Miski Scerif

Anthony P Goldstone

MD Márta Korbonits
email: m.korbonits@qmul.ac.uk

Ghrelin in Obesity and Endocrine Diseases

. Appetite regulation

The hypothalamus is crucial for appetite regulation and energy homeostasis.

Afferent signals from peripheral sites such as the gastrointestinal tract and adipose tissues are integrated by complex neuronal networks to produce efferent responses responsible for food intake and energy metabolism [START_REF] Druce | The regulation of appetite[END_REF]. There are numerous hypothalamic appetite regulators. Orexigenic (appetite-stimulating) compounds include:

neuropeptide Y (NPY), agouti-related peptide (AgRP), ghrelin, orexin and cannabinoids, and anorexigenic (appetite-suppressing) peptides include: pro-opiomelanocortin (POMC)

and cocaine and amphetamine regulatory transcript (CART), thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH).

Ghrelin and appetite regulation

The appetite-stimulating function of ghrelin was identified secondary to its effect on growth hormone (GH) release from somatotroph cells of the anterior pituitary (Korbonits & Grossman, 2004), however, ghrelin is the first known peripheral hormone to display orexigenic effects through its action on the hypothalamic appetite-regulating pathways [START_REF] Nakazato | A role for ghrelin in the central regulation of feeding[END_REF]. In addition, ghrelin is amongst the most powerful of the orexigenic peptides [START_REF] Kojima | Ghrelin: structure and function[END_REF]. While most orexigenic peptides originate from the brain and are only active when injected into the brain, ghrelin is active even with peripheral administration leading to an increase in appetite in rodents and humans (Wren et al., 2001a;Wren et al., 2001b). Ghrelin activates NPY/AgRP neurons [START_REF] Seoane | Agoutirelated peptide, neuropeptide Y, and somatostatin-producing neurons are targets for ghrelin actions in the rat hypothalamus[END_REF] of the hypothalamic arcuate nucleus (ARC) through its receptor,

A c c e p t e d M a n u s c r i p t 3 GHS-R1a, promoting production and secretion of their orexigenic neuropeptides to suppress pro-POMC neuronal activity while promoting food intake [START_REF] Cowley | The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis[END_REF][START_REF] Egecioglu | Hypothalamic gene expression following ghrelin therapy to gastrectomized rodents[END_REF]. Interplay between ghrelin and orexin [START_REF] Toshinai | Ghrelin-induced food intake is mediated via the orexin pathway[END_REF] suggests that arousal and wakefulness may be required for ghrelin's orexigenic effect [START_REF] Olszewski | Ghrelin in the CNS: from hunger to a rewarding and memorable meal?[END_REF]. Mice with double knockout (KO) of NPY/AgRP genes completely lack the orexigenic action of ghrelin, confirming that these neuropeptides mediate and are essential for the orexigenic effect of this peptide hormone [START_REF] Chen | Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein[END_REF].

AMPK and the orexigenic signaling pathway of ghrelin

Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionally well conserved heterotrimeric serine/threonine kinase composed of a catalytic α (alpha) subunit and regulatory β (beta) and γ (gamma) subunits. AMPK is a regulator of cellular and systemic energy homeostasis and acts as a sensor of energy status [START_REF] Kola | Expanding role of AMPK in endocrinology[END_REF][START_REF] Xiao | Structural basis for AMP binding to mammalian AMP-activated protein kinase[END_REF]. Once activated, AMPK leads to a switch from anabolic to catabolic pathways; with the aim of restoring energy balance, ATP production is increased and ATP-utilising pathways are inhibited. Hypothalamic AMPK plays a vital role in the regulation of food intake [START_REF] Kola | Expanding role of AMPK in endocrinology[END_REF][START_REF] Lim | AMPK as a mediator of hormonal signalling[END_REF]. Constitutively active AMPK increases both food intake and body weight; whereas dominant negative AMPK expression in the hypothalamus is sufficient to reduce food intake and body weight [START_REF] Minokoshi | AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus[END_REF].

The peripheral hormones ghrelin [START_REF] Andersson | AMP-activated protein kinase plays a role in the control of food intake[END_REF][START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF] and adiponectin [START_REF] Kadowaki | The physiological and pathophysiological role of adiponectin and adiponectin receptors in the peripheral tissues and CNS[END_REF][START_REF] Kubota | Adiponectin stimulates AMPactivated protein kinase in the hypothalamus and increases food intake[END_REF][START_REF] Wen | Globular adiponectin regulates energy homeostasis through AMP-activated protein kinase-acetyl-CoA carboxylase (AMPK/ACC) pathway in the hypothalamus[END_REF] both have A c c e p t e d M a n u s c r i p t 4 hypothalamic AMPK implicated in their orexigenic effects. Hypothalamic AMPK has also been suggested as the mediator of the orexigenic effects of several other metabolic hormones and neuropeptides/modulators including cannabinoids [START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF] and glucocorticoids [START_REF] Christ-Crain | AMPactivated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome[END_REF]. Evidence that AMPK activity in the hypothalamus is also regulated by anorexigenic hormones: leptin [START_REF] Andersson | AMP-activated protein kinase plays a role in the control of food intake[END_REF][START_REF] Minokoshi | AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus[END_REF] and insulin [START_REF] Minokoshi | AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus[END_REF], further demonstrates that hypothalamic AMPK is directly involved in appetite regulation.

We have shown that rat and mouse hypothalamic AMPK activity is stimulated by both central (intracerebroventricular) and peripheral (intraperitoneal) ghrelin administration [START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF][START_REF] Kola | The orexigenic effect of ghrelin is mediated through central activation of the endogenous cannabinoid system[END_REF]. The central orexigenic effect of ghrelin is AMPK-dependent as compound C, an inhibitor of AMPK, completely prevented ghrelin's orexigenic effect, and adenoviruses harbouring α1/α2 dominant negative AMPK were able to impair the stimulatory effect of ghrelin on food intake [START_REF] Lopez | Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin[END_REF]. The signalling pathway of ghrelin's orexigenic effect in the hypothalamus has been further elucidated with the identification of the endocannabinoid system [START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF][START_REF] Kola | The orexigenic effect of ghrelin is mediated through central activation of the endogenous cannabinoid system[END_REF] and calmodulin kinase kinase 2 (CaMKK2) [START_REF] Anderson | Hypothalamic CaMKK2 contributes to the regulation of energy balance[END_REF] as upstream regulators to AMPK activation. The fatty acid pathways [START_REF] Lopez | Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin[END_REF] and mitochondrial uncoupling protein 2 (UCP2) [START_REF] Andrews | UCP2 mediates ghrelin's action on NPY/AgRP neurons by lowering free radicals[END_REF], have been identified as novel downstream effectors. Combining the recently described data, a pathway by which ghrelin could stimulate appetite has been suggested [START_REF] Kola | Shedding light on the intricate puzzle of ghrelin's effects on appetite regulation[END_REF] (Figure 1).

