} increases activity both of pro-apoptotic Bax with acute treatment and JNK with prolonged treatment. Overall, our results suggest that PKC{delta} mediates cytokine-induced apoptosis via nuclear translocation, cleavage by caspase-3, and upregulation of pro-apoptotic signaling in pancreatic {beta}-cells. Combined with the protective effects of PKC{delta} inhibition with {delta}V1-1, the results of this study will aid in the development of novel therapies to prevent or delay {beta}-cell death and preserve {beta}-cell function in T1D.

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

Small subpopulations of β-cells do not drive islet oscillatory dynamics via gap junction communication

The islets of Langerhans exist as a multicellular network that is important for the regulation of blood glucose levels. The majority of cells in the islet are insulin-producing {beta}-cells, which are excitable cells that are electrically coupled via gap junction channels. {beta}-cells have long been known to display heterogeneous functionality. However, due to gap junction electrical coupling, {beta}-cells show coordinated [Ca2+] oscillations when stimulated with glucose, and global quiescence when unstimulated. Small subpopulations of highly functional {beta}-cells have been suggested to control the dynamics of [Ca2+] and insulin release across the islet. In this study, we investigated the theoretical basis of whether small subpopulations of {beta}-cells can disproportionality control islet [Ca2+] dynamics. Using a multicellular model of the islet, we generated continuous or bimodal distributions of {beta}-cell heterogeneity and examined how islet [Ca2+] dynamics depended on the presence of cells with increased excitability or increased oscillation frequency. We found that the islet was susceptible to marked suppression of [Ca2+] when a [~]10% population of cells with high metabolic activity was hyperpolarized; where hyperpolarizing cells with normal metabolic activity had little effect. However, when these highly metabolic cells were removed from the islet model, near normal [Ca2+] remained. Similarly, when [~]10% of cells with either the highest frequency or earliest elevations in [Ca2+] were removed from the islet, the [Ca2+] oscillation frequency remained largely unchanged. Overall these results indicate that small populations of {beta}-cells with either increased excitability or increased frequency, or signatures of [Ca2+] dynamics that suggest such properties, are unable to disproportionately control islet-wide [Ca2+] via gap junction coupling. As such, we need to reconsider the physiological basis for such small {beta}-cell populations or the mechanism by which they may be acting to control normal islet function. Author summaryMany biological systems can be studied using network theory. How heterogeneous cell subpopulations come together to create complex multicellular behavior is of great value in understanding function and dysfunction in tissues. The pancreatic islet of Langerhans is a highly coupled structure that is important for maintaining blood glucose homeostasis. {beta}-cell electrical activity is coordinated via gap junction communication. The function of the insulin-producing {beta}-cell within the islet is disrupted in diabetes. As such, to understand the causes of islet dysfunction we need to understand how different cells within the islet contribute to its overall function via gap junction coupling. Using a computational model of {beta}-cell electrophysiology, we investigated how small highly functional {beta}-cell populations within the islet contribute to its function. We found that when small populations with greater functionality were introduced into the islet, they displayed signatures of this enhanced functionality. However, when these cells were removed, the islet, retained near-normal function. Thus, in a highly coupled system, such as an islet, the heterogeneity of cells allows small subpopulations to be dispensable, and thus their absence is unable to disrupt the larger cellular network. These findings can be applied to other electrical systems that have heterogeneous cell populations.

biophysics↗

bioRxiv2020

Detecting Insulitis in Type 1 Diabetes with Ultrasound Phase-change Contrast Agents

Type 1 diabetes (T1D) results from immune infiltration and destruction of insulin-producing {beta}-cells within the pancreatic islets of Langerhans (insulitis), resulting in loss of glucose homeostasis. Early diagnosis during pre-symptomatic T1D would allow for therapeutic intervention prior to substantial loss of {beta}-cell mass at T1D onset. There are limited methods to track the progression of insulitis and {beta}-cell mass decline in pre-symptomatic T1D. During insulitis, the islet microvasculature increases permeability, such that sub-micron sized particles can extravasate and accumulate within the islet microenvironment. Ultrasound is a widely deployable and cost-effective clinical imaging modality. However, conventional microbubble contrast agents are restricted to the vasculature. Sub-micron sized nanodroplet (ND) phasechange agents can be vaporized into micron-sized bubbles; serving as a circulating microbubble precursor. We tested if NDs extravasate into the immune-infiltrated islet microenvironment. We performed ultrasound contrast-imaging following ND infusion in NOD mice and NOD;Rag1ko controls, and tracked diabetes development. We measured the biodistribution of fluorescently labeled NDs, with histological analysis of insulitis. Ultrasound contrast signal was elevated in the pancreas of 10w NOD mice following ND infusion and vaporization, but was absent in both the non-infiltrated kidney of NOD mice and pancreas of Rag1ko controls. High contrast elevation also correlated with rapid diabetes onset. In pancreata of NOD mice, infiltrated islets and nearby exocrine tissue were selectively labeled with fluorescent NDs. Thus, contrast ultrasound imaging with ND phase-change agents can detect insulitis prior to diabetes onset. This will be important for monitoring disease progression to guide and assess preventative therapeutic interventions for T1D. SignificanceThere is a need for imaging methods to detect type1 diabetes (T1D) progression prior to clinical diagnosis. T1D is a chronic disease that results from autoreactive T cells infiltrating the islet of Langerhans and destroying insulin-producing {beta}-cells. Overt disease takes years to present and is only diagnosed after significant {beta}-cells loss. As such, the possibility of therapeutic intervention to preserve {beta}-cell mass is hampered by an inability to follow pre-symptomatic T1D progression. There are immunotherapies that can delay T1D development. However identifying at risk individuals, and tracking whether therapeutic interventions are impacting disease progression, prior to T1D onset, is lacking. A method to detect insulitis and {beta}-cell mass decline would present an opportunity to guide therapeutic treatments to prevent T1D.

