Maternal Ghrelin Plays an Important Role in Rat Fetal Development during Pregnancy
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《内分泌学杂志》
Department of Veterinary Physiology (K.N., M.Nakag., Y.B., M.S., N.M.), Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2155, Japan
Third Department of Internal Medicine (K.T., Y.D., M.Nakaz.), Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan
Molecular Genetics (M.K.), Institute of Life Science, Kurume University, Kurume 839-0864, Japan
National Cardiovascular Center Research Institute (M.M., H.K., H.H., K.K.), Osaka 565-8565, Japan
Abstract
Ghrelin, an acylated peptide serving as an endogenous ligand for GH secretagogue receptor (GHS-R), was originally isolated from rat and human stomach. In this study, we report the critical role of maternal ghrelin in fetal development. High levels of ghrelin receptor (GHS-R) mRNA were detected in various peripheral fetal tissues beginning at embryonic d 14 and lasting until birth. Fetal GHS-R expression was also confirmed in fetal tissues by immunohistochemistry. Autoradiography revealed that both des-acyl ghrelin and acyl ghrelin bind to fetal tissues. Chronic treatment of mothers with ghrelin resulted in a significant increase in birth weight in comparison to newborns from saline-treated mothers. Even when maternal food intake after ghrelin treatment was restricted through paired feeding, significant stimulation of fetal development still occurred. Conversely, active immunization of mothers against ghrelin decreased fetal body weight during pregnancy. A single ghrelin injection into the mother increased circulating ghrelin levels in the fetus within 5 min of injection, suggesting that maternal ghrelin transits easily to the fetal circulation. High levels of des-acyl ghrelin were detected in fetal blood and amniotic fluid. Both acylated and des-acyl ghrelin increased [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation of cultured fetal skin cells in a dose-dependent manner, and calcium-imaging analysis revealed that acyl and des-acyl ghrelin increased the Ca2+ influx in discrete cultured fetal skin cells, respectively. These results indicate that maternal ghrelin regulates fetal development during the late stages of pregnancy.
Introduction
GHRELIN, RECENTLY purified from rat and human stomachs as an endogenous ligand for the GH secretagogue receptor (GHS-R), is a 28-amino acid peptide with an n-octanoylation modification at Ser3 (1). This octanoylation is important for the stimulation of GH secretion from the pituitary gland (1). Although cells immunostained for ghrelin are distributed widely in the stomach, hypothalamus, pituitary gland, liver, kidney, pancreas, and placenta, the main source of circulating ghrelin is considered to be the gastrointestinal tract (2, 3, 4, 5, 6). Both acylated and des-acyl ghrelin are observed in the peripheral circulation, the levels of des-acyl ghrelin being higher (7, 8). Several studies on the physiological function of ghrelin have demonstrated that, in addition to stimulating GH secretion, ghrelin also stimulates food intake and body weight gain independent of GH secretion (8, 9, 10, 11, 12, 13). It is likely that the appetite-stimulating effect of peripheral ghrelin is due to action via the afferent vagal nerve (14). In contrast, the central effect is thought to be via neuropeptide Y and agouti-related peptide secretion from the arcuate nucleus in the hypothalamus (11, 15). Administration of ghrelin continuously to rodents resulted in fat deposition and obesity (9). These effects of ghrelin on appetite and fat deposition are counteracted by leptin (11). These results imply that ghrelin may play an important role in the regulation of food intake and energy expenditure.
The ghrelin receptor GHS-R, highly conserved from teleost fish to humans, is expressed widely in both central and peripheral organs, including the brain, pituitary gland, and pancreas (16, 17, 18, 19). The broad distribution of GHS-R suggests that ghrelin may play important roles in addition to those mentioned above. It has been also demonstrated that ghrelin might be involved in stomach motility (10), gastric acid secretion (20), insulin and gastrin release (21), the cardiovascular system, and stress reactions (12). In addition, we demonstrated previously that neonatal rats treated daily with ghrelin for 2 or 3 wk from birth showed faster eye and vaginal opening than those of saline-treated group (22), suggesting that ghrelin may be involved in neonatal development. Therefore, it has been assumed that ghrelin from the maternal stomach or placenta during pregnancy may play a role in fetal development. In the present study, we examined the possible involvement of maternal ghrelin in rat fetal development.
Materials and Methods
Animals
Wistar rats were housed under controlled temperature (23 ± 1 C) and regulated 12-h light 12-h dark conditions (lights on at 0700 h). Female rats were mated on the day of proestrus at approximately 3 months old. The next estrus day was considered to be d 0 of pregnancy. As reported previously, delivery usually occurs in our rat colony during the morning on d 21 of pregnancy (23). The average number (±SEM) of pups per mother at delivery was 13.10 ± 1.78 (n = 122). All procedures were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.
RT-PCR for GHS-R 1a mRNA
Total RNA was extracted from fetal tissues on d 14, 15, and 19 of pregnancy using Trizol reagent (Invitrogen, Carlsbad, CA) as described previously (24). First-strand cDNA was synthesized from 2 μg of total RNA by random primer RT. The resulting cDNA was subjected to PCR amplification using sense and antisense primers specific for GHS-R1a (24). PCR products were electrophoresed on a 2% agarose gel. GAPDH was used as a control housekeeping gene.
Autoradiography for [125I]acyl ghrelin
Fetuses [embryonic d 17 (E17)] were embedded in Tissue-Tec OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and frozen. Sections cut using a cryostat were mounted on gelatin-coated glass slides. Autoradiography was performed as described previously (14) with the following minor modifications. After preincubation for 30 min in incubation buffer at room temperature, sections were incubated for 12 h at 4 C in buffer containing 20 ng/ml rat [125I-Tyr29]acylated rat ghrelin. Nonspecific binding was determined in the presence of excess unlabeled acyl or des-acyl rat ghrelin (10 μg/ml). Sections were then exposed to an IP plate (Fuji Film, Tokyo, Japan) for 12 h and analyzed on BAS-5000 (Fuji Film).
Preparation of anti-GHS-R serum
The [Cys0]-rat GHS-R [342–364] peptide was synthesized using the Fmoc solid-phase method on a peptide synthesizer (433A; Applied Biosystems, Foster City, CA), then purified by reverse phase-HPLC. The synthesized peptide (10 mg) was conjugated to maleimide-activated mariculture keyhole limpet hemocyanin (6 mg) (mcKLH; Pierce, Rockford, IL) in conjugation buffer (Pierce). The conjugate was emulsified with an equal volume of Freund’s complete adjuvant and was used to immunize New Zealand white rabbits by intracutaneous and sc injection. Animals were boosted every 2 wk and bled 7 d after each injection. The specificity of the antisera was confirmed by the immunoreactivity of GHS-R-expressing cells (CHO-GHSR62 cells) and lack thereof in control cells.
