Peter E. Heding

Unraveling the Pathogenesis of Type 1 Diabetes with Proteomics: Present And Future Directions

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

Suppressor of cytokine signalling-3 inhibits tumor necrosis factor-alpha induced apoptosis and signalling in beta cells

Tumor necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine involved in the pathogenesis of several diseases including type 1 diabetes mellitus (T1DM). TNFalpha in combination with interleukin-1-beta (IL-1beta) and/or interferon-gamma (IFNgamma) induces specific destruction of the pancreatic insulin-producing beta cells. Suppressor of cytokine signalling-3 (SOCS-3) proteins regulate signalling induced by a number of cytokines including growth hormone, IFNgamma and IL-1beta which signals via very distinctive pathways. The objective of this study was to investigate the effect of SOCS-3 on TNFalpha-induced signalling in beta cells. We found that apoptosis induced by TNFalpha alone or in combination with IL-1beta was suppressed by expression of SOCS-3 in the beta cell line INSr3#2. SOCS-3 inhibited TNFalpha-induced phosphorylation of the mitogen activated protein kinases ERK1/2, p38 and JNK in INSr3#2 cells and in primary rat islets. Furthermore, SOCS-3 repressed TNFalpha-induced degradation of IkappaB, NFkappaB DNA binding and transcription of the NFkappaB-dependent MnSOD promoter. Finally, expression of Socs-3 mRNA was induced by TNFalpha in rat islets in a transient manner with maximum expression after 1-2h. The ability of SOCS-3 to regulate signalling induced by the three major pro-inflammatory cytokines involved in the pathogenesis of T1DM makes SOCS-3 an interesting therapeutic candidate for protection of the beta cell mass.

Suppressor of cytokine signalling-3 expression inhibits cytokine-mediated destruction of primary mouse and rat pancreatic islets and delays allograft rejection

Aims/hypothesisThe pro-inflammatory cytokines IL-1 and IFNγ 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.MethodsUsing 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.ResultsSocs3 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-κB 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/interpretationSOCS3 inhibits IL-1-induced signalling through the nuclear factor-κB 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.

IL-1β-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

Aims/hypothesisIL-1β released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1β-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 methodsWestern 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 ELISAplus 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.ResultsPre-exposure of INS-1E cells to the FGFR-inhibitor SU5402, but not to the Src-inhibitor PP2, dose-dependently inhibited IL-1β-mediated MAPK activity. A synthetic peptide, C3d, reported to bind NCAM, did not activate MAPK or Akt as reported in neurons but inhibited IL-1β-induced MAPK activity, thereby mimicking the effect of SU5402. Furthermore, C3d inhibited NCAM-induced FGFR phosphorylation and apoptosis induced by IL-1β plus IFN-γ, but did not affect IL-1β-induced NF-κB signalling.Conclusions/interpretationWe suggest that NCAM signalling through FGFR is required for efficient IL-1β pro-apoptotic signalling by facilitating IL-1β-induced MAPK activation downstream of the NF-κB-MAPK branching point. Further, these data identify a novel function of C3d as an inhibitor of NCAM-induced FGFR activity and of IL-1β-induced MAPK activation in beta cells.

No evidence of a functionally significant polymorphism of the BCL2 gene in Danish, Finnish and Basque type 1 diabetes families.

Accumulating evidence has suggested a role for the anti-apoptotic protein BCL2 in the development of autoimmune diseases, including type 1 diabetes mellitus (T1DM). Recently, the first BCL2 polymorphism (Ala43Thr) with association to T1DM in a Japanese population was reported. The polymorphism was found significantly more frequent in control individuals (14.5%) than in T1DM patients (6.8%), and was furthermore found to be functionally relevant, promoting a increased sensitivity to apoptosis when overexpressed in an IL-7 dependent mouse pre-B cell line. To investigate the relevance of the polymorphism in Caucasians, we have genotyped nearly 1400 individuals comprising Danish, Finnish and Basque T1DM family materials, using a PCR-based RFLP assay. In contrast to what was observed in Japanese diabetic/control individuals, we find no evidence for association of the BCL2 Ala43Thr polymorphism to T1DM in Danish, Finnish and Basque Type 1 diabetes families.