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IGF-1 signaling pathways and longevity
Ageing Research Reviews 10 (2011) 201–204
Contents lists available at ScienceDirect
Ageing Research Reviews journal homepage: www.elsevier.com/locate/arr
Review
Genetic variation in insulin/IGF-1 signaling pathways and longevity夽 Elad Ziv ∗ , Donglei Hu Department of Medicine, Institute for Human Genetics and Helen Diller Comprehensive Cancer Center, University of California, 1450 3rd Street 289, Box 3116, San Francisco, CA 94143-3116, United States
a r t i c l e
i n f o
Article history: Received 26 April 2010 Received in revised form 2 September 2010 Accepted 9 September 2010 Available online 22 September 2010 Keywords: Genetics Single nucleotide polymorphisms Insulin Insulin-like growth factor 1 Longevity Association studies
a b s t r a c t The insulin/IGF-1 pathway has been shown to affect lifespan and rate of aging in a variety of animals including worms, flies and mice. Genetic variation in this pathway may also affect human longevity. We review the evidence for the effect of this pathway on longevity with a focus on the genetic studies in humans to date. One gene, FOXO3A, a transcriptional factor homologous to daf-16, has been repeatedly associated with increased lifespan in several studies in different ethnic populations. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In the past two decades, aging researchers have identified numerous single gene mutations in model organisms which extend lifespan and appear to slow characteristics associated with aging. Many of these mutations fall into a single biological pathway that is part of the endocrine system, the insulin/IGF-1 signaling pathway. Several decades of research have demonstrated the critical components of this pathway in relation to their effect on lifespan and have also demonstrated the consistency of the effect in multiple model systems from invertebrates to mammals. Most recently, some evidence from human genetic studies may also corroborate the effect of this pathway in determining human lifespan. Here we review the evidence from invertebrate systems, vertebrates and from human genetic studies and discuss direction for future research. 2. Insulin/IGF1 signaling in aging and longevity in invertebrate models Mutations in genes that are homologous to genes of the mammalian insulin/IGF-1 pathway affect lifespan in yeast, nematodes, and fruit flies (Tatar et al., 2003). The components of this pathway have been well delineated in a series of model organism studies including: the ligand/s, the receptor/s, the insulin receptor
夽 A contribution from the Longevity Consortium, a multi-investigator research initiative supported by NIH contract 5U19AG023122 and AG032498. ∗ Corresponding author. Tel.: +1 415 353 7981; fax: +1 415 353 7932. E-mail address: [email protected] (E. Ziv). 1568-1637/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.arr.2010.09.002
substrate/s the phosphoinositol-3 kinase (PI3K) system, the AKTkinases, and the forkhead transcriptional factors (Fig. 1). Multiple genes in the pathway have been shown to affect aging and longevity and they pathway is well delineated from the ligand to the transcriptional factors. The insulin/IGF-1 pathway was first implicated in aging in the nematode Caenorhabditis elegans (Kenyon et al., 1993; Dorman et al., 1995; Kimura et al., 1997; Lin et al., 1997). Mutations that reduce the function of the nematode ortholog of the IGF1 and insulin receptors, daf-2 result in a phenotype of extended lifespan and stress resistance (Kimura et al., 1997); Mutations in age-1, the C. elegans homologue of mammalian PI3Kinase, which transduces signals from the insulin and IGF-1 receptors (Dorman et al., 1995), also increase lifespan in C. elegans. Homologues of Akt, which also participates in this signaling pathway, have been found to modify the effect of daf-2 mutations on lifespan in C. elegans (Paradis and Ruvkun, 1998), The C. elegans homologue of PDK1, which encodes a protein that phosphorylates Akt, has also been shown to affect the lifespan of some mutants (Paradis and Ruvkun, 1998). Other investigators have shown that daf-18, the C. elegans homologue of PTEN—which negatively regulates akt signaling by decreasing phosphatidylinositol-3,4,5-trisphosphate levels—also affects aging (Bluher et al., 2003). Ultimately, all of the effects of this pathway on aging in C. elegans are mediated daf-16, a gene which encodes a nuclear transcription factor homologous to the mammalian forkhead (FKHR or FOXO) transcription factors (Lin et al., 1997; Ogg et al., 1997). In C. elegans, all of the mutations that increase lifespan – from the ligand to the downstream kinase Akt – reduce the activity of the gene product and thus reduce the phosphorylation of DAF-16. Reduced DAF-16 phosphorylation increases its
