Up-Regulation of Advanced Glycated Products Receptors in the Brain of Diabetic Rats Is Prevented by Antioxidant Treatment
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《内分泌学杂志》
Department of Experimental Medicine and Oncology (M.A., R.M., F.R., O.D.), General Pathology Section, Department of Anatomy, Pharmacology
Forensic Medicine (N.P.)
Department of Analytical Chemistry (C.M.), University of Turin, 10125 Turin
Department of Clinical Pathophysiology (M.G.C., G.B.), University of Turin, 10126 Turin, Italy
Abstract
Diabetics have at least twice the risk of stroke and may show performance deficit in a wide range of cognitive domains. The mechanisms underlying this gradually developing end-organ damage may involve both vascular changes and direct damage to neuronal cells as a result of overproduction of superoxide by the respiratory chain and consequent oxidative stress. The study aimed to assess the role of oxidative stress on the aldose reductase-polyol pathway, on advanced glycated end-product (AGE)/AGE-receptor interaction, and on downstream signaling in the hippocampus of streptozotocin-treated rats. Data show that, in diabetic rats, levels of prooxidant compounds increase, whereas levels of antioxidant compounds fall. Receptor for AGE and galectin-3 content and polyol flux increase, whereas glyceraldehyde-3-phosphate dehydrogenase activity is impaired. Moreover, nuclear factor B (p65) transcription factor levels and S-100 protein are increased in the hippocampus cytosol, suggesting that oxidative stress triggers the cascade of events that finally leads to neuronal damage. Dehydroepiandrosterone, the most abundant hormonal steroid in the blood, has been reported to possess antioxidant properties. When dehydroepiandrosterone was administered to diabetic rats, the improved oxidative imbalance and the marked reduction of AGE receptors paralleled the reduced activation of nuclear factor B and the reduction of S-100 levels, reinforcing the suggestion that oxidative stress plays a role in diabetes-related neuronal damage.
Introduction
THERE IS SUBSTANTIAL epidemiological evidence that, besides the long-term complications of diabetes mellitus, which include accelerated atherosclerosis, retinal microvascular damage, renal failure caused by glomerular injury, and peripheral neuropathy, the disease also has multiple effects on the central nervous system. Diabetic patients have at least twice the risk of stroke (1) and may show performance deficits in a wide range of cognitive domains (2). The mechanisms underlying this gradually developing end-organ damage, known as diabetic encephalopathy, are only partially understood and can involve both vascular changes and direct damage to neuronal cells by glucose (3, 4). Although the high level of glucose in the brain cortex of diabetic rats has been questioned (5), it has recently been reported that glucose levels increase by up to three times in the hippocampus of diabetic rats compared with controls (6). Emerging evidence suggests that the overproduction of superoxide by the respiratory chain and consequent oxidative stress play a role in the pathogenesis of diabetes complications (7). The increase in advanced glycated end-product (AGE) formation and the aldose reductase-polyol pathway flux are among the main mechanisms recruited by oxidative stress and involved in diabetic damage (8, 9). Besides the direct toxic effects on the neurons, AGEs work by interacting with their receptors, a heterogeneous class of molecules (10). AGE/AGE-receptor ligands mediate the long-term effects on key cellular targets, playing a pivotal role in modulating tissue injury in diabetes (11).
Interruption of superoxide overproduction by antioxidants normalizes AGE formation and the polyol pathway flux (7). However, the conventional antioxidants used to prevent oxidative damage in diabetes have failed to achieve substantial results, because these scavenger species react in a stoichiometric manner (11, 12). An emerging compound of physiological origin that possesses multitargeted antioxidant properties is dehydroepiandrosterone (DHEA) (13, 14). This multifunctional steroid, synthesized in the adrenal cortex and brain, prevents tissue damage induced by hyperglycemia in several in vivo and in vitro models (15, 16). In rodents, in contrast to primates, concentrations of circulating DHEA are very low. However, in the brain tissue, remarkable concentrations of DHEA and DHEA sulfate are found confirming that rat brain synthesizes DHEA (17).
The study aimed to assess the role of oxidative stress and the effect of DHEA treatment on the aldose reductase-polyol pathway, on AGE/AGE-receptor interaction, and on downstream signaling in the rat hippocampus, the brain region most involved in cognitive processes and the site of diabetes-mediated impairment of cognitive abilities. Data show that DHEA treatment, preventing activation of the oxidative pathways induced by hyperglycemia, counteracts the enhanced AGE-receptors activation in the hippocampus of streptozotocin (STZ)-treated diabetic rats and normalizes downstream signaling, thus preventing the related events that are initiated by oxidative stress.
Materials and Methods
Animal models
Male Wistar rats (Harlan-Italy, Udine, Italy) weighing 200–220 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 (National Institutes of Health no. 85-23, revised 1985). They were provided with Piccioni pellet diet (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 with Accu-Check Compact kit (Roche Diagnostics GMBH, Mannheim, Germany) on blood collected from the tail vein (50 μl).