Ghrelin in short-and long-term regulation of energy homeostasis

A c c e p t e d M a n u s c r i p t 5

There is evidence to show that ghrelin is important in short-term regulation of appetite and energy balance. The clear pre-prandial rise and post-prandial fall in plasma ghrelin levels support the hypothesis that ghrelin acts as an initiator signal for meal consumption in humans [START_REF] Cummings | A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans[END_REF]. The pre-prandial increase of ghrelin levels was found to initiate meal consumption voluntarily, without time-or food-related cues [START_REF] Cummings | Plasma ghrelin levels and hunger scores in humans initiating meals voluntarily without time-and foodrelated cues[END_REF], whilst the post-prandial ghrelin suppression is proportional to the ingested calorie load [START_REF] Callahan | Postprandial suppression of plasma ghrelin level is proportional to ingested caloric load but does not predict intermeal interval in humans[END_REF]. Ghrelin also appears to be involved in the regulation of long-term energy homeostasis [START_REF] Cummings | Ghrelin and the short-and long-term regulation of appetite and body weight[END_REF]. This peptide hormone has been described as the peripheral counterpart of insulin and leptin, and AMPK also appears to be involved in its peripheral metabolic effects [START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF] (Figure 2).

Ghrelin is able to reverse the down-regulating effect of insulin on mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK), the rate limiting enzyme of gluconeogenesis [START_REF] Murata | Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells[END_REF]. Ghrelin reduces glucose-stimulated insulin secretion and glucose disappearance in healthy humans [START_REF] Tong | Ghrelin suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans[END_REF] thus ghrelin leads to increase in blood glucose levels and impairs glucose tolerance (Korbonits & Grossman, 2004). Ghrelin has been shown to significantly increase sterol-regulatory element binding protein-1 (SREBP1) mRNA levels, as well as several fat storage-related proteins, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and lipoprotein lipase (LPL) leading to stimulation of intracytoplasmic lipid accumulation [START_REF] Rodriguez | Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes[END_REF]. Ghrelin reduces the use of fat as a metabolic fuel and promotes an increase in adipose tissue and body weight [START_REF] Tschop | Ghrelin induces adiposity in rodents[END_REF]. Circulating ghrelin induces abdominal obesity, independently of its central orexigenic activity, via GHS-Rdependent lipid retention [START_REF] Davies | Ghrelin induces abdominal obesity via GHS-R-dependent lipid retention[END_REF]Salome et al., 2009). Obesity is a condition that results from chronic disruption of energy balance; energy intake continuously exceeds energy expenditure and accumulation of body fat follows [START_REF] Hill | Understanding and addressing the epidemic of obesity: an energy balance perspective[END_REF]. The prevalence of obesity is on the rise and the obesity pandemic is arguably amongst the most serious public health challenges in the world today. Obesity is strongly associated with type 2 diabetes mellitus, hyperlipidemia and cardiovascular disease, and provides significant contributions to ill health in both western and developing countries [START_REF] Kopelman | Obesity as a medical problem[END_REF]. Extensive research into the mechanisms of appetite regulation and energy balance has unveiled complex physiological systems behind energy homeostasis, and yielded potential targets for therapeutic intervention in the fight against obesity.

Ghrelin and obesity

Plasma ghrelin levels inversely correlate with body mass index (BMI). Thus, ghrelin levels are reduced in those who are obese compared to normal body weight controls [START_REF] Tschop | Circulating ghrelin levels are decreased in human obesity[END_REF][START_REF] Williams | Overfeeding-induced weight gain suppresses plasma ghrelin levels in rats[END_REF]. Recent evidence suggests that dietinduced obesity causes ghrelin resistance by reducing NPY/AgRP responsiveness to plasma ghrelin and suppressing the neuroendocrine ghrelin axis, in an attempt to limit further food intake [START_REF] Briggs | Diet-induced obesity causes ghrelin resistance in arcuate NPY/AgRP neurons[END_REF]. Ghrelin levels have been shown to negatively correlate with factors which are raised in obesity namely, percentage body fat, insulin and leptin levels [START_REF] Tschop | Circulating ghrelin levels are decreased in human obesity[END_REF]. In one study ghrelin levels were not related to fat mass or intra-abdominal fat content, but showed strong negative correlation with insulin levels A c c e p t e d M a n u s c r i p t 7 and insulin resistance [START_REF] Purnell | Ghrelin levels correlate with insulin levels, insulin resistance, and high-density lipoprotein cholesterol, but not with gender, menopausal status, or cortisol levels in humans[END_REF]. However in an MRI study of non-obese and obese adults, ghrelin was negatively correlated with visceral adiposity, fasting insulin, and homeostasis model insulin resistance index, with visceral adiposity having a stronger inverse correlation with ghrelin than subcutaneous fat depots possibly through hyperinsulinemia, as the negative correlations with insulin resistance were even stronger [START_REF] Goldstone | Elevated fasting plasma ghrelin in prader-willi syndrome adults is not solely explained by their reduced visceral adiposity and insulin resistance[END_REF]. Abnormal glucose homeostasis inversely correlated with, and was found to be an independent determinant of, plasma ghrelin levels in obese children and adolescents [START_REF] Zhu | Plasma ghrelin levels and polymorphisms of ghrelin gene in Chinese obese children and adolescents[END_REF].

It has been shown that selective ghrelin blockade, in diet-induced obese mice, results in reduction of food intake, bodyweight and body fat mass [START_REF] Shearman | Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice[END_REF]. Adult ghrelin-deficient mice, compared to their wild-type littermates, display significantly lower glucose levels and increased insulin sensitivity under conditions of caloric restriction [START_REF] Sun | Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance[END_REF] and are protected from the rapid weight gain induced by early exposure to a high-fat diet [START_REF] Wortley | Absence of ghrelin protects against early-onset obesity[END_REF]. In addition, mice lacking ghrelin receptors resist the development of diet-induced obesity [START_REF] Zigman | Mice lacking ghrelin receptors resist the development of diet-induced obesity[END_REF], and antagonism of the ghrelin receptor results in reduction of food intake and weight gain [START_REF] Asakawa | Antagonism of ghrelin receptor reduces food intake and body weight gain in mice[END_REF]. Therefore, reduced levels of ghrelin activity in obese individuals, as compared with non-obese controls, should provide greater protection from further weight gain. However, obese individuals display smaller post-prandial reductions in serum ghrelin levels, perhaps leading to a reduced feeling of satiety [START_REF] Goldstone | Fasting and postprandial hyperghrelinemia in Prader-Willi syndrome is partially explained by hypoinsulinemia, and is not due to peptide YY3-36 deficiency or seen in hypothalamic obesity due to craniopharyngioma[END_REF][START_REF] Le Roux | Postprandial plasma ghrelin is suppressed proportional to meal calorie content in normalweight but not obese subjects[END_REF]. In an earlier study, food intake failed to suppress ghrelin levels in obese humans [START_REF] English | Food fails to suppress ghrelin levels in obese humans[END_REF]. In addition, weight loss is associated with a A c c e p t e d M a n u s c r i p t 8 simultaneous increase in plasma ghrelin levels (Cummings et al., 2002a;[START_REF] Hansen | Weight loss increases circulating levels of ghrelin in human obesity[END_REF], which may make the maintenance of weight loss more difficult.

Obesity endangers the lives of millions of people worldwide, thus novel approaches to control body weight remain a high priority. Both ghrelin and GHS-R are being investigated as therapeutic targets (Schellekens et al., 2010). In vitro generated biostable RNA-based compounds that specifically bind ghrelin have been used in animal models to inhibit ghrelin-mediated GHS-R activation [START_REF] Helmling | Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer[END_REF]. Zorilla et al. have developed an anti-ghrelin vaccine, providing a novel method to block ghrelin action [START_REF] Zorrilla | Vaccination against weight gain[END_REF]. Exciting prospects lie ahead for an anti-ghrelin vaccine to treat obesity [START_REF] Carlson | Prospects for an anti-ghrelin vaccine to treat obesity[END_REF].

