Orexin Expression and Function: Glucocorticoid Manipulation, Stress, and Feeding Studies
http://www.100md.com
《内分泌学杂志》
Henry Wellcome Laboratory for Integrative Neuroscience and Endocrinology (G.K.F., M.S.H., D.S.J.), University of Bristol, Bristol BS1 3NY, United Kingdom
Department of Biology (D.N.C.J.), Psychiatry Centre of Excellence for Drug Discovery (CEDD) and Discovery Research (S.W.), GlaxoSmithKline, Harlow Essex CM19 5AW, United Kingdom
Metabolic Diseases (K.A.A.-B.), Metabolic and Viral Diseases CEDD, GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Abstract
We investigated the effects of glucocorticoid manipulation on orexin-A-induced feeding and prepro-orexin mRNA levels in the lateral hypothalamic area (LHA) of the rat brain. Adrenalectomy (ADX) reduced orexin-A-induced feeding over 4 h by about 60%, compared with shams, an effect that was reversed by corticosterone (CORT) replacement. ADX had no effect on prepro-orexin mRNA levels in the LHA in either the morning or the evening; however, message was up-regulated by CORT in the morning but not the evening. An increased number of emulsion grains per cell in the LHA suggests that this is a specific increase in prepro-orexin mRNA and is not due to an increased number of cells expressing message. Prepro-orexin mRNA levels in the LHA were elevated 4 h after injection of lipopolysaccharide, compared with saline-injected controls. Partial but not complete abolition of orexin-A-induced feeding by ADX suggests that orexin-A-induced feeding may be mediated through glucocorticoid-dependent and glucocorticoid-independent pathways. In the morning increased prepro-orexin mRNA after CORT replacement demonstrates that orexin expression is sensitive to increased concentrations of glucocorticoids. However, the lack of effect of ADX on prepro-orexin mRNA levels suggests that endogenous glucocorticoids are not involved in tonic regulation of basal prepro-orexin expression. Overall our data constitute a body of evidence for an integrated relationship between central orexin expression, stress, glucocorticoid manipulation, and feeding patterns in the rat.
Introduction
OREXIN (OX)-A AND OX-B (also known as hypocretins) are closely related neuropeptides derived from a single gene and processed from a 130-residue (131 residues in humans) prepro-OX precursor protein (1). OX-A is a 33-residue peptide with identical sequence in humans and rodents, whereas human and rat OX-B differ by two residues. OX-A and OX-B activate two G protein-coupled receptors known as OX-1 and OX-2, the OX-1 receptor having a greater affinity for OX-A over OX-B, whereas the OX-2 receptor has similar affinity for both ligands. Central nervous system (CNS) distribution of orexins is similar in rats and humans, the lateral hypothalamic area (LHA) being the predominant region of synthesis (1).
The LHA has long been implicated in feeding behavior and initial observations highlighted the role of orexins in stimulating appetite (2). However, it is now clear that orexins are also involved in the regulation of sleep, arousal, locomotor activity and neuroendocrine responses (3, 4), and therefore their orexigenic activity may be secondary to other behavioral traits that influence feeding. In behavioral studies, orexins can modulate stress-related behavior such as grooming, and chewing of inedible material (5, 6, 7). Orexin-containing neurones project from the LHA to many CNS areas involved in mediating stress including the paraventricular nucleus (PVN) of the hypothalamus, the arcuate nucleus (ARC), and the locus coeruleus (8, 9, 10). OX-1 and OX-2 receptors are widely and differentially distributed throughout the CNS including the PVN, suprachiasmatic nucleus, supraoptic nucleus, and ARC of the hypothalamus and also the locus coeruleus and hippocampus (11, 12, 13, 14). These areas have individual and synergistic involvement in regulating circadian rhythm and behavioral and neuroendocrine responses to stressors.
The hypothalamo-pituitary-adrenal (HPA) axis is one of the principal pathways mediating neuroendocrine responses to stress. Hypothalamic neuropeptides corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) stimulate the release of the proopiomelanocortin-derived peptide ACTH from the anterior pituitary, with consequent release of glucocorticoids from the adrenal cortex. Corticosterone (CORT), the principal glucocorticoid in rodents, acts through glucocorticoid receptors in the hippocampus and hypothalamus and mineralocorticoid receptors within the hippocampus to negatively regulate HPA axis activity (15). Many studies have demonstrated that orexins can influence HPA axis activity. Plasma ACTH and CORT concentrations are elevated in rats after intracerebroventricular (icv) OX-A administration (5, 16, 17, 18, 19, 20, 21, 22, 23). This is accompanied by the induction of c-fos mRNA (17) and AVP and CRF mRNAs (20) in the parvocellular subdivision of the PVN. In addition, in vitro studies have revealed release of CRF peptide, but not AVP, from hypothalamic explants (19). OX-B has also been reported to stimulate HPA axis activity (16). Orexin expression in the rat LHA is increased after stressors such as immobilization (23), cold stress (23), or hypoglycemia (24). Reduced secretion of plasma ACTH and cortisol is reported in patients with narcolepsy (25), a condition associated with orexin deficiency. Therefore there is considerable evidence that orexins can modulate HPA axis activity and that orexin expression is responsive to alterations in HPA axis activity.
It is well documented that glucocorticoid administration stimulates feeding and conversely that glucocorticoid deficiency results in loss of appetite and weight loss because adrenalectomized (ADX) rats exhibit decreased feeding, which can be reversed by CORT administration (26, 27). Therefore, orexins may not exert a direct effect on appetite regulation within the hypothalamus but may act indirectly to stimulate feeding through increased CORT secretion. To test this hypothesis, we designed experiments to investigate the effect of ADX with or without CORT replacement on OX-A-induced feeding to determine whether endogenous CORT is involved in OX-A-induced feeding behavior. We also investigated the effects of glucocorticoid manipulation on orexin expression in the morning and evening to determine whether orexin expression, which exhibits a diurnal rhythm of expression (28, 29), may be correlated with the diurnal rhythm of HPA axis activity. Finally, we investigated the effects on orexin expression of lipopolysaccharide (LPS), an immunological stressor that elicits a robust and prolonged activation of the HPA axis (30, 31).
Materials and Methods
Animals
Adult male Sprague Dawley rats (200–250 g, Charles River, Margate, Kent, UK) were housed individually under controlled light and temperature (21 ± 2 C), with free access to water and standard rat pelleted chow and maintained on a 12-h light, 12-h dark cycle (lights on 0600 h). All procedures were carried out in accordance with the Animals Scientific Procedures Act (1986) United Kingdom.
Surgical procedures
Intracerebroventricular cannulation.
All rats were anesthetized by im injection of Domitor (medetomidine HCl, 0.04 ml per 100 g; Pfizer, Sandwich, Kent, UK) and an ip injection of Sublimaze (fentanyl, 0.9 ml per 100 g; Janssen-Cilag, High Wycombe, Buckinghamshire, UK). Rats were positioned in a stereotaxic frame and implanted with a 22-gauge stainless steel guide cannula in the left lateral ventricle of the brain under sterile conditions. The cannula was secured on the surface of the skull with jeweler’s screws and dental cement. Stereotaxic coordinates for the left brain ventricle were as follows: 0.8 mm caudal from Bregma, 1.5 mm lateral from the midline, and 4.1 mm vertical from the skull surface, incisor bar at 3.2 mm below zero. On completion of the surgery the anesthesia was reversed by an ip of a 50:50 mixture of Antisedan (atipamezole HCl, 0.02 ml per 100 g; Pfizer) and Nubain (nalbuphine HCl, 0.02 ml per 100 g; DuPont Pharmaceuticals, Stevenage, Hertfordshire, UK). After implantation of the guide cannula, correct placement was checked by using the gravitational flow of a sterile saline filled cannula.
Adrenalectomy.
Rats were bilaterally ADX via a dorsal approach. A small incision was made along the midline of the back just below the rib cage, connective tissue and fat was displaced, and a small hole was made through the muscle either side of the back using blunt cut scissors. The adrenals were excised with curved forceps. Each adrenal was checked that it had remained intact after excision. Only animals that had complete removal of both adrenals were considered ADX. After surgery, all ADX animals were immediately given 0.9% saline to drink to maintain their salt balance. Surgery for sham animals was identical, but the adrenals were not removed. Animals were allowed 7 d postoperative recovery and were handled daily.
Effects of glucocorticoids on OX-A-induced feeding
Thirty-two rats were bilaterally ADX, whereas 16 rats were subjected to sham ADX. Animals were returned to their home cages and all ADX animals were immediately given 0.9% saline, whereas two groups of ADX rats were given 0.9% saline supplemented with CORT (Sigma, Poole, Dorset, UK), either 25 mg/liter (low CORT) or 125 mg/liter (high CORT). These concentrations of CORT were selected as they are within the basal physiological range of endogenous CORT (27). On the morning of experimentation, sham and ADX animals were divided into two groups (n = 9) of equal mean body weights to receive icv injection of either OX-A (30 μg/rat in 5 μl of 0.9% saline) or saline. This dose, equivalent to 8 nmol, falls within the accepted range of 3–30 nmol (2) that has been used extensively. Both low CORT and high CORT replacement groups also received an icv injection of either OX-A (30 μg/rat) or saline. An icv injection cannula with extension tubing, preloaded with drug or vehicle, was inserted into the guide cannula. Injections were given over a period of 1 min. The needle was left in position for a further minute to ensure complete dispersal of the peptide. Injections were given between 0900 and 1000 h. Preweighed food pellets and water bottles were supplied after icv injection. Food intake was determined at 1-, 2-, 3-, and 4-h time points after icv injection. After 4 h, all rats were killed by decapitation, and trunk blood was collected into chilled EDTA tubes on ice for centrifugation to collect plasma. ACTH was measured by RIA after prior extraction from plasma on Seppak columns (32). Assay sensitivity was 10 pg/ml. Total CORT was measured in unextracted plasma by an in-house RIA (33). The radioactive tracer was I125-CORT (ICN Biomedicals, Basingstoke, Hampshire, UK) with a specific activity of 0.37 MBq. The sensitivity of the assay was 5 ng/ml. Both ACTH and CORT primary antisera were kindly supplied by G. Makara (Hungarian Academy of Sciences, Budapest, Hungary). All samples for either ACTH or CORT were processed in the same assay, with intraassay variation less than 10%.
