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Physiological roles of ghrelin on obesity
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Obesity Research & Clinical Practice (2013) xxx, xxx.e1—xxx.e9
REVIEW
Physiological roles of ghrelin on obesity Takahiro Sato a,∗, Takanori Ida b, Yuki Nakamura a, Yuki Shiimura a, Kenji Kangawa c, Masayasu Kojima a,∗ a
Molecular Genetics, Institute of Life Science, Kurume University, Kurume 839-0864, Japan b Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan c Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Suita 565-8565, Japan Received 4 June 2013 ; received in revised form 28 August 2013; accepted 8 October 2013
KEYWORDS Ghrelin; Ghrelin-O-acyl transferase; Prader-Willi syndrome
Summary Ghrelin is a stomach hormone that acts as an endogenous ligand of orphan G-protein coupled receptor. Ghrelin has various physiological functions, such as the stimulation of growth hormone release and of appetite, and fat accumulation. Ghrelin is the only peripheral hormone to transmit satiety signal. Mature ghrelin peptide is consisted of 28 amino acid residues, and is unusual among peptide hormones in that Ser3 is n-octanoylated to obtain. Furthermore, this modification is essential for ghrelin’s activity. In order to add this side chain to acyl ghrelin, it is necessary for the recently discovered enzyme, ghrelin-O-acyl transferase (GOAT). Therefore, to understand of ghrelin’s functions, it is useful to obtain the knowledge on structures and functions of ghrelin, ghrelin receptor and GOAT. Here, we review our current understanding of the structures and functions of ghrelin, and the relation between obesity and ghrelin. Finally, we referred to the ghrelin and related substances as a drug design target for obesity. © 2013 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Contents Introduction.................................................................................................. Structure of ghrelin and of the related substances ........................................................... Ghrelin .................................................................................................. GHS-R ...................................................................................................
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∗
Corresponding authors at: Molecular Genetics, Institute of Life Science, Kurume University, Hyakunen-kouen 1-1, Kurume 839-0864, Japan. Tel.: +81 0942 37 6313; fax: +81 0942 31 5212. E-mail addresses: satou [email protected] (T. Sato), kojima [email protected] (M. Kojima). 1871-403X/$ — see front matter © 2013 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.orcp.2013.10.002
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T. Sato et al.
GOAT .................................................................................................... Obestatin................................................................................................ Distribution, regulation and physiology of ghrelin ............................................................ Methods of ghrelin research .................................................................................. Measurements of ghrelin concentration.................................................................. Cell lines producing ghrelin.............................................................................. Obesity and ghrelin .......................................................................................... Diet-induced obesity and ghrelin ........................................................................ Prader-Willi syndrome (PWS) and ghrelin ................................................................ Genomic variants of ghrelin on obesity .................................................................. Ghrelin system as pharmacological targets ................................................................... Ghrelin as pharmacological targets ...................................................................... GHS-R as pharmacological targets ....................................................................... GOAT as pharmacological targets ........................................................................ Conclusion ................................................................................................... Acknowledgements ......................................................................................... References .................................................................................................
Introduction Today, the increase of obese individuals is the most serious problem on public health in the world today, because obesity causes type 2 diabetes mellitus, hyperlipidemia and hypertension, and finally induces arteriosclerosis. Therefore, the elucidation on energy homeostasis such as the mechanisms of appetite regulation and energy balance is necessary. If some key substances can be identified in a complicated homeostasis regulation system, leading to development of anti-obesity drug is expectable. Ghrelin that is discovered in 1999 as a 28-amino acid peptide from the rat stomach extracts [1] is one of candidate of such a substances. The characteristic structure of ghrelin is to have the side chain of medium-chain fatty acid to Ser3, and this structure is essential for ghrelin functions [1]. Because the ghrelin receptor agonists, growth hormone secretagogues (GHSs), had already been synthesized, some physiological functions of ghrelin were readily predicted [2—5]. Consequently, ghrelin research has advanced rapidly in a short span of time, and several functions of ghrelin was revealed- such as the stimulation of growth hormone (GH) release and of appetite, and fat accumulation [1,6,7]. Therefore, it is necessary to understand the mechanisms of ghrelin secretion because the abnormality of ghrelin secretion induces various metabolic disorder. Recently, the enzyme catalyzed the acylmodification of ghrelin was discovered and named ghrelin-O-acyl transferase (GOAT) [8]. The discovery of GOAT improve our understanding of the biosynthesis and secretion mechanisms of ghrelin because the dynamics of GOAT decide the activity
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of ghrelin. Because the secretory regulation of peptide hormone (ghrelin) by the enzyme of acylmodification (GOAT) is the only system in mammals, it may possible that GOAT became a new drug design target. Thus, structure, physiological function, secretion mechanisms of ghrelin, ghrelin receptor (growth hormone secretagogue receptor; GHS-R) and GOAT have recently become clear. Then, based on the fundamental knowledge about ghrelin, we need to clarify the role of the ghrelin in development and/or maintenance of obesity.
Structure of ghrelin and of the related substances Ghrelin The human ghrelin gene is localized on chromosome 3p25—26, and both human and mouse ghrelin genes comprise five exons [9—11]. The functional ghrelin peptide are encoded in exons 1 and 2. Mature ghrelin peptide is consisted of 28 amino acid residues, and is unusual among peptide hormones in that Ser3 is n-octanoylated [1]. This modification is the first known case in mammals, and is essential for ghrelin’s activity - such as the stimulation of GH secretion and of appetite, and fat accumulation [1,6,7]. Ghrelin has been identified in many species, and the amino acid sequences of ghrelins are well conserved across mammals, including human, rat, mouse, rhesus monkey, mongolian gerbil, cow, pig, goat, sheep and dog [1,11—19]. Specifically, the 10 amino acids in the NH2 termini are identical, strongly suggesting that this
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Physiological roles of ghrelin on obesity NH2 -terminal region is important for the activity of ghrelin. The nonacylated form of ghrelin, des-acyl ghrelin, is also present at significant levels in both stomach and blood [20]. However, des-acyl ghrelin can neither bind GHS-R nor exhibit GH-releasing activity in rats [21]. Because the genome database does not contain another G-protein coupled receptor (GPCR) that resembles GHS-R, it is possible that des-acyl ghrelin acts by mechanisms independent of a GPCR.
