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J Clin Invest. Dec 1, 2008; 118(12): 4014–4024.
Published online Nov 3, 2008. doi:  10.1172/JCI34682
PMCID: PMC2575834

Endogenous prolactin-releasing peptide regulates food intake in rodents

Abstract

Food intake is regulated by a network of signals that emanate from the gut and the brainstem. The peripheral satiety signal cholecystokinin is released from the gut following food intake and acts on fibers of the vagus nerve, which project to the brainstem and activate neurons that modulate both gastrointestinal function and appetite. In this study, we found that neurons in the nucleus tractus solitarii of the brainstem that express prolactin-releasing peptide (PrRP) are activated rapidly by food ingestion. To further examine the role of this peptide in the control of food intake and energy metabolism, we generated PrRP-deficient mice and found that they displayed late-onset obesity and adiposity, phenotypes that reflected an increase in meal size, hyperphagia, and attenuated responses to the anorexigenic signals cholecystokinin and leptin. Hypothalamic expression of 6 other appetite-regulating peptides remained unchanged in the PrRP-deficient mice. Blockade of endogenous PrRP signaling in WT rats by central injection of PrRP-specific mAb resulted in an increase in food intake, as reflected by an increase in meal size. These data suggest that PrRP relays satiety signals within the brain and that selective disturbance of this system can result in obesity and associated metabolic disorders.

Introduction

Classically, feeding is regulated by an alternation between hunger and satiety signals, which include both circulating factors and neurally mediated signals from the gastrointestinal tract (1). These signals act on the peripheral organs and on the CNS, including the hypothalamus and brainstem, to initiate or terminate food intake. One important satiety signal is the brain-gut peptide cholecystokinin (CCK). CCK, released from the gut in response to a meal, has an important regulatory role in gastrointestinal function, inhibiting gastric motility and emptying via a neural reflex within the caudal brainstem or via a reflex loop that ascends to the hypothalamus via a relay in the caudal brainstem (2, 3), but is also involved in the regulation of food intake. Peripheral CCK acts on afferent nerve fibers of the gastric vagus nerve; these vagal neurons project to the caudal brainstem (4), where they activate neurons that project to hypothalamic nuclei involved in appetite regulation (2, 3). In particular, peripheral injections of CCK activate neurons in the nucleus tractus solitarii (NTS) and ventrolateral medulla, including specific subpopulations of the noradrenergic neurons of the A1 and A2 cell groups (5). These noradrenergic neurons comprise several subpopulations, which are anatomically, biochemically, and functionally separate.

Some of these noradrenergic neurons express prolactin-releasing peptide (PrRP; encoded by the Prlh gene) (6). PrRP was discovered as an endogenous ligand for the GPCR GPR10 (encoded by the Prlhr gene) (7), and PrRP-expressing neurons are localized in the NTS and ventrolateral medulla oblongata and in the dorsomedial hypothalamus (8, 9). In the medulla oblongata, PrRP is exclusively colocalized in noradrenergic neurons (6). PrRP has been suggested to be involved in energy metabolism (1015), stress responses (1619), and analgesia (20).

Peripheral administration of CCK activates neurons expressing PrRP (11). There have also been several other indications that PrRP might be involved in the regulation of feeding and energy balance. In particular, central administration of PrRP reduces food intake in rats (10), and PrRP mRNA expression decreases during fasting and is low during lactation, a physiological state of negative energy balance (10). Mice that lack the PrRP receptor GPR10 show adult-onset obesity (15, 21) and, most interestingly, do not reduce their feeding in response to CCK (22).

Here, we examined the role of endogenous PrRP in the control of food intake and energy metabolism by studies in Tg mice deficient in PrRP and by neutralizing the actions of endogenous PrRP in mice and rats using mAb specific for PrRP. We also investigated whether food intake activates PrRP neurons in the caudal brainstem. In this study, we demonstrate that in rodents, PrRP relays satiety signals within the brain and that perturbation of this system can predispose to obesity and associated metabolic disorders.

Results

Generation of PrRP-deficient mice.

To generate mice deficient in PrRP, we designed a targeting vector to disrupt exons 1 and 2 including full coding regions of the prepro-PrRP sequence (Figure (Figure1A).1A). Male chimeric mice transmitted the mutant Prlh allele to their offspring. The heterozygous mice were apparently normal and were intercrossed to obtain homozygous offspring; these mice were viable (Figure (Figure1B).1B). PrRP transcripts and proteins were not detectable in PrRP-deficient mice, confirming the disruption of the Prlh gene (Figure (Figure1,1, C and D).

Figure 1
Generation of PrRP-deficient mice.

Adult-onset obesity in PrRP-deficient mice.

