**1. Introduction**

#### **1.1. General overview of insulin/IGF-signalling**

The IIS cascades are initiated by binding of Insulin or Insulin-like growth factors (IGF-1 and -2) to their receptors, the Insulin receptor (IR), and the two Insulin-like growth factor receptor 1 (IGF-1r) and 2 (IGF-2r) (Fig. 1). While high affinity binding occurs between the cognate ligand-receptor pairs, each ligand binds to the other receptors with lower affinity [2]. IR, IGF-1r and -2r are dimers that occur as homo- but also as heterodimers, the latter of which are studied in various cancer cells [3]. Such hybrid receptors are also found in the central nervous system, however a clear function for them has not emerged as yet [4] although activation of different signalling cascades followed by a different biological effect is a likely scenario [2]. IR, IGF-1r and -2r are tyrosine receptor kinases that phosphorylate themself as well as downstream adaptor proteins like the insulin receptor substrate proteins (IRS-1-4) [5]. Through phosphor‐ ylation, IRS proteins bind to SRC-homology-2 (SH-2) domain-containing proteins like SRC, SRC homology2-B (SH2-B), protein phosphatases like Tyrosine-protein phosphatase nonreceptor type 1 (PTPN1), or the p85 subunit of phosphatidyl inositol 3-kinase (PI3K).

Two major signalling pathways are activated through IIS: the PI3K- and/or the RAS/Mitogenactivated protein kinase- (MAPK) pathways that are implicated in the regulation of a plethora of different cellular processes.

PI3K belongs to a family of lipid kinases that are grouped into three classes. Class IA PI3K are heterodimers of a p110 catalytic and a p85 or p55 regulatory subunit [6]. Binding of PI3K is followed by activation of the p110 catalytic subunit of the kinase, which catalyses the increase

© 2013 Vogel; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Vogel; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of phosphatidylinositol-3,4,5-triphosphate (PtdIns{3,4,5}P3) lipids. PtdIns{3,4,5}P3 induce phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK-1), and the AGC kinase AKT. In mammals, AKT has three different isoforms, AKT-1,-2, and -3. Each of them has two critical sites in their activation domain, Thr308 and Ser473 that need to be phosphory‐ lated both to achieve full kinase activation. Inactive AKT is localised in the cytosol, but it is recruited to the plasma membrane together with PDK-1 through association with phosphati‐ dylinositol-4,5- diphosphosphate (PtdIns{4,5}P2) and PtdIns{3,4,5}P3. As a result, PDK-1 and AKT colocalise at the plasma membrane, which allows PDK-1 to induce phosphorylation of AKT at Thr308. AKT phosphorylation at Ser473 occurs by integrin-linked kinase (ILK) as well as mTORC-2 that are therefore PDK-2s for AKT [7]. Substrates of AKT are numerous, including pro-apoptotic proteins like BAD or anti-apoptotic proteins like BCL-2, NF- κB, and MCL-1, Forkheadbox transcription factors of the FOXO family as well as GSK-3β. AKT also phos‐ phorylates and inhibits the dimer tuberous sclerosis complex-1/-2 (TSC-1/TSC-2), which acts as inhibitory GTPase-activating protein for RHEB. The GTPase RHEB can activate mTORC-1, which has several substrates like p70 ribosomal protein S6 kinase (p70S6K), the translation initiation regulator 4E binding protein (4E-BP), and the proline-rich AKT substrate PRAS40. Through this signalling cascade, IIS activates mTORC-1 to promote cellular growth, transla‐ tion, transcription, and autophagy. mTORC-1 activation initiates a negative feed back loop through active p70S6K that phosphorylates and inhibits IRS, thereby preventing activation of PI3K in response to IIS. As indicated above, mTORC-2 also influences upstream IIS by phosphorylating AKT as PDK-2 and is involved in spatial growth by regulating the actin cytoskeleton. However, little is known about mTORC-2 activation through IIS. Recent data suggest that mTORC-2 activation through IIS relies on a putative PI3K that is insensitive to the negative feed back loop that controls activation of mTORC-1 [8].

IIS triggering the RAS/MAPK pathways can lead to activation of a subset of three downstream kinases, ERK, JNK, and p38 [9] (Fig. 2). Activation of ERK is dependent on the RAS/MAPK pathway, in which IIS results in phosphorylated IRS or SHC that recruit growth factor receptorbound protein 2 (GRB-2). GRB-2 associates with the protein son of sevenless (SOS), which is a guanine nucleotide exchange factor. SOS acts by binding RAS-GTPase and forcing it to release bound GDP and to bind GTP instead, which results in an activated state. Activated RAS phosphorylates RAF, which in turn phosphorylates MEK that is responsible to activate the MAPK ERK-1/-2. Jun aminoterminal kinases (JNK-1/-2) are further members of the MAPK pathway that are as well activated through IIS. JNK activation is dependent on PI3K, whose catalytic subunit does not bind to p85 but to the small RHO-family GTPase CDC42 [10]. This complex activates MKK-4 (or MAP2K-4 (mitogen-activated protein kinase kinase 4), which finally phosphorylates JNK-1/-2. The mechanism of p38 activation via IIS is so far unclear [9].

**2. Insulin/IGF-expression in the central nervous system**

hibitors commony used to study downstream IIS.

