PRL-Releasing Peptide Interacts with Leptin to Reduce Food Intake ...

0013-7227/02/$15.00/0 Printed in U.S.A.

Endocrinology 143(2):368 –374 Copyright © 2002 by The Endocrine Society

PRL-Releasing Peptide Interacts with Leptin to Reduce Food Intake and Body Weight KATE L. J. ELLACOTT, CATHERINE B. LAWRENCE, NANCY J. ROTHWELL, SIMON M. LUCKMAN

AND

School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom PRL-releasing peptide (PrRP) is a novel anorexigen that reduces food intake and body weight gain in rats. In common with other anorexigens, PrRP mRNA expression is reduced during states of negative energy balance, i.e. lactation and fasting in female rats. In this study, we examined the interaction between PrRP and the adiposity signal, leptin, which interacts with a number of peptidergic systems in the brain to regulate energy homeostasis. Intracerebroventricular coadministration of 4 nmol PrRP and 1 ␮g leptin in rats resulted in additive reductions in nocturnal food intake and body weight gain and an increase in core body temperature com-

pared with each peptide alone. We show also, by quantitative in situ hybridization, that PrRP mRNA is reduced in fasted male rats and obese Zucker rats, indicating that PrRP mRNA expression, like that of other anorexigens, may be regulated by leptin. Finally we show, using immunohistochemistry, that greater than 90% of PrRP neurons in all regions where PrRP is expressed contain leptin receptors. Thus, we provide evidence for PrRP neurons forming part of the leptin-sensitive brain circuitry involved in the regulation of food intake and energy homeostasis. (Endocrinology 143: 368 –374, 2002)

P

RL-RELEASING PEPTIDE (PrRP) was identified in 1998 as the endogenous ligand for the orphan GPR10/hGR3 receptor (1). Although it was shown to cause PRL release from cultured pituitary cells (1), the lack of immunoreactive fibers in the external region of the median eminence suggested that it does not act as a typical hypophysiotrophic hormone (2– 6). These findings have been reinforced by a number of studies, which question PrRP’s role in the modulation of PRL release in vivo and in vitro (6 –9). PrRP neurons are located in the dorsal medial hypothalamus (DMH), nucleus of the tractus solitarius (NTS), and ventrolateral medulla (VLM) (2, 10, 11), regions that are known to be involved in the regulation of food intake (for reviews, see Refs. 12 and 13). This suggested to us an alternative role in the regulation of energy balance. This was confirmed when we showed that intracerebroventricular (icv) administration of PrRP reduces normal nocturnal and fast-induced feeding, accompanied by a reduction in body weight gain, without causing taste aversion or disrupting normal feeding behavior (14, 15). Furthermore, PrRP mRNA is down-regulated in two states of negative energy balance in female rats: fasting and lactation (14). Leptin is an adiposity signal, encoded by the obesity (ob) gene (16). It is produced primarily by adipose tissue but is also expressed by gastric parietal (17), skeletal muscle (18), and placental cells (19, 20). Leptin regulates food intake and energy expenditure through interaction with its receptors in the brain (for review, see Ref. 21). There are six known isoforms of the leptin receptor, Ob-Ra-e, but only the long form of the receptor, Ob-Rb, possesses the necessary intracellular domain to enable signaling through transcriptional

pathways. There are two rodent models of defective leptin signaling: the Zucker rat and the db/db mouse, both of which are obese due to defects in Ob-Rb, which severely reduces their ability to respond to leptin (22–24). Ob-R transcripts and immunoreactivity are widely distributed in the rodent brain. In the hypothalamus, strong expression of Ob-R is seen in the arcuate nucleus (ARC), DMH, ventromedial nucleus, premammillary nucleus, supraoptic nucleus, paraventricular nucleus (PVN), lateral hypothalamus, and periventricular nucleus (25–27). In the brainstem, Ob-R mRNA is found in the NTS, medullary reticular nucleus, and the lateral parabrachial nucleus (25, 28). Ob-R is expressed by neurons that contain peptides known to be involved in the regulation of food intake. In the hypothalamus Ob-R is coexpressed with NPY (29), agouti-related peptide (30), POMC (31), cocaineand amphetamine-regulated transcript (32), and orexin/ hypocretin (33, 34), thus providing morphological evidence for an action of leptin on neurons containing these orexigenic and anorexigenic peptides. Leptin has also been shown to interact with a number of these peptides in vivo to modulate food intake or mRNA expression (32, 35–39). In this study, we have examined the interaction between PrRP and leptin in vivo through an examination of their effect on nocturnal food intake, body weight gain, and core body temperature following their coadministration. We have looked at the distribution of PrRP with Ob-R or tyrosine hydroxlase (TH) using double-fluorescence immunohistochemistry in the DMH, NTS, and VLM to ascertain whether there is the morphological basis for an interaction. Also, an acute and a chronic model of energy imbalance, the fasted rat and the obese Zucker rat, have been used to examine the regulation of PrRP gene expression.

