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Aims/hypothesis: Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting., Methods: We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets., Results: Glucagon output decreased three- to fivefold within minutes of shifting from low (3.5 mmol/l) to high (10 mmol/l) glucose (4.0 ± 0.5 pmol/15 min vs 1.3 ± 0.3 pmol/15 min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells., Conclusions/interpretation: Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucose-lowering effects.

Aim/hypothesis: Combination therapy targeting the major actors involved in the immune-mediated destruction of pancreatic beta cells appears to be an indispensable approach to treat type 1 diabetes effectively. We hypothesised that the combination of an orally active pan-histone deacetylase inhibitor (HDACi: givinostat) with subtherapeutic doses of CD3 antibodies may provide ideal synergy to treat ongoing autoimmunity., Methods: NOD mice transgenic for the human CD3ε (also known as CD3E) chain (NOD-huCD3ε) were treated for recent-onset diabetes with oral givinostat, subtherapeutic doses of humanised CD3 antibodies (otelixizumab, 50 μg/day, 5 days, i.v.) or a combination of both drugs. Disease remission, metabolic profiles and autoreactive T cell responses were analysed in treated mice., Results: We demonstrated that givinostat synergised with otelixizumab to induce durable remission of diabetes in 80% of recently diabetic NOD-huCD3ε mice. Remission was obtained in only 47% of mice treated with otelixizumab alone. Oral givinostat monotherapy did not reverse established diabetes but reduced the in situ production of inflammatory cytokines (IL-1β, IL-6, TNF-α). Importantly, the otelixizumab + givinostat combination strongly improved the metabolic status of NOD-huCD3ε mice; the mice recovered the capacity to appropriately produce insulin, control hyperglycaemia and sustain glucose tolerance. Finally, diabetes remission induced by the combination therapy was associated with a significant reduction of insulitis and autoantigen-specific CD8 + T cell responses., Conclusions/interpretation: HDACi and low-dose CD3 antibodies synergised to abrogate in situ inflammation and thereby improved pancreatic beta cell survival and metabolic function leading to long-lasting diabetes remission. These results support the therapeutic potential of protocols combining these two drugs, both in clinical development, to restore self-tolerance and insulin independence in type 1 diabetes.

Aims/hypothesis: Impairment of beta cell mass and function is evident in both type 1 and type 2 diabetes. In healthy physiological conditions pancreatic beta cells adapt to the body's increasing insulin requirements by proliferation and improved function. We hypothesised that during the development of diabetes, there is an increase in the expression of inhibitory factors that prevent the beta cells from adapting to the increased need for insulin. We evaluated the effects of bone morphogenetic protein (BMP) 2 and -4 on beta cells., Methods: The effects of BMP2 and -4 on beta cell proliferation, apoptosis, gene expression and insulin release were studied in isolated islets of Langerhans from rats, mice and humans. The expression of BMPs was analysed by immunocytochemistry and real-time PCR. The role of endogenous BMP was investigated using a soluble and neutralising form of the BMP receptor 1A., Results: BMP2 and -4 were found to inhibit basal as well as growth factor-stimulated proliferation of primary beta cells from rats and mice. Bmp2 and Bmp4 mRNA and protein were expressed in islets and regulated by inflammatory cytokines. Neutralisation of endogenous BMP activity resulted in enhanced proliferation of rodent beta cells. The expression of Id mRNAs was induced by BMP4 in rat and human islets. Finally, glucose-induced insulin secretion was significantly impaired in rodent and human islets pre-treated with BMP4, and inhibition of BMP activity resulted in enhanced insulin release., Conclusions/interpretation: These data show that BMP2 and -4 exert inhibitory actions on beta cells in vitro and suggest that BMPs exert regulatory roles of beta cell growth and function.

