An in vitro model of enhanced stress responsiveness by caloric restriction: A novel method for evaluation of possible mechanisms and candidate caloric restriction mimetics.
R. de Cabo, R. M. Anson, M. Rios, S. Furer, M.A. Lane
Nutritional and Molecular Physiology Unit, National Institute on Aging, NIH
Baltimore, MD USA, 21224
The underlying biological mechanisms that lead to lifespan extension and retardation of aging by caloric restriction (CR) remain elusive. The use of lifespan, disease, or pathological endpoints, though accepted as reliable endpoints for a CR effect, are difficult to manipulate and require long-term studies that are expensive and time-consuming. Such studies therefore limit the ability to investigate rapidly and more mechanistically the metabolic and biochemical pathways that may be involved in the CR response. A limited number of studies have attempted to utilize in vitro models designed to reproduce the effects of CR in culture. These studies have utilized either primary cells isolated from control (CON) and CR animals or to limit nutrients in the culture media. The conditions (nutrients, sera, etc.) used in typical culture methods seriously limit the physiological relevance of these models to study the behavior of cells in the intact organism. This is of particular concern given the numerous endocrine and neuroendocrine changes that are observed in CR. For example, CR is known to have significant effects on insulin, glucocorticoids, IGF-1, growth hormone and other endocrine factors. As a new approach we have designed a cell culture system utilizing autologous serum from CON and CR animals for investigation of possible mechanisms of this intervention and for evaluation of candidate CR mimetics. Not surprisingly, rat hepatoma cells (FaO) grown in serum from CON and CR animals (both rat and monkeys) exhibited reduced growth and proliferation. Interestingly, FaO cells grown in CR serum were better able to withstand challenges with hydrogen peroxide and heat stress as indicated by enhanced survival compared to cells grown in serum from CON animals. Changes in gene expression related to stress responsiveness (HSP70, p21, PPAR, GRP78, GADD153, etc.) were consistent with the enhanced survival of cells grown in CR serum from rats and monkeys. Preliminary experiments in primary hepatocytes isolated from CON and CR F344 rats show that cells obtained from CON rats exhibited an enhanced stress response when grown in CR serum and that the responsiveness of CR cells was reduced when they were grown in serum from CON animals. Combined with our findings in hepatoma cells, these data suggest that factors in serum from CR animals may be responsible for enhanced stress responses of cells in culture and may ultimately be related to enhanced in vivo stress responses in CR animals. This model may be useful for investigations of possible metabolic mechanisms of CR. In addition, our in vitro system provides method for rapid evaluation of compounds that may mimic some of the cellular responses of CR.
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