Isao Shimokawa

Apoptosis in the aging process

Abstract Although many hypotheses have been proposed to explain the aging process, the exact mechanisms are not well defined. Recent accumulating evidence indicates that dysregulation of the apoptotic process may be involved in some aging processes; however, it is still debatable how exactly apoptosis is expressed during aging in vivo. In this review, we discuss recent findings related to apoptosis of individual organs during aging and their significance. We demonstrate that aging enhances apoptosis and susceptibility to apoptosis in several types of intact cells. In contrast, in certain genetically damaged, initiated, and preneoplastic cells, aging suppresses these age-associated apoptotic changes. In various cells, apoptosis enhances the elimination of damaged and dysfunctional cells presumably caused by oxidative stress, glycation, and DNA damage. In these cases, the incidence of apoptosis correlates with the level of accumulated injury. It is concluded that apoptosis plays an important role in the aging process and tumorigenesis in vivo probably as an inherent protective mechanism against age-associated tumorigenesis.

Life span extension by reduction of the growth hormone-insulin-like growth factor-1 axis: relation to caloric restriction.

A reduced growth hormone (GH)‐insulin‐like growth factor (IGF)‐1 axis is associated with an extension of lifespan in laboratory rodents. Several phenotypes of such animal models resemble those induced by caloric restriction (CR). Using a transgenic male Wistar rat model whose GH‐IGF‐1 axis was moderately suppressed by overexpression of the antisense GH transgene (tg), we elucidated a relationship between the effects of a reduced GH‐IGF‐1 axis and CR for some biomarkers of aging, lifespan, and pathologies. Heterozygous (tg/−) rats fed ad libitum (AL) had a dwarf phenotype similar to that of control nontransgenic (−/−) rats subjected to 30% CR from 6 wk of age. Both the reduced GH‐IGF‐1 axis and CR extended lifespan to a similar extent, although the effect of CR seemed to be greater. There was an additive effect of CR to lifespan extension when tg/− rats were subjected to CR. Pathologic analyses indicated that the preventive effect of CR on selected diseases was greater than that of the reduced GH‐IGF‐1 axis. The present study suggests that CR affects aging and longevity by mechanisms other than suppression of the GH‐IGF‐1 axis, although CR might exhibit its effects partly through the reduced GH‐IGF‐1 axis.

Evaluation of adult T‐cell leukemia/lymphoma incidence and its impact on non‐Hodgkin lymphoma incidence in southwestern Japan

The incidence of adult T‐cell leukemia/lymphoma (ATL) and its impact on that of total non‐Hodgkin lymphoma (NHL) were evaluated in Nagasaki, an area in southwestern Japan where human T‐cell lymphotropic virus type I (HTLV‐I) is endemic. The first study area comprised 4 towns located on the K Islands, which had a population of 26,870 in 1990. The overall HTLV‐I seroprevalence estimated from the serologic survey of 18,485 subjects was 16.2%. By using the data from the Nagasaki Prefectural Cancer Registry (NPCR) and reviewing clinical and laboratory information, we identified 40 cases of ATL and 35 cases of other NHL diagnosed between 1985 and 1995. The crude annual incidence of ATL among 100,000 HTLV‐I carriers aged 30 or older was estimated at 137.7 for men and 57.4 for women, with a significant sex difference after adjustment for age (rate ratio = 2.50, 95% confidence interval 1.32–4.73). The cumulative risk from 30 to 79 years of age was estimated at approximately 6.6% for men and 2.1% for women. Among the entire population, ATL accounted for 51 to 59% of the total NHL incidence, showing the strong impact of HTLV‐I infection. The second study area comprised the whole of Nagasaki Prefecture (total population in 1990 = 1.56 million). Between 1985 and 1995, 989 cases of ATL and 1,745 cases of other NHL were registered in the NPCR. The world age‐standardized annual incidence rate of ATL per 100,000 persons aged 30 or older was estimated at 10.5 for men and 6.0 for women, which accounted for approximately 37 to 41% of the total NHL incidence. Int. J. Cancer 85:319–324, 2000. ©2000 Wiley‐Liss, Inc.