Aims/hypothesis: Cytokines may promote or inhibit disease progression in type 1 diabetes. We investigated whether systemic proinflammatory, anti-inflammatory and regulatory cytokines associated differently with fasting and meal-stimulated beta cell function in patients with longer term type 1 diabetes., Methods: The beta cell function of 118 patients with type 1 diabetes of duration of 0.75-4.97 years was tested using a standardised liquid mixed meal test (MMT). Serum samples obtained at -5 to 120 min were analysed by multiplex bead-based technology for proinflammatory (IL-6, TNF-α), anti-inflammatory (IL-1 receptor antagonist [IL-1RA]) and regulatory (IL-10, TGF-β1-3) cytokines, and by standard procedures for C-peptide. Differences in beta cell function between patient groups were assessed using stepwise multiple regression analysis adjusting for sex, age, duration of diabetes, BMI, HbA1c and fasting blood glucose., Results: High fasting systemic concentrations of the proinflammatory cytokines IL-6 and TNF-α were associated with increased fasting and stimulated C-peptide concentrations even after adjustment for confounders (p < 0.03). Interestingly, increased concentrations of anti-inflammatory/regulatory IL-1RA, IL-10, TGF-β1 and TGF-β2 were associated with lower fasting and stimulated C-peptide levels (p < 0.04), losing significance on adjustment for anthropometric variables. During the MMT, circulating concentrations of IL-6 and TNF-α increased (p < 0.001) while those of IL-10 and TGF-β1 decreased (p < 0.02) and IL-1RA and TGF-β2 remained unchanged., Conclusions/interpretation: The association between better preserved beta cell function in longer term type 1 diabetes and increased systemic proinflammatory cytokines and decreased anti-inflammatory and regulatory cytokines is suggestive of ongoing inflammatory disease activity that might be perpetuated by the remaining beta cells. These findings should be considered when designing immune intervention studies aimed at patients with longer term type 1 diabetes and residual beta cell function.

Aims/hypothesis: Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1-11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs mediate inflammatory beta cell damage and how the islet content of these HDACs is regulated in recent-onset type 1 diabetes., Methods: The rat beta cell line INS-1 and dispersed primary islets from rats, either wild type or HDAC1-3 deficient, were exposed to cytokines and HDACi. Molecular mechanisms were investigated using real-time PCR, chromatin immunoprecipitation and ELISA assays. Pancreases from healthy children and children with type 1 diabetes were assessed using immunohistochemistry and immunofluorescence., Results: Screening of 19 compounds with different HDAC selectivity revealed that inhibitors of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine-induced iNos expression but not with altered expression of the pro-inflammatory mediators Il1α, Il1β, Tnfα or Cxcl2. HDAC3 knock-down reduced nuclear factor κB binding to the iNos promoter and HDAC1 knock-down restored insulin secretion. In pancreatic sections from children with type 1 diabetes of recent onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls., Conclusions/interpretation: These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection of beta cell mass and function in clinical islet transplantation and recent-onset type 1 diabetic patients.

Aims/hypothesis: Cytokine-induced beta cell toxicity is abrogated by non-selective inhibitors of lysine deacetylases (KDACs). The KDAC family consists of 11 members, namely histone deacetylases HDAC1 to HDAC11, but it is not known which KDAC members play a role in cytokine-mediated beta cell death. The aim of the present study was to examine the KDAC gene expression profile of the beta cell and to investigate whether KDAC expression is regulated by cytokines. In addition, the protective effect of the non-selective KDAC inhibitor ITF2357 and interdependent regulation of four selected KDACs were investigated., Methods: The beta cell line INS-1 and intact rat and human islets were exposed to cytokines with or without ITF2357. Expression of mRNA was assessed by real-time PCR and selected targets validated at the protein level by immunoblotting. Effects on cytokine-induced toxicity were investigated by in vitro assays., Results: Hdac1 to Hdac11 were expressed and differentially regulated by cytokines in INS-1 cells and rat islets. HDAC1, -2, -6 and -11 were found to be expressed and regulated by cytokines in human islets. ITF2357 protected against cytokine-induced beta cell apoptosis and counteracted cytokine-induced attenuation of basal insulin secretion. In addition, cytokine-induced regulation of Hdac2 and Hdac6, but not Hdac1 and Hdac11, was reduced by KDAC inhibition., Conclusions/interpretation: All classical KDAC genes are expressed by beta cells and differentially regulated by cytokines. Based on the relative expression levels and degree of regulation by cytokines, we propose that HDAC1, -2, -6 and -11 are of particular importance for beta cell function. These observations may help in the design of specific KDAC inhibitors to prevent beta cell destruction in situ and in islet grafts.

Aims/hypothesis: Endothelial progenitor cells (EPC) augment vascular repair and neovascularisation. Patients with type 2 diabetes have reduced EPC and increased risk of cardiovascular disease (CVD), which is reduced by multifactorial intervention. Our aim, therefore, was to evaluate in type 2 diabetic patients whether the numbers of EPC derived from peripheral blood mononuclear cells is influenced by a multifactorial treatment strategy., Methods: We enrolled 28 patients newly referred for initiation of multifactorial treatment, which consisted of improving glycaemic, lipid and blood pressure control, as well as antithrombotic therapy and lifestyle modification. EPC count was assessed by in vitro cultures at baseline and after 90 days of treatment. After 7 days in culture, we identified EPC by fluorescent staining of attached cells. Patients were treated with metformin, aspirin, statins and angiotensin II receptor blockers, and divided accordingly into groups of mono-, dual-, triple- or quadruple therapy., Results: After 90 days of treatment, glycaemic control improved and total cholesterol decreased. Multifactorial intervention for 90 days significantly increased EPC count in cultures by 35% (from 105 [SE 8] to 140 [11] cells per field [p = 0.002]). The change in EPC among patients with quadruple therapy was higher (63%) than in untreated patients (-32%, p = 0.043)., Conclusions/interpretation: Numbers of EPC derived from peripheral blood mononuclear cells increased significantly after multifactorial intervention in type 2 diabetic patients. It remains to be shown whether these changes contribute to the beneficial effects of multifactorial intervention on diabetic micro- and macroangiopathy.

