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Home , Gastrointestinal hormone, Ghrelin, Oxytocin

Endocrine Journal 2015, 62 (2), 107-122

Review The regulation of circulating – with recent updates from cell-based assays

Hiroshi Iwakura1), Kenji Kangawa1), 2) and Kazuwa Nakao1)

1) Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan 2) National Cerebral and Cardiovascular Center Research Institute, Osaka 565-8565, Japan

Abstract. Ghrelin is a -derived orexigenic with a wide range of physiological functions. Elucidation of the regulation of the circulating ghrelin level would lead to a better understanding of control in body energy . Earlier studies revealed that circulating ghrelin levels are under the control of both acute and chronic energy status: at the acute scale, ghrelin levels are increased by fasting and decreased by feeding, whereas at the chronic scale, they are high in obese subjects and low in lean subjects. Subsequent studies revealed that nutrients, , or neural activities can influence circulating ghrelin levels in vivo. Recently developed in vitro assay systems for ghrelin can assess whether and how individual factors affect ghrelin secretion from cells. In this review, on the basis of numerous human, animal, and cell-based studies, we summarize current knowledge on the regulation of circulating ghrelin levels and enumerate the factors that influence ghrelin levels.

Key words: Ghrelin

GHRELIN is a 28-amino-acid hormone [1], on ghrelin secretion. The generation of ghrelin-produc- originally identified from the rat stomach as a natu- ing cell lines [29, 30] and FACS-based isolation of flu- ral ligand for orescently tagged ghrelin-producing cells [30-32] have (GHS-R) [2]. On its third Ser residue, ghrelin has a recently enabled development of in vitro assay sys- unique n-octanoylation modification that is necessary tems for assessing the effects of individual compounds for binding to GHS-R [1]. This acyl modification is on ghrelin secretion and determining the -expres- mediated by a membrane-bound O-acyltransferase, sion profiles (e.g., of encoding receptors or signal ghrelin O-acyltransferase (GOAT) [3, 4]. Ghrelin molecules) of ghrelin-producing cells. exerts a wide range of effects including of In this review, we summarize current knowledge growth hormone secretion [5-10], food intake [11-16], regarding the regulation of circulating ghrelin levels secretion [17, 18], and gastric emptying and ghrelin secretion, with recent updates obtained [19, 20], and suppression of fat utilization [21, 22] and from cell-based assay systems. secretion [23-28]. The regulation of circulating ghrelin levels is the sub- The source of circulating ghrelin: ject of active research. In early studies, plasma ghrelin ghrelin-producing (X/A-like) cells levels were measured under various conditions, provid- ing a basic profile of circulating ghrelin levels and their The majority of circulating ghrelin is derived from the changes in response to specific stimuli. In addition, in stomach [1, 33]. The predominant ghrelin-producing vivo studies using human subjects or animals were con- cell is the X/A-like cell, a type of gastric endocrine cell ducted to determine the effects of various compounds whose physiological role had been previously unknown [34, 35]. X/A-like cells account for 20–25% of the endo- Submitted Sep. 8, 2014; Accepted Sep. 11, 2014 as EJ14-0419 Released online in J-STAGE as advance publication Oct. 2, 2014 crine cells in the oxyntic glands of the stomach [34, 35]. Correspondence to: Hiroshi Iwakura, M.D., Ph.D., Medical Resection of the stomach results in 65–88% reduction of Innovation Center, Kyoto University Graduate School of Medicine, plasma ghrelin levels in rats [35] and humans [36, 37], 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. indicating that the stomach is the primary source of cir- E-mail: [email protected] culating ghrelin. A smaller but still significant amount ©The Japan Endocrine Society 108 Iwakura et al. of ghrelin is produced in the more distal portion of the not be required for the initiation of food