Only rats with blood glucose levels above 18 mmol/liter entered the experimental protocols; normoglycemic rats were used as controls. On the fourth day after injection, hyperglycemic and control rats began DHEA treatment. DHEA was administered for 21 d at 4 mg/d·rat; crystalline DHEA was dissolved in 1 vol 95% ethanol, mixed with 9 vol mineral oil and given daily by gastric intubation. Controls received vehicle alone. After 21 d, control and hyperglycemic rats, with or without DHEA, were anesthetized with ether and killed by decapitation after aortic exsanguination. The blood was collected and the plasma isolated. Glycemia was evaluated as described above. For DHEA analysis, plasma and aliquots of cytosol of hippocampus were extracted with ethyl-ether and evaporated, and the residue was redissolved in isooctane-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 (18). Insulin was measured in plasma samples using a specific enzyme immunoassay (rat-insulin RIA kit; Linco Research Inc., St. Charles, MO).
The hippocampus was isolated from the brain, weighed, and homogenized to obtain different extracts.
Tissue extracts
The cytosolic and nuclear extracts were prepared by the method of Meldrum et al. (19). Briefly, hippocampi were homogenized at 10% (wt/vol) in a Potter Elvehjem homogenizer (Wheaton, Millville, NJ) using a 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, 0.5 mM phenylmethylsulfonyl fluoride, 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 centrifuged at 105,000 x g at 4 C for 40 min to obtain the cytosolic fraction. The pelleted nuclei were resuspended in 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 dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 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 the Bradford assay (20), and samples were stored at –80 C until used.
Biochemical parameters
Reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescein (DCFH) diacetate as a probe. DCFH diacetate is a stable, nonfluorescent molecule that readily crosses the cell membrane and is hydrolyzed by intracellular esterases to nonfluorescent DCFH, which is rapidly oxidized in the presence of peroxides to highly fluorescent 2',7'-dichlorofluorescein, which is then measured fluorometrically (21).
The antioxidant level was evaluated in terms of reduced and oxidized glutathione content by the method of Owens and Belcher (22). A mixture was directly prepared in a cuvette: 0.05 M Na-phosphate buffer (pH 7.0), 1 mM EDTA (pH 7.0), and 10 mM dithionitrobenzoic acid plus an aliquot of the sample. Glutathione-reduced (GSH) content was evaluated after 2 min at 412 nm and expressed as μg/mg protein. Suitable volumes of diluted glutathione reductase and of reduced nicotinamide adenine dinucleotide (NAD) phosphate were then added to evaluate the total glutathione level. The difference between total glutathione and GSH content represents the GSSG (glutathione oxidized) content (expressed as μg/mg protein); the ratio between GSSG content and GSH is considered a good parameter of antioxidant status.
TNF- was determined in plasma samples using a specific enzyme immunoassay (rat TNF- ELISA kit; Diaclone Research, Besancon, France), following the manufacturers’ instructions.
Enzymatic assays
Aldose reductase activity was determined in the cytosol by the method of Wermuth and Von Wartburg (23). The substrate specificity of aldose reductase was determined by kinetic analysis at 340 nm in 0.1 M sodium by kinetic analysis at 340 nm in 0.1 M sodium phosphate (pH 7.0), containing 0.1 mM D-xylose and 0.01 M diphenylhydantoin dissolved in 0.01 M NaOH.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was determined in the cytosol by the method described by Knight et al. (24). A reaction mixture containing 100 mM Tris/HCl (pH 8.6), 1.5 mM NAD, 3 mM dithiotreitol, 5 mM sodium arsenate, 1.5 mM glyceraldehyde-3-phosphate (GAP) and 2–5 μg of protein was monitored at 340 nm during the first 20 sec after addition of substrate. The enzyme activity was expressed as micromoles reduced NADH formed per minute per milligram of protein.
Western blot analyses
Galectin-3 (ABR, Golden, CO), receptor for AGE (RAGE), S-100b, and GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA), were detected in the cytosolic fraction, and nuclear factor (NF)B-p65 (Santa Cruz) was detected both in the cytosol and in nuclear extracts, by the method of Laemmli (25). Aliquots of proteins (25–50 μg) were separated on 10% SDS-polyacrylamide gel, followed by blotting on nitrocellulose membrane (Amersham Biosciences, Braunschweig, Germany). 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 primary antibody, and reacted with the appropriate peroxidase-labeled secondary antibodies in TBS-Tween containing 2% (wt/vol) nonfat dry milk. Immunoreactive proteins were detected with a chemiluminescence assay (ECL; Amersham Biosciences) and subsequent exposure to film for 2–10 min. Anti--actin antibody served as the loading control.
Detection of AGE
Fluorescence was used as a nonspecific marker of AGE production. AGEs were determined through spectrofluorimetric detection following Monnier et al. (26). An aliquot of cytosol was diluted 1:50 with PBS (pH 7.4). Fluorescence intensity was detected at 350 nm excitation and 440 nm emission, and results were expressed as fluorescence units per milligram of proteins.
Statistical analysis
All results are presented as means ± SD. Differences between means were analyzed for significance using one-way ANOVA with the Bonferroni posttest (27).
Results
Biochemical parameters.
The body weight of STZ-diabetic rats was significantly lower than that of controls 21 d after injection of STZ (control group, 239.9 ± 10.4 g; STZ, 217.7 ± 12.4 g). STZ was rapidly eliminated from the body; about 80% appeared in the urine within 6 h (28). DHEA treatment in STZ rats prevented loss of body weight compared with STZ rats (240.32 ± 8.12 g).
