Developmental Changes in the Pattern of Ghrelin’s Acyl Modification and the Levels of Acyl-Modified Ghrelins in Murine Stomach
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内分泌学杂志 2005年第6期
Molecular Genetics, Institute of Life Science (Y.N., H.H., T.S., M.K.), and Institute of Animal Experimentation (H.M.), Kurume University, Fukuoka 839-0861; and Department of Biochemistry, National Cardiovascular Center Research Institute (K.K.), Osaka 565-8565, Japan
Address all correspondence and requests for reprints to: Dr. Masayasu Kojima, Molecular Genetics, Institute of Life Science, Kurume University, Kurume, Fukuoka 839-0861, Japan. E-mail: mkojima@lsi.kurume-u.ac.jp.
Abstract
Ghrelin is an acylated peptide hormone secreted primarily from endocrine cells in the stomach. The major active form of ghrelin is a 28-amino acid peptide with an n-octanoyl modification at Ser3 (n-octanoyl ghrelin), which is essential for its activity. In addition to n-octanoyl ghrelin, other forms of ghrelin peptide exist, including des-acyl ghrelin, which lacks an acyl modification, and other minor acylated ghrelin species, such as n-decanoyl ghrelin, whose Ser3 residue is modified by n-decanoic acid. Multiple reports have identified various physiological functions of ghrelin. However, until now, there have been no reports that explore the process of ghrelin acyl modification, and only a few studies have compared the levels of des-acyl, n-octanoyl, and/or other minor populations of acylated ghrelin peptides. In this study we report that the amount of n-octanoyl ghrelin in murine stomachs increases gradually during the suckling period to a maximal level at 3 wk of age and falls sharply after the initiation of weaning. However, the concentration (picomoles per milligram of wet weight tissue) of total ghrelin, which includes des-acyl and all acylated forms of ghrelin peptides with intact C termini in murine stomach, remains unchanged across this suckling-weaning transition. Prematurely weaned mice exhibited a significant decrease in the amount of n-octanoyl or n-decanoyl ghrelin in the stomach. Orally ingested glyceryl trioctanoate, a medium-chain triacylglyceride rich in milk lipids, significantly increased the level of n-octanoyl-modified ghrelin in murine stomach. Fluctuations in the proportion of this biologically active, acyl-modified ghrelin could contribute to or be influenced by the change in energy metabolism during the suckling-weaning transition.
Introduction
GHRELIN IS AN endogenous ligand for the GH secretagogue (GHS) receptor (GHS-R) (1, 2). GHSs are synthetic substances with potent GH-releasing activity (3, 4, 5). Although ghrelin was initially purified from the stomach, it is also expressed in the brain, pancreas, small intestine, and other tissues (6). In addition to its potent GH-releasing activity (1), ghrelin stimulates appetite and influences adiposity (7, 8). The third amino acid residue of ghrelin, a serine (Ser3), is modified by an acyl group; this modification is essential for ghrelin’s biological activity through the classical ghrelin receptor, GHS-R1a (1, 9). Although the primary acyl chain modifying ghrelin molecules in humans and rodents is an n-octanoyl group (C8:0, an eight-carbon chain containing no double bonds) (1, 10), different acyl groups modify a minor population of ghrelin peptides. These acyl groups include n-decanoyl (C10:0, a 10-carbon chain lacking double bonds) and n-decenoyl (C10:1, a 10-carbon chain containing one double bond) (11, 12, 13). To our knowledge, the acyl modification of ghrelin is the first known example of a fatty acid modification of a peptide hormone.
A number of reports have been published on the regulation of ghrelin secretion in the context of energy metabolism that did not distinguish between acyl-modified and des-acyl ghrelin (14, 15, 16, 17, 18). However, there have been only a few reports comparing the levels of n-octanoyl, des-acyl, and/or total ghrelin, the last of which encompasses des-acyl and all acyl-modified ghrelin peptides with intact C termini (19, 20).
In this study we measured the amount of n-octanoyl and total ghrelin as well as the minor population of n-decanoyl ghrelin in murine stomach throughout the suckling and weaning periods during which energy metabolism is dramatically altered. Furthermore, we investigated the effect of dietary medium-chain triacylglycerides (MCTs) on the stomach levels of n-octanoyl and total ghrelin.
Materials and Methods
Animals
Male C57BL/6J mice and male Wistar rats were used in this experiment. They were maintained under controlled temperature (21–23 C) and light conditions (lights on, 0700–1900 h) with ad libitum access to standard laboratory chow (CE-2, CLEA Co. Ltd., Osaka, Japan) and water. All mice were killed by decapitation, and all rats were killed by exsanguination under anesthesia with an ip injection (30 mg/kg body weight) of sodium pentobarbital (Nembutal injection, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan). All experiments were conducted in accordance with the Kurume University Guide for the Care and Use of Experimental Animals.
RIA for ghrelin
RIAs specific for ghrelin were performed as previously described (6). In brief, two polyclonal antibodies were raised in rabbits against the N terminal (Gly1-Lys11 with O-n-octanoylation at Ser3) or C-terminal (Gln13-Arg28) fragments of rat ghrelin. Both antisera exhibited complete cross-reactivity with human, mouse, and rat ghrelins. The antirat ghrelin-(1–11) antiserum, which specifically recognized the Ser3 n-octanoylated form of ghrelin, does not recognize des-acyl ghrelin, but cross-reacts faintly with other acyl-modified ghrelins. Antirat ghrelin-(13–28) antiserum equally recognizes both des-acyl and all acylated forms of ghrelin with intact C-terminal peptide sequences. In the following sections, the RIA system using the antiserum against the N-terminal fragment of rat ghrelin-(1–11) is termed N-RIA, whereas that specific for the C-terminus [ghrelin-(13–28)] is termed C-RIA. Ghrelin-like immunoreactivity (ghrelin-LI) measured by N-RIA is termed ghrelin N-LI or n-octanoyl ghrelin-LI, and ghrelin-LI measured by C-RIA, which reflects the amount of ghrelin peptides with intact C-termini (regardless of the acyl-modification of Ser3) is termed total ghrelin or ghrelin C-LI.