intake. digestive tract, including the , jejunum, , The mechanism by which circulating ghrelin lev- and cecum [33, 35, 38, 39]. Outside the gastrointesti- els are regulated by food intake remains incompletely nal tract, ghrelin is also detected in the understood. No reduction of ghrelin levels is observed [1, 33, 40, 41], [33, 42-46], [47, 48], upon water intake [21, 61, 67], and pylorus occlusion [49], testis [50-52], and [53], although prevents the reduction of plasma ghrelin levels induced the levels in these tissues are low [33, 54], and it seems by glucose [67], indicating that gastric distension is not unlikely that the ghrelin secreted from these tissues con- the cause of ghrelin suppression after meals, and sug- tributes significantly to the circulating ghrelin level. gesting instead that some post-gastric mechanisms are involved. One plausible factor is absorbed nutrients, Ghrelin-producing cell lines such as glucose, amino acids or fatty acids. Indeed, Ghrelin-producing cell lines were developed by two postprandial ghrelin levels decrease in proportion to independent groups, including ours, using the trans- the amount of calories ingested [68, 69], and meals genic mice that overexpress SV40 T-antigen (TAg) with different nutrient compositions affect plasma in their ghrelin-producing cells [29, 30]. We estab- ghrelin levels differently [39, 70-79]. lished the ghrelin-producing cell line MGN3-1 [29] Other candidate mechanisms are indirect, e.g., gas- from a gastric ghrelinoma derived from ghrelin pro- trointestinal or pancreatic hormones. Meal ingestion moter–SV40 TAg transgenic mice [23]. MGN3-1 stimulates various gastrointestinal and pancreatic hor- cells produce high levels of ghrelin with physiolog- mones, including -like peptide-1 (GLP-1), ical acyl-modification and processing, and preserve gastric inhibitory peptide (GIP), peptide YY (PYY), some aspects of the physiological regulation of ghre- (CCK), and insulin [80]. These hor- lin secretion [55]. Zhao et al. also developed two lines mones play various roles in and absorption of ghrelin-producing cells, PG-1 from pancreas and of nutrients, glucose metabolism, meal termination, SG-1 from a stomach tumor that developed in BAC- and satiety. Moreover, these hormones each affect the transgenic mice overexpressing SV40 TAg in ghre- secretion of other hormones, and are thus regulated by lin cells [30]. Although these cell lines may have lost sophisticated feedback loops. Some of these hormones some of the original characteristics of ghrelin-produc- do indeed affect ghrelin secretion, as discussed below. ing cells, there are several advantages to using these The circadian changes in circulating ghrelin levels cell lines for assays, including their easy handling, may also be controlled to some extent by neural input reproducibility, and cost-effectiveness. from the central nervous system. The circadian pat- tern of plasma ghrelin levels can be observed even if Acute and chronic control of circulating subjects are placed in a 24-hour fasting state [81]. The ghrelin levels peak timing of plasma ghrelin levels differs among sub- jects, reflecting their food-intake intervals [82]. These Circadian rhythms of circulating ghrelin: relation observations cannot be explained simply by nutritional with food intake or hormonal regulations, suggesting the involvement The circulating ghrelin level has a , of a higher level of circadian control. increasing before meals and decreasing after meals [36, 56-58]. Fasting increases plasma ghrelin levels, Chronic regulation of circulating ghrelin levels whereas feeding decreases them [21, 35, 36, 59-61]. reflecting by body energy status These observations, along with the fact that ghre- Besides circadian control, circulating ghrelin lev- lin has a strong orexigenic effect [11-15], led to the els are regulated chronically, and apparently respond assumption that ghrelin is a meal-initiation factor [56]. to body energy status. Ghrelin levels are suppressed in Indeed, plasma ghrelin levels increase along with the [83-91], whereas the levels are elevated in lean score in humans [62], and the plasma ghre- subjects, including patients with nervosa [36, lin rhythm correlates with food intake episodes in rats 90, 92-96] or [97-99]. There is a negative [63]. However, genetic deletion of ghrelin [64, 65] or correlation between circulating ghrelin levels and body ablation of ghrelin cells in adult mice [66] does not mass index [79, 100, 101]. Weight loss increases cir- affect feeding behavior, suggesting that ghrelin may culating ghrelin levels [90, 102-106], whereas weight Regulation of circulating ghrelin 109 gain decreases them [90, 92, 95]. These results suggest acids, calcium-sensing receptor for amino acids) [80]. that ghrelin is not the cause of obesity or leanness, but Some of these mechanisms appear to be utilized in the is rather one aspect of the compensatory mechanisms regulation of ghrelin secretion. that maintain body . The mechanism by which body energy status affects Glucose ghrelin levels is not yet completely understood. Some Oral [61, 67, 79, 93, 118, 119] or intravenous glu- of the factors that reflect body energy status may be cose loading [84, 120, 121] suppresses plasma ghre- responsible for the changes of circulating ghrelin lev- lin level. As mentioned earlier, when the pylorus is els. For example, free fatty acid and insulin levels closed, intra-gastric infusion of glucose dose not sup- increase with fat deposition, and these factors sup- press plasma ghrelin levels in rats [67], suggesting that press ghrelin secretion, as summarized below. Still, it glucose does not directly suppress ghrelin secretion in remains unclear to what extent each factor contributes the stomach. Instead, it is likely that ghrelin secretion to the change in ghrelin levels. is suppressed by absorbed glucose, or indirect factors The increase in circulating ghrelin level during such as insulin induced by glucose. Recently, however, weight loss is believed to hamper maintenance of Sakata et al. reported that high glucose directly sup- reduced weight in obese patients. Ghrelin- or GHS-R– presses ghrelin secretion in primary cultures of gastric knockout mice are resistant to diet-induced obesity [65, mucosal cells [122]. They showed that FACS-sorted 107, 108]. Suppression of ghrelin by either adminis- ghrelin cells from ghrelin-GFP transgenic mice express tration of GOAT inhibitor [109] or vaccination against GLUT1, 4, and 5, glucokinase, Kir6.2, and SUR1. ghrelin [110] also prevents in rodents. 2-deoxy-D-glucose inhibited glucose-induced ghrelin Some researchers claim that the lack of an increase suppression from gastric mucosal cells, suggesting that in ghrelin levels contributes to the long-term mainte- metabolites of glucose in the cells are involved in the nance of weight loss following bariatric surgery [102, regulatory mechanisms. However, neither tolbutamide 111], although others have reported that ghrelin levels nor diazoxide have effects on ghrelin secretion, sug- increase after weight reduction even in patients who gesting that the machinery via K-ATP channel is not have undergone bariatric surgery [112-114]. employed in the ghrelin-producing cells. Because gas- One exceptional case in which ghrelin levels are tric mucosal cell cultures include various other types elevated disproportionally to BMI is Prader-Willi syn- of stomach cells, it remains possible that some indi- drome [115], a genetic disorder characterized by short rect mechanisms were responsible for these findings. stature, mental retardation, dysmorphic features, mus- Currently, it is not clear whether (and, if so, how) glu- cular hypotonia, and obesity [116]. Elevated ghrelin in cose directly affects ghrelin secretion from cells. these patients may contribute to increased appetite and body weight, although suppression of ghrelin by soma- Fatty acids tostatin administration has no effect on their appetite Oral fat intake or intravenous infusion of , [117]. It remains unclear why Prader-Willi syndrome consisting mainly of long-chain fatty acids, suppresses patients have elevated ghrelin levels. plasma ghrelin levels [79, 123-126]. Free fatty-acid receptors are involved