Sweet longevity

September 13, 2008

How sweet is longevity. Insulin signaling's importance in human aging seems to be supported. The pdf-availed (3) below seems to add meat to the previous (1) report using human colon cancer HCT116 cells.

1. s KM, Pennington JD, Bisht KS, Aykin-Burns N, Kim HS, Mishra M, Sun L, Nguyen P, Ahn BH, Leclerc J, Deng CX, Spitz DR, Gius D.SIRT3 interacts with the daf-16 homolog FOXO3a in the Mitochondria, as well as increases FOXO3a Dependent Gene expression.Int J Biol Sci. 2008 Sep 5;4(5):291-9.PMID: 18781224

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis

of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.

2. Research Highlights[No authors listed]Genetics: Sweet longevity.Nature. 2008 Sep 11;455(7210):141. No abstract available. PMID: 18784675

Variations in a gene that mediates responses to insulin are associated with longevity in humans, researchers have found.

Bradley Willcox of the Pacific Health Research Institute in Honolulu, Hawaii, and his colleagues looked for links between longevity and variations in five genes involved in insulin signalling and which had previously been suggested to have a link with ageing. The researchers used samples from more than 600 Japanese-American men: 213 who had lived to at least 95 years of age, and 402 who had died before the age of 81.

Variation within one of the genes, FOXO3A, was associated with longevity. Those with two copies of a particular version of the gene reported fewer health problems and were nearly three times more likely than those with just one copy to live to the age of 98.

3. Genetics:Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, Masaki KH, Willcox DC, B, Curb JD.FOXO3A genotype is strongly associated with human longevity.Proc Natl Acad Sci U S A. 2008 Sep 2. [Epub ahead of print]PMID: 18765803

Human longevity is a complex phenotype with a significant familial component, yet little is known about its genetic antecedents. Increasing evidence from animal models suggests that the insulin/IGF-1 signaling (IIS) pathway is an important, evolutionarily conserved biological pathway that influences aging and longevity. However, to date human data have been scarce. Studies have been hampered by small sample sizes, lack of precise phenotyping, and population stratification, among other challenges.

Therefore, to more precisely assess potential genetic contributions to human longevity from genes linked to IIS signaling, we chose a large, homogeneous, long-lived population of men well-characterized for aging phenotypes, and we performed a nested-case control study of 5 candidate longevity genes.

Genetic variation within the FOXO3A gene was strongly associated with human longevity. The OR for homozygous minor vs. homozygous major alleles between the cases and controls was 2.75 (P = 0.00009; adjusted P = 0.00135). Long-lived men also presented several additional phenotypes linked to healthy aging, including lower prevalence of cancer and cardiovascular disease, better self-reported health, and high physical and cognitive function, despite significantly older ages than controls. Several of these aging phenotypes were associated with FOXO3A genotype. Long-lived men also exhibited several biological markers indicative of greater insulin sensitivity and this was associated with homozygosity for the FOXO3A GG genotype.

Further exploration of the FOXO3A gene, human longevity and other aging phenotypes is warranted in other populations.

Human longevity is a complex phenotype with multiple determinants.While non-genetic factors, including diet, physicalactivity, health habits, and psychosocial factors are important, up to50% of the variation in human lifespan might be explained bygenetic differences (1-5). Several studies suggest that about 25% ofthe variation in human lifespan in average-lived populations can beexplained by genetic factors, but in populations with larger numbersof exceptional survivors, the genetic contribution to lifespan may bemuch higher. For example, family studies of nonagenarians andcentenarians show that sibling relative risk, a common method forassessing potential genetic contribution to a complex phenotype (6),is particularly high and grows with increasing age of the proband(7-10). However, studies of candidate ‘‘longevity-associated’’ genesin humans, hereafter referred to as ‘‘longevity

genes,’’ have generallybeen disappointing. Few replications have been observedacross populations, with the exception of the APOE gene (3).

