Islet Populations Research Articles

Surviving on a Rock, but for How Long? Deviations in the Thermoregulatory Strategy of the Milos Wall Lizard (Podarcis milensis)

Reptiles are unable to generate metabolic heat and regulate body temperature behaviorally depending on environmental conditions. The thermal quality of their habitat is therefore of pivotal importance for their survival. Lizards render themselves as ideal ectothermic models, and their thermal biology has been extensively studied. In this work, we focused on the thermoregulatory performance of the endemic Milos wall lizard (Podarcis milensis) (Milos Archipelago, Aegean Sea, Greece). Applying the same standard methodology, we estimated the effectiveness of thermoregulation (E) taking into account the three main thermal parameters: body (Tb, the temperature of active animals in the field), environmental (Te, the temperature that animals would achieve in the field if passively conform to the environment) and preferred temperatures (Tpref, the temperatures an animal achieves in a laboratory thermal gradient). Here, we compare the thermoregulatory profile of two remote rocky islet populations, Falconera and Velopoula, with the Milos Island population. We collected Tb values from active lizards as well as Te from specially designed copper models, which were appropriately placed in the field so as to cover all possible microhabitats. Lizards were then transported to the laboratory where we assessed their Tprefs. Falconera and Velopoula populations showed the same high thermoregulatory effectiveness as that of Milos Island (EFalconera = 0.97, EVelopoula = 0.95, EMilos = 0.89). However, when we used an alternative evaluation of the thermoregulatory strategy, the E values outlined a much more effective thermoregulation for the islets: de-dbFalconera = 6.97, de-dbVelopoula = 11.54, de-dbMilos = 4.27. The adverse conditions on the islets outline a demanding habitat of low thermal quality that dictates effective thermoregulation. However, the trend of increasing temperatures depicts an even harsher environment for the years to come. Could lizards that have already achieved the highest thermoregulatory effectiveness and cannot escape from the isolated islets they dwell cope with these new conditions? This is the kind of questions to which conservation biology will be called upon to respond.

Unveiling islet heterogeneity using an automated microfluidic imaging system

Islets of Langerhans are a therapeutic target for diabetes and prediabetes. Measurements of therapeutic inhibitory or excitatory concentrations are often performed using large groups of islets, however, the population heterogeneity cannot be observed when examining the ensemble response. Normal islet function and islet response to therapeutic treatment can be affected by islet heterogeneity, influencing the progression of diabetes mellitus. To identify heterogeneity in an islet population, we developed a simple microfluidic device capable of delivering a stimulant to four independent chambers, allowing measurements of individual responses from a population of 20–25 islets. The device enabled accurate delivery of the same stimulant concentration to all four chambers, with an error <1% between chambers. To demonstrate the capability of this system, ensemble and individual EC/IC\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{50}$$\\end{document} measurements of glucose and diazoxide were performed on murine islets. Results showed little heterogeneity of glucose EC\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{50}$$\\end{document} values with all 21 islets within ± 0.6 mM of the ensemble value of 7.4 mM. In contrast, application of diazoxide to 24 islets in the presence of 20 mM glucose resulted in 37% of islets having an IC\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{50}$$\\end{document} greater than 10% from the ensemble value of 10.2 μM. The simple system developed here is amenable to further studies on islet heterogeneity, and is applicable to investigate heterogeneity in other cell types.