in control of various gastrointes- The role of nutrients in the regulation of tinal hormones by fatty acids [80], and that also seems ghrelin secretion to be the case with ghrelin. The long-chain fatty-acid receptor FFAR4 (GPR120), but not FFAR1 (GPR40), Nutrients such as glucose, amino acids, or fatty is expressed in ghrelin-producing cells [32, 127- acids affect secretion. The 129]. Knockdown of FFAR4 attenuates α-linolenic best-known example of this phenomenon is the stim- acid–induced suppression of ghrelin secretion from ulation of insulin secretion by glucose. The eleva- MGN3-1 or SG-1 cells [128, 129]. The FFAR4 ago- tion of glucose-derived ATP levels in β-cells triggers nists GW-9508 and compound B suppress ghrelin insulin secretion via the K-ATP channel. Apart from secretion in vitro [32, 129], but not in gastric mucosal their metabolites, nutrients themselves can stimulate cells of FFAR4-knockout mice [32]. FFAR4-knockout hormone secretion by binding to nutrient-chemosens- mice have higher circulating ghrelin levels than wild- ing receptors on the cell surface (e.g., GPR40 for fatty type animals, suggesting that FFAR4 plays physiologi- 110 Iwakura et al. cal roles in the regulation of ghrelin secretion [32]. studies focused on the relationship between the lev- Ghrelin-producing cells also express short-chain els of ghrelin and other hormones. However, in vivo fatty acid receptors, FFAR2 (GPR43) and FFAR3 studies sometimes provide conflicting results, because (GPR41) [32]. Short-chain fatty acids, such as ace- each hormone may affect other hormones in a manner tate or propionate, suppress ghrelin secretion mainly that depends upon experimental conditions, which in through FFAR2 activation [32]. turn may indirectly affect ghrelin secretion. Cell-based Medium-chain fatty acids have the opposite effect, studies revealed the receptors expressed in the ghre- i.e., they increase acylated ghrelin levels. As noted lin-producing cells [29, 30, 32, 55, 122]. Some hor- above, GOAT mediates the acylation of ghrelin [4] mones directly influence the ghrelin secretion by act- with medium-chain fatty acids, mainly octanoic acid. ing on receptors expressed at high levels in these cells Food-derived medium-chain fatty acids can be used [29, 30, 32, 55, 122]. However, these observations are for this reaction [130], and animals fed a diet rich in confounded by the fact that the expression level of the medium-chain fatty acids exhibit higher circulating mRNA encoding a particular receptor does not always ghrelin levels [131, 132]. Indeed, addition of octanoic predict the action of the ligand on ghrelin secretion [32]. acid to the medium increases acylated ghrelin secretion from ghrelin-producing cell lines in vitro [128]. Insulin Insulin is considered to be one of the major deter- Amino acids minants of the regulation of circulating ghrelin levels. The effects of amino acids on ghrelin secretion have The circadian changes in circulating ghrelin levels are been inconsistent among studies. Knerr et al. reported inverted relative to those of insulin levels [56], which that ingestion of an essential amino-acid mixture are decreased by fasting and increased after meals. increased circulating ghrelin levels in humans [133], Several lines of evidence suggest that this relationship whereas Overduin et al. and Shrestha et al. reported that is causative: when insulin is administered to human an amino-acid mixture suppressed ghrelin levels in rats or rodents, circulating ghrelin levels are reduced [84, [134, 135]. Few studies have examined the effect of sin- 119, 149, 150], and insulin–deficient animals have high gle amino acids on ghrelin secretion. Tryptophan has no plasma ghrelin levels [83, 151]. Glucose-clamp stud- effects in humans [136, 137], whereas in pigs tryptophan ies revealed that hyperinsulinemia suppresses circulat- exerts a stimulatory effect on circulating ghrelin lev- ing ghrelin levels, regardless of blood glucose levels els [138], although the mechanisms remain unknown. [152-158]. These observations indicate that insulin, also seems to have no effect on