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Failure of pancreatic β cells to compensate for insulin resistance is a prerequisite for the development of type 2 diabetes. Sustained elevated circulating levels of free fatty acids and glucose contribute to β-cell failure. Selective inhibition of histone deacetylase ( HDAC)-3 protects pancreatic β cells against inflammatory and metabolic insults in vitro. In the present study, we tested the ability of a selective HDAC3 inhibitor, BRD3308, to reduce hyperglycaemia and increase insulin secretion in a rat model of type 2 diabetes. At diabetes onset, an ambulatory hyperglycaemic clamp was performed. HDAC3 inhibition improved hyperglycaemia over the study period without affecting weight gain. At the end of the hyperglycaemic clamp, circulating insulin levels were significantly higher in BRD3308-treated rats. Pancreatic insulin staining and contents were also significantly higher. These findings highlight HDAC3 as a key therapeutic target for β-cell protection in type 2 diabetes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Type II diabetes is caused by a failure of the pancreatic β-cells to compensate for insulin resistance leading to hyperglycaemia. There is evidence for an essential role of an increased β-cell apoptosis in type II diabetes. High glucose concentrations induce IL-1β production in human β-cells, Fas expression and concomitant apoptosis owing to a constitutive expression of FasL. FASL and FAS map to loci linked to type II diabetes and estimates of insulin resistance, respectively. We have tested two functional promoter polymorphisms, FAS-670 G>A and FASL-844C>T as well as a microsatellite in the 3′ UTR of FASL for association to type II diabetes in 549 type II diabetic patients and 525 normal-glucose-tolerant (NGT) control subjects. Furthermore, we have tested these polymorphisms for association to estimates of β-cell function and insulin resistance in NGT subjects. We found significant association to type II diabetes for the allele distribution of the FASL microsatellite (P-value 0.02, Bonferroni corrected). The FAS-670G>A was associated with homeostasis model assessment insulin resistance index and body mass index (P-values 0.02 and 0.02). We conclude that polymorphisms of FASL and FAS associate with type II diabetes and estimates of insulin resistance in Danish white subjects.Genes and Immunity (2006) 7, 316–321. doi:10.1038/sj.gene.6364300; published online 4 May 2006 [ABSTRACT FROM AUTHOR]

Aims/hypothesis: IL-1β is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1β activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1β-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45β (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study was to investigate if Gadd45b prevents IL-1β-induced beta cell MAPK signalling and apoptosis. Materials: Rat insulinoma INS-1E cells and mouse beta-TC3 cells stably expressing Gadd45b were generated. The effects of Gadd45b expression on signalling by JNK, ERK and p38 were assessed by Western blotting and kinase assays. Apoptosis rate was measured by terminal deoxynucleotidyl-mediated dUTP-biotin nick end-labelling (TUNEL) and an ELISA designed to detect apoptotic nucleosomes. Expression of endogenous Gadd45b mRNA was measured by RT-PCR. Results: In INS-1E and beta-TC3 cells, expression of Gadd45b inhibited IL-1β-induced activation of JNK and ERK, but augmented IL-1β-mediated p38 activity. IL-1β-induced nitric oxide production and decreases in insulin content and secretion were reduced by GADD45β. IL-1β-induced apoptosis was reduced by GADD45β by up to 77%. Although IL-1β stimulated the time-dependent induction of endogenous Gadd45b in INS-1E cells and rat islets, expression levels were lower in these cells than in IL-1β-exposed NIH-3T3 and 3DO T cells. Conclusions/interpretation: Inadequate induction of Gadd45b, which encodes a novel beta cell JNK and ERK inhibitor, may in part explain the pro-apoptotic response of beta cells to IL-1β. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1β, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1β activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and the nuclear factor kappa B (NFκB) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase (iNOS) and for IL-1β-mediated beta cell death. The molecular mechanisms by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFκB activation in beta cells. Materials and methods: The MAPKs ERK, p38 and JNK were inhibited by SB203580, PD98059 or Tat-JNK binding domain or by cells overexpressing the JNK binding domain. The effects of MAPK inhibition on IL-1β-induced iNOS production and kappa B inhibitor protein (IκB) degradation were examined by western blotting. NFκB DNA binding was investigated by electrophoretic mobility shift assay, while NFκB-induced gene transcription was evaluated by gene reporter assays. Results: Inhibition of the MAPKs did not affect IκB degradation or NFκB DNA binding. However, inhibition of ERK reduced NFκB-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NFκB complex seemed critical, as evaluated by amino acid mutation analysis. Conclusions/interpretation: ERK activity is required for NFκB-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NFκB. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase. [ABSTRACT FROM AUTHOR]