A mechanism for slow rhythms in coordinated pancreatic islet activity

Insulin levels in the blood oscillate with a variety of periods, including rapid (5–10 min), ultradian (50–120 min), and circadian (24 h). Oscillations of insulin are beneficial for lowering blood glucose and disrupted rhythms are found in people with type 2 diabetes and their close relatives. These in vivo secretion dynamics imply that the oscillatory activity of individual islets of Langerhans are synchronized, although the mechanism for this is not known. One mechanism by which islets may synchronize is negative feedback of insulin on whole-body glucose levels. In previous work, we demonstrated that a negative feedback loop with a small time delay, to account for the time required for islets to be exposed to a new glucose concentration in vivo, results in small 3–6 islet populations synchronizing to produce fast closed-loop oscillations. However, these same islet populations could also produce slow closed-loop oscillations with periods longer than the natural islet oscillation periods. Here, we investigate the origin of the slow oscillations and the bistability with the fast oscillations using larger islet populations (20–50 islets). In contrast to what was observed earlier, larger islet populations mainly synchronize to longer-period oscillations that are approximately twice the delay time used in the feedback loop. A mean-field model was also used as a proxy for a large islet population to uncover the underlying mechanism for the slow rhythm. The heterogeneous intrinsic oscillation periods of the islets interferes with this rhythm mechanism when islet populations are small, and is similar to adding noise to the mean-field model. Thus, the effect of a time delay in the glucose feedback mechanism is similar to other examples of time-delayed systems in biology and may be a viable mechanism for ultradian oscillations.

Identification of Phytochemicals and Assessment of Hypoglycemic and Haematological Potentials of Terminalia catappa Linn leaf Extract in Alloxan-induced Diabetic Wistar Rats.

Hypoglycemia and anemia are associated with diabetes mellitus. Medicinal plants and orthodox drugs have been used for the management of this disease. This study aimed to validate the ethnomedical claims of Terminalia catappa Linn. leaf extract in reducing hyperglycemia and hematological potentials in alloxan-induced diabetic rats and to identify likely antidiabetic compounds. Ultra-high-performance liquid chromatography was used to identify the various phytochemical constituents. Male Wistar rats were randomly divided into five groups containing 6 rats per group. Group 1 (control) received 0.2 ml/kg of distilled water, group 2 received 130 mg/kg of T. catappa aqueous extract, groups 3-5 were diabetic and received 0.2 ml/g distilled water, 130 mg/kg T. catappa extract and 0.75 IU/kg insulin respectively for 14 days. Hematological parameters were measured and an oral glucose tolerance test was carried out using 2 g/kg body weight glucose. A histological analysis of the pancreas was done. Twenty-five compounds identified as flavonoids, phenolic acids, tannins, and triterpenoids were detected. The blood glucose levels were significantly (p <0.05) elevated in DM groups but were significantly (p <0.05) reduced following Terminalia catappa leaves extract to DM groups. There was s significant (p <0.05) increase in insulin levels improved hematological parameters (RBC, WBC, and platelets), and increased islet population. These results suggest that T. catappa extract has hypoglycemic, insulinogenic, and hematopoietic potentials in diabetic condition and offer protection to the pancreas which could be attributed to the phytochemical constituents thereby justifying its use in traditional therapy.

1735-P: The Dichotomic and Timely Regulation of SerpinB13 Plays a Role in Beta-Cell Neogenesis and Pancreatic Islet Expansion

There has been recent emphasis on the potential role of a functional crosstalk between the exocrine and endocrine pancreas in the development of type 1 diabetes (T1D). Our interest in this was sparked by our observations that, (i) serpinB13, a protease inhibitor of cathepsin L, is expressed in mouse pancreatic ductal cells, and (ii) an antibody response to serpinB13 is associated with better clinical outcomes in mice and humans with T1D. The exact pattern of serpinB13 expression in the human pancreas has, to date, not been examined. Similarly, the exact role of serpinB13 in beta-cell biology and diabetes remains unclear. We aimed to determine the distribution of serpinB13 expression using immunochemistry on pancreatic sections obtained from nPOD. In addition, we developed a mouse serpinB13 genetic knockout model to examine the role of this serpin in regulating pancreatic islet morphology. Visiopharm software was used for an unbiased quantitative image analysis of islet size and cellularity. We found that in healthy humans, serpinB13 expression was confined to the pancreatic ducts, mimicking the pattern we observed previously in rodents. Studies of serpinB13 genetic deficiency revealed that early inhibition of serpinB13 increased the small pancreatic islet population while with time this trend reversed, and a drop in the number of pancreatic islets was observed. Our results demonstrate that serpinB13 is expressed in the exocrine epithelial compartment of the human pancreas. In addition, quite unexpectedly, serpinB13 appears to play a dual role in regulating pancreatic islet mass. More specifically, we postulate that serpinB13 initially hampers the development of additional insulin-producing cells, but with time it promotes expansion of the already formed beta cells. Thus, we propose an approach that first impedes serpinB13, followed by later administration of this serpin, which may lead to an increase in beta-cell mass and improved resistance to T1D. Disclosure Y.Kryvalap: None. S.Z.Meng: None. R.Habte: None. J.Czyzyk: None.