When WT, PrRP-hetero­zygous, and PrRP-deficient mice from heterozygous intercrosses were fed a high-fat diet ad libitum from the age of 5 weeks, PrRP-deficient mice became significantly heavier than either WT mice or PrRP-heterozygous mice by the age of 7 weeks (Figure (Figure2A).2A). When maintained on standard laboratory chow, PrRP-deficient mice weighed significantly more than WT mice at 18 weeks of age (Figure (Figure2B),2B), which was apparently attributable to greater food consumption (Figure (Figure2C).2C). PrRP-deficient mice had substantially more body fat than WT mice (Figure (Figure2D),2D), and this was accompanied by reduced glucose tolerance and increased insulin resistance (Figure (Figure2,2, E and F). Both WAT mass, in either subcutaneous pads or intraabdominal pads (perirenal, mesenteric, epididymal), and brown adipose tissue (BAT) mass were greater in PrRP-deficient mice than in WT mice (Figure (Figure2G),2G), and epididymal adipocytes of PrRP-deficient mice were larger than those in WT mice (Figure (Figure2H).2H). The hepatic tissue of PrRP-deficient mice contained many large fatty vesicles, indicating fatty liver (Figure (Figure2H).2H). Plasma concentrations of insulin, leptin, and triglyceride were higher in PrRP-deficient than in WT mice (insulin: WT, 850 ± 191 pg/ml, PrRP-deficient, 3,001 ± 672 pg/ml; leptin: WT, 11.5 ± 5.9 ng/ml, PrRP-deficient, 40.3 ± 12.9 ng/ml; triglyceride: WT, 71.2 ± 3.5 ng/dl, PrRP-deficient, 95.5 ± 5.7 ng/dl). Hepatic triglyceride concentrations were also higher in PrRP-deficient mice than in WT mice (1.23 ± 0.25 vs. 0.27 ± 0.04 mg/mg protein). The greater BW gain of PrRP-deficient mice was more prominent when they were fed a high-fat diet (Figure (Figure3A),3A), and this was associated with a significantly greater food intake (Figure (Figure3B). 3B).

Figure 2
Late-onset obesity in PrRP-deficient mice.
Figure 3
Energy utilization in PrRP-deficient and WT mice.

Energy expenditure in PrRP-deficient mice.

To examine the pathogenesis of obesity in PrRP-deficient mice, amounts of food supplied were restricted to the mean food intake of age-matched WT mice. Under these conditions, the BWs of pair-fed PrRP-deficient mice did not differ significantly from those of WT mice (Figure (Figure3C).3C). Glucose tolerance and insulin resistance were also not significantly different in WT and pair-fed PrRP-deficient mice (Figure (Figure3,3, D and E).

We found no significant differences between WT and PrRP-deficient mice in locomotor activity in their home cages (Figure (Figure4A)4A) and no significant differences in oxygen consumption (VO2) during 12-hour dark, 12-hour light, and 24-hour periods for mice at the ages of 9, 16, and 51 weeks (Figure (Figure4B).4B). The mean respiratory exchange ratios were also not significantly different in WT and PrRP-deficient mice at the age of 9 (WT, 0.92 ± 0.01, PrRP-deficient, 0.92 ± 0.01), 16 (WT, 0.91 ± 0.01, PrRP-deficient, 0.88 ± 0.01), or 51 weeks (WT, 0.81 ± 0.02, PrRP-deficient, 0.84 ± 0.01). In pair-fed PrRP-deficient mice at the age of 52–54 weeks, neither the total VO2 nor the mean respiratory exchange ratios were significantly different from those in WT mice (Table (Table1). 1).

Figure 4
Energy consumption of PrRP-deficient mice.
Table 1
VO2 and respiratory exchange ratios in pair-fed PrRP-deficient and WT mice

We then examined VO2 in animals whose endogenous PrRP functions were acutely blocked by neutralizing PrRP mAb. We found no significant differences between control animals and animals pretreated with anti-PrRP mAb (Figure (Figure4C). 4C).

Uncoupling proteins are involved in the regulation of thermogenesis and energy expenditure through the sympathetic nervous system. Therefore, we examined body temperature and uncoupling protein 1 (UCP1) mRNA expression in the BAT after exposure to cold. There was no significant difference in core body temperature between WT and PrRP-deficient mice either before (37.5 ± 0.1°C and 37.5 ± 0.1°C, respectively) or after (37.3 ± 0.1 and 37.2°C ± 0.2°C, respectively) exposure to cold. Expression of UCP1 mRNA in the BAT was increased after exposure to cold, but with no significant difference between WT and PrRP-deficient mice (Figure (Figure4D). 4D).

Collectively, these data indicate that the obesity in PrRP-deficient mice mainly reflects hyperphagia rather than lower energy consumption.

Activation of PrRP neurons by food intake.

