Ghrelin acts at different levels to stimulate GH secretion and thus modulate hepatic IGF-1 production (Peino et al., 2000). GHS-R1a is expressed by GHRH arcuate neurons but also by GH cells in the anterior pituitary gland (Kojima et al., 1999). Ghrelin and GHS activate ARC cells (Dickson et al., 1993; Hewson and Dickson, 2000) including neuroendocrine cells in this region (Dickson et al., 1996), notably a sub-population of GHRH neurons (Dickson and Luckman, 1997; Osterstock et al., 2010). Indeed, these compounds acts in synergy with GHRH to induce a greater GH release than would be induced by GHRH alone (Bowers et al., 1984; Arvat et al., 2001; Hataya et al., 2001). This ghrelin-stimulated GH release is dose-dependent and could explain why AN patients have elevated circulating GH levels (Kojima and Kangawa, 2005; Miljic et al., 2006; Misra et al., 2006; Germain et al., 2007, 2010; Estour et al., 2010). Chronic starvation is associated with GH resistance and relatively low IGF-1 levels involving a feedback mechanism, rather than body composition parameters or other circulating factors, e.g., free fatty acid or insulin levels (Støving et al., 1999; Brick et al., 2010). Besides its role in growth, GH stimulates lipolysis through a mechanism independent of IGF-1 (Fazeli et al., 2010a), for which the effects are largely anabolic. The increased GH secretion and the reduction of IGF-1 in starvation may be adaptive since they respectively serve the function of mobilizing fat stores in the setting of reduced energy availability and reduce anabolism. However, the reduction of IGF-1 levels may also have deleterious effects, contributing to bone, and muscle loss in AN women. Even if the mechanisms underlying the development of GH resistance in states of chronic undernutrition are not as well established, ghrelin might strongly participate to such endocrine dysregulation. Studies conducted in rodents support a close link between ghrelin signaling and altered GH/IGF-1 status. In rodents, fasting induced higher expression of GHS-R1a and overexpression of GHS-R1a in female mice provoked higher expression of GH and GHRH (Veldhuis and Bowers, 2010). Furthermore, suppressed ghrelin signaling (using antisense RNA knockdown against GHS-R1a or a GHS-R1a antagonist, BIM-28163), caused a decrease in GH peak pulsatility with or without a decrease in plasma levels of IGF-1 (Zizzari et al., 2005; Veldhuis and Bowers, 2010). Finally, plasma ghrelin concentration is negatively correlated with body weight and subcutaneous, visceral, and total adiposity (reviewed in Veldhuis and Bowers, 2010), probably due to a long term effect on ghrelin in driving pulsatile GH secretion, which is strongly lipolytic.

Ghrelin is perhaps best known as a circulating hunger signal necessary for meal initiation and meal anticipation with a secretion occurring in a pulsatile manner starting with a preprandial rise and postprandial fall 1 h after food intake (Ariyasu et al., 2001; Cummings et al., 2001; Tschöp et al., 2001; Zizzari et al., 2011; Merkestein et al., 2012). Moreover, whatever the route of injection, ghrelin increases food intake both in humans and animals (Wren et al., 2001a,b). In addition, prolonged food reduction or severe caloric restriction causes an increase in plasma ghrelin concentration (Wren et al., 2001b; Méquinion, personal data). In AN patients, ghrelin levels are increased up to twofold and return to normal levels after weight restoration (Otto et al., 2001, 2005; Tolle et al., 2003; Germain et al., 2009; Yi et al., 2011). However, it appears that fluctuations in ghrelin are not always influenced by food intake in AN (Germain et al., 2009) suggesting impairment in its regulation, probably due to a chronic adaptation to long term food restriction (Yi et al., 2011). Mice lacking the gene for ghrelin or its receptor have normal food intake when fed chow, probably due to compensatory adaptation during embryonic development, but show a degree of protection from obesity when fed a high-fat diet, especially from an early age (Sun et al., 2003, 2004; Wortley et al., 2004, 2005).

