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B Activation in Hippocampus of Diabetic Rats
Department of Experimental Medicine and Oncology (M.A., R.M., G.R., M.P., O.D.) General Pathology Section, and Department of Clinical Pathophysiology (E.B., M.C., R.M., G.B.), University of Turin, Turin 10126, Italy
Address all correspondence and requests for reprints to: Prof. Giuseppe Boccuzzi, Department of Clinical Pathophysiology, Via Genova 3, University of Turin, Turin 10126, Italy. E-mail: giuseppe.boccuzzi{at}unito.it.
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Abstract |
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B (NF-B), which in turn activates a variety of target genes linked to the development of diabetic complications. Dehydroepiandrosterone, an adrenal steroid, which possesses a multitargeted antioxidant effects, is also synthesized de novo by the brain. Normoglycemic and streptozotocin-diabetic rats were either treated with dehydroepiandrosterone (DHEA) for 7, 14, or 21 d (4 mg/d per rat) or left untreated. Oxidative state, antioxidant balance and activation of nuclear transcriptional redox-sensitive factor NF-B were evaluated in the hippocampus area. In streptozotocin-treated rats, besides the strong increase in oxygen reactive species, there is also a persistent activation of NF-B. The derangement of the oxidative balance in the brain induced by diabetes improves with DHEA. Moreover, DHEA completely counteracts NF-B activation, measured as DNA binding activity, and hinders the increase of IB-
inhibitory subunit induced by oxidative stress. The time-lag of DHEA’s effects on NF-B activation parallels its effects on oxidative balance. Results indicate that DHEA might protect hippocampus from chronic activation of NF-B-dependent genes by reducing NF-B nuclear translocation. This could result in protection from diabetes-dependent brain damage. |
Introduction |
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A major target of reactive oxygen species is transcription factor nuclear factor-B (NF-B). NF-B exists in the cytoplasm as a dimer in an inactive multisubunit complex bound to one of the inhibitory proteins, called IB. Oxidative stress triggers the dissociation of the IB from the NF-B complex: this involves phosphorylation of the IB- subunit leading to its rapid degradation (4). NF-B activates a variety of target B-dependent genes relevant to the pathophysiology of inflammation, immune response and atherosclerosis, including cytokines, chemokines, and leukocyte adhesion molecules, as well as genes that regulate cell proliferation and mediate cell survival (5).
It has been also reported that NF-B activates the IB- gene, causing the replenish of the cytoplasmatic pool of its own inhibitor (6). Although NF-B activation might be involved in both self-defense and self-damage mechanisms in the brain’s response to injury (7), convincing data have been published showing that persistent NF-B activation induced by long-lasting oxidative stress is responsible for neuronal damage and consequent promotion of cell death (8).
Several studies have pointed out that NF-B activation is inhibited by a variety of antioxidants, such as N-acetyl-cystein, butylated hydroxy-anisole, vitamin E, and lipoic acid (9): these data suggest that antioxidants affect some steps of signaling events leading to phosphorylation, ubiquination and degradation of IB- (4). The role of oxidative stress and NF-B activation on diabetic complications is well documented (10, 11), moreover antioxidant treatment exerts a beneficial effect in experimental models of chronic injury in diabetes and treatment with antioxidants can significantly reduce diabetic complications (12, 13, 14).
Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S), the most abundant adrenal steroids in the blood, are present in the human hippocampus at concentrations higher than those in the plasma (15) and are synthesized de novo by brain neurones and astrocytes (16). Several roles, including increasing neuronal and glial survival and differentiation, have been attributed to these neuroactive steroids (17). Recent evidence indicates that, at concentrations slightly above those found in human tissues, DHEA possesses a multitargeted antioxidant effect (18, 19, 20) and prevents tissue damage induced by acute and chronic hyperglycemia (21, 22, 23), as well as by transient cerebral ischemia (24).
