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Modulation of glutathione and thioredoxin systems by calorie restriction during the aging process
Experimental Gerontology 38 (2003) 539–548 www.elsevier.com/locate/expgero
Modulation of glutathione and thioredoxin systems by calorie restriction during the aging process Chong Gun Choa, Hyon Jeen Kimb, Sang Woon Chungb, Kyung Jin Jungb, Kyung Hee Shimb, Byung Pal Yuc, Junji Yodoid, Hae Young Chungb,* a
b
Department of Molecular Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA Department of Pharmacy, College of Pharmacy, Pusan National University, San 30, Jang-jun-dong, Gumjung-gu, Pusan 609-735, South Korea c Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA d Department of Biological Responses, Institute for Virus Research, Kyoto University,Kyoto 606-8567, Japan Received 26 August 2002; received in revised form 3 January 2003; accepted 9 January 2003
Abstract Accumulating evidence strongly suggests that oxidative stress underlies aging processes and that calorie restriction (CR) retards aging processes, leading to an extended lifespan for various organisms. Recent studies revealed that the anti-aging action of CR depends on its antioxidative mechanism. However, at present, the status of glutathione (GSH) and thioredoxin (Trx) system, two major thiol redox systems in animal cells during aging and its modulation by CR has not fully been explored. The purpose of this study is two-fold: one, to determine whether these two systems in rat kidney are altered as a consequence of aging; two, to determine whether these systems can be modulated by anti-oxidative CR. The results of our study showed that GSH and GSH-related enzyme activities decreased with age in ad libitum (AL)-fed rats, while CR rats consistently showed resistance to decreases in these activities. Data from the present data further showed that while Trx and Trx reductase (TrxR) in cytoplasm decrease with age in AL-fed rats, CR prevents these decreases. In contrast, we also found that the nuclear translocation of the redox regulators, Trx and Ref-1, increase with age, which was suppressed in CR rats. Therefore, increases in nuclear Trx and Ref-1 during aging may result in the up-regulation of redox-sensitive transcription factors, such as NF-kB or AP-1, via the interaction of Ref-1 and Trx in a redox-dependent manner. Our conclusion is that a redox imbalance occurs during aging and that redox changes are minimized through the anti-oxidative action of CR. q 2003 Elsevier Science Inc. All rights reserved. Keywords: Thioredoxin; Glutathione; Aging; Calorie restriction; Ref-1; GSH reductase; GSH peroxidase; GSH S-transferase
1. Introduction Much evidence strongly indicates that most age-related changes are causally linked to deleterious reactive species (RS) generated from aerobic metabolism (Beckman and Ames, 1998; Sohal and Weindruch, 1996). The current oxidative theory of aging proposes that free radical-based oxidative stress is the main culprit causing a net imbalance in an organism’s redox status during aging (Rikans and Hornbrook, 1998). This imbalance or disturbance in the redox status has been associated with aged-related functional loss (Jenkinson et al., 1991). Oxidative stress can cause wide-ranging damage to macromolecules and is * Corresponding author. Tel.: þ 82-51-510-2824; fax: þ82-51-510-2814. E-mail address: [email protected] (H.Y. Chung).
linked to various chronic degenerative disease processes, including vascular diseases (Harman, 1973; Yu and Chung, 2001a,b). A recent review documented sufficient evidence for the oxidative process as a major factor in the inflammatory processes that underpin age-related diseases (Chung et al., 2000). Caloric restriction (CR), the only established anti-aging experimental paradigm, has been investigated extensively and is shown consistently to increase both median and maximum life spans in laboratory animals by retarding the onset and progression of many age-associated, pathologic changes (Yu et al., 1985; Sohal and Weindruch, 1996; Yu and Chung, 2001a,b). Recent research provides strong evidence that CR may exert diverse anti-aging benefits by its ability to modulate age-related oxidative stress (Yu and Chung, 2001a,b; Chung et al., 2001). CR has been shown to
0531-5565/03/$ - see front matter q 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0531-5565(03)00005-6
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attenuate oxidative damage by suppressing RS generation, while maintaining antioxidant defense systems, including major RS neutralizing systems (Sohal and Weindruch, 1996). Several reports highlight the key role of sulphydryl groups (-SH) play in response to oxidative stress (Demple, 1998; Rietsch and Beckwith, 1998). Thiol-disulfide exchange reactions, which are rapid and readily reversible, are ideally suited to the redox control of the protein structural and catalytic function. In many cellular processes, the thiol-disulfide exchange is provided by an interaction between the thioredoxin (Trx) and glutathione systems (Halliwell, 1999), as diagrammed in Fig. 1. The Trx and GSH systems are involved in a variety of redox-dependent pathways. These systems provide reducing equivalents for ribonucleotide reductase and peptide methionine sulfoxide reductase, an antioxidant defense line and regulator of the cellular redox state (Halliwell, 1999; Sies, 1999). More importantly, the Trx and GSH systems regulate the signal transduction activity of various kinases and phosphatases and modulate the redox control of cell growth, death, and the transactivation of redox-sensitive transcription factors (Masutani et al., 2000). The GSH system is also a key component of the overall antioxidant defense system that protects the cell from the deleterious effects of RS (Meister and Anderson, 1983). In addition, GSH detoxifies a variety of exogenous and endogenous substances both non-enzymatically and via enzymatic conjugation catalyzed by GSH S-transferase (GST) (Meister, 1983). GSH peroxidase (GPx) reduces reactive peroxides to alcohols and water at the expense of GSH, which is oxidized to GSSG. GSSG is recycled to GSH by GSH reductase (GR), as shown in Fig. 1. For the agerelated disturbance in GSH metabolism, evidence has been reported by Jenkinson et al. (1991). Recently, Liu and Choi (2000) have reported that activity and gene expression of gglutamylcysteine synthetase, which is the rate-limiting enzyme in GSH synthesis were decreased during aging, providing the molecular insight to the altered GSH content in several tissues.
Fig. 1. Coupling of thioredoxin and glutathione systems. GSH, glutathione; GSSG, oxidized glutathione; Trx, thioredoxin.
Trx is a small, globular ubiquitous protein of 12 kDa with two redox-active half-cysteine redisues in its catalytic active center, having the consensus amino acid sequence, Cys– Gly– Pro – Cys (Holmgren, 1989). The protein exists either in a reduced form with a dithiol or in an oxidized form, in which the half-cysteine residues form an intramolecular disulfide bridge. Trx participates in redox reactions by the reversible oxidation of its active center, dithiol, to disulfide and catalyzes dithiol –disulfide exchange reactions, including signal transduction and gene expression. Trx is maintained in its active reduced form by flavoenzyme thioredoxin reductase (TrxR) in the presence of NADPH, which constitutes the thioredoxin system (Holmgren, 1989). Although Trx is localized in the cytoplasm, it quickly translocates into the nucleus upon stimulation, as seen in response to phorbol myristate acetate (PMA), UV, or other stimuli (Hirota et al., 1999). In the nucleus, Trx forms a physical interaction with the Ref-1 gene product, redox factor-1 (Ref-1) (Hirota et al., 1997). Ref-1 (also designated APE, HAP-1, and APEX) functions as both a nuclear DNA repair enzyme and a reversible regulator of DNA binding activity for several nuclear transcription factors, including AP-1, NF-kB, and HIF-1. The Trx/Ref-1 complex regulates transcriptional activity by altering the redox state of specific cysteine residues located in the basic DNA binding region of these transcription factors (Hirota et al., 1997, 1999). In the present paper, we attempted to establish the changes in the GSH and Trx status that are modulated by CR during the aging process.
2. Experimental procedures 2.1. Animals Rat maintenance procedures for specific-pathogen free status and dietary composition of chow have been previously reported (Yu et al., 1985). Briefly, male, specific pathogen-free Fischer 344 rats were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% a-methionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix, and 3% Solka-Floc. The ad libutum (AL) fed group had free access to both food and water. The animals designated as CR were fed 60% of the food intake of their AL-fed littermates, beginning at 6 weeks of age. Rats at 6, 12, 18, and 24 months of age were sacrificed by decapitation and the kidneys were quickly removed and rinsed in ice-cold buffer (100 mM Tris, 1 mM EDTA, 0.2 mM phenylmethylsulfonylfluoride (PMSF), 1 mM pepstatin, 2 mM leupeptin, 80 mg/L trypsin inhibitor, 20 mM b-glycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate, pH 7.4). The tissue was immediately frozen in liquid nitrogen and stored at 2 80 8C. Histopathological examinations revealed no evidence of nephrotic lesions in soy-protein fed, male Fischer rats even at the advanced age of 24 months, as
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previously reported (Iwasaki et al., 1988). This study complied with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health (Publication No. 85 –23). 2.2. Materials All chemical reagents were obtained from Sigma (MO, USA), except where noted. Western blotting detection reagents were obtained from Amersham (Bucks, UK). RNAzole B was obtained from TEL-TEST, Inc. (TX, USA). Primers for reverse transcriptase –polymerase chain reaction (RT-PCR) were synthesized by Bioneer (Daejeon, Korea). Reverse transcriptase was obtained from Gibco – Bethesda Research Laboratory (MD, USA), Taq DNA polymerase and 10X PCR buffer were from Perkin Elmer (CA, USA). Antibody against Ref-1 was obtained from Santa Cruz Biotechnology (CA, USA). Antibody against TrxR was from Upstate Biotechnology (NY, USA). Antirabbit IgG-horseradish peroxidase-conjugated antibody and anti-mouse IgG-horseradish peroxidase-conjugated antibody were obtained from Amersham (Bucks, UK). Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore Corporation (MA, USA). All other materials were obtained in the highest available grade.
