**6.1 IGF-1 signalling in** *C. elegans*

226 Basic and Clinical Endocrinology Up-to-Date

detoxification of Aβ42 by decreased DAF-2 signalling involves two possible mechanisms. First, HSF-1 regulates disaggregation of toxic oligomers followed by degradation of the resulting fragments. Second, DAF-16 regulates the formation of aggregates with high molecular weight and low toxicity, which are built from aggregates with low molecular weight but high toxicity (Aβ hyperaggregation) (Cohen et al. 2006). Recently, Aβ hyperaggregation has been identified as a mechanism of Aβ detoxification in an IGF-1

Several ways of Aβ clearance from the brain have been discovered contributing to Aβ detoxification. Aβ clearance is achieved via transport over the blood brain barrier, enzymatic degradation and phagocytosis by microglia. Recently, several enzymes have been discovered which degrade Aβ, e.g. insulin degrading enzyme (IDE), endothelin converting enzyme (ECE), neprilysin, and angiotensin converting enzyme (ACE). The expression of IDE is activated by IR and IGF-1R signalling (Zhao et al. 2004). Transport across the blood brain barrier (BBB) is mediated by distinct receptors. This transport is achieved via binding to the low-density lipoprotein receptor related protein (LRP). The binding of Aβ to LRP occurs directly or in complex with APOE (apolipoprotein E) and/or α2-macroglobulin (α2M). After crossing the BBB, Aβ is transported to peripheral tissues for degradation, for example the liver (Tanzi, Moir, and Wagner 2004). It has been proposed that high IGF-1 levels cause degradation or clearance of Aβ. Tg2576 mice present decreased IGF-1 levels compared to wild type mice. The treatment with IGF-1 yield to increased transport of Aβ from the brain, possibly via the choroid plexus (Carro et al. 2002). Accordingly, the inactivation of the IGF-1R signalling in the choroid plexus caused AD-like pathology (Carro et al. 2006). In contrast, studies in rats and Tg2576 mice using acute, subchronic and chronic IGF-1 treatment found no changes in tau phosphorylation and Aβ concentrations (Lanz et al. 2008). Possibly, chronic peripheral treatment with IGF-1 causes downregulation of the IGF-1R signalling pathway as it has been shown for a cohort of individuals with high serum IGF-1 level but low IGF-1R signalling (Suh et al. 2008). This might explain the conflicting results of the different studies. AD mouse models with induced insulin resistance via high fat diet

The different studies dealing with IR/IGF-1R signalling and AD prove a connection between this signalling pathways and AD pathology. However, the exact molecular

IR/IGF-1R signalling is a conserved pathway functioning in development, fertility (Liang et al. 2003; Tatar, Bartke, and Antebi 2003; Broughton et al. 2005), growth (Butler and Le Roith 2001; Ikeya et al. 2002) and metabolic regulation (Saltiel and Kahn 2001). Decreased IR/IGF-1R signalling causes lifespan extension in model organisms like *Drosophila melanogaster*, *Caenorhabditis elegans* and *Mus musculus* (Clancy et al. 2001; Holzenberger et al. 2003; Tatar

The life expenctancy of humans has been increased upon environmental amelioration (Wilmoth 2000). However, this is accompanied by an increase of age associated disorders. Therefore, the study of the molecular mechanism of ageing might lead to identification of disease modifying pathways. One of these pathways is the IR/IGF-1R signalling cascade. Studies investigating temporal or spatial restricted changes of the IR/IGF-1R signalling pathway in different model organisms, using the RU486-induced GAL4/UAS (upstream

resistant mouse model of AD (Cohen et al. 2009).

displayed an exacerbation of amyloid pathology (Ho et al. 2004).

mechanisms need to be elucidated.

et al. 2001).

**6. IGF-1 in the brain and ageing** 

The impact of IR/IGF-1R-like signalling (IIS) pathway on lifespan was first discovered in *C. elegans*. IIS in *C. elegans* is similar to the pathway in mammals (Taguchi and White 2008). It is activated through binding of insulin-like peptides (INS) to DAF-2 (abnormal Dauer formation-2), the IR/IGF-1R in worms (Kenyon et al. 1993). Upon stimulation of DAF-2 an insulin receptor substrate 1 orthologue (IST-1) is recruited to DAF-2 and AGE-1 (AGEing alteration-1), orthologue to human p110. P110, the catalytic subunit of PI3K, promotes the generation of phosphatidylinositide-triphosphate (PI3,4,5P) which then activates AKT kinase family members (Morris, Tissenbaum, and Ruvkun 1996; Kops et al. 1999; Paradis and Ruvkun 1998).

