Effects of Central Infusion of Ghrelin on Food Intake and Plasma Levels of Growth Hormone, Luteinizing Hormone, Prolactin, and Cor
http://www.100md.com
《内分泌学杂志》
Prince Henry’s Institute of Medical Research (J.I., I.J.C.), Clayton, Victoria 3168, Australia
Laboratory of Animal Nutrient Metabolism (Y.K.), Kitasato University, Towada, Aomori 034, Japan
Department of Physiology (B.C.), Monash University, Victoria 3800, Australia
Abstract
Ghrelin is an endogenous ligand for the GH secretagogue/ghrelin receptor (GHS-R) and stimulates feeding behavior and GH levels in rodents and humans. A preprandial increase in plasma ghrelin levels is seen in sheep on programmed feeding, followed by a postprandial rise in plasma GH levels, but effects on food intake and endocrine function are not defined in this ruminant species. We administered ghrelin to female sheep in various modes and measured effects on voluntary food intake (VFI) and plasma levels of GH, LH, prolactin, and cortisol. Whether administered intracerebroventricularly or iv, ghrelin consistently failed to stimulate VFI. On the other hand, ghrelin invariably increased plasma GH levels and ,-diaminopropanoic acid-octanoyl3 human ghrelin was more potent than ovine ghrelin. Bolus injection of ghrelin into the third cerebral ventricle reduced plasma LH levels but did not affect levels of prolactin or cortisol. These findings suggested that the preprandial rise in plasma ghrelin that is seen in sheep on programmed feeding does not influence VFI but is likely to be important in the postprandial rise in GH levels. Thus, ghrelin does not appear to be a significant regulator of ingestive behavior in this species of ruminant but acts centrally to indirectly regulate GH and LH secretion.
Introduction
THE HYPOTHALAMUS is the neuroendocrine center of the brain and also plays an important role in the regulation of ingestive behavior. In particular, cells of the arcuate nucleus that produce neuropeptide Y (NPY), a potent orexigen (1) and the peptides encoded by the pro-opiomelanocortin gene (2) are important in energy homeostasis (1, 2, 3). GHRH is produced in cells of the arcuate nucleus that project to the external zone of the median eminence (4, 5). Secretion of this peptide into the hypophyseal portal system provides a specific stimulus for the release of GH from the pituitary (5). Cells of the arcuate nucleus, and other brain regions, express a GH secretagogue receptor (GHS-R) (6, 7, 8, 9, 10), and exogenous GH-releasing peptides (GHRP) can act centrally (11) to indirectly regulate the secretion of GH from the pituitary gland (12, 13) and other functions, such as food intake (14). GHS-R is also expressed in cells of the pituitary gland, so GHRP can also act directly at this level to stimulate the secretion of GH (12) and other pituitary hormones (12).
Ghrelin is a 28-amino acid peptide that was found in the stomach (15) and is an endogenous ligand for GHS-R (15). Accordingly, ghrelin stimulates GH secretion in humans and rodents (15, 16, 17, 18, 19, 20). Ghrelin has also been implicated in the regulation of other endocrine functions, with evidence of effects in the rat, human, and monkeys on the secretion of prolactin (PRL) (14, 21), adrenocorticotrophic hormone, cortisol (14, 21), and LH (22, 23, 24). Whereas leptin (25), insulin (26), and other peripheral factors act as satiety factors (27, 28), ghrelin stimulates food intake and regulates energy homeostasis (15, 16, 17, 18, 19, 20, 29).
In humans (17, 30), plasma ghrelin levels increase transiently before a scheduled meal and then decline postprandially. Furthermore, administration of exogenous ghrelin causes hyperphagia in rats (19, 20) and humans (18, 31). On the other hand, central administration of ghrelin inhibited food intake in the chicken (32). Date et al. (16) reported that continuous intracerebroventricular infusion of ghrelin to male rats increased plasma GH concentrations after 6 d, but levels were not elevated after 12 d, although effects on food intake were not reported. As in humans and rats, a preprandial rise in plasma ghrelin levels is seen in sheep on programmed feeding (33, 34). Despite these strong indications that ghrelin is important in the regulation of food intake, genetic knockout of ghrelin leads to a minimal phenotype in terms of food intake and body composition (35, 36). Paradoxically, overexpression of the ghrelin gene reduced food intake through hypothalamic mechanisms (37).
Little is known of the role of ghrelin in the regulation of food intake and endocrine function in species other than humans and rodents. The ruminant presents an interesting model because the gut is not emptied between periods of feeding. Despite this, there is a well-discerned rise in plasma ghrelin levels before an expected meal in sheep, with a postprandial rise in plasma GH levels (33, 34) and ghrelin secretion from the stomach may be regulated centrally through cholinergic neurons of the vagus (38). We have investigated the effects of chronic and acute infusion of ghrelin on food intake and on plasma levels of GH, LH, PRL, and cortisol in sheep.
Materials and Methods
Ethics
All animal procedures were conducted in compliance with the Code of Ethics specified by the National Health and Medical Research Council of Australia, and prior approval was obtained from the Animal Experimentation Ethics Committee of Monash Medical Centre and the Victorian Institute of Animal Science.
Animals and surgery
Corriedale ewes with a mean body weight of 44.6 ± 1.2 kg were used during the breeding season. The animals were ovariectomized at least 1 month before surgery to avoid cyclic variations in plasma levels of ovarian steroids. In some cases, the animals were fitted with guide tubes into the third cerebral ventricle (IIIv) as previously described (39) and in other cases, an infusion line was introduced into one lateral cerebral ventricle (LV) (vide infra). Briefly, the animals were introduced to single pens in an experimental facility at least 1 week before experimentation for familarization and adjustment to a standard diet. They were fed an ad libitum diet (2.5 kg Lucerne chaff), once a day, at a time predetermined for each experiment, and were subject to normal environmental variation in temperature and photoperiod. Voluntary food intake (VFI) was recorded by measuring refusals. For the purpose of sampling blood, a dwelling cannula (Tuta Healthcare, Sydney, New South Wales, Australia) was introduced to one external jugular vein and kept patent with heparinized (50 U/ml) normal saline. A manometer line (Smiths Medical Australasia Pty. Ltd., Gold Coast, Queensland, Australia) was attached to the cannula, closed with a three-way tap and attached to the side of the pen so that samples could be taken without disturbing the animal. Blood samples (5 ml) were collected into heparinized tubes and centrifuged at 4 C to obtain plasma, which was stored at –20 C until assayed.
