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Peptides that Regulate Food Intake
School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
Submitted 1 May 2003 ; accepted in final form 30 May 2003
ABSTRACT
Central administration of a single dose of prolactin-releasing peptide (PrRP) causes a reduction in both fast-induced and nocturnal food intake and body weight gain. The aim of this study was to examine the effect of repeated administration of PrRP on energy homeostasis, including a measure of the expression of the mitochondrial uncoupling protein-1 (UCP-1) in brown adipose tissue. Conscious, free-feeding animals received central injections of PrRP (4 nmol icv) or vehicle. A single injection at 1000 caused a sustained hyperthermia over the 4-h test period and an increase in the expression of UCP-1 mRNA. Repeated, twice daily injection caused a reduction in body weight gain greater than that seen in pair-fed animals for the first 48-72 h. After 72 h, the animals became refractory to the actions of PrRP. The pair-fed group showed a reduction in UCP-1 mRNA expression at 48 h, which was reversed by PrRP treatment. This study indicates that PrRP exerts its effects on energy homeostasis in the short-medium term by reducing food intake and increasing energy expenditure.
uncoupling protein-1; food intake
Of the peptide factors known to regulate appetite and body weight, some have actions in the short term to mediate the initiation and termination of feeding incidences, whereas others are involved in longer-term regulation, modulating energy expenditure, and the expression of other regulatory neuropeptides. An example of a peptide that has short-term effects to induce satiety is CCK. CCK causes a reduction in food intake in rats (9) and a number of other species, including humans (5, 8, 13, 25), through its action on vagal sensory afferents from the gut to the brain stem (33). However, chronic administration of CCK in rats results in the rapid development of tolerance (4). Other factors shown to have predominantly short-term effects on energy homeostasis include orexin-A (11, 28, 35), galanin (32), and melanin concentrating hormone (MCH; 27). In contrast, leptin is effective over the longer term by acting on both the brain (36), to reduce food intake and increase energy expenditure, and peripheral tissues. Leptin causes weight loss due to specific depletion of adipose tissue (10), activation of lipid oxidation (30), and apoptosis of adipocytes in rats (26).
There are similarities in the responses to single intracerebroventricular injections of PrRP and leptin, including a reduction in food intake and body weight, accompanied by an increase in core body temperature, a surrogate marker of heightened energy expenditure (6, 16). However, there are also similarities with the behavioral satiating effects of CCK. Indeed, brain stem PrRP neurons are activated by systemic injection of CCK, but not by the nonphysiological anorexigen, lithium chloride (17). The aim of the present study was to examine whether PrRP is able to exert its anorexigenic effects only in the short term as a satiety factor or whether it is effective in the longer term in a similar manner to leptin. Our previous experiments have suggested that PrRP can increase thermogenesis. Thus we predicted an increase in the expression of brown adipose tissue (BAT) uncoupling protein-1 (UCP-1), a marker for activation of the sympathetic nervous system (1, 22).
METHODS
Animals and materials. All experiments were performed using adult male Sprague-Dawley rats (250-300 g, Charles River Laboratories, Sandwich, UK). Animals were kept in a 12:12-h light/dark cycle (lights on 0800-2000) at 21 ± 1°C with 45 ± 10% humidity and free access to food (Beekay International, Hull, UK) and water. All experiments were performed in accordance with the United Kingdom's Animals (Scientific Procedures) Act (1986). Unless stated otherwise, all chemicals were obtained from Sigma-Aldrich (Sigma-Aldrich, Poole, UK).
Animals underwent lateral cerebroventricular cannulation (0.8 mm posterior and 1.5 mm lateral to bregma and 3.0 mm down from dura; coordinates according to Ref. 24) under halothane anesthesia. After 1 wk of recovery, animals were housed individually and left to acclimatize. The free-moving, conscious rats were given intracerebroventricular injections of 4 nmol PrRP1-31 (Peptide Institute, Osaka, Japan) or vehicle (isotonic sterile saline) in a volume of 2 µl at the times stated. At the end of the experiment, animals were killed using a rising concentration of CO2 followed by cervical dislocation. Interscapular BAT and epididymal white adipose tissue (WAT) were collected and weighed.