Diet of the Insular Lizard, Podarcis lilfordi (Günther, 1874): Complementary Morphological and Molecular Approaches.

The diets of insular lizards are extremely varied, depending on the different environmental characteristics of each island population. This is particularly evident in the case of the populations of small coastal islets of the Balearic Islands, where the Balearic lizard, Podarcis lilfordi, is found. The study of trophic ecology carried out by means of traditional tools, such as morphological analysis of feces, has made it possible to detect numerous prey and nutritional elements. However, these methods are clearly insufficient, as some rare groups are not detected. It is also difficult to identify remains of marine subsidies or of foods contributed to these small islands by other predators, such as seabirds. The current study demonstrates the advantages of combining morphological diet analysis with the molecular study of individual feces samples obtained from the same populations. We obtained a greater diversity of prey groups using the combined methodologies, with each method identifying prey items that were not detected using the other method. Particularly, the study of diets at the molecular level identified plant species consumed by lizards that were, occasionally, not identified in morphological analyses. Conversely, the traditional morphological study of an equivalent number of fecal samples allowed for the identification of several prey groups that had not been detected in the molecular study. From this viewpoint, the advantages and disadvantages of each methodology are discussed.

Reduced volume of diabetic pancreatic islets in rodents detected by synchrotron X-ray phase-contrast microtomography and deep learning network

The pancreatic islet is a highly structured micro-organ that produces insulin in response to rising blood glucose. Here we develop a label-free and automatic imaging approach to visualize the islets in situ in diabetic rodents by the synchrotron radiation X-ray phase-contrast microtomography (SRμCT) at the ID17 station of the European Synchrotron Radiation Facility. The large-size images (3.2mm×15.97mm) were acquired in the pancreas in STZ-treated mice and diabetic GK rats. Each pancreas was dissected by 3000 reconstructed images. The image datasets were further analysed by a self-developed deep learning method, AA-Net. All islets in the pancreas were segmented and visualized by the three-dimension (3D) reconstruction. After quantifying the volumes of the islets, we found that the number of larger islets (=>1500 μm3) was reduced by 2-fold (wt 1004±94 vs GK 419±122, P<0.001) in chronically developed diabetic GK rat, while in STZ-treated diabetic mouse the large islets were decreased by half (189±33 vs 90±29, P<0.001) compared to the untreated mice. Our study provides a label-free tool for detecting and quantifying pancreatic islets in situ. It implies the possibility of monitoring the state of pancreatic islets in vivo diabetes without labelling.

1467-P: Chemical Profiles of Acinar Tissue and Pancreatic Islets from Healthy and T2D-Affected Pancreata

Both the endocrine and exocrine pancreas demonstrate morphological and chemical changes associated with T2D development. Despite significant progress, chemical profiles of the islets and acinar tissue following disease progression are not well characterized. In this work we investigated isolated populations of pancreatic islets (n=18) and acinar tissues (n=7) collected from healthy and T2D affected pancreata using several mass spectrometry (MS) approaches. Samples are provided by the ADI IsletCore and the Network for Pancreatic Organ Donors with Diabetes (nPOD) . Both peptide and metabolite profiles were determined and compared via chemical profiling with matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance MS. Not surprisingly, the peptide profiles from T2D islets demonstrated lower signal intensities compared to healthy islets (ROC analysis, AUC=0.9) . Somatostatin, pancreatic icosapeptide, and insulin-related signals are most decreased. Preliminary data for acinar tissues indicate lower peptide levels in samples collected from T2D-affected (n=2) vs. healthy (n=5) pancreata. Several unique peptides are detected specifically in acinar tissue obtained from T2D-affected pancreata. Liquid chromatography-MS reveals a complex endogenous peptide complement of acinar tissue. Analysis of extracellular media for both islet types demonstrated similar differences (AUC=0.7) indicating higher levels of observed peptides in preparations from healthy islets, perhaps suggesting that peptides are released from the islets during shipment. Integrity of received islets was verified using dithizone staining prior to measurement. In contrast, almost no peptide markers of acinar tissue are found in corresponding extracellular media. Our results enhance knowledge on biochemical signature of T2D in the pancreas. Disclosure S.Rubakhin: None. E.V.Romanova: None. J.V.Sweedler: None. Funding American Diabetes Association (1-18-VSN-19)

