2017, 64 (Suppl.), S15-S19

Current knowledge of ghrelin in

Hiroyuki Kaiya1), Kenji Kangawa2) and Mikiya Miyazato1)

1) Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Suita 565-8565, Japan 2) National Cerebral and Cardiovascular Center Research Institute, Suita 565-8565, Japan

Abstract. We are exploring physiological importance of the ghrelin system in vertebrates. This review summarizes current knowledge of the ghrelin system in amphibians. Our study on ghrelin precursor in various amphibians revealed that the third amino acid with acyl modification has changed to threonine (Thr-3) instead of serine (Ser-3) only in the genus, Rana. Functional analyses of the ghrelin receptor in three species of amphibians, Japanese fire belly , American bullfrog and Japanese tree frog revealed that ghrelin and GHS-R1a agonists increase intracellular Ca2+ concentration in HEK293 cells expressing each receptor, and that ligand selectivity of ghrelin with Ser-3 and Thr-3 that expected to see in the bullfrog receptor was not found in the two frog receptors, but in the newt receptor. The brain, gastrointestinal tract, kidney and gonad highly express GHS-R1a mRNA. In frogs and newt, fasting did not increase GHS-R1a mRNA expression in the brain, but in the stomach. However, intraperitoneal (IP) injection of ghrelin did not affect food intake. A dehydration treatment increased GHS-R1a mRNA expression in the brain, stomach and ventral skin in the tree frog. However, intracerebroventricular (ICV) injection of ghrelin did not affect water absorption. Ghrelin did not influence gastrointestinal motility in in vitro studies using smooth muscle strips of the bullfrog and newt in vitro. These results suggest that the ghrelin system is present in various amphibians, but little is known about the physiological functions except hypophyseal hormone secretion.

Key words: Frog, Newt, Ghrelin, GHS-R1a, Function

GHRELIN was firstly identified in rat and human Structure of ghrelin stomachs as a ligand for GHS-R1a in 1999 [1]. The unique n-octanoyl modification of Ser-3 prescribes Rat and human ghrelin precursors are consisted of ghrelin’s activity: the modification is essential for 117-amino acids (AA), and a 28-AA mature ghrelin is binding of ghrelin to GHS-R1a and for eliciting bio- cleaved from the precursor [1]. We identified ghrelin logical activity as ghrelin. At present, the specific from eight species of amphibians, including two species enzyme to modify ghrelin (ghrelin O-acyltransferase, of urodelan Japanese fire belly newt (Cynops pyrrho- GOAT) has been identified, and the mechanism is also gaster) and the Sword-tailed newt (Cynops ensicauda elucidated gradually [2]. Ghrelin or ghrelin-like pep- popeti), and six species of anurans, American bullfrog tide is also present in gastrointestinal tract of vari- (Rana catesbeiana), Japanese tree frog (Hyla japon- ous non-mammalian vertebrates [3]. We are inves- ica), Japanese toad (Bufo japonicus), Black-spotted tigating the physiological significance of the ghrelin pond frog (Rana nigromaculata), Wrinkled frog (Rana system in vertebrates [4]. In this review, we briefly rugosa) and African clawed frog (Xenopus laevis) [3, summarize current knowledge of the structure, recep- 5]. Bullfrog ghrelin precursor was 114-AA residues, tor and physiological functions on the ghrelin system but newly identified ghrelin precursors were consisted in amphibians. of 115-118-AA residues (Fig. 1A). The mature ghrelin was composed of 27 or 28-AA residues. The identity Correspondence to: Hiroyuki Kaiya, Ph.D., Department of of AA sequence of the ghrelin precursor is low in over- Biochemistry, National Cerebral and Cardiovascular Center Research all amphibians, but slightly higher in the same genus Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. (Fig. 1B). This supposes that variation (evolution) of E-mail: [email protected] ghrelin molecule advances among amphibians. ©The Japan Endocrine Society S16 Kaiya et al.

Fig. 1 Deduced amino acid sequence of ghrelin precursor in amphibians (A) The number behind the peptide sequence indicates the number of AA from start, and the number in parenthesis behind the mature peptide sequence shows peptide length. A processing signal of dibasic amino acids (RR) was boxed. (B) Percent identities of multiple comparisons are shown about AA and nucleotide sequences of ghrelin precursor. ghrelin system S17

