Calorie Restriction PromotesMammalian Cell Survival byInducing the SIRT1 DeacetylaseHaim Y. Cohen,1Christine Miller,1Kevin J. Bitterman,1Nathan R. Wall,1Brian Hekking,1Benedikt Kessler,1Konrad T. Howitz,2Myriam Gorospe,3Rafael de Cabo,4David A. Sinclair1*A major cause of aging is thought to result from the cumulative effects of cellloss over time. In yeast, caloric restriction (CR) delays aging by activating theSir2 deacetylase. Here we show that expression of mammalian Sir2 (SIRT1) isinduced in CR rats as well as in human cells that are treated with serum fromthese animals. Insulin and insulin-like growth factor 1 (IGF-1) attenuated thisresponse. SIRT1 deacetylates the DNA repair factor Ku70, causing it to se-quester the proapoptotic factor Bax away from mitochondria, thereby inhibitingstress-induced apoptotic cell death. Thus, CR could extend life-span by inducingSIRT1 expression and promoting the long-term survival of irreplaceable cells.In mammals, caloric restriction (CR) delaysthe onset of numerous age-associated dis-eases including cancer, atherosclerosis, anddiabetes, and can greatly increase life-span(1). The molecular mechanisms underlyingthis effect are not known. In the buddingyeast Saccharomyces cerevisiae,CRex-tends life-span by increasing the activity ofthe Sir2 protein (2–5), a member of theconserved sirtuin family of nicotinamideadenine dinucleotide (NAD⫹)– dependentdeacetylases (6). In yeast and Caenorhab-ditis elegans, life-span is extended by thepresence of extra copies of the SIR2/Sir-2.1gene (7, 8), or by small-molecule sirtuinagonists called STACs (9). In mammals, itis becoming increasingly apparent thatSIRT1 is a key regulator of cell defensesand survival in response to stress (10–14).In response to damage or stress, cells at-tempt to repair and defend themselves, but ifunsuccessful, they often undergo pro-grammed cell death, or apoptosis. Numerousstudies show that aging is associated withincreased rates of stress-induced apoptosis(15), and the cumulative effects of cell losshave been implicated in various diseases in-cluding neurodegeneration, retinal degenera-tion, cardiovascular disease, and frailty (15–17). Consistent with this, rodents subjected toCR and long-lived genetic mutants such asthe p66schknockout mouse are typically lessprone to stress-induced apoptosis (18, 19).A critical step in initiating stress-inducedapoptosis is the relocalization of the Baxprotein from the cytoplasm to the outer mi-tochondrial membrane and the subsequentrelease of cytochrome c. Downstream eventsinclude caspase activation and cleavage ofpoly(ADP-ribose) polymerase (PARP). Un-der normal conditions, Bax is rendered inac-tive in the cytoplasm by its tight associationwith Ku70, a DNA repair factor (20, 21). Inresponse to acute cell damage or stress, twocritical lysines in Ku70 (K539, K542) be-come acetylated by the acetyltransferasesCBP and PCAF, and the Ku70-Bax interac-tion is disrupted, allowing Bax to localize tomitochondria and initiate apoptosis (21).Given the role of yeast Sir2 in life-spanextension by CR (2), we investigated whethermammalian SIRT1 might also be involved inthe CR response. Tissues were extracted from12-month-old rats that had either been fed adlibitum (AL) or a CR diet (60% of AL) sinceweaning. Compared to expression in AL an-imals, SIRT1 expression was higher in manytissues of the CR animals, including brain,visceral fat pads, kidney, and liver (Fig. 1A).To investigate whether SIRT1 might beresponsible for the ability of CR to protect thecells of animals from stress-induced apopto-sis, we used an in vitro cell culture model thatrecapitulates key in vivo proliferative andphenotypic features of CR (22). In this sys-tem, cells are cultured in the presence ofserum from calorically restricted rats, result-ing in the induction of characteristic stress-1Department of Pathology, Harvard Medical School,77 Avenue Louis Pasteur, Boston, MA 02115, USA.2BIOMOL Research Laboratories, 5120 Butler Pike,Plymouth Meeting, PA 19462, USA.3Laboratory ofCellular and Molecular Biology,4Laboratory of Exper-imental Gerontology, Post Office Box 12, Gerontolo-gy Research Center, National Institute on Aging, Na-tional Institutes of Health, 5600 Nathan Shock Drive,Baltimore, MD 21224, USA.*To whom correspondence should be addressed. E-mail: [email protected]. 1. Caloric restriction increas-es SIRT1 expression in a varietyof rat tissues and inhibits Bax-mediated apoptosis. (A) Twelve-month-old male Fisher 344 ratswere fed NIH-31 standard feedad libitum (AL) or a daily foodallotment of 60% of that eatenby the AL animals (CR) immedi-ately after weaning. Extracts oftissues (⬃25 ␮g) from AL and CRanimals were separated bySDS–polyacrylamide gel electro-phoresis (SDS-PAGE) and probedwith a rabbit polyclonal antibodyagainst SIRT1, or monoclonal an-tibody against ␤-actin, whichserved as a loading control (B)HEK 293 cells were grown for 48hours in Dulbecco’s minimumessential medium (DMEM) sup-plemented with serum from ALor CR animals. Total protein ex-tracts (50 ␮g) were separated bySDS-PAGE and probed for SIRT1and ␤-actin. (C) FaO cells weregrown in serum from CR animalssupplemented with insulin (ins,2.2 ng/dl) or IGF-1 (429 ng/dl),or both (22). (D) 293T cells weregrown in DMEM containing 10%serum from either AL rats or CRrats. After 24 hours, cells weretransfected with YFP (1 ␮g),YFP-Bax (1 ␮g), or YFP-Bax (1 ␮g) and Ku70 (2 ␮g). The percentage of YFP-positive cells with apoptoticnuclei was scored 24 hours after transfection. Values are the average of three experiments, and ⬎200cells were scored. Error bars represent the SEM, and statistical significance was assessed usinglogistic regression.R EPORTS16 JULY 2004 VOL 305 SCIENCE www.sciencemag.org390response genes and the attenuation of stress-induced apoptosis (22). SIRT1 expressionwas ⬃twofold higher in human embryonickidney (HEK) 293T cells grown in the pres-ence of CR serum as compared to cells grownin AL serum (Fig. 1B).In worms, flies, and mice, mutations inthe insulin/insulin-like growth factor 1(IGF-1) pathway can extend life-span (6,23). The levels of insulin and IGF-1 are 8-and 1.4-fold lower, respectively, in CR ro-dents as compared to AL rodents (22). Todetermine whether the induction of SIRT1expression by CR serum was due, in part, toreduced levels of these factors, FaO rathepatoma and HEK 293 cells were treatedwith CR serum supplemented with insulinor IGF-1, or both.