Distribution of prepro-OX expression through the lateral hypothalamus
Two male Sprague Dawley rats were killed by decapitation, and brains were removed and stored at –80 C before sectioning. Coronal brain sections (12 μm) were collected through the LHA from the PVN on a cryostat and immediately stored at –80 C. Two sections were collected every four sections, and 48–50 sections were collected from each animal. Prepro-OX expression was investigated using in situ hybridization, which was performed as described previously (34, 35). Sections were warmed to room temperature for 10 min and fixed with 4% formaldehyde for 5 min, washed twice in PBS, and then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl for 10 min. Sections were then dehydrated through graded ethanol washes: 70% (1 min), 80% (1 min), 95% (2 min), and 100% (1 min), and then delipidated in chloroform 100% for 5 min and then rehydrated in 100% (1 min) and 95% (1 min) ethanol before drying in air. The probe used was a 33-mer oligonucleotide complementary to nucleotides 242–275 of the exonic sequence of rat prepro-OX mRNA 5'-TGCCCGCGGCGTGGTTGCCAGCTCCGTGCAACA-3' (GenBank accession no. AF041241). Probes were labeled at the 3' end with 35S-ATP and column purified by NucTrap probe purification columns (Stratagene, UK). Probes were labeled to a specific activity of 1.4x 1018 dpm/mol. Forty-five microliters of hybridization buffer [50% formamide, 4x saline sodium citrate (SSC), 0.5 mg/ml sheared single-stranded salmon sperm DNA, 0.25 mg/ml yeast tRNA, 0.5 x Denhardt’s solution, and 10% dextran sulfate], containing labeled probe (100,000 cpm) was applied to each slide. Hybridization was performed overnight at 37 C. All sections for each experiment were processed at the same time. The sections were washed four times at 55 C in 1x SSC [1x SSC = 0.15 M NaCl/0.015 M sodium citrate (pH 7.0)], followed by two 30-min washes at room temperature in 1x SSC to remove nonspecific binding before two brief rinses in distilled water. The sections were air dried and exposed to Amersham HyperfilmMP (Amersham International, Aylesbury, Buckinghamshire, UK) together with 14C-labeled standard for 14 d. The autoradiographic images of probe bound to brain sections were analyzed using a densitometry method described previously (35), using Image software. The results are presented as arbitrary values of prepro-OX expression through the LHA.
Effect of adrenalectomy and glucocorticoid manipulation on prepro-OX expression in the lateral hypothalamus
Fifteen rats were bilaterally ADX as described above, whereas 12 rats were subjected to sham ADX. Eight animals were given 0.9% saline, whereas seven of the ADX rats and eight of the sham ADX rats were given 0.9% saline supplemented with CORT (high CORT, 125 mg/liter) as described above. The remaining sham ADX animals were given tap water to drink. Animals were placed on this regimen for 7 d and were handled daily. On the morning of the experiment, all rats were killed by decapitation, brains were removed, and trunk blood was collected into chilled EDTA tubes on ice before ACTH and CORT RIAs as described above. Sections were cut on the cryostat from the two LHA areas determined from the distribution study: LHA1 (Bregma –2.30 mm; interaural 6.70 mm) and LHA2 (bregma –2.56 mm; interaural 6.44 mm from the atlas of Paxinos and Watson (36). Nine consecutive coronal lateral hypothalamic brain sections were collected spanning 108 μm and each slide had three sections encompassing 84 μm of LHA to allow for the apparent variation in prepro-OX expression demonstrated to be present through the LHA from the results of the distribution study. Prepro-OX mRNA measurement by in situ hybridization was performed as described above. All ADX and ADX + CORT hybridized sections were dipped into autoradiographic emulsion and exposed at 4 C for 1 wk. All sections were processed at the same time. The slides were then developed, fixed, and counterstained with methyl green.
Seven emulsion-dipped slides per treatment were analyzed at the cellular level, counting the number of labeled cells and silver grains per cell. At least two to five cells per section (minimum eight cells per slide with three sections per slide) were randomly selected under x200 magnification on a microscope (Leica, Heidelberg, Germany) and saved as images. Silver grains were counted by hand from microscope images enlarged by 100% in Photo Editor (Microsoft, Redmond, CA). Silver grains were dotted onto plastic sheets attached to the monitor screen and then counted. Silver grains in each individual cell were counted three times in this manner. Sixty-one to 67 cells were counted for each treatment.
Effect of diurnal variation on prepro-OX expression in ADX rats given glucocorticoid replacement
Thirty-three rats were bilaterally ADX, whereas 13 rats were subjected to sham ADX as described previously. Seventeen ADX rats were given 0.9% saline, whereas 16 were given 0.9% saline supplemented with CORT (125 mg/liter). All sham animals were supplied with tap water to drink. Animals were left on this regimen for 7 d and handled daily. On the morning of experimentation, five sham, eight ADX-only, and seven ADX + CORT rats were killed by decapitation in the morning (0900–1000 h), and the remaining animals were killed in the evening (1800 h). Brains and trunk blood was collected as described previously and prepro-OX measurements and sectioning were performed as described above.
Effects of an LPS injection on prepro-OX mRNA
All animals received an ip injection (0900–1000 h) of either LPS (250 μg/rat in 0.5 ml 0.9% saline) (Escherichia coli, Serotype 055:B5; Sigma) or saline. Animals were killed by decapitation 4 h after injection. Brains and trunk blood were collected and prepro-OX mRNA was measured as described above. Plasma ACTH and CORT were measured by RIAs.
Statistics
Statistical comparisons between multiple groups were made using the Fisher protected least significant difference test after one-way ANOVA. For the LPS study, a Student’s t test was used. All values are expressed as mean ± SEM. P < 0.05 was considered significant. All analyses were generated using Statview.
Results
Effects of glucocorticoids on OX-A-induced feeding
Plasma ACTH and CORT were measured to confirm the success of ADX. Four h after icv injection, all ADX rats had concentrations of CORT at or below the limit of detection (5 ng/ml), compared with the sham + vehicle group (31 ± 7 ng/ml) and sham + OX-A group (24 ± 8 ng/ml). There was no significant difference in plasma CORT concentrations between the sham + OX-A and sham + vehicle groups. Plasma CORT concentrations in the low CORT and high CORT replacement groups were 7.1 ± 1.1 and 7.3 ± 0.6 ng/ml, respectively. Plasma ACTH concentrations were significantly elevated in all ADX groups, compared with sham groups; the increase in ACTH was attenuated by high CORT but not low CORT replacement (Table 1).
After 4 h, food intake was significantly greater in the sham + OX-A (4.1 ± 0.6 g) group, compared with the sham + vehicle group (0.3 ± 0.2 g) (Fig. 1). ADX significantly reduced OX-A-induced food intake (2.1 ± 0.7 g) by about 60%, a reduction that was reversed by high CORT (4.9 ± 0.8 g) but not low CORT replacement. Food intake was significantly greater in the ADX + OX-A rats (2.1 ± 0.7 g), compared with sham + vehicle (0.3 ± 0.2 g) and ADX + vehicle (0.6 ± 0.3 g) groups.
Distribution of prepro-OX expression through the lateral hypothalamus
There was considerable variation in prepro-OX mRNA expression levels throughout the LHA (Fig. 2). However, in sections collected caudal to the PVN area at 1.34 mm from the end of the anterior commissure, the profile of expression tended to peak and trough around similar areas and in some cases prepro-OX levels overlapped between the two animals. Areas LH1 and LH2 were areas of overlap in prepro-OX mRNA between the two brains, which were clearly defined anatomically and therefore permitted greater reproducibility.
Effect of adrenalectomy and glucocorticoid manipulation on prepro-OX expression in the lateral hypothalamus
Concentrations of plasma CORT were measured to confirm the success of ADX. All ADX rats had concentrations of plasma CORT below the limit of assay detection (5 ng/ml), compared with the sham group (21 ± 11 ng/ml). ADX animals with CORT replacement had CORT concentrations of 43 ± 12 ng/ml, compared with the sham + CORT group (126 ± 53 ng/ml).
There was no effect of ADX on prepro-OX mRNA levels in the LHA1 (Fig. 3A) and LHA2 (Fig. 3B) areas, compared with sham animals. Both sham and ADX groups with CORT replacement had significantly elevated levels of prepro-OX mRNA in the LHA1 and LHA2, compared with sham and ADX groups without CORT replacement.
Representative autoradiographic images of prepro-OX mRNA expression and distribution in the LHA2 area are shown in Fig. 4 for each of the treatment groups. Photomicrographs of emulsion dipped slides showing prepro-OX mRNA expression in individual cells are shown in Fig. 5. These images demonstrate that prepro-OX mRNA is expressed in cells of different size. The number of silver grains per cell of individual prepro-OX gene-expressing cells in the emulsion-dipped slides was significantly (P < 0.001) increased in the LHA of ADX rats receiving CORT replacement, compared with ADX rats (Fig. 6). The amount of orexin expression is remarkably consistent for a given treatment with 125 ± 4 grains/cell in the ADX group, compared with 187 ± 6 grains/cell in the ADX + CORT group. There was no significant difference in the number of cells expressing prepro-OX mRNA (ADX = 61 cells and ADX+CORT = 67 cells counted in the selected fields of view).
Effect of diurnal variation on orexin expression in adrenalectomized rats on glucocorticoid replacement
Plasma CORT concentrations in sham animals were significantly higher (P < 0.001) in the evening (111 ± 24 ng/ml), compared with the morning (20 ± 8 ng/ml). Plasma CORT in both morning and evening ADX groups was below the limit of detection (5 ng/ml). CORT replacement in ADX animals resulted in similar morning (43 ± 12 ng/ml) and evening concentrations (36 ± 14 ng/ml), which were not significantly different from morning concentrations in the sham group .
ADX had no effect on morning levels of prepro-OX mRNA in the LHA1, whereas CORT replacement up-regulated orexin mRNA in both sham and ADX groups (Fig. 3A). Consistent with this observation, there was no significant effect of ADX on prepro-OX mRNA expression in the LHA1 area in the morning or evening (Fig. 7). CORT replacement significantly elevated prepro-OX mRNA levels in the morning, compared with sham and ADX groups, but this increase was not observed in the evening (Fig. 7).
Effect of an LPS stress on prepro-OX expression
LPS evoked a 4-fold increase in plasma CORT concentrations (184 ± 44 ng/ml), compared with saline controls (49 ± 17 ng/ml). There was a significant 60% increase in prepro-OX mRNA expression in the LHA1 4 h after LPS injection, compared with saline-injected rats (Fig. 8).
Discussion
Our data reveal an integrated relationship between central orexin expression, endogenous glucocorticoid concentrations, and feeding patterns in the rat. Our observation that central administration of OX-A increases food intake is in agreement with other groups that have demonstrated that OX-A stimulates feeding in the early light phase in intact rats (2, 37, 38). We now demonstrate for the first time that glucocorticoids exert an important influence on OX-A-induced feeding behavior. ADX significantly reduces orexin-A- induced feeding, a phenomenon that is reversible by CORT replacement at physiological concentrations. These data provide evidence that endogenous CORT can exert an important influence on OX-A-induced food intake. However, because ADX does not completely block OX-A-induced feeding but results in about a 60% reduction, this suggests that there is a pathway mediating orexin-induced food intake that is independent of glucocorticoids.
