Insulin VNTR allele-specific effect in type 1 diabetes depends on identity of untransmitted paternal allele. The IMDIAB Group.

The IDDM2 type 1 diabetes susceptibility locus was mapped to and identified as allelic variation at the insulin gene (INS) VNTR regulatory polymorphism. In Caucasians, INS VNTR alleles divide into two discrete size classes. Class I alleles (26 to 63 repeats) predispose in a recessive way to type 1 diabetes, while class III alleles (140 to more than 200 repeats) are dominantly protective. The protective effect may be explained by higher levels of class III VNTR-associated INS mRNA in thymus such that elevated levels of preproinsulin protein enhance immune tolerance to preproinsulin, a key autoantigen in type 1 diabetes pathogenesis. The mode of action of IDDM2 is complicated, however, by parent-of-origin effects and possible allelic heterogeneity within the two defined allele classes. We have now analysed transmission of specific VNTR alleles in 1,316 families and demonstrate that a particular class I allele does not predispose to disease when paternally inherited, suggestive of polymorphic imprinting. But this paternal effect is observed only when the father's untransmitted allele is a class III. This allelic interaction is reminiscent of epigenetic phenomena observed in plants (for example, paramutation; ref. 17) and in yeast (for example, trans-inactivation; ref. 18). If untransmitted chromosomes can have functional effects on the biological properties of transmitted chromosomes, the implications for human genetics and disease are potentially considerable.

Confirmation of novel type 1 diabetes risk loci in families.

AIMS/HYPOTHESIS: Over 50 regions of the genome have been associated with type 1 diabetes risk, mainly using large case/control collections. In a recent genome-wide association (GWA) study, 18 novel susceptibility loci were identified and replicated, including replication evidence from 2,319 families. Here, we, the Type 1 Diabetes Genetics Consortium (T1DGC), aimed to exclude the possibility that any of the 18 loci were false-positives due to population stratification by significantly increasing the statistical power of our family study. METHODS: We genotyped the most disease-predicting single-nucleotide polymorphisms at the 18 susceptibility loci in 3,108 families and used existing genotype data for 2,319 families from the original study, providing 7,013 parent-child trios for analysis. We tested for association using the transmission disequilibrium test. RESULTS: Seventeen of the 18 susceptibility loci reached nominal levels of significance (p < 0.05) in the expanded family collection, with 14q24.1 just falling short (p = 0.055). When we allowed for multiple testing, ten of the 17 nominally significant loci reached the required level of significance (p < 2.8 × 10(-3)). All susceptibility loci had consistent direction of effects with the original study. CONCLUSIONS/INTERPRETATION: The results for the novel GWA study-identified loci are genuine and not due to population stratification. The next step, namely correlation of the most disease-associated genotypes with phenotypes, such as RNA and protein expression analyses for the candidate genes within or near each of the susceptibility regions, can now proceed.

HbA1(c) and mean blood glucose show stronger associations with cardiovascular disease risk factors than do postprandial glycaemia or glucose variability in persons with diabetes: the A1C-Derived Average Glucose (ADAG) study.

AIMS: Increased glucose excursions and postprandial hyperglycaemia have been suggested as unique risk factors for cardiovascular disease (CVD) and mortality in patients with diabetes mellitus. Much of the evidence is based on a single 2 h glucose value after oral glucose tolerance testing in epidemiological studies. We examined the association between various indices of glycaemia measured during everyday activities and metabolic CVD risk factors in the A1C-Derived Average Glucose (ADAG) study. METHODS: Participants (268 with type 1 diabetes, 159 with type 2 diabetes) completed 16 weeks of intensive continuous glucose monitoring (CGM) and self-monitoring of blood glucose (SMBG). From these data, common indices of postprandial glycaemia, overall hyperglycaemia, glucose variability and HbA1(c) were derived. The associations between glycaemic indices and known CVD risk factors (lipids, high-sensitivity C-reactive protein and blood pressure) were explored in linear regression models. RESULTS: For both diabetes types, the overall strongest associations with CVD risk factors were seen for the measures of average glycaemia (mean blood glucose and HbA1(c)). Associations between self-monitored postprandial and fasting glucose and CVD risk factors were weaker, but significant. Measurements of blood glucose variability showed non-significant associations. Overall, calculations based on CGM were not more informative than those based on frequent SMBG. CONCLUSIONS/INTERPRETATION: Mean glycaemia and HbA1(c) show consistent and stronger associations with CVD risk factors than fasting glucose or postprandial glucose levels or measures of glucose variability in patients with diabetes.