Ghrelin also appears to be of importance in the regulation of lipid and glucose metabolism. It has been attributed a role in the maintenance of normal blood glucose levels (Grove and Cowley, 2005). There are indications that ghrelin’s effects on the GH axis may have relevance for glucose homeostasis (and even survival) during chronic food deprivation. Mice lacking acyl ghrelin (due to knockout of GOAT) lose glycemic control and become moribund by 1 week of 60% food deprivation, an effect that can be circumvented by infusion of either acyl ghrelin or GH throughout this 1-week period (Zhao et al., 2010). The work of Sun et al. (2008) on ghrelin homeostasis in ghrelin KO and GHS-R KO mice demonstrate that the ghrelin/GHS-R pathway appears to play an important role in glucose homeostasis by regulating insulin sensitivity and glucose sensing, particularly under conditions of negative energy balance. However, data indicating an action of ghrelin on plasma insulin levels are still controversial (Castañeda et al., 2010; Sangiao-Alvarellos and Cordido, 2010). Intravenous ghrelin injection leads to a decrease in plasma insulin and an increase in blood glucose (Broglio et al., 2001). This result is not found universally and it may be related to the physiological vs. pharmacological doses used (Castañeda et al., 2010). Ghrelin could potentially decrease insulin secretion by altering insulin sensitivity (Castañeda et al., 2010). This is in agreement with the results obtained during insulin and glucose tolerance tests performed in AN patients (Broglio et al., 2004a; Harada et al., 2008). Moreover, under chronic food restriction, fatty acids are mobilized and their oxidation could increase the production of octanoic acid, thereafter used to octanoylate des-acyl ghrelin, leading to a global increase of plasma ghrelin levels. Thus, the greater levels of glucose observed in ABA mice (Méquinion, personal data) or wild type mice during the early phases of the chronic food restriction might be driven by this increase in ghrelin. This might contribute to the adaptive state in the first stages of AN, before a depletion of the supply of free fatty acids and induction of ketosis. Ghrelin acts directly on the liver, to favor glycogenolysis but also on muscle and adipose tissues. Indeed, subcutaneous injection of ghrelin in rats induces an increase of hepatic triglycerides associated with an increase in the gene expression of enzymes involved in lipogenesis like acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS, Barazzoni et al., 2005) as well as increased expression of ACC and FAS mRNA in white visceral adipose tissue (Thompson et al., 2004; Barazzoni et al., 2005). By contrast, subcutaneous ghrelin injection induces a decrease of triglyceride content in muscle without modifying ACC expression and AMPK phosphorylation (Barazzoni et al., 2005). These effects are especially observed in the gastrocnemius muscle, a fast-twitch muscle that is predominantly glycolytic. Similar variations are observed in the food-restricted condition (Samec et al., 2002). Finally, Pardo et al. (2010) describe, in the ABA model, a tissue-specific expression pattern of GHS-R1a receptors in visceral and subcutaneous fat and within the muscle. Indeed, the oxidative-soleus type of muscle appears to be more susceptible to circulating ghrelin levels than the glycolytic-gastrocnemius type under exercise and food restriction situations. All of these modifications could provide a defense mechanism to maintain energy homeostasis in the unbalanced energy state that is found in AN patients.

