ROS are generated in mitochondria throughout the life span, and evidence exists to support the causal involvement of ROS in aging (4
). In this study, we determined the effects of vitamin E, the major lipid antioxidant, on age-related transcriptional alterations in the heart and brain, and also attempted to obtain evidence for unique biological roles of α- and γ-tocopherol. As previously reported (34
), heart aging resulted in induction of genes encoding structural proteins and down-regulation of genes involved in fatty acid metabolism and protein synthesis. An age-related transcriptional shift toward carbohydrate metabolism was also observed. In addition, we observed age-related induction of genes involved in immune and inflammatory responses, which is suggestive of a heightened pro-inflammatory state in aged heart. We also observed induction of genes involved in heat shock response, suggesting increased protein damage with aging. Induction of heat shock protein with aging was previously observed in Caenorhabditis elegans
). Heart aging was associated with a concerted decline of mRNA encoding ribosomal proteins, such as ribosomal protein L10, L10a, L19, L21, S3, S3a, S5 and S28, and translation factors, including several eukaryote translation initiation, elongation and termination factors. Decreased protein biosynthesis with aging in the heart was reported previously in rats (67
), suggesting that this may be a general feature of heart aging in laboratory rodents. Previously, we found no evidence that aging in the heart is associated with a transcriptional profile indicative of an oxidative stress response (34
). In this study, however, several genes involved in the antioxidant response were down-regulated with age, including catalase (0.5-fold), metallothionein 1 (0.6-fold), and metallothionein 2 (0.4-fold). Because these genes are inducible, these observations suggest either a decrease in ROS production in the aged heart, or that the enzymatic defense system for ROS-induced oxidative stress may be compromised.
Vitamin E markedly prevented the age-related up-regulation of genes encoding cellular structural proteins in the heart. Interestingly, we found that vitamin E down-regulates cardiotrophin-1, a cytokine that induces cardiomyocyte hypertrophy (56
) and is induced in congestive heart failure (68
). Based on these observations, it appears that vitamin E has multiple effects that suppress an age-related cardiomyocyte hypertrophy transcriptional program. Vitamin E also markedly suppressed the age-related increase in expression of genes encoding several complement components (complement component 1, q subcompoment, alpha polypeptide, complement component 1, q subcomponent, receptor 1, and complement component 4) (). Complement-related genes are involved in innate immunity and induced in response to ischemia and reperfusion (69
). Our results suggest that vitamin E lowers the expression of immune-related genes, which in turn may confer a cardioprotective effect in the aged heart. In adult hearts, fatty acids are the major energy source, whereas fetal hearts primarily use glucose as metabolic fuel (70
). We show here that the age-related down-regulation of genes involved in fatty acid β-oxidation is not opposed by vitamin E supplementation, a finding in contrast to the our previous observations with caloric restriction in the mouse heart.
Brain aging resulted in a gene expression profile indicative of increased energy metabolism and induced immune and inflammatory responses. We also observed age-related decreases in expression of genes involved in mRNA processing (). There was an age-related up-regulation of genes encoding ribosomal proteins and translation elongation factors in the aged brain. In the aged mouse neocortex, genes involved in base excision repair and DNA-damage responses were significantly decreased in expression, including DNA cross-link repair 1A, PSO2 homolog (0.66-fold), excision repair cross-complementing rodent repair deficiency, complementation group 5 (0.48-fold), and RAD50 homolog (0.54-fold). These results support the hypothesis that a decline in DNA repair activity in the aged brain may be an important mechanism contributing to the age-dependent accumulation of oxidative DNA lesions in brain mitochondria. Interestingly, DNA damage caused by oxidative stress is markedly increased in the promoters of genes that display reduced expression in the aged human cortex, and the base-excision DNA repair is reduced (Lu, 2004 #320). There is tissue-specific regulation of telomerase during aging (71
) and telomere shortening, a marker of cellular senescence, occurs in the rat brain with increasing age (72
). We observed an age-related down-regulation of several genes involved in telomere maintenance in the neocortex. Among them, Poly (ADP-ribose) polymerase family member 1, an enzyme that play a role in base excision repair, has been implicated in the maintenance of telomeres (73
Previous studies demonstrated that several proteins involved in glycolysis and the TCA cycle, and subunits of mitochondrial ATP synthase increased progressively with aging in rat brain (42
). At the mRNA level, we observed age-related induction of genes involved in glycolysis, the TCA cycle and ATP biosynthesis (). Surprisingly, the mixture of α- and γ-tocopherol, but not α-tocopherol alone, prevented most age-related gene expression changes in these functional classes either completely or partially (). As reported previously in brains from mice (33
) and humans (Lu, 2004 #320), age-related increases in expression of genes involved in immune and inflammatory responses occur, which is suggestive of heightened immunity and pro-inflammatory status in aged tissues. Interestingly, transcriptional changes in this class were significantly inhibited by the combination of α- and γ-tocopherol in the heart, but not in the brain ( and ).
