Die Bedeutung zytoprotektiver Enzyme für den Schutz insulinproduzierender Zellen gegenüber einer Schädigung durch Sauerstoffradikale, NO und Zytokine

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Induction of the intrinsic apoptosis pathway in insulin-secreting cells is dependent on oxidative damage of mitochondria but independent of caspase-12 activation

AbstractPro-inflammatory cytokine-mediated beta cell apoptosis is activated through multiple signaling pathways involving mitochondria and endoplasmic reticulum. Activation of organelle-specific caspases has been implicated in the progression and execution of cell death. This study was therefore performed to elucidate the effects of pro-inflammatory cytokines on a possible cross-talk between the compartment-specific caspases 9 and 12 and their differential contribution to beta cell apoptosis. Moreover, the occurrence of ROS-mediated mitochondrial damage in response to beta cell toxic cytokines has been quantified. ER-specific caspase-12 was strongly activated in response to pro-inflammatory cytokines; however, its inhibition did not abolish cytokine-induced mitochondrial caspase-9 activation and loss of cell viability. In addition, there was a significant induction of oxidative mitochondrial DNA damage and elevated cardiolipin peroxidation in insulin-producing RINm5F cells and rat islet cells. Overexpression of the H2O2 detoxifying enzyme catalase effectively reduced the observed cytokine-induced oxidative damage of mitochondrial structures. Taken together, the results strongly indicate that mitochondrial caspase-9 is not a downstream substrate of ER-specific caspase-12 and that pro-inflammatory cytokines cause apoptotic beta cell death through activation of caspase-9 primarily by hydroxyl radical-mediated mitochondrial damage

Optical coherence tomography angiography (OCTA) as a new diagnostic tool in uveitis

Background: The broad spectrum of uveitis disorders requires a multimodal imaging approach in the daily practice of an ophthalmologist. As inflammatory conditions, they have in common an alteration in leukocyte migration. In this context, optical coherence tomography angiography (OCTA) might be of great value for diagnosing or following up patients with these disorders. To date, OCTA has rather been used as an additional tool besides the well-established diagnostic imaging tools, but its complementary diagnostic features become increasingly relevant, to follow disease activity and treatment response and for the understanding of pathomechanisms of various uveitis types. This review summarizes the possible applications of OCTA and its advantages and disadvantages as opposed to dye-based angiographies in uveitic diseases. Main body: Hitherto gold standards in the diagnostic workup of posterior or intermediate uveitis have been angiography on a dye-based method, which is fluorescein or indocyanine green. It gives information about the status of the blood-retinal barrier and the retinal and choroidal vasculature by visualizing diffuse leakage as a state of inflammation or complications as an ischemia or choroidal neovascularization. As noninvasive methods, fundus autofluorescence depicts the status of metabolic activity of the retinal pigment epithelium and OCT or enhanced depth imaging OCT, respectively, as a depth-resolving imaging method can supply additional information. OCTA as a non-invasive, depth-resolution imaging tool of retinal and choroidal vessels adds detailed qualitative and quantitative information of the status of retinal and choroidal vessels and bridges the gap between the mentioned conventional diagnostic tools used in uveitis. It is important, though, to be aware of its limitations, such as its susceptibility to motion artifacts, limited comparability among different devices, and restricted contribution of information regarding the grade of disease activity. Conclusion: OCTA as a non-invasive, depth-resolution imaging tool can give qualitative and quantitative information about the status of retinal and choroidal vessels, but also has certain limitations. Employing OCTA as a complementary rather than exclusive tool, it can give important additional information about the macro- and microvasculature under inflammatory circumstances. Thereby, it also contributes to the understanding of the pathophysiology of various uveitis entities

Modulation of Bcl-2-related protein expression in pancreatic beta cells by pro-inflammatory cytokines and its dependence on the antioxidative defense status. Mol Cell Endocrinol 332: 88–96

a b s t r a c t Pro-inflammatory cytokines are key mediators in the selective and progressive destruction of insulinproducing beta cells during type 1 diabetes development. However, the mechanisms of cytokine-induced beta cell apoptosis are not fully understood. This study demonstrates that pro-inflammatory cytokines strongly modified the expression of the antiapoptotic protein Bcl-2 and the pro-apoptotic BH3-only proteins Bad, Bim, and Bid in primary rat islets and insulin-producing RINm5F cells. Overexpression of mitochondrially located catalase (MitoCatalase) specifically increased basal Bcl-2 and decreased basal Bax expression, suppressed cytokine-mediated reduction of Bcl-2, and thereby prevented the release of cytochrome c, Smac/DIABLO and the activation of caspase-9 and -3. Thus, cytokine-mediated decrease of Bcl-2 expression and the sequentially changed Bax/Bcl-2 ratio are responsible for the release of pro-apoptotic mitochondrial factors, activation of caspase-9, and ultimately caspase-3. These results indicate that activation of the intrinsic/mitochondrial apoptosis pathway is essential for cytokine-induced beta cell death and the mitochondrial generation of reactive oxygen species, in particular mitochondrial hydrogen peroxide, differentially regulates the Bax/Bcl-2 ratio

Cytokine toxicity in insulin-producing cells is mediated by nitro-oxidative stressinduced hydroxyl radical formation in mitochondria

