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Reduced insulin/IGF-1 signalling and human longevity

Reduced insulin/IGF-1 signalling and human longevity <h1>Introduction</h1> Evidence has accumulated that in model organisms aging is hormonally regulated by an evolutionarily conserved insulin/IGF-1 signalling (IIS) pathway (reviewed in Tatar et al ., 2003 ). In the hermaphrodite Caenorhabditis elegans , mutations in components of the IIS pathway were first shown to affect dauer formation. In response to adverse conditions, such as food limitation, juvenile C. elegans can enter a non-feeding, developmentally arrested, reproductively immature, stress-resistant and long-lived dauer state. Upon dauer formation, metabolism shifts to the establishment and utilization of fat reserves. Induction of the dauer state allows the animal to postpone development and reproduction until environmental conditions have become more favourable. More recently, subtle mutations in IIS components were found to affect also reproduction, metabolism, stress response and lifespan in adult C. elegans (reviewed by Kenyon, 2001 ). A reduction in the level of IIS increases the activity of DAF-16, a member of the family of forkhead transcription factors. Changed DAF-16 activity results in a different transcriptional profile and thus coordinately affects different effector genes and pathways. The classes of genes regulated by DAF-16 include those implicated in the cellular stress response, antimicrobial defence, metabolism and hormonal signalling ( Murphy et al ., 2003 ). The link between reduced IIS signalling and longevity originally discovered in C . elegans was subsequently also observed in Drosophila melanogaster . Mutants in the D . melanogaster insulin receptor InR ( Tatar et al ., 2001 ) and in the insulin receptor substrate CHICO ( Clancy et al ., 2001 ) are both long-lived. Strikingly, however, in both cases the long-lived phenotype was only observed for females. In addition to being long-lived, these D. melanogaster females are small, obese and infertile. The IIS pathway has greatly increased in complexity during evolution, leading to different branches and tissue specificity. The IGF1 and INS genes probably derive from a common ancestor, which first evolved into IGF and INS , after which event the IGF gene further diverged into IGF1 and IGF2 ( Patton et al ., 1998 ). GH control of IGF-1 arose relatively late in evolution within vertebrates. The different branches of the mammalian IIS pathway interact at several levels, for example by using the same receptor substrates, such as IRS1. Data from mice indicate that the lifespan regulatory elements of the mammalian IIS pathway are not restricted to one particular component or to one particular branch. For instance, recent data from FIRKO mice, in which the insulin receptor was specifically deleted in fat tissue, provide evidence for a link between longevity and the insulin branch ( Bluher et al ., 2003 ). In addition to being long-lived, FIRKO mice exhibit a reduction in fat mass and lessened age-related loss in insulin sensitivity ( Bluher et al ., 2002 ). Data from other mutant mice models support a link between longevity and the IGF-1 branch of the IIS pathway. The IGF-1 branch acts through growth-hormone-releasing hormone (GHRH) produced by the hypothalamus, GH produced by the anterior pituitary and IGF-1 produced by the liver and peripheral tissues to control mammalian body size. Four dwarf mice models with impeded IGF-1 production, Prop1 df/df ( Brown-Borg et al ., 1996 ), Pit1 dw/dw ( Flurkey et al ., 2001 ), GHRHR lit/lit ( Flurkey et al ., 2001 ) and GHR –/– ( Chandrashekar et al ., 1999 ), all show a long-lived phenotype. However, these four mice models show other hormonal abnormalities besides IGF-1 deficiency ( Carter et al ., 2002 ). Recent results obtained with mice mutated for the IGF-1 receptor hint at a direct role for reduced IGF-1 signalling in mammalian longevity: Igf1r +/– females, but not males, exhibit a long-lived phenotype ( Holzenberger et al ., 2003 ). Interestingly, the long-lived igf1r +/– females show reduced activation of p66Shc and increased resistance to oxidative stress. Possibly, the life extension of igfr1 +/– females occurs through the same mechanism as in long-lived p66shc1 –/– mice ( Migliaccio et al ., 1999 ), in which forkhead transcription activity is up-regulated and oxidative stress resistance is increased ( Nemoto & Finkel, 2002 ). Thus, the link between reduced IIS activity and longevity seems evolutionarily conserved from worms to rodents, with effects on longevity often being stronger in the female sex. In all species analysed, most long-lived IIS mutants also show increased resistance to oxidative stress. Since the discovery of the role of the IIS pathway in longevity in model organisms researchers have speculated on the potential effects of mutations in this pathway in humans. Only few human data have been reported ( Bonafe et al ., 2003 ). Here we report data on the relationship between variation in several components of the IIS pathway and prospective survival in a well-studied Dutch population of elderly people. The IIS pathway genes studied include several components from the evolutionarily complex (or more derived) GH/IGF-1 branch. <h1>Results</h1> For all analysed polymorphisms, genotyping error rates were below 1% and the observed genotype distributions were in Hardy–Weinberg equilibrium. The linkage disequilibrium for the two polymorphisms in the IGF1 gene and the UCP2 gene was relatively weak (respectively D ′ = 0.12 and D ′ = 0.46). Moreover, none of the observed allele frequencies was statistically different between cohort ’87 and cohort ’97 ( Table 1 ). We had selected six polymorphisms from the IIS pathway based on previous reports of their effects on gene expression or protein function. Here, we wanted to determine their effect on two complex phenotypes in the Leiden 85-plus Study, namely body height and longevity. Because we were unaware of the relative impact on each of the two phenotypes (body height and longevity) of the genetic variations selected from the human IIS