Healthy Islets Research Articles

Islet cell spheroids produced by a thermally sensitive scaffold: a new diabetes treatment

The primary issues in treating type 1 diabetes mellitus (T1DM) through the transplantation of healthy islets or islet β-cells are graft rejection and a lack of available donors. Currently, the majority of approaches use cell encapsulation technology and transplant replacement cells that can release insulin to address transplant rejection and donor shortages. However, existing encapsulation materials merely serve as carriers for islet cell growth. A new treatment approach for T1DM could be developed by creating a smart responsive material that encourages the formation of islet cell spheroids to replicate their 3D connections in vivo and controls the release of insulin aggregates. In this study, we used microfluidics to create thermally sensitive porous scaffolds made of poly(N-isopropyl acrylamide)/graphene oxide (PNIPAM/GO). The material was carefully shrunk under near-infrared light, enriched with mouse insulinoma pancreatic β cells (β-TC-6 cells), encapsulated, and cultivated to form 3D cell spheroids. The controlled contraction of the thermally responsive porous scaffold regulated insulin release from the spheroids, demonstrated using the glucose-stimulated insulin release assay (GSIS), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay. Eventually, implantation of the spheroids into C57BL/6 N diabetic mice enhanced the therapeutic effect, potentially offering a novel approach to the management of T1DM.Graphical

Detecting key genes relative expression orderings as biomarkers for machine learning-based intelligent screening and analysis of type 2 diabetes mellitus

Type 2 diabetes (T2D), a prevalent chronic metabolic disease of public health concern, is growing rapidly worldwide. In order to characterize robust transcriptomic differences between T2D and the healthy pancreatic islets, we proposed a novel data analysis approach based on the within-sample relative expression orderings of genes (REOs) and machine learning, which can efficiently integrate data from diverse gene expression datasets and help discover some potential signatures. We first identified the overlapped disease-specific reverse REOs from three bulk islets expression profiles that are substantially distinct. Subsequently, the minimum-Redundancy-Maximum-Relevance (mRMR) combined with incremental feature selection (IFS) strategy was applied to select the optimal reverse REOs by using the support vector machine (SVM). As a result, 7 optimal reverse REOs encompassing 12 genes were found, which could produce encouraging predictive performance on training data, independent testing, and external islets data, showcasing robust prediction capability and superior generalization despite the heterogeneity of data. Furthermore, the analysis of differential expression gene (DEG) in bulk islets data and pseudobulk method in single-cell RNA sequencing (scRNA-seq) islets data reaffirmed the crucial roles of these 7 REOs in islets function and disease progression. In conclusion, these REOs are robust biomarkers in T2D and may also be potential therapeutic targets for T2D. We hope these findings can help elucidate the underlying pathogenic mechanisms of T2D and contribute to the treatment of diabetes. The source code is available at https://github.com/xiexq007/T2D-REO.

Pharmacologic inhibition of somatostatin receptor 2 to restore glucagon counterregulation in diabetes.

Glucose homeostasis is primarily maintained by pancreatic hormones, insulin and glucagon, with an emerging role for a third islet hormone, somatostatin, in regulating insulin and glucagon responses. Under healthy conditions, somatostatin secreted from pancreatic islet δ-cells inhibits both insulin and glucagon release through somatostatin receptor- induced cAMP-mediated downregulation and paracrine inhibition of β- and α-cells, respectively. Since glucagon is the body's most important anti-hypoglycemic hormone, and because glucagon counterregulation to hypoglycemia is lost in diabetes, the study of somatostatin biology has led to new investigational medications now in development that may help to restore glucagon counterregulation in type 1 diabetes. This review highlights the normal regulatory role of pancreatic somatostatin signaling in healthy islet function and how the inhibition of somatostatin receptor signaling in pancreatic α-cells may restore normal glucagon counterregulation in diabetes mellitus.

