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Diabetes is a chronic disease that affects glucose metabolism, either by autoimmune-driven β-cell loss or by the progressive loss of β-cell function, due to continued metabolic stresses. Although both α- and β-cells are exposed to the same stressors, such as proinflammatory cytokines and saturated free fatty acids (e.g., palmitate), only α-cells survive. We previously reported that the abundant expression of BCL-XL, an anti-apoptotic member of the BCL-2 family of proteins, is part of the α-cell defense mechanism against palmitate-induced cell death. Here, we investigated whether BCL-XL overexpression could protect β-cells against the apoptosis induced by proinflammatory and metabolic insults. For this purpose, BCL-XL was overexpressed in two β-cell lines—namely, rat insulinoma-derived INS-1E and human insulin-producing EndoC-βH1 cells—using adenoviral vectors. We observed that the BCL-XL overexpression in INS-1E cells was slightly reduced in intracellular Ca2+ responses and glucose-stimulated insulin secretion, whereas these effects were not observed in the human EndoC-βH1 cells. In INS-1E cells, BCL-XL overexpression partially decreased cytokine- and palmitate-induced β-cell apoptosis (around 40% protection). On the other hand, the overexpression of BCL-XL markedly protected EndoC-βH1 cells against the apoptosis triggered by these insults (>80% protection). Analysis of the expression of endoplasmic reticulum (ER) stress markers suggests that resistance to the cytokine and palmitate conferred by BCL-XL overexpression might be, at least in part, due to the alleviation of ER stress. Altogether, our data indicate that BCL-XL plays a dual role in β-cells, participating both in cellular processes related to β-cell physiology and in fostering survival against pro-apoptotic insults.

Aims/hypothesis Wolfram syndrome is a rare autosomal recessive disorder caused by pathogenic variants in the WFS1 gene. It is characterised by insulin-dependent diabetes mellitus, optic nerve atrophy, diabetes insipidus, hearing loss and neurodegeneration. Considering the unmet treatment need for this orphan disease, this study aimed to evaluate the therapeutic potential of glucagon-like peptide 1 receptor (GLP-1R) agonists under wolframin (WFS1) deficiency with a particular focus on human beta cells and neurons. Methods The effect of the GLP-1R agonists dulaglutide and exenatide was examined in Wfs1 knockout mice and in an array of human preclinical models of Wolfram syndrome, including WFS1-deficient human beta cells, human induced pluripotent stem cell (iPSC)-derived beta-like cells and neurons from control individuals and individuals affected by Wolfram syndrome, and humanised mice. Results Our study shows that the long-lasting GLP-1R agonist dulaglutide reverses impaired glucose tolerance in WFS1-deficient mice, and that exenatide and dulaglutide improve beta cell function and prevent apoptosis in different human WFS1-deficient models including iPSC-derived beta cells from people with Wolfram syndrome. Exenatide improved mitochondrial function, reduced oxidative stress and prevented apoptosis in Wolfram syndrome iPSC-derived neural precursors and cerebellar neurons. Conclusions/interpretation Our study provides novel evidence for the beneficial effect of GLP-1R agonists on WFS1-deficient human pancreatic beta cells and neurons, suggesting that these drugs may be considered as a treatment for individuals with Wolfram syndrome. Graphical abstract

Alternative splicing (AS) within the β-cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We used a computational strategy to prioritize pathogenic splicing events in human islets treated with interleukin-1β plus interferon-γ as an ex vivo model of T1D and coupled this analysis with a k-mer–based approach to predict RNA-binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with a majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to affect protein structure. AS occurred with high frequency in MHC class II–related mRNAs, and targeted quantitative PCR validated reduced inclusion of exon 5 in the MHC class II gene HLA-DMB. Single-molecule RNA fluorescence in situ hybridization confirmed increased HLA-DMB splicing in β-cells from human donors with established T1D and autoantibody positivity. Serine/arginine-rich splicing factor 2 was implicated in 37.2% of potentially pathogenic events, including exon 5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the β-cell response to inflammatory signals during T1D evolution.

