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In commemoration of 100 years since the discovery of glucagon, we review current knowledge about the human α cell. Alpha cells make up 30-40% of human islet endocrine cells and play a major role in regulating whole-body glucose homeostasis, largely through the direct actions of their main secretory product – glucagon – on peripheral organs. Additionally, glucagon and other secretory products of α cells, namely acetylcholine, glutamate, and GLP-1, have been shown to play an indirect role in the modulation of glucose homeostasis through autocrine and paracrine interactions within the islet. Studies of glucagon’s role as a counterregulatory hormone have revealed additional important functions of the α cell, including the regulation of multiple aspects of energy metabolism outside that of glucose. At the molecular level, human α cells are defined by expression of conserved islet-enriched transcription factors and various enriched signature genes, many of which have currently unknown cellular functions. Despite these common threads, notable heterogeneity exists amongst human α cell gene expression and function. Even greater differences are noted at the interspecies level, underscoring the importance of further study of α cell physiology in the human context. Finally, studies on α cell morphology and function in type 1 and type 2 diabetes, as well as other forms of metabolic stress, reveal a key contribution of α cell dysfunction to dysregulated glucose homeostasis in disease pathogenesis, making targeting the α cell an important focus for improving treatment.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs, we developed AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX show the superior performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulated known islet pathobiology and showed differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+T cells infiltration in islets during type 1 diabetes progression.

Recent studies show that cellular neighborhoods play an important role in evolving biological events such as cancer and diabetes. Therefore, it is critical to accurately and efficiently identify cellular neighborhoods from spatially-resolved single-cell transcriptomic data or single-cell resolution tissue imaging data. In this work, we develop CNTools, a computational toolbox for end-to-end cellular neighborhood analysis on annotated cell images, comprising both the identification and analysis steps. It includes state-of-the-art cellular neighborhood identification methods and post-identification smoothing techniques, with our newly proposed Cellular Neighbor Embedding (CNE) method and Naive Smoothing technique, as well as several established downstream analysis approaches. We applied CNTools on three real-world CODEX datasets and evaluated identification methods with smoothing techniques quantitatively and qualitatively. It shows that CNE with Naive Smoothing overall outperformed other methods and revealed more convincing biological insights. Suggestions on how to choose identification methods and smoothing techniques according to input data are provided at last.

Transplantation of insulin-producing cells is an emerging treatment for type 1 diabetes. A recent report in Cell Stem Cell (Aghazadeh et al., 2021) outlines a new approach that accelerates the engraftment and improves the survival and function of such cell transplants by mixing adipose tissue-derived ready-made microvessels with human pancreatic progenitor cells or cadaveric islets prior to transplantation.

Purpose: We tested the hypothesis that a critical biologic determinant of T2D disparities is based on ethnicity-related differences in pancreatic islet function. Methods: Utilizing donor tissues from the NIDDK-funded Human Pancreas Analysis Program (HPAP) , we examined both insulin and glucagon secretion from nondiabetic and diabetic Caucasian and African American donor islets. Initially, a physiological amino acid mixture was used to stimulate glucagon secretion, followed by low and high glucose to stimulate insulin secretion and suppress glucagon secretion. During the high glucose step, IBMX (0.1 mM) is added to potentiate secretion of both hormones. Insulin and glucagon concentration in perifusates and islet extracts was measured by radioimmunoassay or ELISA. Statistical comparisons were drawn by repeated measures ANOVA. Results: Comparing nondiabetic islets, we observed overlapping insulin secretion profiles between both ethnicities; however, glucagon secretion was distinctly greater in both nondiabetic African American (+111%) islets compared to nondiabetic Caucasian islets, under all interventions. In T2D, the reduction in insulin secretion was significantly greater in African American T2D donors (high glucose -76%; IBMX -76%) compared to Caucasian T2D donors (high glucose -47%; IBMX -50%) . More strikingly, glucagon secretion in T2D donors was markedly different based on ethnicity. Whereas Caucasian T2D islets exhibited a similar baseline of glucagon secretion compared to Caucasian controls, there was a marked reduction of glucagon secretion overall in African American T2D donors (-70%) compared to their corresponding controls. The largest detected difference was in IBMX-potentiation of glucagon secretion, which is higher in Caucasian T2D donors (+103%) but significantly decreased in African American T2D donors (-76%) . Preliminary analysis of hormone content in a subset of the donor islet preparations revealed no differences according to ethnicity. Conclusions: We propose that ethnicity-related differences in nondiabetic islet function may contribute to the enhanced risk of T2D. Disclosure N. M. Doliba: None. M. Brissova: None. K. H. Kaestner: None. D. A. Stoffers: Other Relationship; Biomarin, Correlagen. J. Roman: None. A. V. Rozo: None. W. Qin: None. C. Liu: None. A. Naji: None. M. R. Rickels: Advisory Panel; Sernova, Corp., Vertex Pharmaceuticals Incorporated, Zealand Pharma A/S, Consultant; L-Nutra Inc. M. A. Atkinson: None. A. C. Powers: None. Funding This work is supported by 3UC4-112217-01S1 and U01-DK123594-02, UC4-DK-112217, UC4-DK-112232, and U01-DK-123716.

