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Biochemical, Molecular, and Genetic Mechanisms

- and -Tocopherol Prevent Age-Related Transcriptional Alterations in the Heart and Brain of Mice^1–3,

⁴ Department of Genetics and Medical Genetics, and ⁵ Veterans Administration Hospital, Department of Medicine and Wisconsin Primate Research Center, University of Wisconsin, Madison, WI 53706; ⁶ Department of Biostatistics, Section on Statistical Genetics and Clinical Nutrition Research Center, University of Alabama, Birmingham, AL 35294; and ⁷ Vascular Biology Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111

^* To whom correspondence should be addressed. E-mail: taprolla{at}wisc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
We used high-density oligonucleotide arrays to measure transcriptional alterations in the heart and brain (neocortex) of 30-mo-old B6C3F₁ mice supplemented with -tocopherol (T) and -tocopherol (T) since middle age (15 mo). Gene expression profiles were obtained from 5- and 30-mo-old control mice and 30-mo-old mice supplemented with T (1 g/kg) or a mixture of T and T (500 mg/kg of each tocopherol) from middle age (15 mo). In the heart, both tocopherol-supplemented diets were effective in inhibiting the expression of genes previously associated with cardiomyocyte hypertrophy and increased innate immunity. In the brain, induction of genes encoding ribosomal proteins and proteins involved in ATP biosynthesis was observed with aging and was markedly prevented by the mixture of T and T supplementation but not by T alone. These results demonstrate that middle age-onset dietary supplementation with T and T can partially prevent age-associated transcriptional changes and that these effects are tissue and tocopherol specific.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Natural vitamin E consists of tocopherols and tocotrienols. There are 2 major tocopherol forms, -tocopherol (T) and -tocopherol (T), which differ structurally only by a methyl group substitution at the 5-position. T is quantitatively the major form of tocopherol in human and animal tissues, including blood plasma, and is a more potent antioxidant in vitro than T (5). In contrast, T is the most abundant form of vitamin E in foods; however, its bioavailability and bioactivity are lower than that of T (6). Recent studies suggest that T may have biological activities that are not shared by T, such as the inhibition of cyclooxygenase activity (7), detoxification of electrophiles (8), and natriuretic activity (9). A study of the antioxidant activity of T and T suggests that there may be synergism between these tocopherols (10). However, their relative contribution to antioxidant activity in vivo is unknown.

Cardiac tissue is largely postmitotic and relies heavily on mitochondrial oxidative energy metabolism (11). Age-related changes in human and rodent hearts include a reduction in the number of myocytes (12), myocyte hypertrophy (12), cardiac fibrosis (13) and lipofuscin pigment accumulation (14). Increased oxidative stress may contribute to myocardial dysfunction with age (15). Antioxidant vitamins reduce cardiac oxidative stress and apoptosis and also attenuate cardiac dysfunction in pacing-induced congestive heart failure (16).

Aging of the brain is associated with subtle morphological and functional alterations in specific neuronal circuits, rather than large-scale neuronal loss (17). Although there may be several mechanisms involved in brain aging, oxidative damage caused by ROS may contribute to functional impairment in the aged brain (18). The brain is more susceptible to oxidative stress because of its high oxygen consumption per unit weight and scarcity of antioxidant defense systems (19).

