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Liver disease is a major cause of premature death. Oxidative stress in the liver represents a key disease driver. Compounds, such as dimethyl fumarate (DMF), can activate the antioxidant response and are used clinically to treat disease. In this study, we tested the protective properties of DMF before or after paracetamol exposure. Following DMF administration, Nrf2 nuclear translocation was tracked at the single-cell level and target gene transactivation confirmed. Next, the protective properties of DMF were examined following paracetamol exposure. Transcriptomic and biochemical analysis revealed that DMF rescue was underpinned by reduced Nf-kB and TGF-β signaling and cell senescence. Following on from these studies, we employed a Zebrafish model to study paracetamol exposure in vivo . We combined a genetically modified Zebrafish model, expressing green fluorescent protein exclusively in the liver, with automated microscopy. Pre-treatment with DMF, prior to paracetamol exposure, led to reduced liver damage in Zebrafish demonstrating protective properties., Competing Interests: D.C.H is a co-founder and shareholder of Stemnovate Ltd. J.M.-R. and P.J.S.-L. are presently employed at Resolution Therapeutics. E.M.T., E.M.G., H.J., N.B. and A.E.K. are/were employees of Novo Nordisk Ltd. The rest of the authors certify that they have no conflicts of interest in the subject matter or materials discussed in this article. D.C.H. is serving as a guest editor on a special issue for iScience in 2021, and this issue is not related to the current article., (© 2021 The Author(s).)

Recent articles have highlighted the lack of reproducibility of data from scientific publications. Here we would argue that a better way to describe and also tackle this matter is to use the term "lack of robustness," since it points toward potential solutions. Presenting several case reports, we highlight examples with common underlying issues from Novo Nordisk's experience: animal model variability, reagent quality, and inter-lab variability. We discuss means to prevent these issues and argue for increased collaborative work and transparent manuscript revision procedures. Collectively, we believe these measures will help promote a more rapid and efficient self-corrective process in diabetes drug target research., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims/hypothesis: Recent studies suggest that proton pump inhibitor treatment may increase insulin secretion and improve glucose metabolism in type 2 diabetes. In a randomised double-blind prospective placebo-controlled 2 × 2 factorial study, we examined the effect of esomeprazole on insulin secretion, HbA(1c) and cardiovascular risk factors in type 2 diabetes., Methods: Forty-one patients with type 2 diabetes using dietary control or oral glucose-lowering treatment were randomised to receive add-on esomeprazole 40 mg (n = 20) or placebo (n = 21) for 12 weeks. Randomisation was carried out prior to inclusion on the basis of a computer-generated random-number list. The allocation sequence was concealed in sealed envelopes from the researcher enrolling and assessing participants. The study was undertaken at Steno Diabetes Center, Gentofte, Denmark. The primary outcome was change in AUC for insulin levels during a meal test. Secondary outcomes were the levels of HbA(1c) and biochemical markers of cardiovascular risk, including lipids, coagulation factors, inflammation markers, markers of endothelial function and 24 h ambulatory BP measurements., Results: Forty-one participants were analysed. In the esomeprazole-treated group the AUC for insulin did not change (before vs after treatment: 28,049 ± 17,659 vs 27,270 ± 32,004 pmol/l × min (p = 0.838). In the placebo group AUC for insulin decreased from 27,392 ± 14,348 pmol/l × min to 22,938 ± 11,936 pmol/l × min (p = 0.002). Esomeprazole treatment (n = 20) caused a ninefold increase in the AUC for gastrin. HbA(1c) increased from 7.0 ± 0.6% (53 ± 5 mmol/mol) to 7.3 ± 0.8% (56 ± 6 mmol/mol) in the esomeprazole-treated group and from 7.0 ± 0.6% (53 ± 5 mmol/mol) to 7.4 ± 0.8% (57 ± 6 mmol/mol) in the placebo group (n = 21) (p for difference in change >0.05). Except for BP, there were no differences between the groups in the markers of cardiovascular risk (p > 0.05). Monitoring of 24 h ambulatory BP showed a significant decrease in daytime systolic BP, daytime diastolic BP and 24 h diastolic BP in the placebo group (p < 0.05). No change in BP was seen in the patients treated with esomeprazole., Conclusions/interpretation: Treatment with esomeprazole over 12 weeks did not improve insulin secretion, glycaemic control or cardiovascular disease biomarkers in patients with type 2 diabetes.

Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1β induces divalent metal transporter 1 (DMT1) expression correlating with increased β cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, β cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Reg3 peptides INGAP-PP and human Reg3α/β (HIP) have been hypothesized to stimulate β-cell neogenesis in the pancreas. Administration of INGAP-PP has been shown to cause an increase in β-cell mass in multiple animal models, reverse streptozotocin (STZ) induced diabetes in mice and reduces HbA1c levels in type 2 diabetic humans. In this study, we have examined the ability of the INGAP-PP and HIP peptides to induce β-cell formation in vivo in normal mice through short-term administration of the peptides. We assessed the peptides ability to induce an increase in extra-islet insulin-positive cell clusters by looking at β-cell number by point counting morphometry on pancreata that had been randomized using the smooth fractionator principle in non-diabetic NMRI mice after short-term injections of the peptides (5 d). Five day continuous BrdU labeling was used to determine if the new β-cells were derived from replicating β-cells. Real time quantitative RT-PCR and immuno-histochemistry was used to analyze changes in pancreatic transcription factor expression. A 1.5- to 2-fold increase in the volume of small extra-islet insulin-positive clusters post 5 d treatment with INGAP-PP and HIP as compared with mice treated with a non-peptide control or scrambled peptide (p<0.05) (n = 7) was found. Five day continuous BrdU infusion during the 5 d period showed little or no incorporation in islets or small insulin clusters. Five days of treatment with INGAP-PP or HIP, showed a tendency toward increased levels of pancreatic progenitor markers such as Ngn3, Nkx6.1, Sox9 and Ins. These are the first studies to compare and indicate that the human Reg3 α/β (HIP) peptide has similar bioactivity in vivo as INGAP by causing formation of small β-cell clusters in extra-islet pancreatic tissue after only 5 d of treatment. Upregulation of pancreatic transcription factors may be part of the mechanism of action.

