Ryoya Takahashi

Age-associated increases in oxidative stress and nuclear transcription factor κB activation are attenuated in rat liver by regular exercise

The combined effects of aging and regular physical exercise was investigated on the production of reactive oxygen species (ROS), lipid peroxidation, glutathione status, and the activity of nuclear factor‐κB (NF‐κB) in rat liver. A group of 24 male F344 rats was divided into the following categories: adult control (18 months), adult exercised (18 months), and aged control (28 months) and aged exercised (28 months). The ROS formation increased as a function of age and exercise training decreased the rate of ROS formation in the two age groups. Significant positive correlation was found between ROS production and lipid peroxidation (LIPOX). The reduced glutathione (GSH) level was higher and the oxidized glutathione (GSSG) level lower in exercised groups compared with the sedentary controls (P<0.05). An age‐associated increase in NF‐κB activity was attenuated by the regular exercise. The content of p50 and p65 subunits of NF‐κB increased with age and decreased with exercise training. The content of inhibitory factor‐κB was inversely related to NF‐κB activation. Regular exercise‐induced adaptive responses, including attenuation of an increase in ROS production, LIPOX level, NF‐κB activation, and reduced GSH/GSSG ratio, appear to be capable, even in old age, of reducing increases in inflammatory and other detrimental consequences that are often associated with advancing age.

Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice.

Hydrogen is an established anti-oxidant that prevents acute oxidative stress. To clarify the mechanism of hydrogens effect in the brain, we administered hydrogen-rich pure water (H(2)) to senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), also a well-known anti-oxidant. These KO mice were divided into three groups; recipients of H(2), VC, or pure water (H(2)O), administered for 33 days. VC levels in H(2) and H(2)O groups were <6% of those in the VC group. Subsequently, superoxide formation during hypoxia-reoxygenation treatment of brain slices from these groups was estimated by a real-time biography imaging system, which models living brain tissues, with Lucigenin used as chemiluminescence probe for superoxide. A significant 27.2% less superoxide formed in the H(2) group subjected to ischemia-reperfusion than in the H(2)O group. Thus hydrogen-rich pure water acts as an anti-oxidant in the brain slices and prevents superoxide formation.

Carbonylated proteins in aging and exercise: immunoblot approaches

Protein carbonyls were studied in aging and exercise by immunoblot followed by one- or two-dimensional polyacrylamide gel electrophoresis using antibodies against 2,4-dinitrophenylhydrazones. Proteins of rat kidneys exhibited significant age-related increase in the amount of carbonyl while those of the brain and liver did not. Major carbonylated proteins in the kidney included serum albumin. In nematodes in which protein carbonyls increased with age, one of the carbonylated proteins was identified as vitellogenin, an egg-yolk protein. A possible biological significance of this protein present in abundance even after egg-laying stages is discussed in terms of protection against oxidative stress. Exhaustive exercise induced significant increase in the carbonylation of selected but unidentified proteins in the lung. This oxidative stress might be caused by xanthine oxidase in this tissue and hypoxanthine derived from ATP-depleted muscles. Exercise at high altitude caused higher carbonylation of the skeletal muscle proteins, most notably a protein likely to be actin, than that at sea level but no significant difference was observed in lipid peroxidation. These studies emphasize the value of immunoblot analysis of tissue protein carbonyls in a variety of situations where oxidative stress is likely involved.

Implications of Protein Degradation in Aging

Abstract: Aging is characterized by accumulation of potentially harmful altered proteins that could lead to gradual deterioration of cellular functions and eventually result in increased probability of death. Metabolic turnover of proteins thus plays an essential role in maintaining the life of an organism. In this article we summarize our current knowledge on age‐related changes in protein turnover with special reference to degradation. Increase in half‐life of proteins with advancing age is well documented. Qualitative rather than quantitative changes of proteasomes appear to be responsible for this change. Dietary restriction and moderate long‐term exercise seem to restore higher proteasome activity and turnover rate of proteins in aged animals.

Effect of aging and late onset dietary restriction on antioxidant enzymes and proteasome activities, and protein carbonylation of rat skeletal muscle and tendon.

Many studies have shown that lifelong dietary restriction (DR) can retard aging processes. Very few reports, however, are found that examined the effect of late onset DR on biochemical parameters in aging animals [Goto, S., Takahashi, R., Araki, S., Nakamoto, H., 2002b. Dietary restriction initiated in late adulthood can reverse age-related alterations of protein and protein metabolism. Ann. NY Acad. Sci. 959, 50-56]. We studied the effect of every-other-day feeding, initiated at the age of 26.5 months and continued for 3.5 months, on antioxidant enzymes, protein carbonyls, and proteasomes of the gastrocnemius muscle and tendon in rats. Age-related increase in the activity and content of Cu, Zn-SOD and the content of Mn-SOD was attenuated by the DR in both tissues. The same was true for glutathione peroxidase and catalase activities. Significant increase with age in protein reactive carbonyl derivatives (RCD) in the tendon was noted that was partially reversed by the DR. No significant change of RCD, however, was observed in the skeletal muscle. The age-related and DR-induced changes of the RCD in the tendon appeared to be associated with proteasome activity that decreases with age and increases by the DR. It is suggested that the late onset DR can have beneficial effects on the locomotive functions by reducing age-associated potentially detrimental oxidative protein damage in the tendon.

