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Pancreatic β-cell loss induced by saturated free fatty acids (FFAs) is believed to contribute to type 2 diabetes. Previous studies have shown induction of endoplasmic reticulum (ER) stress, increased ubiquitinated proteins, and deregulation of the Bcl-2 family in the pancreas of type 2 diabetic patients. However, the precise mechanism of β-cell death remains unknown. In the present study we demonstrate that the FFA palmitate blocks the ubiquitin-proteasome system (UPS) and causes apoptosis through induction of ER stress and deregulation of Bcl-2 proteins. We found that palmitate and the proteasome inhibitor MG132 induced ER stress in β-cells, resulting in decreased expression of the prosurvival proteins Bcl-2, Mcl-1, and Bcl-XL, and upregulation of the prodeath BH3-only protein PUMA. On the other hand, pharmacological activation of the UPS by sulforaphane ameliorated ER stress, upregulated prosurvival Bcl-2 proteins, and protected β-cells from FFA-induced cell death. Furthermore, transgenic overexpression of Bcl-2 protected islets from FFA-induced cell death in vitro and improved glucose-induced insulin secretion in vivo. Together our results suggest that targeting the UPS and Bcl-2 protein expression may be a valuable strategy to prevent β-cell demise in type 2 diabetes.

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H2O2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.

Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

Environmental factors such as diets rich in saturated fats contribute to dysfunction and death of pancreatic β-cells in diabetes. Endoplasmic reticulum (ER) stress is elicited in β-cells by saturated fatty acids. Here we show that palmitate-induced β-cell apoptosis is mediated by the intrinsic mitochondrial pathway. By microarray analysis, we identified a palmitate-triggered ER stress gene expression signature and the induction of the BH3-only proteins death protein 5 (DP5) and p53-upregulated modulator of apoptosis (PUMA). Knockdown of either protein reduced cytochrome c release, caspase-3 activation, and apoptosis in rat and human β-cells. DP5 induction depends on inositol-requiring enzyme 1 (IRE1)-dependent c-Jun NH₂-terminal kinase and PKR-like ER kinase (PERK)-induced activating transcription factor (ATF3) binding to its promoter. PUMA expression is also PERK/ATF3-dependent, through tribbles 3 (TRB3)-regulated AKT inhibition and FoxO3a activation. DP5(-/-) mice are protected from high fat diet-induced loss of glucose tolerance and have twofold greater pancreatic β-cell mass. This study elucidates the crosstalk between lipotoxic ER stress and the mitochondrial pathway of apoptosis that causes β-cell death in diabetes.

In the course of Type 1 diabetes pro-inflammatory cytokines (e.g., IL-1β, IFN-γ and TNF-α) produced by islet-infiltrating immune cells modify expression of key gene networks in β-cells, leading to local inflammation and β-cell apoptosis. Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding "protective" transcription factors. To this end, we presently evaluated the role of the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) on β-cell apoptosis and production of inflammatory mediators in the rat insulinoma INS-1E cells, in purified primary rat β-cells and in human islets. C/EBPδ is expressed and up-regulated in response to the cytokines IL-1β and IFN-γ in rat β-cells and human islets. Small interfering RNA-mediated C/EBPδ silencing exacerbated IL-1β+IFN-γ-induced caspase 9 and 3 cleavage and apoptosis in these cells. C/EBPδ deficiency increased the up-regulation of the transcription factor CHOP in response to cytokines, enhancing expression of the pro-apoptotic Bcl-2 family member BIM. Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis. Furthermore, C/EBPδ silencing boosted cytokine-induced production of the chemokines CXCL1, 9, 10 and CCL20 in β-cells by hampering IRF-1 up-regulation and increasing STAT1 activation in response to cytokines. These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.

Type 1 diabetes is characterized by local inflammation (insulitis) in the pancreatic islets causing β-cell loss. The mitochondrial pathway of apoptosis is regulated by the balance and interaction between Bcl-2 members. Here we clarify the molecular mechanism of β-cell death triggered by the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The combination of TNF-α + IFN-γ induced DP5, p53 up-regulated modulator of apoptosis (PUMA), and Bim expression in human islets and rodent β-cells. DP5 and PUMA inactivation by RNA interference partially protected against TNF-α + IFN-γ-induced β-cell apoptosis. DP5 knock-out mice had increased β-cell area, and isolated islets from these mice were resistant to cytokine exposure. Bim expression was transcriptionally regulated by STAT1, and its activation triggered cleavage of caspases. Silencing of Bim protected rodent and human β-cells to a large extent against TNF-α + IFN-γ, indicating a major role of this BH3-only activator protein in the mechanism of apoptosis. Our data support a highly regulated and context-dependent modulation of specific Bcl-2 members controlling the mitochondrial pathway of β-cell apoptosis during insulitis.

