**Role of Central Insulin-Like Growth Factor-1 Receptor Signalling in Ageing and Endocrine Regulation**

Lorna Moll, Johanna Zemva and Markus Schubert *Center of Endocrinology, Diabetes and Preventive Medicine Germany* 

#### **1. Introduction**

208 Basic and Clinical Endocrinology Up-to-Date

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regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17 alpha-hydroxylase/c17-20 lyase). *Mol*  Insulin-like growth factors (IGFs) are, besides other mechanisms, controlled by growth hormone (GH) secretion and there are at least two different IGFs: IGF-1 and IGF-2. IGFs regulate various cellular processes e.g. survival, differentiation and proliferation (McMorris et al. 1986; McMorris & Dubois-Dalcq 1988; Mozell & McMorris 1991).

Growth hormone, which induces IGF-1 secretion from the liver, is generated in the anterior pituitary and regulated by the hypothalamus via growth hormone releasing hormone (GHRH) and growth hormone inhibiting hormone (GHIH) (Jansson et al., 1985; Carlsson & Jansson 1990). IGFs in the brain are synthesised de novo or transported across the blood brain barrier (BBB) and seem to induce a variety of effects on the central nervous system (CNS). So far, the exact transport mechanisms of IGFs into the brain are not fully understood (Duffy, Pardridge, and Rosenfeld 1988).

In the rodent brain, IGF-1 is mainly expressed in neuron-dense regions like the hippocampus, striatum, thalamus, hypothalamus and olfactory bulb (Rotwein et al. 1988; Bondy et al. 1990; Garcia-Segura et al. 1991). Accordingly, the insulin-like growth factor-1 receptors (IGF-1Rs) are mainly found in the olfactory bulb, cerebellar cortex and granule cell layer of the dentate gyrus (Rotwein et al. 1988; Bondy et al. 1990; Bondy and Lee 1993; Werther et al. 1990). However, the function of IGF-1R mediated signals in the central nervous system (CNS) is still under investigation.

After binding of IGF-1 to the IGF-1R, a signalling cascade is activated which leads to recruitment and subsequently phosphorylation of intracellular adaptor proteins, the so called insulin receptor substrates (IRS) (Jacobs et al. 1983; Rubin et al., 1983; Sun et al. 1991; Lavan et al. 1997; Lavan et al., 1997). Via these adaptor proteins, the MAP kinase (MAPK, mitogen activated protein kinase) cascade, as well as the phosphatidylinositide(PI)3-kinase signalling pathway, is turned on leading to protein kinase B (PKB/AKT) activation, which phosphorylates forkhead box O transcription factors (FoxO) causing their nuclear export (Stokoe et al. 1997; Alessi et al. 1996; Partridge & Bruning 2008). FoxO mediated transcription is involved in cell proliferation, differentiation and oxidative stress defence (Clark et al. 1993; Brunet et al. 1999; Dijkers et al. 2000; Dijkers, Medema, Pals et al. 2000; Medema et al. 2000) suggesting a function of FoxO during development and cellular stress response.

Role of Central Insulin-Like Growth Factor-1

**Hypothalamus** 

**Pituitary**

Fig. 1. GH/ IGF-1 system

**Liver** 

Receptor Signalling in Ageing and Endocrine Regulation 211

sex steroids interfere with GH secretion (Veldhuis et al. 2008, 2009; Leung et al. 2004). Body growth is influenced more efficiently by GH if it is applicated in a pulsatile fashion than applied continuously (Jansson, Eden, and Isaksson 1985; Clark et al. 1985). This might be due to a higher induction of IGF-1 expression in growth plates and skeletal muscles by pulsatile GH, whereas continuous GH secretion stimulates liver IGF-1 expression and serum IGF-1 levels at least as well as a pulsatile GH release (Isgaard et al. 1988; Bick et al. 1992). Therefore, liver-derived IGF-1 is not essential for body growth itself. This effect might be

**?** 

**IGF-1**

**GHIH \_**

**\_** 

**\_ IGF-1**

**IGFBPs** 

**IGFBPs** 

**\_** 

achieved by locally produced IGF-1 and/or other factors (Ohlsson et al. 2009).

**GH** 

**GH** 

**GHRH**

**+** 

**+** 

Growth hormone releasing hormone (GHRH) and growth hormone inhibiting hormone (GHIH) are produced in neurons of the hypothalamus and transported via the hypothalmopituitary portal vein system to the anterior pituitary where they regulate growth hormone

production in the liver. In addition, free IGF-1 serum levels are regulated by binding to the insulin-like growth factor binding proteins (IGFBPs). A short negative feedback loop of GH, as well as a negative feedback mechanism of IGF-1 on the hypothalamus and possibly on

Mouse models with tissue specific IGF-1 inactivation indicate that normal longitudinal bone growth is maintained by local bone-derived IGF-1 (Sjogren et al. 1999; Yakar et al. 1999). However, several other phenotypes, for example the one observed in the liver IGF-1

(GH) secretion and expression. GH stimulates insulin-like growth factor-1 (IGF-1)

the anterior pituitary, determines serum IGF-1 levels.

Recently, the function of the IGF-1R signalling pathway in neurons has been investigated using different model organisms like worms and flies as well as conventional and conditional mutagenesis in mice. These experiments revealed possible functions of IGF-1 mediated signals in endocrine regulation, longevity, protein turnover and in the pathogenesis for ageing-associated diseases e.g. Alzheimer's Disease (AD). The current article discusses the mechanisms of regulation and the physiological as well as a possible pathophysiological role of IGF-1 mediated signals in the CNS.

#### **2. The GH/insulin-like growth factor system**

#### **2.1 The somatotropic system**

Growth hormone (GH, somatotropin) is produced in the anterior pituitary by certain specified cells and regulated via the hypothalamus by the growth hormone releasing hormone (GHRH, somatoliberin), the growth hormone inhibiting hormone (GHIH, somatostatin), but also by suppression of the short loop negative feedback of GH itself (Jansson, Eden, and Isaksson 1985; Carlsson and Jansson 1990).

The GH-producing cells of the anterior pituitary, also called somatotropes or somatotroph, form 30-40 % of the anterior pituitary (Gage et al. 1996). These cells require transcription factors of the POU-family (Mullis 2010) including the homeoproteins Pit-1 and Prop-1 (prophet of Pit-1) for normal embryonic development and differentiation (Mullis 2005) as well as for GH expression. The expression of these transcription factors and therefore the differentiation of the somatotroph are controlled by GHRH producing neurons of the hypothalamus. Additionally, GH-release in the pituitary is inhibited by serum IGF-1 and enhanced by serum ghrelin (Wortley et al. 2005; Zigman et al. 2005) (Figure 1).

In the periphery, GH mainly influences growth and development via IGF-1. IGF-1 and IGF-2 can act endocrine, paracrine and autocrine (Cohick and Clemmons 1993; Stewart and Rotwein 1996; Mohan, Baylink, and Pettis 1996; Butler and LeRoith 2001). Additionally, there is a truncated form of IGF-1, Des(1-3)IGF-1, which lacks the tripeptide Gly-Pro-Glu at the N-terminus probably resulting from post-translational cleavage of IGF-1. Des(1-3)IGF-1 is mainly expressed in brain and active in lower concentrations than un-truncated IGF-1 (Ballard et al. 1996; Francis et al. 1988; Carlsson-Skwirut et al. 1989; Ogasawara et al. 1989). Studies on hypoxic-ischemic brain injuries in rats suggest that the tripeptide fragment has neuroprotective properties and influences neuronal activity (Guan et al. 1999).

Circulating IGF-1 is mainly produced in the liver, but both IGF-1 and IGF-2 are expressed in many non-hepatic tissues (Cohick and Clemmons 1993; Stewart and Rotwein 1996; Butler and LeRoith 2001). The most important effects of IGFs are cell proliferation and differentiation, skeletal growth and mineralisation as well as the development and function of the reproductive organs and the central nervous system (CNS) (Duan and Xu 2005).