HypothesisThe prevalence of type 2 diabetes is higher in Latino populations compared with other major ancestry groups. Not only has the Latino population been systematically underrepresented in large-scale genetic analyses, but previous studies relied on the imputation of ungenotyped variants based on the 1000 Genomes (1000G) imputation reference panel, which results in suboptimal capture of low-frequency or Latino-enriched variants. The NHLBI Trans-Omics for Precision Medicine (TOPMed) reference panel represents a unique opportunity to analyze rare genetic variations in the Latino population.MethodsWe evaluate the TOPMed imputation performance using genotyping array and whole-exome sequence data in 6 Latino cohorts. To evaluate the ability of TOPMed imputation of increasing the identified loci, we performed a Latino type 2 diabetes GWAS meta-analysis in 8,150 type 2 diabetes cases and 10,735 controls and replicated the results in 6 additional cohorts including whole-genome sequence data from the All of Us cohort.ResultsWe show that, compared to imputation with 1000G, the TOPMed panel improves the identification of rare and low-frequency variants. We identified 26 distinct signals including a novel genome-wide significant variant (minor allele frequency 1.6%, OR=2.0, P=3.4×10−9) near ORC5. A Latino-tailored polygenic score constructed from our data and GWAS data from East Asian and European populations improves the prediction accuracy in a Latino target dataset, explaining up to 7.6% of the type 2 diabetes risk variance.ConclusionsOur results demonstrate the utility of TOPMed imputation for identifying low-frequency variation in understudied populations, leading to the discovery of novel disease associations and the improvement of polygenic scores.

Innate immunity is directly associated with Type 1 diabetes (T1D) . Recently, N6-methyladenosine (m6A) has been shown to modulate human β-cell biology and innate immunity. Here, we focused on the contribution of m6A in modulating T1D development. RNA-seq. in β-cells revealed downregulation of the m6A writers (Mettl3, Mettl14, and Wtap) in pre-diabetic NOD mice. Consistently, pancreases from female NOD mice and human T1D showed downregulation of METTL3 protein levels in insulin-positive cells as the disease progressed. m6A-sequencing in human T1D versus control islets and intersection with differentially expressed genes (DEGs) in T1D β-cells from a single-cell RNA seq. dataset, revealed enriched pathways associated with ER function, T1D, and apoptosis. Pro-inflammatory cytokines are directly linked to β-cell autoimmunity and are considered a useful tool to study T1D onset. We applied m6A-sequencing in cytokine-treated human islet preparations, and in parallel studies, treated EndoC-βH1 cells with cytokines followed by RNA-sequencing. Intersection of m6A-decorated and common DEGs in human islets and EndoC-βH1 cells revealed enrichment in pathways associated with the innate immune response. METTL3 silencing in EndoC-βH1 impacted several innate immune mediators upon cytokine challenge. Incubation of human islets from multiple donors with ER-stress inducers, ROS donors, NO donors, and ROS or NO scavengers revealed that pre-existing ER stress and ROS decrease METTL3, while physiological levels of NO upregulate METTL3. We conclude that ROS drives METTL3 downregulation associated with T1D. Exploring therapeutic relevance by AAV-8 mediated gene delivery showed that β-cell specific upregulation of Mettl3 leads to a decrease in hyperglycemia during early stages of T1D in female NOD mice. Together, these results demonstrate a novel layer of regulation in human β-cells in the context of T1D and point to targeting METTL3 as a therapeutic approach to promote β-cell survival and improve glycemia. Disclosure D.F.De jesus: n/a. D.L.Eizirik: None. R.Kulkarni: Advisory Panel; Biomea, Novo Nordisk, REDD Pharma, Consultant; Relay Therapeutics, Research Support; Inversago, SerPlus. Z.Zhang: Research Support; SinoVac. N.K.Brown: None. G.Basile: None. L.Xiao: None. S.Kahraman: Employee; Boehringer Ingelheim International GmbH. C.E.Mathews: None. A.C.Powers: None. M.A.Atkinson: None. Funding American Diabetes Association (7-21-PDF-140) ; R01 DK067536R01 DK103215R01 DK117639

