2.2. Analytical methods

Anthropometric determinations (weight, height and BMI) were measured as previously reported (Paolisso et al., 1995). Plasma glucose was determined by the glucose oxidase method (Beckman Glucose Autoanalyzer, Fullerton, CA). After centrifugation plasma insulin (Sorin Biomedica, Milan Italy, intra-assay c.v. 3.1 þ 0.3%) were determined by radioimmunoassay.

2.3. Genotype typing

Interestingly, some, but not all, studies have indicated a role for this variant in the pathogenesis of obesity and obesity-associated insulin resistance (Deeb et al., 1998; Ristow et al., 1998; Rosmond et al., 2003a; Koch et al., 1999; Valve et al., 1999; Meirhaeghe et al., 2000; Evans et al., 2000; Altshuler et al., 2000; Clement et al., 2000; Hasstedt et al., 2001; Mancini et al., 1999; Beamer et al., 1998).

A total of 472 individuals were genotypized for codon 12 Pro/Ala locus variability. DNA was extracted from white blood cells by phenol/clorophorm extraction, according to standard procedures (Sambrook et al., 1989).

Genotyping of the CG sequence variation in exon B of PPARg gene was carried out by PCR restriction fragment length polymorphism. Genomic leukocyte DNA (200 ng in a final volume of 10 ml) was amplified by polymerase chain reaction (PCR) using the primers 5-GCC AAT TCA AGC CCA GTC-3 (upstream) and 5-GAT ATG TTT GCA GAC AGT GTA TCA GTG AAG GAA TCG CTT TCC G-3 (downstream), which introduce a BstUI restriction site only when the CG substitution at nucleotide 34 is present (Yen et al., 1997). The amplified product was 270 bp. The PCR conditions were as follows: an initial denaturation step at 94 8C for 3 min, followed by 35 cycles of denaturation at 94 8C for 15 s, annealing at 60 8C for 15 s, and extension at 72 8C for 30 s, with a final extension of 10 min at 72 8C. The PCR reaction product was digested at 60 8C for 3 hours with 5U of Bst UI (New England Biolabs, Beverly, MA, USA), generating two fragments of 227 and 43 bp, respectively, when the G allele is present. The fragments were separated on a 4% agarose gel.

To investigate if the unusual metabolic quantitative phenotypic findings observed in long-lived subjects had a genetic component, the role of genetic variability at Pro/Ala loci of PPARG gene on longevity was studied.

2.1. Subjects

Four hundred and seventy two Caucasian subjects (307 women and 165 men, mean age 75 ^ 26 yrs), living in balance way in north, centrum and south Italy, volunteered for the study.

Yashin et al., 1999), in order to assess the impact of Pro/Ala gene polymorphism on longevity, subjects were then subcategorized in two groups, by splitting the whole sample at the age of 85: healthy people aged ,85 years of age (n ¼ 250; mean age 55 ^ 21 yrs) were grouped under the denomination of ‘aged people’; healthy people aged from 86 to 109 yrs; (n ¼ 222; mean age 97 ^ 4), were collected in the group of ‘long-lived people’.

2.4. Statistical analysis

Insulin resistance (IR) was calculated according to the homeostasis model assessment (HOMA) (Matthews et al., 1985; Bonora et al., 2000): insulin resistance (IR) ¼ FI £ G/22.5, where FI is the fasting insulin (mU/ml) and G the fasting glucose (mmol/l). HOMA method has been recently validated to be a good index of insulin

All subjects were contacted at home or in their and examined by physicians previously trained to a questionnaire that included cognitive and depression All subjects had liver, kidney, and thyroid function

M. Barbieri et al. / Experimental Gerontology 39 (2004) 1095–1100

resistance in subjects with broad range of insulin sensitivity (Matthews et al., 1985) and has a good correlation with the insulin-mediated glucose uptake calculated by euglycemic hyperinsulinemic glucose clamp (Bonora et al., 2000).

Allele and genotype distribution of the PPARG Pro/Ala polymorphism in men ðn ¼ 165Þ

Aged ðn ¼ 71Þ % Long lived ðn ¼ 94Þ % 88.7 79.8 8.5 20.2 2.8 - frequency 93.0 89.9 7.0 10.1

To approximate normal distributions, plasma insulin and IR (HOMA) were logarithmically transformed and used as such in all calculations and back-transformed for result presentations.

The difference in genotype frequency was analyzed by Chi square test. For investigating difference in allele frequency Fisher’s test was used.

Analysis of variance (ANOVA) with Scheffe’s test was used for comparing BMI and HOMA among the genotype groups.

x2 ¼ 6:72; df ¼ 2; p ¼ 0:035 for genotype distribution.

observed was almost similar to those reported in previous studies among Caucasians.

Statistical analyses were performed using SPSS software package. All metabolic parameters are presented as ^ means standard deviation (SD).

The whole sample was then divided in two subgroups ‘aged people’ (median age 60 yrs) and ‘long-lived people’ (median age 99yrs). Gender ratio confirmed a prevalence of females in the two age group.

As expected and in agreement with that previously reported in recent studies on Italian centenarians, long-lived subjects had a significantly lower BMI, fasting plasma glucose and insulin, higher HDL cholesterol and a preserved insulin action compared to aged subjects (Table 1).

3. Results

Clinical characteristics of study group are reported in Table 1. All subjects were old, not obese, in sufficient metabolic control with a greater proportion of female (Table 1).