Abstract Although nitric oxide (NO) and oxidative stress both contribute to proinflammatory cytokine toxicity in pancreatic β-cells during type 1 diabetes mellitus (T1DM) development, the interactions between NO and reactive oxygen species (ROS) in cytokine-mediated β-cell death have not been clarified. Exposure of insulin-producing RINm5F cells to IL-1β generated NO, while exposure to a combination of IL-1β, TNF-α, and IFN-γ, which simulates T1DM conditions, generated both NO and ROS. In theory, two reactions between NO and ROS are possible, one with the superoxide radical yielding peroxynitrite, and the other with hydrogen peroxide (H 2 O 2 ) yielding hydroxyl radicals. Results of the present work exclude peroxynitrite involvement in cytokine toxicity to β-cells because its generation did not correlate with the toxic action of cytokines. On the other hand, we show that H 2 O 2 , produced upon exposure of insulin-producing cell clones and primary rat islet cells to cytokines almost exclusively in the mitochondria, reacted in the presence of trace metal (Fe ++ ) with NO forming highly toxic hydroxyl radicals, thus explaining the severe toxicity that causes apoptotic β-cell death. Expression of the H 2 O 2 -inactivating enzyme catalase in mitochondria protected against cytokine toxicity by preventing hydroxyl radical formation. We therefore conclude that proinflammatory cytokine-mediated β-cell death is due to nitro-oxidative stress-mediated hydroxyl radical formation in the mitochondria

Improvement of the mitochondrial antioxidant defense status prevents cytokine-induced nuclear factor-kappaB activation in insulin-producing cells.

Proinflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha], and gamma-interferon [IFN-gamma]) initiate a variety of signal cascades in pancreatic beta-cells that affect the expression level of genes involved in both the destruction and the protection of the beta-cell. The generation of nitric oxide (NO) via the inducible NO synthase (iNOS) and oxygen free radicals play a key role in cytokine-mediated beta-cell destruction. Within these signal cascades, the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) is crucial, and many cytokine-sensitive genes contain binding sites for this transcription factor in their promoter regions. The aim of this study was to characterize the cytokine-mediated activation of NF-kappaB and the subsequent expression of iNOS protein in insulin-producing RINm5F cells with an improved antioxidant defense status by overexpression of the cytoprotective enzymes catalase (Cat), glutathione peroxidase (Gpx), and the cytoplasmic Cu/Zn superoxide dismutase (Cu/ZnSOD). RINm5F cells with diverse mitochondrial antioxidative defense status were generated by stable overexpression of MnSOD constructs in sense (MnSOD sense) and antisense orientation (MnSOD antisense). Cytokine-induced (IL-1beta or cytokine mix consisting of IL-1beta + TNF-alpha + IFN-gamma) activation of NF-kappaB in RINm5F cells was reduced by >80% through overexpression of MnSOD. The activity of the iNOS promoter remained at basal levels in cytokine-stimulated MnSOD sense cells. In contrast, the suppression of MnSOD gene expression in cytokine-stimulated MnSOD antisense cells resulted in a threefold higher activation of NF-kappaB and a twofold higher activation of the iNOS promoter as compared with control cells. The iNOS protein expression was significantly reduced after a 6- and 8-h cytokine incubation of MnSOD sense cells. The low activity level of MnSOD in RINm5F MnSOD antisense cells increased the iNOS protein expression in particular during the early phase of cytokine-mediated toxicity. Cat, Gpx, and the cytoplasmic Cu/ZnSOD did not affect the activation of NF-kappaB and the iNOS promoter. In conclusion, the overexpression of MnSOD, which inactivates specifically mitochondrially derived oxygen free radicals, significantly reduced the activation of NF-kappaB in insulin-producing cells. As a consequence of this protective effect in the early cytokine signaling pathways, the induction of iNOS, an important event in the beta-cell destruction process, was also significantly reduced. The results provide evidence that mitochondrially derived reactive oxygen species (ROS) play a critical role in the activation of the cytokine-sensitive transcription factor NF-kappaB. Overexpression of MnSOD may thus be beneficial for beta-cell survival through suppression of oxygen free radical formation, prevention of NF-kappaB activation, and iNOS expression.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis.

Pro-inflammatory cytokines involved in the pathogenesis of type 1 diabetes deplete endoplasmic reticulum (ER) Ca2+ stores, leading to ER-stress and beta cell apoptosis. However, the cytokine-induced ER-stress response in beta cells is atypical and characterised by induction of the pro-apoptotic PKR-like ER kinase (PERK)-C/EBP homologous protein (CHOP) branch of the unfolded protein response, but defective X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 activation. The purpose of this study was to overexpress spliced/active Xbp1 (XBP1s) to increase beta cell resistance to cytokine-induced ER-stress and apoptosis.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Additional file 5: Table S1. of TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2

List of plasmids. (PDF 56 kb

Additional file 2: Figure S2. of TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2

The role of R266 in HyPer and TriPer’s reactivity with H2O2. (A) Traces of time-dependent changes to the excitation ratio of recombinant HyPer or TriPer variants with the inactivating R266A mutation. (B) Non-reducing and reducing SDS-PAGE of recombinant TriPerR199A following incubation with increasing concentrations of H2O2 for 15 min. (C) Non-reducing gradient SDS-PAGE (pH 7.3, 4–12%) of samples as in Fig. 2g. (D) Traces of time-dependent changes to the excitation ratio of HyPer and TryPer mutant variants treated as in (B). Note that the variants lacking the ability to form C199-C208 disulfide do not change their excitation ratio upon oxidation. (PDF 93 kb