Human Islet Cells Research Articles

Mitochondrial network expansion and dynamic redistribution during islet morphogenesis in zebrafish larvae.

Mitochondria, organelles critical for energy production, modify their shape and location in response to developmental state and metabolic demands. Mitochondria are altered in diabetes, but the mechanistic basis is poorly defined, due to difficulties in assessing mitochondria within an intact organism. Here, we use invivo imaging in transparent zebrafish larvae to demonstrate filamentous, interconnected mitochondrial networks within islet cells. Mitochondrial movements highly resemble what has been reported for human islet cells invitro, showing conservation in behaviour across species and cellular context. During islet development, mitochondrial content increases with emergence of cell motility, and mitochondria disperse within fine protrusions. Overall, this work presents quantitative analysis of mitochondria within their native environment and provides insights into mitochondrial behaviour during organogenesis.

E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance.

SUMMARYPrevention or amelioration of declining β cell mass is a potential strategy to cure diabetes. Here, we report the pathways utilized by β cells to robustly replicate in response to acute insulin resistance induced by S961, a pharmacological insulin receptor antagonist. Interestingly, pathways that include CENP-A and the transcription factor E2F1 that are independent of insulin signaling and its substrates appeared to mediate S961-induced β cell multiplication. Consistently, pharmacological inhibition of E2F1 blocks β-cell proliferation in S961-injected mice. Serum from S961-treated mice recapitulates replication of β cells in mouse and human islets in an E2F1-dependent manner. Co-culture of islets with adipocytes isolated from S961-treated mice enables β cells to duplicate, while E2F1 inhibition limits their growth even in the presence of adipocytes. These data suggest insulin resistance-induced proliferative signals from adipocytes activate E2F1, a potential therapeutic target, to promote β cell compensation.

In vivo induction of regulatory T cells by anti-CD45RB antibody causes transferable tolerance to discordant human xenogenic islets.

The treatment of diabetes by islet cell transplantation has become an accepted therapy, with transplantation of xenogeneic islet cells an attractive alternative to the problem. Previous studies in mice have demonstrated that anti-CD45RB induce immune tolerance in human pancreatic islet cells. The current study was to define the mechanism of action of anti-CD45RB induced nonspecific immune tolerance to heteroantigens. A total of 1500 IEQ human islets were transplanted to diabetic B6μMT-/- mice, B6 mice, and μMT-/- diabetic mice undergoing thymectomy. These mice were treated short-term with doses of anti-CD45RB. CD4+Foxp3+Tregs were detected in the blood, peripheral lymphatic organs by flow cytometry, and immunohistochemistry. In addition, anti-CD25 mAb was administered to tolerant human islet cells B6μMT-/-mice. Mice then were transplanted with other human islet cells and received CD4+CD25+Tregs isolated from tolerant human islets mice to observe islet destruction. Anti-CD45RB treatment-induced tolerance to islets in both immunocompetent and B-cell-deficient mice (μMT-/- mice) by processes that were dependent on CD25+ Tregs, but not B cells. Anti-CD45RB treatment increased the number of CD4+Foxp3+Tregs cells. Anti-CD45RB treatment-induced immune tolerance that was antigen nonspecific, with Tregs playing an important role. Anti-CD45RB treatment-induced tolerance generated Tregs that could be transferred to another individual to manifest nonspecific immune tolerance. The results of the experiment suggest that anti-CD45RB induced tolerance to human islet xenografts is mediated by the proliferation of Tregs. These tolerogenic Tregs can be transferred to other mice and induce nonspecific immune tolerance.

Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic β cells.

IFNα is a key regulator of the dialogue between pancreatic β cells and the immune system in early type 1 diabetes (T1D). IFNα up-regulates HLA class I expression in human β cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotent-derived islet-like cells to IFNα induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulin-producing EndoC-βH1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation–related genes, and chemokines. NLRC5 also mediates the effects of IFNα on alternative splicing, a generator of β cell neoantigens, suggesting that it is a central player of the effects of IFNα on β cells that contribute to trigger and amplify autoimmunity in T1D.

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.