CSF1R inhibition with PLX5622 affects multiple immune cell compartments and induces tissue-specific metabolic effects in lean mice

Aims/hypothesisColony stimulating factor 1 (CSF1) promotes the proliferation, differentiation and survival of macrophages, which have been implicated in both beneficial and detrimental effects on glucose metabolism. However, the physiological role of CSF1 signalling in glucose homeostasis and the potential therapeutic implications of modulating this pathway are not known. We aimed to study the composition of tissue macrophages (and other immune cells) following CSF1 receptor (CSF1R) inhibition and elucidate the metabolic consequences of CSF1R inhibition.MethodsWe assessed immune cell populations in various organs by flow cytometry, and tissue-specific metabolic effects by hyperinsulinaemic–euglycaemic clamps and insulin secretion assays in mice fed a chow diet containing PLX5622 (a CSF1R inhibitor) or a control diet.ResultsCSF1R inhibition depleted macrophages in multiple tissues while simultaneously increasing eosinophils and group 2 innate lymphoid cells. These immunological changes were consistent across different organs and were sex independent and reversible after cessation of the PLX5622. CSF1R inhibition improved hepatic insulin sensitivity but concomitantly impaired insulin secretion. In healthy islets, we found a high frequency of IL-1β+ islet macrophages. Their depletion by CSF1R inhibition led to downregulation of macrophage-related pathways and mediators of cytokine activity, including Nlrp3, suggesting IL-1β as a candidate insulin secretagogue. Partial restoration of physiological insulin secretion was achieved by injecting recombinant IL-1β prior to glucose stimulation in mice lacking macrophages.Conclusions/interpretationMacrophages and macrophage-derived factors, such as IL-1β, play an important role in physiological insulin secretion. A better understanding of the tissue-specific effects of CSF1R inhibition on immune cells and glucose homeostasis is crucial for the development of targeted immune-modulatory treatments in metabolic disease.Data availabilityThe RNA-Seq dataset is available in the Gene Expression Omnibus (GEO) under the accession number GSE189434 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189434).Graphical

Inhibition of hyaluronan synthesis prevents β-cell loss in obesity-associated type 2 diabetes

Pancreatic β-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low concentrations of HA were present in healthy pancreatic islets. However, HA substantially accumulated in cadaveric islets of T2D patients and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the main HA receptor CD44, preserved glycemic control and insulin concentrations in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserved glycemic control in other settings of β-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we found that 4-MU increased the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on β-cells such that caspase 3 activation did not result in β-cell apoptosis. These data indicated a role for HA accumulation in diabetes pathogenesis and suggested that it may be a viable target to ameliorate β-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Global proteomics reveals insulin abundance as a marker of human islet homeostasis alterations.

The variation in quality between the human islet samples represents a major problem for research, especially when used as control material. The assays assessing the quality of human islets used in research are non-standardized and limited, with many important parameters not being consistently assessed. High-throughput studies aimed at characterizing the diversity and segregation markers among apparently functionally healthy islet preps are thus a requirement. Here, we designed a pilot study to comprehensively identify the diversity of global proteome signatures and the deviation from normal homeostasis in randomly selected human-isolated islet samples. By using Tandem Mass Tag 16-plex proteomics, we focused on the recurrently observed disparity in the detected insulin abundance between the samples, used it as a segregating parameter, and analyzed the correlated changes in the proteome signature and homeostasis by pathway analysis. In this pilot study, we showed that insulin protein abundance is a predictor of human islet homeostasis and quality. This parameter is independent of other quality predictors within their acceptable range, thus being able to further stratify islets samples of apparent good quality. Human islets with low amounts of insulin displayed changes in their metabolic and signaling profile, especially in regard to energy homeostasis and cell identity maintenance. We further showed that xenotransplantation into diabetic hosts is not expected to improve the pre-transplantation signature, as it has a negative effect on energy balance, antioxidant activity, and islet cell identity. Insulin protein abundance predicts significant changes in human islet homeostasis among random samples of apparently good quality.

