Aims/hypothesis The immune-mediated elimination of pancreatic beta cells in type 1 diabetes involves release of cytotoxic cytokines such as IL-1β and IFNγ, which induce beta cell death in vitro by mechanisms that are both dependent and independent of nitric oxide (NO). Nuclear factor kappa B (NFκB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of proapoptotic genes. NFκB 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-1β and IFNγ. Effects on insulin secretion and NO formation were measured by ELISA and Griess reagent, respectively. iNOS levels and NFκB 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-1β induced a bi-phasic phosphorylation of inhibitor protein kappa Bα (IκBα) with the 2nd peak being sensitive to HDAC inhibition. No effect was seen on IκBα degradation and NFκB DNA binding. Conclusions/interpretation HDAC inhibition prevents cytokine-induced beta cell apoptosis and impaired beta cell function associated with a downregulation of NFκB transactivating activity.

IL-1 plays a major role in inflammation and autoimmunity through activation of nuclear factor kappa B (NFkappaB) and MAPKs. Although a great deal is known about the mechanism of activation of NFkappaB and MAPKs by IL-1, much less is known about the down-regulation of this pathway. Suppressor of cytokine signaling (SOCS)-3 was shown to inhibit IL-1-induced transcription and activation of NFkappaB and the MAPKs JNK and p38, but the mechanism is unknown. We show here that SOCS-3 inhibits NFkappaB-dependent transcription induced by overexpression of the upstream IL-1 signaling molecules MyD88, IL-1R-activated kinase 1, TNF receptor-associated factor (TRAF)6, and TGFbeta-activated kinase (TAK)1, but not when the MAP3K MAPK/ERK kinase kinase-1 is used instead of TAK1, indicating that the target for SOCS-3 is the TRAF6/TAK1 signaling complex. By coimmunoprecipitation, it was shown that SOCS-3 inhibited the association between TRAF6 and TAK1 and that SOCS-3 coimmunoprecipitated with TAK1 and TRAF6. Furthermore, SOCS-3 inhibited the IL-1-induced catalytic activity of TAK1. Because ubiquitination of TRAF6 is required for activation of TAK1, we analyzed the role of SOCS-3 on TRAF6 ubiquitination and found that SOCS-3 inhibited ubiquitin modification of TRAF6. These results indicate that SOCS-3 inhibits IL-1 signal transduction by inhibiting ubiquitination of TRAF6, thus preventing association and activation of TAK1.

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

In type 1 diabetes, inflammatory and immunocompetent cells enter the islet and produce proinflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); each contribute to β-cell destruction, mediated in part by nitric oxide. Inhibitors of histone deacetylases (HDAC) are used commonly in humans but also possess antiinflammatory and cytokine-suppressing properties. Here we show that oral administration of the HDAC inhibitor ITF2357 to mice normalized streptozotocin (STZ)-induced hyperglycemia at the clinically relevant doses of 1.25-2.5 mg/kg. Serum nitrite levels returned to nondiabetic values, islet function improved and glucose clearance increased from 14% (STZ) to 50% (STZ + ITF2357). In vitro, at 25 and 250 nmol/L, ITF2357 increased islet cell viability, enhanced insulin secretion, inhibited MIP-1α and MIP-2 release, reduced nitric oxide production and decreased apoptosis rates from 14.3% (vehicle) to 2.6% (ITF2357). Inducible nitric oxide synthase (iNOS) levels decreased in association with reduced islet-derived nitrite levels. In peritoneal macrophages and splenocytes, ITF2357 inhibited the production of nitrite, as well as that of TNFα and IFNγ at an IC 50 of 25-50 nmol/L. In the insulin-producing INS cells challenged with the combination of IL-1β plus IFNγ, apoptosis was reduced by 50% (P < 0.01). Thus at clinically relevant doses, the orally active HDAC inhibitor ITF2357 favors β-cell survival during inflammatory conditions.

Growth hormone and prolactin are important growth factors for pancreatic -cells. The effects exerted by these hormones on proliferation and on insulin synthesis and secretion in -cells are largely mediated through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Suppressors of cytokine signaling (SOCS) proteins are specific inhibitors of the JAK/STAT pathway acting through a negative-feedback loop. To investigate in vivo effects of SOCS-3 in growth hormone (GH)/prolactin signaling in -cells we generated transgenic mice with -cell-specific overexpression of SOCS-3. The relative -cell proliferation and volume in the mice were measured by morphometry. -Cell volume of transgenic female mice was reduced by over 30% compared with -cell volume in wild-type female mice. Stimulation of transgenic islets in vitro with GH showed a reduced tyrosine phosphorylation of STAT-5 when compared with wild-type islets. Transduction of primary islet cultures with adenoviruses expressing various SOCS proteins followed by stimulation with GH or glucagon-like peptide-1 (GLP-1) revealed that SOCS-3 inhibited GH-but not GLP-1-mediated islet cell proliferation, indicating that the decreased -cell volume observed in female transgenic mice could be caused by an inhibition of GH-induced -cell proliferation by SOCS-3. In spite of the reduced -cell volume the transgenic female mice exhibited enhanced glucose tolerance compared with wild-type littermates following an oral glucose-tolerance test. Together these data suggest that SOCS-3 modulates cytokine signaling in pancreatic -cells and therefore potentially could be a candidate target for development of new treatment strategies for diabetes.

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 be 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 following 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 upregulated. 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.

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