Aims/hypothesis: The hormone glucagon-like peptide 1 (GLP-1) is released in response to a meal from the intestinal L-cells, where it is processed from proglucagon by the proconvertase (PC)1/3. In contrast, in the adult islets proglucagon is processed to glucagon by the PC2 enzyme. The aim of the study was to evaluate if, during the development of diabetes, alpha cells produce GLP-1 that, in turn, might trigger beta cell growth., Methods: Beta cell mass, GLP-1 and insulin levels were measured in the gerbil Psammomys obesus (P. obesus), a rodent model of nutritionally induced diabetes. Furthermore, the presence of biologically active forms of GLP-1 and PC1/3 in alpha cells was demonstrated by immunofluorescence, and the release of GLP-1 from isolated P. obesus, mouse and human islets was investigated., Results: During the development of diabetes in P. obesus, a significant increase in GLP-1 was detected in the portal vein (9.8 ± 1.5 vs 4.3 ± 0.7 pmol/l, p < 0.05), and in pancreas extracts (11.4 ± 2.2 vs 5.1 ± 1.3 pmol/g tissue, p < 0.05). Freshly isolated islets from hyperglycaemic animals released more GLP-1 following 24 h culture than islets from control animals (28.2 ± 4.4 pmol/l vs 5.8 ± 2.4, p < 0.01). GLP-1 release was increased from healthy P. obesus islets following culture in high glucose for 6 days (91 ± 9.1 pmol/l vs 28.8 ± 6.6, p < 0.01). High levels of GLP-1 were also found to be released from human islets. PC1/3 colocalised weakly with alpha cells., Conclusions/interpretation: GLP-1 release from alpha cells is upregulated in P. obesus during the development of diabetes. A similar response is seen in islets exposed to high glucose, which supports the hypothesis that GLP-1 released from alpha cells promotes an increase in beta cell mass and function during metabolic challenge such as diabetes.

We investigated the impact of β-cell specific overexpression of suppressor of cytokine signalling-3 (SOCS-3) on the development of multiple low dose streptozotocin (MLDSTZ) induced Type 1 diabetes and the possible mechanisms involved. MLDSTZ treatment was administered to RIP-SOCS-3 transgenic and wild-type (wt) mice and progression of hyperglycemia monitored. Isolated islets from both strains were exposed to human IL-1β (25U/ml) or a combination of human IL-1β (25U/ml) and murine IFN-γ (1000U/ml) for 24h or 48h and we investigated the expression of IL-1 receptor antagonist (IL-1Ra) mRNA in islet cells and secretion of IL-1Ra into culture medium. MLDSTZ treatment caused gradual hyperglycemia both in the wt mice and in the transgenic mice with the latter tending to be more sensitive. In vitro experiments on wt and transgenic islets did not reveal any differences in sensitivity to damaging effects of STZ. Exposure of wt islets to IL-1β or IL-1β+IFN-γ seemed to lead to a failing IL-1Ra response from SOCS-3 transgenic islets. It could be that an increased expression of a possible protective molecule against β-cell destruction may lead to a dampered response of another putative protective molecule. This may have counteracted a protective effect against MLDSTZ in SOCS-3 transgenic mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We retrospectively studied whether treatment with esomeprazole improved HbA₁(c) levels in type 2 diabetic patients. We selected 21 patients who had been treated with esomeprazole for 11 ± 3 months and 21 controls. HbA₁(c) levels decreased in the esomeprazole-treated group. Our data indicate that proton pump inhibitors may improve glycaemic control in type 2 diabetic patients., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: High doses of anti-inflammatory drugs, such as aspirin and salicylates, improve glucose metabolism in insulin resistant and type 2 diabetic patients. It has also been shown that the glucose lowering effect is related to the unspecific ability of these drugs to inhibit inhibitor kinaseβ (IKKβ). In this study we have investigated the effect of a selective IKKβ-inhibitor on beta cell survival and the prevention of diet induced type 2 diabetes in the gerbil Psammomys obesus (P. obesus)., Methodology/principal Findings: P. obesus were fed a diabetes inducing high energy diet for one month in the absence or presence of the IKKβ-inhibitor. Body mass, blood glucose, HbA(1C), insulin production and pancreatic insulin stores were measured. The effects on beta cell survival were also studied in INS-1 cells and primary islets. The cells were exposed to IL-1β and subsequently reactive oxygen species, insulin release and cell death were measured in the absence or presence of the IKKβ-inhibitor. In primary islets and beta cells, IL-1β induced the production of reactive oxygen species, reduced insulin production and increased beta cell death, which were all reversed by pre-treatment with the IKKβ-inhibitor. In P. obesus the IKKβ-inhibitor prevented the development of hyperglycaemia and hyperinsulinaemia, and maintained pancreatic insulin stores with no effect on body weight., Conclusions/significance: Inhibition of IKKβ activity prevents diet-induced diabetes in P. obesus and inhibits IL-1β induced reactive oxygen species, loss of insulin production and beta cell death in vitro.

Aims/hypothesis: Gastrin has been implicated in islet growth/neogenesis, and proton pump inhibitors (PPIs) have been shown to increase endogenous gastrin levels in animals and humans. Therefore, we investigated the effect of PPIs in a model of type 2 diabetes, Psammomys obesus., Methods: P. obesus (morning blood glucose [mBG] 16.9 +/- 0.6 mmol/l) were treated with vehicle or different doses (1-15 mg/kg) of lansoprazole for 17 days., Results: Treatment with lansoprazole resulted in up to ninefold dose-dependent increases in endogenous gastrin levels (p < 0.05 for 10 mg/kg lansoprazole vs vehicle). There was a significant reduction in mBG levels in all animals in the high-dose lansoprazole groups during the 17 day treatment period, whereas there was no significant improvement in mBG in animals in the vehicle groups. The mBG at end of study was 18.2 +/- 2.1, 8.7 +/- 2.2 (p < 0.01), and 6.1 +/- 2.3 (p < 0.001) mmol/l for vehicle and lansoprazole 10 and 15 mg/kg, respectively. The animals treated with 15 mg/kg lansoprazole, compared with vehicle, had a 2.3-fold increase in the intensity of insulin staining in beta cells (p=0.0002) and 50% higher beta cell mass (p=0.04)., Conclusions/interpretations: The PPI lansoprazole had significant glucose-lowering effects in an animal model of type 2 diabetes, an effect that is most likely mediated through an increase in endogenous gastrin levels.

In order to investigate the effect and mechanism of liraglutide and vildagliptin in diabetic Psammomys obesus, we examined proliferation and apoptosis of beta-cells, beta-cell mass (BCM), and pancreatic insulin content after zero, six and fourteen days of treatment compared to control groups. One group of animals was kept on low-energy diet and seven groups were given high-energy diet (HED) that induced diabetes over a four week period. Non-fasting morning blood glucose, body weight, HbA(1C) and pancreatic insulin content were measured and beta cell mass (BCM), proliferation and apoptosis frequencies were determined using stereological point counting. Liraglutide significantly reduced blood glucose and even normalized it in all animals treated for six days and in 11 out of 17 animals treated for fourteen days. HED increased BCM and treatment with liraglutide did not change this. However, compared to the vehicle-treated animals pancreatic insulin content was normalized in animals treated for six and fourteen days with liraglutide. In contrast, vildagliptin, in doses causing full inhibition of plasma DPP-IV activity, neither reduced blood glucose nor altered HED-induced increases in BCM or pancreatic insulin content. These results suggest that liraglutide restores normoglycaemia and improves glycaemic control in P. obesus by increasing their insulin content and improving the function of the beta-cells. In contrast, vildagliptin does not improve glycaemic control in P. obesus nor affect beta-cell insulin content., (Copyright 2009 Elsevier B.V. All rights reserved.)