We examined the expression of phos­phorylated cAMP response element–binding protein (p-CREB) and Fos protein in PrRP neurons after food intake as a marker of neuronal activation. Refeeding following a 24-hour fast increased the percentage of p-CREB–positive PrRP neurons in the NTS (Figure (Figure5A).5A). The number of Fos-positive PrRP neurons in the NTS also significantly increased after refeeding (Figure (Figure5B).5B). By contrast, there were no significant increases in the percentages of p-CREB–positive PrRP neurons either in the ventrolateral medulla oblongata (89.6% ± 3.6% [control] and 90.1% ± 2.1% [refeeding]) or in the dorsomedial hypothalamus following refeeding (3.0% ± 1.5% [control] and 2.4% ± 1.1% [refeeding]). The number of Fos-positive PrRP neurons was increased slightly in the ventrolateral medulla oblongata (0 ± 0 cells/rat [control] and 2.8 ± 1.2 cells/rat [refeeding]; P < 0.05) but not in the dorsomedial hypothalamus following refeeding (1.0 ± 1.0 cells/rat [control] and 5.0 ± 2.0 cells/rat [refeeding]).

Figure 5
Role of endogenous PrRP in food intake.

Food intake after impairment of PrRP signaling.

Up to the age of 14 weeks, the daily food intake of PrRP-deficient mice was not significantly different from that of WT mice. However, even at this young age when no obesity was evident, PrRP-deficient mice ate significantly more after a 24-hour fast than WT mice (Figure (Figure5C),5C), suggesting that PrRP is involved in food intake when food intake is facilitated by fasting. At the age of 24 weeks, PrRP-deficient mice ate more food than WT mice (Figure (Figure5D). 5D).

We then examined food intake in rats whose endogenous PrRP functions were acutely blocked by neutralizing PrRP mAb. Rats pretreated with anti-PrRP mAb ate more during the dark period than control rats, whether anti-PrRP mAb was injected immediately before the dark period or during the light period (Figure (Figure5,5, E and F). Rats injected intracerebroventricularly (i.c.v.) with anti-PrRP mAb ate more after a 24-hour fast as measured either during the dark or the light period (Figure (Figure5,5, G and H). Thus, immunoneutralization of central PrRP facilitates food intake whether meal initiation is triggered by the onset of the dark period or by fasting. Total daily food intake can be considered as the product of meal size and meal frequency. We investigated meal pattern in PrRP-deficient mice (Figure (Figure5I)5I) and in rats injected with anti-PrRP mAb (Figure (Figure5J).5J). The meal size, but not the meal frequency, was higher in PrRP-deficient mice than in WT mice and in rats was increased after i.c.v. injection of anti-PrRP mAb.

PrRP neurons are activated after systemic injections of the peripheral satiety signal CCK (11). We therefore measured the effects of systemic administration of CCK on food intake in mice that had been fasted for 24 hours. CCK had less effect on food intake in PrRP-deficient mice than in WT mice (Figure (Figure5K),5K), suggesting that PrRP mediates a satiety signaling of CCK.

To investigate whether PrRP-deficient mice have enhanced fat preference, mice were tested on 2-choice macronutrient diet; they were given free access to both high-fat and high-carbohydrate diets or to both tallow and standard diet. There was no significant difference between WT and PrRP-deficient mice in the intake ratio of high-fat food (Figure (Figure5L)5L) or tallow (Figure (Figure5M),5M), suggesting that PrRP-deficient mice had no enhanced preference for fat meals.

Food intake–related peptides in the hypothalamus.

We examined the expression of appetite-regulating peptides in the hypothalamus using in situ hybridization histochemistry. In obese PrRP-deficient mice fed a high-fat diet, the expression of mRNA for the anorexigenic peptide corticotrophin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus was not different as compared with that in WT mice (Table (Table2).2). In nonobese PrRP-deficient mice fed standard laboratory chow at the age of 16 weeks, the CRH mRNA expression level was slightly decreased (Table (Table2),2), but the difference was not statistically significant (P = 0.055). We also measured the mRNA expression levels of 2 anorexigenic peptides, proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) and 2 orexigenic peptides, agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus and measured orexin mRNA expression in the lateral hypothalamus; there were no significant differences in any of these expression levels between PrRP-deficient mice and WT mice fed on either standard chow or high-fat diet (Table (Table2). 2).

Table 2
Expression of appetite-related peptides in the hypothalamus

Leptin effects on BW and food intake in PrRP-deficient mice.

An abnormality in satiety signaling alone does not necessarily cause obesity (23), unless it affects fundamental homeostatic mechanisms. As leptin is particularly important for long-term metabolic homeostasis, we examined whether leptin administration activates PrRP neurons. Leptin induced expression of p-STAT3 in PrRP-expressing neurons of the NTS and dorsomedial hypothalamus (Figure (Figure6A).6A). We then examined effects of leptin administration in PrRP-deficient mice. Peripheral or central administration of leptin reduced food intake and BW in WT mice. These effects were much impaired in PrRP-deficient mice (Figure (Figure6,6, B–E), suggesting that PrRP is important for relaying leptin signaling to reduce food intake and BW.

Figure 6
Changes in BW and food intake after leptin administration.