Previously, we performed microarray analyses of gene expression in the aging brain and heart of mice (20,21). Aging in the heart was associated with transcriptional alterations consistent with a metabolic shift from fatty acid to carbohydrate metabolism, increased expression of genes encoding structural proteins, and reduced protein synthesis. The global transcriptional profile of aging in the brain was suggestive of marked immune and inflammatory responses, oxidative stress accompanied by the accumulation of altered or misfolded proteins, and reduced neuronal plasticity and neurotrophic support. In this study, we investigated the effects of T and T on age-related transcriptional changes in the heart and brain of mice on a genomic scale by monitoring mRNA levels using high-density oligonucleotide arrays.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Mice and dietary manipulations. Male B6C3F₁ mice were purchased from Harlan Sprague-Dawley at 6–7 wk of age. Mice were housed singly in a specific pathogen-free facility and consumed acidified water ad libitum. The control groups were fed 410 kJ/wk of AIN-93M diet (22,23), which contained 50 mg/kg of tocopherol as T (Supplemental Table 1). The 2 tocopherol-supplemented groups received either a diet supplemented with 1000 mg/kg of T (T succinate) or a diet containing 500 mg/kg of T and T each, starting at 15 mo of age. Mice were killed at 30 mo of age by rapid cervical dislocation and autopsied to exclude mice showing overt disease. Tissues were immediately flash-frozen in liquid nitrogen and stored at –80°C until use. All aspects of mouse care were approved by the appropriate committees and conformed to institutional guidelines of University of Wisconsin, Madison.

    Measurement of tissue levels of tocopherol supplementation. Tissues were weighed and homogenized in 1.0 mL ethanol containing 1.2% pyrogallol using a PowerGen 125 polytron (Fisher Scientific). The sample was then vortexed for 20 s and heated at 70°C for 2 min. After 100 µL of 30% KOH being added, the sample was vortexed for 20 s and heated at 70°C for 30 min for saponification. After cooling for a few minutes, 1.0 mL distilled water was added, followed by 100 µL internal standard (4 µg/L Tocol in ethanol). Then 2.0 mL hexane containing 0.02% BHT was added to the sample. The sample was then vortexed for 2 min and centrifuged at 1800 x g; 5 min at 4°C. The top hexane layer was transferred to a glass vial where it evaporated under nitrogen gas. The dried sample was reconstituted in 100 µL of ethanol containing 0.02% BHT and injected into HPLC. The concentration of tocopherols was calculated by the HPLC Millennium software by generating a standard curve by injecting known amounts of T and T standard containing the internal standard Tocol. We compared tocopherol measurements of different tissues of mice to tissues of 30-mo-old control mice (OC).

    RNA sample preparation and hybridizations. Total RNA was extracted from frozen tissue and poly A⁺ RNA was purified from total RNA. Double-stranded cDNA synthesized from poly A⁺ RNA was used to make biotin-labeled cRNA as previously described (24). cRNA was then hybridized to the GeneChip Murine Genome Array U74Av2 and GeneChip Mouse Genome 430 2.0 Array for heart and brain tissues, respectively. Following hybridization, the hybridization solutions were removed and the gene chips were installed in a fluidics system for washes and staining. The gene chips were read using a Hewlett Packard GeneArray Scanner (Affymetrix). The averaged images collected from 2 scanned images were used for the analysis. We used 5 mice per group and hybridized each sample to independent DNA chips, because previous work from our laboratory suggested that variability between individuals is higher than variability observed in replicate hybridizations of the same samples (25). The microarray gene expression data are available at http://www.ncbi.nlm.nih.gov/geo/ (accession nos. GSE 8146, 8150, and 8162).

    Quantitative RT-PCR. To validate the results of oligonucleotide arrays, we performed real-time quantitative RT-PCR analyses. Isolated mRNA was reverse transcribed to cDNA and amplified using the TaqMan EZ RT-PCR kit (Applied Biosystems), which uses the 5' nuclease activity of Taq DNA polymerase to generate a real-time quantitative DNA analysis assay. All primer and fluorescent probe sets for assayed genes are shown in Supplemental Table 2. Gene-specific TaqMan probes contain a fluorescent reporter (FAM) at the 5' end of the probe and a quencher dye (TAMRA) at the 3' end of the probe. -Globin and TATA-box binding protein, the expression of which did not change with age, were used as a controls for data normalization. As the PCR cycle progresses, the degradation and release of the fluorescent reporter results in fluorescence at 518 nm. The accumulation of PCR products, therefore, is detected directly by monitoring the increase in fluorescence during the complete amplification process. mRNA quantification was performed using the ABI prism 7000 Sequence Detection system.