Hexacosylidenecyclohexane inhibits enzymatic breakdown of dietary sugars and modulates glucose homeostasis

BackgroundThe quest for benign drug to treat diabetes and its complications is a major scientific search. The investigation evaluates the antidiabetic properties of hexacosylidenecyclohexane purified from Madhuca longifolia by advanced chromatographic approach. MethodsChloroform, methanol and hexane fractions were analysed by TLC and HPLC to isolate the active molecule followed with structure elucidation by LC-MS, FTIR, CHN and NMR spectroscopy. In vitro and in vivo antidiabetic methods were adopted to ascertain the potency of the active molecule. Drug-likeness and ADMET analysis of the molecule were carried out using Molinspiration, SwissADME, DruLiTo software, ADMETlab and admetSAR prediction tools. ResultsBioassay guided fractionation and purification of the crude extract resulted in 25 fractions; MBCF1 to MBCF25 (M. longifolia bark chloroform fraction). MBCF2 showed effective in vitro inhibition of aldose reductase, α-amylase and α-glucosidase with an IC50 value of 85, 30 and 21 µg/mL, respectively. The active molecule, hexacosylidenecyclohexane at a dose of 50 mg /kg b.w., reduced the blood glucose level to 239 from 340 mg /dL on day 14 (experimental period) and at 25 mg /kg b.w., from 348 to 277 mg /dL. It showed significant reversal of lipid profile and bile acids. Histopathological studies revealed the protective effect on cellular population and size of islets. Drug-likeness property was in the acceptable range with no toxic effect as predicted by ADMET analysis. ConclusionThe findings showed that, the active molecule hexacosylidenecyclohexane possesses potential antidiabetic effect with no toxic effect.

Mof acetyltransferase inhibition ameliorates glucose intolerance and islet dysfunction of type 2 diabetes via targeting pancreatic α-cells

BackgroundPreviously, we reported that Mof was highly expressed in α-cells, and its knockdown led to ameliorated fasting blood glucose (FBG) and glucose tolerance in non-diabetic mice, attributed by reduced total α-cell but enhanced prohormone convertase (PC)1/3-positive α-cell mass. However, how Mof and histone 4 lysine 16 acetylation (H4K16ac) control α-cell and whether Mof inhibition improves glucose handling in type 2 diabetes (T2DM) mice remain unknown. MethodsMof overexpression and chromatin immunoprecipitation sequence (ChIP-seq) based on H4K16ac were applied to determine the effect of Mof on α-cell transcriptional factors and underlying mechanism. Then we administrated mg149 to α-TC1-6 cell line, wild type, db/db and diet-induced obesity (DIO) mice to observe the impact of Mof inhibition in vitro and in vivo. In vitro, western blotting and TUNEL staining were used to examine α-cell apoptosis and function. In vivo, glucose tolerance, hormone levels, islet population, α-cell ratio and the co-staining of glucagon and PC1/3 or PC2 were examined. ResultsMof activated α-cell-specific transcriptional network. ChIP-seq results indicated that H4K16ac targeted essential genes regulating α-cell differentiation and function. Mof activity inhibition in vitro caused impaired α-cell function and enhanced apoptosis. In vivo, it contributed to ameliorated glucose intolerance and islet dysfunction, characterized by decreased fasting glucagon and elevated post-challenge insulin levels in T2DM mice. ConclusionMof regulates α-cell differentiation and function via acetylating H4K16ac and H4K16ac binding to Pax6 and Foxa2 promoters. Mof inhibition may be a potential interventional target for T2DM, which led to decreased α-cell ratio but increased PC1/3-positive α-cells.