SUMOylation has long been recognized to regulate multiple biological processes in pancreatic beta cells, but its impact on proinsulin disulfide maturation and endoplasmic reticulum (ER) stress remains elusive. Herein, we conducted comparative proteomic analyses of SUMOylated proteins in primary mouse/human islets following proinflammatory cytokine stimulation. Cytokine challenge rendered beta cells to undergo a SUMOylation turnover manifested by the changes of SUMOylation substrates and SUMOylation levels for multiple substrates. Our data support that SUMOylation may play a crucial role to regulate proinsulin misfolding and ER stress at least by targeting Protein Disulfide Isomerase a3 (Pdia3). SUMOylation regulates Pdia3 enzymatic activity, subcellular localization, and protein binding ability. Furthermore, SUMOylation of Pdia3 exacerbated proinsulin misfolding and ER stress, and repressed Stat3 activation. In contrast, disruption of Pdia3 SUMOylation markedly rescued the outcomes. Collectively, our study expands the understanding how SUMOylation regulates ER stress in beta cells, which shed light on developing potential strategies against beta cell dysfunction., SCOPUS: ar.j, info:eu-repo/semantics/published

It is increasingly appreciated that the pathogenic mechanisms of type 1 diabetes involve both the autoimmune aggressors and their beta cell targets, which engage in a conflicting dialogue within and possibly outside the pancreas. Indeed, autoimmune CD8+ T cells, which are the final mediators of beta cell destruction, circulate at similar frequencies in type 1 diabetic and healthy individuals. Hence a universal state of ‘benign’ islet autoimmunity exists, and we hypothesise that its progression to type 1 diabetes may at least partially rely on a higher vulnerability of beta cells, which play a key, active role in disease development and/or amplification. We posit that this autoimmune vulnerability is rooted in some features of beta cell biology: the stress imposed by the high rate of production of insulin and other granule proteins, their dense vascularisation and the secretion of their products directly into the bloodstream. Gene variants that may predispose individuals to this vulnerability have been identified, e.g. MDA5, TYK2, PTPN2. They interact with environmental cues, such as viral infections, that may drive this genetic potential towards exacerbated local inflammation and progressive beta cell loss. On top of this, beta cells set up compensatory responses, such as the unfolded protein response, that become deleterious in the long term. The relative contribution of immune and beta cell drivers may vary and phenotypic subtypes (endotypes) are likely to exist. This dual view argues for the use of circulating biomarkers of both autoimmunity and beta cell stress for disease staging, and for the implementation of both immunomodulatory and beta cell-protective therapeutic strategies. [Figure not available: see fulltext.], SCOPUS: re.j, info:eu-repo/semantics/published

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells., The cytokine IFNα is expressed in the islets of individuals with type 1 diabetes and contributes to local inflammation and destruction of beta cells. Here, the authors provide a global multiomics view of IFNα-induced changes in human beta cells at the level of chromatin, mRNA and protein expression.

Aims/hypothesis: High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was rediscovered to be a ‘danger signal’ (alarmin) that alerts the immune system once released extracellularly. Therefore, it has been recognised contributing to the pathogenesis of autoimmune diabetes, but its exact impact on the initiation and progression of type 1 diabetes, as well as the related molecular mechanisms, are yet to be fully characterised. Methods: In the current report, we employed NOD mice as a model to dissect the impact of blocking HMGB1 on the prevention, treatment and reversal of type 1 diabetes. To study the mechanism involved, we extensively examined the characteristics of regulatory T cells (Tregs) and their related signalling pathways upon HMGB1 stimulation. Furthermore, we investigated the relevance of our data to human autoimmune diabetes. Results: Neutralising HMGB1 both delayed diabetes onset and, of particular relevance, reversed diabetes in 13 out of 20 new-onset diabetic NOD mice. Consistently, blockade of HMGB1 prevented islet isografts from autoimmune attack in diabetic NOD mice. Using transgenic reporter mice that carry a Foxp3 lineage reporter construct, we found that administration of HMGB1 impairs Treg stability and function. Mechanistic studies revealed that HMGB1 activates receptor for AGE (RAGE) and toll-like receptor (TLR)4 to enhance phosphatidylinositol 3-kinase (PI3K)–Akt–mechanistic target of rapamycin (mTOR) signalling, thereby impairing Treg stability and functionality. Indeed, high circulating levels of HMGB1 in human participants with type 1 diabetes contribute to Treg instability, suggesting that blockade of HMGB1 could be an effective therapy against type 1 diabetes in clinical settings. Conclusions/interpretation: The present data support the possibility that HMGB1 could be a viable therapeutic target to prevent the initiation, progression and recurrence of autoimmunity in the setting of type 1 diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