ghrelin secre- not glucose, is the principal factor responsible for the tion [119, 139, 140]. The effects of other amino acids on meal-related suppression of plasma ghrelin levels. ghrelin secretion have yet to be elucidated. This notion is supported by the fact that meal-related suppression of ghrelin levels is absent in insulin-defi- Lactate cient type 1 diabetes patients [159]. Engelstoft et al. reported that FACS-sorted ghre- Insulin may also be partly responsible for the change lin-producing cells express high levels of the lactate in ghrelin levels in response to chronic energy status. receptor GPR81, and that lactate or the GPR81 ago- Consistent with this idea, circulating ghrelin levels are nist 3-chloro-5-hydroxybenzoic acid (CHBA) sup- suppressed in obesity [83, 84, 86, 89, 160] and insulin presses ghrelin secretion from primary cultured gastric resistance [88, 161, 162]. Furthermore, a strong nega- mucosal cells [32]. Those authors suggested that lac- tive correlation is observed between circulating ghrelin tate-induced ghrelin suppression is related to the sup- levels and insulin levels [73, 76, 101, 163] or indices of pression of ghrelin during exercise observed in some insulin resistance [86, 88, 161, 162, 164]. studies [141-144], although such suppression was not Ghrelin suppression by insulin is mediated at reported in other studies [145-148]. least in part through direct action on ghrelin-produc- ing cells. Perfusion of insulin into isolated rat stom- Hormonal control of ghrelin secretion ach suppresses ghrelin secretion [63, 165]. The ghre- lin-producing cell line MGN3-1 expresses the insulin As with most other endocrine hormones, ghrelin receptor, and insulin directly suppresses ghrelin secre- secretion is influenced by several hormones. Earlier tion from these cells in vitro [29]. Recently, Gagnon Regulation of circulating ghrelin 111 et al. reported that insulin directly suppresses ghrelin Growth hormone secretion in primary cultured neonate rat stomach cells Because ghrelin has a stimulatory effect on growth by activating the PI3K-AKT pathway and suppressing hormone secretion, it has been hypothesized that intracellular cAMP [62]. growth hormone may inhibit circulating ghrelin lev- els via a feedback loop. In line with this notion, acro- megalic patients have lower circulating ghrelin levels Somatostatin is an inhibitory peptide that suppresses [176], and surgical removal of pituitary tumors that many gastrointestinal hormones including , VIP, secrete growth hormone increases ghrelin levels [177]. GIP, , insulin, and glucagon, and also partici- In rats, hypophysectomy increases plasma ghrelin lev- pates in the regulation of growth hormone release. In els, whereas GH administration decreases them [178, the stomach, somatostatin-secreting D cells are located 179]. Furthermore, administration of GH suppresses in close proximity to X/A-like cells [166]. Somatostatin ghrelin secretion from rat gastric explants [172]. or its analog suppresses circulating ghrelin levels in Other studies, however, have reported that GH humans [167-169] and rodents [165, 166, 170-172]. administration has no effect on circulating ghrelin lev- The inhibitory effect of somatostatin on ghrelin secre- els [105, 167, 180]. GH-deficient patients, who should tion is mediated at least in part by direct action on ghre- have high ghrelin levels if this feedback loop exists, lin-producing cells through type have normal [181] or even lower [182, 183] circulat- 2 (SSTR2) [29, 55, 173]. Ghrelin-producing cell line ing ghrelin levels. Furthermore, in vitro studies using MGN3-1 cells express SSTR2 and SSTR5 [29], and ghrelin-producing cell lines showed that GH has no somatostatin directly inhibits ghrelin secretion from direct effects on ghrelin secretion [55]. The reported these cells in vitro [29, 55]. , which has a effects of GH on ghrelin secretion may include indirect high affinity for SSTR2, suppresses ghrelin secre- effects: for example, GH stimulates lipolysis and resul- tion more profoundly than SOM230, which has high tant free fatty acids may suppress ghrelin