Dietary Restriction Initiated in Late Adulthood Can Reverse Age‐related Alterations of Protein and Protein Metabolism

Many reports have been published on the effects of lifelong dietary restriction (DR) on a variety of parameters such as life span, carcinogenesis, immunosenescence, memory function, and oxidative stress. There is, however, limited available information on the effect of late onset DR that might have potential application to intervene in human aging. We have investigated the effect of DR initiated late in life on protein and protein degradation. Two months of DR in 23.5‐month‐old mice significantly reduced heat‐labile altered proteins in the liver, kidney, and brain. DR reversed the age‐associated increase in the half‐life of proteins, suggesting that the dwelling time of the proteins is reduced in DR animals. In accordance with this observation, the activity of proteasome, which is suggested to be responsible for degradation of altered proteins, was found increased in the liver of rats 30 months of age subjected to 3.5 months of DR. Thus, DR can increase turnover of proteins, thereby possibly attenuating potentially harmful consequences by altered proteins. Likewise, DR in old rats reduced carbonylated proteins in liver mitochondria, although the effect was not observed in cytosolic proteins. Fasting induced apoA‐IV synthesis in the liver of young mice for efficient mobilization of stored tissue fats, while it occurred only marginally in the old. DR for 2 months from 23 months of age partially restored inducibility of this protein, suggesting the beneficial effect of DR. Taking all these findings together, it is conceivable that DR conducted in old age can be beneficial not only to retard age‐related functional decline but also to restore functional activity in young rodents. Interestingly, recent evidence that involves DNA array gene expression analysis supports the findings on the age‐related decrease in protein turnover and its reversion by late‐onset DR.

Age-related difference of site-specific histone modifications in rat liver

Aging is associated with decrease in activities of the transcription, replication and DNA repair that can result in deterioration of cellular and tissue functions. Changes of chromatin structures with age are likely major underling mechanisms for the functional decline. Chromatin consists of DNA and histones as well as non-histone proteins. While age-associated change of DNA methylation is well documented, little information is available on site-specific histone modifications in aging. We studied here age-related change of selected modifications of rat liver histone, i.e., histone H3 Lys9 acetylation (H3K9ac), H3 Lys9 methylation (H3K9me), H3 Ser10 phosphorylation (H3S10ph) and H3 Lys14 acetylation (H3K14ac). H3K9ac was decreased and H3S10ph was increased with age significantly. In view of reports indicating that decrease in acetylation and increase in phosphorylation of H3 histones can suppress gene activity, our findings suggest that a mechanism of decreased chromatin functions with age is due to such epigenetic changes.

Modulation of gene expression of SMP-30 by LPS and calorie restriction during aging process

Senescence marker protein-30 (SMP-30) has been proposed as an important aging marker and is now functionally identified as a Ca2+ binding protein. SMP-30 has been shown to blunt cell death caused by intracellular Ca2+ accumulation by enhancing plasma membrane Ca(2+)-pumping activity. Although SMP-30 is reported decrease during aging, at present, neither has the mechanism underlying this decrease been fully defined, nor have the mechanisms related to the modulation of SMP-30 been extensively explored. In the current study, we used the well-known anti-aging action of the calorie restriction (CR) paradigm to explore age-related changes in SMP-30 gene expression. The thrust of our investigation was based on CRs ability to defend against age-related oxidative stress and the inflammatory process. The kidney and liver from Fischer 344 rats at 6, 12, 18 and 24 months of age were utilized for this study. The rats were divided into two groups, ad libitum (AL)-fed and 40% restricted CR. Results showed that SMP-30 expression declined with age and that this decline was clearly blunted by CR. To correlate changes between SMP-30 gene expression and the oxidative status, SMP-30 expression and the production of reactive oxygen species (ROS) during aging and free-radical generating lipopolysaccharide (LPS) were monitored. Our data showed that the down-regulation of SMP-30 was accompanied by increased ROS generation and LPS-induced ROS. The potent anti-aging and anti-oxidative action of CR effectively suppressed the age-related down-regulation of SMP-30 by ROS reduction.

Regular Exercise: An Effective Means to Reduce Oxidative Stress in Old Rats

Abstract: A healthy diet and regular exercise are among the major factors that influence quality of life (QOL) in old age. Exercise is believed to be beneficial to improve QOL, retarding age‐related decline of physiological functions and preventing age‐related diseases. Regular physical exercise can possibly improve age‐related functional decline and delay onset of age‐related diseases by attenuating potentially harmful oxidative damage and suppressing inflammatory processes even in older age.

Alteration of aminoacyl-tRNA synthetase with age: Heat-labilization of the enzyme by oxidative damage

Active oxygens have been suggested to be involved in age-related alterations of organelles and molecules. In this study we investigated the influence of active oxygen on aminoacyl-tRNA synthetases partially purified from rat liver. Treatment of leucyl-tRNA synthetase with Fe3(+)-ascorbate resulted in the increased heat-lability of the enzyme. The inactivation was inhibited by radical scavengers such as mannitol and benzoate, suggesting that hydroxyl radicals are responsible for heat-labilization of the enzyme. On the other hand, a considerable part of tyrosyl-tRNA synthetase was converted to heat-labile forms without added iron and ascorbate under aerobic conditions but not under anaerobic conditions. These and other findings suggested that the heat-labilization of this enzyme is caused by active oxygens probably generated by the reaction of dioxygen and transition metal ions present in the enzyme preparations. Heat-inactivation curves of the enzymes modified as described above were similar to those observed for the enzymes from aged animals in that these enzymes exhibited higher percentages of heat-labile forms than the unmodified enzymes from young animals [Takahashi and Goto, 1987, Arch. Gerontol. Geriatr. 6, 73-82; Takahashi and Goto, 1987, Arch. Biochem. Biophys. 257, 200-206]. The present findings are consistent with the theory that active oxygens are involved in the age-related alterations of enzymes.