The mutations of DAF-2 and AGE-1 cause lifespan extension in a DAF-16 dependent manner which is a forkhead transcription factor in worms (Kenyon et al. 1993; Lin et al. 1997; Ogg et al. 1997; Morris, Tissenbaum, and Ruvkun 1996) and homologue to the mammalian FoxO1 and FoxO3a.

AGE-1 and DAF-2 mutant worms show enhanced thermotaxis learning behaviour during ageing. This might be due to resistance to neuronal diseases and oxidative stress (Murakami 2007).

The insulin-like peptides (INS) are found throughout the whole body of *C. elegans* (Pierce et al. 2001; Li, Kennedy, and Ruvkun 2003). Specific sensory neurons regulate lifespan through DAF-16 (Alcedo and Kenyon 2004). The ablation of olfactory neurons causes lifespan extension being only partially dependent on DAF-16 indicating an involvement of other factors. The regulation of INS is not well analysed, but the sensory neurons seem to be the major source of INS for peripheral tissues (Alcedo and Kenyon 2004).

A study using tissue-specific expression of AGE-1 and DAF-16 revealed that wild-type AGE-1 recovers the extended lifespan of AGE-1 mutants. This was shown for wild-type AGE-1 expressed in neurons and the intestine. In addition, wild-type DAF-16 rescues reduced lifespan of DAF-16/AGE-1 double mutants when expressed in neurons or intestine. Moreover, wild-type DAF-16 exerts its full effect when expressed in neurons and the intestine exclusively. However, DAF-16 expression showed only minor effects when expressed in other tissues (Broughton and Partridge 2009; Iser, Gami, and Wolkow 2007).

#### **6.2 IGF-1 signalling in** *Drosophila melanogaster*

*D. melanogaster* posseses endocrine tissues, which are similar to mammals (Toivonen and Partridge 2009). The median neurosecretory cells (mNSCs) of *Drosophila* are localised in the pars intercerebralis. mNSCs generate three of the seven *Drosophila* insulin-like peptides (DILPs). These mNSCs are functionally similar to β-cells of the pancreas which produce

Role of Central Insulin-Like Growth Factor-1

al. 2006).

animals are discussed.

Simari, and Conover 2006).

Receptor Signalling in Ageing and Endocrine Regulation 229

backgrounds and diets (Coschigano et al. 2003; Bartke and Brown-Borg 2004; Bonkowski et

As described above, GH action is mainly, but not completely, mediated by IGF-1 and circulating levels of IGF-1 are drastically reduced in GH-deficient Ames and Snell dwarf mice as well as in GH-resistant Laron dwarf mice (Brown-Borg et al. 1996; Coschigano et al. 2000; Flurkey et al. 2001). This reduction is primarily due to decreased expression of IGF-1 in the liver, whereas locally derived IGF-1 is, if at all, less affected (Bartke 2011). Normal expression of IGF-1 in the brain, especially in the hippocampus, might explain the maintenance of cognitive function in the long-lived mutants of the GH/IGF-1 system (Kinney et al. 2001). Besides reduced circulating IGF-1, lower insulin levels and enhanced insulin sensitivity are among the most prominent endocrine features shared by Ames dwarf, Snell dwarf and GHRKO mice (Bartke 2006) and have been found to be beneficial for ageing and survival (Bartke 2011). Additionally, increased adiponectin levels, which are associated with improved insulin sensitivity, anti-inflammatory and anti-atherogenic effects, were observed in all three mouse lines (Berryman et al. 2004; Wang et al. 2006). GH-deficient and -resistant mice were also found to have other phenotypic characteristics through which lifespan is thought to be increased, such as resistance to oxidative and other cytotoxic stresses (Bartke et al. 2001; Murakami, Salmon, and Miller 2003), increased activity of antioxidant enzymes (Hauck et al. 2002; Bartke et al. 2001; Romanick, Rakoczy, and Brown-Borg 2004), reduced body temperature (Hunter et al. 1999; Hauck et al. 2001) and reduced

susceptibility to cancer (Yang et al. 1996; Deitel et al. 2002; Ikeno et al. 2003).