Preparation of stomach samples for ghrelin assay
Male C57BL/6J mice (birth to 12 wk of age) were maintained under controlled temperature (21–23 C) and light conditions (lights on, 0700–1900 h) with ad libitum access to a nursing mother, chow, and water. Stomachs collected from mice were washed twice in PBS (pH 7.4). After measuring the wet weights of each sample, the whole stomach tissue was boiled for 5 min in a 10-fold volume of water to inactivate intrinsic proteases. After cooling on ice, boiled samples were adjusted to 1 M acetic acid-20 mM HCl. Peptides were extracted after homogenization with a Polytron mixer (PT 6100, Kinematica AG, Littan-Luzern, Switzerland). Extract supernatants, isolated after a 15-min centrifugation at 15,000 rpm (12,000 x g), were lyophilized and stored at –80 C. The lyophilized samples were dissolved in RIA buffer before ghrelin RIA.
Preparation of plasma samples for ghrelin assay
Plasma samples from rats were prepared as previously described (1, 6). In brief, truncal blood samples obtained by heart puncture were immediately transferred to chilled polypropylene tubes containing EDTA-2Na (1 mg/ml) and aprotinin (1000 kallikrein inactivator units/ml) and centrifuged at 4 C. Immediately after the plasma was separated, HCl was added to the sample to a final concentration of 0.1 N and then diluted with an equal volume of saline. The sample was loaded onto a Sep-Pak C18 cartridge (Waters Corp., Milford, MA) preequilibrated with 0.1% trifluoroacetic acid (TFA) and 0.9% NaCl. The cartridge was washed with 0.9% NaCl and 5% acetonitrile (CH3CN)/0.1% TFA, then eluted with 60% CH3CN/0.1% TFA. The eluate was lyophilized and stored at –80 C. Lyophilized samples were dissolved in RIA buffer before ghrelin RIA.
Analysis of molecular forms of acyl-modified ghrelin in murine stomach
To investigate changes in the pattern of ghrelin’s acyl modification and the levels of des-acyl or acyl-modified ghrelins around the time of initiation of weaning, the molecular properties of stomach ghrelin were analyzed by C18 reverse phase (RP) HPLC (3.9 x 150 mm; Symmetry 300, Waters Corp.). Stomach peptides were extracted from 3- or 4-wk-old male mice under normal weaning (NW) conditions or from prematurely weaned (PW) 3-wk-old male mice. Stomach peptides were also extracted from 3-wk-old male rats under NW or PW conditions. PW was performed in manner described previously by Hahn et al. (21). In brief, eight pups, regardless of sex, were retained from a litter of Wistar rats or C57BL/6J mice on d 2 after birth, weaned to standard laboratory chaw on d 18, and killed on d 21. The stomach or plasma samples from male-only litters were collected and subjected to peptide extraction. Standard laboratory chow (CE-2) derives its caloric content as follows: carbohydrate, 50.3%; protein, 25.4%; and fat, 4.4%. Peptide extracts from stomachs were subjected to RP-HPLC using a linear gradient of 10–60% CH3CN/0.1% TFA at a flow rate of 1.0 ml/min. Five hundred-microliter fractions were collected. The ghrelin peptide content in each fraction was measured by ghrelin C-RIA. The retention times obtained for the extracted ghrelin molecules were compared with those of synthetic des-acyl, n-octanoyl, and n-decanoyl ghrelin.
Concentrations of n-octanoyl and total ghrelin in murine stomach after glyceryl trioctanoate ingestion
Three-week-old male C57BL/6J mice were maintained under controlled temperature (21–23 C) and light (lights on, 0700–1900 h) conditions with ad libitum access to food, water, and their nursing mothers. Glyceryl trioctanoate (Wako Pure Chemical, Osaka, Japan), a MCT rich in rodent milk lipids, was mixed into standard laboratory chow at a concentration of 5% (wt/wt). After 1 wk, whole stomach samples from MCT-fed and control (4-wk-old) mice were collected and assayed for ghrelin N- or C-RIAs as described above.
Statistical analysis
Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA, followed by a post hoc test (Scheffé’s F test). P <0.05 was considered statistically significant.
Results
Concentration of ghrelin-like immunoreactivity in murine stomachs
To elucidate the regulation of ghrelin acyl modification during the developmental stages after birth, the levels of n-octanoyl and total ghrelin immunoreactivity in stomachs of mice from birth to 12 wk of age were measured using ghrelin N- and C-RIAs. The level of n-octanoyl ghrelin-LI in the stomach increased from birth to 3 wk of age and then fell significantly between 3 and 4 wk after birth (Fig. 1A). After mice reached 4 wk of age, the level of stomach n-octanoyl ghrelin remained unchanged. The amount of total ghrelin-LI also increased from birth to 3 wk of age and subsequently remained unchanged from 3–12 wk of age. No significant change in total ghrelin-LI was noted between 3 and 4 wk of age. The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) in stomachs of mice increased throughout the suckling period (from 1–3 wk of age). Thereafter, the N/C ratio fell sharply between 3 and 4 wk of age in proportion to the decline in the amount of n-octanoyl ghrelin-LI. After 4 wk of age, no significant change in the N/C ratio was detected in stomachs of mice until 12 wk of age. Patterns similar to those described above were observed in data from two subsequent experiments using the same protocol.
FIG. 1. Amount of ghrelin-LI in the stomachs of mice from birth to 12 wk of age. Each bar represents means ± SD (n = 6–12/group). A, n-Octanoyl-modified ghrelin-LI concentrations measured by ghrelin N-RIA. B, Total ghrelin-LI (des-acyl and acyl-modified forms of ghrelin with intact C terminus) concentration measured by ghrelin C-RIA. C, The ratio of n-octanoyl/total ghrelin-LI in murine stomach (N/C ratio). Statistical significance is indicated by superscript letters. a, P < 0.001; b, P < 0.05; c, P < 0.01 (vs. the indicated values).
Molecular forms of ghrelin in stomachs from 3- and 4-wk-old mice
To examine whether there was a change in the molecular form of stomach ghrelin peptides before and after the suckling-weaning transition, we fractionated stomach samples from 3- and 4-wk-old mice by RP-HPLC and measured ghrelin levels by C-RIA (Fig. 2). Based on the observed retention times of synthetic ghrelin peptides, peaks a and a' correspond to des-acyl ghrelin, peaks b and b' correspond to n-octanoyl ghrelin modified by an n-octanoyl (C8:0) group, and peaks c and c' corresponded to n-decanoyl ghrelin modified by an n-decanoyl (C10:0) group. Compared with 3-wk-old mice, a decrease in the amount of n-decanoyl ghrelin (Fig. 2, upper panel; peak c: retention time, 24–25 min) was observed in 4-wk-old mice. The level of n-octanoyl ghrelin (peak b': retention time, 20.5–21.5 min) was also decreased in 4-wk-old mice compared with 3-wk-old mice (peak b). In contrast, the level of des-acyl ghrelin (peak a': retention time, 10.5–11.5) in 4-wk-old mouse stomachs was higher than that in 3-wk-old mice (peak a). There was an unidentified peak between peaks b (b') and c (c'), and there were at least two peaks between peaks a (a') and b (b').
