Sci. Aging Knowl. Environ., 9 July 2003
Vol. 2003, Issue 27, p. pe19
[DOI: 10.1126/sageke.2003.27.pe19]


Caloric Restriction in trans

Bruce S. Kristal, and Ugo Paolucci

The authors are with the Dementia Research Service of the Burke Medical Research Institute, White Plains, NY 10605, USA (B.S.K. and U.P.) and the Departments of Biochemistry and Neuroscience at Weill Medical College of Cornell University, New York, NY 10021, USA (B.S.K.). E-mail: bkristal{at};2003/27/pe19

Key Words: caloric restriction • dietary restriction • model systems • mammalian tissue culture • sera


Caloric (or dietary) restriction (CR) is a robust paradigm with established efficacy in delaying age-related morbidity and mortality in at least three species of laboratory mammals (see Masoro Subfield History). Although it has been extensively explored since the 1930s, the biochemical mechanisms through which it acts are not fully understood. A new study by de Cabo and colleagues (1) now shows that mammalian tissue culture can be used as a model system to study aspects of this phenomenon.

Animals maintained on CR regimens are fed a fraction (usually 50 to 70%) of the amount eaten by their ad libitum (AL)-fed counterparts, who have unlimited access to food. CR can be implemented in multiple ways, ranging from harsh methods, such as McCay's original protocols in which animals were fed barely enough to allow growth, to every-other-day feeding, to controlled paradigms that decrease food intake from as little as 20% to 55% or more (2). The restriction of total calories is more important than reducing the intake of specific factors such as fat, proteins, or vitamins and minerals (3, 4).

CR has been shown to extend both mean and maximum life span, reduce age-related morbidity, and delay or prevent most age-associated physiological dysfunction (2, 5). CR also alters many aspects of basic physiology, including metabolism; hormonal balance; and the generation of, detoxification of, and resistance to reactive oxygen species (6). CR extends longevity in essentially all animals (for an exception, see "Not in Medflies") in which it has been tried, including multiple mammalian species (for example, rats, mice, and guinea pigs). Furthermore, promising data suggest that at least some of the benefits of CR, especially those associated with glucose metabolism, also occur in nonhuman primates (7-9), and, perhaps, in humans as well (10) (see Walford et al. abstract and comments). Together, these observations suggest that the CR effect is robust in mammals.

CR affects the frequency and severity of both neoplastic and non-neoplastic disease. As an example, one may consider the effects of CR on breast cancer in laboratory animals (11, 12-15). CR reduces initiation events and slows both tumor promotion and progression (16). In transgenic mice prone to mammary tumors as a result of carrying a mutant v-Ha-ras gene, CR was shown to reduce tumor incidence by 67% (11), indicating that CR is capable of modulating the penetrance (that is, the phenotypic expression of genotype) of genetic predisposition to breast cancer. Furthermore, studies in rats treated with 7,12-dimethyl-benz(a)anthracene (DMBA), a compound that induces mammary tumors, demonstrated that high-fat and high-calorie diets synergistically stimulate tumor formation (12). None of the rats maintained on a 40% CR regimen developed mammary tumors, whereas 60% of AL-fed rats did. Concerns that this effect might have been partially mediated by a reduced availability of lipids needed for tumor growth in the CR diet led to later studies to assess the effects of a CR diet that contained a higher fat content than the AL diet (13). Despite this change, the CR diet reduced the incidence of rats with mammary tumors by 75% and decreased the number of tumors per animal in the tumor-bearing group as compared to the AL diet. CR reduced total tumor yield, average tumor size, and mean tumor burden by 93 to 98%. In other experiments, Sinha et al. demonstrated that even a 20% CR regimen (in this case, not increased in fat content) reduces the frequency of tumors in DMBA-treated rats by 65%, without affecting hormone levels or fertility (14).

The CR-Associated Metabolic Serotype

Consistent with its broad effects on longevity and disease, CR is a systemic phenomenon, and its effects include measurable differences in the concentrations of certain blood constituents relative to those seen in AL-fed animals. Many previous studies have focused on determining changes in the concentrations of a variety of hormones. Observed changes include, for example, alterations in plasma corticosterone patterns and levels (17); alterations in female reproductive hormone levels (18); a 50% reduction in the plasma concentration of cholecystokinin (a peptide hormone that affects digestion and might mediate satiety) (19); a reduction in the concentration of triiodothyronine but not of thyroxine (hormones produced by the thyroid) (20); and a decrease in plasma insulin levels by as much as 60% in some CR models (21). CR also decreases plasma glucose, ascorbate (22-24), and glycohemoglobin levels (22). Discovery-based (data- rather than hypothesis-driven) studies of CR-associated changes in serotype in our laboratory (25, 26) have shown that CR exerts sufficient changes in serotype to distinguish diet group. Targeted studies of known clinical blood markers in animals subjected to CR have been pursued to identify biomarkers of aging (27) (see Miller Perspective). Studies attempting to bridge the gap between human and animal CR studies have shown that three specific markers (body temperature, levels of blood insulin, and levels of the steroid hormone dehyroepiandrosterone-sulphate) are correlated with relative longevity at the population level in humans (28) (see "Monkey in the Middle").