One recent study has investigated the effect of ADX on OX-A-induced feeding (39). In contrast to our findings, these investigators observed no effect of ADX on OX-A-induced food intake over a 2-h period (39). This may be due to the suboptimal doses of OX-A employed over a dose range 1.5–6 nmol, compared with 8 nmol in the present study, the latter dose being considered optimal for OX-A-induced feeding behavior (2). These findings may also be a result of the shorter period of food intake studied (maximum of 2 h, compared with a maximum of 4 h in our study). In the paper by Drazen et al. (39), it seems evident that ADX is associated with decreased food intake at both higher doses of OX-A at the 2-h time point, but the very small increase in food intake in response to OX-A in sham rats does not permit the ADX effect to reach significance. The larger dose of orexin that we used elicited a greater food intake in the sham rats, compared with that observed by Drazen et al. (39), and consequently we were able to observe a significant and reproducible decrease in orexin-induced food intake in ADX rats.
Initially, this experiment was performed with only ADX and sham groups given orexin or saline. Consequent to our observation of a significant decrease in orexin-induced food intake in the ADX group, we repeated the experiment and expanded it to include low CORT and high CORT replacement groups. Our observations were reproducible at all time points when the full study was repeated (40). This, together with the ability of physiological concentrations of CORT to reverse the effects of ADX on orexin-induced food intake, increases our confidence that endogenous glucocorticoids exert physiologically relevant control over orexin-induced feeding behavior.
Our observation that orexin-induced food intake is decreased by about 60% in ADX rats is evidence that a significant component of eating behavior is dependent on endogenous CORT. It is known that CORT can directly stimulate appetite (41), but the indirect effects on appetite of CORT mediated through hypothalamic neuropeptides is not well understood. A number of mechanisms may be invoked to explain the relationship among CORT, OX-A, and food intake. CORT may stimulate endogenous orexin expression through an action at glucocorticoid receptors within the LHA or intermediary nuclei, it may act via orexin peptide release within the PVN/ARC areas of the hypothalamus, or CORT may stimulate food intake itself consequent to orexin activation of HPA axis activity. There is also a possibility that CORT may exert its effect on food intake via other peptides that modulate appetite rather than directly through orexins. There is a well-defined pathway of glucocorticoid feedback on CRF expression in the PVN and release from the median eminence (42). CRF inhibits food intake and therefore increased CORT could increase food intake through its inhibitory action on CRF. Orexin-induced feeding is enhanced by a CRF receptor antagonist, suggesting CRF involvement (43). Neuropeptide Y (NPY) is also a possible mediator of orexin-induced feeding. Glucocorticoid manipulation results in a marked similarity between orexin and NPY effects on feeding. ADX reduced NPY-induced food intake by about 60%, similar to the effects of ADX on orexin-induced feeding, and this was reversed by CORT replacement (44). NPY is the most potent known inducer of food intake (45), and orexin-induced feeding can be blocked by a Y1 receptor antagonist (46). Therefore, it is conceivable that orexin exerts its principal glucocorticoid-dependent actions on food intake through NPY, and it is the direct effects of CORT on NPY, not on orexin expression, which explains our observations. However, we have observed a 40% increase in food intake in response to OX-A in ADX rats in the complete absence of CORT, which is strong evidence for a glucocorticoid-independent central pathway of direct orexin-induced food intake that may not be mediated through NPY.
In situ hybridization histochemistry confirmed that prepro-orexin expression was restricted to the LHA, with extensions to the perifornical nucleus and posterior hypothalamic areas, in agreement with previously reported studies (37). A novel finding from the distribution study was that prepro-OX mRNA expression measured from coronal sequential sections running 1044 μm throughout the LHA shows considerable differences between sections, emphasizing the importance of consistent choice of sections for hybridization in measuring prepro-OX mRNA in the LHA between treatment groups. This observation confirms the need to use anatomical markers to ensure consistency of sectioning between samples.
We observed an up-regulation of morning but not evening prepro-OX mRNA in the LHA after CORT administration. Increased blood CORT may up-regulate orexin expression through the large population of glucocorticoid receptor-positive cells within the LHA (47, 48). Emulsion staining established that there was no significant difference in the number of cells expressing prepro-OX between treatment groups. Therefore, the increases in prepro-OX mRNA that we observed in response to CORT represent increased expression of prepro-OX within cells of the LHA. There was a slight increase in prepro-OX mRNA expression in the sham group in the evening, compared with the morning, although this was not statistically significant. Plasma CORT levels were significantly increased in the evening, compared with the morning, in sham animals in agreement with previous studies demonstrating diurnal variations in HPA axis activity (49). However, it is important to note that CORT levels were similar in both the morning and evening replacement groups and not significantly different from morning levels in the sham animals. It is possible that the increase in evening prepro-OX expression, although not statistically significant, may be a consequence of the circadian increase in endogenous circulating CORT. This increase may be sufficient to mask any further stimulatory effects on orexin expression by exogenous CORT. Further investigations of interrelationships between the circadian cycles of HPA axis activity and orexin expression will undoubtedly shed light on regulatory mechanisms controlling the highly integrated modalities of sleep-wake patterns and feeding behavior.
We found no effect of ADX alone on orexin expression in the LHA, which suggests that basal orexin expression in rats is not under tonic regulation by endogenous glucocorticoids. In contrast, a decrease in prepro-OX mRNA expression 5 d after ADX has been reported (50). However, no data on HPA axis activity were presented in this study, so it is difficult to determine the effectiveness of ADX surgery. In addition, there was no information about where sections were sliced from within the LHA. Given the variability of prepro-OX expression, which we observed throughout the LHA, it is important that the choice of section is justified and well defined. The decrease in orexin expression after ADX was reversed by dexamethasone replacement (50), an observation that is consistent with our data demonstrating CORT up-regulation of orexin expression in sham animals. A decrease in prepro-OX mRNA levels in the LHA 10 d after ADX has also been reported, a phenomenon that could not be reversed by CORT (39).
Discrepancies between our data and published reports on the effects of ADX on prepro-OX mRNA expression in the LHA (39, 50), and lack of agreement on the effects of exogenous glucocorticoids, may be due to methodological differences. Our experiment used a 7-d ADX with CORT replacement, compared with previous studies using 5 d ADX with dexamethasone (50) and 10 d ADX with CORT (39). It is instructive to compare these discrepancies with the literature on NPY expression after ADX. Although there is a general consensus that NPY is up-regulated by glucocorticoids, reports differ on the effects on NPY expression of eliminating endogenous cortisosterone by ADX. Short-term ADX (4 d) did not alter NPY peptide levels in the ARC or PVN (51), whereas the same group observed that long-term ADX (12 d) deceased NPY peptide levels in these areas (52). ADX (7 d) had no effect on NPY immunoreactivity in most hypothalamic areas, one notable exception being the PVN (53). A longer-term ADX (12 d) had no effect on prepro-NPY mRNA in the ARC (54); peptide levels were not measured in this study. Either dexamethasone or CORT replacement up-regulated NPY mRNA, but dexamethasone was much more potent (53). Therefore, any effects of glucocorticoid manipulation on orexin expression, as with NPY, may be dependent on the period of ADX, the concentration and type of glucocorticoid, whether orexin peptide or mRNA is measured, and how specifically defined is the hypothalamic site selected for analysis.
Finally, we have demonstrated that a single injection of LPS, an immunological stressor, can up-regulate prepro-OX mRNA expression in the LHA. Increases in prepro-OX mRNA have been reported in response to other stressors that robustly stimulate the HPA axis such as immobilization and cold stress (23) and hypoglycemia (24). The present study expands this repertoire of stressors to include immune- mediated stress. LPS is a potent stimulator of HPA axis activity and subsequently CORT release, and it is possible that the increase in prepro-OX mRNA may be a result of increasing blood CORT concentrations. Further work is necessary to elucidate whether increased orexin in response to LPS is a direct result of LPS-induced CORT release or indirectly in response to increased cytokines.
We may briefly speculate on the physiological importance of increased orexin expression in response to an immunological stimulus. Orexins have been demonstrated to have analgesic properties in inflammatory-induced hyperalgesia in which the OX-1-receptor antagonist SB-334867-A reduces OX-A-mediated analgesia (55). This suggests that there may a hypothalamic mechanism involving orexin as an analgesic agent during neurogenic pain. It is also possible that, after onset of inflammation in which the acute phase response to LPS plays a crucial role, it is so important to maintain homeostatic control of the peripheral release of inflammatory cytokines through increased secretion of CORT that an immunological stimulus will activate all hypothalamic components that drive HPA axis activity, including orexins.
In conclusion, these data establish that OX-A-induced feeding and prepro-OX mRNA expression are sensitive to glucocorticoid manipulation. Up-regulation of prepro-OX expression in response to CORT may be circadian related because levels were increased in the morning but not the evening. However, further studies will be required to determine the mechanisms underlying these data. These observations highlight the interdependence of orexins and HPA axis activity and provide further insights into the role of glucocorticoids in regulating centrally mediated orexin-induced feeding and orexin expression within the brain.
Footnotes
This work was supported by the Needham Cooper Trust UK, GlaxoSmithKline, and the Neuroendocrine Charitable Trust UK (Ph.D. studentship to G.K.F.).
The results of this work were presented in part at the 85th Annual Meeting of The Endocrine Society, Philadelphia, Pennsylvania, June 2003 (Abstract P3-193).
Abbreviations: ADX, Adrenalectomized; ARC, arcuate nucleus; AVP, arginine vasopressin; CNS, central nervous system; CORT, corticosterone; CRF, corticotropin-releasing factor; HPA, hypothalamo-pituitary-adrenal; icv, intracerebroventricular; LHA, lateral hypothalamic area; LPS, lipopolysaccharide; NPY, neuropeptide Y; OX, orexin; PVN, paraventricular nucleus; SSC, saline sodium citrate.