In contrast, there have been several robust genetic findings inmodel organisms of aging (11-13). For example, variation in singlegenes can result in substantial differences in lifespan in modelorganisms, particularly with genes that are considered part of theinsulin/IGF-1 (IIS) signaling pathway (14-18).

Mutations that increase SIR-2 activity or that decrease insulin/IGF-1 signaling both increase the lifespan of C. elegans by activatingthe DAF-16/FOXO protein (19, 20). In mammalian cells, a Sir2homolog ‘‘SIRT1,’’ influences several downstream transcriptionevents affecting lifespan, including the cellular response to stress.SIRT1 accomplishes this by regulating the FOXO (Forkhead boxtranscription) factors, a family of proteins that function as sensorsin the IIS pathway and influence mammalian longevity (17).Genetic knock-out models in mammals (and other species) havealso supported the IIS hypothesis. For example, mice with afat-specific insulin receptor knockout (FIRKO) have reduced fatmass, protected against age-related obesity, and have extendedlongevity (21). Many other mutations in the IIS pathway appear toimpact longevity in mice. These include mutations in the IGF-1receptor (22),

IRS-1 (22), IRS-2(23), PAPP-A (24), and the AmesDwarf mouse mutation (22).

The basic molecular pathway of insulin signaling is conservedthrough evolution, evidence of which can be seen in yeast, flies,worms, rodents, and humans (25). A key regulator of this pathwayin worms is the transcription factor DAF-16 (abnormal DAuerFormation-16), which is required for the large lifespan extensionproduced in C. elegans by inhibiting insulin/IGF-1 signaling (16). Anumber of factors appear to extend lifespan in C. elegans in aDAF-16 dependent manner, such as AMP kinase (26), 14-3-3proteins (27), the lin-4 microRNA (28), and heat shock factor (29).Homologues of DAF-16 in several species have been linked to agingphenotypes and longevity (30). For example, the stress responsiveJun-N-K terminal kinase (JNK) pathway appears to require FOXOto prolong lifespan in Drosophila (31), and when flies over expressdFOXO, the DAF-16 ortholog, it can increase lifespan (32). Theconvergence of such

a diverse array of signals on DAF-16/FOXOsuggests that this protein may be an important, evolutionarilyconserved ‘‘node’’ in a signaling network that impacts aging andlongevity.

The human homologue of DAF-16 includes four FOXOs:FOXO1, FOXO3, FOXO4 and FOXO6. We hypothesize thatcommon, natural variation in the form of single nucleotide polymorphisms(SNPs) in FOXO and related genes might influencehuman aging and longevity. A connection between insulin, FOXO,oxidative stress, and human longevity would be particularly interestingsince oxidative stress has long been a favorite putativemechanism of aging. Since 1956, the free radical theory of aging hashypothesized that aging results partly from damage to DNA, cells,and tissues from cumulative exposure to reactive oxygen molecules(33) and although not yet universally accepted, supportive evidencehas accumulated over the years (34, 35). Thus, FOXO may providea potential forkhead or bridge between insulin signaling, freeradicals, and human aging/longevity.

There has been some prior work linking genes in the IIS pathwayto human longevity (36, 37) including an interesting recent reportby Suh et al. (38), which links functionally significant IGF-1 receptormutations to exceptional longevity, but we have not found anypublished reports of association between FOXO genes and humanlongevity. Prior studies have found links between FOXO genes andother aging phenotypes, including 4-year survival and stroke risk(39) as well as premature menopause (40).

Human longevity, however, is a complex phenotype that encompassesdisease-specific risks as well as the individual rate of aging.The study of its genetic antecedents is challenging. The study oflongevity may be affected by small genetic effect sizes, populationstratification artifact, population heterogeneity, lack of sufficientnumbers of long-lived study participants, and other problems (3, 4,41). Therefore, to assess potential genetic contributions to humanlongevity from genes linked to IIS signaling, we chose a large,homogeneous, long-lived population of men well-characterized foraging phenotypes, and we performed a nested-case control study of5 candidate longevity genes with links to the IIS pathway. Thesegenes were chosen based on prior associations with aging phenotypesprincipally from gene knockout, transgenic, mutant, andother model organisms (3, 4, 14-17, 36, 42). Priority was for

genesinvolving insulin sensing and glucose (energy) homeostasis.

