Insulin is released from the pancreas in pulses with a period of ~ 5 min. These oscillatory insulin levels are essential for proper liver utilization and perturbed pulsatility is observed in type 2 diabetes. What coordinates the many islets of Langerhans throughout the pancreas to produce unified oscillations of insulin secretion? One hypothesis is that coordination is achieved through an insulin-dependent negative feedback action of the liver onto the glucose level. This hypothesis was tested in an in vitro setting using a microfluidic system where the population response from a group of islets was input to a model of hepatic glucose uptake, which provided a negative feedback to the glucose level. This modified glucose level was then delivered back to the islet chamber where the population response was again monitored and used to update the glucose concentration delivered to the islets. We found that, with appropriate parameters for the model, oscillations in islet activity were synchronized. This approach demonstrates that rhythmic activity of a population of physically uncoupled islets can be coordinated by a downstream system that senses islet activity and supplies negative feedback. In the intact animal, the liver can play this role of the coordinator of islet activity.

Improved methods have recently been developed for assessing islet viability and quantity in human islet preparations for transplantation, and these measurements have proven useful for predicting transplantation outcome. The objectives of this study were to adapt these methods for use with microencapsulated islets, to verify that they provide meaningful quantitative measurements, and to test them with two model systems: (1) barium alginate and (2) barium alginate containing a 70% (w/v) perfluorocarbon (PFC) emulsion, which presents challenges to use of these assays and is of interest in its own right as a means for reducing oxygen supply limitations to encapsulated tissue. Mitochondrial function was assessed by oxygen consumption rate measurements, and the analysis of data was modified to account for the increased solubility of oxygen in the PFC-alginate capsules. Capsules were dissolved and tissue recovered for nuclei counting to measure the number of cells. Capsule volume was determined from alginate or PFC content and used to normalize measurements. After low oxygen culture for 2 days, islets in normal alginate lost substantial viable tissue and displayed necrotic cores, whereas most of the original oxygen consumption rate was recovered with PFC alginate, and little necrosis was observed. All nuclei were recovered with normal alginate, but some nuclei from nonrespiring cells were lost with PFC alginate. Biocompatibility tests revealed toxicity at the islet periphery associated with the lipid emulsion used to provide surfactants during the emulsification process. We conclude that these new assay methods can be applied to islets encapsulated in materials as complex as PFC-alginate. Measurements made with these materials revealed that enhancement of oxygen permeability of the encapsulating material with a concentrated PFC emulsion improves survival of encapsulated islets under hypoxic conditions, but reformulation of the PFC emulsion is needed to reduce toxicity.

Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet's native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation.

Incretins such as glucagon-like peptide 1 (GLP-1) are released from the gut and potentiate insulin release in a glucose-dependent manner. Although this action is generally believed to hinge on cAMP and protein kinase A signaling, up-regulated beta cell intermediary metabolism may also play a role in incretin-stimulated insulin secretion. By employing recombinant probes to image ATP dynamically in situ within intact mouse and human islets, we sought to clarify the role of GLP-1-modulated energetics in beta cell function. Using these techniques, we show that GLP-1 engages a metabolically coupled subnetwork of beta cells to increase cytosolic ATP levels, an action independent of prevailing energy status. We further demonstrate that the effects of GLP-1 are accompanied by alterations in the mitochondrial inner membrane potential and, at elevated glucose concentration, depend upon GLP-1 receptor-directed calcium influx through voltage-dependent calcium channels. Lastly, and highlighting critical species differences, beta cells within mouse but not human islets respond coordinately to incretin stimulation. Together, these findings suggest that GLP-1 alters beta cell intermediary metabolism to influence ATP dynamics in a species-specific manner, and this may contribute to divergent regulation of the incretin-axis in rodents and man.

Tributyltin (TBT) is known as an endocrine disruptor able to interfere with estrogen receptors (ERs) leading to toxic effects on the related endocrine pathways. TBT is an obesogen, reported to disrupt glucose homeostasis leading to diabetes. The aim of this study was to assess the influence of TBT and β-estradiol on the pancreatic islets of Langerhans in simultaneous exposures.