References
Willie JT, Chemelli RM, Sinton CM, Yanagisawa M 2001 To eat or to sleep Orexin in the regulation of feeding and wakefulness. Annu Rev Neurosci 24:429–458
Sakurai T, Amemiya A, Ishi, M, Matsuzak, I, Chemell, RM, Tanaka H, Williams SC, Richardson JA, Kozlowsk, GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M 1998 Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behaviour. Cell 92:573–595
Taheri S, Bloom S 2001 Orexins/hypocretins: waking up the scientific world. Clin Endocrinol (Oxf) 54:421–429
Taylor MM, Samson WK 2003 The other side of the orexins: endocrine and metabolic actions. Am J Physiol Endocrinol Metab 284:E13–E17
Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DNC, Smith MI, Piper DC, Hunter AJ, Porter RA, Upton N 1999 Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci USA 96:10911–10916
Espana RA, Plahn S, Berridge CW 2002 Circadian-dependent and circadian-independent behavioral actions of hypocretin/orexin. Brain Res 94:224–236
Duxon MS, Stretton J, Starr K, Jones DNC, Holland V, Riley G, Jerman J, Brough S, Smart D, Johns A, Chan W, Porter RA, Upton N 2001 Evidence that orexin-A-evoked grooming in the rat is mediated by orexin-1 (OX1) receptors, with downstream 5-HT2C receptor involvement. Psychopharmacology (Berl) 153:203–209
Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M 1999 Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 96:748–753
Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS 1998 Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996–10015
Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, van Den Pol AN 1999 Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J Comp Neurol 415:145–159
Cluderay JE, Harrison DC, Hervieu GJ 2002 Protein distribution of the orexin-2 receptor in the rat central nervous system. Regul Pept 104:131–144
Backberg M, Hervieu G, Wilson S, Meister B 2002 Orexin receptor-1 (OX-R1) immunoreactivity in chemically identified neurons of the hypothalamus: focus on orexin targets involved in control of food and water intake. Eur J Neurosci 15:315–328
Hervieu GJ, Cluderay JE, Harrison DC, Roberts JC, Leslie RA 2001 Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord. Neuroscience 103:777–797
Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM 1998 Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 438:71–75
Buckingham JC, Cowell A-M, Gillies GE, Herbison AE, Steel JH 1997 The neuroendocrine system: anatomy, physiology and responses to stress. In: Buckingham JC, Cowell A-M, Gillies GE, eds. Stress, stress hormones and the immune system. London: John Wiley, Sons; 9–47
Jaszberenyi M, Bujdoso E, Pataki I, Telegdy G 2000 Effects of orexins on the hypothalamic-pituitary-adrenal system. J Neuroendocrinol 12:1174–1178
Kuru M, Ueta Y, Serino R, Nakazato M, Yamamoto Y, Shibuya I, Yamashita H 2000 Centrally administered orexin/hypocretin activates HPA axis in rats. Neuroreport 11:1977–1980
Jones DNC, Gartlon J, Parker F, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Hatcher JP, Johns A, Porter RA, Hagan JJ, Hunter AJ, Upton N 2001 Effects of centrally administered orexin-B and orexin-A: a role for orexin-1 receptors in orexin-B-induced hyperactivity. Psychopharmacology (Berl) 153:210–218
Russell SH, Small CJ, Dakin CL, Abbott CR, Morgan DG, Ghatei MA, Bloom SR 2001 The central effects of orexin-A in the hypothalamic-pituitary-adrenal axis in vivo and in vitro in male rats. J Neuroendocrinol 13:561–566
Al-Barazanji KA, Wilson S, Baker J, Jessop DS, Harbuz MS 2001 Central orexin-A activates hypothalamic-pituitary-adrenal axis and stimulates hypothalamic corticotropin releasing factor and arginine vasopressin neurones in conscious rats. J Neuroendocrinol 13:421–424
Samson WK, Taylor MM, Follwell M, Ferguson AV 2002 Orexin actions in hypothalamic paraventricular nucleus: physiological consequences and cellular correlates. Regul Pept 104:97–103
Brunton PJ, Russell JA 2003 Hypothalamic-pituitary-adrenal responses to centrally administered orexin-A are suppressed in pregnant rats. J Neuroendocrinol 15:633–637
Ida T, Nakahara K, Murakami T, Hanada R, Nakazato M, Murakami N 2000 Possible involvement of orexin in the stress reaction in rats. Biochem Biophys Res Commun 270:318–323
Griffond B, Risold PY, Jacquemard C, Colard C, Fellmann D 1999 Insulin-induced hypoglycemia increases preprohypocretin (orexin) mRNA in the rat lateral hypothalamic area. Neurosci Lett 262:77–80
Kok SW, Roelfsema F, Overeem S, Lammers GJ, Strijers RL, Frolich M, Meinders AE, Pijl H 2002 Dynamics of the pituitary-adrenal ensemble in hypocretin-deficient narcoleptic humans: blunted basal adrenocorticotropin release and evidence for normal time-keeping by the master pacemaker. J Clin Endocrinol Metab 87:5085–5091
Dallman MF, Strack AM, Akana SF, Bradbury MJ, Hanson ES, Scribner KA, Smith M 1993 Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front Neuroendocrinol 14:303–347
Dallman MF, Akana SF, Strack AM, Hanson ES, Sebastian RJ 1995 The neural network that regulates energy balance is responsive to glucocorticoids and insulin and also regulates HPA axis responsivity at a site proximal to CRF neurons. Ann NY Acad Sci 771:730–742
Taheri S, Sunter D, Dakin C, Moyes S, Seal L, Gardiner J, Rossi M, Ghatei, Bloom S 2000 Diurnal variation in orexin A immunoreactivity and prepro-orexin mRNA in the rat central nervous system. Neurosci Lett 279:109–112
Fujiki N, Yoshida Y, Ripley B, Honda K, Mignot E, Nishino S 2001 Changes in CSF hypocretin-1 (orexin A) levels in rats across 24 hours and in response to food deprivation. Neuroreport 12:993–997
Harbuz MS, Lightman SL 1992 Stress and the hypothalamo-pituitary-adrenal axis: acute, chronic and immunological activation. J Endocrinol 134:327–329
Turnbull AV, Rivier CL 1999 Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action. Physiol Rev 79:1–71
Jessop DS, Eckland DJA, Todd K, Lightman SL 1989 Osmotic regulation of hypothalamo-neurointermediate lobe corticotrophin-releasing factor-41 in the rat. J Endocrinol 120:119–124
Harbuz MS, Jessop DS, Lightman SL, Chowdrey HS 1994 The effects of restraint or hypertonic saline stress on corticotrophin-releasing factor, arginine vasopressin, and proenkephalin A mRNAs in the CFY, Sprague-Dawley and Wistar strains of rat. Brain Res 667:6–12
Young 3rd WS, Mezey E, Siegel RE 1986b Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci Lett 70:198–203
Harbuz MS, Lightman SL 1989 Responses of hypothalamic and pituitary mRNA to physical and psychological stress in the rat. J Endocrinol 12:705–711
Paxinos G, Watson C 1998 The rat brain in stereotaxic coordinates. 4th ed. London: Academic Press
Edwards CMB, Abusnana S, Sunter D, Murphy KG, Ghatei MA, Bloom SR 1999 The effect of the orexins on food intake: comparison with neuropeptide Y, melanin-concentrating hormone and galanin. J Endocrinol 160:7–12
Haynes AC, Jackson B, Overend P, Buckingham RE, Wilson S, Tadayyon M, Arch JR 1999 Effects of single and chronic intracerebroventricular administration of the orexins on feeding in the rat. Peptides 20:1099–1105
Drazen DL, Coolen LM, Strader AD, Wortman MD, Woods SC, Seeley RJ 2004 Differential effects of adrenalectomy on melanin-concentrating hormone and orexin A. Endocrinology 145:3404–3412
Ford GK, Al-Barazanji KA, Wilson S, Harbuz MS, Jessop DS 2002 Effects of glucocorticoid manipulation on orexin-A induced food intake in rats. Br J Pharmacol Proc Suppl 135:109P
Tataranni PA, Larson DE, Snitker S, Young JB, Flatt JP, Ravussin E 1996 Effects of glucocorticoids on energy metabolism and food intake in humans. Am J Physiol 271:E317–E325
Dallman MF, Akana SF, Strack AM, Scribner KS, Pecoraro N, La Fleur SE, Houshyar H, Gomez F 2004 Chronic stress-induced effects of corticosterone on brain: direct and indirect. Ann NY Acad Sci 1018:141–150
Ida T, Nakahara K, Kuroiwa T, Fukui K, Nakazato M, Murakami T, Murakami N 2000 Both corticotropin releasing factor and neuropeptide Y are involved in the effect of orexin (hypocretin) on the food intake in rats. Neurosci Lett 293:119–122
Stanley BG, Lanthier D, Chin AS, Leibowitz SF 1989 Suppression of neuropeptide Y-elicited eating by adrenalectomy or hypophysectomy: reversal with corticosterone. Brain Res 501:32–36
Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS 1999 Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20:68–100
Jain MR, Horvath TL, Kalra PS, Kalra SP 2000 Evidence that NPY Y1 receptors are involved in stimulation of feeding by orexins (hypocretins) in sated rats. Regul Pept 87:19–24
Cintra A, Zoli M, Rosen L, Agnati LF, Okret S, Wikstrom AC, Gustaffsson JA, Fuxe K 1994 Mapping and computer assisted morphometry and microdensitometry of glucocorticoid receptor immunoreactive neurons and glial cells in the rat central nervous system. Neuroscience 62:843–897
Morimoto M, Morita N, Ozawa H, Yokoyama K, Kawata M 1996 Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study. Neurosci Res 26:235–269
Watts AG, Tanimura S, Sanchez-Watts G 2004 Corticotropin-releasing hormone and arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of unstressed rats: daily rhythms and their interactions with corticosterone. Endocrinology 145:529–540
Stricker-Krongrad A, Beck B 2002 Modulation of hypothalamic hypocretin/orexin mRNA expression by glucocorticoids. Biochem Biophys Res Commun 296:129–133
Rivet JM, Castagne V, Corder R, Gaillard R, Mormede P 1989 Study of the influence of stress and adrenalectomy on central and peripheral neuropeptide Y levels. Comparison with catecholamines. Neuroendocrinology 50:413–420
Pralong FP, Corder R, Gaillard RC 1993 The effects of chronic glucocorticoid excess, adrenalectomy and stress on neuropeptide Y in individual rat hypothalamic nuclei. Neuropeptides 25:223–231
Akabayashi A, Watanabe Y, Wahlestedt C, McEwen BS, Paez X, Leibowitz SF 1994 Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats. Brain Res 665:201–212
Larsen P, Jessop D, Chowdrey H, Lightman S, Mikkelsen J 1994 Chronic administration of glucocorticoids directly upregulates prepro-neuropeptide Y and Y1-receptor mRNA levels in the arcuate nucleus of the rat. J Neuroendocrinol 6:153–160
Bingham S, Davey PT, Babbs AJ, Irving EA, Sammons MJ, Wyles M, Jeffrey P, Cutler L, Riba I, Johns A, Porter RA, Upton N, Hunter AJ, Parsons AA 2001 Orexin-A, an hypothalamic peptide with analgesic properties. Pain 92:81–90(Gemma K. Ford, Kamal A. A)
Department of Biology (D.N.C.J.), Psychiatry Centre of Excellence for Drug Discovery (CEDD) and Discovery Research (S.W.), GlaxoSmithKline, Harlow Essex CM19 5AW, United Kingdom
Metabolic Diseases (K.A.A.-B.), Metabolic and Viral Diseases CEDD, GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Abstract
We investigated the effects of glucocorticoid manipulation on orexin-A-induced feeding and prepro-orexin mRNA levels in the lateral hypothalamic area (LHA) of the rat brain. Adrenalectomy (ADX) reduced orexin-A-induced feeding over 4 h by about 60%, compared with shams, an effect that was reversed by corticosterone (CORT) replacement. ADX had no effect on prepro-orexin mRNA levels in the LHA in either the morning or the evening; however, message was up-regulated by CORT in the morning but not the evening. An increased number of emulsion grains per cell in the LHA suggests that this is a specific increase in prepro-orexin mRNA and is not due to an increased number of cells expressing message. Prepro-orexin mRNA levels in the LHA were elevated 4 h after injection of lipopolysaccharide, compared with saline-injected controls. Partial but not complete abolition of orexin-A-induced feeding by ADX suggests that orexin-A-induced feeding may be mediated through glucocorticoid-dependent and glucocorticoid-independent pathways. In the morning increased prepro-orexin mRNA after CORT replacement demonstrates that orexin expression is sensitive to increased concentrations of glucocorticoids. However, the lack of effect of ADX on prepro-orexin mRNA levels suggests that endogenous glucocorticoids are not involved in tonic regulation of basal prepro-orexin expression. Overall our data constitute a body of evidence for an integrated relationship between central orexin expression, stress, glucocorticoid manipulation, and feeding patterns in the rat.