The recent major increase in the global incidence of type 2 diabetes suggests that most cases of this disease are caused by changes in environment and lifestyle. All major risk factors for type 2 diabetes (overnutrition, low dietary fibre, sedentary lifestyle, sleep deprivation and depression) have been found to induce local or systemic low-grade inflammation that is usually transient or milder in individuals not at risk for type 2 diabetes. By contrast, inflammatory responses to lifestyle factors are more pronounced and prolonged in individuals at risk of type 2 diabetes and appear to occur also in the pancreatic islets. Chronic low-grade inflammation will eventually lead to overt diabetes if counter-regulatory circuits to inflammation and metabolic stress are compromised because of a genetic and/or epigenetic predisposition. Hence, it is not the lifestyle change per se but a deficient counter-regulatory response in predisposed individuals which is crucial to disease pathogenesis. Novel approaches of intervention may target these deficient defence mechanisms.

Aims/hypothesis: Chemokines recruit activated immune cells to sites of inflammation and are important mediators of insulitis. Activation of the pro-apoptotic receptor Fas leads to apoptosis-mediated death of the Fas-expressing cell. The pro-inflammatory cytokines IL-1beta and IFN-gamma regulate the transcription of genes encoding the Fas receptor and several chemokines. We have previously shown that suppressor of cytokine signalling (SOCS)-3 inhibits IL-1beta- and IFN-gamma-induced nitric oxide production in a beta cell line. The aim of this study was to investigate whether SOCS-3 can influence cytokine-induced Fas and chemokine expression in beta cells., Methods: Using a beta cell line with inducible Socs3 expression or primary neonatal rat islet cells transduced with a Socs3-encoding adenovirus, we employed real-time RT-PCR analysis to investigate whether SOCS-3 affects cytokine-induced chemokine and Fas mRNA expression. The ability of SOCS-3 to influence the activity of cytokine-responsive Fas and Mcp-1 (also known as Ccl2) promoters was measured by reporter analysis., Results: IL-1beta induced a time-dependent increase in Mcp-1 and Mip-2 (also known as Cxcl2) mRNA expression after 6 h of stimulation in insulinoma (INS)-1 and neonatal rat islet cells. This induction was inhibited when Socs3 was expressed in the cells. In INS-1 cells, IL-1beta + IFN-gamma induced a tenfold and eightfold increase of Fas mRNA expression after 6 and 24 h, respectively. This induction was inhibited at both time-points when expression of Socs3 was induced. In promoter studies SOCS-3 significantly inhibited the cytokine-induced activity of Mcp-1 and Fas promoter constructs., Conclusions/interpretation: SOCS-3 inhibits the expression of cytokine-induced chemokine and death-receptor Fas mRNA.

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.

Aims/hypothesis: The aim of the study was to determine whether purified beta cells can replicate in vitro and whether this is enhanced by extracellular matrix (ECM) and growth factors., Methods: Human beta cells were purified by FACS by virtue of their high zinc content using Newport Green, and excluding ductal and dead cells. Rat beta cells were sorted by autofluorescence or using the same method developed for human cells. Cells were plated on poly-L-lysine or ECMs from rat or human bladder carcinoma cells or bovine corneal ECM and incubated in the presence of BrdU with or without growth factors., Results: The newly developed method for sorting human beta cells yields a population containing 91.4 +/- 2.8% insulin-positive cells with a low level of spontaneous apoptosis and a robust secretory response to glucose. Beta cells from 8-week-old rats proliferated in culture and this was increased by ECM. Among growth factors, only human growth hormone (hGH) and the glucagon-like peptide-1 analogue liraglutide enhanced proliferation of rat beta cells, with a significant increase on both poly-L-lysine and ECM. By contrast, sorted adult human beta cells from 16 donors aged 48.9 +/- 14.3 years (range 16-64 years) failed to replicate demonstrably in vitro regardless of the substratum or growth factors used., Conclusions/interpretation: These findings indicate that, in our conditions, the fully differentiated human adult insulin-producing beta cell was unable to proliferate in vitro. This has important implications for any attempt to expand cells from pancreases of donors of this age group. By contrast, the rat beta cells used here were able to divide in vitro, and this was enhanced by ECM, hGH and liraglutide.