We previously showed that α cell function and gene expression in islets from donors with type 1 diabetes (T1D) was significantly compromised. The reason (s) for these intrinsic α cell changes are unknown but may include loss of α-to-β cell contact, chronic hyperglycemia, and/or repeated hypoglycemic events. To test these concurrent hypotheses, we modeled human T1D islets using α cell-enriched (“T1D-like") human pseudoislets. The synchronous acquisition of intracellular Ca2+ and hormone response showed impaired intracellular Ca2+ signaling and glucagon secretion in T1D-like pseudoislets compared to controls containing both α and β cells. For modeling of chronic hyperglycemia, we transplanted pseudoislets into Nod-SCID-IL2Rγnull; RIP-Diphtheria Toxin Receptor mice made diabetic by diphtheria toxin (DT) -induced β cell depletion. In DT-treated mice, α cells in T1D-like pseudoislets maintained expression of MAFB and ARX but began to express the β cell-enriched transcription factor NKX6.1 (control vs. T1D-like, 3.43±2.49 vs. 54.8±20.8% NKX6.1+ α cells; P=0.046, N=3-5 mice, 2 donors/group) . To mimic repeated hypoglycemic events, we intermittently exposed T1D-like pseudoislets to low glucose (1.7 mM) (3 events x 3hr each) in vitro, using those remaining in basal glucose (5 mM) as control. Following multiple low glucose (MLG) exposures, T1D-like pseudoislets had reduced glucagon secretion in the presence of both 1.7 mM (control vs. MLG, 1.51±0.18 vs. 0.79±0.13 % glucagon content; P=0.011, N=4 donors) and 16.7 mM glucose (control vs. MLG, 0.79±0.13 vs. 0.39±0.% glucagon content; P=0.023, N=4) . Native human islets exposed to MLG had similar changes in glucagon response (control vs. MLG, 1.7mM: 0.63±0.vs. 0.22±0.% glucagon content, P=0.007; 16.7mM: 0.18±0.vs. 0.05±0.% glucagon content, P=0.029; N=4 donors) . These data suggest that loss of α-to-β cell contact and repeated exposure to low glucose impair α cell function. Further, exposure to chronic hyperglycemia may lead to changes in α cell identity state after β cell loss. Disclosure Y.D.Pettway: None. C.Dai: None. T.M.Richardson: None. J.T.Walker: None. R.Aramandla: None. G.Poffenberger: None. A.Bradley: None. A.C.Powers: None. M.Brissova: None.

The Integrated Islet Distribution Program (IIDP) is currently the largest source of human islets for research in the U.S. The IIDP Human Islet Phenotyping Program (HIPP) provides standardized islet assessment and makes these data available to researchers. Since 2016, HIPP has phenotyped 337 islet samples from nondiabetic organ donors (40%F / 60%M, 45±12 years, BMI 29.0±5.4; 52% European, 26% Latino, 8% African, 3% Asian, and 11% other ancestry) . To define the islet structure-function relationships, we integrated physiological profiling of hormone secretion with islet cell composition measured by immunohistochemistry­. We found that % β cells varied significantly (range 34 - 92%; CV 21%) and had a strong negative correlation with % α (range 3 - 59%; CV 33%; r -0.95; p Disclosure M. Brissova: None. G. Poffenberger: None. D. C. Saunders: None. A. C. Powers: None. J. C. Niland: None. C. Evans-molina: Advisory Panel; Avotres Inc., DiogenX, Isla Technologies, MaiCell Therapeutics, Provention Bio, Inc., Other Relationship; Astellas Pharma Inc., Dompé, Lilly, Research Support; BMS, Nimbus Therapeutics. H. H. Durai: None. S. Mei: None. A. Coldren: None. C. Davis: None. C. Reihsmann: None. A. L. Hopkirk: None. D. Gibson: None. R. Aramandla: None.