pathway, nor of their relationship, we have chosen first to analyse them as independent covariates in a linear regression model (for body height) and a left-censored Cox proportional hazards model (for longevity). Because, in Drosophila and mice, effects of IIS variants were often more pronounced in females than in males, we performed these analyses stratified for gender. In both males and females, except for the IGF1 CT repeat, the polymorphisms leading to reduced IIS associated with reduced body height, whereas the polymorphism leading to increased IIS associated with increased body height in cohort ’97 of the Leiden 85-plus Study. Generally these effects were small and did not reach statistical significance except for results for the GH1 polymorphism in females ( Table 2 ). Body height was 2.0 cm lower for female carriers of the variant GH1 allele compared with non-carriers ( P = 0.007). We determined the effect of each of these polymorphisms on old age mortality in the combined cohorts of the Leiden 85-plus Study. Here, we observed different and often opposite effects for males and females. In females, the relative mortality risks for carriers of the variant allele were lower for three of the five selected polymorphisms that lead to reduced IIS activity ( GH1 SNP, IGF1 CA repeat and IRS1 SNP) and reached statistical significance for the GH1 polymorphism ( Table 3 ). Our data show that mortality risk (95% confidence interval) is 0.80 (0.67–0.96) reduced ( P = 0.019) for female carriers of the variant GH1 allele compared with non-carriers, with risk estimates being similar in the two independent cohorts [0.79 (0.63–0.99) and 0.86 (0.62–1.20), respectively, for cohorts ’87 and ’97]. The next question we addressed was whether the observed effects were additive. To do this, we calculated a composite IIS6 score, by assigning a value of −1 for carriership of each variant predicted to cause reduced IIS activity ( GHRHR , GH1 , IGF1 CA, IGF1 CT, INS and IRS1 ) and a value of +1 for carriership of the variant predicted to cause increased IIS activity ( IGF1 CT). We then determined the relationship between composite IIS6 score and body height for both sexes in cohort ’97 of the Leiden 85-plus Study. It was expected that lower IIS6 scores would associate with lower body height, and we observed this trend for both men and women ( Table 4 ). The result was statistically significant for women, at P = 0.004 when using IIS6 score as a continuous variable and P = 0.029 when using IIS6 score as a categorical variable, not assuming a linear additive effect. We then determined the relationship between composite IIS6 score and survival for both sexes in the combined cohorts of the Leiden 85-plus Study. In this analysis again a sex difference became apparent ( Table 5 ). Lower composite IIS6 scores were associated with lower old age mortality in women ( P for linear trend = 0.045), but not in men. Assigning one point to each high-risk allele and subtracting one point for each low-risk allele is arbitrary as it ignores obvious and pertinent complications (dominance, epistasis, gene-by-environment interactions among many others). However, we have deliberately chosen the simplest model because the necessary biological information on IIS pathway variants in humans to build more sophisticated models is currently lacking. The results depicted in Table 3 suggest, however, that we may have underestimated the effects of the composite IIS6 score because in females the polymorphisms in GH1, IGF1 CA and IRS1 seemed to contribute most to longevity, although the effect estimates for these three polymorphisms were similar. We therefore also calculated a composite IIS3 score, based on only these three polymorphisms. Within the IIS3 score categories, we detected a highly significant survival benefit in females for the lower IIS3 scores compared with the reference category (IIS3 score 0), the relative mortality risks being 0.92 (0.72–1.18) for IIS3 score −1, 0.74 (0.58–0.96) for IIS3 score −2 and 0.46 (0.27–0.79) for IIS3 score −3, suggesting that the beneficial effects of these three polymorphisms on survival in females are indeed additive. In contrast, in males, no survival benefit was observed for any of the three lower IIS3 scores compared with the reference category. The IIS3 score was also associated with reduced body height in females ( P for linear trend = 0.003) but not in males ( P for linear trend = 0.076). We also determined the effect of a selected number of polymorphisms from genes implicated in the oxidative stress response on mortality in the Leiden 85-plus Study. We detected no differences in relative mortality risk in females or in males for the polymorphisms affecting the oxidative stress response when these polymorphisms were analysed as independent covariates in a left-censored Cox proportional hazards model ( Table 6 ). We also failed to detect an association between composite oxidative stress response (OSR) score and relative mortality risk in the combined cohorts of the Leiden 85-plus Study, the estimates being 1.11 for women ( P for linear trend = 0.14) and 1.03 for men ( P for linear trend = 0.72) for each increase in OSR score. <h1>Discussion</h1> The relationship between IIS signalling and longevity in mammals is complex and controversial ( Rincon et al ., 2004 ). Here we show in a prospective study of older people from the population at large that carriers of combinations of variants that are known to result in decreased IIS signalling are associated with reduced body height and improved old age survival, the effects on survival, however, being confined to women. Of all polymorphisms analysed, these results were most significant for the GH1 SNP, as female carriers of the GH1 SNP were significantly smaller and exhibited a significant benefit in old age survival. How can we explain the sexual dimorphism that we observed in the association of reduced IIS activity and longevity? In mammals, sex differences are known to exist for the activity of the GH/IGF-1 axis. Recently, GH was shown to be the major determinant of sexually dimorphic gene expression in the rat liver ( Ahluwalia et