LV cannulation
Animals were anesthetized with iv injection of Thiopentone (20 mg/kg; Thiobarb Sodium, Jurox Ltd., Rutherford, New South Wales, Australia) and maintained with halothane (1.5–2.5%) inhalation in oxygen. Preoperatively, Rimadyl (4.0 mg/kg; Pfizer Ltd., West Ryde, New South Wales, Australia), was administered iv for analgesia and Oxytet-200 L.A. (1 ml/kg body weight; Troy Laboratories Ltd., Smithfield, New South Wales, Australia) was injected im as an antibiotic. The head was laid on a box containing polystyrene beads and held by a strap over the nose. A midline incision was made from the pole of the head running caudally over the parietal bone. A burr hole was made in the parietal bone 10–15 mm caudal to Bregma and 10–15 mm off midline. A customized apparatus was attached to the skull to hold a length of 18-gauge stainless steel tubing fitted with an obdurator. This was advanced at an angle of approximately 20 degrees, 16–18 mm below the surface of the brain and the obdurator removed; flow of cerebrospinal fluid confirmed placement in the LV. A length of SILASTIC brand tubing (catalog no. 508 003, Dow Corning, Seven Hills, New South Wales, Australia) was introduced to the LV and advanced 10 mm beyond the tip of the guide-tubing. Placement was confirmed by injection of a radio-opaque dye (Omnipaque, Nycomed Australia Pty. Ltd., North Ryde, New South Wales, Australia) and the tip of the cannula was placed at the level of the Foramen of Munro. The SILASTIC brand tubing was secured to the skull with Superglue, coiled under the skin, and exteriorized at the back of the neck. The animals were allowed to recover and kept at pasture until required for experimentation.
Central infusion of ghrelin or vehicle was achieved by connection of IIIv or LV infusion lines to Graseby MS16A infusion pumps (Graseby Medical Ltd., Gold Coast, Queensland, Australia). For IIIv infusion, a 19-gauge stainless steel tubing assembly that was introduced into the IIIv at least 2 mm beyond the end of the guide tube. For LV infusion, the pumps were connected directly to the SILASTIC brand tubing that was exteriorized at the neck. The infusion rate was 60 μl/h. Vehicle for central infusions was artificial cerebrospinal fluid (aCSF; 150 mm NaCl, 1.2 mmCaCl, 1 mm MgCl, and 2.8 mm KCl).
Ghrelin for infusion
Synthetic ovine ghrelin (GSSFLSPEHQKLQ-RKEPKKPSGRLKPR) was synthesized by Peptide Institute Inc. (Osaka, Japan) and [dap (,-diaminopropanoic acid)-octanoyl3]-human ghrelin (catalog no. 031-83) [(DAP-octanoyl3) human ghrelin] was obtained from Phoenix Pharmaceuticals Inc. (Belmont, CA).
Experiment 1: effects of chronic IIIv ovine ghrelin infusion
Part 1.
Groups (n = 5) received either aCSF or ovine ghrelin (5 μg/h) infused into the IIIv for 3 d. On d 1, blood samples were collected at 10-min intervals for 12 h (0600–1800 h) and the infusions commenced at 1200 h. VFI was measured. Plasma samples were assayed for GH, LH, PRL, and cortisol.
Part 2.
Because there were no effects of 5 μg/h ovine ghrelin infusion on VFI, a further experiment was conducted in which animals received infusions of either aCSF (n = 5) or 10 μg/h ovine ghrelin (n = 5) into the IIIv for 2 d, and VFI was measured (as above). Because no effect was seen on VFI, the dose of ovine ghrelin was increased to 20 μg/h for further 24 h. Blood samples were collected (as above) for 6 h immediately before infusion and 6 h immediately after the commencement of infusion (0600–1800 h on d 1) to measure the short-term effect of ghrelin infusion on plasma GH levels. Plasma samples were assayed for GH only.
Experiment 2: dose-response effects of ovine and DAP-octanoyl3 human ghrelin administered to the IIIv
Because ovine ghrelin did not affect VFI in experiment 1, studies were conducted to compare responses to ovine and DAP-octanoyl3 human ghrelin. The animals were prepared as described for experiment 1. To test the short-term effects of central infusion and compare the efficacy of ovine and DAP-octanoyl 3 human ghrelin, doses of 1, 5, 10, or 20 μg of aCSF were injected into the IIIV (n = 4/group) in a volume of 60 μl at 1200 h. The animals were fed 2.5 kg Lucerne chaff/d at 0900 h daily, and VFI was measured. Blood samples were taken at 10-min intervals between 1100 and 1300 h, at 20-min intervals between 1300 and 1500 h and at 30-min intervals between 1500 and 1600 h. Plasma samples were assayed for GH, LH, PRL, and cortisol.
Experiment 3: effects of multiple DAP-octanoyl3 human ghrelin injections into the LV
Animals were randomly divided into two groups (n = 5/group) and fed at 1700 h. They were injected with either 100 μg DAP-octanoyl3 human ghrelin or aCSF (100 μl) into the LV at 0800, 1200, and 1600 h each day for 4 d. VFI was measured.
Experiment 4: effects of iv injection of DAP-octanoyl3 human ghrelin on VFI
Because the chronic infusion of ovine ghrelin and single/multiple central injections of ovine/ DAP-octanoyl3 human ghrelin did not affect VFI, we considered the possibility that ghrelin has a peripheral action that has a secondary effect on the brain to regulate feeding behavior. The animals were fed at 0900 h, and daily VFI was monitored for 5 d and then animals (n = 5/group) received iv injections of either 100 μg of DAP-octanoyl3 human ghrelin or saline at 1200. After injection, VFI was measured for 1, 2, 3, and 12 h after injection.
RIAs
GH assay.
GH concentrations were measured by the method of Thomas et al. (40) using NIDDK-oGH-1–4 as standard and NIDDK-anti-oGH-2 antiserum. Assay sensitivity was 0. 4–1 ng/ml. The intraassay coefficient of variation (CV) was less than 10% between 5 and 42 ng/ml, and the interassay CV was less than 18.5%.
LH assay.
LH concentrations were assayed in duplicate following the method of Lee et al. (41) using NIH-oLH-S18 as standard and NIDDK-anti-oLH-I antiserum. Iodinated ovine LH (125I-NIDDK-AFD-9598B) was used as tracer. Assay sensitivity was 0.1–03 ng/ml. The intraassay CV was less than 10% between 2 and 19 ng/ml and the interassay CV was less than 15.5%.
PRL assay.
Plasma PRL concentrations were measured using Sigma (St. Louis, MO) Lot 114F-0558, NOL-7135 as standard. Intrassay CV was less than 10% between 1 and 47 ng/ml, and the interassay CV was less than 12.6%.
Cortisol assay.
Plasma cortisol concentrations were assayed in duplicate following the method of Broadbear et al. (42) using antiserum no. 3368 (Bioquest Ltd., Melbourne, Victoria, Australia). 125I-labeled cortisol (Amersham Pharmacia Biotech Ltd., Amersham, Buckinghamshire, UK) was used as tracer. The sensitivity of the assay was 0.4 ng/ml, and the intra- and interassay coefficients of variation were 9.9% and 13.8%, respectively.
Statistical analysis
All data are presented as mean (±SEM). The secretory profiles for GH and LH were characterized using pulse analysis as described previously (43, 44). Repeated measures of ANOVA was used to analyze effects of ghrelin/vehicle on VFI and hormone levels, taking account of treatment, group, and order effects. The least significant differences method was used as a post hoc test to compare means.
