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The presence of islet cells double positive for insulin and glucagon (Ins + /Glu + ) has been described in the pancreas from both type 2 (T2D) and type 1 (T1D) diabetic subjects. We studied the role of pro-inflammatory cytokines on the occurrence, trajectory, and characteristics of Ins + /Glu + cells in human pancreatic islets. Pancreas samples, isolated islets, and dispersed islet cells from 3 T1D and 11 non-diabetic (ND) multi-organ donors were studied by immunofluorescence, confocal microscopy, and/or electron microscopy. ND islet cells were exposed to interleukin-1β and interferon-γ for up to 120 h. In T1D islets, we confirmed an increased prevalence of Ins + /Glu + cells. Cytokine-exposed islets showed a progressive increase of Ins + /Glu + cells that represented around 50% of endocrine cells after 120h. Concomitantly, cells expressing insulin granules only decreased significantly over time, whereas those containing only glucagon granules remained stable. Interestingly, Ins + /Glu + cells were less prone to cytokine-induced apoptosis than cells containing only insulin. Cytokine-exposed islets showed down-regulation of β-cell identity genes. In conclusion, pro-inflammatory cytokines induce Ins + /Glu + cells in human islets, possibly due to a switch from a β- to a β-/α-cell phenotype. These Ins + /Glu + cells appear to be resistant to cytokine-induced apoptosis.

The 78-kDa glucose-regulated protein (GRP78) is an ubiquitously expressed endoplasmic reticulum chaperone, with a central role in maintaining protein homeostasis. Recently, an alternative role for GRP78 under stress conditions has been proposed, with stress-induced extracellular secretion and translocation of GRP78 to the cell surface where it acts as a multifunctional signaling receptor. Here we demonstrate translocation of GRP78 to the surface of human EndoC-βH1 cells and primary human islets upon cytokine exposure, in analogy to observations in rodent INS-1E and MIN6 beta cell lines. We show that GRP78 is shuttled via the anterograde secretory pathway, through the Golgi complex and secretory granules, and identify the DNAJ homolog subfamily C member 3 (DNAJC3) as a GRP78-interacting protein that facilitates its membrane translocation. Evaluation of downstream signaling pathways, using N- and C-terminal anti-GRP78 blocking antibodies, demonstrates that both GRP78 signaling domains initiate pro-apoptotic signaling cascades in beta cells. Extracellular GRP78 itself is identified as a ligand for cell surface GRP78 (sGRP78), increasing caspase 3/7 activity and cell death upon binding, which is accompanied by enhanced Chop and Bax mRNA expression. These results suggest that inflammatory cytokines induce a self-destructive pro-apoptotic feedback loop through the secretion and membrane translocation of GRP78. This proapoptotic function distinguishes the role of sGRP78 in beta cells from its reported anti-apoptotic and proliferative role in cancer cells, opening the road for the use of compounds that block sGRP78 as potential beta cell-preserving therapies in type 1 diabetes.

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims/hypothesis: Research on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation., Methods: We compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data., Results: After type 1 diabetes manifestation CD8(+) T cells, CD68(+) macrophages and CD4(+) T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1β and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed., Conclusions/interpretation: With the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

Environmental factors such as diets rich in saturated fats contribute to dysfunction and death of pancreatic β-cells in diabetes. Endoplasmic reticulum (ER) stress is elicited in β-cells by saturated fatty acids. Here we show that palmitate-induced β-cell apoptosis is mediated by the intrinsic mitochondrial pathway. By microarray analysis, we identified a palmitate-triggered ER stress gene expression signature and the induction of the BH3-only proteins death protein 5 (DP5) and p53-upregulated modulator of apoptosis (PUMA). Knockdown of either protein reduced cytochrome c release, caspase-3 activation, and apoptosis in rat and human β-cells. DP5 induction depends on inositol-requiring enzyme 1 (IRE1)-dependent c-Jun NH₂-terminal kinase and PKR-like ER kinase (PERK)-induced activating transcription factor (ATF3) binding to its promoter. PUMA expression is also PERK/ATF3-dependent, through tribbles 3 (TRB3)-regulated AKT inhibition and FoxO3a activation. DP5(-/-) mice are protected from high fat diet-induced loss of glucose tolerance and have twofold greater pancreatic β-cell mass. This study elucidates the crosstalk between lipotoxic ER stress and the mitochondrial pathway of apoptosis that causes β-cell death in diabetes.

Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing β-cell loss. The mitochondrial pathway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of β-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The combination of TNF-α + IFN-γ induced DP5, p53 up-regulated modulator of apoptosis (PUMA), and Bim expression in human islets and rodent β-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-α + IFN-γ-induced β-cell apoptosis. DP5 knock-out mice had increased β-cell area, and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1, and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human β-cells to a large extent against TNF-α + IFN-γ, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context-dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of β-cell apoptosis during insulitis.

Viral infections and local production of cytokines probably contribute to the pathogenesis of Type 1 diabetes. The viral replicative intermediate double-stranded RNA (dsRNA, tested in the form of polyinosinic-polycytidylic acid, PIC), in combination with the cytokine interferon-gamma (IFN-gamma), triggers beta-cell apoptosis. We have previously observed by microarray analysis that PIC induces expression of several mRNAs encoding for genes downstream of Toll-like receptor 3 (TLR3) signaling pathway. In this report, we show that exposure of beta-cells to dsRNA in combination with IFN-alpha, -beta, or -gamma significantly increases apoptosis. Moreover, dsRNA induces TLR3 mRNA expression and activates NF-kappaB and the IFN-beta promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN-beta mRNA expression, and blocking IFN-beta with a specific antibody partially prevents PIC plus IFN-gamma-induced beta-cell death. On the other hand, dsRNA plus IFN-gamma does not induce apoptosis in INS-1E cells, and expression of TLR3 and type I IFNs mRNAs is not detected in these cells. Of note, disruption of the STAT-1 signaling pathway protects beta-cells against dsRNA plus IFN-gamma-induced beta-cell apoptosis. This study suggests that dsRNA plus IFN-gamma triggers beta-cell apoptosis by two complementary pathways, namely TLR3-TRIF-NF-kappaB and STAT-1.

During the development of type 1 diabetes, pancreatic beta-cells are subject to an immune attack, leading to their apoptotic or necrotic cell death. Apoptotic beta-cells are also present during periods of tissue remodeling, such as in early life. Macrophages should clear apoptotic cells silently without production of pro-inflammatory cytokines. The aim of the present study was to investigate the cytokine pattern of NOD macrophages exposed to apoptotic or necrotic cells in vitro. In contrast to the limited response of macrophages from C57BL/6 or NOR mice, NOD macrophages reacted aberrantly to both necrotic and apoptotic cells, with secretion of inappropriately high amounts of IL1beta and TNFalpha. Further exploration of the macrophage behavior showed an excessive response of NOD macrophages when exposed to LPS (high iNOS and IL12p40 levels), accompanied by hyper-activation of NF-kappaB(p65). In contrast, NOD macrophages failed to up-regulate IL1beta and IL12p40 in response to IFNgamma. This failure correlated with low protein levels and a low phosphorylation state of STAT1alpha. We conclude that NOD macrophages have severely aberrant cytokine expression patterns that could contribute to the initiation or continuation of an immune attack towards the pancreatic beta-cells and thus onset and progression of type 1 diabetes., (Copyright 2004 Elsevier Ltd.)