Ghrelin is acylated on its serine 3 residue with octanoate, an eight-carbon fatty acid. This post-translational modification is mediated by ghrelin O-acyltransferase (GOAT) [START_REF] Yang | Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone[END_REF] and is essential for GHS-R1a activation and the endocrine actions of ghrelin [START_REF] Kojima | Ghrelin: structure and function[END_REF]. Consistent with its function, GOAT mRNA is primarily limited to the stomach; the main ghrelin-secreting tissue [START_REF] Yang | Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone[END_REF]. Acylated ghrelin induces body weight gain and adiposity by promoting food intake and decreasing energy expenditure [START_REF] Asakawa | Stomach regulates energy balance via acylated ghrelin and desacyl ghrelin[END_REF][START_REF] Tschop | Ghrelin induces adiposity in rodents[END_REF].

GOAT has been implicated as another potential target for the development of anti-obesity treatment [START_REF] Gualillo | Introducing GOAT: a target for obesity and antidiabetic drugs?[END_REF][START_REF] Yang | Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone[END_REF]. Chronic treatment of mice on a high fat diet with GO-CoA-Tat, a peptide-based GOAT inhibitor, prevented the significant weight gain observed in vehicle-treated controls. Moreover, quantitative magnetic resonance (QMR) measurements showed that, relative to controls, GO-CoA-Tat treated animals displayed significantly lower fat mass, but not lean mass [START_REF] Barnett | Glucose and weight control in mice with a designed ghrelin O-acyltransferase inhibitor[END_REF].

A c c e p t e d M a n u s c r i p t 9

Ghrelin and Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a complex genetic disorder characterised by childhood-onset hyperphagia-associated obesity, mental retardation, GH deficiency (short stature), hypogonadism, sleep and thermoregulation disturbance [START_REF] Goldstone | Prader-Willi syndrome: advances in genetics, pathophysiology and treatment[END_REF]. The PWS genotype is characterized by loss of several paternal genes in the region q11-13 on chromosome 15 [START_REF] Nicholls | Genome organization, function, and imprinting in Prader-Willi and Angelman syndromes[END_REF]. PWS is a human genetic obesity syndrome and the most common cause of syndromic obesity.

In contrast to other forms of obesity, patients with PWS display significantly reduced visceral adiposity, and are protected from the metabolic complications of obesity, namely insulin resistance and hypertriglyceridemia [START_REF] Goldstone | Visceral adipose tissue and metabolic complications of obesity are reduced in Prader-Willi syndrome female adults: evidence for novel influences on body fat distribution[END_REF].

Patients with PWS have high ghrelin levels relative to their obesity, with fasting plasma concentrations up to three-to four-fold higher in PWS than in non-PWS obese controls. [START_REF] Bizzarri | Children with Prader-Willi syndrome exhibit more evident meal-induced responses in plasma ghrelin and peptide YY levels than obese and lean children[END_REF]Cummings et al., 2002b;[START_REF] Gimenez-Palop | A lesser postprandial suppression of plasma ghrelin in Prader-Willi syndrome is associated with low fasting and a blunted postprandial PYY response[END_REF][START_REF] Goldstone | Fasting and postprandial hyperghrelinemia in Prader-Willi syndrome is partially explained by hypoinsulinemia, and is not due to peptide YY3-36 deficiency or seen in hypothalamic obesity due to craniopharyngioma[END_REF]Haqq et al., 2003a;[START_REF] Paik | Correlation between fasting plasma ghrelin levels and age, body mass index (BMI), BMI percentiles, and 24-hour plasma ghrelin profiles in Prader-Willi syndrome[END_REF]. Levels of fasting ghrelin may also even be higher in PWS patients than in lean controls [START_REF] Bizzarri | Children with Prader-Willi syndrome exhibit more evident meal-induced responses in plasma ghrelin and peptide YY levels than obese and lean children[END_REF]Choe et al., 2005a;Cummings et al., 2002b;[START_REF] Delparigi | High circulating ghrelin: a potential cause for hyperphagia and obesity in prader-willi syndrome[END_REF][START_REF] Gimenez-Palop | A lesser postprandial suppression of plasma ghrelin in Prader-Willi syndrome is associated with low fasting and a blunted postprandial PYY response[END_REF]. Postprandial levels of ghrelin do fall in patients with PWS though they remain elevated compared to non-PWS obese controls, with conflicting results as to whether the magnitude of the decrease in ghrelin is blunted [START_REF] Gimenez-Palop | A lesser postprandial suppression of plasma ghrelin in Prader-Willi syndrome is associated with low fasting and a blunted postprandial PYY response[END_REF][START_REF] Goldstone | Fasting and postprandial hyperghrelinemia in Prader-Willi syndrome is partially explained by hypoinsulinemia, and is not due to peptide YY3-36 deficiency or seen in hypothalamic obesity due to craniopharyngioma[END_REF]Haqq et al., 2003b). In addition, the age-related decline in ghrelin is blunted in PWS infants and children [START_REF] Haqq | Ghrelin concentrations in Prader-Willi syndrome (PWS) infants and children: changes during development[END_REF].

A c c e p t e d M a n u s c r i p t 10 Hypothalamic dysfunction has been suggested to underlie the obesity that is typical of PWS however fasting and post-prandial ghrelin levels are not elevated in patients with hypothalamic obesity [START_REF] Goldstone | Fasting and postprandial hyperghrelinemia in Prader-Willi syndrome is partially explained by hypoinsulinemia, and is not due to peptide YY3-36 deficiency or seen in hypothalamic obesity due to craniopharyngioma[END_REF][START_REF] Kanumakala | Fasting ghrelin levels are not elevated in children with hypothalamic obesity[END_REF].

Two-to three-fold increases in the numbers of ghrelin-expressing cells were noted in the gastric body and fundus of PWS patients as compared to lean and obese controls (Choe et al., 2005a). Imprinting of paternal genes in region q11-13 on chromosome 15 may induce the production of excessive amounts of transcription factors that increase ghrelin expression or, loss of a transcription inhibitory factor that normally suppresses ghrelin expression [START_REF] Kojima | Ghrelin: structure and function[END_REF]. Hyperghrelinemia of PWS appears to be partially, but not solely, explained by reduced visceral adiposity [START_REF] Goldstone | Elevated fasting plasma ghrelin in prader-willi syndrome adults is not solely explained by their reduced visceral adiposity and insulin resistance[END_REF] and relative hypoinsulinemia [START_REF] Goldstone | Elevated fasting plasma ghrelin in prader-willi syndrome adults is not solely explained by their reduced visceral adiposity and insulin resistance[END_REF][START_REF] Goldstone | Fasting and postprandial hyperghrelinemia in Prader-Willi syndrome is partially explained by hypoinsulinemia, and is not due to peptide YY3-36 deficiency or seen in hypothalamic obesity due to craniopharyngioma[END_REF]. Other possible causes of the elevated ghrelin levels in PWS include abnormal parasympathetic vagal innervation of the stomach [START_REF] Goldstone | Prader-Willi syndrome: advances in genetics, pathophysiology and treatment[END_REF][START_REF] Higgins | Ghrelin, the peripheral hunger hormone[END_REF] and abnormal sympathetic tone [START_REF] Higgins | Ghrelin, the peripheral hunger hormone[END_REF].

