In Vitro Cellular Adaptations of Indicators of Longevity in Response to Treatment with Serum Collected from Humans on Calorie Restricted Diets Joanne S. Allard 1 , Leonie K. Heilbronn 2 , Carolina Smith 1 , Nicole D. Hunt 1 , Donald K. Ingram 1,2 , Eric Ravussin 2 , Pennington CALERIE Team 2 , Rafael de Cabo 1 * 1 Laboratory of Experimental Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America, 2 Pennington Biomedical Research Center, Baton Rouge, Louisiana, United States of America Abstract Calorie restriction (CR) produces several health benefits and increases lifespan in many species. Studies suggest that alternate-day fasting (ADF) and exercise can also provide these benefits. Whether CR results in lifespan extension in humans is not known and a direct investigation is not feasible. However, phenotypes observed in CR animals when compared to ad libitum fed (AL) animals, including increased stress resistance and changes in protein expression, can be simulated in cells cultured with media supplemented with blood serum from CR and AL animals. Two pilot studies were undertaken to examine the effects of ADF and CR on indicators of health and longevity in humans. In this study, we used sera collected from those studies to culture human hepatoma cells and assessed the effects on growth, stress resistance and gene expression. Cells cultured in serum collected at the end of the dieting period were compared to cells cultured in serum collected at baseline (before the dieting period). Cells cultured in serum from ADF participants, showed a 20% increase in Sirt1 protein which correlated with reduced triglyceride levels. ADF serum also induced a 9% decrease in proliferation and a 25% increase in heat resistance. Cells cultured in serum from CR participants induced an increase in Sirt1 protein levels by 17% and a 30% increase in PGC-1a mRNA levels. This first in vitro study utilizing human serum to examine effects on markers of health and longevity in cultured cells resulted in increased stress resistance and an up-regulation of genes proposed to be indicators of increased longevity. The use of this in vitro technique may be helpful for predicting the potential of CR, ADF and other dietary manipulations to affect markers of longevity in humans. Citation: Allard JS, Heilbronn LK, Smith C, Hunt ND, Ingram DK, et al. (2008) In Vitro Cellular Adaptations of Indicators of Longevity in Response to Treatment with Serum Collected from Humans on Calorie Restricted Diets. PLoS ONE 3(9): e3211. doi:10.1371/journal.pone.0003211 Editor: Stefan Wo ¨ lfl, Universita ¨t Heidelberg, Germany Received April 21, 2008; Accepted August 20, 2008; Published September 15, 2008 Copyright: ß 2008 Allard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging, and also in part by research grant U01 AG20478 from the National Institutes of Health. Competing Interests: The authors have declared that no competing interests exist. * E-mail: decabora@grc.nia.nih.gov Introduction Prolonged calorie restriction (CR) produces beneficial health effects in many species including yeast, worms, flies, spiders, rodents, rabbits, dogs, monkeys and humans [1–4]. These benefits include improved insulin sensitivity, increased neuronal function and neurogenesis, enhanced stress resistance, decreased cancer incidence and in many species increased lifespan [5–8]. Some studies have suggested that alternate methods of energy manip- ulation, including alternate day fasting (ADF) and physical exercise, can produce many of the same health improvements observed with CR [9–14]. However, whether all these effects of CR extend to humans remains speculative. Reports on the dietary habits of the long-lived human population residing in Okinawa, Japan, suggest that CR may lead to an increased lifespan [15]. In addition, a two year study on CR in humans [16] reported many changes consistent with those found in lifespan studies of CR in rodents. Other evidence that CR may increase human lifespan can be found in ongoing studies showing that CR improves markers of disease risk and health in rhesus monkeys [17,18]. Unfortunately, direct studies on the effects of CR on human lifespan are not feasible. However, the identification of biomarkers or indicators of increased longevity is a helpful tool in predicting the potential for CR to increase the lifespan and health-span of humans. One proposed indicator of improved health and longevity is increased resistance to heat and oxidative stresses. With aging, there is a decline in tolerance to stressors such as heat and oxyradicals [19,20]. In addition, there is a reduction in the expression of heat shock proteins [19–22]. Many of the beneficial effects of CR have been proposed to be mediated via heat shock proteins. Increased expression of heat shock proteins is related to increased heat tolerance, while a lack of these proteins induces intolerance to exposure to heat stress [23]. CR prevents these age-related declines in tolerance to heat and oxidative stress [20,24,25] and attenuates the reduction in expression of heat shock proteins [21]. Several important signal transduction pathways have been implicated in the regulation of the physiological processes of CR leading to increased lifespan. These pathways include the Sirt1 (the mammalian homolog of Sir2 (silent mating type information regulation 2)) pathway and the PGC-1a (peroxisome proliferator- activated receptor gamma, coactivator 1 alpha) pathway. In rodents, CR increases the expression of Sirt1 and PGC-1a in many tissues [26]. Sirt1 is an NAD(+)-dependent deacetylase which is considered to be important for the lifespan extending PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3211