Aims/hypothesis: The suppressor of cytokine signalling 1 (SOCS1) is a natural inhibitor of cytokine and insulin signalling pathways and may also play a role in obesity. In addition, SOCS1 is considered a candidate gene in the pathogenesis of both type 1 diabetes (T1D) and type 2 diabetes (T2D). The objective was to perform mutation analysis of SOCS1 and to test the identified variations for association to T2D-related quantitative traits, T2D or T1D., Methods: Mutation scanning was performed by direct sequencing in 27 white Danish subjects. Genotyping was carried out by TaqMan allelic discrimination. A total of more than 8100 individuals were genotyped., Results: Eight variations were identified in the 5' untranslated region (UTR) region. Two of these had allele frequencies below 1% and were not further examined. The six other variants were analysed in groups of T1D families (n = 1461 subjects) and T2D patients (n = 1430), glucose tolerant first-degree relatives of T2D patients (n = 212) and normal glucose tolerant (NGT) subjects. The rs33977706 polymorphism (-820G > T) was associated with a lower body mass index (BMI) (p = 0.004). In a second study (n = 4625 NGT subjects), significant associations of both the rs33977706 and the rs243330 (-1656G > A) variants to obesity were found (p = 0.047 and p = 0.015) respectively. The rs33977706 affected both binding of a nuclear protein to and the transcriptional activity of the SOCS1 promoter, indicating a relationship between this polymorphism and gene regulation., Conclusions/interpretation: This study demonstrates that functional variations in the SOCS1 promoter may associate with alterations in BMI in the general white population.

Background: Beta-cells are extremely rich in zinc and zinc homeostasis is regulated by zinc transporter proteins. beta-cells are sensitive to cytokines, interleukin-1beta (IL-1beta) has been associated with beta-cell dysfunction and -death in both type 1 and type 2 diabetes. This study explores the regulation of zinc transporters following cytokine exposure., Methods: The effects of cytokines IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) on zinc transporter gene expression were measured in INS-1-cells and rat pancreatic islets. Being the more sensitive transporter, we further explored ZnT8 (Slc30A8): the effect of ZnT8 over expression on cytokine induced apoptosis was investigated as well as expression of the insulin gene and two apoptosis associated genes, BAX and BCL2., Results: Our results showed a dynamic response of genes responsible for beta-cell zinc homeostasis to cytokines: IL-1beta down regulated a number of zinc-transporters, most strikingly ZnT8 in both islets and INS-1 cells. The effect was even more pronounced when mixing the cytokines. TNF-alpha had little effect on zinc transporter expression. IFN-gamma down regulated a number of zinc transporters. Insulin expression was down regulated by all cytokines. ZnT8 over expressing cells were more sensitive to IL-1beta induced apoptosis whereas no differences were observed with IFN-gamma, TNF-alpha, or a mixture of cytokines., Conclusion: The zinc transporting system in beta-cells is influenced by the exposure to cytokines. Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.

Aims/hypothesis: The pro-inflammatory cytokines IL-1 and IFNgamma are critical molecules in immune-mediated beta cell destruction leading to type 1 diabetes mellitus. Suppressor of cytokine signalling (SOCS)-3 inhibits the cytokine-mediated destruction of insulinoma-1 cells. Here we investigate the effect of SOCS3 in primary rodent beta cells and diabetic animal models., Methods: Using mice with beta cell-specific Socs3 expression and a Socs3-encoding adenovirus construct, we characterised the protective effect of SOCS3 in mouse and rat islets subjected to cytokine stimulation. In transplantation studies of NOD mice and alloxan-treated mice the survival of Socs3 transgenic islets was investigated., Results: Socs3 transgenic islets showed significant resistance to cytokine-induced apoptosis and impaired insulin release. Neither glucose-stimulated insulin release, insulin content or glucose oxidation were affected by SOCS3. Rat islet cultures transduced with Socs3-adenovirus displayed reduced cytokine-induced nitric oxide and apoptosis associated with inhibition of the IL-1-induced nuclear factor-kappaB and mitogen-activated protein kinase (MAPK) pathways. Transplanted Socs3 transgenic islets were not protected in diabetic NOD mice, but showed a prolonged graft survival when transplanted into diabetic allogenic BALB/c mice., Conclusions/interpretation: SOCS3 inhibits IL-1-induced signalling through the nuclear factor-kappaB and MAPK pathways and apoptosis induced by cytokines in primary beta cells. Moreover, Socs3 transgenic islets are protected in an allogenic transplantation model. SOCS3 may represent a target for pharmacological or genetic engineering in islet transplantation for treatment of type 1 diabetes mellitus.

We recently finemapped a type 1 diabetes (T1D)-linked region on chromosome 21, indicating that one or more T1D-linked genes exist in this region with 33 annotated genes. In the current study, we have taken a novel approach using transcriptional profiling in predicting and prioritizing the most likely candidate genes influencing beta-cell function in this region. Two array-based approaches were used, a rat insulinoma cell line (INS-1alphabeta) overexpressing pancreatic duodenum homeobox 1 (pdx-1) and treated with interleukin 1beta (IL-1beta) as well as human pancreatic islets stimulated with a mixture of cytokines. Several candidate genes with likely functional significance in T1D were identified. Genes showing differential expression in the two approaches were highly similar, supporting the role of these specific gene products in cytokine-induced beta-cell damage. These were genes involved in cytokine signaling, oxidative phosphorylation, defense responses and apoptosis. The analyses, furthermore, revealed several transcription factor binding sites shared by the differentially expressed genes and by genes demonstrating highly similar expression profiles with these genes. Comparable findings in the rat beta-cell line and human islets support the validity of the methods used and support this as a valuable approach for gene mapping and identification of genes with potential functional significance in T1D, within a region of linkage.

The desert gerbil Psammomys obesus, an established model of type 2 diabetes (T2D), has previously been shown to lack pancreatic and duodenal homeobox gene 1 (Pdx-1) expression. Pdx-1 deficiency leads to pancreas agenesis in both mice and humans. We have therefore further examined the pancreas of P. obesus during embryonic development. Using Pdx-1 antisera raised against evolutionary conserved epitopes, we failed to detect Pdx-1 immunoreactivity at any time points. However, at E14.5, Nkx6.1 immunoreactivity marks the nuclei of all epithelial cells of the ventral and dorsal pancreatic buds and the only endocrine cell types found at this time point are glucagon and PYY. At E18.5 the pancreas is well branched and both glucagon- and ghrelin-positive cells are scattered or found in clusters, whereas insulin-positive cells are not found. At E22.5, the acini of the exocrine pancreas are starting to mature, and amylase and carboxypeptidase A immunoreactivity is found scattered and not in all acini. Ghrelin-, glucagon-, PYY-, gastrin-, somatostatin (SS)-, pancreatic polypeptide (PP)-, and insulin-immunoreactive cells are found scattered or in small groups within or lining the developing ductal epithelium as marked by cytokeratin 19. Using degenerate PCR, the P. obesus Neurogenin-3 (Ngn-3) gene was cloned. Nucleotide and amino acid sequences show high homology with known Ngn-3 sequences. Using specific antiserum, we can observe that Ngn-3-immunoreactive cells are rare at E14.5 but readily detectable at E18.5 and E22.5. In conclusion, despite the lack of detection of Pdx-1, the P. obesus pancreas develops similarly to Muridae species, and the Ngn-3 sequence and expression pattern is highly conserved in P. obesus.