GH is secreted pulsatile in rodents as well as in humans (Jansson, Eden, and Isaksson 1985; Frohman and Jansson 1986; Giustina and Veldhuis 1998) and follows a circadian pattern with one major peak after falling asleep. There is a sexual dimorphism in GH secretion, which is more dominant in rodents than in humans (Jansson, Eden, and Isaksson 1985; Tannenbaum and Martin 1976). In females, the GH secretory pattern seems to be less regular compared to males (Giustina and Veldhuis 1998), with higher basal interpulse GH levels, more frequent but lower amplitude pulses and a lower major nocturnal GH pulse (Jaffe et al. 1998; Jessup et al. 2003). Spontaneous and GHRH-stimulated GH secretion is suppressed more efficiently by IGF-1 in men than in women (Ohlsson et al. 2009) suggesting that also

Recently, the function of the IGF-1R signalling pathway in neurons has been investigated using different model organisms like worms and flies as well as conventional and conditional mutagenesis in mice. These experiments revealed possible functions of IGF-1 mediated signals in endocrine regulation, longevity, protein turnover and in the pathogenesis for ageing-associated diseases e.g. Alzheimer's Disease (AD). The current article discusses the mechanisms of regulation and the physiological as well as a possible

Growth hormone (GH, somatotropin) is produced in the anterior pituitary by certain specified cells and regulated via the hypothalamus by the growth hormone releasing hormone (GHRH, somatoliberin), the growth hormone inhibiting hormone (GHIH, somatostatin), but also by suppression of the short loop negative feedback of GH itself

The GH-producing cells of the anterior pituitary, also called somatotropes or somatotroph, form 30-40 % of the anterior pituitary (Gage et al. 1996). These cells require transcription factors of the POU-family (Mullis 2010) including the homeoproteins Pit-1 and Prop-1 (prophet of Pit-1) for normal embryonic development and differentiation (Mullis 2005) as well as for GH expression. The expression of these transcription factors and therefore the differentiation of the somatotroph are controlled by GHRH producing neurons of the hypothalamus. Additionally, GH-release in the pituitary is inhibited by serum IGF-1 and

In the periphery, GH mainly influences growth and development via IGF-1. IGF-1 and IGF-2 can act endocrine, paracrine and autocrine (Cohick and Clemmons 1993; Stewart and Rotwein 1996; Mohan, Baylink, and Pettis 1996; Butler and LeRoith 2001). Additionally, there is a truncated form of IGF-1, Des(1-3)IGF-1, which lacks the tripeptide Gly-Pro-Glu at the N-terminus probably resulting from post-translational cleavage of IGF-1. Des(1-3)IGF-1 is mainly expressed in brain and active in lower concentrations than un-truncated IGF-1 (Ballard et al. 1996; Francis et al. 1988; Carlsson-Skwirut et al. 1989; Ogasawara et al. 1989). Studies on hypoxic-ischemic brain injuries in rats suggest that the tripeptide fragment has

Circulating IGF-1 is mainly produced in the liver, but both IGF-1 and IGF-2 are expressed in many non-hepatic tissues (Cohick and Clemmons 1993; Stewart and Rotwein 1996; Butler and LeRoith 2001). The most important effects of IGFs are cell proliferation and differentiation, skeletal growth and mineralisation as well as the development and function of the reproductive organs and the central nervous system (CNS) (Duan and Xu 2005).GH is secreted pulsatile in rodents as well as in humans (Jansson, Eden, and Isaksson 1985; Frohman and Jansson 1986; Giustina and Veldhuis 1998) and follows a circadian pattern with one major peak after falling asleep. There is a sexual dimorphism in GH secretion, which is more dominant in rodents than in humans (Jansson, Eden, and Isaksson 1985; Tannenbaum and Martin 1976). In females, the GH secretory pattern seems to be less regular compared to males (Giustina and Veldhuis 1998), with higher basal interpulse GH levels, more frequent but lower amplitude pulses and a lower major nocturnal GH pulse (Jaffe et al. 1998; Jessup et al. 2003). Spontaneous and GHRH-stimulated GH secretion is suppressed more efficiently by IGF-1 in men than in women (Ohlsson et al. 2009) suggesting that also

enhanced by serum ghrelin (Wortley et al. 2005; Zigman et al. 2005) (Figure 1).

neuroprotective properties and influences neuronal activity (Guan et al. 1999).

pathophysiological role of IGF-1 mediated signals in the CNS.

(Jansson, Eden, and Isaksson 1985; Carlsson and Jansson 1990).

**2. The GH/insulin-like growth factor system** 

**2.1 The somatotropic system** 

sex steroids interfere with GH secretion (Veldhuis et al. 2008, 2009; Leung et al. 2004). Body growth is influenced more efficiently by GH if it is applicated in a pulsatile fashion than applied continuously (Jansson, Eden, and Isaksson 1985; Clark et al. 1985). This might be due to a higher induction of IGF-1 expression in growth plates and skeletal muscles by pulsatile GH, whereas continuous GH secretion stimulates liver IGF-1 expression and serum IGF-1 levels at least as well as a pulsatile GH release (Isgaard et al. 1988; Bick et al. 1992). Therefore, liver-derived IGF-1 is not essential for body growth itself. This effect might be achieved by locally produced IGF-1 and/or other factors (Ohlsson et al. 2009).

Growth hormone releasing hormone (GHRH) and growth hormone inhibiting hormone (GHIH) are produced in neurons of the hypothalamus and transported via the hypothalmopituitary portal vein system to the anterior pituitary where they regulate growth hormone (GH) secretion and expression. GH stimulates insulin-like growth factor-1 (IGF-1) production in the liver. In addition, free IGF-1 serum levels are regulated by binding to the insulin-like growth factor binding proteins (IGFBPs). A short negative feedback loop of GH, as well as a negative feedback mechanism of IGF-1 on the hypothalamus and possibly on the anterior pituitary, determines serum IGF-1 levels.

Mouse models with tissue specific IGF-1 inactivation indicate that normal longitudinal bone growth is maintained by local bone-derived IGF-1 (Sjogren et al. 1999; Yakar et al. 1999). However, several other phenotypes, for example the one observed in the liver IGF-1

Role of Central Insulin-Like Growth Factor-1

Receptor Signalling in Ageing and Endocrine Regulation 213

those of the IGF-1 receptor (Duan and Xu 2005). Additionally, there are several proteins with lower IGF-1 binding affinity, called IGFBP-related peptides (IGFBP-rPs), which have significant structural homologies with the amino(N)-terminal region of high-affinity IGFBPs (Rechler 1993; Kelley et al. 1996; Rajaram, Baylink, and Mohan 1997; Kim et al. 1997; Poretsky et al. 1999; Baxter 2000). So far, the functions of the IGFBP-rPs, also referred to as IGFBP- 7 to - 10, are not known (Mohan and Baylink 2002). Approximately 75-80 % of IGF is present as a 150 kDa complex, which consists of IGF-1/IGF-2 plus IGFBP-3 (70-75 %) or IGFPB-5 (5-10 %) and an acid-labile subunit (ALS) (Baxter, Meka, and Firth 2002). ALS is mainly produced in the liver (Baxter 1988; Baxter and Martin 1989; Baxter, Martin, and Beniac 1989) and its gene disruption leads to a reduction of circulating IGFs of 80 % (Boisclair et al. 2001). The ternary complex of IGF-1 or -2/ IGFBP-3 or -5/ ALS extends the half-life of IGFs to 15-20 h, compared to a half-life of 20-30 min for free IGF-1 in circulation (Guler, Zapf, and Froesch 1987) suggesting a reservoir function of this complex, which cannot cross the vascular endothelial barrier (Rajaram, Baylink, and Mohan 1997; Baxter 2000). 20-25 % of the IGFs bind to IGFBP-1,- 2,-4 or -6 to form a complex, which is able to cross vascular endothelium (Baxter 2000; Rajaram, Baylink, and Mohan 1997). Thus, the endocrine actions of IGF-1 in serum are regulated by the