Immunohistochemistry for GHS-R
Immunohistochemical analyses for GHS-R were performed on frozen fetuses (E17 and 19) using a modification of a method that has been described previously (25). The fetuses were placed in fixative for 5 d at 4 C and then transferred to 0.1 M phosphate buffer containing 20% sucrose. They were cut into serial, 12-μm-thick sections at –20 C with a cryostat. The sections were incubated for 2 d with a rabbit-anti-GHS-R antibody at 4 C. Slides were then incubated with Alexa-546-labeled goat-antirabbit IgG antibody (Molecular Probes, Inc., Eugene, OR; dilution 1:400). Samples were observed with the aid of an Olympus AX-70 fluorescence microscope (Olympus, Tokyo, Japan). To examine the specificity of GHS-R antibody in tissue sections, the reaction was also performed using GHS-R antibody that had been preabsorbed with excess synthetic GHS-R (10 μg).
Measurement of acyl and des-acyl ghrelin, IGF-I, and corticosterone
Levels of acyl or des-acyl ghrelin were measured by specific ELISA kits for acyl or des-acyl ghrelin (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan). The ELISA can detect each acyl or des-acyl ghrelin using two specific antibodies recognizing only acyl ghrelin (octanoylation modification at [Ser3]-ghrelin [1–11]) or only des-acyl ghrelin (nonoctanoylation modification at [Ser3]-ghrelin [1–11]). Blood collected from pregnant rats and their fetuses was immediately put into chilled polypropylene tubes containing a protease inhibitor, aprotinin (Sigma-Aldrich, St. Louis, MO), and 2Na-EDTA and then centrifuged. We then added a 10% plasma volume of 0.1 N HCl. Maternal blood was taken at 0830 h (satiety phase) at 2-d intervals from d 11–21. Fetal blood and amniotic fluid were collected on d 17, 19, and 21.
To examine the transit of maternal acyl ghrelin to the fetal circulation, acyl ghrelin (0.2 and 20 nmol) or saline was injected into pregnant rats iv under light ether anesthesia on d 19 of pregnancy (n = 12 per group). Blood was then collected from both the mother and fetus at 5, 10, and 30 min after injection.
To determine the effect of maternal treatment with acyl ghrelin on plasma IGF-I and corticosterone levels in the fetal circulation, fetal plasma IGF-I and corticosterone levels were measured by enzyme immunoassay kit (Funakoshi, Tokyo, Japan) and [125I]corticosterone RIA kit (ICN Biomedicals, Costa Mesa, CA), respectively. The limit of assay sensitivity was 5 ng/ml for IGF-I and 20 ng/ml for corticosterone. The intra and interassay coefficients of variation were 5 and 16%, respectively, for IGF-I, and 6 and 12%, respectively, for corticosterone.
Ghrelin administration and neonatal body weights
We sc injected either saline, acyl ghrelin (1.5 or 3.0 nmol), or des-acyl ghrelin (3.0 nmol) three times a day (at 0830, 1330, and 1830 h) from d 14 to delivery, or continuously infused vehicle, acyl ghrelin (0.125 or 0.5 nmol/h) or des-acyl ghrelin (0.5 nmol/h) through an osmotic mini-pump implanted sc from d 15 until delivery (n = 10 per group) (11, 26). We also injected 3 nmol acyl ghrelin three times a day from d 14 to delivery into pair-fed pregnant rats and the effect was compared with saline-treated pregnant rats. Neonatal body weights were measured on the day of delivery. If the pups numbered more than 15 or less than 11 they were excluded from the analyses.
Passive immunization for acyl ghrelin
Rat acyl ghrelin (3 mg) was conjugated to a carrier protein, mcKLH (3 mg), in conjugation buffer (Pierce) (7). Each conjugate was emulsified with an equal volume of Freund’s adjuvant. Immunization, initiated by intradermal injection in 44-d-old female rats, was repeated six times at 2-wk intervals. As a control antigen, carrier protein alone without ghrelin was administered. Rats were mated on d 114 after the fifth immunization. The antibody titers were verified in diluted plasma every 10 d after immunization using [125I]ghrelin binding capacity.
Quantitative RT-PCR of GH mRNA in fetal pituitary
The pituitary gland and blood were collected from E19 and E20 fetuses, isolated from the mothers’ implanted osmotic minipump (acyl ghrelin 0.5 nmol/h and saline). GH mRNA expression was measured by real-time quantitative PCR as described previously (25). Experiments contrasted the relative levels of both GH and GAPDH transcripts in every sample. The total RNA from each tissue was extracted using an RNeasy Micro kit (Qiagen, Valencia, CA) and synthesized into first-strand cDNA using an iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA). An aliquot of the first-strand cDNA (40–100 ng tissue equivalent) was quantified on an iCycler (Bio-Rad Laboratory) using iQ SYBR Green Supermix (Bio-Rad Laboratory) with primers to amplify GAPDH (25) and GH specifically (26).
Incorporation of [3H]thymidine or 5-bromo-2'-deoxyuridine (BrdU) into cultured cells
We assessed the effect of acyl and des-acyl ghrelin administration on the proliferation of fetal skin cells by measuring the incorporation of [3H]thymidine (2 μCi/ml) or BrdU (10 μM). Dispersed fetal skin cells were prepared from E17 fetuses by sequential collagenase treatment, papain digestion, and mechanical desegregation. Dispersed cells were then suspended in MCDB153HAA medium (F-Peptide Co., Ltd., Yamagata, Japan) containing 2% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and 5 ng/ml epidermal growth factor. Cells were seeded in polyethylenimine-coated 48- and 96-well dishes at densities of 5 x 105 per well and 3 x 104 per well for the [3H]thymidine and BrdU experiments, respectively. BrdU was detected using a Cell Proliferation ELISA Kit (Roche Diagnostic GmbH, Mannheim, Germany), as reported by Kusunoki et al. (27).
Statistics
Values are given as means ± SEM. Comparisons between two groups were made by ANOVA with the post hoc Fisher test. Differences at P < 0.05 were accepted as statistically significant.
Results
Expression of GHS-Rs in fetal tissue
GHS-R1a mRNA expression was detected in various fetal tissues with a high density in the spinal cord from E14 until birth (Fig. 1A). GHS-R mRNA expression in the fetal pituitary was also detected at E19. To confirm the expression of GHS-R in fetal tissues at the protein level, we performed immunohistochemistry on E17 fetuses using an antibody specific for GHS-R. Positive cells were distributed extensively in fetal tissues; the skin, bone, intestine, tongue, and muscle being stained particularly strongly (Fig. 1, C–G). Immunoreactivity was not detected in sections that were incubated with GHS-R antiserum that had been preabsorbed with excess synthetic GHS-R (Fig. 1, D-2 and F-2). Although RT-PCR analysis demonstrated the expression of GHS-R mRNA in sections of the brain, pituitary, stomach, and lung, only relatively weak staining was observed in these organs. [125I]Acyl ghrelin autoradiography revealed dense binding to bone, skin, heart, and tongue (Fig. 1H); similar to the immunohistochemistry, the brain and digestive tract bound the isotope only weakly. In addition, excess unlabeled acyl ghrelin (Fig. 1I) and des-acyl ghrelin (Fig. 1J) could displace with [125I]acyl ghrelin binding. More potent replacement was observed in excess unlabeled des-acyl ghrelin treatment (Fig. 1J).