Some studies have shown elevated ghrelin levels in PWS infants and children and thus it appears that hyperghrelinemia may precede hyperphagia in PWS, though this has not been universally seen in all studies [START_REF] Erdie-Lalena | Ghrelin levels in young children with Prader-Willi syndrome[END_REF][START_REF] Feigerlova | Hyperghrelinemia precedes obesity in Prader-Willi syndrome[END_REF][START_REF] Haqq | Ghrelin concentrations in Prader-Willi syndrome (PWS) infants and children: changes during development[END_REF]. Neonatal transgenic deletion PWS mice die from severe neonatal hypoglycemia due to depletion of liver glycogen stores, and ghrelin levels increase reciprocally with the falling glucose levels [START_REF] Stefan | Hormonal and metabolic defects in a prader-willi syndrome mouse model with neonatal failure to thrive[END_REF] Although it has been hypothesised that hyperphagia in PWS may be partly ). High circulating levels of ghrelin can be hypothesised as a potential cause for hyperphagia and obesity in PWS (Cummings et al., 2002b;[START_REF] Delparigi | High circulating ghrelin: a potential cause for hyperphagia and obesity in prader-willi syndrome[END_REF]Haqq et al., 2003a). However, not all PWS patients have elevated ghrelin levels, and pharmacological reduction of ghrelin levels to within normal levels using a somatostatin infusion was unable to reduce acute food intake in PWS adults [START_REF] Tan | Somatostatin infusion lowers plasma ghrelin without reducing appetite in adults with Prader-Willi syndrome[END_REF].

Futhermore, chronic treatment with a long-acting somatostatin analogue led to a sustained decrease in ghrelin concentrations but did not affect weight, eating behaviour or appetite in adolescents with PWS [START_REF] De | Long-acting octreotide treatment causes a sustained decrease in ghrelin concentrations but does not affect weight, behaviour and appetite in subjects with Prader-Willi syndrome[END_REF]. However such interventions may also lower circulating anorexigenic gastroenteropancreatic hormones which may complicate their interpretation [START_REF] De | Long-acting octreotide treatment causes a sustained decrease in ghrelin concentrations but does not affect weight, behaviour and appetite in subjects with Prader-Willi syndrome[END_REF][START_REF] Tan | Somatostatin infusion lowers plasma ghrelin without reducing appetite in adults with Prader-Willi syndrome[END_REF]. Thus although other pathological defects are felt to cause or contribute to hyperphagia in PWS, including reduced circulating anorexigenic pancreatic polypeptide levels [START_REF] Zipf | Pancreatic polypeptide responses to protein meal challenges in obese but otherwise normal children and obese children with Prader-Willi syndrome[END_REF], hypothalamic dysfunction [START_REF] Goldstone | Hypothalamic neuropeptides and regulation of fat mass in Prader-Willi syndrome[END_REF][START_REF] Goldstone | Prader-Willi syndrome: advances in genetics, pathophysiology and treatment[END_REF], defects in brain reward and satiety systems [START_REF] Hinton | Neural representations of hunger and satiety in Prader-Willi syndrome[END_REF][START_REF] Holsen | Neural mechanisms underlying hyperphagia in Prader-Willi syndrome[END_REF][START_REF] Miller | Enhanced activation of reward mediating prefrontal regions in response to food stimuli in Prader-Willi syndrome[END_REF], and decreased brain-derived neurotrophic factor (BDNF) which functions downstream of leptin and POMC/MC4R signalling [START_REF] Han | Lower brain-derived neurotrophic factor in patients with prader-willi syndrome compared to obese and lean control subjects[END_REF], it will need the clinical development of ghrelin antagonists or GOAT inhibitors to be able to definitively assess the role of hyperghrelinemia in the hyperphagia of PWS.

Common genetic variations of GHRL and GHS-R genes in human obesity

Relationships have been described between obesity and genomic variants of the ghrelin (GHRL) [START_REF] Gueorguiev | Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity[END_REF][START_REF] Korbonits | A variation in the ghrelin gene increases weight and decreases insulin secretion in tall, obese children[END_REF][START_REF] Kuzuya | Preproghrelin Leu72Met variant contributes to overweight in middle-aged men of a Japanese large cohort[END_REF][START_REF] Leskela | Fasting plasma total ghrelin concentrations in monozygotic twins discordant for obesity[END_REF][START_REF] Del | Molecular screening of the ghrelin gene in Italian obese children: the Leu72Met variant is associated with an earlier onset of obesity[END_REF][START_REF] Ukkola | Mutations in the preproghrelin/ghrelin gene associated with obesity in humans[END_REF][START_REF] Ukkola | Role of ghrelin polymorphisms in obesity based on three different studies[END_REF] A c c e p t e d M a n u s c r i p t 12 and ghrelin receptor (GHS-R) [START_REF] Baessler | Genetic linkage and association of the growth hormone secretagogue receptor (ghrelin receptor) gene in human obesity[END_REF][START_REF] Gueorguiev | Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity[END_REF][START_REF] Liu | Four missense mutations in the ghrelin receptor result in distinct pharmacological abnormalities[END_REF] genes.

The Leu72Met (C247A) polymorphism of the ghrelin gene was detected in 15.6% (12.5% heterozygote and 3.1% homozygote) of obese subjects and in 12.5% (all heterozygotes) of non-obese control subjects. This polymorphism has been associated

with an earlier onset of obesity [START_REF] Ukkola | Mutations in the preproghrelin/ghrelin gene associated with obesity in humans[END_REF]. We studied the ghrelin gene in obese French children and found a positive correlation between the Leu72Met heterozygote variant with higher BMI and early-onset obesity [START_REF] Korbonits | A variation in the ghrelin gene increases weight and decreases insulin secretion in tall, obese children[END_REF] and a similar significant association between the Leu72Met polymorphism and earlyonset obesity was observed in Italian children [START_REF] Del | Molecular screening of the ghrelin gene in Italian obese children: the Leu72Met variant is associated with an earlier onset of obesity[END_REF]. In addition, the Leu72Met polymorphism has been suggested to contribute to weight gain in middle-aged Japanese men [START_REF] Kuzuya | Preproghrelin Leu72Met variant contributes to overweight in middle-aged men of a Japanese large cohort[END_REF]. However, the above findings could not be reproduced by other studies [START_REF] Bing | Large-scale studies of the Leu72Met polymorphism of the ghrelin gene in relation to the metabolic syndrome and associated quantitative traits[END_REF][START_REF] Hinney | Ghrelin gene: identification of missense variants and a frameshift mutation in extremely obese children and adolescents and healthy normal weight students[END_REF][START_REF] Jo | Preproghrelin Leu72Met polymorphism in obese Korean children[END_REF][START_REF] Zhu | Plasma ghrelin levels and polymorphisms of ghrelin gene in Chinese obese children and adolescents[END_REF]. Furthermore, one study of 3004 subjects even reported the Leu72Met polymorphism to be protective against fat accumulation; the polymorphism was related to lower BMI, fat mass and reduced visceral fat [START_REF] Ukkola | Role of ghrelin polymorphisms in obesity based on three different studies[END_REF]. Another study suggests that Leu72 allele carriers are more prone to weight variability in response to environmental factors given the reduced frequency of Leu72Met polymorphism in monozygotic twins discordant for obesity [START_REF] Leskela | Fasting plasma total ghrelin concentrations in monozygotic twins discordant for obesity[END_REF].