Introduction
OREXIN (OX)-A AND OX-B (also known as hypocretins) are closely related neuropeptides derived from a single gene and processed from a 130-residue (131 residues in humans) prepro-OX precursor protein (1). OX-A is a 33-residue peptide with identical sequence in humans and rodents, whereas human and rat OX-B differ by two residues. OX-A and OX-B activate two G protein-coupled receptors known as OX-1 and OX-2, the OX-1 receptor having a greater affinity for OX-A over OX-B, whereas the OX-2 receptor has similar affinity for both ligands. Central nervous system (CNS) distribution of orexins is similar in rats and humans, the lateral hypothalamic area (LHA) being the predominant region of synthesis (1).
The LHA has long been implicated in feeding behavior and initial observations highlighted the role of orexins in stimulating appetite (2). However, it is now clear that orexins are also involved in the regulation of sleep, arousal, locomotor activity and neuroendocrine responses (3, 4), and therefore their orexigenic activity may be secondary to other behavioral traits that influence feeding. In behavioral studies, orexins can modulate stress-related behavior such as grooming, and chewing of inedible material (5, 6, 7). Orexin-containing neurones project from the LHA to many CNS areas involved in mediating stress including the paraventricular nucleus (PVN) of the hypothalamus, the arcuate nucleus (ARC), and the locus coeruleus (8, 9, 10). OX-1 and OX-2 receptors are widely and differentially distributed throughout the CNS including the PVN, suprachiasmatic nucleus, supraoptic nucleus, and ARC of the hypothalamus and also the locus coeruleus and hippocampus (11, 12, 13, 14). These areas have individual and synergistic involvement in regulating circadian rhythm and behavioral and neuroendocrine responses to stressors.
The hypothalamo-pituitary-adrenal (HPA) axis is one of the principal pathways mediating neuroendocrine responses to stress. Hypothalamic neuropeptides corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) stimulate the release of the proopiomelanocortin-derived peptide ACTH from the anterior pituitary, with consequent release of glucocorticoids from the adrenal cortex. Corticosterone (CORT), the principal glucocorticoid in rodents, acts through glucocorticoid receptors in the hippocampus and hypothalamus and mineralocorticoid receptors within the hippocampus to negatively regulate HPA axis activity (15). Many studies have demonstrated that orexins can influence HPA axis activity. Plasma ACTH and CORT concentrations are elevated in rats after intracerebroventricular (icv) OX-A administration (5, 16, 17, 18, 19, 20, 21, 22, 23). This is accompanied by the induction of c-fos mRNA (17) and AVP and CRF mRNAs (20) in the parvocellular subdivision of the PVN. In addition, in vitro studies have revealed release of CRF peptide, but not AVP, from hypothalamic explants (19). OX-B has also been reported to stimulate HPA axis activity (16). Orexin expression in the rat LHA is increased after stressors such as immobilization (23), cold stress (23), or hypoglycemia (24). Reduced secretion of plasma ACTH and cortisol is reported in patients with narcolepsy (25), a condition associated with orexin deficiency. Therefore there is considerable evidence that orexins can modulate HPA axis activity and that orexin expression is responsive to alterations in HPA axis activity.
It is well documented that glucocorticoid administration stimulates feeding and conversely that glucocorticoid deficiency results in loss of appetite and weight loss because adrenalectomized (ADX) rats exhibit decreased feeding, which can be reversed by CORT administration (26, 27). Therefore, orexins may not exert a direct effect on appetite regulation within the hypothalamus but may act indirectly to stimulate feeding through increased CORT secretion. To test this hypothesis, we designed experiments to investigate the effect of ADX with or without CORT replacement on OX-A-induced feeding to determine whether endogenous CORT is involved in OX-A-induced feeding behavior. We also investigated the effects of glucocorticoid manipulation on orexin expression in the morning and evening to determine whether orexin expression, which exhibits a diurnal rhythm of expression (28, 29), may be correlated with the diurnal rhythm of HPA axis activity. Finally, we investigated the effects on orexin expression of lipopolysaccharide (LPS), an immunological stressor that elicits a robust and prolonged activation of the HPA axis (30, 31).
Materials and Methods
Animals
Adult male Sprague Dawley rats (200–250 g, Charles River, Margate, Kent, UK) were housed individually under controlled light and temperature (21 ± 2 C), with free access to water and standard rat pelleted chow and maintained on a 12-h light, 12-h dark cycle (lights on 0600 h). All procedures were carried out in accordance with the Animals Scientific Procedures Act (1986) United Kingdom.
Surgical procedures
Intracerebroventricular cannulation.
All rats were anesthetized by im injection of Domitor (medetomidine HCl, 0.04 ml per 100 g; Pfizer, Sandwich, Kent, UK) and an ip injection of Sublimaze (fentanyl, 0.9 ml per 100 g; Janssen-Cilag, High Wycombe, Buckinghamshire, UK). Rats were positioned in a stereotaxic frame and implanted with a 22-gauge stainless steel guide cannula in the left lateral ventricle of the brain under sterile conditions. The cannula was secured on the surface of the skull with jeweler’s screws and dental cement. Stereotaxic coordinates for the left brain ventricle were as follows: 0.8 mm caudal from Bregma, 1.5 mm lateral from the midline, and 4.1 mm vertical from the skull surface, incisor bar at 3.2 mm below zero. On completion of the surgery the anesthesia was reversed by an ip of a 50:50 mixture of Antisedan (atipamezole HCl, 0.02 ml per 100 g; Pfizer) and Nubain (nalbuphine HCl, 0.02 ml per 100 g; DuPont Pharmaceuticals, Stevenage, Hertfordshire, UK). After implantation of the guide cannula, correct placement was checked by using the gravitational flow of a sterile saline filled cannula.
Adrenalectomy.
Rats were bilaterally ADX via a dorsal approach. A small incision was made along the midline of the back just below the rib cage, connective tissue and fat was displaced, and a small hole was made through the muscle either side of the back using blunt cut scissors. The adrenals were excised with curved forceps. Each adrenal was checked that it had remained intact after excision. Only animals that had complete removal of both adrenals were considered ADX. After surgery, all ADX animals were immediately given 0.9% saline to drink to maintain their salt balance. Surgery for sham animals was identical, but the adrenals were not removed. Animals were allowed 7 d postoperative recovery and were handled daily.
Effects of glucocorticoids on OX-A-induced feeding
Thirty-two rats were bilaterally ADX, whereas 16 rats were subjected to sham ADX. Animals were returned to their home cages and all ADX animals were immediately given 0.9% saline, whereas two groups of ADX rats were given 0.9% saline supplemented with CORT (Sigma, Poole, Dorset, UK), either 25 mg/liter (low CORT) or 125 mg/liter (high CORT). These concentrations of CORT were selected as they are within the basal physiological range of endogenous CORT (27). On the morning of experimentation, sham and ADX animals were divided into two groups (n = 9) of equal mean body weights to receive icv injection of either OX-A (30 μg/rat in 5 μl of 0.9% saline) or saline. This dose, equivalent to 8 nmol, falls within the accepted range of 3–30 nmol (2) that has been used extensively. Both low CORT and high CORT replacement groups also received an icv injection of either OX-A (30 μg/rat) or saline. An icv injection cannula with extension tubing, preloaded with drug or vehicle, was inserted into the guide cannula. Injections were given over a period of 1 min. The needle was left in position for a further minute to ensure complete dispersal of the peptide. Injections were given between 0900 and 1000 h. Preweighed food pellets and water bottles were supplied after icv injection. Food intake was determined at 1-, 2-, 3-, and 4-h time points after icv injection. After 4 h, all rats were killed by decapitation, and trunk blood was collected into chilled EDTA tubes on ice for centrifugation to collect plasma. ACTH was measured by RIA after prior extraction from plasma on Seppak columns (32). Assay sensitivity was 10 pg/ml. Total CORT was measured in unextracted plasma by an in-house RIA (33). The radioactive tracer was I125-CORT (ICN Biomedicals, Basingstoke, Hampshire, UK) with a specific activity of 0.37 MBq. The sensitivity of the assay was 5 ng/ml. Both ACTH and CORT primary antisera were kindly supplied by G. Makara (Hungarian Academy of Sciences, Budapest, Hungary). All samples for either ACTH or CORT were processed in the same assay, with intraassay variation less than 10%.