Abbreviations: ARC, Arcuate nucleus; AUC, area under the curve; CCK, cholecystokinin; DMH, dorsal medial hypothalamus; icv, intracerebrovintricular(ly); NTS, nucleus of the tractus solitarius; PrRP, PRLrelated peptide; PVN, paraventricular nucleus; TH, tyrosine hydroxlase; VLM, ventrolateral medulla.

Materials and Methods Animals and materials Unless stated otherwise, all experiments were performed on adult, male Sprague Dawley rats (250 –300 g, Charles River Laboratories, Inc.

368

Ellacott et al. • PrRP Interacts with Leptin

Sandwich, UK). Adult, male, obese (fa/fa; 415– 480 g) or lean (fa/⫹; 280 –350 g) Zucker rats were a kind gift from AstraZeneca Plc (Alderley Park, UK). Animals were kept in a 12-h light, 12-h dark cycle (lights on, 0800 –2000 h) at 21 C ⫾ 1 C with 45 ⫾ 10% humidity, with ad libitum access to food and water. All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act (1986). Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich Corp. (Poole, UK).

Coadministration of leptin and PrRP Animals had lateral ventricular cannulae inserted [0.8 mm posterior and 1.5 mm lateral to bregma and 3.0 mm below dura (40)] under 2.5% halothane (AstraZeneca Plc, Macclesfield, UK) anesthesia. During anesthesia, precalibrated, remote radiotelemetry transmitters (TA10TAF40, Data Sciences International, Minneapolis, MN) were implanted into the peritoneal cavity. The animals were allowed to recover and experiments were performed 1 wk later. They were then housed individually at 1000 h and left to acclimatize. During the hour before lights out (2000 h), the conscious, unrestrained animals received one of the following treatments icv, 4 nmol PrRP (1–31) in 2 ␮l sterile water (Peptide Inc., Osaka, Japan) and 2 ␮l isotonic saline (leptin vehicle); 1 ␮g recombinant murine leptin (a kind gift from AstraZeneca Plc) in 2 ␮l isotonic saline and 2 ␮l sterile water (PrRP vehicle); 4 nmol PrRP (2 ␮l) and 1 ␮g leptin (2 ␮l) together; or 2 ␮l of each vehicle. Total injection volume was 4 ␮l (n ⫽ 5– 6 per group). Core body temperature was measured continuously throughout the 12-h dark period. Food and water intake and body weights were measured 12 h after treatments.

In situ hybridization Animals were allowed free access to food, except for the fasted group, before they were killed by cervical dislocation and decapitation (n ⫽ 6 per group for the fed/fasted experiment and n ⫽ 8 per group for the lean/obese Zucker experiment). The fasted group first had food withheld for 48 h. Coronal brain sections (15 ␮m) through the DMH [bregma ⫺3.60 mm to ⫺4.16 mm (40)] and NTS/VLM [bregma ⫺13.30 mm to ⫺14.60 mm (40)] were collected onto Polysine slides (BDH, Poole, UK). Oligonucleotide probes against PrRP (a 1:1 mixture of 5⬘-tag cag cag caa gca cag aag cca cgt ctt cag ggc-3⬘ and 5⬘-tta tcc acg ctg aga gaa ctt ggt gcg tcc atc cag-3⬘) or NPY (5⬘-gga gta gta tct ggc cat gtc ctc tgc tgg-3⬘) were labeled with 35S deoxy-ATP (NEN Life Science Products, Boston, MA). Following hybridization, slides were washed at a final stringency of 1⫻ SSC at 55 C, and developed with photographic emulsion (Ilford Imaging Ltd., Knutsford, UK). The resulting silver grains were quantified using a computer based analysis system, Northern Eclipse (Empix Imaging Inc., Mississauga, Canada). A minimum of four sections per animal for the DMH, and four to six sections for the NTS/VLM, were analyzed. Cells were deemed to be PrRP positive if they contained greater than three times the background number of silver grains (determined from negative control slides hybridized with excess unlabeled probe).

Endocrinology, February 2002, 143(2):368 –374 369

mouse biotinylated IgG (1:200) and 2.6 ␮l/ml streptavidin-Texas Red complex (both from Vector Laboratories, Inc., Burlingame, CA). The specificities of each of the primary antibodies have been tested by their commercial producers and the cellular distributions of their staining in the brain correspond to in situ hybridization data. In addition, staining for the leptin receptor antibody was completely abolished following liquid-phase preabsorption with the peptide immunogen (sc1834P; Santa Cruz Biotechnology, Inc.; results not shown). Double immunohistochemistry was assessed by scoring the number of cells per section that showed specific immunoreactivity for PrRP, with or without Ob-R or TH and the number of neurons that displayed immunoreactivity for both. Only sections in which PrRP neurons were found were included in this analysis.