GHS-R The human ghrelin receptor gene has also been identified on chromosome 3, at position q26—27 [22]. Ghrelin receptor, GHS-R, is a typical GPCR with seven transmembrane domains (7TM); it is expressed as two distinct mRNAs [10]. The first, GHS-R type 1a, encodes a 7TM GPCR with binding and functional properties consistent with its role as the ghrelin receptor. The other GHS-R mRNA, type 1b, is produced by alternative splicing. The GHS-R gene consists of two exons; the first exon encodes TM1—TM5, and the second exon encodes TM6—TM7. Type 1b is derived from the first exon alone, and encodes only five of the seven predicted
xxx.e3 TM domains. The type 1b receptor is thus a COOHterminal truncated form of the type 1a receptor, and is physiologically inactive. Among GPCRs, ghrelin receptor is most similar to the motilin receptor [23,24]. Alignment of the 28-amino acid peptide ghrelin and the 19-amino acid motilin reveals that they share eight identical amino acids. After the discovery of ghrelin, the motilin-related peptide (MTLRP) was identified; the amino acid sequence of MTLRP is identical to that of ghrelin-(1—18) [25]. The ghrelin receptor is well conserved across all vertebrate species examined, including a number of mammals, bird, and fish. This strict conservation suggests that ghrelin and its receptor serve important physiological functions.
GOAT The side chain of acyl ghrelin originates from medium-chain fatty acids (MCFAs) and is posttranslationally attached to ghrelin by the recently discovered enzyme, GOAT [8] (Fig. 1). To catalyze the acyl-modification of ghrelin, the optimum temperature of GOAT is 37—50 ◦ C, and its optimum pH range is pH 7—8 [26]. Although the origin of the modified MCFAs has not been determined, it is known that orally ingested MCFAs are
Figure 1 Ghrelin system as pharmacological targets. Ghrelin is biosynthesized from des-acyl ghrelin and medium-chain fatty acids by the catalyst of GOAT. Ghrelin, ghrelin receptor and GOAT are the candidate of new drug target. Please cite this article in press as: Sato T, et al. Physiological roles of ghrelin on obesity. Obes Res Clin Pract (2013), http://dx.doi.org/10.1016/j.orcp.2013.10.002
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xxx.e4 directly utilized for acyl-modification of ghrelin [27]. Ingestion of either MCFAs or medium-chain triacylglycerols (MCTs) specifically increases production of acyl-modified ghrelin without changing the total (acyl- and des-acyl-) ghrelin level. When mice ingest either MCFAs or MCTs, the acyl group attached to nascent ghrelin molecules corresponds to those of the ingested MCFAs or MCTs [27]. Moreover, n-heptanoyl (C7:0) ghrelin, an unnatural form of ghrelin, can be produced in the stomach of mice following ingestion of n-heptanoic acid or glyceryl triheptanoate [27]. Thus, it is clear that ingested fatty acids are directly utilized for acylmodification of ghrelin.
Obestatin Zhang et al. identified a 23-amino acid peptide derived from the ghrelin peptide precursor by using a bioinformatics technique an named obestatin for its ability to inhibit food intake in mice when injected peripherally or into the brain ventricles [28]. In addition, the authors reported that peripheral injection of obestatin opposes ghrelin’s stimulatory effects on feeding in vivo. Therefore, this discovery of obestatin considered exciting new insights to the gut peptide field. However, their findings could not be reproduced by several groups [29—31], and must therefore be interpreted with caution.
Distribution, regulation and physiology of ghrelin The major source of ghrelin-secreted organ is stomach. Ghrelin is present in X/A-like cells, which account for about 20% of the endocrine cell population in adult oxyntic glands [32]. Ghrelin-immunoreactive cells are also found in the duodenum, jejunum, ileum, and colon. In the intestine, ghrelin concentration gradually decreases from the duodenum to the colon. The hypothalamus and pancreas are also ghrelin-producing organs, and ghrelin mRNA is also expressed in other tissues [33,34]. Ghrelin is only a hunger signals from peripheral tissues. Intravenous and subcutaneous injections of ghrelin increase food intake; likewise, peripherally injected ghrelin stimulates hypothalamic neurons and food intake [6,7,35]. Because the rate at which peripheral ghrelin passes the bloodbrain barrier has shown to be very low, peripheral ghrelin must activate the appropriate hypothalamic regions via an indirect pathway. The GHS-R localizes on vagal
T. Sato et al. afferent neurons in the rat nodose ganglion. Then, ghrelin signals from the stomach are transmitted to the brain via the vagus nerve [36]. In addition, vagotomy inhibits the ability of ghrelin to stimulate food intake [36]. A similar effect is also observed when capsaicin, a specific afferent neurotoxin, is applied to vagus nerve terminals to induce sensory denervation. Moreover, fasting-induced elevation of plasma ghrelin is completely abolished by subdiaphragmatic vagotomy or atropine treatment [37]. Such peripheral ghrelin signaling, which travels to the nucleus tractus solitarius at least in part via the vagus nerve, reaches arcuate nucleus of the hypothalamus [38]. Then, ghrelin is produced primarily in stomach in response to hunger and starvation, circulates in the blood, and serves as a peripheral signal, informing the arcuate nucleus of central nervous system (via vagus nerve) to stimulate feeding. Intracerebroventricular injection of ghrelin also increases cumulative food intake and decreases energy expenditure, resulting in body weight gain. Immunohistochemical analyses indicate that ghrelin-containing neurons also are found in the arcuate nucleus (ARC) of the