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.
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 ß-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.
This work was supported in part by Fondo Investimenti Ricerca di Base (Ministero Istruzione Università 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.
Received June 14, 2005.
Accepted for publication September 6, 2005.