    Statistical methods. For the microarray analysis, we used nonparametric bootstrap hypothesis testing (26) to obtain the most frequent P-value for each gene. Part of our reason to use 5 mice per group is that n = 5 in a 2-group comparison offers a sufficient number (>15,000) of unique bootstrap resamples. We used 10,000 resamples to compute P-values to the level of 10^–4. Gene expression change was considered significant at P < 0.05. In the 2nd step, we used a mixture modeling approach (27) that estimates the posterior probabilities (pp) that a gene is a true positive for each gene. The pp is the Bayesian probability that a gene with a given the most frequent P-value or smaller is true different between the 2 conditions being studied. The mixture modeling approach also provides an omnibus test of whether there is any overall effect of treatment on gene expression levels, an estimate of the total number of genes that have their expression levels altered, and a probabilistic model predicting the effects of treatment on gene expression (27). To classify the gene expression profile according to functional annotation of each gene and test the significance of age-related changes in each category, we used the Web-based Database for Annotation, Visualization and Integrated Discovery (28) and Expression Analysis Systemic Explorer (EASE) (29). The Database for Annotation, Visualization and Integrated Discovery allows us to access a relational database of functional annotation. EASE facilitates the biological interpretation of gene lists derived from the results of microarray and provides statistical methods for discovering enriched biological themes within gene lists. The EASE score, a Fisher Exact P-value for gene-enrichment analysis, ranges from 0 to 1 (29). Gene Ontology annotation categories with Fisher's exact P-values 0.05 were considered significantly enriched.

For tocopherol measurements on tissues and RT-PCR of multiple genes, the significance of the observed mean differences was determined based on ANOVA and adjusted P-values obtained from SAS's least-square mean algorithm. These P-values were adjusted to maintain the family-wise type-1 error rate within each dependent variable despite the multiple comparisons conducted. The method of adjustment used was simulation (30) and was carried out in SAS using the "ADJUST = SIMULATE" option.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Supplementation effects on tissue concentrations. The T and T concentrations were measured in heart, liver, quadriceps, cerebellum, and neocortex of the OC group, 30-mo-old T–supplemented mice (T group), and 30-mo-old - and T–supplemented mice (T group) (Table 1). Tissue levels of T were significantly greater in most tissues of the T and T groups than in the OC group. Tissue levels of T were not significantly increased in the T group. T concentration was significantly higher in the liver, quadriceps, cortex, and cerebellum of the T group.

View this table: [in this window] [in a new window]   TABLE 1 Concentrations of T and T in tissues of unsupplemented mice (OC) and those supplemented with T and T1

In the neocortex, 8523 (20%) of the 45,037 transcripts surveyed displayed significant changes in expression with aging. There were also age-related inductions of genes involved in energy metabolism and cellular immune and inflammatory responses, as observed in heart. However, genes involved in protein synthesis showed opposite age-related expression patterns in the neocortex compared with the heart. mRNA levels of genes encoding ribosomal proteins and translation initiation and elongation factors increased and several genes involved in mRNA processing decreased in expression with aging. In addition, several genes involved in neurotransmitter transport were induced, whereas genes involved in chromosome organization and antiapoptotic activity were reduced in expression with aging in the brain (Supplemental Table 4).

    T and T supplementation reduces many age-related alterations in gene expression in the heart. Tocopherol supplementation prevented many age-related gene expression changes either completely or partially. Of the 1258 genes that changed significantly with aging, 1015 genes (81%) were transcriptionally regulated in the opposite direction as normal aging by T supplementation and 1021 genes (81%) by T and T supplementation (Supplemental Fig. 1A,B). Of these, the number of genes that differed significantly between the OC and tocopherol-supplemented groups was 533 (53%) in the T group and 634 (64%) in the T group. The mean reduction of T supplementation on individual age-related genes was 68.9% and that of the T and T mixture was 55.1%.