Blood glucose levels in STZ-treated rats 3 d after STZ injection was 18–22 mmol/liter. The glucose level remained unchanged during the 21-d period. At the end of the protocol (21 d), glycemia levels were 6.04 ± 0.64 mmol/liter in the control group and 20.72 ± 2.25 mmol/liter in the STZ-treated groups. DHEA treatment did not modify glucose level in STZ-treated rats (Table 1).
DHEA levels after 21 d of treatment reached in plasma values similar to those found in normal humans (Table 1). Hippocampal concentrations of DHEA showed a very high variability, as we have previously reported for other tissues (15).
Insulin levels in STZ rats and in DHEA-treated STZ rats were significantly lower than in control. DHEA treatment in STZ rats did not restore to normal values the insulin levels (Table 1).
RAGEs and AGEs
The most widely studied AGE receptors are RAGE and galectin-3, whose activation is associated with an amplification of damage triggering redox-sensitive signaling pathways (10, 11). RAGE expression was found to be up-regulated in STZ rats, and DHEA reduced this up-regulation (Fig. 1). Galectin-3 expression in STZ rats was also up-regulated (Fig. 1), and again DHEA-treated rats showed less expression of this receptor.
In hyperglycemic conditions, AGEs are not only produced directly by excess glucose but can also arise through other mechanisms, i.e. inhibition of GAPDH (7). Western blot analysis indicated that GAPDH protein was significantly reduced in the hippocampus cytosol of STZ-diabetic rats (Fig. 2), as was GAPDH activity (Table 2). GAPDH inhibition may cause an increase in glyceraldehyde-3-phosphate; its fragmentation leads to methylglyoxal, an AGE product. Moreover, methylglyoxal is degraded by aldose reductase to form acetal, a potent cross-linking agent and AGE product (29). DHEA treatment was found to prevent GAPDH inhibition and to restore GAPDH protein level to control values.
Fluorescence measurement is not all specific for AGEs. However, this marker was markedly increased in cytosol of hippocampus of STZ rats, and it was significantly reduced by DHEA treatment being restored to control values (control, 40.4 ± 1.8; DHEA, 41.5 ± 1.8; STZ, 91.5 ± 19.4; STZ plus DHEA, 51.0 ± 10.2 U fluorescence/mg protein).
Polyol pathway
Aldose reductase activity, the first as well as the rate-limiting enzyme in the polyol pathway, was significantly increased in STZ-diabetic rats (Table 2); DHEA treatment significantly suppressed this increase.
NFB activation
We measured the NFB (p65) transcription factor levels in hippocampus cytosol and nucleus. Figure 3 shows the p65 reduction in the cytosol and its increase in the nucleus, indicating activation of the NFB transcription factor in STZ rats; DHEA treatment reversed the observed activation. We showed previously that the increase of NFB (p65) in the nucleus parallels the increase of both NFB activation and inhibitor of NFB level in the cytosol (30).
S100 level
The content of S100b, a biochemical marker for brain injury in diabetic complications (31), increased in the hippocampus cytosol of STZ rats (Fig. 4); DHEA prevented this increase.
Oxidative stress
Levels of ROS in the hippocampus cytosol of STZ-diabetic rats were double those of normal tissue (Table 2), and consequently, antioxidant levels, as well as those of GSH, also decreased drastically. The GSSG/GSH ratio was increased because the GSSH level was raised, whereas the GSH level was lowered in STZ rats vs. controls. DHEA treatment restored both parameters to a normal redox state (Table 2).
Also, the TNF- concentration in plasma of STZ rats was more than double, and DHEA treatment in STZ rats was able to significantly reduced by 30% the level of this cytokine (Table 1).
Discussion
The study confirms that in the hippocampus of diabetic rats there is an up-regulation of AGE receptors, an increase of glucose flux through the polyol pathway, and an increase in nonspecific markers of AGE levels (32) and demonstrates that DHEA treatment prevents all of these events. It also shows that both GAPDH activity and its protein level are reduced in the hippocampus of diabetic rats. AGEs can arise from the prolonged exposure of proteins to glucose and ribose and have been shown to accumulate in the tissue of diabetic patients (33). Moreover, AGE levels can increase through fragmentation of GAP to methylglyoxal, whose production is dramatically enhanced in diabetes (34), because the activity of GAPDH, an enzyme that catalyzes GAP degradation, is markedly reduced (35). All these mechanisms of AGE formation have been reported to be closely correlated to oxidative stress (7). In accordance with these findings, in the hippocampus of STZ-diabetic rats, ROS levels were found to be double those of normal tissue, and consequently antioxidant levels also decreased drastically.
Besides their well-known direct toxicity, AGEs exert their detrimental effect by interacting and up-regulating their receptors (36, 37). Here we found that DHEA treatment restores RAGE levels, which are markedly increased in the hippocampus of diabetic rats, to those observed in nondiabetic rats. Because ROS and GSH are also normalized by DHEA treatment, we suggest that the effects of DHEA involve its ability to improve the redox balance, acting both on AGE and RAGE levels as well as on downstream pathways activated by RAGE-ligand interaction; the detrimental effect of the AGE-RAGE interaction involves activation of transcription factors, such as NFB, a major target of ROS. Activation of NFB-dependent genes triggers several pathways, i.e. production of proinflammatory cytokines, such as TNF-, leading to tissue damage (38). Besides having a direct detrimental effect on tissues, TNF is an important inducer of the RAGE gene (39); it has been found that TNF stimulates the human RAGE gene to a similar extent as does AGE (40). Independent of NFB-dependent induction of the RAGE gene, tissue damage is amplified by the well-known positive loop between NFB activation and TNF- production, which is mediated by ROS activation (38). Again, the improved oxidative balance induced by DHEA might explain the reduction of TNF- plasma level, and indeed in a previous study we showed that DHEA markedly reduces both ROS and TNF production induced in the kidney by ischemia/reperfusion (41).
