Islet cells in human type 1 diabetes: from recent advances to novel therapies - a symposium-based roadmap for future research.

There is a growing understanding that the early phases of type 1 diabetes (T1D) are characterised by a deleterious dialogue between the pancreatic beta cells and the immune system. This, combined with the urgent need to better translate this growing knowledge into novel therapies, provided the background for the JDRF-DiabetesUK-INNODIA-nPOD symposium entitled 'Islet cells in human T1D: from recent advances to novel therapies', which took place in Stockholm, Sweden, in September 2022. We provide in this article an overview of the main themes addressed in the symposium, pointing to both promising conclusions and key unmet needs that remain to be addressed in order to achieve better approaches to prevent or reverse T1D.

Gene expression signatures of target tissues in type 1 diabetes, lupus erythematosus, multiple sclerosis, and rheumatoid arthritis.

Autoimmune diseases are typically studied with a focus on the immune system, and less attention is paid to responses of target tissues exposed to the immune assault. We presently evaluated, based on available RNA sequencing data, whether inflammation induces similar molecular signatures at the target tissues in type 1 diabetes, systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis. We identified confluent signatures, many related to interferon signaling, indicating pathways that may be targeted for therapy, and observed a high (>80%) expression of candidate genes for the different diseases at the target tissue level. These observations suggest that future research on autoimmune diseases should focus on both the immune system and the target tissues, and on their dialog. Discovering similar disease-specific signatures may allow the identification of key pathways that could be targeted for therapy, including the repurposing of drugs already in clinical use for other diseases.

Modulation of Autophagy Influences the Function and Survival of Human Pancreatic Beta Cells Under Endoplasmic Reticulum Stress Conditions and in Type 2 Diabetes.

Autophagy is the major mechanism involved in degradation and recycling of intracellular components, and its alterations have been proposed to cause beta cell dysfunction. In this study, we explored the effects of autophagy modulation in human islets under conditions associated to endoplasmic reticulum (ER) stress. Human pancreatic islets were isolated by enzymatic digestion and density gradient purification from pancreatic samples of non-diabetic (ND; n = 17; age 65 ± 21 years; gender: 5 M/12 F; BMI 23.4 ± 3.3 kg/m2) and T2D (n = 9; age 76 ± 6 years; 4 M/5 F; gender: BMI 25.4 ± 3.7 kg/m2) organ donors. Nine ND organ donors were treated for hypertension and 1 for both hypertension and hypercholesterolemia. T2D organ donors were treated with metformin (1), oral hypoglycemic agents (2), diet + oral hypoglycemic agents (3), insulin (3) or insulin plus metformin (3) as for antidiabetic therapy and, of these, 3 were treated also for hypertension and 6 for both hypertension and hypercholesterolemia. Two days after isolation, they were cultured for 1-5 days with 10 ng/ml rapamycin (autophagy inducer), 5 mM 3-methyladenine or 1.0 nM concanamycin-A (autophagy blockers), either in the presence or not of metabolic (0.5 mM palmitate) or chemical (0.1 ng/ml brefeldin A) ER stressors. In ND islets palmitate exposure induced a 4 to 5-fold increase of beta cell apoptosis, which was significantly prevented by rapamycin and exacerbated by 3-MA. Similar results were observed with brefeldin treatment. Glucose-stimulated insulin secretion from ND islets was reduced by palmitate (-40 to 50%) and brefeldin (-60 to 70%), and rapamycin counteracted palmitate, but not brefeldin, cytotoxic actions. Both palmitate and brefeldin induced PERK, CHOP and BiP gene expression, which was partially, but significantly prevented by rapamycin. With T2D islets, rapamycin alone reduced the amount of p62, an autophagy receptor that accumulates in cells when macroautophagy is inhibited. Compared to untreated T2D cells, rapamycin-exposed diabetic islets showed improved insulin secretion, reduced proportion of beta cells showing signs of apoptosis and better preserved insulin granules, mitochondria and ER ultrastructure; this was associated with significant reduction of PERK, CHOP and BiP gene expression. This study emphasizes the importance of autophagy modulation in human beta cell function and survival, particularly in situations of ER stress. Tuning autophagy could be a tool for beta cell protection.

JunB protects ß-cells from lipotoxicity via the XBP1-AKT pathway.