Another model for extended healthy lifespan in mammals is the fat-specific insulin receptor knockout (FIRKO) mouse (Bluher, Kahn, and Kahn 2003). These mutant mice maintain low body fat despite normal food intake, have normal lipid metabolism and demonstrate improved glucose tolerance and insulin sensitivity throughout life (Bluher, Kahn, and Kahn 2003). The underlying mechanisms of this model are not well understood and apart from impaired IR signalling, altered inflammatory state and mitochondrial activity in these

A remarkable increase of mean and maximal lifespan has been found in female and male mice lacking PAPP-A. PAPP-A is a metalloproteinase that cleaves inhibitory IGFBPs (as described above), thereby increasing local bioavailability of IGF-1 without altering IGF-1 expression (Lawrence et al. 1999). Conversely, deletion of PAPP-A in PAPP-A-/- mice results in reduced local IGF-1 levels, and thus PAPP-A-/- mice are born as dwarfs (Conover et al. 2004). In these mutants, extension of lifespan was not associated with impaired glucose or fat metabolism and serum IGF-1, insulin and GH levels were unaltered (Conover and Bale 2007). However, these mice have markedly reduced incidence of spontaneous tumors (Conover and Bale 2007) and were found to be resistant to develop experimentally induced neointimal hyperplasia and atherosclerosis (Harrington, Simari, and Conover 2007; Resch,

As described above, homozygous deletion of the IGF-1R or IGF-1 is lethal or produces severe developmental defects, however extended lifespan was reported in mice with heterozygous deletion of the IGF-1R (IGF-1R+/-) (Holzenberger et al. 2003). IGF-1R+/- mice have upregulated serum IGF-1 levels, which might indicate an endocrine compensatory response to the reduced numbers of IGF-1R, and these mice do not develop dwarfism (Holzenberger et al. 2003). Those mutants have no alterations in energy metabolism, physical activity, fertility and reproduction but display greater resistance to oxidative stress (Holzenberger et al. 2003) probably causing the observed lifespan extension, which is

insulin. These neurosecretory cells show an analogue development to the mammalian anterior pituitary (Wang et al. 2007). The ablation of mNSCs causes lifespan extension, because of the depletion of DILP-2, -3 and -5 (Broughton et al. 2005). In a further study using a dominant-negative form of p53 expressed in the CNS of flies, lifespan extension was induced (Bauer et al. 2005; Slee, O'Connor, and Lu 2004). This correlates with decreased DILP-2 expression and reduced PI3K activity in the periphery (Bauer et al. 2007).

In addition to ablation of mNSCs, mutation of the insulin-like receptor (InR) or of its substrate Chico extends lifespan (Clancy et al. 2001; Tu, Epstein, and Tatar 2002; Tatar et al. 2001). The activation of the InR causes phosphorylation of dFOXO, which is equivalent to nematode DAF-16 and mammalian FoxO1 and FoxO3a (Puig et al. 2003; Junger et al. 2003). dFOXO activation in the pericerebral fat body has been shown to regulate ageing. This activation of dFOXO reduces the expression of DLP-2 in mNSCs followed by downregulation of the InR signalling pathway in peripheral fat tissue (Hwangbo et al. 2004) The JNK signalling pathway in neurons enhances stress resistance and lifespan extension partially via dFOXO (Essers et al. 2004; Wang, Bohmann, and Jasper 2005). Oxidative stress induces JNK signalling and subsequently promotes the nuclear localisation of dFOXO and induces expression of antioxidant proteins (Huang and Tindall 2007; Kops et al. 2002). Neurons are prone to oxidative stress based on the high production of ROS and low amount of antioxidant proteins (Lin and Beal 2006). Peroxiredoxin abolishes hydroperoxide using thioredoxin as hydrogen donor, which decreases ROS (reactive oxygen species) (Lim et al. 1993). Mammalians express six peroxiredoxins (I-VI). Peroxiredoxin II is solely expressed in the brain (Jin et al. 2005). Jafrac1, the homologue of the human Peroxiredoxin II (hPrxII), is a downstream protein of JNK signalling and target gene of dFOXO in neurons. Jafrac1 reduces reactive oxygen species (ROS) and extends lifespan (Lee et al. 2009) linking decreased IIS to increased clearance of ROS as possible mechanism for lifespan extension.

#### **6.3 IGF-1 signalling regulates mammalian lifespan**

In mice, it is well known that adult body size is an indicator of GH/IGF-1 actions and negatively correlated to longevity (Rollo 2002; Miller et al. 2002). Furthermore, IGF-1 plasma levels are negatively correlated with median lifespan (Yuan et al. 2009). Up to date, several mouse models of healthy ageing and longevity have been identified and analysed. Most of those long-lived mice have alterations within the IIS, GH or mTOR signalling pathway, indicating their key-role in influencing the process of ageing itself (Broughton and Partridge 2009; Kenyon 2010; Kenyon 2005; Piper et al. 2008; Bluher, Kahn, and Kahn 2003; Kappeler et al. 2008; Selman et al. 2008).