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reductase, and 250 mM H2O2 in a total volume of 250 ml. Samples from the kidney homogenate were mixed with this reaction medium, and absorbance at 340 nm was recorded for 5 min at 37 8C. The GSSG formed by GSH peroxidase activity in the homogenate acting on the hydroperoxide was reduced by GSSG reductase, resulting in the consumption of NADPH. This decrease in NADPH concentration was estimated from the slope of the absorbance by using the appropriate molar extinction coefficient (1 ¼ 6.2 £ 103 M21cm21). GPx activity was measured as the amount of NADPH consumed per minute per milligram of protein. 2.6. GSH S-transferase activity GST activity was estimated by the method of Habig et al. (1974). 1-chloro-2, 4-dinitrobenzene (CDNB) was used as the substrate. The final concentrations of reagents in the assay mixture were 50 mM potassium phosphate buffer (pH 7.0), 1 mM CDNB, 1 mM GSH. The reaction mixture was incubated for 5 min at 25 8C, and then the sample was added. Absorbance was measured at 340 nm and recorded for 5 min. One unit of activity is defined as 1 mmole of 1chloro-2, 4-dinitrobenzene produced. 2.7. GSH reductase activity
2.3. Tissue preparation One gram of kidney was homogenized with 5 ml of 50 mM phosphate homogenate buffer (containing 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM pepstatin, 80 mg/L trypsin inhibitor, pH 7.4) and centrifuged at 900g at 4 8C for 15 min. The supernatants were re-centrifuged at 12; 000g at 4 8C for 15 min to yield sedimented mitochondrial fraction and post-mitochondrial supernatant fraction. The supernatants were centrifuged again at 105; 000g at 4 8C for 1 h to separate cytosol supernatant fraction. 2.4. GSH assay Twenty-five percent meta-phosphoric acid was added to post-mitochondrial fractions, followed by centrifugation at 12; 000g for 10 min for GSH assay in the supernatants. To assay GSH, 1 mM EDTA-50 mM phosphate buffer (pH 8.0) was added to the supernatant followed by o-phthaldehyde. After 20 min at room temperature, the fluorescence was measured at excitation wavelength of 360 nm and emission wavelength of 460 nm (Tauskela et al., 2000). 2.5. GSH peroxidase activity GPx activity was estimated by the indirect coupled assay described by Tappel (1977). The reaction medium consisted of 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 0.15 mM NADPH, 1 mM GSH, 0.24 U GSSG
GSH reductase activity was measured according to the method described by Eklow et al. (1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.6), 1 mM EDTA, 0.1 mM NADPH, 1 mM GSSG in a total volume of 150 ml. The samples from the kidney homogenate (200 ml) were mixed with this reaction medium, and incubated 5 min in at 25 8C. Absorbance at 340 nm was recorded for 2 min. One unit of activity is defined as 1 nmole of NADPH oxidized. 2.8. Western blotting Western blotting was carried out as described previously (Habibet al., 1993). Homogenized samples were boiled for 5 m with a gel-loading buffer (0.125 M Tris –Cl, 4% SDS, 10% 2-mercaptoethanol, pH 6.8, 0.2% bromophenol blue) in ratio of 1:1. Total protein-equivalents (100 mg) for each sample were separated by SDS – PAGE using 10% acrylamide gels as described by Laemmli (1970) and transferred to polyvinylidene difluoride membrane at 15 V for 1 h in a semi-dry transfer system. The membrane was immediately placed into a blocking buffer (1% non-fat milk) in 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20. The blot was allowed to block at room temperature for 1 h. The membrane was incubated with mouse monoclonal anti-Trx (1:1000) for 1 h at 25 8C, and followed by an anti-rabbit IgG-horseradish peroxidase-conjugated antibody (1:5000) for 1 h at 25 8C. Western blotting with rabbit polyclonal anti-TrxR (1:1000) or anti-Ref-1 (1:500) was also
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performed. Antibody labeling was detected using enhanced chemiluminescence per the manufacturer’s instructions. Pre-stained protein markers were used for molecular weight determinations. 2.9. Nuclear extract preparation All solutions, tubes, and centrifuges were maintained at 0 –4 8C. For each nuclear extract preparation, three rat kidney tissues were pooled. The preparation of nuclear extracts was based on previous methods (Hattori et al., 1990). The nuclear extract was frozen at 2 80 8C in aliquots until electrophoretic mobility shift assay (see below) was done. The concentration of total protein in samples was measured with the Sigma protein assay reagent kit containing bicinchoninic acid. 2.10. Isolation of RNA Total RNA was isolated by the method of Puissant and Houdebine (1991). Briefly, tissue samples were homogenized in the presence of RNAzole B (2 ml per 100 mg tissue) with a few strokes in a tissue homogenizer. Aliquots of 0.2 ml chloroform per 2 ml homogenate was added, the samples were shaken vigorously for 15 s, and kept in ice for 5 min. The suspension was centrifuged at 12,000 g for 15 m at 4 8C. The aqueous phase was transferred to the fresh tube, to which an equal volume of isopropanol was added, and the samples were kept at 4 8C for 15 min. Samples were again centrifuged at 12; 000g for 15 min at 4 8C. The supernatant was removed and the RNA pellet was washed once with 75% ethanol by vortexing and subsequent centrifugation at 7500g for 8 min at 4 8C. The pellet was dried for 10 – 15 min. The RNA pellet was dissolved in diethylpyrocarbonate (DEPC)-treated water. 2.11. Assay of RT-PCR (a) Reverse Transcription: The first strand cDNA was synthesized from 2 mg of total RNA. DEPC-treated water and 250 ng random primer were added, incubated for 5 min at 75 8C, and incubated on ice for 5 min. Aliquots of 2 ml 0.1 M DTT, 4 ml 5X buffer, 4 ml 2.5 mM dNTP, 100 U reverse transcriptase and 16.5 U of RNase inhibitor were added and incubated for 2 h at 37 8C. The reaction was stopped by boiling for 2 min at 100 8C, and cDNA was stored at 2 20 8C until use. (b) PCR: The primer pairs for rat Trx were as follows: sense, 50 -ATGGTGAAGCTGATCGAGAG-30 , anti-sense, 50 -GCATGATTAGGCAAACTCCG-30 . The expected product size for rat Trx is 324 bp. The primer pairs for rat Ref-1 were as follows: sense, 50 -GCCAGAGACCAAGAAGAGTA-30 , anti-sense, 50 -TCTGAAGGCTTCATCCCATC-30 . The expected product size for rat Ref-1 is 520 bp. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) primers were used as control for the efficiency of cDNA synthesis in each sample. The primer pairs for GAPDH were as follows: sense, 50 0 GGGTGATGCTGGTGCTGAGTATGT-3 ; anti-sense, 50 AAGAATGGGAG TTGC TGTTGAAGTC-30 . The GAPDH primer set yields a PCR product of 700 bp in length. To carry out the PCR, 25 ml PCR master mix was added to each tube directly. Deoxynucleotides were added to final concentration of 0.2 mM. 10X PCR buffer was diluted in ratio of 1:10 and 1.25 U of Taq DNA polymerase was added. The reaction medium was then added 50 ng of sense and antisense primers for Trx, Ref-1, and GAPDH to each tube, respectively. Reaction conditions consisted of 32 cycles for rat Trx, 25 cycles for rat Ref-1, and 20 cycles for GAPDH at 94 8C for 30 s denaturation, at 54 8C for 30 s annealing, and at 72 8C for 1 min extension. Electrophoresis was performed in 1% agarose gel. After staining in ethidium bromide solution was achieved, the gel was observed under UV transilluminator. 2.12. Statistical analysis The statistical significance of the differences between the treatments (age and diet) was determined by two-factor ANOVA. One-factor ANOVA was conducted to analyse significant differences between all possible age and diet pairs. Differences in the means of individual groups were assessed by the Fischer’s protected LSD post-hoc test. Values of p , 0:05 were considered statistically significant.
3. Results 3.1. GSH status and CR modulation during aging Because GSH is a major anti-oxidant component that battles against oxidative stress, we found it important to assess age-related changes of GSH concentrations. As shown in Fig. 1, we observed a decrease in the level of GSH in the kidney of AL-fed, 18- and 24-month-old rats compared to that of 6-month-old rats. However, CR rats were able to blunt the decrease, as shown in Fig. 2. 3.2. Effect of aging and CR on GSH-related enzyme activity To further investigate individual components of the GSH system, its three key enzymes were monitored. Fig. 3 shows the age-related changes in the activities of GPx (Fig. 3(A)), GST (Fig. 3(B)), and GR (Fig. 3(C)) in four age groups of AL and CR rats. The activities of these three enzymes in old AL rats were lower than those of younger AL rats. Data on CR rats were contrasted by results of higher enzyme activities than those of their AL counterparts. These changes in enzyme activities correlated well with the changes seen in GSH levels.
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Fig. 2. CR suppresses age-related decreases of glutathione content. Glutathione (GSH) content in kidney homogenates was determined as described in the method. Represented are the mean ^ SEM ðn ¼ 6Þ: Results of one-factor ANOVA: p P , 0:05; p p P , 0:01; and p p p P , 0:001 VS. 6-month-old AL rat: # P , 0:05; ## P , 0:01; and ### P , 0:001 VS. age matched AL rats.
3.3. Modulation of Trx and TrxR gene expression by aging and CR We examined age-related modulation in the gene expression of two proteins, Trx and TrxR of the Trx system. Fig. 4(A) shows that cytoplasmic Trx protein levels significantly decreased with age in the AL group. The decrease was so pronounced in 18- and 24-month-old rats that the protein bands were not detectable by Western blotting (AL in Fig. 4(A)). However, Trx protein levels in the CR group were reasonably well maintained even for 24month-old rats (CR in Fig. 4(A)). As shown in Fig. 4(B), TrxR protein levels decreased with age in AL rats, while CR rats showed higher levels than their AL counterparts. Since TrxR is the only class of enzymes known to reduce Trx, it is expected that an alteration in TrxR may affect Trx activity during aging. Further, RT-PCR analysis of Trx RNA to assess Trx transcription expression revealed that the Trx mRNA level interestingly did not change during aging in either AL or CR rats (Fig. 4(D)). 3.4. CR modulates nuclear translocation of Trx and Ref-1 during aging The cytoplasmic Trx is known to be translocated upon stimulation in response to PMA, oxidative stress, UV irradiation, and TNFa. Because the aging process is considered a chronic state of oxidative stress, we sought evidence of the nuclear translocation of Trx protein during aging. As shown in Fig. 5(A), Trx in the nucleus increased with age in AL rats, while CR rats were shown to block this age-related increase. Nucleus-imported Trx interacts with Ref-1 to form a regulatory Trx/Ref-1 complex. A function of this complex is
Fig. 3. CR activates enzyme activities of glutathione peroxidase, glutathione S-transferase, and glutathione reductase. Enzyme activities of glutathione peroxidase (GPx) (A), glutathione S-transferase (GST) (B), and glutathione reductase (GR) (C) were measured in kidney homogenate from AL or CR rats, ages 6, 12, 18, and 24 months old. Values are expressed as mean ^ SEM (n ¼ 10). Results of one-factor ANOVA: p P , 0:05; pp P , 0:01; and p p p P , 0.001 VS. 6-month-old AL rat: # P , 0:05; ## P , 0:01; and ### P , 0:001 VS. age matched AL rats.