Statistical analyses All data are expressed as mean ⫾ sem. Food intake and body weight measurements were analyzed using a one-way ANOVA followed by Tukey’s post hoc test. Core body temperature data were analyzed using the area under the curve (AUC) from 2 h to 12 h [AUC, in degrees Celsius (C)/h] followed by a one-way ANOVA with Dunnett’s multiple comparisons post hoc test. Analysis of core body temperature was performed from 2 h to avoid the transient hypothermia that occurs following administration of PrRP (14). In situ hybridization data were analyzed using a Mann-Whitney U test.

Results The effect of coadministration of PrRP and leptin on nocturnal food intake and body weight

Administration of PrRP (4 nmol; icv) alone caused a 24% reduction in food intake over the 12-h dark period (Fig. 1A; P ⬍ 0.05 compared with vehicle). This reduction in food

Double-fluorescence immunohistochemistry Animals were deeply anesthetized using sodium pentabarbitone (Sagatal, 100 mg/kg; Rhoˆ ne Me´ rieux, Harlow, UK) before undergoing transcardial perfusion with heparinized (100 kU/liter) isotonic saline (0.9% NaCl) followed by 4% paraformaldehyde in 0.1 m phosphate buffer. The brains were postfixed in 4% paraformaldehyde in 0.1 m phosphate buffer for 2 h before cryoprotection in 30% sucrose. Twentymicrometer coronal brain sections were incubated in a rabbit polyclonal antibody raised against the human PrRP sequence (1:1,000; Phoenix Pharmaceuticals, Inc., Belmont, CA), followed by a fluorescein isothiocyanate-labeled donkey antirabbit IgG complex (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The sections then underwent incubation in goat anti-leptin receptor antibody (1:500, M-18; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a Texas Red-labeled donkey antigoat IgG complex (1:500; Jackson Immunoresearch Laboratories, Inc.) The primary antibody recognizes both the long and short forms of the leptin receptor. For TH and PrRP double immunohistochemistry the above procedure was repeated with the Ob-R antibody replaced by mouse anti-TH (1:4000, Mab 318; Chemicon International Inc., Harrow, UK), horse anti-

FIG. 1. The effect of coadministration of PrRP and leptin on (A) food intake and (B) body weight gain over the 12-h dark period in the adult male Sprague Dawley rats. (*, P ⬍ 0.05 compared with vehicle; ***, P ⬍ 0.001 compared with vehicle; #, P ⬍ 0.05 compared with PrRP alone; ␾ P ⬍ 0.05 compared with leptin alone; ␾␾, P ⬍ 0.01 compared with leptin alone; one-way ANOVA.) Data are expressed as mean ⫾ SEM, n ⫽ 5– 6.

370

Endocrinology, February 2002, 143(2):368 –374


intake was accompanied by a 56% reduction in overnight body weight gain (Fig. 1B; P ⬍ 0.05 compared with vehicle). Rats injected with leptin (1 ␮g; icv) ate 18% less food than the control group, but this did not quite reach statistical significance with the post hoc test. However, leptin did produce a significant 55% reduction in overnight body weight gain (Fig. 1B; P ⬍ 0.05 compared with vehicle). Coadministration of PrRP and leptin caused a 54% reduction in food intake indicating an additive effect of the two peptides (Fig. 1A; P ⬍ 0.001 compared with vehicle, P ⬍ 0.01 compared with leptin alone and P ⬍ 0.05 compared with PrRP alone). This was also reflected in the data for body weight gain because animals that received both PrRP and leptin showed a net loss in body weight over the same period (Fig. 1B; P ⬍ 0.001 compared with vehicle and P ⬍ 0.05 compared with leptin alone, but not significantly different when compared with PrRP alone). The effect of coadministration of PrRP and leptin on nocturnal core body temperature