Highest impact on lifespan extension seems to have the ablation of GH signalling, as mutants with impaired GH action demonstrate higher increase of median and maximal lifespan than those with mutations that directly impact IGF-1 signalling (Coschigano et al. 2000; Coschigano et al. 2003; Brown-Borg et al. 1996; Flurkey et al. 2001). Examples for altered GH signalling are the Ames dwarf mice (Prop1def/def), Snell dwarf mice (Pit-1def/def) and Laron dwarf mice (GHR-/-). As the transcription factors Prop1 and Pit-1 are also essential for induction of thyroidea stimulating hormone (TSH) and prolactin, Ames dwarf mice and Snell dwarf mice are not only GH deficient but in addition produce less thyroidea stimulating hormone (TSH) and prolactin. Therefore, it can not be excluded that some of the effects seen in Ames and Snell dwarf mice might arise through deletion of TSH and prolactin. Laron dwarf mice (GHRKO ,GHR-/-) are only resistant to GH signalling, and their longevity phenotype has been reproducible in different laboratories on different genetic

insulin. These neurosecretory cells show an analogue development to the mammalian anterior pituitary (Wang et al. 2007). The ablation of mNSCs causes lifespan extension, because of the depletion of DILP-2, -3 and -5 (Broughton et al. 2005). In a further study using a dominant-negative form of p53 expressed in the CNS of flies, lifespan extension was induced (Bauer et al. 2005; Slee, O'Connor, and Lu 2004). This correlates with decreased

In addition to ablation of mNSCs, mutation of the insulin-like receptor (InR) or of its substrate Chico extends lifespan (Clancy et al. 2001; Tu, Epstein, and Tatar 2002; Tatar et al. 2001). The activation of the InR causes phosphorylation of dFOXO, which is equivalent to nematode DAF-16 and mammalian FoxO1 and FoxO3a (Puig et al. 2003; Junger et al. 2003). dFOXO activation in the pericerebral fat body has been shown to regulate ageing. This activation of dFOXO reduces the expression of DLP-2 in mNSCs followed by downregulation of the InR signalling pathway in peripheral fat tissue (Hwangbo et al. 2004) The JNK signalling pathway in neurons enhances stress resistance and lifespan extension partially via dFOXO (Essers et al. 2004; Wang, Bohmann, and Jasper 2005). Oxidative stress induces JNK signalling and subsequently promotes the nuclear localisation of dFOXO and induces expression of antioxidant proteins (Huang and Tindall 2007; Kops et al. 2002). Neurons are prone to oxidative stress based on the high production of ROS and low amount of antioxidant proteins (Lin and Beal 2006). Peroxiredoxin abolishes hydroperoxide using thioredoxin as hydrogen donor, which decreases ROS (reactive oxygen species) (Lim et al. 1993). Mammalians express six peroxiredoxins (I-VI). Peroxiredoxin II is solely expressed in the brain (Jin et al. 2005). Jafrac1, the homologue of the human Peroxiredoxin II (hPrxII), is a downstream protein of JNK signalling and target gene of dFOXO in neurons. Jafrac1 reduces reactive oxygen species (ROS) and extends lifespan (Lee et al. 2009) linking decreased IIS to increased clearance of ROS as possible mechanism for lifespan extension.

In mice, it is well known that adult body size is an indicator of GH/IGF-1 actions and negatively correlated to longevity (Rollo 2002; Miller et al. 2002). Furthermore, IGF-1 plasma levels are negatively correlated with median lifespan (Yuan et al. 2009). Up to date, several mouse models of healthy ageing and longevity have been identified and analysed. Most of those long-lived mice have alterations within the IIS, GH or mTOR signalling pathway, indicating their key-role in influencing the process of ageing itself (Broughton and Partridge 2009; Kenyon 2010; Kenyon 2005; Piper et al. 2008; Bluher, Kahn, and Kahn 2003; Kappeler

Highest impact on lifespan extension seems to have the ablation of GH signalling, as mutants with impaired GH action demonstrate higher increase of median and maximal lifespan than those with mutations that directly impact IGF-1 signalling (Coschigano et al. 2000; Coschigano et al. 2003; Brown-Borg et al. 1996; Flurkey et al. 2001). Examples for altered GH signalling are the Ames dwarf mice (Prop1def/def), Snell dwarf mice (Pit-1def/def) and Laron dwarf mice (GHR-/-). As the transcription factors Prop1 and Pit-1 are also essential for induction of thyroidea stimulating hormone (TSH) and prolactin, Ames dwarf mice and Snell dwarf mice are not only GH deficient but in addition produce less thyroidea stimulating hormone (TSH) and prolactin. Therefore, it can not be excluded that some of the effects seen in Ames and Snell dwarf mice might arise through deletion of TSH and prolactin. Laron dwarf mice (GHRKO ,GHR-/-) are only resistant to GH signalling, and their longevity phenotype has been reproducible in different laboratories on different genetic

DILP-2 expression and reduced PI3K activity in the periphery (Bauer et al. 2007).

