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NEUROENDOCRINOLOGY |
University of Manchester School of Biological Sciences, Manchester, United Kingdom M13 9PT
Address all correspondence and requests for reprints to: Dr. Simon Luckman, 1.124 Stopford Building, University of Manchester School of Biological Sciences, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: simon.luckman{at}man.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise mechanism of the anabolic actions of GHSs has yet to be fully clarified, although they do not appear to act indirectly via the secretion of GH (12, 15, 17, 18, 21). Previous functional mapping studies have shown that systemic or central administration of GHSs in rodents induce the immediate-early gene c-fos only in the hypothalamic arcuate nucleus (22, 23, 24) despite the relatively wide distribution of the receptor within the brain (5, 6, 7, 8). The highest proportion of arcuate neurons activated by systemic GHS contain NPY (25), and many more neurons are activated if the animal is first fasted, a manipulation known to remove inhibitory influences from NPY neurons (26). Moreover, greater than 90% of arcuate NPY neurons possess GHS-R mRNA, suggesting that they are an important target (27). In support, it has been reported that blocking the action of endogenous NPY inhibits GHS-induced feeding in rodents (15, 16, 17, 18).
Recent studies have demonstrated that additional brain regions can express c-Fos protein after systemic synthetic GHS (28, 29) or central administration of ghrelin (17, 30). However, no systematic quantification of cell number or identification of neuronal phenotype activated by ghrelin in these additional areas has been reported. Furthermore, it has yet to be determined whether the neuronal activation observed after GHSs is secondary to food intake, because the consumption of a large meal may itself lead to c-Fos expression in the brain. For example, a difference in the c-Fos expression pattern is observed between groups of animals allowed access to food or not after central NPY injection (31, 32).
Thus, in this study we compared firstly the effects of central administration of ghrelin and the synthetic GHS, GH-releasing peptide-6 (GHRP-6), on feeding and core body temperature. Secondly, we examined whether the feeding response to ghrelin or GHRP-6 was affected by preadministration of a Y1 NPY receptor-selective antagonist (BIBO3304). Thirdly, we quantified the induction of c-Fos (as a marker for neuronal activation) in the forebrain and brainstem and determined whether any of the neuronal activity observed was secondary to food intake rather than a consequence of the initial stimulus. Finally, as arcuate NPY- containing neurons are implicated in the feeding response of GHSs, and these neurons project to orexin- and melanin-concentrating hormone (MCH)-containing neurons in the lateral hypothalamus (33, 34), we sought to determine whether these latter cell types are activated by GHS treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exp 1: food intake and core body temperature
Animals were injected icv with either vehicle (2 µl saline;
n = 8) or GHRP-6 (2 µg in 2 µl; n = 8;
Bachem, Saffron Walden, UK). In a separate experiment,
groups of animals (n = 6/group) were injected icv with either
vehicle (2 µl saline) or ghrelin (0.01, 0.1, or 1 µg in 2 µl;
Phoenix Pharmaceuticals, Inc., Belmont, CA). Immediately
after injections animals were presented with a preweighed amount of
chow, and food consumption was measured 1, 2, 3, and 4 h later.
Core body temperature was monitored continuously throughout the
experimental procedure.
Exp 2: NPY antagonist pretreatment
In two separate experiments animals (n = 5–13/group) were
injected icv with either vehicle (4 µl water) or BIBO3304 (30 µg in
4 µl; gift from Boehringer-IngelheimPharma KG, Biberach,
Germany). Fifteen minutes later the animals were given vehicle (1 or 2
µl saline), GHRP-6 (2 µg in 2 µl), or ghrelin (1 µg in 1 µl).
Food consumption was measured 1, 2, and 3 h later.