Glucagon secretion is perturbed in both type 1 and type 2 diabetes (T1D, T2D) the pathophysiological changes at the level of individual pancreatic alpha cells are still largely obscure. Using recently-curated single-cell RNA data from human donors with either T1D or T2D and appropriate controls, we leveraged alpha cell transcriptomic alterations consistent with both common and discrete pathways. Firstly, altered expression of genes associated with alpha cell identity (ARX, MAFB) was common to both diseases. In contrast, increased expression of cytokine-regulated genes and genes involved in glucagon biosynthesis and processing were apparent in T1D, whereas mitochondrial genes associated with reactive oxygen species generation (COX7B, NQO2) were dysregulated in alpha cells from T2D patients. Conversely, T1D alpha cells displayed alterations in genes associated with autoimmune-induced ER stress (ERLEC1, HSP90) whilst those from T2D patients showed changes in glycolytic and citrate cycle genes (LDH, PDHB, PDK4) which were unaffected in T1D. These findings suggest that despite some similarities related to loss-of-function, the alterations of alpha cells present important disease-specific signatures, suggesting that they are secondary to the main pathogenic events characteristic to each disease, namely immune-mediated-or metabolic-mediated-stress in respectively T1D and T2D.

A recent discovery effort resulted in identification of novel splice variant and secretory granule antigens within the HLA class I peptidome of human islets and documentation of their recognition by CD8+ T cells from peripheral blood and human islets. In the current study, we applied a systematic discovery process to identify novel CD4+ T cell epitopes derived from these candidate antigens. We predicted 145 potential epitopes spanning unique splice junctions and within conventional secretory granule antigens and measured their in vitro binding to DRB1*04:01. We generated HLA class II tetramers for the 35 peptides with detectable binding and used these to assess immunogenicity and isolate T cell clones. Tetramers corresponding to peptides with verified immunogenicity were then used to label T cells specific for these putative epitopes in peripheral blood. T cells that recognize distinct epitopes derived from a cyclin I splice variant, neuroendocrine convertase 2, and urocortin-3 were detected at frequencies that were similar to those of an immunodominant proinsulin epitope. Cells specific for these novel epitopes predominantly exhibited a Th1-like surface phenotype. Among the three epitopes, responses to the cyclin I peptide exhibited a distinct memory profile. Responses to neuroendocrine convertase 2 were detected among pancreatic infiltrating T cells. These results further establish the contribution of unconventional antigens to the loss of tolerance in autoimmune diabetes.

A sensible control of hormone secretion from pancreatic islets requires concerted inter-cellular communications, but a comprehensive picture of the whole islet interactome is presently missing. Single-cell transcriptomics allows to overcome this and we used here a single-cell dataset from type 2 diabetic (T2D) and non-diabetic (ND) donors to leverage islet interaction networks. The single-cell dataset contains 3046 cells classified in 7 cell types. The interactions across cell types in T2D and ND were obtained and resulting networks analysed to identify high-centrality genes and altered interactions in T2D. The T2D interactome displayed a higher number of interactions (10 787) than ND (9707); 1289 interactions involved beta cells (1147 in ND). High-centrality genes included EGFR, FGFR1 and FGFR2, important for cell survival and proliferation. In conclusion, this analysis represents the first in silico model of the human islet interactome, enabling the identification of signatures potentially relevant for T2D pathophysiology.