Insulin secretion is impaired with increasing age. In this study, we aimed to determine whether aging induces specific transcriptional changes in human islets. Laser capture microdissection was used to extract pancreatic islet tissue from 37 deceased organ donors aged 1-81 years. The transcriptomes of the extracted islets were analysed using Ion AmpliSeq sequencing. 346 genes that co-vary significantly with age were found. There was an increased transcription of genes linked to senescence, and several aspects of the cell cycle machinery were downregulated with increasing age. We detected numerous genes not linked to aging in previous studies likely because earlier studies analysed islet cells isolated by enzymatic digestion which might affect the islet transcriptome. Among the novel genes demonstrated to correlate with age, we found an upregulation of SPP1 encoding osteopontin. In beta cells, osteopontin has been seen to be protective against both cytotoxicity and hyperglycaemia. In summary, we present a transcriptional profile of aging in human islets and identify genes that could affect disease course in diabetes.

It is generally admitted that the endocrine cell organization in human islets is different from that of rodent islets. However, a clear description of human islet architecture has not yet been reported. The aim of this work was to describe our observations on the arrangement of human islet cells.

The islets of Langerhans are critical endocrine micro-organs that secrete hormones regulating energy metabolism in animals. Insulin and glucagon, secreted by beta and alpha cells, respectively, are responsible for metabolic switching between fat and glucose utilization. Dysfunction in their secretion and/or counter-regulatory influence leads to diabetes. Debate in the field centers on the cytoarchitecture of islets, as the signaling that governs hormonal secretion depends on structural and functional factors, including electrical connectivity, innervation, vascularization, and physical proximity. Much effort has therefore been devoted to elucidating which architectural features are significant for function and how derangements in these features are correlated or causative for dysfunction, especially using quantitative network science or graph theory characterizations. Here, we ask if there are non-local features in islet cytoarchitecture, going beyond standard network statistics, that are relevant to islet function. An example is ring structures, or cycles, of α and δ cells surrounding β cell clusters or the opposite, β cells surrounding α and δ cells. These could appear in two-dimensional islet section images if a sphere consisting of one cell type surrounds a cluster of another cell type. To address these issues, we developed two independent computational approaches, geometric and topological, for such characterizations. For the latter, we introduce an application of topological data analysis to determine locations of topological features that are biologically significant. We show that both approaches, applied to a large collection of islet sections, are in complete agreement in the context both of developmental and diabetes-related changes in islet characteristics. The topological approach can be applied to three-dimensional imaging data for islets as well.

The identification of individuals with a high risk of developing type 2 diabetes (T2D) is fundamental for prevention. Here, we used a translational approach and prediction criteria to identify changes in DNA methylation visible before the development of T2D. Islets of Langerhans were isolated from genetically identical 10-week-old female New Zealand Obese mice, which differ in their degree of hyperglycemia and in liver fat content. The application of a semiexplorative approach identified 497 differentially expressed and methylated genes (P = 6.42e-09, hypergeometric test) enriched in pathways linked to insulin secretion and extracellular matrix-receptor interaction. The comparison of mouse data with DNA methylation levels of incident T2D cases from the prospective European Prospective Investigation of Cancer (EPIC)-Potsdam cohort, revealed 105 genes with altered DNA methylation at 605 cytosine-phosphate-guanine (CpG) sites, which were associated with future T2D. AKAP13, TENM2, CTDSPL, PTPRN2, and PTPRS showed the strongest predictive potential (area under the receiver operating characteristic curve values 0.62-0.73). Among the new candidates identified in blood cells, 655 CpG sites, located in 99 genes, were differentially methylated in islets of humans with T2D. Using correction for multiple testing detected 236 genes with an altered DNA methylation in blood cells and 201 genes in diabetic islets. Thus, the introduced translational approach identified novel putative biomarkers for early pancreatic islet aberrations preceding T2D.

No abstract available

Islets of Langerhans are the endocrine tissue within the pancreas that secrete hormones for maintenance of blood glucose homeostasis. A variety of small molecules including classical neurotransmitters are also released from islets. While the roles of most of these small molecules are unknown, some have been hypothesized to play a critical role in islet physiology. To better understand their role on islet function, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify 39 small molecules released from islets. Benzoyl chloride derivatization of analyte molecules was used to impart retention and facilitate electrospray ionization efficiency. Separation was achieved on a 2.1 × 150 mm column packed with 2.7 μm core-shell C18 particles. Calibration curves showed excellent linearity between the concentration and analyte response, with relative standard deviations of the analyte responses below 15% and limits of detection from 0.01-40 nM. The method was applied to examine small molecules released from murine and human islets of Langerhans after static incubation and perfusion with glucose. Results showed a decrease in secretion rates with increasing glucose concentration for most of the analytes. Secretion rates were found to be higher in human islets compared to their murine counterpart. This method will be useful in understanding the roles of small molecules in biological systems.