Appetite, food intake, and energy balance are finely tuned by a complex intercommunication between neural networks and peripheral tissues (Figure 2). Within the CNS, various hypothalamic nuclei containing orexigenic and anorexigenic neurons regulate the different facets of food intake. The ARC cells targeted by ghrelin and its mimetics include the orexigenic neuropeptide Y (NPY) cells (Dickson and Luckman, 1997) that co-express another orexigenic peptide, agouti-related peptide (AgRP). Following administration of ghrelin, these neurons are activated, reflected by the induction of Fos-protein in discrete cell groups (Hewson and Dickson, 2000; Wang et al., 2002), by increased action potential firing (Cowley et al., 2003; Andrews et al., 2008) and by an increased expression of NPY and AgRP mRNA (Kamegai et al., 2000, 2001; Nakazato et al., 2001). Furthermore, the stimulatory effects of ghrelin on NPY/AgRP neurons are complemented by a reduction of the ARC anorexigenic pro-opiomelanocortin (POMC) neuronal activity via inhibitory GABA-ergic inputs from NPY/AgRP neurons (Cowley et al., 2003). Interestingly, sensitivity of the ARC cells to ghrelin appears to be nutritionally regulated as the Fos response was increased up to threefold in fasting rats relative to fed animals (Hewson and Dickson, 2000) an effect that was reversed once again upon refeeding (Luckman et al., 1999). Collectively, these data indicate that ghrelin activates a key orexigenic pathway in the hypothalamic ARC, the NPY/AgRP cells and that this response is metabolically regulated. Consistent with this, stimulation of hypothalamic GHS-R1a results in an anabolic response characterized by an increase in food intake (Wren et al., 2000) and a decrease in energy utilization (Tschöp et al., 2000). The feeding effects ghrelin appear to require normal NPY/AgRP signaling since ablation of NPY or AgRP neurons or the use of NPY receptor antagonists abolish these effects (Chen et al., 2004; Luquet et al., 2007). Conditional deletion of NPY/AgRP co-expressing neurons in the ARC of adult mice (by targeting the human diphtheria toxin to the AgRP locus) caused a rapid starvation to death (Luquet et al., 2005). Moreover, mice homozygous for the anorexia (anx) mutation, characterized by poor food intake and death by 3–5 weeks after birth (Maltais et al., 1984) display a lower density of hypothalamic neuropeptides. The data of Nilsson et al. (2011) support the hypothesis of degeneration of hypothalamic ARC neuron populations; the AgRP system appears to be the first system affected and the POMC system being secondary in this process. Finally, in our models of chronic food restriction (SBA and ABA), we found an alteration of the AgRP signal with an accumulation of this peptide in ARC neurons (Méquinion and Nilsson, personal data). Thus, there are numerous lines of evidence supporting the fact that in chronic food restriction (and probably in AN), a dysregulation of the AgRP system occurs that contributes to deficient ghrelin signaling at the level of the ARC. Recent neuroimaging data obtained from AN patients differing in disease duration showed a significant reduction of total white matter volume and focal gray matter atrophy in various brain areas such as the hypothalamus, especially in patients with shorter food restriction. Collectively, these studies highlight the potential role of endocrine and central (hypothalamic) dysfunction in the altered homeostatic metabolic status in AN, as described in animal models (Boghi et al., 2011).

At the hypothalamic level, ghrelin has also been reported to act directly or indirectly on other nuclei linked to feeding control such as the VMH, PVN, and LHA (López et al., 2008; Mano-Otagiri et al., 2009; Lamont et al., 2012). Although not coupled to c-fos expression, VMN cells exhibit a robust electrical response following bath application of a ghrelin agonist (Hewson et al., 1999). The elegant study of López et al. (2008) showed that, in fasted rats, an elevated ghrelin tone was associated with an increased activation of hypothalamic AMPK and a decreased mRNA expression of enzymes (like FAS) involved in the de novo fatty acid biosynthesis only in the VMH. They concluded that the energy peripheral signals sensed to regulate fatty acid metabolism in the hypothalamus and consequently the feeding behavior, may not be a nutrient, but ghrelin through an action at the level of the VMH.

Ghrelin’s effects in the PVN are also likely linked to feeding control as direct intra-PVN injection of ghrelin induces a robust feeding response that is coupled to neuronal (c-fos) activation (Olszewski et al., 2003). Consistent with this, a reduction of GHS-R1a gene in the PVN using RNA interference in rats, significantly reduced body weight and blood ghrelin levels without affecting food intake (Shrestha et al., 2009). These data reflect a role for ghrelin in the modulation of PVN neuron activity in that is linked to energy homeostasis, but the mechanisms of action remains to be elucidated.