ATP release from β cell secretory vesicles during insulin exocytosis is postulated to modulate islet hormone secretion via purinergic signaling in an autocrine or paracrine manner. Ecto-enzymes, including a family of ectonucleotidases (NTPDase) , control the concentration of extracellular nucleotides. One member of this family, ectonucleotidase triphosphate diphosphohydrolase 3 (NTPDase3) , is specifically expressed in human β cells during postnatal maturation, and its enzymatic activity is present in isolated adult human islets. The role of NTPDase3 in islet hormone secretion is not understood. We hypothesized that NTPDase3 regulates islet hormone secretion by modulating local extracellular nucleotide levels that signal through purinergic receptors expressed by islet cells. To define the cellular distribution of purinergic ecto-enzymes and nucleotide receptors in human islets, we used single-cell RNA sequencing (scRNA-seq) data. In addition to β cell NTPDase3 expression, ecto-enzymes ENPP1, and ENPP2 were abundant in α and ε cells while ecto-5'-NT and ADA were expressed in endothelial and stellate cells. These ecto-enzymes can further hydrolyze β cell-derived ATP to ADP, AMP, and adenosine which all bind purinergic receptors. We found that the ATP receptor, P2RY1, was enriched in β cells, and an adenosine receptor, ADORA2A, was expressed in α cells by both bulk and scRNA-seq. To study the function of this purinergic niche, we further assessed extracellular ATP levels which increased concomitantly (4.2E-12 vs. 4.3E-13 moles/100 IEQ/hour; n=9; p=0.03) with insulin secretion (21 vs. 60 ng/100 IEQ/hour; n=9; p=0.02) upon stimulation with 16.7 mM glucose + IBMX. To directly test NTPDase3 loss of function, we genetically manipulated human pseudoislets using an shRNA knockdown approach, resulting in a 1.7-fold increase of insulin secretion (AUC=3 vs. 5 ng/100 IEQs; n=3) . Overall, these studies provide an insight into how extracellular nucleotides and purinergic signaling mechanisms fine-tune human islet hormone secretion. Disclosure T. M. Richardson: None. D. C. Saunders: None. K. C. Coate: None. S. C. Robson: Advisory Panel; eGenesis, Consultant; Puretech, Synlogic, Research Support; Tizona Inc, Stock/Shareholder; EPurines, Purinomia. A. C. Powers: None. M. Brissova: None. C. Dai: None. R. Aramandla: None. H. H. Durai: None. C. Reihsmann: None. C. Davis: None. S. Mei: None. D. Gibson: None. S. Shrestha: None.

This protocol describes the imaging workflow currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group for CO-Detection by indEXing (CODEX®, now PhenoCycler™; Akoya Biosciences). See also CODEX® Multiplexed Imaging | Modality overview.

This protocol describes the staining and pre-imaging preparation currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group prior to performing multiplexed imaging of the human pancreas using the CO-Detection by indEXing (CODEX®) platform (now PhenoCycler™; Akoya Biosciences). See also CODEX® Multiplexed Imaging | Modality overview.

This protocol describes the antibody conjugation and validation processes for the CODEX® (now PhenoCycler™) system by Akoya Biosciences. For the comprehensive multiplexed imaging workflow currently in use at the Vanderbilt Diabetes Research Center, please see CODEX® Multiplexed Imaging | Modality overview.

Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor’s diabetes. Examination of the donor’s pancreatic tissue found substantial but reduced β-cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor’s clinical insulin deficiency was most likely the result autoimmune-mediated β-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.