Exp 3: c-Fos immunohistochemistry
Rats (n = 5/group) were given icv injections of GHRP-6 (2
µg), ghrelin (0.1 µg), or the equivalent volume of vehicle (2 µl
saline), followed by a preweighed amount of food. Ninety minutes after
peptide administration food intake was measured, and the rats were
anesthetized with sodium pentobarbitone (90 mg/kg, ip; Sagatal,
Rhône-Mérieux, Harlow, UK) and perfused transcardially with
fixative (4% paraformaldehyde). Equivalent 30-µm forebrain or
brainstem sections from each brain, 90 µm apart, were cut on a sledge
microtome. Free-floating sections were incubated with a rabbit
polyclonal anti-c-Fos antibody (1:1000; Oncogene Research Products,
Cambridge, MA) and then with a peroxidase-labeled goat antirabbit IgG
antibody (1:500; Vector Laboratories, Inc., Burlingame,
CA). Nuclear c-Fos was visualized using a nickel-intensified
diaminobenzidine reaction to produce a black precipitate. The number of
neurons expressing c-Fos was counted bilaterally in nuclei defined by
the atlas of Paxinos and Watson (35). The number of
immunopositive neurons per section was assessed for each animal. These
values were then averaged to determine a group mean for each area of
the brain.
Exp 4: effect of food intake on c-Fos immunohistochemistry
Rats (n = 5/6/group) were given an icv injection of either
vehicle (2 µl saline) or GHRP-6 (2 µg). After injections animals
were either allowed access to food or food was withheld. Ninety minutes
later food intake was measured (where appropriate), and transcardial
perfusion followed by immunohistochemistry for c-Fos protein was
carried out as described above (see Exp 3).
Exp 5: c-Fos and orexin or MCH immunohistochemistry
Rats were injected icv with either vehicle (2 µl saline;
n = 6) or GHRP-6 (2 µg; n = 6). After injections animals
were allowed access to food, which was measured 90 min later, and
transcardial perfusion was performed. Two sets of forebrain sections
were taken through the level of the lateral hypothalamus (-2.12 to
-4.52 mm to bregma) (35), and immunohistochemistry for
c-Fos protein was carried out as described above (see Exp 3). Sections
then were incubated sequentially in either a rabbit polyclonal antibody
raised against orexin A (1:100; Oncogene Research Products), or MCH
(1:1000; Phoenix Pharmaceuticals, Inc.), biotinylated
antirabbit IgG (1: 200; Vector Laboratories, Inc.), and
then a streptavidin-biotin-peroxidase complex (1:200; Amersham Pharmacia Biotech, Little Chalfont, UK). Cytoplasmic orexin or
MCH staining was visualized by a normal diaminobenzidine reaction to
yield a brown precipitate. The number of neurons expressing c-Fos
and/or orexin or MCH was counted bilaterally in the lateral
hypothalamus as defined by the atlas of Paxinos and Watson
(35).
Statistical analyses
All data are presented as the mean ± SEM. Core
body temperatures were plotted as the mean change from the point of
injection (time zero). Statistical analyses were carried out using
two-tailed t tests for two group experiments and ANOVA
followed by post-hoc Tukey multiple comparisons test for
experiments containing three or more groups. For the analysis of c-Fos
expression, when SD values were not equal between
groups, these parametric tests were replaced by nonparametric
Mann-Whitney U tests or Kruskal-Wallis ANOVA, followed by
Dunn’s multiple comparisons test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The effect on food intake began
within the first hour postinjection, was almost complete at the 2
h point, and was increased 30-fold at 4 h relative to the control.
Intracerebroventricular injection of GHRP-6 also caused a rapid
reduction in core body temperature, which reached a nadir 30 min after
injection (change in temperature at 30 min: control, +0.3 ± 0.1
C; GHRP-6, -1.2 ± 0.1 C; P < 0.001) and had
returned to control values by 120 min (Fig. 1B).
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). However, results of ANOVA and
post-hoc tests showed significant increases in food intake
only after 1 µg ghrelin at 1 h and 0.1 and 1 µg at 2, 3, and
4 h after injection. Ghrelin (0.1 and 1 µg) also caused a rapid
hypothermic response that was temporally similar to that observed after
GHRP-6 injection, but the magnitude of the change was smaller (change
in temperature at 30 min: control, +0.1 ± 0.1 C; 0.01 µg,
+0.1 ± 0.1 C; 0.1 µg, -0.6 ± 0.2 C; 1 µg, -0.8
± 0.2 C; P < 0.05 and P < 0.01
vs. control for 0.1 and 1 µg, respectively).
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). Similarly,
ghrelin (1 µg)-induced food intake was significantly inhibited at 1,
2, and 3 h postinjection by BIBO3304 (30 µg; Fig. 3B). Central
administration of vehicle or BIBO3304 alone in either experiment had no
effect on feeding.