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Fig. 4. CR upregulates age-related decreases of cytosolic Trx and TrxR. Western blot analysis was performed to detect renal Trx (A) and TrxR (B) protein levels in cytoplasmic extracts (50 mg protein) from AL or CR rats, ages 6, 12, 18, and 24 months old. Trx, thioredoxin; TrxR, thioredoxin reductase (C) b-Actin protein level is loading control for western blot. (D) RT-PCR analysis of renal Trx was performed from RNA isolated from rat kidney. GAPDH primers were used as control for the efficiency of cDNA synthesis in each sample. One representative result of each is shown from three experiments that yielded similar results, respectively. The data were quantified by densitometry, and presented as a percentage of 6-month-old AL rat. Results of one-factor ANOVA: p P , 0:05; pp P , 0:01; and ppp P , 0:001 VS. 6-month-old AL rat: # P , 0:05; ## P , 0:01; and ### P , 0:001 VS. age matched AL rats.
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to modulate the transcriptional activities of redox-sensitive transcription factors, such as NF-kB, AP-1, and HIF-1 by keeping cysteine residues reduced. We examined changes in nuclear Ref-1 protein levels with age (Fig. 5(B)) in AL and CR rats, which showed age-related changes with the nuclear translocation of Trx (shown in Figs. 4(A) and 5(A)). We recently obtained evidence that NF-kB (Kim et al., 2002 a,c, d),), AP-1 Kim et al., 2002b, and HIF-1 (data not shown, manuscription in preparation) were activated during aging, suggesting that an age-related increase in nuclear Trx and Ref-1 may contribute to the activation of these transcription factors during aging.
4. Discussion
Fig. 5. CR suppresses nuclear translocation of nuclear Trx and Ref-1. Trx (A) Ref-1 (B), and HIF-1b (C, as loading control) protein levels in nuclear extracts (30 mg protein) were measured by Western blot analysis. Trx, thioredoxin; Ref-1, redox factor-1. In the upper panel, one representative blot of nuclear Trx and Ref-1 is shown from three experiments that yielded similar results, respectively. The levels of each protein were quantified by densitometry. The data shown in bottom are presented as the percentage relative to 6-month-old AL rat.
The purpose of the current study was to explore at the molecular level two major disulfide-reducing systems, GSH and Trx, and their modulation by the anti-oxidative, lifeprolonging action of CR during aging. Data show that GSH and GSH-related enzymes are decreased in the kidney of 24month-old rats. Similar findings were reported by Nakata et al. (1996), showing a large decrease in the hepatic level of GSH in 24-month-old mice, and Sasaki et al. (2001) who reported increased oxidized GSH with age in rat brain. However, findings on the age effect of the GSH status are not unanimous: some reported that GSH levels in rats remain unchanged (Richie, 1992), while others found that GSH levels increase during aging (Quiroga et al., 1990; Jenkinson et al., 1991). A recent data reported by Liu and Choi (2000) shed further evidence that the age-related changes in GSH may depend on the tissue or organ. The authors found that the GSH content in rat liver, kidney and lung decreased roughly 20– 37% between the age of 12– 24 months, while no detectable decrease was noticed in heart and diaphragm. This organ specificity was further substantiated by the age-related decrease in gene expression of g-glutamylcysteine synthetase in the corresponding organs (Liu and Choi, 2000). A literature search reveals that the status of the Trx system during aging has not been studied extensively and that existing data are conflicting. For instance, it was demonstrated that TrxR activity in livers of male and female rats were unchanged with aging (Rikans and Hornbrook, 1998), but data reported by Santa Maria and Machado (1986a and b) showed decreased activity of TrxR activity during aging in kidney and lung. The latter report is in agreement with our present findings showing an age-related decrease in Trx system activity. The tissue-specific modulation of Trx activity may be related to the functional state of the organ under oxidative stress. Recently, Carmel-Harel et al. (2001) reported that decreased TrxR diminished the capacity to detoxify oxidants and to repair oxidative stress-induced damage. These findings provide additional evidence that decreases in cytoplasmic Trx and
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Fig. 6. A schematic presentation of protein interaction and nuclear translocation of Trx and Ref-1. Trx, thioredoxin; Ref-1, Redox factor-1.
TrxR activity certainly compromise the organism’s ability to guard against cellular oxidative forces during aging. In the current work, we observed that the translocation of Trx into the nucleus with aging was facilitated (Fig. 6). Recently, our laboratory showed increased DNA binding activity of redox-sensitive transcription factors such as NFkB (Kim et al., 2002 a,c,d) and AP-1 (Kim et al., 2002b) with age. We also showed that the activated MAPK signaling pathway is likely caused by a compromised agerelated redox status (Kim et al., 2002a,b,c). The essential role that the thiol groups play in the intricate regulation of defense mechanisms against oxidative stress is well established (Halliwell, 1999; Sies, 1999). The crucial function of thiols in the modulation of redox-sensitive proteins to oxidative stress in cell membranes also has been reported (Freeman and Crapo, 1982). These investigators reported that high contents of GSH and Trx protect thiol oxidation in structural proteins and enzymes for optimal biological functions. Recently, Lou et al. (1999) reported that the size of GSH pool was diminished in aged lenses under oxidative stress, and that some protein thiols are being S-thiolated by oxidized nonprotein thiols to form protein –thiol mixed disulfides. The authors concluded that thiol oxidation is the result of a cascade of events, starting with protein disulfide crosslinking, protein solubility loss, and eventual lens opacification. GSH, one of the most biologically versatile antioxidant systems, is known to affect cellular functions under the altered redox state as found in low GSH contents in several pathological conditions, including alcoholic liver disease (MacDonald et al., 1977), acquired immunodeficiency syndrome (Staal et al., 1992), xenobiotic-induced oxidative stress and toxicity (DiSimplicio et al., 1984) and precancerous lesions (Kumar et al., 1995). Based on these findings, the maintenance of optimal GSH levels and a proper redox status is crucial to the modulation of various pathogenic conditions and the course of diseases. Thus, low GSH levels may be a risk factor for aging.
The roles of Trx seem to be diverse and often contradictory. Trx has been shown to assume the role of an antioxidant in the tumor-preventing system (Halliwell, 1999; Sies, 1999) and have cytoprotective effects against oxidative stress. However, the beneficial role of Trx ceases once a tumor becomes established, purportedly promoting the growth of variety of cells (Iwata et al., 1994; Gasdaska et al., 1995). Its role is not limited to the detoxification of reactive oxygen metabolites but also is seen in cellular signaling processes. For instance, several studies examining reactive oxygen species (ROS) production in cancer (Shimoda et al., 1994; Toyokuni et al., 1995) found that the cellular level of Trx increases in response to oxidative stress, suggesting ROS production as a possible reason for elevated Trx in cancer. Trx is also shown to promote DNA binding of redox-sensitive, pro-inflammatory transcription factors such as NFkB, AP-1 and is implicated in inflammatory diseases, such as Sjo¨gren’s syndrome and rheumatoid arthritis (Yoshida et al., 1999). Furthermore, decreased Trx in Alzheimer’s disease brain led to the loss of protection against amyloid b-peptide toxicity (Lovell et al., 2000). Some evidence provides strong support for the roles of the Trx and GSH systems in determining the thiol – disulfide balance in the cytoplasm. Although each of these two systems has their own specific actions, their interaction is substantial (Miranda-Vizuete et al., 1996; Prinz et al., 1997). For example, a small but consistent increase in Trx was observed in the absence of GSH-coupled glutaredoxin 1 (Grx1); but an over production of one component was shown to decrease the level of the other (Miranda-Vizuete et al., 1996). These results suggest the existence of the interactive network in which the balance between Trx and Grx1 is closely regulated. It should be noted, however, that these studies were performed with prokaryote and that the roles of Trx and GSH remain to be tested in mammalian cells. A recent report by Casgrande et al. (2002) highlighted possible crosstalk between the GSH and Trx systems in human T cell.
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Extensive gerontological research has confirmed that CR imposed on laboratory rodents extends mean and maximum lifespans (Yu et al., 1985; Yu, 1996). Research has also established that CR retards age-associated pathologies such as nephropathy, cardiomyopathy, gastric ulcer, cataract, and cancer (Yu et al., 1985; Yu, 1996; Sohal and Weindruch, 1996). A recent proposal on the anti-oxidative role of CR is well supported by many studies in its life-prolonging effects (Yu, 1996). In the present study, we presented molecular evidence explaining the modulating effects of CR and its anti-oxidative mechanism, as shown in Figs. 2 –4. In summary, CR was shown to up-regulate GSH and GSH-related enzymes throughout aging process and to maintain cytoplasmic Trx levels even in 24-month-old rats, while neither was detectable in AL rats at 18 and 24 months. If aging is due to the result of a long-term cellular redox imbalance as proposed (Yu, 1996; Beckman and Ames, 1998), then the anti-oxidative action of CR and its ability to maintain a proper redox state are likely the most powerful antiaging measures for lifespan extension. In addition to the cytoplasmic regulation of Trx and Ref-1, our data indicate that CR prevents the increase of Trx and Ref-1 levels in the nucleus. These findings are important in view of our previous data showing CR’s prevention of NF-kB transactivation in aged animals (Kim et al., 2002a,b,c), because Trx and related molecules (Tanaka et al., 2001) are responsible for the maintenance of the cellular reducing environment with the help of GSH system. On the other hand, mRNA levels were shown to have no agerelated changes and not to be modulated by CR. This discrepancy in the age-related changes might be related to the possibility that enhanced protease degradation facilitates translocation of Trx into the nulcleus, while CR suppresses the process. In conclusion, the present study documents age-related decreases of GSH and Trx levels in the cytoplasm, which results in an altered redox balance. Our data further demonstrate the ability of CR to correct the imbalance. Our report showed for the first time the down-regulation of Trx and Ref-1 by aging and their attenuation by CR. The protective effects of CR on the thiol-reducing system are important to the regulation of redox balance and the promotion of an optimally maintained organism during aging. (Yu and Chung, 2001a,b).
Acknowledgements This work was supported by a grant from the Korean Research Foundation (FS0004). We are grateful to the ‘Aging Tissue Bank’ granted by KOSEF and National Institute on Aging (AG01188) for the supply of the aging tissue.
