Gonadal Steroids Differentially Regulate the Messenger Ribonucleic ...

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Endocrinology 144(4):1219 –1225 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-0030

Gonadal Steroids Differentially Regulate the Messenger Ribonucleic Acid Expression of Pituitary Orexin Type 1 Receptors and Adrenal Orexin Type 2 Receptors ¨ HREN, NORBERT BRU ¨ GGEMANN, ANDREAS DENDORFER, OLAF JO

AND

PETER DOMINIAK

Institute of Experimental and Clinical Pharmacology and Toxicology, University Clinic Lu¨beck, D-23538 Lu¨beck, Germany Hypothalamic prepro-orexin as well as pituitary and adrenal orexin receptors are gender-specifically expressed. To assess the regulation by gonadal steroids, we investigated the effect of 17␤-estradiol in female and of testosterone in male rats on prepro-orexin and orexin receptor mRNA expression. Rats were either sham-operated or gonadectomized and subsequently treated with placebo, 17␤-estradiol, or testosterone for 21 d. Tissue mRNA levels of prepro-orexin, orexin type-1 (OX1), and orexin type-2 (OX2) receptors were measured using quantitative real-time RT-PCR. In female rats, pituitary OX1 receptor mRNA levels were increased 12-fold after ovariectomy compared with shamoperated rats. The increase of pituitary OX1 receptor mRNA was inhibited by treatment with 17␤-estradiol. Adrenal mRNA levels of OX2 receptors in ovariectomized rats were increased

O

REXIN A AND OREXIN B were isolated as endogenous ligands for an orphan G protein-coupled receptor (1). Both peptides derive from a common precursor, prepro-orexin, which is identical with the independently cloned prepro-hypocretin (2). Prepro-orexin is selectively expressed in the lateral hypothalamic area, a region involved in the control of feeding behavior (1, 2). Consequently, orexins were shown to stimulate food intake in rats when injected into the brain ventricles and prepro-orexin mRNA was upregulated in the lateral hypothalamus by fasting (1). Orexin (hypocretin)-containing neurons project to numerous areas throughout the brain and the spinal cord, suggesting the involvement of orexins in the regulation of multiple physiological functions besides their effect on feeding behavior (3–7). These areas include the cerebral cortex, limbic system, locus coeruleus, and brain stem regions but also the hypothalamus itself. Accordingly, orexins were shown to regulate autonomic functions like heart rate and blood pressure (8, 9), sleep-wake behavior and arousal (10 –12), and various neuroendocrine systems (13–16). Two subtypes of orexin receptors, namely OX1 and OX2 receptors, were cloned (1). Their expression patterns in the rat brain are in good agreement with the widespread projections of orexin immunoreactive neurons (17, 18). Within the hypothalamus, OX1 receptor mRNA was detected in areas like the medial preoptic nucleus, the anterior hypothalamic nucleus, and the ventromedial nucleus, whereas Abbreviations: CT, Cycle threshold; E2, 17␤-estradiol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; icv, intracerebroventricular; ORX, orchidectomized; OVX, ovariectomized; OX1, orexin type-1; OX2, orexin type-2; Plac, placebo; Rn, fluorescence threshold; T, testosterone.

2-fold compared with sham-operated rats and were also reduced by treatment with 17␤-estradiol. In male rats, orchidectomy increased the mRNA levels of pituitary OX1 receptors compared with sham-operated rats. In contrast, adrenal OX2 receptor mRNA was reduced after orchidectomy. Testosterone treatment reversed the effect of orchidectomy on pituitary OX1 and adrenal OX2 receptors. In the hypothalamus, no differences were found in the mRNA levels of prepro-orexin, OX1, and OX2 receptors between sham-operated, placebotreated, and steroid-treated female or male rats. Our results indicate that gonadal steroids differentially regulate pituitary OX1 receptors and adrenal OX2 receptors in male and female rats and may contribute to specific sexdependent neuroendocrine and endocrine actions of orexins. (Endocrinology 144: 1219 –1225, 2003)

OX2 receptor mRNA was found in regions such as the paraventricular and the arcuate nucleus (18). Therefore, the effects of intracerebroventricularly (icv) injected orexins on plasma hormones such as ACTH, prolactin, LH, and GH (13–16) may be mediated by affecting the synthesis and/or liberation of releasing hormones. In addition, orexins may directly influence pituitary hormones because orexinergic nerve fibers are present in the median eminence and pituitary gland and orexin receptors were detected in the pituitary gland (4, 19, 20). Indeed, orexins were shown to stimulate CRH and GnRH release from the hypothalamus (21) but also to inhibit releasing hormone-stimulated ACTH and LH release from the pituitary (22, 23). The inhibitory action of orexin on GnRH-stimulated pituitary LH release was observed only in proestrous female rats and not in male rats (22). Moreover, in female rats the effect of icv-injected orexins was dependent on the status of ovarian steroids (16). Furthermore, whereas in ovariectomized (OVX) rats treated with 17␤-estradiol (E2) and progesterone icv orexin increased plasma LH levels, in untreated OVX rats orexin decreased plasma LH levels (16). One explanation for this bimodal action of orexins on LH release is a possible regulation of orexin receptors by estrogens. Interestingly, we have recently detected a sexually dimorphic expression of peripheral orexin receptors with higher mRNA levels of OX1 receptors in the pituitary and of OX2 receptors in adrenal glands of male rats when compared with female rats (20). To clarify the effect of gonadal steroids on the orexin system, we analyzed the mRNA expression of prepro-orexin as well as OX1 and OX2 receptors in the hypothalamus, pituitary, and adrenal glands of sham-operated and gona-

