Stephan Lortz

Relation Between Antioxidant Enzyme Gene Expression and Antioxidative Defense Status of Insulin-Producing Cells

Antioxidant enzyme expression was determined in rat pancreatic islets and RINm5F insulin-producing cells on the level of mRNA, protein, and enzyme activity in comparison with 11 other rat tissues. Although superoxide dismutase expression was in the range of 30% of the liver values, the expression of the hydrogen peroxide-inactivating enzymes catalase and glutathione per-oxidase was extremely low, in the range of 5% of the liver. Pancreatic islets but not RINm5F cells expressed an additional phospholipid hydroperoxide glutathione peroxidase that exerted protective effects against lipid peroxidation of the plasma membrane. Regression analysis for mRNA and protein expression and enzyme activities from 12 rat tissues revealed that the mRNA levels determine the enzyme activities of the tissues. The induction of cellular stress by high glucose, high oxygen, and heat shock treatment did not affect antioxidant enzyme expression in rat pancreatic islets or in RINm5F cells. Thus insulin-producing cells cannot adapt the low antioxidant enzyme activity levels to typical situations of cellular stress by an upregulation of gene expression. Through stable transfection, however, we were able to increase catalase and glutathione peroxidase gene expression in RINm5F cells, resulting in enzyme activities more than 100-fold higher than in nontransfected controls. Catalase-transfected RINm5F cells showed a 10-fold greater resistance toward hydrogen peroxide toxicity, whereas glutathione peroxidase overexpression was much less effective. Thus inactiva-tion of hydrogen peroxide through catalase seems to be a step of critical importance for the removal of reactive oxygen species in insulin-producing cells. Overexpression of catalase may therefore be an effective means of preventing the toxic action of reactive oxygen species.

Protection against the co-operative toxicity of nitric oxide and oxygen free radicals by overexpression of antioxidant enzymes in bioengineered insulin-producing RINm5F cells

Aims/hypothesis. The importance of different antioxidative enzymes for the defence of insulin-producing cells against the toxicity of nitric oxide (NO) was characterised in bioengineered RINm5F cells. Methods. RINm5F insulin-producing cells stably overexpressing glutathione peroxidase (GPX), catalase (CAT) or Cu/Zn superoxide dismutase (SOD) were exposed to S-nitroso-N-acetyl-d,l-penicillamine (SNAP), sodium nitroprusside (SNP) and 3 morpholinosydnonimine (SIN-1), which generate both NO and reactive oxygen species, and to the polyamine/NO, complex DETA/NO which generates NO alone. The viability of the cells was tested by the MTT assay. Results. Overexpression of antioxidant enzymes provided significant protection against the toxicity of SNAP, SNP and SIN-1, with an individual specificity related to their chemical characteristics, but was without effect upon the toxicity of DETA/NO. Cells overexpressing GPX were well protected against SNP and SNAP, while CAT was most effective against SIN-1. SOD overexpression provided less protection against the toxicity of SNAP and SNP than overexpression of GPX but was more effective in protecting against SIN-1. Co-incubation of cells with NO donors and hydrogen peroxide or hypoxanthine and xanthine oxidase showed an overadditive synergism of toxicity. Conclusion/interpretation. The results emphasise the importance of a synergism between NO and reactive oxygen species for pancreatic beta-cell death. Such a synergism has also been observed after exposure of beta cells to cytokines. The component of the toxicity that is mediated by oxygen radicals can be suppressed effectively through overexpression of CAT, GPX or SOD or both. [Diabetologia (1999) 42: 849–855]

Sequential inactivation of reactive oxygen species by combined overexpression of SOD isoforms and catalase in insulin-producing cells

Insulin-producing cells show very low activity levels of the cytoprotective enzymes catalase, glutathione peroxidase, and superoxide dismutase. This weak antioxidative defense status has been considered a major feature of the poor resistance against oxidative stress. Therefore, we analyzed the protective effect of a combined overexpression of Cu,ZnSOD or MnSOD together with different levels of catalase. Catalase alone was able to increase the resistance of transfected RINm5F insulin-producing tissue culture cells against H(2)O(2) and HX/XO, but no protection was seen in the case of menadione. In combination with an increase of the MnSOD or Cu,ZnSOD expression, the protective action of catalase overexpression could be further increased and extended to the toxicity of menadione. Thus, optimal protection of insulin-producing cells against oxidative stress-mediated toxicity requires a combined overexpression of both superoxide- and hydrogen peroxide-inactivating enzymes. This treatment can compensate for the constitutively low level of antioxidant enzyme expression in insulin-producing cells and may provide an improved protection in situations of free radical-mediated destruction of pancreatic beta cells in the process of autoimmune diabetes development.

The LEW.1AR1/Ztm-iddm rat: a new model of spontaneous insulin-dependent diabetes mellitus.

