Regulation of corticotropin-releasing factor and its types 1 and 2 receptors by leptin in rats subjected to treadmill running-induced stress

in Journal of Endocrinology
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Qingling Huang Laval Hospital Research Center and Merck-Frosst/IRSC Obesity Research Chair, 2725 chemin Sainte-Foy, Québec, Canada G1V 4G5

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Elena Timofeeva Laval Hospital Research Center and Merck-Frosst/IRSC Obesity Research Chair, 2725 chemin Sainte-Foy, Québec, Canada G1V 4G5

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Denis Richard Laval Hospital Research Center and Merck-Frosst/IRSC Obesity Research Chair, 2725 chemin Sainte-Foy, Québec, Canada G1V 4G5

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(Requests for offprints should be addressed to D Richard; Email: denis.richard@crhl.ulaval.ca)
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The present study was conducted to investigate the long-term effects of subchronic elevation of central leptin levels on the expression of corticotropin-releasing factor (CRF) and its types 1 and 2 receptors in the brain of rats subjected to treadmill running-induced stress. PBS or recombinant murine leptin was infused continuously for a period of 5 days into the third ventricle of rats with the aid of osmotic minipumps at a delivery rate of 2 μg/day. On the fifth day of infusion, rats were killed under resting conditions or after a session of treadmill running, which is known to induce a stress response in rats. Leptin treatment significantly decreased food intake, body weight, white adipose tissue weight, glucose and insulin plasma contents, and blunted the treadmill running-induced elevation in plasma levels of corticosterone. Leptin infusion prevented stress-induced de novo synthesis of CRF in the paraventricular hypothalamic nucleus (PVN), which was measured using the intronic probe for CRF heteronuclear RNA. The induction of the type 1 CRF receptor (CRF1R) in the PVN and supraoptic nucleus in running rats was also significantly blunted by leptin. In contrast, leptin treatment strongly increased the expression of type 2 CRF receptor (CRF2R) in the ventromedial hypothalamic nucleus (VMH). The present results suggest that subchronic elevation of central levels of leptin blunts treadmill running-induced activation of the hypothalamic–pituitary–adrenal axis through the inhibition of activation of the CRFergic PVN neurons, and potentially enhances the anorectic CRF effects via the stimulation of expression of CRF2R in the VMH.

Abstract

The present study was conducted to investigate the long-term effects of subchronic elevation of central leptin levels on the expression of corticotropin-releasing factor (CRF) and its types 1 and 2 receptors in the brain of rats subjected to treadmill running-induced stress. PBS or recombinant murine leptin was infused continuously for a period of 5 days into the third ventricle of rats with the aid of osmotic minipumps at a delivery rate of 2 μg/day. On the fifth day of infusion, rats were killed under resting conditions or after a session of treadmill running, which is known to induce a stress response in rats. Leptin treatment significantly decreased food intake, body weight, white adipose tissue weight, glucose and insulin plasma contents, and blunted the treadmill running-induced elevation in plasma levels of corticosterone. Leptin infusion prevented stress-induced de novo synthesis of CRF in the paraventricular hypothalamic nucleus (PVN), which was measured using the intronic probe for CRF heteronuclear RNA. The induction of the type 1 CRF receptor (CRF1R) in the PVN and supraoptic nucleus in running rats was also significantly blunted by leptin. In contrast, leptin treatment strongly increased the expression of type 2 CRF receptor (CRF2R) in the ventromedial hypothalamic nucleus (VMH). The present results suggest that subchronic elevation of central levels of leptin blunts treadmill running-induced activation of the hypothalamic–pituitary–adrenal axis through the inhibition of activation of the CRFergic PVN neurons, and potentially enhances the anorectic CRF effects via the stimulation of expression of CRF2R in the VMH.

Introduction

Corticotropin-releasing factor (CRF) is a 41 amino acid peptide widely distributed throughout the brain, and particularly concentrated in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN; Vale et al. 1981, Sawchenko & Swanson 1985). The CRF neurons of the parvocellular PVN are predominantly involved in the control of the hypothalamic–pituitary–adrenal (HPA) axis. Activation of all components of the HPA axis, including the stimulation of PVN CRFergic neurons and the significant increase in adrenocorticotropin (ACTH) and corticosterone plasma levels, is seen in rats subjected to stressful conditions such as treadmill running (Richard et al. 1996, Timofeeva et al. 2003). After a session of treadmill running, about all PVN CRFergic neurons express the immediate early gene c-fos, a marker of persisting neuronal activity (Timofeeva et al. 2003). Besides its stress-related hypophysiotropic effects, CRF blunts energy balance through concomitantly reducing energy intake and augmenting energy expenditure (Richard et al. 2002). Central administration of CRF decreases food intake, as well as food hoarding, and increases thermogenesis (Gosnell et al. 1983, Rothwell 1990, Cabanac & Richard 1995, Buwalda et al. 1997).

The central effects of CRF are mediated through two distinct high-affinity membrane receptors, which are referred to as CRF types 1 (CRF1R; Perrin et al. 1993) and 2 (CRF2R; Lovenberg et al. 1995) receptors. The CRF1R is widely distributed throughout the brain and the anterior and intermediate lobes of the pituitary. Within the PVN, CRF1R mRNA is barely detectable under basal conditions, but can be readily induced by stressful stimuli (Rivest et al. 1995). The CRF1R has been suggested to mediate the corticotropic effects of CRF and assure basal and stress-induced HPA axis activity (Desouza et al. 1991). The expression of the CRF2R is restricted to the particular limbic regions and the ventromedial hypothalamic nucleus (VMH). The VMH is thought to mediate the anorectic effects of CRF through the CRF2R (Vaughan et al. 1995), and lesions of VMH increase food intake and lead to obesity (Egawa et al. 1993, Dube et al. 1999). The expression of the CRF2R in the VMH is significantly lower in genetically obese rats than in lean rats (Richard et al. 1996, Timofeeva & Richard 1997). There is evidence that the central effects of leptin may be mediated at least partially through the VMH, where the leptin receptors are expressed (Elmquist et al. 1998a,b, Nishiyama et al. 1999).