Results
Ghrelin effect on VFI
In experiment 1, continuous infusion of 5 μg/h ovine ghrelin into the IIIV for 3 d had no effect on VFI (Fig. 1A) and neither did continuous infusion of 10 μg/h for 2 d followed by 20 μg/h for 1 d, have any effect (Fig. 1B). In experiment 2, single injection of either 1, 5, 10, and 20 μg of ovine or DAP-octanoyl3 human ghrelin into the IIIv 3 h after feeding also failed to affect VFI (Fig. 2, A–D). In experiment 3, multiple injections of 100 μg DAP-octanoyl3 human ghrelin into the LV at 0800, 1200, and 1600 h for 4 d did not stimulate either short-term or long term VFI, when the programmed feeding time was 1700 h (Fig. 3). In experiment 4, a single iv injection of 100 μg DAP-octanoyl3 human ghrelin had no effect on VFI, when scheduled feeding was at 0900 h and the ghrelin was administered at 1200 h (Fig. 4, A and B).
Ghrelin effect on plasma GH concentrations
In experiment 1, infusion of 5 μg/h of ovine ghrelin into the IIIv significantly (P < 0. 0.05) increased plasma GH levels and GH pulse amplitude within the period immediately after commencement of infusion, but the GH interpulse interval was unaltered (Fig. 5A and Table 1). When the dose was increased to 10 μg/h of ovine ghrelin a significant (P < 0.05), an increase in plasma GH concentrations was also seen immediately after commencement of infusion (Fig. 5B). In experiment 2, single bolus injection of DAP-octanoyl3 human ghrelin (Fig. 6A) or ovine (Fig. 6B) ghrelin into the IIIv significantly (P < 0.05 and P < 0.001) elicited an immediate GH response. The response to all doses of DAP-octanoyl3 human ghrelin was greater than the response to ovine ghrelin (Table 2).
Ghrelin effect on plasma LH concentrations
In experiment 1, continuous infusion of 5 μg/h of ovine ghrelin into the IIIv for 3 d did not affect mean plasma LH levels, pulse frequency or pulse amplitude (Table 3), within the period immediately after commencement of infusion or after 3 d of infusion. In experiment 2, DAP-octanoyl 3 human ghrelin was found to be more potent than ovine ghrelin as a stimulator of GH secretion (vide supra), so the samples from animals that received the former were assayed to analyze effects on plasma LH levels. Bolus IIIv injection of 1, 5, or 20 μg (but not 10 μg) of DAP-octanoyl3 human ghrelin suppressed the plasma LH levels to the same extent (Fig. 7, A and B).
Ghrelin effect on plasma PRL and cortisol levels
In experiment 1, administration of 5 μg/h of ovine ghrelin into the IIIv for 3 d did not affect the plasma PRL levels (data not shown), neither did bolus IIIv injection of 1, 5, 10, and 20 μg of DAP-octanoyl3 human ghrelin (experiment 2). A slight, albeit significant (P < 0.05), increase in plasma PRL levels was seen with injection of 5 μg (data not shown). No effects on plasma cortisol levels were seen with either infusion or bolus injection of ghrelin (data not shown).
Discussion
In a ruminant species, and using a variety of experimental paradigms, we have shown that ghrelin has consistent effects to stimulate GH secretion, but we have not observed any effect on food intake. This contrasts with results obtained in other species and suggests that ruminants may not be responsive to the orexigenic properties of ghrelin. This is surprising because clear preprandial elevation of ghrelin levels in plasma are seen in the ovine species (33, 34) as in other species (17, 30).
Ghrelin and food intake
Ghrelin has a rapid effect to stimulate food intake in satiated rats and reduces locomotor activity, leading to increased adiposity and body weight (17, 18, 19, 20, 29, 45, 46, 47, 48, 49, 50, 51). These effects are mediated via NPY neurons in the arcuate nucleus (46, 47, 52), which possess GHS-R (8), but orexin cells in the lateral hypothalamic area are also implicated (50). Ghrelin effects are indirectly transmitted to pro-opiomelanocortin neurons via NPY/agouti-related peptide neurons (53). Other mechanisms are clearly involved, however, because NPY knockout animals eat and grow normally (54). Ghrelin also enhanced appetite and increased food intake in humans that were fasted overnight (18). The predominant effect of ghrelin on food intake is thought to be via the arcuate nucleus, although injection into various other hypothalamic nuclei also stimulated food intake (19) and ghrelin-receptor agonists induce Fos protein expression in the area postrema (55). Reduction in GHS-R in the arcuate nucleus by production of transgenic rats expressing GHS-R antisense mRNA under the control of a tyrosine hydroxylase promoter led to reduced food intake, reduced adiposity and lowered body weight (56), suggesting that GHS-R action constitutively maintains feeding. Constitutive signaling, even in the absence of ligand has been proposed for the ghrelin receptor based on in vitro studies (57), and this could be operative in vivo in the ruminant. If this were the case, then inverse agonists would be expected to affect food intake in species such as the sheep. On the other hand, the phenotype of the ghrelin knockout mouse, which has no reduction in growth or body weight would argue against this (35). Species differences may be important in this regard, and the effects of receptor antagonists in the ruminant would be instructive.
Contrary to the observations made in rodents and humans (vide supra), central administration of ghrelin inhibited food intake in the neonatal chicken (32). Thus, the effects of ghrelin on the appetite regulating systems of the brain is not the same across species. In this paper, we report that VFI in a ruminant species is not influenced by ghrelin. In the first instance we administered ghrelin by constant intracerebroventricular infusion over 3 d, but this had no effect. This contrasts with the effect of leptin on food intake in sheep because third ventricular infusion of this anorexigenic hormone caused significant reduction in food intake within 24 h (44). We tested whether ghrelin might be more efficacious if given as a bolus injection or a series of bolus injections, but VFI was not affected in either case. Data from rodent species show that ghrelin affects short-term food intake and not necessarily long term food intake (17, 30, 37) Accordingly, we measured food intake at hourly intervals around central administration of ghrelin (experiment 3) and did not find any significant effect. Finally, we determined whether systemic (iv) injection of ghrelin might affect VFI, but no effect was seen. It is important to note that with both bolus injections and infusions, ghrelin stimulated GH secretion, indicating that the administered material was bioactive.
One difference between ruminant and monogastric species is the location of ghrelin-producing cells. In the monogastric, including the human, ghrelin-producing cells are found in the proximal region of the stomach (58), whereas ghrelin is produced in cells of the abomasum in the ruminant (59). The rumen, or first stomach, of the ruminant functions to grind food and houses a variety of microorganisms that digest cellulose; it is not emptied between meals. Furthermore, in the field situation, ruminants graze without defined feeding times, so the gastrointestinal tract is not emptied and ghrelin levels may not vary substantially due to the continuous presence of food. Nevertheless, when placed on programmed feeding regimens, sheep do show preprandial elevation and postprandial decrements in plasma ghrelin levels (33, 34), suggesting that it is not the relative level of gut-fill that determines excursions in ghrelin levels, but the anticipation of feeding. Ghrelin secretion in the sheep appears to be regulated by the autonomic nervous system (38). The sheep in our studies were on programmed feeding regimens, so the preprandial excursions in ghrelin were preserved. Substantial overlay of ghrelin (by continuous infusion) (experiment 2) or supplemental excursions (bolus injection) between feeding times (experiment 3), did not increase food intake, although GH secretion was stimulated. We conclude, therefore, that ghrelin does not have an acute or chronic effect to stimulate food intake in sheep.