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References Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 78, 547 –581. Carmel-Harel, O., Stearman, R., Gasch, A.P., Botstein, D., Brown, P.O., Storz, G., 2001. Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae. Mol. Microbiol. 39, 595–605. Casgrande, S., Bonetto, V., Fratelli, M., Gianazza, E., Eberini, I., Massignan, T., Salmona, M., Chang, G., Holmgren, A., Ghezzi, P., 2002. Glutathionylation of human thioredoxin: a possible crosstalk between the glutatione and thioredoxin systems. Proc. Natl Acad. Sci. USA 99, 9745–9749. Chung, H.Y., Kim, H.J., Jung, K.J., Yoon, J.S., Yoo, M.A., Kim, K.W., Yu, B.P., 2000. The inflammatory process in aging. Rev. Clin. Gerontol. 10, 207– 222. Chung, H.Y., Kim, H.J., Kim, J.W., Yu, B.P., 2001. The inflammation hypothesis of aging: molecular modulation by calorie restriction. Ann. NY Acad. Sci. 928, 327–335. Demple, B., 1998. A bridge to control. Science 279, 1655–1656. DiSimplicio, P., Dolara, P., Lodovici, M., 1984. Blood glutathione as a measure of exposure to toxic compounds. J. Appl. Toxicol. 4, 227– 229. Eklow, L., Moldeus, P., Orrenius, S., 1984. Oxidation of glutathione during hydroperoxide metabolism. Eur. J. Biochem. 138, 459– 463. Freeman, B.A., Crapo, J.D., 1982. Free radical and tissue injury. Lab. Invest. 47, 412–426. Gasdaska, J.R., Berggren, M., Powis, G., 1995. Cell growth stimulation by the redox protein thioredoxin occurs by a novel helper mechanism. Cell Growth Differ. 6, 1643–1650. Habib, A., Creminon, C., Frobert, Y., Grassi, J., Pradelles, P., Maclouf, J., 1993. Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2. J. Biol. Chem. 268, 23448–23454. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Halliwell, B., 1999. Antioxidant defence mechanisms: from the beginning to the end (of the beginning). Free Radical Res. 31, 261 –272. Harman, D., 1973. Free radical theory of aging. Triangle 12, 153–158. Hattori, M., Tugores, A., Veloz, L., Karin, M., Brenner, D.A.A., 1990. Simplified method for the preparation of transcriptionally active liver nuclear extracts. DNA Cell Biol. 9, 777–781. Hirota, K., Matsui, M., Iwata, S., Nishiyama, A., Mori, K., Yodoi, J., 1997. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc. Natl Acad. Sci. USA 94, 3633–3638. Hirota, K., Murata, M., Sachi, Y., Nakamura, H., Takeuchi, J., Mori, K., Yodoi, J., 1999. Distinct roles of thioredoxin in the cytoplasm and in the nucleus. J. Biol. Chem. 274, 27891–27897. Holmgren, A., 1989. Thioredoxin and glutaredoxin systems. J. Biol. Chem. 264, 13963–13966. Iwasaki, K., Gleiser, C.A., Masoro, E.J., McMahan, C.A., Seo, E.J., Yu, B.P., 1988. The influence of dietary protein source on longevity and age-related disease processes of Fischer rats. J. Gerontol. 43, B5 –B12. Iwata, S., Hori, T., Sato, N., Ueda-Taniguchi, Y., Yamabe, T., Nakamura, H., Masutani, H., Yodoi, J., 1994. Thiol-mediated redox regulation of lymphocyte proliferation. Possible involvement of adult T cell leukemia-derived factor and glutathione in transferring receptor expression. J. Immunol. 152, 5633–5642. Jenkinson, S.G., Duncan, C.A., Bryan, C.L., Lawrence, R.A., 1991. Effects of age on rat glutathione metabolism. Am. J. Med. Sci. 302, 347–352. Kim, H.J., Jung, K.J., Yu, B.P., Cho, C.G., Chung, H.Y., 2002a. Influence of aging and calorie restriction on MAPKs activity in rat kidney. Exp. Gerontol. 37, 1041–1053.
548
C.G. Cho et al. / Experimental Gerontology 38 (2003) 539–548
Kim, H.J., Jung, K.J., Seo, A.Y., Choi, J.S., Yu, B.P., Chung, H.Y., 2002b. Calorie restriction modulates redox-sensitive AP-1 during the aging process. J. Amer. Aging Assoc. 25, 123– 130. Kim, H.J., Yu, B.P., Chung, H.Y., 2002c. Molecular exploration of agerelated NF-kappaB/IKK downregulation by calorie restriction in rat kidney. Free Radical Biol. Med. 32, 991–1005. Kim, J.W., No, J.K., Ikeno, Y., Yu, B.P., Choi, J.S., Yokozawa, T., Chung, H.Y., 2002d. Age-related changes in redox status of rat serum. Arch. Gerontol. Geriatr. 34, 9–17. Kumar, A., Sharma, S., Pundir, C.S., Sharma, A., 1995. Decreased plasma glutathione in cancer of the uterine cervix. Cancer Lett. 94, 107– 111. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685. Liu, R.M., Choi, J., 2000. Age-associated decline in g-glutamylcysteine synthetase gene expression in rats. Free Radical Biol. Med. 28, 566– 574. Lou, M.F., Dickerson, J.E. Jr., Tung, W.H., Wolfe, J.K., Chylack, L.T. Jr., 1999. Correlation of nuclear color and opalescence with protein Sthiolation in human lenses. Exp. Eye Res. 68, 547–552. Lovell, M.A., Xie, C., Gabbita, S.P., Markesbery, W.R., 2000. Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer’s disease brain. Free Radical Biol. Med. 28, 418–427. MacDonald, C.M., Dow, J., Moore, M.R., 1977. A possible protective role for sulphydryl compounds in acute alcoholic liver injury. Biochem. Pharmacol. 26, 1529–1531. Masutani, H., Ueno, M., Ueda, S., Yodori, J., 2000. Role of thioredoxin and redox regulation in oxidative response and signaling. In: Sen, C.H., Sies, H., Baeuerle, P.A. (Eds.), Antioxidant and Redox Regulation of Gense, Academic Press, San Diego, pp. 297 –310. Meister, A.M., 1983. Selective modification of glutathione metabolism. Science 220, 472–477. Meister, A., Anderson, M.E., 1983. Glutathione. Annu. Rev. Biochem. 52, 711– 760. Miranda-Vizuete, A., Rodriguez-Ariza, A., Toribio, F., Holmgren, A., Lopez-Barea, J., Pueyo, C., 1996. The levels of ribonucleotide reductase, thioredoxin, glutaredoxin 1, and GSH are balanced in Escherichia coli K12. J. Biol. Chem. 271, 19099–19103. Nakata, K., Kawase, M., Ogino, S., Kinoshita, C., Murat, H., Sakaue, T., Ogat, K., Ohmori, S., 1996. Effect if age on levels of cysteine, glutathione and related enzyme activities in livers of mice and rats and an attempt to replenish hepatic glutathione level of mouse with cysteine derivatives. Mech. Ageing Dev. 90, 95–207. Prinz, W., Aslund, F., Holmgren, A., Beckwith, J., 1997. The role of the thioredoxin and the glutaredoxin pathways in reducing protein disulphide bonds in the Escherichia coli cytoplasm. J. Biol. Chem. 272, 15611–15667. Puissant, C., Houdebine, L., 1991. An Improvement of the single-step method of RNA isolation by acid guanidium thiocyanate–phenol– chloroform extraction. Biotechniques 8, 148 –149. Quiroga, G.B., Pe´rez-Campo, R., Torres, M.L., 1990. Anti-oxidant defences and eroxidation in liver and brain of aged rats. Biochem. J. 272, 247–250.
Richie, J.P. Jr., 1992. The role of glutathione in aging and cancer. Exp. Gerontol. 27 (5–6), 615 –626. Rietsch, A., Beckwith, J., 1998. The genetics of disulfide bond metabolism. Annu. Rev. Genet. 32, 63–184. Rikans, L.E., Hornbrook, K.R., 1998. Thiol–disulfide exchange systems in the liver of aging Fischer 344 rats. Gerontology 44, 72 –77. Santa Maria, C., Machado, A., 1986a. Effects of development and ageing on pulmonary NADPH-cytochrome c reductase, glutathione peroxidase, glutathione reductase and thioredoxin reductase activities in male and female rats. Mech. Ageing Dev. 37, 183 –195. Santa Maria, C., Machado, A., 1986b. Age and sex related differences in some rat renal NADPH-consuming detoxification enzymes. Arch. Gerontol. Geriatr. 5, 235– 247. Sasaki, T., Senda, M., Kim, S., Kojima, S., Kubodera, A., 2001. Age-related changes of glutathione content, glucose transport and metabolism, and mitochondrial electron transfer function in mouse brain. Nucl. Med. Biol. 28, 25 –31. Shimoda, R., Nagashima, M., Sakamoto, M., Yamaguchi, N., Hirohashi, S., Yokota, J., Kasai, H., 1994. Increased formation of oxidative DNA damage 8-hydroxydeoxyguanosine, in human livers with chronic hepatitis. Cancer Res. 54, 3171–3172. Sies, H., 1999. Glutathione and its role in cellular functions. Free Radical Biol. Med. 27, 916–921. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59–63. Staal, F.J., Ela, S.W., Roederer, M., Anderson, M.T., Herzenberg, L.A., 1992. Glutathione deficiency and human immunodeficiency virus infection. Lancet 339, 909– 912. Tanaka, T., Nakamura, H., Nishiyama, A., Hosoi, F., Masutani, H., Wada, H., Yodoi, J., 2001. Redox regulation by thioredoxin super family; protection against oxidative stress and aging. Free Radical Res. 33, 851 –855. Tappel, A.L., 1977. Glutathione peroxidase and hydroperoxides. Meth. Enzymol. 52, 506– 513. Tauskela, J.S., Hewitt, K., Kang, L.P., Comas, T., Gendron, T., Hakim, A., Hogan, M., Durkin, J., Morley, P., 2000. Evaluation of glutathionesensitive fluorescent dyes in cortical culture. Glia 30, 329– 341. Toyokuni, S., Okamoto, K., Yodoi, J., Hirai, H., 1995. Persistent oxidative stress in cancer. FEBS Lett. 358, 1–3. Yoshida, S., Katoh, T., Tetsuka, T., Uno, K., Matsui, N., Okamoto, T., 1999. Involvement of thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF-alpha-induced production of IL-6 and IL-8 from cultured synovial fibroblasts. J. Immunol. 163, 351–358. Yu, B.P., 1996. Aging and oxidative stress: modulation by dietary restriction. Free Radical Biol. Med 21, 651–668. Yu, B.P., Chung, H.Y., 2000a. Stress resistance of calorie restriction for longevity. Ann. N.Y. Acad. Sci. 928, 39–47. Yu, B.P., Chung, H.Y., 2001b. Oxidative stress and vascular aging. Diabetes Res. Clin. Pract. 54 (Suppl. 2), S73–S80. Yu, B.P., Masoro, E.J., McMahan, C.A., 1985. Nutritional influences on aging of Fischer 344 rats: I. physical, metabolic, and longevity characteristics. J. Gerontol. 40, 657–670.