The galectin-3 receptor level was also up-regulated in the hippocampus of diabetic rats and returned to normal after DHEA treatment. Galectin-3, a soluble -galactoside with various biological functions that binds lectin, has been reported to be linked to atherosclerosis (42) and may contribute to expansion of the mesangium and macrophage activation (43). Although it has been observed that galectin-3 speeds up removal of the enhanced amounts of AGE formed during chronic hyperglycemia (44), the AGE-galectin-3 interaction has been listed among the mechanisms of macroangiopathy in diabetes.
Activity of aldose reductase, which is also doubled in the hippocampus of diabetic rats, is normalized by DHEA treatment. Two mechanisms cooperate in cell damage induced by hyperactivity of this enzyme: besides causing damage induced by sorbitol accumulation, which may disrupt cell integrity and function by imposing osmotic stress (45), the enzyme also accelerates the degradation of methylglyoxal, promoting the formation of the acetal, a potent cross-linking agent (46). The latter detrimental effect is amplified by the elevated tissue levels of methylglyoxal, whose production is dramatically enhanced in diabetes (7). Because it is well known that many enzymes have maximal activities in excess of that required in vivo, it should not be assumed that partial inhibition of an enzyme will result in increased substrate levels. Impairment of GAPDH may hamper the correct metabolism of GAP, which can therefore be used to produce methylglyoxal. Here we show that both GAPDH activity and its protein level, which are reduced in the hippocampus of diabetic rats, are restored to normal values by DHEA treatment. The mechanisms by which DHEA restores both aldose reductase and GAPDH activities involve its effect on the redox balance. It has been reported that aldose reductase activity is stimulated by imbalance of the redox state and by hydroxynonenal, an end-product of lipid oxidation (47, 48) and that GAPDH activity is reversibly inhibited by ROS (7). In line with these observations, and as we report elsewhere, 4-hydroxy-2,3-trans-nonenal, which is produced in large quantities in the hippocampus of diabetic rats as a consequence of enhanced lipid peroxidation (47), is reduced by DHEA.
In the hippocampus of diabetic rats, we observed an increase in S-100 protein, taken as a marker of brain injury. S-100 has been reported to act as a cytokine with neurotrophic and neurite-extending activity (49), and it might be implicated in the pathobiology of diabetes by interacting with RAGE (50). S-100 protein levels are also normalized by DHEA treatment, an effect that likewise might be related to DHEA’s antioxidant properties; a similar reduction of S-100 protein levels has been found in the hippocampus and cortex of diabetic rats after vitamin E treatment (51).
The finding that the ROS level is reduced and antioxidant defenses are restored after DHEA treatment (52, 53) strongly suggests that DHEA’s mechanism of action involves its antioxidant properties. This is also showed by the time course of the DHEA effect on NFB activation and on markers of damage, which both parallel the DHEA effect on oxidative stress (52, 53). Several options have been proposed (54, 55, 56) to explain the multitargeted antioxidant effects of DHEA, including its effect on catalase expression (30), fatty acid composition of cellular membranes, and TNF- production. However, the precise mechanisms still remain to be fully defined, and additional non-antioxidant effects, as have been reported for vitamin E (57), cannot be excluded. Specific receptor and non-receptor-mediated effects interfering with glucose uptake and cellular metabolism (58) and a possible involvement of peroxisome proliferator-activated receptor- and - as transcriptional activators of numerous genes (59, 60) have been described, suggesting that DHEA might exert its beneficial effect indirectly by blocking the catabolic consequence of uncontrolled diabetes in addition to its direct neuronal action on oxidative balance. In addition, other mechanisms, involving the fall of proinsulin C-peptide in diabetes might occur in hippocampal neuronal loss and spatial learning and memory deficit (61).
In conclusion, the results of this study show for the first time that an endogenous steroid directly synthesized in the brain, whatever the mechanism involved, counteracts the enhanced AGE-receptor activation in the hippocampus of STZ-diabetic rats, restores downstream signaling to normal, and thus prevents the related events that lead to cellular damage. Additional study on the potential benefits of DHEA in preventing diabetic neuronal damage is indicated.
Footnotes
This work was supported in part by Fondo Investimenti Ricerca di Base (Ministero Istruzione Universita e Ricerca) and the Regione Piemonte.
First Published Online September 15, 2005
Abbreviations: AGE, Advanced glycated end-product; DCFH, 2',7'-dichlorofluorescein; DHEA, dehydroepiandrosterone; GAP, glyceraldehyde-3-phosphate; GAPDH, GAP dehydrogenase; GSH, glutathione-reduced; GSSG, glutathione oxidized; NAD, nicotinamide adenine dinucleotide; NFB, nuclear factor B; RAGE, receptor for AGE; ROS, reactive oxygen species; STZ, streptozotocin.