The third AA of ghrelin is modified with n-octanoic Ghrelin of the tree frog and newt has Ser-3, so that we acid [1], which is Ser in all vertebrates examined so far expected that GHS-R1a in each is able to rec- [3]. However, that of bullfrog ghrelin has substituted ognize Thr-3 or Ser-3 ghrelin. However, GHS-R1a to Thr [5]. It had missed for a long time that the change of bullfrog and the tree frog did not show any ligand is specific or general among amphibians. We examined selectivity. In contrast, the newt receptor preferably five genera of amphibians, and found that the third AA bound Ser-3 ghrelin, and the affinity of Thr-3 ghrelin is Thr only in the genus Rana, and is Ser in other gen- was 10-times lower than that of Ser-3 ghrelin. It has era Bufo, Hyla, Xenopus and Cynops [3]. In addition, been unclear yet why bullfrog receptor did not select the AA alteration possibly occurs a single base substi- Thr-3 ghrelin. tution from AGC to ACC. These results suggest that We examined expression level of the GHS-R1a Thr-3 is not specific in overall amphibians, although mRNA. In general, the mRNA expression is high in there are many genera not to check yet. the brain, intestine and kidney in all species exam- The type of fatty acid modification in the bullfrog ined, although considerable levels of expression was ghrelin was n-octanoic or n-decanoic acid [5]. We seen in other tissues; the gall bladder and ventral skin newly determined the type of fatty acid modification in in tree frog, and in the lung, gonad and skins in the Japanese toad and Sword-tailed newt. The third AA of newt. As described earlier, bullfrog ghrelin stimulates the two species is Ser, and the major ghrelin was modi- GH and PRL from the pituitary cells [5], suggesting fied by unsaturated decanoic acid in the toad, and by presence of GHS-R1a in the pituitary. Curiously, n-octanoic acid in the newt. This suggests that simi- however, mRNA expression of the identified receptor lar mechanisms play for modifying ghrelin in amphib- was not detected in the bullfrog or tree frog pituitary, ians, too. but found in the newt pituitary. These results suggest that another GHS-R, which expresses in the pituitary, Ghrelin receptor may exit in frogs. We are exploring the receptor but have not yet been identified. We have explored the structure, molecular evolu- tion and functionality of GHS-R1a in various verte- Physiological functions brates [6, 7], and revealed that the ghrelin receptor is roughly classified into two groups, GHS-Ra and Ghrelin has known to be a multifunctional hormone GHS-R1a-like receptor (GHS-R1a-LR) [6], having to participate in the regulation of hypophyseal hor- possibilities to accord with the evolution of the lungs, mone release, glucose metabolism, feeding, cardiovas- although the details have been still unclear [7]. cular function, reproductive function, gastrointestinal We determined GHS-R1a in three species of amphib- function, endocrine and exocrine functions [10-12]. ians [8, 9]. GHS-R1a in the bullfrog or Japanese tree In amphibians, it has been reported that bullfrog frog is consisted of 374- and 371-AA, respectively, and or rat ghrelin is capable of stimulating GH and PRL the sequence identity of the two receptors was 85%. In release from dispersed bullfrog pituitary cells [5]. the newt, two lengths of the receptor were identified: Similar releasing effects have been observed by intra- a short type is consisted of 362-AA, and a long type venous injection of bullfrog ghrelin into juvenile bull- was the receptor that 16-aa elongated at the N-terminus frogs (our unpublished observation). of the short-type receptor. The short-type receptor We have established a radioimmunoassay (RIA) showed 80 and 78% identity to bullfrog and tree frog for measurement of total (acylated and unacylated GHS-R1a, respectively. ghrelin) bullfrog ghrelin [13]. Total ghrelin concen- All identified receptor proteins that expressed in tration in bullfrog plasma is approximately 150 fmol/ mammalian HEK 293 cells are functional, and intra- mL, and octanoylated ghrelin concentration is only cellular Ca2+ concentrations increase after treatment 2.5 fmol/mL. Total ghrelin concentration in the stom- of ghrelin or GHS-R1a agonists. We attracted atten- ach is in ranges from 82 to 135 fmol/mg tissue, and tion to the ligand selectivity of the bullfrog GHS-R1a most of them are active forms of ghrelin [13]. Plasma because bullfrog ghrelin exhibited much stronger and gastric ghrelin level increase 10 days after fast- activity in the release of GH and PRL from the bull- ing, suggesting a role of ghrelin in energy homeosta- frog pituitary cells when compared to rat ghrelin [5]. sis in the bullfrog. S18 Kaiya et al.

Fasting for 10 days increases receptor mRNA weight up-regulated receptor mRNA expression in the expression in the stomach of the bullfrog and tree frog brain, stomach and ventral skin [8]. Ghrelin exhib- [8]. Ghrelin regulates feeding: stimulatory in mam- its an anti-dipsogenic effect in eels, chickens and rats mals (human and rodents), and fish (goldfish and tila- [3, 4, 10]. However, in the tree frog, ICV injection of pia), and inhibitory in birds (chickens and quails) and rat ghrelin did not show any effect on water absorp- fish (rainbow trout) [3, 4, 10]. Our preliminary stud- tion from the ventral skin. Gastrointestinal tract more ies using Japanese fire belly showed that IP expresses ghrelin receptor mRNA compared with the injection of ghrelin did not affect feed consumption brain. However, ghrelin does not influence on con- although fasting increased receptor mRNA expression traction of smooth muscle strips of the gastrointesti- (Fig. 2). Dehydration with 20 % decrease in body nal tract of the bullfrog and newt [14].

Fig. 2 Changes in mRNA expression of ghrelin and GHS-R1a in Japanese fire belly newt after 4 days and 2 weeks fasting Ghrelin mRNA in the brain (A), pituitary (B) and stomach (C). GHS-R1a in the brain (D), pituitary (E) and stomach (F). The values are expressed the mean + SEM (n = 4-5). Amphibian ghrelin system S19

Perspective ghrelin system. We could know the distribution of the ghrelin receptor, and consider various possibilities of In the point of vertebrate evolution, amphibians the function. However, little is known about physio- are interesting , and are important for under- logical functions at present. We hope that much more standing the functional and structural evolution of the researchers challenge this theme.

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