Aims/hypothesis: The immune-mediated elimination of pancreatic beta cells in type 1 diabetes involves release of cytotoxic cytokines such as IL-1beta and IFNgamma, which induce beta cell death in vitro by mechanisms that are both dependent and independent of nitric oxide (NO). Nuclear factor kappa B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone deacetylases (HDAC), and positive effects of HDAC inhibition have been obtained in several inflammatory diseases. Thus, the aim of this study was to investigate whether HDAC inhibition protects beta cells against cytokine-induced toxicity., Materials and Methods: The beta cell line, INS-1, or intact rat islets were precultured with HDAC inhibitors suberoylanilide hydroxamic acid or trichostatin A in the absence or presence of IL-1beta and IFNgamma. Effects on insulin secretion and NO formation were measured by ELISA and Griess reagent, respectively. iNOS levels and NFkappaB activity were measured by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone-DNA complex ELISA., Results: HDAC inhibition reduced cytokine-mediated decrease in insulin secretion and increase in iNOS levels, NO formation and apoptosis. IL-1beta induced a bi-phasic phosphorylation of inhibitor protein kappa Balpha (IkappaBalpha) with the 2nd peak being sensitive to HDAC inhibition. No effect was seen on IkappaBalpha degradation and NFkappaB DNA binding., Conclusions/interpretation: HDAC inhibition prevents cytokine-induced beta cell apoptosis and impaired beta cell function associated with a downregulation of NFkappaB transactivating activity.

Aims/hypothesis: IL-1beta released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1beta-induced pro-apoptotic signalling in beta cells. Pancreatic beta cells express NCAM, but its biological effects in these cells are unclear. The aim of this study was to investigate whether there is cross-talk between NCAM signalling and cytokine-induced pro-apoptotic signalling., Materials and Methods: Western blotting was used to investigate levels of NCAM and inducible nitric oxide synthase, phosphorylation of Src and MAPKs, and cleavage of caspase-3. MAPK activity was investigated with an in vitro kinase assay. Apoptosis was detected by cleaved caspase-3 and a Cell Death Detection ELISA(plus) assay. NCAM-induced fibroblast growth factor receptor (FGFR) activation was investigated in NCAM(-/-) Trex293 cells where FGFR phosphorylation was measured by Western blotting after NCAM transfection., Results: Pre-exposure of INS-1E cells to the FGFR-inhibitor SU5402, but not to the Src-inhibitor PP2, dose-dependently inhibited IL-1beta-mediated MAPK activity. A synthetic peptide, C3d, reported to bind NCAM, did not activate MAPK or Akt as reported in neurons but inhibited IL-1beta-induced MAPK activity, thereby mimicking the effect of SU5402. Furthermore, C3d inhibited NCAM-induced FGFR phosphorylation and apoptosis induced by IL-1beta plus IFN-gamma, but did not affect IL-1beta-induced NF-kappaB signalling., Conclusions/interpretation: We suggest that NCAM signalling through FGFR is required for efficient IL-1beta pro-apoptotic signalling by facilitating IL-1beta-induced MAPK activation downstream of the NF-kappaB-MAPK branching point. Further, these data identify a novel function of C3d as an inhibitor of NCAM-induced FGFR activity and of IL-1beta-induced MAPK activation in beta cells.

Aims/hypothesis: IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta 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-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study was to investigate if Gadd45b prevents IL-1beta-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-1beta-induced activation of JNK and ERK, but augmented IL-1beta-mediated p38 activity. IL-1beta-induced nitric oxide production and decreases in insulin content and secretion were reduced by GADD45beta. IL-1beta-induced apoptosis was reduced by GADD45beta by up to 77%. Although IL-1beta 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-1beta-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-1beta.