The present study investigates the effect of chronic DHEA treatment on oxidative stress and on activation of nuclear transcriptional redox-sensitive factor NF-B induced by chronic hyperglycemia in the hippocampus area of streptozotocin (STZ)-treated rats, a district of the brain among the most susceptible to oxidative insult. The results show that DHEA treatment markedly reduces activation of redox-sensitive nuclear factor NF-B induced by chronic hyperglycemia in rat hippocampus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ATP and poly(deoxyinosine-deoxycytidine) were from Amersham UK Ltd. (Buckinghamshire, UK). T4 polynucleotide kinase, buffer 10 x kinase, NF-B oligonucleotide, recombinant human NF-B (p50) were from Promega Corp. (Madison, WI). QIAquick Nucleotide Removal Kit (50) was from QIAGEN GmbH (Hilden, Germany). IB- (C-21), NF-B (p65) and ß-actin antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Unless specifically indicated, all the other reagents were from Sigma (St. Louis, MO).
Experimental protocol
Male Wistar rats (Harlan-Italy, Udine, Italy) weighing 250–280 g were cared for in compliance with the Italian Ministry of Health Guidelines (no. 86/609/EEC) and with the Principles of Laboratory Animal Care (NIH no. 85-23, revised 1985). They were provided with a pellet diet (Piccioni no. 48, Gessate Milanese, Italy) and water ad libitum. Hyperglycemia was induced through a single injection of STZ (50 mg/kg) in the tail vein. Three days later, glycemia was measured (Sigma kit, catalog no. 635) on blood collected from the heart (1 ml). Only rats with blood glucose levels above 20 mmol/liter entered the experimental protocols; normoglycemic rats were used as controls. On d 4 post injection, hyperglycemic and control rats began DHEA treatment.
DHEA was given for 7, 14, or 21 d at 4 mg/d per rat. Crystalline DHEA was dissolved in 1 vol of 95% ethanol, mixed with 9 vol of mineral oil, and given daily by gastric intubation. Controls received vehicle alone. After 7, 14, or 21 d, control rats and hyperglycemic rats, with or without DHEA, were anesthetized with ether and killed by decapitation after aortic exsanguination. Blood was collected and plasma isolated. Cerebral hemispheres were dissected and hippocampus rapidly isolated, weighed and homogenized to extract different fractions. Glycemia was evaluated with o-toluidine reagent (Sigma kit, catalog no. 635). For isolation of cytosolic fraction, the homogenates were centrifuged at 15,000 x g at 4 C for 18 min, then again at 105,000 x g at 4 C for 40 min.
Biochemical determination on cytosolic fraction
DHEA content.
Plasma and aliquots of cytosol of hippocampus were extracted with ethyl-ether, evaporated and the residue redissolved in iso-octane-ethylacetate (94:4, vol:vol) and chromatographed on celite:ethyleneglycol (2:1, vol:vol) microcolumns, using isooctane-benzene (94:4, vol:vol) as mobile phase. RIA was performed on the resulting DHEA chromatographic fraction (25).
Pro-oxidant species.
Redox state was determined by monitoring generation of hydrogen peroxide (H2O2) adding peroxidase from horseradish and acetylated ferrocytochrome c to cytosolic fractions. H2O2 release was evaluated as the increase of the acetylated ferrocytochrome c oxidation rate and was monitored at 550 nm minus 540 nm using an absorption coefficient of 19.9 mmol·l-1·cm-1 as described by Zoccarato et al. (26).
Antioxidant pattern.
Glutathione (GSH) levels were evaluated by the Owens and Belcher method (27). A mixture was directly prepared in cuvette: 0.05 M Na-phosphate buffer, pH 7.0; 1 mM EDTA, pH 7.0; 10 mM dithiobis-nitrobenzoic acid and an aliquot of the sample; the mixture was monitored at 412 nm for 2 min (total thiol), and then a suitable volume of diluted GSH reductase and nicotinamide adenine dinucleotide phosphate reduced form were added (total GSH). Catalase activity was evaluated following Aebi’s method (28). A mixture was directly prepared in cuvette: 0.05 M Na-phosphate buffer and an aliquot of the sample were added and monitored at 240 nm for 1 min. Cu-Zn superoxide dismutase activity was assayed by the method described by Flohè and Otting (29). A solution of 5 mmol of xanthine in 0.001 N sodium hydroxide and 2 mmol of cytochrome c was mixed with 50 mM phosphate buffer (pH 7.8) containing 0.1 mM EDTA. Because the activity of xanthine-oxidase may vary, sufficient enzyme should be used to produce a rate of cytochrome c reduction of at least 0.025 absorbance units/min in the assay without superoxide dismutase. GSH-peroxidase Se-dependent activity was assayed by the method described by Flohè and Gunzler (30), using hydrogen peroxide as substrate and by kinetic analysis at 340 nm in 0.1 M K-phosphate buffer, pH 7.0, containing 1 mM EDTA.