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separate receptors. These pathways are more complex than in nematodes and fruitflies and have a certain degree of redundancy. Thus, translating the results from Drosophila and C. elegans is not straightforward. Nevertheless, a series of genetic manipulations in the mouse have provided evidence that this pathway affects longevity and aging in mammals. Mutations that result in growth hormone deficiency or mutations in growth hormone receptors cause reduced size, lower insulin levels, increased stress resistance and longer lifespan (Bartke, 2008). The activity of growth hormone activity is mediated through IGF-1 and, therefore, the effect on lifespan in these mice may also be mediated by this pathway. Heterozygous deletion of the IGF-1 receptor (IGF1R) gene in mice causes a modest reduction in size but a similar phenotype of improved stress resistance and extended lifespan in females only (Holzenberger et al., 2003). Disruption of the insulin receptor (INSR) gene globally or in most tissues leads to insulin resistance and shortened lifespan (Okamoto and Accili, 2003); however, mice homozygous for INSR deletion specific to fat cells have extended lifespan in both sexes (Bluher et al., 2003). These findings indicate that tissue-specific effects are important for these pathways. Most recently, a knockout of the downstream signaling adapter protein insulin receptor substrate 1 (IRS1) also resulted in increased longevity (Selman et al., 2008). There is also indirect evidence that IGF-1 signaling regulates life span in dogs. The life span among dog breeds has a strong inverse correlation with body size (Patronek et al., 1997). Recently, body size in dogs was mapped to a single haplotype at the IGF-1 locus that explained body size variation both within and between breeds (Sutter et al., 2007). Although, there is no direct evidence yet that the variation in lifespan is due to the same allele or to variation in IGF-1 activity, the fact that this haplotype explains the majority of variation in size in dogs, suggests that it accounts for the correlation between body size and longevity at least in part. 4. Potential mechanisms of insulin/IGF-1 signaling effect on longevity
Fig. 1. Schematic of the insulin/IGF-1 pathway. GH: growth hormone; GHR: growth hormone receptor; IGF-1: insulin like growth factor 1; IGF1R: IGF1 receptor; INSR: insulin receptor; IRS: insulin related substrate; PI3K: phosphoinositide 3-kinase; PIP3: phosphatidylinositol (3,4,5)-trisphosphate.
translocation to the nucleus and its activity as a transcription factor. Similarly, in the fruit fly, Drosophila melanogaster, a series of mutations have been identified that affect longevity. Mutations in the insulin receptor homolog and the insulin receptor substrate, chico (Clancy et al., 2001), both of which decrease activity of the gene products, lead to prolonged lifespan in Drosophila (Hwangbo et al., 2004). Similar effects are seen after ablation of insulinproducing cells in the fly (Giannakou et al., 2007). Mutations in the daf-16/foxo homologue that decrease transcription factor activity lead to decreased lifespan, consistent with the C. elegans findings. Thus, in summary, the evidence from C. elegans and Drosophila demonstrates convincingly that decreased activity of the insulin/IGF1 insulin/IGF-1 pathway leading to increased daf16/foxo activity extend lifespans.
In vitro studies have demonstrated that the insulin/IGF-1 may affect aging through several mechanisms. First, DAF-16/FOXO directly stimulates the transcription of genes that affect stress resistance such as heat shock proteins (Murphy et al., 2003). In addition, mammalian FOXO3A can act as a brake on cell cycle progression and a stimulus to activate DNA repair mechanisms thus potentially limit tissue aging due to cell loss and perhaps decrease the risk of malignancy (Tran et al., 2002). Thus, one possible explanation of the effect of FOXO3A is that it acts as a transcription factor on multiple stress-resistance pathways in response to decreased insulin/IGF-1 signaling and thus affects lifespan. In addition, there may be a complex interaction between FOXO3A and sirtuins (Daitoku et al., 2004; Wang et al., 2007). Sirtuins are histone deacetylases which extend lifespan when overexpressed (Giannakou and Partridge, 2004). In C. elegans, the lifespan extension of sirtuins requires daf-16 (Tissenbaum and Guarente, 2001). In mammalian cells SIRT1, one of the seven sirtuin homologues in mammals, deacetylates FOXO3a (Brunet et al., 2004) and modulates its response to oxidative stress (Brunet et al., 2004). SIRT2 may also increase FOXO binding to DNA targets (Wang et al., 2007). 5. Polymorphisms in insulin/IGF1 and human longevity
3. Insulin/IGF1 signaling in aging and longevity in mammalian models In mammalian systems, growth and glucose metabolism is regulated by two parallel pathways, with IGF-1 and insulin having
Human genetic studies have several inherent differences compared to model organism studies. First, most model organism studies are done in either isogenic background or in inbred stocks, where genetic heterogeneity is reduced which may enhance the
E. Ziv, D. Hu / Ageing Research Reviews 10 (2011) 201–204 Table 1 Association studies with FOXO3A. Note
Report
Top SNPs associated with longevity
Willcox et al. (2008)
rs2802292 rs276426 rs13217795
Flachsbart et al. (2009)
rs9400239 rs6911407 rs2802288
R2 a between rs9400239 and rs2802292 = 1
Pawlikowska et al. (2009)
rs1935949
R2 a between rs1935949 and rs2802292 = 0.91
Anselmi et al. (2009)
rs2802292 rs2802288
Li et al. (2009)
rs2802292 rs4946936 rs2253310
a R2 is a measure of correlation between SNPs on a scale of 0–1 with 1 denoting perfect correlation).