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Endocrinology, April 2003, 144(4):1219 –1225

dectomized male and female rats and the influence of subsequent E2 or T treatment using quantitative real-time PCR. Materials and Methods Animals and treatments OVX and sham-operated female Wistar rats (8 wk old) as well as orchidectomized (ORX) and sham-operated male rats (8 wk old) were purchased from Charles River (Sulzfeld, Germany) after they had been operated at the facilities of the supplier. Rats were kept under controlled conditions with a 12-h dark, 12-h light cycle with free access to standard diet and water. One week after surgery, rats were anesthetized with pentobarbital (40 mg kg⫺1, ip) and implanted sc with 21 d-release hormone pellets (Innovative Research of America, Sarasota, FL). Shamoperated rats received placebo (Plac) pellets (Sham ⫹ Plac, n ⫽ 8). OVX female rats received either Plac pellets (OVX ⫹ Plac, n ⫽ 8) or E2 pellets (OVX ⫹ E2, n ⫽ 8) with concentrations of 0.1 mg E2/pellet. ORX male rats received either Plac pellets (ORX ⫹ Plac, n ⫽ 8) or testosterone (T) pellets (ORX ⫹ T, n ⫽ 8) with concentrations of 5.0 mg T/pellet. Three weeks after the pellet implantation, the rats were weighed and killed by decapitation. Trunk blood was collected on ice, serum was processed from blood, aliquoted and stored at –20 C until assayed for orexin A, E2, or T levels. Brains, pituitaries, and adrenal glands were isolated, frozen immediately in isopentane (–30 C), and stored at – 80 C until use. The hypothalami were dissected according to Palkovits and Brownstein (24). All animal protocols complied with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Ministerium fu¨ r Umwelt, Natur und Forsten of Schleswig-Holstein, Germany (Animal Protocol No. 9/z/01).

RNA isolation and cDNA synthesis Total RNA was isolated from tissue homogenates by the guanidinium isothiocyanate method and purified using silica-gel-based spin columns (RNeasy Kit, QIAGEN GmbH, Hilden, Germany) after digestion of genomic DNA by treatment with deoxyribonuclease I (QIAGEN). Firststrand cDNA was synthesized from 1 ␮g of total RNA in the presence of 5 mm MgCl2, 10 mm Tris-HCl (pH 9.0), 50 mm KCl, 0.1% Triton X-100, 1 mm deoxy-NTPs, 1 U/␮l RNasin, 0.5 ␮g oligo-(deoxythymidine)15 primer, and 15 U avian myeloblastosis virus reverse transcriptase (Promega GmbH, Mannheim, Germany). To validate successful deoxyribonuclease I treatment, the reverse transcriptase was omitted in control reactions. The absence of PCR-amplified DNA fragments in these samples indicated the isolation of RNA free of genomic DNA.

Quantitative real-time PCR To amplify prepro-orexin, OX1 and OX2 receptor, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, sense and antisense oligonucleotide primers were designed based on the published cDNA sequences (1, 25) using the Primer3 software by S. Rozen and H. J. Skaletsky (code available at http://www-genome.wi.mit.edu/genome_ software/other/primer3.html). Oligonucleotides were obtained from Invitrogen GmbH (Karlsruhe, Germany). The sequences of the primers were as follows: 5⬘-GCC GTC TCT ACG AAC TGT TG-3⬘ (prepro-orexin sense), 5⬘-CGA GGA GAG GGG AAA GTT AG-3⬘ (prepro-orexin antisense), 5⬘-CCC TCA ACT CCA GTC CTA GC-3⬘ (OX1 sense), 5⬘-CAG GGA GGG CCT ATA ATT GA-3⬘ (OX1 antisense), 5⬘-CAA TGT TGT TGG GGT GCT TA-3⬘ (OX2 sense), 5⬘-TCC CCC TCT CAT AAA CTT GG-3⬘ (OX2 antisense), 5⬘-CTC CCT CAA GAT TGT CAG CA-3⬘ (GAPDH sense), and 5⬘-GTT CAG CTC TGG GAT GAC CT-3⬘ (GAPDH antisense). Quantitative measurement of prepro-orexin, OX1 and OX2 receptor, and GAPDH cDNA was performed by kinetic PCR using SYBR green I as fluorescent dye (Eurogentec SA, Seraing, Belgium) as described previously (20). Each sample was analyzed in triplicate on the GeneAmp 5700 sequence detection system (PE Applied Biosystems, Weiterstadt, Germany) along with specific standards and no template controls to monitor contaminating DNA. Each reaction consisted of 100 ng cDNA, 0.3 ␮m primers, 10 mm Tris-HCl, 50 mm KCl, 3 mm MgCl2, 0.2 mm deoxy-NTPs, and 1.25 U Hot GoldStar DNA Polymerase (Eurogentec) in a final volume of 50 ␮l. After denaturation at 95 C for 10 min, the