Modulation of glutathione and thioredoxin systems by calorie restriction during the aging process Chong Gun Choa, Hyon Jeen Kimb, Sang Woon Chungb, Kyung Jin Jungb, Kyung Hee Shimb, Byung Pal Yuc, Junji Yodoid, Hae Young Chungb,* a
b
Department of Molecular Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA Department of Pharmacy, College of Pharmacy, Pusan National University, San 30, Jang-jun-dong, Gumjung-gu, Pusan 609-735, South Korea c Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA d Department of Biological Responses, Institute for Virus Research, Kyoto University,Kyoto 606-8567, Japan Received 26 August 2002; received in revised form 3 January 2003; accepted 9 January 2003
Abstract Accumulating evidence strongly suggests that oxidative stress underlies aging processes and that calorie restriction (CR) retards aging processes, leading to an extended lifespan for various organisms. Recent studies revealed that the anti-aging action of CR depends on its antioxidative mechanism. However, at present, the status of glutathione (GSH) and thioredoxin (Trx) system, two major thiol redox systems in animal cells during aging and its modulation by CR has not fully been explored. The purpose of this study is two-fold: one, to determine whether these two systems in rat kidney are altered as a consequence of aging; two, to determine whether these systems can be modulated by anti-oxidative CR. The results of our study showed that GSH and GSH-related enzyme activities decreased with age in ad libitum (AL)-fed rats, while CR rats consistently showed resistance to decreases in these activities. Data from the present data further showed that while Trx and Trx reductase (TrxR) in cytoplasm decrease with age in AL-fed rats, CR prevents these decreases. In contrast, we also found that the nuclear translocation of the redox regulators, Trx and Ref-1, increase with age, which was suppressed in CR rats. Therefore, increases in nuclear Trx and Ref-1 during aging may result in the up-regulation of redox-sensitive transcription factors, such as NF-kB or AP-1, via the interaction of Ref-1 and Trx in a redox-dependent manner. Our conclusion is that a redox imbalance occurs during aging and that redox changes are minimized through the anti-oxidative action of CR. q 2003 Elsevier Science Inc. All rights reserved. Keywords: Thioredoxin; Glutathione; Aging; Calorie restriction; Ref-1; GSH reductase; GSH peroxidase; GSH S-transferase
1. Introduction Much evidence strongly indicates that most age-related changes are causally linked to deleterious reactive species (RS) generated from aerobic metabolism (Beckman and Ames, 1998; Sohal and Weindruch, 1996). The current oxidative theory of aging proposes that free radical-based oxidative stress is the main culprit causing a net imbalance in an organism’s redox status during aging (Rikans and Hornbrook, 1998). This imbalance or disturbance in the redox status has been associated with aged-related functional loss (Jenkinson et al., 1991). Oxidative stress can cause wide-ranging damage to macromolecules and is * Corresponding author. Tel.: þ 82-51-510-2824; fax: þ82-51-510-2814. E-mail address: [email protected] (H.Y. Chung).
linked to various chronic degenerative disease processes, including vascular diseases (Harman, 1973; Yu and Chung, 2001a,b). A recent review documented sufficient evidence for the oxidative process as a major factor in the inflammatory processes that underpin age-related diseases (Chung et al., 2000). Caloric restriction (CR), the only established anti-aging experimental paradigm, has been investigated extensively and is shown consistently to increase both median and maximum life spans in laboratory animals by retarding the onset and progression of many age-associated, pathologic changes (Yu et al., 1985; Sohal and Weindruch, 1996; Yu and Chung, 2001a,b). Recent research provides strong evidence that CR may exert diverse anti-aging benefits by its ability to modulate age-related oxidative stress (Yu and Chung, 2001a,b; Chung et al., 2001). CR has been shown to
0531-5565/03/$ - see front matter q 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0531-5565(03)00005-6
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attenuate oxidative damage by suppressing RS generation, while maintaining antioxidant defense systems, including major RS neutralizing systems (Sohal and Weindruch, 1996). Several reports highlight the key role of sulphydryl groups (-SH) play in response to oxidative stress (Demple, 1998; Rietsch and Beckwith, 1998). Thiol-disulfide exchange reactions, which are rapid and readily reversible, are ideally suited to the redox control of the protein structural and catalytic function. In many cellular processes, the thiol-disulfide exchange is provided by an interaction between the thioredoxin (Trx) and glutathione systems (Halliwell, 1999), as diagrammed in Fig. 1. The Trx and GSH systems are involved in a variety of redox-dependent pathways. These systems provide reducing equivalents for ribonucleotide reductase and peptide methionine sulfoxide reductase, an antioxidant defense line and regulator of the cellular redox state (Halliwell, 1999; Sies, 1999). More importantly, the Trx and GSH systems regulate the signal transduction activity of various kinases and phosphatases and modulate the redox control of cell growth, death, and the transactivation of redox-sensitive transcription factors (Masutani et al., 2000). The GSH system is also a key component of the overall antioxidant defense system that protects the cell from the deleterious effects of RS (Meister and Anderson, 1983). In addition, GSH detoxifies a variety of exogenous and endogenous substances both non-enzymatically and via enzymatic conjugation catalyzed by GSH S-transferase (GST) (Meister, 1983). GSH peroxidase (GPx) reduces reactive peroxides to alcohols and water at the expense of GSH, which is oxidized to GSSG. GSSG is recycled to GSH by GSH reductase (GR), as shown in Fig. 1. For the agerelated disturbance in GSH metabolism, evidence has been reported by Jenkinson et al. (1991). Recently, Liu and Choi (2000) have reported that activity and gene expression of gglutamylcysteine synthetase, which is the rate-limiting enzyme in GSH synthesis were decreased during aging, providing the molecular insight to the altered GSH content in several tissues.
Fig. 1. Coupling of thioredoxin and glutathione systems. GSH, glutathione; GSSG, oxidized glutathione; Trx, thioredoxin.
Trx is a small, globular ubiquitous protein of 12 kDa with two redox-active half-cysteine redisues in its catalytic active center, having the consensus amino acid sequence, Cys– Gly– Pro – Cys (Holmgren, 1989). The protein exists either in a reduced form with a dithiol or in an oxidized form, in which the half-cysteine residues form an intramolecular disulfide bridge. Trx participates in redox reactions by the reversible oxidation of its active center, dithiol, to disulfide and catalyzes dithiol –disulfide exchange reactions, including signal transduction and gene expression. Trx is maintained in its active reduced form by flavoenzyme thioredoxin reductase (TrxR) in the presence of NADPH, which constitutes the thioredoxin system (Holmgren, 1989). Although Trx is localized in the cytoplasm, it quickly translocates into the nucleus upon stimulation, as seen in response to phorbol myristate acetate (PMA), UV, or other stimuli (Hirota et al., 1999). In the nucleus, Trx forms a physical interaction with the Ref-1 gene product, redox factor-1 (Ref-1) (Hirota et al., 1997). Ref-1 (also designated APE, HAP-1, and APEX) functions as both a nuclear DNA repair enzyme and a reversible regulator of DNA binding activity for several nuclear transcription factors, including AP-1, NF-kB, and HIF-1. The Trx/Ref-1 complex regulates transcriptional activity by altering the redox state of specific cysteine residues located in the basic DNA binding region of these transcription factors (Hirota et al., 1997, 1999). In the present paper, we attempted to establish the changes in the GSH and Trx status that are modulated by CR during the aging process.
2. Experimental procedures 2.1. Animals Rat maintenance procedures for specific-pathogen free status and dietary composition of chow have been previously reported (Yu et al., 1985). Briefly, male, specific pathogen-free Fischer 344 rats were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% a-methionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix, and 3% Solka-Floc. The ad libutum (AL) fed group had free access to both food and water. The animals designated as CR were fed 60% of the food intake of their AL-fed littermates, beginning at 6 weeks of age. Rats at 6, 12, 18, and 24 months of age were sacrificed by decapitation and the kidneys were quickly removed and rinsed in ice-cold buffer (100 mM Tris, 1 mM EDTA, 0.2 mM phenylmethylsulfonylfluoride (PMSF), 1 mM pepstatin, 2 mM leupeptin, 80 mg/L trypsin inhibitor, 20 mM b-glycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate, pH 7.4). The tissue was immediately frozen in liquid nitrogen and stored at 2 80 8C. Histopathological examinations revealed no evidence of nephrotic lesions in soy-protein fed, male Fischer rats even at the advanced age of 24 months, as
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previously reported (Iwasaki et al., 1988). This study complied with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health (Publication No. 85 –23). 2.2. Materials All chemical reagents were obtained from Sigma (MO, USA), except where noted. Western blotting detection reagents were obtained from Amersham (Bucks, UK). RNAzole B was obtained from TEL-TEST, Inc. (TX, USA). Primers for reverse transcriptase –polymerase chain reaction (RT-PCR) were synthesized by Bioneer (Daejeon, Korea). Reverse transcriptase was obtained from Gibco – Bethesda Research Laboratory (MD, USA), Taq DNA polymerase and 10X PCR buffer were from Perkin Elmer (CA, USA). Antibody against Ref-1 was obtained from Santa Cruz Biotechnology (CA, USA). Antibody against TrxR was from Upstate Biotechnology (NY, USA). Antirabbit IgG-horseradish peroxidase-conjugated antibody and anti-mouse IgG-horseradish peroxidase-conjugated antibody were obtained from Amersham (Bucks, UK). Polyvinylidene difluoride (PVDF) membranes were obtained from Millipore Corporation (MA, USA). All other materials were obtained in the highest available grade.