Insulin, Igf-1 and -2 as well as the three corresponding receptors are widely expressed in the developing and mature central nervous system (CNS) (for a recent review refer to [1]). Expression of Insulin and Igfs is in part under the control of Growth hormone (GH), but action of several tissue- and developmental-specific transcription factors are also involved in Insulin/ Igf expression as is the nutritional status (our own unpublished observations and [12-14]). Coordination and regulation of the biological activity of Igf-1 and -2, but not Insulin, is not only achieved by transcriptional or translational control but also through a set of proteins that have the ability to bind these ligands, namely the Igf-binding proteins (Igfbp). Upto date there

**Figure 1.** General overview of PI3K-dependent and mTor-driven IIS cascade and points of interference of serveral in‐

Insulin/IGF-Signalling in Embryonic and Adult Neural Proliferation and Differentiation in the Mammalian Central...

http://dx.doi.org/10.5772/54946

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PI3K- and RAS/MAPK-pathways also converge on some downstream molecules like FOXO proteins. Phosphorylation of FOXO through AKT leads to nuclear exclusion and interference with target gene expression. Several other sites are phosphorylated through ERK and p38 MAPK. Interference with these posttranslational modifications leads to decreased promoter binding together with ETS-1 transcription factor (TF) [11].

Insulin/IGF-Signalling in Embryonic and Adult Neural Proliferation and Differentiation in the Mammalian Central... http://dx.doi.org/10.5772/54946 39

of phosphatidylinositol-3,4,5-triphosphate (PtdIns{3,4,5}P3) lipids. PtdIns{3,4,5}P3 induce phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK-1), and the AGC kinase AKT. In mammals, AKT has three different isoforms, AKT-1,-2, and -3. Each of them has two critical sites in their activation domain, Thr308 and Ser473 that need to be phosphory‐ lated both to achieve full kinase activation. Inactive AKT is localised in the cytosol, but it is recruited to the plasma membrane together with PDK-1 through association with phosphati‐ dylinositol-4,5- diphosphosphate (PtdIns{4,5}P2) and PtdIns{3,4,5}P3. As a result, PDK-1 and AKT colocalise at the plasma membrane, which allows PDK-1 to induce phosphorylation of AKT at Thr308. AKT phosphorylation at Ser473 occurs by integrin-linked kinase (ILK) as well as mTORC-2 that are therefore PDK-2s for AKT [7]. Substrates of AKT are numerous, including pro-apoptotic proteins like BAD or anti-apoptotic proteins like BCL-2, NF- κB, and MCL-1, Forkheadbox transcription factors of the FOXO family as well as GSK-3β. AKT also phos‐ phorylates and inhibits the dimer tuberous sclerosis complex-1/-2 (TSC-1/TSC-2), which acts as inhibitory GTPase-activating protein for RHEB. The GTPase RHEB can activate mTORC-1, which has several substrates like p70 ribosomal protein S6 kinase (p70S6K), the translation initiation regulator 4E binding protein (4E-BP), and the proline-rich AKT substrate PRAS40. Through this signalling cascade, IIS activates mTORC-1 to promote cellular growth, transla‐ tion, transcription, and autophagy. mTORC-1 activation initiates a negative feed back loop through active p70S6K that phosphorylates and inhibits IRS, thereby preventing activation of PI3K in response to IIS. As indicated above, mTORC-2 also influences upstream IIS by phosphorylating AKT as PDK-2 and is involved in spatial growth by regulating the actin cytoskeleton. However, little is known about mTORC-2 activation through IIS. Recent data suggest that mTORC-2 activation through IIS relies on a putative PI3K that is insensitive to

38 Trends in Cell Signaling Pathways in Neuronal Fate Decision

the negative feed back loop that controls activation of mTORC-1 [8].

binding together with ETS-1 transcription factor (TF) [11].

IIS triggering the RAS/MAPK pathways can lead to activation of a subset of three downstream kinases, ERK, JNK, and p38 [9] (Fig. 2). Activation of ERK is dependent on the RAS/MAPK pathway, in which IIS results in phosphorylated IRS or SHC that recruit growth factor receptorbound protein 2 (GRB-2). GRB-2 associates with the protein son of sevenless (SOS), which is a guanine nucleotide exchange factor. SOS acts by binding RAS-GTPase and forcing it to release bound GDP and to bind GTP instead, which results in an activated state. Activated RAS phosphorylates RAF, which in turn phosphorylates MEK that is responsible to activate the MAPK ERK-1/-2. Jun aminoterminal kinases (JNK-1/-2) are further members of the MAPK pathway that are as well activated through IIS. JNK activation is dependent on PI3K, whose catalytic subunit does not bind to p85 but to the small RHO-family GTPase CDC42 [10]. This complex activates MKK-4 (or MAP2K-4 (mitogen-activated protein kinase kinase 4), which finally phosphorylates JNK-1/-2. The mechanism of p38 activation via IIS is so far unclear [9].

PI3K- and RAS/MAPK-pathways also converge on some downstream molecules like FOXO proteins. Phosphorylation of FOXO through AKT leads to nuclear exclusion and interference with target gene expression. Several other sites are phosphorylated through ERK and p38 MAPK. Interference with these posttranslational modifications leads to decreased promoter

**Figure 1.** General overview of PI3K-dependent and mTor-driven IIS cascade and points of interference of serveral in‐ hibitors commony used to study downstream IIS.