Secretagogin is a newly identified calcium-binding protein selectively expressed in neuroendocrine tissue and pancreatic beta-cells. The function of secretagogin is unknown, but it has been suggested in beta-cells to influence calcium-influx, insulin secretion and proliferation, and has been observed downregulated in diabetes-prone BB rat islets exposed to cytokines. In the present study, we identified and characterized promoter activity of a human 1498 bp sequence upstream the transcription start site. The promoter sequence showed subtle but significant regulation by glucose within the normo-physiological range. Glucose also led to changes in expression of secretagogin protein in INS-1e cells, but not in primary cells from non-diabetes-prone Wistar Furth rats. No effects of cytokines neither on promoter activity nor protein expression were observed. The promoter region was furthermore screened by direct sequencing, and 11 polymorphisms were identified. Genotyping in a large homogenous Type 1 diabetes (T1D) family collection did not reveal association with T1D.

Type 1 diabetes (T1D) is characterized by selective autoimmune destruction of the insulin producing beta-cells in the islets of Langerhans. When the beta-cells are destroyed exogenous administration of insulin is necessary for maintenance of glucose homeostasis. Allogeneic islet transplantation has been used as a means to circumvent the need for insulin administration and has in some cases been able to restore endogenous insulin production for years. However, long life immunosuppression is needed to prevent the graft from being rejected and destroyed. Changes in protein expression pattern during spontaneous diabetes development in the diabetes prone BioBreeding rat (BB-DP) have previously been described. In the present study, we have investigated if any of the changes seen in the protein expression pattern during spontaneous diabetes development are also present during allograft rejection of BB-DP rat islets. Two hundred neonatal islets were syngeneically transplanted under the kidney capsule of 30 day old BB-DP rats and removed prior to and at onset of diabetes. Allogeneically transplanted islets from BB-DP rats were removed before onset of allograft rejection and at maximal islet graft inflammation (rejection). The protein expression profiles of the transplants were visualised by two-dimensional gel (2-DG) electrophoresis, analysed and compared. In total, 2590 protein spots were visualised and of these 310 changed expression (p < 0.01) in syngeneic islet transplants in the BB-DP rats from 7 days after transplantation until onset of diabetes. In BB-DP islets transplanted to WK rats 53 protein spots (p < 0.01) showed changes in expression when comparing islet grafts removed 7 days after transplantation with islet grafts removed 12 days after transplantation where mononuclear cell infiltration is at its maximum. Only four protein spots (1%) were significantly changed in both syngeneic (autoimmune) and allogeneic islet destruction. When comparing protein expression changes in syngeneic BB-DP islet transplants from 37 days after transplantation to onset of diabetes with protein expression changes in allografts from day 7 to 12 after transplantation only three spot were found to commonly change expression in both situations. In conclusion, a large number of protein expression changes were detected in both autoimmune islet destruction and allogeneic islet rejection, only two overlaps were detected, suggesting that autoimmune islet destruction and allogeneic islet rejection may result from different target cell responses to signals induced by the cellular infiltrate. Whether this reflects activation of distinct signalling pathways in islet cells is currently unknown and need to be further investigated.

Pro-apoptotic cytokines are toxic to the pancreatic beta-cells and have been associated with the pathogenesis of Type 1 diabetes (T1D). Proteome analysis of IL-1beta exposed isolated rat islets identified galectin-3 (gal-3) as the most up-regulated protein. Here analysis of human and rat islets and insulinoma cells confirmed IL-1beta regulated gal-3 expression of several gal-3 isoforms and a complex in vivo expression profile during diabetes development in rats. Over-expression of gal-3 protected beta-cells against IL-1beta toxicity, with a complete blockage of JNK phosphorylation, essential for IL-1-mediated apoptosis. Mutation scanning of regulatory and coding regions of the gal-3 gene (LGALS3) identified six polymorphisms. A haplotype comprising three cSNPs showed significantly increased transmission to unaffected offspring in 257 T1D families and replicated in an independent set of 170 T1D families. In summary, combined proteome-transcriptome-genome and functional analyses identify gal-3 as a candidate gene/protein in T1D susceptibility that may prove valuable in future intervention/prevention strategies.

The proinflammatory cytokines interleukin-1beta (IL-1beta), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) are toxic to pancreatic beta-cells and are implicated in the pathogenesis of type 1 diabetes. We have previously found that GH and prolactin (PRL) stimulate both proliferation and insulin production in pancreatic beta-cells and rat insulin-producing INS-1 cells. Here we report that human (h) GH can prevent the apoptotic effects of IL-1beta, IFN-gamma and TNF-alpha in INS-1 and INS-1E cells. Using adenovirus-mediated gene transfer, we found that the anti-apoptotic effect of hGH is abrogated by expression of a dominant negative signal transducer and activator of transcription (STAT5) mutant in INS-1E cells. hGH and the cytotoxic cytokines was found to additively increase suppressor of cytokine signalling-3 mRNA expression after 4 h of exposure. In order to identify possible targets for the STAT5-mediated protection of INS-1E cells, we studied the effect of hGH on activation of the transcription factors STAT1 and nuclear factor-kappaB (NF-kappaB) by IFN-gamma and IL-1beta+TNF-alpha respectively. Gel retardation experiments showed that hGH affects neither IFN-gamma+TNF-alpha-induced STAT1 DNA binding nor IL-1beta and IFN-gamma+TNF-alpha-induced NFkappaB DNA binding. The lack of influence of hGH on cytokine-mediated activation of STAT1 and NFkappaB is in accordance with the finding that hGH had only a minor effect on cytokine-induced inducible nitric oxide synthase (iNOS) gene expression and in fact augmented the IL-1beta-stimulated nitric oxide production. As the anti-apoptotic Bcl-xL gene has been shown to harbour a STAT5-binding element we measured the expression of Bcl-xL as well as the pro-apoptotic Bax. We found that hGH increased the Bcl-xL/Bax ratio both in the absence and in the presence of cytotoxic cytokines. In conclusion, these results suggested that GH and PRL protect beta-cells against cytotoxic cytokines via STAT5-dependent mechanisms distal to iNOS activation possibly at the level of Bcl-xL.

The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1beta-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1beta-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca(2+) channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse betaTC3 cells. Ca(2+) influx, specifically via L-type but not T-type channels, contributed to IL-1beta activation of JNK. Activation of p38 and ERK in response to IL-1beta was also dependent on L-type Ca(2+) influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca(2+) ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin-secreting cells.

