The discovery of ghrelin was rooted in the search for. Ghrelin for the Gastroenterologist: History and Potential

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1 GASTROENTEROLOGY 2003;125: Ghrelin for the Gastroenterologist: History and Potential CHARLES D. R. MURRAY,* MICHAEL A. KAMM,* STEPHEN R. BLOOM, and ANTON V. EMMANUEL* *St. Mark s Hospital, Harrow, Middlesex; and Department of Metabolic Medicine, Hammersmith Hospital, London, England Ghrelin, a novel 28 amino acid orexigenic peptide discovered in 1999, has given us further insights into the control of energy homeostasis and growth hormone secretion. As a natural endogenous ligand of the growth hormone secretagogue receptor, it potently stimulates growth hormone release but is also implicated in many other homeostatic mechanisms. Released from the stomach, it stimulates lactotroph and corticotroph secretion, increases appetite and adiposity, has beneficial hemodynamic effects, has prokinetic and gastric acid secretory functions in the stomach, and may even be implicated in sleep. As advances in the understanding of appetite and obesity are made, it is timely to reviewthe possibly central role of ghrelin in these physiological and pathophysiological states. This reviewwill discuss the recent literature concerning this exciting novel neuropeptide and discuss the possible therapeutic possibilities it may open up to us. The discovery of ghrelin was rooted in the search for an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R). This quest received a new lead from a surprising source. In an effort to develop less potent and addictive narcotics in the late 1970s, it was observed in an enkephalin analogue program that some novel synthetic opioid derivatives had weak GHreleasing activity. 1,2 In turn, this led to the synthesis of many peptide derivatives in search of more potent GH secretagogues (GHSs), including GH-releasing peptide-6, which was shown to be a potent stimulant of GH release, both in vitro and in vivo, in all animal species tested. 3 The characterization, in 1982, of GH-releasing hormone (GHRH) led to the focus of further research being directed to the interactions of GHRH and somatostatin, and it became accepted that GH secretion was the net result of a balance between the stimulatory and inhibitory effects, respectively, of these 2 hypothalamic peptides. On investigation of the first nonpeptide GHSs in 1993, however, it became clear that they were acting via different pathways. 4 GHRH acts on the GHRH receptor to increase intracellular adenosine 3,5 -cyclic monophosphate, whereas GHSs act on a different receptor to increase the intracellular Ca 2 concentration via inositol 1,4,5-triphosphate signal transduction. 5 The GHS-R was identified by Smith et al. 6 in 1996 by expression cloning and was found to be a classic G protein coupled receptor, containing 7 putative -helical membrane spanning segments and 3 intracellular and extracellular loops. The search for endogenous ligands for this receptor was undertaken next, and initial identification was by Kojima et al. 7 They adopted an orphan receptor strategy to discover the ligand, constructing a cell line that expressed rat GHS-R and monitoring changes in intracellular calcium concentration induced by tissue extracts from rat brain, lung, heart, kidney, intestine, and stomach. Unexpectedly, the highest activity was found in the stomach, from which was purified a 28 amino acid peptide. This peptide was named ghrelin, from ghre, the Proto-Indo-European word root for grow. After the amino acid sequence of this peptide was ascertained, synthetic peptide could be produced. However, unlike the endogenous peptide, the synthetic peptide did not increase intracellular calcium levels and on high-performance liquid chromatography was eluted earlier than native ghrelin. Furthermore, on mass spectrometric analysis, the molecular weight of purified ghrelin was found to be 126 daltons greater than the synthetic peptide. Further analysis showed that there was an unusual hydrophobic substituent on Ser3 and that the most probable modification was the addition of an acyl group, n-octanoic acid (Figure 1). This modification is unique to ghrelin and is required for biological activity. In their initial report of the discovery of ghrelin in 1999, Kojima et al. 7 went on to show that it stimulated GH secretion both in vitro and in vivo. Using rat ghrelin complementary DNA (cdna), they then isolated human Abbreviations used in this paper: AUC, area under the curve; BMI, body mass index; GH, growth hormone; GHRH, growth hormone releasing hormone; GHS, growth hormone secretagogue; GHS-R, growth hormone secretagogue receptor; ICV, intracerebroventricular by the American Gastroenterological Association /03/$30.00 doi: /s (03)