Immunological parameters in hyperglycemic COVID19 patients of wasit-Iraq

Background: A number of studies have also reported that preexisting diabetes as well as newly diagnosed diabetes with a first glucose measurement on hospital admission are both associated with an increased risk of all-cause mortality. This study aimed to investigate the immunological characteristics of patients with severe COVID-19 with diabetes mellitus. Methods: This study was carried out in a period between November 2021 and March 2022 in Iraq. A total of samples were subjected to laboratory examinations including Human Glutamic acid decarboxylase (GAD) ELISA Kit, Tumor necrosis factor α using specific kit ELISA Kit, Human Islet cell antibody (ICA)ELISA Kit and Human C-Peptide ELISA Kit using specific ELISA Kit. Results: Regarding mean difference of variables among diabetic and non-diabetic COVID-19 patients all the investigated parameters has been showed significant differences between diabetic and non-diabetic patients with a P-value less than 0.001 except for CRP, correlation between age and COVID-19 patients immunological parameters showed that there was no significant correlation between age and the studied variables, mean differences of COVID-19 patients between males and females did not revealed a significant differences,,,,

Virus Safety of Xenotransplantation.

The practice of xenotransplantation using pig islet cells or organs is under development to alleviate the shortage of human donor islet cells or organs for the treatment of diabetes or organ failure. Multiple genetically modified pigs were generated to prevent rejection. Xenotransplantation may be associated with the transmission of potentially zoonotic porcine viruses. In order to prevent this, we developed highly sensitive PCR-based, immunologicals and other methods for the detection of numerous xenotransplantation-relevant viruses. These methods were used for the screening of donor pigs and xenotransplant recipients. Of special interest are the porcine endogenous retroviruses (PERVs) that are integrated in the genome of all pigs, which are able to infect human cells, and that cannot be eliminated by methods that other viruses can. We showed, using droplet digital PCR, that the number of PERV proviruses is different in different pigs (usually around 60). Furthermore, the copy number is different in different organs of a single pig, indicating that PERVs are active in the living animals. We showed that in the first clinical trials treating diabetic patients with pig islet cells, no porcine viruses were transmitted. However, in preclinical trials transplanting pig hearts orthotopically into baboons, porcine cytomegalovirus (PCMV), a porcine roseolovirus (PCMV/PRV), and porcine circovirus 3 (PCV3), but no PERVs, were transmitted. PCMV/PRV transmission resulted in a significant reduction of the survival time of the xenotransplant. PCMV/PRV was also transmitted in the first pig heart transplantation to a human patient and possibly contributed to the death of the patient. Transmission means that the virus was detected in the recipient, however it remains unclear whether it can infect primate cells, including human cells. We showed previously that PCMV/PRV can be eliminated from donor pigs by early weaning. PERVs were also not transmitted by inoculation of human cell-adapted PERV into small animals, rhesus monkey, baboons and cynomolgus monkeys, even when pharmaceutical immunosuppression was applied. Since PERVs were not transmitted in clinical, preclinical, or infection experiments, it remains unclear whether they should be inactivated in the pig genome by CRISPR/Cas. In summary, by using our sensitive methods, the safety of xenotransplantation can be ensured.

The Protective Action of Metformin against Pro-Inflammatory Cytokine-Induced Human Islet Cell Damage and the Mechanisms Involved.

Metformin, a drug widely used in type 2 diabetes (T2D), has been shown to protect human β-cells exposed to gluco- and/or lipotoxic conditions and those in islets from T2D donors. We assessed whether metformin could relieve the human β-cell stress induced by pro-inflammatory cytokines (which mediate β-cells damage in type 1 diabetes, T1D) and investigated the underlying mechanisms using shotgun proteomics. Human islets were exposed to 50 U/mL interleukin-1β plus 1000 U/mL interferon-γ for 48 h, with or without 2.4 µg/mL metformin. Glucose-stimulated insulin secretion (GSIS) and caspase 3/7 activity were studied, and a shotgun label free proteomics analysis was performed. Metformin prevented the reduction of GSIS and the activation of caspase 3/7 induced by cytokines. Proteomics analysis identified more than 3000 proteins in human islets. Cytokines alone altered the expression of 244 proteins (145 up- and 99 down-regulated), while, in the presence of metformin, cytokine-exposure modified the expression of 231 proteins (128 up- and 103 downregulated). Among the proteins inversely regulated in the two conditions, we found proteins involved in vesicle motility, defense against oxidative stress (including peroxiredoxins), metabolism, protein synthesis, glycolysis and its regulation, and cytoskeletal proteins. Metformin inhibited pathways linked to inflammation, immune reactions, mammalian target of rapamycin (mTOR) signaling, and cell senescence. Some of the changes were confirmed by Western blot. Therefore, metformin prevented part of the deleterious actions of pro-inflammatory cytokines in human β-cells, which was accompanied by islet proteome modifications. This suggests that metformin, besides use in T2D, might be considered for β-cell protection in other types of diabetes, possibly including early T1D.