Accepted for publication September 6, 2005.
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Forensic Medicine (N.P.)
Department of Analytical Chemistry (C.M.), University of Turin, 10125 Turin
Department of Clinical Pathophysiology (M.G.C., G.B.), University of Turin, 10126 Turin, Italy
Abstract
Diabetics have at least twice the risk of stroke and may show performance deficit in a wide range of cognitive domains. The mechanisms underlying this gradually developing end-organ damage may involve both vascular changes and direct damage to neuronal cells as a result of overproduction of superoxide by the respiratory chain and consequent oxidative stress. The study aimed to assess the role of oxidative stress on the aldose reductase-polyol pathway, on advanced glycated end-product (AGE)/AGE-receptor interaction, and on downstream signaling in the hippocampus of streptozotocin-treated rats. Data show that, in diabetic rats, levels of prooxidant compounds increase, whereas levels of antioxidant compounds fall. Receptor for AGE and galectin-3 content and polyol flux increase, whereas glyceraldehyde-3-phosphate dehydrogenase activity is impaired. Moreover, nuclear factor B (p65) transcription factor levels and S-100 protein are increased in the hippocampus cytosol, suggesting that oxidative stress triggers the cascade of events that finally leads to neuronal damage. Dehydroepiandrosterone, the most abundant hormonal steroid in the blood, has been reported to possess antioxidant properties. When dehydroepiandrosterone was administered to diabetic rats, the improved oxidative imbalance and the marked reduction of AGE receptors paralleled the reduced activation of nuclear factor B and the reduction of S-100 levels, reinforcing the suggestion that oxidative stress plays a role in diabetes-related neuronal damage.
Introduction
THERE IS SUBSTANTIAL epidemiological evidence that, besides the long-term complications of diabetes mellitus, which include accelerated atherosclerosis, retinal microvascular damage, renal failure caused by glomerular injury, and peripheral neuropathy, the disease also has multiple effects on the central nervous system. Diabetic patients have at least twice the risk of stroke (1) and may show performance deficits in a wide range of cognitive domains (2). The mechanisms underlying this gradually developing end-organ damage, known as diabetic encephalopathy, are only partially understood and can involve both vascular changes and direct damage to neuronal cells by glucose (3, 4). Although the high level of glucose in the brain cortex of diabetic rats has been questioned (5), it has recently been reported that glucose levels increase by up to three times in the hippocampus of diabetic rats compared with controls (6). Emerging evidence suggests that the overproduction of superoxide by the respiratory chain and consequent oxidative stress play a role in the pathogenesis of diabetes complications (7). The increase in advanced glycated end-product (AGE) formation and the aldose reductase-polyol pathway flux are among the main mechanisms recruited by oxidative stress and involved in diabetic damage (8, 9). Besides the direct toxic effects on the neurons, AGEs work by interacting with their receptors, a heterogeneous class of molecules (10). AGE/AGE-receptor ligands mediate the long-term effects on key cellular targets, playing a pivotal role in modulating tissue injury in diabetes (11).
Interruption of superoxide overproduction by antioxidants normalizes AGE formation and the polyol pathway flux (7). However, the conventional antioxidants used to prevent oxidative damage in diabetes have failed to achieve substantial results, because these scavenger species react in a stoichiometric manner (11, 12). An emerging compound of physiological origin that possesses multitargeted antioxidant properties is dehydroepiandrosterone (DHEA) (13, 14). This multifunctional steroid, synthesized in the adrenal cortex and brain, prevents tissue damage induced by hyperglycemia in several in vivo and in vitro models (15, 16). In rodents, in contrast to primates, concentrations of circulating DHEA are very low. However, in the brain tissue, remarkable concentrations of DHEA and DHEA sulfate are found confirming that rat brain synthesizes DHEA (17).
The study aimed to assess the role of oxidative stress and the effect of DHEA treatment on the aldose reductase-polyol pathway, on AGE/AGE-receptor interaction, and on downstream signaling in the rat hippocampus, the brain region most involved in cognitive processes and the site of diabetes-mediated impairment of cognitive abilities. Data show that DHEA treatment, preventing activation of the oxidative pathways induced by hyperglycemia, counteracts the enhanced AGE-receptors activation in the hippocampus of streptozotocin (STZ)-treated diabetic rats and normalizes downstream signaling, thus preventing the related events that are initiated by oxidative stress.
Materials and Methods
Animal models
Male Wistar rats (Harlan-Italy, Udine, Italy) weighing 200–220 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 (National Institutes of Health no. 85-23, revised 1985). They were provided with Piccioni pellet diet (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 with Accu-Check Compact kit (Roche Diagnostics GMBH, Mannheim, Germany) on blood collected from the tail vein (50 μl).