secretion. In affinities for SSTR1 and SSTR5 [170]. The SSTR2- addition, GH causes insulin resistance and elevated selective agonist S346-011 inhibits ghrelin secretion in insulin may also suppress ghrelin secretion. the rats, and the SSTR2a antagonist S-406-028 attenu- ates ghrelin suppression after abdominal surgery [173]. Glucagon The effects of glucagon on ghrelin secretion have and AVP been inconsistent among reports. Most human studies We found that oxytocin and AVP stimulate ghrelin [184-187] except for one [119] have reported that glu- secretion from MGN3-1 cells. Oxytocin and AVP are cagon suppresses circulating ghrelin levels, whereas structurally similar, with differences in only two out of in rats glucagon stimulates ghrelin secretion [63, nine amino-acid residues, and AVP can act on the oxy- 188, 189]. tocin receptor as a partial agonist [174]. The stimula- The glucagon receptor is coupled with Gs to tory effects seem to be mediated by , increase intracellular cAMP levels, which may poten- as MGN3-1 cells express oxytocin receptor but not AVP tially stimulate ghrelin secretion [30]. However, receptors, and the oxytocin blocks two recent studies using FACS-purified GFP-tagged AVP-stimulated ghrelin secretion [55]. Recently, how- ghrelin cells suggested that the glucagon receptor is ever, Engelstoft et al. reported that FACS-sorted GFP- expressed at low levels in ghrelin-producing cells [32, Tagged ghrelin–producing cells express high level of 127]. No effects have been observed in in vitro studies AVPR1B but a negligible level of oxytocin receptor, and using ghrelin-producing cell lines [30, 55] or FACS- that AVP stimulates ghrelin secretion from primary gas- purified GFP-tagged ghrelin cells [127]. These dis- tric mucosal cells [32]. The discrepancy between the crepancies may result from the indirect effects of glu- expression patterns of AVP and oxytocin receptors may cagon on ghrelin secretion. For instance, glucagon be due to primer design or differences between FACS- strongly stimulates insulin secretion, which in turn sorted cells and cell lines. In an in vivo study, Vila et suppresses ghrelin secretion; Soule et al. reported that al. reported that oxytocin suppresses circulating ghrelin ghrelin levels dropped, while insulin levels rose, fol- levels in normal healthy subjects [175]. No studies have lowing glucagon injection [186]. reported the effects of AVP on ghrelin secretion. 112 Iwakura et al.

Glucagon-like peptide 1, 2 failed to demonstrate any effects of CCK and gastrin on Administration of glucagon-like peptide 1 (GLP-1) ghrelin secretion, suggesting that the reported effects or its analog exendin-4 suppressed circulating ghrelin of these on plasma ghrelin levels are indirect. levels in some studies [190, 191], whereas another study reported no effect [192]. GLP-2 also suppresses ghre- lin secretion [193]; Lu et al. reported the expression of Leptin is a fat-derived hormone with strong anorexic GLP-1 receptor in the FACS-purified ghrelin-produc- effects that stimulates energy expenditure to maintain ing cells [127], whereas Engelstoft et al. reported that body weight [63, 198, 199]. Because the actions of GLP-2 receptor, but not GLP-1 receptor, is expressed in leptin are contrary to those of ghrelin, it is tempting such cells [32]. In vitro studies using ghrelin-produc- to hypothesize that leptin has direct effects on ghre- ing cell lines, FACS-sorted ghrelin-producing cells, or lin secretion. However, the reported results are incon- primary cultured gastric mucosal cells failed to demon- sistent. Plasma ghrelin and leptin levels were nega- strate any effect of GLP-1 on ghrelin secretion [32, 55, tively correlated in some studies [95, 200], but not in 127], suggesting that the inhibitory action of GLP-1 on others [163, 201]. Furthermore, leptin administration ghrelin secretion is indirect. At least some of the inhib- suppressed ghrelin secretion in rat studies [63, 199, itory action of GLP-1 on ghrelin seems to be mediated 202, 203], but not in a human study [200]. Recent in by elevation of insulin levels [190]. vitro studies