SMNDC1 links chromatin remodeling and splicing to regulate pancreatic hormone expression.

Insulin expression is primarily restricted to the pancreatic β cells, which are physically or functionally depleted in diabetes. Identifying targetable pathways repressing insulin in non-β cells, particularly in the developmentally related glucagon-secreting α cells, is an important aim of regenerative medicine. Here, we perform an RNA interference screen in a murine α cell line to identify silencers of insulin expression. We discover that knockdown of the splicing factor Smndc1 triggers a global repression of α cell gene-expression programs in favor of increased β cell markers. Mechanistically, Smndc1 knockdown upregulates the β cell transcription factor Pdx1 by modulating the activities of the BAF and Atrx chromatin remodeling complexes. SMNDC1's repressive role is conserved in human pancreatic islets, its loss triggering enhanced insulin secretion and PDX1 expression. Our study identifies Smndc1 as a key factor connecting splicing and chromatin remodeling to the control of insulin expression in human and mouse islet cells.

Reduced Retinoic Acid Receptor Beta (Rarβ) Affects Pancreatic β-Cell Physiology.

Simple SummaryVitamin A is one of the important micronutrients involved in various biological functions. Numerous studies have suggested a link between vitamin A (retinoic acid) and type 2 diabetes (T2D). However, the functional role of vitamin A receptors (RARα, β, and γ) in β-pancreatic cells is not clear yet. In this study, we performed bioinformatics and functional experiments in human islets and INS-1 cells in order to evaluate the potential role of RARβ on insulin secretion and pancreatic β-cell function.Various studies have suggested a link between vitamin A (VA), all-trans-retinol, and type 2 diabetes (T2D). However, the functional role/expression of vitamin A receptors (Rarα, β, and γ) in pancreatic β-cells is not clear yet. Accordingly, we performed a series of bioinformatics, molecular and functional experiments in human islet and INS-1 cells to evaluate the role of Rarβ on insulin secretion and pancreatic β-cell function. Microarray and RNA-sequencing (RAN-seq) expression analysis showed that RARα, β, and γ are expressed in human pancreatic islets. RNA-seq expression of RARβ in diabetic/hyperglycemic human islets (HbA1c ≥ 6.3%) revealed a significant reduction (p = 0.004) compared to nondiabetic/normoglycemic cells (HbA1c < 6%). The expression of RARβ with INS and PDX1 showed inverse association, while positive correlations were observed with INSR and HbA1c levels. Exploration of the T2D knowledge portal (T2DKP) revealed that several genetic variants in RARβ are associated with BMI. The most associated variant is rs6804842 (p = 1.2 × 10−25). Silencing of Rarβ in INS-1 cells impaired insulin secretion without affecting cell viability or apoptosis. Interestingly, reactive oxygen species (ROS) production levels were elevated and glucose uptake was reduced in Rarβ-silenced cells. mRNA expression of Ins1, Pdx1, NeuroD1, Mafa, Snap25, Vamp2, and Gck were significantly (p < 0.05) downregulated in Rarβ-silenced cells. For protein levels, Pro/Insulin, PDX1, GLUT2, GCK, pAKT/AKT, and INSR expression were downregulated considerably (p < 0.05). The expression of NEUROD and VAMP2 were not affected. In conclusion, our results indicate that Rarβ is an important molecule for β-cell function. Hence, our data further support the potential role of VA receptors in the development of T2D.

A Novel Intron-Encoded Neuropilin-1 Isoform in Pancreatic Islets Associated With Very Young Age of Onset of Type 1 Diabetes.

