In a subset of 211 individuals, direct sequencing of a 203-bp region ott aii (position 4703 267) of the PIK3CB promoter was performed   (CEQ2000, Beckman) using primers 5 -GAA AAACTCAGCCTAA- GAAGC-3(cid:3) (upstream) and 5(cid:3) -AATCCATACCACCAACTAAAG-3(cid:3) iwt—3 (downstream) to detect a polymorphism A3G at 303 bp upstream as described by Kossila et al. (24). This procedure allowed confirmation of   the data on the site T3C located at 359 bp and proved that no other polymorphic loci were present in that DNA region.

The region of exon I surrounding the G to A transition at codon 972 ofIRS-1(chromosome2q35-q36.1,GI:5031804)wasamplifiedbyprimers Vis5! 1,  5 -GCAGCCTGGCAGGAGAG-3 (upstream) and 5 -CTCACCTC- NIN CTCTGCAGC-3 (downstream). The 220-bp PCR product was digested with BstNI (New England Biolabs, Inc., Beverly, MA). The presence of the restriction site (allele A) generated two fragments of 164 and 56 bp, which were run on a 4.0% agarose gel stained with ethidium bromide.

The fragment of 296 bp spanning the T to C transition at position +9 97347 in intron I of the FOXO1A gene (GenBank GI:17476061, chro-   mosome 13q14.1) was amplified by primers 5 -CCTGGTAAAACCG- muir   GAAGGT-3 (upstream) and 5 -CCCTTCCTTTGGAATGACTG-3 (downstream) and digested with SspI (New England Biolabs, Inc.). The presence of the T allele allowed cutting of the amplified region in two fragments of 186 and 110 bp.

Subjects and Methods

Subjects

Four hundred ninety-six Caucasian subjects (132 men and 364 wom- en), from 18–109 yr of age, equally distributed in northern, central, and southern Italy, volunteered for this study. On the basis of literature data (18–20), to assess the impact of genes on longevity, subjects were then subcategorized into two groups by splitting the sample at the age of 85 aban=!  yr: healthy people 17–85 yr of age (n 278; mean age, 54.8 21.5; male/female ratio, 76/202) were grouped as “young people,” and    healthy people from 86–109 yr of age (n 218; mean age, 98.0 4.31 male/female ratio, 56/162) were grouped as “long-lived people.” All subjects were contacted at home or their institutions and examined by physicians previously trained to administer a questionnaire that in- cludedcognitiveanddepressiontests.Allsubjectshadliver,kidney,and thyroid function tests within the normal range. According to American Diabetes Association criteria (21), no subject was diabetic or affected by impaired fasting glucose. No subject used drugs affecting insulin se- cretion and/or action or plasma lipid levels. In particular, long-lived subjects represented a group of carefully selected individuals free of major age-related diseases. After a clear explanation of the potential risk of the study, all subjects (as well as the relatives of the long-lived people) gave informed consent to participate in the study, which was approved by the ethical committee of our institutions.

Calculations and statistical analyses

Thedegreeofinsulinresistance(IR)wasestimatedbythehomeostatic model assessment (HOMA) method (25, 26). The HOMA method has been recently validated to be a good index of insulin resistance (IR) in subjects with a broad range of insulin sensitivity (25) and has a good correlation with the insulin-mediated glucose uptake calculated by the euglycemic hyperinsulinemic glucose clamp (26). To approximate nor- mal distributions, plasma insulin and IR (HOMA) were logarithmically transformed and used in all calculations and were back-transformed for resultpresentations.Thedifferenceingenotypefrequencywasanalyzed by (cid:1)2 test. For investigating the difference in allele frequency, Fisher’s test was used. ANOVA with Scheffe´’s test was used for comparing IGF-I plasma levels among the genotype groups. A linear general model analysis was used to test the independent contributions of the interac- tions between the different genotypes as well as the effect of individual genotype on plasma IGF-I levels. A t test with a Bonferroni-adjusted ‘yrS) aN (p ) value was used when indicated. Statistical analyses were per- adj formed using the SPSS software package (SPSS, Inc., Chicago, IL). All  metabolic parameters are presented as the mean sd.

Analytical methods

Anthropometric determinations (weight, height, and BMI) were mea- sured as previously reported (22). Plasma glucose was determined by theglucoseoxidasemethod(GlucoseAutoanalyzer,Beckman,Fullerton, CA). Subsequently centrifugation plasma insulin (Sorin Biomedica, Mi- suse,L + ( lan Italy; intraassay coefficient of variation, 3.1 0.3%) and free plasma IGF-I levels (Diagnostic Systems Laboratories, Webster, TX; intraassay demeee5 + | coefficients of variation, 3.8 0.4%) were determined by RIA.

Results

In the entire population, free IGF-I plasma levels displayed an age-related decrease (Fig. 1) through all ages and were ee  negatively correlated with the degree of IR (HOMA; , 0.132; S6He<< “YY ior<<

Genotype typing

In all study groups, the genotype distributions were in the se>| 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 uni- variate 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.

GI:11068002) containing the G to A transition at nucleotide 3174  1013)inexon16wasamplifiedbyprimers5 ure3"  TCC-3 (upstream) and 5 -GGAACTTTCTCTTACCAC ATG-3 stream) and was digested with 10 U MnlI (New England Biolabs, G yielded to four restriction products of 132, 80, 23, and 20 bp, were separated on 4.5% agarose gels and visualized with bromide.