FIG. 2. Molecular forms of ghrelin in the stomachs of 3- and 4-wk-old mice. Peptide extracts from fresh stomachs were fractionated by C18 RP-HPLC, and the ghrelin immunoreactivity of the extract was measured by C-RIA (ghrelin C-LI). An assay tube contained equivalent quantities of peptide extract derived from 0.2 mg stomach tissue. , Ghrelin C-LI in each HPLC fraction. Arrows indicate the elution of synthetic des-acyl ghrelin (I), n-octanoyl ghrelin (II), and n-decanoyl ghrelin (III). Based on the retention times of synthetic ghrelin peptides, peaks a and a' correspond to des-acyl ghrelin, peaks b and b' corresponded to n-octanoyl ghrelin, and peaks c and c' correspond to n-decanoyl ghrelin.
Concentrations of des-acyl, n-octanoyl, and n-decanoyl ghrelin in stomachs of growing mice
To confirm in detail the change in stomach concentrations of des-acyl and acyl-modified ghrelins between 3- and 4-wk-old mice and to check the influence of diet composition on the stomach concentrations of ghrelin molecules, the levels of des-acyl and acyl-modified ghrelins in 3- and 4-wk-old mice under NW and PW conditions (n = 8 each) were measured and statistically analyzed after HPLC fractionation (Fig. 3). Compared with 3-wk-old mice, the amounts of n-octanoyl and n-decanoyl ghrelin were significantly decreased in 4-wk-old mice (Fig. 3, B and C). No significant change was observed in the stomach concentration of des-acyl ghrelin between 3- and 4-wk-old mice (Fig. 3A). Compared with NW mice, the amounts of n-octanoyl and n-decanoyl ghrelin in stomachs of PW mice were significantly decreased (Fig. 3, B and C). No significant change was observed in the stomach concentration of des-acyl ghrelin between NW and PW mice (Fig. 3A). The average body weights of the PW mice just before (d 18) and after (d 21) the treatment were 7.6 ± 0.6 and 9.1 ± 0.5 g (n = 8), respectively. The average body weights of NW mice on d 18 and 21 were 7.6 ± 0.6 and 8.2 ± 0.3 g (n = 8), respectively. A significant increase in body weight on d 21 was observed in PW mice compared with NW mice (P < 0.01).
FIG. 3. Concentrations of des-acyl, n-octanoyl, and n-decanoyl ghrelin peptides in stomachs of 3-wk-old NW and PW mice and 4-wk-old NW mice. The stomach amounts of des-acyl, n-octanoyl, and n-decanoyl ghrelin-LI were measured by ghrelin C-RIA after HPLC fractionation. A, Des-acyl ghrelin; B, n-octanoyl ghrelin; C, n-decanoyl ghrelin concentrations in murine stomach. Each bar represents mean ± SD of samples obtained by HPLC fractionation (n = 8). *, P < 0.05; **, P < 0.01 (vs. the indicated values).
Plasma and stomach concentrations of n-octanoyl and total ghrelin from normally weaned and prematurely weaned rats
To clarify whether the proportion of n-octanoyl ghrelin in the circulation is also influenced by diet composition, plasma concentrations of n-octanoyl and total ghrelin from 3-wk-old NW and PW rats were measured by ghrelin N- and C-RIAs. Stomach concentrations of n-octanoyl and total ghrelin were measured in the same rats. As shown in Fig. 4A, the plasma n-octanoyl ghrelin levels were significantly lower in PW rats compared with NW rats. The plasma total ghrelin concentration was increased slightly, but not significantly, in PW compared with NW rats (Fig. 4B). The amount of n-octanoyl ghrelin in the stomachs of PW rats was lower than that in NW rats (Fig. 4D). However, the level of total ghrelin in the stomachs of PW rats was significantly higher than that in NW rats (Fig. 4E). Consequently, the proportions of n-octanoyl ghrelin to total ghrelin in both the circulation and the stomach were decreased in prematurely weaned rats (Fig. 4, C and F). The average body weights of the PW rats just before (d 18) and after (d 21) the treatment were 41.3 ± 2.8 and 54.9 ± 3.3 g (n = 8), respectively. The average body weights of the NW rats on d 18 and 21 were 41.7 ± 2.8 and 55.6 ± 2.6 g (n = 8), respectively.
FIG. 4. Concentrations of plasma and stomach ghrelin in NW and PW rats. A, Concentrations of plasma n-octanoyl ghrelin measured by ghrelin N-RIA (ghrelin N-LI). B, Plasma total ghrelin concentrations measured by ghrelin C-RIA (ghrelin C-LI). C, The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) in plasma calculated from plasma ghrelin N- and C-LI. D, Concentrations of stomach n-octanoyl ghrelin measured by ghrelin N-RIA (ghrelin N-LI). E, Concentrations of stomach total ghrelin measured by ghrelin C-RIA (ghrelin C-LI). F, The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) in stomach calculated from stomach ghrelin N- and C-LI. Data represent the mean ± SD of samples (n = 8). *, P < 0.05; **, P < 0.01 (vs. the indicated values).
Stomach concentrations of n-octanoyl and total ghrelin after glyceryl trioctanoate ingestion
To examine whether the ingestion of MCTs, which are found in high levels in milk lipids, influences the acyl modification of ghrelin, the stomach concentrations of n-octanoyl and total ghrelin were measured by N- and C-RIAs after feeding chow mixed with 5% glyceryl trioctanoate (C8:0-MCT) to 3-wk-old mice for 1 wk (from 3–4 wk after birth; n = 8). Compared with control mice fed a standard laboratory chow, a significant increase in the stomach concentration of n-octanoyl ghrelin was observed (Fig. 5). The proportion of n-octanoyl ghrelin to total ghrelin was also increased, because there was a slight, but not significant, decline in the level of total ghrelin in the stomachs of these mice. There was no significant difference between the body weights of C8:0-MCT-fed (19.1 ± 2.4 g) and control (standard CE-2 laboratory chow-fed; 19.6 ± 2.0 g) mice on the day of ghrelin RIA.
FIG. 5. Stomach concentrations of n-octanoyl and total ghrelin in 4-wk-old mice that ingested glyceryl trioctanoate (C8:0-MCT) for 1 wk (from 3–4 wk of age). A, n-Octanoyl ghrelin concentrations measured by ghrelin N-RIA (n = 8). B, Total ghrelin concentrations measured by ghrelin C-RIA (n = 8). C, The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) calculated from the ghrelin N- and C-LI (n = 8). *, P < 0.01 (vs. the indicated values).
