1 Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany
2 Centre of Anatomy, Hannover Medical School, Hannover, Germany
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ABSTRACT |
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Pancreatic islets and insulin-producing tissue culture cells are known for their extremely low antioxidative defense status and their unusual susceptibility to reactive oxygen species (ROS) and nitric oxide (NO). In particular, the expression level of the H2O2-inactivating enzymes catalase and glutathione peroxidase is very low (1,2). Cytokine toxicity, as well as oxidative stress, is generally accepted to play an important role in the autoimmune destruction of pancreatic ß-cells during the development of type 1 diabetes (35). It has been shown that overexpression of antioxidant enzymes provides protection against the toxicity of ROS and proinflammatory cytokines (610). However, overexpression of the H2O2-inactivating enzymes catalase and glutathione peroxidase in the cytoplasmic compartment only gave a limited protection of insulin-producing RINm5F cells against cytokine-mediated cell damage (9). Recent studies, however, have provided evidence for a crucial role of the mitochondrial antioxidative defense status for the susceptibility of insulin-producing cells to cytokine toxicity (1113).
Superoxide radicals produced by the respiratory chain through the reduction of molecular oxygen are physiologically inactivated by the mitochondrial superoxide dismutase (MnSOD) to H2O2. This toxic compound can be further reduced by the thioredoxin-dependent peroxiredoxin III and H2O2-inactivating enzymes (14), which, however, are only weakly expressed in insulin-producing cells (1,2). As cytokines can enhance superoxide radical generation and induce MnSOD expression (15), thereby increasing the capacity for superoxide radical inactivation, the capacity for subsequent H2O2 inactivation may become rate limiting under these circumstances (16). Therefore, in this study, we have investigated whether it is possible to improve the cellular antioxidative defense status through targeted overexpression of the H2O2-inactivating enzyme catalase in the mitochondria.
To compare the importance of the intramitochondrial antioxidative defense capacity relative to that of the cytoplasm, we included in this investigation three additional chemical compounds that differ in their specific kinetic behavior, the radicals they release, and, in particular, the sides of their ROS generation: 1) H2O2, which mimics the accumulation of this compound from superoxide dismutation, 2) the hypoxanthine/xanthine oxidase (HX/XO) system (17), which releases superoxide radicals and H2O2 extracellularly and continuously in a long-term manner, and 3) menadione, which generates superoxide radicals in the mitochondrial compartment (18).
In this study, we show that mitochondrial catalase overexpression protects insulin-producing cells and is particularly efficient against toxic chemical compounds, which generate ROS preferentially in the mitochondria as well as against proinflammatory cytokines.
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RESEARCH DESIGN AND METHODS |
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Tissue culture of RINm5F cells.
Insulin-producing RINm5F tissue culture cells (passage no. 75-85) were cultured as described (2) in RPMI-1640 medium, supplemented with 10 mmol/l glucose, 10% (vol/vol) FCS, penicillin, and streptomycin in a humidified atmosphere at 37°C and 5% CO2.
Targeted overexpression of catalase in RINm5F cells.
The pCMV/myc/mito-plasmid was used for overexpression of the mitochondrially targeted human catalase cDNA. At the 5' end, a BssHII restriction site and the 3' end of the mitochondrial leader sequence were added (forward-primer: 5'-GCCGCGCGCCAAGATCCATTCGTTGGCTGACAGCCGGGATCCC-3') by PCR. The 3' end was equipped with an additional NotI restriction site for subcloning (reverse-primer: 5'-TACAGCGGCCGCTCACAGATTTGCCTTCTCCCTT-3'). This BssHII/NotI-cut catalase construct was subcloned in frame with the mitochondrial targeting signal into the precut vector by standard techniques. For the cytoplasmic catalase overexpression, cDNA for human catalase was subcloned into the pcDNA3 expression vector as described in detail earlier (7). RINm5F cells were transfected with the vector DNA by the use of lipofectamine (Invitrogen). Positive clones were selected through resistance against G 418 (250 µg/ml) (Invitrogen) and verified by Northern and Western blot analyses and measurement of enzyme activity. A total of 1520 clones were obtained in each transfection. Clones with expression levels comparable to those in liver were selected for the present study. The cells transfected with the pCMV/myc/mito vector lacking insert were used as the control cells. In control experiments, it was confirmed that transfection with the pCMV/myc/mito vector lacking insert did not affect the expression of catalase. Transfection with the mitochondrial catalase cDNA did not affect insulin content and basal and KCl-induced insulin secretion. The insulin content and basal and KCl-induced insulin secretion were as follows in the control and catalase-overexpressing RINm5F cells clones, respectively: insulin content (in ng/µg DNA): control, 4.3 ± 0.2; cyto, 3.9 ± 0.2; mito 1, 4.1 ± 0.2; mito 2, 3.8 ± 0.2; basal insulin secretion (in pg · µg DNA 1 · h1): control, 156 ± 37; cyto, 169 ± 28; mito 1, 189 ± 29; mito 2, 136 ± 18; KCl-induced insulin secretion (in ng · µg DNA 1 · h1): control, 3.3 ± 0.3; cyto, 2.9 ± 0.2; mito 1, 3.3 ± 0.2; mito 2, 2.8 ± 0.2.