The adipocyte-derived hormone leptin circulates in the blood in proportion to body adiposity (Zhang et al. 1994, Campfield et al. 1995) and has a marked potential in reducing energy deposition by decreasing food intake and increasing energy expenditure (Halaas et al. 1995, Pelleymounter et al. 1995, Levin et al. 1996, Huang et al. 1998). Leptin may affect the HPA axis at different levels. Plasma leptin is inversely correlated with diurnal variations in glucocorticoid levels (Korbonits et al. 1997, Licinio et al. 1997). Leptin administration inhibits adrenal glucocorticoid synthesis and release (Bornstein et al. 1997, Kruse et al. 1998), and may reduce the ACTH stress-induced secretion (Trottier et al. 1998, Oates et al. 2000, Wilson et al. 2005). The effects of leptin on the synthesis and secretion of CRF from the PVN remain largely controversial. In vitro studies have demonstrated a stimulatory (Costa et al. 1997, Raber et al. 1997) as well as an inhibitory (Heiman et al. 1997) effect of leptin on CRF release. Acute administration of leptin may induce the expression of c-fos (Van Dijk et al. 1996, Elmquist et al. 1997, 1998a, Badoer & Ryan 1999, Elias et al. 2000) and increases the CRF mRNA levels (Nishiyama et al. 1999) in the PVN. However, chronic administration of leptin reduces ACTH and glucocorticoids secretion and inhibits stress-induced Fos expression in the PVN neurons (Huang et al. 1998, Oates et al. 2000).

The present study was designed to investigate the long-term effects of chronically elevated central leptin levels on the expression of the primary CRF transcript and CRF types 1 and 2 receptors in the brain of rats stressed by treadmill running. The results of this study suggest that daily leptin infusion inhibits stress-induced activation of PVN CRFergic neurons by blunting de novo CRF synthesis as well as the induction of CRF1R. In addition, the present results confirm the ability of centrally infused leptin to increase the expression of VMH CRF2R, which could potentially enhance the anorectic effects of CRF.

Materials and Methods

Animals and treatments

Thirty-two adult male Wistar rats were purchased from the Canadian Breeding Laboratories (St Constant, Quebec, Canada). All rats were cared for and handled according to the Canadian Guide for the Care and Use of Laboratory Animals and the present protocol was approved by our institutional animal care committee. The rats were individually housed in wire-bottom cages that were suspended above absorbent paper, and a stock diet was available ad libitum (Charles River Rodent Animal Diet, distributed by Ralston Products, Woodstock, Ontario, Canada). They were subjected to a 12 h light:12 h darkness cycle (light on 0645 h) and kept under an ambient temperature of 23 ± 1 °C.

On the tenth day after arrival, all rats were subjected to brain surgery to implant a guide cannula into the third ventricle. Rats were first anesthetized intraperitoneally with 1.5 ml mixture containing 20 mg/ml ketamine and 2.5 mg/ml xylazine and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). Then, a 22 gauge guide cannula was aimed at the third ventricle using the following stereotaxic coordinates: 2.3 mm posterior to bregma at the midline and 8.4 mm ventral to the brain surface (Paxinos & Watson 1997). The guide cannula was secured with screws and dental cement (Dentsply International, York, PA, USA) and was directly connected to the Alzet osmotic minipump (Model 2001; ALZA Pharmaceuticals, Palo Alto, CA, USA; 1.0 μl/h), which contained PBS (pH 7.4) or recombinant murine leptin (2 μg/day per rat; high purity, endotoxin level < 0.1 ng/μg; US Biological, Swampscott, MA, USA). PBS or leptin was continuously infused into the third ventricle for 5 days. Thereafter, the rats were killed either (i) at rest (PBS-resting; leptin-resting), (ii) immediately following a treadmill running session lasting 60 min (PBS-running; leptin-running), or (iii) at rest following a recovery period of 120 min after 60-min running (PBS-post-running; leptin-post-running). The running session took place between 0730 and 0910 h. During running, the speed of the motor-driven treadmill was raised from 10 to 25 m/min by increase of 5 m/min every 15 min. The rats were forced to run continuously for 60 min. Each experimental group included five to six rats.

Body weight, food intake, and tissue weight

Measurements of body weight and food intake were performed every morning between 0700 and 0800 h. Rats weighed between 270 and 290 g at the time of surgery. Epididymal white adipose tissue (eWAT) was quickly dissected out during intracardial perfusion with saline.

Plasma determinations

An intracardial blood sample was taken from anesthetized rats immediately before the intracardial perfusion. Serum corticosterone was determined by a competitive protein-binding assay (sensitivity 0.058 nmol/l; inter-assay coefficient of variation 9.0%) using plasma from a dexamethasone-treated female rhesus monkey as the source of transcortin (Murphy 1967).

Brain preparation

Rats were anesthetized with 1.5 ml mixture containing 20 mg/ml ketamine and 2.5 mg/ml xylazine. Without delay, they were intracardially perfused with 50 ml ice-cold isotonic saline followed by 500 ml paraformaldehyde (4%) solution. Brains were removed and kept in paraformaldehyde for an additional period of 7 days. They were then transferred overnight to a solution containing paraformaldehyde and sucrose (10%) and sliced using a sliding microtome (Histoslide 2000, Reichert-Jung, Heidelberger, Germany). Brain sections were taken from the olfactory bulb to the brain stem. Thirty micrometer thick sections of brains were collected and stored at −30 °C in a cold cryoprotecting solution containing sodium phosphate buffer (50 mM), ethylene glycol (30%), and glycerol (20%).