In the LHA, ghrelin is thought to mediate hyperphagia through orexin neurons. Indeed, central administration of ghrelin or a ghrelin mimetic induces Fos expression in orexin-containing, but not melanin-concentrating hormone-containing, neurons (Lawrence et al., 2002; Olszewski et al., 2003), and activates glucose responding neurons (Chen et al., 2005a) in this area. Furthermore, ghrelin-induced feeding is suppressed in orexin KO mice (Toshinai et al., 2003). The role of orexin neurons to simulate feeding behavior (appetite/metabolism) is now well established although they are also especially important for sleep and wakefulness (España and Scammell, 2011; Gao, 2012) and play important roles in the stress response, in analgesia and reward/addiction (see Kukkonen, 2013). Moreover, ghrelin’s effects to increase the reward value of a high-fat diet appear to involve a LHA-VTA orexin pathway (Perello et al., 2010).

The action of ghrelin to increase food intake and associated appetitive behaviors involves an integrated neurobiological response exerted at many levels, not only via the hypothalamus. For example, structures located in the caudal brainstem also express GHS-R1a. In particular, ghrelin receptors are found in all three components of the dorsal vagal complex with a highest expression within the area postrema, a moderately dense signal in the nucleus of the solitary tract and a low density signal in the dorsal motor nucleus of the vagus (Zigman et al., 2006). Peripheral injection of ghrelin and, prior to its discover, ghrelin mimetics, induces c-Fos induction in the nucleus of the solitary tract and area postrema (Bailey et al., 2000; Lawrence et al., 2002). The effects of ghrelin on food intake/behavior are similar when injected into the fourth as for the third ventricle, in terms of the amount of food eaten, the number of meals and meal size during the first few hours after treatment (Faulconbridge et al., 2003). The effects of ghrelin in the dorsal vagal complex might be more related to autonomic effects such as on the cardiovascular system. However, since the hypothalamus is strongly connected with the nucleus solitary tract, we cannot exclude an indirect effect of the ghrelin to hypothalamic structures through an activation of brainstem areas, although appears not to include a noradrenergic pathway (Bailey et al., 2000; see infra).