Gut dysbiosis promotes islet-autoimmunity by increasing T-cell attraction in islets via CXCL10 chemokine

CXCL10 is an IFNγ-inducible chemokine implicated in the pathogenesis of type 1 diabetes. T-cells attracted to pancreatic islets produce IFNγ, but it is unclear what attracts the first IFNγ -producing T-cells in islets. Gut dysbiosis following administration of pathobionts induced CXCL10 expression in pancreatic islets of healthy non-diabetes-prone (C57BL/6) mice and depended on TLR4-signaling, and in non-obese diabetic (NOD) mice, gut dysbiosis induced also CXCR3 chemokine receptor in IGRP-reactive islet-specific T-cells in pancreatic lymph node. In amounts typical to low-grade endotoxemia, bacterial lipopolysaccharide induced CXCL10 production in isolated islets of wild type and RAG1 or IFNG−receptor-deficient but not type-I–IFN–receptor-deficient NOD mice, dissociating lipopolysaccharide-induced CXCL10 production from T-cells and IFNγ. Although mostly myeloid-cell dependent, also β-cells showed activation of innate immune signaling pathways and Cxcl10 expression in response to lipopolysaccharide indicating their independent sensitivity to dysbiosis. Thus, CXCL10 induction in response to low levels of lipopolysaccharide may allow islet-specific T-cells imprinted in pancreatic lymph node to enter in healthy islets independently of IFN-g, and thus link gut dysbiosis to early islet-autoimmunity via dysbiosis-associated low-grade endotoxemia.

5-LB: Rescuing Alpha-Cell Function in Type 1 Diabetes

Increased glucagon secretion from the pancreatic alpha cell is the first and most important defense against hypoglycemia. In T1D this defense mechanism is lost. We propose that the loss of the neighboring beta cells causes, and in turn the loss of inhibitory signals they generated, is a key factor in the inability of alpha cells to respond appropriately to blood sugar levels. The sudden cessation of this inhibitory input during hypoglycemia is a necessary signal for the glucagon response. We hypothesize that reactivation of endogenous paracrine and autocrine signaling might restore the alpha cell’s ability to respond to hypoglycemia in T1D. We used isolated islets and tissue slices from non-diabetic organ donors and donors with T1D and measured dynamic hormone secretion and calcium recordings. We found that alpha cell responses are briefer than the sustained beta cell responses. If not reset by inhibitory input, the alpha cell is not able to respond again. Glucagon secretion can be recovered by activation of paracrine signals to inhibit the alpha cell. We found full glucagon recovery if reset for 15 min or longer using high glucose in healthy islets (no reset 0.51 +/- 0.09 vs. 5 min reset 0.99 +/- 0.12 vs. 15 min 1.63 +/- 0.31, fold change to baseline +/- SEM). In tissues from T1D donors, alpha cells failed to respond to decreases in glucose concentration despite normal glucagon content and responses to KCl depolarization. Baseline glucagon secretion was significantly elevated compared to healthy individuals (mean 25.93 +/- SEM 8.55 pM in T1D vs 7.49 +/- 2.21, p<0.05). Furthermore, we found severely diminished Ca2+ responses to both lowering glucose concentration and glutamate receptor stimulation. We demonstrate that alpha cells from T1D donors cannot mount an efficient glucagon response due to deficient glutamate receptor signaling and loss of paracrine inhibitory input. Our results suggest that restoring both signals rescues glucagon secretion. Disclosure J. Panzer: None. A. Pugliese: Consultant; Provention Bio, Inc. Funding American Diabetes Association (4-22-PDFPM-12 to J.P.); The Leona M. and Harry B. Helmsley Charitable Trust (2018PG-T1D053, G-2108-04793)

1775-P: Liraglutide Corrects Impaired Insulin Secretion in 14-3-3-ζ Overexpressing Mice