SummaryMultiple islet autoantibodies (AAb) predict type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in just ∼15% of single AAb+ (generally against glutamic acid decarboxylase, GADA+) individuals; hence the single GADA+ state may represent an early stage of T1D amenable to interventions. Here, we functionally, histologically, and molecularly phenotype human islets from non-diabetic, GADA+ and T1D donors. Similar to the few remaining beta cells in T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, alpha cell glucagon secretion was dysregulated in both T1D and GADA+ islets with impaired glucose suppression of glucagon secretion. Single cell RNA sequencing (scRNASeq) of GADA+ alpha cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of PKIB, providing a molecular basis for the loss of glucose suppression and the increased effect of IBMX observed in GADA+ donor islets. The striking observation of a distinct early defect in alpha cell function that precedes beta cell loss in T1D suggests that not only overt disease, but also the progression to T1D itself, is bihormonal in nature.

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells, and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing β cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.

Procuring and preparing human pancreas for paraffin-embedding, fixed cryosections, flash frozen sections, and RNA isolation. This protocol was developed in collaboration with Dr. Rita Bottino of Imagine Pharma and Allegheny Health Network and is based on protocols developed by Dr. Marcela Brissova of Vanderbilt University.

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled >40,000 cells from normal human islets by scRNA-seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells co-expressingARX/MAFB(α cells) andMAFA/MAFB(β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-seq,MAFA/MAFBco-expressing β cells showed enhanced electrophysiological activity. Thus, these results indicate combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.

Dopamine (DA) and norepinephrine (NE) are catecholamines primarily studied in the central nervous system that also act in the pancreas as peripheral regulators of metabolism. Pancreatic catecholamine signaling has also been increasingly implicated as a mechanism responsible for the metabolic disturbances produced by antipsychotic drugs (APDs). Critically, however, the mechanisms by which catecholamines modulate pancreatic hormone release are not completely understood. We show that human and mouse pancreatic α- and β-cells express the catecholamine biosynthetic and signaling machinery, and that α-cells synthesize DA de novo. This locally-produced pancreatic DA signals via both α- and β-cell adrenergic and dopaminergic receptors with different affinities to regulate glucagon and insulin release. Significantly, we show DA functions as a biased agonist at α2A-adrenergic receptors, preferentially signaling via the canonical G protein-mediated pathway. Our findings highlight the interplay between DA and NE signaling as a novel form of regulation to modulate pancreatic hormone release. Lastly, pharmacological blockade of DA D2-like receptors in human islets with APDs significantly raises insulin and glucagon release. This offers a new mechanism where APDs act directly on islet α- and β-cell targets to produce metabolic disturbances.

Reports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet β cells. This would require binding and entry of SARS-CoV-2 into host β cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. ACE2 and TMPRSS2 transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in α or β cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet β cells through these cell entry proteins.

This review focuses on the human pancreatic islet—including its structure, cell composition, development, function, and dysfunction. After providing a historical timeline of key discoveries about human islets over the past century, we describe new research approaches and technologies that are being used to study human islets and how these are providing insight into human islet physiology and pathophysiology. We also describe changes or adaptations in human islets in response to physiologic challenges such as pregnancy, aging, and insulin resistance and discuss islet changes in human diabetes of many forms. We outline current and future interventions being developed to protect, restore, or replace human islets. The review also highlights unresolved questions about human islets and proposes areas where additional research on human islets is needed.

Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.

Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.

As part of studies to understand human pancreatic islet pathophysiology in type 1 diabetes (T1D), we discovered normal β cell mass in a 33-year-old deceased organ donor with T1D for 17 years. Further analysis revealed the donor’s diabetes resulted from a causative variant in hepatic nuclear factor 1-alpha (HNF1A), associated with Maturity-onset Diabetes of the Young-3 (MODY3) (J Clin Invest 129:246, 2019), which can be treated with sulfonylureas. Review of the donor’s redacted medical chart suggested a family history of diabetes consistent with the known autosomal dominant pattern of inheritance of MODY3. With this clinically-actionable information, we desired to inform the donor’s family. However, de-identified samples are not considered “human subject” research by Institutional Review Boards (IRB), so the family’s identity was unknown with no defined pathway for communicating these findings. Working with a Human Tissue and Organ Research Resource and an organ procurement organization, we developed a new, IRB-approved communication path between those involved in tissue collection, scientific investigation, and the donor’s family and physician. This allowed targeted genetic testing of potentially affected family members, which revealed five living relatives of the donor who carried the HNF1A variant: three adults diagnosed with diabetes before the age of 25 currently treated with insulin and two children (ages 6 months and 3 years) who did not have clinical diabetes. Since none of the identified individuals were known to have MODY3, they will likely benefit from appropriate clinical care and surveillance. This experience demonstrates the need for the scientific community to develop guidelines to address handling, reporting, and use of research findings from de-identified tissue collected for research. Defining ethical avenues for donor family-investigator communications will benefit for both organ donor families and scientific research. Disclosure D. Sparling: None. R. Haliyur: None. J. Lindner: None. L. Weber: None. A. Ramirez: None. M. Brissova: None. A.C. Powers: None.