hypothalamus, a region involved in appetite regulation [1]. This localization suggests a role of hypothalamic ghrelin in controlling food intake. In the ARC, these ghrelin-containing neurons send efferent fibers onto neuropeptide Y (NPY)- and agouti-related protein (AgRP)-expressing neurons in order to stimulate the release of the orexigenic peptides, and onto proopiomelanocortin neurons in order to suppress the release of the anorexigenic peptide [39]. The ARC is also a target of leptin, an appetitesuppressing hormone produced in adipose tissues [40]. Appetite-stimulating effects of NPY and AgRP are inhibited directly by leptin. At least part of the orexigenic effect of ghrelin is mediated by upregulating the genes encoding these potent appetite stimulants. Ghrelin has been shown to inhibit insulin secretion in some experiments and to stimulate insulin release in others [33,41—43]. Both plasma ghrelin and insulin levels are affected by blood glucose level. At high glucose suppresses ghrelin secretion and stimulates insulin release [33]. It was reported that ghrelin stimulates insulin release in the presence of high levels of glucose (8.3 mM) that could independently cause insulin release from cultured islet cells [33]. In contrast, ghrelin had no effect on insulin release in the context of a basal level of glucose (2.8 mM). In humans, ghrelin induces hyperglycemia and reduces insulin secretion [44]. Antagonism of the ghrelin function can enhance insulin release to meet increased demand for insulin in high-fat diet-induced obesity of mice
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Physiological roles of ghrelin on obesity [45]. Thus, ghrelin originating from pancreatic islets is a physiological regulator of glucose-induced insulin release. Consistent with its function, GOAT mRNA is primarily limited to the stomach; the main ghrelin-secreting tissue. Fasting and satiation could modulate the activity of GOAT as ghrelin levels [46]. Dietary lipids are critical for the activation of GOAT, and consequently ghrelin acylation to wildtype mice. Indeed, GOAT knock-out mice submitted to a diet containing 10% medium-chain triglyceride exhibited lower body weight that can be explained by lower fat mass compared to wild-type mice [47]. In addition, GOAT transgenic mice only fed with a medium-chain triglycerides supplementation produced large amounts of acyl ghrelin [47].
Methods of ghrelin research Measurements of ghrelin concentration To obtain the correct data of plasma ghrelin concentration and tissue ghrelin contents, we recommended next methods. The acyl-modification of ghrelin is easily cleaved during sample extraction, and peptide samples are easily digested by a wide range of cellular proteases. Furthermore, in order to correctly measure the plasma concentration of ghrelin, it is necessary to use EDTA and aprotinin when collecting blood samples, and plasma must be collected into 1/10 volume of 1 N HCl [48]. If the treated plasma samples are kept at −20 to −80 ◦ C, they are stable for at least 6 to 12 months. In human, the normal ghrelin concentration of plasma samples is 10—20 fmol/ml for n-octanoyl ghrelin and 100—150 fmol/ml for total ghrelin, including both acyl-modified and des-acyl ghrelins [46,49]. When measuring the tissue concentration of ghrelin, it is sufficient to inactivate proteases by boiling the tissues in water for 5—10 min [50,51]. In rats, ghrelin concentration in the stomach is 377.3 ± 55.8 fmol/ml for n-octanoyl ghrelin and 1779.8 ± 533.9 fmol/ml for total ghrelin [20]. Thus, the concentration of n-octanoyl ghrelin is 10—20% that of des-acyl ghrelin.
Cell lines producing ghrelin Several cell lines express ghrelin. TT cells, a human thyroid medullary carcinoma cell line, produced ghrelin mRNA; both conditioned medium and cellular extracts of TT cells contain ghrelin peptides [52]. Cellular extracts of TT cells also contain
xxx.e5 both n-octanoyl ghrelin and des-acyl ghrelin. Other cultured cells that express ghrelin include the kidney-derived cell line NRK-49F, gastric carcinoid ECC10 cells, and the cardiomyocyte cell line HL-1 [53—55]. However, the most useful tool for ghrelin research is MGN3-1 cells, although several cell lines as above have established. Recently, Iwakura et al. established a ghrelin-producing cell line MGN3-1 from a gastric ghrelin-producing cell tumor derived from transgenic mice in which SV40 Large T antigen was expressed under control of the ghrelin promoter [56]. MGN3-1 cells produce a substantial amount of ghrelin at levels approximately 5000 times higher than in TT cells. In addition, MGN3-1 cells express two key enzymes: GOAT, for acyl modification, and prohormone convertase 1/3 which is required for maturation of ghrelin. Moreover, MGN3-1 cells maintain physiological regulation of ghrelin secretion, at least in regard to the suppression by somatostatin and insulin, which has been well established in in vivo studies. This cell line will be a useful tool for studying both production and secretion of ghrelin, as well as for screening of ghrelin-modulating drugs [56].
Obesity and ghrelin Diet-induced obesity and ghrelin Obesity is controlled by the balances of between energy intake and expenditure. Anabolic hormone, ghrelin, are reduced in those who are obese compared to normal body weight controls. Ghrelin levels is low in visceral adiposity than in subcutaneous adiposity [57]. One of mechanism of the diet-induced obesity is that ghrelin resistance arises by reducing NPY/AgRP responsiveness to plasma ghrelin and suppressing the neuroendocrine ghrelin axis [58]. Then, obese people attempt to further food intake. Thus, diet-induced obesity may induce by hyperphagia occured by ghrelin resistance.