Exposure to chemical compounds.
Control and catalase-overexpressing RINm5F cells were seeded at a concentration of 5 x 104 cells/well in 100 µl culture medium in 96-well microplates and allowed to attach for a period of 24 h before they were incubated at 37°C with the different test compounds. Cells exposed to serial concentrations of H2O2 were incubated for 2 h in Hepes (20 mmol/l) supplemented Krebs-Ringer bicarbonate medium with 5 mmol/l glucose and, after removal of H2O2, for another 18 h in RPMI-1640 medium. Cells exposed to a mixture of HX/XO or menadione were incubated with these compounds in RPMI-1640 medium for 18 h. After the overnight incubation period, the viability of the cells was determined using a microtiter platebased MTT assay (19). Viability was expressed as the percentage of untreated samples.
Cytokine exposure.
For MTT assays, control and catalase-overexpressing RINm5F cells were seeded at a concentration of 6,000 cells/well in 100 µl culture medium in 96-well microplates and allowed to attach for a period of 24 h before 600 units/ml human interleukin-1ß (IL-1ß) (PromoCell, Heidelberg, Germany) or a combination of cytokines (called here 1 x cytokine mixture or 0.5 x cytokine mixture) were added for a 72-h time period. The 1 x cytokine mixture consisted of 60 units/ml IL-1ß, 185 units/ml human tumor necrosis factor- (TNF-
), and 14 units/ml rat
-interferon (IFN-
) (PromoCell). After the incubation period, the viability of the cells was determined using a microtiter platebased MTT assay (19). Viability was expressed as the percentage of untreated samples. Vital dye staining using Hoechst dye and propidium iodide revealed >95% attached viable cells in all RINm5F clones in the absence of cytokines or chemical generators of free radicals. The absolute optical density (OD550) absorbance rates of the MTT assay at 72 h were as follows in the control and catalase-overexpressing RINm5F cell clones, respectively: control, 486 ± 55 mOD550; cyto, 525 ± 58 mOD550; mito 1,509 ± 42 mOD550; mito 2, 537 ± 40 mOD550 (n = 10 in each group). The proliferation rate was determined by BrdU incorporation and detection with a specific enzyme-linked immunosorbent assay (ELISA) from Roche (Mannheim, Germany) as described earlier (9). The cells were incubated for 24 h with 600 units/ml human IL-1ß or with a cytokine mixture for Western blot analyses, the measurement of accumulated nitrite, and the measurement of inducible NO synthase (iNOS) promoter activation or 72 h for ultrastructural analyses. Glucose oxidation in the different cell clones after a 72-h incubation with 600 units/ml human IL-1ß or with the 1 x cytokine mixture was determined at 10 mmol/l [U-14C]glucose (1 Ci/mol) as described earlier (20).
Tissue fractionation.
The cells were homogenized in 500 µl of ice-cold H-medium (70 mmol/l sucrose, 210 mmol/l mannitol, 20 mmol/l HEPES, 0.5 mmol/l EGTA, pH 7.4) using a Potter-Elvehjem homogenizer with a teflon pestle and maintained on ice. Fractions were obtained through differential centrifugation. Supernatant after centrifugation for 10 min at 100g was taken as whole-cell extract and used for Western blot analyses and enzyme activity measurements. The sediment of cell debris was discarded. The mitochondrial fraction was obtained through centrifugation for 10 min at 8,000g at 4°C. The supernatant was then centrifuged for another 90 min at 100,000g at 4°C to separate the microsomal and the cytoplasmic fraction (21).