Distribution of prepro-OX expression through the lateral hypothalamus
Two male Sprague Dawley rats were killed by decapitation, and brains were removed and stored at –80 C before sectioning. Coronal brain sections (12 μm) were collected through the LHA from the PVN on a cryostat and immediately stored at –80 C. Two sections were collected every four sections, and 48–50 sections were collected from each animal. Prepro-OX expression was investigated using in situ hybridization, which was performed as described previously (34, 35). Sections were warmed to room temperature for 10 min and fixed with 4% formaldehyde for 5 min, washed twice in PBS, and then incubated in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl for 10 min. Sections were then dehydrated through graded ethanol washes: 70% (1 min), 80% (1 min), 95% (2 min), and 100% (1 min), and then delipidated in chloroform 100% for 5 min and then rehydrated in 100% (1 min) and 95% (1 min) ethanol before drying in air. The probe used was a 33-mer oligonucleotide complementary to nucleotides 242–275 of the exonic sequence of rat prepro-OX mRNA 5'-TGCCCGCGGCGTGGTTGCCAGCTCCGTGCAACA-3' (GenBank accession no. AF041241). Probes were labeled at the 3' end with 35S-ATP and column purified by NucTrap probe purification columns (Stratagene, UK). Probes were labeled to a specific activity of 1.4x 1018 dpm/mol. Forty-five microliters of hybridization buffer [50% formamide, 4x saline sodium citrate (SSC), 0.5 mg/ml sheared single-stranded salmon sperm DNA, 0.25 mg/ml yeast tRNA, 0.5 x Denhardt’s solution, and 10% dextran sulfate], containing labeled probe (100,000 cpm) was applied to each slide. Hybridization was performed overnight at 37 C. All sections for each experiment were processed at the same time. The sections were washed four times at 55 C in 1x SSC [1x SSC = 0.15 M NaCl/0.015 M sodium citrate (pH 7.0)], followed by two 30-min washes at room temperature in 1x SSC to remove nonspecific binding before two brief rinses in distilled water. The sections were air dried and exposed to Amersham HyperfilmMP (Amersham International, Aylesbury, Buckinghamshire, UK) together with 14C-labeled standard for 14 d. The autoradiographic images of probe bound to brain sections were analyzed using a densitometry method described previously (35), using Image software. The results are presented as arbitrary values of prepro-OX expression through the LHA.
Effect of adrenalectomy and glucocorticoid manipulation on prepro-OX expression in the lateral hypothalamus
Fifteen rats were bilaterally ADX as described above, whereas 12 rats were subjected to sham ADX. Eight animals were given 0.9% saline, whereas seven of the ADX rats and eight of the sham ADX rats were given 0.9% saline supplemented with CORT (high CORT, 125 mg/liter) as described above. The remaining sham ADX animals were given tap water to drink. Animals were placed on this regimen for 7 d and were handled daily. On the morning of the experiment, all rats were killed by decapitation, brains were removed, and trunk blood was collected into chilled EDTA tubes on ice before ACTH and CORT RIAs as described above. Sections were cut on the cryostat from the two LHA areas determined from the distribution study: LHA1 (Bregma –2.30 mm; interaural 6.70 mm) and LHA2 (bregma –2.56 mm; interaural 6.44 mm from the atlas of Paxinos and Watson (36). Nine consecutive coronal lateral hypothalamic brain sections were collected spanning 108 μm and each slide had three sections encompassing 84 μm of LHA to allow for the apparent variation in prepro-OX expression demonstrated to be present through the LHA from the results of the distribution study. Prepro-OX mRNA measurement by in situ hybridization was performed as described above. All ADX and ADX + CORT hybridized sections were dipped into autoradiographic emulsion and exposed at 4 C for 1 wk. All sections were processed at the same time. The slides were then developed, fixed, and counterstained with methyl green.
Seven emulsion-dipped slides per treatment were analyzed at the cellular level, counting the number of labeled cells and silver grains per cell. At least two to five cells per section (minimum eight cells per slide with three sections per slide) were randomly selected under x200 magnification on a microscope (Leica, Heidelberg, Germany) and saved as images. Silver grains were counted by hand from microscope images enlarged by 100% in Photo Editor (Microsoft, Redmond, CA). Silver grains were dotted onto plastic sheets attached to the monitor screen and then counted. Silver grains in each individual cell were counted three times in this manner. Sixty-one to 67 cells were counted for each treatment.
Effect of diurnal variation on prepro-OX expression in ADX rats given glucocorticoid replacement
Thirty-three rats were bilaterally ADX, whereas 13 rats were subjected to sham ADX as described previously. Seventeen ADX rats were given 0.9% saline, whereas 16 were given 0.9% saline supplemented with CORT (125 mg/liter). All sham animals were supplied with tap water to drink. Animals were left on this regimen for 7 d and handled daily. On the morning of experimentation, five sham, eight ADX-only, and seven ADX + CORT rats were killed by decapitation in the morning (0900–1000 h), and the remaining animals were killed in the evening (1800 h). Brains and trunk blood was collected as described previously and prepro-OX measurements and sectioning were performed as described above.
Effects of an LPS injection on prepro-OX mRNA
All animals received an ip injection (0900–1000 h) of either LPS (250 μg/rat in 0.5 ml 0.9% saline) (Escherichia coli, Serotype 055:B5; Sigma) or saline. Animals were killed by decapitation 4 h after injection. Brains and trunk blood were collected and prepro-OX mRNA was measured as described above. Plasma ACTH and CORT were measured by RIAs.
Statistics
Statistical comparisons between multiple groups were made using the Fisher protected least significant difference test after one-way ANOVA. For the LPS study, a Student’s t test was used. All values are expressed as mean ± SEM. P < 0.05 was considered significant. All analyses were generated using Statview.
Results
Effects of glucocorticoids on OX-A-induced feeding
Plasma ACTH and CORT were measured to confirm the success of ADX. Four h after icv injection, all ADX rats had concentrations of CORT at or below the limit of detection (5 ng/ml), compared with the sham + vehicle group (31 ± 7 ng/ml) and sham + OX-A group (24 ± 8 ng/ml). There was no significant difference in plasma CORT concentrations between the sham + OX-A and sham + vehicle groups. Plasma CORT concentrations in the low CORT and high CORT replacement groups were 7.1 ± 1.1 and 7.3 ± 0.6 ng/ml, respectively. Plasma ACTH concentrations were significantly elevated in all ADX groups, compared with sham groups; the increase in ACTH was attenuated by high CORT but not low CORT replacement (Table 1).
After 4 h, food intake was significantly greater in the sham + OX-A (4.1 ± 0.6 g) group, compared with the sham + vehicle group (0.3 ± 0.2 g) (Fig. 1). ADX significantly reduced OX-A-induced food intake (2.1 ± 0.7 g) by about 60%, a reduction that was reversed by high CORT (4.9 ± 0.8 g) but not low CORT replacement. Food intake was significantly greater in the ADX + OX-A rats (2.1 ± 0.7 g), compared with sham + vehicle (0.3 ± 0.2 g) and ADX + vehicle (0.6 ± 0.3 g) groups.
Distribution of prepro-OX expression through the lateral hypothalamus
There was considerable variation in prepro-OX mRNA expression levels throughout the LHA (Fig. 2). However, in sections collected caudal to the PVN area at 1.34 mm from the end of the anterior commissure, the profile of expression tended to peak and trough around similar areas and in some cases prepro-OX levels overlapped between the two animals. Areas LH1 and LH2 were areas of overlap in prepro-OX mRNA between the two brains, which were clearly defined anatomically and therefore permitted greater reproducibility.
Effect of adrenalectomy and glucocorticoid manipulation on prepro-OX expression in the lateral hypothalamus
Concentrations of plasma CORT were measured to confirm the success of ADX. All ADX rats had concentrations of plasma CORT below the limit of assay detection (5 ng/ml), compared with the sham group (21 ± 11 ng/ml). ADX animals with CORT replacement had CORT concentrations of 43 ± 12 ng/ml, compared with the sham + CORT group (126 ± 53 ng/ml).
There was no effect of ADX on prepro-OX mRNA levels in the LHA1 (Fig. 3A) and LHA2 (Fig. 3B) areas, compared with sham animals. Both sham and ADX groups with CORT replacement had significantly elevated levels of prepro-OX mRNA in the LHA1 and LHA2, compared with sham and ADX groups without CORT replacement.
Representative autoradiographic images of prepro-OX mRNA expression and distribution in the LHA2 area are shown in Fig. 4 for each of the treatment groups. Photomicrographs of emulsion dipped slides showing prepro-OX mRNA expression in individual cells are shown in Fig. 5. These images demonstrate that prepro-OX mRNA is expressed in cells of different size. The number of silver grains per cell of individual prepro-OX gene-expressing cells in the emulsion-dipped slides was significantly (P < 0.001) increased in the LHA of ADX rats receiving CORT replacement, compared with ADX rats (Fig. 6). The amount of orexin expression is remarkably consistent for a given treatment with 125 ± 4 grains/cell in the ADX group, compared with 187 ± 6 grains/cell in the ADX + CORT group. There was no significant difference in the number of cells expressing prepro-OX mRNA (ADX = 61 cells and ADX+CORT = 67 cells counted in the selected fields of view).
Effect of diurnal variation on orexin expression in adrenalectomized rats on glucocorticoid replacement
Plasma CORT concentrations in sham animals were significantly higher (P < 0.001) in the evening (111 ± 24 ng/ml), compared with the morning (20 ± 8 ng/ml). Plasma CORT in both morning and evening ADX groups was below the limit of detection (5 ng/ml). CORT replacement in ADX animals resulted in similar morning (43 ± 12 ng/ml) and evening concentrations (36 ± 14 ng/ml), which were not significantly different from morning concentrations in the sham group .
ADX had no effect on morning levels of prepro-OX mRNA in the LHA1, whereas CORT replacement up-regulated orexin mRNA in both sham and ADX groups (Fig. 3A). Consistent with this observation, there was no significant effect of ADX on prepro-OX mRNA expression in the LHA1 area in the morning or evening (Fig. 7). CORT replacement significantly elevated prepro-OX mRNA levels in the morning, compared with sham and ADX groups, but this increase was not observed in the evening (Fig. 7).
Effect of an LPS stress on prepro-OX expression
LPS evoked a 4-fold increase in plasma CORT concentrations (184 ± 44 ng/ml), compared with saline controls (49 ± 17 ng/ml). There was a significant 60% increase in prepro-OX mRNA expression in the LHA1 4 h after LPS injection, compared with saline-injected rats (Fig. 8).
Discussion
Our data reveal an integrated relationship between central orexin expression, endogenous glucocorticoid concentrations, and feeding patterns in the rat. Our observation that central administration of OX-A increases food intake is in agreement with other groups that have demonstrated that OX-A stimulates feeding in the early light phase in intact rats (2, 37, 38). We now demonstrate for the first time that glucocorticoids exert an important influence on OX-A-induced feeding behavior. ADX significantly reduces orexin-A- induced feeding, a phenomenon that is reversible by CORT replacement at physiological concentrations. These data provide evidence that endogenous CORT can exert an important influence on OX-A-induced food intake. However, because ADX does not completely block OX-A-induced feeding but results in about a 60% reduction, this suggests that there is a pathway mediating orexin-induced food intake that is independent of glucocorticoids.