Type 2 diabetes (T2D) has been considered a relentlessly worsening disease, due to the progressive deterioration of the pancreatic beta cell functional mass. Recent evidence indicates, however, that remission of T2D may occur in variable proportions of patients after specific treatments that are associated with recovery of beta cell function. Here we review the available information on the recovery of beta cells in (a) non-diabetic individuals previously exposed to metabolic stress; (b) T2D patients following low-calorie diets, pharmacological therapies or bariatric surgery; (c) human islets isolated from non-diabetic organ donors that recover from “lipo-glucotoxic” conditions; and (d) human islets isolated from T2D organ donors and exposed to specific treatments. The improvement of insulin secretion reported by these studies and the associated molecular traits unveil the possibility to promote T2D remission by directly targeting pancreatic beta cells.

In type 1 diabetes, autoimmune β-cell destruction may be favored by neo-antigens harboring post-translational modifications such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in type 1 diabetic and healthy donors and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidyl-arginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T-cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, post-translational modifications may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Remission of type 2 diabetes (T2D) may occur after very low-calorie diets or bariatric surgery, and is associated with improved pancreatic beta cell function. Here, we evaluated if T2D beta cell dysfunction can be rescued ex-vivo and which are the molecular mechanisms involved. Islets from 19 T2D donors were studied after isolation (“basal”) and following culture at 5.5 or 11.1 mmol/l glucose (“cultured”). We evaluated glucose-stimulated insulin secretion (GSIS) and transcriptomes by RNA sequencing, correlated insulin secretion changes (“cultured” vs “basal”) to global gene expression, and searched for potential therapeutic gene targets and compounds that mimic gene signatures of recovered beta cell function in T2D islets. GSIS improved in 12 out of 19 islet preparations from T2D donors after culture at 5.5 mmol/l glucose (insulin stimulation index increased from 1.4±0.1 to 2.3±0.2, p

Type 1 diabetes (T1D) is an autoimmune disease characterized by a progressive destruction of β-cells and several candidate genes associated with T1D have been linked to innate immunity. Recently, N6-methyladenosine (m6A) has been shown to modulate human β-cell biology and to target type I interferons. Here, we focused on the contribution of m6A mRNA methylation in modulating innate immunity and β-cell destruction. Immune cells contribute to β-cell apoptosis by the release of pro-inflammatory cytokines during the onset of T1D. We assessed whether m6A abundance or expression of m6A modulators was altered in human islets and EndoC-βH1 cells treated with cytokines to mimick T1D onset. LC-MS/MS analyses of human islets showed that treatment with IL-1β plus IFN-α for 48h increased m6A levels. Consistently, IL-1β plus IFN-α treatment of human islets and EndoC-βH1 induced upregulation of the m6A writer METTL3. Next, we applied m6A-sequencing in cytokine-treated human islet preparations, and in parallel studies, treated EndoC-βH1 cells followed by RNA-sequencing. Intersection of m6A-decorated genes in cytokine-treated human islets with common differentially expressed genes in human islets and EndoC-βH1 cells revealed enrichment in pathways associated with the innate immune response, negative regulation of immune response, and extrinsic apoptotic signaling pathway. Differentially expressed and m6A decorated genes included genes associated with T1D that are involved in innate immunity. Re-analyses of RNA-seq. data in sorted-β-cells from T1D patients revealed downregulation of METTL3. These data suggest that while healthy β-cells are able to upregulate m6A levels and METTL3 early in T1D (e.g., when they are exposed to cytokines), this response is lost in established T1D that allows an exacerbated immune response and β-cell death. Together, these results point to a possible therapeutic approach in targeting METTL3 to modulate innate immunity and promote β-cell survival. Disclosure D. F. De jesus: None. C. He: None. R. Kulkarni: None. Z. Zhang: None. N. K. Brown: None. J. Hu: None. S. Kahraman: None. C. E. Mathews: None. A. C. Powers: None. M. A. Atkinson: None. D. L. Eizirik: None. Funding National Institutes of Health (1UC4DK116278)