Northern blot analysis of catalase expression.
Total RNA from RINm5F cells was isolated by a combined water-saturated phenol-chloroform-isoamyl alcohol extraction according to Chomczynski and Sacchi (22). Ten micrograms total RNA per lane were separated through electrophoresis on denaturing formamide/formaldehyde 1% agarose gels and transferred to nylon membranes. Hybridization was performed, as described before (7), using a digoxigenin-labeled cRNA probe coding for human catalase. The digoxigenin-labeled hybrids were detected by an enzyme-linked immunoassay followed by chemiluminescence detection.
Western blot analyses.
RINm5F cell subcellular fractions (for catalase detection) or whole-cell extracts (for MnSOD detection) were sonified in ice-cold PBS on ice for 15 s at 60 W with a Braun-Sonic 125 sonifier. Protein content was determined by the bicinchoninic acid assay (Pierce, Rockford, IL). SDS-PAGE was used to fractionate 10 µg (for catalase) or 20 µg (for MnSOD) of protein, which was then transferred to polyvinylidene fluoride membranes. Nonspecific binding sites of the membranes were blocked by 5% nonfat dry milk overnight at 4°C. Then the blots were incubated with a specific primary antibody against bovine liver catalase at a dilution of 1:5,000 for 1 h at room temperature or with a specific primary antibody against rat MnSOD at a dilution of 1:10,000 for 1 h at room temperature followed by a 1-h incubation period with a peroxidase-labeled secondary antibody at a dilution of 1:15,000 at room temperature. The protein bands were visualized by chemiluminescence using the enhanced chemiluminescence detection system (Amersham Bioscience, Freiburg, Germany). The intensity of the bands was quantified through densitometry with the Gel-Pro Analyzer 4.0 program (Media Cybernetics, Silver Spring, MD).
Catalase enzyme activity measurements.
Catalase enzyme activity in homogenates and subcellular fractions was measured by ultraviolet spectroscopy, monitoring the decomposition of H2O2 at 240 nm as described previously (7). One unit of catalase activity was defined as 1 µmol H2O2/min at 25°C.
Ultrastructural characterization of cell viability and integrity.
For electron microscopy, cell pellets were fixed in 2% paraformaldehyde and 2% glutaraldehyde, 0.1 mol/l cacodylate buffer, pH 7.3, postfixed in 1% OsO4, and finally embedded in Epon. Thin sections were contrast stained with saturated solutions of lead citrate and uranyl acetate and viewed in an electron microscope (23).
Nitrite measurements and iNOS promoter reporter gene assay.
Nitrite accumulation after incubation with IL-1ß or with a cytokine mixture was determined spectrophotometrically at 562 nm by the Griess reaction as described earlier (9). For the iNOS promoter reporter gene assay, 2 x 104 cells/well were seeded in 96-well plates 24 h before transient transfection was performed and 48 h before the incubation with IL-1ß or with a cytokine mixture. The pSEAP-iNOS construct was used as described in detail before (13).
Statistical analyses.
Data are expressed as means ± SE. Unless stated otherwise, statistical analyses were performed using ANOVA plus Bonferroni test for multiple comparisons. Half-maximal concentration (EC50) values were calculated from nonlinear regression analyses using least square algorithms of the Prism analysis program (Graphpad, San Diego, CA).
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RESULTS |
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Effects of targeted catalase overexpression on the toxicity of chemical compounds in the MTT test
Toxicity of H2O2.
H2O2 caused a concentration-dependent loss of cell viability in the MTT assay, in control cells, and in cell clones overexpressing catalase in the cytoplasmic (cyto) and mitochondrial (mito 1 and mito 2) compartment (Fig. 2A). In control RINm5F cells, the EC50 of H2O2, that is, the concentration of the toxin at which 50% of the of viability of the cells was lost, was 52 µmol/l (Table 2). In the clones mito 1 and 2, which overexpressed catalase preferentially in the mitochondrial compartment, the EC50 values were 129 and 231 µmol/l, respectively, and thus significantly (P < 0.01) higher than in control cells (Table 2). In the cyto clone, with preferential catalase overexpression in the cytoplasmic compartment, the EC50 value was 346 µmol/l for the H2O2 toxicity, and thus even significantly (P < 0.01) higher than in the mito 1 and 2 clones (Table 2). Therefore, the protection against H2O2 toxicity was significantly better in the cyto clone than in the clones with a preferential mitochondrial catalase overexpression (Table 2).