Remote radiotelemetry was used to measure the core body temperature continuously through the dark period of the animals used in the feeding study (values at 30-min intervals are plotted in Fig. 2). Injections were made just before lights out; thus, a normal, nocturnal rise in core body temperature is seen in both vehicle- and hormone-treated animals. Administration of leptin alone caused a slight hyperthermic response for the duration of the dark phase, compared with vehicle-treated controls, although this was not statistically significant in this experiment (Fig. 2A; AUC 2–12 h, vehicle 5.6 ⫾ 0.6 C/h; leptin 9.0 ⫾ 1.4 C/h, P ⬎ 0.05). Administration of PrRP caused an initial rapid hypothermic response characteristic of PrRP (14), which reached a nadir at 30 min, after which time the animals displayed a small but significant hyperthermia (Fig. 2B; AUC 2–12 h, 9.6 ⫾ 0.5 C/h, P ⬍ 0.05 compared with vehicle-treated animals). Animals that received PrRP and leptin showed the hypothermic response followed by an elevation in core body temperature compared with vehicle-treated animals, which was greater than for administration either PrRP or leptin alone (Fig. 2C; AUC 2–12 h, 10.9 ⫾ 1.3 C/h, P ⬍ 0.05). The expression of PrRP mRNA in fasted and Zucker rats

In the brain, PrRP mRNA was expressed exclusively in the DMH, NTS, and VLM of fed- and fasted-male rats. The negative control (excess unlabeled PrRP probe in addition to the radiolabeled probe) resulted in only background levels of silver grains, indicating that the signal was due to specific binding of the PrRP probe (results not shown). These data do not indicate absolute numbers of PrRP mRNA transcripts, but the number of silver grains does represent relative abundance. In each brain region where PrRP mRNA was detected, fasted animals showed a reduction in the number of silver grains per cell compared with ad libitum-fed controls (see Table 1A and Fig. 3. DMH; 32% reduction, NTS; 34% reduction and VLM; 55% reduction), though this reduction was statistically significant only in the case of the DMH (P ⬍ 0.05). Fasted animals also showed a significantly reduced number of detectable cells per section expressing PrRP mRNA compared with ad libitum-fed control animals in each area (Table

FIG. 2. The effect of (A) leptin, (B) PrRP, or (C) leptin and PrRP on nocturnal changes in core body temperature in adult, male Sprague Dawley rats. Core body temperature (C) was recorded continuously by remote radiotelemetry and the temperature change from basal values, calculated at 30 min intervals, were plotted over the 12-h dark phase. For ease of comparison, the trace for the control group has been included in each panel with one treatment group (mean ⫾ SEM, n ⫽ 5– 6 in each group). See text for details of statistical analysis.

1A, DMH 58% reduction, P ⬍ 0.01; NTS 33% reduction, P ⬍ 0.05; VLM 64% reduction, P ⬍ 0.05). Expression of mRNA for the orexigen NPY in the ARC was used as a positive control. In agreement with the literature (41), fasted animals showed an increased level of NPY mRNA with respect to the number of grains per cell (fed 49 ⫾ 5 grains per cell; fasted 69 ⫾ 4 grains per cell; P ⬍ 0.05) and the number of detectable cells per section (fed 61 ⫾ 5 cells per section; fasted 112 ⫾ 10 cells per section; P ⬍ 0.01) compared with ad libitum-fed animals. In obese Zucker rats, there was a reduction in the number of silver grains per cell in all regions examined (Table 1B and Fig. 3; DMH 43% reduction, P ⬍ 0.01; NTS 82% reduction, P ⬍ 0.001; VLM 68% reduction, P ⬍ 0.001). There was also a reduction in the number of detectable PrRP cells per section in obese compared with lean animals (Table 1B; DMH 59% reduction, P ⬍ 0.01, NTS 30% reduction, P ⬍ 0.05; VLM 45% reduction, P ⬍ 0.05). Expression of ARC NPY mRNA was used as a positive control again and, in agreement with the



literature (42), showed an increased level of NPY mRNA with respect to the amount per cell (lean 35 ⫾ 3 grains per cell; obese 50 ⫾ 3 grains per cell; P ⬍ 0.001) and the number of detectable cells per section (lean 39 ⫾ 3 cells per section; obese 56 ⫾ 6 cells per section; P ⬍ 0.05) in obese compared with lean animals. TABLE 1. PrRP mRNA expression in male rats A. Fed and fasted Sprague-Dawley rats Nuclei