Although blood components that display significant changes in concentration in animals subjected to CR compared to AL-fed animals have been treated simply as markers by some researchers, others have looked to these data for possible insight into the mechanism(s) of CR. Such studies include, for example, the work done on insulin-like growth factor 1 (IGF-1) (29, 30). Interest in IGF-1 has been augmented by the concordance of evidence from human epidemiological studies that links higher levels of IGF-1 with increased risks of premenopausal breast cancer, advanced colorectal cancer, and prostate cancer (31-33), and from laboratory studies showing that IGF-1 is a powerful growth factor and may thus directly stimulate tumor cell growth (29). (For a discussion of the IGF-1 pathway and life span, see "One for All".) These studies from disparate systems tie together well with the finding that plasma IGF-1 levels are greatly reduced in animals subjected to CR (29, 30).

A Model for the CR Phenotype in Cultured Cells: de Cabo et al.

Now, de Cabo and colleagues tie together these two areas of sera research--markers and mechanisms--by showing that cells cultured in sera derived from male rats and rhesus monkeys undergoing CR (referred to as CR sera) display different properties than those cultured in sera obtained from AL-fed animals (1). In the main line of experiments, the sera were added to FaO rat hepatoma cells (as 10% of media), and the cells were then tested for proliferation, oxidant (hydrogen peroxide) resistance, and resistance to heat shock. Previously, CR has been shown to increase resistance to several forms of stress, including heat and oxidants, in intact organisms [see (1) and references therein]. Proliferation was lower in cells supplemented with CR sera, and such cells were more resistant to heat and oxidant challenge, as compared to cells supplemented with AL sera. Cell viability was monitored and the level of expression of a heat shock protein (Hsp70) was determined by Western analysis after exposure to high temperature (45°C) in several tissue culture cell lines, including B16 mouse melanoma cells, HeLa human cervical carcinoma cells, and RKO human colorectal carcinoma cells, as well as the FaO cells. The level of induction of Hsp70 after heat shock was increased in cells exposed to CR sera as compared to those exposed to AL sera, as was cell viability. Microarray studies, in which global gene expression was analyzed in cells treated with either CR or AL sera, suggested that a number of other stress-response genes display enhanced expression in cells treated with CR sera.

The studies described above indicate that CR serum contains altered levels of certain factor(s) that mediate cell proliferation and response to stress. The dramatically reduced levels of insulin and IGF-1 in sera derived from animals undergoing CR (1, 29, 30) suggest that these hormones might be responsible for the difference. In support of this idea, de Cabo et al. found that supplementation of CR sera with IGF-1 and insulin so that their concentrations were equivalent to the concentrations in AL sera restored proliferation and partially restored oxidant and heat sensitivity to cells treated with CR sera (1).

These studies provide a new type of evidence to support the theory that some of the effects of CR are exerted in trans; in other words, that phenotypic changes may occur in cells and tissues (in this case, the cultured cells) distinct from those that initially sense and react to the change in caloric intake (in the animal subjected to CR). Other evidence in support of this idea was provided by the work of Pashko and Schwartz (34) that showed that adrenalectomy abolishes CR-mediated protection against skin tumors. This and subsequent reports addressed a leading hypothesis in the field, providing support to the idea that glucocorticoids (steroid hormones produced by the adrenal gland) play an important role in some aspects of the antineoplastic effects of CR. The work of Pashko and Schwartz rests on a foundation of decades of work by others, and it was only this foundation that allowed the critical experiments to be done.

Although the Pashko and Schwartz work provided important in vivo support for a proposed mechanism of the antineoplastic actions of CR, the de Cabo et al. paper provides a new model that can be used more broadly to determine the multiple actions of CR in trans. In part, de Cabo and colleagues address this issue through well-established experimental approaches, in that they add a factor (serum) to cells to examine an effect. They then use supplementation approaches to argue for the presence of proximate mediator(s). This provides a standard "nice complete story" linking insulin and IGF-1 to some of the trans effects observed in CR. Perhaps more important, however, the de Cabo et al. paper provides a new model for the study of CR.

Model Systems in Biology and the Study of CR

In general, the use of different models in biology reflects the different strengths of those models. Viruses, for example, mutate frequently and can hijack all cellular machinery to synthesize relatively few RNAs and proteins. These properties facilitate biochemical studies of gene expression and protein folding. Bacteria are easy and inexpensive to grow, mutate frequently, exchange plasmids, and display basic gene regulation. These properties made them one of the most important workhorses of the advent of molecular biology. Yeast offers facile genetics and an entrance to the eukaryotic cell. Drosophila provided Morgan and his scientific progeny with an organism having readily observable phenotypic variability, a short generation time, and polytene chromosomes. Later, the work on P-element transfer offered a powerful genetic tool. The nematode Caenorhabditis elegans provides a powerful model of development and cell death. Rodents offer a mammalian form that can be easily housed and handled, and that has relatively fast generation times and sufficient large tissues for biochemical and physiological studies. The diversity of inbred mouse strains contributes greatly to the field of molecular genetics, and led to the ability to examine how a variety of manipulations play out in the context of different genetic backgrounds. Successively higher organisms provide, at least theoretically, greater and greater relevance to humans.