Prader-Willi syndrome (PWS) and ghrelin PWS is a complex genetic disorder characterized by hypotoina, hypogonadism, hypomentia and obesity [59]. PWS occurs owing to a loss of several paternal genes in the q11—13 region of chromosome 15 or a genomic imprinting disomy of maternal chromosome [59]. PWS is highly correlated with ghrelin secretion. Patients with PWS have high ghrelin levels relative to non-PWS obesity [60]. In fasting, plasma ghrelin levels in PWS patients is
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xxx.e6 higher than in lean controls, and is higher three- to four-fold than in non-PWS obese controls. In molecular level, imprinting of paternal genes in region q11—13 on chromosome 15 may induce the production of excessive amounts of transcription factors that increase ghrelin gene expression or loss of a transcription inhibitory factor that normally suppresses ghrelin gene expression [21]. The age-related decline in ghrelin level is blunted in PWS infants and children [61]. In addition, the number of ghrelin-expressing cells in stomach of PWS patients were increased two- to three-fold as compared to lean and obese controls [62]. Post-prandial levels of ghrelin do decrease in adult PWS patient but is still high level than lean control [46]. Therefore, it is possible that hyperphagia and obesity in PWS is correlated with high circulating levels of ghrelin. However, science not all PWS patients have elevated ghrelin levels, further research is required.
Genomic variants of ghrelin on obesity There may be a relationship between genomic variation in the ghrelin gene and obesity [63—65]. In humans, two polymorphisms have been reported: Arg51Gln and Leu72Met [63—65]. For both polymorphisms, allelic frequencies are similar between obese patients and controls [66]. However, obese patients with the Met72 allele became obese earlier than patients homozygous for the wild-type Leu72 allele, suggesting that the polymorphism may affect ghrelin’s activity [66]. The Leu72Met polymorphism of the ghrelin gene was detected in 15.6% of obese subjects whereas in 12.5% of non-obese control subjects [65]. The Arg51Gln mutation changes the sequence of the COOHterminal processing site of the ghrelin peptide, within its precursor protein, from Pro-Arg to ProGln; this mutation prevents the normal cleavage necessary to produce full-length mature ghrelin [65]. This variant of the ghrelin gene was found in 6.3% obese subjects but was not found in nonobese controls. Thus, the polymorphism of ghrelin is correlated to obesity [65].
Ghrelin system as pharmacological targets Ghrelin as pharmacological targets Ghrelin and its related substances are possible to be useful targets against obesity. Neutralization of ghrelin by the specific antibody reduced body
T. Sato et al. weight gain with the reduction of fat mass in rats [67]. Spiegelmers, antisense polyethylene glycolmodified L-oligonucleotides, have the ability of specifically binding a target molecule. Inhibitory effect of ghrelin-induced GH release in rats were exerted by NOX-B11-3 [68]. However, this spiegelmers did not block the fasting-induced neuronal activation in the ARC [69]. In conclusion, it may be useful the vaccination against ghrelin and the use of ghrelin spiegelmers in the treatment of obesity.
GHS-R as pharmacological targets GHS-R is also an useful target for the drugs to obesity. For many years, several classes of GHS-R1a antagonists have been developed. Representative GHS-R1a antagonists is [D-Lys3 -]GHRP-6. By treatment of [D-Lys3 -]GHRP-6, food intake is decreased and weight gain is reduced in lean and obese mice [70,71]. Piperidine-substituted quinazolinone derivatives were identified as a novel class of small GHS-R1a antagonists molecules [72]. A piperidinesubstituted quinazolinone derivative, YIL-781, improved glucose-stimulated insulin secretion and reduced food intake and weight loss in diet-induced obese mice [73]. Thus, a certain results on obesity is expected from GHS-R1a antagonists. For the treatment of obesity, inverse GHS-R1a agonist may be one of the candidate. Recently, it has come to be known that an inverse agonist is one of candidate to regulates ghrelin action, such as [D-Arg1 , D-Phe5 , D-Trp7,9, Leu11] substance P [74]. Reduction of the GHS-R1a constitutive activity by an inverse agonist could increase the sensitivity to anorexigenic hormones like leptin, and then, inhibit food intake. Thus, inverse GHR-R1a agonists will have unique pharmacological roles.
GOAT as pharmacological targets GOAT has been implicated as another attractive and potential target for the development of anti-obesity treatment. Recently, Barnett et al. described the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT [75]. GO-CoA-Tat potently inhibits GOAT in vitro, in both cultured cells and mice [75]. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type but not ghrelin-deficient mice [75]; thus, its beneficial metabolic effects are due specifically to GOAT inhibition. Moreover, quantitative magnetic resonance measurements showed that, relative to controls, GO-CoA-Tat treated animals displayed significantly
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Physiological roles of ghrelin on obesity lower fat mass, but not lean mass [75]. GOAT is therefore a potentially useful target for future development of therapeutic compounds.
Conclusion After the discovery of ghrelin, the unusual structure of ghrelin represented a new finding in biochemistry. In addition, the newly discovered enzyme that catalyzes the acyl-modification of ghrelin, GOAT, provides for the secretory machinery of the ghrelin, suggesting that the GOAT is new candidate of new drug target (Fig. 1). On the other hand, the physiological roles of ghrelin indicate the control of obesity by ghrelin. Although the energy metabolic system is complicated, the further study of ghrelin will be able to provide against obesity and the related syndrome.
Acknowledgements This work was supported by a MEXT-Supported Program for the Stratefic Research Foundation at Private Universities, 2010—2014 (to M.K.), by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.K.), by Research on Measures for Intractable Diseases from the Health and Labor Sciences Research Grants (to M.K.), by the Vehicle Racing Commemorative Foundation (to M.K.), and by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) (to T.S.).