Net synthesis of pancreatic β-cells peaks before 2 years of life. β-Cell mass is set within the first 5 years of life. In-frame translational readthrough of the NRP1 gene exon 9 into intron 9 generates a truncated neuropilin-1 protein lacking downstream sequence necessary for binding VEGF that stimulates β-cell replication. VEGF is critical for developing but not adult islet neogenesis. Herein we show that cells in human pancreatic islets containing the full-length neuropilin-1 possess insulin but cells that contain the truncated neuropilin-1 are devoid of insulin. Decreased insulin cells increases susceptibility to onset of type 1 diabetes at a younger age. We also show that the frequency of a genetic marker in NRP1 intron 9 is higher among patients with onset of type 1 diabetes before age 4 years (31.8%), including those with onset at 0.67-2.00 and 2-4 years, compared with that in patients with onset at 4-8 years, at 8-12 years, and after 16 years (16.1%) with frequency equal to that in subjects without diabetes (16.0%). Decreased insulin cells plus the genetic data are consistent with a low effect mechanism that alters the onset of type 1 diabetes to a very young age in some patients, thus supporting the endotype concept that type 1 diabetes is a heterogeneous disease.

Heparan Sulfate Mimicking Glycopolymer Prevents Pancreatic β Cell Destruction and Suppresses Inflammatory Cytokine Expression in Islets under the Challenge of Upregulated Heparanase.

Diabetes is a chronic disease in which the levels of blood glucose are too high because the body does not effectively produce insulin to meet its needs or is resistant to insulin. β Cells in human pancreatic islets produce insulin, which signals glucogen production by the liver and causes muscles and fat to uptake glucose. Progressive loss of insulin-producing β cells is the main cause of both type 1 and type 2 diabetes. Heparan sulfate (HS) is a ubiquitous polysaccharide found at the cell surface and in the extracellular matrix (ECM) of a variety of tissues. HS binds to and assembles proteins in ECM, thus playing important roles in the integrity of ECM (particularly basement membrane), barrier function, and ECM-cell interactions. Islet HS is highly expressed by the pancreatic β cells and critical for the survival of β cells. Heparanase is an endoglycosidase and cleaves islet HS in the pancreas, resulting in β-cell death and oxidative stress. Heparanase could also accelerate β-cell death by promoting cytokine release from ECM and secretion by activated inflammatory and endothelial cells. We demonstrate that HS-mimicking glycopolymer, a potent heparanase inhibitor, improves the survival of cultured mouse pancreatic β cells and protects HS contents under the challenge of heparanase in human pancreatic islets. Moreover, this HS-mimicking glycopolymer reduces the expression levels of cytokines (IL8, IL1β, and TNFα) and the gene encoding Toll-like Receptor 2 (TLR2) in human pancreatic islets.

1459-P: Human Islet Cell Transcriptome Analysis Using Single Nucleus RNA-seq Reveals New Beta-Cell Markers and Define Distinct Beta-Cell Subpopulations with Different Transcriptional Activity

Single-cell RNA sequencing (scRNA-seq) of human islets requires cell dissociation and does not provide information on the transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and provides abundant information on intronic sequences that can be used to identify actively transcribed genes. Based on this, we seek to compare scRNA-seq and snRNA-seq approaches in human islets to determine: 1) whether similar cell clusters could be detected; 2) whether new gene markers could be discovered in human islet endocrine cells using intronic reads; 3) whether human beta cell subpopulations could be identified based on INS expression dynamics; and 4) whether snRNA-seq could be used in human islet grafts transplanted in immunosuppressed mice. We performed scRNA-seq and snRNA-seq on three pairs of human islets obtained from three healthy adult human donors using exon only or exon plus intron reads, respectively. Analysis of integrated data revealed similar human islet cell clusters. In the snRNA-seq data, however, top differentially expressed genes were identified as new markers of human endocrine cells such as ZNF385D (β-cells) , PTPRT (α-cells) , LRFN5 (δ-cells) and CACNA2D3 (PP cells) . These markers also accurately define endocrine cell populations in human islet grafts. Additionally, we distinguished several beta cell sub-clusters- INS rich cluster, HNRNPA2B1 (vesicle) rich cluster and active INS transcribing cluster. In conclusion, snRNA-seq analysis of human islet cells is a previously unrecognized tool for the identification of human islet cell types in samples where nuclear RNA processing is required. By comparing INS expression between scRNA-seq and snRNA-seq data sets, we can detect different beta cell sub-populations with distinct gene expression patterns representing different biological dynamic states. Disclosure R.Kang: None. Y.Li: None. C.Rosselot: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. G.Lu: None. Funding NIH DK105015

229-LB: G6PC2, a GWAS Locus for Fasting Glucose and HbA1c, Regulates Glucose-Suppression of Glucagon Secretion in a-Cells