Interruption of glucagon signaling by glucagon receptor gene inactivation or treatment with a glucagon receptor blocking monoclonal antibody (GCGR-Ab) stimulates alpha cell proliferation and expansion. Surprisingly, pancreas weight is also increased in mice with GCG gene inactivation. To investigate mechanisms linking glucagon signaling and pancreas size, we treated C57BL/6J mice with GCGR-Ab or a control IgG. Eight weeks of treatment (N=4-15 mice/treatment) increased absolute pancreas weight (0.42±0.013 vs. 0.32±0.008; p Disclosure C. Dai: None. A. Bradley: None. K.C. Coate: None. G. Poffenberger: None. E. Spears: None. J.J. Wright: None. S. Shrestha: None. D. Dean: None. M. Brissova: None. A.C. Powers: None. Funding National Institutes of Health (DK117147)

The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell–enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that MafB expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express MafB in adult mouse β-cells using MafA transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.

Type 1 diabetes (T1D) is an autoimmune disease where pancreatic β-cells are destroyed by islet-infiltrating T cells. Although a role for β-cell defects has been suspected, β-cell abnormalities are difficult to demonstrate. We show a β-cell DNA damage response (DDR), presented by activation of the 53BP1 protein and accumulation of p53, in biopsy and autopsy material from patients with recently diagnosed T1D as well as a rat model of human T1D. The β-cell DDR is more frequent in islets infiltrated by CD45+ immune cells, suggesting a link to islet inflammation. The β-cell toxin streptozotocin (STZ) elicits DDR in islets, both in vivo and ex vivo, and causes elevation of the proinflammatory molecules IL-1β and Cxcl10. β-Cell–specific inactivation of the master DNA repair gene ataxia telangiectasia mutated (ATM) in STZ-treated mice decreases the expression of proinflammatory cytokines in islets and attenuates the development of hyperglycemia. Together, these data suggest that β-cell DDR is an early event in T1D, possibly contributing to autoimmunity.

SUMMARY Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia., In Brief Brissova et al. find that β cells in the type 1 diabetic (T1D) pancreas maintain several functional and molecular features, but α cells have impaired glucagon secretion and an altered gene expression profile. These findings provide insight into the mechanism of α cell dysfunction in T1D.

In recent years, the need for enhanced rigor, reproducibility, and transparency in biomedical research has been the topic of much discussion. The National Institutes of Health (NIH) led the way in this conversation with issuance of the NIH guidelines for rigor and reproducibility, which have been mandated for all grant applications since 2016 (1,2). In support of this initiative, a recent review article by Hart and Powers, published in Diabetologia (3), with an accompanying editorial copublished in Diabetes and Diabetologia by Poitout et al. (4,5) highlighted the need for improved standardization of human islet data and its reporting in scientific publications. Diabetologia and Diabetes have initiated a checklist that is now required at the time of manuscript submission. At a minimum, the source and isolation center, a unique identifier number for each human islet preparation, and key demographic/clinical data must be included. Although not mandatory, the editorial encourages the inclusion of additional data, such as the cause of donor death, measurements of islet purity and viability, functional measures, ischemia duration, and culture time. Since 2004, the Integrated Islet Distribution Program (IIDP) has served as the main source of human islets for research in the U.S. (6–8). Directed by Joyce C. Niland at the City of Hope in Duarte, CA, the IIDP is funded by the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK) and the JDRF. To date, the IIDP has distributed over 250 million islets, serving more than 400 islet studies across nine different countries, with the resulting research yielding more than 700 publications. Thus, the IIDP is a …

SUMMARYG-protein-coupled-receptors (GPCRs) modulate insulin secretion from β cells and glucagon secretion from α cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. We studied the Gi and Gq GPCR pathways by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles and showed that the dual effects for Gq signaling occur through changes in intracellular Ca2+. By combining pseudoislets with a microfluidic system, we co-registered intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.