In situ hybridization histochemistry

In situ hybridization histochemistry was used to localize CRF heteronuclear (hn) RNA, CRF1R mRNA, and CRF2R mRNA on the brain sections taken from the entire brain. The protocol used was largely adapted from the technique described by Simmons et al.(1989). Briefly, one out of every five brain sections were mounted onto poly-l-lysine coated slides. The sections were then successively fixed in paraformaldehyde (4%), digested with proteinase K (10 μg/ml in 100 mM Tris–HCl containing 50 mM EDTA (pH 8.0)), acetylated with acetic anhydride (0.25% in 0.1 M triethanolamine (pH 8.0)), and dehydrated through graded concentrations (50, 70, 95, and 100%) of alcohol. Thereafter, 90 μl hybridization mixture, which contains an antisense 35S-labeled cRNA probe (107 c.p.m./ml), were spotted on each slide. The slides were sealed with the coverslips and incubated overnight at 58 °C in a slide warmer. The next day, the coverslips were removed and the slides were rinsed four times with SSC (0.6 M NaCl, 60 mM trisodium citrate buffer (pH 7.0)), digested with RNAse A (20 μg/ml in 10 mM Tris–500 mM NaCl containing 1 mM EDTA), washed in descending concentrations of SSC (2×, 10 min; 1×, 5 min; 0.5×, 5 min; 0.1×, 30 min at 60 °C) and dehydrated through graded concentrations of alcohol. Slides were exposed to an X-ray film (Kodak). Once removed from the autoradiography cassettes, the slides were defatted in xylene, dipped in NTB2 nuclear emulsion (Eastman Kodak), and exposed from 7 to 21 days, before being developed in D 19 developer and fixed in rapid fixer (Kodak). Finally, tissues were rinsed in distilled water and counterstained with thionin (0.25%).

Antisense 35S-labeled cRNA probes

The hn CRF cRNA probe was generated from 530 bp fragment of the CRF intron 1 (Dr S Watson, University of Michigan, Ann Arbor, MI, USA) subcloned into pGem-3 plasmid (Stratagene, La Jolla, CA, USA), and linearized with HindIII and EcoRI (Pharmacia Biotech, Inc.) for antisense and sense probes respectively. The CRF1R cRNA was generated from a 1.3 kb PstI fragment of the rat CRF1R cDNA (Dr W Vale, Peptide Biology Laboratory, The Salk Institute, La Jolla, CA, USA) subcloned into pBluescript-SKII plasmid (Stratagene), and linearized with BamHI and EcoRI (Pharmacia Biotech, Inc.) for antisense and sense probes respectively. The rat CRF2R cRNA probe was prepared from a 275 bp fragment of the 5′-region cDNA of the CRF receptor (Dr T W Lovenberg, Neurocrine Biosciences, Inc., San Diego, CA, USA) subcloned into a pBluescript SK+ vector (Stratagene), and linearized with EcoRI and RamHI for antisense and sense probes respectively. The radioactive riboprobes were synthesized, extracted, and precipitated as described elsewhere (Timofeeva et al. 2003, 2005). The specificity of each probe was confirmed by the absence of positive signal in sections hybridized with sense probe.

Quantitative analysis of the hybridization signals

The hybridization signals revealed on NTB2 dipped nuclear emulsion slides were analyzed and quantified under a light microscope (Olympus, BX50) equipped with a black and white video camera (Sony, XC-77) coupled to a Macintosh computer (Power PC 7100/66) using Image software (version 1.55 non-FPU; Wayne Rasband, NIH, Bethesda, MD, USA). The optical density (OD) of the hybridization signal was measured under dark field illumination. Brain sections were matched for rostrocaudal levels as closely as possible according to the atlas of the rat brain (Paxinos & Watson 1997). The OD for each specific region was corrected for the average background signal, which was determined by sampling an unlabeled area outside the areas of interest. When no hybridization signal was visible under dark field illumination, the brain structures of interest were outlined under bright field illumination and then subjected to densitometric analysis under dark field illumination. Saturation of the hybridization signal was avoided by the creation of the calibration density profile plot for the strongest hybridization signals sampled for each region in every series. The luminosity of the system was then corrected, so that for the strongest hybridization signals, the reading OD did not exceed the half of the pixel value maximum, and this luminosity was conserved throughout the analysis of the entire series. The mean of OD for individual animals was used to determine the mean ± s.e.m. for each group.

Statistical analysis

A 2×3 ANOVA was used to examine the main and interaction effects of infusion (I; PBS, leptin) and activity (A; resting, running, and post-running) on the various dependent variables measured in this study. In the cases of significant I×A interactions, a posteriori comparisons were performed using the Bonferroni–Dunn multiple comparison procedure.

Results

Body weight, weight of white fat tissue, and food intake

Figure 1 and Table 1 demonstrate the effects of an intracerebroventricular infusion of leptin on body weight, food intake, and weight of eWAT. Infusion of leptin led to a substantial decrease in food intake and body weight. The effect of leptin on food intake and body weight was already significant on the first or the second day of infusion. Cumulative food intake measured upon five consecutive days of infusion was two times higher in PBS-treated rats than in leptin-infused animals (Table 1). The weight of the eWAT was also affected by leptin. Five days of leptin infusion produced a twofold reduction in eWAT weight compared with PBS-treated animals (Table 1). Acute treadmill running did not noticeably affect body weight or eWAT weight.

Plasma glucose, insulin, and corticosterone

Table 1 presents the plasma levels of glucose, insulin, and corticosterone in PBS- and leptin-infused rats in resting, running, or post-running conditions. Plasma glucose and insulin levels were significantly lower in leptin-treated rats compared with PBS controls. Plasma corticosterone levels were not apparently different between resting leptin- and PBS-treated rats and noticeably increased in running rats in both the PBS- and leptin-infused animals. It is noteworthy that the increase in plasma corticosterone levels induced by running was apparently blunted by leptin infusion; increases in corticosterone response to running were 22.9 vs 14.5 times above basal levels in PBS and leptin rats respectively.

Expression of the CRF hnRNA in the PVN

The effects of leptin treatment and running stress on the expression of the CRF gene were evaluated by measuring the expression of CRF hnRNA using an intronic probe complementary to the intron 1 of the rat CRF gene. Intronic probe allows the rapid detection of the primary transcript of CRF (Rivest et al. 1995, Lacroix et al. 1996). The expression of CRF hnRNA increased during treadmill running in PBS, but not in leptin-treated rats (Figs 2 and 3). The levels of hnCRF transcript in PBS-treated rats returned to the basal values 2 h after running (Figs 2 and 3).