The Arg51Gln (G185A) variant of the ghrelin gene was found in six (all heterozygotes) obese subjects (6.3%) but was not found in non-obese controls [START_REF] Ukkola | Mutations in the preproghrelin/ghrelin gene associated with obesity in humans[END_REF]. Obese subjects with the Arg51Gln variant had lower body weights than obese subjects with the wild-type Arg51Arg variant [START_REF] Ukkola | Mutations in the preproghrelin/ghrelin gene associated with obesity in humans[END_REF]. Although studies A c c e p t e d M a n u s c r i p t 13 have showed correlation between Arg51Gln and lower ghrelin levels [START_REF] Poykko | Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes[END_REF][START_REF] Ukkola | Role of ghrelin polymorphisms in obesity based on three different studies[END_REF], another study was unable to confirm this finding [START_REF] Vivenza | Ghrelin gene polymorphisms and ghrelin, insulin, IGF-I, leptin and anthropometric data in children and adolescents[END_REF].

Direct sequencing identified 12 GHRL single-nucleotide polymorphisms (SNPs) and 8 GHS-R SNPs [START_REF] Gueorguiev | Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity[END_REF]. The GHRL polymorphism A265T and the GHS-R polmorphism G477A have been associated with obesity in a French cohort of 263 families, while the GHRL variant Leu72Met (C247A) and GHS-R variant C60T may modulate eating patterns. However, after correction for multiple comparisons and testing in a German cohort with early-onset extreme obesity, it appears that these variants of GHRL and GHS-R genes are not strong genetic factors determining the susceptibility to obesity [START_REF] Gueorguiev | Association studies on ghrelin and ghrelin receptor gene polymorphisms with obesity[END_REF]. (1,418 Caucasians individuals). The linkage disequilibrium analysis revealed a disequilibrium block consisting of five SNPs, which showed significant association with obesity in both study cohorts [START_REF] Baessler | Genetic linkage and association of the growth hormone secretagogue receptor (ghrelin receptor) gene in human obesity[END_REF]. However, Wang et al. studied SNPs of the GHS-R in German children and adolescents and failed to find conclusive evidence for the involvement of the GHS-R gene in body weight regulation [START_REF] Wang | Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normal-weight and underweight students, and children with short normal stature[END_REF].

This is supported by a systematic study of common GHS-R variants in three large population-based UK cohort studies comprising 5807 adults and 843 children, which found no association with body weight or BMI [START_REF] Garcia | Ghrelin receptor gene polymorphisms and body size in children and adults[END_REF]. In addition, a

A c c e p t e d M a n u s c r i p t 14 population-based study of 15,854 Danish subjects, in which seven common GHS-R SNPs were genotyped, failed to show association between the GHS-R variants and obesity [START_REF] Gjesing | Family and population-based studies of variation within the ghrelin receptor locus in relation to measures of obesity[END_REF]. Further clinical studies are required to better define the frequency and the aetiological role of GHS-R isoforms in obesity [START_REF] Liu | Four missense mutations in the ghrelin receptor result in distinct pharmacological abnormalities[END_REF].

GHS-R mutations in patients

Ala204Glu [START_REF] Pantel | Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature[END_REF][START_REF] Wang | Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normal-weight and underweight students, and children with short normal stature[END_REF] and Phe279Leu [START_REF] Wang | Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normal-weight and underweight students, and children with short normal stature[END_REF] are rare GHS-R mutations which lead to loss of constitutive activity of GHS-R and subsequent short stature and obesity [START_REF] Holst | Ghrelin receptor mutations--too little height and too much hunger[END_REF]. Obesity would be unexpected in loss-of-function mutations of GHS-R, however compensatory changes in related pathways may account for the obese phenotype in subjects with the inactivating mutations [START_REF] Holst | Ghrelin receptor mutations--too little height and too much hunger[END_REF]. Four missense mutations of the GHS-R: Ala204Glu, to French reference values) was found to carry the Ala204Glu mutation in the homozygous state; whilst the parents were heterozygous for this mutation (maternal and paternal height was noted to be 2.7SD and 3.7SD below the mean, respectively). In addition, several members of family 1 were overweight or obese -BMI of the 13 year old proband was 26.2kg/m 2 [START_REF] Pantel | Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature[END_REF]. In family 2, the 11 year old female proband was found to carry the same mutation in the heterozygous state, and the affected father (height 2SD below mean) was also heterozygous for the mutation [START_REF] Pantel | Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature[END_REF].

In contrast to the Ala204Glu mutation which has been implicated in short stature with dominant transmission; recessive GHS-R mutations have also been reported [START_REF] Pantel | Recessive isolated growth hormone deficiency and mutations in the ghrelin receptor[END_REF]. The proband was born to unrelated parents of normal phenotype, presented with episodes of abdominal pain, vomiting, hypoglycemia, and ketosis and required glucose infusions. He showed reduced height, weight, BMI and delayed bone age.

Endocrine investigations revealed markedly low insulin-like growth factor 1 (IGF-1) levels and the patient displayed a low growth hormone (GH) response to provocative tests. GHS-R sequencing revealed that the patient was compound heterozygous for two new defects: a mutation predicting a premature stop codon (Trp2X) inherited from his unaffected father, therefore strongly arguing against haploinsufficiency as a disease mechanism, and a missense mutation (Arg237Trp), inherited from his healthy mother [START_REF] Pantel | Recessive isolated growth hormone deficiency and mutations in the ghrelin receptor[END_REF].

In a study of 82 Danish obese probands and 28 lean subjects, a rare mutation in the promoter region (-151 C/T) of GHS-R was identified in one obese individual. The -A c c e p t e d M a n u s c r i p t 16 expression of GHS-R and increased potential for constitutive activity. This mutation cosegregated with obesity in the pedigree of the identified proband. The proband, her son, and sister had all been overweight or obese since the onset of puberty and her mother since her early twenties. Screening for the -151C/T GHS-R mutation was also performed in 289 obese Czech children, and an additional heterozygous carrier of the -151C/T mutation was identified. Co-segregation was seen for all obese individuals in the Czech pedigree; however two members of this pedigree were carriers of this mutation but not obese, possibly due to incomplete penetrance. The -151C/T mutation was subsequently genotyped in a population-based study of 15,854 Danish subjects; however in this study no association was found between the -151C/T mutation and obesity [START_REF] Gjesing | Family and population-based studies of variation within the ghrelin receptor locus in relation to measures of obesity[END_REF].

Ghrelin and bariatric surgery

Gastric bypass operations, the most common and most effective form of bariatric surgery, provide patients with morbid obesity an option of long-term sustained weight loss [START_REF] Buchwald | Bariatric surgery worldwide 2003[END_REF][START_REF] Fobi | Surgical treatment of obesity: a review[END_REF]. Data regarding the alteration of ghrelin levels following gastric bypass surgery are inconsistent [START_REF] Higgins | Ghrelin, the peripheral hunger hormone[END_REF][START_REF] Pournaras | The effect of bariatric surgery on gut hormones that alter appetite[END_REF][START_REF] Pournaras | Ghrelin and metabolic surgery[END_REF][START_REF] Vincent | Changes in gut hormones after bariatric surgery[END_REF].