1144-P: Chemical Assessment of Populations of Human Pancreatic Islets Isolated for Transplantation

Clinical transplantation of human cadaveric islets is an important therapy for the treatment of T1D. Such transplantation has demonstrated safety and efficacy in academic and clinical trials. Quality control during all stages of islet isolation, handling, and transplantation is critical for success of the therapy. A variety of tests are used to assess cellular integrity, islet population purity, and potency. Unfortunately, most of applied tests are not evaluating individual pancreatic islet chemical compositions which are indicative of overall islets functional and health state. Here we describe the results of mass spectrometric (MS) assessment of populations of individual pancreatic islets received from seven donors including four T2D cases. Random sampling and MS analysis of 232 islet-like structures detected 150 islets (based on size, morphology, and visual inspection). Our findings correlate (R2=0.9) with data on sample purity for different batches provided by the ADI IsletCore. The classic pancreatic islet hormones are observed in the majority of islets. Peptide profiles are mostly dominated by insulin, although we observe a greater variability of insulin peaks than somatostatin- and glucagon-related signals by mass spectrometry. No significant differences in our results are observed between healthy and T2D-affected islets. Fast mass-spectrometry-based assessment allowed selection of representative individual islets for liquid chromatography-MS (LC-MS), which provides an in-depth chemical coverage. Using LC-MS, a number of histone-related peptides are detected significantly expanding analyte coverage. To increase the throughput of direct MS analysis of islets, we use our optically guided mass spectrometry approach to automate the localization and analysis of larger islet populations. Using this technology and MS detection, hundreds of islets can be profiled for presence of a variety of metabolites, lipids, and peptides during a several-hour timespan. Disclosure S. Rubakhin: None. E. V. Romanova: None. J. V. Sweedler: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-18-VSN-19 to J.V.S.)

T cells accumulate in non-diabetic islets during ageing

BackgroundThe resident immune population of pancreatic islets has roles in islet development, beta cell physiology, and the pathology of diabetes. These roles have largely been attributed to islet macrophages, comprising 90% of islet immune cells (in the absence of islet autoimmunity), and, in the case of type 1 diabetes, to infiltrating autoreactive T cells. In adipose, tissue-resident and recruited T and B cells have been implicated in the development of insulin resistance during diet-induced obesity and ageing, but whether this is paralleled in the pancreatic islets is not known. Here, we investigated the non-macrophage component of resident islet immune cells in islets isolated from C57BL/6 J male mice during ageing (3 to 24 months of age) and following similar weight gain achieved by 12 weeks of 60% high fat diet. Immune cells were also examined by flow cytometry in cadaveric non-diabetic human islets.ResultsImmune cells comprised 2.7 ± 1.3% of total islet cells in non-diabetic mouse islets, and 2.3 ± 1.7% of total islet cells in non-diabetic human islets. In 3-month old mice on standard diet, B and T cells each comprised approximately 2–4% of the total islet immune cell compartment, and approximately 0.1% of total islet cells. A similar amount of T cells were present in non-diabetic human islets. The majority of islet T cells expressed the αβ T cell receptor, and were comprised of CD8-positive, CD4-positive, and regulatory T cells, with a minor population of γδ T cells. Interestingly, the number of islet T cells increased linearly (R2 = 0.9902) with age from 0.10 ± 0.05% (3 months) to 0.38 ± 0.11% (24 months) of islet cells. This increase was uncoupled from body weight, and was not phenocopied by a degree similar weight gain induced by high fat diet in mice.ConclusionsThis study reveals that T cells are a part of the normal islet immune population in mouse and human islets, and accumulate in islets during ageing in a body weight-independent manner. Though comprising only a small subset of the immune cells within islets, islet T cells may play a role in the physiology of islet ageing.