Hydroxynonenal concentration.
HNE concentration was also determined on the fresh cytosolic fractions by the method of Esterbauer et al. (31). An aliquot of cytosol (0.5 ml) was added to an equal volume of acetonitrile:acetic acid (96:4, vol:vol). After centrifugation at 250 x g for 20 min at 4 C, the supernatant was directly injected into an HPLC (Waters Associated, Milford, MA) Symmetry C18 column (5 mm, 3.9 x 150 mm). The mobile phase used was acetonitrile:bidistilled water (42%, vol:vol). The HNE concentration was calculated by comparison with a standard solution of HNE of established concentration.
TNF-immunoassay.
TNF- in cytosol from hippocampus was determined using an enzyme immunoassay specific for rat TNF- (QuantiKine M, R&D Systems, Inc., Minneapolis, MN), following manufacturer’s instructions.
Preparation of nuclear extracts
Nuclear extracts were prepared following Meldrum et al. (32). Briefly, between 90–120 mg of hippocampus tissue were homogenized at 10% (wt/vol) in a Dounce tissue homogenizer (Wheaton, Millville, NJ) using an homogenization buffer containing 20 mM HEPES, pH 7.9; 1 mM MgCl2; 0.5 mM EDTA; 1% Nonidet P-40; 1 mM EGTA; 1 mM dithiothreitol (DTT); 0.5 mM phenylmethylsulfonyl fluoride (PMSF); 5 µg/ml aprotinin; and 2.5 µg/ml leupeptin.
Homogenates were centrifuged at 1000 x g for 5 min at 4 C. Supernatants were removed and pelleted nuclei were resuspended in 300–400 µ l of extraction buffer containing 20 mM HEPES, pH 7.9; 1.5 mM MgCl2; 300 mM NaCl; 0.2 mM EDTA; 20% glycerol; 1 mM EGTA; 1 mM DTT; 0.5 mM PMSF; 5 µg/ml aprotinin; and 2.5 µg/ml leupeptin. The suspensions were incubated on ice for 30 min for high-salt extraction followed by centrifugation at 15,000 x g for 20 min at 4 C.
The resulting supernatants, containing DNA-binding proteins, were carefully removed; protein content was determined using Bradford assay (33) and samples were then stored at -80 C until use.
EMSA
EMSA was performed by the method of Pahl et al. (34). NF-B consensus oligonucleotides (5'-AGTTGAGGGGACTTTCCCAGG-3') were labeled with 32P ATP using T4 polynucleotide kinase and purified on QIAquick Nucleotide Removal Kit. For the EMSA, 50 µg of nuclear proteins were used. Briefly, the samples were incubated with 100,000 cpm of 32P-labeled NF-B oligonucleotide probe for 30 min at room temperature in binding buffer containing 35 mM HEPES-KOH, pH 7.8; 0.5 mM EDTA; 0.5 mM DTT; 10% glycerol; 0.25 mM Spermidine; and 0.1 µg/µl poly(deoxyinosine-deoxycytidine) in a final vol of 40 µl.
Protein-DNA complexes were resolved by electrophoresis through 5% native polyacrylamide gels containing 10% glycerol and 1x Tris-glycine buffer.
Gels were dried under vacuum and exposed for 48–72 h to Amersham Hyperfilms (Amersham Pharmacia Biotech, Braunschweig, Germany) at -80 C with intensifying screens.
Specificity of binding was ascertained by competition with a 25-fold molar excess of unlabeled oligonucleotides. Recombinant human NF-B incubating with the radiolabeled NF-B-probe served as a positive control. NF-B-specific bands were quantified by densitometry using an analytic software (Bio-Rad Laboratories, Inc. MultiAnalyst).
