Endocrine Journal 2015, 62 (2), 107-122 REVIEW The regulation of circulating ghrelin – 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 stomach-derived orexigenic hormone with a wide range of physiological functions. Elucidation of the regulation of the circulating ghrelin level would lead to a better understanding of appetite control in body energy homeostasis. 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, hormones, or neural activities can influence circulating ghrelin levels in vivo. Recently developed in vitro assay systems for ghrelin secretion 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 peptide 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 growth hormone secretagogue receptor 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 gene-expres- mediated by a membrane-bound O-acyltransferase, sion profiles (e.g., of genes encoding receptors or signal ghrelin O-acyltransferase (GOAT) [3, 4]. Ghrelin molecules) of ghrelin-producing cells. exerts a wide range of effects including stimulation of In this review, we summarize current knowledge growth hormone secretion [5-10], food intake [11-16], regarding the regulation of circulating ghrelin levels gastric acid 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. insulin 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 duodenum, jejunum, ileum, 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 hypothalamus understood. No reduction of ghrelin levels is observed [1, 33, 40, 41], pancreas [33, 42-46], thyroid [47, 48], upon water intake [21, 61, 67], and pylorus occlusion kidney [49], testis [50-52], and placenta [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 glucagon-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- cholecystokinin (CCK), and insulin [80]. These hor- lin secretion [55]. Zhao et al. also developed two lines mones play various roles in digestion 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 circadian rhythm, 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 obesity [83-91], whereas the levels are elevated in lean hunger score in humans [62], and the plasma ghre- subjects, including patients with anorexia nervosa [36, lin rhythm correlates with food intake episodes in rats 90, 92-96] or cachexia [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 energy homeostasis. 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