Expression of the CRF1R mRNA in the PVN and supraoptic nucleus (SON)

The CRF1R transcript was barely detectable in resting rats in both the PVN and SON. However, CRF1R mRNA was significantly induced in these structures in PBS-treated rats after treadmill running. The increase in the CRF1R mRNA expression in both the PVN and SON in PBS-infused animals was not observed immediately after running, but during the post-running period, as shown on the histogram illustrating the ODs of the hybridization signal of the CRF1R mRNA in both structures (Fig. 4), and on the photomicrographs taken at the level of the PVN (Fig. 5) and SON (Fig. 6). The chronic leptin infusion significantly blunted the induction of expression of the CRF1R mRNA during the post-running period in the PVN and SON (Figs 4–6). In both structures, the increase in CRF1R mRNA expression after running was significantly low in leptin-infused rats compared with PBS-treated animals.

Expression of the CRF2R mRNA in the VMH

The levels of expression of the CRF2R mRNA in the VMH were significantly higher in leptin-infused rats compared with the PBS-treated animals. The analysis of the ODs of the hybridization signal of CRF2R mRNA is shown in Fig. 7. The photomicrographs in Fig. 8 depict the positive signal for CRF2R transcript in the VMH of resting rats infused with PBS or leptin. Resting, running, and post-running leptin-infused rats demonstrated higher levels of CRF2R mRNA than PBS-treated animals, but within the leptin- or PBS-treated groups, the expression of CRF2R mRNA did not change during or after a session of treadmill running (Fig. 7).

Discussion

The present study demonstrates that the subchronic brain infusion of leptin alters the stress response induced by treadmill running in rats (Richard et al. 1996, Timofeeva et al. 2003). Leptin lessens corticosterone secretion as well as the expression of CRF and the CRF1R mRNA in rats subjected to 1 h treadmill running. Our results also confirm the stimulatory effects of leptin on the expression of the CRF2R mRNA in the VMH (Nishiyama et al. 1999).

Although, daily intracerebroventricular leptin infusion did not affect plasma levels of corticosterone in resting animals, it considerably blunted running-induced hypercorticosteronemia. This finding is consonant with previous results that demonstrated the ability of leptin to reduce the HPA axis activity in response to stress through actions exerted on the various components of the pituitary–adrenal system. Leptin has been reported to inhibit both the synthesis and release of adrenal glucocorticoids (Bornstein et al. 1997, Kruse et al. 1998), and reduce the magnitude and duration of stress-induced ACTH secretion (Trottier et al. 1998, Oates et al. 2000). In addition, it has been demonstrated that leptin may prevent the response of the pituitary–adrenal axis to insulin administration in rat (Giovambattista et al. 2000) and attenuate the elevation in plasma cortisol and ACTH following an unpredictable stress in rhesus monkeys (Wilson et al. 2005).

The present results also suggest that leptin may reduce the hypothalamic drive exerted by the hypophysiotropic CRF neurons on the HPA axis. Leptin infusion suppressed the expression of CRF in the PVN neurons during stress. In fact, while treadmill-induced expression of CRF hnRNA was readily detected in the parvocellular PVN of PBS control rats, it was barely apparent in rats chronically infused with leptin. The use of the intronic probe allows for the detection of the CRF primary transcript, whose brain constitutive expression is low but rapidly increases in the PVN in response to stressful conditions (Rivest et al. 1995, Lacroix et al. 1996). Whether the leptin effect on stress-induced activation of the CRF gene is due to a direct leptin action on the PVN neurons or involves the inhibition of larger assemblies of stress-related circuits has yet to be untangled. Leptin might directly affect the CRF-producing parvocellular PVN neurons as these cells moderately express the leptin receptor (Elmquist et al. 1998b). Leptin could increase the inhibitory tone on the hypo-thalamic neurons (Jo et al. 2005).

The PVN induction of CRF hnRNA after treadmill running was accompanied by the induction of the CRF1R gene, which was not only seen in the PVN but also in the SON. Several lines of evidence emerging from studies using CRF1R transgenic/knockout mice, CRF1R antisense oligonucleotide, and CRF1R agonists/antagonists have suggested that CRF1R mediates the endocrine, autonomic, and behavioral effects of CRF during stress (for review, see Behan et al. 1996, Takahashi 2001, Bale & Vale 2004). In resting conditions, the CRF1R transcript is barely detectable in the PVN, but it is readily induced by stress (Rivest et al. 1995). The induction of CRF1R in the PVN has indeed been shown in several stress paradigms (Desouza et al. 1991, Rivest et al. 1995, Timofeeva & Richard 1997, Timofeeva et al. 2003, Wang et al. 2004) and has to be seen as a valid marker of stress. The present study reports for the first time that leptin may blunt the stress-induced expression of CRF1R in both the PVN and SON. The mechanism underlying this action remains to be delineated. The observation that the PVN expression of CRF1R followed that of CRF hnRNA nonetheless suggests that the reduced expression of CRF1R following leptin might be a consequence of a leptin-induced reduction in the CRF tone, all the more since CRF has been reported to trigger the expression of its own type 1 receptor (Mansi et al. 1996). The CRF1R seemingly favors further activation of CRF PVN neurons via an ultrashort positive feedback loop (Imaki et al. 2001). It is noteworthy that the repressing effect of leptin on CRF1R mRNA was also seen in the SON, which is not recognized to markedly express CRF (Kawata et al. 1983, Merchenthaler 1984). Furthermore, the possibility for a direct effect of leptin on SON CRF1R expression is weak due to the reported absence of the leptin receptors in this hypothalamic structure (Elmquist et al. 1998b).