Discussion

PrRP is expressed in subpopulations of neurons in the NTS, the ventrolateral medulla, and the dorsomedial hypothalamus and has been implicated in several physiological processes (24, 25), including satiety signaling (1013, 22), stress-induced release of ACTH and oxytocin (16, 19, 26, 27), and nociception (20). In the present study, most of the PrRP neurons in the NTS were activated after initiation of food intake, with no significant activation of PrRP neurons in the hypothalamus. Perturbation of PrRP signaling either by gene KO or by administration of neutralizing PrRP mAb increased amounts of food intake per meal but not meal frequencies. These data suggest that PrRP release from NTS neurons is a central satiety signal.

PrRP-deficient mice showed adult-onset hyperphagia and obesity and exhibited an accompanying impaired glucose tolerance and insulin resistance. These data are consistent with reports that mice deficient in GPR10, the PrRP receptor, become obese in adulthood (15, 21). Although PrRP administration has been reported previously to increase energy consumption (13), VO2 was not significantly changed in PrRP-deficient mice in the present study. Neither body temperature changes during exposure to a cold environment nor cold exposure–induced increase in UCP1 mRNA were different in WT and PrRP-deficient mice. Locomotor activity was also unchanged in PrRP-deficient mice. Furthermore, pair feeding abolished the induction of obesity in PrRP-deficient mice. These data suggest that PrRP-deficient mice gain more weight than WT mice as a result of the increased food intake rather than reduced energy expenditure.

In the present study, blocking PrRP signaling in rats by administration of anti-PrRP mAb facilitated food intake only when food intake was initiated by the onset of the dark period or by fasting, suggesting that blocking PrRP actions does not itself initiate feeding but instead that feeding continues for longer once initiated. This suggests that PrRP might be involved in satiety signaling — a conclusion supported by the observation that CCK-induced reduction in food intake was impaired in PrRP-deficient mice. These findings are consistent with reports that GPR10-deficient mice have impaired responses to CCK (22). As peripheral administration of CCK activates most of the PrRP neurons in the medulla oblongata (11), these data suggest that PrRP/GPR10 systems mediate the satiety-inducing effects of peripheral CCK. We also found that PrRP-deficient mice showed an increase in meal size rather than meal frequency, similar to observations in CCKA receptor–deficient mice (23), indicating that CCK/PrRP signaling is important for determining meal size.

PrRP is localized in the A2 noradrenergic neurons of the NTS, in the A1 noradrenergic neurons of the ventrolateral medulla, and in neurons in the dorsomedial hypothalamus (6, 8, 9). Meal consumption activates neurons in the NTS (28, 29), including in particular noradrenergic neurons (30). In part, this reflects activation of satiety pathways arising from the gastric vagus, which are responsive to CCK. Thus, peripheral administration of CCK activates noradrenergic neurons in the NTS (5), and these neurons are critical for CCK signaling (31, 32). Here we show that food intake rapidly activates PrRP neurons in the NTS. The data suggest that PrRP neurons in the NTS mediate satiety signaling and support the view that satiety circuits are activated coincidentally with food intake (30). At present, the precise mechanisms by which PrRP reduces food intake remain to be clarified. PrRP may convey satiety signals to the hypothalamus (22), but microinjection of PrRP into the dorsal motor nucleus of the vagus also modulates gastric motor function (33). It is thus also possible that PrRP may inhibit food intake via an action on gastric emptying.

Mice deficient in CCKA receptors do not show obesity (23), so the defects in CCKA receptor signaling in PrRP-deficient or GPR10-deficient mice might not fully explain the obesity observed in PrRP-deficient mice. Leptin administration induces nuclear translocation of STAT3 in the dorsomedial hypothalamus (34) and p-STAT3 expression in the NTS (35), where PrRP neurons are localized. PrRP neurons express leptin receptors (25), and leptin administration increases PrRP mRNA (36), suggesting that the activity of PrRP neurons is under the control of leptin. Here we showed that leptin induces p-STAT3 expression in PrRP neurons, especially in the dorsomedial hypothalamus, and that the leptin-induced reduction in food intake and BW is impaired in PrRP-deficient mice. These data suggest that PrRP neurons, especially those in the dorsomedial hypothalamus, might be an important target of leptin’s actions to restrain food intake or that leptin modulates the effects of PrRP by its actions on the neuronal targets of PrRP.

GPR10-deficient mice show reduced energy expenditure (15), and as acute administration of PrRP increases VO2 (13), these data suggest that PrRP plays a role in energy expenditure. However, in the present study, VO2 was not different in WT and PrRP-deficient mice, and acute blockade of PrRP signaling by anti-PrRP mAb did not increase VO2. Furthermore, the obesity observed in PrRP-deficient mice could be fully reversed by pair feeding. These data suggest that endogenous PrRP does not play an important role in energy expenditure in basal conditions. The apparent discrepancies concerning energy expenditure between GPR10-deficient and PrRP-deficient mice remain to be clarified.