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Obesity Research & Clinical Practice (2013) xxx, xxx.e1—xxx.e9
REVIEW
Physiological roles of ghrelin on obesity Takahiro Sato a,∗, Takanori Ida b, Yuki Nakamura a, Yuki Shiimura a, Kenji Kangawa c, Masayasu Kojima a,∗ a
Molecular Genetics, Institute of Life Science, Kurume University, Kurume 839-0864, Japan b Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan c Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Suita 565-8565, Japan Received 4 June 2013 ; received in revised form 28 August 2013; accepted 8 October 2013
KEYWORDS Ghrelin; Ghrelin-O-acyl transferase; Prader-Willi syndrome
Summary Ghrelin is a stomach hormone that acts as an endogenous ligand of orphan G-protein coupled receptor. Ghrelin has various physiological functions, such as the stimulation of growth hormone release and of appetite, and fat accumulation. Ghrelin is the only peripheral hormone to transmit satiety signal. Mature ghrelin peptide is consisted of 28 amino acid residues, and is unusual among peptide hormones in that Ser3 is n-octanoylated to obtain. Furthermore, this modification is essential for ghrelin’s activity. In order to add this side chain to acyl ghrelin, it is necessary for the recently discovered enzyme, ghrelin-O-acyl transferase (GOAT). Therefore, to understand of ghrelin’s functions, it is useful to obtain the knowledge on structures and functions of ghrelin, ghrelin receptor and GOAT. Here, we review our current understanding of the structures and functions of ghrelin, and the relation between obesity and ghrelin. Finally, we referred to the ghrelin and related substances as a drug design target for obesity. © 2013 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
Contents Introduction.................................................................................................. Structure of ghrelin and of the related substances ........................................................... Ghrelin .................................................................................................. GHS-R ...................................................................................................
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∗
Corresponding authors at: Molecular Genetics, Institute of Life Science, Kurume University, Hyakunen-kouen 1-1, Kurume 839-0864, Japan. Tel.: +81 0942 37 6313; fax: +81 0942 31 5212. E-mail addresses: satou [email protected] (T. Sato), kojima [email protected] (M. Kojima). 1871-403X/$ — see front matter © 2013 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.orcp.2013.10.002
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GOAT .................................................................................................... Obestatin................................................................................................ Distribution, regulation and physiology of ghrelin ............................................................ Methods of ghrelin research .................................................................................. Measurements of ghrelin concentration.................................................................. Cell lines producing ghrelin.............................................................................. Obesity and ghrelin .......................................................................................... Diet-induced obesity and ghrelin ........................................................................ Prader-Willi syndrome (PWS) and ghrelin ................................................................ Genomic variants of ghrelin on obesity .................................................................. Ghrelin system as pharmacological targets ................................................................... Ghrelin as pharmacological targets ...................................................................... GHS-R as pharmacological targets ....................................................................... GOAT as pharmacological targets ........................................................................ Conclusion ................................................................................................... Acknowledgements ......................................................................................... References .................................................................................................
Introduction Today, the increase of obese individuals is the most serious problem on public health in the world today, because obesity causes type 2 diabetes mellitus, hyperlipidemia and hypertension, and finally induces arteriosclerosis. Therefore, the elucidation on energy homeostasis such as the mechanisms of appetite regulation and energy balance is necessary. If some key substances can be identified in a complicated homeostasis regulation system, leading to development of anti-obesity drug is expectable. Ghrelin that is discovered in 1999 as a 28-amino acid peptide from the rat stomach extracts [1] is one of candidate of such a substances. The characteristic structure of ghrelin is to have the side chain of medium-chain fatty acid to Ser3, and this structure is essential for ghrelin functions [1]. Because the ghrelin receptor agonists, growth hormone secretagogues (GHSs), had already been synthesized, some physiological functions of ghrelin were readily predicted [2—5]. Consequently, ghrelin research has advanced rapidly in a short span of time, and several functions of ghrelin was revealed- such as the stimulation of growth hormone (GH) release and of appetite, and fat accumulation [1,6,7]. Therefore, it is necessary to understand the mechanisms of ghrelin secretion because the abnormality of ghrelin secretion induces various metabolic disorder. Recently, the enzyme catalyzed the acylmodification of ghrelin was discovered and named ghrelin-O-acyl transferase (GOAT) [8]. The discovery of GOAT improve our understanding of the biosynthesis and secretion mechanisms of ghrelin because the dynamics of GOAT decide the activity
00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00
of ghrelin. Because the secretory regulation of peptide hormone (ghrelin) by the enzyme of acylmodification (GOAT) is the only system in mammals, it may possible that GOAT became a new drug design target. Thus, structure, physiological function, secretion mechanisms of ghrelin, ghrelin receptor (growth hormone secretagogue receptor; GHS-R) and GOAT have recently become clear. Then, based on the fundamental knowledge about ghrelin, we need to clarify the role of the ghrelin in development and/or maintenance of obesity.
Structure of ghrelin and of the related substances Ghrelin The human ghrelin gene is localized on chromosome 3p25—26, and both human and mouse ghrelin genes comprise five exons [9—11]. The functional ghrelin peptide are encoded in exons 1 and 2. Mature ghrelin peptide is consisted of 28 amino acid residues, and is unusual among peptide hormones in that Ser3 is n-octanoylated [1]. This modification is the first known case in mammals, and is essential for ghrelin’s activity - such as the stimulation of GH secretion and of appetite, and fat accumulation [1,6,7]. Ghrelin has been identified in many species, and the amino acid sequences of ghrelins are well conserved across mammals, including human, rat, mouse, rhesus monkey, mongolian gerbil, cow, pig, goat, sheep and dog [1,11—19]. Specifically, the 10 amino acids in the NH2 termini are identical, strongly suggesting that this
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Physiological roles of ghrelin on obesity NH2 -terminal region is important for the activity of ghrelin. The nonacylated form of ghrelin, des-acyl ghrelin, is also present at significant levels in both stomach and blood [20]. However, des-acyl ghrelin can neither bind GHS-R nor exhibit GH-releasing activity in rats [21]. Because the genome database does not contain another G-protein coupled receptor (GPCR) that resembles GHS-R, it is possible that des-acyl ghrelin acts by mechanisms independent of a GPCR.