Besides the homeostatic ghrelin sensitive pathways, ghrelin also appears to target mesolimbic circuits linked to reward. Hedonic (non-homeostatic) brain pathways are also involved in feeding control and are modulated by circulating energy balance signals such as ghrelin, thereby influencing the evaluation of the pleasure derived from the taste, smell, or texture of food. Many regions of the corticolimbic and mesolimbic brain are thus involved in learning, memory, emotion, and reward processing associated with food. Among these complex feeding networks (for review, see Van Vugt, 2010; Figure 3), the VTA-Acc (dopaminergic) pathway plays a pivotal role in conferring reward from a wide range of reinforcers, from chemical drugs of abuse to natural rewards such as food (Nestler, 1996; Corwin et al., 2011). Using optogenetic techniques, Adamantidis et al. (2011) demonstrated that phasic activation dopaminergic VTA neurons is associated with reward-predicting cues and facilitates the development of positive reinforcement during reward-seeking and behavioral flexibility. As reviewed in Carr (2011), burst firing of VTA dopaminergic neurons may operate as a “teaching signal.” For example, in the case of food intake, when rats are presented with a highly palatable food for the first time, this triggers dopamine release in the Acc (shell), whereas repeated exposure to the same palatable food blunts the dopamine response despite avid consumption. Interestingly, food restriction has been described to sustain the Acc (shell) dopamine release in this model. Moreover, simply delivering cues linked previously to the food reward can be sufficient to reinstate the Acc (shell) dopamine response, indicating transference of the dopamine response from the reinforcer to the cue (for review, see Volkov et al. (2011). Ghrelin has emerged as one important modulator of the VTA-NAcc (dopaminergic) reward pathway (for review, see Skibicka and Dickson, 2011). More than 50% of the dopaminergic VTA neurons express GHS-R (Zigman et al., 2006), although it is also expressed on other cell types in this area (Abizaid et al., 2006). Whether administered peripherally, into the brain ventricles or into the VTA, ghrelin administration triggers a robust Acc dopamine response (Abizaid et al., 2006; Jerlhag et al., 2006, 2007) that is accompanied by an increased feeding response in rodents (Naleid et al., 2005; Egecioglu et al., 2010) and an increase in food-motivated behavior (Skibicka and Dickson, 2011; Skibicka et al., 2012). Central ghrelin signaling, via GHS-R1A, appears to be important not only for food reward (Egecioglu et al., 2010), but also for the reward associated with artificial rewards like alcohol, cocaine, and amphetamine (Wellman et al., 2005; Jerlhag et al., 2009, 2010). As an example, the locomotor stimulating effect of cocaine is decreased in ghrelin KO mice compared to their wild type littermates (Abizaid et al., 2010). Furthermore, in rodents, ghrelin elevates the motivation to obtain high-sugar or high-fat reward (Perello et al., 2010; Skibicka and Dickson, 2011; Skibicka et al., 2012). In particular, both peripheral and central injections of ghrelin augment the food-motivated behavior of a satiated rat to get sugar whereas blockade of ghrelin signaling reduces the operant responding of an hungry rat to the level of a satiated rat (Skibicka et al., 2012). These data strongly support the involvement of ghrelin in behaviors related to food reward. Thereby one can suggest that ghrelin could be considered as a key internal cue, available during period of energy deficit to motivate adequate food intake behavior (Skibicka and Dickson, 2011). These data reinforce the now well-documented role of ghrelin in food reward, considering that the shell region of the Acc is described to process unpredicted rewards and motivational states to reinforce food intake behaviors, but also use of drugs of abuse. However, the action of circulating ghrelin upon GHS-R1A-expressing cells in the VTA, located mostly on dopaminergic neurons, begs the question of how this hormone reaches deep brain structures that are far away from circumventricular organs (see infra). Finally, one study shows that presence of food is necessary to induce dopamine release (Kawahara et al., 2009). Chronic ghrelin treatment also modifies the expression of dopaminergic receptors in Acc, more specially D1 and D3 (Skibicka et al., 2012), which are described to be involved in obesity, food reward (D1 receptors) and inhibition of reward behavior (D3 receptors). Similar data were obtained from human imaging studies that emphasize the role of ghrelin in food reward. Indeed, intravenous ghrelin injection to human subjects increases activity in brain areas involved in the evaluation of the reward value attributed to food and food cues including the striatum, amygdala, insula, and orbitofrontal cortex (Malik et al., 2008).

Almost 6000 articles have been published on ghrelin, since its discovery in Kojima et al. (1999) and it is not surprising that its biological effects extend beyond feeding and energy balance, effects that may also be relevant for AN. These include peripheral effects (e.g., gastric motility, bone homeostasis, cardiovascular system, glucose homeostasis, reproduction, immune system) as well as CNS effects (mood disorder, sleep disturbance).

There is now increasing evidence that ghrelin stimulates motor activity in the gastrointestinal tract (gastric motility and emptying). In fact, ghrelin shares high homology degree with motilin (Kojima and Kangawa, 2005), a hormone released by endocrine cells of duodenum and jejunum during fasting and which increases gastric motility after feeding (Sanger, 2008). Ghrelin has been described to use both central and local pathways to exert its effects on the gut through receptors located on vagal afferents, in the nodose ganglion and myenteric plexus. In fact in normal rodents, central pathways are operational whereas after vagotomy, ghrelin is able to exert effects via the myenteric plexus (see review of Peeters, 2003). Moreover, in mice, central administration of ghrelin accelerates gastric emptying (Asakawa et al., 2001) and changes the excitability of neurons located in the PVN identified as receiving ascending afferent signals from mechanoreceptors in the stomach (Zhao et al., 2003). Clinically, it has been reported that the intravenous administration of ghrelin accelerates the rate of gastric emptying and induces gastrointestinal contraction in healthy volunteers (Fujitsuka et al., 2012). Since the plasma levels of ghrelin are high in AN patients, one can hypothesize an alteration of the signaling both at central and peripheral levels that may worsen the outcome of the patients. These findings suggest that ghrelin could provide a therapeutic target for disorders related to gastrointestinal discomfort.