2 November 2003 GHRELIN FOR THE GASTROENTEROLOGIST 1493 Figure 1. The structure of human ghrelin. ghrelin cdna from a stomach cdna library. Human ghrelin differs from rat ghrelin by only 2 amino acids and circulates at a plasma concentration of approximately 150 fmol/ml. At much the same time, Tomasetto et al. 8 identified what they named motilin-related peptide, which was later shown to have the same amino acid sequence as ghrelin, without the n-octanoylation of the Ser3 residue. They isolated and sequenced a novel cdna, and its expression was examined in mouse tissue. The novel peptide was mainly expressed by the secretory granules of the stomach enteroendocrine cells. However, without a biological end point, the significance of their findings becomes clear only when combined with the data from Kojima et al. 7 Distribution and Localization In their original experiments, Kojima et al. 7 showed that ghrelin was present in large amounts in rat stomach. Taking this together with the fact that ghrelin, when injected intravenously, induces GH release, they proposed that ghrelin was secreted in the stomach and circulated in the bloodstream to act on the pituitary gland. They also showed ghrelin-immunoreactive neurons in the hypothalamic arcuate nucleus and suggested that ghrelin in the arcuate nucleus may either act on the hypothalamus or be transported to the anterior pituitary. Ghrelin-producing endocrine cells in human digestive tracts are most abundant in the oxyntic cells. 9 The oxyntic mucosa is rich in endocrine cells, which are made up of 3 types: enterochromaffin-like cells, A-like cells, and somatostatin-containing cells, representing 65% 70%, 20% 25%, and 10% 15% of the endocrine cells, respectively. Ghrelin-immunoreactive cells were found to be localized to A-like cells. 9,10 Removal of the acidproducing part of the stomach decreased circulating ghrelin by 80%, in keeping with the oxyntic mucosa being the major source of circulating ghrelin. 11 Infusion of gastrin or hypergastrinemia induced by the proton pump inhibitor omeprazole had no effect on serum ghrelin levels or on ghrelin messenger RNA (mrna) levels in the oxyntic mucosa. 10 This suggests that, unlike enterochromaffin-like cells, ghrelin-containing A-like cells do not operate under gastrin control. Recent data suggest that although patients who are Helicobacter pylori positive do not have higher ghrelin levels than control subjects, 12 the effect of eradication is to increase serum ghrelin. 13 These data may argue that chronic Helicobacter pylori infection is associated with persisting gastric inflammation and down-regulation of ghrelin release, which then increases after eradication of the bacterium. Another group identified ghrelin cells in the rat gastrointestinal tract in the stomach, duodenum, ileum, cecum, and colon and found them to be both open- and closed-type endocrine cells, with progressively more closed-type cells in the distal gut. 14 Open-type cells are in contact with the glandular lumen, whereas closed-type endocrine cells do not have a luminal connection. The presence of distinct cell types with ghrelin immunoreactivity raises the possibility that ghrelin release is dependent on the origin of the stimulus, with circulating triggers acting via closed-type cells and luminal ones via open-type cells. In humans, the stomach seems to be the most abundant source of ghrelin. Ghrelin mrna is most highly expressed in the stomach when compared with other tissues. 15 Plasma ghrelin levels decrease by as much as 65% after gastrectomy, 15 and this is consistent with the findings of decreased plasma levels found after gastric bypass surgery. 11 Ghrelin mrna has been identified in rat and human placenta, with expression increasing in mid pregnancy and decreasing thereafter, suggesting a possible physiological role. 16 In young rats, stomach expression of ghrelin mrna increases with age but is not sex dependent, and levels are not affected by gonadal hormones. 17 An alternative strategy to study the distribution of ghrelin action is to identify the sites of GHS-R localization. The highest concentration of the GHS-R has been reported in the pituitary and hypothalamus, 5 and a comparison has been made between the tissue distribution of GHS-R with ghrelin mrna in humans. 