Type 1 diabetes (T1D) is an autoimmune disease in genetically predisposed individuals characterised by selective destruction of the beta-cells. Development of diabetes is in the asymptomatic pre-diabetic period characterised by impaired first-phase insulin response and the first clinical symptom is elevated blood glucose (BG). It is still uncertain whether stress or incidental hyperglycaemia can be regarded as predictors for development of T1D or not, even when immunologic and genetic markers for T1D are considered. The aim of this study was to investigate if there was any relationship between elevated BG in 30-day-old anaesthetised pre-diabetic diabetes-prone Bio Breeding (BB-DP) rats and later development of diabetes. Rats anaesthetised by intraperitoneal (ip) injection for islet transplantation displayed significantly higher BG values (Delta1.27 mmol/l, p=8.27x10(-12)) compared to non-anaesthetised non-transplanted rats, indicating that ip injection and/or anaesthesia induce a higher BG level. Linear regression analysis of BG and time of onset of diabetes in transplanted and non-transplanted BB-DP rats revealed no correlation (R(2) at 0.0075 and 0.0324 and p-values at 0.56 and 0.23 respectively). We were not able to identify any association or correlation between the induced temporary hyperglycaemia in 30-day-old BB-DP rats and later development of diabetes.

We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the beta-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9-39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining beta-cell mass in both type 1 and type 2 diabetic patients.

Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the beta-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 beta in particular, are present in the islets during beta-cell destruction and are able to modulate beta-cell function and induce beta-cell death. In beta-cells exposed to IL-1 beta, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies of differentiating beta-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in beta-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the beta-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed beta-cell to dynamic instability and eventually to beta-cell destruction.

Malignant insulinoma is a critical cancer form with a poor prognosis. Because cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse betaTC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N-terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin, also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability, and induced apoptosis in betaTC3 and rat INS-1E cells. Both ALLN and lactacystin caused a marked decrease in the cellular amount of the JNK scaffold protein JNK-interacting protein 1/islet-brain-1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion but had no impact on cell viability and even partially protected against the toxic effect of proinflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess antitumorigenic and antiinsulinogenic effects on insulinoma cells.

Aim/hypothesis: Maturation of the beta cells in the islets of Langerhans is dependent upon sequential activation of different transcription factors such as Pdx-1 and Nkx6.1. This maturation is associated with an acquired sensitivity to cytokines and may eventually lead to type 1 diabetes. The aims of this study were to characterise changes in mRNA expression during beta cell maturation as well as after interleukin-1beta (IL-1beta) exposure., Methods: Transcriptome analyses were performed on two phenotypes characterised as a glucagon-producing pre-beta-cell phenotype (NHI-glu), which matures to an IL-1beta-sensitive insulin-producing beta cell phenotype (NHI-ins). Beta cell lines over-expressing Pdx-1 or Nkx6.1, respectively, were used for functional characterisation of acquired IL-1beta sensitivity., Results: During beta cell maturation 98 fully annotated mRNAs changed expression levels. Of these, 50 were also changed after 24 h of IL-1beta exposure. In addition, 522 and 197 fully annotated mRNAs, not affected by maturation, also changed expression levels following IL-1beta exposure of the beta cell and the pre-beta-cell phenotype, respectively. Beta cell maturation was associated with an increased expression of Nkx6.1, whereas both Pdx-1 and Nkx6.1 expression were decreased following IL-1beta exposure. Over-expression of Nkx6.1 or Pdx-1 in cell lines resulted in a significantly increased sensitivity to IL-1beta., Conclusions/interpretation: These results suggest that the final beta cell maturation accompanied by increased IL-1beta sensitivity is, in part, dependent upon the expression of genes regulated by Pdx-1 and Nkx6.1. Future classification of the genes regulated by these transcription factors and changed during beta cell maturation should elucidate their role in the acquired sensitivity to IL-1beta and may be helpful in identifying new targets for intervention/prevention strategies.

Aims/hypothesis: The proinflammatory cytokine IL-1beta induces apoptosis in pancreatic beta cells via pathways dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein kinase, and protein kinase C. We recently showed suppressor of cytokine signalling (SOCS)-3 to be a natural negative feedback regulator of IL-1beta- and IFN-gamma-mediated signalling in rat islets and beta cell lines, preventing their deleterious effects. However, the mechanisms underlying SOCS-3 inhibition of IL-1beta signalling and prevention against apoptosis remain unknown., Methods: The effect of SOCS-3 expression on the global gene-expression profile following IL-1beta exposure was microarray-analysed using a rat beta cell line (INS-1) with inducible SOCS-3 expression. Subsequently, functional analyses were performed., Results: Eighty-two known genes and several expressed sequence tags (ESTs) changed expression level 2.5-fold or more in response to IL-1beta alone. Following 6 h of IL-1beta exposure, 23 transcripts were up-regulated. Of these, several, including all eight transcripts relating to immune/inflammatory response pathways, were suppressed by SOCS-3. Following 24 h of IL-1beta exposure, secondary response genes were detected, affecting metabolism, energy generation, protein synthesis and degradation, growth arrest, and apoptosis. The majority of these changes were prevented by SOCS-3 expression. Multiple IL-1beta-induced NF-kappaB-dependent proapoptotic early response genes were inhibited by SOCS-3 expression, suggesting that SOCS-3 inhibits NF-kappaB-mediated signalling. These observations were experimentally confirmed in functional analyses., Conclusions/interpretation: This study suggests that there is an unexpected cross-talk between the SOCS/IFN and the IL-1beta pathways of signalling in pancreatic beta cells, which could lead to a novel perspective of blocking two important proapoptotic pathways in pancreatic beta cells by influencing a single signalling molecule, namely SOCS-3.

Mortalin has been found to be up-regulated by 2D-protein gel analysis in isolated rodent islets exposed to cytokines. In islets from two rat strains with different sensitivity to the toxic effects of cytokines we observed a significant difference in IL-1beta mediated mortalin expression. Constitutive over-expression of rat mortalin in NIH3T3 cells reduced cellular survival in accordance with mortalin being associated to cellular senescence. Hence we consider the gene encoding for mortalin at chromosome 5q31.1 a putative candidate gene in cytokine induced beta-cell destruction. We scanned the human mortalin gene for polymorphisms and identified three novel polymorphisms. Neither the SNPs individually nor as constructed haplotypes showed disease association tested by (E)TDT in a Danish type 1 diabetes (T1DM) population. Furthermore, we tested the D5S500 microsatelite located close to 5q31.1 without finding linkage to (T1DM). In conclusion, the functional data identifying a difference in mortalin expression in IL-1beta stimulated islets between two rat strains and over-expression of mortalin in NIH3T3 cells associated with decreased viability suggests a functional role for mortalin in cytokine mediated beta cell destruction; however, the identified polymorphisms did not reveal any association in the presence of linkage disequilibrium of mortalin to T1DM in the Danish population.

Aims/hypothesis: Beta cell loss in Type 1 and Type 2 diabetes mellitus may result from apoptosis and necrosis induced by inflammatory mediators. The suppressor of cytokine signalling (SOCS)-3 is a natural inhibitor of cytokine signalling and also influences insulin signalling. SOCS3 could therefore be a candidate gene in the development of Type 1 and Type 2 diabetes mellitus., Methods: Mutation analysis of the SOCS3 gene was performed in 21 patients with Type 1 diabetes mellitus and in seven healthy subjects. An identified promoter variant was examined in (i) 250 families with Type 1 diabetic family members (1097 individuals); (ii) 212 glucose-tolerant first-degree relatives of Type 2 diabetic patients; and (iii) 370 population-based young, healthy subjects who were unrelated., Results: Three mutations were identified in the promoter region, but none in the coding region or the 3'UTR. Two of the three mutations had allele frequencies below 1% whereas the C -920-->A substitution had a minor allele frequency of 8%. In the group of young healthy subjects the insulin sensitivity index was higher among homozygous carriers of the A-allele than among heterozygous and wild-type subjects ( p=0.027, uncorrected). The same trend was found in the group of first-degree relatives of Type 2 diabetic patients. No association or linkage was found to Type 1 diabetes mellitus., Conclusions/interpretation: Homozygosity for the A-allele of the C -920-->A promoter polymorphism of the SOCS3 gene may be associated with increased whole-body insulin sensitivity, but deserves further investigation.