Discussion
In this study we demonstrated that the level of n-octanoyl ghrelin in mouse stomach increased gradually throughout the suckling period and then decreased sharply after weaning. In contrast, the total ghrelin concentration in mouse stomach, measured by ghrelin C-RIA, did not show a significant change during the suckling-weaning transition. Consequently, the proportion of n-octanoyl ghrelin to total ghrelin exhibited a significant decrease after weaning.
To date, four reports have been published about developmental changes in stomach ghrelin production (14, 22, 23, 24). It has been difficult, however, to compare these previous findings with our current data, because the methods used to calculate and analyze stomach ghrelin concentrations are different from those we employed, and none of these previous reports recorded the change in the ratio of acyl-modified ghrelin to total ghrelin in the stomach.
Because it was difficult to obtain large plasma samples from mice, we used Wistar rats to measure changes in the level of circulating n-octanoyl ghrelin during the suckling-weaning transition. Stomach n-octanoyl ghrelin levels were found to decline at a similar rate in PW rats and PW mice. We observed a low ratio of plasma n-octanoyl ghrelin to total ghrelin in 3-wk-old PW rats compared with NW rats of the same age. Our findings largely corroborate those of Raff et al. (24), who reported recently that the plasma concentration of active, n-octanoyl-modified ghrelin after the weaning period, at 35 d of age, was significantly lower than that seen at 7 d, during the suckling period (7 d of age) in rats. However, in our study we also observed a slight increase in the amount of plasma total ghrelin together with a significant increase in the stomach total ghrelin concentration in PW rats. Because the circulating ghrelin level is determined by a delicate balance among the secretion rate (mainly from stomach), the degradation rate (des-acylation by plasma esterase and/or degradation of peptide by plasma proteases), and the clearance rate (capture by ghrelin receptor or urinary clearance) of this acyl-modified peptide, it is unlikely that the concentration of n-octanoyl or total ghrelin in the circulation simply reflects the stomach concentration of each form of ghrelin under all metabolic conditions.
In addition to its functions within the endocrine system, ghrelin exerts biological effects using autocrine, paracrine (25, 26), and/or neuroendocrine (27, 28, 29, 30) pathways. Therefore, it is possible that the change in the level of n-octanoyl ghrelin in murine stomach during the suckling-weaning transition could act through pathways independent of circulating ghrelin.
Despite a significant decrease in the proportion of stomach n-octanoyl ghrelin, 21-d-old PW mice exhibited a significantly larger increase in body weight compared with NW mice. This result is counterintuitive in light of ghrelin’s close association with adiposity (8, 31). However, the amount of stomach n-octanoylated ghrelin measured in the PW mice may not truly reflect the physiological ghrelin balance in these animals. First, the rapid development that occurs after birth and the concomitant negative energy balance may accelerate the secretion of n-octanoyl ghrelin from the stomach to the circulation, thus aberrantly lowering its stomach levels even though the production of stomach ghrelin (including both acylated and nonacylated forms) remains high. Furthermore, plasma esterases quickly deacylate ghrelin peptides upon their entry into the circulation (32), thus lowering the plasma concentration of acylated ghrelin. In these ways, rapid turnover of acylated ghrelin could mask an increase in its production, giving rise to the unexpected correlation of decreased stomach acylated ghrelin and increased body weight in PW mice. This hypothesis is partly supported by our data for stomach and plasma concentrations of n-octanoyl and total ghrelin in PW rats, although there was no significant difference between the average body weights of the PW and NW rats a short time (3 d) after treatment.
Throughout the suckling period, over 60% of a rodent’s energy is derived from milk lipids, primarily triglycerides (33, 34). After weaning, there is a progressive rise in the proportion of energy supplied by carbohydrates (35). During the suckling-weaning transition, energy metabolism as well as the secretion and production of ghrelin are dramatically altered under the influence of various components of the diet. Lee et al. (14) demonstrated that in rats, the rate of ghrelin’s production, as indicated by the stomach ghrelin mRNA level, as well as its secretion, as indicated by the plasma ghrelin level, are both increased under low protein and high carbohydrate diets. In contrast, the rates of production and secretion of ghrelin were both decreased by high fat diets. These diet-related changes in ghrelin production and secretion rates might influence the proportion of acyl-modified ghrelin in murine stomach.
During the suckling-weaning transition, the production and secretion rates of hormones that influence energy metabolism are also dramatically changed in conjunction with the change in energy source. The concentration of serum T4 gradually rises after birth to maximal levels just before the initiation of weaning and remains high until the fourth week of life (36). The concentration of plasma glucagon declines (37, 38), and plasma insulin (39) and corticosterone levels (40) rise after the initiation of weaning. These changes in hormone secretion together with the secretion of ghrelin, in turn, influence the activity of energy-metabolizing enzymes in liver and peripheral tissues (35).
Our finding that the stomach concentration of n-octanoyl ghrelin rises significantly after the ingestion of glyceryl trioctanoate is interesting, because there is a high proportion of MCTs and medium-chain fatty acids (MCFAs) in the milk lipids of rodents (41, 42). A significant decrease in the stomach levels of n-octanoyl and n-decanoyl ghrelin after the initiation of weaning (4 wk of age) implies that MCTs and MCFAs in milk lipids may be sources for acyl groups that can attach to the Ser3 residue of ghrelin during the suckling period.
Orally ingested MCFAs and those hydrolyzed from ingested MCTs are directly absorbed from gastric and intestinal mucosa (43), where ghrelin-producing cells are found. Therefore, it appears possible that the orally ingested MCFAs and those hydrolyzed from ingested MCTs can diffuse into the ghrelin-producing cells and serve as a nutrient signal by interfering with the acyl modification of ghrelin. It is also possible that endogenously synthesized MCFAs and their derivatives that are produced upon lipid metabolism are transferred to the ghrelin-producing cells and used in the acyl modification of ghrelin.
Additional study is required to elucidate in detail the relationship between the acyl modification of ghrelin and energy metabolism. It also remains to be determined whether ingested MCTs directly modulate the Ser3 residue of ghrelin peptide or indirectly stimulate the acyl modification of ghrelin through the induction of an acyl-modifying enzyme. Our new findings described here provide important clues to enhance our understanding of the regulation of ghrelin acyl modification during the development of the murine stomach.