Diets rich in saturated fats may contribute to the loss of pancreatic ß-cells in type 2 diabetes. JunB, a member of the activating protein 1 (AP-1) transcription factor family, promotes ß-cell survival and mediates part of the beneficial effects of GLP-1 agonists. In this study we interrogated the molecular mechanisms involved in JunB-mediated ß-cell protection from lipotoxicity. The saturated fatty acid palmitate decreased JunB expression, and this loss may contribute to ß-cell apoptosis, as overexpression of JunB protected cells from lipotoxicity. Array analysis of JunB-deficient ß-cells identified a gene expression signature of a downregulated endoplasmic reticulum (ER) stress response and inhibited AKT signaling. JunB stimulates XBP1 expression via the transcription factor c/EBPδ during ER stress, and forced expression of XBP1s rescued the viability of JunB-deficient cells, constituting an important antiapoptotic mechanism. JunB silencing inhibited AKT activation and activated the proapoptotic Bcl-2 protein BAD via its dephosphorylation. BAD knockdown reversed lipotoxic ß-cell death potentiated by JunB siRNA. Interestingly, XBP1s links JunB and AKT signaling as XBP1 knockdown also reduced AKT phosphorylation. GLP-1 agonists induced cAMP-dependent AKT phosphorylation leading to ß-cell protection against palmitate-induced apoptosis. JunB and XBP1 knockdown or IRE1 inhibition decreased AKT activation by cAMP, leading to ß-cell apoptosis. In conclusion, JunB modulates the ß-cell ER stress response and AKT signaling via the induction of XBP1s. The activation of the JunB gene network and the crosstalk between the ER stress and AKT pathway constitute a crucial defense mechanism by which GLP-1 agonists protect against lipotoxic ß-cell death. These findings elucidate novel ß-cell-protective signal transduction in type 2 diabetes.

Discovery of molecular pathways mediating 1,25-dihydroxyvitamin D3 protection against cytokine-induced inflammation and damage of human and male mouse islets of Langerhans.

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced ß-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to ß-cell protection against cytokine-induced ß-cell dysfunction and death. Human and mouse islets were exposed to IL-1ß and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and ß-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in ß-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic ß-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced ß-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.

Candidate genes for type 1 diabetes modulate pancreatic islet inflammation and ß-cell apoptosis.

Genome-wide association studies (GWAS) have identified more than 50 loci associated with genetic risk of type 1 diabetes (T1D). Several T1D candidate genes have been suggested or identified within these regions, but the molecular mechanisms by which they contribute to insulitis and ß-cell destruction remain to be clarified. More than 60% of the T1D candidate genes are expressed in human pancreatic islets, suggesting that they contribute to T1D by regulating at least in part pathogenic mechanisms at the ß-cell level. Recent studies by our group indicate that important genetically regulated pathways in ß-cells include innate immunity and antiviral activity, involving RIG-like receptors (particularly MDA5) and regulators of type I IFNs (i.e. PTPN2 and USP18), and genes related to ß-cell phenotype and susceptibility to pro-apoptotic stimuli (i.e. GLIS3). These observations reinforce the concept that the early pathogenesis of T1D is characterized by a dialogue between the immune system and pancreatic ß-cells. This dialogue is probably influenced by polymorphisms in genes expressed at the ß-cell and/or immune system level, leading to inadequate responses to environmental cues such as viral infections. Further studies are needed to clarify how these disease-associated variants affect pancreatic ß-cell responses to inflammation and the subsequent triggering of autoimmune responses and progressive ß-cell loss.

Endoplasmic reticulum stress sensitizes pancreatic beta cells to interleukin-1ß-induced apoptosis via Bim/A1 imbalance.

We have recently shown that the crosstalk between mild endoplasmic reticulum (ER) stress and low concentrations of the pro-inflammatory cytokine interleukin (IL)-1ß exacerbates beta cell inflammatory responses via the IRE1α/XBP1 pathway. We presently investigated whether mild ER stress also sensitizes beta cells to cytokine-induced apoptosis. Cyclopiazonic acid (CPA)-induced ER stress enhanced the IL-1ß apoptosis in INS-1E and primary rat beta cells. This was not prevented by XBP1 knockdown (KD), indicating the dissociation between the pathways leading to inflammation and cell death. Analysis of the role of pro- and anti-apoptotic proteins in cytokine-induced apoptosis indicated a central role for the pro-apoptotic BH3 (Bcl-2 homology 3)-only protein Bim (Bcl-2-interacting mediator of cell death), which was counteracted by four anti-apoptotic Bcl-2 (B-cell lymphoma-2) proteins, namely Bcl-2, Bcl-XL, Mcl-1 and A1. CPA+IL-1ß-induced beta cell apoptosis was accompanied by increased expression of Bim, particularly the most pro-apoptotic variant, small isoform of Bim (BimS), and decreased expression of A1. Bim silencing protected against CPA+IL-1ß-induced apoptosis, whereas A1 KD aggravated cell death. Bim inhibition protected against cell death caused by A1 silencing under all conditions studied. In conclusion, mild ER stress predisposes beta cells to the pro-apoptotic effects of IL-1ß by disrupting the balance between pro- and anti-apoptotic Bcl-2 proteins. These findings link ER stress to exacerbated apoptosis during islet inflammation and provide potential mechanistic targets for beta cell protection, namely downregulation of Bim and upregulation of A1.