18 Previous localization of the GHS-R by radioreceptor assay with [ 125 I]Tyr-Ala-hexarelin, a GHS, suggested peripheral expression mainly in the myocardium, but with binding in many other tissues. Negligible binding was found in the stomach and colon. 19 Other studies using reverse-transcription polymerase chain reaction have identified the GHS-R in stomach, small intestine, colon, liver, kidney, pancreas, kidney, and T cells. 9,20 23 A variety of techniques to localize ghrelin and its receptor (such as reverse-transcription polymerase chain reaction, Northern blot, and functional receptor assay) have reported widespread distribution of small amounts of the peptide and

3 1494 MURRAY ET AL. GASTROENTEROLOGY Vol. 125, No. 5 its receptor. 7,9,15,17,23 The widespread occurrence of ghrelin suggests that it may have multiple physiological roles. Nevertheless, the high concentrations in endocrine cells in the stomach and the effect of gastrectomy on circulatory levels of ghrelin have focused attention on its potential roles in the control of appetite and digestive processes. Ghrelin and Growth Hormone Secretion Having identified the natural ligand for GHS-R and having shown stimulation of GH secretion by ghrelin in the rat, investigators showed the potency of the peptide on GH release in the same species Human studies have shown a dose-dependent stimulation of GH release with both intravenous infusions and boluses of ghrelin There were no significant adverse effects with doses of up to 500 g. 27 As well as GH release, there were increases in prolactin, 27,28 adrenocorticotropic hormone, and cortisol. 28 The effect of ghrelin on GH release is significantly more potent than the effect of GHRH 28 and is observed in a variety of other species. 29,30 The mechanism by which GH release is stimulated is not entirely clear. Smith et al. 31 proposed that GHS acts on the arcuate nucleus in the hypothalamus, up-regulating the GHRH neurons there, and thus acts as a functional somatostatin antagonist. However, work with GH-releasing peptide-2 (a ghrelin receptor agonist) in patients with GHRH mutations still leads to an increase in GH, 32 suggesting an additional locus of action. Bowers 33 proposed that GHS stimulates an unknown hypothalamic u-factor with GH-releasing capabilities. Yamazaki et al. 34 used an in vitro perfusion system to examine the effects of ghrelin alone and in combination with GHRH and somatostatin on GH secretion from rat anterior pituitary cells. They found a dose-dependent increase in GH peak caused by ghrelin in a dose-dependent manner, different from that caused by GHRH. GH secretion caused by ghrelin declined rapidly to basal levels after the peak, whereas the GH response profile by GHRH showed a relatively long duration. They also found that the combination of ghrelin and GHRH had neither a synergistic nor an additive effect on GH release. Pretreatment with somatostatin abolished the GH release caused by ghrelin with or without GHRH. However, other groups have found additive effects in vitro with GHRH and other GHSs. 35 The reason for the differences is not clear. One possibility is that some of these GHSs act on a yet-unidentified GHS-R. Alternatively, ghrelin may have different affinities and effects at different GHS-Rs. In vivo experiments have shown synergism between GHRH and GHS, suggesting that exogenous GHS may induce the release of another hypothalamic factor. 36,37 These findings are consistent with the in vivo experiments with ghrelin in which combined GHRH and ghrelin clearly led to a synergistic increase in GH release. 38,39 Indeed, such was the synergism found by one group that they suggested that this phenomenon could be used advantageously for a provocative test to diagnose GH deficiency. 39 Patients with a genetic GHRH deficiency present an opportunity to examine GH secretory dynamics in the selective absence of GHRH input. Maheshwari et al. 40 looked at subjects with this condition and found that there was residual GH secretion. GH secretion was pulsatile, with normal pulse frequency but severely reduced amplitude. Nocturnal enhancement of GH secretion remained, which they hypothesize was secondary to a decrease in somatostatin tone ; a role for ghrelin in this phenomenon cannot be ruled out, especially because the diurnal rhythm of ghrelin increases throughout the day and peaks between midnight and 2:00 AM. 41 In humans, increases in serum ghrelin during fasting are followed by similar quantitative changes in serum GH levels (enhanced GH secretion and amplified secretion rhythm), suggesting that ghrelin could drive GH secretion during fasting in humans. 