Subclinical, low-grade systemic inflammation has been observed in patients with type 2 diabetes and in those at increased risk of the disease. This may be more than an epiphenomenon. Alleles of genes encoding immune/inflammatory mediators are associated with the disease, and the two major environmental factors the contribute to the risk of type 2 diabetes—diet and physical activity—have a direct impact on levels of systemic immune mediators. In animal models, targeting of immune genes enhanced or suppressed the development of obesity or diabetes. Obesity is associated with the infiltration and proinflammatory activity of macrophages in adipose tissue, and immune mediators may be important regulators of insulin resistance, mitochondrial function, ectopic lipid storage and beta cell dysfunction or death. Intervention studies targeting these pathways would help to determine the contribution of an activated innate immune system to the development of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. The proinflammatory cytokine IL-1β induces apoptosis in pancreatic beta cells via pathways dependent on nuclear factor-κB (NF-κB), mitogen-activated protein kinase, and protein kinase C. We recently showed suppressor of cytokine signalling (SOCS)-3 to he a natural negative feedback regulator of IL-1β- and IFN-γ-mediated signalling in rat islets and beta cell lines, preventing their deleterious effects. However, the mechanisms underlying SOCS-3 inhibition of IL-1β signalling and prevention against apoptosis remain unknown. Methods. The effect of SOCS-3 expression on the global gene-expression profile knowing IL-1β 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-1β alone. Following 6 h of IL-1β 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-1β 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-1β-induced NF-κB-dependent proapoptotic early response genes were inhibited by SOCS-3 expression, suggesting that SOCS-3 inhibits NF-κB-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-1β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1β (IL-1β) 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-1β 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-1β exposed Wistar Furth (WF) rat islets. Methods. The 82 protein spots, that changed expression after IL-1β 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-1β 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-1β. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Type I (insulin-dependent) diabetes mellitus is the result of a T-cell regulated selective destruction of pancreatic beta cells. There is evidence that the apoptosis inducing T-cell effector, Fas ligand (FasL) could be involved in the pathogenesis of Type I diabetes, probably because FasL-mediated apoptosis is important in maintaining peripheral self-tolerance and in down-regulating an immune response. We therefore evaluated the human FasL gene FASL on chromosome 1q23 as a candidate susceptibility gene for Type I diabetes. Methods: The entire FASL (promoter, exons 1–4 and 3'UTR) was scanned for polymorphisms using single strand conformational polymorphism-heteroduplex analysis and direct sequencing. Results: We identified two novel polymorphisms, a g-C843T and a g-A475T, in a negative regulatory region of the promoter. A Danish Type I diabetes family collection of 1143 subjects comprising 257 families (420 affected and 252 unaffected offspring) was typed for the g-C843T polymorphism and for a FASL microsatellite. Haplotypes were established and data were analysed using the extended transmission disequilibrium test. Conclusion/interpretation: We found no overall evidence for linkage in the presence of association of the FASL polymorphism to Type I diabetes and conclude that FASL does not contribute to the genetic susceptibility to Type I diabetes. [Diabetologia (2002) 45: 134–139] [ABSTRACT FROM AUTHOR]