Circulating ghrelin levels during late pregnancy
We measured the circulating levels of acyl and des-acyl ghrelin in pregnant rats and their fetuses, respectively. The levels of acyl ghrelin in maternal plasma exhibited a gradual but not significant decline in late pregnancy (Fig. 2A). In contrast, des-acyl ghrelin increased significantly during late pregnancy (Fig. 2A). Both ghrelin forms, acyl and des-acyl ghrelin, were also present in the fetal circulation; these levels decreased gradually as the time for delivery approached (Fig. 2B). We noticed a significant difference in des-acyl ghrelin levels when compared between the maternal and fetal plasma: the fetal levels of des-acyl ghrelin were 5- to 10-fold higher than the maternal levels (Fig. 2, A and B). On d 17 and 19 of pregnancy, we detected a large quantity of des-acyl ghrelin in the amniotic fluid (Fig. 2C). Acyl ghrelin levels increased rapidly in fetal blood within 5 min of administration of either 0.2 or 20 nmol acyl ghrelin (iv) into the mother (Fig. 2D). In the case of the 20-nmol dose, although maternal trunk ghrelin levels declined 30 min after injection, fetal trunk ghrelin levels were still increased at the sampling time.
Effect of chronic ghrelin treatment on fetal body weight at birth
We examined the effect of prolonged maternal treatment with ghrelin, beginning at d 14 or 15 of pregnancy and lasting until delivery, on neonatal body weight at birth. Chronic treatment with acyl ghrelin, either by injection three times per day (Fig. 3A) or constant infusion through an osmotic mini-pump (Fig. 3B), significantly increased the average neonatal body weight at birth in comparison to that of neonates delivered by a saline-treated group. We observed more than a 10% body weight gain, and the increase was dose-dependent. No significant changes were observed after treatment with des-acyl ghrelin.
We investigated the effect of acyl ghrelin injection on food intake of pregnant females. Daily treatment with acyl ghrelin significantly increased daily maternal food intake (Fig. 3C). However, a paired feeding study demonstrated that even when pregnant females treated with acyl ghrelin consumed the same amount of food as saline-treated pregnant females, neonatal body weight was significantly greater in the ghrelin-treated group (Fig. 3D).
To examine the effect of endogenous maternal ghrelin on fetal development, we compared the birth weight of pups born to mothers passive-immunized against a complex of acyl ghrelin and mcKLH (carrier protein) with that of pups born to mothers passive-immunized against mcKLH. After six immunizations at 2-wk intervals beginning at 44 d after birth, rats were mated when the relative ghrelin binding titer was maximally increased (Fig. 3E). Although body weight gain was temporarily lower, it was not significantly so. The body weights of ghrelin-immunized females recovered gradually to normal levels at 104 d of age (Fig. 3E). The body weights of neonates born to mothers passive-immunized against acyl ghrelin were lower than those of neonates born to saline-treated mothers (Fig. 3F).
Effect of ghrelin on GH mRNA levels in fetal pituitary tissue, and IGF-I and corticosterone levels in fetal plasma
If GH, prolactin, or corticosterone secretions from fetal pituitary or adrenal tissues were stimulated by maternal ghrelin, the released hormone might stimulate fetal development. We examined the effect on fetal pituitary GH mRNA levels and fetal plasma IGF-I or corticosterone levels by administering acyl ghrelin to pregnant females. However, pituitary GH mRNA at E19 and E20 was not affected by this treatment (Fig. 4A). In addition, fetal plasma IGF-I and corticosterone concentrations at E19 and E20 were not affected by maternal ghrelin treatment (Fig. 4, B and C). We found no significant change in fetal prolactin levels (data not shown).
Effect of ghrelin on proliferation of cultured fetal skin cells
To examine a possibility of direct effect of circulating ghrelin on fetal development, we examined the fetal cell proliferation by ghrelin using [3H]thymidine and BrdU incorporation. We used primary cultured fetal skin cells at E17, because abundant cells at this stage were easy to collect. Both [3H]thymidine (Fig. 5A) and BrdU (Fig. 5, B–E) incorporation increased significantly after treatment with acyl ghrelin in a dose-dependent or time-dependent manner. Des-acyl ghrelin was more potent than acyl ghrelin at stimulating the proliferation of fetal skin cells (Fig. 5E). The GHS-R antagonist [D-Lys3]-GHRP-6 inhibited acyl ghrelin- and des-acyl ghrelin-stimulated cell proliferation (Fig. 5E).
Calcium-imaging analysis revealed two types of fetal skin cells (Fig. 5F): one type responding to des-acyl ghrelin, but not to acyl ghrelin, and the other responding to acyl ghrelin, but not to des-acyl ghrelin. No. 21 and 23 cells were shown as examples, respectively.
Discussion
The present study clearly demonstrated that maternal ghrelin would play an important role in fetal development during pregnancy; first, exogenous chronic treatment of the mother with ghrelin increased fetal body weight at birth; second, mothers immunized against ghrelin delivered fetuses with a lower body weight; and third, proliferation of cultured fetal skin cells was stimulated by ghrelin. Both GHS-R1a mRNA expression and GHS-R protein were detected in various fetal tissues. Autoradiography using [125I]acyl ghrelin also demonstrated dense binding to the bone, skin, heart, and tongue. This distribution of functional GHS-R throughout peripheral fetal tissues suggests that ghrelin acts on such fetal peripheral tissues. Surprisingly, excess unlabeled des-acyl ghrelin could displace completely with [125I]acyl ghrelin binding, suggesting that the acyl modification is dispensable for ghrelin function in binding site of fetal tissues. Because des-acyl ghrelin does not bind to GHS-R (1), we presume that fetal tissues may express a GHS-R subtype for des-acyl ghrelin. In support of this supposition (28, 29), it has been shown that the increases in plasma glucose and decreases in insulin, but not increases in GH secretion, induced by acyl ghrelin administration can be counteracted by coadministration of des-acyl ghrelin (28). In addition, ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and phosphatidylinositol 3-kinase/AKTJ (30).