Unraveling the effects of 1,25OH2D3 on global gene expression in pancreatic islets.

INTRODUCTION: Vitamin D deficiency has been linked to type 1 and 2 diabetes, whereas supplementation may prevent both diseases. However, the extent of the effects of vitamin D or its metabolites directly on pancreatic islets is still largely unknown. The aim of the present study was to investigate how active vitamin D, 1,25(OH)2D3, affects beta cells directly by establishing its effects on global gene expression in healthy murine islets. MATERIALS AND METHODS: Pancreatic islets were isolated from 2 to 3 week old C57BL/6 mice and cultured in vitro with 1,25(OH)2D3 or vehicle for 6 and 24h. Total RNA was extracted from the islets and the effects on global gene expression were analyzed using Affymetrix microarrays. RESULTS AND DISCUSSION: Exposure to 1,25(OH)2D3 compared to vehicle resulted in 306 and 151 differentially expressed genes after 6 and 24h, respectively (n=4, >1.3-fold, p<0.02). Of these 220 were up-regulated, whereas 86 displayed a decreased expression after 6h. Furthermore, expression levels were increased for 124 and decreased for 27 genes following 24h of exposure. Formation of intercellular junctions, cytoskeletal organization, and intracellular trafficking as well as lipid metabolism and ion transport were among the most affected gene classes. Effects on several genes already identified as being part of vitamin D signaling in other cell types were observed along with genes known to affect insulin release, although with our assay we were not able to detect any effects of 1,25(OH)2D3 on glucose-stimulated insulin release from healthy pancreatic islets. CONCLUSION: The effects of 1,25(OH)2D3 on the expression of cytoskeletal and intracellular trafficking genes along with genes involved in ion transport may influence insulin exocytosis. However, an effect of 1,25(OH)2D3 on insulin release could not be detected for healthy islets in contrast to islets subjected to pathological conditions such as cytokine exposure and vitamin D deficiency as suggested by other studies. Thus, in addition to previously identified tolerogenic effects on the immune system, 1,25(OH)2D3 may affect basic functions of pancreatic beta cells, with the potential to render them more resistant to the detrimental conditions encountered during type 1 and 2 diabetes. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Signalling danger: endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation.

Protein synthesis is increased by several-fold in stimulated pancreatic beta cells. Synthesis and folding of (pro)insulin takes place in the endoplasmic reticulum (ER), and beta cells trigger the unfolded protein response (UPR) to upgrade the functional capacity of the ER. Prolonged or excessive UPR activation contributes to beta cell dysfunction and death in type 2 diabetes, but there is another side of the UPR that may be of particular relevance for autoimmune type 1 diabetes, namely, the cross-talk between the UPR and innate immunity/inflammation. Recent evidence, discussed in this review, indicates that both saturated fats and inflammatory mediators such as cytokines trigger the UPR in pancreatic beta cells. The UPR potentiates activation of nuclear factor κB, a key regulator of inflammation. Two branches of the UPR, namely IRE1/XBP1s and PERK/ATF4/CHOP, mediate the UPR-induced sensitisation of pancreatic beta cells to the proinflammatory effects of cytokines. This can contribute to the upregulation of local inflammatory mechanisms and the aggravation of insulitis. The dialogue between the UPR and inflammation may provide an explanation for the parallel increase in the prevalence of childhood obesity and type 1 diabetes.

USP18 is a key regulator of the interferon-driven gene network modulating pancreatic beta cell inflammation and apoptosis.