Apoptosis is likely to be the main form of beta-cell death in immune-mediated diabetes mellitus in rodents and possibly in humans. Clarification of the regulation of beta-cell death could indicate novel sites for therapeutic intervention in Type I (insulin-dependent) diabetes mellitus. We review the molecular effectors and signal transduction of immune-mediated beta-cell apoptosis. [ABSTRACT FROM AUTHOR]

Type 1 (insulin-dependent) diabetes is a complex trait. The region harboring the ICAM1 gene on 19p13 links to type 1 diabetes, and a growing body of evidence indicates that intercellular adhesion molecule-1 (ICAM-1) could play a role in type 1 diabetes development. Recently, association studies of an ICAM-1 K469E polymorphism in type 1 diabetes populations have reported conflicting results. Hence, we performed a transmission disequilibrium test analysis of the ICAM-1 K469E variations in 253 Danish type 1 diabetes families. Linkage and association was not found between the ICAM-1 K469E variation and type 1 diabetes in Danish patients (Ptdt≥0.48), and our data did not indicate an interaction between ICAM1 and IDDM1 in predisposition to type 1 diabetes in Danes (P=0.78). We did not observe significant association with late-onset type 1 diabetes (Ptdt≥0.12) or differences in transmission patterns between groups of affected offspring stratified for age at onset (P≥0.19), as suggested in Japanese patients. Combined analysis of the present and previously reported transmission data comprising 728 affected offspring of Romanian, Finnish, and Danish ancestry suggested association between the ICAM-1 E469 allele and type 1 diabetes (Ptdt=0.013), but association was not found in the combined Scandinavian material. In conclusion, we found no association of the ICAM-1 K469E polymorphism with type 1 diabetes or its subsets stratified for age at onset and HLA risk in Danish patients. Analysis of ICAM-1 K469E transmissions reported in three populations suggested association to type 1 diabetes, but also demonstrated heterogeneity between populations. [ABSTRACT FROM AUTHOR]

The effects of dietary supplementation with ω-3-polyunsaturated fatty acids (ω-3-PUFA) on the proliferative response of PBMC and on the secretion of monokines and arachidonic acid metabolites from PBMC and monocytes (Mo) from healthy subjects and patients with recent-onset insulin-dependent diabetes mellitus (IDDM) were examined. Three groups of eight to nine healthy individuals were randomized to either 2.0 g/day or 4.0 g/day of ω-3-PUFA devoid of vitamins A and D, or an isocaloric amount of placebo. Furthermore, eight patients with recentonset IDDM received 4.0 g/day of ω-3-PUFA. IL-lβ production and TNF-α secretion was determined before and after 7 weeks of treatment, and 10 weeks after withdrawal of treatment. Significant increases in platelet and PBMC membrane eicosapentaenoic acid was found in ω-3- PUFA-treated individuals. ω-3-PUFA treatment significantly reduced the content of IL-Iβ in lysates of PBMC, but did not affect PBMC or Mo secretion of IL-1β, TNF-α or prostaglandin E2 (PGE2) or PBMC leukotriene B4 (LTB4) secretion in healthy subjects or in IDDM patients. A significant inhibition of the PHA-stimulated, but not the spontaneous or PPD-stimulated, proliferative response of PBMC was observed in healthy and diabetic subjects treated with ω-3- PUFA. No correlation was found between PHA-stimulated PBMC proliferation and PBMC secretion of TNF-α and IL-lβ. There were no significant differences in the spontaneous or the PPD- or PHA-stimulated proliferative responses of PBMC between diabetic and healthy individuals at entry. We conclude that although dietary supplementation with 4.0 g/day of ω-3- PUFA inhibits the proliferation of PBMC and reduces IL-1β immunoreactivity in PBMC and Mo, it does not alter monokine, PGE2 or LTB4, secretion in healthy or IDDM subjects. [ABSTRACT FROM AUTHOR]

Examines whether CD4+ t-cells play a role in diabetes pathogenesis. T-cell response to antigen presentation; Evidence qualifying the CD4 gene as a candidate gene for diabetes; Transmission disequilibrium study; Contribution of the CD4 gene polymorphism to familial clustering.