chi-square test. For investigating difference in allele fre- quency, Fisher’s test was used. The chi-square test was used to compare the expected genotypic frequencies based on the hardy–Weinberg equilibrium with the actual frequencies ob- served in the long-lived and control groups separately. Insu- lin resistance (homeostasis Model Assessment [hOMA]-IR), insulin sensitivity (hOMA IS), and ß-cell function (hO- MA-B cell) were calculated by the hOMA2 calculator v2.2 (www.dtu.ox.ac.uk/homa). To approximate normal distribu- tions, plasma insulin, hOMA-IR, and hOMA-B cell were logarithmically transformed for use in all calculations and back-transformed for result presentations. All metabolic pa- rameters were presented as mean ± SD. Analysis of variance with Scheffe’s test was used for comparing the mean body mass index (BMI), hOMA2%B, and hOMA2 IR between control and long-lived male and female participants. A logis- tic regression analysis was used to test the association of IRS2 gene polymorphism with longevity (being LL vs C) independently of multiple covariates. Furthermore, in order to estimate the odds ratio, for different IRS2 genotypes, of reaching the extreme old age independently of multiple co- variates (sex, hOMA-IR, hOMA%B, BMI, cholesterol, triglycerides, and hDL), participants were subdivided by age into 10 percentiles and used as ordinate dependent vari- ables in multinomial regression with a logit link function. Such analysis expands the general linear model analysis so that the dependent variable is linearly related to the factors and covariates via a specified link function. The cumulative logit, f(x) = ln(x/(1 − x)), applied to the cumulative probabil- ity of each category of the response. The odds ratio, 95% Wald confidence intervals, and the p value with the Bonfer-

For the enrollment of long-lived participants, a written permission with project explanation was submitted to large communities in the Italian regions in order to obtain a de- mographic list of participants born between 1908 and 1924. All participants were contacted at home or in their institu- tion and examined by trained physicians. Control partici- pants were selected from Italian geriatric medicine, internal medicine, or geriatric rehabilitation structures in regimen of Day hospital, ordinary admission or outpatient office. After a clear explanation of the aims and the potential risk of the study, all participants gave informed consent to participate in the study, which was approved by the ethical Committee at the University of Naples.

All participants were normotensive and had liver, kidney, and thyroid function tests within normal range. According to American Diabetes Association criteria (16), all partici- pants were affected neither by diabetes nor by impaired fasting glucose. No participant used drugs affecting insulin secretion and/or action or plasma lipid levels. Participants with cancer, obesity, and major cardiovascular or endocrine diseases were excluded.

Analytical Methods

years old) or controls (C; <85 years old). Anthropometric determinations were measured as previously reported (17). Blood samples were collected in the morning after the participants had been fasting for at least 8 hours. Plasma glucose levels were determined by the glucose oxidase method (Beckman Glucose Autoanalyzer; Beckman Instruments, Fullerton, CA). Commercial enzymatic tests were used to determine serum total and high-density lipoprotein (hDL) cholesterol and triglycerides levels (Roche Diagnostics, Gmbh, Mannheim, Germany). After centrifugation, plasma insulin concentrations were determined by enzyme-linked immunosorbent assay (Mercodia AB, Uppsala, Sweden).

Results

Our experimental population included 677 people aged between 16 and 104 years—289 males and 388 females— with metabolic characteristics in the normal range (Table 1). Long-lived participants displayed significantly lower BMI than controls (Figure 1A). Similar to IRS+/− mice (2), long- lived participants displayed greater insulin sensitivity and reduced b-cell function compared with the controls (Figure 1B and C). The allele and genotype frequencies of the Gly1057-Asp IRS2 gene polymorphism in our experi- mental population were determined by PCR–restriction fragment length polymorphism.

Genotyping

A total of 677 individuals were genotyped for Gly1057Asp- IRS2 locus variability (Cluster Report number: rs1805097). Genomic DNA was obtained from blood lymphocytes col- lected into ethylenediaminetetraacetic acid–containing tubes using a commercial DNA extraction kit (Illustra; Ge health- care, Uppsala, Sweden). Genotyping was carried out by polymerase chain reaction (PCR)–restriction fragment length polymorphism. Details of primer sequences and experimen- tal protocol are available upon request.

In order to assess the potential effect of population stratification, we compared genotype and allele frequency distributions of IRS2 gene variants between the three geo- graphically different Italian regions. Indeed, no differences in allele and genotype frequency distributions of IRS2 gene polymorphism between northern, central, or southern Italy were found (data not shown). The allele and genotype

Table 1. Clinical Characteristics of Study Population (n = 677)

(≥90 percentile, 96–104 years). As expected, women were more likely to be associated with the oldest participants, whereas a high BMI or hOMA2%B reduced significantly the odds of reaching the 90 percentile (Table 3). however, controlling for these covariates, the odds of reaching ex- treme old age was increased 2-fold for IRS2Asp/Asp partici- pants and 1.5-fold for IRS2Gly/Asp participants (Table 3).