bioengineering↗

bioRxiv2020

Dynamic changes in β-cell electrical activity and regulates NFATc3 activation and downstream gene transcription

Diabetes results from insufficient insulin secretion as a result of dysfunction to {beta}-cells within the islet of Langerhans. Elevated glucose causes {beta}-cell membrane depolarization and action potential generation, voltage gated Ca2+ channel activation and oscillations in free-Ca2+ activity ([Ca2+]), triggering insulin release. Nuclear Factor of Activated T-cell (NFAT) is a transcription factor that is regulated by increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems, and within the {beta}-cell regulates proliferation and insulin granule biogenesis. However the link between the regulation of {beta}-cell electrical activity and oscillatory [Ca2+], with NFAT activation and downstream transcription is poorly understood. In this study we tested whether dynamic changes to {beta}-cell electrical activity and [Ca2+] regulates NFAT activation and downstream transcription. In cell lines, mouse islets and human islets, including those from donors with type2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate {beta}-cell electrical activity. Both glucose-induced membrane depolarization and optogenetic-stimulation triggered NFAT activation, and increased transcription of NFAT targets and intermediate early genes (IEGs). Importantly only conditions in which slow sustained [Ca2+] oscillations were generated led to NFAT activation and downstream transcription. In contrast in human islets from donors with type2 diabetes NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. Thus, we gain insight into the specific patterns of electrical activity that regulate NFAT activation and gene transcription and how this is disrupted in diabetes.

biophysics↗

bioRxiv2020

Contrast-enhanced ultrasound measurement of pancreatic blood flow dynamics predicts type1 diabetes therapeutic reversal in preclinical models

In type 1 diabetes (T1D) immune-cell infiltration into the islets of Langerhans (insulitis) and {beta}-cell decline occurs many years before diabetes presents. Non-invasively detecting insulitis and {beta}-cell decline would allow diagnosis of eventual diabetes and provide a means to monitor the efficacy of therapeutic intervention. However, there is a lack of validated clinical approaches for non-invasively imaging disease progression leading to T1D. Islets have a dense microvasculature that reorganizes during diabetes. We previously demonstrated contrast-enhanced ultrasound measurements of pancreatic blood-flow dynamics could predict disease progression in T1D pre-clinical models. Here we test whether these measurements can predict successful therapeutic prevention of T1D. We performed destruction-reperfusion measurements using a small-animal ultrasound machine and size-isolated microbubbles, in NOD-scid mice receiving an adoptive transfer of diabetogenic splenocytes (AT mice). Mice received vehicle control or either of the following treatments: 1) antiCD4 to deplete CD4+ T cells; 2) antiCD3 to block T cell activation, 3) Verapamil to reduce {beta}-cell apoptosis and 4) TUDCA to reduce ER stress. We compared measurements of pancreas blood-flow dynamics with subsequent progression to diabetes. In AT mice blood-flow dynamics were altered >2 weeks after splenocyte transfer. AntiCD4, antiCD3 and verapamil provided a significant delay in diabetes development. Treated AT mice with delayed or absent diabetes development showed significantly altered blood flow dynamics compared to untreated AT mice. Conversely, treated AT mice that developed diabetes, despite therapy, showed similar blood-flow dynamics to untreated AT mice. Thus, contrast-enhanced ultrasound measurement of pancreas blood-flow dynamics can predict the successful or unsuccessful delay or prevention of diabetes upon therapeutic treatments that target both immune activity or {beta}-cell protection. This strategy may provide a clinically deployable predictive marker for disease progression and therapeutic reversal in asymptomatic T1D.

bioengineering↗

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↗

bioRxiv2020

Engineering Functional Pseudo-Islets of Defined Sizes from Primary Murine Cells Using PEG Microwell Devices

A major limitation of islet transplantation as a therapy for treating Type 1 Diabetes is eventual graft failure, which can be partially attributed to islet cell death. When cultured in vitro, cells in the center of large islets show increased necrosis and exhibit decreased viability and insulin secretion compared to smaller islets. Given the necessity of {beta}-cell-to-{beta}-cell coupling for the physiological response to glucose, a technique to re-aggregate primary islet cells or cells derived from progenitor cells into small clusters of defined sizes may prove advantageous for promoting function upon transplantation. Here, hydrogel microwell arrays were utilized to generate 3-dimensional pseudo-islets from primary murine islets. Pseudo-islets ranged from 50 to 100 m in diameter as controlled through the microwell dimensions, and contained {beta}-, -, and {delta}-cells with ratios similar to those in whole murine islets. Over two weeks in culture, pseudo-islets remained highly viable and responsive to glucose. Intracellular calcium flux showed more robust and coordinated dynamics at high glucose and decreased activity at low glucose compared to age-matched wild-type islets. Therefore, microwell devices can control the aggregation of cells isolated from primary islets to produce islet-like clusters that are functionally similar to freshly isolated islets, and may provide a technique to create improved cellular therapies for Type 1 Diabetes.

bioengineering↗