Type 1 diabetes (T1D) is an autoimmune disease targeting pancreatic beta cells. Genome-wide association studies and gene expression analysis identified interferon (IFN)-driven gene networks as crucial pathways in the pathogenesis of T1D. IFNs are linked to the response to viral infections and might contribute to the initiation of the autoimmune process in T1D. We presently analyzed the role of ubiquitin-specific peptidase 18 (USP18), an interferon-stimulated gene 15-specific protease, on IFN-induced pancreatic beta cell inflammation and apoptosis. Our findings indicate that USP18 inhibition induces inflammation by increasing the STAT signaling and exacerbates IFN-induced beta cell apoptosis by the mitochondrial pathway of cell death. USP18 regulates activation of three BH3-only proteins, namely, DP5, Bim and PUMA in pancreatic beta cells, suggesting a direct link between regulators of the type I IFN signaling pathway and members of the BCL-2 family. USP18 depletion increases the expression of the T1D candidate gene MDA5, leading to an upregulation of double-stranded RNA-induced chemokine production. These data suggest a cross talk between the type I IFN signaling pathway and a candidate gene for T1D to increase pro-inflammatory responses in beta cells. The present study shows that USP18 is a key regulator of IFN signaling in beta cells and underlines the importance of this pathway in beta cell inflammation and death.

Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.

AIMS/HYPOTHESIS: Endoplasmic reticulum (ER) stress may play a role in cytokine-mediated beta cell death in type 1 diabetes, but it remains controversial whether ER stress markers are present in islets from type 1 diabetic individuals. Therefore, we evaluated by immunostaining the expression of markers of the three main branches of the ER stress response in islets from 13 individuals with and 15 controls without type 1 diabetes (eight adults and seven children). METHODS: Antibodies against the ER stress markers C/EBP homologous protein (CHOP), immunoglobulin heavy chain (BIP) and X-box binding protein 1 (XBP-1) were validated using HeLa cells treated with the ER stressor thapsigargin. These antibodies were then used to stain serial sections of paraffin-embedded pancreas from type 1 diabetic and non-diabetic individuals; samples were also immunostained for CD45, insulin and glucagon. Immunostaining intensities of the ER stress markers were quantified using a software-based, unbiased quantitative approach. RESULTS: Islets from individuals with type 1 diabetes showed increased levels of CHOP and, at least for insulitis-positive and beta cell-containing islets, BIP. XBP-1 expression was not, however, increased. CONCLUSIONS/INTERPRETATION: Islet cells from individuals with type 1 diabetes display a partial ER stress response, with evidence of the induction of some, but not all, components of the unfolded protein response.

C/EBP homologous protein contributes to cytokine-induced pro-inflammatory responses and apoptosis in ß-cells.

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic ß-cells. Pro-inflammatory cytokines are early mediators of ß-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in ß-cells, but the role for CHOP overexpression in cytokine-induced ß-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating ß-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting ß-cell death: (1) CHOP directly contributes to cytokine-induced ß-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.

Differential usage of NF-κB activating signals by IL-1ß and TNF-α in pancreatic beta cells.

The cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α induce ß-cell death in type 1 diabetes via NF-κB activation. IL-1ß induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in ß-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1ß and TNF-α in ß-cells. While TNF-α uses IKK complexes containing both IKKα and IKKß, IL-1ß induces complexes with IKKα only; this effect is achieved by induction of IKKß degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1ß-induced IKKß degradation.

Obstacles on the way to the clinical visualisation of beta cells: looking for the Aeneas of molecular imaging to navigate between Scylla and Charybdis.

For more than a decade, researchers have been trying to develop non-invasive imaging techniques for the in vivo measurement of viable pancreatic beta cells. However, in spite of intense research efforts, only one tracer for positron emission tomography (PET) imaging is currently under clinical evaluation. To many diabetologists it may remain unclear why the imaging world struggles to develop an effective method for non-invasive beta cell imaging (BCI), which could be useful for both research and clinical purposes. Here, we provide a concise overview of the obstacles and challenges encountered on the way to such BCI, in both native and transplanted islets. We discuss the major difficulties posed by the anatomical and cell biological features of pancreatic islets, as well as the chemical and physical limits of the main imaging modalities, with special focus on PET, SPECT and MRI. We conclude by indicating new avenues for future research in the field, based on several remarkable recent results.

Deletion of C/EBP homologous protein (Chop) in C57Bl/6 mice dissociates obesity from insulin resistance.