42 Ghrelin is, however, not involved in the acute exercise-induced stimulation of GH release. 43 Other Endocrine Activities As well as the marked increase in GH secretion elicited by ghrelin, there are effects on corticotroph and lactotroph secretion, 26,28,38,39 and ghrelin may play a role in glucose homeostasis and insulin secretion. 20,44 46 Ghrelin administration leads to an increase in prolactin, adrenocorticotropic hormone, cortisol, and aldosterone through mechanisms that are not yet clear It has been suggested that some of the adrenocorticotropic hormone release may depend on a central nervous system mediated mechanism via arginine vasopressin. 50 In support of this model, Ishizaki et al. 52 showed that central administration of ghrelin potently stimulated arginine vasopressin release in conscious rats. There seems to be no effect of ghrelin infusion on plasma concentrations of luteinizing hormone or follicle-stimulating hormone in humans, 28 although intracerebroventricular (ICV) administration of ghrelin suppresses pulsatile luteinizing hormone secretion in ovariectomized rats. 53 The effects on thyroid-stimulating hormone release are more complex. States of negative energy balance are associated with an increase in ghrelin levels, and

4 November 2003 GHRELIN FOR THE GASTROENTEROLOGIST 1495 thyroidectomy in rats has been shown to lead to a small, but significant, increase in stomach ghrelin expression. 46 Wren et al. 26 noted an inhibition of thyroid-stimulating hormone by ICV ghrelin in rats and proposed that this may represent an adaptive response to promote feeding and reduce energy expenditure at times of limited nutrition. The exact relationship between ghrelin and insulin in body weight regulation remains controversial. There is some evidence supporting a reciprocal negative feedback relationship between these 2 hormones, with insulin administration reducing ghrelin concentration and ghrelin administration reducing insulin secretion. 54,55 Although insulin is anabolic in the periphery, it is a central hormone of catabolism. Selective elimination of insulin receptors from the central nervous system leads to hyperphagia and fat accumulation, 56 in support of a reciprocal relationship with ghrelin in energy homeostasis, because ghrelin is anabolic. However, in contradiction to these findings, Caixas et al. 57 have shown that physiological doses of insulin do not suppress ghrelin secretion in human subjects. Furthermore, Egido et al. 45 have shown that in isolated rat pancreatic preparations, the addition of ghrelin reduces the insulin response to known secretagogues; this argues for a tonic inhibitory effect of ghrelin on -cell function. Ghrelin may therefore have effects on glucose and insulin metabolism other than those related to adiposity. A recent study has reported ghrelin to affect the insulin-signaling system, implicating insulin-antagonistic peripheral actions of ghrelin in glucose homeostasis. 22 In addition, GH is known to counteract the action of insulin, and ghrelin may also play a role in glucose metabolism via its somatotrophic effects. The question of whether ghrelin has an indirect or direct effect on pancreatic endocrine function has also been investigated. Ghrelin mrna and peptide have been found in the cells of rat and human pancreas. It was proposed that ghrelin stimulates insulin secretion, whereas ghrelin secretion from the cells may be regulated by the plasma concentrations of glucose, insulin, or both. 20 Volante et al. 58 localized expression of ghrelin and GHS-R to the pancreatic islet cells and related endocrine tumors. Their findings suggested that ghrelin was produced by pancreatic cells, but in this case by the cells. Other work with isolated and in vivo pancreatic models has concentrated on the effects on pancreatic exocrine secretion. Ghrelin was found to be a potent inhibitor of pancreatic exocrine secretion in anaesthetised rats in vivo, but not in pancreatic lobules in vitro. 