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Toxicity of HX/XO.
The HX/XO free radicalgenerating system caused a concentration-dependent loss of cell viability in the MTT assay (Fig. 2C). However, in comparison with control cells, protection against HX/XO toxicity was significant only in the cells with the preferential cytoplasmic catalase overexpression and in the cells with a high level of preferential mitochondrial catalase overexpression, as documented by the significantly (P < 0.01) higher EC50 values of 4.0 and 5.8 mU/ml, respectively (Table 2). So, significant protection against HX/XO toxicity requires a high catalase enzyme activity in the cytoplasmic compartment.
Effects of targeted catalase overexpression on the toxicity of cytokines in the MTT test
IL-1ß.
Incubation of control RINm5F cells with IL-1ß (600 units/ml) caused a loss of cell viability in the MTT assay of 40% (Table 3). In the cyto clone, with a preferential cytoplasmic overexpression, and in the mito 1 clone, with a preferential mitochondrial overexpression of catalase, IL-1ß (600 units/ml) caused a reduction in the cell viability of about one-third in both clones, which was not significantly different from the viability loss in the control clone (Table 3). In the mito 2 clone, with the highest catalase activity in the mitochondrial compartment, however, this protective effect of catalase overexpression was significant (P < 0.01), as evidenced by a <20% reduction of the cell viability as compared with control cells (Table 3). This protection was also significantly better than that observed in the cyto clone (Table 3). Thus, the protection of RINm5F cells against IL-1ß toxicity crucially depends on the level of catalase activity in the mitochondrial compartment.
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Differences measured in the MTT assay between the different catalase-overexpressing cell clones were not caused by differences in the proliferation rate. Only the proliferation rate of nontransfected controls was significantly lower (P < 0.05), while all catalase-transfected clones did not differ in their proliferation rate. The proliferation rate (percentage of untreated cells) after a 72-h incubation was as follows in the control and catalase-overexpressing RINm5F clones. Incubation with 600 units/ml IL-1ß: control cells, 85 ± 2%; cyto clone, 96 ± 2%; mito 1 clone, 91 ± 4%; mito 2 clone, 97 ± 2%. Incubation with a 1 x cytokine mixture: control cells, 59 ± 4%; cyto clone, 89 ± 2%; mito 1 clone, 91 ± 2%; mito 2 clone, 94 ± 2% (means ± SE of four experiments). The absolute proliferation values under control conditions did not differ among nontransfected, cyto, and mito RINm5F cell clones (data not shown). Therefore, the differences in the MTT assay do not represent different replication rates in the different catalase-overexpressing clones.
Catalase enzyme activity in the different catalase-overexpressing clones was not differentially affected by cytokine exposure. After incubation with 600 units/ml IL-1ß or the 1 x cytokine mixture, all clones showed a reduction in catalase enzyme activity; however, there were no significant differences between the different clones. After an IL-1ß incubation, the remaining enzyme activities were as follows: cyto clone, 78 ± 10%; mito 1 clone, 81 ± 17%; mito 2 clone, 77 ± 16% (means ± SE of four experiments). After a cytokine mixture incubation, the remaining enzyme activities were as follows: cyto clone, 75 ± 3%; mito 1 clone, 92 ± 8%; mito 2 clone, 85 ± 14% (means ± SE of three to four experiments).
Effects of targeted catalase overexpression on glucose oxidation after cytokine exposure
IL-1ß.
Cytoplasmic but not mitochondrial catalase overexpression was accompanied by a slight reduction of glucose oxidation under control conditions (Table 4). Incubation of control RINm5F cells with IL-1ß (600 units/ml) caused a decrease of the glucose oxidation rate by 60% (Table 4). A comparable reduction of the glucose oxidation rate that was not significantly lower than in the control cell clone was observed in the cyto and the mito 1 clones (Table 4). No inhibitory effect of IL-1ß, on the other hand, was seen in the mito 2 clone, with the highest catalase activity in the mitochondrial compartment. Rather, the glucose oxidation rate was found to be higher than under control conditions (Table 4).