Jo¨ hren et al. • Orexin Receptor Regulation by Sex Steroids

cDNA products were amplified with 40 cycles, each cycle consisting of denaturation at 95 C for 30 sec, annealing and extension at 60 C for 60 sec. The accumulating DNA products were monitored by the GeneAmp 5700 sequence detection system, and data were stored continuously during the reaction. Product purity was confirmed by dissociation curve analysis and agarose gel electrophoresis in the presence of ethidium bromide. The identity of the amplified cDNA fragments was confirmed by restriction analysis. Furthermore, the amplified prepro-orexin, OX1 and OX2 receptor, and GAPDH cDNA fragments were cloned using a TA cloning kit (Invitrogen) and the identities of their nucleotide sequences were confirmed. The calculations of the initial mRNA copy numbers in each sample were made according to the cycle threshold (CT) method (26). Dilutions of known amounts of cloned prepro-orexin, OX1 and OX2 receptor cDNA fragments were used to generate standard curves (20). The CT for each sample was calculated at a fluorescence threshold (Rn) of 0.3 using the GeneAmp 5700 sequence detection system software with an automatic baseline setting. The detection limit for the gene of interest was about 20 copies per 100 ng cDNA. The copy numbers of prepro-orexin, OX1 and OX2 receptor mRNA were normalized using GAPDH mRNA levels. No differences were observed between treatment groups in the GAPDH mRNA levels.

RIAs Concentrations of plasma E2 or T were measured using 125I RIA kits (ICN Biomedicals, Inc., Eschwege, Germany). After two extractions of 200 ␮l serum with 1 ml ether and evaporation, each sample was reconstituted in 100 ␮l RIA buffer, and 25 ␮l of the samples were assayed in duplicate according to the manufacturer’s instructions. Intraassay and interassay variations were less than 6% and 10%, respectively. Plasma orexin A was extracted by adsorption to phenyl-silica (Isolute SPE, International Sorbent Technology, Mid Glamorgan, UK). After elution in 60% acetonitrile and 1% trifluoroacetic acid, the samples were concentrated by lyophilization. Orexin A concentrations were determined by a specific RIA according to the manufacturer’s instructions (Peninsula Laboratories, Inc., Belmont, CA). The detection limit of the assay was 3 pg/tube, based on the amount of orexin A that produced at least 10% tracer displacement. All samples were analyzed within the same assay. The intraassay variability was less than 10%.

Statistics Data are presented as mean ⫾ sem. Differences between treatment groups were estimated by one-way variance analysis (ANOVA) followed by Bonferroni’s posttest using the GraphPad Software, Inc. (San Diego, CA) Prism Software. P ⬍ 0.05 indicated statistically significant differences.

Results Specific amplification of prepro-orexin, OX1, and OX2 receptor mRNA in the hypothalamus-pituitary-adrenal axis by real-time PCR

Amplification of cDNA from the hypothalamus of male and female rats with prepro-orexin, OX1, and OX2 receptorspecific primers resulted in PCR products of the expected size (Fig. 1). No amplification products were observed in no-template controls (H2O), which were analyzed simultaneously in all assays indicating the absence of any contaminating DNA (Fig. 1). Restriction analysis of the amplified cDNA fragments confirmed their identity with the published cDNA clones of prepro-orexin, and OX1 or OX2 receptors (Fig. 1). The size and restriction pattern of the amplified cDNA of OX1 and OX2 receptors was also confirmed for the pituitary and adrenal glands (not shown). Amplification of prepro-orexin, OX1, and OX2 receptors in the hypothalamus, pituitary, and adrenal gland was achieved within the exponential phase of the PCR (Fig. 2). Background fluorescence in


no-template control samples typically did not surpass the Rn value of 0.3 at less than 35 cycles (Fig. 2). Expression prepro-orexin mRNA in the hypothalamus of male and female rats and effect of gonadectomy and hormone treatment

Very high mRNA levels of prepro-orexin were found in hypothalamus of female (Fig. 3A) and male (Fig. 3B) rats by quantitative real-time PCR using the CT method. No differences were detected in the mRNA levels of hypothalamic prepro-orexin between sham-operated control rats, gonadectomized rats treated with Plac, and steroid-treated female or male rats (Fig. 3). Expression of OX1 and OX2 receptor mRNA in female rats and effect of ovariectomy and E2 treatment

High mRNA levels of OX1 and OX2 receptors were found in hypothalamus of female rats (Fig. 4). In the pituitary gland of sham-operated female rats, significant amounts of OX1 receptor mRNA were present (Fig. 4A), whereas only very low mRNA levels of OX2 receptors were detected (Fig. 4B).