59 This set of experiments supports the concept that ghrelin inhibits pancreatic secretion via a mechanism involving the intrapancreatic nervous system. The effect was indirect, because the response is only seen in vivo, not in vitro. Effects of Ghrelin on Feeding and Energy Homeostasis During study of synthetic GHSs, it was noted that there was a short-lived increase in appetite after their acute administration, 60,61 and weight gain was noted with chronic systemic administration to immature rodents. 3 Several investigators showed that either ICV or intraperitoneal ghrelin stimulated food intake as well as GH secretion in rats 25,26,62 ; the orexigenic effect was comparable with that of neuropeptide Y (NPY). 63,64 Later experiments in volunteers by Wren et al. 65 showed that intravenous ghrelin enhanced appetite and increased food intake: subjects consumed a mean of 28% additional calories from an unlimited buffet after administration. For these experiments, a dose of 5 pmol kg 1 min 1 was used as a continuous infusion. This dose was found on previous studies to be the approximately halfmaximal GH-stimulating dose. The lowest infusion rate of ghrelin to significantly stimulate GH was 3.2 pmol kg 1 min 1. Cummings et al. 41 showed a preprandial increase in ghrelin by an average of 78%, suggesting a role in meal initiation in humans. They measured serum levels of ghrelin, leptin, and insulin throughout a 24-hour period in 10 healthy subjects who were provided with meals on a fixed schedule. Plasma ghrelin levels increased to nearly 2-fold shortly before each meal and decreased to trough levels within 1 hour of eating. 41 This postprandial decrease in levels was also shown in another study. 66 Ghrelin levels taken between meals showed a diurnal rhythm over the 24-hour period that was exactly in phase with that of leptin. 41 In patients who have had a gastric bypass, the meal-related fluctuations and diurnal rhythm of ghrelin are absent. 11 Despite a 36% weight loss in these patients, the 24-hour area under the curve (AUC) ghrelin profile was 77% lower than in matched obese controls. In contrast, a diet-induced weight loss of 17% was associated with a 24% increase in the 24-hour AUC ghrelin profile. 11 This diet-induced increase in ghrelin level is consistent with the hypothesis that ghrelin has a role in the regulation of body weight. The changes in ghrelin levels after a Roux-en-Y gastric bypass require further explanation. Despite massive weight loss, there was a large decrease in ghrelin levels. Compared with patients who undergo other forms of

5 1496 MURRAY ET AL. GASTROENTEROLOGY Vol. 125, No. 5 obesity surgery (such as vertical banded gastroplasty), these patients are also more likely to maintain this weight loss What is interesting is why these patients have decreased ghrelin levels and whether this indirectly or directly contributes to the degree and duration of weight loss. Additionally, the size of the gastric pouch may restrict the size of postoperative meals and the speed of onset of satiety. However, one would expect these patients to then eat smaller meals more frequently. This does not seem to be the case. It is important to note that there seems to be a voluntary restriction in highcalorie foods, despite there being no effect on subjective feelings about different food groups after surgery. 70 Similar changes in ghrelin levels are not seen in the other forms of antiobesity surgery. This would suggest that the reduced ghrelin levels are, in some way, related to the ghrelin-secreting part of the stomach being out of touch with the normal flow of nutrients. This situation would mimic starvation, in which high levels of ghrelin would be expected. There is perhaps, therefore, a possible override inhibition in this situation in which ghrelin secretion is ultimately suppressed, 71 although the real mechanism underlying suppression of ghrelin levels after gastric bypass is still unknown. 71 Tschop et al. 25 showed that ghrelin not only stimulated weight gain in rodents, but did so specifically through an increase in adiposity. Daily subcutaneous ghrelin led to an increase in fat mass, but no change in lean or bone mass, as shown on dual-emission x-ray absorptiometry. There was also an increase in respiratory quotient (the ratio of the volume of CO 2 produced and the volume of oxygen consumed). These findings suggest that ghrelin administration leads to a switch toward glycolysis and away from fatty acid oxidation for energy expenditure, favoring fat deposition. It is interesting to note that there was no demonstrable hyperphagia after subcutaneous ghrelin administration, although this may have been due to the short half-life of ghrelin when it is given once daily. This was shown in another study in which the dosing was more regular. 