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Effects of targeted catalase overexpression on the toxicity of cytokines in ultrastructural analysis
IL-1ß.
To compare the results obtained in the MTT assay and in the glucose oxidation experiments with morphological features of cell death, we performed a quantitative ultrastructural analysis through electron microscopy (Table 5). IL-1ß (600 units/ml) alone had a weak toxic effect in RINm5F control cells, with 19% of cells destroyed (Table 5). Overexpression of catalase resulted in a significant protection against IL-1ß toxicity in the cyto and mito clones. The greatest protection was evident in the mito 2 clone (Table 5).
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The induction of the iNOS promoter after a 24-h incubation with IL-1ß (600 units/ml) or with the 1 x cytokine mixture is expressed as the percentage induction of the iNOS promoter activity under nonstimulated control conditions. The data are given as means ± SE from at least six individual experiments (control, IL-1ß: 850 ± 53%, 1 x cytokine mixture: 805 ± 70%; cyto, IL-1ß: 945 ± 69%, 1 x cytokine mixture: 938 ± 115%; mito 1, IL-1ß: 700 ± 102%, 1 x cytokine mixture: 689 ± 71%; mito 2, IL-1ß: 1,063 ± 70%, 1 x cytokine mixture: 922 ± 67%).
Accumulated nitrite (in pmol/µg protein) after a 24-h incubation was as follows: control, nonstimulated: 0.28 ± 0.05, IL-1ß: 1.70 ± 0.54, 1 x cytokine mixture: 1.61 ± 0.28; cyto, nonstimulated: 0.28 ± 0.17, IL-1ß: 1.71 ± 0.24, 1 x cytokine mixture: 1.61 ± 0.29; mito 1, nonstimulated: 0.25 ± 0.10, IL-1ß: 1.90 ± 0.36, 1 x cytokine mixture: 1.38 ± 0.50; mito 2, nonstimulated: 0.26 ± 0.16, IL-1ß: 1.28 ± 0.51, 1 x cytokine mixture: 1.11 ± 0.31. The data are given as means ± SE from four individual experiments.
Effects of targeted catalase overexpression on MnSOD protein expression after exposure to IL-1ß alone or a cytokine mixture
Incubation of RINm5F cells, both with IL-1ß (600 units/ml) alone and with the cytokine mixture (60 units/ml IL-1ß, 185 units/ml TNF-, 14 units/ml IFN-
), resulted in a significant increase of MnSOD protein expression (Fig. 4). This increase of MnSOD expression was not significantly affected by catalase overexpression in the cyto and the mito 1 and 2 clones (Fig. 4).
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DISCUSSION |
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H2O2-inactivating enzymes are typically considered to reside in the cytoplasmic compartment of the cell (24). In the present study, we also observed a minor fraction of catalase expression in the mitochondrial compartment of insulin-producing cells, thus confirming corresponding observations in other cell types (2527). However, the expression level of the H2O2-inactivating enzymes glutathione peroxidase and catalase, both of which convert H2O2 into H2O, is extremely low in insulin-producing cells, comprising only 1% of the expression level in liver (1,2). This represents a particular challenge for the antioxidative defense situation of the mitochondria in insulin-producing cells because these organelles are physiologically the major site of superoxide radical formation in the cell (28,29). The imbalance between superoxide radical and H2O2-inactivating enzymes in the mitochondrial compartment makes pancreatic ß-cells particularly vulnerable to oxidative stress.
To determine the importance of the intramitochondrial antioxidative defense status for cellular survival, we bioengineered, through targeted expression, insulin-producing RINm5F cells that stably overexpressed catalase preferentially in the mitochondria or in the cytoplasm. In these cell clones, we studied the protective effects of catalase overexpression against the toxic action of a number of ROS-generating chemical compounds as well as cytokines.
Catalase overexpression in the cytoplasmic or in the mitochondrial compartment protected insulin-producing RINm5F cells against the toxicity of ROS, which is in agreement with observations made in HepG2 hepatoma cells (30). Interestingly, however, it was only the high mitochondrial overexpression of catalase that provided significant protection against cell death in the case of menadione toxicity, a compound that preferentially generates superoxide radicals intramitochondrially (31,32). In contrast, catalase overexpression in the cytoplasmic cell compartment was not effective, thus confirming an earlier observation (7).