Energy homeostasis is regulated by various orexigenic and anorexigenic peptides in the hypothalamus. Administration of PrRP activates CRH neurons in the hypothalamus (37), and PrRP-induced anorexia is attenuated by a CRH receptor antagonist (13). However, in the present study, there was no significant decrease in CRH mRNA expression in the paraventricular nucleus, suggesting that changes in CRH neuronal activity may not be the main cause of obesity in PrRP-deficient mice. We also found that the expression levels of mRNAs encoding appetite-regulating peptides, AgRP, NPY, orexin, CART, and POMC, were normal in PrRP-deficient mice, indicating that the hyperphagia observed in PrRP-deficient mice is unlikely to be the result of a grossly perturbed functioning of these hypothalamic neuronal populations.

Both PrRP-deficient mice and GPR10-deficient mice show adult-onset obesity (15, 21), but there are several differences. Hyperphagia is not obvious in GPR10-deficient mice (22) but is clear in PrRP-deficient mice, and a high-fat diet can induce obesity in PrRP-deficient but not in GPR10-deficient mice (21). It is possible that PrRP acts on not only GPR10 but also other receptors, including neuropeptide FF receptor 2 (38). Administration of PrRP reduces food intake in rats with a naturally occurring polymorphism of the Prlhr gene that abolishes its ability to bind PrRP (39), suggesting that receptors other than GPR10 also play a role in the action of PrRP on feeding behavior. It is also possible that ligands other than PrRP might exist for GPR10. In conclusion, this study indicates that PrRP is a potent satiety peptide in the rodent CNS that contributes to the determination of meal size and that perturbation of PrRP signaling can predispose to obesity, mainly due to hyperphagia.

Methods

Generation of PrRP-deficient mice.

To construct the targeting vector, a Prlh genomic clone was isolated from a 129/Svj-derived RW-4 ES cell genomic library. The Prlh gene targeting vector was designed to replace a 2.0-kb BamHI-HpaI fragment including exons 1 and 2 constituting the entire coding region, with a neomycin phosphotransferase resistance cassette (neo). A 2.1-kb VspI-BamHI fragment was used as the 5′ homology region and a 5.0-kb HpaI-EcoRI fragment was used as the 3′ homology region. We linearized the targeting vector and electroporated it into RW-4 ES cells (Genome Systems Inc.). We selected G418-resistant clones and screened them by Southern blot analysis. Heterozygous ES cell clones were injected into blastocysts of C57BL/6N mice (Charles River Laboratories) and transferred into uteri of pseudopregnant females. Chimeric males were mated to C57BL/6N females, and the resulting heterozygous mice were intercrossed. Their offspring were screened by Southern blot analysis. PrRP-deficient mice backcrossed into C57BL/6N background for 8 generations were used in this study. Permission of Takeda Chemical Industries Ltd. is necessary for the distribution of PrRP-deficient mice.

Animals.

Animals were housed in a temperature- (22 ± 2°C) and humidity-controlled (40%–70%) room with a 12-hour light/12-hour dark cycle (light on 7:30–19:30). Food and water were available ad libitum. All animal procedures were approved by the Judging Committee of Experimental Animal Ethics of Jichi Medical University and were in accordance with Japanese legislation concerning animal experiments.

Intraperitoneal glucose tolerance test and insulin tolerance test.

For the glucose tolerance test, male WT and PrRP-deficient mice (39–40 weeks old) were fasted for 14 hours (23:00–13:00) and injected i.p. with glucose (2 g/kg BW). For the insulin tolerance test, male mice (40–41 weeks old) were fasted for 4 hours (9:00–13:00) and then injected i.p. with insulin (1 U/kg BW; Novolin; Novo Nordisk). For both tests, blood samples were taken from the tail vein at 0, 15, 30, 60, and 120 minutes after loading. Blood glucose concentrations were determined by an enzymatic method using a GLUCOCARD DIA meter (ARKRAY).

High-fat diet.

Male WT mice or PrRP-deficient mice were given either standard laboratory chow (CE-2; CLEA Japan) or a high-fat diet (Quick Fat; CLEA Japan) from the age of 5 weeks or for 4 weeks from the age of 12 weeks. The standard laboratory chow contains 12% fat, 29% protein, and 59% carbohydrates, with a caloric density of 3.45 kcal/g. The high-fat diet has 31% fat, 24% protein, and 45% carbohydrates, with a caloric density of 4.11 kcal/g.

Pair feeding.

Male WT and PrRP-deficient mice were housed individually from 6 weeks of age, provided with food ad libitum, and weighed weekly. Pair feeding of standard laboratory chow or high-fat diet was started at the age of 12 weeks.

Surgery and i.c.v. injection of an anti-PrRP mAb.