Ghrelin is secreted from the stomach into plasma in acylated and des-acylated forms (60). The acylated (n-octanoyl at serine in position 3) ghrelin is involved in the regulation of GH, energy balance, and other biological activities, whereas des-acylated ghrelin is devoid of endocrine activities (61), despite significantly higher plasma levels of the des-acyl form (60). Recent studies in the rodents have shown that, in contrast to ghrelin, the des-acyl form decreases food intake and delays gastric emptying (37). This is substantiated by studies in humans, which showed that iv administration of des-acyl ghrelin counterbalances the metabolic effects of ghrelin (21, 62). In the present studies, ghrelin consistently affected GH secretion without effect on food intake, so it is a possibility that, in the ovine species, des-acyl ghrelin is more important than ghrelin in the regulation of appetite.
Ghrelin effects on GH secretion
In the adult animal, GH plays an important role in metabolic regulation (63, 64, 65) and plasma levels are affected by the level of adiposity [reviewed by Henry (66)]. GH secretion from the anterior pituitary somatotropes is regulated by the hypothalamic factors GHRH and somatostatin (SOM) [reviewed in McMahon et al. (5) and Muller et al. (67)], but ghrelin also appears to play a role. Certainly, there is good evidence of the direct action of GH secretagogues and ghrelin on the pituitary somatotropes (68). In humans, increased adiposity leads to low levels of ghrelin and GH (14), although this relationship does not hold for the sheep because plasma ghrelin levels were higher in relatively obese animals despite reduced plasma GH levels (69). The means by which ghrelin affects GH secretion could be through central action on GHRH and SOM cells or on other neuronal systems that regulate these hypophysiotropic systems (5, 11, 67). Indeed, data from sheep show that synthetic ligands of the ghrelin receptor (GH secretagogues) stimulate GHRH secretion in the sheep (70). Within the brain, GHS-R localize to GHRH and NPY cells in the arcuate nucleus (7, 8, 10), and NPY stimulates GH secretion (5). Thus, central effects of ghrelin to regulate the GH axis could be directly upon GHRH cells, indirectly via NPY cells that convey signals to GHRH cells, or on SOM cells. NPY and GHRH also colocalize in cells of the arcuate nucleus of rodents (71), humans (72), and sheep (73). Certainly, ghrelin activates NPY cells in the arcuate nucleus of the rat (52, 74), as demonstrated by Fos immunohistochemistry, and it is possible that this response is confined to a subset of NPY or NPY/GHRH cells that selectively regulate the GH axis, rather than food intake. In the sheep, it is unlikely that NPY-producing, non-GHRH cells of the arcuate nucleus directly affect the GHRH cells because the former do not appear to provide input to the latter (73).
Ghrelin administration also causes a Fos response in orexin-producing cells and increases SOM mRNA levels (50, 74). ORX receptors are also found in SOM cells (75), perhaps providing a conduit for regulation of the GH axis. In the sheep, SOM cells receive input from GHRH, galanin, NPY, and ORX, and GHRH cells receive input from ENK and SOM cells (73), but the extent to which various cell types express GHS-R in the ovine brain is not known. In the present study, a major objective was to determine effects of ghrelin on food intake, so most of our treatment paradigms involved central administration. The consistent GH responses obtained with these treatments strongly support the notion that ghrelin acts via central mechanisms to regulate this axis. It is unlikely that the mechanisms by which ghrelin acts centrally on the GH axis in the ovine is the same as that in the rat, based on the observation that altered body weight (by food restriction or fasting) causes opposite effects on GH secretion in the two species [reviewed in Henry (66)]. Furthermore, plasma ghrelin levels are similar in normal and food-restricted sheep (69), whereas levels are lower in obese humans (14) and higher in lean (30) humans. GH responses to DAP-octanoyl3 human ghrelin were greater than those to ovine ghrelin. This is most likely due to the greater stability of the DAP-octanoyl3 human ghrelin (Bowers, C. Y., Tulane University Health Sciences Center, New Orleans, LA; results published on www.phoenixpeptide.com).
Ghrelin effects on LH secretion
Chronic infusion of ovine ghrelin into the IIIv did not affect LH plasma levels, but bolus injection of various doses of DAP-octanoyl3 human ghrelin did so, which is most likely due to the greater stability of the DAP-octanoyl3 human ghrelin (vide supra). Infusion of 1, 5, and 20 μg human ghrelin significantly reduced LH secretion. There was no significant effect at the 10 μg dose, which is an obvious inconsistency. Nevertheless, the effects seen with the three other doses and the clear effect that is observed in the plasma profiles of treated animals (FIG. 7A) suggest that ghrelin does affect LH secretion as in rodents (22, 23) and monkeys (24). On the other hand, in vivo and in vitro studies in the goldfish, showed that ghrelin elevates LH levels (76) in this species. No effect of ghrelin was seen on plasma LH levels in the human (77). The present data are, therefore, consistent with those obtained in rats and monkeys.
Ghrelin effects on PRL and cortisol secretion
Most studies show that administration of synthetic GHS stimulates the HPA axis and PRL secretion in humans (21, 77, 78, 79, 80), although one (14) failed to show an effect on PRL levels. Studies in the rodents have shown that ip or central administration of ghrelin inhibits PRL secretion, whereas in vitro treatment of pituitary cells with ghrelin did not inhibit PRL secretion (81). In the present study, neither infusion of ovine ghrelin into the IIIv nor bolus injections of various doses of DAP-octanoyl3 human ghrelin into the IIIv had any substantial effect on plasma PRL levels, although a small increase was observed in animals that received a dose of 5 μg DAP-octanoyl3 human ghrelin. This suggests that ghrelin does not act via central mechanisms to regulate PRL secretion in this species, although a direct effect on the pituitary lactotropes cannot be ruled out. Ghrelin also stimulates the ACTH and cortisol secretion in humans (14, 21, 77, 79, 82), but we did not observe a similar effect in sheep with central administration. In rodents, iv injection of ghrelin did not stimulate PRL and ACTH secretion (83). We conclude that any effects of ghrelin on the secretion of PRL or ACTH in sheep are likely to be at the level of the pituitary gland.
In conclusion, this study demonstrates that, despite the well-demonstrated preprandial rise in plasma ghrelin levels in sheep, this hormone does not appear to enhance food intake in this species. It is possible that ghrelin is important in the initiation of ingestive behavior, but it does not increase the amount of food that animals eat. On the other hand, we consistently observed acute effects of ghrelin to stimulate GH secretion and to inhibit LH secretion. No effects were seen on the secretion of PRL or the HPA axis. Thus, ghrelin has specific central effects to regulate the GH axis and the reproductive axis in this species.
Acknowledgments
We thank Bruce Doughton, Lynda Morrish, and Karen Briscoe for animal care and Ms. Isabella Retamal for cortisol assays. We also gratefully acknowledge the supply of assay reagents by National Hormone and Peptide Program and Dr. A. Parlow, Harbor, UCLA (Torrance, CA).
Footnotes
Present address for I.C.: Department of Physiology, P.O. Box 13F, Monash University, Victoria 3800, Australia.
J.I. received support from Prince Henry’s Institute of Medical Research.
First Published Online October 6, 2005
Abbreviations: aCSF, Artificial cerebrospinal fluid; CV, coefficient of varation; DAP, ,-diaminopropanoic acid; GHS-R, GH secretagogue receptor; IIIv, third cerebral ventricle; LV, lateral cerebral ventricle; NPY, neuropeptide Y; PRL, prolactin; SOM, somatostatin; VFI, voluntary food intake.