Results

The baseline characteristics of the HHP/HAAS study population atthe 1991-1993 examination are presented in supporting information(SI) Table S1. The mean age was 77.9 years and 100% of thepopulation was male and of Japanese ethnicity. Biological characteristics,general health status, disease prevalence, and functionalstatus are presented.

From this 1991-1993 baseline population, we selected all participantswho, by 2007, had survived to age 95 years or more as‘‘longevity’’ cases (n=213). We then selected all participants whodied before the age of 81 years as ‘‘average-lived’’ controls (n=402). Baseline characteristics of the cases and controls are presentedin Table 1. In terms of biological characteristics, the longlivedcases were older, leaner (lower waist:hip ratio), had lowertriglycerides (borderline), lower glucose, lower insulin levels, andhigher prevalence of the FOXO3A3 allele at the baseline examination.The cases also had better self-rated health and lowerprevalence of cardiovascular disease [coronary heart disease(CHD) and stroke] and cancer. Functionally, they appeared betterable to walk but had lower grip strength. There was no differencein cognitive score (43).

Table 1. Baseline characteristics by case-control status.=========================================================Variables at baseline examination (1991-1993)---Average lived phenotype (mean attained age 78.5 years) (n=402)---Longevity phenotype (mean attained age 97.9 years)* (n=213)--- ---Mean±SD Min-Max Mean±SD Min-Max---P¶=========================================================Biological, fasting valuesAge at baseline exam, years 74.63±2.05 71-79 85.62±3.12 80-93 <.0001Body mass index, kg/m2 23.4±3.17 15.89-32.33 23.0±2.91 15.4-31.1 0.1272Waist/hip ratio 0.95±0.06 0.78-1.15 0.93±0.06 0.73-1.07 0.0008Total cholesterol, mg/dl 187.96±34.6 98-303

185.36±32.16 95-304 0.3680HDL, mg/dl 50.82±14.17 21-129 51.29±13.54 27-100 0.6911Triglycerides, mg/dl 154.72±118.72 46-1369 140.32±82.23 38-649 0.1178Log triglycerides¶¶ 4.88±0.51 3.83-7.22 4.81±0.50 3.64-6.48 0.0965Glucose, mg/dl 117.83±35.9 69-323 108.98±22.55 77-298 0.0012Insulin, mIU/liter 25.54±82.89 3.3-1164 13.8±11.39 1.5-104 0.0421Log Insulin¶¶ 2.69±0.74 1.19-7.06 2.44±0.58 0.41-4.64 <0.0001FOXO3A3 MAF (rs2802292)§ 0.255 - 0.371 - <0.0001----------------------------------------------------General Health StatusSelf-rated "poor" health, % 41.92 - 31.07 - 0.0163Disease prevalenceCHD, % 26.37 - 7.55 - <0.0001Stroke, % 7.46 - 3.3 - 0.0394Cancer, % 20.15 - 13.68 - 0.0468Diabetes, % 60.55 - 59.81 - 0.8587Physical/cognitive functionLower body (difficulty walking), % 30.59 - 16.83 - 0.0002Upper body (grip strength), kg 29.85±7.54 0-47 26.37±5.53 8-45

<0.0001Cognitive score (CASI)§ 80.96±19.48 0-100 78.54±13.85 12-98 0.1088========================================================= * Cases (longevity phenotype) consisted of all HHP/HAAS participants with DNA samples (living and dead) who had reached the age of 95 years by Aug. 2007: Gp 1: Alive, n=37, mean age 98.7, range 97-106 years; Gp 2: dead, n=166, mean death age 97.5, range 95-106 y). ¶ P value from Student’s t test for continuous variables and chi^2 for categorical variables. ¶¶ Log transformation performed for variables not normally distributed. § MAF, minor allele frequency; CASI, cognitive abilities screening instrument.