Male animals were anesthetized with Avertin (200 mg/kg BW, i.p.; tribromoethanol; Sigma-Aldrich) and placed in a stereotaxic frame. Stainless steel guide cannulae (25-gauge [mice] or 23-gauge [rats]) were inserted into the right lateral cerebral ventricle (mice: 0.4 mm caudal to bregma, 1.0 mm lateral to the midline, and 2.0 mm below the skull; rats: 0.6 mm caudal to bregma, 1.6 mm lateral to the midline, and 4.5 mm below the skull) and secured to the skull with screws and dental cement. The position of the tip was verified with methylene blue injected through the cannulae after experiments. Animals were allowed to recover for 1 week. Animals were injected i.c.v. with anti-PrRP mAb (2 μl [mice] or 8 μl [rats]; 4.9 mg/ml P2L-1C; Takeda Pharmaceutical Co.) (8, 19, 26, 37) or control IgG (4.9 mg/ml mouse IgG; Sigma-Aldrich) via inner cannulae (32-gauge [mice] or 30-gauge [rats]). Permission of Takeda Chemical Industries Ltd. is necessary for the distribution of anti-PrRP mAb (P2L-1C).

Locomotor activity and VO2.

Spontaneous motor activity was recorded in the home cage by the activity sensor unit (O’hara and Co.) (40). VO2 and carbon dioxide production (VCO2) were measured simultaneously for 4 animals using an indirect calorimeter (Oxymax Equal Flow System; Columbus Instruments) with an airflow of 0.8 l/min (mice) or 2 l/min (rats). Animals were placed in individual calorimeter chambers. After a 2-hour acclimation period, VO2 (ml/kg BW0.75 per hour) was measured every 5 minutes for 24 hours. In the experiments with WT and PrRP-deficient mice, the measurements were conducted in male mice with free access to food (9, 16, and 51 weeks old) or under pair-fed conditions (52–54 weeks old). In the experiments with an i.c.v. injection, anti-PrRP mAb or mouse IgG was injected i.c.v. into male mice (10–11 weeks old; C57BL/6N; Charles River Laboratories) or male rats (12 weeks old, std:Wistar; SLC Japan) after a 1-hour baseline measurement of VO2, and the measurements were conducted in animals with no access to food. The VO2 values from 12 consecutive readings, encompassing a 1-hour period, were averaged for analysis. The respiratory exchange ratio was calculated as VCO2/VO2.

Exposure to a cold environment.

Male WT or PrRP-deficient mice (24 weeks old) were placed in an incubator at 2°C for 6 hours. Body temperature was measured with a rectal temperature probe (model BDT-100; BRC). For measurements of Ucp1 mRNA, mice were deeply anesthetized i.p. with pentobarbital (50 mg/kg BW; Nembutal; Dainippon Pharmaceutical) 24 hours after the cold exposure, and the BAT was obtained. The total RNA of the BAT was isolated using TRIzol (Invitrogen) and treated with RQ1-DNase (Promega) to remove residual contamination with DNA. First-strand cDNA synthesis was completed using ReverTra Ace (Toyobo). Primers for quantitative RT-PCR were first examined by HotStarTaq DNA polymerase (94°C for 15 seconds, 60°C for 20 seconds, and 72°C for 20 seconds × 30 cycles; QIAGEN) and agarose gel electrophoresis for correct product size and absence of primer-dimer formation. Using a QuantiTect SYBR Green PCR kit (QIAGEN), quantitative RT-PCRs (95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 20 seconds × 40 cycles) were performed in an ABI-Prism 7700 sequence detector (Applied Biosystems). Product accumulation was measured in real time, and the mean Ct was determined for replicate samples on the same plate. Different cDNA samples were normalized using primer sets to Gapdh. Primers for Gapdh were 5′-GGCACAGTCAAGGCTGAGAATG-3′ and 5′-ATGGTGGTGAAGACGCCAGTA-3′; those for Ucp1 were 5′-CGACAACTTCCGAAGTGCAAC-3′ and 5′-TGGATCTGAAGGCGGACTTT-3′.

Detection of activated PrRP neurons after food intake.