effects of a single mutation. Second, transgenic experiments allow very large manipulations of genetic activity (knockouts or knockins under strong promoters) whereas studies in human populations are presumably searching for small effects. Third, in human studies environmental factors cannot be artificially controlled and only those environmental factors that are known to have a strong impact on lifespan in humans and are adequately measured, can be statistically adjusted for. Finally, study design for longevity studies in human populations are a particular challenge since prospective studies are not easily available. Many investigators use a modified “case-control” design in which “cases” are people with exceptional longevity (not always defined in the same way). However, since controls are usually living, they are a different birth cohort compared to cases. Some of the limitations may be compensated for by matching cases and controls by ethnic and cultural background. An alternative approach may be to use genetic markers to infer the genetic ancestry of each individual in the study and to adjust for differences in genetic ancestry between cases and controls as a confounding variable using multivariate regression methods (Hu and Ziv, 2008). Several studies have demonstrated associations between human aging phenotypes and genetic variants in genes in the insulin/IGF-1 signaling pathway, including FOXO3A. Bonafe and colleagues identified a genotype in IGF-1 that was associated with lower serum levels of the protein and over-represented among long-lived people (Bonafe et al., 2003). A study in Ashkenazi Jewish centenarians found that rare non-synonymous mutations in the IGF1R gene were significantly more common in female centenarians (Suh et al., 2008). Female centenarians carrying those mutations had lower IGF1R levels and decreased IGF-1 signaling compared with female centenarians without those mutations (Suh et al., 2008). In a study of long lived Italians, individuals carrying a variant allele at the IGF1R locus had lower plasma IGF-1 levels than those without the allele; this allele was over-represented in longlived Caucasians (Bonafe et al., 2003). A variant allele of a GH1 SNP was associated with lower body height and reduced mortality in the population-based Leiden 85-plus study (van Heemst et al., 2005). In the same study, haplotypes of FOXO1A and FOXO3A were associated with an increase in all-cause mortality and mortality caused by diabetes and cardiovascular diseases (Kuningas et al., 2007), but no single SNP associations with mortality were identified. A genomewide association scan in the Framingham study showed association between age at death with two FOXO1A SNPs (Lunetta et al., 2007) The strongest evidence for association any gene in this pathway is with FOXO3A (Table 1). A recent case-control association study in elderly Japanese men in Hawaii found an association between 3
203
SNPs in FOXO3A and longevity (Willcox et al., 2008). Several subsequent studies have confirmed and extended these results. First, a similar but smaller effect was seen in a study of a German population (van Heemst et al., 2005). Then, our group confirmed this more modest but significant effect in a study that included a metaanalysis of three populations in the US (Pawlikowska et al., 2009). A recent Italian study has demonstrated the same effect (Anselmi et al., 2009). In addition, a recent paper in a Chinese population also confirmed an effect with several SNPs in FOXO3A (Li et al., 2009). The population-based Leiden 85-plus study (van Heemst et al., 2005) also found a FOXO3A haplotype, but no single SNPs, associated with an increase in mortality (Kuningas et al., 2007) in Caucasians. Many of these papers have genotyped different polyrmorphisms; however, several of the SNPs typed across different studies are in perfect or near-perfect linkage disequilibrium and appear to have the same effect (Table 1). This suggests that the effect may be due to a single polymorphism. Further studies with precise fine mapping are needed to help to clarify the causative polymorphism/s in this region. While the FOXO3A results are compelling, it is important to note that candidate gene association studies have led to numerous false positive results. Publication bias, the tendency for positive studies to be reported and for negative studies to go unreported, may lead to false conclusions from the literature (Ioannidis and Trikalinos, 2005). Thus, it is crucial that both positive and negative results be systematically published in order to firmly confirm or refuse the effect of FOXO3A polymorphisms. The insulin-IGF1 pathway was systematically investigated by testing