Real-life glycaemic profiles in non-diabetic individuals with low fasting glucose and normal HbA1c: the A1C-Derived Average Glucose (ADAG) study.

AIMS/HYPOTHESIS: Real-life glycaemic profiles of healthy individuals are poorly studied. Our aim was to analyse to what extent individuals without diabetes exceed OGTT thresholds for impaired glucose tolerance (IGT) and diabetes. METHODS: In the A1C-Derived Average Glucose (ADAG) study, 80 participants without diabetes completed an intensive glucose monitoring period of 12 weeks. From these data, we calculated the average 24 h glucose exposure as time spent above different plasma glucose thresholds. We also derived indices of postprandial glucose levels, glucose variability and HbA(1c). RESULTS: We found that 93% of participants reached glucose concentrations above the IGT threshold of 7.8 mmol/l and spent a median of 26 min/day above this level during continuous glucose monitoring. Eight individuals (10%) spent more than 2 h in the IGT range. They had higher HbA(1c), fasting plasma glucose (FPG), age and BMI than those who did not. Seven participants (9%) reached glucose concentrations above 11.1 mmol/l during monitoring. CONCLUSIONS/INTERPRETATION: Even though the non-diabetic individuals monitored in the ADAG study were selected on the basis of a very low level of baseline FPG, 10% of these spent a considerable amount of time at glucose levels considered to be 'prediabetic' or indicating IGT. This highlights the fact that exposure to moderately elevated glucose levels remains under-appreciated when individuals are classified on the basis of isolated glucose measurements.

Overview of the Type I Diabetes Genetics Consortium.

The Type I Diabetes Genetics Consortium (T1DGC) is an international, multicenter research program with two primary goals. The first goal is to identify genomic regions and candidate genes whose variants modify an individual's risk of type I diabetes (T1D) and help explain the clustering of the disease in families. The second goal is to make research data available to the research community and to establish resources that can be used by, and that are fully accessible to, the research community. To facilitate the access to these resources, the T1DGC has developed a Consortium Agreement (http://www.t1dgc.org) that specifies the rights and responsibilities of investigators who participate in Consortium activities. The T1DGC has assembled a resource of affected sib-pair families, parent-child trios, and case-control collections with banks of DNA, serum, plasma, and EBV-transformed cell lines. In addition, both candidate gene and genome-wide (linkage and association) studies have been performed and displayed in T1DBase (http://www.t1dbase.org) for all researchers to use in their own investigations. In this supplement, a subset of the T1DGC collection has been used to investigate earlier published candidate genes for T1D, to confirm the results from a genome-wide association scan for T1D, and to determine associations with candidate genes for other autoimmune diseases or with type II diabetes that may be involved with beta-cell function.

Current status and the future for the genetics of type I diabetes.

The Type I Diabetes Genetics Consortium (T1DGC) is an international collaboration whose primary goal is to identify genes whose variants modify an individual's risk of type I diabetes (T1D). An integral part of the T1DGC's mission is the establishment of clinical and data resources that can be used by, and that are fully accessible to, the T1D research community (http://www.t1dgc.org). The T1DGC has organized the collection and analyses of study samples and conducted several major research projects focused on T1D gene discovery: a genome-wide linkage scan, an intensive evaluation of the human major histocompatibility complex, a detailed examination of published candidate genes, and a genome-wide association scan. These studies have provided important information to the scientific community regarding the function of specific genes or chromosomal regions on T1D risk. The results are continually being updated and displayed (http://www.t1dbase.org). The T1DGC welcomes all investigators interested in using these data for scientific endeavors on T1D. The T1DGC resources provide a framework for future research projects, including examination of structural variation, re-sequencing of candidate regions in a search for T1D-associated genes and causal variants, correlation of T1D risk genotypes with biomarkers obtained from T1DGC serum and plasma samples, and in-depth bioinformatics analyses.