The G6PC2 locus, encoding the catalytic subunit of the islet-specific glucose-6-phosphatase enzyme, is an established risk locus for HbA1c and fasting glucose levels. A critical role of glycolytic flux in regulating glucagon secretion was established through manipulation of the activity and expression of the glycolytic enzyme glucokinase (GCK) which controls the setpoint for glucose-suppression of glucagon secretion (GSGS) . G6PC2 opposes the action of GCK by hydrolyzing glucose-6-phosphate back to glucose and creating a futile substrate cycle. Previous studies from germline and β-cell-specific G6PC2 null mice have demonstrated that G6PC2 ablation reduces glucose-6-phosphatase activity, glucose cycling, and glycolytic flux, resulting in a leftward shift of dose-response curve for glucose-stimulated insulin secretion. Through cell type specific chromatin accessibility maps of sorted human islet cells, we established that G6PC2 risk SNPs are in open chromatin regions in human α-cells, suggesting that they might not only affect β-cell but also α-cell expression of this critical gene. To test whether α-cell G6PC2 impacts glycemic control, we derived a new mouse line that induces ablation of the first three exons of G6PC2 after Cre activation – resulting in a null allele (G6pc2 loxP/loxP; Gcg-CreERT2; referred to as “α-G6PC2KO”) . Assessment of glucagonemia during glucose tolerance tests revealed a decrease in glucagon levels in α-G6PC2KO mice relative to controls. Intriguingly, fasting blood glucose levels were significantly lower in α-G6PC2KO mice. α-G6PC2KO mice recovered more slowly from hypoglycemia during an insulin tolerance test, and presented lower glucagon levels relative to control mice following the insulin bolus. α-G6PC2KO islets secreted less glucagon relative to control islets when incubated in low-glucose media, independent of alterations in insulin secretion or islet hormone content. Collectively, our data demonstrate that the G6PC2 locus impacts glycemic control via its action in α-cells. Disclosure V. Bahl: None. C. L. May: None. Human pancreas analysis program: n/a. K. H. Kaestner: None.

14-OR: Harmine and Exenatide Combination Increases Human ß-Cell Survival

GLP1R agonists such as Exendin-4 (E) together with DYRK1A inhibitors such as Harmine (H) significantly increase human β-cell replication in vitro and in vivo. Importantly, 3-month treatment with H+E combination markedly increases human beta cell mass (∼7-fold) in islets transplanted in immunosuppressed mice, beyond the increase induced by proliferation. Therefore, we addressed whether H+E also enhances β-cell survival and that contributes to the increased human β-cell mass expansion in vivo. Treatment with H+E significantly decreased β-cell apoptosis in dispersed primary human islet cells treated in vitro with either thapsigargin (ER stress) , cytokines (inflammation) and H2O2 (ROS) , while the drugs alone or vehicle (V) did not induce such effect. Importantly, H+E treatment for 7 days significantly reduced β-cell apoptosis in human islet grafts transplanted in immunosuppressed mice assessed by insulin and TUNEL staining. Human β-cell mass analysis of these grafts by iDISCO+ revealed a significant ∼50% increase in H+E-treated mice compared with mice treated with drugs alone or V. Human α-cell mass was unchanged. To address the signaling pathways modulated by H+E after 7-day treatment, we performed RNA-seq analysis of these human islet grafts. We identified 29 differentially expressed human genes (&amp;gt; 2-fold, p&amp;lt;0.05) in H+E-treated human islet grafts. GSEA analysis revealed cell adhesion, survival, vascularization and secretion as the main pathways induced by H+E. Among the upregulated genes, VGF (VGF Nerve Growth Factor Inducible) is known to regulate insulin secretion and β-cell survival. We found that H+E treatment of human islets in vitro increases 2-to-3-fold VGF mRNA and secretion. We are now determining VGF involvement in H+E-induced human β-cell survival. In conclusion, we have uncovered a novel prosurvival function of H+E in human β-cells with the potential implication of VGF in these effects. These studies can lead to the discovery of future β-cell protection and regeneration therapies for diabetes. Disclosure C.Rosselot: None. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Y.Li: None. D.Guevara: None. K.A.Beliard: None. R.Kang: None. P.Wang: None. K.Thakkar: None. G.Lu: None. R.J.Devita: None. Funding DYRK Inhibitors for Human Beta Cell Expansion RDK105015