Quantification of UCP-1 mRNA levels in BAT. RNA was extracted from BAT using Trizol (Invitrogen, Paisley, UK) according to the manufacturer's instructions. One microgram RNA was treated with 1 U DNase 1 (Invitrogen) to minimize DNA contamination. Reverse transcription was performed using the Taqman reverse transcription reagents kit (Perkin-Elmer/NEN Life Science, Hounslow, UK): 0.5 µg DNase 1-treated RNA was incubated with 1x Taqman reverse transcriptase buffer, 5.5 mM MgCl2, 500 µM of each dNTP, 2.5 µM random hexamers, 0.4 U/µl RNase inhibitor ± 1.25 U/µl Multiscribe reverse transcriptase in a total volume of 50 µl. The reverse transcription reaction was performed in a thermocycler under the following conditions: 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. PCR was performed in a total volume of 25 µl in a 96-well plate; 5 µl cDNA and 12.5 µl Taqman universal PCR master mix containing 300 nM forward and reverse primers and 200 nM probe made up to 25 µl with nuclease-free water. The sequence of the primers and probe for UCP-1 and the housekeeping gene, human hypoxanthine phosphoribosyl transferase (HPRT), were as follows: UCP-1, forward: 5'-GAAACGCCTGCCTCTTTGG-3', reverse: 5'-GTGAAGGTCAGAATGCAAGCAC-3', probe: 5'-AAGCAAGATCTCGGCTGGCTTGATGA-3'; and HPRT, forward: 5'-CGAGCCGACCGGTTCTG-3', reverse: 5' CATAACCTGGTTCATCACTAATCAC-3', probe: 5'-CATGTCGACCCTCAGTCCCAGCG-3'. The probe had two dyes bound, a reporter dye, FAM (6-carboxy-fluorescien), and a quencher dye, TAMRA (6-carboxy-tetramethyl-rhodamine), at the 5'-and 3'-ends, respectively. The reaction was carried out in a sequence detector with the following cycling conditions: an initial denaturation at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The housekeeping gene, HPRT, and mitochondrial UCP-1 were examined in triplicate. Each plate included a no-reverse transcriptase control for each sample to check for DNA contamination.
Experiment 1. For the first experiment, rats were given a single injection at 1000. The 4-nmol dose was chosen because we showed previously that, although producing reductions in nighttime or fast-induced feeding, it dose not disrupt the behavioral satiety sequence or support conditioned taste aversion (17). Tissues were collected at 4 h because this is when the thermogenic effect of PrRP is most apparent. Satiated rats do not eat at this time of the light/dark cycle (1000-1400) and, therefore, because food intake cannot be measured, the effectiveness of the injection was demonstrated by measuring core body temperature. A temperature-sensitive, precalibrated radiotelemetery transmitter (TA10TA-F40, Data Sciences International, Minneapolis, MN) was implanted into the peritoneal cavity at the same time as the ventricular cannulation. The core body temperature of the animals was measured continuously throughout the 4-h experimental period.
Experiments 2 and 3. The animals were given their first intracerebroventricular injection immediately before lights out (1900-2000). The injections were then repeated at 0800-0900 and 1900-2000 for 48 h (experiment 2) or for 5 days (experiment 3). The initial aim of experiment 3 was to treat rats with PrRP twice daily until they became unresponsive to its effects or until such a time that the degree of weight loss was detrimental to the welfare of the animals. Food and water intake and body weights were measured every 24 h. Vehicle-treated animals, which received the same amount of food as was consumed by the PrRP-treated animals (i.e., pair-fed groups), were also included in these studies.
Statistical analyses. Food intake, body weight gain, and UCP-1 mRNA expression were analyzed using a parametric one-way analysis of variance with Dunnett's multiple comparisons post hoc test for analysis of three groups, and an unpaired Student's t-test was used for the analysis of two groups. For experiment 1, core body temperature was expressed as change from mean basal values and was analyzed by calculating area under the curve (AUC;°C/h) by the trapezoid method for the period of hyperthermia (0.5-4 h after injection).