72 Gastric distention with saline had no effect on ghrelin levels, whereas filling with 50% dextrose decreased them, suggesting that it is caloric intake rather than gastric distention that affects ghrelin levels. 25 Of note, the increase in weight was maintained in GH-deficient dwarf rats, suggesting a different mode of action from its GH-secreting capabilities. 25,73 Human studies of macronutrient dietary composition have compared the effect of low- and high-fat diets on ghrelin and leptin plasma AUC over 24 hours. 74 A low-fat (15%), high-carbohydrate (65%) diet of approximately 2200 kcal/day did not differentially affect either the ghrelin or leptin AUC. However, when a 12-week similar diet was consumed with no calorie enforcement (ad libitum), weight loss was observed, and there was a decrease in the leptin AUC and no effect on the ghrelin AUC, without a compensatory increase in food intake. It is important to note that this diet reduced postprandial ghrelin release compared with an isocaloric high-fat (35%) diet. 74 Taken together, this suggests a role for ghrelin in modulating appetite in low-fat high-carbohydrate weight-loss diets. Changes in ghrelin levels after gastrectomy or smallbowel resection indicate that approximately two thirds and one third of circulating ghrelin are secreted from stomach and small bowel, respectively. 15,75 It is not clear how much of the variation in ghrelin levels can be attributed to stomach or intestinal mucosal contact with nutrients. With the findings of a preprandial increase and a postprandial decrease in ghrelin levels, together with the demonstration of weight gain and increased appetite after central and peripheral administration, it is important to consider the role of the peptide in energy homeostasis. Additionally, ghrelin levels correlate inversely with measures of body adiposity 76 and are altered in a compensatory manner by changes in body weight. 11 After administration of ghrelin or synthetic GHSs, there is an increase in c-fos mrna expression (a marker for neuronal activation) in the arcuate nucleus of the hypothalamus. 77,78 The highest proportion of arcuate neurons activated by systemic GHS contain NPY, 77 and more than 90% of NPY neurons possess GHS-R mrna. 79 NPY is known to be a potent orexigenic peptide, and it was thought probable that ghrelin exerted its orexigenic action via activation of NPY neurons in the arcuate nucleus. When administered systemically, ghrelin leads to c-fos-like immunoreactivity in the arcuate nucleus only, although ICV administration leads to activation in other areas, including the area postrema and nucleus tractus solitarius. 62,80 NPY and agouti-related peptide are colocalized in the neurons of the medial arcuate nucleus, 79 and they show c-fos-like immunoreactivity after administration of ghrelin or GHS by the intravenous or central routes. 62,73,77 79,81,82 More specifically, antagonism at the NPY-Y1 receptor abolishes the orexigenic effects of ghrelin, 62,63,72,73 as do antibodies against NPY and agouti-related peptide. 62 However, these orexigenic effects of ghrelin are not all mediated through NPY. When ghrelin is administered to NPY knockout mice, they still put on weight, 25 and when GH-releasing peptide-2 (a ghrelin receptor agonist) is given to NPY knockout mice, there is evidence of

6 November 2003 GHRELIN FOR THE GASTROENTEROLOGIST 1497 increased agouti-related peptide mrna in the hypothalamus, 83 suggesting that NPY is not obligatory for the orexigenic effects of ghrelin. Leptin is an adipocyte-derived satiety factor that acts directly on the hypothalamus, analogous but reciprocal to ghrelin, an orexigenic peptide produced predominantly in the periphery with central effects. 84 Leptin inhibits appetite and augments energy expenditure. 85 Ghrelin and leptin are both released in a pulsatile manner in both the fasted and fed state, and ghrelin and leptin levels are inversely correlated. 85 As with ghrelin, it is likely that the hypothalamic NPY neurons also mediate many of the actions of leptin. 86 In fact, ICV leptin in rats leads to a decrease in hypothalamic NPY mrna expression induced by fasting, which is the opposite of that seen with ghrelin. 82,87,88 When leptin is administered to rats, ICV ghrelin reverses the leptin-induced inhibition of food intake in a dose-dependent manner. 