**6.3 IGF-1 signalling regulates mammalian lifespan** 

et al. 2008; Selman et al. 2008).

backgrounds and diets (Coschigano et al. 2003; Bartke and Brown-Borg 2004; Bonkowski et al. 2006).

As described above, GH action is mainly, but not completely, mediated by IGF-1 and circulating levels of IGF-1 are drastically reduced in GH-deficient Ames and Snell dwarf mice as well as in GH-resistant Laron dwarf mice (Brown-Borg et al. 1996; Coschigano et al. 2000; Flurkey et al. 2001). This reduction is primarily due to decreased expression of IGF-1 in the liver, whereas locally derived IGF-1 is, if at all, less affected (Bartke 2011). Normal expression of IGF-1 in the brain, especially in the hippocampus, might explain the maintenance of cognitive function in the long-lived mutants of the GH/IGF-1 system (Kinney et al. 2001). Besides reduced circulating IGF-1, lower insulin levels and enhanced insulin sensitivity are among the most prominent endocrine features shared by Ames dwarf, Snell dwarf and GHRKO mice (Bartke 2006) and have been found to be beneficial for ageing and survival (Bartke 2011). Additionally, increased adiponectin levels, which are associated with improved insulin sensitivity, anti-inflammatory and anti-atherogenic effects, were observed in all three mouse lines (Berryman et al. 2004; Wang et al. 2006). GH-deficient and -resistant mice were also found to have other phenotypic characteristics through which lifespan is thought to be increased, such as resistance to oxidative and other cytotoxic stresses (Bartke et al. 2001; Murakami, Salmon, and Miller 2003), increased activity of antioxidant enzymes (Hauck et al. 2002; Bartke et al. 2001; Romanick, Rakoczy, and Brown-Borg 2004), reduced body temperature (Hunter et al. 1999; Hauck et al. 2001) and reduced susceptibility to cancer (Yang et al. 1996; Deitel et al. 2002; Ikeno et al. 2003).

Another model for extended healthy lifespan in mammals is the fat-specific insulin receptor knockout (FIRKO) mouse (Bluher, Kahn, and Kahn 2003). These mutant mice maintain low body fat despite normal food intake, have normal lipid metabolism and demonstrate improved glucose tolerance and insulin sensitivity throughout life (Bluher, Kahn, and Kahn 2003). The underlying mechanisms of this model are not well understood and apart from impaired IR signalling, altered inflammatory state and mitochondrial activity in these animals are discussed.

A remarkable increase of mean and maximal lifespan has been found in female and male mice lacking PAPP-A. PAPP-A is a metalloproteinase that cleaves inhibitory IGFBPs (as described above), thereby increasing local bioavailability of IGF-1 without altering IGF-1 expression (Lawrence et al. 1999). Conversely, deletion of PAPP-A in PAPP-A-/- mice results in reduced local IGF-1 levels, and thus PAPP-A-/- mice are born as dwarfs (Conover et al. 2004). In these mutants, extension of lifespan was not associated with impaired glucose or fat metabolism and serum IGF-1, insulin and GH levels were unaltered (Conover and Bale 2007). However, these mice have markedly reduced incidence of spontaneous tumors (Conover and Bale 2007) and were found to be resistant to develop experimentally induced neointimal hyperplasia and atherosclerosis (Harrington, Simari, and Conover 2007; Resch, Simari, and Conover 2006).

