Results

Height and IGFI Levels in the Offspring of Centenarians. Because plasma levels of IGFI in centenarians do not reflect their levels at a younger age, we compared the levels of IGFI in offspring of centenarians to controls without a family history of longevity to identify a potential alteration in the IGF signaling pathway, which may contribute to exceptional longevity. Remarkably, ath female offspring (n 105) showed 35% higher serum IGFI   levels (P 0.01) compared with female controls (n 67), suggesting a possible alteration in the GH/IGF pathway in the longevity of females (Fig. 1A). This phenomenon was gender-   specific, as IGFI levels in male offspring (n 92) and male

Author contributions: Y.S., N.B., and P.C. designed research; G.A., M.-O.C., D.H., and B.L. performed research; G.A. and D.J.L. contributed new reagents/analytic tools; Y.S., G.A., M.-O.C., D.J.L. and P.C. analyzed data; and Y.S., G.A., N.B., and P.C. wrote the paper.

Author contributions: Y.S., N.B., and P.C. designed research; G.A., M.-O.C., D.H., and performed research; G.A. and D.J.L. contributed new reagents/analytic tools; Y.S., M.-O.C., D.J.L. and P.C. analyzed data; and Y.S., G.A., N.B., and P.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. ¶To whom correspondence should be addressed. E-mail: barzilai@aecom.yu.edu. This article contains supporting information online at 0705467105/DC1.

Despite the substantial parallels between the insulin/IGFI in invertebrates and mammals in laboratory settings, the

© 2008 by The National Academy of Sciences of the USA

           A = Control 0 Offspring B mControl 1 Offspring300~ * 185z 250> 175= 200 =.3 & 165 #e% 150 z= D© 100 2 15550 + 145 +Female Male Female MaleC12 D E4 R407H ControlIGFIR 8 IGF-1-(AU) induced DAT i a —4 A Phosfotal 2 «AKT ratioi0 v 0 IGF-I - *- - Sd Non-carrier Carrier Non-carrier Carrier (100ng/ml)

Fig. 1. Phenotypes and chemotypes of the IGF system in the centenarian cohort. (A) Female offspring of centenarians (n   105) have higher serum IGFI levels comparedwithage-matchedfemalecontrolswithoutafamilyhistoryofunusuallongevity(n(cid:1)67).IGFIlevelsinmaleoffspring(n(cid:1)92)andmalecontrols(n(cid:1) 42) were identical. IGFI levels in serum were measured by ELISA (*, P ‘yu>< ( 0.01). Results are reported as mean   SD. (B) Female offspring are shorter than controls asmeasuredbymaximalreportedheight(**,P(cid:2)0.001).Resultsarereportedasmean(cid:4)SD.(C)Immortalizedlymphocytesfromthefemalecentenarianscarrying mutations (Carrier) in IGF1R (Ala-37–Thr, Arg-407–His, and Thr-470–Thr) show significant reductions in IGFIR levels compared with immortalized lymphocytes fromfemalecentenarianswithnomutations(Noncarrier,n(cid:1)10)asmeasuredbyELISA((cid:3),P(cid:2)0.03).(D)IGFsignalingisdefectiveintheIGF1Rmutationcarriers (Carrier)offemalecentenariansascomparedwithfemalecentenarianswithnomutations(Noncarrier,n(cid:1)10)asmeasuredbyimmunoblotanalysisoftheratio of phosphorylated to total AKT in response to IGFI treatment in immortalized lymphocytes. (E) A representative immunoblot for total and phosphorylated AKT in immortalized lymphocytes from a centenarian carrying the Arg-407–His mutation and a control centenarian without the mutation.

 controls (n 42) were identical (Fig. 1A). To distinguish whether the high IGFI is caused by increased production or is a result of IGF insensitivity, we assessed maximal reported height, a necessary parameter because of the height shrinkage that occurs in the elderly. Female offspring were 2.5 cm shorter AM>< compared with controls (P 0.001), whereas the height of males was identical in offspring and controls (Fig. 1B). Thus, a likely explanation for this finding is that the higher IGFI levels represent a compensatory response to reduced IGFIR signaling, which would also be associated with a small decrease in maximal height, (although a less bioactive IGFI molecule is also a possibility). Remarkably, this combination of a female-specific effect on longevity, higher IGFI levels, with a small reduction in auxological growth, is highly reminiscent of a report on female mutant mice haploinsufficient for the IGFIR, which display nearly identical features, including gender-specific lifespan ex- tension (15).

among centenarians compared with controls was statistically ene Ae significant (P 0.04). Genotyping of the offspring of the centenarian mutation carriers shows that none of the offspring harbor these rare mutations.

Genotyping the IGF1RMutations in the Entire Cohort. We genotyped

the entire cohort (384 centenarians and 312 controls) for the i>A presence of the two nonsynonymous mutations, i.e., 244G A yas3>A (Ala-37–Thr) and 1355G A (Arg-407–His). We found nine AMR centenarians (2.3%) who carried either the Ala-37–Thr (n 2)  or Arg-407–His (n 7) mutation, whereas only one control (0.3%) was found to be a carrier of the Arg-407–His mutation and no control carried the Ala-37–Thr mutation. These results indicate that the nonsynonymous mutations were significantly ALay more common in centenarians compared with controls (P 0.02). Of the nine centenarian carriers, IGFI levels and height were available for six carriers. When we compared the mutation achaindll nM“ carriers (n 6, 66% female) vs. noncarriers (n 163, 67% female) among centenarians, the mutation carriers had signifi- cantly higher IGFI levels (165 tly4 21 vs. 121   6 ng/ml, P   0.04) than noncarriers after adjustment for gender and age (Table 2). Although the mutation carriers were slightly shorter (162  2.8 vs. 165 Se | 0.8 cm, P   0.41) than noncarriers, this difference was not statistically significant.

Screening of the IGF1and IGF1RGenes for Mutations Associated with