Many studies have shown gastric bypass operations to be associated with significant reduction of ghrelin levels (Cummings et al., 2002a;[START_REF] Geloneze | Ghrelin: a gut-brain hormone: effect of gastric bypass surgery[END_REF][START_REF] Lin | The effects of gastric surgery on systemic ghrelin levels in the morbidly obese[END_REF][START_REF] Roth | Ghrelin and obestatin levels in severely obese women before and after weight loss after Roux-en-Y gastric bypass surgery[END_REF][START_REF] Stylopoulos | Changes in serum ghrelin predict weight loss after Roux-en-Y gastric bypass in rats[END_REF][START_REF] Suzuki | Changes in GI hormones and their effect on gastric emptying and transit times after Roux-en-Y gastric bypass in rat model[END_REF]. In addition, the normal, meal-related fluctuation and the diurnal rhythm of ghrelin level was found to be absent after gastric bypass surgery (Cummings et al., 2002a). Ghrelin levels A c c e p t e d M a n u s c r i p t 17 were significantly suppressed in diet-induced obese rats post-gastric bypass as compared with both sham-operated obese and lean control rats [START_REF] Shin | Meal-induced hormone responses in a rat model of Roux-en-Y gastric bypass surgery[END_REF]. GHS-R1a protein expression in the hypothalamus was found to be much lower in rats following gastric bypass as compared to sham-operated controls [START_REF] Wang | Combination of bypassing stomach and vagus dissection in high-fat dietinduced obese rats-a long-term investigation[END_REF].

The suppression of ghrelin levels following gastric bypass surgery may contribute to reduced hunger and the weight-reducing effect of the procedure. However, the mechanism for the decrease in plasma ghrelin levels following gastric bypass is not yet known. It may be explained by major loss of gastric mucosa, thus ghrelin-producing endocrine cells. Alternatively, direct contact between gastric mucosa with food may be important for the production and secretion of ghrelin [START_REF] Beckman | Changes in gastrointestinal hormones and leptin after Roux-en-Y gastric bypass procedure: a review[END_REF][START_REF] Kojima | Ghrelin: structure and function[END_REF].

In contrast, ghrelin levels have been found to remain unchanged after gastric bypass surgery in several studies [START_REF] Pournaras | Ghrelin and metabolic surgery[END_REF][START_REF] Vincent | Changes in gut hormones after bariatric surgery[END_REF].

Increases in ghrelin levels post-gastric bypass have also been seen [START_REF] Garcia-Fuentes | Different effect of laparoscopic Roux-en-Y gastric bypass and open biliopancreatic diversion of Scopinaro on serum PYY and ghrelin levels[END_REF][START_REF] Holdstock | Ghrelin and adipose tissue regulatory peptides: effect of gastric bypass surgery in obese humans[END_REF][START_REF] Perez-Romero | Effects of two variants of Roux-en-Y Gastric bypass on metabolism behaviour: focus on plasma ghrelin concentrations over a 2-year followup[END_REF][START_REF] Ybarra | Persistent correlation of ghrelin plasma levels with body mass index both in stable weight conditions and during gastric-bypass-induced weight loss[END_REF]; however this may be associated with active weight loss rather than the surgery itself [START_REF] Faraj | Plasma acylation-stimulating protein, adiponectin, leptin, and ghrelin before and after weight loss induced by gastric bypass surgery in morbidly obese subjects[END_REF]. Several explanations have been proposed for the conflicting findings regarding ghrelin levels following gastric bypass surgery including differences in pre-operative insulin resistance, surgical methods and post-operative vagal nerve dysfunction [START_REF] Pournaras | Ghrelin and metabolic surgery[END_REF][START_REF] Vincent | Changes in gut hormones after bariatric surgery[END_REF].

Weight loss following gastric banding is independent of circulating plasma ghrelin, as an increase in fasting ghrelin is accompanied by a paradoxical decrease in hunger, but relies on changes in eating behaviour induced by gastric restriction (Schindler , 2004). In sleeve gastrectomy, the fundus of the stomach, the main location of ghrelin-producing cells, is excluded and studies have confirmed a decrease in fasting ghrelin levels after the procedure [START_REF] Cohen | Ghrelin levels and sleeve gastrectomy in super-super-obesity[END_REF][START_REF] Langer | Sleeve gastrectomy and gastric banding: effects on plasma ghrelin levels[END_REF][START_REF] Wang | Plasma ghrelin modulation in gastric band operation and sleeve gastrectomy[END_REF]. In a recent review, Beckman et al. suggest that the stronger, better designed studies indicate greatly reduced ghrelin levels in the post-gastric bypass state as compared to the obese or lean state, or when weight loss is achieved through procedures other than gastric bypass or through lifestyle modification [START_REF] Beckman | Changes in gastrointestinal hormones and leptin after Roux-en-Y gastric bypass procedure: a review[END_REF].

Ghrelin and endocrine diseases

The role of ghrelin in growth hormone deficiency, acromegaly, diabetes mellitus as well as in gastrointestinal neuroendocrine tumours will be discussed elsewhere in this special issue, but we will summarise data on Cushing's syndrome, thyroid disease, pheochromocytoma and insulinoma.

Ghrelin and hypothalamic-pituitary-adrenal axis

Ghrelin shows stimulatory effects on the corticotropic system [START_REF] Giordano | Ghrelin, hypothalamus-pituitary-adrenal (HPA) axis and Cushing's syndrome[END_REF]. Systemic administration of ghrelin, or the synthetic hexarelin, has been shown to increase ACTH and cortisol levels in healthy subjects [START_REF] Arvat | Endocrine activities of ghrelin, a natural growth hormone secretagogue (GHS), in humans: comparison and interactions with hexarelin, a nonnatural peptidyl GHS, and GHreleasing hormone[END_REF]Korbonits et al., 1999a). A hypothalamic site of action of ghrelin on the hypothalamic-pituitaryadrenal (HPA) axis has been proposed, as transection of the hypophyseal stalk dramatically decreased cortisol responses to synthetic growth hormone secretagogue (GHS) treatment [START_REF] Hickey | Mediation by the central nervous system is critical to the in vivo activity of the GH secretagogue L-692,585[END_REF]. Furthermore these compounds fail to increase ACTH release from pituitary tissue in vitro [START_REF] Shimon | Human fetal pituitary expresses functional growth hormone-releasing peptide receptors[END_REF]. There are data to suggest that ghrelin stimulates the HPA axis at a hypothalamic level, at least in part via arginine vasopressin (AVP) and CRH [START_REF] Coiro | Adrenocorticotropin/cortisol and arginine-vasopressin secretory patterns in response to ghrelin in normal men[END_REF]Korbonits et al., 1999a;[START_REF] Mozid | Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin[END_REF]. It has been shown that NPY can stimulate CRH and AVP release from the rat hypothalamus (Korbonits et al., 1999b), therefore the effect of ghrelin on CRH and AVP could occur through direct action on CRH and AVP neurons in the paraventricular nucleus, or may occur indirectly via NPY [START_REF] Mozid | Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin[END_REF]. Ghrelin does not have direct stimulatory effects on adrenal hormone secretion [START_REF] Andreis | Ghrelin and growth hormone secretagogue receptor are expressed in the rat adrenal cortex: Evidence that ghrelin stimulates the growth, but not the secretory activity of adrenal cells[END_REF][START_REF] Barreiro | Expression and homologous regulation of GH secretagogue receptor mRNA in rat adrenal gland[END_REF]. It appears that a feedback loop may exist between ghrelin levels and the HPA axis. While ghrelin stimulates the HPA axis, hypercortisolemia is associated with reduced ghrelin levels [START_REF] Otto | Endogenous and exogenous glucocorticoids decrease plasma ghrelin in humans[END_REF].