Plasma total ghrelin levels have been measured in pregnant women, rats, and human fetuses (31, 32, 33, 34). In pregnant rats, plasma total ghrelin, determined with an antibody recognizing the C-terminal region, was shown to decrease at around the middle to late stage of pregnancy (31). Total ghrelin increases at around mid-gestation in human pregnancy (32, 33). Human fetuses exhibit levels of total ghrelin in umbilical venous blood that are not correlated with either gestational age or maternal ghrelin levels (34). In addition, ghrelin mRNA expression has been observed in the placenta and ovary of pregnant rats, and in the fetal pancreas (3, 35, 36). It has also been reported that ghrelin might play an important role in the regulation of blood pressure and the development of preimplantation embryos (37, 38). In the present study, both acyl and des-acyl ghrelin were present in the maternal and fetal circulations during the last half of pregnancy, and there was a significant difference in des-acyl ghrelin levels between the maternal and fetal plasma. The fetal levels of plasma des-acyl ghrelin were 5- to 10-fold higher than the maternal levels. In addition, we detected a large quantity of des-acyl ghrelin in the amniotic fluid. As demonstrated previously, ghrelin-positive cells were not evident in the fetal stomach until E19 by immunohistochemistry using an antibody recognizing the N-terminal of acyl ghrelin, suggesting that fetal plasma ghrelin originates from the maternal placenta and/or the maternal blood (3, 22). Indeed, acyl ghrelin levels in fetal plasma increased rapidly within 5 min after administration of acyl ghrelin to the mother, indicating that maternal ghrelin easily transits to the fetal circulation. Although maternal trunk ghrelin levels declined 30 min after injection, fetal trunk ghrelin increased at the time, probably resulting from a longer half-life of ghrelin in fetuses than in adults, and high levels of des-acyl ghrelin might accumulate in the fetal circulation. The existence of GHS-R and an additional GHS-R subtype in fetal tissues, combined with both acyl ghrelin and large quantities of des-acyl ghrelin in the fetal circulation and amniotic fluid, supports the hypothesis that maternal ghrelin plays a critical role in fetal development.
Fetal growth is mainly influenced by the nutrition provided by the mother through the arteria umbilicalis (39, 40). Decreases in the amount of food given to pregnant mothers during the gestational period tend to decrease the size of their neonatal pups in comparison with pups born to mothers fed ad libitum. Daily treatment with acyl ghrelin significantly increased daily maternal food intake. The stimulation of fetal growth by maternal ghrelin injection would result from increased nutrition provided by the mother. However, a paired feeding study demonstrated that even when pregnant females treated with acyl ghrelin consumed the same amount of food as saline-treated pregnant females, neonatal body weight was significantly greater in the ghrelin-treated group. This result indicates that maternal ghrelin affected fetal development through a mechanism independent of increased nutrition.
In rats, a rapid increase in fetal body weight occurs during the last quarter of pregnancy. The somatotroph, a GH-secreting cell, appears in the fetal pituitary near E18 (41). Pituitary GH mRNA at E19 and E20 was not altered by ghrelin treatment, indicating that maternal ghrelin-induced fetal development is not due to increased release of fetal GH. The stimulation of maternal GH secretion by daily treatment of ghrelin, leading to the transition of maternal GH to fetal circulation, may stimulate fetal development. Garcia-Aragon and colleagues (42) provided evidence for the wide distribution of GH receptor in the mid-late gestation of rat fetus. The receptor expression markedly increased between E12 and E18; the receptor was present in all major organ systems at E18. Genetically manipulated model mice, Laron dwarfs, with inactivating GH receptor mutations, were shorter in length than normal at birth. Congenitally GH-deficient newborn babies are also much shorter (43, 44). In contrast, the fetuses of GH-deficient dwarf rats were proportionately smaller in size (45). However, we previously reported that continuous infusion of ghrelin to rats stimulated GH secretion for several days, but that the effect decreased after prolonged administration (26). Levels of GH mRNA within the pituitary were also decreased by these treatments (26), probably due to transcriptional down-regulation. In addition, fetal plasma IGF-I levels were not affected by maternal treatment with ghrelin. We found no significant change in fetal circulating levels of corticosterone and prolactin during maternal ghrelin administration. Therefore, the stimulation of fetal development by maternal ghrelin administration is probably not due to the maternal GH and fetal circulating IGF-I and corticosterone levels.
Both [3H]thymidine and BrdU incorporation increased significantly after treatment with acyl ghrelin in a dose-dependent and time-dependent manner. Interestingly, des-acyl ghrelin stimulated proliferation more potently than acyl ghrelin. The GHS-R antagonist [D-Lys3]-GHRP-6 inhibited acyl ghrelin- and des-acyl ghrelin-stimulated cell proliferation. These results clearly indicate that both acyl ghrelin and des-acyl ghrelin stimulate proliferation of fetal skin cells. Acyl ghrelin induces neurogenesis in the dorsal motor nucleus (46) and stimulates bone formation (47). During pregnancy, maternal ghrelin is likely transferred to the fetal circulation, and then would prompt fetal growth through stimulation of cell proliferation. Calcium-imaging analysis revealed that two types of cells exist in cultured fetal skin cells: one responds only to des-acyl ghrelin, and the other one responds only to acyl ghrelin. These results strongly suggest that fetal skin cells have different type of receptors: one is a classical receptor for acyl ghrelin, GHS-R 1a, and the other is a novel receptor for des-acyl ghrelin that mediates intracellular calcium mobilization.
In this study, we detected high levels of des-acyl ghrelin in the fetal circulation and amniotic fluid. These findings suggest that amniotic fluid serves, in part, as an incubation medium to provide des-acyl ghrelin to the fetus. In this way, des-acyl ghrelin may act on fetal development by direct stimulation of proliferation. If this is true, however, the lack of an effect of des-acyl ghrelin treatment on neonatal body weight at birth (Fig. 3) remains to be explained. We speculate that, late in pregnancy, high endogenous quantities of des-acyl ghrelin in the fetal circulation and amniotic fluid saturate the GHS-R 1a subtype des-acyl ghrelin receptors, effectively preventing the exogenous des-acyl ghrelin from exerting an effect. It has been reported that ghrelin knockout mice do not exhibit any changes in development (probably including fetal development) (13). We do not know the reason for the discrepancy of neonatal body weights between mothers passive-immunized against acyl ghrelin and ghrelin knockout mice. Further studies are required to elucidate this discrepancy.
In conclusion, the present study has demonstrated that maternal ghrelin is easily transferred to the fetal circulation, and then prompts fetal growth through stimulation of cell proliferation during the late half of pregnancy. Recent reports that ghrelin directly stimulates bone formation (47) also supports this hypothesis. These findings may have implications for the clinical application of ghrelin for pregnant subjects.
Footnotes
This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan (to N.M. and K.N.), Mishima Kaiun Memorial Foundation (to K.N.), by the Program for Promotion of Basic Research Activities for Innovative Bioscience, Mitsubishi Foundation, and a Grant-in-Aid for the Promotion of Evolutional Science and Technology in Miyazaki Prefecture (to N.M.).
First Published Online December 8, 2005
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; E, embryonic day; GHS-R, GH secretagogue receptor; mcKLH, mariculture keyhole limpet hemocyanin.