AIMS/HYPOTHESIS: Individuals with longstanding and recent-onset type 1 diabetes have a smaller pancreas. Since beta cells represent a very small portion of the pancreas, the loss of pancreas volume in diabetes is primarily due to the loss of pancreatic exocrine mass. However, the structural changes in the exocrine pancreas in diabetes are not well understood. METHODS: To characterise the pancreatic endocrine and exocrine compartments in diabetes, we studied pancreases from adult donors with type 1 diabetes compared with similarly aged donors without diabetes. Islet cell mass, islet morphometry, exocrine mass, acinar cell size and number and pancreas fibrosis were assessed by immunohistochemical staining. To better understand possible mechanisms of altered pancreas size, we measured pancreas size in three mouse models of insulin deficiency. RESULTS: Pancreases from donors with type 1 diabetes were approximately 45% smaller than those from donors without diabetes (47.4±2.6 vs 85.7±3.7 g), independent of diabetes duration or age of onset. Diabetic donor pancreases had decreased beta cell mass (0.061±0.025 vs 0.94±0.21 g) and reduced total exocrine mass (42.0±4.9 vs 96.1±6.5 g). Diabetic acinar cells were similar in size but fewer in number compared with those in pancreases from non-diabetic donors (63.7±8.1×10(9) vs 111.5±12.2×10(9) cells/pancreas), likely accounting for the difference in pancreas size. Within the type 1 diabetes exocrine tissue, there was a greater degree of fibrosis. The pancreases in three mouse models of insulin deficiency were similar in size to those in control mice. CONCLUSIONS/INTERPRETATION: Pancreases from donors with type 1 diabetes are smaller than normal donor pancreases because exocrine cells are fewer in number rather than smaller in size; these changes occur early in the disease process. Our mouse data suggest that decreased pancreas size in type 1 diabetes is not directly caused by insulin deficiency, but the precise mechanism responsible remains unclear.

Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes.

Efforts to advance our understanding of human islet biology are hindered by a paucity of approaches to effectively manipulate gene expression in human islets. To address this, we developed a pseudoislet system of human islet dispersion and reaggregation via modified hanging drop with 2500 cells/drop to generate 150-200 μm diameter pseudoislets (n=9 normal donors; 26-73 years). Pseudoislet morphology, dithizone uptake, and expression of markers of α and/or β cell identity (PDX1, NKX6.1, ARX, MAFB, NKX2.2, and PAX6) were similar to native islets; in paired comparisons from the same donor, pseudoislets had slightly fewer β cells and more α cells (53% β, 44% α, 3% δ cells) than native islets (62% β, 35% α, 3% δ cells; p90% β cell transduction) formed functioning pseudoislets similar to untransduced controls. Using this approach, we virally expressed hM3Dq, a Gq-coupled designer receptor exclusively activated by a designer drug (DREADD), which selectively responds to the otherwise inert ligand clozapine-n-oxide (CNO) and signals through PLC/IP3. In low glucose (2mM G + 10μM CNO), hM3Dq pseudoislets secreted more insulin than control mCherry pseudoislets (19.2 vs. 1.0 ng/100IEQ/hr; p0.05), indicating that activation of Gq signaling does not potentiate insulin secretion in high glucose. These data demonstrate how manipulation of gene expression in pseudoislets can provide insight into human islet biology and suggest that activation of Gq signaling in β cells elicits robust insulin secretion that appears to be uncoupled from glucose metabolism. Disclosure J.T. Walker: None. R. Haliyur: None. R. Aramandla: None. G. Poffenberger: None. H.A. Nelson: None. J. Luchsinger: None. P. Wang: None. A. Garcia-Ocaña: None. M. Brissova: None. A.C. Powers: None.