CD4 is a candidate gene in autoimmune diseases, including Type 1 diabetes mellitus (T1DM), because the CD4 receptor is crucial for appropriate antigen responses of CD4(+) T cells. We previously found linkage between a CD4-1188(TTTTC)(5-14) promoter polymorphism and T1DM. In the present study, we screened the human CD4 promoter for mutations and identified three frequent single nucleotide polymorphisms (SNPs): CD4-181C/G, CD4-521C/G and CD4-1050T/C. The SNPs are in strong linkage disequilibrium (LD) and association with the CD4-1188(TTTTC)(5-14) alleles, and we observed nine CD4 promoter haplotypes, of which four are frequent. We genotyped the SNPs in 253 Danish T1DM families (1129 individuals) and found evidence for linkage and association of a CD4 (A4(-1188)T(-1050)G(-521)C(-181)) haplotype to T1DM. In reporter studies, we show that (1) the T1DM-associated CD4 haplotype encodes high constitutive promoter activity and (2) the CD4-181G variant encodes higher stimulated promoter activity than the CD4-181C variant. This difference is in part neutralized in the frequently occurring CD4 promoter haplotypes by the more upstream genetic variants. Thus, we report functional impact of a novel CD4-181C/G SNP on stimulated CD4 promoter activity and the identification of a novel CD4 haplotype with high constitutive promoter activity that is linked and associated with T1DM.

Aims/hypothesis: Type 1 diabetes mellitus is a multifactorial autoimmune disease characterised by selective destruction of beta cells in the islets of Langerhans. We have previously shown that IL-1 beta modulates beta cell function, causes beta cell death and induces expression changes in 82 out of 1815 protein spots detected by two-dimensional gel electrophoresis (2-DGE) in diabetes-prone bio-breeding (BB-DP) rat islets in vitro. The aim of this study was to describe the relevance of these proteins in the development of diabetes in vivo., Methods: Syngeneic neonatal islets ( n=200) were transplanted under the kidney capsule of 30-day-old BB-DP and control rats, removed to different time points after transplantation or at the onset of diabetes, and metabolically labelled with S(35)-methionine for 2-DGE. The 82 proteins were re-localised and followed. In addition, transplants were examined for expression of IL-1 beta mRNA by in situ hybridisation., Results: All 82 proteins could be re-localised in all syngeneic transplants from BB-DP and control rats. A total of 60 of the 82 proteins were changed during development of diabetes. Of the 82 proteins, 32 were changed in expression at the onset of diabetes compared to non-diabetic BB-DP rats, and 25 of these were changed as by IL-1 beta in vitro. Highest expression of IL-1 beta mRNA was found at the onset of diabetes., Conclusions/interpretation: IL-1 beta-induced protein expression changes in islets in vitro also occur in vivo and change in a complex pattern during the development of diabetes in the BB-DP rat. No single protein seems to be responsible for the development of diabetes, but rather the cumulative numbers of changes seem to interfere with the intracellular stability of the beta cell.

Complex traits like type 1 diabetes mellitus (T1DM) are generally taken to be under the influence of multiple genes interacting with each other to confer disease susceptibility and/or protection. Although novel methods are being developed, analyses of whole-genome scans are most often performed with multipoint methods that work under the assumption that multiple trait loci are unrelated to each other; that is, most models specify the effect of only one locus at a time. We have applied a novel approach, which includes decision-tree construction and artificial neural networks, to the analysis of T1DM genome-scan data. We demonstrate that this approach (1) allows identification of all major susceptibility loci identified by nonparametric linkage analysis, (2) identifies a number of novel regions as well as combinations of markers with predictive value for T1DM, and (3) may be useful in characterizing markers in linkage disequilibrium with protective-gene variants. Furthermore, the approach outlined here permits combined analyses of genetic-marker data and information on environmental and clinical covariates.

Aim/hypothesis: Type 1 diabetes mellitus (T1DM) is caused by specific destruction of the pancreatic beta cells in the islets of Langerhans. Increased sensitivity to cytokines, in particular to interleukin-1beta (IL-1beta) seems to be an acquired trait during beta-cell maturation. In response to cytokines both protective and deleterious mechanisms are induced in beta cells, and when the deleterious prevail, T1DM develops. The aims of this study were to identify perturbation in protein patterns (PiPP) associated with beta-cell maturation, and compare these changes to previous analyses of IL-1beta exposed rat islets. For this purpose, proteome analyses were carried out using a cell-line, which matures from a glucagon-producing pre-beta-cell phenotype (NHI-glu) to an insulin-producing beta-cell phenotype (NHI-ins). We have previously shown that this maturation is accompanied by acquired sensitivity to the toxic effects of IL-1beta., Methods: 2D-gel electrophoresis was used to separate the proteins and MALDI-MS and database searches were performed to identify the proteins., Results: During beta-cell maturation 135 protein spots out of 2239 detectable changed expression levels. Of these, 74 were down-regulated, 44 up-regulated, 16 were suppressed and 1 was expressed de novo. Using MALDI-MS, positive identification was obtained for 93 out of the 135 protein-spots revealing 97 different proteins. Of these, 22 proteins were in common with changes identified in previous proteome analysis of perturbation in protein pattern in IL-1beta exposed rat islets. Several of the proteins were present in more than one spot suggesting post-translational modification., Conclusion/interpretation: Several proteins and protein modifications were identified that could be critically involved in beta-cell maturation, insulin-gene expression and the acquired IL-1beta sensitivity.

The stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) is a central signal for interleukin-1beta (IL-1beta)-induced apoptosis in insulin-producing beta-cells. The cell-permeable peptide inhibitor of JNK (JNKI1), that introduces the JNK binding domain (JBD) of the scaffold protein islet-brain 1 (IB1) inside cells, effectively prevents beta-cell death caused by this cytokine. To define the molecular targets of JNK involved in cytokine-induced beta-cell apoptosis we investigated whether JNKI1 or stable expression of JBD affected the expression of selected pro- and anti-apoptotic genes induced in rat (RIN-5AH-T2B) and mouse (betaTC3) insulinoma cells exposed to IL-1beta. Inhibition of JNK significantly reduced phosphorylation of the specific JNK substrate c-Jun (p<0.05), IL-1beta-induced apoptosis (p<0.001), and IL-1beta-mediated c-fos gene expression. However, neither JNKI1 nor JBD did influence IL-1beta-induced NO synthesis or iNOS expression or the transcription of the genes encoding mitochondrial manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase rho (GSTrho), heat shock protein (HSP) 70, IL-1beta-converting enzyme (ICE), caspase-3, apoptosis-inducing factor (AIF), Bcl-2 or Bcl-xL. We suggest that the anti-apoptotic effect of JNK inhibition by JBD is independent of the transcription of major pro- and anti-apoptotic genes, but may be exerted at the translational or posttranslational level.