DMH NTS VLM

Number of cells per section

Number of grains per cell Fed

Fasted

37 ⫾ 3 164 ⫾ 22 65 ⫾ 5

25 ⫾ 3 108 ⫾ 18 30 ⫾ 14 a

Fed

Fasted

9⫾1 19 ⫾ 1 3⫾0

4 ⫾ 1b 13 ⫾ 2a 1 ⫾ 1a

B. Obese (fa/fa) and lean (fa/⫹) Zucker rats Nuclei

DMH NTS VLM

Number of grains per cell

Number of cells per section

Lean

Obese

Lean

Obese

20 ⫾ 2 365 ⫾ 120 110 ⫾ 10

11 ⫾ 1b 68 ⫾ 9c 35 ⫾ 13c

5⫾1 14 ⫾ 2 14 ⫾ 2

2 ⫾ 0b 10 ⫾ 1a 8 ⫾ 2a

PrRP mRNA levels were measured by radioactive in situ hybridization. Bound probe was detected by coating slides with photographic emulsion and quantified by counting the resulting silver grains. Data are expressed as mean ⫾ SEM of the number of grains per cell and the number of detectable cells per section expressing PrRP mRNA. n ⫽ 6 for fed and fasted groups (Table 1A) and n ⫽ 8 for obese and lean Zucker groups (Table 1B). a , P ⬍ 0.05; b, P ⬍ 0.01; c, P ⬍ 0.001; Mann-Whitney U test.

PrRP and leptin receptor (Ob-R) localization by doublefluorescence immunohistochemistry

In agreement with our in situ hybridization studies, PrRPimmunoreactive neurons were identified exclusively in the DMH, NTS, and VLM. Figure 4 shows the localization of Ob-R visualized using a Texas Red-labeled secondary antibody and PrRP visualized using a fluorescein isothiocyanatelabeled secondary antibody. In the DMH, there were a large number of cells expressing Ob-R and relatively few expressing PrRP. Almost all of the PrRP neurons in this region contained Ob-R, whereas the PrRP neurons accounted for less than one percent of the leptin-receptive neurons (see Table 2). In the brainstem, most PrRP neurons were found at the level of the area postrema or more caudally. No PrRPimmunopositive neurons were found rostral to the area postrema. Ob-R-expressing neurons were more abundant than PrRP-expressing neurons particularly around the level of the area postrema. Overall, the majority of PrRP-immunopositive neurons in the NTS contained Ob-R immunoreactivity (see Table 2 and Fig. 4). Conversely, just under a fifth of the Ob-R-containing neurons on these sections also contained PrRP immunoreactivity. In the VLM, nearly all of the PrRP neurons expressed Ob-R. In these sections, Ob-R expression was less widespread and approximately half of Ob-Rexpressing neurons were found to contain PrRP immunoreactivity. Single immunocytochemsitry studies were performed for all regions and demonstrated that the colocalization seen was not due to antibody cross-reactivity (data not shown).

FIG. 3. An example of radioactive in situ hybridization for PrRP mRNA in the NTS of fed (A) and fasted (B) adult male Sprague Dawley rats and lean (C) and obese (D) Zucker rats. cc, Central canal. Insets are higher-magnification micrographs to show silver grains over individual cells. Scale bars, 300 ␮m.

372



FIG. 4. Examples of double-fluorescence immunohistochemistry for Ob-R and PrRP in DMH (A and B), NTS (C and D), and VLM (E and F). Ob-R is shown in the upper panels (A, C, E) and PrRP in the lower panels (B, D, F). White arrows indicate examples of neurons that show colocalization. The gray arrow indicates an example of a PrRP-positive neuron that does not show colocalization with Ob-R. cc, Central canal; 3V, third ventricle. Scale bars, 150 ␮m. TABLE 2. PrRP and Ob-R immunoreactivity in rat brain sections Nuclei

% PrRP neurons containing Ob-R

% Ob-R neurons containing PrRP

DMH NTS VLM

97 ⫾ 2 92 ⫾ 3 97 ⫾ 1

⬍1 16 ⫾ 1 54 ⫾ 8

PrRP and Ob-R immunoreactivity was assessed using doublefluorescence immunohistochemistry. The number of neurons on each section expressing PrRP or Ob-R immunoreactivity was scored, as was the number of neurons expressing both. Data are expressed as mean ⫾ SEM, n ⫽ 4 –5.

PrRP and TH localization by double-fluorescence immunohistochemistry

PrRP neurons in the NTS and VLM also express TH immunoreactivity (3, 10, 43– 45). In the present study, the majority of PrRP neurons in the NTS and VLM contained TH immunoreactivity (see Table 3 and Fig. 5). In sections of the NTS and VLM, at the same level as PrRP-immunoreactive neurons, approximately two-thirds of the TH-positive neurons also contained PrRP immunoreactivity. Discussion

The results of this study indicate a similarity in the actions of acutely administered PrRP and leptin and provides evidence for an interaction between the two peptides. Studies in

TABLE 3. PrRP and TH immunoreactivity in rat brain sections Nuclei

% PrRP neurons containing TH

% TH neurons containing PrRP

NTS VLM

87 ⫾ 4 97 ⫾ 1

67 ⫾ 3 60 ⫾ 4

PrRP and TH immunoreactivity was assessed using doublefluorescence immunohistochemistry. The number of neurons on each section expressing PrRP or TH immunoreactivity was scored, as was the number of neurons expressing both. Data are expressed as mean ⫾ SEM, n ⫽ 3–5.