On the other hand, the cell clones with mitochondrial catalase overexpression were less well protected against cell damage than cells with catalase overexpression in the cytoplasmic compartment when exposed to H2O2. This is not surprising because catalase in the cytoplasmic compartment is better suited to inactivate H2O2 entering the cell from the extracellular space, thus inactivating H2O2 before causing damage to the cytoplasmic matrix and before reaching the mitochondria. This confirms the results of earlier studies (7), and it is also in agreement with the observation that exogenous addition of catalase to the incubation medium provided complete protection against H2O2 toxicity (7).
When cells are incubated with HX/XO, H2O2 will arise extracellularly through an enzymatic reaction at a relatively lower concentration than in the case of the incubation with H2O2 (7,17), but it will be generated continuously over a prolonged period. Comparing the cell clones cyto and mito 1 with the same level of total catalase activity, the better protection was observed in the cyto clone with the preferential cytoplasmic catalase overexpression rather than in the mito 1 clone with the preferential mitochondrial catalase overexpression. This is not surprising, as in analogy to the situation in the experiments with H2O2 incubation, catalase in the cytoplasmic compartment is better suited to inactivate H2O2 entering the cell from the extracellular space. Nevertheless, a further improvement of the protection was obtained through an additional increase of the catalase activity in the mitochondrial compartment, as is evident from the comparison of the defense situation in the mito 2 clone, which showed an even better protection with a comparable level of cytoplasmic catalase overexpression but a higher level of catalase activity in the mitochondria than the cyto clone.
Thus, it can be concluded from the experiments with ROS-generating compounds that targeted overexpression of catalase in the mitochondria provide particularly effective protection against cell death when ROS are generated intramitochondrially.
Using these catalase-overexpressing cell clones, we addressed the question of whether an enhanced intramitochondrial H2O2-inactivating capacity might also improve the resistance against cytokine-mediated toxicity, since ROS have also been considered to be mediators of the proinflammatory action of cytokines (3,4,33,34).
Both IL-1ß and mixtures of proinflammatory cytokines favor the intramitochondrial generation of H2O2 through induction of MnSOD expression (3538), an observation that we confirmed in the present study for insulin-producing RINm5F cells. Though there was no difference in the degree of MnSOD expression among the different catalase-overexpressing clones, the protection against cytokine-induced cell death, as analyzed in the MTT assay, was significantly better in the clones overexpressing catalase preferentially in the mitochondria. In line with previous observations, cytoplasmic overexpression of catalase provided less protection (9,39).
The observed high rate of cell death after exposure to a cytokine mixture composed of IL-1ß, TNF-, and IFN-
when compared with the weaker effect of IL-1ß alone may be due to TNF-
induced ROS formation in the mitochondria. A number of recent reports have shown direct ROS generation after exposure of cells to TNF-
(4043), emphasizing a major role of ceramide as a mediator of superoxide radical formation at the ubisemiquinone site (41,44,45). An enhancement of the toxicity of IL-1ß by an additional induction of MnSOD gene expression by TNF-
(E.G., unpublished observations) provides additional support for increased H2O2 generation (16). IL-1ß toxicity is enhanced in the presence of TNF-
and IFN-
probably by a signaling pathways cross talk between these cytokines (4648). In addition, the induction of iNOS and the subsequent production of NO through cytokines is able to reduce catalase enzyme activity by binding to the iron of the catalase haem groups (49). Although the mitochondrial localization of catalase could theoretically decrease this inactivation reaction, this was not the case. Thus, differences in NO-mediated inhibition of catalase enzyme activity cannot account for the better protection of mitochondrial catalase overexpression against cytokine toxicity.
The iNOS promoter activity and nitrite accumulation after exposure to IL-1ß alone or to a cytokine mixture were not significantly affected both in the clones overexpressing catalase in the cytoplasm and in the mitochondria. This is in contrast to MnSOD suppression, which significantly increases iNOS promoter activity and confirms earlier observations made with RINm5F cells overexpressing catalase in the cytoplasm (9,13).