To detect p-CREB in PrRP neurons, male rats (9 weeks old, std:Wistar; SLC Japan) were anesthetized with Avertin, and polyethylene catheters (PE50; BD) were inserted into the right jugular vein. Seven days later, the rats were fasted for 24 hours (19:30–19:30). One group of rats was refed for 30 minutes after the fast, anesthetized with pentobarbital (60 mg/kg BW, i.v.), and perfused transcardially with heparinized saline (20 U/ml), followed by 3.75% acrolein and 2% PFA in 0.1 M phosphate buffer (pH 7.4). The brains were removed, postfixed in 3.75% acrolein and 2% PFA overnight, and transferred to 30% sucrose solution in 0.1 M phosphate buffer until tissue sank. The brains were frozen on dry ice and stored at –80°C. Coronal sections were cut at 30 μm with a freezing sledge microtome. Every fourth section of the hypothalamus and the medulla oblongata was collected and processed for immunohistochemistry. Sections were pretreated with 0.5% sodium metaperiodate for 20 minutes and with 1% sodium borohydride for 20 minutes. The sections were incubated for 15 minutes with 1.5% H2O2 solution to block endogenous peroxidase, with 10% normal goat serum for 1 hour, and then with a rabbit polyclonal Ab against p-CREB (diluted 1:200; Ser133; Cell Signaling Technology) for 48 hours at 4°C, followed by incubation with biotinylated goat anti-rabbit IgG (2 μg/ml BA-1000; Vector Laboratories) for 2 hours and then with avidin-biotinylated horseradish peroxidase complex (VECTASTAIN Elite ABC Kit; Vector Laboratories) for 30 minutes at room temperature. p-CREB immunoreactivity was visualized as a black nuclear precipitate using a glucose oxidase–based, nickel-intensified, 3,3′-diaminobenzidine procedure. p-CREB immunolabeled sections were treated with 1.5% H2O2 and 10% normal horse serum and then incubated with a mouse anti-PrRP mAb (5 μg/ml P2L-1T; Takeda Pharmaceutical Co.) for 48 hours at 4°C (6), followed by incubation with biotinylated horse anti-mouse IgG (1 μg/ml BA-2001; Vector Laboratories) and then with avidin-biotinylated horseradish peroxidase complex for 30 minutes at room temperature. PrRP immunoreactivity was visualized as a brown cytoplasmic precipitate using a 3,3′-diaminobenzidine procedure. For detection of Fos protein in PrRP neurons, male rats (9 weeks old, std:Wistar; SLC Japan) were fasted for 24 hours (10:00–10:00). One group of rats was refed for 2 hours after the fast, anesthetized i.p. with pentobarbital, and perfused with acrolein and PFA. Coronal brain sections were cut at 30 μm with a freezing sledge microtome. Every fourth section of the hypothalamus and the medulla oblongata was collected and processed for immunohistochemistry. Following treatments with sodium metaperiodate, sodium borohydride, and H2O2, the sections were incubated with 10% normal goat serum and incubated with a rabbit polyclonal Ab against Fos protein (diluted 1:10,000; Oncogene) for 48 hours at 4°C. The sections were incubated overnight with peroxidase-labeled goat anti-rabbit IgG (1 μg/ml PI-1000; Vector Laboratories) at 4°C. Fos immunoreactivity was visualized as a black nuclear precipitate using a glucose oxidase–based, nickel-intensified, 3,3′-diaminobenzidine procedure. Fos-immunolabeled sections were treated for detection of PrRP immunoreactivity, as described above. The number of PrRP-positive cells that were positive or negative for p-CREB or Fos protein was determined, and the percentage of PrRP-positive cells expressing p-CREB was calculated for each rat. Fourteen sections of the caudal brainstem (between –1,560 μm and 0 μm relative to the obex) were examined in each rat. For the dorsomedial hypothalamus, cells were counted in 3 sections per rat at a 120-μm interval to give a mean value for each rat; these values were averaged to determine a group mean. Permission of Takeda Chemical Industries, Ltd. is necessary for the distribution of anti-PrRP mAb (P2L-1T).

Measurements of food intake after i.c.v. injection of an anti-PrRP mAb.

Seven days after cannula implantation, male rats (10 weeks old, std:Wistar; SLC Japan) were injected i.c.v. with anti-PrRP mAb or control IgG, and cumulative food intake was measured by an automatic food counter (O’hara and Co.). An i.c.v. injection was performed at the beginning of the dark period or 3 hours after the onset of the light period. Half of the rats were fasted for 24 hours until the i.c.v. administration.

Measurements of meal size and meal frequency.

Food intake was recorded every minutes by an automatic food counter (O’hara and Co.). For male WT and PrRP-deficient mice (14 weeks old), meal initiation was defined as more than 50 mg pellets within 10 minutes. Once a meal was initiated, meal termination was defined as the onset of a 10-minute interval with no intake. For male rats (10 weeks old, std:Wistar; SLC Japan), meal initiation was defined as a minimum of 100 mg pellets within 10 minutes.

Administration of CCK.

Male WT and PrRP-deficient mice (19–21 weeks old) were individually housed, deprived of food for 24 hours, and injected i.p. at 11:00 with saline or CCK-octapeptide26–33 (sulfated form) (20 μg/kg BW; Peptide Institute). Cumulative food intake was measured before and after injection.

Two-choice macronutrient diet test.

Male WT and PrRP-deficient mice at the age of 34–37 weeks had free access to both high-fat diet (Quick Fat) and high-carbohydrate diet (63.7% of calories from carbohydrate and 9.6% of calories from fat, 360.7 kcal/100 g; KBT Oriental Co.). Daily food intake was measured for 9 days. Other mice had free access to both standard laboratory chow (CE-2) and tallow. The daily calories from the high-fat diet or tallow were calculated as a ratio of total caloric intake and averaged for the last 2 days.

