In 2003, de Cabo and colleagues described an in vitro
technique demonstrating that many of the features of CR including reduced cellular proliferation, enhanced stress responsiveness and changes in gene expression could be reproduced in cells cultured in media supplemented with serum from animals on CR diets 
. These findings suggested that several effects of CR are mediated through circulating factors in the sera of the animals subjected to the dietary regimen. In fact, studies have shown that changes in hormones and nutrients that are constituents of serum including insulin, glucose, IGF-1 and fatty acids, modulate gene expression pathways involved in stress responsiveness and longevity 
. Since it is unlikely that long-term studies determining the effect of CR or other dietary manipulations on human lifespan will be conducted, this in vitro
technique is extremely useful for the investigation of changes in biomarkers of aging and longevity, which may be predictive of effects on lifespan. Therefore, using this in vitro
technique, we examined the effect of human serum samples collected from FEAST and CALERIE participants on cellular proliferation, stress resistance and protein expression.
We found that changes in stress resistance as well as mRNA and protein levels of genes considered to be important for the beneficial effects of CR can be induced in cells incubated in media supplemented by serum collected from human participants who had been adhering to different CR regimens when compared to cells incubated with serum from the same individuals before the dietary manipulation. Serum collected at the end of the diet period from participants on the ADF regimen induced significant decreases in proliferation, increases in heat shock resistance and increased Sirt1 levels compared to baseline serum, while serum samples from participants on the CR regimen also induced significant increases in Sirt1 levels in addition to significant increases in PGC-1α mRNA expression compared to baseline serum. These findings are supportive of improved health and longevity.
Long term CR is known to decrease cellular proliferation in many tissues, which is associated with lower cancer risk 
. In addition, CR has been shown to have protective effects against oxidative and heat stress 
. In this study, we observed that human hepatoma cells, cultured with blood serum collected after 21 days of ADF, have a decreased rate of cellular proliferation and an increased resistance to heat-induced stress, but not to oxidative stress induced by H2
, when compared to cells cultured with blood serum collected at baseline. Similar findings were reported previously which demonstrated that the treatment of cells cultured in sera collected from rats and monkeys fed CR diets cause reduced cell proliferation, enhanced tolerance to heat and oxidative stress and an increased expression of stress response genes when compared to cells cultured in AL sera 
. Cells treated with serum from CALERIE participants on CR and CREX regimens for 3 months, however, showed no significant changes in cellular proliferation or heat shock resistance compared to cells cultured in the baseline serum. Although FEAST participants were instructed to eat twice their usual intake on feast days in order to maintain their bodyweight, subjects lost an average of 2.0% of their initial bodyweight by the end of the 3 week period. In comparison, CR and CREX subjects lost respective averages of 7.4% and 5.8% of their initial bodyweight after 3 months on their diet regimens. Therefore, overall weight loss was not the necessary impetus that led to these effects on proliferation and heat stress resistance. Instead, it may be that the short, regular intervals of complete caloric deprivation, as practiced by FEAST participants, provided a more potent stimulus for triggering the necessary changes in serum constituents than either the CR or CREX regimens practiced by the CALERIE participants. Indeed, other studies in rodents have shown that ADF results in increased stress protection, sometimes surpassing the protective effect seen with CR 
. In addition, FEAST subjects showed several phenotypes, typical of CR, which may play a role in the stress response, including decreased insulin and triglyceride levels 
. A surprising outcome was the small but significant increase in survival after heat shock treatment seen in cells cultured with 3 month serum from CALERIE control subjects compared to baseline serum treated cells. Further analyses will be needed to provide a possible explanation for this outcome since control subjects showed no changes in the neuroendocrine measurements taken that may provide a plausible explanation. Changes in other factors that were not measured in CALERIE participants may have contributed to this unexpected effect. Although control group participants were not calorie restricted, their diet during the study provided a healthy 30% of calories from fat with the recommended daily allowance of nutrients. This regimen may have been very different from their usual diet and therefore may have induced changes leading to our finding. Also due to their healthier diet, they showed a tendency toward less DNA damage as reported previously 
. The increase in heat shock resistance seen in cells cultured with FEAST serum was not accompanied by increased HSP70 protein levels as anticipated. However, other heat shock proteins not examined in this study (due to limited availability of serum samples) such as HSP90 or HSP72 may have been responsible for this protective effect. In addition, changes in resistance to oxidative stress were not seen in any of the human serum treated cells. Increased length of time on diet may be needed to induce the changes necessary to enhance resistance to oxidative stress in cells treated with the serum.