Despite the variety of model systems available, relatively little work on CR has been done outside the small laboratory mammal. Indeed, mouse and rat studies account for most or all analyses of implementation strategies, mechanisms, genetic and environmental influences, phenomenology, pathology, and biomarkers. Although studies have been conducted in dogs, C. elegans, and insects such as Drosophila, such work has been arguably more useful to show the robust nature of CR-associated effects than to shed light on how CR works in mammals. Studies in nonhuman primates are beginning to suggest that CR exerts many of the same effects in these close human relatives as it does in laboratory rodents, but these studies will require decades to complete and involve small numbers of animals. These studies are also complicated by the need to develop, maintain, and monitor diets without being able to continually add cohorts (because of the expense and time involved), as has been done with rodent models. Studies in humans are limited by practical considerations, in that they can only be carried out for limited time periods, and by the general complexity inherent in controlling human nutrition and nutritional studies. There have been few attempts to use phylogenetically lower species to probe mechanisms responsible for CR-associated effects. Studies of aging in C. elegans (35) and CR in Drosophila (36) implicate aspects of insulin-dependent pathways (see Sonntag Perspective and "The Road More Traveled"). Other studies in yeast and Drosophila provide support for a role of altered histone deacetylase activity in the response to CR (37, 38) (see Kaeberlein Perspective, "High-Octane Endurance--Yeast in the Metabolic Fast Lane Live Longer", and "Domino Effect"). Whether these observations hold true in higher organisms clearly needs to be determined. It is here that the model developed by de Cabo et al. might pay large dividends.

The model of de Cabo et al. should enable researchers to probe the effects of CR at the level of the mechanism(s) of trans-acting effects. One of the great advantages of cell culture systems is that they allow scientists to investigate which effects are primary to the system in question (the cell or the sera) and which are not. An example of the power of this approach can be seen in Alzheimer's disease (AD) research, where investigators have used skin fibroblasts to show that some cellular defects (for example, in Ca2+ handling) are present in cells derived from anatomical areas that do not exhibit AD-associated pathology and which are maintained after the influence of any drugs given to the patient should have been diluted out (39). As another example of this potential in action, the work of de Cabo et al. already has shown that decreased flux through the energy pathways of the cells in question is not necessary to display at least some of the effects of CR. Given that changes in this flux are often considered one aspect of free radical/Maillard-based models of aging and disease (40, 41) (see Obrenovich Perspective and "The Two Faces of Oxygen"), these new data provide support for a well-accepted model that CR involves essential effects at multiple levels.

The power of the de Cabo et al. model is that it is inherently straightforward, easily modified, and relatively easy to scale (within limits). The model is inherently simple in that it requires only tissue culture cells and sera, which can be readily obtained under highly controlled conditions. The model can be easily modified in that changes can be made in the serum donor (for example, with respect to age, sex, diet, or degree of CR); the target cells (possibilities include cell lines, primary cells, secreting cells, cells that become cancerous, cells of metabolic interest such as insulin-secreting cells, muscle cells, or cells derived from adipose tissue); treatment paradigms [for example, duration, sera concentration, or pretreatment (such as exposure to oxidants or proteases to inactivate specific components)]; and methods of analysis, including high-data-density studies suitable for discovery-based research (such as transcriptomics, proteomics, and metabolomics). Although sera may become limiting for very large-scale experiments, the system clearly can be sufficiently scaled up for basic biochemical measurements and factor purification or scaled down for high-throughput screens (for example, for CR mimetics, as suggested by the authors).

It is reasonable to expect that the de Cabo et al. model will also share weaknesses with other cell culture systems. Some potential limitations include false positive and false negative results ensuing from the consequences of inappropriate choices of cell lines or conditions, difficulty in observing effects of short-lived sera factors, and lack of correct physiological interactions. In addition, results will require eventual confirmation in intact systems. These limitations are, however, generally well appreciated and should not be considered to detract substantially from the potential power of the model.

In summary, de Cabo and colleagues provide an initial report showing the development of an in vitro model of CR that can be used to examine the ability of this phenomenon to exert effects in trans, and offers the potential to identify new directions for further studies into the mechanisms of CR-mediated protection.

July 9, 2003
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Citation: B. S. Kristal, U. Paolucci, Caloric Restriction in trans. Sci. SAGE KE 2003, pe19 (9 July 2003);2003/27/pe19

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