Surprisingly, ghrelin is also involved in sleep-wakefulness regulation. Indeed, experiments conducted in adult male rats demonstrate that repeated intravenously administrations of ghrelin stimulate wakefulness, decrease slow-wave sleep, and reduce the duration of rapid eye movement sleep (Tolle et al., 2002). This action could involve a reduction in the release of acetylcholine from the dorsal tegmental nucleus (LDTg) on neurons expressing somatotropin release-inhibiting factor known to indirectly regulate the rapid eye movement sleep periods (Tolle et al., 2002). In fact, among the various neurochemical systems involved in wakefulness (acetylcholine, norepinephrine, dopamine, serotonin, histamine), the hypothalamic orexigenic neurons are crucial promoters of wakefulness since deficiency in the orexin system leads to disorders such as narcolepsy (Modirrousta et al., 2005). The status of activity in orexin neurons is closely related with the nutritional and behavioral state of animals. Moreover, Lamont et al. (2012) observed that both GHS-R and ghrelin KO mice had fewer orexin-immunoreactive cells than their wild type littermates. Their data support the synergistic relationship between ghrelin and orexin in the coordination of metabolism, reward and arousal to adopt the adapted behavior for food seeking and restoration of energy deficiency. In humans, AN patients exhibit sleep disorders. As an example, AN adolescents have an increase in wakefulness after sleep onset, a fragmentation of sleep as well as a reduction of slow-wave sleep and slow-wave activity during their total sleep time (Lauer and Krieg, 2004; Nobili et al., 2004). Even if deepening of nocturnal sleep follows a partial weight restoration, the neurobiological mechanisms linking starvation, mood disorders, and sleep disturbance remain to be elucidated.

The impact of ghrelin on anxiety behaviors remains controversial: studies show an anxiolytic effect under caloric restriction or after subcutaneous ghrelin injection (Lutter et al., 2008) while anxiogenic effects are observed in others (acute) studies with intracerebroventricular or intraperitoneal ghrelin injection (Asakawa et al., 2001; see review Chuang and Zigman, 2010). Interestingly, chronic central ghrelin treatment was found to increase anxiety-like behavior in rats (Hansson et al., 2011). Recently, one study investigating the amygdala as a target for ghrelin found that acute ghrelin injection at this site elicits behaviors consist with a reduction in anxiety-like behavior, but only in rats that were not allowed access to food during the initial hour after injection. It was concluded that ghrelin, acting at the level of the amygdala, may provide an especially important signal to suppress anxiety-like behaviors that would otherwise prohibit the animal from finding food (Alvarez-Crespo et al., 2012). It is not yet known whether the ghrelin system regulates anxiety behavior associated with AN. One study found that a SNP in the preproghrelin gene was associated with panic disorder in a small patient group (Hansson et al., 2011).

Among the disorders described in AN, osteopenia/osteoporosis is also one major problem that cause long term outcomes with in particular a strong increase of the bone fracture incidence (Lucas et al., 1999). Ghrelin has and ghrelin mimetics have been shown to increase bone mineral density (Svensson et al., 2000; Fukushima et al., 2005; Delhanty et al., 2006) by a mechanism that appears to include the promotion of both proliferation and differentiation of osteoblasts (cells involved in bone formation), involving GHS-R1a and GHS-R1b receptors (Fukushima et al., 2005; Delhanty et al., 2006). The etiopathogenesis of bone disease in AN is complex and multifaceted. Indeed, the low bone mineral density (Legroux-Gerot et al., 2005, 2008; Legroux-Gérot et al., 2007; Estour et al., 2010) is usually linked with alteration of multiple factors (Tables 3– 6) that are thought to contribute to the “uncoupling” of bone turnover, leading to increased bone resorption, and decreased bone formation (see Howgate et al., 2013). However, it has been demonstrated that ghrelin affects bone metabolism by operating in an autocrine/paracrine mode, independent of the GH/IGF-1 axis (see Nikolopoulos et al., 2010). Weight recovery is associated with partial recovery of bone mineral density. There is currently no approved effective therapy that completely reverses the bone mineral density deficit. The most convincing results were obtained with a treatment of recombinant human IGF-1 alone or in combination with the oral contraceptive pills (see Misra and Klibanski, 2011). The link between ghrelin and estrogen on bone metabolism is always matter of debate even if it is established that ghrelin suppresses pulsatile LH and FSH pulsatility (Meczekalski et al., 2008; Kluge et al., 2012).