Crosstalk between α-cells and β-cells is an important regulator of healthy islet function. α-cells display heterogeneity in their hormone profile. α-cells typically produce glucagon but, under certain conditions, can produce glucagon-like peptide-1 (GLP-1). However, the mechanisms regulating α-cell GLP-1 production are poorly understood. We found that increased β-cell GLP-1R signaling via the GLP-1 analog, liraglutide, activates α-cell GLP-1 production through a paracrine signaling pathway. We recently identified the protein 14-3-3-ζ as a key downstream mediator of this pathway. Specifically, liraglutide downregulates β-cell 14-3-3-ζ expression, which activates α-cell GLP-1 production. Consistent with this, we found that β-cell 14-3-3-ζ ablation enhances glucose-stimulated insulin secretion (GSIS). Furthermore, mice with 14-3-3-ζ overexpression (OE) have impaired GSIS. Therefore, we tested the hypothesis that liraglutide can correct the effect of 14-3-3-ζ OE to impair GSIS. Male and female wild-type (WT) or 14-3-3-ζ OE mice maintained on high-fat diet underwent an oral glucose tolerance test (OGTT) after 2 weeks of liraglutide (200 μg/kg sc twice daily) or saline treatment. Liraglutide improved glucose tolerance in WT mice and tended to improve glucose tolerance in OE mice. Saline-treated 14-3-3-ζ OE mice exhibited lower GSIS compared with WT (P<0.05). Liraglutide treatment of 14-3-3-ζ OE mice normalized GSIS to match that of liraglutide-treated WT mice (Insulin secretion index [Insulin AUC/Glucose AUC] *10-9: Saline WT = 38 ± 4, Lira WT = 37 ± 5, Saline OE = 25 ± 4, Lira OE = 39 ± 7; n=7-8 per group, P<0.05 Saline WT vs. Saline OE, P=0.06 Saline OE vs. Lira OE). Overall, these data suggest that liraglutide can rescue deficits in GSIS induced by 14-3-3-ζ OE. Disclosure R.Shishani: None. M.Tucker: None. R.Moreno: None. J.M.Bustamante: None. G.E.Lim: None. B.Cummings: None. Funding Hartwell Foundation; National Institute of Diabetes and Digestive and Kidney Diseases (R56DK124853)

Disrupting of family with sequence similarity 105, member A (Fam105a) deteriorates pancreatic β-cell physiology and insulin secretion in INS-1 cells

The role of “Family with sequence similarity 105, member A” (FAM105A) in pancreatic β-cell function in relation to type 2 diabetes mellitus (T2D) is not fully understood. To address this issue, various molecular and functional experiments were conducted on primary human islets and INS-1 cells. RNA-seq expression analysis showed that FAM105A is highly expressed in human islets and its expression is reduced in diabetic islets compared to healthy islets. FAM105A expression correlated negatively with HbA1c levels and body mass index (BMI). Co-expression analysis showed a significant correlation between FAM105A with PDX1, GCK, GLUT1 and INSR, but not the INS gene. Silencing of Fam105a impaired insulin release, content, glucose uptake, and mitochondria ATP content but did not affect cell viability, reactive oxygen species (ROS) or apoptosis levels. Silencing of Fam105a was associated with reduced Pdx1 and Glut2 expression at mRNA and protein levels. RNA-seq analysis of dysregulated genes in Fam105a-silenced cells showed an overall downregulation of gene expression in β-cells and insulin secretion pathway. Disrupting Pdx1 did not affect Fam105a expression in INS-1 cells. Overall, the results suggest that FAM105A plays an important role in pancreatic β-cells biology and may be involved in the development of T2D.

A selective nonpeptide somatostatin receptor 5 agonist effectively decreases insulin secretion in hyperinsulinism