Western blot analyses
IB- and NF-B were detected on cytosolic extracts by the method of Laemmli (35). Aliquots of proteins (25 µg) were separated into 10% sodium dodecyl sulfate-polyacrylamide gels, followed by blotting on nitrocellulose membranes (Amersham Pharmacia Biotech). The membranes were blocked with 5% (wt/vol) nonfat dry milk in 5 mM Tris-HCl, pH 7.4, containing 200 mM NaCl and 0.05% (vol/vol) Tween 20 (TBS-Tween) for 1 h at 25 C, incubated with antibody against IB- and NF-B (p65), respectively, and reacted with peroxidase-labeled antirabbit immunoglobulin (Bio-Rad Laboratories, Inc.) in TBS-Tween containing 2% (w/vol) nonfat dry milk. Immunoreactive proteins were detected with the chemiluminescence assay (Amersham Pharmacia Biotech) and subsequent exposure to film for 2–10 min.
Anti-ß-actin antibody served as the loading control for the cytosolic extract.
Statistical analysis
All results are presented as means ± SD. Differences between means were analyzed for significance using the one-way ANOVA test with the Bonferroni posttest (36).
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Results |
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shows the H2O2 content in the hippocampus cytosol isolated from control and from diabetic rats treated or not with DHEA 4 mg/d for different times: 7, 14, and 21 d. The content of H2O2, increased in diabetic rats after 7, 14, and 21 d. The value of hydrogen peroxide in DHEA-treated STZ rats was markedly reduced with respect to diabetic rats only after 21 d of DHEA treatment (P < 0.05).
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. Diabetic rats, already on d 7, showed a dramatic and statistically significant reduction of levels of these parameters. DHEA treatment (4 mg/d) for 7 d did not prevent the loss of antioxidant defenses induced by hyperglycemia. When DHEA treatment in diabetic rats was extended to 14 d, loss of GSH-peroxydase, and catalase activities was significantly prevented, whereas GSH content remained low. After 21 d of DHEA treatment, hyperglycemic rats again had normal antioxidant defenses. Moreover, DHEA treatment in normoglycemic rats determined a time-dependent, transient induction of the activity of the two enzymes. Both these antioxidant enzymes, after 7 and 14 d of DHEA treatment, showed higher activities (by 15–30%) than those of control group. This stimulatory effect of DHEA treatment did not affect GSH content. In all samples, TNF- levels were undetectable.
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B. EMSA was performed with nuclear extracts of hippocampus and 32P-labeled oligonucleotides containing NF-B binding site. Nuclear extracts of hippocampus were prepared from control and STZ-treated rats with or without DHEA. Figure 2 (top panel) shows DNA-binding activity of NF-B. Nuclear translocation of NF-B in diabetic rats already occurred at 7 d and persisted until the end of the protocol (21 d). Nuclear extracts from the hippocampus of normoglycemic rats treated with DHEA, at 7, 14, and 21 d, did not show any activation of NF-B binding activity. In diabetic rats treated with DHEA for 7 d, the DNA binding activity of NF-B was not affected by the steroid treatment: NF-B signal remained strongly activated as those observed in untreated STZ-rats. After 14 d, DHEA produced a considerable reduction of NF-B activation in diabetic rats, which become even more evident at 21 d. Densitometric analysis of NF-B specific bands was performed (Fig. 2, middle panel). NF-B protein level in the cytosol of hippocampus of rats was determined by Western blot analysis (Fig. 2, bottom panel). Diabetic rats showed a marked reduction of the content of NF-B protein in the cytosol justified by its translocation in the nucleus. DHEA treatment in STZ-rats modulates NF-B translocation into nucleus: in fact, NF-B protein level that was low after 7 d of DHEA treatment, increased in the cytosol at later time points, data consistent with a reduction of DNA binding activity in the nuclear extract of the same animals. Results related to the cytosolic dissociation of NF-B/IB- complex are consistent with these observations. The increased level of isoform IB- (Figure 3) in STZ-treated rats seems to be correlated to rapid nuclear translocation of NF-B, possibly as result of a direct trans-activation of IB- gene by NF-B (37). Immunoblot analysis of cytoplasmic extracts of the hippocampus of diabetic rats showed IB- protein levels to be greatly increased at 7, 14, and 21 d. In normoglycemic rats treated with DHEA, IB- content in the cytosol was not modified compared with control values. When DHEA was given to diabetic rats, responses differed throughout the experimental protocol. At d 7 of DHEA treatment in STZ-rats, the level of IB- protein was high, close to those observed in diabetic rats: DHEA did not show any protective effect on NF-B activation. IB- protein level was significantly decreased after 14 d after DHEA treatment and, as expected, the level of IB- protein after 21 d of DHEA treatment reached levels similar to unstimulated controls. This result confirmed the lack of activation of NF-B at later experimental time points.