Aims/hypothesis: We studied the impact of genetic versus pre- and postnatal environmental factors on the plasma levels of IL6, TNF and the soluble TNF receptor superfamily, member 1A (TNFRSF1A, previously known as TNF receptor type1 [TNFR1])., Materials and Methods: Using the hyperinsulinaemic-euglycaemic clamp, we assessed the association between cytokine levels and both peripheral insulin sensitivity and hepatic glucose production. Fasting plasma IL6, TNF and soluble TNFRSF1A levels were measured using ELISA in 55 young and 43 elderly twin pairs., Results: Plasma IL6 and TNF were influenced by genetic factors in young and elderly twins respectively, while no significant impact of genetics was found for soluble TNFRSF1A. Significant intra-twin pair correlations between birthweight and both IL6 and soluble TNFRSF1A levels were found. In addition to the effect of birthweight on IL6, age and fat % also significantly influenced IL6 levels, whereas soluble TNFRSF1A levels were significantly influenced by zygosity, age, fat % and sex. Plasma soluble TNFRSF1A was associated with higher plasma NEFA and to some extent with reduced insulin sensitivity. Plasma IL6 was associated positively with basal NEFA. TNF level was not associated with in vivo glucose or fat metabolism after correction for known confounders., Conclusions/interpretation: Plasma IL6 and soluble TNFRSF1A are influenced by the intrauterine environment. We found some evidence of a genetic component for TNF and IL6 in young and elderly twins respectively. Plasma IL6 may influence basal lipolysis, but neither plasma TNF nor IL6 levels are independently associated with hepatic or peripheral insulin action. Nevertheless, plasma soluble TNFRSF1A may play some role in the control of insulin action.

Aims/hypothesis: The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1beta, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1beta 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 (NFkappaB) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase (iNOS) and for IL-1beta-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 NFkappaB 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-1beta-induced iNOS production and kappa B inhibitor protein (IkappaB) degradation were examined by western blotting. NFkappaB DNA binding was investigated by electrophoretic mobility shift assay, while NFkappaB-induced gene transcription was evaluated by gene reporter assays., Results: Inhibition of the MAPKs did not affect IkappaB degradation or NFkappaB DNA binding. However, inhibition of ERK reduced NFkappaB-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NFkappaB complex seemed critical, as evaluated by amino acid mutation analysis., Conclusions/interpretation: ERK activity is required for NFkappaB-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NFkappaB. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase.

Aims/hypothesis: Pro-inflammatory cytokines cause beta cell secretory dysfunction and apoptosis--a process implicated in the pathogenesis of type 1 diabetes. Cytokines induce the expression of inducible nitric oxide (NO) synthase (iNOS) leading to NO production. NO contributes to cytokine-induced apoptosis, but the underlying mechanisms are unclear. The aim of this study was to investigate whether NO modulates signalling via mitogen-activated protein kinases (MAPKs) and Akt., Materials and Methods: MAPK activities in INS-1 cells and isolated islets were determined by immunoblotting and in vitro kinase assay. Apoptosis was determined by ELISA measurement of histone-DNA complexes present in cytoplasm., Results: Apoptosis in INS-1 cells induced by IL-1beta plus IFNgamma was dependent on NO production as demonstrated by the use of the NOS blocker NG-methyl-L-arginine. Accordingly, an NO donor (S-nitroso-N-acetyl-D, L-penicillamine, SNAP) dose-dependently caused apoptosis in INS-1 cells. SNAP activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but suppressed the activity of extracellular signal-regulated kinase MAPK. In rat islets, NOS inhibition decreased JNK and p38 activities induced by a 6-h exposure to IL-1beta. Likewise, IL-1beta-induced JNK and p38 activities were lower in iNOS(-/-) mouse islets than in wild-type islets. In human islets, SNAP potentiated IL-1beta-induced JNK activation. The constitutive level of active, Ser473-phosphorylated Akt in INS-1 cells was suppressed by SNAP. IGF-I activated Akt and protected against SNAP-induced apoptosis. The anti-apoptotic effect of IGF-I was not associated with reduced JNK activation., Conclusions/interpretation: We suggest that NO contributes to cytokine-induced apoptosis via potentiation of JNK activity and suppression of Akt.

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.

Aims/hypothesis: The cytokine interleukin 6 (IL-6) is an essential regulator of the acute phase response associated with insulin-resistant states including type 2 diabetes and obesity. Three polymorphisms at positions -597, -572, and -174 of the IL6 promoter have been reported to influence IL6 transcription. The aim of this study was to investigate whether the IL6 promoter polymorphisms were associated with features of the WHO-defined metabolic syndrome and related quantitative traits in 7,553 Caucasian Danes., Methods: Using analysis of PCR-generated primer extension products by mass spectrometry we examined -597 G/A, -572 G/C, and -174 G/C IL6 variants in the population-based Inter99 study cohort of middle-aged people (n=6,164) and in a group of type 2 diabetic patients (n=1,389)., Results: The -174 G/C and -597 G/A polymorphisms were in strong linkage disequilibrium (R(2)=0.95). In the Inter99 cohort the -174 G-allele was associated with insulin resistance (p<0.02) and dyslipidaemia (p<0.007) whereas the C-allele of the -572 polymorphism was associated with increased serum insulin release during an OGTT (p<0.0005). Composite genotype or haplotype analyses of all 3 IL6 promoter variants showed associations with type 2 diabetes (p<0.002), obesity (p<0.02), and the metabolic syndrome (p<0.01)., Conclusions: The present studies suggest that single-nucleotide polymorphisms and composite genotypes or haplotypes of the IL6 promoter may be associated with several features of the metabolic syndrome in Caucasians.

