Deucravacitinib, a tyrosine kinase 2 pseudokinase inhibitor, protects human EndoC-ßH1 ß-cells against proinflammatory insults.

Introduction: Type 1 diabetes is characterized by pancreatic islet inflammation and autoimmune-driven pancreatic ß-cell destruction. Interferon-α (IFNα) is a key player in early human type 1 diabetes pathogenesis. IFNα activates the tyrosine kinase 2 (TYK2)-signal transducer and activator of transcription (STAT) pathway, leading to inflammation, HLA class I overexpression, endoplasmic reticulum (ER) stress, and ß-cell apoptosis (in synergy with IL-1ß). As TYK2 inhibition has raised as a potential therapeutic target for the prevention or treatment of type 1 diabetes, we investigated whether the selective TYK2 inhibitor deucravacitinib could protect ß-cells from the effects of IFNα and other proinflammatory cytokines (i.e., IFNγ and IL-1ß). Methods: All experiments were performed in the human EndoC-ßH1 ß-cell line. HLA class I expression, inflammation, and ER stress were evaluated by real-time PCR, immunoblotting, and/or immunofluorescence. Apoptosis was assessed by the DNA-binding dyes Hoechst 33342 and propidium iodide or caspase 3/7 activity. The promoter activity was assessed by luciferase assay. Results: Deucravacitinib prevented IFNα effects, such as STAT1 and STAT2 activation and MHC class I hyperexpression, in a dose-dependent manner without affecting ß-cell survival and function. A comparison between deucravacitinib and two Janus kinase inhibitors, ruxolitinib and baricitinib, showed that deucravacitinib blocked IFNα- but not IFNγ-induced signaling pathway. Deucravacitinib protected ß-cells from the effects of two different combinations of cytokines: IFNα + IL-1ß and IFNγ + IL-1ß. Moreover, this TYK2 inhibitor could partially reduce apoptosis and inflammation in cells pre-treated with IFNα + IL-1ß or IFNγ + IL-1ß. Discussion: Our findings suggest that, by protecting ß-cells against the deleterious effects of proinflammatory cytokines without affecting ß-cell function and survival, deucravacitinib could be repurposed for the prevention or treatment of early type 1 diabetes.

BCL-XL Overexpression Protects Pancreatic ß-Cells against Cytokine- and Palmitate-Induced Apoptosis.

Diabetes is a chronic disease that affects glucose metabolism, either by autoimmune-driven ß-cell loss or by the progressive loss of ß-cell function, due to continued metabolic stresses. Although both α- and ß-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 ß-cells against the apoptosis induced by proinflammatory and metabolic insults. For this purpose, BCL-XL was overexpressed in two ß-cell lines-namely, rat insulinoma-derived INS-1E and human insulin-producing EndoC-ßH1 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-ßH1 cells. In INS-1E cells, BCL-XL overexpression partially decreased cytokine- and palmitate-induced ß-cell apoptosis (around 40% protection). On the other hand, the overexpression of BCL-XL markedly protected EndoC-ßH1 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 ß-cells, participating both in cellular processes related to ß-cell physiology and in fostering survival against pro-apoptotic insults.

G protein-coupled estrogen receptor activation by bisphenol-A disrupts the protection from apoptosis conferred by the estrogen receptors ERα and ERß in pancreatic beta cells.

17ß-estradiol protects pancreatic ß-cells from apoptosis via the estrogen receptors ERα, ERß and GPER. Conversely, the endocrine disruptor bisphenol-A (BPA), which exerts multiple effects in this cell type via the same estrogen receptors, increased basal apoptosis. The molecular-initiated events that trigger these opposite actions have yet to be identified. We demonstrated that combined genetic downregulation and pharmacological blockade of each estrogen receptor increased apoptosis to a different extent. The increase in apoptosis induced by BPA was diminished by the pharmacological blockade or the genetic silencing of GPER, and it was partially reproduced by the GPER agonist G1. BPA and G1-induced apoptosis were abolished upon pharmacological inhibition, silencing of ERα and ERß, or in dispersed islet cells from ERß knockout (BERKO) mice. However, the ERα and ERß agonists PPT and DPN, respectively, had no effect on beta cell viability. To exert their biological actions, ERα and ERß form homodimers and heterodimers. Molecular dynamics simulations together with proximity ligand assays and coimmunoprecipitation experiments indicated that the interaction of BPA with ERα and ERß as well as GPER activation by G1 decreased ERαß heterodimers. We propose that ERαß heterodimers play an antiapoptotic role in beta cells and that BPA- and G1-induced decreases in ERαß heterodimers lead to beta cell apoptosis. Unveiling how different estrogenic chemicals affect the crosstalk among estrogen receptors should help to identify diabetogenic endocrine disruptors.