Reproducible association with type 1 diabetes in the extended class I region of the major histocompatibility complex.

The high-risk human leukocyte antigen (HLA)-DRB1, DQA1 and DQB1 alleles cannot explain the entire type 1 diabetes (T1D) association observed within the extended major histocompatibility complex. We have earlier identified an association with D6S2223, located 2.3 Mb telomeric of HLA-A, on the DRB1(*)03-DQA1(*)0501-DQB1(*)0201 haplotype, and this study aimed to fine-map the associated region also on the DRB1(*)0401-DQA1(*)03-DQB1(*)0302 haplotype, characterized by less extensive linkage disequilibrium. To exclude associations secondary to DRB1-DQA1-DQB1 haplotypes, 205 families with at least one parent homozygous for these loci, were genotyped for 137 polymorphisms. We found novel associations on the DRB1(*)0401-DQA1(*)03-DQB1(*)0302 haplotypic background with eight single nucleotide polymorphisms (SNPs) located within or near the PRSS16 gene. In addition, association at the butyrophilin (BTN)-gene cluster, particularly the BTN3A2 gene, was observed by multilocus analyses. We replicated the associations with SNPs in the PRSS16 region and, albeit weaker, to the BTN3A2 region, in an independent material of 725 families obtained from the Type 1 Diabetes Genetics Consortium. It is important to note that these associations were independent of the HLA-DRB1-DQA1-DQB1 genes, as well as of associations observed at HLA-A, -B and -C. Taken together, our results identify PRSS16 and BTN3A2, two genes thought to play important roles in regulating the immune response, as potentially novel susceptibility genes for T1D.

Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans.

Activated mononuclear cells appear to be important effector cells in autoimmune beta cell destruction leading to insulin-dependent (type 1) diabetes mellitus. Conditioned medium from activated mononuclear cells (from human blood) is cytotoxic to isolated rat and human islets of Langerhans. This cytotoxic activity was eliminated from crude cytokine preparations by adsorption with immobilized, purified antibody to interleukin-1 (IL-1). The islet-inhibitory activity and the IL-1 activity (determined by its comitogenic effect on thymocytes) were recovered by acid wash. Purified natural IL-1 and recombinant IL-1 derived from the predominant pI 7 form of human IL-1, consistently inhibited the insulin response. The pI 6 and pI 5 forms of natural IL-1 were ineffective. Natural and recombinant IL-1 exhibited similar dose responses in their islet-inhibitory effect and their thymocyte-stimulatory activity. Concentrations of IL-1 that inhibited islet activity were in the picomolar range. Hence, monocyte-derived pI 7 IL-1 may contribute to islet cell damage and therefore to the development of insulin-dependent diabetes mellitus.

Functional SOCS1 polymorphisms are associated with variation in obesity in whites.

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.

Fine mapping of a region on chromosome 21q21.11-q22.3 showing linkage to type 1 diabetes.

