Results In the entire population, free IGF-I plasma levels displayed an age-related decrease (Fig. 1) through all ages and were negatively correlated with the degree of IR (HOMA; -0.132). 

Genotype typing In all study groups, the genotype distributions were in Hardy-Weinberg equilibrium (P > 0.05). DNA was extracted from white blood cells by phenol/chloroform extraction according to standard procedures (23). The impact of genetic variability at IGF-IR, PIK3CB, IRS-1, and FOXO1A loci on plasma IGF-I levels was tested by univariate analysis (Table 1). IGF-IR and PIK3CB genotypes affected IGF-I plasma levels, whereas no change in IGF-I plasma levels was observed according to genotypes of IRS-1 and FOXO1A loci. The gene polymorphism due to a T to C transition located 359 bp upstream from the starting codon of PIK3CB (chromosome 3q22.3, GI: 5453894) was analyzed by the allele refractory method. 

Accordingly, the effects of IGF-IR and PIK3CB polymorphisms on IGF-I plasma levels were assessed by means of a general linear model ANOVA, including age, sex, IR (HOMA), and BMI as covariates (Table 2). The analysis revealed lower free IGF-I plasma levels in IGF1R A allele subjects (AG and AA genotypes) than in G allele subjects (GG genotype) (2.95 2.62 vs. 2.21 2.34 ng/ml; F=6.56; P=0.011) as well as in PIK3CB T allele individuals (TT and TC genotypes) compared with T allele individuals (CC genotypes) (2.22 2.22 vs. 3.02 3.11; F=6.017; P=0.015). Inasmuch as the interaction (IGF-IR PIK3CB) was significant, a univariate two-factor general linear model ANOVA was performed, which revealed that IGF-IR/PIK3CB A/T individuals had the highest IGF-I levels in the population; this was independent of age and gender (A/T vs. all other groups, 4.25 4.03 vs. 2.17 2.31; t=3.63; p=0.005).

The genotype frequency distributions at IGF-IR, PIK3CB, IRS-1, and FOXO1A loci in young people and long-lived people were studied in the two groups. Carriers of the A allele (A subjects) at the IGF-IR locus were more represented among long-lived people than in young people (Table 3). No significant differences were found when PIK3CB, IRS-1, and FOXO1A gene polymorphisms were examined.

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, individuals bearing at least one A allele at the IGF-IR locus (IGF-IR A) have lower plasma IGF-I levels than the rest of the population. 2) IGF-IR A allele subjects are found in increased proportion in long-livedPlasma levels (with A/T individuals having the highest free IGF-I plasma levels), as well as longevity, showed that the proportion of A/T subjects significantly increased among long-lived individuals. 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 co-regulated by overlapping sets of genes is in line with previous findings on the genetics of longevity in animal models. Indeed, 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 lifespan (912, 27), whereas animals with elevated plasma IGF-I levels display a shortened lifespan (13). In humans, 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 diseases, such as cancer (31, 32), but the local expression (bioavailability) 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 (13). It has been proposed that DAF-2 mutations lead to the up-regulation of genes involved in heat stress, UV, and antioxidant responses, i.e., pathways that are expected to affect the lifespan in all species (33). Furthermore, it has recently been shown that IGF-IR inactivation prolongs the lifespan 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.