plasma insulin levels as well as CHD, cancer, and Type 2 diabetes prevalence. This is consistent with a known role for FOXO as a mediator of the effects of insulin and insulin-like growth factors on diverse physiological functions, including cell proliferation, apopto- sis, and metabolism (17, 54). Genetic studies in C. elegans and Drosophila have shown that FOXO proteins are ancient targets of insulin-like signaling that regulate metabolism and longevity. Ad- ditional work in mammalian cells has shown that FOXO proteins are the targets of protein kinases, influence cell cycle progression, and regulate resistance to oxidative stress in vitro (54). In vivo studies have shown that FOXO modifies hepatic glucose output in response to insulin and mediates other metabolic actions (54). This strengthens the evidence that FOXO proteins may mediate insulin effects on metabolism and influence longevity in humans.

Table 6. Prevalence of aging-related phenotypes in relation to FOXO3A3 genotype

FOXO3A in human health, aging, and longevity. The association of FOXO with diverse aging phenotypes, including insulin sensitivity, CHD, cancer, type 2 diabetes, and longevity, is suggestive of a ‘‘gatekeeper’’ role in the IIS pathway. An important downstream mechanism whereby FOXO3A might influence human aging is through modification of oxidative stress—a long held theory of how we age (33), although we have no direct evidence for this in the current study. However, since FOXO genes are the closest human homologues of C. elegans DAF-16, which protects cells from oxidative stress, this is a plausible mechanism of action for modi- fication of human aging (17). In C. elegans, DAF-16 increases the expression of manganese superoxide dismutase (SOD2), which converts superoxide to less damaging hydrogen peroxide and is a potent endogenous protector against free radicals (55), among other ‘‘anti-aging’’ effects. In vivo studies show that oxidative lesions in DNA, proteins, and other tissues accumulate with age and feeding calorically restricted diets (a potent insulin sensitizer) to rodents (56) and humans (57) mitigates this damage.

TT TG GG P CHD prevalence, % Average-lived 32.3 18.3 23.1 0.010 Long-lived 7.4 7.6 7.7 0.998 All 25.7 14.0 15.4 0.002 Stroke prevalence, % Average-lived 6.7 8.5 7.7 0.813 Long-lived 4.9 1.9 3.8 0.510 All 6.3 5.8 5.8 0.974 Cancer prevalence, % Average-lived 22.4 18.3 11.5 0.326 Long-lived 17.3 12.4 7.7 0.400 All 21.1 15.9 9.6 0.075 Diabetes prevalence, % Average-Lived 60.6 62.3 50.0 0.498 Long-Lived 57.5 64.1 50.0 0.368 All 59.8 63.0 50.0 0.212

While FOXO was clearly associated with longevity we did not observe a strong effect of genotype on insulin sensitivity in cases— onlyincontrols.However,theGGgenotypedemonstratedsimilarly

P values based on (cid:1)2 test comparing frequency of GG genotype to other genotypesforaveragelivedcontrols(n(cid:1)402),longlivedcases(n(cid:1)213),and all subjects (n   615).

adjacent SNPs in the FOXO3A gene. Third, the findings are biologically plausible and support the prior findings in animal models and also support the limited prior human studies. Fourth, the case-control associations with longevity were detected using a nested case-control analysis with a high event rate (deaths) during a long period of follow-up. Fifth, the HHP cohort is highly homogenous and no population stratification was detected.

a modulating effect of genotype on insulin levels in both groups. It is tempting to speculate that since the cases showed greater insulin sensitivity no matter what their genotype, they have multiple mechanisms to maintain insulin sensitivity other than FOXO. This would be consistent with the hypothesis that most longevity genes havemodestorsmalleffectsizes.Itisalsopossiblethatsmallsample size limited our ability to detect differences in the cases. On the other hand, long-lived mice carrying mutations in either IRS-1 (58) or IRS-2 (23) are actually insulin resistant, so insulin sensitivity is not a necessary condition for mutations in the IIS pathway to be able to confer greater longevity.

A possible drawback is that since the cases and controls had an average age difference of 11 years we cannot exclude birth cohort as a confounder. But this is unlikely since there was a maximum 19-year difference in birth years between participants. Also, suba- nalyses revealed no differences in education and occupation (data not shown) between cases and controls. Moreover, it was the participants who were older at baseline who were more likely to have lived to 95-plus years and thus obtain the longevity phenotype. Most cohort effects show health advantages for younger cohorts.

However, it is interesting to note that in C. elegans, several genes that by themselves may have small effects on lifespan are influenced by the transcription regulating ‘‘master gene’’ DAF-16 (59). Small differences in FOXO3A that may be otherwise difficult to detect, could theoretically modify several downstream genes related to DNA binding, protein–protein interactions, cell cycle progression, apoptosis, and metabolism. In this manner, a small modifying effect by FOXO3A potentially has larger, additive downstream effects on aging phenotypes and longevity.

In summary, we found that common, natural genetic variation within the FOXO3A gene was strongly associated with human longevity and was also associated with several phenotypes of healthy aging. Further study of FOXO genes and aging phenotypes is warranted in other populations.