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.

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.

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.

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.

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. Data obtained on non-obese diabetic (NOD) mice suggest that macrophages and CD4+ T-cells are the main cellular effectors, whereas CD8+ T-cells are more important initiators of the immune process leading to beta-cell death. Perforin could be the effector molecule utilized by CD8+ T-cell initiation, whereas CD4+ mediated beta-cell destruction is mostly dependent on Fas/FasL and the cytokines IFNgamma and TNF-alpha. The macrophage cytokine IL-1beta in combination with IFN-gamma and TNF-alpha, plays an important role for beta-cell dysfunction and death. Signal transduction by these cytokines involves: (i) binding to specific receptors, (ii) signal transduction by cytosolic kinases (especially the so-called mitogen- and stress-activated protein kinases) and/or phosphatases, (iii) mobilization of diverse transcription factors - with nuclear factor kappaB (NF-kappaB), AP-1 and STAT-1 probably playing key roles for beta-cell apoptosis; (iv) up-regulation or down-regulation of gene transcription. Recent data obtained by microarray and proteomic analysis suggest that the process of beta-cell apoptosis depends on the parallel and/or sequential up-regulation and down-regulation of considerable numbers of genes, which can be grouped in gene modules or patterns according to their functions. A detailed characterization of these "gene modules", and of the signalling pathways and transcription factors regulating them could allow us to understand the ultimate mechanisms leading to beta-cell apoptosis.

Aims/hypothesis: Interleukin-1 beta (IL-1beta) in synergy with tumour necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) is cytotoxic to pancreatic beta cells. Mitogen-activated protein kinase (MAPK) activity that is induced by interleukin-1 beta has been suggested to signal nitric oxide-dependent as well as nitric oxide-independent beta-cell destructive pathways. The aim of this study was to investigate if TNFalpha and IFNgamma signal through mitogen-activated protein kinases in isolated rat islets of Langerhans and if they potentiate mitogen-activated protein kinase activity induced by IL-1beta., Methods: Islets of Langerhans were isolated from 5- to 7-day-old Wistar rats and precultured for 7 days before stimulation with IL-1beta, TNFalpha and/or IFNgamma for 20 min followed by lysis. Kinase activity was measured with a whole cell lysate kinase assay and after immunoprecipitation of the kinase using immunocomplex kinase assay., Results: Exposure to IL-1beta or TNFalpha significantly increased mitogen-activated protein kinase activity, whereas IFNgamma tended to decrease extracellular-signal-regulated kinase activity. Further, TNFalpha and IFNgamma were found to synergistically increase mitogen-activated protein kinase activity induced by IL-1beta., Conclusion/interpretation: We hypothesise that the synergistic effect of IL-1beta, TNFalpha and IFNgamma in the functional inhibition and induction of cell death in pancreatic beta cells is signalled through a synergistic activation of mitogen-activated protein kinase activity.

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 (P(tdt)> or =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 (P(tdt)> or =0.12) or differences in transmission patterns between groups of affected offspring stratified for age at onset (P> or =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 (P(tdt)=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.

Aims/hypothesis: The human Fas gene (FAS) on chromosome 10q24.1 encoding a cell surface receptor involved in apoptosis was evaluated as a candidate susceptibility gene for Type I (insulin-dependent) diabetes mellitus. Apoptosis mediated by Fas is important in maintaining peripheral self-tolerance and in down-regulating the immune response and could have a role in immune-mediated beta-cell destruction., Methods: We did a molecular scan of the entire human FAS (promoter, exons 1-9 including exon-intron boundaries and the 3'UTR) using single strand conformational polymorphism-heteroduplex analysis., Results: We identified 15 mutations, of which 11 are new. Of these a g-1194A-->T and a g-295Ains give rise to alterations of transcription-factor-binding consensus sequences for c-Myb, SP-1 and NF-kappa B, respectively. A total of 1068 people from a Danish family collection comprising 138 Type I diabetic sib-pair families (289 affected and 121 unaffected offspring) and 103 Type I diabetic parent-offspring multiplex families (103 affected and 112 unaffected offspring) were typed for the three most frequent polymorphisms with high heterozygosity indices and for a FAS microsatellite. Haplotypes were established and data analysed using the extended transmission disequilibrium test, ETDT., Conclusion/interpretation: We found no overall evidence for linkage of the FAS polymorphisms to Type I diabetes. We conclude that it is unlikely that the Fas gene does contribute to genetic susceptibility for Type I diabetes.

