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Age-related changes in glycogen concentration in the heart of SAMP8, a substrain of senescence-accelerated prone mouse, were compared with that in SAMR1, a substrain of senescence-accelerated resistant mouse. The decrease by about 50% in the glycogen concentration in the hearts of 4-8 weeks old SAMP8 was observed in comparison with that in age-matched SAMR1, whereas there was no difference in adult hearts between the two substrains. Additionally, glycogen phosphorylase activity was significantly increased (1.3-fold) in the heart of 4–8 weeks old SAMP8 without the change in glycogen synthase activity between the two substrains. At the same age, the lipid peroxide concentration was also increased (1.4-fold) in the hearts of SAMP8, suggesting that oxidative stress was involved in the early damage to the hearts of SAMP8. Furthermore, the present findings that the cardiac glycogen concentration in SAMP8 decreases more quickly than that in SAMR1 may indicate the possibility that an accelerated aging of SAMP8 is related to the early changes in the energy sources in the heart.

The effect of insulin on the translocation of GLUT4 (glucose transporter isoform 4) from the intracellular membranes to the plasma membranes was compared in the hearts of 4-8 week old SAMP8, a substrain of senescence-accelerated prone mouse and those of SAMR1, a substrain of senescence-accelerated resistant mouse. After 20 min of the intravenous injection of insulin, the blood glucose levels in SAMR1 and SAMP8 were decreased by 50 and 68%, respectively. Under this condition, the concentrations of GLUT4 protein in the plasma membranes in the hearts of SAMR1 and SAMP8 were increased 1.8- and 2.1-fold, respectively. Concomitantly, the concentrations of the GLUT4 protein in the intracellular membranes in the hearts of SAMR1 and SAMP8 were decreased by about 70 and 50%, respectively. These results suggest that the heart of 4-8 week old SAMP8 is more sensitive to insulin than that of age-matched SAMR1.

SAMP8, a substrain of senescence-accelerated mouse (SAM), has been characterized by several age-related deficits in the brain. Previously, we reported that the contents of lipid peroxides and protein carbonyl, and net generation of H2O2 were increased in the cerebral cortex of SAMP8 at 4–8 weeks of age in comparison with those in age-matched SAMR1 substrain used as a control. To study the cause of these increases, we compared the activities of antioxidative enzymes in the cerebral cortex between the two substrains. The catalase activity was decreased by 75% in SAMP8 at 4–8 weeks of age, whereas neither superoxide dismutase nor glutathione peroxidase activities were changed. The change in the catalase activity was seen only in the cerebral cortex where oxidative stress was increased in SAMP8. On the contrary, the activity of acyl-CoA oxidase, a microperoxisomal H2O2-producing enzyme, in the cerebral cortex of SAMP8 was increased 1.6 fold in comparison with that in age-matched SAMR1 without change in the activity of D-amino acid oxidase. Furthermore, the changes in the activities of catalase and acyl-CoA oxidase with age were paralleled with those observed in oxidative stress in SAMP8. These results suggest that the abnormality of activities in two microperoxisomal enzymes, catalase and acyl-CoA oxidase, may be one of the causes of the early increase in oxidative stress observed in the cerebral cortex of 4–8 weeks old SAMP8.

Age-related changes in oxidative stress in the cerebral cortex of SAMP8, a substrain of senescence-accelerated mouse (SAM), were investigated in comparison with those in SAMR1, which were used as a control. The lipid peroxide and protein carbonyl contents were transiently increased in SAMP8 from 4- to 8-weeks of age. The increases in lipid peroxide were seen only in the cerebral cortex and not in other regions of the cerebrum. Furthermore, the net generation of reactive oxygen species in cerebral cells was also increased in SAMP8. In addition, the activity of glutamine synthetase, which is known as an enzyme-highly sensitive to reactive oxygen, was decreased in the cerebral cortex of SAMP8 from 4- to 8-weeks of age. These results suggest that oxidative stress may be induced in the cerebral cortex of SAMP8 from 4- to 8-weeks of age, preceding the appearance of distinctive deficits in the brain of SAMP8.

A human diploid fibroblast strain, TIG-1, ceased to proliferate at about 60–62 population doubling level. The percentage of nucleic incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxy-uridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.

When quiescent young or senescent human diploid cells, TIG-1, were metabolically labeled with 32 P i and stimulated with 10% fetal bovine serum, the phosphorylation of ribosomal S6 protein was enhanced in young cells but not in senescent cells while that of some other proteins were increased in both cells. Inability to stimulate the phosphorylation of S6 protein in senescent cells after serum addition may be the primary cause of the failure of enhancement in protein synthesis followed by the block of prereplicative events dependent on protein synthesis and thus of the failure of cells to enter S phase. However, when the cell-free preparation from serum-stimulated senescent cells was incubated with [γ- 32 P] ATP, S6-kinase activity was stimulated and S6 in ribosomal fraction was susceptible to phosphorylation as observed in young cells. Differences in S6 phosphorylation of senescent cells between in vivo and in vitro was discussed.

Responses of human diploid cells, TIG-1, were examined with respect to their ability to initiate DNA synthesis under the influence of various growth factors and their combinations. The following agents stimulated DNA synthesis in quiescent TIG-1 cells at 37-49 PDL (population doubling level) (66-79% of lifespan completed): fetal bovine serum; tumor-derived DNA synthesis factors such as those from rat rhodamine fibrosarcoma, human adenoma and from the conditioned medium of cultured human pituitary cells; human and mouse epidermal growth factors; tumor promotors such as 12-O-tetradecanoylphorbol 13-acetate and teleocidin; microtubule-disrupting agents as colchicine, vinblastine, podophyllotoxin and TN-16; melittin; and dexamethasone. Cells at 58-60 PDL (94-97% of lifespan completed) were stimulated to synthesize DNA by fetal bovine serum, tumor-derived DNA synthesis factors and epidermal growth factors, but not by other agents. Finally, in senescent cells at 62 PDL (100% of lifespan completed), any of these growth factors and of their combinations failed to induce DNA synthesis at all. These senescent cells, however, still retained the ability to initiate DNA synthesis following infection with SV40 as reported previously [Exp. Cell Res., 143 (1983) 343-349].

When senescent human diploid cells, TIG-1, were stimulated with serum at the end of their proliferative life span, such biochemical events as uptakes of 2-deoxyglucose and uridine, and expression of c-myc, were enhanced. However, RNA synthesis, polyamine accumulation, thymidine uptake and DNA synthesis were not enhanced at all. Protein synthesis increased only moderately as compared with that observed in younger cells. These results indicated that the events in prereplicative phase known to be independent on protein synthesis are induced in senescent cells after the stimulation with serum, whereas those required protein synthesis failed to increse to the same extent as seen in young cells.

When human diploid fibroblasts, TIG-1, reached the end of their proliferative lifespan in vitro , they still could synthesize proteins with molecular weight of about 70 000, 85 000 and 116 000 following the heat shock at 46°C for 10 min as well as young cells did so.