Congenital hyperinsulinism (HI), a beta cell disorder most commonly caused by inactivating mutations of beta cell KATP channels, results in dysregulated insulin secretion and persistent hypoglycemia. Children with KATP-HI are unresponsive to diazoxide, the only FDA-approved drug for HI, and utility of octreotide, the second line therapy, is limited because of poor efficacy, desensitization, and somatostatin receptor type 2 (SST2)-mediated side effects. Selective targeting of SST5, an SST receptor associated with potent insulin secretion suppression, presents a new avenue for HI therapy. Here, we determined that CRN02481, a highly selective nonpeptide SST5 agonist, significantly decreased basal and amino acid-stimulated insulin secretion in both Sur1-/- (a model for KATP-HI) and wild type mouse islets. Oral administration of CRN02481 significantly increased fasting glucose and prevented fasting hypoglycemia compared to vehicle in Sur1-/- mice. During a glucose tolerance test, CRN02481 significantly increased glucose excursion in both WT and Sur1-/- mice compared to control. CRN02481 also reduced glucose- and tolbutamide-stimulated insulin secretion from healthy, control human islets similar to the effects observed with SS14 and peptide somatostatin analogs. Moreover, CRN02481 significantly decreased glucose- and amino acid-stimulated insulin secretion in islets from two infants with KATP-HI and one with Beckwith-Weideman Syndrome-HI. Taken together, these data demonstrate that a potent and selective SST5 agonist effectively prevents fasting hypoglycemia and suppresses insulin secretion not only in a KATP-HI mouse model, but also in healthy human islets and islets from HI patients.

Chromatin accessibility differences between alpha, beta, and delta cells identifies common and cell type-specific enhancers

BackgroundHigh throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity.ResultsHere, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise.ConclusionsBoth alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.

Treatment with semaglutide, a GLP-1 receptor agonist, improves extracellular matrix remodeling in the pancreatic islet of diet-induced obese mice

AimsThe extracellular matrix (ECM) is fundamental for the normal endocrine functions of pancreatic islet cells and plays key roles in the pathophysiology of type 2 diabetes. Here we investigated the turnover of islet ECM components, including islet amyloid polypeptide (IAPP), in an obese mouse model treated with semaglutide, a glucagon-like peptide type 1 receptor agonist. Main methodsMale one-month-old C57BL/6 mice were fed a control diet (C) or a high-fat diet (HF) for 16 weeks, then treated with semaglutide (subcutaneous 40 μg/kg every three days) for an additional four weeks (HFS). The islets were immunostained and gene expressions were assessed. Key findingsComparisons refer to HFS vs HF. Thus, IAPP immunolabeling and beta-cell-enriched beta-amyloid precursor protein cleaving enzyme (Bace2, −40 %) and heparanase immunolabeling and gene (Hpse, −40 %) were mitigated by semaglutide. In contrast, perlecan (Hspg2, +900 %) and vascular endothelial growth factor A (Vegfa, +420 %) were enhanced by semaglutide. Also, semaglutide lessened syndecan 4 (Sdc4, −65 %) and hyaluronan synthases (Has1, −45 %; Has2, −65 %) as well as chondroitin sulfate immunolabeling, and collagen type 1 (Col1a1, −60 %) and type 6 (Col6a3, −15 %), lysyl oxidase (Lox, −30 %) and metalloproteinases (Mmp2, −45 %; Mmp9, −60 %). SignificanceSemaglutide improved the turnover of islet heparan sulfate proteoglycans, hyaluronan, chondroitin sulfate proteoglycans, and collagens in the islet ECM. Such changes should contribute to restoring a healthy islet functional milieu and should reduce the formation of cell-damaging amyloid deposits. Our findings also provide additional evidence for the involvement of islet proteoglycans in the pathophysiology of type 2 diabetes.

Functional characteristics of hub and wave-initiator cells in β cell networks

Islets of Langerhans operate as multicellular networks in which several hundred β cells work in synchrony to produce secretory pulses of insulin, a hormone crucial for controlling metabolic homeostasis. Their collective rhythmic activity is facilitated by gap junctional coupling and affected by their functional heterogeneity, but the details of this robust and coordinated behavior are still not fully understood. Recent advances in multicellular imaging and optogenetic and photopharmacological strategies, as well as in network science, have led to the discovery of specialized β cell subpopulations that were suggested to critically determine the collective dynamics in the islets. In particular hubs, i.e., β cells with many functional connections, are believed to significantly enhance communication capacities of the intercellular network and facilitate an efficient spreading of intercellular Ca2+ waves, whereas wave-initiator cells trigger intercellular signals in their cohorts. Here, we determined Ca2+ signaling characteristics of these two β cell subpopulations and the relationship between them by means of functional multicellular Ca2+ imaging in mouse pancreatic tissue slices in combination with methods of complex network theory. We constructed network layers based on individual Ca2+ waves to identify wave initiators, and functional correlation-based networks to detect hubs. We found that both cell types exhibit a higher-than-average active time under both physiological and supraphysiological glucose concentrations, but also that they differ significantly in many other functional characteristics. Specifically, Ca2+ oscillations in hubs are more regular, and their role appears to be much more stable over time than for initiator cells. Moreover, in contrast to wave initiators, hubs transmit intercellular signals faster than other cells, which implies a stronger intercellular coupling. Our research indicates that hubs and wave-initiator cell subpopulations are both natural features of healthy pancreatic islets, but their functional roles in principle do not overlap and should thus not be considered equal.