Pancreatic islet cells develop mature physiological responses to glucose and other fuels postnatally. In this study, we used fluorescence imaging techniques to measure changes in intracellular calcium ([Ca2+]i) to compare islets isolated from mice on postnatal days 0, 4, and 12 with islets from adult CD-1 mice. In addition, we used publicly available RNA-sequencing data to compare expression levels of key genes in β-cell physiology with [Ca2+]i data across these ages. We show that islets isolated from mice on postnatal day 0 displayed elevated [Ca2+]i in basal glucose (≤4 mM) but lower [Ca2+]i responses to stimulation by 12-20 mM glucose compared to adult. Neonatal islets displayed more adult-like [Ca2+]i in basal glucose by day 4 but continued to show lower [Ca2+]i responses to 16 and 20 mM glucose stimulation up to at least day 12. A right shift in glucose sensing (EC50) correlated with lower fragment-per-kilobase-of-transcript-per-million-reads-mapped (FPKM) of Slc2a2 (glut2) and Actn3 and increased FPKM for Galk1 and Nupr1. Differences in [Ca2+]i responses to additional stimuli were also observed. Calcium levels in the endoplasmic reticulum were elevated on day 0 but became adult-like by day 4, which corresponded with reduced expression in Atp2a2 (SERCA2) and novel K+-channel Ktd17, increased expression of Pml, Wfs1, Thada, and Herpud1, and basal [Ca2+]i maturing to adult levels. Ion-channel activity also matured rapidly, but RNA sequencing data mining did not yield strong leads. In conclusion, the maturation of islet [Ca2+]i signaling is complex and multifaceted; several possible gene targets were identified that may participate in this process.

Type-2 diabetes (T2D) is a complex disease characterized by insulin resistance in target tissues and impaired insulin release from pancreatic beta cells. As central tissue of glucose homeostasis, the pancreatic islet continues to be an important focus of research to understand the pathophysiology of the disease. The increased access to human pancreatic islets has resulted in improved knowledge of islet function, and together with advances in RNA sequencing and related technologies, revealed the transcriptional and epigenetic landscape of human islet cells. The discovery of thousands of long non-coding RNA (lncRNA) transcripts highly enriched in the pancreatic islet and/or specifically expressed in the beta-cells, points to yet another layer of gene regulation of many hitherto unknown mechanistic principles governing islet cell functions. Here we review fundamental islet physiology and propose functional implications of the lncRNAs in islet development and endocrine cell functions. We also take into account important differences between rodent and human islets in terms of morphology and function, and suggest how species-specific lncRNAs may partly influence gene regulation to define the unique phenotypic identity of an organism and the functions of its constituent cells. The implication of primate-specific lncRNAs will be far-reaching in all aspects of diabetes research, but most importantly in the identification and development of novel targets to improve pancreatic islet cell functions as a therapeutic approach to treat T2D.

Biomarkers for the measurement of islets of Langerhans could help elucidate the etiology of diabetes. Synaptic vesicle glycoprotein 2 A (SV2A) is a potential marker reported to be localized in the endocrine pancreas. [11C]UCB-J is a novel positron emission tomography (PET) radiotracer that binds to SV2A and was previously evaluated as a synaptic marker in the central nervous system. Here, we evaluated whether [11C]UCB-J could be utilized as a PET tracer for the islets of Langerhans in the pancreas by targeting SV2A. The mRNA transcription of SV2A was evaluated in human isolated islets of Langerhans and exocrine tissue. In vitro autoradiography was performed on pancreas and brain sections from rats and pigs, and consecutive sections were immunostained for insulin. Sprague-Dawley rats were examined with PET-MRI and ex vivo autoradiography at baseline and with administration of levetiracetam (LEV). Similarly, pigs were examined with dynamic PET-CT over the pancreas and brain after administration of [11C]UCB-J at baseline and after pretreatment with LEV. In vivo radioligand binding was assessed using a one-compartment tissue model. The mRNA expression of SV2A was nearly 7 times higher in endocrine tissue than in exocrine tissue (p < 0.01). In vitro autoradiography displayed focal binding of [11C]UCB-J in the pancreas of rats and pigs, but the binding pattern did not overlap with the insulin-positive areas or with ex vivo autoradiography. In rats, pancreas binding was higher than that in negative control tissues but could not be blocked by LEV. In pigs, the pancreas and brain exhibited accumulation of [11C]UCB-J above the negative control tissue spleen. While brain binding could be blocked by pretreatment with LEV, a similar effect was not observed in the pancreas. Transcription data indicate SV2A to be a valid target for imaging islets of Langerhans, but [11C]UCB-J does not appear to have sufficient sensitivity for this application.