Individuals with longstanding type 1 diabetes (T1D) have smaller pancreata. Recent studies have found that pancreas volume is less at the time of T1D diagnosis and slightly decreased in auto-antibody positive, nondiabetic (ND) individuals. The reduced pancreas size in T1D cannot be due solely to β cell loss, since islets make up only ~2% of total pancreas mass. To better understand the T1D pancreas, we studied the pancreatic endocrine and exocrine compartments from T1D donors (N=23; age 18-62 years; duration 10-45 years) and age-matched, ND controls (N= 20; age 18-55 years). During islet isolation from T1D pancreata, standard approaches and enzymes were quite inadequate to free most islets or disperse the exocrine tissue. We also noted that T1D pancreata were more firm in physical consistency and had reduced relative pancreas weight (RPW, gm pancreas per kg body weight; 0.63 ± 0.04 in T1D vs. 1.1 ± 0.1 in ND, p Disclosure J.J. Wright: None. D.C. Saunders: None. J. Lindner: None. M. Bogdani: None. M. Brissova: None. R. Bottino: Research Support; Self; Imagine Pharma. A.C. Powers: None.

Impaired counterregulatory response of glucagon to hypoglycemia is a common complication of type 1 diabetes (T1D). One proposed contributor to abnormal glucagon secretion is decreased sympathetic innervation in T1D islets. To systematically assess the innervation of human pancreatic tissues, we examined samples from donors with recent-onset T1D (10 years, age 27-63 years, n = 3), and nondiabetic controls (age 10-52 years, n = 4). Islet and exocrine tissue innervation was visualized by pan-neuronal marker tubulin β-3 and analyzed by a 2-D morphometry and 3-D rendering. By quantifying the nerve fiber length (1.9±0.4 nm/μm2) and density (646±94 fibers/mm2), we found that innervation in control human islets was nearly 20-fold less than previously reported in mouse islets. In contrast to mouse, human exocrine tissue was far more innervated than islets with fiber length of 6.8±0.4 nm/μm2(p0.05), and so was the 3-D arrangement of nerve fibers in exocrine tissue and islets. Regardless of T1D duration, the islet fiber length (recent-onset: 1.5±0.2 nm/μm2; long-standing: 1.0±0.2 nm/μm2) and density (recent-onset: 623±61 fibers/mm2; longstanding: 486±49 fibers/mm2), as well as exocrine fiber length (recent-onset: 5.2±.5 nm/μm2; longstanding: 5.8±0.4 nm/μm2) and density (recent-onset: 1119±77 fibers/mm2; longstanding: 1537±72 fibers/mm2) did not differ from controls (p>0.05). These data indicate that neuronal patterning of human pancreas is significantly different from that of mouse and islet innervation does not appear to be altered in T1D. Disclosure T.M. Richardson: None. R. Haliyur: None. R. Aramandla: None. R. Bottino: Research Support; Self; Imagine Pharma. A.C. Powers: None. M. Brissova: None.

Single cell RNA-sequencing (scRNA-seq) approaches are continuously improving at the point of data collection and analysis to simultaneously profile all cell types within islet microorgans to better understand heterogeneity and phenotype-function relationships and how these change with aging and disease. To better understand the advantages and limitations of scRNA-seq, we used the ChromiumTMplatform to profile human islets with a robust insulin and glucagon secretion by perifusion (5 nondiabetic donors, ages 14-66 years) and compared these results to scRNA-seq analysis of purified α and β cells. In context of the same donor (n=2), purified α and β cells (using NTPDase3 marker) showed high correlation (r=0.99) with the Seurat-identified α and β subsets originating from dispersed whole islets. Based on the analysis of a very large pool of single cells (79,881 cells; 2316 genes/cell), the Seurat-guided analysis identified cell types that segregated into eight different clusters (17% β, 66% α, 4% δ, 4% ductal, 4% mesenchymal, 2% acinar, 2% endothelial, and 1% immune). Interstingly, in this large single cell context, we did not identify the four β cell subsets previously reported on the basis of alternative cell surface markers, but found a β cell cluster with highly expressed ER stress-associated genes and an α cell cluster with higher cell cycle regulation marker expression. We concurrently investigated expression of functionally important MAFA and MAFB genes, which are expressed only in a fraction of β cells by histological analysis, and found that PERK-mediated UPR transcripts were enriched in the MAFB-expressing β cells, while the MAFA-expressing β cells differentially expressed genes associated with β cell function. Overall, our study indicates high fidelity of α and β cell clustering in dispersed and sorted islet cells and points to a different pattern of β cell heterogeneity when β cells are purified using NTPDase3 expression compared to other cell surface markers. Disclosure S. Shrestha: None. D.C. Saunders: None. J.T. Walker: None. R. Haliyur: None. G. Poffenberger: None. R. Aramandla: None. A. Jones: None. N. Prasad: None. S.E. Levy: None. A.C. Powers: None. M. Brissova: None.