Investigation of the Chemical Composition, Antihyperglycemic and Antilipidemic Effects of Bassia eriophora and Its Derived Constituent, Umbelliferone on High-Fat Diet and Streptozotocin-Induced Diabetic Rats.

This study was designed to investigate the chemical profile, antihyperglycemic and antilipidemic effect of total methanolic extract (TME) of Bassia eriophora and isolated pure compound umbelliferone (UFN) in high-fat diet (HFD)- and streptozotocin (STZ)- induced diabetic rats. TME was subjected to various techniques of chromatography to yield UFN. Diabetes was induced after eight weeks of HFD by administration of STZ (40 mg/kg) intraperitoneally, and experimental subjects were divided into five groups. The diabetic control showed an increase in levels of blood glucose throughout the experiment. Treatments were initiated in the other four groups with glibenclamide (GLB) (6 mg/kg), TME (200 mg/kg and 400 mg/kg) and isolated UFN (50 mg/kg) orally. The effect on blood glucose, lipid profile and histology of the pancreatic and adipose tissues was assessed. Both 200 and 400 mg/kg of TME produced a comparably significant decrease in blood glucose levels and an increase in insulin levels with GLB. UFN began to show a better blood sugar-lowering effect after 14 days of treatment, comparatively. However, both 400 mg/kg TME and UFN significantly returned blood glucose levels in diabetic rats compared to normal rats. Analysis of the lipid profile showed that while HFD + STZ increased all lipid profile parameters, TME administration produced a significant decrease in their levels. Histopathological examinations showed that treatment with TME and UFN revealed an improved cellular architecture, with the healthy islets of Langerhans and compact glandular cells for pancreatic cells distinct from damaged cells in non-treated groups. Conversely, the adipose tissue displayed apparently normal polygonal fat cells. Therefore, these results suggest that TME has the potential to ameliorate hyperglycemia conditions and control lipid profiles in HFD + STZ-induced diabetic rats.

A transcriptional cross species map of pancreatic islet cells

ObjectivePancreatic islets of Langerhans secrete hormones to regulate systemic glucose levels. Emerging evidence suggests that islet cells are functionally heterogeneous to allow a fine-tuned and efficient endocrine response to physiological changes. A precise description of the molecular basis of this heterogeneity, in particular linking animal models to human islets, is an important step towards identifying the factors critical for endocrine cell function in physiological and pathophysiological conditions. MethodsIn this study, we used single-cell RNA sequencing to profile more than 50′000 endocrine cells isolated from healthy human, pig and mouse pancreatic islets and characterize transcriptional heterogeneity and evolutionary conservation of those cells across the three species. We systematically delineated endocrine cell types and α- and β-cell heterogeneity through prior knowledge- and data-driven gene sets shared across species, which altogether capture common and differential cellular properties, transcriptional dynamics and putative driving factors of state transitions. ResultsWe showed that global endocrine expression profiles correlate, and that critical identity and functional markers are shared between species, while only approximately 20% of cell type enriched expression is conserved. We resolved distinct human α- and β-cell states that form continuous transcriptional landscapes. These states differentially activate maturation and hormone secretion programs, which are related to regulatory hormone receptor expression, signaling pathways and different types of cellular stress responses. Finally, we mapped mouse and pig cells to the human reference and observed that the spectrum of human α- and β-cell heterogeneity and aspects of such functional gene expression are better recapitulated in the pig than mouse data. ConclusionsHere, we provide a high-resolution transcriptional map of healthy human islet cells and their murine and porcine counterparts, which is easily queryable via an online interface. This comprehensive resource informs future efforts that focus on pancreatic endocrine function, failure and regeneration, and enables to assess molecular conservation in islet biology across species for translational purposes.