vivo show that coadministration of PrRP and leptin caused an additive effect on reductions in food intake and body weight (Fig. 1). We have demonstrated previously, using pair-feeding experiments, that the reduction in body weight caused by PrRP cannot be explained solely by the reduction in food intake, and is likely to include an effect on energy expenditure (14). Here we show that the hyperthermic responses to PrRP and leptin, which are an indirect measure of increased energy expenditure, were also additive (Fig. 2). The interaction between these peptides appears to be additive rather than synergistic as the effect induced by coadministration of the two peptides was equivalent to the sum of the peptides administered alone. This additivity demonstrates that the effects of one peptide are not mediated by the other but does not define the level of interaction. At the present time, there are no known antagonists to the GPR10/hGR3 or leptin


FIG. 5. Examples of double-fluorescence immunohistochemistry for PrRP and TH in the NTS (A and B) and the VLM (C and D). The left panels show TH (A, C) and the right panels PrRP (B, D). White arrows indicate examples of neurons that show colocalization. cc, Central canal. Scale bars, 150 ␮m.

receptors, which would enable further clarification of this interaction. The reduction in PrRP mRNA expression in fasted, male rats provides further evidence that PrRP is an anorexigen. This confirms our previous result in fasted female rats (14) and removes the complication of hormonal fluctuations because some studies report variations in PrRP mRNA levels according to the stage of the estrous cycle (46). We report also that PrRP mRNA is reduced in obese Zucker rats in each of the three areas of the brain where PrRP is expressed. This phenomenon has been reported for a number of other anorexigenic peptides including cocaine- and amphetamineregulated transcript (32), POMC (47), and CRH (48). The reduction in PrRP mRNA in the models of energy imbalance used in this study may indicate that PrRP mRNA expression is regulated by leptin. We have localized leptin receptor immunoreactivity in PrRP neurons, which demonstrates that the regulation of PrRP mRNA could be achieved by a direct action of leptin on PrRP neurons. In addition to containing Ob-R, PrRP neurons of the NTS and VLM also contain TH, as has been shown previously by others (3, 10, 43– 45). The location of the PrRP neurons mainly in the caudal brainstem makes it likely that they are noradrenergic, rather than adrenergic, neurons. It has been reported recently that almost all TH-positive cells in the A1 and A2 regions express Ob-R immunoreactivity (49). This, combined with our findings indicating high levels of colocalization of PrRP with Ob-R and TH, suggests the presence of a subpopulation of noradrenergic neurons that express PrRP and Ob-R. Retrograde tracing studies have shown that the PrRP neurons of the NTS and VLM project to the hypothalamic PVN (44), where PrRP-immunoreactive fibers are de-


tected (2). The PVN contains a major population of neurons containing the anorexigen, CRH, which has been reported to mediate some of the effects of leptin (50, 51). Thus, CRH neurons of the PVN may act as a point of convergence between various leptin-sensitive systems. We have shown that the peripheral satiety signal, cholecystokinin (CCK), activates PrRP neurons in the brainstem (15). Recent interest has focused on the ability of leptin to enhance the effects of CCK and vice versa (52–54). Although some of this cooperativity occurs at the level of the gut (55, 56), our results suggest that PrRP neurons in the brainstem may be another point of interaction of leptin on the CCK-mediated satiety pathway. In summary, our studies suggest that PrRP interacts with leptin in the regulation of food intake and metabolism. The expression of PrRP mRNA is reduced in obese Zucker rats and fasted rats, indicating that leptin may be regulating PrRP’s expression. In addition, leptin receptors are present on PrRP neurons in the DMH, NTS, and VLM, providing morphological evidence for a direct interaction. As with PrRP, a number of peptides that are coexpressed with Ob-R in hypothalamic neurons have been shown also to interact with leptin (29 –34). Together, these data support the notion that leptin acts throughout the entire brain circuitry involved in appetite and body weight regulation. Acknowledgments Received July 26, 2001. Accepted October 5, 2001. Address all correspondence and requests for reprints to: Dr. Simon M. Luckman, 1.124 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail: [email protected]. This work was supported by the Biotechnology and Biological Sciences Research Council and AstraZeneca Plc (with whom K.L.J.E. is a Medical Research Council Industrial Collaborative Student).