Accepted for publication November 23, 2005.
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Third Department of Internal Medicine (K.T., Y.D., M.Nakaz.), Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan
Molecular Genetics (M.K.), Institute of Life Science, Kurume University, Kurume 839-0864, Japan
National Cardiovascular Center Research Institute (M.M., H.K., H.H., K.K.), Osaka 565-8565, Japan
Abstract
Ghrelin, an acylated peptide serving as an endogenous ligand for GH secretagogue receptor (GHS-R), was originally isolated from rat and human stomach. In this study, we report the critical role of maternal ghrelin in fetal development. High levels of ghrelin receptor (GHS-R) mRNA were detected in various peripheral fetal tissues beginning at embryonic d 14 and lasting until birth. Fetal GHS-R expression was also confirmed in fetal tissues by immunohistochemistry. Autoradiography revealed that both des-acyl ghrelin and acyl ghrelin bind to fetal tissues. Chronic treatment of mothers with ghrelin resulted in a significant increase in birth weight in comparison to newborns from saline-treated mothers. Even when maternal food intake after ghrelin treatment was restricted through paired feeding, significant stimulation of fetal development still occurred. Conversely, active immunization of mothers against ghrelin decreased fetal body weight during pregnancy. A single ghrelin injection into the mother increased circulating ghrelin levels in the fetus within 5 min of injection, suggesting that maternal ghrelin transits easily to the fetal circulation. High levels of des-acyl ghrelin were detected in fetal blood and amniotic fluid. Both acylated and des-acyl ghrelin increased [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation of cultured fetal skin cells in a dose-dependent manner, and calcium-imaging analysis revealed that acyl and des-acyl ghrelin increased the Ca2+ influx in discrete cultured fetal skin cells, respectively. These results indicate that maternal ghrelin regulates fetal development during the late stages of pregnancy.
Introduction
GHRELIN, RECENTLY purified from rat and human stomachs as an endogenous ligand for the GH secretagogue receptor (GHS-R), is a 28-amino acid peptide with an n-octanoylation modification at Ser3 (1). This octanoylation is important for the stimulation of GH secretion from the pituitary gland (1). Although cells immunostained for ghrelin are distributed widely in the stomach, hypothalamus, pituitary gland, liver, kidney, pancreas, and placenta, the main source of circulating ghrelin is considered to be the gastrointestinal tract (2, 3, 4, 5, 6). Both acylated and des-acyl ghrelin are observed in the peripheral circulation, the levels of des-acyl ghrelin being higher (7, 8). Several studies on the physiological function of ghrelin have demonstrated that, in addition to stimulating GH secretion, ghrelin also stimulates food intake and body weight gain independent of GH secretion (8, 9, 10, 11, 12, 13). It is likely that the appetite-stimulating effect of peripheral ghrelin is due to action via the afferent vagal nerve (14). In contrast, the central effect is thought to be via neuropeptide Y and agouti-related peptide secretion from the arcuate nucleus in the hypothalamus (11, 15). Administration of ghrelin continuously to rodents resulted in fat deposition and obesity (9). These effects of ghrelin on appetite and fat deposition are counteracted by leptin (11). These results imply that ghrelin may play an important role in the regulation of food intake and energy expenditure.
The ghrelin receptor GHS-R, highly conserved from teleost fish to humans, is expressed widely in both central and peripheral organs, including the brain, pituitary gland, and pancreas (16, 17, 18, 19). The broad distribution of GHS-R suggests that ghrelin may play important roles in addition to those mentioned above. It has been also demonstrated that ghrelin might be involved in stomach motility (10), gastric acid secretion (20), insulin and gastrin release (21), the cardiovascular system, and stress reactions (12). In addition, we demonstrated previously that neonatal rats treated daily with ghrelin for 2 or 3 wk from birth showed faster eye and vaginal opening than those of saline-treated group (22), suggesting that ghrelin may be involved in neonatal development. Therefore, it has been assumed that ghrelin from the maternal stomach or placenta during pregnancy may play a role in fetal development. In the present study, we examined the possible involvement of maternal ghrelin in rat fetal development.
Materials and Methods
Animals
Wistar rats were housed under controlled temperature (23 ± 1 C) and regulated 12-h light 12-h dark conditions (lights on at 0700 h). Female rats were mated on the day of proestrus at approximately 3 months old. The next estrus day was considered to be d 0 of pregnancy. As reported previously, delivery usually occurs in our rat colony during the morning on d 21 of pregnancy (23). The average number (±SEM) of pups per mother at delivery was 13.10 ± 1.78 (n = 122). All procedures were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.
RT-PCR for GHS-R 1a mRNA
Total RNA was extracted from fetal tissues on d 14, 15, and 19 of pregnancy using Trizol reagent (Invitrogen, Carlsbad, CA) as described previously (24). First-strand cDNA was synthesized from 2 μg of total RNA by random primer RT. The resulting cDNA was subjected to PCR amplification using sense and antisense primers specific for GHS-R1a (24). PCR products were electrophoresed on a 2% agarose gel. GAPDH was used as a control housekeeping gene.
Autoradiography for [125I]acyl ghrelin
Fetuses [embryonic d 17 (E17)] were embedded in Tissue-Tec OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and frozen. Sections cut using a cryostat were mounted on gelatin-coated glass slides. Autoradiography was performed as described previously (14) with the following minor modifications. After preincubation for 30 min in incubation buffer at room temperature, sections were incubated for 12 h at 4 C in buffer containing 20 ng/ml rat [125I-Tyr29]acylated rat ghrelin. Nonspecific binding was determined in the presence of excess unlabeled acyl or des-acyl rat ghrelin (10 μg/ml). Sections were then exposed to an IP plate (Fuji Film, Tokyo, Japan) for 12 h and analyzed on BAS-5000 (Fuji Film).
Preparation of anti-GHS-R serum
The [Cys0]-rat GHS-R [342–364] peptide was synthesized using the Fmoc solid-phase method on a peptide synthesizer (433A; Applied Biosystems, Foster City, CA), then purified by reverse phase-HPLC. The synthesized peptide (10 mg) was conjugated to maleimide-activated mariculture keyhole limpet hemocyanin (6 mg) (mcKLH; Pierce, Rockford, IL) in conjugation buffer (Pierce). The conjugate was emulsified with an equal volume of Freund’s complete adjuvant and was used to immunize New Zealand white rabbits by intracutaneous and sc injection. Animals were boosted every 2 wk and bled 7 d after each injection. The specificity of the antisera was confirmed by the immunoreactivity of GHS-R-expressing cells (CHO-GHSR62 cells) and lack thereof in control cells.