RESULTS
Experiment 1: acute effect of PrRP. A single, intracerebroventricular injection of 4 nmol PrRP caused a rapid and transient hypothermia (nadir 10 min after injection), followed by a small, but extended, hyperthermia as reported after nighttime PrRP injection (6, 16). This hyperthermia was apparent from 30 min after the injection to the end of the 4-h recording period and was significantly different from vehicle-treated animals (AUC: vehicle 0.5 ± 0.05°C/h, PrRP 1.0 ± 0.1°C/h; P < 0.05, Fig. 1A). The increase in BAT UCP-1 expression was significant using a one-tailed Student's t-test (relative expression: vehicle 1.0 ± 0.1, PrRP 1.6 ± 0.3; P < 0.05; Fig. 1B).
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Experiment 2: effect of repeated administration of PrRP (48 h). Administration of PrRP twice daily produced a reduction in cumulative food intake in the 24 h after the first injection, but this reduction was not statistically significant compared with vehicle-treated animals (vehicle 27.1 ± 1.4 g, PrRP 23.1 ± 1.9 g; P > 0.05; Fig. 2A). However, there was a significant reduction in cumulative body weight gain over the 24 h after the first injection (vehicle 10.1 ± 1.7 g, PrRP 0.6 ± 2.7 g; P < 0.01; Fig. 2B). Pair-fed animals that received the same amount of food as was consumed by the PrRP-treated group, gained significantly less body weight in the first 24 h after the initial injection than vehicle-treated animals, but this reduction was not as great as that for the PrRP-treated group (pair-fed 4.7 ± 1.6 g; P < 0.05 vs. vehicle, P > 0.05 vs. PrRP; Fig. 2B). Water intake was unaffected by any of the treatments (results not shown).
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Food intake in PrRP-treated animals remained lower than that of the control group during the 48-h period, although again this did not reach statistical significance (vehicle 55.0 ± 1.6 g, PrRP 48.4 ± 3.4 g; P > 0.05; Fig. 2A). However, there was again a significant difference in body weight gain (vehicle 21.3 ± 1.1 g, PrRP 11.0 ± 3.0 g; P < 0.05; Fig. 2B). Pair-fed animals showed a significantly reduced body weight gain compared with vehicle, but not PrRP-treated animals (pair-fed 13.4 ± 2.1 g; P < 0.01 vs. vehicle, P > 0.05 vs. PrRP; Fig. 2B).
There was no significant difference in the interscapular BAT (vehicle 0.06 ± 0.005%, PrRP 0.06 ± 0.005%, and pair-fed 0.07 ± 0.005%; P > 0.05) or epididymal WAT (vehicle 0.41 ± 0.04%, PrRP 0.37 ± 0.03%, and pair-fed 0.36 ± 0.02%; P > 0.05) mass, expressed as percentage of total body weight, between groups after 48 h of twice daily administration of PrRP. UCP-1 mRNA expression was significantly reduced in the pair-fed animals (vehicle 1.0 ± 0.17, pair-fed 0.38 ± 0.07; P < 0.05 compared with vehicle-treated group; Fig. 2C). PrRP-treated animals did not show this decrease (PrRP 0.95 ± 0.12; P > 0.05 and P < 0.01 compared with vehicle-treated and pair-fed groups, respectively; Fig. 2C).
Experiment 3: effect of repeated administration of PrRP (5 days). Animals again received twice daily administration of PrRP, and the experiment was continued until the animals became tolerant to the actions of PrRP. As with the previous experiment, there was no significant difference in cumulative food intake between groups at any time point (Fig. 3A). Tolerance to the effect of PrRP appeared to occur at around 72 h after the first injection. Forty-eight hours after the initial injection, animals treated with PrRP showed a 32.5 ± 8.4% reduction in overnight food intake compared with vehicle-treated animals. The reduction in overnight food intake in the PrRP-treated group did not quite reach statistical significance at 72 h (P = 0.053) and, by 96 h, the overnight food intake of the PrRP-treated animals was indistinguishable from vehicle-treated animals and remained so for the duration of the experiment.