Life Span Extension by Reduction in Growth Hormone-Insulin-Like Growth Factor-1 Axis in a Transgenic Rat Model

The longer life span in dwarf mice suggests that a reduction in the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis retards aging and extends the life span in mammals. We tested this hypothesis in a transgenic strain of rats whose GH gene was suppressed by an anti-sense GH transgene. Male rats homozygous for the transgene (tg/tg) had a reduced number of pituitary GH cells, a lower plasma concentration of IGF-1, and a dwarf phenotype. Heterozygous rats (tg/-) had an intermediate phenotype in plasma IGF-1, food intake, and body weight between tg/tg and control (-/-) rats. The life span of tg/tg rats was 5 to 10% shorter than -/- rats. In contrast, the life span of tg/- rats was 7 to 10% longer than -/- rats. Pathological analysis suggested that neoplasms caused earlier death in tg/tg rats; in contrast, tg/- rats had reduced nonneoplastic diseases and a prolonged life span. Immunological analysis revealed a smaller population and lower activity of splenic natural killer cells in tg/tg rats. The results of the present study support the hypothesis, but suggest that there is an optimal level of the GH-IGF-1 axis to maximize survival in mammals.

Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47

Abstract To explore the possible role of heat shock protein (HSP) 47 in the age-related renal changes in Fischer 344 (F 344) rats, the expression of collagen-binding HSP47 with various proteins implicated in phenotypic modulation (α-smooth muscle actin, desmin, and vimentin) and fibrosis (type I, type III, and type IV collagens) was examined in young and old F 344 rat kidneys. Male F 344 rats often develop spontaneous nephropathy in old age. Kidneys obtained from 24-month-old F 344 rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while no significant histological alteration was found in the kidneys of 6-month-old rats. Immunohistochemical analysis showed an increased accumulation of type I, type III, and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. In kidneys of young rats, collagen-binding HSP47 expression was weak in the glomeruli and occasionally seen in the interstitial cells. In contrast, strong immunostaining for HSP47 was noted in the glomeruli, tubular epithelial cells, and interstitial cells in kidneys of old rats. In addition, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for α-smooth muscle actin), glomerular epithelial cells (immunopositive for desmin), and tubular epithelial cells (immunopositive for vimentin) were found in the kidneys of old F 344 rats. Double immunostaining showed that all these phenotypically altered renal cells express HSP47 and that increased expression of HSP47 was always associated with increased expression of collagens in the old rat kidneys. From the above observations, it is concluded that overexpression of HSP47 by phenotypically altered renal cells might play an important role in the excessive assembly of collagens and could thereby contribute to the glomerulosclerosis and interstitial fibrosis found in kidneys of aged F 344 rats.

LEU-7 IMMUNOREACTIVITY IN HUMAN AND RAT EMBRYONIC HEARTS, WITH SPECIAL REFERENCE TO THE DEVELOPMENT OF THE CONDUCTION TISSUE

SummaryThe distribution pattern of Leu-7 (HNK-1) in developing human embryonic hearts and rat hearts was studied by immunohistochemistry. Human and rat embryos at Streeters stages XIII ∼ XX and fetus stage I were used. Leu-7, which is absent in the newborn rat heart, is expressed transiently in the embryo and fetus I stages. The earliest embryonic heart shows two incomplete circular structures with immunoreactivity in the myocardium along the primitive atrioventricular cushion and bulboventricular canal. These two structures become localized topographically in the definitive atrioventricular node and atrioventricular bundle after rearrangement and partial disappearance during embryonic development. At Streeters stages XVIII ∼ XX, Leu-7 immunoreactivity appears to localize topographically in almost all the pathways of the conduction system, although some discontinuities are observed in the atrioventricular junction and atrial internodal tracts. Thereafter, immunoreactivity decreases gradually and differentially by site and stage. The precise nature of Leu-7 immunoreactive cells, that is, whether or not they are neurogenic or myogenic, is not revealed by this study. The present observations are discussed in connection with the hypothesis that specialized ring tissue is the primordium of the conduction system.

FoxO1 is involved in the antineoplastic effect of calorie restriction

The FoxO transcription factors may be involved in the antiaging effect of calorie restriction (CR) in mammals. To test the hypothesis, we used FoxO1 knockout heterozygotic (HT) mice, in which the FoxO1 mRNA level was reduced by 50%, or less, of that in wild‐type (WT) mouse tissues. The WT and HT mice were fed ad libitum (AL) or 30% CR diets from 12 weeks of age. Aging‐ and CR‐related changes in body weight, food intake, blood glucose, and insulin concentrations were similar between the WT and HT mice in the lifespan study. The response to oxidative stress, induced by intraperitoneal injection of 3‐nitropropionic acid (3‐NPA), was evaluated in the liver and hippocampus at 6 months of age. Several of the selected FoxO1‐target genes for cell cycle arrest, DNA repair, apoptosis, and stress resistance were up‐regulated in the WT‐CR tissues after 3‐NPA injection, while the effect was mostly diminished in the HT‐CR tissues. Of these gene products, we focused on the nuclear p21 protein level in the liver and confirmed its up‐regulation only in the WT‐CR mice in response to oxidative stress. The lifespan did not differ significantly between the WT and HT mice in AL or CR conditions. However, the antineoplastic effect of CR, as indicated by reduced incidence of tumors at death in the WT‐CR mice, was mostly abrogated in the HT‐CR mice. The present results suggest a role for FoxO1 in the antineoplastic effect of CR through the induction of genes responsible for protection against oxidative and genotoxic stress.