Rapid liquid chromatography-mass spectrometry quantitation of glucose-regulating hormones from human islets of Langerhans

Glucose homeostasis is maintained through the secretion of peptide hormones, such as insulin, somatostatin, and glucagon, from islets of Langerhans, clusters of endocrine cells found in the pancreas. This report describes an LC-MS method using multiple reaction monitoring for quantitation of insulin, C-peptide, glucagon, and somatostatin secretion from human islet populations. For rapid analysis, a 5 min separation was achieved using a 2.1 × 30 mm (i.d. x length) C18 column with 2.7 µm diameter core shell particles. A sacrificial protein hydrolysate was used with the sample and found to improve signal magnitude, repeatability, and to reduce carryover between runs. At optimized gradient conditions, the gradient run time was 4.55 min producing an average peak width of 0.3 min, a minimum resolution of 1.2, and a peak capacity of 20. As a proof of concept, the method was used to measure secretions from static incubations of human islets from 2 donors. Insulin and C-peptide were quantified and matched well with literature values of these hormones. We expect that this antibody-free quantitation of multiple hormones secreted from islets will provide insights into the temporal relationships of these peptides in the future.

Fluorescence lifetime metabolic mapping of hypoxia-induced damage in pancreatic pseudo-islets.

Pancreatic islet isolation from donor pancreases is an essential step for the transplantation of insulin-secreting β-cells as a therapy to treat type 1 diabetes mellitus. This process however damages islet basement membranes, which can lead to islet dysfunction or death. Posttransplantation, islets are further stressed by a hypoxic environment and immune reactions that cause poor engraftment and graft failure. The current standards to assess islet quality before transplantation are destructive procedures, performed on a small islet population that does not reflect the heterogeneity of large isolated islet batches. In this study, we incorporated fluorescence lifetime imaging microscopy (FLIM) into a pancreas-on-chip system to establish a protocol to noninvasively assess the viability and functionality of pancreatic β-cells in a three-dimensional in vitro model (= pseudo-islets). We demonstrate how (pre-) hypoxic β-cell-composed pseudo-islets can be discriminated from healthy functional pseudo-islets according to their FLIM-based metabolic profiles. The use of FLIM during the pretransplantation pancreatic islet selection process has the potential to improve the outcome of β-cell islet transplantation.

Inter- and intra-site differences in wild goat (Capra aegagrus) habitat quality detected through male horn size comparisons in Greek islands

In caprid taxa, horn size is correlated with body mass, being a potential indicator of habitat quality. In this study, horn length and circumference measurements of, genetically similar, native island (Samaria National Forest Park, Crete) and introduced islet (a Game Breeding Station, and a Controlled Hunting Zone) populations of the Vulnerable agrimi were compared and evaluated with respect to population size information. Horns belonged to male animals that died between 1975 and 2016. There were significant age-related differences in inner horn length (annual increments and total) and annulus circumference between the three areas reflecting differences in food resources. Longest horns currently grow in the Controlled Hunting Zone. Length appeared negatively associated with population density. Results, also, showed that, in the short term, forage improvement can reverse horn size decrease trends. To support conservation efforts, it is suggested that native range and islet managers should establish horn measuring in monitoring practices.

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human isletsamples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