241.3: Generation of Prevascularized Endocrine Constructs to Treat Type 1 Diabetes

Introduction: The goal of our study was to generate a functional, prevascularized endocrine constructs utilizing amniotic membrane derived hydrogel, islets and blood outgrowth endothelial cells (BOECs) to be transplanted in diabetic hosts. Methods: Human amniotic membranes were dissected from placentas decellularized, lyophilized, and solubilized to obtain hydrogels. DNA quantification and scanning electron microscopy showed complete removal of cellular components and preserved extracellular matrix. Characterization studies demonstrated widespread distribution of all essential structural proteins, including collagen, GAGs and laminin. The islets and BOECs were admixed in amnion derived hydrogels and cultured in specially designed media to enhance endothelial cell assembly into tubular, vascular-like structures. Constructs were kept under culture for 3 days. Glucose stimulated insulin secretion (GSIS) tests showed an adequate insulin secretion in response to high glucose stimulation, confirming the functional activity of the prevascularized endocrine constructs. Results: To test in vivo biocompatibility and function, the vascularized constructs generated from 500 IEQ rat islets and 2× 105 human derived BOECs were implanted under the skin of the diabetic NSG mice. Transplantation of the constructs led to rapid and long-term (90 days) normalization of blood glucose levels. Removal of the grafts led to recurrence of hyperglycemia within 24 hours, indicating that the transplanted constructs was responsible for normalized glucose levels. Histological analysis of the explanted grafts revealed healthy islet morphology and perfect revascularization. Grafts stained positive for insulin and human specific CD 31 indicating presence of human endothelial cells in newly formed intra-islet micro vessels. Conclusion: Our findings show that amnion derived hydrogel seeded with endocrine pancreatic tissue and endothelial cells could be used for functional, preveascularized endocrine construct bioengineering. This work is supported by grants from the European Commission (Horizon 2020 Framework Program; VANGUARD grant 874700), the European Foundation for the Study of Diabetes (EFSD), the Juvenile Diabetes Research Foundation (JDRF; grant 3-SRA-2020-926-S-B) and the Shota Rustaveli National Science Foundation (grant FR-19-19760).

D-Amino Acids and Classical Neurotransmitters in Healthy and Type 2 Diabetes-Affected Human Pancreatic Islets of Langerhans.

The pancreatic islets of Langerhans are clusters of cells that function as endocrine units synthesizing and releasing insulin and a range of additional peptide hormones. The structural and chemical characteristics of islets change during type 2 diabetes development. Although a range of metabolites including neurotransmitters has been reported in rodent islets, the involvement of these cell-to-cell signaling molecules within human pancreatic islets in the pathophysiology of type 2 diabetes is not well known, despite studies suggesting that these molecules impact intra- and inter-islet signaling pathways. We characterize the enigmatic cell-to-cell signaling molecules, d-serine (d-Ser) and d-aspartate (d-Asp), along with multiple classical neurotransmitters and related molecules, in healthy versus type 2 diabetes-affected human islets using capillary electrophoresis separations. Significantly reduced d-Ser percentage and gamma-aminobutyric acid (GABA) levels were found in type 2 diabetes-affected islets compared to healthy islets. In addition, the negative correlations of many of the signaling molecules, such as d-Ser percentage (r = −0.35), d-Asp (r = −0.32), serotonin (r = −0.42), and GABA (r = −0.39) levels, with hemoglobin A1c (HbA1c) levels and thus with the progression of type 2 diabetes further demonstrate the disruption in intra- or inter-islet signaling pathways and suggest that these cell-to-cell signaling molecules may be potential therapeutic targets.

Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of β-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse β-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non–GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

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)