Bisphenol-S and Bisphenol-F alter mouse pancreatic ß-cell ion channel expression and activity and insulin release through an estrogen receptor ERß mediated pathway.

Bisphenol-S (BPS) and Bisphenol-F (BPF) are current Bisphenol-A (BPA) substitutes. Here we used pancreatic ß-cells from wild type (WT) and estrogen receptor ß (ERß) knockout (BERKO) mice to investigate the effects of BPS and BPF on insulin secretion, and the expression and activity of ion channels involved in ß-cell function. BPS or BPF rapidly increased insulin release and diminished ATP-sensitive K+ (KATP) channel activity. Similarly, 48 h treatment with BPS or BPF enhanced insulin release and decreased the expression of several ion channel subunits in ß-cells from WT mice, yet no effects were observed in cells from BERKO mice. PaPE-1, a ligand designed to preferentially trigger extranuclear-initiated ER pathways, mimicked the effects of bisphenols, suggesting the involvement of extranuclear-initiated ERß pathways. Molecular dynamics simulations indicated differences in ERß ligand-binding domain dimer stabilization and solvation free energy among different bisphenols and PaPE-1. Our data suggest a mode of action involving ERß whose activation alters three key cellular events in ß-cell, namely ion channel expression and activity, and insulin release. These results may help to improve the hazard identification of bisphenols.

Pancreatic alpha-cell mass in the early-onset and advanced stage of a mouse model of experimental autoimmune diabetes.

Most studies in type 1 diabetes (T1D) have focused on the loss of the pancreatic beta-cell population. However, despite the involvement of the alpha-cell in the aetiology and complications of T1D, little is known about the regulation of the pancreatic alpha-cell mass in this disease. The need for a better understanding of this process is further emphasized by recent findings suggesting that alpha-cells may constitute a potential reservoir for beta-cell regeneration. In this study, we characterized the pancreatic alpha-cell mass and its regulatory processes in the transgenic RIP-B7.1 mice model of experimental autoimmune diabetes (EAD). Diabetic mice presented insulitis, hyperglycaemia, hypoinsulinemia and hyperglucagonemia along with lower pancreatic insulin content. While alpha-cell mass and pancreatic glucagon content were preserved at the early-onset of EAD, both parameters were reduced in the advanced phase. At both stages, alpha-cell size, proliferation and ductal neogenesis were up-regulated, whereas apoptosis was almost negligible. Interestingly, we found an increase in the proportion of glucagon-containing cells positive for insulin or the beta-cell transcription factor PDX1. Our findings suggest that pancreatic alpha-cell renewal mechanisms are boosted during the natural course of EAD, possibly as an attempt to maintain the alpha-cell population and/or to increase beta-cell regeneration via alpha-cell transdifferentiation.

Oestrogen receptor ß mediates the actions of bisphenol-A on ion channel expression in mouse pancreatic beta cells.

AIMS/HYPOTHESIS: Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and ß (ERß). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. METHODS: Microarray gene profiling of isolated islets from vehicle- and BPA-treated (100 µg/kg per day for 4 days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm 'robust multi-array average'. Whole islets or dispersed islets from C57BL/6J or oestrogen receptor ß (ERß) knockout (Erß-/-) mice were treated with vehicle or BPA (1 nmol/l) for 48 h. Whole-cell patch-clamp recordings were used to measure Na+ and K+ currents. mRNA expression was evaluated by quantitative real-time PCR. RESULTS: Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na+ and K+ channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na+ and K+ currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K+ channels (Kv2.1/2.2 channels) and large-conductance Ca2+-activated K+ channels (KCa1.1 channels), which agrees with BPA's effects on gene expression. Beta cells from ERß-/- mice did not present BPA-induced changes, suggesting that ERß mediates BPA's effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential half-width, leading to alteration in beta cell electrical activity. CONCLUSIONS/INTERPRETATION: Our data suggest that BPA modulates the expression and function of Na+ and K+ channels via ERß in mouse pancreatic islets. Furthermore, BPA alters beta cell electrical activity. Altogether, these BPA-induced changes in beta cells might play a role in the diabetogenic action of BPA described in animal models.

DEXI, a candidate gene for type 1 diabetes, modulates rat and human pancreatic beta cell inflammation via regulation of the type I IFN/STAT signalling pathway.