al ., 2004 ). Entropy analysis has revealed that in humans a large gender difference exists in the regularity of GH pulsatility, with irregularity being greater in the female sex. Oestrogen increases the irregularity of GH release, and in both sexes the growth spurt that occurs at puberty is sex hormone driven (reviewed in Giustina & Veldhuis, 1998 ). Given the existence of sex differences in GH/IGF-1 signalling, how can we explain the association observed in females between reduced GH/IGF-1 signalling and longevity? When interpreting the effects of GH/IGF-1 signalling on longevity two factors should be taken into account; cumulative exposure to GH/IGF-1 over an individual's lifetime ( Parr, 1996 ) and the effect of GH/IGF-1 at different time points in an individual's life history ( Holzenberger, 2004 ). There are several indications that increased cumulative exposure to GH/IGF-1 has adverse effects. In mice, overexpression of GH is associated with a reduced lifespan and increased insulin resistance ( Steger et al ., 1993 ). In humans, states of GH excess are correlated with insulin resistance (reviewed in Dominici & Turyn, 2002 ), which is associated with the development of diabetes mellitus type II and metabolic syndrome, conditions that reduce life expectancy. Moreover, a continuously higher exposure to the mitogenic effects of IGF-1 could influence the development of cancer. In female mice overexpressing GH the incidence of mammary tumours was increased ( Steger et al ., 1993 ). However, reduced GH/IGF-1 signalling may also have adverse effects, especially in old age, as increased induction of apoptosis is associated with growth factor withdrawal. Throughout adulthood, serum GH concentration and pulsatile GH secretion rates fall steadily, with an estimated 14% per decade in men, a phenomenon known as somatopause ( Iranmanesh et al ., 1991 ). The reduced GH secretion is believed to contribute to many age-related diseases, such as loss of muscle mass, increased adiposity, reduced bone mineral density and a decline in cognition (reviewed in Bartke et al ., 2003 ). In these parameters too, sex differences have been found. Premenopausal women are significantly better protected than men from the negative impact of increasing age on GH levels (reviewed in Giustina & Veldhuis, 1998 ). Our results suggest that in females but not in males the beneficial effects of reduced cumulative exposure to GH/IGF-1 outweigh the possible detrimental effects of lower GH/IGF-1 levels at old age. Of the polymorphisms analysed, the association with body height and longevity is most significant for the GH1 SNP (in female carriers), although a similar trend was observed for two of the other polymorphisms tested (the IGF1 CA repeat and the IRS1 SNP). This could be caused by differences in the effects of the genetic variants on gene expression or protein function or by differences in the impact of these effects. Because the IIS pathway is a hierarchical network, effects of variation in IIS components may depend on their position within the hierarchy. We speculate that effects of variation in regulatory components that arose relatively late in evolution, such as GH1 , may be of greater importance for human lifespan than variation in core components, such as INS or IGF1 . One of the mechanisms postulated for the life-extending effect of reduced signalling via the GH/IGF-1 axis is increased resistance to oxidative stress. In mice, administration of GH or IGF-1 in vitro as well as in vivo significantly suppresses enzymes implicated in the antioxidative stress response ( Brown-Borg & Rakoczy, 2003 ). We therefore looked at the effect of polymorphisms in genes implicated in the antioxidative stress response on longevity. One mechanism by which forkhead transcription factor FOXO3a (also known as FKHR-L1), the closest mammalian homologue of C. elegans DAF-16, protects quiescent cells from oxidative stress is increasing the expression of manganese superoxide dismutase (SOD2) ( Kops et al ., 2002 ), which converts superoxide to hydrogen peroxide. Mild uncoupling of oxidative phosphorylation that decreases mitochondrial reactive oxygen species (ROS) production through up-regulation of uncoupling proteins, such as UCP2 ( Walder et al ., 1998 ), is another part of the antioxidative stress response. However, none of the tested polymorphisms in SOD2 or UCP2 was associated with lifespan in our analyses. This result is in line with recent data from C. elegans ( Murphy et al ., 2003 ) . In C. elegans , multiple effector genes are coordinately regulated by DAF-16 but probably act in a cumulative manner to influence lifespan, as they were found to have only small effects on lifespan by themselves when compared with the effect of DAF-16. In our analyses in humans, where the effects of key components from the IIS pathway on longevity being modest, the influence of downstream effector genes may be extremely difficult to detect. Moreover, this analysis does not rule out the possibility that the GH1 allele exerts its effect (partly) via increased antioxidant defence. The strengths of this study are several fold. First, the analysed polymorphisms were selected on the basis of known effects on gene expression. Second, the effects on longevity were detected in a prospective analysis, which contained a high number of events (deaths) during the time of follow-up. Third, these effects were detected despite the fact that gene-by-environment interactions are important and human studies are uncontrolled with respect to environmental exposure. A first weakness of the study is that the results have not been replicated in another cohort of elderly, as cohorts of similarly aged people are not readily available. A second weakness is that we cannot completely exclude the possibility that differences in birth cohorts have confounded our results. However, such problems are of greater importance in cross-sectional studies than in prospective studies, whereas allele frequencies were not statistically different between cohort ’87 (birth cohorts 1883–1902) and cohort ’97 (birth cohorts 1912–1914). A third drawback is related to our selection