Accepted for publication September 29, 2005.
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Laboratory of Animal Nutrient Metabolism (Y.K.), Kitasato University, Towada, Aomori 034, Japan
Department of Physiology (B.C.), Monash University, Victoria 3800, Australia
Abstract
Ghrelin is an endogenous ligand for the GH secretagogue/ghrelin receptor (GHS-R) and stimulates feeding behavior and GH levels in rodents and humans. A preprandial increase in plasma ghrelin levels is seen in sheep on programmed feeding, followed by a postprandial rise in plasma GH levels, but effects on food intake and endocrine function are not defined in this ruminant species. We administered ghrelin to female sheep in various modes and measured effects on voluntary food intake (VFI) and plasma levels of GH, LH, prolactin, and cortisol. Whether administered intracerebroventricularly or iv, ghrelin consistently failed to stimulate VFI. On the other hand, ghrelin invariably increased plasma GH levels and ,-diaminopropanoic acid-octanoyl3 human ghrelin was more potent than ovine ghrelin. Bolus injection of ghrelin into the third cerebral ventricle reduced plasma LH levels but did not affect levels of prolactin or cortisol. These findings suggested that the preprandial rise in plasma ghrelin that is seen in sheep on programmed feeding does not influence VFI but is likely to be important in the postprandial rise in GH levels. Thus, ghrelin does not appear to be a significant regulator of ingestive behavior in this species of ruminant but acts centrally to indirectly regulate GH and LH secretion.
Introduction
THE HYPOTHALAMUS is the neuroendocrine center of the brain and also plays an important role in the regulation of ingestive behavior. In particular, cells of the arcuate nucleus that produce neuropeptide Y (NPY), a potent orexigen (1) and the peptides encoded by the pro-opiomelanocortin gene (2) are important in energy homeostasis (1, 2, 3). GHRH is produced in cells of the arcuate nucleus that project to the external zone of the median eminence (4, 5). Secretion of this peptide into the hypophyseal portal system provides a specific stimulus for the release of GH from the pituitary (5). Cells of the arcuate nucleus, and other brain regions, express a GH secretagogue receptor (GHS-R) (6, 7, 8, 9, 10), and exogenous GH-releasing peptides (GHRP) can act centrally (11) to indirectly regulate the secretion of GH from the pituitary gland (12, 13) and other functions, such as food intake (14). GHS-R is also expressed in cells of the pituitary gland, so GHRP can also act directly at this level to stimulate the secretion of GH (12) and other pituitary hormones (12).
Ghrelin is a 28-amino acid peptide that was found in the stomach (15) and is an endogenous ligand for GHS-R (15). Accordingly, ghrelin stimulates GH secretion in humans and rodents (15, 16, 17, 18, 19, 20). Ghrelin has also been implicated in the regulation of other endocrine functions, with evidence of effects in the rat, human, and monkeys on the secretion of prolactin (PRL) (14, 21), adrenocorticotrophic hormone, cortisol (14, 21), and LH (22, 23, 24). Whereas leptin (25), insulin (26), and other peripheral factors act as satiety factors (27, 28), ghrelin stimulates food intake and regulates energy homeostasis (15, 16, 17, 18, 19, 20, 29).
In humans (17, 30), plasma ghrelin levels increase transiently before a scheduled meal and then decline postprandially. Furthermore, administration of exogenous ghrelin causes hyperphagia in rats (19, 20) and humans (18, 31). On the other hand, central administration of ghrelin inhibited food intake in the chicken (32). Date et al. (16) reported that continuous intracerebroventricular infusion of ghrelin to male rats increased plasma GH concentrations after 6 d, but levels were not elevated after 12 d, although effects on food intake were not reported. As in humans and rats, a preprandial rise in plasma ghrelin levels is seen in sheep on programmed feeding (33, 34). Despite these strong indications that ghrelin is important in the regulation of food intake, genetic knockout of ghrelin leads to a minimal phenotype in terms of food intake and body composition (35, 36). Paradoxically, overexpression of the ghrelin gene reduced food intake through hypothalamic mechanisms (37).
Little is known of the role of ghrelin in the regulation of food intake and endocrine function in species other than humans and rodents. The ruminant presents an interesting model because the gut is not emptied between periods of feeding. Despite this, there is a well-discerned rise in plasma ghrelin levels before an expected meal in sheep, with a postprandial rise in plasma GH levels (33, 34) and ghrelin secretion from the stomach may be regulated centrally through cholinergic neurons of the vagus (38). We have investigated the effects of chronic and acute infusion of ghrelin on food intake and on plasma levels of GH, LH, PRL, and cortisol in sheep.
Materials and Methods
Ethics
All animal procedures were conducted in compliance with the Code of Ethics specified by the National Health and Medical Research Council of Australia, and prior approval was obtained from the Animal Experimentation Ethics Committee of Monash Medical Centre and the Victorian Institute of Animal Science.
Animals and surgery
Corriedale ewes with a mean body weight of 44.6 ± 1.2 kg were used during the breeding season. The animals were ovariectomized at least 1 month before surgery to avoid cyclic variations in plasma levels of ovarian steroids. In some cases, the animals were fitted with guide tubes into the third cerebral ventricle (IIIv) as previously described (39) and in other cases, an infusion line was introduced into one lateral cerebral ventricle (LV) (vide infra). Briefly, the animals were introduced to single pens in an experimental facility at least 1 week before experimentation for familarization and adjustment to a standard diet. They were fed an ad libitum diet (2.5 kg Lucerne chaff), once a day, at a time predetermined for each experiment, and were subject to normal environmental variation in temperature and photoperiod. Voluntary food intake (VFI) was recorded by measuring refusals. For the purpose of sampling blood, a dwelling cannula (Tuta Healthcare, Sydney, New South Wales, Australia) was introduced to one external jugular vein and kept patent with heparinized (50 U/ml) normal saline. A manometer line (Smiths Medical Australasia Pty. Ltd., Gold Coast, Queensland, Australia) was attached to the cannula, closed with a three-way tap and attached to the side of the pen so that samples could be taken without disturbing the animal. Blood samples (5 ml) were collected into heparinized tubes and centrifuged at 4 C to obtain plasma, which was stored at –20 C until assayed.
LV cannulation
Animals were anesthetized with iv injection of Thiopentone (20 mg/kg; Thiobarb Sodium, Jurox Ltd., Rutherford, New South Wales, Australia) and maintained with halothane (1.5–2.5%) inhalation in oxygen. Preoperatively, Rimadyl (4.0 mg/kg; Pfizer Ltd., West Ryde, New South Wales, Australia), was administered iv for analgesia and Oxytet-200 L.A. (1 ml/kg body weight; Troy Laboratories Ltd., Smithfield, New South Wales, Australia) was injected im as an antibiotic. The head was laid on a box containing polystyrene beads and held by a strap over the nose. A midline incision was made from the pole of the head running caudally over the parietal bone. A burr hole was made in the parietal bone 10–15 mm caudal to Bregma and 10–15 mm off midline. A customized apparatus was attached to the skull to hold a length of 18-gauge stainless steel tubing fitted with an obdurator. This was advanced at an angle of approximately 20 degrees, 16–18 mm below the surface of the brain and the obdurator removed; flow of cerebrospinal fluid confirmed placement in the LV. A length of SILASTIC brand tubing (catalog no. 508 003, Dow Corning, Seven Hills, New South Wales, Australia) was introduced to the LV and advanced 10 mm beyond the tip of the guide-tubing. Placement was confirmed by injection of a radio-opaque dye (Omnipaque, Nycomed Australia Pty. Ltd., North Ryde, New South Wales, Australia) and the tip of the cannula was placed at the level of the Foramen of Munro. The SILASTIC brand tubing was secured to the skull with Superglue, coiled under the skin, and exteriorized at the back of the neck. The animals were allowed to recover and kept at pasture until required for experimentation.