Furthermore, IGFBPs are located in the extracellular matrix or on the cell surface where they either inhibit (mainly IGFBP-4 and -6) or potentiate (mainly IGFBP-3 and -5) IGF-1 binding to its receptors (Rajaram, Baylink, and Mohan 1997; Mohan et al. 1995; Qin et al. 1998; Jones and Clemmons 1995; Firth and Baxter 2002; Rechler and Clemmons 1998). Functions of the IGFBPs might vary indicated by the inhibiting or potentiating effect of IGFBP-1, -2, -3 and -5

The actions of IGFBPs are, in turn, modulated by IGFBP proteases that are further dependent on activators and inhibitors (Mohan et al. 2002). Those IGFBP proteases are able to cleave IGFBPs, thereby reducing their affinity for IGF-1. Some of them are relatively specific for a given IGFBP. For example, pregnancy-associated plasma protein-A (PAPP-A), which is produced by a variety of cell types, cleaves specifically IGFBP-4 (Lawrence et al. 1999; Byun et al. 2001; Conover et al. 2001), whereas complement C1s (Busby et al. 2000) and a disintigrin and metalloprotease-like (ADAM)-9 (Mohan et al. 2002) were identified to be relatively specific for IGFBP-5. Additionally, a number of other serum proteases were shown to be capable of cleaving IGFBPs, e.g. plasmin, cathepsin D and prostate specific antigen (Conover 1995; Fowlkes et al. 1995; Rajah et al. 1995; Rajaram, Baylink, and Mohan 1997; Maile and Holly 1999). Therefore, the binding affinity and hence the bio-availability of IGF-1

IGFBPs also have IGF independent functions, for example IGFBP-2 and -3 can induce direct cellular effects (Firth and Baxter 2002; Oh et al. 1993; Yamanaka et al. 1999; Schutt et al. 2004) and it has been shown that IGFBP-3 at least partially mediates these effects by binding to the cell surface, possibly to specific receptors (Mohseni-Zadeh and Binoux 1997; Rechler and Clemmons 1998). IGFBP-2, -3 and -5 contain sequences for nuclear localisation (Schutt et al. 2004; Radulescu 1994; Schedlich et al. 1998; Hoeflich et al. 2004) and might as well influence

Insulin and IGF-1 receptors are receptor tyrosine kinases. Receptor tyrosine kinases contain a membrane-bound domain with tyrosine kinase activity which phosphorylates tyrosineresidues of downstream signalling proteins. Typical members of the receptor tyrosine kinase

on IGF-1 action depending on the experimental conditions (Yin, Xu, and Duan 2004).

is regulated via the degradation of IGFBPs through the IGFBP protease system.

**2.3 The insulin- and insulin-like growth factor-1 signalling pathway** 

gene expression.

IGFBPs determining how much IGF is bio-available to the local tissues.

deficient (LID) mouse, suggest that locally derived IGF-1 cannot fully replace liver-derived IGF-1 function and vice versa.

A lacking negative feedback of liver-derived IGF-1 on GH secretion leads to a compensatory increase in serum GH levels in mice (Yakar et al. 1999; Sjogren et al. 1999) and also in humans (Veldhuis et al. 2001). Hence, the phenotype of the LID mice might either arise directly or is mediated by the elevated GH-levels. LID mice show an increased expression of pituitary GHRH- and ghrelin-receptors indicating that at least some action of up-regulating GH secretion influences the pituitary (Ohlsson et al. 2009). However, the target sites of IGF-1 for regulating GH-release remain unclear and might either be located in the hypothalamus and/ or the pituitary. Recently, a cell-specific knockout mouse in which the IGF-1 receptor (IGF-1R) was ablated from the somatotroph, the so called somatotroph IGF-1 receptor knockout (SIGFRKO) mouse, has been generated (Romero et al. 2010). The phenotype of the SIGFRKO mouse includes increased GH expression and secretion as well as increased serum IGF-1 levels (Romero et al. 2010). Feedback mechanisms in the hypothalamus resulted in decreased GHRH and increased GHIH mRNA levels (Romero et al. 2010). Furthermore, decreased growth hormone releasing hormone receptor (GHRH-R) expression was observed in the anterior pituitary (Romero et al. 2010). However, these changes were not able to reduce GH secretion in the SIGFRKO mouse indicating a role of IGF-1R signalling in the pituitary in addition to the hypothalamus in regulating GH secretion (Romero et al. 2010).

IGF-1 influences glucose metabolism directly, e.g. by inducing insulin-like effects on glucose-uptake in tissues expressing IGF-1-receptors, but also by suppressing the GHrelease from the pituitary. GH is known to have diabetogenic effects (Yakar et al. 2004) by decreasing insulin-sensitivity in skeletal muscle, liver and fat. Consequently, LID mice show an impaired insulin-sensitivity. At the same time, those mice are protected against the increase in body fat mass that occurs in older age (Sjogren et al. 2001; Tang et al. 2005). This might be explained by elevated GH-levels in these mice (Ohlsson et al. 2009) as decreased GH secretion in both rodents and humans has been proposed to contribute to age-related obesity (Sonntag et al. 1980; Rudman et al. 1990).

The inactivation of IGF-1 or its receptor during early development reduces brain size including a reduction of the granule cell layer in the dentate gyrus and the number of oligodendrocytes and myelinated axons (Beck et al. 1995; Holzenberger et al. 2001; Vicario-Abejon et al. 2003) demonstrating the importance of IGF-1 signalling during brain development. LID mice show a milder phenotype with reduced exploratory activity (Svensson et al. 2005; Bohlooly et al. 2001), as well as impaired spatial learning and memory, suggesting that liver-derived IGF-1 enhances brain functions (Svensson et al. 2006). Circulating IGF-1 is essential for mediating exercise induced effects on the adult brain by promoting the numbers of newly generated neurons in the adult hippocampus, brain vessel growth, spatial learning and reducing anxiety (Trejo, Carro, and Torres-Aleman 2001; Trejo, Llorens-Martin, and Torres-Aleman 2008; Lopez-Lopez, LeRoith, and Torres-Aleman 2004). Additionally, liver-derived IGF-1 might enhance the clearance of brain amyloid-β (Aβ), whose aggregation is one of the hallmarks of AD (see 5.1).

#### **2.2 IGF-binding proteins**

Only a small amount of IGF-1 (~1 %) circulates "free" within the blood. The remaining 99 % are bound to IGF-binding proteins (IGFBPs). IGFBPs are a family of at least six proteins (IGFBP-1 to IGFBP-6), which bind IGF-1 with high affinities that are equal to or greater than

deficient (LID) mouse, suggest that locally derived IGF-1 cannot fully replace liver-derived