of analysed polymorphisms on the basis of published effects on gene expression. This approach implies that we may have missed other existing (but not yet published) functional polymorphisms and that therefore our analyses are not exhaustive. In conclusion, we present evidence that in females but not in males reduced IIS activity is associated with longevity. Of the analysed polymorphisms, results were most pronounced for the GH1 SNP. The detected associations with longevity are in line with corresponding associations with body height, and the observed sex difference can be explained by biologically determined sex differences in the regulation of the GH/IGF-1 axis. <h1>Experimental procedures</h1> <h2>Subjects</h2> For the prospective follow-up study, we made use of the population-based Leiden 85-plus Study ( Table 1 ), which consists of two cohorts of inhabitants of Leiden, The Netherlands ( Slagboom et al ., 2000 ; Bootsma-van der Wiel et al ., 2002 ). For cohort ’87, inhabitants of Leiden aged 85 years and over were enrolled between 1987 and 1989 and followed for mortality until May 2001. For cohort ’97, inhabitants of Leiden of exactly 85 years were enrolled between 1997 and 1999 and followed for mortality until April 2004. There were no exclusion criteria related to health. In cohort ’87, almost 14 years after the start of the study 704 had died (98%). In cohort ’97, almost 7 years after the start of the study 308 had died (51%). Body height was measured using a tape measure for participants of cohort ’97 at age 85 years when standing upright without shoes. The Leiden 85-plus Study was approved by the medical ethics committee of the Leiden University Medical Centre. Informed consent was obtained from all participants after the nature and possible consequences of the studies were explained. <h2>Selected polymorphisms</h2> On the basis of sequence and/or functional homology with components of the IIS pathway and/or oxidative stress response pathway that were associated with longevity in C. elegans , D. melanogaster and/or mice, we constructed a list of human candidate genes from these signalling pathways. We next searched PubMed and dbSNP for variations in these candidate genes occurring at a frequency of at least 5% that were previously associated with changes in the expression level of the gene and/or the activity of the encoded protein. Such polymorphims were found for GHRHR, GH1, IGF1, INS and IRS1 (IIS pathway; see supplementary Table S1) and SOD2 and UCP2 (oxidative stress response; see supplementary Table S2). <h2>Statistical analyses</h2> Linear regression was used to model the relation of genotypes with body height. For the sex-stratified mortality analyses, we used left-censored Cox regression to calculate proportional hazard ratios, as subjects from the cohort ’87 entered the risk set at a different age ( Kurtzke, 1989 ). For cohort ’87, the age of entry varied between 85 years and 103 years, whereas for cohort ’97, the age of entry was 85 years for all subjects. Because low variant allele frequencies resulted in a very small group size of homozygous variant allele carriers, we combined heterozygous and homozygous variant allele carriers to increase the power to detect dominant effects. The hazard ratio indicates the relative mortality risk of variant allele carriers vs. the homozygous wild-type allele carriers, the latter being the reference group. To test for combined effects of the selected polymorphisms, a composite (sub)pathway score was calculated for each participant by assigning a score of +1 for carriership of a variant causing increased signalling and a score of −1 for carriership of each variant causing decreased signalling. Alternatively, the polymorphisms belonging to the same (sub)pathway (IIS pathway or oxidative stress response) were tested simultaneously as independent covariates in the above-mentioned models. We used multivariate analyses to adjust for the effects of the other functional variants from the same pathway. SPSS and STATA were used for the statistical analyses and Thesias was used for calculation of linkage disequilibrium between the two polymorphisms in IGF1 and the two polymorphisms in UCP2 . <h2>Genotyping</h2> DNA was available for 1245 subjects of the combined cohorts of the Leiden 85-plus Study. The genotypes for the five SNPs were determined with Taqman assays (Applied Biosystems, Nieuwerkerk, The Netherlands). Assay by Design was used as recommended, except for the following modifications. A qPCR core kit was used (Eurogentec, Maastricht, The Netherlands) with one-third of the recommended amount of assay mix. PCRs were performed on an ABI9700 and post-PCR fluorescence measurements on an ABI7900 (both Applied Biosystems). The IGF1 CA repeat ( Weber & May, 1989 ), the IGF1 CT repeat ( Arends et al ., 2002 ) and a 45-bp insertion/deletion polymorphism in UCP2 ( Esterbauer et al ., 2001 ) were analysed by fragment analysis using a ABI3700 (Applied Biosystems) as described. For determination of the INS VNTR, all samples were genotyped for the −23/ Hph I restriction fragment polymorphism, as this polymorphism is in very tight linkage disequilibrium with INS VNTR class alleles in populations of European descent (99.6% concordance between −23/ Hph I and INS VNTR alleles) ( Bennett & Todd, 1996 ). As a quality check, for each polymorphism, 10% of the samples were genotyped twice. <h1>Supplementary material</h1> The following tables are available as supplementary material at http://www.blackwellpublishing.com/products/journals/suppmat/ACE/ACE148/ACE148sm.htm Table 1 Polymorphisms from the IIS pathway selected on the basis of previously described functionality and their expected effect on IIS activity. Table 2 Polymorphisms from the oxidative stress response selected on the basis of previously described functionality and their expected effect on the oxidative stress response pathway. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aging Cell Wiley

Reduced insulin/IGF-1 signalling and human longevity

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Publisher
Wiley
Copyright
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2005