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In the 24 h after the first injection, PrRP-treated animals gained 6.1 ± 1.8 g compared with vehicle-treated animals, which gained 10.7 ± 1.0 g (P < 0.05; Fig. 3B). By 48 h after the start of the experiment, animals treated with PrRP showed an overall loss in cumulative body weight, -1.5 ± 4.4 g, whereas animals that were treated with vehicle continued show an increase in cumulative body weight, 17.4 ± 1.9 g. The pair-fed animals that received the same amount of food as the PrRP-treated animals did not show the same degree of weight loss as the PrRP-treated animals and overall gained 11.9 ± 1.2 g in weight at 48 h, which was significantly different from the PrRP-treated group (P < 0.05; Fig. 3B). After 72 h, there was still a significant difference in cumulative body weight gain between PrRP- and vehicle-treated groups (vehicle 25.8 ± 1.5 g, PrRP 5.5 ± 5.6 g; P < 0.05; Fig. 3B), but at this time point there was no significant difference when compared with pair-fed animals (pair-fed 11.6 ± 1.6 g; P > 0.05). After 96 h and for the duration of the experiment there was no significant difference in cumulative body weight gain between groups (Fig. 3B).
There was no significant difference in the BAT (vehicle 0.10 ± 0.07%, PrRP 0.08 ± 0.008%, and pair-fed 0.09 ± 0.009%; P > 0.05) and WAT (vehicle 0.38 ± 0.02%, PrRP 0.36 ± 0.03%, and pair-fed 0.38 ± 0.01%; P < 0.05) mass, expressed as percentage of total body weight, between groups. After 5 days of twice daily PrRP administration, there was no significant difference in the expression in BAT UCP-1 mRNA, compared with vehicle-treated or pair-fed animals (P < 0.05; Fig. 3C).
DISCUSSION
We previously showed that PrRP has a transient effect on food intake, measurable in the first hours after injection (6, 16). Although no effect on cumulative food intake is measured at 24 h, a single injection does appear to have a longer-lasting effect on body weight gain. As demonstrated here and previously with the pair-feeding groups, PrRP had additional effects on body weight that could not be attributed purely to its anorectic action. We have confirmed that PrRP has an apparent thermogenic effect, which is independent of whether the animal is eating. As during normal free feeding and fast-induced refeeding (6, 16), daytime injection of PrRP caused a long-lasting hyperthermia in satiated rats (experiment 1). Raised core body temperature is an indirect indication that energy expenditure has increased. More direct evidence for this is the fact that PrRP increases oxygen consumption (C. B. Lawrence, unpublished observations) and, as demonstrated herein, acutely increases the expression of UCP-1 mRNA in BAT. This suggests that PrRP may mediate changes in body weight gain via the modulation of UCP-1 expression, which in turn alters energy expenditure. Although changes in UCP-1 mRNA levels occurred after administration of PrRP, this does not necessarily reflect a change in UCP-1 protein expression or mitochondrial activity. There have been reports in the literature to suggest that under certain circumstances changes in UCP mRNA levels are not reflected by changes in protein expression (31). It will be important to confirm that there are consistent changes in mitochondrial activity.
The main aim of the study was to examine the effects of chronic administration of PrRP on energy homeostasis. Vergoni and colleagues (34) reported an experiment whereby rats were injected twice daily with PrRP. Surprisingly, they saw no effect on acute feeding on the first day, but did see a reduction on days 2 and 3. Unfortunately, they did not extend their experiment beyond this point. Experiment 2 in this study shows a significant effect of PrRP on body weight gain at 24 and 48 h of repeated injection. Thus we decided to carry out a further study whereby the animals were to be treated until they became unresponsive to PrRP's effects or until such a time that the degree of weight loss was detrimental to their welfare (experiment 3). In fact, the animals became refractory to the anorectic and body weight actions of PrRP after 72 h of repeated injections. This finding indicates that PrRP has effects on energy homeostasis in the short to medium term. Tolerance to the effects of chronic administration has been reported in response to a number of other anorectic and orectic peptides, including CCK (4), orexin-A (11, 28, 35), galanin (32), and MCH (27). This result supports our hypothesis that PrRP mediates some of the effects of CCK on satiety (17), although other possibilities need consideration. First, it is possible that the apparent tolerance is secondary to other endocrine changes following weight loss, such as reduced plasma leptin and the activation of counter-regulatory mechanisms. However, if this were the case, then we might expect to see a rebound hyperphagia. Second, repeated injections may lead to a gradual increase of stress to the animals that would mask any small effects of PrRP on feeding at later time points. This would seem unlikely, because, if stress were a major factor, then we would expect to see increasing weight loss in the control group.