FIG. 1. Amplification and restriction analysis of cDNA fragments of the expected size of 303 bp for prepro-orexin (A), 260 bp for the OX1 receptor (B), and 314 bp for the OX2 receptor (C). PvuII cuts the prepro-orexin cDNA fragments into 136- and 167-bp fragments (A). Alu I cuts the OX1 receptor cDNA fragments into four fragments of 20 bp (not visible in the gel), 51 bp, 59 bp, and 130 bp (B), and the OX2 receptor cDNA fragments into two fragments with a size of 89 bp and 225 bp (C). ⫹, Treatment with restriction enzyme. M, DNA size marker as indicated in base pairs on the left.

Endocrinology, April 2003, 144(4):1219 –1225 1221

In the adrenal gland of female rats, we found low mRNA levels of OX1 receptors and high mRNA levels of OX2 receptors (Fig. 4). In the hypothalamus of female rats, no differences were detected in the mRNA levels of OX1 or OX2 receptors between sham-operated control rats, OVX rats, and OVX rats treated with E2 for 21 d (Fig. 4). In the pituitary gland of Plac-treated OVX rats, OX1 receptor mRNA levels were significantly higher compared with Plac-treated sham-operated rats (Fig. 4A). Treatment of OVX rats with E2 completely reversed the OVX-induced increase of the mRNA levels of pituitary OX1 receptors (Fig. 4A). As in control rats, low amounts of OX2 receptor mRNA were found in the pituitary glands of Plac- or E2-treated OVX rats (Fig. 4B). In adrenal glands, there were no substantial differences of the mRNA levels of OX1 receptors between the treatment groups (Fig. 4A). However, OX2 receptor mRNA levels were significantly higher in Plac-treated OVX rats when compared with Plactreated sham-operated rats and significantly reduced again by E2 treatment in OVX rats (Fig. 4B).

FIG. 3. Effects of OVX and E2 treatment (A) or ORX and T treatment (B) on hypothalamic prepro-orexin mRNA levels. Shown are means ⫾ SEM (n ⫽ 8). Sham, Sham-operated rats.

FIG. 2. Amplification plots of prepro-orexin (A), OX1 receptor mRNA (B), and OX2 receptor mRNA (C) in the hypothalamus, pituitary, and adrenal gland of male rats (n ⫽ 8). Fluorescence was measured during PCR after every cycle using the GeneAmp 5700 sequence detection system. For each sample, the CT was determined automatically, at which an Rn of 0.3 was reached. No template controls (H2O) were used to estimate background amplification. Because in the pituitary and adrenal no significant amounts of prepro-orexin mRNA were previously detected (20), real-time PCR for prepro-orexin was not performed in these organs.

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FIG. 4. Effects of OVX and E2 treatment on hypothalamic, pituitary, and adrenal OX1 and OX2 receptor mRNA levels in female rats. Shown are means ⫾ SEM (n ⫽ 8). Note the different scales. *, P ⬍ 0.05; ***, P ⬍ 0.001.

Expression of OX1 and OX2 receptor mRNA in male rats and effect of ORX and T treatment

OX1 and OX2 receptor mRNA was detected at high levels in the hypothalamus of male rats (Fig. 5). In the pituitary gland, high mRNA levels of OX1 receptors and only very low mRNA levels of OX2 receptors were present in male rats (Fig. 5). In contrast to the low levels of adrenal OX1 receptor mRNA (Fig. 5A), very high mRNA levels of OX2 receptors were detected in the adrenal gland of male rats (Fig. 5B). No differences were detected between the amounts of OX1 or OX2 receptor mRNA in the hypothalamus of male shamoperated control rats, ORX rats, and ORX rats treated with T for 21 d (Fig. 5). Pituitary mRNA levels of OX1 receptors were significantly up-regulated, and the mRNA levels of adrenal OX2 receptors were significantly down-regulated in Plac-treated ORX rats when compared with sham-operated rats (Fig. 5). The effects of orchidectomy in both pituitary and adrenal glands were reversed by treatment of ORX rats with T (Fig. 5). No differences were observed in the low amounts of pituitary OX2 and adrenal OX1 receptor mRNA between the treatment groups in male rats (Fig. 5). Effect of gonadectomy and hormone treatment on plasma hormones and body weight

Ovariectomy reduced and subsequent treatment of OVX rats with E2 significantly increased plasma E2 levels (Table

