SKAP2, a Candidate Gene for Type 1 Diabetes, Regulates ß-Cell Apoptosis and Glycemic Control in Newly Diagnosed Patients.

The single nucleotide polymorphism rs7804356 located in the Src kinase-associated phosphoprotein 2 (SKAP2) gene is associated with type 1 diabetes (T1D), suggesting SKAP2 as a causal candidate gene. The objective of the study was to investigate if SKAP2 has a functional role in the ß-cells in relation to T1D. In a cohort of children with newly diagnosed T1D, rs7804356 predicted glycemic control and residual ß-cell function during the 1st year after diagnosis. In INS-1E cells and rat and human islets, proinflammatory cytokines reduced the content of SKAP2. Functional studies revealed that knockdown of SKAP2 aggravated cytokine-induced apoptosis in INS-1E cells and primary rat ß-cells, suggesting an antiapoptotic function of SKAP2. In support of this, overexpression of SKAP2 afforded protection against cytokine-induced apoptosis, which correlated with reduced nuclear content of S536-phosphorylated nuclear factor-κB (NF-κB) subunit p65, lower nitric oxide production, and diminished CHOP expression indicative of decreased endoplasmic reticulum stress. Knockdown of CHOP partially counteracted the increase in cytokine-induced apoptosis caused by SKAP2 knockdown. In conclusion, our results suggest that SKAP2 controls ß-cell sensitivity to cytokines possibly by affecting the NF-κB-inducible nitric oxide synthase-endoplasmic reticulum stress pathway.

The non-canonical NF-κB pathway and its contribution to ß-cell failure in diabetes.

The prevalence of diabetes has reached 8.8% in worldwide population and is predicted to increase up to 10.4% by 2040. Thus, there is an urgent need for the development of means to treat or prevent this major disease. Due to its role in inflammatory responses, several studies demonstrated the importance of the transcription factor nuclear factor-κB (NF-κB) in both type 1 diabetes (T1D) and type 2 diabetes (T2D). The two major NF-κB pathways are the canonical and the non-canonical. The later pathway is activated by the NF-κB-inducing kinase (NIK) that triggers p100 processing into p52, which forms with RelB its main dimer. Cytokines mediating the activation of this pathway are present in the serum of T1D and T2D patients. Conversely, limited information is available regarding the role of the alternative pathway on diabetes development and ß-cell fate. In the present review, we will briefly describe the involvement of NF-κB on diabetes pathology and discuss new studies indicating an important role for the non-canonical NF-κB activation in ß-cell function and survival. The non-canonical NF-κB pathway is emerging as a novel potential target for the development of therapeutic strategies to treat or prevent diabetes.

Dysfunctional autophagy following exposure to pro-inflammatory cytokines contributes to pancreatic ß-cell apoptosis.

Type 1 diabetes (T1D) results from ß-cell destruction due to concerted action of both innate and adaptive immune responses. Pro-inflammatory cytokines, such as interleukin-1ß and interferon-γ, secreted by the immune cells invading islets of Langerhans, contribute to pancreatic ß-cell death in T1D. Cytokine-induced endoplasmic reticulum (ER) stress plays a central role in ß-cell demise. ER stress can modulate autophagic response; however, no study addressed the regulation of autophagy during the pathophysiology of T1D. In this study, we document that cytokines activate the AMPK-ULK-1 pathway while inhibiting mTORC1, which stimulates autophagy activity in an ER stress-dependent manner. On the other hand, time-course analysis of LC3-II accumulation in autophagosomes revealed that cytokines block the autophagy flux in an ER stress independent manner, leading to the formation of large dysfunctional autophagosomes and worsening of ER stress. Cytokines rapidly impair lysosome function, leading to lysosome membrane permeabilization, Cathepsin B leakage and lysosomal cell death. Blocking cathepsin activity partially protects against cytokine-induced or torin1-induced apoptosis, whereas blocking autophagy aggravates cytokine-induced CHOP overexpression and ß-cell apoptosis. In conclusion, cytokines stimulate the early steps of autophagy while blocking the autophagic flux, which aggravate ER stress and trigger lysosomal cell death. Restoration of autophagy/lysosomal function may represent a novel strategy to improve ß-cell resistance in the context of T1D.

MCL-1 Is a Key Antiapoptotic Protein in Human and Rodent Pancreatic ß-Cells.

Induction of endoplasmic reticulum stress and activation of the intrinsic apoptotic pathway is widely believed to contribute to ß-cell death in type 1 diabetes (T1D). MCL-1 is an antiapoptotic member of the BCL-2 protein family, whose depletion causes apoptosis in rodent ß-cells in vitro. Importantly, decreased MCL-1 expression was observed in islets from patients with T1D. We report here that MCL-1 downregulation is associated with cytokine-mediated killing of human ß-cells, a process partially prevented by MCL-1 overexpression. By generating a ß-cell-specific Mcl-1 knockout mouse strain (ßMcl-1KO), we observed that, surprisingly, MCL-1 ablation does not affect islet development and function. ß-Cells from ßMcl-1KO mice were, however, more susceptible to cytokine-induced apoptosis. Moreover, ßMcl-1KO mice displayed higher hyperglycemia and lower pancreatic insulin content after multiple low-dose streptozotocin treatment. We found that the kinase GSK3ß, the E3 ligases MULE and ßTrCP, and the deubiquitinase USP9x regulate cytokine-mediated MCL-1 protein turnover in rodent ß-cells. Our results identify MCL-1 as a critical prosurvival protein for preventing ß-cell death and clarify the mechanisms behind its downregulation by proinflammatory cytokines. Development of strategies to prevent MCL-1 loss in the early stages of T1D may enhance ß-cell survival and thereby delay or prevent disease progression.

Endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation.

Insulin-secreting pancreatic ß-cells are extremely dependent on their endoplasmic reticulum (ER) to cope with the oscillatory requirement of secreted insulin to maintain normoglycemia. Insulin translation and folding rely greatly on the unfolded protein response (UPR), an array of three main signaling pathways designed to maintain ER homeostasis and limit ER stress. However, prolonged or excessive UPR activation triggers alternative molecular pathways that can lead to ß-cell dysfunction and apoptosis. An increasing number of studies suggest a role of these pro-apoptotic UPR pathways in the downfall of ß-cells observed in diabetic patients. Particularly, the past few years highlighted a cross talk between the UPR and inflammation in the context of both type 1 (T1D) and type 2 diabetes (T2D). In this article, we describe the recent advances in research regarding the interplay between ER stress, the UPR, and inflammation in the context of ß-cell apoptosis leading to diabetes.

The non-canonical NF-κB pathway is induced by cytokines in pancreatic beta cells and contributes to cell death and proinflammatory responses in vitro.

AIMS/HYPOTHESIS: Activation of the transcription factor nuclear factor (NF)-κB by proinflammatory cytokines plays an important role in beta cell demise in type 1 diabetes. Two main signalling pathways are known to activate NF-κB, namely the canonical and the non-canonical pathways. Up to now, studies on the role of NF-κB activation in beta cells have focused on the canonical pathway. The aim of this study was to investigate whether cytokines activate the non-canonical pathway in beta cells, how this pathway is regulated and the consequences of its activation on beta cell fate. METHODS: NF-κB signalling was analysed by immunoblotting, promoter reporter assays and real-time RT-PCR, after knockdown or overexpression of key genes/proteins. INS-1E cells, FACS-purified rat beta cells and the human beta cell line EndoC-ßH1 exposed to cytokines were used as models. RESULTS: IL-1ß plus IFN-γ induced stabilisation of NF-κB-inducing kinase and increased the expression and cleavage of p100 protein, culminating in the nuclear translocation of p52, the hallmark of the non-canonical signalling. This activation relied on different crosstalks between the canonical and non-canonical pathways, some of which were beta cell specific. Importantly, cytokine-mediated activation of the non-canonical pathway controlled the expression of 'late' NF-κB-dependent genes, regulating both pro-apoptotic and inflammatory responses, which are implicated in beta cell loss in early type 1 diabetes. CONCLUSIONS/INTERPRETATION: The atypical activation of the non-canonical NF-κB pathway by proinflammatory cytokines constitutes a novel 'feed-forward' mechanism that contributes to the particularly pro-apoptotic effect of NF-κB in beta cells.

A20 Inhibits ß-Cell Apoptosis by Multiple Mechanisms and Predicts Residual ß-Cell Function in Type 1 Diabetes.

Activation of the transcription factor nuclear factor kappa B (NFkB) contributes to ß-cell death in type 1 diabetes (T1D). Genome-wide association studies have identified the gene TNF-induced protein 3 (TNFAIP3), encoding for the zinc finger protein A20, as a susceptibility locus for T1D. A20 restricts NF-κB signaling and has strong antiapoptotic activities in ß-cells. Although the role of A20 on NF-κB inhibition is well characterized, its other antiapoptotic functions are largely unknown. By studying INS-1E cells and rat dispersed islet cells knocked down or overexpressing A20 and islets isolated from the ß-cell-specific A20 knockout mice, we presently demonstrate that A20 has broader effects in ß-cells that are not restricted to inhibition of NF-κB. These involves, suppression of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK), activation of survival signaling via v-akt murine thymoma viral oncogene homolog (Akt) and consequently inhibition of the intrinsic apoptotic pathway. Finally, in a cohort of T1D children, we observed that the risk allele of the rs2327832 single nucleotide polymorphism of TNFAIP3 predicted lower C-peptide and higher hemoglobin A1c (HbA1c) levels 12 months after disease onset, indicating reduced residual ß-cell function and impaired glycemic control. In conclusion, our results indicate a critical role for A20 in the regulation of ß-cell survival and unveil novel mechanisms by which A20 controls ß-cell fate. Moreover, we identify the single nucleotide polymorphism rs2327832 of TNFAIP3 as a possible prognostic marker for diabetes outcome in children with T1D.

Heterozygous inactivation of plasma membrane Ca(2+)-ATPase in mice increases glucose-induced insulin release and beta cell proliferation, mass and viability.

AIMS/HYPOTHESIS: Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca(2+) extrusion--the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. METHODS: Biological and morphological methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. RESULTS: Pmca (isoform 2) heterozygous inactivation increased intracellular Ca(2+) stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. CONCLUSIONS/INTERPRETATION: The present data indicates that inhibition of Ca(2+) extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca(2+) extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.