GHS-R The human ghrelin receptor gene has also been identified on chromosome 3, at position q26—27 [22]. Ghrelin receptor, GHS-R, is a typical GPCR with seven transmembrane domains (7TM); it is expressed as two distinct mRNAs [10]. The first, GHS-R type 1a, encodes a 7TM GPCR with binding and functional properties consistent with its role as the ghrelin receptor. The other GHS-R mRNA, type 1b, is produced by alternative splicing. The GHS-R gene consists of two exons; the first exon encodes TM1—TM5, and the second exon encodes TM6—TM7. Type 1b is derived from the first exon alone, and encodes only five of the seven predicted
xxx.e3 TM domains. The type 1b receptor is thus a COOHterminal truncated form of the type 1a receptor, and is physiologically inactive. Among GPCRs, ghrelin receptor is most similar to the motilin receptor [23,24]. Alignment of the 28-amino acid peptide ghrelin and the 19-amino acid motilin reveals that they share eight identical amino acids. After the discovery of ghrelin, the motilin-related peptide (MTLRP) was identified; the amino acid sequence of MTLRP is identical to that of ghrelin-(1—18) [25]. The ghrelin receptor is well conserved across all vertebrate species examined, including a number of mammals, bird, and fish. This strict conservation suggests that ghrelin and its receptor serve important physiological functions.
GOAT The side chain of acyl ghrelin originates from medium-chain fatty acids (MCFAs) and is posttranslationally attached to ghrelin by the recently discovered enzyme, GOAT [8] (Fig. 1). To catalyze the acyl-modification of ghrelin, the optimum temperature of GOAT is 37—50 ◦ C, and its optimum pH range is pH 7—8 [26]. Although the origin of the modified MCFAs has not been determined, it is known that orally ingested MCFAs are
Figure 1 Ghrelin system as pharmacological targets. Ghrelin is biosynthesized from des-acyl ghrelin and medium-chain fatty acids by the catalyst of GOAT. Ghrelin, ghrelin receptor and GOAT are the candidate of new drug target. Please cite this article in press as: Sato T, et al. Physiological roles of ghrelin on obesity. Obes Res Clin Pract (2013), http://dx.doi.org/10.1016/j.orcp.2013.10.002
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xxx.e4 directly utilized for acyl-modification of ghrelin [27]. Ingestion of either MCFAs or medium-chain triacylglycerols (MCTs) specifically increases production of acyl-modified ghrelin without changing the total (acyl- and des-acyl-) ghrelin level. When mice ingest either MCFAs or MCTs, the acyl group attached to nascent ghrelin molecules corresponds to those of the ingested MCFAs or MCTs [27]. Moreover, n-heptanoyl (C7:0) ghrelin, an unnatural form of ghrelin, can be produced in the stomach of mice following ingestion of n-heptanoic acid or glyceryl triheptanoate [27]. Thus, it is clear that ingested fatty acids are directly utilized for acylmodification of ghrelin.
Obestatin Zhang et al. identified a 23-amino acid peptide derived from the ghrelin peptide precursor by using a bioinformatics technique an named obestatin for its ability to inhibit food intake in mice when injected peripherally or into the brain ventricles [28]. In addition, the authors reported that peripheral injection of obestatin opposes ghrelin’s stimulatory effects on feeding in vivo. Therefore, this discovery of obestatin considered exciting new insights to the gut peptide field. However, their findings could not be reproduced by several groups [29—31], and must therefore be interpreted with caution.
Distribution, regulation and physiology of ghrelin The major source of ghrelin-secreted organ is stomach. Ghrelin is present in X/A-like cells, which account for about 20% of the endocrine cell population in adult oxyntic glands [32]. Ghrelin-immunoreactive cells are also found in the duodenum, jejunum, ileum, and colon. In the intestine, ghrelin concentration gradually decreases from the duodenum to the colon. The hypothalamus and pancreas are also ghrelin-producing organs, and ghrelin mRNA is also expressed in other tissues [33,34]. Ghrelin is only a hunger signals from peripheral tissues. Intravenous and subcutaneous injections of ghrelin increase food intake; likewise, peripherally injected ghrelin stimulates hypothalamic neurons and food intake [6,7,35]. Because the rate at which peripheral ghrelin passes the bloodbrain barrier has shown to be very low, peripheral ghrelin must activate the appropriate hypothalamic regions via an indirect pathway. The GHS-R localizes on vagal
T. Sato et al. afferent neurons in the rat nodose ganglion. Then, ghrelin signals from the stomach are transmitted to the brain via the vagus nerve [36]. In addition, vagotomy inhibits the ability of ghrelin to stimulate food intake [36]. A similar effect is also observed when capsaicin, a specific afferent neurotoxin, is applied to vagus nerve terminals to induce sensory denervation. Moreover, fasting-induced elevation of plasma ghrelin is completely abolished by subdiaphragmatic vagotomy or atropine treatment [37]. Such peripheral ghrelin signaling, which travels to the nucleus tractus solitarius at least in part via the vagus nerve, reaches arcuate nucleus of the hypothalamus [38]. Then, ghrelin is produced primarily in stomach in response to hunger and starvation, circulates in the blood, and serves as a peripheral signal, informing the arcuate nucleus of central nervous system (via vagus nerve) to stimulate feeding. Intracerebroventricular injection of ghrelin also increases cumulative food intake and decreases energy expenditure, resulting in body weight gain. Immunohistochemical analyses indicate that ghrelin-containing neurons also are found in the arcuate nucleus (ARC) of the