In addition to binding to the CRF1R, CRF also binds to the CRF2R, which could be involved in the anorectic effects of CRF (Vaughan et al. 1995, Richard et al. 2000, 2002). The present study demonstrates an increased expression of CRF2R mRNA in the VMH after leptin infusion into the brain. The levels of the CRF2R transcript were independent of the running activity, but were about two times higher in leptin-infused rats compared with PBS-treated animals. These data are in agreement with the reported increase in the VMH CRF2R expression after systemic administration of leptin (Nishiyama et al. 1999) and the positive correlation between the levels of VMH CRF2R expression and plasma leptin concentrations (Makino et al. 1998). We have observed a decrease in the expression of CRF2R in the VMH of food-deprived rats and in rats with uncontrolled diabetes, which exhibit decreases in plasma leptin levels (Timofeeva & Richard 1997, Huang et al. 2006). Leptin signaling deficiency due to the lack of leptin as in ob/ob mice, or to the dysfunctional leptin receptor as in fa/fa Zucker rats, also leads to a decrease in CRF2R expression in the VMH (Richard et al. 1996, Timofeeva & Richard 1997). Leptin may directly affect the VMH neurons, which express the leptin receptor and exhibit leptin-induced Fos expression (Elmquist et al. 1997, 1998b, Elias et al. 2000). Through the expression of CRF2R in the VMH, leptin may potentiate the anorectic effects of CRF.

In conclusion, the results of the present study provide evidence for the inhibitory effect of continuous central leptin infusion on the treadmill running-induced activation of the CRF hypophysiotropic system. Chronic leptin infusion prevented stress-induced de novo synthesis of CRF, and significantly decreased the induction of expression of CRF1R in the PVN. In addition, leptin infusion increased the expression of the CRF2R mRNA in the VMH. These results provide evidence that chronic increase in central leptin signaling may blunt the HPA axis response to stress and increase the anorectic effects of CRF.

Table 1

Final body weight (BW), cumulative food intake, epididymal white adipose tissue (eWAT), and plasma variables of the PBS- and leptin-infused Wistar rats before (resting), during (60-min; running), and after (120-min; post-running) treadmill running session. Values are presented as means ± s.e.m.

RestRunningPost-runningANOVA (P)
PBS (n = 5)Leptin (n = 5)PBS (n = 5)Leptin (n = 6)PBS (n = 5)Leptin (n = 6)Infusion (I)Activity (A)Interaction (I×A)
Values were compared by 2×3 ANOVA. Significant I×A interactions were further analyzed using Bonferroni–Dunn multiple comparison procedure. *Significantly different from the PBS-infused rats within the same activity. Significantly different from the resting rats within the same infusion, P < 0.05.
Final BW (g)305.2 ± 6.4252.8 ± 5.8293.6 ± 2.9252.6 ± 6.9290.9 ± 4.4249.5 ± 7.9< 0.00010.37000.6049
Cumulative food intake (g)126.3 ± 4.864.9 ± 4.1123.7 ± 3.563.3 ± 7.5119.586 ± 6.668.9 ± 7.6< 0.00010.94450.6410
eWAT (g)1.122 ± 0.0570.550 ± 0.1031.144 ± 0.1020.822 ± 0.0981.111 ± 0.1100.673 ± 0.079< 0.00010.30170.4285
Plasma
    Glucose (mmol/l)8.758 ± 0.3037.770 ± 0.64610.823 ± 0.2668.371 ± 0.4418.569 ± 0.2747.705 ± 0.2640.00010.00130.0935
    Insulin (nmol/l)0.458 ± 0.0710.091 ± 0.018*0.356 ± 0.0500.269 ± 0.0520.210 ± 0.0230.083 ± 0.013*< 0.00010.00160.0071
    Corticosterone (μmol/l)0.065 ± 0.0550.073 ± 0.0411.491 ± 0.1161.058 ± 0.0640.501 ± 0.1660.339 ± 0.1810.0587< 0.00010.2079
Figure 1
Figure 1

Daily measurements of (A) body weight and (B) food intake in rats infused intracerebroventricularly with PBS or recombinant murine leptin (2 μg/day per rat). Values are means ± s.e.m. *Significantly different from PBS-infused rats. Arrow indicates start of infusion.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

Figure 2
Figure 2

The optical densities (ODs) of the hybridization signals of the CRF heteronuclear (hn) RNA in the paraventricular hypothalamic nucleus (PVN) in resting, running, and post-running rats injected with PBS or leptin. The main and interaction effects of infusion (I; PBS, leptin) and activity (A; resting, running, or post-running) were determined using 2×3 ANOVA, followed by a posteriori Bonferroni–Dunn multiple comparison procedure. *Significantly different from the PBS-infused rats within the same activity; significantly different from the resting rats within the same infusion, P < 0.05.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

Figure 3
Figure 3

Dark field photomicrographs of coronal brain sections taken from the hypothalamic paraventricular nucleus (PVN), illustrating positive hybridization signal of the CRF heteronuclear (hn) RNA. The brain sections (30 μm thick) were obtained from PBS (top panels) or leptin (bottom panels) infused rats before (resting), during (running), or after (120-min post-running) treadmill running. The scale bar corresponds to 200 μm.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

Figure 4
Figure 4

The optical densities (ODs) of the hybridization signals of the CRF1R mRNA in the paraventricular hypothalamic nucleus (PVN; A) and the supraoptic nucleus (SON; B) in resting, running, and post-running rats injected with PBS or leptin. The main and interaction effects of infusion (I; PBS, leptin) and activity (A; resting, running, or post-running) were determined using 2×3 ANOVA, followed by a posteriori Bonferroni–Dunn multiple comparison procedure. *Significantly different from the PBS-infused rats within the same activity; significantly different from the resting rats within the same infusion, P < 0.05.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

Figure 5
Figure 5

Dark field photomicrographs of coronal brain sections taken from the paraventricular hypothalamic nucleus (PVN) illustrating effects of leptin on CRF1R mRNA before (resting), during (running), or after (120-min post-running) treadmill running. The scale bar corresponds to 200 μm.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

Figure 6
Figure 6

Dark field photomicrographs of coronal brain sections taken from the supraoptic nucleus (SON) illustrating effects of leptin on CRF1R mRNA before (resting), during (running), or after (120-min post-running) treadmill running. The scale bar corresponds to 200 μm. OX, optic chiasm.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

Figure 7
Figure 7

The optical densities (ODs) of the hybridization signals of the CRF2R mRNA in the ventromedial hypothalamic nucleus (VMH) in resting, running, and post-running rats infused with PBS or leptin. The main and interaction effects of infusion (I; PBS, leptin) and activity (A; resting, running, or post-running) were determined using 2×3 ANOVA.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

Figure 8
Figure 8

Film autoradiograms of coronal brain sections demonstrating the effects of leptin on CRF2R transcript at the level of the ventromedial hypothalamic nucleus (VMH). The brain sections (30 μm thick) were obtained from resting rats infused with PBS (A) or leptin (B). The scale bar corresponds to 1 mm.

Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06906

This work was supported by the Canadian Institutes of Health Research grant (to D R). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

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  • Bale TL & Vale WW 2004 CRF and CRF receptors: role in stress responsivity and other behaviors. Annual Review of Pharmacology and Toxicology 44 525–557.

  • Behan DP, Grigoriadis DE, Lovenberg T, Chalmers D, Heinrichs S, Liaw C & Souza EB 1996 Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders. Molecular Psychiatry 1 265–277.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bornstein SR, Uhlmann K, Haidan A, Ehrhart-Bornstein M & Scherbaum WA 1997 Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly. Diabetes 46 1235–1238.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Buwalda B, de Boer SF, Van Kalkeren AA & Koolhaas JM 1997 Physiological and behavioral effects of chronic intracerebroventricular infusion of corticotropin-releasing factor in the rat. Psychoneuroendocrinology 22 297–309.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cabanac M & Richard D 1995 Acute intra-ventricular CRF lowers the hoarding threshold in male rats. Physiology and Behavior 57 705–710.

  • Campfield LA, Smith FJ, Guisez Y, Devos R & Burn P 1995 Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269 546–549.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Costa A, Poma A, Martignoni E, Nappi G, Ur E & Grossman A 1997 Stimulation of corticotrophin-releasing hormone release by the obese (ob) gene product, leptin, from hypothalamic explants. Neuroreport 8 1131–1134.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Desouza EB, Grigoriadis DE & Webster EL 1991 Role of brain, pituitary and spleen corticotropin-releasing factor receptors in the stress response. In Methods and Achievements in Experimental Pathology, pp 23–44. Ed. G Jasmin. Basel: S Karger.

    • PubMed
    • Export Citation
  • Dube MG, Xu B, Kalra PS, Sninsky CA & Kalra SP 1999 Disruption in neuropeptide Y and leptin signaling in obese ventromedial hypothalamic-lesioned rats. Brain Research 816 38–46.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Egawa M, Inoue S, Satoh S, Takamura Y, Nagai K & Nakagawa H 1993 Acute and chronic effects of VMH lesions on circadian rhythms in food intake and metabolites. Brain Research Bulletin 31 293–299.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elias CF, Kelly JF, Lee CE, Ahima RS, Drucker DJ, Saper CB & Elmquist JK 2000 Chemical characterization of leptin-activated neurons in the rat brain. Journal of Comparative Neurology 423 261–281.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elmquist JK, Ahima RS, Maratos-Flier E, Flier JS & Saper CB 1997 Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology 138 839–842.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elmquist JK, Ahima RS, Elias CF, Flier JS & Saper CB 1998a Leptin activates distinct projections from the dorsomedial and ventromedial hypothalamic nuclei. PNAS 95 741–746.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elmquist JK, Bjorbaek C, Ahima RS, Flier JS & Saper CB 1998b Distributions of leptin receptor mRNA isoforms in the rat brain. Journal of Comparative Neurology 395 535–547.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Giovambattista A, Chisari AN, Gaillard RC & Spinedi E 2000 Food intake-induced leptin secretion modulates hypothalamo–pituitary–adrenal axis response and hypothalamic Ob-Rb expression to insulin administration. Neuroendocrinology 72 341–349.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gosnell BA, Morley JE & Levine ASA 1983 Comparison of the effects of corticotropin releasing factor and sauvagine on food intake. Pharmacology, Biochemistry and Behavior 19 771–775.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK & Friedman JM 1995 Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269 543–546.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW & Flier JS 1997 Leptin inhibition of the hypothalamic–pituitary–adrenal axis in response to stress. Endocrinology 138 3859–3863.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Huang Q, Rivest R & Richard D 1998 Effects of leptin on corticotropin-releasing factor (CRF) synthesis and CRF neuron activation in the paraventricular hypothalamic nucleus of obese (ob/ob) mice. Endocrinology 139 1524–1532.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Huang Q, Timofeeva E & Richard D 2006 Corticotropin-releasing factor and its receptors in the brain of rats with insulin and corticosterone deficits. Journal of Molecular Endocrinology 37 1–15.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Imaki T, Katsumata H, Miyata M, Naruse M, Imaki J & Minami S 2001 Expression of corticotropin-releasing hormone type 1 receptor in paraventricular nucleus after acute stress. Neuroendocrinology 73 293–301.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jo YH, Chen YJ, Chua SC Jr, Talmage DA & Role LW 2005 Integration of endocannabinoid and leptin signaling in an appetite-related neural circuit. Neuron 48 1055–1066.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kawata M, Hashimoto K, Takahara J & Sano Y 1983 Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus. Cell and Tissue Research 230 239–246.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Korbonits M, Trainer PJ, Little JA, Edwards R, Kopelman PG, Besser GM, Svec F & Grossman AB 1997 Leptin levels do not change acutely with food administration in normal or obese subjects, but are negatively correlated with pituitary–adrenal activity. Clinical Endocrinology 46 751–757.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kruse M, Bornstein SR, Uhlmann K, Paeth G & Scherbaum WA 1998 Leptin down-regulates the steroid producing system in the adrenal. Endocrine Research 24 587–590.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lacroix S, Vallieres L & Rivest S 1996 C-fos mRNA pattern and corticotropin-releasing factor neuronal activity throughout the brain of rats injected centrally with a prostaglandin of E2 type. Journal of Neuroimmunology 70 163–179.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Levin N, Nelson C, Gurney A, Vandlen R & Desauvage F 1996 Decreased food intake does not completely account for adiposity reduction after ob protein infusion. PNAS 93 1726–1730.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Licinio J, Mantzoros C, Negrao AB, Cizza G, Wong ML, Bongiorno PB, Chrousos GP, Karp B, Allen C, Flier JS et al.1997 Human leptin levels are pulsatile and inversely related to pituitary–adrenal function. Nature Medicine 3 575–579.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lovenberg TM, Liaw CW, Grigoriadis DF, Clevenger W, Chalmers DT, DeSouza EB & Olsterdorf T 1995 Cloning and characterization of functionally distinct corticotropin-releasing factor receptor subtype from rat brain. PNAS 92 836–840.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Makino S, Nishiyama M, Asaba K, Gold PW & Hashimoto K 1998 Altered expression of type 2 CRH receptor mRNA in the VMH by glucocorticoids and starvation. American Journal of Physiology 275 R1138–R1145.