Immunohistochemistry for GHS-R
Immunohistochemical analyses for GHS-R were performed on frozen fetuses (E17 and 19) using a modification of a method that has been described previously (25). The fetuses were placed in fixative for 5 d at 4 C and then transferred to 0.1 M phosphate buffer containing 20% sucrose. They were cut into serial, 12-μm-thick sections at –20 C with a cryostat. The sections were incubated for 2 d with a rabbit-anti-GHS-R antibody at 4 C. Slides were then incubated with Alexa-546-labeled goat-antirabbit IgG antibody (Molecular Probes, Inc., Eugene, OR; dilution 1:400). Samples were observed with the aid of an Olympus AX-70 fluorescence microscope (Olympus, Tokyo, Japan). To examine the specificity of GHS-R antibody in tissue sections, the reaction was also performed using GHS-R antibody that had been preabsorbed with excess synthetic GHS-R (10 μg).
Measurement of acyl and des-acyl ghrelin, IGF-I, and corticosterone
Levels of acyl or des-acyl ghrelin were measured by specific ELISA kits for acyl or des-acyl ghrelin (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan). The ELISA can detect each acyl or des-acyl ghrelin using two specific antibodies recognizing only acyl ghrelin (octanoylation modification at [Ser3]-ghrelin [1–11]) or only des-acyl ghrelin (nonoctanoylation modification at [Ser3]-ghrelin [1–11]). Blood collected from pregnant rats and their fetuses was immediately put into chilled polypropylene tubes containing a protease inhibitor, aprotinin (Sigma-Aldrich, St. Louis, MO), and 2Na-EDTA and then centrifuged. We then added a 10% plasma volume of 0.1 N HCl. Maternal blood was taken at 0830 h (satiety phase) at 2-d intervals from d 11–21. Fetal blood and amniotic fluid were collected on d 17, 19, and 21.
To examine the transit of maternal acyl ghrelin to the fetal circulation, acyl ghrelin (0.2 and 20 nmol) or saline was injected into pregnant rats iv under light ether anesthesia on d 19 of pregnancy (n = 12 per group). Blood was then collected from both the mother and fetus at 5, 10, and 30 min after injection.
To determine the effect of maternal treatment with acyl ghrelin on plasma IGF-I and corticosterone levels in the fetal circulation, fetal plasma IGF-I and corticosterone levels were measured by enzyme immunoassay kit (Funakoshi, Tokyo, Japan) and [125I]corticosterone RIA kit (ICN Biomedicals, Costa Mesa, CA), respectively. The limit of assay sensitivity was 5 ng/ml for IGF-I and 20 ng/ml for corticosterone. The intra and interassay coefficients of variation were 5 and 16%, respectively, for IGF-I, and 6 and 12%, respectively, for corticosterone.
Ghrelin administration and neonatal body weights
We sc injected either saline, acyl ghrelin (1.5 or 3.0 nmol), or des-acyl ghrelin (3.0 nmol) three times a day (at 0830, 1330, and 1830 h) from d 14 to delivery, or continuously infused vehicle, acyl ghrelin (0.125 or 0.5 nmol/h) or des-acyl ghrelin (0.5 nmol/h) through an osmotic mini-pump implanted sc from d 15 until delivery (n = 10 per group) (11, 26). We also injected 3 nmol acyl ghrelin three times a day from d 14 to delivery into pair-fed pregnant rats and the effect was compared with saline-treated pregnant rats. Neonatal body weights were measured on the day of delivery. If the pups numbered more than 15 or less than 11 they were excluded from the analyses.
Passive immunization for acyl ghrelin
Rat acyl ghrelin (3 mg) was conjugated to a carrier protein, mcKLH (3 mg), in conjugation buffer (Pierce) (7). Each conjugate was emulsified with an equal volume of Freund’s adjuvant. Immunization, initiated by intradermal injection in 44-d-old female rats, was repeated six times at 2-wk intervals. As a control antigen, carrier protein alone without ghrelin was administered. Rats were mated on d 114 after the fifth immunization. The antibody titers were verified in diluted plasma every 10 d after immunization using [125I]ghrelin binding capacity.
Quantitative RT-PCR of GH mRNA in fetal pituitary
The pituitary gland and blood were collected from E19 and E20 fetuses, isolated from the mothers’ implanted osmotic minipump (acyl ghrelin 0.5 nmol/h and saline). GH mRNA expression was measured by real-time quantitative PCR as described previously (25). Experiments contrasted the relative levels of both GH and GAPDH transcripts in every sample. The total RNA from each tissue was extracted using an RNeasy Micro kit (Qiagen, Valencia, CA) and synthesized into first-strand cDNA using an iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA). An aliquot of the first-strand cDNA (40–100 ng tissue equivalent) was quantified on an iCycler (Bio-Rad Laboratory) using iQ SYBR Green Supermix (Bio-Rad Laboratory) with primers to amplify GAPDH (25) and GH specifically (26).
Incorporation of [3H]thymidine or 5-bromo-2'-deoxyuridine (BrdU) into cultured cells
We assessed the effect of acyl and des-acyl ghrelin administration on the proliferation of fetal skin cells by measuring the incorporation of [3H]thymidine (2 μCi/ml) or BrdU (10 μM). Dispersed fetal skin cells were prepared from E17 fetuses by sequential collagenase treatment, papain digestion, and mechanical desegregation. Dispersed cells were then suspended in MCDB153HAA medium (F-Peptide Co., Ltd., Yamagata, Japan) containing 2% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and 5 ng/ml epidermal growth factor. Cells were seeded in polyethylenimine-coated 48- and 96-well dishes at densities of 5 x 105 per well and 3 x 104 per well for the [3H]thymidine and BrdU experiments, respectively. BrdU was detected using a Cell Proliferation ELISA Kit (Roche Diagnostic GmbH, Mannheim, Germany), as reported by Kusunoki et al. (27).
Statistics
Values are given as means ± SEM. Comparisons between two groups were made by ANOVA with the post hoc Fisher test. Differences at P < 0.05 were accepted as statistically significant.
Results
Expression of GHS-Rs in fetal tissue
GHS-R1a mRNA expression was detected in various fetal tissues with a high density in the spinal cord from E14 until birth (Fig. 1A). GHS-R mRNA expression in the fetal pituitary was also detected at E19. To confirm the expression of GHS-R in fetal tissues at the protein level, we performed immunohistochemistry on E17 fetuses using an antibody specific for GHS-R. Positive cells were distributed extensively in fetal tissues; the skin, bone, intestine, tongue, and muscle being stained particularly strongly (Fig. 1, C–G). Immunoreactivity was not detected in sections that were incubated with GHS-R antiserum that had been preabsorbed with excess synthetic GHS-R (Fig. 1, D-2 and F-2). Although RT-PCR analysis demonstrated the expression of GHS-R mRNA in sections of the brain, pituitary, stomach, and lung, only relatively weak staining was observed in these organs. [125I]Acyl ghrelin autoradiography revealed dense binding to bone, skin, heart, and tongue (Fig. 1H); similar to the immunohistochemistry, the brain and digestive tract bound the isotope only weakly. In addition, excess unlabeled acyl ghrelin (Fig. 1I) and des-acyl ghrelin (Fig. 1J) could displace with [125I]acyl ghrelin binding. More potent replacement was observed in excess unlabeled des-acyl ghrelin treatment (Fig. 1J).
