In accordance with previous studies (16–18, 22), remark- ably low IR as well as BMI were found in our sample of long-lived people, confirming that these individuals show metabolic characteristics different from those in the rest of aging population. To date no genetics have been found that can be associated with these parameters (18); it cannot be excluded that BMI and IR (HOMA) as well as plasma IGF-I (whose variance was explained by only 13% by the variables used in this report) could be influenced by the peculiar nu- tritional regimen of these long-lived people, which could, in fact, share some similarities with moderate caloric restriction. In this regard, the preserved insulin pathway efficiency ob- served in long-lived subjects might be indirectly due to an enhanced tissue response to IGF-I secondary to an increase in IGF-I receptor number/activity (30). Our previous study (14) demonstrated that centenarians had a plasma IGF-I/ IGFBP-3 molar ratio greater than that of aged subjects. This finding appears to contradict the present study, because the greater molar ratio was due to the lower IGFBP-3 present in long-lived subjects rather than to higher IGF-I plasma levels (no significant difference in total plasma IGF-I levels was found among long-lived and aged subjects), thus suggesting that in centenarians the bioavailability of IGF-I is enhanced despite its lower levels. The significance of plasma IGF-I remains to be established. It could be hypothesized that the decrease in plasma IGF-I observed in long-lived subjects might minimize the generalized mitogenic stimulus to tis- sues and contribute to the reduction in age-related pathol- ogies due to the effect of IGF-I on cellular replication. Such

Discussion

The major findings of this investigation are the following: 1) IGF-I plasma levels, which decrease significantly with age, are affected by the polymorphisms at IGF-IR and PI3CKB genes, both alone and in combination. In particular, indi- viduals bearing at least one allele A at the IGF-IR locus wee\ +) (IGF-IR A ) have lower plasma IGF-I levels than the rest of the population. 2) IGF-IR A(cid:5) subjects are found in increased proportion in long-lived individuals. 3) Genotype combina- tions of an A allele at the IGF-IR locus and a T allele at the PI3CKB locus (A(cid:5) /T(cid:5) subjects) affect IGF-I plasma levels (having A(cid:4) /T(cid:4) individuals the highest free IGF-I plasma levels), as well as longevity, and the proportion of A(cid:5) /T(cid:5) subjects significantly increased among long-lived individuals.

Collectively, these data suggest that genetic variants of genes involved in the intracellular response to IGF-I (IGF-IR and PI3CKB) jointly affect two phenotypes: free IGF-I plasma levels and longevity. The fact that the two traits are coregu- lated by overlapping sets of genes is in line with previous findings on the genetics of longevity in animal models. In- deed, in both invertebrates (C. elegans) and mammals (Mus musculus), the down-regulation of the IGF-I pathway or IGF-I plasma levels is associated with an extension of the life span (9–12, 27), whereas animals with elevated plasma IGF-I lev- els display a shortened life span (13). Indeed, in humans there

TABLE 3. Allele and genotype distribution of different gene polymorphisms involved in insulin signaling pathway

Young Long lived IGF1R A/G polymorphism (n   248) (n   162) G/G 0.399 (n are 99) 0.296 (n  48) (cid:1)2   6.04; P  0.04 A/G 0.407 (n   101) 0.426 (n   69) A/A 0.194 (n  148) 0.278 (n  45) Allele frequency g 0.603 0.509 a 0.397 0.491 A(cid:5) subjects 0.601 (n   149) 0.704 (n   114) Fisher’s test: P  0.03 IRS-1 G/A polymorphism (n ~ ia 197) (n   153) G/G 0.858 (n +q 169) 0.862 (n  132) G/A 0.137 (n  27) 0.131 (n  20) A/A 0.005 (n  1) 0.007 (n  1) Allele frequency g 0.926 0.928 a 0.074 0.072 A(cid:5) subjects 0.142 (n  28) 0.137 (n  21) PI3KCB T/C polymorphism (n  245) (n   179) T/T 0.351 (n  86) 0.335 (n  60) T/C 0.384 (n  94) 0.447 (n  80) C/C 0.265 (n   65) 0.218 (n  39) Allele frequency t 0.543 0.559 c 0.457 0.441 C(cid:5) subjects 0.649 (n   159) 0.665 (n   119) FOXO1A T/C polymorphism (n   198) (n   153) T/T 0.934 (n avy 185) 0.948 (n  145) T/C 0.061 (n  12) 0.052 (n  8) CC 0.005 (n  1) – Allele frequency t 0.965 0.974 c 0.035 0.026 C(cid:5) subjects 0.066 (n   13) 0.052 (n   8)

When not reported, (cid:1)2 and Fisher’s tests were not significant (P  0.05).

the insulin/IGF-I signaling response pathway plays a role in human longevity, indicating that the impact of these genes on species longevity is an evolutionarily conserved property. We hope that our data will be the starting point for future longitudinal studies, including the insulin response pathway genes as a possible trigger for longevity.

a hormone is also linked to the development of several dis- eases, such as cancer (31, 32), but the local expression (bio- availability) of IGF-I may be an important factor contributing to the maintenance of normal tissue function.

To our knowledge the results reported here are the first findings supporting the hypothesis that genetic variability in the genes responsible for IGF-I regulation plays a role in human longevity. The ortholog of human IGF-IR, DAF-2, is one of the best characterized longevity genes in C. elegans (1–3). It has been proposed that DAF-2 mutations lead to the up-regulation of genes involved in heat stress, UV, and an- tioxidant responses, i.e. pathways that are expected to affect the life span in all species (33). Furthermore, it has been recently shown that IGF-IR inactivation prologs the life span in mice and increases their resistance to oxidative stress (34). However, the functional effects of the structural variation in IGF-IR due to the polymorphism studied here are not known.

Acknowledgments