Acknowledgments
We thank Prof. H. Nawata and Dr. T. Yanase for reading and reviewing the draft of our manuscript, K. Shirouzu and Y. Yamashita for their technical assistance, and Drs. H. Hosoda and H. Kaiya for providing us antibodies and tracers for ghrelin RIA.
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Address all correspondence and requests for reprints to: Dr. Masayasu Kojima, Molecular Genetics, Institute of Life Science, Kurume University, Kurume, Fukuoka 839-0861, Japan. E-mail: mkojima@lsi.kurume-u.ac.jp.
Abstract
Ghrelin is an acylated peptide hormone secreted primarily from endocrine cells in the stomach. The major active form of ghrelin is a 28-amino acid peptide with an n-octanoyl modification at Ser3 (n-octanoyl ghrelin), which is essential for its activity. In addition to n-octanoyl ghrelin, other forms of ghrelin peptide exist, including des-acyl ghrelin, which lacks an acyl modification, and other minor acylated ghrelin species, such as n-decanoyl ghrelin, whose Ser3 residue is modified by n-decanoic acid. Multiple reports have identified various physiological functions of ghrelin. However, until now, there have been no reports that explore the process of ghrelin acyl modification, and only a few studies have compared the levels of des-acyl, n-octanoyl, and/or other minor populations of acylated ghrelin peptides. In this study we report that the amount of n-octanoyl ghrelin in murine stomachs increases gradually during the suckling period to a maximal level at 3 wk of age and falls sharply after the initiation of weaning. However, the concentration (picomoles per milligram of wet weight tissue) of total ghrelin, which includes des-acyl and all acylated forms of ghrelin peptides with intact C termini in murine stomach, remains unchanged across this suckling-weaning transition. Prematurely weaned mice exhibited a significant decrease in the amount of n-octanoyl or n-decanoyl ghrelin in the stomach. Orally ingested glyceryl trioctanoate, a medium-chain triacylglyceride rich in milk lipids, significantly increased the level of n-octanoyl-modified ghrelin in murine stomach. Fluctuations in the proportion of this biologically active, acyl-modified ghrelin could contribute to or be influenced by the change in energy metabolism during the suckling-weaning transition.
Introduction
GHRELIN IS AN endogenous ligand for the GH secretagogue (GHS) receptor (GHS-R) (1, 2). GHSs are synthetic substances with potent GH-releasing activity (3, 4, 5). Although ghrelin was initially purified from the stomach, it is also expressed in the brain, pancreas, small intestine, and other tissues (6). In addition to its potent GH-releasing activity (1), ghrelin stimulates appetite and influences adiposity (7, 8). The third amino acid residue of ghrelin, a serine (Ser3), is modified by an acyl group; this modification is essential for ghrelin’s biological activity through the classical ghrelin receptor, GHS-R1a (1, 9). Although the primary acyl chain modifying ghrelin molecules in humans and rodents is an n-octanoyl group (C8:0, an eight-carbon chain containing no double bonds) (1, 10), different acyl groups modify a minor population of ghrelin peptides. These acyl groups include n-decanoyl (C10:0, a 10-carbon chain lacking double bonds) and n-decenoyl (C10:1, a 10-carbon chain containing one double bond) (11, 12, 13). To our knowledge, the acyl modification of ghrelin is the first known example of a fatty acid modification of a peptide hormone.
A number of reports have been published on the regulation of ghrelin secretion in the context of energy metabolism that did not distinguish between acyl-modified and des-acyl ghrelin (14, 15, 16, 17, 18). However, there have been only a few reports comparing the levels of n-octanoyl, des-acyl, and/or total ghrelin, the last of which encompasses des-acyl and all acyl-modified ghrelin peptides with intact C termini (19, 20).
In this study we measured the amount of n-octanoyl and total ghrelin as well as the minor population of n-decanoyl ghrelin in murine stomach throughout the suckling and weaning periods during which energy metabolism is dramatically altered. Furthermore, we investigated the effect of dietary medium-chain triacylglycerides (MCTs) on the stomach levels of n-octanoyl and total ghrelin.
Materials and Methods
Animals
Male C57BL/6J mice and male Wistar rats were used in this experiment. They were maintained under controlled temperature (21–23 C) and light conditions (lights on, 0700–1900 h) with ad libitum access to standard laboratory chow (CE-2, CLEA Co. Ltd., Osaka, Japan) and water. All mice were killed by decapitation, and all rats were killed by exsanguination under anesthesia with an ip injection (30 mg/kg body weight) of sodium pentobarbital (Nembutal injection, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan). All experiments were conducted in accordance with the Kurume University Guide for the Care and Use of Experimental Animals.
RIA for ghrelin
RIAs specific for ghrelin were performed as previously described (6). In brief, two polyclonal antibodies were raised in rabbits against the N terminal (Gly1-Lys11 with O-n-octanoylation at Ser3) or C-terminal (Gln13-Arg28) fragments of rat ghrelin. Both antisera exhibited complete cross-reactivity with human, mouse, and rat ghrelins. The antirat ghrelin-(1–11) antiserum, which specifically recognized the Ser3 n-octanoylated form of ghrelin, does not recognize des-acyl ghrelin, but cross-reacts faintly with other acyl-modified ghrelins. Antirat ghrelin-(13–28) antiserum equally recognizes both des-acyl and all acylated forms of ghrelin with intact C-terminal peptide sequences. In the following sections, the RIA system using the antiserum against the N-terminal fragment of rat ghrelin-(1–11) is termed N-RIA, whereas that specific for the C-terminus [ghrelin-(13–28)] is termed C-RIA. Ghrelin-like immunoreactivity (ghrelin-LI) measured by N-RIA is termed ghrelin N-LI or n-octanoyl ghrelin-LI, and ghrelin-LI measured by C-RIA, which reflects the amount of ghrelin peptides with intact C-termini (regardless of the acyl-modification of Ser3) is termed total ghrelin or ghrelin C-LI.
Preparation of stomach samples for ghrelin assay
Male C57BL/6J mice (birth to 12 wk of age) were maintained under controlled temperature (21–23 C) and light conditions (lights on, 0700–1900 h) with ad libitum access to a nursing mother, chow, and water. Stomachs collected from mice were washed twice in PBS (pH 7.4). After measuring the wet weights of each sample, the whole stomach tissue was boiled for 5 min in a 10-fold volume of water to inactivate intrinsic proteases. After cooling on ice, boiled samples were adjusted to 1 M acetic acid-20 mM HCl. Peptides were extracted after homogenization with a Polytron mixer (PT 6100, Kinematica AG, Littan-Luzern, Switzerland). Extract supernatants, isolated after a 15-min centrifugation at 15,000 rpm (12,000 x g), were lyophilized and stored at –80 C. The lyophilized samples were dissolved in RIA buffer before ghrelin RIA.