73 Hence, ghrelin and leptin seem to have counter-regulatory roles in energy homeostasis. A recent animal study has raised the possibility that leptin regulates ghrelin levels and hence exerts its influence on body weight regulation. 89 However, the exact nature of the relationship between ghrelin and leptin remains controversial. 74,90 The fact that ghrelin levels are decreased by caloric load to the stomach, rather than gastric distention alone, suggests a role for gastric chemosensory afferents. 25 Asakawa et al. 63 found that the potent orexigenic activity after ghrelin administration was lost after vagotomy and that ghrelin decreased gastric vagal afferent discharge, in contrast to other anorexigenic peptides that increased it. Date et al. 91 showed that blockade of the gastric vagal afferents by both vagotomy and capsaicin decreased ghrelin-induced GH secretion and abolished ghrelin-induced feeding and activation of NPY neurons. Ghrelin administration reduced firing of vagal afferents. They also showed that ghrelin receptors are synthesized in the vagal afferent cell bodies in the nodose ganglia and transported to the periphery. Relatively little is known about ghrelin-induced efferent signals, although it would seem that the hypothalamic-pituitary axes have a role to play, because hypophysectomy abolishes the adiposity induced by ghrelin administration. 92 Obesity and Eating Disorders Taken cumulatively, these findings suggest that ghrelin is up-regulated under conditions of negative energy balance and down-regulated in the setting of positive energy balance. The fact that ghrelin plasma levels are high in anorexia nervosa and low in obesity has led to speculation regarding a possible role in the pathophysiology of obesity. 93 Several studies have established that plasma ghrelin levels are inversely correlated with body mass index (BMI), 76,93 96 suggesting ghrelin levels are down-regulated in obesity. Weight loss in obese patients leads to a significant increase in circulating levels of ghrelin, and the increase in ghrelin levels is positively correlated with the extent of weight loss, 96,97 whereas food fails to suppress ghrelin levels as well in obese patients as in lean individuals. Similarly, English et al. 95 showed no change in circulating ghrelin levels in an obese group after a fast. Studies in twins do not support a role for plasma ghrelin in the etiology of common obesity. Two sets of monozygotic twins were compared: one was overfed, and the other was kept in negative energy balance. 98 The authors hypothesized that the subjects with higher ghrelin levels would gain more weight in response to overfeeding and lose less weight when in negative energy balance. However, there was no significant difference between the 2 groups. Whether or not obesity can be linked to mutations in the ghrelin gene has been studied. Hinney et al. 99 identified missense variants and a frameshift mutation in the ghrelin gene, but none of these seemed to influence weight regulation. Ukkola et al. 100 suggested, however, that sequence variation in the preproghrelin genes could play a role in the etiology of obesity. An Arg51Gln mutation in the preproghrelin gene was associated with lower plasma ghrelin, but not with obesity. However, carrier status of an alternative mutation, Met72, seems to be protective against fat accumulation and associated metabolic morbidity. 101 Cummings et al. 94 showed that obesity associated with the Prader Willi syndrome is associated with high ghrelin levels. Prader Willi syndrome is the most common form of syndromic obesity, and in these subjects ghrelin levels were uniformly above the regression line of ghrelin vs. BMI for non-prader Willi subjects. Hence, unlike in simple obesity, where ghrelin levels are negatively correlated with BMI, there seems to be a role for hyperghrelinemia in the pathogenesis of hyperphagia in this syndrome. Patients with anorexia have very high levels of circulating ghrelin, which return to the reference range after treatment and weight gain. 93,102 It is interesting to note that patients with bulimia nervosa also show higher circulating levels of ghrelin when compared with controls, although their BMIs are not significantly differ-

