Ilir Mehmeti

Modulation of Bcl-2-related protein expression in pancreatic beta cells by pro-inflammatory cytokines and its dependence on the antioxidative defense status.

Pro-inflammatory cytokines are key mediators in the selective and progressive destruction of insulin-producing 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 anti-apoptotic 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 stress-induced hydroxyl radical formation in mitochondria

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 (H2O2) 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 H2O2, 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 H2O2-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.

The H2O2-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol–disulfide milieu

Oxidative protein folding in the endoplasmic reticulum (ER) is associated with the formation of native disulfide bonds, which inevitably results in the formation of hydrogen peroxide (H(2)O(2)). Particularly in pancreatic β-cells with their high secretory activity and extremely low antioxidant capacity, the H(2)O(2) molecules generated during oxidative protein folding could represent a significant oxidative burden. Therefore this study was conducted to elucidate the H(2)O(2) generation during disulfide bond formation in insulin-producing RINm5F cells by targeting and specifically expressing the H(2)O(2)-sensitive biosensor HyPer in the ER (ER-HyPer). In addition the influence of overexpression of the H(2)O(2)-metabolizing ER-resident peroxiredoxin IV (PRDXIV) on H(2)O(2) levels was examined. The ER-HyPer fluorescent protein was completely oxidized, whereas HyPer expressed in cytosol, peroxisomes, and mitochondria was prevalently in the reduced state, indicating a high basal H(2)O(2) concentration in the ER lumen. These results could also be confirmed in non-insulin-producing COS-7 cells. Overexpression of PRDXIV in RINm5F cells effectively protected against H(2)O(2)-mediated toxicity; however, it did not affect the fluorescence signal of ER-HyPer. Moreover, the inhibition of de novo protein synthesis and the associated H(2)O(2) generation by cycloheximide had no influence on the ER-HyPer redox state. Taken together, these findings strongly suggest that the H(2)O(2)-sensitive biosensor reflects exclusively the oxidative milieu in the ER and not the H(2)O(2) concentration in the ER lumen.

Overexpression of the nuclear factor-κB subunit c-Rel protects against human islet cell death in vitro.

The transcription factor nuclear factor (NF)-κB is known to modulate rates of apoptosis and may therefore play a role in the increased β-cell death that occurs in type 1 and type 2 diabetes. The aim of the present investigation was to study the expression of NF-κB subunits in human islet cells and whether overexpression of the NF-κB subunit c-Rel affects islet cell survival. We detected expression of p65, Rel-B, p50, p105, p52, and the ribosomal protein S3 (rpS3) in human islet cells. Among these, only p65 and rpS3 were translocated from the cytosolic to the nuclear fraction in response to cytokines. Interestingly, rpS3 participated in p65 binding to the κB-element in gel shift analysis experiments. We observed cytoplasmic c-Rel expression in vivo in 6J mice, and signs of nuclear translocation in β-cells of infiltrated nonobese diabetic islets. Human islet cells were also dispersed by trypsin treatment and transduced with a c-Rel adenoviral vector. This resulted in increased expression of c-Rel and inhibitory factor κB, increased κB-binding activity, and augmented protein levels of Bcl-X(L,) c-IAP2, and heat shock protein 72. c-Rel expression in human islet cells protected against cytokine-induced caspase 3 activation and cell death. c-Rel protected also against streptozotocin- and H(2)O(2)-induced cell death, in both intact rat islets and human islet cells. We conclude that rpS3 participates in NF-κB signaling and that a genetic increase in the activity of the NF-κB subunit c-Rel results in protection against cell death in human islets.

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

Pro-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 H(2)O(2) 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.

ERO1-independent production of H2O2 within the endoplasmic reticulum fuels Prdx4-mediated oxidative protein folding

Tracking the kinetics of equilibration of H2O2 between compartments reveals unexpected isolation of the endoplasmic reticulum and hints at a hitherto unsuspected local source of peroxide.

Peroxiredoxin 4 improves insulin biosynthesis and glucose-induced insulin secretion in insulin-secreting INS-1E cells.