An alternative strategy to using repeated injections would have been to use osmotic minipumps that continuously infuse into the ventricle. These pumps remove the need for repeated handling of the animals and produce a constant elevation of peptide, which may or may not create a more physiologically relevant effect depending on the mode of action of the peptide under study. In the case of PrRP, the peptide may not be stable for long periods of time at 37°C, making the pumps impractical. However, the main disadvantage of this alternative system is that the pumps are active immediately after implantation and, thus, do not allow the animals to recover body weight lost due to the surgery before the start of the experiment. We opted for repeated injection because it probably reflects better the mode of action of a satiety peptide and because it also allows the data to be compared directly with acute studies.
The mechanism by which the tolerance to PrRP is mediated is not known, for example, whether it has a physiological or a pharmacological basis, or both. Crawley and colleagues (4) proposed a number of possible mechanisms for the behavioral tolerance that develops in response to chronic administration of CCK. At a physiological level, they proposed that tolerance might develop to the conditioned satiety component of gastric distension. At a pharmacological level, it was postulated that tolerance might occur due to the development of receptor subsensitivity, a change in receptor number, tolerance to second messengers, or receptor desensitization. These pharmacological mechanisms could also account for the tolerance seen after repeated administration of PrRP. Whatever the mechanism of the tolerance, this result would argue against PrRP having long-term actions on body weight like, for example, leptin, although these two peptides do have very similar acute effects (6).
Animals that undergo fasting, or food restriction in a relevant pair-fed group, show a reduction in BAT UCP-1 mRNA that contributes to the fall in energy expenditure (31). PrRP treatment for 48 h prevented the reduction in BAT UCP-1 mRNA caused by food restriction seen in the pair-fed group. This reversal of effects of pair-feeding on UCP-1 is well documented in the literature and is seen after administration of other anorectic factors, including leptin (31), urocortin (15), and the melanocortin-receptor agonist, MTII (2). After 5 days of treatment the animals were no longer responding to PrRP, and both the PrRP-treated and pair-fed animals had food intake and body weight equivalent to vehicle-treated controls. Thus, as expected, UCP-1 mRNA expression was not different among the three groups at this time point.
In summary, the data presented here suggest that PrRP exerts its effects on energy homeostasis in the short-medium term by inhibiting food intake and reducing body weight gain by increasing thermogenesis. The latter is indicated by an increase in the expression of UCP-1 mRNA and presumably mitochondrial activity in BAT. In rats, the main neural projections to BAT originate from the brain stem and hypothalamus, particularly the paraventricular nucleus (PVN), lateral hypothalamic area, perifornical area, and retrochiasmatic nucleus. Neurochemical characterization of paraventricular hypothalamic neurons projecting to preganglionic efferent cells indicates that some are immunoreactive for CRH or oxytocin (23). PrRP neurons of the brain stem project to the PVN (21), where they form synaptic contact with CRH- and oxytocin-containing cells (18, 19). This suggests that PrRP may be exerting an effect on BAT via connections with oxytocin- and CRH-expressing neurons in the PVN.
DISCLOSURES
The authors acknowledge the support of the Biotechnology and Biosciences Research Council and AstraZeneca. K. L. J. Ellacott was a UK Medical Research Council/AstraZeneca-funded postgraduate student.
FOOTNOTES
Address for reprint requests and other correspondence: S. Luckman, 1.124 Stopford Bldg., School of Biological Sciences, Univ. of Manchester, Oxford Road, Manchester, M13 9PT UK (E-mail: simon.luckman{at}man.ac.uk).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
This article has been cited by other articles:
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  S. P. Kelly and R. E. Peter Prolactin-releasing peptide, food intake, and hydromineral balance in goldfish Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2006; 291(5): R1474 - R1481. [Abstract] [Full Text] [PDF]
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 S. Stanley, K. Wynne, B. McGowan, and S. Bloom Hormonal Regulation of Food Intake Physiol Rev, October 1, 2005; 85(4): 1131 - 1158. [Abstract] [Full Text] [PDF]
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W. A. Cupples Physiological regulation of food intake Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1438 - R1443. [Full Text] [PDF]
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C. B. Lawrence, Y.-L. Liu, M. J. Stock, and S. M. Luckman Anorectic actions of prolactin-releasing peptide are mediated by corticotropin-releasing hormone receptors Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2004; 286(1): R101 - R107. [Abstract] [Full Text]
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