BACKGROUND: Results of a Scandinavian genome scan in type 1 diabetes mellitus (T1D) have recently been reported. Among the novel, not previously reported chromosomal regions showing linkage to T1D was a region on chromosome 21. OBJECTIVE: To fine map this region on chromosome 21. METHODS AND RESULTS: The linked region was initially narrowed by linkage analysis typing microsatellite markers. Linkage was significantly increased, with a peak NPL score of 3.61 (p = 0.0002), suggesting the presence of one or several T1D linked genes in the region. The support interval for linkage of 6.3 Mb was then studied by linkage disequilibrium (LD) mapping with gene based single nucleotide polymorphisms (SNPs). Thirty two candidate genes were identified in this narrowed region, and LD mapping was carried out with SNPs in coding regions (cSNPs) of all these genes. However, none of the SNPs showed association to T1D in the complete material, whereas some evidence for association to T1D of variants of the TTC3, OLIG2, KCNE1, and CBR1 genes was observed in conditioned analyses. The disease related LD was further assessed by a haplotype based association study, in which several haplotypes showed distorted transmission to diabetic offspring, substantiating a possible T1D association of the region. CONCLUSIONS: Although a single gene variant responsible for the observed linkage could not be identified, there was evidence for several combinations of markers, and for association of markers in conditioned analyses, supporting the existence of T1D susceptibility genes in the region.

Restriction fragment length polymorphism of the insulin gene in diabetes mellitus.

Variant DNA sequences flanking the human insulin gene were found in the Danish population using restriction endonucleases (restriction fragment length polymorphism). The frequencies of these DNA sequences were determined in 47 non-insulin-dependent diabetics and 93 control individuals. We report an association between a restriction fragment length polymorphism of the insulin gene and NIDDM.

Direct effects of cyclosporin A on human pancreatic beta-cells.

Cyclosporin A (CyA) may induce clinical remission in newly diagnosed insulin-dependent diabetes mellitus patients. Recently, however, adverse effects of high doses of CyA on rodent islets have been reported in vivo and in vitro. The possible direct effects of CyA on the human endocrine pancreas were therefore evaluated. Islets isolated from eight necrokidney donors were cultured in the presence of a therapeutically relevant dose of CyA, i.e., 100 ng/ml. During a 5-day culture period the release of insulin was reduced by 36% (range 7-61%), whereas the islets' content of insulin was increased by 59% (range 3-268%). The glucagon content was not affected. Cyclosporin G inhibited the insulin release, whereas dihydrocyclosporin D had no consistent effects. Glucose-stimulated insulin release from perifused islets was markedly depressed in CyA-treated islets. This effect was not fully reversed 48 h after removal of the drug. We concluded that CyA has a direct inhibitory effect on insulin release from human pancreatic islets with a concomitant increase in the residual insulin content. If applicable to the in vivo condition, CyA may therefore, in addition to its immunosuppressive effect, have direct effects on the endocrine pancreas, which may be relevant for clinical application of the drug.

Declining incidence of persistent proteinuria in type I (insulin-dependent) diabetic patients in Denmark.

The decreasing relative mortality among type I (insulin-dependent) diabetic patients during the last 50 years might be related to the incidence of clinical diabetic nephropathy. We therefore followed 2890 type I diabetic patients (1607 males and 1283 females) diagnosed between 1933 and 1972 and before the age of 31, from admission to death, emigration, or January 1, 1984. All patients had been admitted to the Steno Memorial Hospital. Information on development of proteinuria was obtained in 2658 patients (92%). Five hundred twenty-five patients developed proteinuria due to diabetes and 49 developed nondiabetic proteinuria. When comparing patients diagnosed between 1933 and 1942 with those diagnosed between 1953 and 1962, the incidence of proteinuria decreased by 30% (P less than .03). This might explain the decrease of relative mortality in type I diabetic patients. The incidence decreased with increasing age at onset but was always highest in males. Insulin dose (U/kg) and diabetes duration at admission did not influence the incidence of proteinuria. The incidence peaked after 15-17 yr of diabetes duration independent of sex, age at diagnosis, or calendar year of diagnosis. However, the majority of patients did not develop proteinuria during 40 yr of diabetes. This suggests individual renal susceptibility to the deleterious effect of hyperglycemia.

HLA, islet cell antibodies, and types of diabetes mellitus.

