Polymorphic variants of Insulin-Like Growth Factor I (IGF-I) Receptor and Phosphoinositide 3-Kinase genes affect IGF-I plasma levels and human longevity: Cues for an evolutionarily conserved mechanism of life span control. Massimiliano Bonafe, Michelangela Barbieri, Francesca Marchegiani, Fabiola Olivieri, Emilia Ragno, Claudia Giampieri, Elena Mugianesi, Matteo Centurelli, Claudio Franceschi, Giuseppe Paolisso and the Department of Experimental Pathology, University of Bologna, 40126 Bologna, Italy; Department of Geriatric Medicine and Metabolic Diseases II, University of Naples (B.M., R.E., P.G.), 80138 Naples, Italy; and Italian National Research Centers on Aging (M.F., O.F., G.C., M.E., C.M.), 60100 Ancona, Italy. Current literature indicates that abrogation of the IGF-I response pathway affects longevity in Caenorhabditis elegans. The down-regulation of IGF-I gene expression is associated with an extension of the lifespan in mice. In this paper, we tested the hypothesis that polymorphic variants of IGF-I response pathway genes, namely IGF-IR (IGF-I receptor; G/A, codon 1013), PI3KCB (phosphoinositide 3-kinase; T/C, 359 bp; A/G, 303 bp), IRS-1 (insulin receptor substrate-1; G/A, codon 972), and FOXO1A (T/C, 97347 bp), play a role in systemic IGF-I regulation and human longevity. The major finding of this investigation was that subjects carrying at least an A allele at IGF-IR have low levels of free plasma IGF-I and are more represented among long-lived people. Moreover, genotype combinations at IGF-IR and PI3KCB genes affect free IGF-I plasma levels and longevity. These findings represent the first indication that free IGF-I plasma levels and human longevity are co-regulated by an overlapping set of genes, contributing to the hypothesis that the impact of the IGF-I/insulin pathway on longevity is a property that has been evolutionarily conserved throughout the animal kingdom. (J Clin Endocrinol Metab 88: 3299-3304, 2003). AKT1/AKT2 kinases affect the lifespan of yeast (8). Based on these data, it has been predicted that genes involved in the insulin/IGF-I response pathway play a role in longevity throughout evolution (9). Mutations in genes that share similarities with human genes involved in the insulin/IGF-I signal response pathway are responsible for an impairment of IGF-I/insulin signaling and for an extension of the Caenorhabditis elegans lifespan (1). In particular, the best-characterized genes of this ancestral IGF-I/insulin response pathway are: DAF-2, ortholog to the human IGF-I/insulin receptor family; AGE-1, ortholog to human p110 catalytic subunit of phosphoinositide 3-kinase, which is involved in IGF-I/insulin signal transduction; and DAF-16, ortholog to the human FOXO family of Forkhead transcription factors, which regulate IGF-I/insulin-induced changes in gene transcription (25).

A major role of IGF-I in mammalian longevity has been suggested by studies on dwarf mice carrying mutations in transcription factors PIT-1 and PROP-1, which are characterized by a long lifespan and reduced IGF-I plasma levels (10-12). Conversely, transgenic animals with elevated plasma IGF-I levels have a short lifespan (13). In humans, there is evidence that long-lived subjects have decreased plasma IGF-I levels (14, 15) and preserved insulin action compared with aged subjects, thus indicating that an efficient insulin response has an impact on human longevity (16-18). Further recent studies have shown that mutations in the Drosophila melanogaster INR gene (similar to the human insulin receptor) and in the CHICO gene, the ortholog to human insulin receptor substrate-1 (IRS-1), the protein associated with the IGF-I/insulin receptor, affect the lifespan, confirming the hypothesis that the insulin/IGF-I response pathway plays a role in the regulation of invertebrate longevity (6, 7).

Based on the above-mentioned literature, we investigated the role of genetic variability at human loci that share similarities with the genes that regulate the insulin/IGF-I response in C. elegans (14) and D. melanogaster. In particular, polymorphisms at IGF-IR (IGF-I receptor; 34% protein sequence identityplasma levels independent of age and gender.

Result presentations. The difference in genotype frequency was analyzed by t-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 interactions between the different genotypes as well as the effect of individual genotype on plasma IGF-I levels. A t-test with a Bonferroni-adjusted p-value was used when indicated. Statistical analyses were performed 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 measured as previously reported (22). Plasma glucose was determined by the glucose oxidase method (Glucose Autoanalyzer, Beckman, Fullerton, CA). Subsequently, centrifugation plasma insulin (Sorin Biomedica, Misuse, Italy; intraassay coefficient of variation, 3.1 0.3%) and free plasma IGF-I levels (Diagnostic Systems Laboratories, Webster, TX; intraassay 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 negatively correlated with the degree of IR (HOMA; -0.132).