A lacking negative feedback of liver-derived IGF-1 on GH secretion leads to a compensatory increase in serum GH levels in mice (Yakar et al. 1999; Sjogren et al. 1999) and also in humans (Veldhuis et al. 2001). Hence, the phenotype of the LID mice might either arise directly or is mediated by the elevated GH-levels. LID mice show an increased expression of pituitary GHRH- and ghrelin-receptors indicating that at least some action of up-regulating GH secretion influences the pituitary (Ohlsson et al. 2009). However, the target sites of IGF-1 for regulating GH-release remain unclear and might either be located in the hypothalamus and/ or the pituitary. Recently, a cell-specific knockout mouse in which the IGF-1 receptor (IGF-1R) was ablated from the somatotroph, the so called somatotroph IGF-1 receptor knockout (SIGFRKO) mouse, has been generated (Romero et al. 2010). The phenotype of the SIGFRKO mouse includes increased GH expression and secretion as well as increased serum IGF-1 levels (Romero et al. 2010). Feedback mechanisms in the hypothalamus resulted in decreased GHRH and increased GHIH mRNA levels (Romero et al. 2010). Furthermore, decreased growth hormone releasing hormone receptor (GHRH-R) expression was observed in the anterior pituitary (Romero et al. 2010). However, these changes were not able to reduce GH secretion in the SIGFRKO mouse indicating a role of IGF-1R signalling in the pituitary in addition to the hypothalamus in regulating GH secretion (Romero et al. 2010). IGF-1 influences glucose metabolism directly, e.g. by inducing insulin-like effects on glucose-uptake in tissues expressing IGF-1-receptors, but also by suppressing the GHrelease from the pituitary. GH is known to have diabetogenic effects (Yakar et al. 2004) by decreasing insulin-sensitivity in skeletal muscle, liver and fat. Consequently, LID mice show an impaired insulin-sensitivity. At the same time, those mice are protected against the increase in body fat mass that occurs in older age (Sjogren et al. 2001; Tang et al. 2005). This might be explained by elevated GH-levels in these mice (Ohlsson et al. 2009) as decreased GH secretion in both rodents and humans has been proposed to contribute to age-related

The inactivation of IGF-1 or its receptor during early development reduces brain size including a reduction of the granule cell layer in the dentate gyrus and the number of oligodendrocytes and myelinated axons (Beck et al. 1995; Holzenberger et al. 2001; Vicario-Abejon et al. 2003) demonstrating the importance of IGF-1 signalling during brain development. LID mice show a milder phenotype with reduced exploratory activity (Svensson et al. 2005; Bohlooly et al. 2001), as well as impaired spatial learning and memory, suggesting that liver-derived IGF-1 enhances brain functions (Svensson et al. 2006). Circulating IGF-1 is essential for mediating exercise induced effects on the adult brain by promoting the numbers of newly generated neurons in the adult hippocampus, brain vessel growth, spatial learning and reducing anxiety (Trejo, Carro, and Torres-Aleman 2001; Trejo, Llorens-Martin, and Torres-Aleman 2008; Lopez-Lopez, LeRoith, and Torres-Aleman 2004). Additionally, liver-derived IGF-1 might enhance the clearance of brain amyloid-β (Aβ),

Only a small amount of IGF-1 (~1 %) circulates "free" within the blood. The remaining 99 % are bound to IGF-binding proteins (IGFBPs). IGFBPs are a family of at least six proteins (IGFBP-1 to IGFBP-6), which bind IGF-1 with high affinities that are equal to or greater than

IGF-1 function and vice versa.

obesity (Sonntag et al. 1980; Rudman et al. 1990).

whose aggregation is one of the hallmarks of AD (see 5.1).

**2.2 IGF-binding proteins** 

those of the IGF-1 receptor (Duan and Xu 2005). Additionally, there are several proteins with lower IGF-1 binding affinity, called IGFBP-related peptides (IGFBP-rPs), which have significant structural homologies with the amino(N)-terminal region of high-affinity IGFBPs (Rechler 1993; Kelley et al. 1996; Rajaram, Baylink, and Mohan 1997; Kim et al. 1997; Poretsky et al. 1999; Baxter 2000). So far, the functions of the IGFBP-rPs, also referred to as IGFBP- 7 to - 10, are not known (Mohan and Baylink 2002). Approximately 75-80 % of IGF is present as a 150 kDa complex, which consists of IGF-1/IGF-2 plus IGFBP-3 (70-75 %) or IGFPB-5 (5-10 %) and an acid-labile subunit (ALS) (Baxter, Meka, and Firth 2002). ALS is mainly produced in the liver (Baxter 1988; Baxter and Martin 1989; Baxter, Martin, and Beniac 1989) and its gene disruption leads to a reduction of circulating IGFs of 80 % (Boisclair et al. 2001). The ternary complex of IGF-1 or -2/ IGFBP-3 or -5/ ALS extends the half-life of IGFs to 15-20 h, compared to a half-life of 20-30 min for free IGF-1 in circulation (Guler, Zapf, and Froesch 1987) suggesting a reservoir function of this complex, which cannot cross the vascular endothelial barrier (Rajaram, Baylink, and Mohan 1997; Baxter 2000). 20-25 % of the IGFs bind to IGFBP-1,- 2,-4 or -6 to form a complex, which is able to cross vascular endothelium (Baxter 2000; Rajaram, Baylink, and Mohan 1997). Thus, the endocrine actions of IGF-1 in serum are regulated by the IGFBPs determining how much IGF is bio-available to the local tissues.

Furthermore, IGFBPs are located in the extracellular matrix or on the cell surface where they either inhibit (mainly IGFBP-4 and -6) or potentiate (mainly IGFBP-3 and -5) IGF-1 binding to its receptors (Rajaram, Baylink, and Mohan 1997; Mohan et al. 1995; Qin et al. 1998; Jones and Clemmons 1995; Firth and Baxter 2002; Rechler and Clemmons 1998). Functions of the IGFBPs might vary indicated by the inhibiting or potentiating effect of IGFBP-1, -2, -3 and -5 on IGF-1 action depending on the experimental conditions (Yin, Xu, and Duan 2004).

The actions of IGFBPs are, in turn, modulated by IGFBP proteases that are further dependent on activators and inhibitors (Mohan et al. 2002). Those IGFBP proteases are able to cleave IGFBPs, thereby reducing their affinity for IGF-1. Some of them are relatively specific for a given IGFBP. For example, pregnancy-associated plasma protein-A (PAPP-A), which is produced by a variety of cell types, cleaves specifically IGFBP-4 (Lawrence et al. 1999; Byun et al. 2001; Conover et al. 2001), whereas complement C1s (Busby et al. 2000) and a disintigrin and metalloprotease-like (ADAM)-9 (Mohan et al. 2002) were identified to be relatively specific for IGFBP-5. Additionally, a number of other serum proteases were shown to be capable of cleaving IGFBPs, e.g. plasmin, cathepsin D and prostate specific antigen (Conover 1995; Fowlkes et al. 1995; Rajah et al. 1995; Rajaram, Baylink, and Mohan 1997; Maile and Holly 1999). Therefore, the binding affinity and hence the bio-availability of IGF-1 is regulated via the degradation of IGFBPs through the IGFBP protease system.

IGFBPs also have IGF independent functions, for example IGFBP-2 and -3 can induce direct cellular effects (Firth and Baxter 2002; Oh et al. 1993; Yamanaka et al. 1999; Schutt et al. 2004) and it has been shown that IGFBP-3 at least partially mediates these effects by binding to the cell surface, possibly to specific receptors (Mohseni-Zadeh and Binoux 1997; Rechler and Clemmons 1998). IGFBP-2, -3 and -5 contain sequences for nuclear localisation (Schutt et al. 2004; Radulescu 1994; Schedlich et al. 1998; Hoeflich et al. 2004) and might as well influence gene expression.

#### **2.3 The insulin- and insulin-like growth factor-1 signalling pathway**

Insulin and IGF-1 receptors are receptor tyrosine kinases. Receptor tyrosine kinases contain a membrane-bound domain with tyrosine kinase activity which phosphorylates tyrosineresidues of downstream signalling proteins. Typical members of the receptor tyrosine kinase

Role of Central Insulin-Like Growth Factor-1

IRS-2 remains unclear.

**2.3.2 PI3K signalling** 

Phosphatase(SHP)2 (Figure 2).