Abstract.Aims/hypothesis: We describe a new Type I (insulin-dependent) diabetes mellitus rat model (LEW.1AR1/Ztm-iddm) which arose through a spontaneous mutation in a congenic Lewis rat strain with a defined MHC haplotype (RT1.Aa B/Du Cu).Methods: The development of diabetes was characterised using biochemical, immunological and morphological methods. Results: Diabetes appeared in the rats with an incidence of 20 % without major sex preference at 58 ± 2 days. The disease was characterised by hyperglycaemia, glycosuria, ketonuria and polyuria. In peripheral blood, the proportion of T lymphocytes was in the normal range expressing the RT6.1 differentiation antigen. Islets were heavily infiltrated with B and T lymphocytes, macrophages and NK cells with beta cells rapidly destroyed through apoptosis in areas of insulitis. Conclusion/interpretation: This Type I diabetic rat develops a spontaneous insulin-dependent autoimmune diabetes through beta cell apoptosis. It could prove to be a valuable new animal model for clarifying the mechanisms involved in the development of autoimmune diabetes. [Diabetologia (2001) 44: 1189–1196]

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

Aims/hypothesisPro-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.MethodsXbp1s was overexpressed using adenoviruses and knocked down using small interference RNA in rat islet cells. In selected experiments, Xbp1 was also knocked down in FACS-purified rat beta cells and rat fibroblasts. Expression and production of XBP1s and key downstream genes and proteins was measured and beta cell function and viability were evaluated.ResultsAdenoviral-mediated overproduction of Xbp1s resulted in increased XBP1 activity and induction of several XBP1s target genes. Surprisingly, XBP1s overexpression impaired glucose-stimulated insulin secretion and increased beta cell apoptosis, whereas it protected fibroblasts against cell death induced by ER-stress. mRNA expression of Pdx1 and Mafa was inhibited in cells overproducing XBP1s, leading to decreased insulin expression. XBP1s knockdown partially restored cytokine/ER-stress-driven insulin and Pdx1 inhibition but had no effect on cytokine-induced ER-stress and apoptosis.Conclusions/interpretationXBP1 has a distinct inhibitory role in beta cell as compared with other cell types. Prolonged XBP1s production hampers beta cell function via inhibition of insulin, Pdx1 and Mafa expression, eventually leading to beta cell apoptosis.

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.

Effects of metformin on SGLT1, GLUT2, and GLUT5 hexose transporter gene expression in small intestine from rats

The effect of the antihyperglycaemic agent metformin was studied on gene expression of the energy-dependent sodium-hexose cotransporter (SGLT1) and the facilitative hexose transporters GLUT2 and GLUT5 in rat intestine. Metformin treatment (125 mg/kg body wt. twice daily for a period of 3 days) significantly increased SGLT1 gene expression in duodenum and jejunum. GLUT5 gene expression was increased by metformin treatment only in the jejunum. Gene expression of GLUT2 in the intestine was not significantly affected by metformin treatment. This increase in transporter gene expression offers the potential for increases in hexose uptake at the brush border membrane, and may compensate for other effects of the drug that have been suggested to decrease glucose uptake by SGLT1, as well as for metformin stimulation of glucose utilization by the intestinal mucosa.

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 antioxidant enzyme catalase does not interfere with the glucose responsiveness of insulin-secreting INS-1E cells and rat islets

Aims/hypothesisHydrogen peroxide (H2O2)-inactivating enzymes such as catalase are produced in extraordinarily low levels in beta cells. Whether this low expression might be related to a signalling function of H2O2 within the beta cell is unknown. A high level of H2O2-inactivating enzymes could potentially be incompatible with glucose-induced insulin secretion. Therefore the effect of catalase overexpression on mitochondrial function and physiological insulin secretion was studied in insulin-secreting INS-1E and primary islet cells.MethodsINS-1E and rat islet cells were lentivirally transduced to overexpress catalase in the cytosol (CytoCat) or in mitochondria (MitoCat). Cell viability and caspase-3 activation were assessed after cytokine incubation and hypoxia. Insulin secretion was quantified and expression of the gene encoding the mitochondrial uncoupling protein 2 (Ucp2) was measured in parallel to mitochondrial membrane potential and reactive oxygen species (ROS) formation.ResultsThe ability to secret insulin in a glucose-dependent manner was not suppressed by catalase overexpression, although the glucose-dependent increase in the mitochondrial membrane potential was attenuated in MitoCat cells along with an increased Ucp2 expression and reduced mitochondrial ROS formation. In addition, MitoCat overexpressing cells were significantly more resistant against pro-inflammatory cytokines and hypoxia than CytoCat and control cells.Conclusions/interpretationThe results demonstrate that an improved antioxidative defence status of insulin-secreting cells allowing efficient H2O2 inactivation is not incompatible with proper insulin secretory responsiveness to glucose stimulation and provide no support for a signalling role of H2O2 in insulin-secreting cells. Interestingly, the results also document for the first time that the decreased ROS formation with increasing glucose concentrations is of mitochondrial origin.