ISSN
1474-9718
eISSN
1474-9726
DOI
10.1111/j.1474-9728.2005.00148.x
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15771611
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Abstract

<h1>Introduction</h1> Evidence has accumulated that in model organisms aging is hormonally regulated by an evolutionarily conserved insulin/IGF-1 signalling (IIS) pathway (reviewed in Tatar et al ., 2003 ). In the hermaphrodite Caenorhabditis elegans , mutations in components of the IIS pathway were first shown to affect dauer formation. In response to adverse conditions, such as food limitation, juvenile C. elegans can enter a non-feeding, developmentally arrested, reproductively immature, stress-resistant and long-lived dauer state. Upon dauer formation, metabolism shifts to the establishment and utilization of fat reserves. Induction of the dauer state allows the animal to postpone development and reproduction until environmental conditions have become more favourable. More recently, subtle mutations in IIS components were found to affect also reproduction, metabolism, stress response and lifespan in adult C. elegans (reviewed by Kenyon, 2001 ). A reduction in the level of IIS increases the activity of DAF-16, a member of the family of forkhead transcription factors. Changed DAF-16 activity results in a different transcriptional profile and thus coordinately affects different effector genes and pathways. The classes of genes regulated by DAF-16 include those implicated in the cellular stress response, antimicrobial defence, metabolism and hormonal signalling ( Murphy et al ., 2003 ). The link between reduced IIS signalling and longevity originally discovered in C . elegans was subsequently also observed in Drosophila melanogaster . Mutants in the D . melanogaster insulin receptor InR ( Tatar et al ., 2001 ) and in the insulin receptor substrate CHICO ( Clancy et al ., 2001 ) are both long-lived. Strikingly, however, in both cases the long-lived phenotype was only observed for females. In addition to being long-lived, these D. melanogaster females are small, obese and infertile. The IIS pathway has greatly increased in complexity during evolution, leading to different branches and tissue specificity. The IGF1 and INS genes probably derive from a common ancestor, which first evolved into IGF and INS , after which event the IGF gene further diverged into IGF1 and IGF2 ( Patton et al ., 1998 ). GH control of IGF-1 arose relatively late in evolution within vertebrates. The different branches of the mammalian IIS pathway interact at several levels, for example by using the same receptor substrates, such as IRS1. Data from mice indicate that the lifespan regulatory elements of the mammalian IIS pathway are not restricted to one particular component or to one particular branch. For instance, recent data from FIRKO mice, in which the insulin receptor was specifically deleted in fat tissue, provide evidence for a link between longevity and the insulin branch ( Bluher et al ., 2003 ). In addition to being long-lived, FIRKO mice exhibit a reduction in fat mass and lessened age-related loss in insulin sensitivity ( Bluher et al ., 2002 ). Data from other mutant mice models support a link between longevity and the IGF-1 branch of the IIS pathway. The IGF-1 branch acts through growth-hormone-releasing hormone (GHRH) produced by the hypothalamus, GH produced by the anterior pituitary and IGF-1 produced by the liver and peripheral tissues to control mammalian body size. Four dwarf mice models with impeded IGF-1 production, Prop1 df/df ( Brown-Borg et al ., 1996 ), Pit1 dw/dw ( Flurkey et al ., 2001 ), GHRHR lit/lit ( Flurkey et al ., 2001 ) and GHR –/– ( Chandrashekar et al ., 1999 ), all show a long-lived phenotype. However, these four mice models show other hormonal abnormalities besides IGF-1 deficiency ( Carter et al ., 2002 ). Recent results obtained with mice mutated for the IGF-1 receptor hint at a direct role for reduced IGF-1 signalling in mammalian longevity: Igf1r +/– females, but not males, exhibit a long-lived phenotype ( Holzenberger et al ., 2003 ). Interestingly, the long-lived igf1r +/– females show reduced activation of p66Shc and increased resistance to oxidative stress. Possibly, the life extension of igfr1 +/– females occurs through the same mechanism as in long-lived p66shc1 –/– mice ( Migliaccio et al ., 1999 ), in which forkhead transcription activity is up-regulated and oxidative stress resistance is increased ( Nemoto & Finkel, 2002 ). Thus, the link between reduced IIS activity and longevity seems evolutionarily conserved from worms to rodents, with effects on longevity often being stronger in the female sex. In all species analysed, most long-lived IIS mutants also show increased resistance to oxidative stress. Since the discovery of the role of the IIS pathway in longevity in model organisms researchers have speculated on the potential effects of mutations in this pathway in humans. Only few human data have been reported ( Bonafe et al ., 2003 ). Here we report data on the relationship between variation in several components of the IIS pathway and prospective survival in a well-studied Dutch population of elderly people. The IIS pathway genes studied include several components from the evolutionarily complex (or more derived) GH/IGF-1 branch. <h1>Results</h1> For all analysed polymorphisms, genotyping error rates were below 1% and the observed genotype distributions were in Hardy–Weinberg equilibrium. The linkage disequilibrium for the two polymorphisms in the IGF1 gene and the UCP2 gene was relatively weak (respectively D ′ = 0.12 and D ′ = 0.46). Moreover, none of the observed allele frequencies was statistically different between cohort ’87 and cohort ’97 ( Table 1 ). We had selected six polymorphisms from the IIS pathway based on previous reports of their effects on gene expression or protein function. Here, we wanted to determine their effect on two complex phenotypes in the Leiden 85-plus Study, namely body height and longevity. Because we were unaware of the relative impact on each of the two phenotypes (body height and longevity) of the