Central infusion of ghrelin or vehicle was achieved by connection of IIIv or LV infusion lines to Graseby MS16A infusion pumps (Graseby Medical Ltd., Gold Coast, Queensland, Australia). For IIIv infusion, a 19-gauge stainless steel tubing assembly that was introduced into the IIIv at least 2 mm beyond the end of the guide tube. For LV infusion, the pumps were connected directly to the SILASTIC brand tubing that was exteriorized at the neck. The infusion rate was 60 μl/h. Vehicle for central infusions was artificial cerebrospinal fluid (aCSF; 150 mm NaCl, 1.2 mmCaCl, 1 mm MgCl, and 2.8 mm KCl).
Ghrelin for infusion
Synthetic ovine ghrelin (GSSFLSPEHQKLQ-RKEPKKPSGRLKPR) was synthesized by Peptide Institute Inc. (Osaka, Japan) and [dap (,-diaminopropanoic acid)-octanoyl3]-human ghrelin (catalog no. 031-83) [(DAP-octanoyl3) human ghrelin] was obtained from Phoenix Pharmaceuticals Inc. (Belmont, CA).
Experiment 1: effects of chronic IIIv ovine ghrelin infusion
Part 1.
Groups (n = 5) received either aCSF or ovine ghrelin (5 μg/h) infused into the IIIv for 3 d. On d 1, blood samples were collected at 10-min intervals for 12 h (0600–1800 h) and the infusions commenced at 1200 h. VFI was measured. Plasma samples were assayed for GH, LH, PRL, and cortisol.
Part 2.
Because there were no effects of 5 μg/h ovine ghrelin infusion on VFI, a further experiment was conducted in which animals received infusions of either aCSF (n = 5) or 10 μg/h ovine ghrelin (n = 5) into the IIIv for 2 d, and VFI was measured (as above). Because no effect was seen on VFI, the dose of ovine ghrelin was increased to 20 μg/h for further 24 h. Blood samples were collected (as above) for 6 h immediately before infusion and 6 h immediately after the commencement of infusion (0600–1800 h on d 1) to measure the short-term effect of ghrelin infusion on plasma GH levels. Plasma samples were assayed for GH only.
Experiment 2: dose-response effects of ovine and DAP-octanoyl3 human ghrelin administered to the IIIv
Because ovine ghrelin did not affect VFI in experiment 1, studies were conducted to compare responses to ovine and DAP-octanoyl3 human ghrelin. The animals were prepared as described for experiment 1. To test the short-term effects of central infusion and compare the efficacy of ovine and DAP-octanoyl 3 human ghrelin, doses of 1, 5, 10, or 20 μg of aCSF were injected into the IIIV (n = 4/group) in a volume of 60 μl at 1200 h. The animals were fed 2.5 kg Lucerne chaff/d at 0900 h daily, and VFI was measured. Blood samples were taken at 10-min intervals between 1100 and 1300 h, at 20-min intervals between 1300 and 1500 h and at 30-min intervals between 1500 and 1600 h. Plasma samples were assayed for GH, LH, PRL, and cortisol.
Experiment 3: effects of multiple DAP-octanoyl3 human ghrelin injections into the LV
Animals were randomly divided into two groups (n = 5/group) and fed at 1700 h. They were injected with either 100 μg DAP-octanoyl3 human ghrelin or aCSF (100 μl) into the LV at 0800, 1200, and 1600 h each day for 4 d. VFI was measured.
Experiment 4: effects of iv injection of DAP-octanoyl3 human ghrelin on VFI
Because the chronic infusion of ovine ghrelin and single/multiple central injections of ovine/ DAP-octanoyl3 human ghrelin did not affect VFI, we considered the possibility that ghrelin has a peripheral action that has a secondary effect on the brain to regulate feeding behavior. The animals were fed at 0900 h, and daily VFI was monitored for 5 d and then animals (n = 5/group) received iv injections of either 100 μg of DAP-octanoyl3 human ghrelin or saline at 1200. After injection, VFI was measured for 1, 2, 3, and 12 h after injection.
RIAs
GH assay.
GH concentrations were measured by the method of Thomas et al. (40) using NIDDK-oGH-1–4 as standard and NIDDK-anti-oGH-2 antiserum. Assay sensitivity was 0. 4–1 ng/ml. The intraassay coefficient of variation (CV) was less than 10% between 5 and 42 ng/ml, and the interassay CV was less than 18.5%.
LH assay.
LH concentrations were assayed in duplicate following the method of Lee et al. (41) using NIH-oLH-S18 as standard and NIDDK-anti-oLH-I antiserum. Iodinated ovine LH (125I-NIDDK-AFD-9598B) was used as tracer. Assay sensitivity was 0.1–03 ng/ml. The intraassay CV was less than 10% between 2 and 19 ng/ml and the interassay CV was less than 15.5%.
PRL assay.
Plasma PRL concentrations were measured using Sigma (St. Louis, MO) Lot 114F-0558, NOL-7135 as standard. Intrassay CV was less than 10% between 1 and 47 ng/ml, and the interassay CV was less than 12.6%.
Cortisol assay.
Plasma cortisol concentrations were assayed in duplicate following the method of Broadbear et al. (42) using antiserum no. 3368 (Bioquest Ltd., Melbourne, Victoria, Australia). 125I-labeled cortisol (Amersham Pharmacia Biotech Ltd., Amersham, Buckinghamshire, UK) was used as tracer. The sensitivity of the assay was 0.4 ng/ml, and the intra- and interassay coefficients of variation were 9.9% and 13.8%, respectively.
Statistical analysis
All data are presented as mean (±SEM). The secretory profiles for GH and LH were characterized using pulse analysis as described previously (43, 44). Repeated measures of ANOVA was used to analyze effects of ghrelin/vehicle on VFI and hormone levels, taking account of treatment, group, and order effects. The least significant differences method was used as a post hoc test to compare means.
Results
Ghrelin effect on VFI
In experiment 1, continuous infusion of 5 μg/h ovine ghrelin into the IIIV for 3 d had no effect on VFI (Fig. 1A) and neither did continuous infusion of 10 μg/h for 2 d followed by 20 μg/h for 1 d, have any effect (Fig. 1B). In experiment 2, single injection of either 1, 5, 10, and 20 μg of ovine or DAP-octanoyl3 human ghrelin into the IIIv 3 h after feeding also failed to affect VFI (Fig. 2, A–D). In experiment 3, multiple injections of 100 μg DAP-octanoyl3 human ghrelin into the LV at 0800, 1200, and 1600 h for 4 d did not stimulate either short-term or long term VFI, when the programmed feeding time was 1700 h (Fig. 3). In experiment 4, a single iv injection of 100 μg DAP-octanoyl3 human ghrelin had no effect on VFI, when scheduled feeding was at 0900 h and the ghrelin was administered at 1200 h (Fig. 4, A and B).