References 1. Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M, Fukusumi S, Kitada C, Masuo Y, Asano T, Matsumoto H, Sekiguchi M, Kurokawa T, Nishimura O, Onda H, Fujino M 1998 A prolactin-releasing peptide in the brain. Nature 393:272–276 2. Maruyama M, Matsumoto H, Fujiwara K, Kitada C, Hinuma S, Onda H, Fujino M, Inoue K 1999 Immunocytochemical localization of prolactinreleasing peptide in the rat brain. Endocrinology 140:2326 –2333 3. Iijima N, Kataoka Y, Kakihara K, Bamba H, Tamada Y, Hayashi S, Matsuda T, Tanaka M, Honjyo H, Hosoya M, Hinuma S, Ibata Y 1999 Cytochemical study of prolactin-releasing peptide (PrRP) in the rat brain. Neuroreport 10: 1713–1716 4. Yamakawa K, Kudo K, Kanba S, Arita J 1999 Distribution of prolactinreleasing peptide-immunoreactive neurons in the rat hypothalamus. Neurosci Lett 267:113–116 5. Matsumoto H, Murakami Y, Horikoshi Y, Noguchi J, Habata Y, Kitada C, Hinuma S, Onda H, Fujino M 1999 Distribution and characterization of immunoreactive prolactin-releasing peptide in rat tissue and plasma. Biochem Biophys Res Commun 257:264 –268 6. Jarry H, Heuer H, Schomburg L, Bauer K 2000 Prolactin-releasing peptides do not stimulate prolactin release in vivo. Neuroendocrinology 71:262–267 7. Watanobe H, Schio¨th HB, Wikberg JE, Suda T 2000 Evaluation of the role for prolactin-releasing peptide in prolactin secretion induced by ether stress and suckling in the rat: comparison with vasoactive intestinal peptide. Brain Res 865:91–96 8. Samson WK, Resch ZT, Murphy TC, Chang JK 1998 Gender-biased activity of the novel prolactin releasing peptides: comparison with thyrotropin releasing hormone reveals only pharmacologic effects. Endocrine 9:289 –291 9. Takahashi K, Abe T, Matsumoto K, Tomita M 2000 Does prolactin releasing peptide receptor regulate prolactin-secretion in human pituitary adenomas? Neurosci Lett 291:159 –162 10. Chen C, Dun SL, Dun NJ, Chang JK 1999 Prolactin-releasing peptide-immunoreactivity in A1 and A2 noradrenergic neurons of the rat medulla. Brain Res 822:276 –279

374


11. Lee Y, Yang SP, Soares MJ, Voogt JL 2000 Distribution of prolactin-releasing peptide mRNA in the rat brain. Brain Res Bull 51:171–176 12. Elmquist JK, Elias CF, Saper CB 1999 From lesions to leptin: hypothalamic control of food intake and body weight. Neuron 22:221–232 13. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS 1999 Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20:68 –100 14. Lawrence CB, Celsi F, Brennand J, Luckman SM 2000 Alternative role for prolactin-releasing peptide in the regulation of food intake. Nat Neurosci 3:645– 646 15. Lawrence CB, Ellacott KLJ, Luckman SM 2002 PRL-releasing peptide reduces food intake and may mediate satiety signaling. Endocrinology 143:360 –367 16. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425– 432 17. Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, Moizo L, Lehy T, Guerre-Millo M, Marchand-Brustel Y, Lewin MJ 1998 The stomach is a source of leptin. Nature 394:790 –793 18. Wang J, Liu R, Hawkins M, Barzilai N, Rossetti L 1998 A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature 393: 684 – 688 19. Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, Nishimura H, Yoshimasa Y, Tanaka I, Mori T, Nakao K 1997 Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 3:1029 –1033 20. Hoggard N, Hunter L, Duncan JS, Williams LM, Trayhurn P, Mercer JG 1997 Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta. Proc Natl Acad Sci USA 94:11073–11078 21. Ahima RS, Flier JS 2000 Leptin. Annu Rev Physiol 62:413– 437 22. Phillips MS, Liu Q, Hammond HA, Dugan V, Hey PJ, Caskey CJ, Hess JF 1996 Leptin receptor missense mutation in the fatty Zucker rat. Nat Genet 13:18 –19 23. Seeley RJ, van Dijk G, Campfield LA, Smith FJ, Burn P, Nelligan JA, Bell SM, Baskin DG, Woods SC, Schwartz MW 1996 Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats. Horm Metab Res 28:664 – 668 24. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP 1996 Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491– 495 25. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P 1996 Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett 387:113–116 26. Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB 1998 Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 395:535–547 27. Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B 1998 Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 18:559 –572 28. Mercer JG, Moar KM, Hoggard N 1998 Localization of leptin receptor (Ob-R) messenger ribonucleic acid in the rodent hindbrain. Endocrinology 139:29 –34 29. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Morgan PJ, Trayhurn P 1996 Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 8: 733–735 30. Wilson BD, Bagnol D, Kaelin CB, Ollmann MM, Gantz I, Watson SJ, Barsh GS 1999 Physiological and anatomical circuitry between Agouti-related protein and leptin signaling. Endocrinology 140:2387–2397 31. Cheung CC, Clifton DK, Steiner RA 1997 Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology 138:4489 – 4492 32. Kristensen P, Judge ME, Thim L, Ribel U, Christjansen KN, Wulff BS, Clausen JT, Jensen PB, Madsen OD, Vrang N, Larsen PJ, Hastrup S 1998 Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393:72–76 33. Hakansson M-L, de Lecea L, Sutcliffe JG, Yanagisawa M, Meister B 1999 Leptin receptor- and STAT3-immunoreactivities in hypocretin/orexin neurons of the lateral hypothalamus. J Neuroendocrinol 11:653– 663 34. Funahashi H, Hori T, Shimoda Y, Mizushima H, Ryushi T, Katoh S, Shioda