hypothalamus, a region involved in appetite regulation [1]. This localization suggests a role of hypothalamic ghrelin in controlling food intake. In the ARC, these ghrelin-containing neurons send efferent fibers onto neuropeptide Y (NPY)- and agouti-related protein (AgRP)-expressing neurons in order to stimulate the release of the orexigenic peptides, and onto proopiomelanocortin neurons in order to suppress the release of the anorexigenic peptide [39]. The ARC is also a target of leptin, an appetitesuppressing hormone produced in adipose tissues [40]. Appetite-stimulating effects of NPY and AgRP are inhibited directly by leptin. At least part of the orexigenic effect of ghrelin is mediated by upregulating the genes encoding these potent appetite stimulants. Ghrelin has been shown to inhibit insulin secretion in some experiments and to stimulate insulin release in others [33,41—43]. Both plasma ghrelin and insulin levels are affected by blood glucose level. At high glucose suppresses ghrelin secretion and stimulates insulin release [33]. It was reported that ghrelin stimulates insulin release in the presence of high levels of glucose (8.3 mM) that could independently cause insulin release from cultured islet cells [33]. In contrast, ghrelin had no effect on insulin release in the context of a basal level of glucose (2.8 mM). In humans, ghrelin induces hyperglycemia and reduces insulin secretion [44]. Antagonism of the ghrelin function can enhance insulin release to meet increased demand for insulin in high-fat diet-induced obesity of mice
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Physiological roles of ghrelin on obesity [45]. Thus, ghrelin originating from pancreatic islets is a physiological regulator of glucose-induced insulin release. Consistent with its function, GOAT mRNA is primarily limited to the stomach; the main ghrelin-secreting tissue. Fasting and satiation could modulate the activity of GOAT as ghrelin levels [46]. Dietary lipids are critical for the activation of GOAT, and consequently ghrelin acylation to wildtype mice. Indeed, GOAT knock-out mice submitted to a diet containing 10% medium-chain triglyceride exhibited lower body weight that can be explained by lower fat mass compared to wild-type mice [47]. In addition, GOAT transgenic mice only fed with a medium-chain triglycerides supplementation produced large amounts of acyl ghrelin [47].
Methods of ghrelin research Measurements of ghrelin concentration To obtain the correct data of plasma ghrelin concentration and tissue ghrelin contents, we recommended next methods. The acyl-modification of ghrelin is easily cleaved during sample extraction, and peptide samples are easily digested by a wide range of cellular proteases. Furthermore, in order to correctly measure the plasma concentration of ghrelin, it is necessary to use EDTA and aprotinin when collecting blood samples, and plasma must be collected into 1/10 volume of 1 N HCl [48]. If the treated plasma samples are kept at −20 to −80 ◦ C, they are stable for at least 6 to 12 months. In human, the normal ghrelin concentration of plasma samples is 10—20 fmol/ml for n-octanoyl ghrelin and 100—150 fmol/ml for total ghrelin, including both acyl-modified and des-acyl ghrelins [46,49]. When measuring the tissue concentration of ghrelin, it is sufficient to inactivate proteases by boiling the tissues in water for 5—10 min [50,51]. In rats, ghrelin concentration in the stomach is 377.3 ± 55.8 fmol/ml for n-octanoyl ghrelin and 1779.8 ± 533.9 fmol/ml for total ghrelin [20]. Thus, the concentration of n-octanoyl ghrelin is 10—20% that of des-acyl ghrelin.
Cell lines producing ghrelin Several cell lines express ghrelin. TT cells, a human thyroid medullary carcinoma cell line, produced ghrelin mRNA; both conditioned medium and cellular extracts of TT cells contain ghrelin peptides [52]. Cellular extracts of TT cells also contain
xxx.e5 both n-octanoyl ghrelin and des-acyl ghrelin. Other cultured cells that express ghrelin include the kidney-derived cell line NRK-49F, gastric carcinoid ECC10 cells, and the cardiomyocyte cell line HL-1 [53—55]. However, the most useful tool for ghrelin research is MGN3-1 cells, although several cell lines as above have established. Recently, Iwakura et al. established a ghrelin-producing cell line MGN3-1 from a gastric ghrelin-producing cell tumor derived from transgenic mice in which SV40 Large T antigen was expressed under control of the ghrelin promoter [56]. MGN3-1 cells produce a substantial amount of ghrelin at levels approximately 5000 times higher than in TT cells. In addition, MGN3-1 cells express two key enzymes: GOAT, for acyl modification, and prohormone convertase 1/3 which is required for maturation of ghrelin. Moreover, MGN3-1 cells maintain physiological regulation of ghrelin secretion, at least in regard to the suppression by somatostatin and insulin, which has been well established in in vivo studies. This cell line will be a useful tool for studying both production and secretion of ghrelin, as well as for screening of ghrelin-modulating drugs [56].
Obesity and ghrelin Diet-induced obesity and ghrelin Obesity is controlled by the balances of between energy intake and expenditure. Anabolic hormone, ghrelin, are reduced in those who are obese compared to normal body weight controls. Ghrelin levels is low in visceral adiposity than in subcutaneous adiposity [57]. One of mechanism of the diet-induced obesity is that ghrelin resistance arises by reducing NPY/AgRP responsiveness to plasma ghrelin and suppressing the neuroendocrine ghrelin axis [58]. Then, obese people attempt to further food intake. Thus, diet-induced obesity may induce by hyperphagia occured by ghrelin resistance.