Circulating ghrelin levels during late pregnancy
We measured the circulating levels of acyl and des-acyl ghrelin in pregnant rats and their fetuses, respectively. The levels of acyl ghrelin in maternal plasma exhibited a gradual but not significant decline in late pregnancy (Fig. 2A). In contrast, des-acyl ghrelin increased significantly during late pregnancy (Fig. 2A). Both ghrelin forms, acyl and des-acyl ghrelin, were also present in the fetal circulation; these levels decreased gradually as the time for delivery approached (Fig. 2B). We noticed a significant difference in des-acyl ghrelin levels when compared between the maternal and fetal plasma: the fetal levels of des-acyl ghrelin were 5- to 10-fold higher than the maternal levels (Fig. 2, A and B). On d 17 and 19 of pregnancy, we detected a large quantity of des-acyl ghrelin in the amniotic fluid (Fig. 2C). Acyl ghrelin levels increased rapidly in fetal blood within 5 min of administration of either 0.2 or 20 nmol acyl ghrelin (iv) into the mother (Fig. 2D). In the case of the 20-nmol dose, although maternal trunk ghrelin levels declined 30 min after injection, fetal trunk ghrelin levels were still increased at the sampling time.
Effect of chronic ghrelin treatment on fetal body weight at birth
We examined the effect of prolonged maternal treatment with ghrelin, beginning at d 14 or 15 of pregnancy and lasting until delivery, on neonatal body weight at birth. Chronic treatment with acyl ghrelin, either by injection three times per day (Fig. 3A) or constant infusion through an osmotic mini-pump (Fig. 3B), significantly increased the average neonatal body weight at birth in comparison to that of neonates delivered by a saline-treated group. We observed more than a 10% body weight gain, and the increase was dose-dependent. No significant changes were observed after treatment with des-acyl ghrelin.
We investigated the effect of acyl ghrelin injection on food intake of pregnant females. Daily treatment with acyl ghrelin significantly increased daily maternal food intake (Fig. 3C). However, a paired feeding study demonstrated that even when pregnant females treated with acyl ghrelin consumed the same amount of food as saline-treated pregnant females, neonatal body weight was significantly greater in the ghrelin-treated group (Fig. 3D).
To examine the effect of endogenous maternal ghrelin on fetal development, we compared the birth weight of pups born to mothers passive-immunized against a complex of acyl ghrelin and mcKLH (carrier protein) with that of pups born to mothers passive-immunized against mcKLH. After six immunizations at 2-wk intervals beginning at 44 d after birth, rats were mated when the relative ghrelin binding titer was maximally increased (Fig. 3E). Although body weight gain was temporarily lower, it was not significantly so. The body weights of ghrelin-immunized females recovered gradually to normal levels at 104 d of age (Fig. 3E). The body weights of neonates born to mothers passive-immunized against acyl ghrelin were lower than those of neonates born to saline-treated mothers (Fig. 3F).
Effect of ghrelin on GH mRNA levels in fetal pituitary tissue, and IGF-I and corticosterone levels in fetal plasma
If GH, prolactin, or corticosterone secretions from fetal pituitary or adrenal tissues were stimulated by maternal ghrelin, the released hormone might stimulate fetal development. We examined the effect on fetal pituitary GH mRNA levels and fetal plasma IGF-I or corticosterone levels by administering acyl ghrelin to pregnant females. However, pituitary GH mRNA at E19 and E20 was not affected by this treatment (Fig. 4A). In addition, fetal plasma IGF-I and corticosterone concentrations at E19 and E20 were not affected by maternal ghrelin treatment (Fig. 4, B and C). We found no significant change in fetal prolactin levels (data not shown).
Effect of ghrelin on proliferation of cultured fetal skin cells
To examine a possibility of direct effect of circulating ghrelin on fetal development, we examined the fetal cell proliferation by ghrelin using [3H]thymidine and BrdU incorporation. We used primary cultured fetal skin cells at E17, because abundant cells at this stage were easy to collect. Both [3H]thymidine (Fig. 5A) and BrdU (Fig. 5, B–E) incorporation increased significantly after treatment with acyl ghrelin in a dose-dependent or time-dependent manner. Des-acyl ghrelin was more potent than acyl ghrelin at stimulating the proliferation of fetal skin cells (Fig. 5E). The GHS-R antagonist [D-Lys3]-GHRP-6 inhibited acyl ghrelin- and des-acyl ghrelin-stimulated cell proliferation (Fig. 5E).
Calcium-imaging analysis revealed two types of fetal skin cells (Fig. 5F): one type responding to des-acyl ghrelin, but not to acyl ghrelin, and the other responding to acyl ghrelin, but not to des-acyl ghrelin. No. 21 and 23 cells were shown as examples, respectively.
Discussion
The present study clearly demonstrated that maternal ghrelin would play an important role in fetal development during pregnancy; first, exogenous chronic treatment of the mother with ghrelin increased fetal body weight at birth; second, mothers immunized against ghrelin delivered fetuses with a lower body weight; and third, proliferation of cultured fetal skin cells was stimulated by ghrelin. Both GHS-R1a mRNA expression and GHS-R protein were detected in various fetal tissues. Autoradiography using [125I]acyl ghrelin also demonstrated dense binding to the bone, skin, heart, and tongue. This distribution of functional GHS-R throughout peripheral fetal tissues suggests that ghrelin acts on such fetal peripheral tissues. Surprisingly, excess unlabeled des-acyl ghrelin could displace completely with [125I]acyl ghrelin binding, suggesting that the acyl modification is dispensable for ghrelin function in binding site of fetal tissues. Because des-acyl ghrelin does not bind to GHS-R (1), we presume that fetal tissues may express a GHS-R subtype for des-acyl ghrelin. In support of this supposition (28, 29), it has been shown that the increases in plasma glucose and decreases in insulin, but not increases in GH secretion, induced by acyl ghrelin administration can be counteracted by coadministration of des-acyl ghrelin (28). In addition, ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and phosphatidylinositol 3-kinase/AKTJ (30).