The type 1 diabetes mellitus (T1DM) candidate gene SNP IL6-174G/C was genotyped in 253 Danish T1DM families (1129 individuals). TDT analysis demonstrated linkage in the presence of association between the IL6-174C allele and T1DM in the 416 T1DM offspring, P(tdt)=0.04. Gender conditioned TDT analyses revealed that linkage and association with T1DM were present in females exclusively; P(tdt)=6.5 x 10(-4) and P(tdt)=2.4 x 10(-4), respectively. Random transmission of the IL6-174C/G alleles was found in T1DM males, non-T1DM males and non-T1DM females; all P(tdt)>/=0.37. Heterogeneity analyses (T1DM versus non-T1DM females) excluded preferential meiotic segregation in females, P=4.6 x 10(-3), and demonstrated differences in the transmission patterns between female and male T1DM offspring, P=5.1 x 10(-3). The IL6-174 CC genotype was associated with younger age at onset of T1DM in females (P=0.002). The impact of 17beta-estradiol (E(2)) on the IL6-174G/C variants was investigated by reporter studies. The PMA stimulated activity of the T1DM risk IL6-174C variant exceeded that of the T1DM protective IL6-174G variant by approximately 70% in the absence of E(2) (P(c)=0.004), but not with E(2) present (P(c)=0.12). The PMA stimulated activity of the IL6-174G variant was repressed without E(2) present, but was derepressed by addition of E(2), P(c)=0.024. In contrast, the PMA stimulated IL6-174C activity was unaffected by E(2) as were the constitutive activities of the IL6-174G/C variants. In conclusion, higher IL6 promoter activity may confer risk to T1DM in very young females. This excess risk is negated with increasing age, possibly by the increasing E(2) levels in puberty.

The free radical nitric oxide (NO) has been implicated in cytokine mediated destruction of rat beta-cells in islets of Langerhans. Cytokine mediated NO production is associated with increased expression of the inducible nitric oxide synthase (iNOS). We have previously shown a strain dependent difference between Wistar Kyoto (WKY) and Brown Norway (BN) rats of IL-1beta mediated destruction of islets of Langerhans to be related to expression levels of iNOS and NO production. The aim of the present study was to clone and screen the iNOS gene promoter region from WKY and BN rats for polymorphisms and to functionally test such nucleotide differences. Within the total 2077 bp sequenced from both rat strains we identified three polymorphisms in two separate areas: (i) a GT-repeat polymorphism linked to (ii) a C/T polymorphisms, leading to a WT1 binding site approximately 1650bp upstream the BN iNOS promoter and (iii) a G/A SNP in exon 1. Apart from these polymorphisms the homology between all published rat iNOS sequences including the presently described are about 96%. Promoter activity was detected for both genes in a luciferase assay followed cloning of 2012 bp fragments and transient transfection into RIN cells. For both strains IL-1beta induced dose-dependent activity and strain dependent iNOS promoter activity was demonstrated when WT1 was co-expressed. To our knowledge, this is the first demonstration of functional WT1/iNOS promoter interaction. We conclude that the iNOS promoter is strain-dependently regulated which may relate to quantitatively as well as qualitatively strain dependent differences in transcription factor expression, in this study exemplified by WT1.

Unlabelled: Type 1 Diabetes Mellitus is characterized by selective destruction of the pancreatic beta-cells in the islets of Langerhans and insulitis. Subcutaneous insulin injections given to diabetes prone BioBreeding (BB-DP) rats reduce diabetes incidence. The underlying mechanism(s) are not known in detail. Previously, we showed that transplantation of 200 syngeneic neonatal islets under the kidney capsule is useful for studying molecular events during diabetes development in BB-DP rats. In the present study we tested if prophylactic insulin treatment of syngeneically transplanted BB-DP rats would protect both islets in situ and transplanted islets from destruction., Methods: BB-DP rats received transplants of 200 syngeneic neonatal islets under the kidney capsule at 30 days of age. They were given a subcutaneous insulin or placebo implant and were compared to control rats. Blood glucose was measured three times weekly. In total, 193 rats were transplanted and rats were sacrificed 7, 23, 50, 90 days post-transplantation or at onset of diabetes. Pancreatic and transplant sections were stained for insulin and mononuclear cell infiltration and insulitis was graded., Results: Eight (19%) rats developed diabetes in the insulin-treated group and 19 (63%) and 19 (65%) rats in the control and placebo, respectively (p = 0.0002 and p = 0.0001). Onset of diabetes in the insulin treated group was delayed compared to control and placebo, (102, 77 and 81 days of age, respectively (p = 0.0001 and p = 0.0001)). Insulin treatment diminished mononuclear cell infiltration in the islets at day 50 after transplantation compared to placebo. Infiltration pattern in islets in situ correlates with infiltration in transplants (r is 0.9076 and p < 0.001)., Conclusion/interpretation: These results suggest that insulin-treatment of syngeneically transplanted BB-DP rats considerably decreases the incidence of diabetes and that this model is well suited for studying molecular changes in the islet transplants.

The cytokine IL-1beta suppresses rodent islets of Langerhans in vitro. Presently we used inhibitors of the proteasome to investigate if these compounds could counteract the suppressive effects of the cytokine. Thus, isolated rat islets were cultured and pre-treated with proteasome inhibitors and subsequently exposed for 48 h to 25 U/ml human IL-1beta. After this period functional tests were carried out. The rate of glucose oxidation (pmol/10 islets x 90 min) was suppressed by IL-1beta (115 +/- 17 vs. control 380 +/- 57). Pre-treatment with 10 microM of the proteasome inhibitor MG115 (N-carbobenzoxyl-leu-leu-norvalinal) and 100 microM of the calpain inhibitor norLEU (N-acetyl-leu-leu-norleucinal; known to affect proteasome activity) counteracted the suppressive effects (253 +/- 17 and 262 +/- 10 respectively). The calpain inhibitor alIMET (N-acetyl-leu-leu-methional) had no effect. MG115 (10 microM) and norLEU (100 microM) blocked nitric oxide formation induced by IL-1beta, while alIMET was without effect. We also investigated if IL-1beta could influence the expression of two inducible proteasome subunits, namely LMP2 and LMP7, and found that the cytokine increased the mRNA expression of the proteasome subunit LMP2 in islets, and that the proteasome inhibitor MG115 prevented this increase. In conclusion our study shows that IL-1beta increases the transcription of the proteasome subunit LMP2, and that the proteasome is involved in IL-1beta induced suppression of islet function. Moreover, the observation that inhibitors of the proteasome protect islets against IL-1beta induced inhibition of glucose metabolism, suggests that these compounds might be worthwile to explore in future therapies against the development of type 1 diabetes.