the association between longevity and polymorphisms in 27 genes in the insulin and IGF1 canonical signaling pathway from the ligands to the transcriptional FOXO transcription factors that are the homologues of daf-16 (Pawlikowska et al., 2009; Hu et al., 2009). The authors identified an additional polymorphism in the AKT1 gene associated with longevity. Thus, in summary there is substantial but not conclusive evidence of an effect in some genes in this pathway on achievement of exceptional longevity. The strongest evidence to date is in the FOXO3A gene. Additional studies of this gene including more replication and fine mapping are needed to clarify its effect. In addition, more studies that comprehensively test all of genes in the pathway are needed. 6. Future directions 6.1. Refining the “longevity” phenotype While human studies to date have focused on identification of variants associated with exceptional longevity, animal models suggest that the phenotype of “longevity” is associated with delayed onset of degenerative diseases and a variety of other traits. Human studies that focus on expanding and refining the longevity “phenotype” may help to enhance the power of genetic studies. 6.2. “Missing heritability” and rare variants A series of genome wide association studies focusing on common variants have recently identified numerous loci associated with numerous complex traits. However, these studies have identified polymorphisms and loci that account for only a small fraction of the overall heritability of these complex traits. Thus, assuming that most of the rest of the heritability is due to DNA variants (as opposed to epigenetic inherited traits), there are likely to be many more variants associated with complex traits. These may include rare single nucleotide changes, rare small deletions/insertions and larger copy number variants that are not in linkage disequilibrium with common SNPs. Thus, there is also increased interest among
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longevity researchers in expanding the search for rare variants and copy number variants in the insulin/IGF-1 pathway and their effect on human longevity. 7. Summary/conclusions The insulin/IGF-1pathway has been shown to affect lifespan of yeast, worms, flies and mice. Recent studies in humans also suggest an association but are not yet conclusive. Additional studies of common variants in larger samples will be required to detect modest effects. In addition, new sequencing technologies may help detect rare variants in this pathway associated with longevity in humans. References Anselmi, C.V., et al., 2009. Association of the FOXO3A locus with extreme longevity in a southern Italian centenarian study. Rejuvenat. Res. 12 (2), 95–104. Bartke, A., 2008. Impact of reduced insulin-like growth factor-1/insulin signaling on aging in mammals: novel findings. Aging Cell 7 (3), 285–290. Bluher, M., Kahn, B.B., Kahn, C.R., 2003. Extended longevity in mice lacking the insulin receptor in adipose tissue. Science 299 (5606), 572–574. Bonafe, M., et al., 2003. Polymorphic variants of insulin-like growth factor I (IGFI) receptor and phosphoinositide 3-kinase genes affect IGF-I plasma levels and human longevity: cues for an evolutionarily conserved mechanism of life span control. J. Clin. Endocrinol. Metab. 88 (7), 3299–3304. Brunet, A., et al., 2004. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303 (5666), 2011–2015. Clancy, D.J., et al., 2001. Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292 (5514), 104–106. Daitoku, H., et al., 2004. Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity. Proc. Natl. Acad. Sci. U.S.A. 101 (27), 10042–10047. Dorman, J.B., et al., 1995. The age-1 and daf-2 genes function in a common pathway to control the lifespan of Caenorhabditis elegans. Genetics 141 (4), 1399–1406. Flachsbart, F., et al., 2009. Association of FOXO3A variation with human longevity confirmed in German centenarians. Proc. Natl. Acad. Sci. U.S.A. 106 (8), 2700–2705. Giannakou, M.E., Partridge, L., 2004. The interaction between FOXO and SIRT1: tipping the balance towards survival. Trends Cell Biol. 14 (8), 408–412. Giannakou, M.E., et al., 2007. Dynamics of the action of dFOXO on adult mortality in Drosophila. Aging Cell 6 (4), 429–438. Holzenberger, M., et al., 2003. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature 421 (6919), 182–187. Hu, D., Ziv, E., 2008. Confounding in genetic association studies and its solutions. Methods Mol. Biol. 448, 31–39. Hu, D., et al., 2009. Serum insulin-like growth factor-1 binding proteins 1 and 2 and mortality in older adults: the health, aging, and body composition study. J. Am. Geriatr. Soc. 57 (7), 1213–1218.