Upregulation of many procollagens observed in normal aging in the heart was completely suppressed in both the T and T groups (Table 2). Tocopherol supplementation suppressed age-related increases in mRNA levels of genes involved in cytoskeleton organization, including some actin proteins and myosin light chains, as well as age-related increases in gene expression for genes encoding junctional proteins, such as claudin 5 and the gap junction membrane channel protein 1. Many age-related diseases are associated with a chronic inflammatory state, including local infiltration of inflammatory cells and higher circulatory levels of proinflammatory cytokines, complement components, and cell adhesion molecules (31). Age-related upregulation of genes involved in the complement cascade and histocompatibility complex factors was largely prevented in both the T and T groups (Table 2). Three key enzymes involved in the metabolic shift toward carbohydrate metabolism, pyruvate dehydrogenase kinase 4, uncoupling protein 3, and malonyl CoA decarboxylase, were downregulated in the hearts of the OC group. Supplementation had no or marginal effect in preventing age-related decreases in expression of these 3 genes (Table 2). Age-related change in expression of other genes involved in fatty acid metabolism was only marginally prevented.

View this table: [in this window] [in a new window]   TABLE 2 The effects of T and a mixture of T and T supplementation on age-related transcriptional changes in mouse heart1

    T and T inhibit several age-related transcriptional changes in the neocortex.

Many genes involved in glycolysis and the tricarboxylic acid (TCA) cycle were induced in the aged neocortex (Table 3). Compared with age-matched controls, only the mixture of T and T suppressed this induction. There was an age-associated increase in expression of genes encoding the mitochondrial F₀F₁-ATP synthase complex and most of these transcriptional inductions were prevented in the T group. We also observed a concerted induction of the complement cascade genes and histocompatibility genes involved in innate immunity in the brain (Table 3). Neither experimental diet showed significant inhibitory effect on age-related changes in expression of these genes.

View this table: [in this window] [in a new window]   TABLE 3 The effects of T and a mixture of T and T supplementation on age-related transcriptional changes in mouse brain1

    T and T supplementation alters expression levels of genes involved in apoptosis, the antioxidant response, and DNA repair in the aged heart.

View this table: [in this window] [in a new window]   TABLE 4 Genes altered in expression in mouse heart by supplementation with T or a mixture of T and T1

    T and T supplementation modulates the expression of genes involved in apoptosis, heat shock response, and lipid biosynthesis in the aged brain. Tocopherol supplementation reduced expression of proapoptotic factors and induced antiapoptotic factors in the brain (Table 5). These included genes encoding proteins having antiapoptotic activity, such as Bcl2, BCL2/adenovirus E1B 19kDa-interacting protein 1, and BCL2-associated athanogene 4, and genes involved in the initiation of apoptosis, including Bcl10, Bcl2-binding component 3, capase 3, and programmed cell death proteins. Our data suggest that tocopherol supplementation may decrease oxidative stress in the aged brain as a result of its antioxidant activity. A state of lowered endogenous oxidative stress in T and T groups is also suggested by reduced expression of several heat shock proteins that did not change in expression in the OC group (Table 5).

View this table: [in this window] [in a new window]   TABLE 5 Genes altered in expression in mouse brain by supplementation with T or a mixture of T and T1

    RT-PCR validation of the oligonucleotide array data and induction of p53, p16, and cardiotrophin. Quantitative RT-PCR was used to independently evaluate a subset of genes whose expression changes significantly with age and for a few other genes of interest (Fig. 1). In the heart, the age-related increase in expression of actin, 1, mitochondrial ribosomal protein L34, and procollagen type XVIII, 1, which were significantly prevented by tocopherol supplementation as determined by the microarray analysis (Table 2), were also markedly prevented as determined by quantitative RT-PCR (Fig. 1A). Phenylalanine hydroxylase demonstrated the largest fold-change (FC) in normal aging in the heart (7.7-fold) and the alteration was partially prevented (46%) by T as determined by both DNA microarray and quantitative RT-PCR data. Cardiotrophin 1 (Ctf1), a molecular marker of cardiac hypertrophy (34), did not change in expression levels with normal aging (P = 0.485) but showed decreased expression in both T and T groups as determined by DNA microarray analysis (P < 0.05) and quantitative RT-PCR (Fig. 1A).