An age-related increase in the basal level of DNA damage and DNA vulnerability to oxygen radicals in the individual hepatocytes of male F344 rats

Previous biochemical studies on pooled hepatocytes have provided a wealth of information concerning age-related changes in DNA damage and DNA vulnerability (susceptibility) to oxygen radicals and related oxidants, but these studies focused on the whole liver and not on individual hepatocytes. The present study was designed to clarify the DNA damage and DNA vulnerability to hydrogen peroxide in individual hepatocytes using single cell gel electrophoresis (comet assay), a method that measures DNA single-stranded breaks/alkali-labile sites in individual cells. Hepatocytes were prepared from the liver of young (6-11 months) and old (26-29 months) male Fischer 344 rats. DNA damage was induced by exposure to hydrogen peroxide (3 x 10(-5), 3 x 10(-3)%). Observation of each comet image (migration length of DNA (MLD)) was performed in a non-exposure status (basal level) and after exposure. The mean value of MLD was significantly increased (approximately 1.5-fold) in the old rats at the basal level (P = 0.009). Moreover, the proportion of highly DNA-damaged hepatocytes (MLD > 80 microns) increased significantly (approximately 2.5-fold) in advanced age (P = 0.02). The mean value of MLD after exposure to hydrogen peroxide was increased with its concentration, but no significant difference was observed in DNA vulnerability to hydrogen peroxide between young and old rats. However, the proportion of hepatocytes showing a markedly high DNA vulnerability (MLD > 140 microns) to hydrogen peroxide was significantly higher in the old rats than in the young rats. It is suggested that the age-related increase in DNA vulnerability to oxygen radicals and/or related oxidants in some subpopulations causes the increase in DNA damage in advanced age in the liver as a whole.

Manipulation of caloric content but not diet composition, attenuates the deficit in learning and memory of senescence-accelerated mouse strain P8

Calorie restriction (CR) is an experimental intervention in laboratory animals that attenuates age-associated increases in morbidity, mortality, and functional impairment. It is characterized by mild ketosis, hypoinsulinemia and hypoglycemia. In this study, we examined whether metabolic simulation of CR by a diet of isocaloric ketogenic or hypoinsulinemic diets ameliorated the learning and memory deficit in a strain of senescence-accelerated prone mice (SAMP8), a mouse model of age-dependent impairments in learning and memory. Male SAMP8 mice were fed high carbohydrate (CHO), high fat (FAT), or high protein (PRO) diets after weaning, and calorie intake was adjusted to 95% (sub ad libitum, sAL) or 70% (CR) of the mean calorie intake of control mice. At 28 weeks of age, we found CR ameliorated the performance defects of SAMP8 mice in a passive avoidance task. Neither FAT nor PRO diets affected performance of the task when fed sAL level, although a diet of these compositions partially mimicked the serum parameters of CR mice. These results suggest restriction of calorie intake is important for the prevention of learning and memory deficits, and that the simulation of serum changes induced by CR is not sufficient to prevent the cognitive defects of SAMP8 mice.

Leptin signaling and aging: insight from caloric restriction

Organisms have evolved neuroendocrine and metabolic response systems to enhance survival during periods of food shortage, which occur frequently in nature. The anti-aging effect of caloric restriction (CR) might derive from these adaptive responses to maximize organism survival. The present article discusses the potential role for leptin, a hormone secreted from adipocytes, as a key signal that induces the adaptive responses relevant to CR. Evidence indicates that a CR-induced reduction of the plasma leptin concentration suppresses the gonadal, somatotropic, and thyroidal axes, and activates the adrenal axis. Metabolic adaptation, a shift in fuel utilization mainly conducted in the liver, seems to require leptin signaling. Although alternative signaling pathways might also mediate the anti-aging effects of CR, leptin signaling could be a substantial pathway involved in these effects. Molecular dissection of the mechanisms underlying the effects of CR will contribute to a better understanding of the aging process, leading to the extension of a healthy lifespan in humans.