Ghrelin effect on plasma GH concentrations
In experiment 1, infusion of 5 μg/h of ovine ghrelin into the IIIv significantly (P < 0. 0.05) increased plasma GH levels and GH pulse amplitude within the period immediately after commencement of infusion, but the GH interpulse interval was unaltered (Fig. 5A and Table 1). When the dose was increased to 10 μg/h of ovine ghrelin a significant (P < 0.05), an increase in plasma GH concentrations was also seen immediately after commencement of infusion (Fig. 5B). In experiment 2, single bolus injection of DAP-octanoyl3 human ghrelin (Fig. 6A) or ovine (Fig. 6B) ghrelin into the IIIv significantly (P < 0.05 and P < 0.001) elicited an immediate GH response. The response to all doses of DAP-octanoyl3 human ghrelin was greater than the response to ovine ghrelin (Table 2).
Ghrelin effect on plasma LH concentrations
In experiment 1, continuous infusion of 5 μg/h of ovine ghrelin into the IIIv for 3 d did not affect mean plasma LH levels, pulse frequency or pulse amplitude (Table 3), within the period immediately after commencement of infusion or after 3 d of infusion. In experiment 2, DAP-octanoyl 3 human ghrelin was found to be more potent than ovine ghrelin as a stimulator of GH secretion (vide supra), so the samples from animals that received the former were assayed to analyze effects on plasma LH levels. Bolus IIIv injection of 1, 5, or 20 μg (but not 10 μg) of DAP-octanoyl3 human ghrelin suppressed the plasma LH levels to the same extent (Fig. 7, A and B).
Ghrelin effect on plasma PRL and cortisol levels
In experiment 1, administration of 5 μg/h of ovine ghrelin into the IIIv for 3 d did not affect the plasma PRL levels (data not shown), neither did bolus IIIv injection of 1, 5, 10, and 20 μg of DAP-octanoyl3 human ghrelin (experiment 2). A slight, albeit significant (P < 0.05), increase in plasma PRL levels was seen with injection of 5 μg (data not shown). No effects on plasma cortisol levels were seen with either infusion or bolus injection of ghrelin (data not shown).
Discussion
In a ruminant species, and using a variety of experimental paradigms, we have shown that ghrelin has consistent effects to stimulate GH secretion, but we have not observed any effect on food intake. This contrasts with results obtained in other species and suggests that ruminants may not be responsive to the orexigenic properties of ghrelin. This is surprising because clear preprandial elevation of ghrelin levels in plasma are seen in the ovine species (33, 34) as in other species (17, 30).
Ghrelin and food intake
Ghrelin has a rapid effect to stimulate food intake in satiated rats and reduces locomotor activity, leading to increased adiposity and body weight (17, 18, 19, 20, 29, 45, 46, 47, 48, 49, 50, 51). These effects are mediated via NPY neurons in the arcuate nucleus (46, 47, 52), which possess GHS-R (8), but orexin cells in the lateral hypothalamic area are also implicated (50). Ghrelin effects are indirectly transmitted to pro-opiomelanocortin neurons via NPY/agouti-related peptide neurons (53). Other mechanisms are clearly involved, however, because NPY knockout animals eat and grow normally (54). Ghrelin also enhanced appetite and increased food intake in humans that were fasted overnight (18). The predominant effect of ghrelin on food intake is thought to be via the arcuate nucleus, although injection into various other hypothalamic nuclei also stimulated food intake (19) and ghrelin-receptor agonists induce Fos protein expression in the area postrema (55). Reduction in GHS-R in the arcuate nucleus by production of transgenic rats expressing GHS-R antisense mRNA under the control of a tyrosine hydroxylase promoter led to reduced food intake, reduced adiposity and lowered body weight (56), suggesting that GHS-R action constitutively maintains feeding. Constitutive signaling, even in the absence of ligand has been proposed for the ghrelin receptor based on in vitro studies (57), and this could be operative in vivo in the ruminant. If this were the case, then inverse agonists would be expected to affect food intake in species such as the sheep. On the other hand, the phenotype of the ghrelin knockout mouse, which has no reduction in growth or body weight would argue against this (35). Species differences may be important in this regard, and the effects of receptor antagonists in the ruminant would be instructive.
Contrary to the observations made in rodents and humans (vide supra), central administration of ghrelin inhibited food intake in the neonatal chicken (32). Thus, the effects of ghrelin on the appetite regulating systems of the brain is not the same across species. In this paper, we report that VFI in a ruminant species is not influenced by ghrelin. In the first instance we administered ghrelin by constant intracerebroventricular infusion over 3 d, but this had no effect. This contrasts with the effect of leptin on food intake in sheep because third ventricular infusion of this anorexigenic hormone caused significant reduction in food intake within 24 h (44). We tested whether ghrelin might be more efficacious if given as a bolus injection or a series of bolus injections, but VFI was not affected in either case. Data from rodent species show that ghrelin affects short-term food intake and not necessarily long term food intake (17, 30, 37) Accordingly, we measured food intake at hourly intervals around central administration of ghrelin (experiment 3) and did not find any significant effect. Finally, we determined whether systemic (iv) injection of ghrelin might affect VFI, but no effect was seen. It is important to note that with both bolus injections and infusions, ghrelin stimulated GH secretion, indicating that the administered material was bioactive.
One difference between ruminant and monogastric species is the location of ghrelin-producing cells. In the monogastric, including the human, ghrelin-producing cells are found in the proximal region of the stomach (58), whereas ghrelin is produced in cells of the abomasum in the ruminant (59). The rumen, or first stomach, of the ruminant functions to grind food and houses a variety of microorganisms that digest cellulose; it is not emptied between meals. Furthermore, in the field situation, ruminants graze without defined feeding times, so the gastrointestinal tract is not emptied and ghrelin levels may not vary substantially due to the continuous presence of food. Nevertheless, when placed on programmed feeding regimens, sheep do show preprandial elevation and postprandial decrements in plasma ghrelin levels (33, 34), suggesting that it is not the relative level of gut-fill that determines excursions in ghrelin levels, but the anticipation of feeding. Ghrelin secretion in the sheep appears to be regulated by the autonomic nervous system (38). The sheep in our studies were on programmed feeding regimens, so the preprandial excursions in ghrelin were preserved. Substantial overlay of ghrelin (by continuous infusion) (experiment 2) or supplemental excursions (bolus injection) between feeding times (experiment 3), did not increase food intake, although GH secretion was stimulated. We conclude, therefore, that ghrelin does not have an acute or chronic effect to stimulate food intake in sheep.