Only rats with blood glucose levels above 18 mmol/liter entered the experimental protocols; normoglycemic rats were used as controls. On the fourth day after injection, hyperglycemic and control rats began DHEA treatment. DHEA was administered for 21 d at 4 mg/d·rat; crystalline DHEA was dissolved in 1 vol 95% ethanol, mixed with 9 vol mineral oil and given daily by gastric intubation. Controls received vehicle alone. After 21 d, control and hyperglycemic rats, with or without DHEA, were anesthetized with ether and killed by decapitation after aortic exsanguination. The blood was collected and the plasma isolated. Glycemia was evaluated as described above. For DHEA analysis, plasma and aliquots of cytosol of hippocampus were extracted with ethyl-ether and evaporated, and the residue was redissolved in isooctane-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 (18). Insulin was measured in plasma samples using a specific enzyme immunoassay (rat-insulin RIA kit; Linco Research Inc., St. Charles, MO).
The hippocampus was isolated from the brain, weighed, and homogenized to obtain different extracts.
Tissue extracts
The cytosolic and nuclear extracts were prepared by the method of Meldrum et al. (19). Briefly, hippocampi were homogenized at 10% (wt/vol) in a Potter Elvehjem homogenizer (Wheaton, Millville, NJ) using a 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, 0.5 mM phenylmethylsulfonyl fluoride, 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 centrifuged at 105,000 x g at 4 C for 40 min to obtain the cytosolic fraction. The pelleted nuclei were resuspended in 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 dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 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 the Bradford assay (20), and samples were stored at –80 C until used.
Biochemical parameters
Reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescein (DCFH) diacetate as a probe. DCFH diacetate is a stable, nonfluorescent molecule that readily crosses the cell membrane and is hydrolyzed by intracellular esterases to nonfluorescent DCFH, which is rapidly oxidized in the presence of peroxides to highly fluorescent 2',7'-dichlorofluorescein, which is then measured fluorometrically (21).
The antioxidant level was evaluated in terms of reduced and oxidized glutathione content by the method of Owens and Belcher (22). A mixture was directly prepared in a cuvette: 0.05 M Na-phosphate buffer (pH 7.0), 1 mM EDTA (pH 7.0), and 10 mM dithionitrobenzoic acid plus an aliquot of the sample. Glutathione-reduced (GSH) content was evaluated after 2 min at 412 nm and expressed as μg/mg protein. Suitable volumes of diluted glutathione reductase and of reduced nicotinamide adenine dinucleotide (NAD) phosphate were then added to evaluate the total glutathione level. The difference between total glutathione and GSH content represents the GSSG (glutathione oxidized) content (expressed as μg/mg protein); the ratio between GSSG content and GSH is considered a good parameter of antioxidant status.
TNF- was determined in plasma samples using a specific enzyme immunoassay (rat TNF- ELISA kit; Diaclone Research, Besancon, France), following the manufacturers’ instructions.
Enzymatic assays
Aldose reductase activity was determined in the cytosol by the method of Wermuth and Von Wartburg (23). The substrate specificity of aldose reductase was determined by kinetic analysis at 340 nm in 0.1 M sodium by kinetic analysis at 340 nm in 0.1 M sodium phosphate (pH 7.0), containing 0.1 mM D-xylose and 0.01 M diphenylhydantoin dissolved in 0.01 M NaOH.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was determined in the cytosol by the method described by Knight et al. (24). A reaction mixture containing 100 mM Tris/HCl (pH 8.6), 1.5 mM NAD, 3 mM dithiotreitol, 5 mM sodium arsenate, 1.5 mM glyceraldehyde-3-phosphate (GAP) and 2–5 μg of protein was monitored at 340 nm during the first 20 sec after addition of substrate. The enzyme activity was expressed as micromoles reduced NADH formed per minute per milligram of protein.
Western blot analyses
Galectin-3 (ABR, Golden, CO), receptor for AGE (RAGE), S-100b, and GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA), were detected in the cytosolic fraction, and nuclear factor (NF)B-p65 (Santa Cruz) was detected both in the cytosol and in nuclear extracts, by the method of Laemmli (25). Aliquots of proteins (25–50 μg) were separated on 10% SDS-polyacrylamide gel, followed by blotting on nitrocellulose membrane (Amersham Biosciences, Braunschweig, Germany). 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 primary antibody, and reacted with the appropriate peroxidase-labeled secondary antibodies in TBS-Tween containing 2% (wt/vol) nonfat dry milk. Immunoreactive proteins were detected with a chemiluminescence assay (ECL; Amersham Biosciences) and subsequent exposure to film for 2–10 min. Anti--actin antibody served as the loading control.
Detection of AGE
Fluorescence was used as a nonspecific marker of AGE production. AGEs were determined through spectrofluorimetric detection following Monnier et al. (26). An aliquot of cytosol was diluted 1:50 with PBS (pH 7.4). Fluorescence intensity was detected at 350 nm excitation and 440 nm emission, and results were expressed as fluorescence units per milligram of proteins.
Statistical analysis
All results are presented as means ± SD. Differences between means were analyzed for significance using one-way ANOVA with the Bonferroni posttest (27).
Results
Biochemical parameters.
The body weight of STZ-diabetic rats was significantly lower than that of controls 21 d after injection of STZ (control group, 239.9 ± 10.4 g; STZ, 217.7 ± 12.4 g). STZ was rapidly eliminated from the body; about 80% appeared in the urine within 6 h (28). DHEA treatment in STZ rats prevented loss of body weight compared with STZ rats (240.32 ± 8.12 g).