Both IL-1ß alone and the cytokine mixture significantly inhibited glucose oxidation and overexpression of catalase in the cyto clone and, in the mito 1 clone, did not prevent this inhibition, thus confirming earlier observations (50). Only the high catalase expression level in the mitochondria in the mito 2 clone was able to prevent the cytokine-induced inhibition of glucose oxidation. These results indicate that a high mitochondrial capacity for H2O2 inactivation is obligatory to maintain mitochondrial function, as documented by both preservation of dehydrogenase enzyme activity and glucose oxidation capacity.
The observations made in the present study in the MTT assay and the glucose oxidation studies that targeted overexpression of catalase in the mitochondria, providing particularly effective protection, were confirmed in the present study on the ultrastructural level. Nearly all cells of the mito 2 clone remained intact after cytokine exposure. Ultrastructural analyses additionally revealed well-preserved mitochondria, even in the few cells of the mito 2 clone in which the plasma membrane and the membranes of intracellular organelles other than mitochondria had been damaged through the cytokine action. This was different from the situation observed in the damaged cells of the cyto clone, in which virtually no intact mitochondria could be detected.
While the toxic effect of IL-1ß alone was somewhat more pronounced in the MTT assay, in the case of the incubation with the cytokine mixture the proportion of destroyed cells was larger on the ultrastructural level. This indicates that a combination of IL-1ß, TNF-, and IFN-
compared with IL-1ß alone decreased the cellular membrane integrity more than IL-1ß alone, which had a proportionally larger effect on mitochondrial function. This may be related to an activation of additional pathways of cytokine signaling by the cytokine mixture (42,56).
The results clearly indicate that mitochondrial overexpression of catalase provides superior protection against cytokine-mediated cell destruction and death. The protection achieved through catalase overexpression in the mitochondria is apparently a result of the inactivation of the toxic H2O2, which is produced in increased amounts (16,42) through cytokine-induced MnSOD upregulation (13,36,52) in conjunction with cytokine-induced superoxide radical production (44,45). A high level of catalase expression in the mitochondria guarantees a fast and efficient inactivation of the toxic H2O2 and at the same time prevents formation of highly toxic hydroxyl radicals in the Haber-Weiss reaction (53). While the initial mitochondrial damage is likely to be caused by H2O2 itself, progressive destruction of the proteins in the mitochondria may facilitate the release of Fe2+ and other trace metals, which subsequently amplify the destructive potential through hydroxyl radical formation via the Haber-Weiss reaction (53). Thus, it can be concluded that increased intramitochondrial H2O2 generation contributes significantly to cytokine-induced cell death. This is particularly unfavorable in insulin-producing cells because of the extremely low constitutive level of the H2O2-inactivating enzymes and thus provides a plausible explanation for the particular sensitivity of pancreatic ß-cells against ROS-mediated cytokine toxicity (54,55).
A further aggravation can be anticipated in situations of increased and prolonged hyperglycemia when, during autoimmune attack in a developing diabetic metabolic state, the remaining pancreatic ß-cells are under particular functional workload (56,57). The resulting increased rate of glucose metabolism and oxygen consumption in insulin-producing cells (51,58) will foster the generation of superoxide radicals in the respiratory chain (59). This vicious cycle will ultimately reduce the chances of survival of the remaining ß-cells under autoimmune attack (6064). This scenario may also help to explain the causes underlying glucotoxicity in situations of prolonged hyperglycemia (6567).
Our results provide evidence that the mitochondrial targeting of catalase is an attractive strategy to protect insulin-producing cells against oxidative injury and cytokine toxicity. This may also open new perspectives for antioxidative gene therapy of type 1 diabetes.
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ACKNOWLEDGMENTS |
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The authors thank Dr. K. Asayama (Kitakyushu, Japan) for the antibody against MnSOD and Dr. R. Munday (Hamilton, New Zealand) for helpful advice and discussion.
Address correspondence and reprint requests to Prof. Sigurd Lenzen, Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany
Received for publication January 12, 2004 and accepted in revised form May 21, 2004
ELISA, enzyme-linked immunosorbent assay; HX/XO, hypoxanthine/xanthine oxidase; IFN-,
-interferon; IL-1ß, interleukin-1ß; iNOS, inducible nitric oxide synthase; MnSOD, mitochondrial superoxide dismutase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; ROS, reactive oxygen species; SEAP, secreted alkaline phosphatase; TNF-
, tumor necrosis factor-
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References |
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