Ghrelin is secreted from the stomach into plasma in acylated and des-acylated forms (60). The acylated (n-octanoyl at serine in position 3) ghrelin is involved in the regulation of GH, energy balance, and other biological activities, whereas des-acylated ghrelin is devoid of endocrine activities (61), despite significantly higher plasma levels of the des-acyl form (60). Recent studies in the rodents have shown that, in contrast to ghrelin, the des-acyl form decreases food intake and delays gastric emptying (37). This is substantiated by studies in humans, which showed that iv administration of des-acyl ghrelin counterbalances the metabolic effects of ghrelin (21, 62). In the present studies, ghrelin consistently affected GH secretion without effect on food intake, so it is a possibility that, in the ovine species, des-acyl ghrelin is more important than ghrelin in the regulation of appetite.
Ghrelin effects on GH secretion
In the adult animal, GH plays an important role in metabolic regulation (63, 64, 65) and plasma levels are affected by the level of adiposity [reviewed by Henry (66)]. GH secretion from the anterior pituitary somatotropes is regulated by the hypothalamic factors GHRH and somatostatin (SOM) [reviewed in McMahon et al. (5) and Muller et al. (67)], but ghrelin also appears to play a role. Certainly, there is good evidence of the direct action of GH secretagogues and ghrelin on the pituitary somatotropes (68). In humans, increased adiposity leads to low levels of ghrelin and GH (14), although this relationship does not hold for the sheep because plasma ghrelin levels were higher in relatively obese animals despite reduced plasma GH levels (69). The means by which ghrelin affects GH secretion could be through central action on GHRH and SOM cells or on other neuronal systems that regulate these hypophysiotropic systems (5, 11, 67). Indeed, data from sheep show that synthetic ligands of the ghrelin receptor (GH secretagogues) stimulate GHRH secretion in the sheep (70). Within the brain, GHS-R localize to GHRH and NPY cells in the arcuate nucleus (7, 8, 10), and NPY stimulates GH secretion (5). Thus, central effects of ghrelin to regulate the GH axis could be directly upon GHRH cells, indirectly via NPY cells that convey signals to GHRH cells, or on SOM cells. NPY and GHRH also colocalize in cells of the arcuate nucleus of rodents (71), humans (72), and sheep (73). Certainly, ghrelin activates NPY cells in the arcuate nucleus of the rat (52, 74), as demonstrated by Fos immunohistochemistry, and it is possible that this response is confined to a subset of NPY or NPY/GHRH cells that selectively regulate the GH axis, rather than food intake. In the sheep, it is unlikely that NPY-producing, non-GHRH cells of the arcuate nucleus directly affect the GHRH cells because the former do not appear to provide input to the latter (73).
Ghrelin administration also causes a Fos response in orexin-producing cells and increases SOM mRNA levels (50, 74). ORX receptors are also found in SOM cells (75), perhaps providing a conduit for regulation of the GH axis. In the sheep, SOM cells receive input from GHRH, galanin, NPY, and ORX, and GHRH cells receive input from ENK and SOM cells (73), but the extent to which various cell types express GHS-R in the ovine brain is not known. In the present study, a major objective was to determine effects of ghrelin on food intake, so most of our treatment paradigms involved central administration. The consistent GH responses obtained with these treatments strongly support the notion that ghrelin acts via central mechanisms to regulate this axis. It is unlikely that the mechanisms by which ghrelin acts centrally on the GH axis in the ovine is the same as that in the rat, based on the observation that altered body weight (by food restriction or fasting) causes opposite effects on GH secretion in the two species [reviewed in Henry (66)]. Furthermore, plasma ghrelin levels are similar in normal and food-restricted sheep (69), whereas levels are lower in obese humans (14) and higher in lean (30) humans. GH responses to DAP-octanoyl3 human ghrelin were greater than those to ovine ghrelin. This is most likely due to the greater stability of the DAP-octanoyl3 human ghrelin (Bowers, C. Y., Tulane University Health Sciences Center, New Orleans, LA; results published on www.phoenixpeptide.com).
Ghrelin effects on LH secretion
Chronic infusion of ovine ghrelin into the IIIv did not affect LH plasma levels, but bolus injection of various doses of DAP-octanoyl3 human ghrelin did so, which is most likely due to the greater stability of the DAP-octanoyl3 human ghrelin (vide supra). Infusion of 1, 5, and 20 μg human ghrelin significantly reduced LH secretion. There was no significant effect at the 10 μg dose, which is an obvious inconsistency. Nevertheless, the effects seen with the three other doses and the clear effect that is observed in the plasma profiles of treated animals (FIG. 7A) suggest that ghrelin does affect LH secretion as in rodents (22, 23) and monkeys (24). On the other hand, in vivo and in vitro studies in the goldfish, showed that ghrelin elevates LH levels (76) in this species. No effect of ghrelin was seen on plasma LH levels in the human (77). The present data are, therefore, consistent with those obtained in rats and monkeys.
Ghrelin effects on PRL and cortisol secretion
Most studies show that administration of synthetic GHS stimulates the HPA axis and PRL secretion in humans (21, 77, 78, 79, 80), although one (14) failed to show an effect on PRL levels. Studies in the rodents have shown that ip or central administration of ghrelin inhibits PRL secretion, whereas in vitro treatment of pituitary cells with ghrelin did not inhibit PRL secretion (81). In the present study, neither infusion of ovine ghrelin into the IIIv nor bolus injections of various doses of DAP-octanoyl3 human ghrelin into the IIIv had any substantial effect on plasma PRL levels, although a small increase was observed in animals that received a dose of 5 μg DAP-octanoyl3 human ghrelin. This suggests that ghrelin does not act via central mechanisms to regulate PRL secretion in this species, although a direct effect on the pituitary lactotropes cannot be ruled out. Ghrelin also stimulates the ACTH and cortisol secretion in humans (14, 21, 77, 79, 82), but we did not observe a similar effect in sheep with central administration. In rodents, iv injection of ghrelin did not stimulate PRL and ACTH secretion (83). We conclude that any effects of ghrelin on the secretion of PRL or ACTH in sheep are likely to be at the level of the pituitary gland.
In conclusion, this study demonstrates that, despite the well-demonstrated preprandial rise in plasma ghrelin levels in sheep, this hormone does not appear to enhance food intake in this species. It is possible that ghrelin is important in the initiation of ingestive behavior, but it does not increase the amount of food that animals eat. On the other hand, we consistently observed acute effects of ghrelin to stimulate GH secretion and to inhibit LH secretion. No effects were seen on the secretion of PRL or the HPA axis. Thus, ghrelin has specific central effects to regulate the GH axis and the reproductive axis in this species.
Acknowledgments
We thank Bruce Doughton, Lynda Morrish, and Karen Briscoe for animal care and Ms. Isabella Retamal for cortisol assays. We also gratefully acknowledge the supply of assay reagents by National Hormone and Peptide Program and Dr. A. Parlow, Harbor, UCLA (Torrance, CA).
Footnotes
Present address for I.C.: Department of Physiology, P.O. Box 13F, Monash University, Victoria 3800, Australia.
J.I. received support from Prince Henry’s Institute of Medical Research.
First Published Online October 6, 2005
Abbreviations: aCSF, Artificial cerebrospinal fluid; CV, coefficient of varation; DAP, ,-diaminopropanoic acid; GHS-R, GH secretagogue receptor; IIIv, third cerebral ventricle; LV, lateral cerebral ventricle; NPY, neuropeptide Y; PRL, prolactin; SOM, somatostatin; VFI, voluntary food intake.
Accepted for publication September 29, 2005.
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