7 1498 MURRAY ET AL. GASTROENTEROLOGY Vol. 125, No. 5 ent, 103 and it has been hypothesized that frequent vomiting can cause an increase in ghrelin levels. 103 In the markedly catabolic state of cancer cachexia, ghrelin levels are increased when compared with healthy volunteers and those with cancer without cachexia. 104 This increased ghrelin level may represent a compensatory anabolic mechanism. In support of this, in a cancer cachexia mouse model, ghrelin administration increased food intake and adiposity. 105 In summary, ghrelin levels seem to correlate inversely with a positive energy balance in humans. Whether ghrelin has a role in the pathophysiology of common obesity is not clear. Cardiovascular and Other Effects of Ghrelin Previous work with GH and GHS has suggested beneficial hemodynamic effects. GH and insulin-like growth factor-i are essential for both skeletal and myocardial growth 106 and may be beneficial in some patients with cardiac failure. 107 GHS-R is abundantly expressed in many tissues, including the heart and blood vessels, 5,19,108 and stimulation of GHS-R prevents cardiac damage after ischemia reperfusion injury in hypophysectomized rats. 109 Hence, it seemed likely that ghrelin would have beneficial hemodynamic effects. Nagaya et al. 110 showed that, as well as leading to secretion of GH, a single dose of intravenous ghrelin in healthy volunteers decreases mean arterial pressure and increases cardiac output, with no change in pulse rate. These beneficial effects were maintained in rats and humans with chronic heart failure, and endogenous ghrelin levels are increased in this condition. 111,112 Because hexarelin, a GHS, has been shown to increase the left ventricular ejection fraction in rats, it may be that some of the beneficial effects are via direct action at the GHS-R. 113 The beneficial effects on cardiac output, despite hypotension, are likely to be secondary to a decrease in vascular resistance. In a recent study, 114 ghrelin was found to be a physiological antagonist of endothelin-1, a potent and longlasting vasoconstrictor. When human internal mammary artery muscle rings were constricted with endothelin-1, ghrelin potently dilated them. Effect of Ghrelin on Gastric Motility and Gastric Acid Secretion After the initial discovery of ghrelin, Tomasetto et al. 8 named the peptide the motilin-related peptide because of its structural homology with motilin. The aim of their study was to look for new proteins with expression restricted to the gastric epithelium that may provide insight to the differentiation and function of the gastric unit. They identified a novel mouse cdna, m46, that encodes a peptide of 117 amino acids whose primary sequence is weakly similar to that of prepromotilin (22% identity and 47% similarity). It is not, therefore, surprising that beyond its effect on appetite and weight gain, ghrelin seems to have gastric prokinetic properties. Gastric emptying 115 and gastric acid secretion 115,116 were found to be increased in several studies after administration of ghrelin in rats, and this intervention has been effective in reversing postoperative gastric ileus. 117 In vitro studies have shown an increase in neuronally mediated contractility in mouse stomach. 118 Human data are lacking, although one study showed no increase in gastric emptying when measured by the rather insensitive means of the paracetamol absorption test. 65 The literature regarding ghrelin s effects on gastric motility and secretion is somewhat controversial. Overall, the current weight of evidence suggests a small stimulatory effect on both of these parameters. Conclusions The discovery of ghrelin as an endogenous ligand for the GHS-R and the subsequent work described previously has widened our understanding of the control of GH secretion and energy homeostasis. Ghrelin increases feeding and weight gain and may have a role to play in the treatment and prevention of obesity; it may also offer a novel therapeutic strategy for the treatment of cancer cachexia. Its role as a prokinetic is not well established in humans, but further research may lead to advances in the treatment of conditions such as gastroparesis, postoperative ileus, and functional dyspepsia. The beneficial hemodynamic effects shown may also lead to novel therapies for cardiac disease, especially in the setting of chronic heart failure. In short, ghrelin has given us new insight into possible pathophysiological mechanisms in energy homeostasis and may lead to a host of future therapeutic targets. References 1. Bowers CY, Reynolds GA, Chang D, Hong A, Chang K, Momany F. A study on the regulation of growth hormone release from the pituitaries of rats in vitro. Endocrinology 1981;108: Bowers CY, Reynolds GA, Momany FA. 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