A model of the pathogenesis of insulin-dependent diabetes mellitus, i.e. the initial phase of beta-cell destruction, is proposed: in a cascade-like fashion efficient antigen presentation, unbalanced cytokine, secretion and poor beta-cell defence result in beta-cell destruction by toxic free radicals (O2- and nitric oxide) produced by the beta cells themselves. This entire process is under polygenetic control.

The monokines interleukin-1 alpha and -beta have been implicated as effector molecules in the immune-mediated pancreatic beta-cell destruction leading to insulin-dependent diabetes mellitus. Here we investigated the effects of interleukin-1 receptor antagonism on insulin and glucagon release of rat, mouse and human islets exposed to recombinant human interleukin-1 beta, and on interleukin-1 beta induced changes in blood glucose, serum insulin and serum glucagon levels in Wistar Kyoto rats. The interleukin-1 receptor antagonist reduced the co-mitogenic effect of interleukin-1 beta on mouse and rat thymocytes with a 50% inhibitory concentration of 10- and 100-fold molar excess, respectively. Complete inhibition was obtained with a 100-1,000-fold molar excess. However, at a 100-fold molar excess the interleukin-1 receptor antagonist did not antagonise the potentiating effect of interleukin-1 beta on rat islet insulin accumulation during 3 and 6 h of exposure or of interleukin-1 beta-induced inhibition of insulin release after 24 h. In contrast, interleukin-1 beta-stimulated islet glucagon release was completely antagonised by a 100-fold molar excess of interleukin-1 receptor antagonist. A 10,000-fold molar excess of interleukin-1 receptor antagonist was needed to antagonise interleukin-1 beta stimulatory and inhibitory effects on rat beta-cell function in vitro. A 100-fold excess of interleukin-1 receptor antagonist could not counteract interleukin-1 beta effects on mouse and human beta cells, excluding species difference in the efficacy of the human interleukin-1 receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

It is unknown whether interleukin-1 exerts a bimodal effect on Beta-cell function in vivo, and whether interleukin-1 has a diabetogenic action in normal animals. We therefore studied: (a) acute effects 2 h after an intraperitoneal bolus injection of 4 micrograms of recombinant human interleukin-1 beta per kg body weight on blood glucose, plasma levels of insulin, glucagon and corticosterone in Wistar Kyoto rats, either untreated or pre-treated with 4 micrograms/kg of interleukin-1 daily for 3 or 5 days; (b) the cumulative effects of repetitive intraperitoneal injections of 4 micrograms/kg interleukin-1 on blood glucose, glucose tolerance, plasma levels of insulin, glucagon and corticosterone, pancreatic insulin content and pancreatic ultrastructure; and (c) blood glucose and plasma concentrations of insulin, glucagon and corticosterone 10 h after the last of five intraperitoneal injections of interleukin-1, at which time point the inhibitory effect of short-term interleukin-1 exposure on insulin secretion reaches its nadir in vitro. A single injection of 4 micrograms/kg of interleukin-1 caused a slight, but significant lowering of blood glucose 2 h after interleukin-1 injection with no significant changes in plasma insulin and in spite of increases in plasma glucagon and corticosterone. A lowering of blood glucose 2 h after interleukin-1 administration was reproduced with 40, but not 0.4 micrograms/kg of interleukin-1, and was also seen in interleukin-1 pre-treated rats. Two hours after the fifth injection of interleukin-1, intraperitoneal glucose tolerance was impaired with elevated plasma insulin and corticosterone levels and increased pancreatic insulin content, indicating a state of insulin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

