Network approach reveals preferential T-cell and macrophage association with α-linked ß-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 hinder 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 the 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 that interact with immune cells first". This study utilized the non-obese diabetic mouse model of type one diabetes and examined the interplay among α-, ß-, T-cells, myeloid cells, and macrophages in pancreatic islets during the progression of insulitis. Disease evolution was tracked based on the T/ß cell ratio in individual islets. In the early stage, we found that immune cells are preferentially interacting with α-cell-rich regions of an islet. At the islet periphery α-linked ß-cells were found to be targeted significantly more compared to those without α-cell neighbors. Additionally, network analysis revealed increased T-myeloid, and T-macrophage interactions with all ß-cells.

Network approach reveals preferential T-cell and macrophage association with α-linked ß-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 α-, ß-, T-cells, myeloid cells, and macrophages in pancreatic islets during the progression of insulitis. Disease evolution was tracked based on T/ß cell ratio in individual islets. In the early stage, we found that immune cells are preferentially interacting with α-cell-rich regions of an islet. At the islet periphery α-linked ß-cells were found to be targeted significantly more compared to those without α-cell neighbors. Additionally, network analysis revealed increased T-myeloid, and T-macrophage interactions with all ß-cells.

Network representation of multicellular activity in pancreatic islets: Technical considerations for functional connectivity analysis.

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.

ß-cell intrinsic dynamics rather than gap junction structure dictates subpopulations in the islet functional network.

Diabetes is caused by the inability of electrically coupled, functionally heterogeneous ß-cells within the pancreatic islet to provide adequate insulin secretion. Functional networks have been used to represent synchronized oscillatory [Ca2+] dynamics and to study ß-cell subpopulations, which play an important role in driving islet function. The mechanism by which highly synchronized ß-cell subpopulations drive islet function is unclear. We used experimental and computational techniques to investigate the relationship between functional networks, structural (gap junction) networks, and intrinsic ß-cell dynamics in slow and fast oscillating islets. Highly synchronized subpopulations in the functional network were differentiated by intrinsic dynamics, including metabolic activity and KATP channel conductance, more than structural coupling. Consistent with this, intrinsic dynamics were more predictive of high synchronization in the islet functional network as compared to high levels of structural coupling. Finally, dysfunction of gap junctions, which can occur in diabetes, caused decreases in the efficiency and clustering of the functional network. These results indicate that intrinsic dynamics rather than structure drive connections in the functional network and highly synchronized subpopulations, but gap junctions are still essential for overall network efficiency. These findings deepen our interpretation of functional networks and the formation of functional subpopulations in dynamic tissues such as the islet.

Restoring connexin-36 function in diabetogenic environments precludes mouse and human islet dysfunction.

The secretion of insulin from ß-cells in the islet of Langerhans is governed by a series of metabolic and electrical events, which can fail during the progression of type 2 diabetes (T2D). ß-cells are electrically coupled via connexin-36 (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 in vitro 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 pro-inflammatory cytokines (TNF-α, IL-1ß, IFN-É£) 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 pro-inflammatory 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 pro-inflammatory 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: Connexin-36 (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 glucose-stimulated insulin secretion in human T2D and mouse models of T2D.

Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response.

Insulin-secreting ß-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. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (ß-GCamP6s) that show ß-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of ß-cells in the islet was spatially organized, dependent on the cell's distance to the first responder cell, and consistent over time up to approximately 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 ß-cell state, critical for the islet first-phase response to glucose.

The physiological role of ß-cell heterogeneity in pancreatic islet function.