Ghrelin and Cushing's syndrome

The complications of Cushing's syndrome include central obesity, impaired glucose tolerance, insulin resistance, dyslipidemia, hepatic steatosis and systemic arterial hypertension (Arnaldi et al., 2003a) and eventually the metabolic syndrome [START_REF] Anagnostis | Clinical review: The pathogenetic role of cortisol in the metabolic syndrome: a hypothesis[END_REF]Arnaldi et al., 2003a). Ghrelin levels are decreased in patients with Cushing's syndrome [START_REF] Libe | Ghrelin and adiponectin in patients with Cushing's disease before and after successful transsphenoidal surgery[END_REF][START_REF] Otto | Endogenous and exogenous glucocorticoids decrease plasma ghrelin in humans[END_REF]. This may be explained by the associated hyperinsulinemia and insulin resistance [START_REF] Goldstone | Elevated fasting plasma ghrelin in prader-willi syndrome adults is not solely explained by their reduced visceral adiposity and insulin resistance[END_REF][START_REF] Goldstone | Fasting and postprandial hyperghrelinemia in Prader-Willi syndrome is partially explained by hypoinsulinemia, and is not due to peptide YY3-36 deficiency or seen in hypothalamic obesity due to craniopharyngioma[END_REF][START_REF] Poykko | Low plasma ghrelin is associated with insulin resistance, hypertension, and the prevalence of type 2 diabetes[END_REF]. In Cushing's syndrome, the decrease in plasma ghrelin concentration is not associated with ACTH levels. Both endogenous hypercortisolemia, in which ACTH levels are reduced, and Cushing's disease which results from elevated ACTH secretion, lead to reduced ghrelin levels [START_REF] Otto | Endogenous and exogenous glucocorticoids decrease plasma ghrelin in humans[END_REF]. Ghrelin levels significantly increased after successful surgical treatment of Cushing's disease (Libe et , 2005;[START_REF] Otto | Endogenous and exogenous glucocorticoids decrease plasma ghrelin in humans[END_REF]. These findings make it unlikely that ghrelin has a causative role in the unfavourable energy balance and characteristic body composition of Cushing's syndrome [START_REF] Otto | Endogenous and exogenous glucocorticoids decrease plasma ghrelin in humans[END_REF], but rather it appears that ghrelin levels in Cushing's syndrome are altered as a consequence of the metabolic disturbance.

The ghrelin-induced secretion of ACTH and cortisol is enhanced in Cushing's disease patients as compared to healthy controls [START_REF] Correa-Silva | Decreased GH secretion and enhanced ACTH and cortisol release after ghrelin administration in Cushing's disease: comparison with GH-releasing peptide-6 (GHRP-6) and GHRH[END_REF][START_REF] Leal-Cerro | Ghrelin is no longer able to stimulate growth hormone secretion in patients with Cushing's syndrome but instead induces exaggerated corticotropin and cortisol responses[END_REF][START_REF] Miljic | ACTH and cortisol responses to ghrelin and DDAVP in patients with Cushing's disease (CD) and adrenal enlargement[END_REF]. GHS-R overexpression has been demonstrated in pituitary and ectopic ACTH-secreting tumours and in other neuroendocrine tumours (Arnaldi et al., 2003b;[START_REF] Korbonits | The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors[END_REF]. In patients with an ACTH-secreting tumour, ghrelin may further stimulate ACTH levels, and subsequently cortisol levels, through direct action on tumoral GHS-R [START_REF] Correa-Silva | Decreased GH secretion and enhanced ACTH and cortisol release after ghrelin administration in Cushing's disease: comparison with GH-releasing peptide-6 (GHRP-6) and GHRH[END_REF]. Ghrelin has been shown to act directly on corticotrope tumour cells derived from patients with Cushing's disease and this supports the suggestion of a cellular basis for the exaggerated ACTH response to ghrelin in Cushing's disease [START_REF] Martinez-Fuentes | Ghrelin is produced by and directly activates corticotrope cells from adrenocorticotropin-secreting adenomas[END_REF]. In addition, ghrelin has been shown in vitro to stimulate ACTH secretion by human ACTH-secreting pituitary adenomas [START_REF] Pecori | Ghrelin stimulates adrenocorticotrophic hormone (ACTH) secretion by human ACTHsecreting pituitary adenomas in vitro[END_REF]. After one month of ketoconazole treatment and potent inhibition of steroidogenesis, patients with Cushing's disease showed significant reduction in cortisol responses to ghrelin [START_REF] Correa-Silva | Effect of one month ketoconazole treatment on GH, cortisol and ACTH release after ghrelin, GHRP-6 and GHRH administration in patients with Cushing's disease[END_REF].

Ghrelin and thyroid disease

Graves' disease patients have been reported to display increased appetite and food intake as compared to healthy subjects and this hyperphagia may be a response to the excessive energy expenditure which characterises hyperthyroidism. Interestingly, A c c e p t e d M a n u s c r i p t 21 normalisation of appetite occurs after pharmacological therapy [START_REF] Cugini | Hunger sensation in Graves' disease before and after pharmacological therapy[END_REF]). It appears that the hyperphagia of hyperthyroidism is not mediated by circulating ghrelin [START_REF] Katergari | Ghrelin in pathological conditions[END_REF]. Circulating plasma ghrelin levels are reduced in hyperthyroidism as compared to the euthyroid state (Altinova et al., 2006a), exhibit a positive correlation with thyroid stimulating hormone (TSH) levels (Sawicka et al., 2010) and rise to normal levels with pharmacological therapy of hyperthyroidism [START_REF] Braclik | Serum leptin and ghrelin levels in premenopausal women with stable body mass index during treatment of thyroid dysfunction[END_REF][START_REF] Gimenez-Palop | Circulating ghrelin in thyroid dysfunction is related to insulin resistance and not to hunger, food intake or anthropometric changes[END_REF][START_REF] Riis | Hyperthyroidism is associated with suppressed circulating ghrelin levels[END_REF][START_REF] Rojdmark | Hunger-satiety signals in patients with Graves' thyrotoxicosis before, during, and after long-term pharmacological treatment[END_REF][START_REF] Theodoropoulou | Ghrelin response to oral glucose load in hyperthyroidism, before and after treatment with antithyroid drugs[END_REF].

Total ghrelin levels were found to be significantly lower in overt than in subclinical hyperthyroid patients, with a trend to a reduction also in acylated ghrelin levels [START_REF] Tanda | Plasma total and acylated Ghrelin concentrations in patients with clinical and subclinical thyroid dysfunction[END_REF]. Insulin resistance is increased in hyperthyroidism [START_REF] Gimenez-Palop | Circulating ghrelin in thyroid dysfunction is related to insulin resistance and not to hunger, food intake or anthropometric changes[END_REF] and hyperinsulinaemia has been shown to suppress ghrelin levels [START_REF] Saad | Insulin regulates plasma ghrelin concentration[END_REF] regardless of thyroid status [START_REF] Riis | Hyperthyroidism is associated with suppressed circulating ghrelin levels[END_REF].

In hyperthyroid rats increased ghrelin-immunoreactivity in gastric mucosa has been observed along with the decreased plasma ghrelin concentration and this suggests that thyroxine may inhibit ghrelin secretion [START_REF] Dadan | Estimation of gastric ghrelinpositive cells activity in hyperthyroid rats[END_REF].

There are variable data on ghrelin in hypothyroidism. A rat model of hypothyroidism showed increased circulating ghrelin and gastric ghrelin mRNA levels [START_REF] Caminos | Influence of thyroid status and growth hormone deficiency on ghrelin[END_REF]. Following thyroxine replacement in hypothyroid patients, serum ghrelin levels showed a significant reduction [START_REF] Braclik | Serum leptin and ghrelin levels in premenopausal women with stable body mass index during treatment of thyroid dysfunction[END_REF][START_REF] Gjedde | Serum ghrelin levels are increased in hypothyroid patients and become normalized by L-thyroxine treatment[END_REF].