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A distinctive pattern of neuronal activation, as assessed by
immunohistochemistry for the protein product of the immediate-early
gene, c-fos, was noted after the injection of ghrelin or
GHRP-6 compared with the control. Significant increases in the number
of c-Fos-positive neuronal profiles were recorded in the hypothalamic
arcuate nucleus, the hypothalamic paraventricular nucleus, the
dorsomedial hypothalamus, the lateral hypothalamus, and the nucleus of
the tractus solitarius and the area postrema of the
brainstem (Figs. 4 and 5). The induction of c-Fos in the
hypothalamic arcuate nucleus was mostly in the medial regions and was
relatively strong compared with staining in other regions. There was
also a small increase in the average number of c-Fos-positive profiles
in the ventromedial hypothalamus, which was statistically significant
only in the GHRP-6-treated animals. No c-Fos staining above background
was recorded in the hypothalamic supraoptic nucleus or the brainstem
dorsal motor nucleus of the vagus nerve (results not shown). Generally,
there were no major differences noted between the pattern of response
between injection of ghrelin and GHRP-6, although there were
significantly more c-Fos-positive cells in the nucleus of the tractus
solitarius and the area postrema after GHRP-6 injection.
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The pattern of c-Fos-positive profiles induced by icv injection of
GHRP-6 in animals allowed access to food was comparable to that
reported in Exp 3. Furthermore, equivalent pattern and number of cells
expressing c-Fos were observed in GHRP-6-treated animals that had food
withheld (Fig. 6). Significant increases
in the induction of c-Fos were observed in both groups of animals given
GHRP-6 (plus or minus food) compared with their appropriate control in
the hypothalamic arcuate nucleus, hypothalamic paraventricular nucleus,
dorsomedial hypothalamus (only in the minus food group), lateral
hypothalamus, and nucleus of the tractus solitarius and the area
postrema of the brainstem.
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, A and B). However there was no
significant difference in the percentage of MCH neurons expressing
c-Fos between control and GHRP-6-treated animals (control, 6 ±
1%; GHRP-6, 5 ± 1%; P > 0.05; Fig. 7, C and
D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 2000, Tschop and colleagues (21) reported that the
orexigenic effects of ghrelin are present in NPY-deficient mice, thus
questioning the role of NPY neurons in this response. However, this
knockout mouse may not be an ideal model. It does not have the expected
lean phenotype (36) and can respond to other stimuli
thought to be mediated by these neurons (37). Other
transmitters, present in NPYergic neurons (e.g.
agouti-related protein and 
-aminobutyric acid) and other parallel
pathways (e.g. 
MSH in POMC neurons), may not be affected.
The results presented here and previously (15, 16, 17, 18) using
NPY receptor antagonists are more supportive of a key role for central
NPY pathways in mediating the effects of GHSs on food intake.
Additional evidence includes the location of GHS-R mRNA in arcuate NPY
neurons (27) as well as the activation of these neurons by
systemic or central injections of GHSs (17, 25). Two
caveats concerning the hypothesis that the major target for GHSs is
arcuate NPY neurons need mention. First, the use of receptor
antagonists does not distinguish between NPY derived from neurons
in the arcuate nucleus and those in the dorsomedial hypothalamic
nucleus that may also form part of the pathway. Second, antagonism
of signaling through Y1 NPY receptors may cause anxiety in rodents (for
review, see Ref. 38); thus, the specificity of receptor
antagonism should be viewed cautiously.
Using immunohistochemistry for the protein product of the immediate-early gene, c-fos, central administration of GHRP-6 was reported previously to activate only cells of the arcuate nucleus (23). However, more recently, central administration of ghrelin has been shown to activate other regions in both the forebrain (17) and the brainstem (30), although no quantification was carried out. Here, we demonstrate a more extensive neuronal activation by icv injection of GHSs (ghrelin and GHRP-6), with significant increases in the expression of c-Fos observed in the arcuate, paraventricular, dorsomedial, and lateral hypothalamic nuclei, and in two regions of the brainstem, the nucleus of the tractus solitarius and the area postrema. The pattern and magnitude of c-Fos induction described above were similar in animals injected with doses of ghrelin or GHRP-6 that caused comparable food consumption. An exception was that more Fos-positive cells were noted in the nucleus of the tractus solitarius and the area postrema in animals treated with GHRP-6 compared with ghrelin injection.