Receptor Signalling in Ageing and Endocrine Regulation 215

Only IRS-2 contains a domain which binds to the phosphorylated kinase regulatory loop of the β-subunit of the IR. This domain is called the KLRB domain (Sawka-Verhelle et al. 1997; Sawka-Verhelle et al. 1996). However, the physiological function of the KLRB domain of

Insulin induces tyrosine and serine phosphorylation of IRS-1 (Gual, Le Marchand-Brustel, and Tanti 2005). These phosphorylations lead to specific regulation of downstream signalling. The phosphorylation of serine residues of IRS-1 contribute to positive or negative regulation of IRS-1 action (Weigert et al. 2005; Weigert et al. 2008). Important for this regulation are the particular phosphorylation sites (Herschkovitz et al. 2007) as well as the timing of phosphorylation (Weigert et al. 2005; Weigert et al. 2008). Currently, the serine phosphorylation sites with positive effect on IRS-1 action are regarded to be phosphorylated at first to support IRS-1 activity protecting from phosphorylation at residues with inhibitory effect (Weigert et al. 2005; Weigert et al. 2008; Gual, Le Marchand-Brustel, and Tanti 2005; Luo et al. 2007). In addition, serine phosphorylation with activating effect might prevent the association of IRS-1 with tyrosine phosphatases (Luo et al. 2005). The serine residues with inhibitory effect are located near the PTB domain. These residues are phosphorylated later than the residues with positive effect upon insulin stimulation or other signals. The phosphorylation of serine sites near the PTB domain causes disruption of the binding between IRS-1 and the IR followed by degradation of IRS-1. The phosphorylation of inhibitory serine residues in the C-terminus of IRS-1 disturb their interaction with the phosphatidylinositide (PI)3-kinase (Figure 2) (Gual, Le Marchand-Brustel, and Tanti 2005; Boura-Halfon and Zick 2009). Serine sites with inhibitory effect are phosphorylated by serine kinases like the mammalian target of rapamycin (mTor), PKCzeta and p70S6 (S6K) kinase (Boura-Halfon and Zick 2009; Herschkovitz et al. 2007; Gual et al. 2003). Insulin and IGF-1 resistance might be induced via kinases like c-Jun N-terminal kinase (JNK), mTor/S6K, inhibotory-κB kinase β (IKKβ), SIK-2 and extracellular signal regulated kinases (ERK) promoting the phosphorylation of the inhibitory sites of the IRS proteins (Boura-Halfon and Zick 2009; Herschkovitz et al. 2007). The IRS-2 serine phosphorylation sites are still under investigation. It is known that JNK phosphorylates Thr348 of IRS-2 which is located near the PTB domain (Solinas et al. 2006) and might cause disruption of the binding between IRS-2 and the receptor. Furthermore, JNK phosphorylates Ser488 of IRS-2 promoting the phosphorylation at Ser484 by glycogen synthase kinase (GSK)-3β and thereby

inhibiting the signalling pathway (Sharfi and Eldar-Finkelman 2008).

The mammalian phosphatidylinositide(PI)3-kinases are subdivided into three classes, class I-III. Class I is further divided into Ia and Ib (Vanhaesebroeck et al. 2005). These classes catalyse the phosphorylation of the 3´hydroxyl position of phosphatidyl-myo-inositol lipids. The PI3K of the insulin and IGF-1 signalling pathway belongs to the class Ia kinases (Fruman, Meyers, and Cantley 1998). These kinases display a heterodimeric structure containing a catalytic subunit of 110 kDa. This subunit is non-covalently associated with a 50-, 55- or 85 kDa regulatory subunit. Following activation of the insulin receptor and IRS binding, the PI3K is recruited to the membrane via the p85 regulatory subunit. Other recruited factors are the growth factor receptor binding protein (GRB)-2 and the SH2-

The activated PI3K, in turn, phosphorylates phosphatidylinositide-diphosphate (PI4,5P) to produce phosphatidylinositide-triphosphate (PI3,4,5P). This event is reversible by PTEN (the

family are the epidermal growth factor receptor (EGFR), the nerve growth factor receptor (NGFR) and the insulin receptor (IR). The IR was discovered in 1974 and its tyrosine kinase was found in 1982 (Kasuga, Karlsson, and Kahn 1982; Kasuga et al. 1982). Later, in addition to the IR, the insulin-like growth factor-1 receptor (IGF-1R) was discovered as a tyrosine kinase activity containing receptor (Jacobs et al. 1983; Rubin, Shia, and Pilch 1983).

The IR and IGF-1R are heterotetrameric structures. The different subunits are linked by disulfide bonds. The α-subunits are exclusively localised extracellular (Van Obberghen et al. 1981; Ullrich et al. 1986). The β-subunits consist of a short extracellular part, a transmembrane domain and an intracellular part with ATP-binding motifs, autophosphorylation sites and tyrosine-specific protein kinase activity, which is activated after binding of insulin or IGF-1 to their receptors (Chou et al. 1987).

The insulin receptor gene consists of 22 exons and 21 introns (Seino et al. 1989). Alternative splicing of exon 11, which codes for 12 amino acids, causes two different isoforms: A, which lacks the 12 amino acids, and B. This alternative splicing does not exist for the IGF-1 receptor, because this receptor contains no equivalent to exon 11 of the IR. The A- and Bisoforms both bind to insulin with similar affinity (McClain 1991). The A-isoform has a higher affinity to IGF-1 (Yamaguchi et al. 1991) and IGF-2 (Frasca et al. 1999) compared to the B-isoform. The A-isoform is expressed in hematopoietic cells, fetal tissue and the adult nervous system. The B-isoform of the IR is mainly present in liver, adipose tissue and muscle (Seino and Bell 1989; Moller et al. 1989; Goldstein and Kahn 1989; Mosthaf et al. 1990). The selective binding of insulin or IGF-1 is also dependent on the assembly of the receptors (Pandini et al. 2002). The hybrid of IGF-1 receptor and the A-isoform of the IR binds IGF-1, IGF-2 and insulin with similar affinity. The hybrid of the IGF-1R and the Bisoform of the IR only binds IGF-1 (Louvi, Accili, and Efstratiadis 1997).

## **2.3.1 Insulin receptor substrates**

The binding of IGF-1 or IGF-2 to the IGF-1R causes a conformational change of the receptor, which induces autophosphorylation. This leads to the recruitment of insulin receptor substrates (IRS) to the autophosphorylated receptor tyrosine kinase, which in turn phosphorylates the tyrosine residues of the IRS proteins (Figure 2). The IRS protein family consists at least of four proteins, IRS-1 to IRS-4 (Sun et al. 1991; Lavan et al. 1997; Lavan, Lane, and Lienhard 1997).

The IRS proteins show different expression patterns. IRS-1 and -2 are ubiquitously expressed, but IRS-3 is only present in rodent adipose tissue. IRS-4 mainly occurs in thymus, hypothalamus, kidney and heart. All IRS proteins share the same structural characteristics and have similar functions (Giovannone et al. 2000; Schubert et al. 2003).