Five genes were investigated (ADIPOQ, FOXO1A, FOXO3A,SIRT1, and COQ7). Minor allele frequencies and other relatedgenetic information for the cases and controls are presented inTable 2. However, only FOXO3A genotype was associated withlongevity using an initial cut-off value of P <0.05.

Table 2. Candidate genes for human longevity and the MAF in cases and controls.===============================================Gene name Symbol SNP ID Variable name---MAF--- ---Cases Controls---P*===============================================Adipo,¶ C1Q, CDC ADIPOQ rs1063539 ADIPOQ_1 0.297 0.263 0.20rs182052 ADIPOQ_2 0.455 0.493 0.22rs266729 ADIPOQ_3 0.195 0.239 0.08Forkhead Box O1A FOXO1A rs2755209 FOXO1A1 0.272 0.291 0.48rs2721069 FOXO1A2 0.293 0.307 0.62rs2755213 FOXO1A3 0.350 0.358 0.77Forkhead Box O3A FOXO3A rs2764264 FOXO3A1 0.347 0.248 0.0002rs13217795 FOXO3A2 0.340 0.248 0.0006rs2802292 FOXO3A3 0.371 0.255 <0.0001Sirtuin 1 SIRT1 rs7069102 SIRT1_1 0.185 0.181

0.84rs10823112 SIRT1_2 0.337 0.360 0.44rs1885472 SIRT1_3 0.188 0.179 0.71Coenzyme Q7 COQ7 rs8051232 COQ7_1 0.147 0.150 0.90rs11074359 COQ7_2 0.153 0.171 0.43rs7192898 COQ7_3 0.162 0.170 0.73=============================================== * Comparing MAF between cases and controls with chi^2 test. ¶ Adipocyte, C1Q, and collagen domain containing

Further investigation comparing the genotype frequencies ofFOXO3A3 between cases and controls revealed a highly significantdifference P=0.00009 for the Pearson’s exact chi^2 statistic (Table 3).Five loci with 3 SNPs within each allele were tested (Table 2).Bonferroni adjustment for multiple comparisons resulted in acorrected p value of 15 0.00009=0.00135. Due to the high LDbetween the 3 SNPs of FOXO3A, we further investigated theFOXO3A3 SNP only (rs 2802292). The OR for homozygous minorvs. homozygous major alleles for FOXO3A3 between the cases andcontrols was 2.75 (95% CI: 1.51 5.02, P=0.0007), and the ORfor heterozygous vs. homozygous major alleles between the casesand controls was 1.91 (95% CI: 1.34 2.72, P=0.0003). Theseresults suggest an additive effect on longevity.

Table 3. FOXO3A3 genotype by case-control status.====================================================Case-Control Status---FOXO 3A3 Genotype (rs 2802292) ---TT TG GG====================================================Average-Lived Phenotype* 223 (55%) 153 (38%) 26 (6%)Longevity Phenotype¶ 81 (38%) 106 (50%) 26 (12%)p value for Pearson Exact test¶¶ 0.000091p value after Bonferroni adjustment 0.00135==================================================== *Number and percentage of subjects from n=402 "average-lived" decreased controls (mean attained age 78.5 years). ¶ Number and percent of subjects from n=213 "long-lived" cases (mean attained age 97.9 years). ¶¶ From the exact Pearson chi^2 test comparing the genotype frequencies in the cases and

controls.

To understand more about the longevity phenotype at youngerages, we compared the proportion of people who were healthy atthe baseline examination (1991-1993) for each of the threeFOXO3A genotype groups using the definition of healthy survivalfrom Willcox et al. (44). The differences were highly significant(Table 4). Those who possessed one or more G alleles were morelikely to be healthy at baseline than those who were homozygous forthe major (TT) allele; 75% of those homozygous for the minorallele were healthy at the baseline examination vs. 57% of thosehomozygous for the major allele. After adjusting for case-controlstatus, the differences were still marginally significant. This suggestsremaining association of the allele with health status in cases andcontrols.