In situ hybridization.

The brains of mice fed either standard laboratory chow or a high-fat diet for 4 weeks from the age of 12 weeks were analyzed for mRNA levels of food intake–related peptides. Semiquantitative in situ hybridization was performed using antisense probes for AgRP, NPY, orexin, CART, CRH, and POMC mRNAs as described in detail previously (4143). We analyzed the images in an MCID imaging analyzer (Imaging Research Inc.).

Leptin injection and detection of activated PrRP neurons after leptin injection.

Murine recombinant leptin (Peprotech) was dissolved in 5 mM sodium citrate buffer (pH 4) and stored at –80°C. Immediately before use, the pH of the solution was adjusted to 7.0–7.5 by addition of sodium hydroxide. For measurement of BWs and food intake, male WT and PrRP-deficient mice (i.p., 16 weeks old; i.c.v., 18–19 weeks old) were housed individually and injected i.p. at 8:00 and at 18:00 or i.c.v. at 11:00 with vehicle (mixture solution of sodium citrate, pH 4, and 0.01 M sodium hydroxide; final pH, 7.2) or leptin (i.p., 2.5 mg/kg BW; i.c.v., 0.2 μg/2 μl/mouse) for 3 days. For detection of PrRP neurons activated after leptin injection, male mice (11 weeks old, C57BL/6N mice (Charles River Laboratories) were injected i.c.v. with leptin (5 μg/2 μl/mouse) and perfused with acrolein and PFA. Coronal brain sections were cut at 30 μm with a freezing sledge microtome. Every second section of the hypothalamus and the medulla oblongata was collected and processed for immunohistochemistry. After treatments with sodium metaperiodate, sodium borohydride, and H2O2, the sections were blocked in M.O.M Mouse Ig Blocking Reagent (Vector Laboratories), incubated with normal goat serum and then with normal goat serum and P2L-1T for 48 hours at 4°C, followed by treatment with a peroxidase-labeled polymer conjugated to goat anti-mouse immunoglobulins (EnVision+ system; Dako) for 2 hours at room temperature. PrRP immunoreactivity was visualized as a brown cytoplasmic precipitate using a 3,3′-diaminobenzidine procedure. PrRP-immunolabeled sections were treated with H2O2 and normal goat serum and then incubated with a rabbit mAb against p-STAT3 (diluted 1:100; Tyr705; Cell Signaling Technology) for 48 hours at 4°C, followed by treatment with a peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins (EnVision+ system; Dako) for 2 hours at room temperature. p-STAT3 immunoreactivity was visualized as a black nuclear precipitate using a glucose oxidase–based, nickel-intensified, 3,3′-diaminobenzidine procedure. The number of PrRP-positive cells that were positive or negative for p-STAT3 was determined, and the percentage of PrRP-positive cells expressing p-STAT3 was calculated for each mouse. Twenty sections of the caudal brainstem (between –1,140 μm and 0 μm relative to the obex) were examined in each mouse. For the dorsomedial hypothalamus, cells were counted in 11 sections per mouse (60-μm intervals) to give a mean value for each mouse; these values were averaged to determine a group mean.

Statistics.

Changes in food intake, BW, blood glucose concentrations, motor activity, and VO2 were analyzed by repeated-measures ANOVA. Other data were analyzed by 1-way ANOVA followed by Scheffé’s multiple comparison test or 2-tailed t test. A P value of less than 0.05 was considered statistically significant.

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research (C) (18590223, 20590237 to T. Onaka) from the Japan Society for the Promotion of Science; and a Grant-in-Aid for Young Scientists (B) (18790172, 20790194 to Y. Takayanagi) and a Grant-in-Aid for Scientific Research on Priority Areas–System Study on Higher-Order Brain Functions (18020025, 20020023 to T. Onaka) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are grateful to Mitsugu Nakata and Takashi Yano (Takeda Pharmaceutical Co. Ltd.) for cooperation in producing the PrRP-deficient mice.

Footnotes

Nonstandard abbreviations used: AgRP, agouti-related protein; BAT, brown adipose tissue; CART, cocaine- and amphetamine-regulated transcript; CCK, cholecystokinin; CRH, corticotrophin-releasing hormone; i.c.v., intracerebroventricular(ly); NPY, neuropeptide Y; NTS, nucleus tractus solitarii; p-CREB, phosphorylated cAMP response element–binding protein; POMC, proopiomelanocortin; PrRP, prolactin-releasing peptide; UCP1, uncoupling protein 1; VO2, oxygen consumption.

Conflict of interest: Hirokazu Matsumoto, Shoji Fukusumi, and Shuji Hinuma are employees of Takeda Chemical Industries Ltd.

Citation for this article: J. Clin. Invest. 118:4014–4024 (2008). doi:10.1172/JCI34682

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