RETRACTED ARTICLE: Calcium-dependent transcriptional changes in human pancreatic islet cells reveal functional diversity in islet cell subtypes

Aims/hypothesisPancreatic islets depend on cytosolic calcium (Ca2+) to trigger the secretion of glucoregulatory hormones and trigger transcriptional regulation of genes important for islet response to stimuli. To date, there has not been an attempt to profile Ca2+-regulated gene expression in all islet cell types. Our aim was to construct a large single-cell transcriptomic dataset from human islets exposed to conditions that would acutely induce or inhibit intracellular Ca2+ signalling, while preserving biological heterogeneity.MethodsWe exposed intact human islets from three donors to the following conditions: (1) 2.8 mmol/l glucose; (2) 16 mmol/l glucose and 40 mmol/l KCl to maximally stimulate Ca2+ signalling; and (3) 16 mmol/l glucose, 40 mmol/l KCl and 5 mmol/l EGTA (Ca2+ chelator) to inhibit Ca2+ signalling, for 1 h. We sequenced 68,650 cells from all islet cell types, and further subsetted the cells to form an endocrine cell-specific dataset of 59,373 cells expressing INS, GCG, SST or PPY. We compared transcriptomes across conditions to determine the differentially expressed Ca2+-regulated genes in each endocrine cell type, and in each endocrine cell subcluster of alpha and beta cells.ResultsBased on the number of Ca2+-regulated genes, we found that each alpha and beta cell cluster had a different magnitude of Ca2+ response. We also showed that polyhormonal clusters expressing both INS and GCG, or both INS and SST, are defined by Ca2+-regulated genes specific to each cluster. Finally, we identified the gene PCDH7 from the beta cell clusters that had the highest number of Ca2+-regulated genes, and showed that cells expressing cell surface PCDH7 protein have enhanced glucose-stimulated insulin secretory function.Conclusions/interpretationHere we use our large-scale, multi-condition, single-cell dataset to show that human islets have cell-type-specific Ca2+-regulated gene expression profiles, some of them specific to subpopulations. In our dataset, we identify PCDH7 as a novel marker of beta cells having an increased number of Ca2+-regulated genes and enhanced insulin secretory function.Data availabilityA searchable and user-friendly format of the data in this study, specifically designed for rapid mining of single-cell RNA sequencing data, is available at https://lynnlab.shinyapps.io/Human_Islet_Atlas/. The raw data files are available at NCBI Gene Expression Omnibus (GSE196715).Graphical abstract

Characterizing the Role of the Neuroregulatory Protein Piccolo in Secretion of Insulin from Beta Pancreatic Islet Cells

Secretion of insulin from pancreatic beta‐cells is required for glucose homeostasis; however, the molecular mechanisms of insulin secretion have not been fully elucidated. In pancreatic beta‐cells, glucose‐induced insulin secretion is synergistically potentiated through a calcium (Ca2+)‐mediated Epac2‐Rim2‐Piccolo signaling response mechanism. Both Piccolo and Rim2 were originally identified in neurons as active zone proteins that regulate neurotransmitter secretion. It has been demonstrated that Piccolo is downregulated in human pancreatic islet cells isolated from patients with type 2 diabetes (T2D). We hypothesize that Piccolo plays a key role in regulated insulin secretion and further study of Piccolo in pancreatic islets cells may provide valuable insights into the etiology of T2D. The C‐terminus of Piccolo contains a Ca2+‐binding C2A domain and we hypothesize that in pancreatic beta‐cells, Piccolo may regulate insulin secretion by serving as a Ca2+‐sensor. Alternative splicing within the coding region of the C2A domain results in an exon‐skipping event generating either a splice variant that lacks the 27‐nucleotide exon 16 (e.g., short C2A) or a variant that retains this exon (e.g., long C2A). Due to this splicing event, the short C2A domain lacks 9 amino acids resulting in enhanced Ca2+‐binding affinity. In this study, we sought to examine the differential roles of the Piccolo splice variants in Ca2+‐mediated insulin secretion using a rat pancreatic beta‐cell line (INS‐832/13). We demonstrate that INS‐832/13 cells express both variants containing either the short or long C2A domains. We also demonstrate that these cells express an additional splice variant called Piccolino previously thought to be only expressed in neurons associated with the eye and ear. To further assess the role of Piccolo in regulating insulin secretion, we plan to use a gene knockdown approach employing multiple shRNAs expressed in lentivirus that target Piccolo RNA sequences. To assess the effect of knockdown of Piccolo, we will stimulate ß pancreatic islet cells with increasing glucose concentrations to quantify changes in insulin secretion.