Table 4. Difference in health status between genotype groups at baseline.======================================== Healthy at baseline,* %===P value for trend Homo. Major---Heter.---Homo. Minor===Unadjusted Adj. for Case-Control Stat========================================FOXO3A1 57.41 69.48 75.51 0.01 0.065FOXO3A2 57.37 69.35 77.08 0.01 0.035FOXO3A3 57.89 68.34 75.00 0.02 0.097======================================== *"Healthy" is defined as absence of 6 major chronic diseases (CHD, stroke, cancer, PD, COPD and treated type 2 diabetes; high physical function (can walk one-half mile) and high cognitive function (CASI score >74).

To assess whether there was a relation between insulin sensitivity,a potential intermediate phenotype of longevity, and genotype, wetested the relation between fasting insulin, glucose, HOMA andgenotype (Table 5). For non-normally distributed variables we usedlog conversion to a normal distribution. There was a significantrelation between insulin, log insulin, HOMA and genotype. Homozygosityfor the G allele was associated with markedly lowerinsulin, log insulin and HOMA score, but in controls only.

Table 5. Insulin sensitivity phenotypes according to FOXO3A genotype.================================================== FOXO3A genotype (rs 2802292)--- TT TG GG---P*==================================================Fasting glucose, mg/dlAverage-lived 118.4±34.0 117.4±38.0 115.9±40.1 0.80Long-lived 108.3±20.7 109.1±23.7 110.5±24.1 0.73Fasting insulin, mIU/literAverage-lived 23.7±81.2 30.4±91.9 13.2±5.9 0.004Long-lived 13.5±9.0 14.1±13.4 13.3±9.3 0.77Log fasting insulin, mIU/literAverage-lived 2.68±0.67 2.73±0.85 2.47±0.48 0.03Long-lived 2.45±0.55 2.43±0.61 2.44±0.52 0.99HOMA IR ScoreAverage-lived 9.1±53.0 10.0±32.2 3.8±2.4 0.03Long-lived 3.7±2.8 4.0±4.3 3.6±2.2 0.55================================================== * P value for Student’s t test comparing mean values

between GG genotype and other groups within cases and controls.

We also tested for a relation between lifetime prevalence ofseveral chronic diseases and FOXO3A genotype (Table 6). Asignificant protective relation was found for homozygosity for theGallele with regard to prevalence of CHD and a borderline relationfor cancer. Finally, we assessed the FOXO3A3MAF distribution bymaximum attained age in all participants combined. The MAFincreased markedly with age (Table 7).

Table 6. Prevalence of aging-related phenotypes in relation to FOXO3A3 genotype.==================================== FOXO3A3 Genotype TT TG GG P====================================CHD prevalence, %Average-lived 32.3 18.3 23.1 0.010Long-lived 7.4 7.6 7.7 0.998All 25.7 14.0 15.4 0.002Stroke prevalence, %Average-lived 6.7 8.5 7.7 0.813Long-lived 4.9 1.9 3.8 0.510All 6.3 5.8 5.8 0.974Cancer prevalence, %Average-lived 22.4 18.3 11.5 0.326Long-lived 17.3 12.4 7.7 0.400All 21.1 15.9 9.6 0.075Diabetes prevalence, %Average-Lived 60.6 62.3 50.0 0.498Long-Lived 57.5 64.1 50.0 0.368All 59.8 63.0 50.0 0.212==================================== P values based on chi^2 test comparing frequency of GG genotype to other genotypes for average lived controls (n=402), long lived cases (n=213), and all

subjects (n=615).

Table 7. Genotype distribution by maximum attained age.===================================Age at death (years)* n MAF of FOXO3A3===================================72-74 17 0.2175-79 277 0.2580-81 108 0.2695-99 185 0.37100-106 28 0.39=================================== * Thirty-seven cases were still alive; mean age 98.7 years (range 97-106).