Hwangbo, D.S., et al., 2004. Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429 (6991), 562–566. Ioannidis, J.P., Trikalinos, T.A., 2005. Early extreme contradictory estimates may appear in published research: the Proteus phenomenon in molecular genetics research and randomized trials. J. Clin. Epidemiol. 58 (6), 543–549. Kenyon, C., et al., 1993. A C. elegans mutant that lives twice as long as wild type. Nature 366 (6454), 461–464. Kimura, K.D., et al., 1997. daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277 (5328), 942–946. Kuningas, M., et al., 2007. Haplotypes in the human Foxo1a and Foxo3a genes; impact on disease and mortality at old age. Eur. J. Hum. Genet. 15 (3), 294–301. Li, Y., et al., 2009. Genetic association of FOXO1A and FOXO3A with longevity trait in Han Chinese populations. Hum. Mol. Genet. 18 (24), 4897–4904. Lin, K., et al., 1997. daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278 (5341), 1319– 1322. Lunetta, K.L., et al., 2007. Genetic correlates of longevity and selected age-related phenotypes: a genome-wide association study in the Framingham study. BMC Med. Genet. 8 (Suppl. 1), S13. Murphy, C.T., et al., 2003. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424 (6946), 277–283. Ogg, S., et al., 1997. The fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389 (6654), 994–999. Okamoto, H., Accili, D., 2003. In vivo mutagenesis of the insulin receptor. J. Biol. Chem. 278 (31), 28359–28362. Paradis, S., Ruvkun, G., 1998. Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 12 (16), 2488–2498. Patronek, G.J., Waters, D.J., Glickman, L.T., 1997. Comparative longevity of pet dogs and humans: implications for gerontology research. J. Gerontol. A Biol. Sci. Med. Sci. 52 (3), B171–B178. Pawlikowska, L., et al., 2009. Association of common genetic variation in the insulin/IGF1 signaling pathway with human longevity. Aging Cell 8 (4), 460– 472. Selman, C., et al., 2008. Evidence for lifespan extension and delayed age-related biomarkers in insulin receptor substrate 1 null mice. Faseb. J. 22 (3), 807–818. Suh, Y., et al., 2008. Functionally significant insulin-like growth factor I receptor mutations in centenarians. Proc. Natl. Acad. Sci. U.S.A. 105 (9), 3438–3442. Sutter, N., et al., 2007. A single IGF1 allele is a major determinant of small size in dogs. Science 316, 112–115. Tatar, M., Bartke, A., Antebi, A., 2003. The endocrine regulation of aging by insulinlike signals. Science 299 (5611), 1346–1351. Tissenbaum, H.A., Guarente, L., 2001. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410 (6825), 227–230. Tran, H., et al., 2002. DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein. Science 296 (5567), 530–534. van Heemst, D., et al., 2005. Reduced insulin/IGF-1 signalling and human longevity. Aging Cell 4 (2), 79–85. Wang, F., et al., 2007. SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction. Aging Cell 6 (4), 505–514. Willcox, B.J., et al., 2008. FOXO3A genotype is strongly associated with human longevity. Proc. Natl. Acad. Sci. U.S.A. 105 (37), 13987–13992.
Contents lists available at ScienceDirect
Ageing Research Reviews journal homepage: www.elsevier.com/locate/arr
Review
Genetic variation in insulin/IGF-1 signaling pathways and longevity夽 Elad Ziv ∗ , Donglei Hu Department of Medicine, Institute for Human Genetics and Helen Diller Comprehensive Cancer Center, University of California, 1450 3rd Street 289, Box 3116, San Francisco, CA 94143-3116, United States
a r t i c l e
i n f o
Article history: Received 26 April 2010 Received in revised form 2 September 2010 Accepted 9 September 2010 Available online 22 September 2010 Keywords: Genetics Single nucleotide polymorphisms Insulin Insulin-like growth factor 1 Longevity Association studies
a b s t r a c t The insulin/IGF-1 pathway has been shown to affect lifespan and rate of aging in a variety of animals including worms, flies and mice. Genetic variation in this pathway may also affect human longevity. We review the evidence for the effect of this pathway on longevity with a focus on the genetic studies in humans to date. One gene, FOXO3A, a transcriptional factor homologous to daf-16, has been repeatedly associated with increased lifespan in several studies in different ethnic populations. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In the past two decades, aging researchers have identified numerous single gene mutations in model organisms which extend lifespan and appear to slow characteristics associated with aging. Many of these mutations fall into a single biological pathway that is part of the endocrine system, the insulin/IGF-1 signaling pathway. Several decades of research have demonstrated the critical components of this pathway in relation to their effect on lifespan and have also demonstrated the consistency of the effect in multiple model systems from invertebrates to mammals. Most recently, some evidence from human genetic studies may also corroborate the effect of this pathway in determining human lifespan. Here we review the evidence from invertebrate systems, vertebrates and from human genetic studies and discuss direction for future research. 2. Insulin/IGF1 signaling in aging and longevity in invertebrate models Mutations in genes that are homologous to genes of the mammalian insulin/IGF-1 pathway affect lifespan in yeast, nematodes, and fruit flies (Tatar et al., 2003). The components of this pathway have been well delineated in a series of model organism studies including: the ligand/s, the receptor/s, the insulin receptor