Design, Synthesis, and Characterization of a Novel Fluoroprobe for Live Human Islet Cell Imaging of Serotonin 5-HT1A Receptor.

Mounting evidence suggests that the serotonin system serves in signal transmission to regulate insulin secretion in pancreatic islets of Langerhans. Among the 5-HT receptor subtype found in pancreatic islets, serotonin receptor 1A (5-HT1A ) demonstrates a unique ability to inhibit β-cell insulin secretion. We report the design, synthesis, and characterization of two novel fluorescent probes for the 5-HT1A receptor. The compounds were prepared by conjugating the scaffold of the 5-HT1A receptor agonist 8-OH-DPAT with two fluorophores suitable for live-cell imaging. Compound 5a {5-(dimethylamino)-N-[5-[(8-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino]pentyl]naphtalen-1-sulfonammide} showed high affinity for the 5-HT1A receptor (Ki =1.8 nM). Fluoroprobe 5a was able to label the 5-HT1A receptor in pancreatic islet cell cultures in a selective manner, as the fluorescence emission was significantly attenuated by co-administration of the 5-HT1A receptor antagonist WAY-100635. Thus, fluoroprobe 5a showed useful properties to further characterize this unique receptor's role.

Analysis of Half a Billion Datapoints Across Ten Machine-Learning Algorithms Identifies Key Elements Associated With Insulin Transcription in Human Pancreatic Islet Cells.

Machine learning (ML)-workflows enable unprejudiced/robust evaluation of complex datasets. Here, we analyzed over 490,000,000 data points to compare 10 different ML-workflows in a large (N=11,652) training dataset of human pancreatic single-cell (sc-)transcriptomes to identify genes associated with the presence or absence of insulin transcript(s). Prediction accuracy/sensitivity of each ML-workflow was tested in a separate validation dataset (N=2,913). Ensemble ML-workflows, in particular Random Forest ML-algorithm delivered high predictive power (AUC=0.83) and sensitivity (0.98), compared to other algorithms. The transcripts identified through these analyses also demonstrated significant correlation with insulin in bulk RNA-seq data from human islets. The top-10 features, (including IAPP, ADCYAP1, LDHA and SST) common to the three Ensemble ML-workflows were significantly dysregulated in scRNA-seq datasets from Ire-1αβ-/- mice that demonstrate dedifferentiation of pancreatic β-cells in a model of type 1 diabetes (T1D) and in pancreatic single cells from individuals with type 2 Diabetes (T2D). Our findings provide direct comparison of ML-workflows in big data analyses, identify key elements associated with insulin transcription and provide workflows for future analyses.

Novel diabetes gene discovery through comprehensive characterization and integrative analysis of longitudinal gene expression changes.

Type 2 diabetes is a complex, systemic disease affected by both genetic and environmental factors. Previous research has identified genetic variants associated with type 2 diabetes risk; however, gene regulatory changes underlying progression to metabolic dysfunction are still largely unknown. We investigated RNA expression changes that occur during diabetes progression using a two-stage approach. In our discovery stage, we compared changes in gene expression using two longitudinally collected blood samples from subjects whose fasting blood glucose transitioned to a level consistent with type 2 diabetes diagnosis between the time points against those who did not with a novel analytical network approach. Our network methodology identified 17 networks, one of which was significantly associated with transition status. This 822-gene network harbors many genes novel to the type 2 diabetes literature but is also significantly enriched for genes previously associated with type 2 diabetes. In the validation stage, we queried associations of genetically determined expression with diabetes-related traits in a large biobank with linked electronic health records. We observed a significant enrichment of genes in our identified network whose genetically determined expression is associated with type 2 diabetes and other metabolic traits and validated 31 genes that are not near previously reported type 2 diabetes loci. Finally, we provide additional functional support, which suggests that the genes in this network are regulated by enhancers that operate in human pancreatic islet cells. We present an innovative and systematic approach that identified and validated key gene expression changes associated with type 2 diabetes transition status and demonstrated their translational relevance in a large clinical resource.