1). Plasma T levels were undetectable after orchidectomy and restored after treatment with T (Table 2). No differences in plasma orexin A levels were observed between sham-operated, OVX, and E2-treated female rats (Table 1). Male rats showed a slight but significant reduction of plasma orexin A levels after ORX when compared with sham-operated rats. Treatment of ORX rats with T restored the plasma orexin A levels (Table 2). Body weight was significantly increased in female rats after ovariectomy, whereas treatment with E2 for 21 d suppressed weight gain (Table 1). No differences in body weight were observed between male sham-operated control rats, ORX rats, and ORX rats treated with T for 21 d (Table 2). Discussion

We have recently found profound differences in the mRNA levels of pituitary OX1 and adrenal OX2 receptors between male and female rats (20). In addition, the mRNA expression of hypothalamic prepro-orexin was higher in female rats (27). Our present results confirm the expression of OX1 receptors in the pituitary and OX2 receptors in the adrenal gland (20, 28 –31). Furthermore, we show for the first time that gonadal steroids affect the mRNA expression of OX1 and OX2 receptors in the pituitary and adrenal glands. The up-regulation by ovariectomy and down-regulation by E2 of pituitary OX1 and adrenal OX2 receptors in female rats



FIG. 5. Effects of ORX and T treatment on hypothalamic, pituitary, and adrenal OX1 and OX2 receptor mRNA levels in male rats. Shown are means ⫾ SEM (n ⫽ 8). Note the different scales. *, P ⬍ 0.05; ***, P ⬍ 0.001.

TABLE 2. Plasma T concentrations, plasma orexin A concentrations, and body weights in sham-operated and ORX male rats treated with placebo or T

TABLE 1. Plasma E2 concentrations, plasma orexin A concentrations, and body weights in sham-operated and OVX female rats treated with placebo or E2 Sham ⫹ placebo

OVX ⫹ placebo

OVX ⫹ E2

Sham ⫹ placebo

ORX ⫹ placebo

ORX ⫹ T

1.8 ⫾ 0.3 53.4 ⫾ 1.7 417 ⫾ 3

nd 45.4 ⫾ 2.3a,b 415 ⫾ 4ns,b

1.1 ⫾ 0.2 53.7 ⫾ 2.7a,c 404 ⫾ 8ns,c

Plasma E2 (pg/ml) 32.9 ⫾ 9.2 9.3 ⫾ 2.8a,c 127.9 ⫾ 25.2b,d,e Plasma orexin A (pg/ml) 50.5 ⫾ 4.2 49.6 ⫾ 2.7ns,c 48.8 ⫾ 3.1ns,d Body weight (g) 234 ⫾ 4 281 ⫾ 3b,c 212 ⫾ 2b,d,e

Plasma T (ng/ml) Plasma orexin A (pg/ml) Body weight (g)

Shown are means ⫾ SEM (n ⫽ 8). a, P ⬍ 0.05; b, P ⬍ 0.001; significant differences. c Compared with Plac-treated Sham rats. d Compared with Plac-treated OVX rats. e Compared with sham-operated rats.

Shown are means ⫾ SEM (n ⫽ 8). a, P ⬍ 0.001; differences; nd, not detectable. b Compared with Plac-treated Sham rats. c Compared with Plac-treated ORX rats.

ns

, no

is in accordance with the much lower mRNA levels of these receptors in intact female rats compared with male rats (20). In addition, T up-regulated adrenal OX2 receptor mRNA in male rats, whereas pituitary OX1 receptor mRNA was downregulated. Thus, the lower pituitary OX1 receptor mRNA levels in female rats (20) may be explained by inhibitory actions of E2 in female rats. In the adrenal, both inhibitory actions of E2 in female rats and stimulatory actions of T in male rats may contribute to the differences of OX2 receptor mRNA levels between female and male rats (20). In addition to their effects on energy homeostasis, there is considerable evidence that orexins can regulate the activity of the hypothalamo-pituitary-gonadal axis (16, 22, 32–34). Icv

ns

, no significant

injected orexins increased plasma LH concentrations in ovarian steroid-treated female rats, whereas in untreated OVX rats plasma LH was reduced as initially shown by Pu et al. (16). This bimodal response appears, in part, to be mediated by GnRH because orexin stimulates GnRH release from hypothalamic explants of male and proestrous female rats (21, 22). In addition, orexin containing neurons project to several hypothalamic nuclei including areas involved in the regulation of GnRH where orexin receptors are present (3, 4, 35, 36). However, because orexin receptors are also present in the pituitary gland, a direct effect of orexins can be assumed. In ovarian steroid-treated rats, the stimulatory effect of icv orexins on pituitary LH release was observed already at low doses, whereas a high dose of orexin was necessary to sup-