GWAS have identified more than 700 genetic signals associated with type 2 diabetes (T2D). To gain insight into the underlying molecular mechanisms, we created the Translational human pancreatic Islet Genotype tissue-Expression Resource (TIGER), aggregating >500 human islet RNA-seq and genotyping datasets. We imputed genotypes using 4 reference panels and meta-analyzed cohorts to improve coverage of expression quantitative trait loci (eQTL) and developed a method to combine allele-specific expression across samples (cASE). We identified >1 million islet eQTLs (56% novel), of which 53 colocalize with T2D signals (60% novel). Among them, a low-frequency allele that reduces T2D risk by half increases CCND2 expression. We identified 8 novel cASE colocalizations, among which an SLC30A8 T2D associated variant. We make all the data available through the open-access TIGER portal (http://tiger.bsc.es), which represents a comprehensive human islet genomic data resource to elucidate how genetic variation affects islet function and translate this into therapeutic insight and precision medicine for T2D.

Loss of functional β-cell mass is a key factor contributing to the poor glycaemic control in type 2 diabetes. However, therapies that directly target these underlying processes remains lacking. Here we demonstrate that gene expression of neuropeptide Y1 receptor and its ligand, neuropeptide Y, was significantly upregulated in human islets from subjects with type 2 diabetes. Importantly, the reduced insulin secretion in type 2 diabetes was associated with increased neuropeptide Y and Y1 receptor expression in human islets. Consistently, pharmacological inhibition of Y1 receptors by BIBO3304 significantly protected β-cells from dysfunction and death under multiple diabetogenic conditions in islets. In a preclinical study, Y1 receptor antagonist BIBO3304 treatment improved β-cell function and preserved functional β-cell mass, thereby resulting in better glycaemic control in both high-fat-diet/multiple low-dose streptozotocin- and db/db type 2 diabetic mice. Collectively, our results uncovered a novel causal link of increased islet NPY-Y1 receptor signaling to β-cell dysfunction and failure in human type 2 diabetes. These results further demonstrate that inhibition of Y1 receptor by BIBO3304 represents a novel and effective β-cell protective therapy for improving functional β-cell mass and glycaemic control in type 2 diabetes.

Chronic exercise protects pancreatic beta cells from diabetogenic stress, reducing insulin secretion through unknown mechanisms. We tested the hypothesis that the IL-6/mitochondrial STAT3 (pS-STAT3) axis plays a role in this protective effect. C57BL/6N mice were subjected to endurance training ahead of pancreatic islet isolation and functional analysis. Similar in vitro experiments were performed using insulin-producing INS-1E cells and islets from untrained mice, cultured with serum from trained animals and treated with or without an IL-6 receptor (IL-6R) inhibitor. Then, IL-6R/pS-STAT3 pathway activation and its effects on mitochondrial function and insulin secretion were assessed. Exercise-induced down-regulation of insulin secretion was prevented by inhibition of IL-6R signaling and following STAT3 knockdown. IL-6R activation promoted STAT3 phosphorylation and translocation to the mitochondria, increasing oxygen consumption. Accordingly, lower H2O2 content was reported in islets from trained mice and beta cells exposed to exercise-conditioned serum, while exposure to exogenous H2O2 blocked the down-regulatory effect of training on insulin secretion. Similar findings were observed in islets from obese-trained mice. Together, these findings suggest that the IL-6R/pS-STAT3 axis mediates exercise-induced down-regulation of insulin secretion through modulation of the mitochondrial redox state.

Pancreatic islet β-cell failure is key to the onset and progression of type 2 diabetes (T2D). The advent of single-cell RNA sequencing (scRNA-seq) has opened the possibility to determine transcriptional signatures specifically relevant for T2D at the β-cell level. Yet, applications of this technique have been underwhelming, as three independent studies failed to show shared differentially expressed genes in T2D β-cells. We performed an integrative analysis of the available datasets from these studies to overcome confounding sources of variability and better highlight common T2D β-cell transcriptomic signatures. After removing low-quality transcriptomes, we retained 3046 single cells expressing 27 931 genes. Cells were integrated to attenuate dataset-specific biases, and clustered into cell type groups. In T2D β-cells (

In type 1 diabetes, autoimmune β-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8(+) T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8(+) T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8(+) T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8(+) T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-βH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| We identified a total of 264 genes stably expressed in EndoC-βH1 cells and human islets following cytokine- or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.