Because heat shock proteins have been shown to play a critical role in protecting cells from hyperthermia and other types of physiological stresses, it was of interest to determine what effect age and caloric restriction have on the ability of cells to regulate the expression of heat shock protein 70 (hsp70), the most prominent and most evolutionarily conserved of the heat shock proteins. Caloric restriction is the only experimental manipulation known to retard aging and increase survival of mammals. The ability of hepatocytes isolated from young/adult (4-to 7-month-old) and old (22-to 28-month-old) male Fischer F344 rats fed ad libitum or a caloric restriction diet (60% of the content of the ad libitum diet) to express hsp70 was determined after a mild heat shock (42.5°C for 30 min). We found that the induction of hsp70 synthesis and mRNA levels by heat shock was 40 to 50S% lower in hepatocytes isolated from old rats than in hepatocytes isolated from young rats. Using in situ hybridization, we found that essentially all hepatocytes from the young/adult and old rats expressed hsp70 in response to heat shock; therefore, the age-related decrease in the induction of hsp70 expression was not due to an age-related accumulation of cells that do not respond to heat shock. Measurements of hsp70 mRNA stability and hsp70 transcription demonstrated that the age-related decline in hsp70 expression arose from a decline in hsp70 transcription. Interestingly, the age-related decline in the induction of hsp70 expression was reversed by caloric restriction; e.g., the induction of hsp70 synthesis, mRNA levels, and nuclear transcription were significantly higher in hepatocytes isolated from old rats fed the caloric restricted diet than in hepatocytes isolated from old rats fed ad libitum. The levels of the heat shock transcription factor in nuclear extracts isolated from heat-shocked hepatocytes were measured in a gel shift assay. Binding of the heat shock transcription factor to the heat shock element decreased with age and was significantly higher in hepatocyte extracts isolated from old rats fed the caloric restriction diet than in those from old rats fed ad libitum. Thus, our study demonstrates that the ability of hepatocytes to respond to hyperthermia and express hsp70 decreases significantly with age and that this decrease occurs at the transcriptional level. In addition, caloric restriction, which retards aging, reversed the age-related decline in the induction of hsp70 transcription in hepatocytes.Organisms ranging from bacteria to humans and plants respond to hyperthermia by synthesizing a group of proteins known as heat shock proteins (hsps). Because the synthesis of hsps is induced not only by hyperthermia but also by a variety of other stresses (ethanol, amino acid analogs, heavy metal4, free radicals, etc.), hsps also have been referred to as stress-induced proteins (26). The number of hsps varies from organism to organism and even between cell types in an organism (9). However, all organisms (e.g., archaebacteria and...

Objective: Human adenovirus Ad-36 causes adiposity in animal models and enhances differentiation and lipid accumulation in human and 3T3-L1 preadipocytes, which may, in part, explain the adipogenic effect of Ad-36. We determined the consequences of Ad-36 infection on leptin and glucose metabolism in fat cells. Design: 3T3-L1 preadipocytes were used to determine the effect of infection by human adenoviruses Ad-36, Ad-2, Ad-9 and Ad-37 on leptin secretion and lipid accumulation. Rat primary adipocytes were used to determine the effect of Ad-36 infection on leptin secretion and glucose uptake in vitro. Furthermore, the effect of Ad-36 on expressions of leptin and selected genes of de novo lipogenesis pathway of visceral adipose tissue were compared ex vivo, between Ad-36 infected and uninfected control rats. Results: Ad-36 suppressed the expression of leptin mRNA in 3T3-L1 cells by approximately 58 and 52% on days 3 and 5 postinfection, respectively. Leptin release normalized to cellular lipid content was 51% lower (Po0.002) in the Ad-36 infected 3T3-L1 cells. Lipid accumulation was significantly greater and leptin secretion was lower for the 3T3-L1 cells infected with other human adenoviruses Ad-9, Ad-36, or Ad-37. Whereas, human adenovirus Ad-2 did not influence cellular lipid accumulation or the leptin release. In rat primary adipocytes, Ad-36 reduced leptin release by about 40% in presence of 0.48 (Po0.01) or 1.6 nM insulin (Po0.05) and increased glucose uptake by 93% (Po0.001) or 18% (Po0.05) in presence of 0 or 0.48 nM insulin, respectively. Next, the adipose tissue of Ad-36 infected rats showed two to fivefold lower leptin mRNA expression, and 1.6-to 21-fold greater expressions for acetyl Co-A carboxylase-1 and 1.2-to 6.3-fold greater expressions for fatty acid synthase, key genes of de novo lipogenesis, compared to the uninfected weight and adiposity matched controls. Conclusion: The in vitro and ex vivo studies show that Ad-36 modulates adipocyte differentiation, leptin production and glucose metabolism. Whether such a modulation contributes to enhanced adipogenesis and consequent adiposity in Ad-36 infected animals or humans needs to be determined.

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The biological mechanisms responsible for aging remain poorly understood. We propose that increases in DNA damage and mutations that occur with age result from a reduced ability to repair DNA damage. To test this hypothesis, we have measured the ability to repair DNA damage in vitro by the base excision repair (BER) pathway in tissues of young (4-month-old) and old (24-month-old) C57BL/6 mice. We find in all tissues tested (brain, liver, spleen and testes), the ability to repair damage is significantly reduced (50-75%; P<0.01) with age, and that the reduction in repair capacity seen with age correlates with decreased levels of DNA polymerase beta (beta-pol) enzymatic activity, protein and mRNA. To determine the biological relevance of this age-related decline in BER, we measured spontaneous and chemically induced lacI mutation frequency in young and old animals. In line with previous findings, we observed a three-fold increase in spontaneous mutation frequency in aged animals. Interestingly, lacI mutation frequency in response to dimethyl sulfate (DMS) does not significantly increase in young animals whereas identical exposure in aged animals results in a five-fold increase in mutation frequency. Because DMS induces DNA damage processed by the BER pathway, it is suggested that the increased mutagenicity of DMS with age is related to the decline in BER capacity that occurs with age. The inability of the BER pathway to repair damages that accumulate with age may provide a mechanistic explanation for the well-established phenotype of DNA damage accumulation with age.

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