Aims/hypothesis: Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1beta (IL-1beta) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1beta exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1beta exposed Wistar Furth (WF) rat islets., Methods: The 82 protein spots, that changed expression after IL-1beta exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification., Results: Forty-five different proteins were identified from 51 spots and grouped according to function: (i) energy transduction and redox potentials; (ii) glycolytic and Krebs cycle enzymes; (iii) protein, DNA and RNA synthesis, chaperoning and protein folding; (iv) signal transduction, regulation, differentiation and apoptosis; (v) cellular defence; and (vi) other functions. Comparison of IL-1beta exposed BB-DP and WF islets showed common changes in 14 proteins and several proteins influencing similar pathways, suggesting that similar routes in the two strains lead to beta-cell destruction., Conclusion/interpretation: We demonstrate that proteome analysis is a powerful tool to identify proteins and pathways in BB-DP rat islets exposed to IL-1beta.

GH is an important regulator of cell growth and metabolism. In the pancreas, GH stimulates mitogenesis as well as insulin production in beta-cells. The cellular effects of GH are exerted mainly through activation of the Janus kinase-signal transducer and activator of transcription (STAT) pathway. Recently it has been found that suppressors of cytokine signaling (SOCS) proteins are able to inhibit GH-induced signal transduction. In the present study, the role of SOCS-3 in GH signaling was investigated in the pancreatic beta-cell lines RIN-5AH and INS-1 by means of inducible expression systems. Via stable transfection of the beta-cell lines with plasmids expressing SOCS-3 under the control of an inducible promoter, a time- and dose-dependent expression of SOCS-3 in the cells was obtained. EMSA showed that SOCS-3 is able to inhibit GH-induced DNA binding of both STAT3 and STAT5 in RIN-5AH cells. Furthermore, using Northern blot analysis it was shown that SOCS-3 can completely inhibit GH-induced insulin production in these cells. Finally, 5-bromodeoxyuridine incorporation followed by fluorescence-activated cell sorting analysis showed that SOCS-3 inhibits GH-induced proliferation of INS-1 cells. These findings support the hypothesis that SOCS-3 is a major regulator of GH signaling in insulin-producing cells.

Type 1 diabetes mellitus (T1DM) is a multifactorial disorder characterized by a specific destruction of the insulin-producing beta cells in the islets of Langerhans. Cells from the immune system infiltrate the islet during the pathogenesis, releasing a mixture of cytokines demonstrated to be specifically toxic to the beta cells within the islets. The goal is to understand the molecular mechanisms responsible for this specific beta-cell toxicity, which will allow the design of novel intervention strategies for T1DM. The proteome approach provides a detailed picture of the beta-cell proteins changing expression pattern during cytokine-mediated beta-cell destruction. Combining the information from this proteome approach with genetic studies makes us believe that it is possible to reach this goal., (Copyright 2002 Wiley-Liss, Inc.)

Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-gamma signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and IFN-gamma. IL-1beta and IFN-gamma alone, or potentiated by TNF-alpha, are cytotoxic to the insulin producing pancreatic beta-cells and beta-cell lines in vitro and suggested to contribute to the specific beta-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat beta-cell line INS-1, we demonstrate that the toxic effect of both IL-1beta or IFN-gamma at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-gamma signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1beta signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1beta-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1beta revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of beta-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.

Unlabelled: Nitric oxide (NO) may be a necessary but not sufficient mediator of cytokine-mediated, selective beta-cell destruction. Previously, we have described a difference in NO-dependent IL-1beta sensitivity in vivo and in vitro of pancreatic islets from two rat strains, Brown Norway (BN) and Wistar Kyoto (WK), the latter being the more sensitive strain. Here we investigated whether strain-dependent, differential islet iNOS expression was associated with differences in islet expression of the IL-1 receptor type 1(IL-1RI) or interferon regulating factor 1 (IRF-1), and/or caused differences in HSP70 expression, a marker of cell defence against oxidative stress., Methods: isolated islets from both rat strains were exposed to increasing concentrations of IL-1beta (0-150 pg/ml) for 24 hours or for varying culture periods (4-48 hours) to 15 pg/ml of IL-1beta., Measurements: accumulated insulin and nitrite release into incubation medium, and islet mRNA and protein expression of iNOS, IL-1RI, IRF-1 and HSP70 by semi-quantitative RT-PCR and Western blotting., Results: Higher insulin and lower nitrite release into the incubation medium were seen for BN compared to WK rats islets in both dose- and time-response experiments. IRF-1 expression preceded iNOS expression and was more pronounced in WK than in BN islets. No strain differences were observed for islet expression of IL-1RI. A strain-dependent HSP70 expression in response to IL-1beta with the highest levels in WK rat islets following iNOS expression was seen., Conclusion: There was a strain-dependent difference in iNOS expression which was associated with IRF-1 and HSP70 expression.

Exposure of human pancreatic islets to a mixture of cytokines induces expression of the inducible nitric oxide synthase (iNOS), impairs beta-cell function, and induces apoptosis. We performed a mutational scanning of all 27 exons of the human NOS2 gene and linkage transmission disequilibrium testing of identified NOS2 polymorphisms in a Danish nationwide type 1 diabetes mellitus (IDDM) family collection. Mutational screening was performed using PCR-amplified exons, followed by single stranded conformation polymorphism and verification of potential polymorphisms by sequencing. The transmission disequilibrium test was performed in an IDDM family material comprising 257 Danish families; 154 families were affected sibling pair families, and 103 families were simplex families. In total, 10 polymorphisms were identified in 8 exons, of which 4 were tested in the family material. A C/T single nucleotide polymorphism in exon 16 resulting in an amino acid substitution, Ser(608)Leu, showed linkage to IDDM in human leukocyte antigen DR3/4-positive affected offspring (P = 0.008; corrected P = 0.024). No other distorted transmission patterns were found for any other tested single nucleotide polymorphism or constructed haplotypes with the exception of those including data from exon 16. In conclusion, linkage of the human NOS2 gene to IDDM in a subset of patients supports a pathogenic role of nitric oxide in human IDDM.

The intracellular molecular events involved in the beta-cell death process are complex but poorly understood. Cytokines, e.g., interleukin (IL)-1beta, may play a crucial role in inducing this process. Protein synthesis is necessary for the deleterious effect of IL-1, and induction of both protective and deleterious proteins has been described. To characterize the rather complex pattern of islet protein expression in rat islets in response to IL-1, we have attempted to identify proteins of altered expression level after IL-1 exposure by 2D gel electrophoresis and mass spectrometry. Of 105 significantly changed (i.e., up- or downregulated or de novo-induced) protein spots, we obtained positive protein identification for 60 protein spots. The 60 identifications corresponded to 57 different proteins. Of these, 10 proteins were present in two to four spots, suggesting that posttranslatory modifications had occurred. In addition, 11 spots contained more than one protein. The proteins could be classified according to their function into the following groups: 1) energy transduction; 2) glycolytic pathway; 3) protein synthesis, chaperones, and protein folding; and 4) signal transduction, regulation, differentiation, and apoptosis. In conclusion, valuable information about the molecular mechanisms involved in cytokine-mediated beta-cell destruction was obtained by this approach.