Aims/hypothesis: The streptococcal wall component, OK432, prevents diabetes in NOD mice and BB rats by elimination of effector cells. Based on the knowledge of a link between autoimmunity and resistance of immune cells to elimination by apoptosis, we investigated whether OK432 treatment restored the sensitivity of NOD lymphocytes to apoptotic signals centrally (thymus) or peripherally (spleen) or both and we examined the pathways for the enhanced apoptosis rate., Methods: We treated NOD mice with OK432 (0.1 mg/kg i.p. weekly from 21 to 70 days). Apoptosis was measured by TUNEL 16 h after cyclophosphamide (70 mg/kg) and 24 h after dexamethasone (0.2 mg/mouse). Real time quantitative RT-PCR was used to investigate changes in gene expression., Results: Thymocyte apoptosis levels after cyclophosphamide were restored by OK432 treatment to levels observed in C57BL/6 mice: in NOD males apoptosis increased from 8 +/- 1% to 18 +/- 5% (p < 0.05) compared with 20 +/- 4% in C57BL/6 males, and in NOD females from 6+/- 2% to 11 +/- 2% (p < 0.05) compared with 12 +/- 2% in C57BL/6 females. The dexamethasone-induced thymocyte apoptosis rate was equally restored by OK432 treatment (58 +/- 4% vs 41 +/- 3% in control males (p < 0.0005) and 39 +/- 5% vs 26 +/- 3% in control females (p < 0.05)]. No change in apoptosis levels was on the contrary observed in splenocytes after OK432 treatment. By RT-PCR analysis of a panel of apoptosis-related genes in thymocytes we showed a down-regulation of anti-apoptotic Bcl-xL and c-myc by OK432 treatment., Conclusions/interpretation: Our data suggest that OK432 prevents diabetes in NOD mice by better elimination of effector cells through increased sensitivity to apoptotic signals centrally in the thymus.

Combinations of cytokines, including interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma), induce nitric oxide (NO) production and cell death in pancreatic islet cells. We have previously shown that these events are preceded by increased expression of the transcription factor interferon regulatory factor-1 (IRF-1). We utilized an IRF-1 knockout mouse (IRF-1-/-) to investigate the role of IRF-1 in cytokine-induced islet- and beta-cell gene expression and cell death. For this purpose, pancreatic islets or FACS-purified beta-cells were isolated from wild type (wt) or IRF-1-/- mice. These cells were exposed for different time points to IL-1beta (50 U/mI), IFN-gamma (1,000 U/ml) and/or TNF-alpha (1,000 U/ml) before being harvested for determination of viability (by nuclear dyes) and mRNA expression (by RT-PCR with specific primers). Following a 24 hours exposure to IL-1beta or IL-1beta + IFN-gamma, pancreatic islets isolated from IRF-1-/- mice presented a 30-50% reduction in medium nitrite accumulation and inducible NO-synthase (iNOS) expression. Interestingly, both wt and IRF-1-/- purified beta-cells failed to produce NO in response to IL-1beta alone, but presented a similar increase in nitrite accumulation and iNOS expression following exposure to IL-1beta + IFN-gamma. The basal expression of MHC class I mRNA was lower in IRF-1-/- islet cells (30% reduction), but there was a similar 2-4 fold-increase in MHC expression in islet cells from both strains following cytokine exposure. IL-1beta induced serine protease inhibitor-3 (SPI-3; a putative cellular "defense" protein) mRNA expression in both wt and IRF-1-/- islets or beta-cells. IFN-gamma decreased the IL-1beta-induced SPI-3 expression in wt islets or beta-cells, but induced a 5-fold increase in the expression of this mRNA in IRF-1-/- islets cells, suggesting that IRF-1 mediates an inhibitory effect of IFN-gamma on SPI-3 expression. Treatment of whole islets for 3 days with IL-1beta + IFN-gamma induced significantly more islet cell death in wt than in IRF-1-/- mice (respectively 85 +/- 3% versus 31 +/- 4% dead cells). On the other hand, prolonged exposure (3-9 days) of FACS-purified beta-cells to the same cytokines, or a combination of 3 cytokines, led to a similar increase in cell death in both IRF-1-/- and wt islets. In conclusion, IRF-1 contributes to cytokine-induced islet iNOS expression and cell death. These effects are absent in purified beta-cells, suggesting that IRF-1 may mediate its effects on whole islets via activation of non-endocrine cells (e.g. macrophages and ductal cells) present in these preparations.