genetic variations selected from the human IIS pathway, nor of their relationship, we have chosen first to analyse them as independent covariates in a linear regression model (for body height) and a left-censored Cox proportional hazards model (for longevity). Because, in Drosophila and mice, effects of IIS variants were often more pronounced in females than in males, we performed these analyses stratified for gender. In both males and females, except for the IGF1 CT repeat, the polymorphisms leading to reduced IIS associated with reduced body height, whereas the polymorphism leading to increased IIS associated with increased body height in cohort ’97 of the Leiden 85-plus Study. Generally these effects were small and did not reach statistical significance except for results for the GH1 polymorphism in females ( Table 2 ). Body height was 2.0 cm lower for female carriers of the variant GH1 allele compared with non-carriers ( P = 0.007). We determined the effect of each of these polymorphisms on old age mortality in the combined cohorts of the Leiden 85-plus Study. Here, we observed different and often opposite effects for males and females. In females, the relative mortality risks for carriers of the variant allele were lower for three of the five selected polymorphisms that lead to reduced IIS activity ( GH1 SNP, IGF1 CA repeat and IRS1 SNP) and reached statistical significance for the GH1 polymorphism ( Table 3 ). Our data show that mortality risk (95% confidence interval) is 0.80 (0.67–0.96) reduced ( P = 0.019) for female carriers of the variant GH1 allele compared with non-carriers, with risk estimates being similar in the two independent cohorts [0.79 (0.63–0.99) and 0.86 (0.62–1.20), respectively, for cohorts ’87 and ’97]. The next question we addressed was whether the observed effects were additive. To do this, we calculated a composite IIS6 score, by assigning a value of −1 for carriership of each variant predicted to cause reduced IIS activity ( GHRHR , GH1 , IGF1 CA, IGF1 CT, INS and IRS1 ) and a value of +1 for carriership of the variant predicted to cause increased IIS activity ( IGF1 CT). We then determined the relationship between composite IIS6 score and body height for both sexes in cohort ’97 of the Leiden 85-plus Study. It was expected that lower IIS6 scores would associate with lower body height, and we observed this trend for both men and women ( Table 4 ). The result was statistically significant for women, at P = 0.004 when using IIS6 score as a continuous variable and P = 0.029 when using IIS6 score as a categorical variable, not assuming a linear additive effect. We then determined the relationship between composite IIS6 score and survival for both sexes in the combined cohorts of the Leiden 85-plus Study. In this analysis again a sex difference became apparent ( Table 5 ). Lower composite IIS6 scores were associated with lower old age mortality in women ( P for linear trend = 0.045), but not in men. Assigning one point to each high-risk allele and subtracting one point for each low-risk allele is arbitrary as it ignores obvious and pertinent complications (dominance, epistasis, gene-by-environment interactions among many others). However, we have deliberately chosen the simplest model because the necessary biological information on IIS pathway variants in humans to build more sophisticated models is currently lacking. The results depicted in Table 3 suggest, however, that we may have underestimated the effects of the composite IIS6 score because in females the polymorphisms in GH1, IGF1 CA and IRS1 seemed to contribute most to longevity, although the effect estimates for these three polymorphisms were similar. We therefore also calculated a composite IIS3 score, based on only these three polymorphisms. Within the IIS3 score categories, we detected a highly significant survival benefit in females for the lower IIS3 scores compared with the reference category (IIS3 score 0), the relative mortality risks being 0.92 (0.72–1.18) for IIS3 score −1, 0.74 (0.58–0.96) for IIS3 score −2 and 0.46 (0.27–0.79) for IIS3 score −3, suggesting that the beneficial effects of these three polymorphisms on survival in females are indeed additive. In contrast, in males, no survival benefit was observed for any of the three lower IIS3 scores compared with the reference category. The IIS3 score was also associated with reduced body height in females ( P for linear trend = 0.003) but not in males ( P for linear trend = 0.076). We also determined the effect of a selected number of polymorphisms from genes implicated in the oxidative stress response on mortality in the Leiden 85-plus Study. We detected no differences in relative mortality risk in females or in males for the polymorphisms affecting the oxidative stress response when these polymorphisms were analysed as independent covariates in a left-censored Cox proportional hazards model ( Table 6 ). We also failed to detect an association between composite oxidative stress response (OSR) score and relative mortality risk in the combined cohorts of the Leiden 85-plus Study, the estimates being 1.11 for women ( P for linear trend = 0.14) and 1.03 for men ( P for linear trend = 0.72) for each increase in OSR score. <h1>Discussion</h1> The relationship between IIS signalling and longevity in mammals is complex and controversial ( Rincon et al ., 2004 ). Here we show in a prospective study of older people from the population at large that carriers of combinations of variants that are known to result in decreased IIS signalling are associated with reduced body height and improved old age survival, the effects on survival, however, being confined to women. Of all polymorphisms analysed, these results were most significant for the GH1 SNP, as female carriers of the GH1 SNP were significantly smaller and exhibited a significant benefit in old age survival. How can we explain the sexual dimorphism that we observed in the association of reduced IIS activity and longevity? In mammals, sex differences are known to exist for the activity of the GH/IGF-1 axis. Recently, GH was shown to be the major determinant of sexually dimorphic