The four IRS family members contain an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain and a C-terminal tail containing multiple tyrosine phosphorylation sites. The phosphotyrosine motifes of the IRS proteins are binding sites for Src homology(SH)2 domain-containing proteins (Yenush and White 1997). The PH domain binds to lipids and with high affinity to phosphoinositides (Fruman, Rameh, and Cantley 1999). The PTB domain of IRS binds to phosphotyrosine residues of other proteins including the IR and IGF-1R. In more detail, the PTB domain binds to the phosphorylated NPXP motif at the juxtamembrane domain of the receptor after binding of insulin or IGF-1. Following binding to this motif, the IRS proteins are tyrosinephosphorylated (Cheatham and Kahn 1995; White 2002).

family are the epidermal growth factor receptor (EGFR), the nerve growth factor receptor (NGFR) and the insulin receptor (IR). The IR was discovered in 1974 and its tyrosine kinase was found in 1982 (Kasuga, Karlsson, and Kahn 1982; Kasuga et al. 1982). Later, in addition to the IR, the insulin-like growth factor-1 receptor (IGF-1R) was discovered as a tyrosine

The IR and IGF-1R are heterotetrameric structures. The different subunits are linked by disulfide bonds. The α-subunits are exclusively localised extracellular (Van Obberghen et al. 1981; Ullrich et al. 1986). The β-subunits consist of a short extracellular part, a transmembrane domain and an intracellular part with ATP-binding motifs, autophosphorylation sites and tyrosine-specific protein kinase activity, which is activated

The insulin receptor gene consists of 22 exons and 21 introns (Seino et al. 1989). Alternative splicing of exon 11, which codes for 12 amino acids, causes two different isoforms: A, which lacks the 12 amino acids, and B. This alternative splicing does not exist for the IGF-1 receptor, because this receptor contains no equivalent to exon 11 of the IR. The A- and Bisoforms both bind to insulin with similar affinity (McClain 1991). The A-isoform has a higher affinity to IGF-1 (Yamaguchi et al. 1991) and IGF-2 (Frasca et al. 1999) compared to the B-isoform. The A-isoform is expressed in hematopoietic cells, fetal tissue and the adult nervous system. The B-isoform of the IR is mainly present in liver, adipose tissue and muscle (Seino and Bell 1989; Moller et al. 1989; Goldstein and Kahn 1989; Mosthaf et al. 1990). The selective binding of insulin or IGF-1 is also dependent on the assembly of the receptors (Pandini et al. 2002). The hybrid of IGF-1 receptor and the A-isoform of the IR binds IGF-1, IGF-2 and insulin with similar affinity. The hybrid of the IGF-1R and the B-

The binding of IGF-1 or IGF-2 to the IGF-1R causes a conformational change of the receptor, which induces autophosphorylation. This leads to the recruitment of insulin receptor substrates (IRS) to the autophosphorylated receptor tyrosine kinase, which in turn phosphorylates the tyrosine residues of the IRS proteins (Figure 2). The IRS protein family consists at least of four proteins, IRS-1 to IRS-4 (Sun et al. 1991; Lavan et al. 1997; Lavan,

The IRS proteins show different expression patterns. IRS-1 and -2 are ubiquitously expressed, but IRS-3 is only present in rodent adipose tissue. IRS-4 mainly occurs in thymus, hypothalamus, kidney and heart. All IRS proteins share the same structural characteristics

The four IRS family members contain an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain and a C-terminal tail containing multiple tyrosine phosphorylation sites. The phosphotyrosine motifes of the IRS proteins are binding sites for Src homology(SH)2 domain-containing proteins (Yenush and White 1997). The PH domain binds to lipids and with high affinity to phosphoinositides (Fruman, Rameh, and Cantley 1999). The PTB domain of IRS binds to phosphotyrosine residues of other proteins including the IR and IGF-1R. In more detail, the PTB domain binds to the phosphorylated NPXP motif at the juxtamembrane domain of the receptor after binding of insulin or IGF-1. Following binding to this motif, the IRS proteins are tyrosinephosphorylated (Cheatham and Kahn

kinase activity containing receptor (Jacobs et al. 1983; Rubin, Shia, and Pilch 1983).

after binding of insulin or IGF-1 to their receptors (Chou et al. 1987).

isoform of the IR only binds IGF-1 (Louvi, Accili, and Efstratiadis 1997).

and have similar functions (Giovannone et al. 2000; Schubert et al. 2003).

**2.3.1 Insulin receptor substrates** 

Lane, and Lienhard 1997).

1995; White 2002).

Only IRS-2 contains a domain which binds to the phosphorylated kinase regulatory loop of the β-subunit of the IR. This domain is called the KLRB domain (Sawka-Verhelle et al. 1997; Sawka-Verhelle et al. 1996). However, the physiological function of the KLRB domain of IRS-2 remains unclear.

Insulin induces tyrosine and serine phosphorylation of IRS-1 (Gual, Le Marchand-Brustel, and Tanti 2005). These phosphorylations lead to specific regulation of downstream signalling. The phosphorylation of serine residues of IRS-1 contribute to positive or negative regulation of IRS-1 action (Weigert et al. 2005; Weigert et al. 2008). Important for this regulation are the particular phosphorylation sites (Herschkovitz et al. 2007) as well as the timing of phosphorylation (Weigert et al. 2005; Weigert et al. 2008). Currently, the serine phosphorylation sites with positive effect on IRS-1 action are regarded to be phosphorylated at first to support IRS-1 activity protecting from phosphorylation at residues with inhibitory effect (Weigert et al. 2005; Weigert et al. 2008; Gual, Le Marchand-Brustel, and Tanti 2005; Luo et al. 2007). In addition, serine phosphorylation with activating effect might prevent the association of IRS-1 with tyrosine phosphatases (Luo et al. 2005). The serine residues with inhibitory effect are located near the PTB domain. These residues are phosphorylated later than the residues with positive effect upon insulin stimulation or other signals. The phosphorylation of serine sites near the PTB domain causes disruption of the binding between IRS-1 and the IR followed by degradation of IRS-1. The phosphorylation of inhibitory serine residues in the C-terminus of IRS-1 disturb their interaction with the phosphatidylinositide (PI)3-kinase (Figure 2) (Gual, Le Marchand-Brustel, and Tanti 2005; Boura-Halfon and Zick 2009). Serine sites with inhibitory effect are phosphorylated by serine kinases like the mammalian target of rapamycin (mTor), PKCzeta and p70S6 (S6K) kinase (Boura-Halfon and Zick 2009; Herschkovitz et al. 2007; Gual et al. 2003). Insulin and IGF-1 resistance might be induced via kinases like c-Jun N-terminal kinase (JNK), mTor/S6K, inhibotory-κB kinase β (IKKβ), SIK-2 and extracellular signal regulated kinases (ERK) promoting the phosphorylation of the inhibitory sites of the IRS proteins (Boura-Halfon and Zick 2009; Herschkovitz et al. 2007). The IRS-2 serine phosphorylation sites are still under investigation. It is known that JNK phosphorylates Thr348 of IRS-2 which is located near the PTB domain (Solinas et al. 2006) and might cause disruption of the binding between IRS-2 and the receptor. Furthermore, JNK phosphorylates Ser488 of IRS-2 promoting the phosphorylation at Ser484 by glycogen synthase kinase (GSK)-3β and thereby inhibiting the signalling pathway (Sharfi and Eldar-Finkelman 2008).

#### **2.3.2 PI3K signalling**

The mammalian phosphatidylinositide(PI)3-kinases are subdivided into three classes, class I-III. Class I is further divided into Ia and Ib (Vanhaesebroeck et al. 2005). These classes catalyse the phosphorylation of the 3´hydroxyl position of phosphatidyl-myo-inositol lipids. The PI3K of the insulin and IGF-1 signalling pathway belongs to the class Ia kinases (Fruman, Meyers, and Cantley 1998). These kinases display a heterodimeric structure containing a catalytic subunit of 110 kDa. This subunit is non-covalently associated with a 50-, 55- or 85 kDa regulatory subunit. Following activation of the insulin receptor and IRS binding, the PI3K is recruited to the membrane via the p85 regulatory subunit. Other recruited factors are the growth factor receptor binding protein (GRB)-2 and the SH2- Phosphatase(SHP)2 (Figure 2).

The activated PI3K, in turn, phosphorylates phosphatidylinositide-diphosphate (PI4,5P) to produce phosphatidylinositide-triphosphate (PI3,4,5P). This event is reversible by PTEN (the

Role of Central Insulin-Like Growth Factor-1

Kitamura et al. 2005) to regulate FoxO mediated transcription.