Background: Peroxiredoxin 4 facilitates de novo disulfide bond formation by metabolizing hydrogen peroxide. Results: Overexpression of peroxiredoxin 4 improved insulin synthesis and glucose-induced insulin secretion. Conclusion: Increasing the constitutively low expression of peroxiredoxin 4 enhances the ER folding capacity and improves β-cell function. Significance: Peroxiredoxin 4 contributes to the preservation of β-cell function under conditions of high insulin requirement. Oxidative folding of (pro)insulin is crucial for its assembly and biological function. This process takes place in the endoplasmic reticulum (ER) and is accomplished by protein disulfide isomerase and ER oxidoreductin 1β, generating stoichiometric amounts of hydrogen peroxide (H2O2) as byproduct. During insulin resistance in the prediabetic state, increased insulin biosynthesis can overwhelm the ER antioxidative and folding capacity, causing an imbalance in the ER redox homeostasis and oxidative stress. Peroxiredoxin 4 (Prdx4), an ER-specific antioxidative peroxidase can utilize luminal H2O2 as driving force for reoxidizing protein disulfide isomerase family members, thus efficiently contributing to disulfide bond formation. Here, we examined the functional significance of Prdx4 on β-cell function with emphasis on insulin content and secretion during stimulation with nutrient secretagogues. Overexpression of Prdx4 in glucose-responsive insulin-secreting INS-1E cells significantly metabolized luminal H2O2 and improved the glucose-induced insulin secretion, which was accompanied by the enhanced proinsulin mRNA transcription and insulin content. This β-cell beneficial effect was also observed upon stimulation with the nutrient insulin secretagogue combination of leucine plus glutamine, indicating that the effect is not restricted to glucose. However, knockdown of Prdx4 had no impact on H2O2 metabolism or β-cell function due to the fact that Prdx4 expression is negligibly low in pancreatic β-cells. Moreover, we provide evidence that the constitutively low expression of Prdx4 is highly susceptible to hyperoxidation in the presence of high glucose. Overall, these data suggest an important role of Prdx4 in maintaining insulin levels and improving the ER folding capacity also under conditions of a high insulin requirement.

Susceptibility of brown adipocytes to pro-inflammatory cytokine toxicity and reactive oxygen species

Pro-inflammatory cytokine-induced brown adipocyte dysfunction and consecutive cell death is mediated by suppression of the mitochondrial uncoupling protein 1 and concomitant generation of reactive oxygen species.

Increased Hsp70 expression attenuates cytokine-induced cell death in islets of Langerhans from Shb knockout mice.

Type 1 diabetes may depend on cytokine-induced beta-cell death and therefore the current investigation was performed in order to elucidate this response in Shb-deficient islets. A combination of interleukin-1beta and interferon-gamma caused a diminished beta-cell death response in Shb null islets. Furthermore, the induction of an unfolded protein response (UPR) by adding cyclopiazonic acid did not increase cell death in Shb-deficient islets, despite simultaneous expression of UPR markers. The heat-shock protein Hsp70 was more efficiently induced in Shb knockout islets, providing an explanation for the decreased susceptibility of Shb-deficient islets to cytokines. It is concluded that islets deficient in the Shb protein are less susceptible to cytotoxic conditions, and that this partly depends on their increased ability to induce Hsp70 under such circumstances. Interference with Shb signaling may provide means to improve beta-cell viability under conditions of beta-cell stress.

Sensitivity profile of the human EndoC-βH1 beta cell line to proinflammatory cytokines

Aims/hypothesisThe aim of this study was to perform a detailed analysis of cytokine toxicity in the new human EndoC-βH1 beta cell line.MethodsThe expression profile of the antioxidative enzymes in the new human EndoC-βH1 beta cells was characterised and compared with that of primary beta cells in the human pancreas. The effects of proinflammatory cytokines on reactive oxygen species formation, insulin secretory responsiveness and apoptosis of EndoC-βH1 beta cells were determined.ResultsEndoC-βH1 beta cells were sensitive to the toxic action of proinflammatory cytokines. Glucose-dependent stimulation of insulin secretion and an increase in the ATP/ADP ratio was abolished by proinflammatory cytokines without induction of IL-1β expression. Cytokine-mediated caspase-3 activation was accompanied by reactive oxygen species formation and developed more slowly than in rodent beta cells. Cytokines transiently increased the expression of unfolded protein response genes, without inducing endoplasmic reticulum stress-marker genes. Cytokine-mediated NFκB activation was too weak to induce inducible nitric oxide synthase expression. The resultant lack of nitric oxide generation in EndoC-βH1 cells, in contrast to rodent beta cells, makes these cells dependent on exogenously generated nitric oxide, which is released from infiltrating immune cells in human type 1 diabetes, for full expression of proinflammatory cytokine toxicity.Conclusions/interpretationEndoC-βH1 beta cells are characterised by an imbalance between H2O2-generating and -inactivating enzymes, and react to cytokine exposure in a similar manner to primary human beta cells. They are a suitable beta cell surrogate for cytokine-toxicity studies.