As described above, homozygous deletion of the IGF-1R or IGF-1 is lethal or produces severe developmental defects, however extended lifespan was reported in mice with heterozygous deletion of the IGF-1R (IGF-1R+/-) (Holzenberger et al. 2003). IGF-1R+/- mice have upregulated serum IGF-1 levels, which might indicate an endocrine compensatory response to the reduced numbers of IGF-1R, and these mice do not develop dwarfism (Holzenberger et al. 2003). Those mutants have no alterations in energy metabolism, physical activity, fertility and reproduction but display greater resistance to oxidative stress (Holzenberger et al. 2003) probably causing the observed lifespan extension, which is

Role of Central Insulin-Like Growth Factor-1

Fig. 4. GH/IGF-1 in ageing

diabetes.

serum levels

Receptor Signalling in Ageing and Endocrine Regulation 231

population studies (Bartke 2011) and serum IGF-1 levels correlate with cancer mortality in elderly men (Major et al. 2010). Therefore, the decline of GH secretion in the process of ageing might not simply reflect a progressive failure of the hypothalamus-somatotrope axis, but might be protective for the development of insulin resistance and cancer (Bartke 2003; Shechter et al. 2007). GH/IGF-1 therapy in elderly and GH deficient patients improves the ratio of lean and fat body mass, the lipid profile, protein synthesis, bone density, immune functions and memory (Khan et al. 2001), but certainly bears adverse side-effects, as GH is known to be diabetogenic and IGF-1 might increase the risk of cancer (Juul 1999; Khan et al.

Serum levels of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) decrease during ageing contributing to increased fat mass and decreased lean mass and might influence cognitive function negatively. Serum insulin levels increase with age indicating age-associated insulin insensitivity possibly leading to impaired glucose tolerance and

lean mass **↓**

insulin

IGF-1 GH

cognitive function **↓**?

adipose tissue **↑**

glucose tolerance **↓** cognitive function **↓**?

resulting changes

Recently, a study on a cohort of Ecuadorian patients carrying mutations in the GHR gene was published after 22 years of monitoring. Patients demonstrated severe GH resistance and reduced circulating IGF-1 and IGF-2 levels resulting in short stature (Guevara-Aguirre et al. 2011). No extension in lifespan was found in this cohort, which is in contrast to the GHR deficient mouse model (Guevara-Aguirre et al. 2011). However, causes of death were different in unaffected relatives compared to GHR deficient subjects, which died much more frequently from accidents, alcohol-related diseases and convulsive disorders, which might be the reason for unchanged lifespan (Guevara-Aguirre et al. 2011). Cancer accounted for 17 % and diabetes for 5 % of all diseases in non-affected relatives, whereas only one nonlethal malignancy and no cases of diabetes, probably due to improved insulin sensitivity, were reported for the individuals with GHR mutations (Guevara-Aguirre et al. 2011). Importantly, GHR deficient subjects appear to have no increased mortality from vascular diseases compared to their relatives (30 % of deaths verses 33 % of deaths in the nonaffected relatives), however the proportion of strokes (3 % verses 12 %) and cardiac disease

(27 % verses 21 %) was slightly different (Guevara-Aguirre et al. 2011).

2001; Pollak, Schernhammer, and Hankinson 2004; Major et al. 2010).

age

predominantly seen in females. As described earlier, the selective reduction of IGF-1 signalling in the brain of bIGF1R+/- mice leads to an increase in median but not maximal lifespan (Kappeler et al. 2008).

The knockout models for IRS-proteins provide evidence that interference with IGF-1/insulin signalling downstream of the IGF-1 and insulin receptor influences lifespan in mice. Female IRS-1 knockout (IRS-1-/-) mice were reported to be long-lived and resistant to a number of conditions related to neurological and neuromuscular, immune, skin and bone disease (Selman et al. 2008). In these animals, body weight including fat mass was reduced, but endocrine function of the pituitary as well as circulating IGF-1 levels were preserved (Selman et al. 2008). Additionally, female IRS-1-/- mice were hyperinsulinemic and IGF-1/insulin resistant and rectal temperature was significantly elevated (Selman et al. 2008). All these characteristics are in contrast to the long-lived GH-deficient and GH-resistant mice, however female IRS-1-/- mutants have similar alterations in expression of genes involved in oxidative stress defence and DNA repair (Selman et al. 2008). In the same study, no significant increase of lifespan was found for male IRS-1-/- mice, as well as for IRS-1+/- and IRS-2+/- mice of both sexes, and IRS-2-/- mice were even short-lived (Selman et al. 2008).

IRS-2+/- mice were found to be long-lived, slightly larger and more insulin sensitive and glucose tolerant than wild-type mice (Taguchi, Wartschow, and White 2007). However, the animals studied by Selman et al. (2008) were fed a 'standard' rodent diet with a fat content of 5 %, while those studied by Taguchi et al. (2007) were on a 'high energy' diet with a fat content of 9 %, hence indicating that the IRS-2+/- mice might be protected from the harming effects of a high-fat diet (Bartke 2008). In addition, Taguchi et al. (2007) reported that brain specific alterations of IRS-2 in bIRS-2+/- and bIRS-2-/- mice result in increased lifespan despite glucose and insulin resistance (Taguchi, Wartschow, and White 2007). Further work will be needed to clarify and reconcile the observations made in those two laboratories (Bartke 2008).