One recent study has investigated the effect of ADX on OX-A-induced feeding (39). In contrast to our findings, these investigators observed no effect of ADX on OX-A-induced food intake over a 2-h period (39). This may be due to the suboptimal doses of OX-A employed over a dose range 1.5–6 nmol, compared with 8 nmol in the present study, the latter dose being considered optimal for OX-A-induced feeding behavior (2). These findings may also be a result of the shorter period of food intake studied (maximum of 2 h, compared with a maximum of 4 h in our study). In the paper by Drazen et al. (39), it seems evident that ADX is associated with decreased food intake at both higher doses of OX-A at the 2-h time point, but the very small increase in food intake in response to OX-A in sham rats does not permit the ADX effect to reach significance. The larger dose of orexin that we used elicited a greater food intake in the sham rats, compared with that observed by Drazen et al. (39), and consequently we were able to observe a significant and reproducible decrease in orexin-induced food intake in ADX rats.
Initially, this experiment was performed with only ADX and sham groups given orexin or saline. Consequent to our observation of a significant decrease in orexin-induced food intake in the ADX group, we repeated the experiment and expanded it to include low CORT and high CORT replacement groups. Our observations were reproducible at all time points when the full study was repeated (40). This, together with the ability of physiological concentrations of CORT to reverse the effects of ADX on orexin-induced food intake, increases our confidence that endogenous glucocorticoids exert physiologically relevant control over orexin-induced feeding behavior.
Our observation that orexin-induced food intake is decreased by about 60% in ADX rats is evidence that a significant component of eating behavior is dependent on endogenous CORT. It is known that CORT can directly stimulate appetite (41), but the indirect effects on appetite of CORT mediated through hypothalamic neuropeptides is not well understood. A number of mechanisms may be invoked to explain the relationship among CORT, OX-A, and food intake. CORT may stimulate endogenous orexin expression through an action at glucocorticoid receptors within the LHA or intermediary nuclei, it may act via orexin peptide release within the PVN/ARC areas of the hypothalamus, or CORT may stimulate food intake itself consequent to orexin activation of HPA axis activity. There is also a possibility that CORT may exert its effect on food intake via other peptides that modulate appetite rather than directly through orexins. There is a well-defined pathway of glucocorticoid feedback on CRF expression in the PVN and release from the median eminence (42). CRF inhibits food intake and therefore increased CORT could increase food intake through its inhibitory action on CRF. Orexin-induced feeding is enhanced by a CRF receptor antagonist, suggesting CRF involvement (43). Neuropeptide Y (NPY) is also a possible mediator of orexin-induced feeding. Glucocorticoid manipulation results in a marked similarity between orexin and NPY effects on feeding. ADX reduced NPY-induced food intake by about 60%, similar to the effects of ADX on orexin-induced feeding, and this was reversed by CORT replacement (44). NPY is the most potent known inducer of food intake (45), and orexin-induced feeding can be blocked by a Y1 receptor antagonist (46). Therefore, it is conceivable that orexin exerts its principal glucocorticoid-dependent actions on food intake through NPY, and it is the direct effects of CORT on NPY, not on orexin expression, which explains our observations. However, we have observed a 40% increase in food intake in response to OX-A in ADX rats in the complete absence of CORT, which is strong evidence for a glucocorticoid-independent central pathway of direct orexin-induced food intake that may not be mediated through NPY.
In situ hybridization histochemistry confirmed that prepro-orexin expression was restricted to the LHA, with extensions to the perifornical nucleus and posterior hypothalamic areas, in agreement with previously reported studies (37). A novel finding from the distribution study was that prepro-OX mRNA expression measured from coronal sequential sections running 1044 μm throughout the LHA shows considerable differences between sections, emphasizing the importance of consistent choice of sections for hybridization in measuring prepro-OX mRNA in the LHA between treatment groups. This observation confirms the need to use anatomical markers to ensure consistency of sectioning between samples.
We observed an up-regulation of morning but not evening prepro-OX mRNA in the LHA after CORT administration. Increased blood CORT may up-regulate orexin expression through the large population of glucocorticoid receptor-positive cells within the LHA (47, 48). Emulsion staining established that there was no significant difference in the number of cells expressing prepro-OX between treatment groups. Therefore, the increases in prepro-OX mRNA that we observed in response to CORT represent increased expression of prepro-OX within cells of the LHA. There was a slight increase in prepro-OX mRNA expression in the sham group in the evening, compared with the morning, although this was not statistically significant. Plasma CORT levels were significantly increased in the evening, compared with the morning, in sham animals in agreement with previous studies demonstrating diurnal variations in HPA axis activity (49). However, it is important to note that CORT levels were similar in both the morning and evening replacement groups and not significantly different from morning levels in the sham animals. It is possible that the increase in evening prepro-OX expression, although not statistically significant, may be a consequence of the circadian increase in endogenous circulating CORT. This increase may be sufficient to mask any further stimulatory effects on orexin expression by exogenous CORT. Further investigations of interrelationships between the circadian cycles of HPA axis activity and orexin expression will undoubtedly shed light on regulatory mechanisms controlling the highly integrated modalities of sleep-wake patterns and feeding behavior.
We found no effect of ADX alone on orexin expression in the LHA, which suggests that basal orexin expression in rats is not under tonic regulation by endogenous glucocorticoids. In contrast, a decrease in prepro-OX mRNA expression 5 d after ADX has been reported (50). However, no data on HPA axis activity were presented in this study, so it is difficult to determine the effectiveness of ADX surgery. In addition, there was no information about where sections were sliced from within the LHA. Given the variability of prepro-OX expression, which we observed throughout the LHA, it is important that the choice of section is justified and well defined. The decrease in orexin expression after ADX was reversed by dexamethasone replacement (50), an observation that is consistent with our data demonstrating CORT up-regulation of orexin expression in sham animals. A decrease in prepro-OX mRNA levels in the LHA 10 d after ADX has also been reported, a phenomenon that could not be reversed by CORT (39).
Discrepancies between our data and published reports on the effects of ADX on prepro-OX mRNA expression in the LHA (39, 50), and lack of agreement on the effects of exogenous glucocorticoids, may be due to methodological differences. Our experiment used a 7-d ADX with CORT replacement, compared with previous studies using 5 d ADX with dexamethasone (50) and 10 d ADX with CORT (39). It is instructive to compare these discrepancies with the literature on NPY expression after ADX. Although there is a general consensus that NPY is up-regulated by glucocorticoids, reports differ on the effects on NPY expression of eliminating endogenous cortisosterone by ADX. Short-term ADX (4 d) did not alter NPY peptide levels in the ARC or PVN (51), whereas the same group observed that long-term ADX (12 d) deceased NPY peptide levels in these areas (52). ADX (7 d) had no effect on NPY immunoreactivity in most hypothalamic areas, one notable exception being the PVN (53). A longer-term ADX (12 d) had no effect on prepro-NPY mRNA in the ARC (54); peptide levels were not measured in this study. Either dexamethasone or CORT replacement up-regulated NPY mRNA, but dexamethasone was much more potent (53). Therefore, any effects of glucocorticoid manipulation on orexin expression, as with NPY, may be dependent on the period of ADX, the concentration and type of glucocorticoid, whether orexin peptide or mRNA is measured, and how specifically defined is the hypothalamic site selected for analysis.
Finally, we have demonstrated that a single injection of LPS, an immunological stressor, can up-regulate prepro-OX mRNA expression in the LHA. Increases in prepro-OX mRNA have been reported in response to other stressors that robustly stimulate the HPA axis such as immobilization and cold stress (23) and hypoglycemia (24). The present study expands this repertoire of stressors to include immune- mediated stress. LPS is a potent stimulator of HPA axis activity and subsequently CORT release, and it is possible that the increase in prepro-OX mRNA may be a result of increasing blood CORT concentrations. Further work is necessary to elucidate whether increased orexin in response to LPS is a direct result of LPS-induced CORT release or indirectly in response to increased cytokines.
We may briefly speculate on the physiological importance of increased orexin expression in response to an immunological stimulus. Orexins have been demonstrated to have analgesic properties in inflammatory-induced hyperalgesia in which the OX-1-receptor antagonist SB-334867-A reduces OX-A-mediated analgesia (55). This suggests that there may a hypothalamic mechanism involving orexin as an analgesic agent during neurogenic pain. It is also possible that, after onset of inflammation in which the acute phase response to LPS plays a crucial role, it is so important to maintain homeostatic control of the peripheral release of inflammatory cytokines through increased secretion of CORT that an immunological stimulus will activate all hypothalamic components that drive HPA axis activity, including orexins.
In conclusion, these data establish that OX-A-induced feeding and prepro-OX mRNA expression are sensitive to glucocorticoid manipulation. Up-regulation of prepro-OX expression in response to CORT may be circadian related because levels were increased in the morning but not the evening. However, further studies will be required to determine the mechanisms underlying these data. These observations highlight the interdependence of orexins and HPA axis activity and provide further insights into the role of glucocorticoids in regulating centrally mediated orexin-induced feeding and orexin expression within the brain.
Footnotes
This work was supported by the Needham Cooper Trust UK, GlaxoSmithKline, and the Neuroendocrine Charitable Trust UK (Ph.D. studentship to G.K.F.).
The results of this work were presented in part at the 85th Annual Meeting of The Endocrine Society, Philadelphia, Pennsylvania, June 2003 (Abstract P3-193).
Abbreviations: ADX, Adrenalectomized; ARC, arcuate nucleus; AVP, arginine vasopressin; CNS, central nervous system; CORT, corticosterone; CRF, corticotropin-releasing factor; HPA, hypothalamo-pituitary-adrenal; icv, intracerebroventricular; LHA, lateral hypothalamic area; LPS, lipopolysaccharide; NPY, neuropeptide Y; OX, orexin; PVN, paraventricular nucleus; SSC, saline sodium citrate.