夽 A contribution from the Longevity Consortium, a multi-investigator research initiative supported by NIH contract 5U19AG023122 and AG032498. ∗ Corresponding author. Tel.: +1 415 353 7981; fax: +1 415 353 7932. E-mail address: [email protected] (E. Ziv). 1568-1637/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.arr.2010.09.002
substrate/s the phosphoinositol-3 kinase (PI3K) system, the AKTkinases, and the forkhead transcriptional factors (Fig. 1). Multiple genes in the pathway have been shown to affect aging and longevity and they pathway is well delineated from the ligand to the transcriptional factors. The insulin/IGF-1 pathway was first implicated in aging in the nematode Caenorhabditis elegans (Kenyon et al., 1993; Dorman et al., 1995; Kimura et al., 1997; Lin et al., 1997). Mutations that reduce the function of the nematode ortholog of the IGF1 and insulin receptors, daf-2 result in a phenotype of extended lifespan and stress resistance (Kimura et al., 1997); Mutations in age-1, the C. elegans homologue of mammalian PI3Kinase, which transduces signals from the insulin and IGF-1 receptors (Dorman et al., 1995), also increase lifespan in C. elegans. Homologues of Akt, which also participates in this signaling pathway, have been found to modify the effect of daf-2 mutations on lifespan in C. elegans (Paradis and Ruvkun, 1998), The C. elegans homologue of PDK1, which encodes a protein that phosphorylates Akt, has also been shown to affect the lifespan of some mutants (Paradis and Ruvkun, 1998). Other investigators have shown that daf-18, the C. elegans homologue of PTEN—which negatively regulates akt signaling by decreasing phosphatidylinositol-3,4,5-trisphosphate levels—also affects aging (Bluher et al., 2003). Ultimately, all of the effects of this pathway on aging in C. elegans are mediated daf-16, a gene which encodes a nuclear transcription factor homologous to the mammalian forkhead (FKHR or FOXO) transcription factors (Lin et al., 1997; Ogg et al., 1997). In C. elegans, all of the mutations that increase lifespan – from the ligand to the downstream kinase Akt – reduce the activity of the gene product and thus reduce the phosphorylation of DAF-16. Reduced DAF-16 phosphorylation increases its
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E. Ziv, D. Hu / Ageing Research Reviews 10 (2011) 201–204
separate receptors. These pathways are more complex than in nematodes and fruitflies and have a certain degree of redundancy. Thus, translating the results from Drosophila and C. elegans is not straightforward. Nevertheless, a series of genetic manipulations in the mouse have provided evidence that this pathway affects longevity and aging in mammals. Mutations that result in growth hormone deficiency or mutations in growth hormone receptors cause reduced size, lower insulin levels, increased stress resistance and longer lifespan (Bartke, 2008). The activity of growth hormone activity is mediated through IGF-1 and, therefore, the effect on lifespan in these mice may also be mediated by this pathway. Heterozygous deletion of the IGF-1 receptor (IGF1R) gene in mice causes a modest reduction in size but a similar phenotype of improved stress resistance and extended lifespan in females only (Holzenberger et al., 2003). Disruption of the insulin receptor (INSR) gene globally or in most tissues leads to insulin resistance and shortened lifespan (Okamoto and Accili, 2003); however, mice homozygous for INSR deletion specific to fat cells have extended lifespan in both sexes (Bluher et al., 2003). These findings indicate that tissue-specific effects are important for these pathways. Most recently, a knockout of the downstream signaling adapter protein insulin receptor substrate 1 (IRS1) also resulted in increased longevity (Selman et al., 2008). There is also indirect evidence that IGF-1 signaling regulates life span in dogs. The life span among dog breeds has a strong inverse correlation with body size (Patronek et al., 1997). Recently, body size in dogs was mapped to a single haplotype at the IGF-1 locus that explained body size variation both within and between breeds (Sutter et al., 2007). Although, there is no direct evidence yet that the variation in lifespan is due to the same allele or to variation in IGF-1 activity, the fact that this haplotype explains the majority of variation in size in dogs, suggests that it accounts for the correlation between body size and longevity at least in part. 4. Potential mechanisms of insulin/IGF-1 signaling effect on longevity
Fig. 1. Schematic of the insulin/IGF-1 pathway. GH: growth hormone; GHR: growth hormone receptor; IGF-1: insulin like growth factor 1; IGF1R: IGF1 receptor; INSR: insulin receptor; IRS: insulin related substrate; PI3K: phosphoinositide 3-kinase; PIP3: phosphatidylinositol (3,4,5)-trisphosphate.