Regardless the criticism on the reproducibility of the observations made by Holzenberger (Kappeler et al. 2008) and Taguchi (2007), they provide evidence that selective disruption of the IIS pathway in neurons might not only extend lifespan in *C.elegans* and *Drosophila* (Wolkow et al. 2000; Kenyon 2005) but also in mammals. These findings may indirectly be verified by other studies that demonstrate a protection from AD pathology in a transgenic AD mouse model via deletion of IGF-1R or IRS-2 (Cohen et al. 2009; Freude et al. 2009; Killick et al. 2009).

#### **6.4 IGF-1 signalling and its relevance to human longevity**

The IIS pathway is highly conserved throughout evolution from nematodes and flies to mammals. In the last years, an increasing body of data suggests that this pathway is important for human longevity as well.

GH secretion and IGF-1 levels decrease with age (Rudman et al. 1981), which contributes to ageing-associated changes in body composition such as increased adipose tissue and reduced lean body mass leading to impaired insulin sensitivity and cardiovascular disease. The same observations are made in patients with GH deficiency (Khan et al. 2001) and mutation in the GHR gene: Low serum IGF-1 levels, short stature, obesity, glucose intolerance and possible mental retardation but no decrease in life expectancy (Rincon, Rudin, and Barzilai 2005). Conversely, pathologic GH excess in acromegaly leads to reduced life expectancy due to cardiovascular disease, diabetes and malignancies (Orme et al. 1998). Additionally, body height has been identified as a cancer risk in a number of large

predominantly seen in females. As described earlier, the selective reduction of IGF-1 signalling in the brain of bIGF1R+/- mice leads to an increase in median but not maximal

The knockout models for IRS-proteins provide evidence that interference with IGF-1/insulin signalling downstream of the IGF-1 and insulin receptor influences lifespan in mice. Female IRS-1 knockout (IRS-1-/-) mice were reported to be long-lived and resistant to a number of conditions related to neurological and neuromuscular, immune, skin and bone disease (Selman et al. 2008). In these animals, body weight including fat mass was reduced, but endocrine function of the pituitary as well as circulating IGF-1 levels were preserved (Selman et al. 2008). Additionally, female IRS-1-/- mice were hyperinsulinemic and IGF-1/insulin resistant and rectal temperature was significantly elevated (Selman et al. 2008). All these characteristics are in contrast to the long-lived GH-deficient and GH-resistant mice, however female IRS-1-/- mutants have similar alterations in expression of genes involved in oxidative stress defence and DNA repair (Selman et al. 2008). In the same study, no significant increase of lifespan was found for male IRS-1-/- mice, as well as for IRS-1+/- and IRS-2+/- mice of both sexes, and IRS-2-/- mice were even short-lived (Selman et al. 2008). IRS-2+/- mice were found to be long-lived, slightly larger and more insulin sensitive and glucose tolerant than wild-type mice (Taguchi, Wartschow, and White 2007). However, the animals studied by Selman et al. (2008) were fed a 'standard' rodent diet with a fat content of 5 %, while those studied by Taguchi et al. (2007) were on a 'high energy' diet with a fat content of 9 %, hence indicating that the IRS-2+/- mice might be protected from the harming effects of a high-fat diet (Bartke 2008). In addition, Taguchi et al. (2007) reported that brain specific alterations of IRS-2 in bIRS-2+/- and bIRS-2-/- mice result in increased lifespan despite glucose and insulin resistance (Taguchi, Wartschow, and White 2007). Further work will be needed to clarify and reconcile the observations made in those two laboratories

Regardless the criticism on the reproducibility of the observations made by Holzenberger (Kappeler et al. 2008) and Taguchi (2007), they provide evidence that selective disruption of the IIS pathway in neurons might not only extend lifespan in *C.elegans* and *Drosophila* (Wolkow et al. 2000; Kenyon 2005) but also in mammals. These findings may indirectly be verified by other studies that demonstrate a protection from AD pathology in a transgenic AD mouse model via deletion of IGF-1R or IRS-2 (Cohen et al. 2009; Freude et al. 2009;

The IIS pathway is highly conserved throughout evolution from nematodes and flies to mammals. In the last years, an increasing body of data suggests that this pathway is

GH secretion and IGF-1 levels decrease with age (Rudman et al. 1981), which contributes to ageing-associated changes in body composition such as increased adipose tissue and reduced lean body mass leading to impaired insulin sensitivity and cardiovascular disease. The same observations are made in patients with GH deficiency (Khan et al. 2001) and mutation in the GHR gene: Low serum IGF-1 levels, short stature, obesity, glucose intolerance and possible mental retardation but no decrease in life expectancy (Rincon, Rudin, and Barzilai 2005). Conversely, pathologic GH excess in acromegaly leads to reduced life expectancy due to cardiovascular disease, diabetes and malignancies (Orme et al. 1998). Additionally, body height has been identified as a cancer risk in a number of large

**6.4 IGF-1 signalling and its relevance to human longevity** 

important for human longevity as well.

lifespan (Kappeler et al. 2008).