Insulin-dependent diabetes mellitus, in contrast to non-insulin-dependent diabetes mellitus, is associated with HLA factors B8, BW15, and B18. Recent studies have shown the association to be even stronger with HLA, DW3, and DW4 and have produced evidence for the existence of two "diabetogenic" genes predisposing to insulin-dependent diabetes in different ways. Evidence to suggest the existence of a gene--associated with DW2--that protects against the disease is accumulating. Islet cell antibodies are a feature of insulin-dependent diabetes mellitus and can be seen, in most cases, at the time of diagnosis.

Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes.

Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated beta-cell destruction. In insulin-producing RINm5F cells, overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined whether overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Zn superoxide dismutase (SOD) can provide protection for bioengineered RINm5F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F control cells to interleukin-1beta (IL-1beta) alone or a combination of IL-1beta, tumor necrosis factor-alpha, and gamma-interferon resulted in a time- and concentration-dependent decrease of cell viability in the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1beta alone caused only a moderate reduction of viability in the range of 25%, the cytokine mixture induced a significant loss of viability of >75%. This increased toxicity of the cytokine mixture compared with that of IL-1beta alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic synergism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn SOD protected against toxicity of the cytokine mixture but not against that of IL-1beta alone. The reduction of cytokine-mediated toxicity was evident also because of an increased proliferation rate and a drastic decrease in the cell death rate. The improved antioxidant defense status did not prevent the activation of iNOS after cytokine exposure. However, RINm5F cells overexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn SOD overexpression was apparently because of an inactivation of ROS generated in the signal cascades of the cytokines. Overexpression of cytoprotective enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.

Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1 beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide.

Interleukin (IL)-1 beta-mediated damage to beta-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 beta-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 beta has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 beta-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 beta-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 beta, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 beta-, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 beta-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 beta +/- nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial beta-cell destruction in insulin-dependent diabetes mellitus.

Glutamic acid decarboxylase (GAD65) autoantibodies in prediction of beta-cell function and remission in recent-onset IDDM after cyclosporin treatment. The Canadian-European Randomized Control Trial Group.

We have investigated whether glutamic acid decarboxylase (GAD) autoantibodies (GAD65 Ab) were affected by cyclosporin therapy and were related to subsequent non-insulin-requiring remission and loss of glucagon-stimulated C-peptide response in 132 recent-onset insulin-dependent diabetes mellitus (IDDM) patients treated with cyclosporin or placebo for 12 months. GAD65 Ab were detected in a quantitative radioligand assay using as tracer recombinant, in vitro translated, human islet [35S]methionine-labeled GAD65. GAD65 Ab were found at onset in 66% (87 of 132) of IDDM patients and in 1% (1 of 100) of healthy control subjects. The prevalence of GAD65 Ab and median GAD65 Ab levels did not change in serum samples taken 3, 6, 9, and 12 months after study entry in either the cyclosporin- or the placebo-treated groups. The presence or absence of GAD65 Ab at study entry did not predict non-insulin-requiring remission in either cyclosporin- or placebo-treated patients. However, the relative (compared with 0 months) glucagon-stimulated C-peptide response was more than 30% lower in GAD65 Ab+ patients receiving placebo at 9 and 12 months compared with the GAD65 Ab- placebo patients (P < 0.035). Islet cell cytoplasmic antibody (ICA) and GAD65 Ab+ placebo-treated patients showed no significant differences in stimulated C-peptide levels compared with those who were ICA- and GAD65 Ab+, suggesting that ICA was not independently associated with loss of beta-cell function.(ABSTRACT TRUNCATED AT 250 WORDS)

Onset of type 1 diabetes: a dynamical instability.