View larger version (32K): [in this window] [in a new window]   FIGURE 1  Quantitative RT-PCR analysis of a subset of genes in the heart (A) and brain (B) of 5-mo-old control mice (YC), OC, T, and T mice. The ANOVA tests comparing the OC group to other groups showed significant differences (P < 0.05) in all genes tested, except p53 in brain, P = 0.082. Values are means ± SEM, n = 3–5. •Significantly different from OC group (P < 0.05).

Increasing evidence suggests that p53-induced effects play a key role in organismal aging and longevity (37). There was an age-related upregulation of p53 in the heart (P < 0.05) and this age-related induction was completely prevented in the T and T groups (Fig. 1A). However, the expression of p53 did not change with aging (P = 0.542) or tocopherol in the aged neocortex (Fig. 1B). p16, one of the downstream components of p53 and a marker of senescence (38), displayed an age-related increase in expression in both heart and brain and was partially inhibited in the T and T groups (Fig. 1).

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
ROS are generated in mitochondria throughout the life span and evidence exists to support the causal involvement of ROS in aging (39,40). In this study, we determined the effects of supplementation of T and T on age-related transcriptional alterations in the heart and brain and also attempted to obtain evidence for unique biological roles of T and T. Tocopherol supplementation markedly prevented the age-related upregulation of genes encoding cellular structural proteins in the heart. Interestingly, we found that supplementation downregulates Ctf1, a cytokine that induces cardiomyocyte hypertrophy (34) and is induced in congestive heart failure (41). Supplementation with either T or the combination of T and T also markedly suppressed the age-related increase in expression of genes encoding several complement components (Table 2). Complement-related genes are involved in innate immunity and induced in response to ischemia and reperfusion (42). In adult hearts, fatty acids are the major energy source, whereas fetal hearts primarily use glucose as metabolic fuel (43). We show here that the age-related downregulation of genes involved in fatty acid β-oxidation is not opposed by tocopherol supplementation, a finding contrary to the effects of energy restriction in the mouse heart (20). The T and T groups did not differ in the overall, global effect on aging. These results suggest that in the mouse heart, supplemental T does not enhance the overall beneficial effects that result from T supplementation.

Unlike heart tissue, the brain was influenced by T supplementation. The mixture of T and T prevented the age-related transcriptional alterations in some specific transcriptional classes (Table 3). For example, the T group displayed marked prevention of the age-related upregulation of genes involved in glycolysis, the TCA cycle, and ATP biosynthesis. As reported previously in brains from mice (21) and humans (44), age-related increases in expression of genes involved in immune and inflammatory responses occur, which is suggestive of heightened immunity and proinflammatory status in aged tissues. Interestingly, transcriptional changes in this class were significantly inhibited by the combination of T and T in the heart but not in the brain (Tables 2 and 3). Compared with age-matched controls, only the mixture of T and T showed a significant aging-retarding effect on functional classes of genes in brain (Table 3). Because it has been observed that T has higher activity in trapping reactive nitrogen species than T (45), our observations suggest that reactive nitrogen species may induce certain aspects of aging in the brain.