gene expression in the rat liver ( Ahluwalia et al ., 2004 ). Entropy analysis has revealed that in humans a large gender difference exists in the regularity of GH pulsatility, with irregularity being greater in the female sex. Oestrogen increases the irregularity of GH release, and in both sexes the growth spurt that occurs at puberty is sex hormone driven (reviewed in Giustina & Veldhuis, 1998 ). Given the existence of sex differences in GH/IGF-1 signalling, how can we explain the association observed in females between reduced GH/IGF-1 signalling and longevity? When interpreting the effects of GH/IGF-1 signalling on longevity two factors should be taken into account; cumulative exposure to GH/IGF-1 over an individual's lifetime ( Parr, 1996 ) and the effect of GH/IGF-1 at different time points in an individual's life history ( Holzenberger, 2004 ). There are several indications that increased cumulative exposure to GH/IGF-1 has adverse effects. In mice, overexpression of GH is associated with a reduced lifespan and increased insulin resistance ( Steger et al ., 1993 ). In humans, states of GH excess are correlated with insulin resistance (reviewed in Dominici & Turyn, 2002 ), which is associated with the development of diabetes mellitus type II and metabolic syndrome, conditions that reduce life expectancy. Moreover, a continuously higher exposure to the mitogenic effects of IGF-1 could influence the development of cancer. In female mice overexpressing GH the incidence of mammary tumours was increased ( Steger et al ., 1993 ). However, reduced GH/IGF-1 signalling may also have adverse effects, especially in old age, as increased induction of apoptosis is associated with growth factor withdrawal. Throughout adulthood, serum GH concentration and pulsatile GH secretion rates fall steadily, with an estimated 14% per decade in men, a phenomenon known as somatopause ( Iranmanesh et al ., 1991 ). The reduced GH secretion is believed to contribute to many age-related diseases, such as loss of muscle mass, increased adiposity, reduced bone mineral density and a decline in cognition (reviewed in Bartke et al ., 2003 ). In these parameters too, sex differences have been found. Premenopausal women are significantly better protected than men from the negative impact of increasing age on GH levels (reviewed in Giustina & Veldhuis, 1998 ). Our results suggest that in females but not in males the beneficial effects of reduced cumulative exposure to GH/IGF-1 outweigh the possible detrimental effects of lower GH/IGF-1 levels at old age. Of the polymorphisms analysed, the association with body height and longevity is most significant for the GH1 SNP (in female carriers), although a similar trend was observed for two of the other polymorphisms tested (the IGF1 CA repeat and the IRS1 SNP). This could be caused by differences in the effects of the genetic variants on gene expression or protein function or by differences in the impact of these effects. Because the IIS pathway is a hierarchical network, effects of variation in IIS components may depend on their position within the hierarchy. We speculate that effects of variation in regulatory components that arose relatively late in evolution, such as GH1 , may be of greater importance for human lifespan than variation in core components, such as INS or IGF1 . One of the mechanisms postulated for the life-extending effect of reduced signalling via the GH/IGF-1 axis is increased resistance to oxidative stress. In mice, administration of GH or IGF-1 in vitro as well as in vivo significantly suppresses enzymes implicated in the antioxidative stress response ( Brown-Borg & Rakoczy, 2003 ). We therefore looked at the effect of polymorphisms in genes implicated in the antioxidative stress response on longevity. One mechanism by which forkhead transcription factor FOXO3a (also known as FKHR-L1), the closest mammalian homologue of C. elegans DAF-16, protects quiescent cells from oxidative stress is increasing the expression of manganese superoxide dismutase (SOD2) ( Kops et al ., 2002 ), which converts superoxide to hydrogen peroxide. Mild uncoupling of oxidative phosphorylation that decreases mitochondrial reactive oxygen species (ROS) production through up-regulation of uncoupling proteins, such as UCP2 ( Walder et al ., 1998 ), is another part of the antioxidative stress response. However, none of the tested polymorphisms in SOD2 or UCP2 was associated with lifespan in our analyses. This result is in line with recent data from C. elegans ( Murphy et al ., 2003 ) . In C. elegans , multiple effector genes are coordinately regulated by DAF-16 but probably act in a cumulative manner to influence lifespan, as they were found to have only small effects on lifespan by themselves when compared with the effect of DAF-16. In our analyses in humans, where the effects of key components from the IIS pathway on longevity being modest, the influence of downstream effector genes may be extremely difficult to detect. Moreover, this analysis does not rule out the possibility that the GH1 allele exerts its effect (partly) via increased antioxidant defence. The strengths of this study are several fold. First, the analysed polymorphisms were selected on the basis of known effects on gene expression. Second, the effects on longevity were detected in a prospective analysis, which contained a high number of events (deaths) during the time of follow-up. Third, these effects were detected despite the fact that gene-by-environment interactions are important and human studies are uncontrolled with respect to environmental exposure. A first weakness of the study is that the results have not been replicated in another cohort of elderly, as cohorts of similarly aged people are not readily available. A second weakness is that we cannot completely exclude the possibility that differences in birth cohorts have confounded our results. However, such problems are of greater importance in cross-sectional studies than in prospective studies, whereas allele frequencies were not statistically different between cohort ’87 (birth cohorts 1883–1902) and cohort ’97 (birth cohorts 