Type 1 diabetes is a disease characterized by progressive loss of beta-cell function due to an autoimmune reaction affecting the islets of Langerhans. It is now generally accepted that cytokines are implicated in the pathogenesis of autoimmune diseases. Animal studies have shown that interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma affect type 1 diabetes development profoundly. It has been suggested that beta-cells are destroyed by cytokine-induced free radical formation before cytotoxic T-helper (Th)-lymphocytes and/or autoantibody-mediated cytolysis. This hypothesis is known as the "Copenhagen model." We introduce a mathematical model encompassing the various processes within this framework. The model is expressed in rate equations describing the changes in numbers of beta-cells, macrophages, and Th-lymphocytes. Being concerned with the earliest events, we explore the conditions necessary to maintain self-sustained beta-cell elimination based on the feedback between immune cells and insulin-producing cells. The motivation for this type of analysis becomes clear when we consider the multifactorial and complicated nature of the disease. Indeed, recent research has provided detailed information about the different factors that contribute to the development of the disease, stressing the importance of incorporating these findings into a more general picture. A mathematical formalism allows for a more comprehensive description of the biological problem and can reveal nonintuitive properties of the dynamics. Despite the rather complicated structure of the equations, our main conclusion is simple: onset of type 1 diabetes is due to a collective, dynamical instability, rather than being caused by a single etiological factor.

Beta-cell maturation leads to in vitro sensitivity to cytotoxins.

Pancreatic beta-cells are more sensitive to several toxins (e.g., streptozotocin, alloxan, cytokines) than the other three endocrine cell types in the islets of Langerhans. Cytokine-induced free radicals in beta-cells may be involved in beta-cell-specific destruction in type 1 diabetes. To investigate if this sensitivity represents an acquired trait during beta-cell maturation, we used two in vitro cultured cell systems: 1) a pluripotent glucagon-positive pre-beta-cell phenotype (NHI-glu) that, after in vivo passage, matures into an insulin-producing beta-cell phenotype (NHI-ins) and 2) a glucagonoma cell-type (AN-glu) that, after stable transfection with pancreatic duodenal homeobox factor-1 (PDX-1), acquires the ability to produce insulin (AN-ins). After exposure to interleukin (IL)-1beta, both of the insulin-producing phenotypes were significantly more susceptible to toxic effects than their glucagon-producing counterparts. Nitric oxide (NO) production was induced in both NHI phenotypes, and inhibition with 0.5 mmol/l N(G)-monomethyl-L-arginine (NMMA) fully protected the cells. In addition, maturation into the NHI-ins phenotype was associated with an acquired dose-dependent sensitivity to the toxic effect of streptozotocin. Our results support the hypothesis that the exquisite sensitivity of beta-cells to IL-1beta and streptozotocin is an acquired trait during beta-cell maturation. These two cell systems will be useful tools for identification of molecular mechanisms involved in beta-cell maturation and sensitivity to toxins in relation to type 1 diabetes.

Nicotinamide prevents interleukin-1 effects on accumulated insulin release and nitric oxide production in rat islets of Langerhans.

Nicotinamide (NA) prevents macrophage- and interleukin-1 (IL-1)-mediated beta-cell damage in vitro as well as diabetes development in animal models of insulin-dependent diabetes mellitus (IDDM). IL-1 beta-mediated inhibition of insulin release and damage to beta-cells are associated with intracellular production of nitric oxide (NO) radicals. Therefore, we studied whether NA prevented IL-1 beta-induced islet NO production, measured as nitrite release from isolated rat islets, and, if so, whether this action was associated with prevention of IL-1 beta-mediated inhibition of insulin release. NA dose- and time-dependently inhibited and delayed IL-1 beta-induced islet NO production. Light microscopy detected that 25 mM of NA protected against IL-1 beta-induced islet damage. Five to 50 mM of NA dose-dependently reduced inhibition of accumulated islet insulin release induced by 150 pg/ml of IL-1 beta. NA was not able to reverse the reduced ability of IL-1 beta-treated islets to respond to an acute glucose challenge. NO or nitrite did not interact directly with NA, because NA did not reduce sodium nitroprusside-generated nitrite. No-synthase inhibition with L-arginine depletion abolished NO production but only partially reduced IL-1 beta-induced inhibition of accumulated insulin release. Complete inhibition of IL-1 beta effects could not be obtained by adding L-arginine analogues to L-arginine-depleted medium, indicating that an NO-independent action of IL-1 beta on islet insulin release may exist.(ABSTRACT TRUNCATED AT 250 WORDS)