Interestingly, compared to aged subjects, healthy long-lived humans display energy expenditure parame- ters that are closer to the values of healthy middle-aged adults (Rizzo et al. 2005). It is likely that variants of UCP2 gene might help to explain such peculiar phenotype found in the long-lived subject. Indeed, although no difference in relative mortality risk among UCP2 gene variant was observed in a Dutch cohort (van Heemst et al. 2005), the possibility that UCP2 gene has an influence on human physiology and life span, directly or through the interaction with other genes could not be ruled out. Interaction with a different genetic and/or environmental background may, in fact, differently modulate the effect of a given gene in different populations.

The purpose of the present study was to investigate the role of genetic variability at human loci of UCP2 gene on human longevity. Thus, the role of interchro- mosomal interactions among IGF1R, IRS2 and UCP2 genes on the probability to reach the extreme of old age was also evaluated. In particular, the G/A-IGF1R, Gly/Asp-IRS2 and Ala/Val-UCP2 allele combination was tested for association with longevity, metabolic profile and energy expenditure parameters in 722

Analytical methods

Weight and height were measured by using a standard beam balance scale. BMI was calculated as body weight (kilograms) divided by height squared (meters). Waist circumference was measured at the midpoint between the lower rib margin and the iliac crest and hip circumference at trochanter level. The

restriction analysis with AhdI enzyme of PCR product ′ obtained using the following primers: F5 - ′ ′ GGGCCAGTGCGACCTACAG-3 and R5 -ATGC ′ GGACAGAGGCAAAGC-3 and identified on 4% − agarose gel. The 866 polymorphism was evaluated using restriction analysis with MLUI enzyme of PCR ′ product obtained using the following primers: F5 - ′ ′ CACGCTGCTTCTGCCAGGAC-3 and R5 - ′ − AGGCGTCAGGAGATGGACCG-3 . The 45 ins/ del polymorphism was evaluated using PCR analysis and identified on 4% agarose gel.

ratio between them provided the WHR (waist/hip ratio). Blood samples were collected in the morning after the participants had been fasting for at least 8 h. Plasma glucose levels were determined by the glucose oxidase method (Beckman Glucose Autoanalyzer, Fullerton, CA). Commercial enzymatic tests were used to determine serum lipids (Roche Diagnostics, GmbH, Mannheim, Germany) levels. After centrifu- gation, plasma insulin concentrations were deter- mined by enzyme-linked immunoassay (ELISA; Mercodia AB, Uppsala, Sweden).

The 255-bp fragment of the IGF-1R (Chromosome 15q25-q26, rs2229765) containing the G to A transition at codon 1043 (position 3179) in exon 16 ′ was amplified by primers (upstream) 5 - ′ TCTTCTCCAGTGTACGTTCC-3 and (downstream) ′ ′ 5 -GGAACTTTCTCTTACCAC ATG-3 , and was digested with 10 U MnlI (New England Biolabs). Genotyping of Gly1057Asp IRS2 variant (rs 1805097, chromosome 13q34) was carried out by PCR restriction analysis with HAEII enzyme of PCR ′ product obtained using the following primers: F5 - ′ ′ AGCTCCCCCAAGTCTCCTAA-3 and R5 - ′ CACACCAAAAGCCAT CTCG-3 and identified on 4% agarose gel.

Energy expenditure analysis

In a subset of 260 subjects, energy expenditure analysis was performed. Resting metabolic rate (RMR) was assessed by a portable indirect calorimeter (Cosmed K4 b2, Cosmed, Rome, Italy) for 60 min. The device is a lightweight system that measures the total volume of expired air (TV), oxygen volume (VO ) consumed in 2 liters per minute, carbon dioxide volume (VCO ) 2 expired in liters per minute, Rq as the ratio of VCO 2 to VO , heart rate, and respiratory frequency. It consists 2 of a face mask, a portable unit, an electrode to record heart rate, and a battery pack. Each morning before the RMR measurements, K4 was calibrated by using a calibration gas mixture with the known composition (16% O ; 5% CO ). The test was made in a comfortable 2 2 supine position, with an environmental temperature of – 22 23 C. All measurements were done in the morning after a 12-h fast and a minimum of 8 h sleep. Abstention (12 h) from any strenuous exercise before the RMR and Rq measurements was also requested for aged and adult subjects. The last 30 min of steady state of each experiment was kept for measurement.

Statistical analysis

Insulin resistance (HOMA-IR) and B cell function (HOMA-B cell) were calculated according to the – homeostasis model assessment (HOMA) (31 32): insulin resistance (IR)=FI×G/22.5 and B cell function: − 20×FI/G 3.5, where FI=fasting insulin (mU/ml) and G=fasting glucose (mmol/l; Matthews et al. 1985; Bonora et al. 2000).

To approximate normal distributions, plasma insulin, HOMA-IR, and HOMA-B cell were logarithmically transformed and used in all calculations and back- transformed for result presentations.

Genetic analysis

Genomic DNA was obtained from blood lympho- cytes collected into EDTA-containing tubes using a commercial DNA extraction kit (Illustra, GE Healthcare UK Limited, Buckinghamshire, HP7 9 NA, UK).

For each single-nucleotide polymorphism (SNP), the genotypic and allelic frequencies were calculated. The Chi-square test was used to compare the expected genotypic frequencies with the actual frequencies – observed based on the Hardy Weinberg equilibrium. The difference in genotype frequency between cases and controls was analyzed by standard contingency Chi-square test.

In all subjects, the following UCP2 gene poly- morphisms on Chromosome 11 were evaluated using PCR and restriction analysis: Ala55Val in exon 4 (rs − ′ − 660339), 866 G/A in 5 region (rs 659366), 45 ins/del in position 173247 of the AC019121. In particular, Ala55val gene polymorphism was evaluated using

’ Analysis of variance (ANOVA) with Scheffe s test was used for comparing plasma insulin levels, BMI,