Another criterion used to characterize AN patients is amenorrhea. Indeed, in negative energy balance conditions like in AN, the increase of plasma ghrelin is associated with decrease of LH secretion (Table 6). Evidence is mounting that ghrelin may operate as a pleitropic modulator of gonadal function and reproduction (Tena-Sempere, 2008; Muccioli et al., 2011; Repaci et al., 2011). Notably, most of the actions of ghrelin upon the reproductive axis reported to date are inhibitory. Ghrelin can suppress not only LH, but also FSH secretion in male and female rats (Fernández-Fernández et al., 2005; Martini et al., 2006). Such effects are also described in humans (Kluge et al., 2012). Centrally, ghrelin exerts a predominant action directly at the level of the GnRH pulse generator by inhibiting directly GnRH release (Fernández-Fernández et al., 2005; Muccioli et al., 2011) or by an indirect modulation of other neuronal pathways. For example, Forbes et al. (2009) recently showed the ability of ghrelin to decrease Kiss1 mRNA expression in the medial preoptic area. Given the importance of the kisspeptin system to control the reproductive axis, these data provide new hypothesis for ghrelin-induced suppression of pulsatile LH secretion. Once again, in the AN, ghrelin might dynamically mediate the suppressive effect of energy deficit on the onset of puberty, gonadal function, and fertility. Here, the effects of ghrelin on the gonadal axis might protect females in a condition of strong energy insufficiency to develop a reproductive behavior that can be deleterious for her and her progeny.

Finally, ghrelin is also involved in other physiological functions that are more or less affected in AN like cardiovascular function or immune system. Among the cardiovascular effects, this hormone improves left ventricular contractility and cardiac output in healthy humans (Enomoto et al., 2003; Tesauro et al., 2010) and lowers blood pressure in mice concomitantly with a decrease in sympathetic nerve activity that is not caused by a direct action on blood vessels (Callaghan et al., 2012). In AN, the neuroendocrine alterations are also accompanied by autonomic dysfunctions like lower blood pressure values, lack of circadian variation of blood pressure and bradycardia (Oswiecimska et al., 2007). Thus, ghrelin might participate in AN to the cardiovascular complications observed in AN (Casiero and Frishman, 2006; Jáuregui-Garrido and Jáuregui-Lobera, 2012), but no studies currently display any correlation between these cardiovascular risks and the high plasma levels of ghrelin.

The role of ghrelin on the immune system remains unclear. However, Taub (2008) describes its implication in the regulation of immune factors, by inhibiting inflammatory cytokine production, more specifically in mediating anti-inflammatory effects on IL-1, TNF-α, and IL-6 cytokine expression by T-cells and mononuclear cells via GHS-R, and promoting thymic function. In AN, data related to the evaluation of circulating pro-inflammatory or inflammatory cytokines or in adipocytes are still a matter of debate and, as underlined by Nova and Marcos (2006), “controversial findings have been published regarding some aspects of the immune system that are otherwise impaired in more typical types of malnutrition.”

Concerning des-acyl ghrelin, its role in food intake has been much debated. The recent paper of Delhanty et al. (2012) gives numerous arguments supporting des-acyl ghrelin as an hormone that can be metabolically active, when co-administrated with acyl ghrelin, by counteracting the effects of acyl ghrelin on insulin secretion and glucose metabolism. Des-acyl ghrelin appears to be increased in AN patients (Harada et al., 2008; Germain et al., 2009). Kojima et al. (1999) showed that des-acyl ghrelin was not able to bind to GHS-R1a. Although the des-acyl ghrelin receptor remains unknown, the increasing data suggesting that des-acyl ghrelin is a biologically active molecular, indicate that a dedicated receptor may exist.