The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

Type 1 diabetes is an autoimmune disorder characterized by chronic inflammation and pancreatic beta-cell loss. Here, we demonstrate that the proinflammatory cytokine interleukin-1beta, combined with interferon-gamma, induces the expression of the Bcl-2 homology 3 (BH3)-only activator PUMA (p53 up-regulated modulator of apoptosis) in beta-cells. Transcriptional activation of PUMA is regulated by nuclear factor-kappaB and endoplasmic reticulum stress but is independent of p53. PUMA activation leads to mitochondrial Bax translocation, cytochrome c release, and caspase-3 cleavage resulting in beta-cell demise. The antiapoptotic Bcl-XL protein is localized mainly at the mitochondria of the beta-cells and antagonizes PUMA action, but Bcl-XL is inactivated by the BH3-only sensitizer DP5/Hrk in cytokine-exposed beta-cells. Moreover, a pharmacological mimic of the BH3-only sensitizer Bad, which inhibits Bcl-XL and Bcl-2, induces PUMA-dependent beta-cell death and potentiates cytokine-induced apoptosis. Our data support a hierarchical activation of BH3-only proteins controlling the intrinsic pathway of beta-cell apoptosis in the context of inflammation and type 1 diabetes.

Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic beta-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1beta+IFN-gamma and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in beta-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for beta-cell apoptosis after different stimuli, and that it can act up- and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1beta+IFN-gamma induce beta-cell death and the nature of the downstream signals by which ER stress 'convinces' beta-cells to trigger apoptosis.

Objectives: Cyclin E1 is expressed during the late G1 phase of the cell cycle and mediates the initiation of DNA synthesis by activating cyclin-dependent kinases 2 (CDK2). Abnormally high levels of cyclin E1 expression have frequently been found in cancer cells. Here, we investigate the effect of cyclin E1 knockdown on cancer cells., Methods: RNA interference, expressed from a DNA-based retroviral vector, was used to knockdown cyclin E1 in adenocarcinoma (HeLa), breast (MDA-MB-31) and glioblastoma (U-373-MG) cell lines and an explant from one glioma patient (GB-LP-2)., Results: We have obtained very efficient depletion of cyclin E1 protein (over 80%) and considerable apoptotic induction (50-70%) after 96 hours post-infection. The ability of U-373-MG cells to induce tumor growth in nude mice was also abolished after cyclin E1 knockdown., Discussion: Our results indicate that retrovirus carrying the DNA to be transcribed into a short hairpin RNA (shRNA) against cyclin E1 could be used as a therapeutic agent for cancer therapy.

Objective: CD4 T-cells secreting interleukin (IL)-17 are implicated in several human autoimmune diseases, but their role in type 1 diabetes has not been defined. To address the relevance of such cells, we examined IL-17 secretion in response to β-cell autoantigens, IL-17A gene expression in islets, and the potential functional consequences of IL-17 release for β-cells.Research Design and Methods: Peripheral blood CD4 T-cell responses to β-cell autoantigens (proinsulin, insulinoma-associated protein, and GAD65 peptides) were measured by IL-17 enzyme-linked immunospot assay in patients with new-onset type 1 diabetes (n = 50). mRNA expression of IL-17A and IFNG pathway genes was studied by qRT-PCR using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease, respectively, and from matched control subjects. IL-17 effects on the function of human islets, rat β-cells, and the rat insulinoma cell line INS-1E were examined.Results: A total of 27 patients (54%) showed IL-17 reactivity to one or more β-cell peptides versus 3 of 30 (10%) control subjects (P = 0.0001). In a single case examined close to diagnosis, islet expression of IL17A, RORC, and IL22 was detected. It is noteworthy that we show that IL-17 mediates significant and reproducible enhancement of IL-1β/interferon (IFN)-γ-induced and tumor necrosis factor (TNF)-α/IFN-γ-induced apoptosis in human islets, rat β-cells, and INS-1E cells, in association with significant upregulation of β-cell IL17RA expression via activation of the transcription factors STAT1 and nuclear factor (NF)-κB.Conclusions: Circulating IL-17(+) β-cell-specific autoreactive CD4 T-cells are a feature of type 1 diabetes diagnosis. We disclose a novel pathway to β-cell death involving IL-17 and STAT1 and NF-κB, rendering this cytokine a novel disease biomarker and potential therapeutic target. [ABSTRACT FROM AUTHOR]