reversal of the obesity in db/db mice having transgenic replacement of LepRb in the ARC (Coppari et al. 2005), indicating that other leptin-responsive regions also play a role.

Thus, aside from the well-recognised role of the ARC, it is becoming increasingly apparent that other brain regions are of importance in the regulation of energy balance. Numerous studies have now demonstrated roles for alternative sites of leptin action. Neurons within both the VMH and LHA have been shown to respond directly to leptin administration (Elmquist et al. 1998a; Dhillon et al. 2006; Leinninger et al. 2009). Extra-hypothalamic sites, particularly the brainstem, also contain LepRs (Elmquist et al. 1998b; Mercer et al. 1998), and LepR-containing neurons within the nucleus tractus solitarii (NTS) and area postrema have been demonstrated to be leptin responsive (Bjorbaek and Kahn 2004; Hayes et al. 2011). In addition to the reception of circulating factors, NTS neurons also receive neural inputs from gastrointestinal afferents (e.g., stomach distension that occurs with feeding), which play a role in the termination of feeding (Grill et al. 2002). It has also been shown recently that there are interactions between leptin and signals of gastrointestinal distension, for example, a potentiation of afferent neural signals by hindbrain leptin delivery (Huo et al. 2007). These findings indicate that leptin is involved both directly and indirectly in the regulation of appetite and feeding behaviour, and that the sites at which leptin acts are widely dispersed throughout the brain.

4 Regulation of Leptin Production and Receptor Levels

As mentioned above, leptin is mainly expressed in adipose tissue, and circulating concentrations in the fed state are highly correlated with the degree of adiposity, at least in adults (Maffei et al. 1995). Additional tissues have been shown to express leptin mRNA, including the stomach, placenta, mammary epithelium, pituitary and hypothalamus (Bado et al. 1998; Smith-Kirwin et al. 1998; Hoggard et al. 2001). Circulating leptin concentrations are acutely modulated by nutritional state, with levels being markedly decreased in response to fasting (Ahren et al. 1997a, b) or cold exposure (Hardie et al. 1996). This fall in leptin mediates a number of physiological adaptations to disrupted energy homeostasis, including a stimulation of feeding behaviour and reduction in energy expenditure, as well as suppression of the reproductive axis. Indeed, administration of leptin during fasting is able to ameliorate a number of these neuroendocrine adaptations (Ahima et al. 1996). Thus, leptin is considered a key integrator of metabolic and behavioural adaptation to a reduction in food availability and depletion of energy stores.

As with many ligand-receptor interactions, LepR levels are regulated in response to changes in circulating hormone concentrations. In the case of ob/ob mice, these animals exhibit a marked upregulation of hypothalamic LepR in response to the complete absence of hormone, which can be countered with administration of exogenous leptin (Mercer et al. 1997) (Fig. 2, below). Similarly,

Fig. 2 Leptin receptor gene expression in ob/ob mouse and a lean (+/?) littermate control, as demonstrated by in situ hybridisation

cold exposure or food restriction, and the accompanying rapid reduction in circulating leptin that occurs in these states, also result in significant upregulation of LepR within the hypothalamus (Lin and Huang 1997; Mercer et al. 1997) (Fig. 1). These changes in LepR gene expression would presumably enhance leptin signalling in the face of low circulating levels of ligand.

In situations of hyperleptinaemia (as in the db/db mouse model or during obesity progression), reduced LepR expression and impaired downstream signalling are observed (Malik and Young 1996; Wilsey and Scarpace 2004). These are discussed in more detail below, in the context of leptin resistance occurring in the face of chronically elevated leptin levels.

5 Leptin Receptor Signalling

The leptin receptor is a member of the class I cytokine-receptor family, having an extra-cellular ligand-binding domain, a transmembrane domain and a cytoplasmic signalling domain. To date, six LepR isoforms have been identified (LepRa-f; reviewed in (Fruhbeck 2006)), resulting from alternative mRNA splicing and/or proteolytic processing. Each of these isoforms shares common extra-cellular and transmembrane domains but differs in their intra-cellular C-terminal sequences. LepRa, c, d and f possess relatively short cytoplasmic domains (and are referred to as "short" isoforms), and LepRe is a secreted form of the leptin receptor. A degree of intra-cellular signalling has been demonstrated for the short isoforms (Bjorbaek et al. 1997); however, only LepRb, with an extended intra-cellular C-terminal region of ~300 amino acids, is capable of complete intra-cellular signal transduction (Baumann et al. 1996). Indeed, it is a mutation in the cytoplasmic region of LepRb that confers the leptin insensitivity in db/db mice, as well as in the Zucker fatty rat (Chua et al. 1996). Furthermore, LepRb has been shown to be critical in mediating leptin's actions in terms of body weight regulation, as there is no discernable phenotypic difference between mice which lack all leptin receptor isoforms (db3//db3/), db/db mice (which lack only LepRb function) and leptin-deficient ob/ob animals (Chua et al. 1996; Tartaglia 1997).