The NOD mouse, a model for type 1 diabetes, is characterized by resistance to apoptosis in immunocytes. The aim of this study was to investigate a link between apoptosis in NOD thymocytes and autoimmunity. First, we demonstrated that the sexual dimorphism in diabetes incidence in NOD mice (females are more diabetes-prone than males) is reflected by differences in apoptosis. Apoptosis in NOD thymocytes, 24 h after dexamethasone, was decreased in both sexes compared with C57B1/6, but it was lower in female mice (26 +/- 2%) than in male mice (50 +/- 3%, P < 0.001). Further, we demonstrated that sex hormones themselves play a central role in this difference, since castration of NOD male mice, which increases diabetes incidence, decreased apoptosis levels (32 +/- 2%), while treatment of NOD female mice with dihydrotestosterone, which protects against diabetes, restored apoptosis to male levels (42 +/- 1.5%). Finally, we demonstrated that 1,25-dihydroxyvitamin D3, a steroid hormone that prevents diabetes in NOD mice, restored apoptosis levels to C57B1/6 reference levels. This improved apoptosis was seen in male (68 +/- 1 vs. 50 +/- 3% in untreated NOD mice, P < 0.001) but especially in female NOD mice (51 +/- 5 vs. 26 +/- 2% in untreated NOD mice, P < 0.001). Fluorescence-activated cell sorter analysis of thymocyte subsets revealed marked differences, especially in CD4+CD8+ and CD4+ cells. We conclude that the sexual dimorphism in diabetes incidence in NOD mice is paralleled by a dimorphism in resistance to apoptotic signals in NOD thymocytes. This resistance to apoptosis is driven by sex hormones and is corrected by 1,25-dihydroxyvitamin D3.

Pancreatic insulin-producing β-cells have traditionally been viewed as a quiescent cell population. However, several recent lines of evidence indicated that like most tissues the β-cell mass is dynamically regulated with ongoing β-cell regeneration throughout life to replenish lost or damaged β-cells. In type 1 diabetes ( T1D), this fine-tuned balance between β-cell death and β-cell renewal in the endocrine pancreas is lost and the deficit in β-cell mass is largely caused by autoimmune-mediated apoptosis. Currently, the concept that a cure for T1D will require both re-establishment of immunological tolerance along with replacement or regeneration of a functional β-cell mass in T1D patients is generally accepted. In this study our current understanding of the events directing β-cell replication, β-cell reprogramming from different cell types and β-cell regeneration is reviewed, in view of the results of various immunomodulatory strategies aiming at blocking autoimmune responses against pancreatic β-cells and at improving β-cell mass and function in subjects with T1D. [ABSTRACT FROM AUTHOR]

The differentiation of embryonic mesenchymal cells into chondrocytes and the subsequent formation of a cartilaginous scaffold that enables the formation of long bones are hallmarks of endochondral ossification. During this process, chondrocytes undergo a remarkable sequence of events involving proliferation, differentiation, hypertrophy and eventually apoptosis. Forkhead Box O (FoxO) transcription factors (TFs) are well-known regulators of such cellular processes. Although FoxO3a was previously shown to be regulated by 1,25-dihydroxyvitamin D 3 in osteoblasts, a possible role for this family of TFs in chondrocytes during endochondral ossification remains largely unstudied. By crossing Collagen2-Cre mice with FoxO1 lox/lox ;FoxO3a lox/lox ;FoxO4 lox/lox mice, we generated mice in which the three main FoxO isoforms were deleted in growth plate chondrocytes (chondrocyte triple knock-out; CTKO). Intriguingly, CTKO neonates showed a distinct elongation of the hypertrophic zone of the growth plate. CTKO mice had increased overall body and tail length at eight weeks of age and suffered from severe skeletal deformities at older ages. CTKO chondrocytes displayed decreased expression of genes involved in redox homeostasis. These observations illustrate the importance of FoxO signaling in chondrocytes during endochondral ossification. [ABSTRACT FROM AUTHOR]