Discussion

The rapid aging of the population will place unprecedented challengeson society due to increased prevalence of chronic disease anddisability (45). Better understanding of mechanisms of aging,including biological pathways that may have widespread influenceon how we age, could have important implications for lowering ourrisk for age-related disease and disability. There are many biologicallyplausible candidate genes for human longevity but only onefinding has so far been widely replicated in multiple populations,that of the APOE gene (3). This gene has widespread effects onaging phenotypes, particularly cardiovascular disease and dementia,and as such influences the ability to achieve a long and healthy life.

To find other such genes, it may be helpful to use modelorganisms to identify a priori potential candidates before conducting human studies. Therefore, we chose to study several candidate genes within the human insulin/IGF-1 signaling pathway and/or oxidative stress response system on the basis of sequence and/or functional homology with model organisms of aging or prior human studies.We constructed a list of human candidate genes (Table S2) from these signaling pathways and assessed variations in these genes occurring at a frequency of <10% in the Japanese population. Three SNPs were chosen from each gene for analysis. SNPs were selected mainly from regions with linkage disequilibrium (LD) for maximal coverage of each gene.

Analysis of five candidate genes demonstrated that one gene clearly stood out from the others in terms of a potential human longevity gene-FOXO3A. That this gene might be important to human longevity is supported by several lines of evidence. First, in nested case-control analyses, variation within this gene was strongly associated with longevity. Furthermore, two copies of the G allele conferred about twice the protective effect (suggesting an additive effect), roughly tripling the odds of living close to a century. The minor allele frequency also rose markedly from septuagenarian to centenarian ages (Table 7).

Second, all three SNPs that we assessed in the FOXO3A gene, which were in tight LD, were strongly correlated with the longevity phenotype. This indicates that the finding was unlikely to be due to chance. Third, carriers of the minor (G) alleles were healthier at the baseline examination, 15 years prior (Table 4).

In fact, the baseline examination suggested that cases were markedly healthier than controls despite the fact that cases were, on average, 11 years older. The cases possessed significantly less age-related disease, including less prevalent CHD, stroke, and cancer. They also had better self-rated health and generally had high physical function, including less difficulty walking. Interestingly, despite being more than a decade older than controls, the longevity cases had similar levels of cognitive function. This supports the existence of a ‘‘healthy aging’’ phenotype where individuals somehow delay or avoid major clinical disease and disability until late in life. The healthy aging phenotype that we observed in cases is similar to the healthy aging phenotypes reported in centenarians at younger ages when compared to their age-matched birth cohorts (46-48) and in centenarian offspring (49). Long-lived cases also had metabolic profiles

suggesting higher insulin sensitivity at younger ages, with lower waist to hip ratio, lower glucose, insulin, and HOMA values (Tables 1 and 5). Several phenotypes were associated with variation in FOXO3A genotype.

Surprisingly, there was no significant difference in diabetes prevalence between cases and controls. However, since the cases were more than a decade older than controls, and diabetes tends to increase markedly with age, it is noteworthy that prevalence of diabetes was not significantly different. In fact, both cases and controls had a high prevalence of diabetes (near 60%), despite relatively low BMI. Why Type 2 diabetes tends to be more prevalent in Japanese at a relatively low BMI is not completely understood (50). However, there may be metabolic differences in Japanese (and some other Asians) with higher visceral fat in Asians at lower BMI than in whites and blacks (51, 52). Indeed, Japan national guidelines reflect such population differences and consider Japanese obese at a BMI of 25 (53). Other contributing factors to the high prevalence of diabetes in the HHP/HAAS cohort are that participants were tested for diabetes by several clinical

methods and at several examinations making detection more likely.

Of note, the FOXO3A genotype was significantly associated with 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, apoptosis, 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. Additional 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.

Overall, the totality of the evidence supports a potential role of 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 modification 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.

While FOXO was clearly associated with longevity we did not observe a strong effect of genotype on insulin sensitivity in cases-only in controls. However, theGGgenotype demonstrated similarly low plasma insulin levels in both cases and controls, consistent with 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 have modest or small effect sizes. It is also possible that small sample 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.