Microscopic analysis of molecules and physiology in living cells and systems is a powerful tool in life sciences. While in vivo subcellular microscopic analysis of healthy and diseased human organs remains impossible, zebrafish larvae allow studying pathophysiology of many organs using in vivo microscopy. Here, we review the potential of the larval zebrafish pancreas in the context of islets of Langerhans and Type 1 diabetes. We highlight the match of zebrafish larvae with the expanding toolbox of fluorescent probes that monitor cell identity, fate and/or physiology in real time. Moreover, fast and efficient modulation and localization of fluorescence at a subcellular level, through fluorescence microscopy, including confocal and light sheet (single plane illumination) microscopes tailored to in vivo larval research, is addressed. These developments make the zebrafish larvae an extremely powerful research tool for translational research. We foresee that living larval zebrafish models will replace many cell line-based studies in understanding the contribution of molecules, organelles and cells to organ pathophysiology in whole organisms.

Traditionally plant-based remedies such as Commiphora myrrha (CM) have been used as an ayurvedic medicine to treat diabetes mellitus in some region of Arabia and Africa. Previous reports have shown that CM reduced blood glucose levels and increased insulin concentrations in animal models of diabetes in vivo. However, the exact mechanisms by which CM improved glycemic control in these animals are not fully understood. We hypothesized that CM may have a direct insulinotropic activity on β-cells to increase insulin secretion.

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.

The discovery of Langerhans and microscopic description of islets in the pancreas were crucial steps in the discovery of insulin. Over the past 150 years, many discoveries in islet biology and type 1 diabetes have been made using powerful microscopic techniques. In the past decade, combination of new probes, animal and tissue models, application of new biosensors and automation of light and electron microscopic methods and other (sub)cellular imaging modalities have proven their potential in understanding the beta cell under (patho)physiological conditions. The imaging evolution, from fluorescent jellyfish to real-time intravital functional imaging, the revolution in automation and data handling and the increased resolving power of analytical imaging techniques are now converging. Here, we review innovative approaches that address islet biology from new angles by studying cells and molecules at high spatiotemporal resolution and in live models. Broad implementation of these cellular imaging techniques will shed new light on cause/consequence of (mal)function in islets of Langerhans in the years to come.

Diabetes mellitus (DM) is a complex disease with high prevalence of comorbidity and mortality. DM is predicted to reach more than 700 million people by 2045. In recent years, several advanced in vitro models and analytical tools were developed to investigate the pancreatic tissue response to pathological situations and identify therapeutic solutions. Of all the in vitro promising models, cell culture in microfluidic biochip allows the reproduction of in-vivo-like micro-environments. Here, we cultured rat islets of Langerhans using dynamic cultures in microfluidic biochips. The dynamic cultures were compared to static islets cultures in Petri. The islets' exometabolomic signatures, with and without GLP1 and isradipine treatments, were characterized by GC-MS. Compared to Petri, biochip culture contributes to maintaining high secretions of insulin, C-peptide and glucagon. The exometabolomic profiling revealed 22 and 18 metabolites differentially expressed between Petri and biochip on Day 3 and 5. These metabolites illustrated the increase in lipid metabolism, the perturbation of the pentose phosphate pathway and the TCA cycle in biochip. After drug stimulations, the exometabolome of biochip culture appeared more perturbed than the Petri exometabolome. The GLP1 contributed to the increase in the levels of glycolysis, pentose phosphate and glutathione pathways intermediates, whereas isradipine led to reduced levels of lipids and carbohydrates.

Glucagon is a 29-amino acid peptide released from α-cells within pancreatic islets of Langerhans to help raise blood glucose levels. While a plethora of methodologies have been developed for quantitative measurement of insulin released from islets, such methods are not well developed for glucagon despite its importance in blood sugar regulation. In this work, a simple yet robust microfluidic device was developed for holding human pancreatic islets and perfuse them with glucose. The perfusate was collected into 2 min fractions and glucagon quantified using a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay. Simulation of fluid flow within the microfluidic device indicated the device produced low amounts of shear stress on islets, and characterization of the flow with standard glucagon solutions revealed response times within 2 fractions (<4 min). Results with human islets from multiple donors demonstrated either a "burst" of glucagon or a "sustained" glucagon release across the entire period of stimulation. The simplicity, yet robustness, of the device and method is expected to appeal to a number of researchers examining pancreatic islet physiology.