AIMS/HYPOTHESIS: The initial stages of type 1 diabetes are characterised by an aberrant islet inflammation that is in part regulated by the interaction between type 1 diabetes susceptibility genes and environmental factors. Chromosome 16p13 is associated with type 1 diabetes and CLEC16A is thought to be the aetiological gene in the region. Recent gene expression analysis has, however, indicated that SNPs in CLEC16A modulate the expression of a neighbouring gene with unknown function named DEXI, encoding dexamethasone-induced protein (DEXI). We therefore evaluated the role of DEXI in beta cell responses to 'danger signals' and determined the mechanisms involved. METHODS: Functional studies based on silencing or overexpression of DEXI were performed in rat and human pancreatic beta cells. Beta cell inflammation and apoptosis, driven by a synthetic viral double-stranded RNA, were evaluated by real-time PCR, western blotting and luciferase assays. RESULTS: DEXI-silenced beta cells exposed to a synthetic double-stranded RNA (polyinosinic:polycytidylic acid [PIC], a by-product of viral replication) showed reduced activation of signal transducer and activator of transcription (STAT) 1 and lower production of proinflammatory chemokines that was preceded by a reduction in IFNß levels. Exposure to PIC increased chromatin-bound DEXI and IFNß promoter activity. This effect on IFNß promoter was inhibited in DEXI-silenced beta cells, suggesting that DEXI is implicated in the regulation of IFNß transcription. In a mirror image of knockdown experiments, DEXI overexpression led to increased levels of STAT1 and proinflammatory chemokines. CONCLUSIONS/INTERPRETATION: These observations support DEXI as the aetiological gene in the type 1 diabetes-associated 16p13 genomic region, and provide the first indication of a link between this candidate gene and the regulation of local antiviral immune responses in beta cells. Moreover, our results provide initial information on the function of DEXI.

Timing of Exposure and Bisphenol-A: Implications for Diabetes Development.

Bisphenol-A (BPA) is one of the most widespread endocrine disrupting chemicals (EDCs). It is used as the base compound in the production of polycarbonate and other plastics present in many consumer products. It is also used as a building block in epoxy can coating and the thermal paper of cash register receipts. Humans are consistently exposed to BPA and, in consequence, this compound has been detected in the majority of individuals examined. Over the last decade, an enlarging body of evidence has provided a strong support for the role of BPA in the etiology of diabetes and other metabolic disorders. Timing of exposure to EDCs results crucial since it has important implications on the resulting adverse effects. It is now well established that the developing organisms are particularly sensitive to environmental influences. Exposure to EDCs during early life may result in permanent adverse consequences, which increases the risk of developing chronic diseases like diabetes in adult life. In addition to that, developmental abnormalities can be transmitted from one generation to the next, thus affecting future generations. More recently, it has been proposed that gestational environment may also program long-term susceptibility to metabolic disorders in the mother. In the present review, we will comment and discuss the contributing role of BPA in the etiology of diabetes. We will address the metabolic consequences of BPA exposure at different stages of life and comment on the final phenotype observed in different whole-animal models of study.

PDL1 is expressed in the islets of people with type 1 diabetes and is up-regulated by interferons-α and-γ via IRF1 induction.

BACKGROUND: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. METHODS: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-ßH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. FINDINGS: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1ß, induced PDL1 expression in vitro in human islet cells and EndoC-ßH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction. INTERPRETATION: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.

IFN-α induces a preferential long-lasting expression of MHC class I in human pancreatic beta cells.

AIMS/HYPOTHESIS: IFN-α, a cytokine expressed in human islets from individuals affected by type 1 diabetes, plays a key role in the pathogenesis of diabetes by upregulating inflammation, endoplasmic reticulum (ER) stress and MHC class I overexpression, three hallmarks of islet histology in early type 1 diabetes. We tested whether expression of these mediators of beta cell loss is reversible upon IFN-α withdrawal or IFN-α pathway inhibition. METHODS: IFN-α-induced MHC class I overexpression, ER stress and inflammation were evaluated by flow cytometry, immunofluorescence and real-time PCR in human EndoC-ßH1 cells or human islets exposed to IFN-α with or without the presence of Janus kinase (JAK) inhibitors. Protein expression was evaluated by western blot. RESULTS: IFN-α-induced expression of inflammatory and ER stress markers returned to baseline after 24-48 h following cytokine removal. In contrast, MHC class I overexpression at the cell surface persisted for at least 7 days. Treatment with JAK inhibitors, when added with IFN-α, prevented MHC class I overexpression, but when added 24 h after IFN-α exposure these inhibitors failed to accelerate MHC class I return to baseline. CONCLUSIONS/INTERPRETATION: IFN-α mediates a long-lasting and preferential MHC class I overexpression in human beta cells, which is not affected by the subsequent addition of JAK inhibitors. These observations suggest that IFN-α-stimulated long-lasting MHC class I expression may amplify beta cell antigen presentation during the early phase of type 1 diabetes and that IFN-α inhibitors might need to be used at very early stages of the disease to be effective.