AIMS/HYPOTHESIS: Endoplasmic reticulum (ER) stress has been implicated in the development of type 2 diabetes, via effects on obesity, insulin resistance and pancreatic beta cell health. C/EBP homologous protein (CHOP) is induced by ER stress and has a central role in apoptotic execution pathways triggered by ER stress. The aim of this study was to characterise the role of CHOP in obesity and insulin resistance. METHODS: Metabolic studies were performed in Chop ( -/- ) and wild-type C57Bl/6 mice, and included euglycaemic-hyperinsulinaemic clamps and indirect calorimetry. The inflammatory state of liver and adipose tissue was determined by quantitative RT-PCR, immunohistology and macrophage cultures. Viability and absence of ER stress in islets of Langerhans was determined by electron microscopy, islet culture and quantitative RT-PCR. RESULTS: Systemic deletion of Chop induced abdominal obesity and hepatic steatosis. Despite marked obesity, Chop ( -/- ) mice had preserved normal glucose tolerance and insulin sensitivity. This discrepancy was accompanied by lower levels of pro-inflammatory cytokines and less infiltration of immune cells into fat and liver. CONCLUSIONS/INTERPRETATION: These observations suggest that insulin resistance is not induced by fat accumulation per se, but rather by the inflammation induced by ectopic fat. CHOP may play a key role in the crosstalk between excessive fat deposition and induction of inflammation-mediated insulin resistance.

Pancreatic ß-cells activate a JunB/ATF3-dependent survival pathway during inflammation.

Destruction of insulin-producing pancreatic ß-cells by local autoimmune inflammation is a hallmark of type 1 diabetes. Histochemical analysis of pancreases from non-obese diabetic mice indicated activation of the transcription factor JunB/AP-1 (activator protein-1) after autoimmune infiltration of the islets. In vitro studies demonstrated that the cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ induce JunB expression as a protective mechanism against apoptosis in both human and rodent ß-cells. The gene network affected was studied by microarray analysis showing that JunB regulates nearly 20% of the cytokine-modified ß-cell genes, including the transcription factor ATF3. Direct transcriptional induction of ATF3 by JunB is a key event for ß-cell survival after TNF-α+IFN-γ treatment. Moreover, pharmacological upregulation of JunB/ATF3 via increased cAMP protected rodent primary ß-cells and human islet cells against pro-inflammatory mediators. These results were confirmed in genetically modified islets derived from Ubi-JunB transgenic mice. Our findings identify ATF3 as a novel downstream target of JunB in the survival mechanism of ß-cells under inflammatory stress.

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to ß-cell apoptosis.

Pancreatic ß-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of ß-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in ß-cells following exposure to well-defined ß-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the ß-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to ß-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.

Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes.

AIMS/HYPOTHESIS: Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate. METHODS: Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes. RESULTS: Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1beta, TNF-alpha, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1beta signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor kappaB (NF-kappaB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-kappaB activation similar to that occurring with palmitate. CONCLUSIONS/INTERPRETATION: Saturated-fatty-acid-induced NF-kappaB activation and endoplasmic reticulum stress may contribute to IL-1beta production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.

A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)gammaa as a pancreatic beta cell-specific biomarker.

AIMS/HYPOTHESIS: Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers. METHODS: We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays. RESULTS: After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na(+)-K(+)-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2gammaa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2gammaa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2gammaa and loss of insulin-positive cells. CONCLUSIONS/INTERPRETATION: We propose human FXYD2gammaa as a novel beta cell-specific biomarker.

Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines involved in the pathogenesis of type 1 diabetes deplete endoplasmic reticulum (ER) Ca2+ stores, leading to ER-stress and beta cell apoptosis. However, the cytokine-induced ER-stress response in beta cells is atypical and characterised by induction of the pro-apoptotic PKR-like ER kinase (PERK)-C/EBP homologous protein (CHOP) branch of the unfolded protein response, but defective X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 activation. The purpose of this study was to overexpress spliced/active Xbp1 (XBP1s) to increase beta cell resistance to cytokine-induced ER-stress and apoptosis. METHODS: Xbp1s was overexpressed using adenoviruses and knocked down using small interference RNA in rat islet cells. In selected experiments, Xbp1 was also knocked down in FACS-purified rat beta cells and rat fibroblasts. Expression and production of XBP1s and key downstream genes and proteins was measured and beta cell function and viability were evaluated. RESULTS: Adenoviral-mediated overproduction of Xbp1s resulted in increased XBP1 activity and induction of several XBP1s target genes. Surprisingly, XBP1s overexpression impaired glucose-stimulated insulin secretion and increased beta cell apoptosis, whereas it protected fibroblasts against cell death induced by ER-stress. mRNA expression of Pdx1 and Mafa was inhibited in cells overproducing XBP1s, leading to decreased insulin expression. XBP1s knockdown partially restored cytokine/ER-stress-driven insulin and Pdx1 inhibition but had no effect on cytokine-induced ER-stress and apoptosis. CONCLUSIONS/INTERPRETATION: XBP1 has a distinct inhibitory role in beta cell as compared with other cell types. Prolonged XBP1s production hampers beta cell function via inhibition of insulin, Pdx1 and Mafa expression, eventually leading to beta cell apoptosis.