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press the release of LH in the absence of ovarian steroids (16, 32). It could be possible that orexins, when injected icv at high doses, reach the pituitary gland by diffusion into the portal circulation and stimulate pituitary OX1 receptors, which are up-regulated in the absence of ovarian steroids as shown by our results. Although, there was no significant effect of orexin on basal LH release from dispersed pituitary cells, orexin inhibited GnRH-induced release of LH from isolated pituitary cells in proestrous female rats (22). Thus, orexins may differentially regulate the activity of the hypothalamopituitary gonadal axis by exerting contrary effects on the hypothalamus and pituitary gland. These estrogen-dependent bimodal actions of orexins on LH release in female rats may be caused by the up-regulation of OX1 receptors at low E2 levels. Such a receptor-dependent regulation of hormonerelease is well established for other peptides such as angiotensin II (37, 38). In addition to the effect on GnRH-induced LH release, other direct actions of orexin on the pituitary gland are possible. Although no effects of orexins on basal ACTH were observed in male and female rats, Samson and Taylor (23) reported an inhibitory effect of orexins on CRH-stimulated ACTH release in vitro. This finding is supported by the localization of orexin receptors in human corticotrope pituitary cells (30). Interestingly, the ACTH response to stress depends on gonadal steroids in both male and female rats (39, 40). The role of orexins in these responses remains unclear. However, our findings of a strong regulation of pituitary OX1 receptors by E2 and, to a lesser degree, by T, imply a so far undefined hormonal regulation of orexin actions in the pituitary gland of male and female rats. In the adrenal gland, we found very high mRNA levels of OX2 receptors in accordance with our previous findings which also showed much higher amounts of OX2 receptor mRNA in male rats compared with female rats (20). Interestingly, gonadal hormones differentially affected the mRNA levels of adrenal OX2 receptors. E2 reduced the mRNA levels of OX2 receptors, whereas T increased the mRNA levels of OX2 receptors. Therefore, both E2 in female rats and T in male rats may contribute to the observed sex differences in adrenal OX2 receptor mRNA expression. Inconsistent data exist regarding the localization of adrenal orexin receptors. In rats, OX1 and OX2 receptor like immunoreactivity was found in the adrenal medulla but not in the cortex (28). In contrast, quantitative PCR showed much higher levels of OX2 receptor mRNA, which was selectively localized in the adrenal cortex of the rat by in situ hybridization (20). These results are supported by findings of Randeva et al. (31), who found OX2 receptors but not OX1 receptors selectively in the cortex of human adrenals using RT-PCR, in situ hybridization and immunostaining. Others, however, have described OX1 receptors in the cortex and OX2 receptors in the medulla of the human adrenal (41). Because orexins stimulate adrenal glucocorticoid release in rats and humans (42, 43) and glucocorticoids play an unique role in energy homeostasis, it is intriguing to speculate that in male rats orexins may regulate glucose metabolism via regulation of glucocorticoids. Interestingly, we found increased levels of plasma orexin A by T in male rats in contrast to female rats where plasma orexin A levels were unchanged by E2. Further exper-


iments are needed to clarify possible T-dependent functions of the adrenal orexin system. Thus far, it is unclear whether, in rats, adrenal OX2 receptors are stimulated by circulating orexins or whether orexins are locally produced in the adrenal gland as described by Randeva et al. (31) in humans. The presence of orexin in plasma has been described before in rats and humans and circulating orexins may activate adrenal orexin receptors (20, 44). Although the source of plasma orexins is currently uncertain, orexin-producing neurons project to the median eminence and may therefore contribute to plasma orexins (29). In contrast to the lower mRNA levels of orexin receptors in the pituitary and adrenal glands of female rats compared with male rats, prepro-orexin and OX1 receptor mRNA levels are elevated in the hypothalamus of female rats (20, 27). In our present study, we could not reveal changes of hypothalamic prepro-orexin or orexin receptor mRNA after gonadectomy or hormonal treatment in male and female rats. Our results in female rats are in accordance with findings of Russell et al. (22), showing no effect of estrogens on hypothalamic prepro-orexin mRNA in female rats. In addition, in male sheep, E2 did not affect prepro-orexin mRNA levels (45). Thus, other factors may be responsible for the observed differences between male and female rats, and it may be concluded that the central (hypothalamic) expression of prepro-orexin and orexin receptors is not under the control of ovarian steroids. However, because orexin receptor subtypes are distinctively expressed in hypothalamic nuclei (18) discrete changes of orexin receptor mRNA levels in specific hypothalamic nuclei might not be detectable by analyzing whole hypothalamic extracts using RT-PCR and cannot completely ruled out by our results. Estrogens and T have divergent effects on food intake, energy expenditure, and body weight and plasma levels of estrogens correlate positively, whereas plasma levels of T correlate negatively with body fat mass (46). Our findings of weight gain after ovariectomy is in accordance with previous observations (47). The reduced weight in female rats after E2 replacement may be attributed to the somewhat higher plasma E2 levels in E2-treated female rats in comparison to sham-operated female rats (47). Studies by Bernardis et al. (48) showed that OVX could influence body weight independently of food intake and separately from central mechanisms. In our study, the regulation of orexin receptors by gonadal steroids appears to be limited to peripheral organs. Thus, the peripheral orexin system and particularly adrenal OX2 receptors may contribute to sex specific regulations of energy homeostasis. In summary, our results show that E2 and T differentially regulate the mRNA expression of pituitary OX1 and adrenal OX2 receptors in female and male rats. Thus, in addition to the ability of orexins to regulate the reproductive system, sex steroids may affect the actions of orexins. Like other peptides regulating energy homeostasis and reproductive functions (49) orexins may play an important role in coordinating these systems but may also sex dependently influence other orexin-related functions like the autonomic system and sleeping behavior.