    • PubMed
    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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  • Figure 1

    Daily measurements of (A) body weight and (B) food intake in rats infused intracerebroventricularly with PBS or recombinant murine leptin (2 μg/day per rat). Values are means ± s.e.m. *Significantly different from PBS-infused rats. Arrow indicates start of infusion.

  • Figure 2

    The optical densities (ODs) of the hybridization signals of the CRF heteronuclear (hn) RNA in the paraventricular hypothalamic nucleus (PVN) in resting, running, and post-running rats injected with PBS or leptin. The main and interaction effects of infusion (I; PBS, leptin) and activity (A; resting, running, or post-running) were determined using 2×3 ANOVA, followed by a posteriori Bonferroni–Dunn multiple comparison procedure. *Significantly different from the PBS-infused rats within the same activity; significantly different from the resting rats within the same infusion, P < 0.05.

  • Figure 3

    Dark field photomicrographs of coronal brain sections taken from the hypothalamic paraventricular nucleus (PVN), illustrating positive hybridization signal of the CRF heteronuclear (hn) RNA. The brain sections (30 μm thick) were obtained from PBS (top panels) or leptin (bottom panels) infused rats before (resting), during (running), or after (120-min post-running) treadmill running. The scale bar corresponds to 200 μm.

  • Figure 4

    The optical densities (ODs) of the hybridization signals of the CRF1R mRNA in the paraventricular hypothalamic nucleus (PVN; A) and the supraoptic nucleus (SON; B) in resting, running, and post-running rats injected with PBS or leptin. The main and interaction effects of infusion (I; PBS, leptin) and activity (A; resting, running, or post-running) were determined using 2×3 ANOVA, followed by a posteriori Bonferroni–Dunn multiple comparison procedure. *Significantly different from the PBS-infused rats within the same activity; significantly different from the resting rats within the same infusion, P < 0.05.

  • Figure 5

    Dark field photomicrographs of coronal brain sections taken from the paraventricular hypothalamic nucleus (PVN) illustrating effects of leptin on CRF1R mRNA before (resting), during (running), or after (120-min post-running) treadmill running. The scale bar corresponds to 200 μm.

  • Figure 6

    Dark field photomicrographs of coronal brain sections taken from the supraoptic nucleus (SON) illustrating effects of leptin on CRF1R mRNA before (resting), during (running), or after (120-min post-running) treadmill running. The scale bar corresponds to 200 μm. OX, optic chiasm.

  • Figure 7

    The optical densities (ODs) of the hybridization signals of the CRF2R mRNA in the ventromedial hypothalamic nucleus (VMH) in resting, running, and post-running rats infused with PBS or leptin. The main and interaction effects of infusion (I; PBS, leptin) and activity (A; resting, running, or post-running) were determined using 2×3 ANOVA.

  • Figure 8

    Film autoradiograms of coronal brain sections demonstrating the effects of leptin on CRF2R transcript at the level of the ventromedial hypothalamic nucleus (VMH). The brain sections (30 μm thick) were obtained from resting rats infused with PBS (A) or leptin (B). The scale bar corresponds to 1 mm.

  • Badoer E & Ryan A 1999 Effects of leptin on spinally projecting neurons in the PVN of the hypothalamus. Brain Research 844 210–215.

  • Bale TL & Vale WW 2004 CRF and CRF receptors: role in stress responsivity and other behaviors. Annual Review of Pharmacology and Toxicology 44 525–557.

  • Behan DP, Grigoriadis DE, Lovenberg T, Chalmers D, Heinrichs S, Liaw C & Souza EB 1996 Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders. Molecular Psychiatry 1 265–277.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bornstein SR, Uhlmann K, Haidan A, Ehrhart-Bornstein M & Scherbaum WA 1997 Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly. Diabetes 46 1235–1238.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Buwalda B, de Boer SF, Van Kalkeren AA & Koolhaas JM 1997 Physiological and behavioral effects of chronic intracerebroventricular infusion of corticotropin-releasing factor in the rat. Psychoneuroendocrinology 22 297–309.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cabanac M & Richard D 1995 Acute intra-ventricular CRF lowers the hoarding threshold in male rats. Physiology and Behavior 57 705–710.

  • Campfield LA, Smith FJ, Guisez Y, Devos R & Burn P 1995 Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269 546–549.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Costa A, Poma A, Martignoni E, Nappi G, Ur E & Grossman A 1997 Stimulation of corticotrophin-releasing hormone release by the obese (ob) gene product, leptin, from hypothalamic explants. Neuroreport 8 1131–1134.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Desouza EB, Grigoriadis DE & Webster EL 1991 Role of brain, pituitary and spleen corticotropin-releasing factor receptors in the stress response. In Methods and Achievements in Experimental Pathology, pp 23–44. Ed. G Jasmin. Basel: S Karger.