Gene expression patterns in synchronized islet populations

ABSTRACTIn vivo levels of insulin are oscillatory with a period of ~5–10 minutes, indicating that the islets of Langerhans within the pancreas are synchronized. While the synchronizing factors are still under investigation, one result of this behavior is expected to be coordinated and oscillatory intracellular factors, such as intracellular Ca2+ levels, throughout the islet population. In other cell types, oscillatory intracellular signals, like intracellular Ca2+, have been shown to affect specific gene expression. To test how the gene expression landscape may differ between a synchronized islet population with its reproducible intracellular oscillations and an unsynchronized islet population with heterogeneous oscillations, gene set enrichment analysis (GSEA) was used to compare an islet population that had been synchronized using a glucose wave with a 5-min period, and an unsynchronized islet population. In the population exposed to the glucose wave, 58/62 islets showed synchronization as evidenced by coordinated intracellular Ca2+ oscillations with an average oscillation period of 5.1 min, while in the unsynchronized population 29/62 islets showed slow oscillations with an average period of 5.2 min. The synchronized islets also had a significantly smaller drift of their oscillation period during the experiment as compared to the unsynchronized population. GSEA indicated that the synchronized population had reduced expression of gene sets related to protein translation, protein turnover, energy expenditure, and insulin synthesis, while those that were related to maintenance of cell morphology were increased.

Synchronization of pancreatic islets by periodic or non-periodic muscarinic agonist pulse trains.

Pulsatile insulin secretion into the portal vein from the many pancreatic islets of Langerhans is critical for efficient glucose homeostasis. The islets are themselves endogenous oscillators, but since they are not physically coupled it is not obvious how their oscillations are synchronized across the pancreas. It has been proposed that synchronization of islets is achieved through periodic activity of intrapancreatic ganglia, and indeed there are data supporting this proposal. Postganglionic nerves are cholinergic, and their product, acetylcholine, can influence islet β-cells through actions on M3 muscarinic receptors which are coupled to Gq type G-proteins. In addition, the neurons secrete several peptide hormones that act on β-cell receptors. The data supporting synchronization via intrapancreatic ganglia are, however, limited. In particular, it has not been shown that trains of muscarinic pulses are effective at synchronizing islets in vitro. Also, if as has been suggested, there is a ganglionic pacemaker driving islets to a preferred frequency, no neural circuitry for this pacemaker has been identified. In this study, both points are addressed using a microfluidic system that allows for the pulsed application of the muscarinic agonist carbachol. We find that murine islets are entrained and synchronized over a wide range of frequencies when the carbachol pulsing is periodic, adding support to the hypothesis that ganglia can synchronize islets in vivo. We also find that islet synchronization is very effective even if the carbachol pulses are applied at random times. This suggests that a neural pacemaker is not needed; all that is required is that islets receive occasional coordinated input from postganglionic neurons. The endogenous rhythmic activity of the islets then sets the frequency of the islet population rhythm, while the input from ganglia acts only to keep the islet oscillators in phase.

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

ObjectiveIslets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. MethodsWe have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). ResultsBy increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 μm2 area (∼210 μm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. ConclusionsWe have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research.

Insulin secretion kinetics from single islets reveals distinct subpopulations

Type II diabetes progresses with inadequate insulin secretion and prolonged elevated circulating glucose levels. Also, pancreatic islets isolated for transplantation or tissue engineering can be exposed to glucose over extended timeframe. We hypothesized that isolated pancreatic islets can secrete insulin over a prolonged period of time when incubated in glucose solution and that not all islets release insulin in unison. Insulin secretion kinetics was examined and modeled from single mouse islets in response to chronic glucose exposure (2.8-20 mM). Results with single islets were compared to those from pools of islets. Kinetic analysis of 58 single islets over 72 h in response to elevated glucose revealed distinct insulin secretion profiles: slow-, fast-, and constant-rate secretors, with slow-secretors being most prominent (ca., 50%). Variations in the temporal response to glucose therefore exist. During short-term (<4 h) exposure to elevated glucose few islets are responding with sustained insulin release. The model allowed studying the influence of islet size, revealing no clear effect. At high-glucose concentrations, when secretion is normalized to islet volume, the tendency is that smaller islets secrete more insulin. At high-glucose concentrations, insulin secretion from single islets is representative of islet populations, while under low-glucose conditions pooled islets did not behave as single ones. The characterization of insulin secretion over prolonged periods complements studies on insulin secretion performed over short timeframe. Further investigation of these differences in secretion profiles may resolve open-ended questions on pre-diabetic conditions and transplanted islets performance. This study deliberates the importance of size of islets in insulin secretion. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1059-1068, 2018.