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reductase, and 250 mM H2O2 in a total volume of 250 ml. Samples from the kidney homogenate were mixed with this reaction medium, and absorbance at 340 nm was recorded for 5 min at 37 8C. The GSSG formed by GSH peroxidase activity in the homogenate acting on the hydroperoxide was reduced by GSSG reductase, resulting in the consumption of NADPH. This decrease in NADPH concentration was estimated from the slope of the absorbance by using the appropriate molar extinction coefficient (1 ¼ 6.2 £ 103 M21cm21). GPx activity was measured as the amount of NADPH consumed per minute per milligram of protein. 2.6. GSH S-transferase activity GST activity was estimated by the method of Habig et al. (1974). 1-chloro-2, 4-dinitrobenzene (CDNB) was used as the substrate. The final concentrations of reagents in the assay mixture were 50 mM potassium phosphate buffer (pH 7.0), 1 mM CDNB, 1 mM GSH. The reaction mixture was incubated for 5 min at 25 8C, and then the sample was added. Absorbance was measured at 340 nm and recorded for 5 min. One unit of activity is defined as 1 mmole of 1chloro-2, 4-dinitrobenzene produced. 2.7. GSH reductase activity
2.3. Tissue preparation One gram of kidney was homogenized with 5 ml of 50 mM phosphate homogenate buffer (containing 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM pepstatin, 80 mg/L trypsin inhibitor, pH 7.4) and centrifuged at 900g at 4 8C for 15 min. The supernatants were re-centrifuged at 12; 000g at 4 8C for 15 min to yield sedimented mitochondrial fraction and post-mitochondrial supernatant fraction. The supernatants were centrifuged again at 105; 000g at 4 8C for 1 h to separate cytosol supernatant fraction. 2.4. GSH assay Twenty-five percent meta-phosphoric acid was added to post-mitochondrial fractions, followed by centrifugation at 12; 000g for 10 min for GSH assay in the supernatants. To assay GSH, 1 mM EDTA-50 mM phosphate buffer (pH 8.0) was added to the supernatant followed by o-phthaldehyde. After 20 min at room temperature, the fluorescence was measured at excitation wavelength of 360 nm and emission wavelength of 460 nm (Tauskela et al., 2000). 2.5. GSH peroxidase activity GPx activity was estimated by the indirect coupled assay described by Tappel (1977). The reaction medium consisted of 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 0.15 mM NADPH, 1 mM GSH, 0.24 U GSSG
GSH reductase activity was measured according to the method described by Eklow et al. (1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.6), 1 mM EDTA, 0.1 mM NADPH, 1 mM GSSG in a total volume of 150 ml. The samples from the kidney homogenate (200 ml) were mixed with this reaction medium, and incubated 5 min in at 25 8C. Absorbance at 340 nm was recorded for 2 min. One unit of activity is defined as 1 nmole of NADPH oxidized. 2.8. Western blotting Western blotting was carried out as described previously (Habibet al., 1993). Homogenized samples were boiled for 5 m with a gel-loading buffer (0.125 M Tris –Cl, 4% SDS, 10% 2-mercaptoethanol, pH 6.8, 0.2% bromophenol blue) in ratio of 1:1. Total protein-equivalents (100 mg) for each sample were separated by SDS – PAGE using 10% acrylamide gels as described by Laemmli (1970) and transferred to polyvinylidene difluoride membrane at 15 V for 1 h in a semi-dry transfer system. The membrane was immediately placed into a blocking buffer (1% non-fat milk) in 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20. The blot was allowed to block at room temperature for 1 h. The membrane was incubated with mouse monoclonal anti-Trx (1:1000) for 1 h at 25 8C, and followed by an anti-rabbit IgG-horseradish peroxidase-conjugated antibody (1:5000) for 1 h at 25 8C. Western blotting with rabbit polyclonal anti-TrxR (1:1000) or anti-Ref-1 (1:500) was also
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performed. Antibody labeling was detected using enhanced chemiluminescence per the manufacturer’s instructions. Pre-stained protein markers were used for molecular weight determinations. 2.9. Nuclear extract preparation All solutions, tubes, and centrifuges were maintained at 0 –4 8C. For each nuclear extract preparation, three rat kidney tissues were pooled. The preparation of nuclear extracts was based on previous methods (Hattori et al., 1990). The nuclear extract was frozen at 2 80 8C in aliquots until electrophoretic mobility shift assay (see below) was done. The concentration of total protein in samples was measured with the Sigma protein assay reagent kit containing bicinchoninic acid. 2.10. Isolation of RNA Total RNA was isolated by the method of Puissant and Houdebine (1991). Briefly, tissue samples were homogenized in the presence of RNAzole B (2 ml per 100 mg tissue) with a few strokes in a tissue homogenizer. Aliquots of 0.2 ml chloroform per 2 ml homogenate was added, the samples were shaken vigorously for 15 s, and kept in ice for 5 min. The suspension was centrifuged at 12,000 g for 15 m at 4 8C. The aqueous phase was transferred to the fresh tube, to which an equal volume of isopropanol was added, and the samples were kept at 4 8C for 15 min. Samples were again centrifuged at 12; 000g for 15 min at 4 8C. The supernatant was removed and the RNA pellet was washed once with 75% ethanol by vortexing and subsequent centrifugation at 7500g for 8 min at 4 8C. The pellet was dried for 10 – 15 min. The RNA pellet was dissolved in diethylpyrocarbonate (DEPC)-treated water. 2.11. Assay of RT-PCR (a) Reverse Transcription: The first strand cDNA was synthesized from 2 mg of total RNA. DEPC-treated water and 250 ng random primer were added, incubated for 5 min at 75 8C, and incubated on ice for 5 min. Aliquots of 2 ml 0.1 M DTT, 4 ml 5X buffer, 4 ml 2.5 mM dNTP, 100 U reverse transcriptase and 16.5 U of RNase inhibitor were added and incubated for 2 h at 37 8C. The reaction was stopped by boiling for 2 min at 100 8C, and cDNA was stored at 2 20 8C until use. (b) PCR: The primer pairs for rat Trx were as follows: sense, 50 -ATGGTGAAGCTGATCGAGAG-30 , anti-sense, 50 -GCATGATTAGGCAAACTCCG-30 . The expected product size for rat Trx is 324 bp. The primer pairs for rat Ref-1 were as follows: sense, 50 -GCCAGAGACCAAGAAGAGTA-30 , anti-sense, 50 -TCTGAAGGCTTCATCCCATC-30 . The expected product size for rat Ref-1 is 520 bp. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) primers were used as control for the efficiency of cDNA synthesis in each sample. The primer pairs for GAPDH were as follows: sense, 50 0 GGGTGATGCTGGTGCTGAGTATGT-3 ; anti-sense, 50 AAGAATGGGAG TTGC TGTTGAAGTC-30 . The GAPDH primer set yields a PCR product of 700 bp in length. To carry out the PCR, 25 ml PCR master mix was added to each tube directly. Deoxynucleotides were added to final concentration of 0.2 mM. 10X PCR buffer was diluted in ratio of 1:10 and 1.25 U of Taq DNA polymerase was added. The reaction medium was then added 50 ng of sense and antisense primers for Trx, Ref-1, and GAPDH to each tube, respectively. Reaction conditions consisted of 32 cycles for rat Trx, 25 cycles for rat Ref-1, and 20 cycles for GAPDH at 94 8C for 30 s denaturation, at 54 8C for 30 s annealing, and at 72 8C for 1 min extension. Electrophoresis was performed in 1% agarose gel. After staining in ethidium bromide solution was achieved, the gel was observed under UV transilluminator. 2.12. Statistical analysis The statistical significance of the differences between the treatments (age and diet) was determined by two-factor ANOVA. One-factor ANOVA was conducted to analyse significant differences between all possible age and diet pairs. Differences in the means of individual groups were assessed by the Fischer’s protected LSD post-hoc test. Values of p , 0:05 were considered statistically significant.
3. Results 3.1. GSH status and CR modulation during aging Because GSH is a major anti-oxidant component that battles against oxidative stress, we found it important to assess age-related changes of GSH concentrations. As shown in Fig. 1, we observed a decrease in the level of GSH in the kidney of AL-fed, 18- and 24-month-old rats compared to that of 6-month-old rats. However, CR rats were able to blunt the decrease, as shown in Fig. 2. 3.2. Effect of aging and CR on GSH-related enzyme activity To further investigate individual components of the GSH system, its three key enzymes were monitored. Fig. 3 shows the age-related changes in the activities of GPx (Fig. 3(A)), GST (Fig. 3(B)), and GR (Fig. 3(C)) in four age groups of AL and CR rats. The activities of these three enzymes in old AL rats were lower than those of younger AL rats. Data on CR rats were contrasted by results of higher enzyme activities than those of their AL counterparts. These changes in enzyme activities correlated well with the changes seen in GSH levels.
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Fig. 2. CR suppresses age-related decreases of glutathione content. Glutathione (GSH) content in kidney homogenates was determined as described in the method. Represented are the mean ^ SEM ðn ¼ 6Þ: Results of one-factor ANOVA: p P , 0:05; p p P , 0:01; and p p p P , 0:001 VS. 6-month-old AL rat: # P , 0:05; ## P , 0:01; and ### P , 0:001 VS. age matched AL rats.
3.3. Modulation of Trx and TrxR gene expression by aging and CR We examined age-related modulation in the gene expression of two proteins, Trx and TrxR of the Trx system. Fig. 4(A) shows that cytoplasmic Trx protein levels significantly decreased with age in the AL group. The decrease was so pronounced in 18- and 24-month-old rats that the protein bands were not detectable by Western blotting (AL in Fig. 4(A)). However, Trx protein levels in the CR group were reasonably well maintained even for 24month-old rats (CR in Fig. 4(A)). As shown in Fig. 4(B), TrxR protein levels decreased with age in AL rats, while CR rats showed higher levels than their AL counterparts. Since TrxR is the only class of enzymes known to reduce Trx, it is expected that an alteration in TrxR may affect Trx activity during aging. Further, RT-PCR analysis of Trx RNA to assess Trx transcription expression revealed that the Trx mRNA level interestingly did not change during aging in either AL or CR rats (Fig. 4(D)). 3.4. CR modulates nuclear translocation of Trx and Ref-1 during aging The cytoplasmic Trx is known to be translocated upon stimulation in response to PMA, oxidative stress, UV irradiation, and TNFa. Because the aging process is considered a chronic state of oxidative stress, we sought evidence of the nuclear translocation of Trx protein during aging. As shown in Fig. 5(A), Trx in the nucleus increased with age in AL rats, while CR rats were shown to block this age-related increase. Nucleus-imported Trx interacts with Ref-1 to form a regulatory Trx/Ref-1 complex. A function of this complex is
Fig. 3. CR activates enzyme activities of glutathione peroxidase, glutathione S-transferase, and glutathione reductase. Enzyme activities of glutathione peroxidase (GPx) (A), glutathione S-transferase (GST) (B), and glutathione reductase (GR) (C) were measured in kidney homogenate from AL or CR rats, ages 6, 12, 18, and 24 months old. Values are expressed as mean ^ SEM (n ¼ 10). Results of one-factor ANOVA: p P , 0:05; pp P , 0:01; and p p p P , 0.001 VS. 6-month-old AL rat: # P , 0:05; ## P , 0:01; and ### P , 0:001 VS. age matched AL rats.