References
Willie JT, Chemelli RM, Sinton CM, Yanagisawa M 2001 To eat or to sleep Orexin in the regulation of feeding and wakefulness. Annu Rev Neurosci 24:429–458
Sakurai T, Amemiya A, Ishi, M, Matsuzak, I, Chemell, RM, Tanaka H, Williams SC, Richardson JA, Kozlowsk, GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M 1998 Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behaviour. Cell 92:573–595
Taheri S, Bloom S 2001 Orexins/hypocretins: waking up the scientific world. Clin Endocrinol (Oxf) 54:421–429
Taylor MM, Samson WK 2003 The other side of the orexins: endocrine and metabolic actions. Am J Physiol Endocrinol Metab 284:E13–E17
Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DNC, Smith MI, Piper DC, Hunter AJ, Porter RA, Upton N 1999 Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci USA 96:10911–10916
Espana RA, Plahn S, Berridge CW 2002 Circadian-dependent and circadian-independent behavioral actions of hypocretin/orexin. Brain Res 94:224–236
Duxon MS, Stretton J, Starr K, Jones DNC, Holland V, Riley G, Jerman J, Brough S, Smart D, Johns A, Chan W, Porter RA, Upton N 2001 Evidence that orexin-A-evoked grooming in the rat is mediated by orexin-1 (OX1) receptors, with downstream 5-HT2C receptor involvement. Psychopharmacology (Berl) 153:203–209
Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M 1999 Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 96:748–753
Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS 1998 Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996–10015
Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, van Den Pol AN 1999 Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J Comp Neurol 415:145–159
Cluderay JE, Harrison DC, Hervieu GJ 2002 Protein distribution of the orexin-2 receptor in the rat central nervous system. Regul Pept 104:131–144
Backberg M, Hervieu G, Wilson S, Meister B 2002 Orexin receptor-1 (OX-R1) immunoreactivity in chemically identified neurons of the hypothalamus: focus on orexin targets involved in control of food and water intake. Eur J Neurosci 15:315–328
Hervieu GJ, Cluderay JE, Harrison DC, Roberts JC, Leslie RA 2001 Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord. Neuroscience 103:777–797
Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM 1998 Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 438:71–75
Buckingham JC, Cowell A-M, Gillies GE, Herbison AE, Steel JH 1997 The neuroendocrine system: anatomy, physiology and responses to stress. In: Buckingham JC, Cowell A-M, Gillies GE, eds. Stress, stress hormones and the immune system. London: John Wiley, Sons; 9–47
Jaszberenyi M, Bujdoso E, Pataki I, Telegdy G 2000 Effects of orexins on the hypothalamic-pituitary-adrenal system. J Neuroendocrinol 12:1174–1178
Kuru M, Ueta Y, Serino R, Nakazato M, Yamamoto Y, Shibuya I, Yamashita H 2000 Centrally administered orexin/hypocretin activates HPA axis in rats. Neuroreport 11:1977–1980
Jones DNC, Gartlon J, Parker F, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Hatcher JP, Johns A, Porter RA, Hagan JJ, Hunter AJ, Upton N 2001 Effects of centrally administered orexin-B and orexin-A: a role for orexin-1 receptors in orexin-B-induced hyperactivity. Psychopharmacology (Berl) 153:210–218
Russell SH, Small CJ, Dakin CL, Abbott CR, Morgan DG, Ghatei MA, Bloom SR 2001 The central effects of orexin-A in the hypothalamic-pituitary-adrenal axis in vivo and in vitro in male rats. J Neuroendocrinol 13:561–566
Al-Barazanji KA, Wilson S, Baker J, Jessop DS, Harbuz MS 2001 Central orexin-A activates hypothalamic-pituitary-adrenal axis and stimulates hypothalamic corticotropin releasing factor and arginine vasopressin neurones in conscious rats. J Neuroendocrinol 13:421–424
Samson WK, Taylor MM, Follwell M, Ferguson AV 2002 Orexin actions in hypothalamic paraventricular nucleus: physiological consequences and cellular correlates. Regul Pept 104:97–103
Brunton PJ, Russell JA 2003 Hypothalamic-pituitary-adrenal responses to centrally administered orexin-A are suppressed in pregnant rats. J Neuroendocrinol 15:633–637
Ida T, Nakahara K, Murakami T, Hanada R, Nakazato M, Murakami N 2000 Possible involvement of orexin in the stress reaction in rats. Biochem Biophys Res Commun 270:318–323
Griffond B, Risold PY, Jacquemard C, Colard C, Fellmann D 1999 Insulin-induced hypoglycemia increases preprohypocretin (orexin) mRNA in the rat lateral hypothalamic area. Neurosci Lett 262:77–80
Kok SW, Roelfsema F, Overeem S, Lammers GJ, Strijers RL, Frolich M, Meinders AE, Pijl H 2002 Dynamics of the pituitary-adrenal ensemble in hypocretin-deficient narcoleptic humans: blunted basal adrenocorticotropin release and evidence for normal time-keeping by the master pacemaker. J Clin Endocrinol Metab 87:5085–5091
Dallman MF, Strack AM, Akana SF, Bradbury MJ, Hanson ES, Scribner KA, Smith M 1993 Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front Neuroendocrinol 14:303–347
Dallman MF, Akana SF, Strack AM, Hanson ES, Sebastian RJ 1995 The neural network that regulates energy balance is responsive to glucocorticoids and insulin and also regulates HPA axis responsivity at a site proximal to CRF neurons. Ann NY Acad Sci 771:730–742
Taheri S, Sunter D, Dakin C, Moyes S, Seal L, Gardiner J, Rossi M, Ghatei, Bloom S 2000 Diurnal variation in orexin A immunoreactivity and prepro-orexin mRNA in the rat central nervous system. Neurosci Lett 279:109–112
Fujiki N, Yoshida Y, Ripley B, Honda K, Mignot E, Nishino S 2001 Changes in CSF hypocretin-1 (orexin A) levels in rats across 24 hours and in response to food deprivation. Neuroreport 12:993–997
Harbuz MS, Lightman SL 1992 Stress and the hypothalamo-pituitary-adrenal axis: acute, chronic and immunological activation. J Endocrinol 134:327–329
Turnbull AV, Rivier CL 1999 Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action. Physiol Rev 79:1–71
Jessop DS, Eckland DJA, Todd K, Lightman SL 1989 Osmotic regulation of hypothalamo-neurointermediate lobe corticotrophin-releasing factor-41 in the rat. J Endocrinol 120:119–124
Harbuz MS, Jessop DS, Lightman SL, Chowdrey HS 1994 The effects of restraint or hypertonic saline stress on corticotrophin-releasing factor, arginine vasopressin, and proenkephalin A mRNAs in the CFY, Sprague-Dawley and Wistar strains of rat. Brain Res 667:6–12
Young 3rd WS, Mezey E, Siegel RE 1986b Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci Lett 70:198–203
Harbuz MS, Lightman SL 1989 Responses of hypothalamic and pituitary mRNA to physical and psychological stress in the rat. J Endocrinol 12:705–711
Paxinos G, Watson C 1998 The rat brain in stereotaxic coordinates. 4th ed. London: Academic Press
Edwards CMB, Abusnana S, Sunter D, Murphy KG, Ghatei MA, Bloom SR 1999 The effect of the orexins on food intake: comparison with neuropeptide Y, melanin-concentrating hormone and galanin. J Endocrinol 160:7–12
Haynes AC, Jackson B, Overend P, Buckingham RE, Wilson S, Tadayyon M, Arch JR 1999 Effects of single and chronic intracerebroventricular administration of the orexins on feeding in the rat. Peptides 20:1099–1105
Drazen DL, Coolen LM, Strader AD, Wortman MD, Woods SC, Seeley RJ 2004 Differential effects of adrenalectomy on melanin-concentrating hormone and orexin A. Endocrinology 145:3404–3412
Ford GK, Al-Barazanji KA, Wilson S, Harbuz MS, Jessop DS 2002 Effects of glucocorticoid manipulation on orexin-A induced food intake in rats. Br J Pharmacol Proc Suppl 135:109P
Tataranni PA, Larson DE, Snitker S, Young JB, Flatt JP, Ravussin E 1996 Effects of glucocorticoids on energy metabolism and food intake in humans. Am J Physiol 271:E317–E325
Dallman MF, Akana SF, Strack AM, Scribner KS, Pecoraro N, La Fleur SE, Houshyar H, Gomez F 2004 Chronic stress-induced effects of corticosterone on brain: direct and indirect. Ann NY Acad Sci 1018:141–150
Ida T, Nakahara K, Kuroiwa T, Fukui K, Nakazato M, Murakami T, Murakami N 2000 Both corticotropin releasing factor and neuropeptide Y are involved in the effect of orexin (hypocretin) on the food intake in rats. Neurosci Lett 293:119–122
Stanley BG, Lanthier D, Chin AS, Leibowitz SF 1989 Suppression of neuropeptide Y-elicited eating by adrenalectomy or hypophysectomy: reversal with corticosterone. Brain Res 501:32–36
Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS 1999 Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20:68–100
Jain MR, Horvath TL, Kalra PS, Kalra SP 2000 Evidence that NPY Y1 receptors are involved in stimulation of feeding by orexins (hypocretins) in sated rats. Regul Pept 87:19–24
Cintra A, Zoli M, Rosen L, Agnati LF, Okret S, Wikstrom AC, Gustaffsson JA, Fuxe K 1994 Mapping and computer assisted morphometry and microdensitometry of glucocorticoid receptor immunoreactive neurons and glial cells in the rat central nervous system. Neuroscience 62:843–897
Morimoto M, Morita N, Ozawa H, Yokoyama K, Kawata M 1996 Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study. Neurosci Res 26:235–269
Watts AG, Tanimura S, Sanchez-Watts G 2004 Corticotropin-releasing hormone and arginine vasopressin gene transcription in the hypothalamic paraventricular nucleus of unstressed rats: daily rhythms and their interactions with corticosterone. Endocrinology 145:529–540
Stricker-Krongrad A, Beck B 2002 Modulation of hypothalamic hypocretin/orexin mRNA expression by glucocorticoids. Biochem Biophys Res Commun 296:129–133
Rivet JM, Castagne V, Corder R, Gaillard R, Mormede P 1989 Study of the influence of stress and adrenalectomy on central and peripheral neuropeptide Y levels. Comparison with catecholamines. Neuroendocrinology 50:413–420
Pralong FP, Corder R, Gaillard RC 1993 The effects of chronic glucocorticoid excess, adrenalectomy and stress on neuropeptide Y in individual rat hypothalamic nuclei. Neuropeptides 25:223–231
Akabayashi A, Watanabe Y, Wahlestedt C, McEwen BS, Paez X, Leibowitz SF 1994 Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats. Brain Res 665:201–212
Larsen P, Jessop D, Chowdrey H, Lightman S, Mikkelsen J 1994 Chronic administration of glucocorticoids directly upregulates prepro-neuropeptide Y and Y1-receptor mRNA levels in the arcuate nucleus of the rat. J Neuroendocrinol 6:153–160
Bingham S, Davey PT, Babbs AJ, Irving EA, Sammons MJ, Wyles M, Jeffrey P, Cutler L, Riba I, Johns A, Porter RA, Upton N, Hunter AJ, Parsons AA 2001 Orexin-A, an hypothalamic peptide with analgesic properties. Pain 92:81–90(Gemma K. Ford, Kamal A. A)