We recently reported a potentiating effect of recombinant human interleukin-1 beta on glucose-stimulated insulin release from the isolated perfused pancreas. With the aim of determining whether the stimulatory effect of recombinant interleukin-1 beta on the B cell in the intact gland was modulated by varying the concentration, time of exposure to recombinant interleukin-1 beta or B-cell activity, and to elucidate a possible mechanism of action, we measured in the perfused rat pancreas the release of insulin, glucagon and/or prostaglandin E2 according to the following three different protocols: (1) perfusion with 20 ng/ml of recombinant interleukin-1 beta for 92 min at 5 and 20 mmol/l D-glucose (2) perfusion with varying concentrations of recombinant interleukin-1 beta ranging from 0.1 x 10(-3) ng/ml to 100 ng/ml at 5 and 20 mmol/l D-glucose (3) perfusion with 20 ng/ml of recombinant interleukin-1 beta at 5, 11 or 20 mmol/l D-glucose. Furthermore, in a separate set of experiments we examined the influence of the cytokine on the morphology of the endocrine pancreas. Interleukin-1 beta stimulated insulin secretion at 11 and 20 mmol/l D-glucose and potentiated first as well as second phase insulin release in a dose-dependent fashion, with decreasing effect at higher concentrations. Glucagon secretion was also stimulated by recombinant interleukin-1 beta, irrespective of increasing glucose (5, 11, 20 mmol/l) and insulin concentrations. The potentiating effect of recombinant interleukin-1 beta on insulin secretion was evident even after discontinued perfusion with the cytokine, suggesting a priming effect on B-cell function.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood was drawn from 74 children, 3-16 years old, at diagnosis of Type 1 (insulin-dependent) diabetes and before the first insulin injection. Insulin autoantibodies were detected with a polyethylen-glycol-method in 27/74 (36.4%) and with an immuno-electrophoretic method in 6/74 (8.1%). Islet cell cytoplasmic antibodies detected by indirect immuno-fluorescence were found in 49/74 patients (66.2%), who included as many as 23 of the 27 patients with insulin autoantibodies determined with the polyethylen-glycol-method (p less than 0.01). The proportion of insulin autoantibody-positive patients who developed insulin antibodies during the first 9 months of insulin treatment was not significantly greater (51.8%) than that of insulin autoantibody-negative patients (44.6%), but patients with both islet cell antibodies and insulin autoantibodies at diagnosis produced more insulin antibodies during the first 9 months (p less than 0.05). There was no difference in fasting or meal stimulated serum C-peptide after 3, 9 or 18 months as related to occurrence of insulin autoantibodies and/or islet cell antibodies. The correlation between insulin autoantibodies and islet cell antibodies indicates that both types of autoantibodies reflect the same immunological process, although the lack of correlation to C-peptide may indicate that they play a minor causal role. In addition, the results show that patients with an active autoimmune process evidently tend to produce more insulin antibodies during the first months of insulin treatment, but the islet cell antibodies and insulin autoantibodies-positive patients had at least as good residual B-cell function as patients without autoantibodies at diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play an important role in the molecular mechanisms underlying autoimmune B-cell destruction leading to Type 1 (insulin-dependent) diabetes mellitus.

Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro) insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.

A highly polymorphic locus flanking the human insulin gene contains two major size classes of DNA restriction fragments, which segregate in families as stable genetic elements. The L-allele, i.e. fragments with an average size of about 600 base-pairs seems to be a weak genetic marker for Type 1 (insulin-dependent) diabetes mellitus, whereas the U-allele, i.e. fragments of an average size of about 2500 base-pairs hitherto has been associated with Type 2 (non-insulin-dependent) diabetes mellitus and diabetic hypertriglyceridaemia. The most recent reports on this subject do not confirm an association between the U-allele and Type 2 diabetes. Our own studies indicate that the U-allele is a fairly strong marker for the development of atherosclerosis (relative risk for U-carriers 3.36). The putative functions of the polymorphic region in atherogenesis and the relation of this region to other genetic markers for atherosclerosis are not known.

A cohort of 82 patients with Type 1 (insulin-dependent) diabetes was followed prospectively for 24 months, and 54 of them for 30 months, to study the relationship between fasting levels of immunoreactive C-peptide and titres of islet cell antibodies. After diagnosis, fasting C-peptide rose temporarily for 1-6 months of insulin therapy and declined continuously thereafter. While islet cell antibodies were present among 55% of the newly diagnosed patients, only 31% remained positive at 30 months. Their antibody titres decreased from 1:81 at diagnosis to 1:3. Only 3 patients (4%) who were islet cell antibody negative at diagnosis became positive later. The median C-peptide values among the persistently islet cell antibody positive patients decreased from 0.11 pmol/ml at 18 months, to 0.09 pmol/ml at 24 months, to 0.06 pmol/ml at 30 months compared to 0.18 (p = 0.04), 0.15 (p = 0.05) and 0.16 (p less than 0.003) pmol/ml, respectively, for the islet cell antibody negative patients. The median slope for the latter was -0.09 compared to -0.19 for the islet cell antibody positive patients (p = 0.01). These differences were reflected in increasing dosages of insulin, since patients remaining antibody-positive for 30 months were given 1.3-1.4 times more insulin (p = 0.01-0.004) than the antibody negative patients. This study demonstrates that islet cell antibodies may be a useful marker for predicting an increased rate by which endogenous B cell function is lost in Type 1 diabetes.