Preparation of plasma samples for ghrelin assay
Plasma samples from rats were prepared as previously described (1, 6). In brief, truncal blood samples obtained by heart puncture were immediately transferred to chilled polypropylene tubes containing EDTA-2Na (1 mg/ml) and aprotinin (1000 kallikrein inactivator units/ml) and centrifuged at 4 C. Immediately after the plasma was separated, HCl was added to the sample to a final concentration of 0.1 N and then diluted with an equal volume of saline. The sample was loaded onto a Sep-Pak C18 cartridge (Waters Corp., Milford, MA) preequilibrated with 0.1% trifluoroacetic acid (TFA) and 0.9% NaCl. The cartridge was washed with 0.9% NaCl and 5% acetonitrile (CH3CN)/0.1% TFA, then eluted with 60% CH3CN/0.1% TFA. The eluate was lyophilized and stored at –80 C. Lyophilized samples were dissolved in RIA buffer before ghrelin RIA.
Analysis of molecular forms of acyl-modified ghrelin in murine stomach
To investigate changes in the pattern of ghrelin’s acyl modification and the levels of des-acyl or acyl-modified ghrelins around the time of initiation of weaning, the molecular properties of stomach ghrelin were analyzed by C18 reverse phase (RP) HPLC (3.9 x 150 mm; Symmetry 300, Waters Corp.). Stomach peptides were extracted from 3- or 4-wk-old male mice under normal weaning (NW) conditions or from prematurely weaned (PW) 3-wk-old male mice. Stomach peptides were also extracted from 3-wk-old male rats under NW or PW conditions. PW was performed in manner described previously by Hahn et al. (21). In brief, eight pups, regardless of sex, were retained from a litter of Wistar rats or C57BL/6J mice on d 2 after birth, weaned to standard laboratory chaw on d 18, and killed on d 21. The stomach or plasma samples from male-only litters were collected and subjected to peptide extraction. Standard laboratory chow (CE-2) derives its caloric content as follows: carbohydrate, 50.3%; protein, 25.4%; and fat, 4.4%. Peptide extracts from stomachs were subjected to RP-HPLC using a linear gradient of 10–60% CH3CN/0.1% TFA at a flow rate of 1.0 ml/min. Five hundred-microliter fractions were collected. The ghrelin peptide content in each fraction was measured by ghrelin C-RIA. The retention times obtained for the extracted ghrelin molecules were compared with those of synthetic des-acyl, n-octanoyl, and n-decanoyl ghrelin.
Concentrations of n-octanoyl and total ghrelin in murine stomach after glyceryl trioctanoate ingestion
Three-week-old male C57BL/6J mice were maintained under controlled temperature (21–23 C) and light (lights on, 0700–1900 h) conditions with ad libitum access to food, water, and their nursing mothers. Glyceryl trioctanoate (Wako Pure Chemical, Osaka, Japan), a MCT rich in rodent milk lipids, was mixed into standard laboratory chow at a concentration of 5% (wt/wt). After 1 wk, whole stomach samples from MCT-fed and control (4-wk-old) mice were collected and assayed for ghrelin N- or C-RIAs as described above.
Statistical analysis
Data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA, followed by a post hoc test (Scheffé’s F test). P <0.05 was considered statistically significant.
Results
Concentration of ghrelin-like immunoreactivity in murine stomachs
To elucidate the regulation of ghrelin acyl modification during the developmental stages after birth, the levels of n-octanoyl and total ghrelin immunoreactivity in stomachs of mice from birth to 12 wk of age were measured using ghrelin N- and C-RIAs. The level of n-octanoyl ghrelin-LI in the stomach increased from birth to 3 wk of age and then fell significantly between 3 and 4 wk after birth (Fig. 1A). After mice reached 4 wk of age, the level of stomach n-octanoyl ghrelin remained unchanged. The amount of total ghrelin-LI also increased from birth to 3 wk of age and subsequently remained unchanged from 3–12 wk of age. No significant change in total ghrelin-LI was noted between 3 and 4 wk of age. The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) in stomachs of mice increased throughout the suckling period (from 1–3 wk of age). Thereafter, the N/C ratio fell sharply between 3 and 4 wk of age in proportion to the decline in the amount of n-octanoyl ghrelin-LI. After 4 wk of age, no significant change in the N/C ratio was detected in stomachs of mice until 12 wk of age. Patterns similar to those described above were observed in data from two subsequent experiments using the same protocol.
FIG. 1. Amount of ghrelin-LI in the stomachs of mice from birth to 12 wk of age. Each bar represents means ± SD (n = 6–12/group). A, n-Octanoyl-modified ghrelin-LI concentrations measured by ghrelin N-RIA. B, Total ghrelin-LI (des-acyl and acyl-modified forms of ghrelin with intact C terminus) concentration measured by ghrelin C-RIA. C, The ratio of n-octanoyl/total ghrelin-LI in murine stomach (N/C ratio). Statistical significance is indicated by superscript letters. a, P < 0.001; b, P < 0.05; c, P < 0.01 (vs. the indicated values).
Molecular forms of ghrelin in stomachs from 3- and 4-wk-old mice
To examine whether there was a change in the molecular form of stomach ghrelin peptides before and after the suckling-weaning transition, we fractionated stomach samples from 3- and 4-wk-old mice by RP-HPLC and measured ghrelin levels by C-RIA (Fig. 2). Based on the observed retention times of synthetic ghrelin peptides, peaks a and a' correspond to des-acyl ghrelin, peaks b and b' correspond to n-octanoyl ghrelin modified by an n-octanoyl (C8:0) group, and peaks c and c' corresponded to n-decanoyl ghrelin modified by an n-decanoyl (C10:0) group. Compared with 3-wk-old mice, a decrease in the amount of n-decanoyl ghrelin (Fig. 2, upper panel; peak c: retention time, 24–25 min) was observed in 4-wk-old mice. The level of n-octanoyl ghrelin (peak b': retention time, 20.5–21.5 min) was also decreased in 4-wk-old mice compared with 3-wk-old mice (peak b). In contrast, the level of des-acyl ghrelin (peak a': retention time, 10.5–11.5) in 4-wk-old mouse stomachs was higher than that in 3-wk-old mice (peak a). There was an unidentified peak between peaks b (b') and c (c'), and there were at least two peaks between peaks a (a') and b (b').