In contrast, other studies have found ghrelin levels in hypothyroid patients to be similar to those of healthy subjects and there was no significant change in ghrelin levels after medical treatment of hypothyroidism [START_REF] Gimenez-Palop | Circulating ghrelin in thyroid dysfunction is related to insulin resistance and not to hunger, food intake or anthropometric changes[END_REF][START_REF] Sadegholvad | Serum ghrelin changes in thyroid dysfunction[END_REF][START_REF] Tanda | Plasma total and acylated Ghrelin concentrations in patients with clinical and subclinical thyroid dysfunction[END_REF]. Anti-parietal cell antibodies and atrophic gastritis has been detected in patients with autoimmune thyroid disease [START_REF] Centanni | Atrophic body gastritis in patients with autoimmune thyroid disease: an underdiagnosed association[END_REF].

Furthermore, patients with Hashimoto's thyroiditis have displayed reduced ghrelin levels as compared to euthyroid subjects, and those with high thyroid peroxidase antibody titres showed greater reduction in ghrelin levels (Altinova et al., 2006b). Thus, thyroid peroxidase antibodies may have a ghrelin lowering effect. In autoimmune thyroid disease there may be reduced production of ghrelin due to destructive antibody-mediated action on the gastric mucosa.

Ghrelin and GHS-R1a mRNA expression has been demonstrated in normal human thyroid by RT-PCR [START_REF] Gnanapavan | The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans[END_REF]. Another study found expression of ghrelin in fetal but not in infant or adult thyroid [START_REF] Volante | Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effects on tumor growth[END_REF], and ghrelin may operate as a regulator of thyroid cell differentiation during normal development. Ghrelin appears to be re-expressed in thyroid tumours [START_REF] Volante | Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effects on tumor growth[END_REF] and ghrelin immunoreactivity has been demonstrated in medullary, follicular and papillary thyroid carcinomas [START_REF] Raghay | Ghrelin localization in rat and human thyroid and parathyroid glands and tumours[END_REF]. Human medullary thyroid carcinoma surgical specimens showed higher ghrelin content than normal thyroid tissues [START_REF] Kanamoto | Substantial production of ghrelin by a human medullary thyroid carcinoma cell line[END_REF]. However, ghrelin levels were reduced in thyroid tissue of patients with papillary thyroid carcinoma, as compared with normal thyroid tissue [START_REF] Karaoglu | Expression of obestatin and ghrelin in papillary thyroid carcinoma[END_REF].

Ghrelin [START_REF] Volante | Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effects on tumor growth[END_REF] and growth hormone secretagogues [START_REF] Cassoni | Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue[END_REF] have been shown to inhibit in vitro cell proliferation of thyroid carcinoma cell lines, and may have a role in regulating thyroid tumour growth. Thus, high levels of ghrelin in thyroid tumours may be a mechanism by which there is an attempt to prevent tumour expansion.

A c c e p t e d M a n u s c r i p t 23

Pheochromocytoma

Pheochromocytomas are rare catecholamine-secreting tumours that arise from chromaffin cells of the adrenal medulla. Ghrelin immunoreactivity has been demonstrated in the medulla of human adrenal glands and in human pheochromocytomas [START_REF] Raghay | Ghrelin localization in the medulla of rat and human adrenal gland and in pheochromocytomas[END_REF]. Ghrelin has been shown to inhibit basal dopamine release and may have an effect in reducing the catecholamine secretion of pheochromocytomas [START_REF] Nanmoku | Effect of Ghrelin on catecholamine secretion in rat pheochromocytoma PC12 cells[END_REF]. The expression of ghrelin mRNA was found to be significantly less in pheochromocytomas as compared to normal adrenal tissue [START_REF] Ueberberg | Differential expression of ghrelin and its receptor (GHS-R1a) in various adrenal tumors and normal adrenal gland[END_REF], thus reduced ghrelin levels may have a role in the increased catecholamine secretion of pheochromocytomas.

Insulinoma

The role of ghrelin in beta-cell function is discussed elsewhere is this special issue. Expression of ghrelin and GHS-R1a mRNA has been detected in human insulinomas [START_REF] Ekeblad | Co-expression of ghrelin and its receptor in pancreatic endocrine tumours[END_REF][START_REF] Volante | Expression of ghrelin and of the GH secretagogue receptor by pancreatic islet cells and related endocrine tumors[END_REF]. Acylated ghrelin, via the GHS-R1a, was found to stimulate insulin secretion in INS-1E rat insulinoma cells. Unacylated ghrelin also stimulated insulin secretion but independently of GHS-R1a [START_REF] Gauna | Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin releasing factor 2 receptor[END_REF]. In contrast, ghrelin has recently been shown to inhibit insulin secretion in MIN6 insulinoma cells partly via the AMPK-UCP pathway (Wang et al., 2010).

Conclusions

There is currently much research into the numerous physiological and pathophysiological roles of ghrelin. Ghrelin plays a crucial function in appetite inducing effect of ghrelin [START_REF] Kola | Shedding light on the intricate puzzle of ghrelin's effects on appetite regulation[END_REF].

  explained by rapid gastric emptying due to increased gastric motility caused by elevated ghrelin levels, in fact patients with PWS have delayed gastric emptying (Choe et al., A c c e p t e d M a n u s c r i p t 11 2005b

  Linkage and association of SNPs and haplotypes within the GHS-R gene region and human obesity has been demonstrated. Baessler et al. performed analysis of the entire GHS-R gene region (99.3 kb) in 178 pedigrees with multiple obese members (1,095 Caucasian individuals) and in an independent sample of the general population

Phe279Leu,

  Ile134Thr and Val160Met, have been reported andLiu et al. have shown that all these mutations affect the constitutive activity of the GHS-R, thus may have physiological consequences and/or pharmacogenomic implications[START_REF] Liu | Four missense mutations in the ghrelin receptor result in distinct pharmacological abnormalities[END_REF]. Wang et al. studied 746 obese German children and adolescents, 232 underweight students, 96 normal-weight students and 43 children with short stature, and identified two individuals with novel missense mutations of the GHS-R. The Ala204Glu mutation was detected in a child with obesity and the Phe279Leu mutation was found in a child with short stature (Wang et al., 2004). Pantel et al. studied 2 unrelated families of Moroccan origin and identified a GHS-R missense mutation that segregates with short stature. The Ala204Glu mutation was not observed in a control group of Moroccan individuals with normal stature, indicating that it is not a frequent polymorphism. In family 1, the 13 year A c c e p t e d M a n u s c r i p t 15 old female proband with significant height reduction (3.7SD below the mean, according

  well as short-and long-term energy homeostasis. The current obesity pandemic is a serious public health challenge. Antagonism of ghrelin and its signalling pathway may in future have a powerful role to play in the management of obesity.Continued research into PWS, genetic variations of ghrelin and GHS-R genes and gastric bypass and other forms of bariatric surgery will provide further insight into the role of ghrelin in obesity. Most likely as a result of secondary insulin resistance, altered ghrelin levels have also been observed in Cushing's syndrome and thyroid disease.

  Figure 1

Figure 1 :

 1 Figure 1: Flow diagram showing the proposed molecules involved in the appetite-

  Figure 1 Caption

Figure 2

 2 Figure 2

Conflict of interest

The authors declare that there is no conflict of interest. [START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF][START_REF] Kola | Expanding role of AMPK in endocrinology[END_REF].