To find possible mechanisms involved in the effect of supplementation with the tocopherols in the aging process, we analyzed genes that showed significant alterations in the tocopherol-supplemented groups but were not affected by aging. Several genes involved in the inhibition of apoptosis were upregulated by tocopherol supplementation, whereas genes involved in the activation of apoptosis were downregulated compared with the control group (Tables 4 and 5). Phaneuf et al. (46) first demonstrated that cytochrome C release from mitochondria and alterations in the level of Bcl2, an antiapoptotic 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 aging in the brain (47). Qin et al. (16) observed that tocopherol supplementation attenuates myocyte apoptosis in rabbits. These results support the role of ROS-induced apoptosis in aging tissues and suggest that prevention of apoptosis in normal aging by supplementation with T and T may play a role in preventing some aspects of aging.

A recent gene expression profiling study supports the hypothesis that the underlying molecular mechanism of neuroprotection by T supplementation is associated with alterations in hormone metabolism and apoptosis (48). It is also known that T supplementation represses the expression of genes involved in de novo synthesis of cholesterol in testes and adrenal glands of growing rats (49). In our study, several genes involved in steroid biosynthesis were induced by T (Table 5), suggesting that neuroprotective activity of tocopherol supplementation in the brain may be partially due to the increased biosynthesis of steroids.

Recently, biological functions of T that are independent of its antioxidant activity have been reported, including inhibition of cell proliferation, platelet aggregation, and monocyte adhesion (50). At the transcriptional level, a number of genes may be regulated through a nonantioxidant activity of T, including -tropomyosin (51) and collagenase (52). Long-term T supplementation modified serum hormone levels, including androstenedione and testosterone, which might contribute to the reduced risk of prostate cancer of T treatment (53). Therefore, it appears likely that some effects of T supplementation in this study may be due to biological processes unrelated to antioxidant activity.

In summary, supplementation with either T or a mixture of T and T can alter the transcriptional profiles associated with aging in the heart and this effect of tocopherol may be mediated through several mechanisms. First, supplementation reduces the expression of structural proteins in the aging heart and thus may oppose age-related cardiomyocyte hypertrophy. Increased cardiomyocyte apoptosis associated with aging may be prevented by tocopherol supplementation, because it upregulates genes encoding antiapoptotic proteins and downregulates genes involved in the induction of apoptosis in the aging heart (Table 4). In the brain, both forms of tocopherol supplementation increased the expression of antiapoptotic proteins and decreased the mRNA levels of proapoptotic proteins (Table 5). Surprisingly, only the mixture of T and T prevents age-related transcriptional alterations in the brain (Supplemental Fig. 1D and Supplemental Table 3). Therefore, the effect of tocopherol supplementation in aging is likely to be tissue and tocopherol specific. Extension of this study to other postmitotic or regenerating tissues should result in the identification of common mechanisms of action, facilitating an understanding of the role of oxidative stress and the effect of antioxidant supplementation in the aging process. Our general finding that supplementation with either T or a mixture of T and T can modulate the aging process in 2 critical organs, if applicable to humans, may have major public health implications.

	ACKNOWLEDGMENTS

 
We thank Raymond E. McCubrey at the University of Alabama at Birmingham for statistical assistance.

	FOOTNOTES

 
¹ Supported by NIH grant R01AG020681.

² Author disclosures: S.-K. Park, G. P. Page, K. Kim, D. B. Allison, M. Meydani, R. Weindruch, and T. A. Prolla, no conflicts of interest.

³ Supplemental Figure 1 and Supplemental Tables 1–4 are available with the online posting of this paper at jn.nutrition.org.

⁸ Abbreviations used: T, -tocopherol; T, -tocopherol; T group, 30-mo-old -tocopherol–supplemented mice; T group, 30-mo-old - and -tocopherol–supplemented mice; ApoD, apolipoprotein D; Ctf1, cardiotrophin 1; EASE, Expression Analysis Systemic Explorer; FC, fold-change; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; OC, 30-mo-old control mice; pp, posterior probability; ROS, reactive oxygen species; SOD, superoxide dismutase; TCA, tricarboxylic acid; YC, 5-mo-old control mice.

Manuscript received 7 February 2007. Initial review completed 22 March 2007. Revision accepted 14 March 2008.

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