    • PubMed
    • Export Citation
  • Dube MG, Xu B, Kalra PS, Sninsky CA & Kalra SP 1999 Disruption in neuropeptide Y and leptin signaling in obese ventromedial hypothalamic-lesioned rats. Brain Research 816 38–46.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Egawa M, Inoue S, Satoh S, Takamura Y, Nagai K & Nakagawa H 1993 Acute and chronic effects of VMH lesions on circadian rhythms in food intake and metabolites. Brain Research Bulletin 31 293–299.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elias CF, Kelly JF, Lee CE, Ahima RS, Drucker DJ, Saper CB & Elmquist JK 2000 Chemical characterization of leptin-activated neurons in the rat brain. Journal of Comparative Neurology 423 261–281.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elmquist JK, Ahima RS, Maratos-Flier E, Flier JS & Saper CB 1997 Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology 138 839–842.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elmquist JK, Ahima RS, Elias CF, Flier JS & Saper CB 1998a Leptin activates distinct projections from the dorsomedial and ventromedial hypothalamic nuclei. PNAS 95 741–746.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Elmquist JK, Bjorbaek C, Ahima RS, Flier JS & Saper CB 1998b Distributions of leptin receptor mRNA isoforms in the rat brain. Journal of Comparative Neurology 395 535–547.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Giovambattista A, Chisari AN, Gaillard RC & Spinedi E 2000 Food intake-induced leptin secretion modulates hypothalamo–pituitary–adrenal axis response and hypothalamic Ob-Rb expression to insulin administration. Neuroendocrinology 72 341–349.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gosnell BA, Morley JE & Levine ASA 1983 Comparison of the effects of corticotropin releasing factor and sauvagine on food intake. Pharmacology, Biochemistry and Behavior 19 771–775.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK & Friedman JM 1995 Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269 543–546.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Heiman ML, Ahima RS, Craft LS, Schoner B, Stephens TW & Flier JS 1997 Leptin inhibition of the hypothalamic–pituitary–adrenal axis in response to stress. Endocrinology 138 3859–3863.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Huang Q, Rivest R & Richard D 1998 Effects of leptin on corticotropin-releasing factor (CRF) synthesis and CRF neuron activation in the paraventricular hypothalamic nucleus of obese (ob/ob) mice. Endocrinology 139 1524–1532.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Huang Q, Timofeeva E & Richard D 2006 Corticotropin-releasing factor and its receptors in the brain of rats with insulin and corticosterone deficits. Journal of Molecular Endocrinology 37 1–15.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Imaki T, Katsumata H, Miyata M, Naruse M, Imaki J & Minami S 2001 Expression of corticotropin-releasing hormone type 1 receptor in paraventricular nucleus after acute stress. Neuroendocrinology 73 293–301.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Jo YH, Chen YJ, Chua SC Jr, Talmage DA & Role LW 2005 Integration of endocannabinoid and leptin signaling in an appetite-related neural circuit. Neuron 48 1055–1066.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kawata M, Hashimoto K, Takahara J & Sano Y 1983 Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus. Cell and Tissue Research 230 239–246.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Korbonits M, Trainer PJ, Little JA, Edwards R, Kopelman PG, Besser GM, Svec F & Grossman AB 1997 Leptin levels do not change acutely with food administration in normal or obese subjects, but are negatively correlated with pituitary–adrenal activity. Clinical Endocrinology 46 751–757.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kruse M, Bornstein SR, Uhlmann K, Paeth G & Scherbaum WA 1998 Leptin down-regulates the steroid producing system in the adrenal. Endocrine Research 24 587–590.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lacroix S, Vallieres L & Rivest S 1996 C-fos mRNA pattern and corticotropin-releasing factor neuronal activity throughout the brain of rats injected centrally with a prostaglandin of E2 type. Journal of Neuroimmunology 70 163–179.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Levin N, Nelson C, Gurney A, Vandlen R & Desauvage F 1996 Decreased food intake does not completely account for adiposity reduction after ob protein infusion. PNAS 93 1726–1730.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Licinio J, Mantzoros C, Negrao AB, Cizza G, Wong ML, Bongiorno PB, Chrousos GP, Karp B, Allen C, Flier JS et al.1997 Human leptin levels are pulsatile and inversely related to pituitary–adrenal function. Nature Medicine 3 575–579.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lovenberg TM, Liaw CW, Grigoriadis DF, Clevenger W, Chalmers DT, DeSouza EB & Olsterdorf T 1995 Cloning and characterization of functionally distinct corticotropin-releasing factor receptor subtype from rat brain. PNAS 92 836–840.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Makino S, Nishiyama M, Asaba K, Gold PW & Hashimoto K 1998 Altered expression of type 2 CRH receptor mRNA in the VMH by glucocorticoids and starvation. American Journal of Physiology 275 R1138–R1145.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Mansi JA, Rivest S & Drolet G 1996 Regulation of corticotropin-releasing factor type 1 (CRF1) receptor messenger ribonucleic acid in the paraventricular nucleus of rat hypothalamus by exogenous CRF. Endocrinology 137 4619–4629.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Merchenthaler I 1984 Corticotropin releasing factor (CRF)-like immunoreactivity in the rat central nervous system. Extrahypothalamic distribution. Peptides 5 53–69.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Murphy BEP 1967 Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassays. Journal of Clinical Endocrinology and Metabolism 27 973–990.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nishiyama M, Makino S, Asaba K & Hashimoto K 1999 Leptin effects on the expression of type-2 CRH receptor mRNA in the ventromedial hypothalamus in the rat. Journal of Neuroendocrinology 11 307–314.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Oates M, Woodside B & Walker CD 2000 Chronic leptin administration in developing rats reduces stress responsiveness partly through changes in maternal behavior. Hormones and Behavior 37 366–376.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Paxinos G & Watson C 1997 The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press.

    • PubMed
    • Export Citation
  • Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T & Collins F 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269 540–543.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Perrin MH, Donaldson CJ, Chen R, Lewis KA & Vale WW 1993 Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor. Endocrinology 133 3058–3061.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Raber J, Chen S, Mucke L & Feng L 1997 Corticotropin-releasing factor and adrenocorticotrophic hormone as potential central mediators of OB effects. Journal of Biological Chemistry 272 15057–15060.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Richard D, Rivest R, Naïmi N, Timofeeva E & Rivest S 1996 Expression of corticotropin-releasing factor and its receptors in the brain of lean and obese Zucker rats. Endocrinology 137 4786–4795.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Richard D, Huang Q & Timofeeva E 2000 The corticotropin-releasing hormone system in the regulation of energy balance in obesity. International Journal of Obesity and Related Metabolic Disorders 24 S36–S39.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Richard D, Lin Q & Timofeeva E 2002 The corticotropin-releasing factor family of peptides and CRF receptors: their roles in the regulation of energy balance. European Journal of Pharmacology 440 189–197.

    • PubMed
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