Protective Role of Complement C3 Against Cytokine-Mediated ß-Cell Apoptosis.

Type 1 diabetes is a chronic autoimmune disease characterized by pancreatic islet inflammation and ß-cell destruction by proinflammatory cytokines and other mediators. Based on RNA sequencing and protein-protein interaction analyses of human islets exposed to proinflammatory cytokines, we identified complement C3 as a hub for some of the effects of cytokines. The proinflammatory cytokines interleukin-1ß plus interferon-γ increase C3 expression in rodent and human pancreatic ß-cells, and C3 is detected by histology in and around the islets of diabetic patients. Surprisingly, C3 silencing exacerbates apoptosis under both basal condition and following exposure to cytokines, and it increases chemokine expression upon cytokine treatment. C3 exerts its prosurvival effects via AKT activation and c-Jun N-terminal kinase inhibition. Exogenously added C3 also protects against cytokine-induced ß-cell death and partially rescues the deleterious effects of inhibition of endogenous C3. These data suggest that locally produced C3 is an important prosurvival mechanism in pancreatic ß-cells under a proinflammatory assault.

Interferon-α mediates human beta cell HLA class I overexpression, endoplasmic reticulum stress and apoptosis, three hallmarks of early human type 1 diabetes.

AIMS/HYPOTHESIS: Three hallmarks of the pancreatic islets in early human type 1 diabetes are overexpression of HLA class I, endoplasmic reticulum (ER) stress and beta cell apoptosis. The mediators of these phenomena remain to be defined. The type I interferon IFNα is expressed in human islets from type 1 diabetes patients and mediates HLA class I overexpression. We presently evaluated the mechanisms involved in IFNα-induced HLA class I expression in human beta cells and determined whether this cytokine contributes to ER stress and apoptosis. METHODS: IFNα-induced inflammation, ER stress and apoptosis were evaluated by RT-PCR, western blot, immunofluorescence and nuclear dyes, and proteins involved in type I interferon signalling were inhibited by small interfering RNAs. All experiments were performed in human islets or human EndoC-ßH1 cells. RESULTS: IFNα upregulates HLA class I, inflammation and ER stress markers in human beta cells via activation of the candidate gene TYK2, and the transcription factors signal transducer and activator of transcription 2 and IFN regulatory factor 9. Furthermore, it acts synergistically with IL-1ß to induce beta cell apoptosis. CONCLUSIONS/INTERPRETATION: The innate immune effects induced by IFNα may induce and amplify the adaptive immune response against human beta cells, indicating that IFNα has a central role in the early phases of diabetes.

Pancreatic Beta Cell Survival and Signaling Pathways: Effects of Type 1 Diabetes-Associated Genetic Variants.

Type 1 diabetes (T1D) is a complex autoimmune disease in which pancreatic beta cells are specifically destroyed by the immune system. The disease has an important genetic component and more than 50 loci across the genome have been associated with risk of developing T1D. The molecular mechanisms by which these putative T1D candidate genes modulate disease risk, however, remain poorly characterized and little is known about their effects in pancreatic beta cells. Functional studies in in vitro models of pancreatic beta cells, based on techniques to inhibit or overexpress T1D candidate genes, allow the functional characterization of several T1D candidate genes. This requires a multistage procedure comprising two major steps, namely accurate selection of genes of potential interest and then in vitro and/or in vivo mechanistic approaches to characterize their role in pancreatic beta cell dysfunction and death in T1D. This chapter details the methods and settings used by our groups to characterize the role of T1D candidate genes on pancreatic beta cell survival and signaling pathways, with particular focus on potentially relevant pathways in the pathogenesis of T1D, i.e., inflammation and innate immune responses, apoptosis, beta cell metabolism and function.

Differential cell autonomous responses determine the outcome of coxsackievirus infections in murine pancreatic α and ß cells.

Type 1 diabetes (T1D) is an autoimmune disease caused by loss of pancreatic ß cells via apoptosis while neighboring α cells are preserved. Viral infections by coxsackieviruses (CVB) may contribute to trigger autoimmunity in T1D. Cellular permissiveness to viral infection is modulated by innate antiviral responses, which vary among different cell types. We presently describe that global gene expression is similar in cytokine-treated and virus-infected human islet cells, with up-regulation of gene networks involved in cell autonomous immune responses. Comparison between the responses of rat pancreatic α and ß cells to infection by CVB5 and 4 indicate that α cells trigger a more efficient antiviral response than ß cells, including higher basal and induced expression of STAT1-regulated genes, and are thus better able to clear viral infections than ß cells. These differences may explain why pancreatic ß cells, but not α cells, are targeted by an autoimmune response during T1D.