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Fig. 4. CR upregulates age-related decreases of cytosolic Trx and TrxR. Western blot analysis was performed to detect renal Trx (A) and TrxR (B) protein levels in cytoplasmic extracts (50 mg protein) from AL or CR rats, ages 6, 12, 18, and 24 months old. Trx, thioredoxin; TrxR, thioredoxin reductase (C) b-Actin protein level is loading control for western blot. (D) RT-PCR analysis of renal Trx was performed from RNA isolated from rat kidney. GAPDH primers were used as control for the efficiency of cDNA synthesis in each sample. One representative result of each is shown from three experiments that yielded similar results, respectively. The data were quantified by densitometry, and presented as a percentage of 6-month-old AL rat. Results of one-factor ANOVA: p P , 0:05; pp P , 0:01; and ppp P , 0:001 VS. 6-month-old AL rat: # P , 0:05; ## P , 0:01; and ### P , 0:001 VS. age matched AL rats.
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to modulate the transcriptional activities of redox-sensitive transcription factors, such as NF-kB, AP-1, and HIF-1 by keeping cysteine residues reduced. We examined changes in nuclear Ref-1 protein levels with age (Fig. 5(B)) in AL and CR rats, which showed age-related changes with the nuclear translocation of Trx (shown in Figs. 4(A) and 5(A)). We recently obtained evidence that NF-kB (Kim et al., 2002 a,c, d),), AP-1 Kim et al., 2002b, and HIF-1 (data not shown, manuscription in preparation) were activated during aging, suggesting that an age-related increase in nuclear Trx and Ref-1 may contribute to the activation of these transcription factors during aging.
4. Discussion
Fig. 5. CR suppresses nuclear translocation of nuclear Trx and Ref-1. Trx (A) Ref-1 (B), and HIF-1b (C, as loading control) protein levels in nuclear extracts (30 mg protein) were measured by Western blot analysis. Trx, thioredoxin; Ref-1, redox factor-1. In the upper panel, one representative blot of nuclear Trx and Ref-1 is shown from three experiments that yielded similar results, respectively. The levels of each protein were quantified by densitometry. The data shown in bottom are presented as the percentage relative to 6-month-old AL rat.
The purpose of the current study was to explore at the molecular level two major disulfide-reducing systems, GSH and Trx, and their modulation by the anti-oxidative, lifeprolonging action of CR during aging. Data show that GSH and GSH-related enzymes are decreased in the kidney of 24month-old rats. Similar findings were reported by Nakata et al. (1996), showing a large decrease in the hepatic level of GSH in 24-month-old mice, and Sasaki et al. (2001) who reported increased oxidized GSH with age in rat brain. However, findings on the age effect of the GSH status are not unanimous: some reported that GSH levels in rats remain unchanged (Richie, 1992), while others found that GSH levels increase during aging (Quiroga et al., 1990; Jenkinson et al., 1991). A recent data reported by Liu and Choi (2000) shed further evidence that the age-related changes in GSH may depend on the tissue or organ. The authors found that the GSH content in rat liver, kidney and lung decreased roughly 20– 37% between the age of 12– 24 months, while no detectable decrease was noticed in heart and diaphragm. This organ specificity was further substantiated by the age-related decrease in gene expression of g-glutamylcysteine synthetase in the corresponding organs (Liu and Choi, 2000). A literature search reveals that the status of the Trx system during aging has not been studied extensively and that existing data are conflicting. For instance, it was demonstrated that TrxR activity in livers of male and female rats were unchanged with aging (Rikans and Hornbrook, 1998), but data reported by Santa Maria and Machado (1986a and b) showed decreased activity of TrxR activity during aging in kidney and lung. The latter report is in agreement with our present findings showing an age-related decrease in Trx system activity. The tissue-specific modulation of Trx activity may be related to the functional state of the organ under oxidative stress. Recently, Carmel-Harel et al. (2001) reported that decreased TrxR diminished the capacity to detoxify oxidants and to repair oxidative stress-induced damage. These findings provide additional evidence that decreases in cytoplasmic Trx and
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Fig. 6. A schematic presentation of protein interaction and nuclear translocation of Trx and Ref-1. Trx, thioredoxin; Ref-1, Redox factor-1.
TrxR activity certainly compromise the organism’s ability to guard against cellular oxidative forces during aging. In the current work, we observed that the translocation of Trx into the nucleus with aging was facilitated (Fig. 6). Recently, our laboratory showed increased DNA binding activity of redox-sensitive transcription factors such as NFkB (Kim et al., 2002 a,c,d) and AP-1 (Kim et al., 2002b) with age. We also showed that the activated MAPK signaling pathway is likely caused by a compromised agerelated redox status (Kim et al., 2002a,b,c). The essential role that the thiol groups play in the intricate regulation of defense mechanisms against oxidative stress is well established (Halliwell, 1999; Sies, 1999). The crucial function of thiols in the modulation of redox-sensitive proteins to oxidative stress in cell membranes also has been reported (Freeman and Crapo, 1982). These investigators reported that high contents of GSH and Trx protect thiol oxidation in structural proteins and enzymes for optimal biological functions. Recently, Lou et al. (1999) reported that the size of GSH pool was diminished in aged lenses under oxidative stress, and that some protein thiols are being S-thiolated by oxidized nonprotein thiols to form protein –thiol mixed disulfides. The authors concluded that thiol oxidation is the result of a cascade of events, starting with protein disulfide crosslinking, protein solubility loss, and eventual lens opacification. GSH, one of the most biologically versatile antioxidant systems, is known to affect cellular functions under the altered redox state as found in low GSH contents in several pathological conditions, including alcoholic liver disease (MacDonald et al., 1977), acquired immunodeficiency syndrome (Staal et al., 1992), xenobiotic-induced oxidative stress and toxicity (DiSimplicio et al., 1984) and precancerous lesions (Kumar et al., 1995). Based on these findings, the maintenance of optimal GSH levels and a proper redox status is crucial to the modulation of various pathogenic conditions and the course of diseases. Thus, low GSH levels may be a risk factor for aging.
The roles of Trx seem to be diverse and often contradictory. Trx has been shown to assume the role of an antioxidant in the tumor-preventing system (Halliwell, 1999; Sies, 1999) and have cytoprotective effects against oxidative stress. However, the beneficial role of Trx ceases once a tumor becomes established, purportedly promoting the growth of variety of cells (Iwata et al., 1994; Gasdaska et al., 1995). Its role is not limited to the detoxification of reactive oxygen metabolites but also is seen in cellular signaling processes. For instance, several studies examining reactive oxygen species (ROS) production in cancer (Shimoda et al., 1994; Toyokuni et al., 1995) found that the cellular level of Trx increases in response to oxidative stress, suggesting ROS production as a possible reason for elevated Trx in cancer. Trx is also shown to promote DNA binding of redox-sensitive, pro-inflammatory transcription factors such as NFkB, AP-1 and is implicated in inflammatory diseases, such as Sjo¨gren’s syndrome and rheumatoid arthritis (Yoshida et al., 1999). Furthermore, decreased Trx in Alzheimer’s disease brain led to the loss of protection against amyloid b-peptide toxicity (Lovell et al., 2000). Some evidence provides strong support for the roles of the Trx and GSH systems in determining the thiol – disulfide balance in the cytoplasm. Although each of these two systems has their own specific actions, their interaction is substantial (Miranda-Vizuete et al., 1996; Prinz et al., 1997). For example, a small but consistent increase in Trx was observed in the absence of GSH-coupled glutaredoxin 1 (Grx1); but an over production of one component was shown to decrease the level of the other (Miranda-Vizuete et al., 1996). These results suggest the existence of the interactive network in which the balance between Trx and Grx1 is closely regulated. It should be noted, however, that these studies were performed with prokaryote and that the roles of Trx and GSH remain to be tested in mammalian cells. A recent report by Casgrande et al. (2002) highlighted possible crosstalk between the GSH and Trx systems in human T cell.
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Extensive gerontological research has confirmed that CR imposed on laboratory rodents extends mean and maximum lifespans (Yu et al., 1985; Yu, 1996). Research has also established that CR retards age-associated pathologies such as nephropathy, cardiomyopathy, gastric ulcer, cataract, and cancer (Yu et al., 1985; Yu, 1996; Sohal and Weindruch, 1996). A recent proposal on the anti-oxidative role of CR is well supported by many studies in its life-prolonging effects (Yu, 1996). In the present study, we presented molecular evidence explaining the modulating effects of CR and its anti-oxidative mechanism, as shown in Figs. 2 –4. In summary, CR was shown to up-regulate GSH and GSH-related enzymes throughout aging process and to maintain cytoplasmic Trx levels even in 24-month-old rats, while neither was detectable in AL rats at 18 and 24 months. If aging is due to the result of a long-term cellular redox imbalance as proposed (Yu, 1996; Beckman and Ames, 1998), then the anti-oxidative action of CR and its ability to maintain a proper redox state are likely the most powerful antiaging measures for lifespan extension. In addition to the cytoplasmic regulation of Trx and Ref-1, our data indicate that CR prevents the increase of Trx and Ref-1 levels in the nucleus. These findings are important in view of our previous data showing CR’s prevention of NF-kB transactivation in aged animals (Kim et al., 2002a,b,c), because Trx and related molecules (Tanaka et al., 2001) are responsible for the maintenance of the cellular reducing environment with the help of GSH system. On the other hand, mRNA levels were shown to have no agerelated changes and not to be modulated by CR. This discrepancy in the age-related changes might be related to the possibility that enhanced protease degradation facilitates translocation of Trx into the nulcleus, while CR suppresses the process. In conclusion, the present study documents age-related decreases of GSH and Trx levels in the cytoplasm, which results in an altered redox balance. Our data further demonstrate the ability of CR to correct the imbalance. Our report showed for the first time the down-regulation of Trx and Ref-1 by aging and their attenuation by CR. The protective effects of CR on the thiol-reducing system are important to the regulation of redox balance and the promotion of an optimally maintained organism during aging. (Yu and Chung, 2001a,b).
Acknowledgements This work was supported by a grant from the Korean Research Foundation (FS0004). We are grateful to the ‘Aging Tissue Bank’ granted by KOSEF and National Institute on Aging (AG01188) for the supply of the aging tissue.