Blood glucose levels in STZ-treated rats 3 d after STZ injection was 18–22 mmol/liter. The glucose level remained unchanged during the 21-d period. At the end of the protocol (21 d), glycemia levels were 6.04 ± 0.64 mmol/liter in the control group and 20.72 ± 2.25 mmol/liter in the STZ-treated groups. DHEA treatment did not modify glucose level in STZ-treated rats (Table 1).
DHEA levels after 21 d of treatment reached in plasma values similar to those found in normal humans (Table 1). Hippocampal concentrations of DHEA showed a very high variability, as we have previously reported for other tissues (15).
Insulin levels in STZ rats and in DHEA-treated STZ rats were significantly lower than in control. DHEA treatment in STZ rats did not restore to normal values the insulin levels (Table 1).
RAGEs and AGEs
The most widely studied AGE receptors are RAGE and galectin-3, whose activation is associated with an amplification of damage triggering redox-sensitive signaling pathways (10, 11). RAGE expression was found to be up-regulated in STZ rats, and DHEA reduced this up-regulation (Fig. 1). Galectin-3 expression in STZ rats was also up-regulated (Fig. 1), and again DHEA-treated rats showed less expression of this receptor.
In hyperglycemic conditions, AGEs are not only produced directly by excess glucose but can also arise through other mechanisms, i.e. inhibition of GAPDH (7). Western blot analysis indicated that GAPDH protein was significantly reduced in the hippocampus cytosol of STZ-diabetic rats (Fig. 2), as was GAPDH activity (Table 2). GAPDH inhibition may cause an increase in glyceraldehyde-3-phosphate; its fragmentation leads to methylglyoxal, an AGE product. Moreover, methylglyoxal is degraded by aldose reductase to form acetal, a potent cross-linking agent and AGE product (29). DHEA treatment was found to prevent GAPDH inhibition and to restore GAPDH protein level to control values.
Fluorescence measurement is not all specific for AGEs. However, this marker was markedly increased in cytosol of hippocampus of STZ rats, and it was significantly reduced by DHEA treatment being restored to control values (control, 40.4 ± 1.8; DHEA, 41.5 ± 1.8; STZ, 91.5 ± 19.4; STZ plus DHEA, 51.0 ± 10.2 U fluorescence/mg protein).
Polyol pathway
Aldose reductase activity, the first as well as the rate-limiting enzyme in the polyol pathway, was significantly increased in STZ-diabetic rats (Table 2); DHEA treatment significantly suppressed this increase.
NFB activation
We measured the NFB (p65) transcription factor levels in hippocampus cytosol and nucleus. Figure 3 shows the p65 reduction in the cytosol and its increase in the nucleus, indicating activation of the NFB transcription factor in STZ rats; DHEA treatment reversed the observed activation. We showed previously that the increase of NFB (p65) in the nucleus parallels the increase of both NFB activation and inhibitor of NFB level in the cytosol (30).
S100 level
The content of S100b, a biochemical marker for brain injury in diabetic complications (31), increased in the hippocampus cytosol of STZ rats (Fig. 4); DHEA prevented this increase.
Oxidative stress
Levels of ROS in the hippocampus cytosol of STZ-diabetic rats were double those of normal tissue (Table 2), and consequently, antioxidant levels, as well as those of GSH, also decreased drastically. The GSSG/GSH ratio was increased because the GSSH level was raised, whereas the GSH level was lowered in STZ rats vs. controls. DHEA treatment restored both parameters to a normal redox state (Table 2).
Also, the TNF- concentration in plasma of STZ rats was more than double, and DHEA treatment in STZ rats was able to significantly reduced by 30% the level of this cytokine (Table 1).
Discussion
The study confirms that in the hippocampus of diabetic rats there is an up-regulation of AGE receptors, an increase of glucose flux through the polyol pathway, and an increase in nonspecific markers of AGE levels (32) and demonstrates that DHEA treatment prevents all of these events. It also shows that both GAPDH activity and its protein level are reduced in the hippocampus of diabetic rats. AGEs can arise from the prolonged exposure of proteins to glucose and ribose and have been shown to accumulate in the tissue of diabetic patients (33). Moreover, AGE levels can increase through fragmentation of GAP to methylglyoxal, whose production is dramatically enhanced in diabetes (34), because the activity of GAPDH, an enzyme that catalyzes GAP degradation, is markedly reduced (35). All these mechanisms of AGE formation have been reported to be closely correlated to oxidative stress (7). In accordance with these findings, in the hippocampus of STZ-diabetic rats, ROS levels were found to be double those of normal tissue, and consequently antioxidant levels also decreased drastically.
Besides their well-known direct toxicity, AGEs exert their detrimental effect by interacting and up-regulating their receptors (36, 37). Here we found that DHEA treatment restores RAGE levels, which are markedly increased in the hippocampus of diabetic rats, to those observed in nondiabetic rats. Because ROS and GSH are also normalized by DHEA treatment, we suggest that the effects of DHEA involve its ability to improve the redox balance, acting both on AGE and RAGE levels as well as on downstream pathways activated by RAGE-ligand interaction; the detrimental effect of the AGE-RAGE interaction involves activation of transcription factors, such as NFB, a major target of ROS. Activation of NFB-dependent genes triggers several pathways, i.e. production of proinflammatory cytokines, such as TNF-, leading to tissue damage (38). Besides having a direct detrimental effect on tissues, TNF is an important inducer of the RAGE gene (39); it has been found that TNF stimulates the human RAGE gene to a similar extent as does AGE (40). Independent of NFB-dependent induction of the RAGE gene, tissue damage is amplified by the well-known positive loop between NFB activation and TNF- production, which is mediated by ROS activation (38). Again, the improved oxidative balance induced by DHEA might explain the reduction of TNF- plasma level, and indeed in a previous study we showed that DHEA markedly reduces both ROS and TNF production induced in the kidney by ischemia/reperfusion (41).