35.

36. 37. 38. 39. 40. 41. 42. 43.

44. 45. 46.

47. 48. 49. 50. 51. 52. 53. 54. 55. 56.

S 2000 Morphological evidence for neural interactions between leptin and orexin in the hypothalamus. Regul Pept 92:31–35 Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A 1995 The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377: 530 –532 Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG 1997 Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119 –2123 Sahu A 1998 Leptin decreases food intake induced by melanin-concentrating hormone (MCH), galanin (GAL) and neuropeptide Y (NPY) in the rat. Endocrinology 139:4739 – 4742 Beck B, Richy S 1999 Hypothalamic hypocretin/orexin and neuropeptide Y: divergent interaction with energy depletion and leptin. Biochem Biophys Res Commun 258:119 –122 Sahu A, Carraway RE, Wang YP 2001 Evidence that neurotensin mediates the central effect of leptin on food intake in rat. Brain Res 888:343–347 Paxinos G, Watson C 1986 The rat brain in stereotaxic coordinates. San Diego: Academic Press Brady LS, Smith MA, Gold PW, Herkenham M 1990 Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinology 52:441– 447 Beck B, Burlet A, Nicolas JP, Burlet C 1990 Hyperphagia in obesity is associated with a central peptidergic dysregulation in rats. J Nutr 120:806 – 811 Roland BL, Sutton SW, Wilson SJ, Luo L, Pyati J, Huvar R, Erlander MG, Lovenberg TW 1999 Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery. Endocrinology 140:5736 –5745 Morales T, Hinuma S, Sawchenko PE 2000 Prolactin-releasing peptide is expressed in afferents to the endocrine hypothalamus, but not in neurosecretory neurones. J Neuroendocrinol 12:131–140 Maruyama M, Matsumoto H, Fujiwara K, Noguchi J, Kitada C, Fujino M, Inoue K 2001 Prolactin-releasing peptide as a novel stress mediator in the central nervous system. Endocrinology 142:2032–2038 Kataoka Y, Iijima N, Yano T, Kakihara K, Hayashi S, Hinuma S, Honjo H, Hayashi S, Tanaka M, Ibata Y 2001 Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat. Mol Brain Res 87:42– 47 Kim EM, O’Hare E, Grace MK, Welch CC, Billington CJ, Levine AS 2000 ARC POMC mRNA and PVN ␣-MSH are lower in obese relative to lean zucker rats. Brain Res 862:11–16 Richard D, Rivest R, Naimi 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 Hay-Schmidt A, Helboe L, Larsen PJ 2001 Leptin receptor immunoreactivity is present in ascending serotonergic and catecholaminergic neurons of the rat. Neuroendocrinology 73:215–226 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 Gardner JD, Rothwell NJ, Luheshi GN 1998 Leptin affects food intake via CRF-receptor-mediated pathways. Nat Neurosci 1:103 Barrachina MD, Martinez V, Wang L, Wei JY, Tache Y 1997 Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice. Proc Natl Acad Sci USA 94:10455–10460 Matson CA, Wiater MF, Kuijper JL, Weigle DS 1997 Synergy between leptin and cholecystokinin (CCK) to control daily caloric intake. Peptides 18:1275– 1278 Emond M, Schwartz GJ, Ladenheim EE, Moran TH 1999 Central leptin modulates behavioral and neural responsivity to CCK. Am J Physiol 276: R1545–R1549 Wang YH, Tache Y, Sheibel AB, Go VL, Wei JY 1997 Two types of leptinresponsive gastric vagal afferent terminals: an in vitro single-unit study in rats. Am J Physiol 273:R833–R837 Yuan CS, Attele AS, Dey L, Xie JT 2000 Gastric effects of cholecystokinin and its interaction with leptin on brainstem neuronal activity in neonatal rats. J Pharmacol Exp Ther 295:177–182