PGC-1α mRNA levels were examined in cells incubated with serum from CALERIE participants after 3 months on their diet regimens compared to cells cultured in baseline serum. Only cells cultured in serum from subjects on the CR regimen showed significant increases in PGC-1α mRNA expression. This result falls in line with those of Lopez-Lloyd and colleagues who reported that other members of the PGC-1α pathway including peroxisome proliferation-activated receptor (PPAR)α and nuclear respiratory factors (NRFs), showed increased expression in primary rat hepatoma cells and Fao cells cultured in serum collected from CR-fed rats compared to cells cultured in serum from AL-fed rats
. The increase in PGC-1α levels and other proteins of the pathway is associated with increased mitochondrial biogenesis and metabolic efficiency
. An increase in PGC-1α levels may have been expected to occur in cells treated with CREX serum since exercise is known to increase PGC-1α levels 
. However, although exercise has been shown to be a potent stimulant for increased PGC-1α levels in muscle, it is not certain whether exercise also increases expression of this gene in other tissues including liver. If the changes in blood serum required to induce increases in PGC-1α in liver are capable of being induced by CR but not exercise, then the 12.5% CR in the CREX group may not have been adequate stimulus to produce the endocrine changes required to invoke PGC-1α.
Our finding that Sirt1 protein levels were increased in cells incubated with serum from participants on ADF and CR regimens is particularly interesting. Increases in Sirt1 protein is thought to be a major contributing factor for many of the health benefits associated with CR. Increased Sir2 levels have been associated with increased lifespan in yeast, flies, and worms 
and Sirt1is a leading contender for proteins responsible for the longevity effect of CR in rodents 
. Sirt1 is an NAD-dependent deacetylase that activates and deactivates several proteins that regulate stress reactions, growth, and metabolism
. In rodent models with extended lifespan through CR, an increase in Sirt1 levels is seen in many tissues including muscle and liver. Transgenic mice over-expressing Sirt1 mimic the physiologic responses of CR 
and the Sirt1 activating molecule, resveratrol, has been shown to extend the lifespan of mice fed a high fat diet 
. The increase in Sirt1 expression in cells cultured with serum from FEAST and CR CALERIE participants strongly corresponds with the increase in Sirt1 mRNA expression found in muscle biopsies from a subset of FEAST 
and CR CALERIE participants 
The increased Sirt1 expression in our cells may suggest that ADF and CR induce changes in blood serum constituents that lead to increased resistance to stress. Previous studies have suggested that increased Sirt1 levels may be related to decreased fasting glucose and insulin levels and decreased body temperature 
. In support of our findings with Sirt1, CR participants showed a decrease in core body temperature and fasting insulin levels 
. FEAST participants showed decreases in fasting insulin and triglyceride levels but no changes in surface body temperature, although core temperature was not measured. In this study the observed changes in Sirt1 were significantly correlated to changes in serum triglyceride levels.
These in vitro
results provide supporting evidence for the possibility of extending human lifespan and health-span through diet manipulations such as CR and ADF. In fact, our results indicate that ADF may have a more powerful effect than CR in inducing the changes in neuroendocrine factors that lead to increases in stress protection and Sirt1 protein levels. These are changes thought to be associated with improved health and increased longevity. In addition and importantly, this study lends support to the proposal that at least some of the effects of CR are partially due to changes in the hormonal and nutritive milieu of blood serum. In fact, a recent study investigating the age associated variations in immunity, showed that incubation of isolated peritoneal macrophages from young rats in sera collected from old rats results in a significant increase in inflammatory cytokines 
. This and other similar in vitro
studies further validate the use of this in vitro
method to analyze changes in known and proposed indicators of health and longevity as well as the mechanisms behind the beneficial effect of CR. This in vitro
method may also prove useful for predicting the potential of CR, ADF and other dietary treatments to affect human health and lifespan.