The galectin-3 receptor level was also up-regulated in the hippocampus of diabetic rats and returned to normal after DHEA treatment. Galectin-3, a soluble -galactoside with various biological functions that binds lectin, has been reported to be linked to atherosclerosis (42) and may contribute to expansion of the mesangium and macrophage activation (43). Although it has been observed that galectin-3 speeds up removal of the enhanced amounts of AGE formed during chronic hyperglycemia (44), the AGE-galectin-3 interaction has been listed among the mechanisms of macroangiopathy in diabetes.
Activity of aldose reductase, which is also doubled in the hippocampus of diabetic rats, is normalized by DHEA treatment. Two mechanisms cooperate in cell damage induced by hyperactivity of this enzyme: besides causing damage induced by sorbitol accumulation, which may disrupt cell integrity and function by imposing osmotic stress (45), the enzyme also accelerates the degradation of methylglyoxal, promoting the formation of the acetal, a potent cross-linking agent (46). The latter detrimental effect is amplified by the elevated tissue levels of methylglyoxal, whose production is dramatically enhanced in diabetes (7). Because it is well known that many enzymes have maximal activities in excess of that required in vivo, it should not be assumed that partial inhibition of an enzyme will result in increased substrate levels. Impairment of GAPDH may hamper the correct metabolism of GAP, which can therefore be used to produce methylglyoxal. Here we show that both GAPDH activity and its protein level, which are reduced in the hippocampus of diabetic rats, are restored to normal values by DHEA treatment. The mechanisms by which DHEA restores both aldose reductase and GAPDH activities involve its effect on the redox balance. It has been reported that aldose reductase activity is stimulated by imbalance of the redox state and by hydroxynonenal, an end-product of lipid oxidation (47, 48) and that GAPDH activity is reversibly inhibited by ROS (7). In line with these observations, and as we report elsewhere, 4-hydroxy-2,3-trans-nonenal, which is produced in large quantities in the hippocampus of diabetic rats as a consequence of enhanced lipid peroxidation (47), is reduced by DHEA.
In the hippocampus of diabetic rats, we observed an increase in S-100 protein, taken as a marker of brain injury. S-100 has been reported to act as a cytokine with neurotrophic and neurite-extending activity (49), and it might be implicated in the pathobiology of diabetes by interacting with RAGE (50). S-100 protein levels are also normalized by DHEA treatment, an effect that likewise might be related to DHEA’s antioxidant properties; a similar reduction of S-100 protein levels has been found in the hippocampus and cortex of diabetic rats after vitamin E treatment (51).
The finding that the ROS level is reduced and antioxidant defenses are restored after DHEA treatment (52, 53) strongly suggests that DHEA’s mechanism of action involves its antioxidant properties. This is also showed by the time course of the DHEA effect on NFB activation and on markers of damage, which both parallel the DHEA effect on oxidative stress (52, 53). Several options have been proposed (54, 55, 56) to explain the multitargeted antioxidant effects of DHEA, including its effect on catalase expression (30), fatty acid composition of cellular membranes, and TNF- production. However, the precise mechanisms still remain to be fully defined, and additional non-antioxidant effects, as have been reported for vitamin E (57), cannot be excluded. Specific receptor and non-receptor-mediated effects interfering with glucose uptake and cellular metabolism (58) and a possible involvement of peroxisome proliferator-activated receptor- and - as transcriptional activators of numerous genes (59, 60) have been described, suggesting that DHEA might exert its beneficial effect indirectly by blocking the catabolic consequence of uncontrolled diabetes in addition to its direct neuronal action on oxidative balance. In addition, other mechanisms, involving the fall of proinsulin C-peptide in diabetes might occur in hippocampal neuronal loss and spatial learning and memory deficit (61).
In conclusion, the results of this study show for the first time that an endogenous steroid directly synthesized in the brain, whatever the mechanism involved, counteracts the enhanced AGE-receptor activation in the hippocampus of STZ-diabetic rats, restores downstream signaling to normal, and thus prevents the related events that lead to cellular damage. Additional study on the potential benefits of DHEA in preventing diabetic neuronal damage is indicated.
Footnotes
This work was supported in part by Fondo Investimenti Ricerca di Base (Ministero Istruzione Universita e Ricerca) and the Regione Piemonte.
First Published Online September 15, 2005
Abbreviations: AGE, Advanced glycated end-product; DCFH, 2',7'-dichlorofluorescein; DHEA, dehydroepiandrosterone; GAP, glyceraldehyde-3-phosphate; GAPDH, GAP dehydrogenase; GSH, glutathione-reduced; GSSG, glutathione oxidized; NAD, nicotinamide adenine dinucleotide; NFB, nuclear factor B; RAGE, receptor for AGE; ROS, reactive oxygen species; STZ, streptozotocin.
Accepted for publication September 6, 2005.
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