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shows HNE concentrations in the hippocampus cytosol of control and diabetic rats treated or not with DHEA for 7, 14, and 21 d. DHEA significantly inhibited formation of the aldehyde after 14 and 21 d of treatment. The low levels of HNE in DHEA-treated STZ rats confirmed the ability of the steroid to prevent damage to polyunsaturated lipids in the hippocampus of diabetic rats.
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Discussion |
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B, mediated by oxidative stress. Moreover, we show that DHEA treatment counteracts both oxidative derangement and DNA binding activity of NF-B induced by chronic hyperglycemia. Reactive oxygen species, acting as intracellular mediators of IB phosphorylation and ubiquitination, activate NF-B, which in turn translocates from cytoplasm to nucleus. In the nucleus, it up-regulates the transcription of several genes possessing a B motif in promoter regions and able to codify for cytokines, adhesion molecules and chemokines closely involved in neuronal death and vascular brain damage.
A dual action of NF-B in the tissues, which may either be protective or detrimental depending on the onset and duration of its activation, has been described (8, 38, 39). However, convincing data show that persistent NF-B activation induced by long-lasting oxidative stress promotes cell death and neuronal degeneration: mice with targeted deletion of the p50 protein gene exhibit reduced infarction when subjected to focal ischemia, and inhibition of NF-B activation by proteasome inhibition dramatically reduces infarct size after middle cerebral artery occlusion (40, 41). Our results, showing that in diabetic rats DNA-binding activity of NF-B is constantly elevated until the end of the experiment, are in keeping with the hypothesis that persistent NF-B activation is detrimental for the brain and might contribute to the death of hippocampus neurons and to the disruption of synaptic function that has been recently described in diabetic rats (8).
A dramatic increase of the IB- mRNA and protein synthesis has been reported to be concomitant to NF-B activation (6). Signals such oxidizing agents as well as many other stimuli that led to an induction of NF-B activity result in the phosphorylation of IB. This phosphorylation facilitates the subsequent formation of an ubiquitin-ligase complex responsible for adding ubiquitin groups to IB- on specific lysine residues. The ubiquinated form of IB- is then targeted to the 26S proteoasome and degraded. This IB- degradation leads to the release of NF-B, which translocates to the nucleus and promotes gene transcription (42). However, NF-B activation causes a rapid up-regulation of IB- mRNA levels due to the presence of NF-B sites in the IB- promoter. The newly synthesized IB- mRNA is translated and the accumulated IB- protein guarantees that activation of NF-B terminates through an autoregolatory negative feedback mechanism and ensures that the activated cells return to a quiescent state (43). In vitro studies suggest that this autoregolatory mechanism may play a key role in self-defense of the nervous system, allowing early activation of NF-B-dependent antiapoptotic genes (44) and interrupting the activation of proinflammatory cytokine genes.
Here we show that in STZ-treated rats, in spite of a persistent increase of IB-, NF-B activation remained elevated for at least 21 d. We suggest that in diabetic rats, persistent oxidative stress induced by chronic hyperglycemia overcomes NF-B/IB- autoregolatory mechanism leading to transactivation of key genes involved in brain damage and accelerated vascular dysfunction observed in diabetes (45).
The use of antioxidants has been reported to improve some of the features of diabetes in experimental models (12, 13). The reported effect of DHEA in preventing disruption of ionic homeostasis with consequent neurodegeneration via its multitargeted antioxidant effect, led us to focus our attention on the role of DHEA in preventing NF-B activation. Here we show that DHEA treatment prevents the persistent rise of the DNA-binding activity of NF-B in the hippocampus of STZ-treated rats. We also found that DHEA can counteract the increase of H2O2 levels and the decrease of antioxidant defenses induced by diabetes, as previously demonstrated (22, 23, 24). These results confirm that an oxidative imbalance occurs in the hippocampus of diabetic rats and that this event can be prevented by DHEA treatment.