translocation to the nucleus and its activity as a transcription factor. Similarly, in the fruit fly, Drosophila melanogaster, a series of mutations have been identified that affect longevity. Mutations in the insulin receptor homolog and the insulin receptor substrate, chico (Clancy et al., 2001), both of which decrease activity of the gene products, lead to prolonged lifespan in Drosophila (Hwangbo et al., 2004). Similar effects are seen after ablation of insulinproducing cells in the fly (Giannakou et al., 2007). Mutations in the daf-16/foxo homologue that decrease transcription factor activity lead to decreased lifespan, consistent with the C. elegans findings. Thus, in summary, the evidence from C. elegans and Drosophila demonstrates convincingly that decreased activity of the insulin/IGF1 insulin/IGF-1 pathway leading to increased daf16/foxo activity extend lifespans.
In vitro studies have demonstrated that the insulin/IGF-1 may affect aging through several mechanisms. First, DAF-16/FOXO directly stimulates the transcription of genes that affect stress resistance such as heat shock proteins (Murphy et al., 2003). In addition, mammalian FOXO3A can act as a brake on cell cycle progression and a stimulus to activate DNA repair mechanisms thus potentially limit tissue aging due to cell loss and perhaps decrease the risk of malignancy (Tran et al., 2002). Thus, one possible explanation of the effect of FOXO3A is that it acts as a transcription factor on multiple stress-resistance pathways in response to decreased insulin/IGF-1 signaling and thus affects lifespan. In addition, there may be a complex interaction between FOXO3A and sirtuins (Daitoku et al., 2004; Wang et al., 2007). Sirtuins are histone deacetylases which extend lifespan when overexpressed (Giannakou and Partridge, 2004). In C. elegans, the lifespan extension of sirtuins requires daf-16 (Tissenbaum and Guarente, 2001). In mammalian cells SIRT1, one of the seven sirtuin homologues in mammals, deacetylates FOXO3a (Brunet et al., 2004) and modulates its response to oxidative stress (Brunet et al., 2004). SIRT2 may also increase FOXO binding to DNA targets (Wang et al., 2007). 5. Polymorphisms in insulin/IGF1 and human longevity
3. Insulin/IGF1 signaling in aging and longevity in mammalian models In mammalian systems, growth and glucose metabolism is regulated by two parallel pathways, with IGF-1 and insulin having
Human genetic studies have several inherent differences compared to model organism studies. First, most model organism studies are done in either isogenic background or in inbred stocks, where genetic heterogeneity is reduced which may enhance the
E. Ziv, D. Hu / Ageing Research Reviews 10 (2011) 201–204 Table 1 Association studies with FOXO3A. Note
Report
Top SNPs associated with longevity
Willcox et al. (2008)
rs2802292 rs276426 rs13217795
Flachsbart et al. (2009)
rs9400239 rs6911407 rs2802288
R2 a between rs9400239 and rs2802292 = 1
Pawlikowska et al. (2009)
rs1935949
R2 a between rs1935949 and rs2802292 = 0.91
Anselmi et al. (2009)
rs2802292 rs2802288
Li et al. (2009)
rs2802292 rs4946936 rs2253310
a R2 is a measure of correlation between SNPs on a scale of 0–1 with 1 denoting perfect correlation).