suggest that the effect of leptin on ghrelin secretion, if it exists, is indirect [30, 55, 127]. Gastric inhibitory polypeptide Gastric inhibitory polypeptide (GIP) receptor is PYY, NPY, PP expressed in FACS-purified ghrelin-producing cells Batterham et al. reported that PYY3-36 infusion [32, 127], but its effects on ghrelin secretion have been to obese or lean subjects suppresses circulating ghre- inconsistent among studies. GIP stimulated ghrelin lin levels [204], whereas Ito et al. observed no effects secretion in studies using primary gastric cell culture of PYY3-36 infusion on plasma ghrelin levels in pigs [32] or isolated rat stomach [165], whereas no effects [205]. Takahashi et al. reported that PP administration were observed in a human study [140] or in studies to sheep suppresses circulating ghrelin levels [206]. No using cell lines [55], gastric microdialysis [171], or effects of PYY, NPY, or PP on ghrelin secretion were FACS-purified ghrelin-producing cells [127]. observed in a microdialysis study using rat stomach [171] or in vitro studies [32], and neither NPY Y1 nor Cholecystokinin (CCK) and Gastrin Y2 receptor is detectable in ghrelin-producing cells [32]. Ghrelin-producing cells express CCK-A [32, 194] and CCK-B (gastrin) receptors [32, 195]. The CCK-A Other peptide hormones receptor has a higher affinity for CCK than for gas- In a microdialysis study using rat stomach, De la trin, whereas CCK-B receptor has an almost identi- Cour et al. reported that ghrelin secretion is stimulated cal affinity for both peptides. Conflicting results have by secretin and endothelin stimulate, but inhibited by been reported regarding the effects of CCK, as well as gastrin-releasing peptide (GRP) and [171]. gastrin, on ghrelin secretion. Administration of CCK Engelstoft et al. also observed stimulation of secretin suppresses plasma ghrelin levels in humans [192, 196], on ghrelin secretion in primary cultured gastric muco- but increases circulating ghrelin levels in rats [59, sal cells [32], although other in vitro studies reported 134]. Murakami et al. and Fukumoto et al. reported no effect [30, 55, 122]. The effects of endothelin, GRP, that administration of gastrin to rats increases plasma and bombesin have not yet been confirmed by other ghrelin levels [59, 195], and Zhao et al. reported sim- groups [30, 122]. ilar results in cattle [197]. On the other hand, Lippl Engelstoft et al. reported the expression of calci- et al. reported that gastrin inhibits ghrelin secretion in tonin receptor–like receptor and receptor activity– the isolated rat stomach [165]. Gastrin/CCK [194] or modifying (RAMP) 1, which constitute the CCK-AR/BR double knockout mice have lower cir- gene related peptide (CGRP) receptor, in culating ghrelin levels, supporting the idea that these ghrelin-producing cells, as well as a stimulatory effect peptides stimulate ghrelin secretion. However, in vitro of α-CGRP on ghrelin secretion [32]. However, this studies [32, 55] or a gastric microdialysis study [171] effect was not observed in a microdialysis study [171]. Regulation of circulating ghrelin 113

They also reported that αMSH stimulated ghrelin Neural control of ghrelin secretion secretion, which was inhibited by MC4R antagonist PG901 [32]. Thus far, no other reports have described Secretion of many hormones is under neural con- the effects of αMSH. trol, and this is also true for ghrelin. Ghrelin secretion The peptide hormones reported to have no effects is regulated by the autonomic nervous system, which is on ghrelin secretion in microdialysis or in vitro studies predominantly controlled by sympathetic nerves. The are as follows: , atrial , bra- electrical or pharmacological activation of gastric sym- dykinin, calcitonin, c-type natriuretic peptide, cortico- pathetic nerves increases ghrelin secretion in rats [224]. tropin-releasing factor, , β-endorphin, enter- Systemic administration of isoproterenol increases ostatin, , , met-, , plasma ghrelin levels, whereas atenolol and reserpine neuromedin C, , , pituitary suppress fasting-induced elevation of ghrelin [30, 225]. adenylate