1912–1914). A third drawback is related to our selection of analysed polymorphisms on the basis of published effects on gene expression. This approach implies that we may have missed other existing (but not yet published) functional polymorphisms and that therefore our analyses are not exhaustive. In conclusion, we present evidence that in females but not in males reduced IIS activity is associated with longevity. Of the analysed polymorphisms, results were most pronounced for the GH1 SNP. The detected associations with longevity are in line with corresponding associations with body height, and the observed sex difference can be explained by biologically determined sex differences in the regulation of the GH/IGF-1 axis. <h1>Experimental procedures</h1> <h2>Subjects</h2> For the prospective follow-up study, we made use of the population-based Leiden 85-plus Study ( Table 1 ), which consists of two cohorts of inhabitants of Leiden, The Netherlands ( Slagboom et al ., 2000 ; Bootsma-van der Wiel et al ., 2002 ). For cohort ’87, inhabitants of Leiden aged 85 years and over were enrolled between 1987 and 1989 and followed for mortality until May 2001. For cohort ’97, inhabitants of Leiden of exactly 85 years were enrolled between 1997 and 1999 and followed for mortality until April 2004. There were no exclusion criteria related to health. In cohort ’87, almost 14 years after the start of the study 704 had died (98%). In cohort ’97, almost 7 years after the start of the study 308 had died (51%). Body height was measured using a tape measure for participants of cohort ’97 at age 85 years when standing upright without shoes. The Leiden 85-plus Study was approved by the medical ethics committee of the Leiden University Medical Centre. Informed consent was obtained from all participants after the nature and possible consequences of the studies were explained. <h2>Selected polymorphisms</h2> On the basis of sequence and/or functional homology with components of the IIS pathway and/or oxidative stress response pathway that were associated with longevity in C. elegans , D. melanogaster and/or mice, we constructed a list of human candidate genes from these signalling pathways. We next searched PubMed and dbSNP for variations in these candidate genes occurring at a frequency of at least 5% that were previously associated with changes in the expression level of the gene and/or the activity of the encoded protein. Such polymorphims were found for GHRHR, GH1, IGF1, INS and IRS1 (IIS pathway; see supplementary Table S1) and SOD2 and UCP2 (oxidative stress response; see supplementary Table S2). <h2>Statistical analyses</h2> Linear regression was used to model the relation of genotypes with body height. For the sex-stratified mortality analyses, we used left-censored Cox regression to calculate proportional hazard ratios, as subjects from the cohort ’87 entered the risk set at a different age ( Kurtzke, 1989 ). For cohort ’87, the age of entry varied between 85 years and 103 years, whereas for cohort ’97, the age of entry was 85 years for all subjects. Because low variant allele frequencies resulted in a very small group size of homozygous variant allele carriers, we combined heterozygous and homozygous variant allele carriers to increase the power to detect dominant effects. The hazard ratio indicates the relative mortality risk of variant allele carriers vs. the homozygous wild-type allele carriers, the latter being the reference group. To test for combined effects of the selected polymorphisms, a composite (sub)pathway score was calculated for each participant by assigning a score of +1 for carriership of a variant causing increased signalling and a score of −1 for carriership of each variant causing decreased signalling. Alternatively, the polymorphisms belonging to the same (sub)pathway (IIS pathway or oxidative stress response) were tested simultaneously as independent covariates in the above-mentioned models. We used multivariate analyses to adjust for the effects of the other functional variants from the same pathway. SPSS and STATA were used for the statistical analyses and Thesias was used for calculation of linkage disequilibrium between the two polymorphisms in IGF1 and the two polymorphisms in UCP2 . <h2>Genotyping</h2> DNA was available for 1245 subjects of the combined cohorts of the Leiden 85-plus Study. The genotypes for the five SNPs were determined with Taqman assays (Applied Biosystems, Nieuwerkerk, The Netherlands). Assay by Design was used as recommended, except for the following modifications. A qPCR core kit was used (Eurogentec, Maastricht, The Netherlands) with one-third of the recommended amount of assay mix. PCRs were performed on an ABI9700 and post-PCR fluorescence measurements on an ABI7900 (both Applied Biosystems). The IGF1 CA repeat ( Weber & May, 1989 ), the IGF1 CT repeat ( Arends et al ., 2002 ) and a 45-bp insertion/deletion polymorphism in UCP2 ( Esterbauer et al ., 2001 ) were analysed by fragment analysis using a ABI3700 (Applied Biosystems) as described. For determination of the INS VNTR, all samples were genotyped for the −23/ Hph I restriction fragment polymorphism, as this polymorphism is in very tight linkage disequilibrium with INS VNTR class alleles in populations of European descent (99.6% concordance between −23/ Hph I and INS VNTR alleles) ( Bennett & Todd, 1996 ). As a quality check, for each polymorphism, 10% of the samples were genotyped twice. <h1>Supplementary material</h1> The following tables are available as supplementary material at http://www.blackwellpublishing.com/products/journals/suppmat/ACE/ACE148/ACE148sm.htm Table 1 Polymorphisms from the IIS pathway selected on the basis of previously described functionality and their expected effect on IIS activity. Table 2 Polymorphisms from the oxidative stress response selected on the basis of previously described functionality and their expected effect on the oxidative stress response pathway.

Journal

Aging Cell – Wiley

Published: Apr 1, 2005

Keywords: human; insulin/IGF-1 signalling; longevity; polymorphisms; prospective analysis

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