Plasma total ghrelin levels have been measured in pregnant women, rats, and human fetuses (31, 32, 33, 34). In pregnant rats, plasma total ghrelin, determined with an antibody recognizing the C-terminal region, was shown to decrease at around the middle to late stage of pregnancy (31). Total ghrelin increases at around mid-gestation in human pregnancy (32, 33). Human fetuses exhibit levels of total ghrelin in umbilical venous blood that are not correlated with either gestational age or maternal ghrelin levels (34). In addition, ghrelin mRNA expression has been observed in the placenta and ovary of pregnant rats, and in the fetal pancreas (3, 35, 36). It has also been reported that ghrelin might play an important role in the regulation of blood pressure and the development of preimplantation embryos (37, 38). In the present study, both acyl and des-acyl ghrelin were present in the maternal and fetal circulations during the last half of pregnancy, and there was a significant difference in des-acyl ghrelin levels between the maternal and fetal plasma. The fetal levels of plasma des-acyl ghrelin were 5- to 10-fold higher than the maternal levels. In addition, we detected a large quantity of des-acyl ghrelin in the amniotic fluid. As demonstrated previously, ghrelin-positive cells were not evident in the fetal stomach until E19 by immunohistochemistry using an antibody recognizing the N-terminal of acyl ghrelin, suggesting that fetal plasma ghrelin originates from the maternal placenta and/or the maternal blood (3, 22). Indeed, acyl ghrelin levels in fetal plasma increased rapidly within 5 min after administration of acyl ghrelin to the mother, indicating that maternal ghrelin easily transits to the fetal circulation. Although maternal trunk ghrelin levels declined 30 min after injection, fetal trunk ghrelin increased at the time, probably resulting from a longer half-life of ghrelin in fetuses than in adults, and high levels of des-acyl ghrelin might accumulate in the fetal circulation. The existence of GHS-R and an additional GHS-R subtype in fetal tissues, combined with both acyl ghrelin and large quantities of des-acyl ghrelin in the fetal circulation and amniotic fluid, supports the hypothesis that maternal ghrelin plays a critical role in fetal development.
Fetal growth is mainly influenced by the nutrition provided by the mother through the arteria umbilicalis (39, 40). Decreases in the amount of food given to pregnant mothers during the gestational period tend to decrease the size of their neonatal pups in comparison with pups born to mothers fed ad libitum. Daily treatment with acyl ghrelin significantly increased daily maternal food intake. The stimulation of fetal growth by maternal ghrelin injection would result from increased nutrition provided by the mother. However, a paired feeding study demonstrated that even when pregnant females treated with acyl ghrelin consumed the same amount of food as saline-treated pregnant females, neonatal body weight was significantly greater in the ghrelin-treated group. This result indicates that maternal ghrelin affected fetal development through a mechanism independent of increased nutrition.
In rats, a rapid increase in fetal body weight occurs during the last quarter of pregnancy. The somatotroph, a GH-secreting cell, appears in the fetal pituitary near E18 (41). Pituitary GH mRNA at E19 and E20 was not altered by ghrelin treatment, indicating that maternal ghrelin-induced fetal development is not due to increased release of fetal GH. The stimulation of maternal GH secretion by daily treatment of ghrelin, leading to the transition of maternal GH to fetal circulation, may stimulate fetal development. Garcia-Aragon and colleagues (42) provided evidence for the wide distribution of GH receptor in the mid-late gestation of rat fetus. The receptor expression markedly increased between E12 and E18; the receptor was present in all major organ systems at E18. Genetically manipulated model mice, Laron dwarfs, with inactivating GH receptor mutations, were shorter in length than normal at birth. Congenitally GH-deficient newborn babies are also much shorter (43, 44). In contrast, the fetuses of GH-deficient dwarf rats were proportionately smaller in size (45). However, we previously reported that continuous infusion of ghrelin to rats stimulated GH secretion for several days, but that the effect decreased after prolonged administration (26). Levels of GH mRNA within the pituitary were also decreased by these treatments (26), probably due to transcriptional down-regulation. In addition, fetal plasma IGF-I levels were not affected by maternal treatment with ghrelin. We found no significant change in fetal circulating levels of corticosterone and prolactin during maternal ghrelin administration. Therefore, the stimulation of fetal development by maternal ghrelin administration is probably not due to the maternal GH and fetal circulating IGF-I and corticosterone levels.
Both [3H]thymidine and BrdU incorporation increased significantly after treatment with acyl ghrelin in a dose-dependent and time-dependent manner. Interestingly, des-acyl ghrelin stimulated proliferation more potently than acyl ghrelin. The GHS-R antagonist [D-Lys3]-GHRP-6 inhibited acyl ghrelin- and des-acyl ghrelin-stimulated cell proliferation. These results clearly indicate that both acyl ghrelin and des-acyl ghrelin stimulate proliferation of fetal skin cells. Acyl ghrelin induces neurogenesis in the dorsal motor nucleus (46) and stimulates bone formation (47). During pregnancy, maternal ghrelin is likely transferred to the fetal circulation, and then would prompt fetal growth through stimulation of cell proliferation. Calcium-imaging analysis revealed that two types of cells exist in cultured fetal skin cells: one responds only to des-acyl ghrelin, and the other one responds only to acyl ghrelin. These results strongly suggest that fetal skin cells have different type of receptors: one is a classical receptor for acyl ghrelin, GHS-R 1a, and the other is a novel receptor for des-acyl ghrelin that mediates intracellular calcium mobilization.
In this study, we detected high levels of des-acyl ghrelin in the fetal circulation and amniotic fluid. These findings suggest that amniotic fluid serves, in part, as an incubation medium to provide des-acyl ghrelin to the fetus. In this way, des-acyl ghrelin may act on fetal development by direct stimulation of proliferation. If this is true, however, the lack of an effect of des-acyl ghrelin treatment on neonatal body weight at birth (Fig. 3) remains to be explained. We speculate that, late in pregnancy, high endogenous quantities of des-acyl ghrelin in the fetal circulation and amniotic fluid saturate the GHS-R 1a subtype des-acyl ghrelin receptors, effectively preventing the exogenous des-acyl ghrelin from exerting an effect. It has been reported that ghrelin knockout mice do not exhibit any changes in development (probably including fetal development) (13). We do not know the reason for the discrepancy of neonatal body weights between mothers passive-immunized against acyl ghrelin and ghrelin knockout mice. Further studies are required to elucidate this discrepancy.
In conclusion, the present study has demonstrated that maternal ghrelin is easily transferred to the fetal circulation, and then prompts fetal growth through stimulation of cell proliferation during the late half of pregnancy. Recent reports that ghrelin directly stimulates bone formation (47) also supports this hypothesis. These findings may have implications for the clinical application of ghrelin for pregnant subjects.
Footnotes
This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan (to N.M. and K.N.), Mishima Kaiun Memorial Foundation (to K.N.), by the Program for Promotion of Basic Research Activities for Innovative Bioscience, Mitsubishi Foundation, and a Grant-in-Aid for the Promotion of Evolutional Science and Technology in Miyazaki Prefecture (to N.M.).
First Published Online December 8, 2005
Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; E, embryonic day; GHS-R, GH secretagogue receptor; mcKLH, mariculture keyhole limpet hemocyanin.
Accepted for publication November 23, 2005.
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