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.

Supportive evidence is beginning to accumulate for a role of insulin-signaling in human aging and longevity, but the genes that might mediate these effects are not known. Prior studies have found over or under representation of single nucleotide polymorphisms (SNPs) from the insulin-IGF-1signaling pathway in long-lived humans of Italian (36), Japanese (37, 42), Dutch (60), and Ashkenazi Jewish (38) ethnicity, with links to several aging phenotypes. While some of these findings have been limited by small effect sizes and marginal statistical significance, the study by Suh et al. (38) also demonstrated that functionally significant mutations in the IGF-1 receptor exist in some long-lived humans, such as centenarians.

To date, there has been little study of FOXO genes and phenotypes of aging in humans. Two recent studies suggest that FOXO genes deserve further scrutiny. First, a longitudinal study of elderly Dutch men and women found that a FOXO1A haplotype predicted 4-year survival and that a FOXO3A haplotype predicted stroke risk (39). Second, the Framingham Study, in a genome-wide association analysis, found that a FOXO3A SNP was strongly associated with age at natural menopause in women (P=0.00003). However, the Dutch findings were not statistically significant when accounting for multiple comparisons and both studies need replication. The present study is supportive and extends the associations of FOXO3A to human longevity and insulin sensitivity.

One of the major advantages of the current study is that it used a nested case-control design. This study design selects cases and controls from an ongoing cohort study with longitudinally collected data. Therefore, several phenotypes of interest (e.g., disease prevalence, health status, function) were obtained by direct clinical examination when the participants were younger, making the data less subject to recall bias.

Indeed, studies of exceptional survivors, such as centenarians, that have found evidence for phenotypes suggestive of slower aging (46-48) could potentially suffer from significant recall bias. That is, older participants may not recall precisely their past medical history and their past functional status. However, in the current study, major diseases were adjudicated by a morbidity and mortality committee and performance-based measures of physical and cognitive function were used to supplement self-reports, and evidence was found for such a healthy aging phenotype. This lends prospective support to previous retrospective work.

There are several other strengths to this study. First, the candidate genes selected for analysis were chosen a priori based on hypothesis-driven criteria. That is, studies of models organisms of aging employing various methods, particularly knockouts, have shown that the IIS pathway is important for aging and longevity. And many functions appear to be evolutionarily conserved. Second, the findings are strong, highly significant, and include several 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 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, sub-analyses 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. 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.

Subjects and Methods

Study Population.

This nested-case control study was conducted as part of the Hawaii Lifespan Study, an embedded cohort study of healthy aging drawn from the original population of the Honolulu Heart Program (HHP) and Honolulu Asia Aging Study (HAAS). The HHP is a population-based, prospective study of cardiovascular disease among 8006 Japanese Americanmen that began in 1965. The HHP participants were recruited from 9877 men with valid contact information who were born from 1900-1919 and living on the island of Oahu (61).

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-- Al Pater, alpater@...

2. Research Highlights[No authors listed]Genetics: Sweet longevity.Nature. 2008 Sep 11;455(7210):141. No abstract available. PMID: 18784675

Variations in a gene that mediates responses to insulin are associated with longevity in humans, researchers have found.

Bradley Willcox of the Pacific Health Research Institute in Honolulu, Hawaii, and his colleagues looked for links between longevity and variations in five genes involved in insulin signalling and which had previously been suggested to have a link with ageing. The researchers used samples from more than 600 Japanese-American men: 213 who had lived to at least 95 years of age, and 402 who had died before the age of 81.

Variation within one of the genes, FOXO3A, was associated with longevity. Those with two copies of a particular version of the gene reported fewer health problems and were nearly three times more likely than those with just one copy to live to the age of 98.

2. Genetics:Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, Masaki KH, Willcox DC, B, Curb JD.FOXO3A genotype is strongly associated with human longevity.Proc Natl Acad Sci U S A. 2008 Sep 2. [Epub ahead of print]PMID: 18765803