Acknowledgments The authors wish to thank C. Eichholz for real-time PCR assistance and G. Vierke for RIA assistance. Received November 12, 2002. Accepted December 18, 2002. Address all correspondence and requests for reprints to: Olaf Jo¨ hren, Ph.D., Institute of Experimental and Clinical Pharmacology and Toxicology, University Clinic Lu¨ beck, Ratzeburger Allee 160, D-23538 Lu¨ beck, Germany. E-mail: [email protected].

22. 23. 24. 25.

References

26.

1. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski 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 behavior. Cell 92:573–585 2. de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG 1998 The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 95:322–327 3. 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 4. 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 5. van den Pol AN 1999 Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J Neurosci 19:3171–3182 6. Harrison TA, Chen CT, Dun NJ, Chang JK 1999 Hypothalamic orexin Aimmunoreactive neurons project to the rat dorsal medulla. Neurosci Lett 273:17–20 7. Cutler DJ, Morris R, Sheridhar V, Wattam TA, Holmes S, Patel S, Arch JR, Wilson S, Buckingham RE, Evans ML, Leslie RA, Williams G 1999 Differential distribution of orexin-A and orexin-B immunoreactivity in the rat brain and spinal cord. Peptides 20:1455–1470 8. Samson WK, Gosnell B, Chang JK, Resch ZT, Murphy TC 1999 Cardiovascular regulatory actions of the hypocretins in brain. Brain Res 831:248 –253 9. Shirasaka T, Nakazato M, Matsukura S, Takasaki M, Kannan H 1999 Sympathetic and cardiovascular actions of orexins in conscious rats. Am J Physiol 277:R1780 –R1785 10. Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ, Nishino S, Mignot E 1999 The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98:365–376 11. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M 1999 Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98:437– 451 12. Peyron C, Faraco J, Rogers W, Ripley B, Overeem S, Charnay Y, Nevsimalova S, Aldrich M, Reynolds D, Albin R, Li R, Hungs M, Pedrazzoli M, Padigaru M, Kucherlapati M, Fan J, Maki R, Lammers GJ, Bouras C, Kucherlapati R, Nishino S, Mignot E 2000 A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains. Nat Med 6:991–997 13. 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 DN, 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 14. 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 15. 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 16. Pu S, Jain MR, Kalra PS, Kalra SP 1998 Orexins, a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone secretion in an ovarian steroid-dependent manner. Regul Pept 78:133–136 17. 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 18. Marcus JN, Aschkenasi CJ, Lee CE, Chemelli RM, Saper CB, Yanagisawa M, Elmquist JK 2001 Differential expression of orexin receptors 1 and 2 in the rat brain. J Comp Neurol 435:6 –25 19. Nambu T, Sakurai T, Mizukami K, Hosoya Y, Yanagisawa M, Goto K 1999 Distribution of orexin neurons in the adult rat brain. Brain Res 827:243–260 20. Jo¨hren O, Neidert SJ, Kummer M, Dendorfer A, Dominiak P 2001 Preproorexin and orexin receptor mRNAs are differentially expressed in peripheral tissues of male and female rats. Endocrinology 142:3324 –3331 21. Russell SH, Kim MS, Small CJ, Abbott CR, Morgan DG, Taheri S, Murphy KG, Todd JF, Ghatei MA, Bloom SR 2000 Central administration of orexin A

27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

42.

43.

44. 45. 46. 47. 48. 49.