Several studies show controversial effects of des-acyl ghrelin on food intake that are either inhibitory (Asakawa et al., 2005; Chen et al., 2005b) or stimulatory (Toshinai et al., 2006) in rodents. These results can be due to the different methods used like the type of injection, the dose used, the time of injection (light or dark period), the nutritional status, fed, or fasted. However, the overexpression of des-acyl ghrelin in a transgenic mouse model results in a small phenotype, associated with a reduction of food intake and body fat mass, reduced IGF-1 plasma levels without significant changes in circulating GH and also higher des-acyl ghrelin with no change in total ghrelin plasma levels (Ariyasu et al., 2005). In these mice, no significant differences have been noticed for glycemia and insulinemia (Ariyasu et al., 2005; Asakawa et al., 2005) while studies other studies have shown that des-acyl ghrelin inhibits glucose release in vivo and in vitro (Broglio et al., 2004b; Gauna et al., 2005; Qader et al., 2008). Moreover, it appears that ghrelin and des-acyl ghrelin do not have the same blood concentration in systemic and in portal circulations suggesting that liver could be involved in ghrelin regulation (Goodyear et al., 2010).

Concerning lipid metabolism, in vivo studies showed that des-acyl ghrelin as well as ghrelin increase bone marrow adipogenesis in rat shinbone (Thompson et al., 2004) and both forms enhance lipid accumulation in visceral tissue in humans (Rodríguez et al., 2009). The mechanisms involved remain unclear. Similarly, acute or chronic des-acyl ghrelin injections in adult male rats cause an inhibition of LH secretion like ghrelin (Martini et al., 2006). By contrast, transgenic mice overexpressing des-acyl ghrelin do not display any changes in LH and FSH levels (Ariyasu et al., 2005).

Des-acyl ghrelin may also have a role in gastric motility. Indeed, intracerebroventricular or intravenous injections alter motor activity in the antrum with a decrease of antrum activity only in fasted rats (Chen et al., 2005b). Mice overexpressing des-acyl ghrelin exhibit a decrease in gastric emptying (Asakawa et al., 2005). Other studies are necessary to understand the mechanisms involved since vagotomy does not disrupt the (intravenous) des-acyl ghrelin effect (Chen et al., 2005b).

Studies about the effects of des-acyl ghrelin on the cardiovascular system are rare. Nevertheless, like acyl ghrelin, it promotes bradycardia and hypotension (Tsubota et al., 2005). Moreover, ghrelin and des-acyl ghrelin display vasodilator effect and no inotropic effects when they are applied on human artery in vitro (Kleinz et al., 2006).

Similarly, concerning obestatin, it remains again an open question whether this peptide is a physiologically relevant peptide to regulate energy homeostasis, food intake and gastric motility (Gourcerol et al., 2007). Obestatin binds to GRP 39, a receptor of the same subfamily than ghrelin receptor, to decrease food intake and body weight in an opposite manner to ghrelin (Stengel et al., 2009; Hassouna et al., 2010; Mokrosinski and Holst, 2010; Veldhuis and Bowers, 2010). Subsequent studies failed to show activation of this receptor and only few studies have reproduced the obestatin effects under specific conditions. Such results should be interpreted with caution since variations are observed according to the kits and conditions applied for obestatin assays (Hassouna et al., 2010). Due to their potential functions, it should be interesting to measure ghrelin/obestatin ratio to better understand their roles in the alteration of energy balance. It seems that AN affects obestatin blood levels with a lower ghrelin/obestatin ratio in AN patients of restrictive type compared to constitutional thin women (Germain et al., 2009, 2010). Moreover, other functions are attributed to this hormone such as the inhibition of thirst, gastric motility, cell survival, pancreatic hormone secretion, sleep, thermoregulation, memory, and anxiety (Szentirmai et al., 2009; Hassouna et al., 2010; Veldhuis and Bowers, 2010).