<sup>P</sup> PI3K

Mnk eIF4A eIF4G eIF4E

P

P

Transation initiation

eEF2K eEF2

protein kinase) and phosphatidylinositide(PI)3-kinase (PI3K) pathway.

eEF2K

P <sup>P</sup> <sup>P</sup>

The binding of Insulin or IGF-1 to the IR/IGF-1R causes autophosphorylation and activation of the receptor. Insulin receptor substrates (1-4) are recruited to the activated receptor and IRS phosphorylation results in activation of the MAP kinase (MAPK, mitogen activated

P110

PI4,5P PI3,4,5P

PTEN SHP2

PP2A GSK3β

GTP GDP mTOR GAP

p70S6K

RHEB

P

14-3-3 GAB-1

PDK1/2

TSC1/2 TSC1/2

P

AKT Foxo1

Foxo1

P

Transcription Stop

P85

IR/IGF-1R

Mnk

Tranlsation elongation

eIF4A eIF4G

Transcription eEF2

IRS1-4

P

4E-BP

4E-BP eIF4E

P

GRB2

SOS

RAS

CRAF

P

P

P

MEK

ERK1/2

P P

Fig. 2. IR/IGF-R signalling

ELK-1 FOS

Receptor Signalling in Ageing and Endocrine Regulation 217

translocation out of the nucleus terminating FoxO mediated transcription (Figure 2) (Brunet et al. 1999). Additionally, FoxOs are phosphorylated by other kinases depending on the stimulus (Huang and Tindall 2007). Furthermore, FoxOs are regulated by ubiquitylation. Ubiquitylation is dependent on phosphorylation of Ser256 of FoxO1 via AKT (Huang et al. 2005). FoxO1 and FoxO3a need to be polyubiquitylated for degradation. In contrast, FoxO4 requires monoubiquitylation to be degraded (van der Horst et al. 2006). Another regulatory mechanism for FoxO transcription is acetylation. CBP and p300 with their associated proteins, for example CBP- and p300-associated factor (PCAF), display intrinsic histone acetyl-transferase activity. These proteins promote transcription via histone acetylation and they directly regulate transcription via acetylation of particular transcription factors (Li et al. 2002). It has been shown that CBP acetylates FoxO transcription factors and inhibits their action (Daitoku et al. 2004). Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide(NAD)-dependent histone deacetylase, which forms a complex with acetylated FoxOs upon stress stimuli and deacetylates the transcription factors (Brunet et al. 2004;

phosphatase and tensin homolog deleted on chromosome ten). The generation of PI3,4,5P causes activation of the downstream signalling proteins like phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB, AKT). PDK is present in two isoforms, PDK-1 and PDK-2. PDK-1 phosphorylates AKT at Thr308, which partially activates AKT. To completely activate AKT, phosphorylation of Ser473 is necessary (Alessi et al. 1996; Lawlor and Alessi 2001; Stokoe et al. 1997). AKT is a serine/threonine kinase with a size of 57 kDa. It contains a PH domain and there are three isoformes, AKT-1, AKT-2 and AKT-3. These isoforms display a conserved domain structure: a kinase domain, a PH-domain at the N- as well as a regulatory subunit at the C-terminus (Hresko, Murata, and Mueckler 2003). AKT phosphorylates tuberin 2 (TSC-2). TSC-1 and -2 form a heterodimer with GTPase activity that inhibits the GTPase RHEB (RAS homolog enriched in brain). The phosphorylation via AKT causes the accumulation of the RHEB-GTP complex which activates mTOR (Astrinidis and Henske 2005; Hay and Sonenberg 2004). Furthermore, S6K is activated by phosphorylation of PDK-1 and mTOR (Figure 2). The regulation of protein synthesis via IGF-1 occurs through controlling the intrinsic activity and/or binding properties of specific translation initiation and elongation factors called eIFs and eEFs. mTOR phosphorylates 4E-BP (4E binding protein). This causes the release of eIF4E (eukaryotic initiation factor 4E) to form an active complex, which promotes translation initiation and also activation of S6K. S6K phosphorylates the eEF2 (eukaryotic elongation factor 2) kinase which releases eEF2 and initiates elongation (Figure 2) (Nojima et al. 2003; Oshiro et al. 2004).

Other proteins, which are regulated via IR and IGF-1R signalling, are the glycogen synthase kinase(GSK)-3β, a major tau kinase, and BAD (Bcl-2/Bcl-X-associated death promoter), a proapoptotic factor. These proteins are inactivated via the IR/IGF-1R signalling cascade (Song, Ouyang, and Bao 2005). BAD interacts with the apoptosis suppressors Bcl-2 and more intense with Bcl-XL (Yang et al. 1995). BAD directly binds to Bcl-XL with its BH3 homology domain (Zha et al. 1997). This interaction is regulated by the phosphorylation state of BAD. Therefore, the IGF-1R signalling is a potent inhibitor of neuronal apoptosis (Schubert et al. 2003).

#### **2.3.3 Forkhead box O transcription factor**

AKT phosphorylates the Forkhead box O transcription factors (FoxOs). This induces the binding to 14-3-3 and nuclear exclusion of FoxOs and thereby inactivation of FoxOmediated transcription. FoxOs regulate transcription of genes, which are involved in apoptosis, metabolism, growth, ageing and development (Partridge and Bruning 2008).

The mammalian FoxO protein family consists of 4 members: FoxO1, FoxO3a, FoxO4 and FoxO6. These transcription factors contain a conserved DNA binding domain, the forkhead domain (FKHR) (Clark et al. 1993). FoxO1 and FoxO3a are ubiquitously expressed, whereas FoxO6 is exclusively found in the brain, and FoxO4 has yet not been detected in the brain (Furuyama et al. 2000; Jacobs et al. 2003). The expression pattern of the different FoxOs in the adult mouse brain is distinct. FoxO1 is predominantly expressed in the striatum, dentate gyrus and ventral hippocampus and FoxO3a in the cortex, cerebellum and hippocampus. FoxO6 is expressed in amygdala, hippocampus and cingulite cortex (Hoekman et al. 2006).

The FoxO transription factors are regulated by post-translational modifications. One major modification is the phosphorylation of the FoxOs. FoxO1 is phosphorylated by AKT at Thr24, Ser256 and Ser319 (Biggs et al. 1999; Brunet et al. 1999; Kops et al. 1999; Rena et al. 1999; Tang et al. 1999). Phosphorylation triggers binding to 14-3-3 and subsequently

phosphatase and tensin homolog deleted on chromosome ten). The generation of PI3,4,5P causes activation of the downstream signalling proteins like phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB, AKT). PDK is present in two isoforms, PDK-1 and PDK-2. PDK-1 phosphorylates AKT at Thr308, which partially activates AKT. To completely activate AKT, phosphorylation of Ser473 is necessary (Alessi et al. 1996; Lawlor and Alessi 2001; Stokoe et al. 1997). AKT is a serine/threonine kinase with a size of 57 kDa. It contains a PH domain and there are three isoformes, AKT-1, AKT-2 and AKT-3. These isoforms display a conserved domain structure: a kinase domain, a PH-domain at the N- as well as a regulatory subunit at the C-terminus (Hresko, Murata, and Mueckler 2003). AKT phosphorylates tuberin 2 (TSC-2). TSC-1 and -2 form a heterodimer with GTPase activity that inhibits the GTPase RHEB (RAS homolog enriched in brain). The phosphorylation via AKT causes the accumulation of the RHEB-GTP complex which activates mTOR (Astrinidis and Henske 2005; Hay and Sonenberg 2004). Furthermore, S6K is activated by phosphorylation of PDK-1 and mTOR (Figure 2). The regulation of protein synthesis via IGF-1 occurs through controlling the intrinsic activity and/or binding properties of specific translation initiation and elongation factors called eIFs and eEFs. mTOR phosphorylates 4E-BP (4E binding protein). This causes the release of eIF4E (eukaryotic initiation factor 4E) to form an active complex, which promotes translation initiation and also activation of S6K. S6K phosphorylates the eEF2 (eukaryotic elongation factor 2) kinase which releases eEF2

and initiates elongation (Figure 2) (Nojima et al. 2003; Oshiro et al. 2004).