suppresses basal and domperidone stimulated plasma prolactin. J Neuroendocrinol 12:1213–1218 Russell SH, Small CJ, Kennedy AR, Stanley SA, Seth A, Murphy KG, Taheri S, Ghatei MA, Bloom SR 2001 Orexin A interactions in the hypothalamopituitary gonadal axis. Endocrinology 142:5294 –5302 Samson WK, Taylor MM 2001 Hypocretin/orexin suppresses corticotroph responsiveness in vitro. Am J Physiol Regul Integr Comp Physiol 281:R1140 –R1145 Palkovits M, Brownstein MJ 1988 Maps and guide to microdissection of the rat brain. New York: Elsevier Tso JY, Sun XH, Kao TH, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:2485–2502 Higuchi R, Fockler C, Dollinger G, Watson R 1993 Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11:1026 –1030 Jo¨hren O, Neidert SJ, Kummer M, Dominiak P 2002 Sexually dimorphic expression of prepro-orexin mRNA in the rat hypothalamus. Peptides 23:1177–1180 Lopez M, Senaris R, Gallego R, Garcia-Caballero T, Lago F, Seoane L, Casanueva F, Dieguez C 1999 Orexin receptors are expressed in the adrenal medulla of the rat. Endocrinology 140:5991–5994 Date Y, Mondal MS, Matsukura S, Ueta Y, Yamashita H, Kaiya H, Kangawa K, Nakazato M 2000 Distribution of orexin/hypocretin in the rat median eminence and pituitary. Brain Res Mol Brain Res 76:1– 6 Blanco M, Lopez M, Garcia-Caballero T, Gallego R, Vazquez-Boquete A, Morel G, Senaris R, Casanueva F, Dieguez C, Beiras A 2001 Cellular localization of orexin receptors in human pituitary. J Clin Endocrinol Metab 86:1616 –1619 Randeva HS, Karteris E, Grammatopoulos D, Hillhouse EW 2001 Expression of orexin-A and functional orexin type 2 receptors in the human adult adrenals: implications for adrenal function and energy homeostasis. J Clin Endocrinol Metab 86:4808 – 4813 Tamura T, Irahara M, Tezuka M, Kiyokawa M, Aono T 1999 Orexins, orexigenic hypothalamic neuropeptides, suppress the pulsatile secretion of luteinizing hormone in ovariectomized female rats. Biochem Biophys Res Commun 264:759 –762 Kohsaka A, Watanobe H, Kakizaki Y, Suda T, Schioth HB 2001 A significant participation of orexin-A, a potent orexigenic peptide, in the preovulatory luteinizing hormone and prolactin surges in the rat. Brain Res 898:166 –170 Furuta M, Funabashi T, Kimura F 2002 Suppressive action of orexin A on pulsatile luteinizing hormone secretion is potentiated by a low dose of estrogen in ovariectomized rats. Neuroendocrinology 75:151–157 van den Pol AN, Gao XB, Obrietan K, Kilduff TS, Belousov AB 1998 Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J Neurosci 18:7962–7971 Iqbal J, Pompolo S, Sakurai T, Clarke IJ 2001 Evidence that orexin-containing neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus. J Neuroendocrinol 13:1033–1041 Steele MK, McCann SM, Negro-Vilar A 1982 Modulation by dopamine and estradiol of the central effects of angiotensin II on anterior pituitary hormone release. Endocrinology 111:722–729 Jo¨hren O, Sanvitto GL, Egidy G, Saavedra JM 1997 Angiotensin II AT1A receptor mRNA expression is induced by estrogen-progesterone in dopaminergic neurons of the female rat arcuate nucleus. J Neurosci 17:8283– 8292 Viau V, Meaney MJ 1991 Variations in the hypothalamic-pituitary-adrenal response to stress during the estrous cycle in the rat. Endocrinology 129:2503–2511 Handa RJ, Nunley KM, Lorens SA, Louie JP, McGivern RF, Bollnow MR 1994 Androgen regulation of adrenocorticotropin and corticosterone secretion in the male rat following novelty and foot shock stressors. Physiol Behav 55:117–124 Blanco M, Garcia-Caballero T, Fraga M, Gallego R, Cuevas J, Forteza J, Beiras A, Dieguez C 2002 Cellular localization of orexin receptors in human adrenal gland, adrenocortical adenomas and pheochromocytomas. Regul Pept 104: 161–165 Malendowicz LK, Tortorella C, Nussdorfer GG 1999 Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade. J Steroid Biochem Mol Biol 70:185–188 Mazzocchi G, Malendowicz LK, Gottardo L, Aragona F, Nussdorfer GG 2001 Orexin A stimulates cortisol secretion from human adrenocortical cells through activation of the adenylate cyclase-dependent signaling cascade. J Clin Endocrinol Metab 86:778 –782 Arihara Z, Takahashi K, Murakami O, Totsune K, Sone M, Satoh F, Ito S, Mouri T 2001 Immunoreactive orexin-A in human plasma. Peptides 22:139 –142 Archer ZA, Findlay PA, Rhind SM, Mercer JG, Adam CL 2002 Orexin gene expression and regulation by photoperiod in the sheep hypothalamus. Regul Pept 104:41– 45 Mystkowski P, Schwartz MW 2000 Gonadal steroids and energy homeostasis in the leptin era. Nutrition 16:937–946 Wade GN 1975 Some effects of ovarian hormones on food intake and body weight in female rats. J Comp Physiol Psychol 88:183–193 Bernardis LL, Bellinger LL, Goldman JK, MacKenzie R 1981 Dorsomedial hypothalamic lesions at weaning and ovariectomy after maturity: somatic and metabolic changes. Physiol Behav 26:91–98 Kalra SP, Kalra PS 1996 Nutritional infertility: the role of the interconnected hypothalamic neuropeptide Y-galanin-opioid network. Front Neuroendocrinol 17:371– 401