It has been observed that γ-tocopherol has higher activity in trapping reactive nitrogen species (RNS) than α-tocopherol (75
). According to the nitric oxide hypothesis of brain aging (76
), accumulation of oxidative damage caused by RNS may lead to degeneration of brain cells. Compared with age-matched control, only the mixture of α- and γ-tocopherol showed significant aging-retarding effect on functional classes of genes in brain (). These observations suggest that γ-tocopherol can prevent the age-related changes in expression in brain, independent of α-tocopherol, and may provide evidence to support the nitric oxide hypothesis of brain aging. It was also reported recently that the protective effect of γ-tocopherol against neurodegenerative toxicity is stronger than that of α-tocopherol (78
In order to find possible mechanisms involved in the effect of vitamin E on the aging process, we analyzed genes that showed significant alterations in the vitamin E-supplemented mice, but were not affected by aging. Several genes involved in the inhibition of apoptosis were up-regulated by vitamin E supplementation, whereas genes involved in the activation of apoptosis were down-regulated as compared with the control group (Table 7
and ). Phaneuf et al.
) first demonstrated that cytochrome C release from mitochondria and alterations in the level of Bcl2, an anti-apoptotic protein, were correlated with cardiomyocyte apoptosis in the aging heart. Experimental evidence suggests that neuronal loss due to oxidative stress induced apoptosis may play a central role during normal brain aging (28
). Qin et al.
) observed that vitamin E feeding attenuates myocyte apoptosis in rabbits. These results support the role of ROS-induced apoptosis in aging tissues and suggest prevention of apoptosis in normal aging by vitamin E supplementation. We report here that vitamin E supplementation induced the up-regulation of several genes involved in the antioxidant response, including Cu/Zn-SOD and glutathione peroxidases, in the aging heart (). Thus, vitamin E may suppress the damaging effects of increased ROS in the aging heart not only through its own antioxidant activity, but also through its ability to increase the expression of genes encoding antioxidant genes. However, supplementation of a relatively high dose of α-tocopherol acted as a prooxidant showing no attenuation of oxidative stress in rat skeletal muscle (79
). Therefore, it is possible that the induction of antioxidant genes by vitamin E may be due to its potential to become a pro-oxidant in the oxidative milieu of the heart. Vitamin E supplementation was also associated with a reduction in the expression of genes involved in DNA repair in the heart(), a finding consistent with reduced endogenous DNA damage. Our finding of a cardioprotective role of vitamin E contrasts with the recent finding of an apparent association between vitamin E intake and heart failure in diabetics (27
Steroid hormones act on neurons and glial cells, regulating differentiation, survival and neuronal connectivity. A recent gene expression profiling study showed that the underlying molecular mechanisms of neuroprotection by vitamin E are associated with hormone metabolism and apoptosis (80
). In our study, several genes involved in steroid biosynthesis were found to be induced by vitamin E supplementation (), suggesting that neuroprotective activity of vitamin E in brain may be partially contributed by the increased biosynthesis of steroids.
Recently biological functions of α-tocopherol that are independent of its antioxidant activity have been reported, including inhibition of cell proliferation, platelet aggregation and monocyte adhesion (81
). At transcriptional level, a number of genes have been characterized to be regulated by the non-antioxidant activity of α-tocopherol. A non-antioxidant activity of α-tocopherol induces α-tropomyosin expression in muscle (83
) and prevents age-related increase of collagenase expression in human skin fibroblasts (84
). A non-antioxidant function of γ-tocopherol was also observed recently (85
). Therefore, some effects of vitamin E supplementation observed in this study might be accounted for by non-antioxidant activities of vitamin E.
In summary, we report here that vitamin E can alter the transcriptional profiles associated with aging, and that this effect of vitamin E may be mediated through several mechanisms. First, vitamin E reduces the expression of structural proteins in the aging heart and thus could oppose age-related cardiomyocyte hypertrophy. Increased cardiomyocyte apoptosis associated with aging may be prevented by vitamin E, since it up-regulates genes encoding anti-apoptotic proteins and down-regulates genes involved in the induction of apoptosis in the aging heart (). Enhanced apoptosis in aged cardiomyocytes may be further attenuated by vitamin E through the suppression of the p53-induced apoptotic response (). In brain, both forms of vitamin E increased the expression of anti-apoptotic proteins and decreased the mRNA levels of pro-apoptotic proteins (). Surprisingly, only the mixture of α- and γ-tocopherol prevents age-related transcriptional alterations in the brain ( and ). Genes involved in steroid biosynthesis were also changed in expression by vitamin E supplementation. Importantly, we observed a beneficial effect of γ-tocopherol on the transcriptional profile of brain aging, which was not observed in the aging heart. Therefore, the effect of vitamin E in aging is likely to be tissue and tocopherol-specific. Taken as a whole, the data from the present study demonstrate that the middle age-onset vitamin E supplementation can prevent age-related transcriptional alterations in post-mitotic tissues. Extension of this study to other post-mitotic or regenerating tissues should result in the identification of common mechanisms of action, facilitating the understanding the role of oxidative stress and the effect antioxidant supplementation in the aging process. Our general findings that vitamin E can modulate the aging process in two critical organs, if applicable to humans, may have major public health implications.