(Schubert et al. 2003).

**2.3.3 Forkhead box O transcription factor** 

cingulite cortex (Hoekman et al. 2006).

Other proteins, which are regulated via IR and IGF-1R signalling, are the glycogen synthase kinase(GSK)-3β, a major tau kinase, and BAD (Bcl-2/Bcl-X-associated death promoter), a proapoptotic factor. These proteins are inactivated via the IR/IGF-1R signalling cascade (Song, Ouyang, and Bao 2005). BAD interacts with the apoptosis suppressors Bcl-2 and more intense with Bcl-XL (Yang et al. 1995). BAD directly binds to Bcl-XL with its BH3 homology domain (Zha et al. 1997). This interaction is regulated by the phosphorylation state of BAD. Therefore, the IGF-1R signalling is a potent inhibitor of neuronal apoptosis

AKT phosphorylates the Forkhead box O transcription factors (FoxOs). This induces the binding to 14-3-3 and nuclear exclusion of FoxOs and thereby inactivation of FoxOmediated transcription. FoxOs regulate transcription of genes, which are involved in apoptosis, metabolism, growth, ageing and development (Partridge and Bruning 2008). The mammalian FoxO protein family consists of 4 members: FoxO1, FoxO3a, FoxO4 and FoxO6. These transcription factors contain a conserved DNA binding domain, the forkhead domain (FKHR) (Clark et al. 1993). FoxO1 and FoxO3a are ubiquitously expressed, whereas FoxO6 is exclusively found in the brain, and FoxO4 has yet not been detected in the brain (Furuyama et al. 2000; Jacobs et al. 2003). The expression pattern of the different FoxOs in the adult mouse brain is distinct. FoxO1 is predominantly expressed in the striatum, dentate gyrus and ventral hippocampus and FoxO3a in the cortex, cerebellum and hippocampus. FoxO6 is expressed in amygdala, hippocampus and

The FoxO transription factors are regulated by post-translational modifications. One major modification is the phosphorylation of the FoxOs. FoxO1 is phosphorylated by AKT at Thr24, Ser256 and Ser319 (Biggs et al. 1999; Brunet et al. 1999; Kops et al. 1999; Rena et al. 1999; Tang et al. 1999). Phosphorylation triggers binding to 14-3-3 and subsequently translocation out of the nucleus terminating FoxO mediated transcription (Figure 2) (Brunet et al. 1999). Additionally, FoxOs are phosphorylated by other kinases depending on the stimulus (Huang and Tindall 2007). Furthermore, FoxOs are regulated by ubiquitylation. Ubiquitylation is dependent on phosphorylation of Ser256 of FoxO1 via AKT (Huang et al. 2005). FoxO1 and FoxO3a need to be polyubiquitylated for degradation. In contrast, FoxO4 requires monoubiquitylation to be degraded (van der Horst et al. 2006). Another regulatory mechanism for FoxO transcription is acetylation. CBP and p300 with their associated proteins, for example CBP- and p300-associated factor (PCAF), display intrinsic histone acetyl-transferase activity. These proteins promote transcription via histone acetylation and they directly regulate transcription via acetylation of particular transcription factors (Li et al. 2002). It has been shown that CBP acetylates FoxO transcription factors and inhibits their action (Daitoku et al. 2004). Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide(NAD)-dependent histone deacetylase, which forms a complex with acetylated FoxOs upon stress stimuli and deacetylates the transcription factors (Brunet et al. 2004; Kitamura et al. 2005) to regulate FoxO mediated transcription.

#### Fig. 2. IR/IGF-R signalling

The binding of Insulin or IGF-1 to the IR/IGF-1R causes autophosphorylation and activation of the receptor. Insulin receptor substrates (1-4) are recruited to the activated receptor and IRS phosphorylation results in activation of the MAP kinase (MAPK, mitogen activated protein kinase) and phosphatidylinositide(PI)3-kinase (PI3K) pathway.

Role of Central Insulin-Like Growth Factor-1

**3.3 IGF receptor expression in the brain** 

Valentino, Ocrant, and Rosenfeld 1990).

of insulin and possibly IGF-1 across the BBB.

**4. IGF-1 action in the developing brain** 

**4.1 Brain growth and myelination** 

Porte, and Baskin 1991).

might transmit some intracellular signalling (Chen et al. 2011).

Receptor Signalling in Ageing and Endocrine Regulation 219

2 but M6P-containing ligands. This receptor functions in the mediation of endocytosis and lysosomal enzyme trafficking and regulation of apoptotic/ mitogenic effects (Morgan et al. 1987; von Figura and Hasilik 1986; Ghahary et al. 2000). Recent studies have demonstrated that IGF-2 influences memory enhancement via the IGF-2R suggesting that the IGF-2R

IGF-1R mRNA is widely expressed in the developing CNS and persists at high levels in the mature brain especially in neuron-rich regions such as the olfactory bulb, the granule cell layer of the dentate gyrus and cerebellar cortex (Rotwein et al. 1988; Bondy et al. 1990; Bondy and Lee 1993; Werther et al. 1990). Glia cells have a lower IGF-1R expression than neurons and therefore regions mainly containing those cells, like white matter zones, show low IGF-1R mRNA levels (Bondy and Lee 1993). Some neurons, which are still in the process of developing, express increasing IGF-1R mRNA levels postnatal until they have reached maturity, for example Purkinje cells (Bondy et al. 1992). In the adult brain, high expression levels of IGF-1R mRNA are also found in the choroid plexus, meninges and vascular sheaths (Bondy et al. 1992; Bohannon et al. 1988; Werther et al. 1989; Matsuo et al. 1989; Marks,

The IGF-2R is abundantly expressed in the CNS, especially in the pyramidal cell layers of the hippocampus, the granule cell layer of the dentate gyrus, olfactory bulbus, choroid plexus as well as in the microvasculature, retina, pituitary, brainstem and spinal cord (Hawkes and Kar 2004; Couce, Weatherington, and McGinty 1992; Wilczak et al. 2000;

In addition to the IGF-1R and the IGF-2R, IR and IGF-1R hybrids are expressed in the brain, through which IGFs and insulin induce intracellular signalling. IRs are mainly expressed in regions that are linked to olfaction, appetite and autonomic functions, such as the olfactory bulb, limbic system and hypothalamus (Werther et al. 1987; Unger, Livingston, and Moss 1991) and seem to play a key-role in controlling feeding, body weight and reproduction (Bruning et al. 2000). Furthermore, IR expression is also present in remarkable concentration in the choroid plexus, circumventricular organs and brain microvessels (van Houten and Posner 1979, 1981; Werther et al. 1987) suggesting the IRs might contribute to the transport

Different mouse models indicated the essential actions of IGF-1 signalling for normal brain development. Homozygous IGF-1 knockout mice (IGF-1-/-), homozygous IGF-2 knockout mice (IGF-2-/-) as well as doubly deficient mutants are viable but have small brains (Beck et al. 1995; Liu et al. 1993). The majority of homozygous IGF-1-/- mice die perinatally and those which survive (< 5 %) demonstrate severe growth retardation with reduction in brain weight of 38 % distributed evenly over all major brain areas (Beck et al. 1995). However, certain cell-types and brain-regions were especially affected by the IGF-1 gene disruption such as white matter, striatum and hippocampus (Beck et al. 1995). The reduction of white matter was due to a net loss of axons and an additional shift from myelinated to unmyelintated fibres indicating the role of IGF-1 in axonal growth and/or maturation and its effect on the amount of oligodendrocytes and axon-myelination (Beck et al. 1995). These findings are in line with many *in vitro* studies, which demonstrated a stimulating effect of