(Bartke 2008).

Killick et al. 2009).

population studies (Bartke 2011) and serum IGF-1 levels correlate with cancer mortality in elderly men (Major et al. 2010). Therefore, the decline of GH secretion in the process of ageing might not simply reflect a progressive failure of the hypothalamus-somatotrope axis, but might be protective for the development of insulin resistance and cancer (Bartke 2003; Shechter et al. 2007). GH/IGF-1 therapy in elderly and GH deficient patients improves the ratio of lean and fat body mass, the lipid profile, protein synthesis, bone density, immune functions and memory (Khan et al. 2001), but certainly bears adverse side-effects, as GH is known to be diabetogenic and IGF-1 might increase the risk of cancer (Juul 1999; Khan et al. 2001; Pollak, Schernhammer, and Hankinson 2004; Major et al. 2010).

#### Fig. 4. GH/IGF-1 in ageing

Serum levels of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) decrease during ageing contributing to increased fat mass and decreased lean mass and might influence cognitive function negatively. Serum insulin levels increase with age indicating age-associated insulin insensitivity possibly leading to impaired glucose tolerance and diabetes.

Recently, a study on a cohort of Ecuadorian patients carrying mutations in the GHR gene was published after 22 years of monitoring. Patients demonstrated severe GH resistance and reduced circulating IGF-1 and IGF-2 levels resulting in short stature (Guevara-Aguirre et al. 2011). No extension in lifespan was found in this cohort, which is in contrast to the GHR deficient mouse model (Guevara-Aguirre et al. 2011). However, causes of death were different in unaffected relatives compared to GHR deficient subjects, which died much more frequently from accidents, alcohol-related diseases and convulsive disorders, which might be the reason for unchanged lifespan (Guevara-Aguirre et al. 2011). Cancer accounted for 17 % and diabetes for 5 % of all diseases in non-affected relatives, whereas only one nonlethal malignancy and no cases of diabetes, probably due to improved insulin sensitivity, were reported for the individuals with GHR mutations (Guevara-Aguirre et al. 2011). Importantly, GHR deficient subjects appear to have no increased mortality from vascular diseases compared to their relatives (30 % of deaths verses 33 % of deaths in the nonaffected relatives), however the proportion of strokes (3 % verses 12 %) and cardiac disease (27 % verses 21 %) was slightly different (Guevara-Aguirre et al. 2011).

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In a cohort of Ashkenazi Jewish centenarians, a gender-specific increase in serum IGF-1 associated with smaller stature in female offspring was found to be due to heterozygous mutations in the IGF-1R gene (Suh et al. 2008). These rare mutations were significantly more common in female centenarians and overrepresented compared to controls (Suh et al. 2008). These observations are in line with the IGF-1R+/- mouse model, in which female mice show growth retardation and extended lifespan (Holzenberger et al. 2003) but IGF-1 resistance.

Further studies on human centenarians, mainly screening selectively genes of the IIS pathway, identified certain genes in the insulin/IGF-1 pathway to be important for human longevity. Several cohorts with different genetic background have reported an association of FOXO3A single nucleotide polymorphisms (SNPs) with longevity (Willcox et al. 2008; Flachsbart et al. 2009; Anselmi et al. 2009; Pawlikowska et al. 2009; Li et al. 2009). Interestingly, the study on long-lived Han Chinese revealed a SNP of FOXO1 associated with female longevity exclusively (Li et al. 2009).

Furthermore, in the Leiden 85-plus Study an association of a certain FOXO1 haplotype with higher HbA1c levels, higher prevalence of diabetes, myocardial infarction as well as increased mortality was observed (Kuningas et al. 2007). In addition, a certain haplotype of the FOXO3A gene revealed increased risks for stroke and mortality (Kuningas et al. 2007). These findings indicate that alterations within the FOXO genes might be causative involved in age-associated diseases and regulation of lifespan (Flachsbart et al. 2009).