cyclase–activating peptide, parathyroid hor- Ghrelin-producing cells express the α1 and β1 adrener- mone, , and vasoactive intestinal peptide gic receptors [30, 32, 55], and and noradrena- [30, 32, 55, 122, 171]. line directly stimulate ghrelin secretion via the β1 recep- tor [30, 55, 62, 171, 188]. Activation of the β1 receptor Thyroid hormone results in elevation of intracellular cAMP, followed by Circulating ghrelin levels are reduced in hyperthy- Ca2+ release from ER and granular . EPAC roid patients [157, 207-211]. On the other hand, ghre- seems to mediate between cAMP elevation and Ca2+ lin levels were elevated in hypothyroid patients in some release, whereas the role of PKA is controversial [226]. reports [212, 213], but not in others [207, 211, 214]. In addition to adrenaline and noradrenaline, we Aminotriazole treatment increases gastric ghrelin mRNA found that relatively high doses of can stim- level, whereas l-thyroxine decreases it in rats [215]. ulate ghrelin secretion from a ghrelin-producing cell Because addition of thyroxine to a ghrelin-producing cell line; this response is mediated by the D1a receptor line does not affect ghrelin secretion [55], the observed [55]. However, no effects were observed in other stud- changes in ghrelin levels in thyroid dysfunction may be ies that used smaller doses [32, 171]. indirect, or at least not mediated by TSH [216]. Several reports have suggested the involvement of the in the meal-related regulation of ghre- Sex steroids lin [123, 227, 228]. Administration of There are gender differences in plasma ghrelin lev- or muscarinic receptor agonists increase ghrelin secre- els, which are high in women and low in men [79, tion in human or rats [134, 225, 229], whereas mus- 169, 217]. These differences may result, at least in carinic receptor antagonists suppress it [225, 229- part, from differences in the levels of sex hormones. 231]. However, recent studies indicate that muscarinic administration to anorectic women [218] receptor is expressed at low levels in ghrelin-produc- or post-menopausal women [219] increases plasma ing cells [32], and no direct effects of acetylcholine on ghrelin levels, and the effects of estrogen on ghrelin ghrelin secretion were observed in in vitro studies [32, secretion appear to be direct. Ghrelin-producing cells 55, 171], suggesting that the role of the vagus nerve express estrogen receptor α [220] or G protein–coupled in the regulation of ghrelin secretion may be indirect. estrogen receptor [32], and ex–vivo estrogen treatment No other , including , sero- of gastric mucosa results in upregulation of ghrelin tonin, GABA, or glutamate, have been shown to regulate mRNA [220] and ghrelin secretion [32]. ghrelin secretion in cell-based studies [32, 55, 171]. Androgens also seem to increase circulating ghrelin levels. Circulating ghrelin levels are reduced in hypo- Conclusion gonadal men, and plasma levels are posi- tively correlated with circulating ghrelin levels [221, Ghrelin secretion is influenced by various fac- 222]. Testosterone treatment of normal or hypogo- tors including nutrients, hormones, and neural con- nadal men increases plasma ghrelin levels [221, 223]. trol. These factors may influence each other directly Thus, androgens may not be responsible for the sex dif- or indirectly, and ultimately the circulating ghrelin ference in circulating ghrelin levels. level is determined by the sum of their activities. Cell- based assays have resolved some of this complexity 114 Iwakura et al. and enabled us to assess the effects of individual fac- ies will be required in order to elucidate the physiologi- tors on ghrelin secretion. However, it remains unclear cal importance of each factor involved in ghrelin reg- exactly which factors, either alone or in combination, ulation. The results of such work will contribute to a are responsible for the meal-related changes in ghrelin greater understanding of the physiological role of ghre- levels or changes in body energy status. Further stud- lin in body energy homeostasis.

References

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