Prader-Willi syndrome (PWS) and ghrelin PWS is a complex genetic disorder characterized by hypotoina, hypogonadism, hypomentia and obesity [59]. PWS occurs owing to a loss of several paternal genes in the q11—13 region of chromosome 15 or a genomic imprinting disomy of maternal chromosome [59]. PWS is highly correlated with ghrelin secretion. Patients with PWS have high ghrelin levels relative to non-PWS obesity [60]. In fasting, plasma ghrelin levels in PWS patients is
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xxx.e6 higher than in lean controls, and is higher three- to four-fold than in non-PWS obese controls. In molecular level, imprinting of paternal genes in region q11—13 on chromosome 15 may induce the production of excessive amounts of transcription factors that increase ghrelin gene expression or loss of a transcription inhibitory factor that normally suppresses ghrelin gene expression [21]. The age-related decline in ghrelin level is blunted in PWS infants and children [61]. In addition, the number of ghrelin-expressing cells in stomach of PWS patients were increased two- to three-fold as compared to lean and obese controls [62]. Post-prandial levels of ghrelin do decrease in adult PWS patient but is still high level than lean control [46]. Therefore, it is possible that hyperphagia and obesity in PWS is correlated with high circulating levels of ghrelin. However, science not all PWS patients have elevated ghrelin levels, further research is required.
Genomic variants of ghrelin on obesity There may be a relationship between genomic variation in the ghrelin gene and obesity [63—65]. In humans, two polymorphisms have been reported: Arg51Gln and Leu72Met [63—65]. For both polymorphisms, allelic frequencies are similar between obese patients and controls [66]. However, obese patients with the Met72 allele became obese earlier than patients homozygous for the wild-type Leu72 allele, suggesting that the polymorphism may affect ghrelin’s activity [66]. The Leu72Met polymorphism of the ghrelin gene was detected in 15.6% of obese subjects whereas in 12.5% of non-obese control subjects [65]. The Arg51Gln mutation changes the sequence of the COOHterminal processing site of the ghrelin peptide, within its precursor protein, from Pro-Arg to ProGln; this mutation prevents the normal cleavage necessary to produce full-length mature ghrelin [65]. This variant of the ghrelin gene was found in 6.3% obese subjects but was not found in nonobese controls. Thus, the polymorphism of ghrelin is correlated to obesity [65].
Ghrelin system as pharmacological targets Ghrelin as pharmacological targets Ghrelin and its related substances are possible to be useful targets against obesity. Neutralization of ghrelin by the specific antibody reduced body
T. Sato et al. weight gain with the reduction of fat mass in rats [67]. Spiegelmers, antisense polyethylene glycolmodified L-oligonucleotides, have the ability of specifically binding a target molecule. Inhibitory effect of ghrelin-induced GH release in rats were exerted by NOX-B11-3 [68]. However, this spiegelmers did not block the fasting-induced neuronal activation in the ARC [69]. In conclusion, it may be useful the vaccination against ghrelin and the use of ghrelin spiegelmers in the treatment of obesity.
GHS-R as pharmacological targets GHS-R is also an useful target for the drugs to obesity. For many years, several classes of GHS-R1a antagonists have been developed. Representative GHS-R1a antagonists is [D-Lys3 -]GHRP-6. By treatment of [D-Lys3 -]GHRP-6, food intake is decreased and weight gain is reduced in lean and obese mice [70,71]. Piperidine-substituted quinazolinone derivatives were identified as a novel class of small GHS-R1a antagonists molecules [72]. A piperidinesubstituted quinazolinone derivative, YIL-781, improved glucose-stimulated insulin secretion and reduced food intake and weight loss in diet-induced obese mice [73]. Thus, a certain results on obesity is expected from GHS-R1a antagonists. For the treatment of obesity, inverse GHS-R1a agonist may be one of the candidate. Recently, it has come to be known that an inverse agonist is one of candidate to regulates ghrelin action, such as [D-Arg1 , D-Phe5 , D-Trp7,9, Leu11] substance P [74]. Reduction of the GHS-R1a constitutive activity by an inverse agonist could increase the sensitivity to anorexigenic hormones like leptin, and then, inhibit food intake. Thus, inverse GHR-R1a agonists will have unique pharmacological roles.
GOAT as pharmacological targets GOAT has been implicated as another attractive and potential target for the development of anti-obesity treatment. Recently, Barnett et al. described the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT [75]. GO-CoA-Tat potently inhibits GOAT in vitro, in both cultured cells and mice [75]. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type but not ghrelin-deficient mice [75]; thus, its beneficial metabolic effects are due specifically to GOAT inhibition. Moreover, quantitative magnetic resonance measurements showed that, relative to controls, GO-CoA-Tat treated animals displayed significantly
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Physiological roles of ghrelin on obesity lower fat mass, but not lean mass [75]. GOAT is therefore a potentially useful target for future development of therapeutic compounds.
Conclusion After the discovery of ghrelin, the unusual structure of ghrelin represented a new finding in biochemistry. In addition, the newly discovered enzyme that catalyzes the acyl-modification of ghrelin, GOAT, provides for the secretory machinery of the ghrelin, suggesting that the GOAT is new candidate of new drug target (Fig. 1). On the other hand, the physiological roles of ghrelin indicate the control of obesity by ghrelin. Although the energy metabolic system is complicated, the further study of ghrelin will be able to provide against obesity and the related syndrome.
Acknowledgements This work was supported by a MEXT-Supported Program for the Stratefic Research Foundation at Private Universities, 2010—2014 (to M.K.), by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.K.), by Research on Measures for Intractable Diseases from the Health and Labor Sciences Research Grants (to M.K.), by the Vehicle Racing Commemorative Foundation (to M.K.), and by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS) (to T.S.).
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