FIG. 2. Molecular forms of ghrelin in the stomachs of 3- and 4-wk-old mice. Peptide extracts from fresh stomachs were fractionated by C18 RP-HPLC, and the ghrelin immunoreactivity of the extract was measured by C-RIA (ghrelin C-LI). An assay tube contained equivalent quantities of peptide extract derived from 0.2 mg stomach tissue. , Ghrelin C-LI in each HPLC fraction. Arrows indicate the elution of synthetic des-acyl ghrelin (I), n-octanoyl ghrelin (II), and n-decanoyl ghrelin (III). Based on the retention times of synthetic ghrelin peptides, peaks a and a' correspond to des-acyl ghrelin, peaks b and b' corresponded to n-octanoyl ghrelin, and peaks c and c' correspond to n-decanoyl ghrelin.
Concentrations of des-acyl, n-octanoyl, and n-decanoyl ghrelin in stomachs of growing mice
To confirm in detail the change in stomach concentrations of des-acyl and acyl-modified ghrelins between 3- and 4-wk-old mice and to check the influence of diet composition on the stomach concentrations of ghrelin molecules, the levels of des-acyl and acyl-modified ghrelins in 3- and 4-wk-old mice under NW and PW conditions (n = 8 each) were measured and statistically analyzed after HPLC fractionation (Fig. 3). Compared with 3-wk-old mice, the amounts of n-octanoyl and n-decanoyl ghrelin were significantly decreased in 4-wk-old mice (Fig. 3, B and C). No significant change was observed in the stomach concentration of des-acyl ghrelin between 3- and 4-wk-old mice (Fig. 3A). Compared with NW mice, the amounts of n-octanoyl and n-decanoyl ghrelin in stomachs of PW mice were significantly decreased (Fig. 3, B and C). No significant change was observed in the stomach concentration of des-acyl ghrelin between NW and PW mice (Fig. 3A). The average body weights of the PW mice just before (d 18) and after (d 21) the treatment were 7.6 ± 0.6 and 9.1 ± 0.5 g (n = 8), respectively. The average body weights of NW mice on d 18 and 21 were 7.6 ± 0.6 and 8.2 ± 0.3 g (n = 8), respectively. A significant increase in body weight on d 21 was observed in PW mice compared with NW mice (P < 0.01).
FIG. 3. Concentrations of des-acyl, n-octanoyl, and n-decanoyl ghrelin peptides in stomachs of 3-wk-old NW and PW mice and 4-wk-old NW mice. The stomach amounts of des-acyl, n-octanoyl, and n-decanoyl ghrelin-LI were measured by ghrelin C-RIA after HPLC fractionation. A, Des-acyl ghrelin; B, n-octanoyl ghrelin; C, n-decanoyl ghrelin concentrations in murine stomach. Each bar represents mean ± SD of samples obtained by HPLC fractionation (n = 8). *, P < 0.05; **, P < 0.01 (vs. the indicated values).
Plasma and stomach concentrations of n-octanoyl and total ghrelin from normally weaned and prematurely weaned rats
To clarify whether the proportion of n-octanoyl ghrelin in the circulation is also influenced by diet composition, plasma concentrations of n-octanoyl and total ghrelin from 3-wk-old NW and PW rats were measured by ghrelin N- and C-RIAs. Stomach concentrations of n-octanoyl and total ghrelin were measured in the same rats. As shown in Fig. 4A, the plasma n-octanoyl ghrelin levels were significantly lower in PW rats compared with NW rats. The plasma total ghrelin concentration was increased slightly, but not significantly, in PW compared with NW rats (Fig. 4B). The amount of n-octanoyl ghrelin in the stomachs of PW rats was lower than that in NW rats (Fig. 4D). However, the level of total ghrelin in the stomachs of PW rats was significantly higher than that in NW rats (Fig. 4E). Consequently, the proportions of n-octanoyl ghrelin to total ghrelin in both the circulation and the stomach were decreased in prematurely weaned rats (Fig. 4, C and F). The average body weights of the PW rats just before (d 18) and after (d 21) the treatment were 41.3 ± 2.8 and 54.9 ± 3.3 g (n = 8), respectively. The average body weights of the NW rats on d 18 and 21 were 41.7 ± 2.8 and 55.6 ± 2.6 g (n = 8), respectively.
FIG. 4. Concentrations of plasma and stomach ghrelin in NW and PW rats. A, Concentrations of plasma n-octanoyl ghrelin measured by ghrelin N-RIA (ghrelin N-LI). B, Plasma total ghrelin concentrations measured by ghrelin C-RIA (ghrelin C-LI). C, The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) in plasma calculated from plasma ghrelin N- and C-LI. D, Concentrations of stomach n-octanoyl ghrelin measured by ghrelin N-RIA (ghrelin N-LI). E, Concentrations of stomach total ghrelin measured by ghrelin C-RIA (ghrelin C-LI). F, The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) in stomach calculated from stomach ghrelin N- and C-LI. Data represent the mean ± SD of samples (n = 8). *, P < 0.05; **, P < 0.01 (vs. the indicated values).
Stomach concentrations of n-octanoyl and total ghrelin after glyceryl trioctanoate ingestion
To examine whether the ingestion of MCTs, which are found in high levels in milk lipids, influences the acyl modification of ghrelin, the stomach concentrations of n-octanoyl and total ghrelin were measured by N- and C-RIAs after feeding chow mixed with 5% glyceryl trioctanoate (C8:0-MCT) to 3-wk-old mice for 1 wk (from 3–4 wk after birth; n = 8). Compared with control mice fed a standard laboratory chow, a significant increase in the stomach concentration of n-octanoyl ghrelin was observed (Fig. 5). The proportion of n-octanoyl ghrelin to total ghrelin was also increased, because there was a slight, but not significant, decline in the level of total ghrelin in the stomachs of these mice. There was no significant difference between the body weights of C8:0-MCT-fed (19.1 ± 2.4 g) and control (standard CE-2 laboratory chow-fed; 19.6 ± 2.0 g) mice on the day of ghrelin RIA.
FIG. 5. Stomach concentrations of n-octanoyl and total ghrelin in 4-wk-old mice that ingested glyceryl trioctanoate (C8:0-MCT) for 1 wk (from 3–4 wk of age). A, n-Octanoyl ghrelin concentrations measured by ghrelin N-RIA (n = 8). B, Total ghrelin concentrations measured by ghrelin C-RIA (n = 8). C, The ratio of n-octanoyl ghrelin-LI to total ghrelin-LI (N/C-ratio) calculated from the ghrelin N- and C-LI (n = 8). *, P < 0.01 (vs. the indicated values).