The differences between the present and a previous study using central administration of GHRP-6 (23) could be due to the relative weakness of c-Fos protein immunostaining in the other brain regions compared with that in the arcuate nucleus. Whether the weaker c-Fos staining was due to these neurons being synaptically, rather than directly, activated cannot be determined from our results, and further anatomical studies, involving tract tracing, and electrophysiology will be required. GHS-R mRNA is located in several brain regions where c-Fos expression was observed, including the arcuate and paraventricular hypothalamic nuclei and the brainstem (including the area postrema) (5, 6, 7), suggesting that these neurons may be directly activated. Furthermore, a more recent publication has detected low levels of GHS-R mRNA in the lateral and dorsomedial hypothalamic areas (8). c-Fos is useful only as a marker for electrically activated neurons, and it is clear that GHSs are equally capable of directly inhibiting hypothalamic neurons (39), which may include anorexigenic mediators. This fact may explain some of the discrepancies between levels of c-Fos induction and the presence of GHS-R, for example in the ventromedial nucleus.
Satiety signals from the gastrointestinal tract are detected by vagal nerve afferents that project to the brainstem nucleus of the tractus solitarius (40, 41). Central processing of these signals can be detected as the induction of c-Fos in brainstem structures (42), suggesting that some of the c-Fos detected may be secondary to the rats consuming a large meal. However, the present study illustrates that the GHS, GHRP-6, activated neurons independently of food intake, as the pattern and extent of c-Fos expression were similar in animals that had food withheld. Thus, the current findings demonstrate that cell activation in the brain after GHS is not due to satiety signaling from the gastrointestinal tract. Instead, some of the activity may be related to efferent signaling from the brain back down to the gut, although we found no evidence of activity in the dorsal motor nucleus of the vagus nerve.
The mediators of the actions of GHS on feeding, perhaps downstream of arcuate NPY neurons, remain to be clarified. In the present study GHSs activated additional hypothalamic nuclei, for example the lateral hypothalamus, an area that contains other orexigenic peptides, including orexin and MCH. As NPY-containing neurons of the arcuate hypothalamic nucleus project directly to lateral hypothalamic orexin- and MCH-containing neurons (33, 34), we sought to determine the phenotype of the cells activated by GHS in the lateral hypothalamus. Our data clearly show that central GHSs activated orexin, but not MCH, neurons. It is possible, therefore, that orexin-containing neurons of the lateral hypothalamus are activated indirectly via arcuate NPY neurons and that GHSs may selectively activate certain orexigenic pathways.
The major source of ghrelin is the stomach (4), and the level of circulating ghrelin fluctuates through the day according to food intake, with starvation increasing ghrelin and meals suppressing it (21). There is only one published report on the presence of ghrelin in the hypothalamic arcuate nucleus determined by immunohistochemistry (4). Until this is confirmed, one might question whether the fluctuations of ghrelin measured in plasma are sufficient to stimulate a feeding response under physiological conditions. It may be more likely that circulating ghrelin can access the brain at the arcuate nucleus to modulate anabolic and catabolic pathways in an opposing way to other hormones, such as leptin and insulin. However, pharmacological activation of GHS-R by central administration of GHS is a useful method for the study of orexigenic pathways.
In summary, we have shown that ghrelin and a synthetic GHS, GHRP-6, are potent inducers of food intake when injected centrally. We hypothesize that NPY neurons of the arcuate nucleus are a primary target for this action of GHSs, and that the stimulation of central NPYergic pathways may lead to further neuronal activation of orexigenic circuits. An additional opposing effect on anorexigenic pathways is likely.
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Footnotes |
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Abbreviations: GHRP-6, GH-releasing peptide-6; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; icv, intracerebroventricular; MCH, melanin-concentrating hormone.
Received July 26, 2001.
Accepted for publication September 10, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or
2 µl saline (control; 
) on cumulative food intake (A) and change
in core body temperature (B) in satiated rats (1000 h injection). All
data are the mean ± SEM (n = 8 in each group).
*, P < 0.05; ***, P < 0.001
(vs. control).
), or 1 µg (
) ghrelin or 2 µl saline (control; 