Numerous compounds stimulate rodent β-cell proliferation; however, translating these findings to human β-cells remains a challenge. To examine human β-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human β-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled β-cells with high specificity using both nuclear and cytoplasmic markers. β-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1β signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ β-cells, whereas treatment with other compounds had limited to no effect on human β-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human β-cell proliferation, thus allowing for increased testing of candidate human β-cell mitogens.

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

Summary Human tissue phenotyping generates complex spatial information from numerous imaging modalities, yet images typically become static figures for publication, and original data and metadata are rarely available. While comprehensive image maps exist for some organs, most resources have limited support for multiplexed imaging or have non-intuitive user interfaces. Therefore, we built a Pancreatlas resource that integrates several technologies into a unique interface, allowing users to access richly annotated web pages, drill down to individual images, and deeply explore data online. The current version of Pancreatlas contains over 800 unique images acquired by whole-slide scanning, confocal microscopy, and imaging mass cytometry, and is available at https://www.pancreatlas.org. To create this human pancreas-specific biological imaging resource, we developed a React-based web application and Python-based application programming interface, collectively called Flexible Framework for Integrating and Navigating Data (FFIND), which can be adapted beyond Pancreatlas to meet countless imaging or other structured data-management needs., Graphical Abstract, Highlights • Human organ phenotyping databases benefit from intuitive user interfaces • Pancreatlas resource enables exploration of bioimaging data from human pancreas • The front-end framework of Pancreatlas, FFIND, is modular and easily adaptable • FFIND provides structured data-exploration capabilities across countless domains, The Bigger Picture Scientists need cost-effective yet fully featured database solutions that facilitate large dataset sharing in a structured and easily digestible manner. Flexible Framework for Integrating and Navigating Data (FFIND) is a data-agnostic web application that is designed to easily connect existing databases with data-browsing clients. We used FFIND to build Pancreatlas, an online imaging resource containing datasets linking imaging data with clinical data to facilitate advances in the understanding of diabetes, pancreatitis, and pancreatic cancer. FFIND architecture, which is available as open-source software, can be easily adapted to meet other field- or project-specific needs; we hope it will help data scientists reach a broader audience by reducing the development life cycle and providing familiar interactivity in communicating data and underlying stories., Human tissue phenotyping generates complex imaging data that is difficult to share in publications, and many organ-specific databases lack intuitive user interfaces or have limited support for multiplexed imaging. Therefore, we built a Pancreatlas resource (https://www.pancreatlas.org) that integrates several technologies into a unique interface, allowing users to access richly annotated web pages. To create this imaging resource, we developed a data-agnostic, React-based web application and Python-based application programming interface, collectively called Flexible Framework for Integrating and Navigating Data (FFIND; https://github.com/Powers-Brissova-Research-Group/FFIND).

Although cellular stress response is important for maintaining function and survival, overactivation of late-stage stress effectors cause dysfunction and death. We show that the myelin transcription factors (TFs) Myt1 (Nzf2), Myt2 (Myt1l, Nztf1, and Png-1), and Myt3 (St18 and Nzf3) prevent such overactivation in islet β cells. Thus, we found that co-inactivating the Myt TFs in mouse pancreatic progenitors compromised postnatal β cell function, proliferation, and survival, preceded by upregulation of late-stage stress-response genes activating transcription factors (e.g., Atf4) and heat-shock proteins (Hsps). Myt1 binds putative enhancers of Atf4 and Hsps, whose overexpression largely recapitulated the Myt-mutant phenotypes. Moreover, Myt(MYT)-TF levels were upregulated in mouse and human β cells during metabolic stress-induced compensation but downregulated in dysfunctional type 2 diabetic (T2D) human β cells. Lastly, MYT knockdown caused stress-gene overactivation and death in human EndoC-βH1 cells. These findings suggest that Myt TFs are essential restrictors of stress-response overactivity.