The time course of DHEA’s effects on NF-B activation parallels its effects on oxidative balance, suggesting that the block of oxidative stress contributes to the reduced NF-B activation induced by DHEA treatment in STZ-treated rats. The results show that an appreciable reduction of both NF-B and IB- levels appears after 14 d of treatment and becomes consistent by the end of the experiment, thus excluding a direct interception of free radical species by the steroid. However, DHEA might act on the oxidative balance and consecutive DNA-binding activity of NF-B in several ways. It has been shown that moderate levels of overexpression of catalase have a major inhibitory effect on NF-B activation (46). Here we demonstrate that DHEA treatment normalizes the level of this enzyme in the hippocampus of STZ-rats. This might be due to a direct effect of the steroid on the enzyme or, more likely, to the reduction of H2O2 and other reactive species observed during DHEA treatment, as our observation of the early increase of catalase activity in DHEA-treated control rats seems to suggest. An increase of platelet superoxide dismutase activity, a superoxide degrading enzyme, after DHEA treatment has been described in hypercholesterolemic rabbits (47). However, many other mechanisms could explain the effect of DHEA: in astrocytes, DHEA selectively inhibits TNF-, a cytokine that activates NF-B pathway (48). Moreover, it counteracts the loss of arachidonic acid content of the synaptosomal membrane, induced by diabetes and by transient cerebral ischemia, making the membrane more resistant to oxidative stress (24). Intercalation of DHEA into lipid membranes has been suggested as the mechanism responsible for the change in shape induced in vitro by DHEA in human red blood cells (49). Moreover, DHEA has been reported to change fatty acid composition of mitochondrial membrane phospholipids in rats (50). The question of whether the antioxidant effect of DHEA is due to DHEA itself, to its metabolites or to a combination of both is still open, because the relevant molecule(s) and mechanism(s) are largely unknown. Nevertheless, we showed previously that DHEA, but not a variety of steroids, including androstendione, 17ß-estradiol, 5-en-androsten-3ß,17ß-diol and dihydrotestosterone, was able to protect bovine retinal capillary pericytes against glucose-induced lipid peroxidation (21). Whatever the active molecule(s) and the mechanism(s) involved, our data clearly demonstrate that DHEA supplementation can protect hippocampus of diabetic rats from activation of a variety of target genes relevant to the pathophysiology of diabetic cerebrovascular disease. Oxidative stress induced by chronic hyperglycemia directly can damage ionic homeostasis and membrane transport systems in the brain (24) and, through up-regulation of NF-B dependent genes including cell surface expression of vascular cell adhesion molecule-1 and other adhesion proteins (51) as well as proinflammatory cytokines, can elicit multiple pathways contributing to vascular dysfunction. Both events, i.e. oxidative derangement and chronic NF-B activation, are counteracted by DHEA treatment.
The use of NF-B inhibitors in neurodegenerative diseases has recently been questioned in the light of the emerging concept that the NF-B/IB system may serve innate functions of host defense and response. A primary goal of therapeutic approach should be to separate beneficial responses from those that may lead to cell damage. Administration of DHEA, which is physiologically produced by the brain, at doses able to maintain its brain concentration similar to the one of young adults might contribute to regulate NF-B/IB- system, to block events triggered by persistent NF-B activation and to reduce the severity of brain damage induced by diabetes.
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Acknowledgments |
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Footnotes |
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Abbreviations: DHEA, Dehydroepiandrosterone; DHEA-S, DHEA sulfate; DTT, dithiothreitol; GSH, glutathione; HNE, 4-hydroxynonenal; IB, inhibitory protein; NF-B, nuclear factor-B; PMSF, phenylmethylsulfonyl fluoride; STZ, streptozotocin.
Received February 13, 2002.
Accepted for publication May 1, 2002.
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, P < 0.01 vs. C; 
, P < 0.05 vs. STZ.