Endocrine cells within the pancreatic islets of Langerhans are heterogeneous in terms of transcriptional profile, protein expression and the regulation of hormone release. Even though this heterogeneity has long been appreciated, only within the past 5 years have detailed molecular analyses led to an improved understanding of its basis. Although we are beginning to recognize why some subpopulations of endocrine cells are phenotypically different to others, arguably the most important consideration is how this heterogeneity affects the regulation of hormone release to control the homeostasis of glucose and other energy-rich nutrients. The focus of this Review is the description of how endocrine cell heterogeneity (and principally that of insulin-secreting ß-cells) affects the regulation of hormone secretion within the islets of Langerhans. This discussion includes an overview of the functional characteristics of the different islet cell subpopulations and describes how they can communicate to influence islet function under basal and glucose-stimulated conditions. We further discuss how changes to the specific islet cell subpopulations or their numbers might underlie islet dysfunction in type 2 diabetes mellitus. We conclude with a discussion of several key open questions regarding the physiological role of islet cell heterogeneity.

Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient ß cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among ß cells, yet testing this in vivo in the intact pancreas is challenging. Robo ßKO mice, in which the genes Robo1 and Robo2 are deleted selectively in ß cells, provide a unique model of altered islet spatial architecture without loss of ß cell differentiation or islet damage from diabetes. Combining Robo ßKO mice with intravital microscopy, we show here that Robo ßKO islets have reduced synchronized intra-islet Ca2+ oscillations among ß cells in vivo. We provide evidence that this loss is not due to a ß cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Optogenetic stimulation of cholinergic fibers for the modulation of insulin and glycemia.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion, expected concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools, however, provide the potential for cell-type specific neural stimulation using genetic targeting and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting parasympathetic (cholinergic) axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to (1) ultrasound image-guided optical stimulation of axon terminals in the pancreas or (2) optical stimulation of axons of the cervical vagus nerve. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and cervical vagal stimulation, while a rapid reduction in glycemic levels were observed under pancreatic stimulation. Additionally, ultrasound-based measurements of blood flow in the pancreas were increased under pancreatic stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest its therapeutic potential for the control of insulin secretion and glucose homeostasis.

Caloric restriction recovers impaired ß-cell-ß-cell gap junction coupling, calcium oscillation coordination, and insulin secretion in prediabetic mice.

Caloric restriction can decrease the incidence of metabolic diseases, such as obesity and Type 2 diabetes mellitus. The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis 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 whether 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 mo, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to 1 mo of 40% caloric restriction (2 g/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.

From the Transcriptome to Electrophysiology: Searching for the Underlying Cause of Diabetes.

Cells within the islet of Langerhans are heterogeneous. Camunas-Soler et al. (2020) implement a patch-seq technique to collect both transcriptomic and electrophysiological data from the same cell. By doing so, they discover new genes that correlate with functional heterogeneity and find that shifts in these correlations indicate ß cell compensation in type 2 diabetes.

Imaging of Biological Cells Using Luminescent Silver Nanoparticles.

The application of luminescent silver nanoparticles as imaging agents for neural stem and rat basophilic leukemia cells was demonstrated. The experimental size dependence of the extinction and emission spectra for silver nanoparticles were also studied. The nanoparticles were functionalized with fluorescent glycine dimers. Spectral position of the resonance extinction and photoluminescence emission for particles with average diameters ranging from 9 to 32 nm were examined. As the particle size increased, the spectral peaks for both extinction and the intrinsic emission of silver nanoparticles shifted to the red end of the spectrum. The intrinsic photoluminescence of the particles was orders of magnitude weaker and was spectrally separated from the photoluminescence of the glycine dimer ligands. The spectral position of the ligand emission was independent of the particle size; however, the quantum yield of the nanoparticle-ligand system was size-dependent. This was attributed to the enhancement of the ligand's emission caused by the local electric field strength's dependence on the particle size. The maximum quantum yield determined for the nanoparticle-ligand complex was (5.2 ± 0.1) %. The nanoparticles were able to penetrate cell membranes of rat basophilic leukemia and neural stem cells fixed with paraformaldehyde. Additionally, toxicity studies were performed. It was found that towards rat basophilic leukemia cells, luminescent silver nanoparticles had a toxic effect in the silver atom concentration range of 10-100 µM.