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References Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 78, 547 –581. Carmel-Harel, O., Stearman, R., Gasch, A.P., Botstein, D., Brown, P.O., Storz, G., 2001. Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae. Mol. Microbiol. 39, 595–605. Casgrande, S., Bonetto, V., Fratelli, M., Gianazza, E., Eberini, I., Massignan, T., Salmona, M., Chang, G., Holmgren, A., Ghezzi, P., 2002. Glutathionylation of human thioredoxin: a possible crosstalk between the glutatione and thioredoxin systems. Proc. Natl Acad. Sci. USA 99, 9745–9749. Chung, H.Y., Kim, H.J., Jung, K.J., Yoon, J.S., Yoo, M.A., Kim, K.W., Yu, B.P., 2000. The inflammatory process in aging. Rev. Clin. Gerontol. 10, 207– 222. Chung, H.Y., Kim, H.J., Kim, J.W., Yu, B.P., 2001. The inflammation hypothesis of aging: molecular modulation by calorie restriction. Ann. NY Acad. Sci. 928, 327–335. Demple, B., 1998. A bridge to control. Science 279, 1655–1656. DiSimplicio, P., Dolara, P., Lodovici, M., 1984. Blood glutathione as a measure of exposure to toxic compounds. J. Appl. Toxicol. 4, 227– 229. Eklow, L., Moldeus, P., Orrenius, S., 1984. Oxidation of glutathione during hydroperoxide metabolism. Eur. J. Biochem. 138, 459– 463. Freeman, B.A., Crapo, J.D., 1982. Free radical and tissue injury. Lab. Invest. 47, 412–426. Gasdaska, J.R., Berggren, M., Powis, G., 1995. Cell growth stimulation by the redox protein thioredoxin occurs by a novel helper mechanism. Cell Growth Differ. 6, 1643–1650. Habib, A., Creminon, C., Frobert, Y., Grassi, J., Pradelles, P., Maclouf, J., 1993. Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2. J. Biol. Chem. 268, 23448–23454. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Halliwell, B., 1999. Antioxidant defence mechanisms: from the beginning to the end (of the beginning). Free Radical Res. 31, 261 –272. Harman, D., 1973. Free radical theory of aging. Triangle 12, 153–158. Hattori, M., Tugores, A., Veloz, L., Karin, M., Brenner, D.A.A., 1990. Simplified method for the preparation of transcriptionally active liver nuclear extracts. DNA Cell Biol. 9, 777–781. Hirota, K., Matsui, M., Iwata, S., Nishiyama, A., Mori, K., Yodoi, J., 1997. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc. Natl Acad. Sci. USA 94, 3633–3638. Hirota, K., Murata, M., Sachi, Y., Nakamura, H., Takeuchi, J., Mori, K., Yodoi, J., 1999. Distinct roles of thioredoxin in the cytoplasm and in the nucleus. J. Biol. Chem. 274, 27891–27897. Holmgren, A., 1989. Thioredoxin and glutaredoxin systems. J. Biol. Chem. 264, 13963–13966. Iwasaki, K., Gleiser, C.A., Masoro, E.J., McMahan, C.A., Seo, E.J., Yu, B.P., 1988. The influence of dietary protein source on longevity and age-related disease processes of Fischer rats. J. Gerontol. 43, B5 –B12. Iwata, S., Hori, T., Sato, N., Ueda-Taniguchi, Y., Yamabe, T., Nakamura, H., Masutani, H., Yodoi, J., 1994. Thiol-mediated redox regulation of lymphocyte proliferation. Possible involvement of adult T cell leukemia-derived factor and glutathione in transferring receptor expression. J. Immunol. 152, 5633–5642. Jenkinson, S.G., Duncan, C.A., Bryan, C.L., Lawrence, R.A., 1991. Effects of age on rat glutathione metabolism. Am. J. Med. Sci. 302, 347–352. Kim, H.J., Jung, K.J., Yu, B.P., Cho, C.G., Chung, H.Y., 2002a. Influence of aging and calorie restriction on MAPKs activity in rat kidney. Exp. Gerontol. 37, 1041–1053.
548
C.G. Cho et al. / Experimental Gerontology 38 (2003) 539–548
Kim, H.J., Jung, K.J., Seo, A.Y., Choi, J.S., Yu, B.P., Chung, H.Y., 2002b. Calorie restriction modulates redox-sensitive AP-1 during the aging process. J. Amer. Aging Assoc. 25, 123– 130. Kim, H.J., Yu, B.P., Chung, H.Y., 2002c. Molecular exploration of agerelated NF-kappaB/IKK downregulation by calorie restriction in rat kidney. Free Radical Biol. Med. 32, 991–1005. Kim, J.W., No, J.K., Ikeno, Y., Yu, B.P., Choi, J.S., Yokozawa, T., Chung, H.Y., 2002d. Age-related changes in redox status of rat serum. Arch. Gerontol. Geriatr. 34, 9–17. Kumar, A., Sharma, S., Pundir, C.S., Sharma, A., 1995. Decreased plasma glutathione in cancer of the uterine cervix. Cancer Lett. 94, 107– 111. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685. Liu, R.M., Choi, J., 2000. Age-associated decline in g-glutamylcysteine synthetase gene expression in rats. Free Radical Biol. Med. 28, 566– 574. Lou, M.F., Dickerson, J.E. Jr., Tung, W.H., Wolfe, J.K., Chylack, L.T. Jr., 1999. Correlation of nuclear color and opalescence with protein Sthiolation in human lenses. Exp. Eye Res. 68, 547–552. Lovell, M.A., Xie, C., Gabbita, S.P., Markesbery, W.R., 2000. Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer’s disease brain. Free Radical Biol. Med. 28, 418–427. MacDonald, C.M., Dow, J., Moore, M.R., 1977. A possible protective role for sulphydryl compounds in acute alcoholic liver injury. Biochem. Pharmacol. 26, 1529–1531. Masutani, H., Ueno, M., Ueda, S., Yodori, J., 2000. Role of thioredoxin and redox regulation in oxidative response and signaling. In: Sen, C.H., Sies, H., Baeuerle, P.A. (Eds.), Antioxidant and Redox Regulation of Gense, Academic Press, San Diego, pp. 297 –310. Meister, A.M., 1983. Selective modification of glutathione metabolism. Science 220, 472–477. Meister, A., Anderson, M.E., 1983. Glutathione. Annu. Rev. Biochem. 52, 711– 760. Miranda-Vizuete, A., Rodriguez-Ariza, A., Toribio, F., Holmgren, A., Lopez-Barea, J., Pueyo, C., 1996. The levels of ribonucleotide reductase, thioredoxin, glutaredoxin 1, and GSH are balanced in Escherichia coli K12. J. Biol. Chem. 271, 19099–19103. Nakata, K., Kawase, M., Ogino, S., Kinoshita, C., Murat, H., Sakaue, T., Ogat, K., Ohmori, S., 1996. Effect if age on levels of cysteine, glutathione and related enzyme activities in livers of mice and rats and an attempt to replenish hepatic glutathione level of mouse with cysteine derivatives. Mech. Ageing Dev. 90, 95–207. Prinz, W., Aslund, F., Holmgren, A., Beckwith, J., 1997. The role of the thioredoxin and the glutaredoxin pathways in reducing protein disulphide bonds in the Escherichia coli cytoplasm. J. Biol. Chem. 272, 15611–15667. Puissant, C., Houdebine, L., 1991. An Improvement of the single-step method of RNA isolation by acid guanidium thiocyanate–phenol– chloroform extraction. Biotechniques 8, 148 –149. Quiroga, G.B., Pe´rez-Campo, R., Torres, M.L., 1990. Anti-oxidant defences and eroxidation in liver and brain of aged rats. Biochem. J. 272, 247–250.
Richie, J.P. Jr., 1992. The role of glutathione in aging and cancer. Exp. Gerontol. 27 (5–6), 615 –626. Rietsch, A., Beckwith, J., 1998. The genetics of disulfide bond metabolism. Annu. Rev. Genet. 32, 63–184. Rikans, L.E., Hornbrook, K.R., 1998. Thiol–disulfide exchange systems in the liver of aging Fischer 344 rats. Gerontology 44, 72 –77. Santa Maria, C., Machado, A., 1986a. Effects of development and ageing on pulmonary NADPH-cytochrome c reductase, glutathione peroxidase, glutathione reductase and thioredoxin reductase activities in male and female rats. Mech. Ageing Dev. 37, 183 –195. Santa Maria, C., Machado, A., 1986b. Age and sex related differences in some rat renal NADPH-consuming detoxification enzymes. Arch. Gerontol. Geriatr. 5, 235– 247. Sasaki, T., Senda, M., Kim, S., Kojima, S., Kubodera, A., 2001. Age-related changes of glutathione content, glucose transport and metabolism, and mitochondrial electron transfer function in mouse brain. Nucl. Med. Biol. 28, 25 –31. Shimoda, R., Nagashima, M., Sakamoto, M., Yamaguchi, N., Hirohashi, S., Yokota, J., Kasai, H., 1994. Increased formation of oxidative DNA damage 8-hydroxydeoxyguanosine, in human livers with chronic hepatitis. Cancer Res. 54, 3171–3172. Sies, H., 1999. Glutathione and its role in cellular functions. Free Radical Biol. Med. 27, 916–921. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59–63. Staal, F.J., Ela, S.W., Roederer, M., Anderson, M.T., Herzenberg, L.A., 1992. Glutathione deficiency and human immunodeficiency virus infection. Lancet 339, 909– 912. Tanaka, T., Nakamura, H., Nishiyama, A., Hosoi, F., Masutani, H., Wada, H., Yodoi, J., 2001. Redox regulation by thioredoxin super family; protection against oxidative stress and aging. Free Radical Res. 33, 851 –855. Tappel, A.L., 1977. Glutathione peroxidase and hydroperoxides. Meth. Enzymol. 52, 506– 513. Tauskela, J.S., Hewitt, K., Kang, L.P., Comas, T., Gendron, T., Hakim, A., Hogan, M., Durkin, J., Morley, P., 2000. Evaluation of glutathionesensitive fluorescent dyes in cortical culture. Glia 30, 329– 341. Toyokuni, S., Okamoto, K., Yodoi, J., Hirai, H., 1995. Persistent oxidative stress in cancer. FEBS Lett. 358, 1–3. Yoshida, S., Katoh, T., Tetsuka, T., Uno, K., Matsui, N., Okamoto, T., 1999. Involvement of thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF-alpha-induced production of IL-6 and IL-8 from cultured synovial fibroblasts. J. Immunol. 163, 351–358. Yu, B.P., 1996. Aging and oxidative stress: modulation by dietary restriction. Free Radical Biol. Med 21, 651–668. Yu, B.P., Chung, H.Y., 2000a. Stress resistance of calorie restriction for longevity. Ann. N.Y. Acad. Sci. 928, 39–47. Yu, B.P., Chung, H.Y., 2001b. Oxidative stress and vascular aging. Diabetes Res. Clin. Pract. 54 (Suppl. 2), S73–S80. Yu, B.P., Masoro, E.J., McMahan, C.A., 1985. Nutritional influences on aging of Fischer 344 rats: I. physical, metabolic, and longevity characteristics. J. Gerontol. 40, 657–670.