The main intra-cellular events following the binding of leptin to LepRb involve receptor dimerisation and activation of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway (Banks et al. 2000). Heterodimers of LepRa and LepRb appear to be incapable of signalling since the short LepRa isoform lacks the residues Leu896 and Phe897 that are critical for dimerisation

(Bahrenberg et al. 2002). Like many cytokine receptors, LepRs do not possess any intrinsic kinase activity, and, as such, signalling requires interaction with and activation of cytoplasmic non-receptor tyrosine kinases (JAKs). The JAK proteins are non-covalently attached through a proline-rich "box1" motif located in the juxtamembrane region of the intra-cellular domain. LepRb is associated preferentially with JAK2 (Kloek et al. 2002), and upon leptin binding and receptor dimerisation, JAK2 is autophosphorylated on several tyrosine residues and at the same time phosphorylates a number of tyrosine residues on the LepRb intra-cellular domain. These phosphorylated tyrosines provide sites for recruitment of intra-cellular proteins that contain phosphotyrosine-binding domains, such as Src homol-ogy 2 (SH2) domains. The transcription factor STAT3, containing a specific SH2 domain, is one of the key intra-cellular signalling pathways activated by leptin signalling through LepRb. Recruited STAT3 proteins are phosphorylated and dimerised, and these active pSTAT-3 dimers translocate to the nucleus where they bind target genes to bring about changes in gene transcription (Vaisse et al. 1996). In addition to JAK/STAT signalling, activation of LepRb results in the activation of extra-cellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) pathways (Fruhbeck 2006). Leptin signalling by LepRb is also under negative feedback regulation, through suppressors of cytokine signalling (SOCS) proteins, specifically SOCS3, which functions to inhibit tyrosine phosphorylation of LepR (Munzberg et al. 2003), and thus attenuates further signalling (Fig. 3).

At least three important tyrosine residues have been identified within the LepRb cytoplasmic domain (Tyr985, Tyr1077 and Tyr1138). The Tyr985 and Tyr1077 residues are considered critical sites for SOCS3 recruitment, and therefore negative feedback of leptin signalling (Eyckerman et al. 2000). Tyr1138 has been shown to be crucial in mediating the activation of STAT3 pathways, as mice in which this tyrosine was replaced with a serine residue fail to activate STAT3 and exhibit hyperphagia and early-onset obesity, similar to the db/db mice (Bates et al. 2003). Furthermore, through the use of these mice (termed "s/s" mice), it has been elegantly demonstrated that although Tyr1138-STAT3 signalling is critical for the actions of leptin on energy balance regulation, disruption does not result in infertility or linear growth impairments. s/s mice are reproductively functional without the need for exogenous leptin and can attain a normal body length. Within the ARC, s/s mice have reduced POMC and increased AgRP gene expression, similar to db/db mice, but levels of NPY mRNA are relatively normal. Thus, these studies show that downstream of the leptin receptor, intra-cellular actions of leptin are mediated by distinct components of the leptin signalling pathway. The normalised levels of NPY gene expression in the s/s model and resultant reversal of the hypothala-mic-gonadal axis are consistent with earlier reports of ob/ob/NPY~/~ mice, in which obesity is attenuated and animals have improved fertility and growth profiles (Erickson et al. 1996). More recently, attention has turned to the role of other leptin signalling pathways (PI3K and ERK), as it is clear that not all leptin actions require altered gene expression through STAT3 transcriptional alterations. Leptin can bring about rapid changes in membrane potential, within minutes of application, indicating non-genomic (and therefore pSTAT3-independent) actions (Cowley


log. rwuroiwWm. S0CS3)

Fig. 3 Simplified diagrammatic overview of main intracellular pathway activated following leptin binding to LepRb. Upon leptin binding, JAK2 undergoes autophosphorylation and

0 0

Post a comment