Discussion
In this study we demonstrated that the level of n-octanoyl ghrelin in mouse stomach increased gradually throughout the suckling period and then decreased sharply after weaning. In contrast, the total ghrelin concentration in mouse stomach, measured by ghrelin C-RIA, did not show a significant change during the suckling-weaning transition. Consequently, the proportion of n-octanoyl ghrelin to total ghrelin exhibited a significant decrease after weaning.
To date, four reports have been published about developmental changes in stomach ghrelin production (14, 22, 23, 24). It has been difficult, however, to compare these previous findings with our current data, because the methods used to calculate and analyze stomach ghrelin concentrations are different from those we employed, and none of these previous reports recorded the change in the ratio of acyl-modified ghrelin to total ghrelin in the stomach.
Because it was difficult to obtain large plasma samples from mice, we used Wistar rats to measure changes in the level of circulating n-octanoyl ghrelin during the suckling-weaning transition. Stomach n-octanoyl ghrelin levels were found to decline at a similar rate in PW rats and PW mice. We observed a low ratio of plasma n-octanoyl ghrelin to total ghrelin in 3-wk-old PW rats compared with NW rats of the same age. Our findings largely corroborate those of Raff et al. (24), who reported recently that the plasma concentration of active, n-octanoyl-modified ghrelin after the weaning period, at 35 d of age, was significantly lower than that seen at 7 d, during the suckling period (7 d of age) in rats. However, in our study we also observed a slight increase in the amount of plasma total ghrelin together with a significant increase in the stomach total ghrelin concentration in PW rats. Because the circulating ghrelin level is determined by a delicate balance among the secretion rate (mainly from stomach), the degradation rate (des-acylation by plasma esterase and/or degradation of peptide by plasma proteases), and the clearance rate (capture by ghrelin receptor or urinary clearance) of this acyl-modified peptide, it is unlikely that the concentration of n-octanoyl or total ghrelin in the circulation simply reflects the stomach concentration of each form of ghrelin under all metabolic conditions.
In addition to its functions within the endocrine system, ghrelin exerts biological effects using autocrine, paracrine (25, 26), and/or neuroendocrine (27, 28, 29, 30) pathways. Therefore, it is possible that the change in the level of n-octanoyl ghrelin in murine stomach during the suckling-weaning transition could act through pathways independent of circulating ghrelin.
Despite a significant decrease in the proportion of stomach n-octanoyl ghrelin, 21-d-old PW mice exhibited a significantly larger increase in body weight compared with NW mice. This result is counterintuitive in light of ghrelin’s close association with adiposity (8, 31). However, the amount of stomach n-octanoylated ghrelin measured in the PW mice may not truly reflect the physiological ghrelin balance in these animals. First, the rapid development that occurs after birth and the concomitant negative energy balance may accelerate the secretion of n-octanoyl ghrelin from the stomach to the circulation, thus aberrantly lowering its stomach levels even though the production of stomach ghrelin (including both acylated and nonacylated forms) remains high. Furthermore, plasma esterases quickly deacylate ghrelin peptides upon their entry into the circulation (32), thus lowering the plasma concentration of acylated ghrelin. In these ways, rapid turnover of acylated ghrelin could mask an increase in its production, giving rise to the unexpected correlation of decreased stomach acylated ghrelin and increased body weight in PW mice. This hypothesis is partly supported by our data for stomach and plasma concentrations of n-octanoyl and total ghrelin in PW rats, although there was no significant difference between the average body weights of the PW and NW rats a short time (3 d) after treatment.
Throughout the suckling period, over 60% of a rodent’s energy is derived from milk lipids, primarily triglycerides (33, 34). After weaning, there is a progressive rise in the proportion of energy supplied by carbohydrates (35). During the suckling-weaning transition, energy metabolism as well as the secretion and production of ghrelin are dramatically altered under the influence of various components of the diet. Lee et al. (14) demonstrated that in rats, the rate of ghrelin’s production, as indicated by the stomach ghrelin mRNA level, as well as its secretion, as indicated by the plasma ghrelin level, are both increased under low protein and high carbohydrate diets. In contrast, the rates of production and secretion of ghrelin were both decreased by high fat diets. These diet-related changes in ghrelin production and secretion rates might influence the proportion of acyl-modified ghrelin in murine stomach.
During the suckling-weaning transition, the production and secretion rates of hormones that influence energy metabolism are also dramatically changed in conjunction with the change in energy source. The concentration of serum T4 gradually rises after birth to maximal levels just before the initiation of weaning and remains high until the fourth week of life (36). The concentration of plasma glucagon declines (37, 38), and plasma insulin (39) and corticosterone levels (40) rise after the initiation of weaning. These changes in hormone secretion together with the secretion of ghrelin, in turn, influence the activity of energy-metabolizing enzymes in liver and peripheral tissues (35).
Our finding that the stomach concentration of n-octanoyl ghrelin rises significantly after the ingestion of glyceryl trioctanoate is interesting, because there is a high proportion of MCTs and medium-chain fatty acids (MCFAs) in the milk lipids of rodents (41, 42). A significant decrease in the stomach levels of n-octanoyl and n-decanoyl ghrelin after the initiation of weaning (4 wk of age) implies that MCTs and MCFAs in milk lipids may be sources for acyl groups that can attach to the Ser3 residue of ghrelin during the suckling period.
Orally ingested MCFAs and those hydrolyzed from ingested MCTs are directly absorbed from gastric and intestinal mucosa (43), where ghrelin-producing cells are found. Therefore, it appears possible that the orally ingested MCFAs and those hydrolyzed from ingested MCTs can diffuse into the ghrelin-producing cells and serve as a nutrient signal by interfering with the acyl modification of ghrelin. It is also possible that endogenously synthesized MCFAs and their derivatives that are produced upon lipid metabolism are transferred to the ghrelin-producing cells and used in the acyl modification of ghrelin.
Additional study is required to elucidate in detail the relationship between the acyl modification of ghrelin and energy metabolism. It also remains to be determined whether ingested MCTs directly modulate the Ser3 residue of ghrelin peptide or indirectly stimulate the acyl modification of ghrelin through the induction of an acyl-modifying enzyme. Our new findings described here provide important clues to enhance our understanding of the regulation of ghrelin acyl modification during the development of the murine stomach.
Acknowledgments
We thank Prof. H. Nawata and Dr. T. Yanase for reading and reviewing the draft of our manuscript, K. Shirouzu and Y. Yamashita for their technical assistance, and Drs. H. Hosoda and H. Kaiya for providing us antibodies and tracers for ghrelin RIA.
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