effects of a single mutation. Second, transgenic experiments allow very large manipulations of genetic activity (knockouts or knockins under strong promoters) whereas studies in human populations are presumably searching for small effects. Third, in human studies environmental factors cannot be artificially controlled and only those environmental factors that are known to have a strong impact on lifespan in humans and are adequately measured, can be statistically adjusted for. Finally, study design for longevity studies in human populations are a particular challenge since prospective studies are not easily available. Many investigators use a modified “case-control” design in which “cases” are people with exceptional longevity (not always defined in the same way). However, since controls are usually living, they are a different birth cohort compared to cases. Some of the limitations may be compensated for by matching cases and controls by ethnic and cultural background. An alternative approach may be to use genetic markers to infer the genetic ancestry of each individual in the study and to adjust for differences in genetic ancestry between cases and controls as a confounding variable using multivariate regression methods (Hu and Ziv, 2008). Several studies have demonstrated associations between human aging phenotypes and genetic variants in genes in the insulin/IGF-1 signaling pathway, including FOXO3A. Bonafe and colleagues identified a genotype in IGF-1 that was associated with lower serum levels of the protein and over-represented among long-lived people (Bonafe et al., 2003). A study in Ashkenazi Jewish centenarians found that rare non-synonymous mutations in the IGF1R gene were significantly more common in female centenarians (Suh et al., 2008). Female centenarians carrying those mutations had lower IGF1R levels and decreased IGF-1 signaling compared with female centenarians without those mutations (Suh et al., 2008). In a study of long lived Italians, individuals carrying a variant allele at the IGF1R locus had lower plasma IGF-1 levels than those without the allele; this allele was over-represented in longlived Caucasians (Bonafe et al., 2003). A variant allele of a GH1 SNP was associated with lower body height and reduced mortality in the population-based Leiden 85-plus study (van Heemst et al., 2005). In the same study, haplotypes of FOXO1A and FOXO3A were associated with an increase in all-cause mortality and mortality caused by diabetes and cardiovascular diseases (Kuningas et al., 2007), but no single SNP associations with mortality were identified. A genomewide association scan in the Framingham study showed association between age at death with two FOXO1A SNPs (Lunetta et al., 2007) The strongest evidence for association any gene in this pathway is with FOXO3A (Table 1). A recent case-control association study in elderly Japanese men in Hawaii found an association between 3
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SNPs in FOXO3A and longevity (Willcox et al., 2008). Several subsequent studies have confirmed and extended these results. First, a similar but smaller effect was seen in a study of a German population (van Heemst et al., 2005). Then, our group confirmed this more modest but significant effect in a study that included a metaanalysis of three populations in the US (Pawlikowska et al., 2009). A recent Italian study has demonstrated the same effect (Anselmi et al., 2009). In addition, a recent paper in a Chinese population also confirmed an effect with several SNPs in FOXO3A (Li et al., 2009). The population-based Leiden 85-plus study (van Heemst et al., 2005) also found a FOXO3A haplotype, but no single SNPs, associated with an increase in mortality (Kuningas et al., 2007) in Caucasians. Many of these papers have genotyped different polyrmorphisms; however, several of the SNPs typed across different studies are in perfect or near-perfect linkage disequilibrium and appear to have the same effect (Table 1). This suggests that the effect may be due to a single polymorphism. Further studies with precise fine mapping are needed to help to clarify the causative polymorphism/s in this region. While the FOXO3A results are compelling, it is important to note that candidate gene association studies have led to numerous false positive results. Publication bias, the tendency for positive studies to be reported and for negative studies to go unreported, may lead to false conclusions from the literature (Ioannidis and Trikalinos, 2005). Thus, it is crucial that both positive and negative results be systematically published in order to firmly confirm or refuse the effect of FOXO3A polymorphisms. The insulin-IGF1 pathway was systematically investigated by testing the association between longevity and polymorphisms in 27 genes in the insulin and IGF1 canonical signaling pathway from the ligands to the transcriptional FOXO transcription factors that are the homologues of daf-16 (Pawlikowska et al., 2009; Hu et al., 2009). The authors identified an additional polymorphism in the AKT1 gene associated with longevity. Thus, in summary there is substantial but not conclusive evidence of an effect in some genes in this pathway on achievement of exceptional longevity. The strongest evidence to date is in the FOXO3A gene. Additional studies of this gene including more replication and fine mapping are needed to clarify its effect. In addition, more studies that comprehensively test all of genes in the pathway are needed. 6. Future directions 6.1. Refining the “longevity” phenotype While human studies to date have focused on identification of variants associated with exceptional longevity, animal models suggest that the phenotype of “longevity” is associated with delayed onset of degenerative diseases and a variety of other traits. Human studies that focus on expanding and refining the longevity “phenotype” may help to enhance the power of genetic studies. 6.2. “Missing heritability” and rare variants A series of genome wide association studies focusing on common variants have recently identified numerous loci associated with numerous complex traits. However, these studies have identified polymorphisms and loci that account for only a small fraction of the overall heritability of these complex traits. Thus, assuming that most of the rest of the heritability is due to DNA variants (as opposed to epigenetic inherited traits), there are likely to be many more variants associated with complex traits. These may include rare single nucleotide changes, rare small deletions/insertions and larger copy number variants that are not in linkage disequilibrium with common SNPs. Thus, there is also increased interest among
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