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Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Ibaraki 300-2611, Japan
Address all correspondence and requests for reprints to: Hisashi Iwaasa, Ph.D., Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan. E-mail: hisashi_iwaasa{at}merck.com.
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Abstract |
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Introduction |
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A novel 43-amino acid RFamide was recently discovered using a bioinformatics approach and was identified as a ligand of the G protein-coupled receptor GPR103 (2, 3). This RFamide was designated QRFP (also known as P518/26RFa), because it begins from N-terminal pyroglutamic acid and ends at the C-terminal arginine-phenylalanine-amide peptide. The cDNA encoding QRFP precursor has been cloned in human, bovine, rat, mouse, and frog (2, 3, 4). In mammals, mRNAs of prepro-QRFP and its receptor (GPR103) are abundantly expressed in the brain, particularly in the hypothalamus (2, 3, 4). In addition, a recent study demonstrated that intracerebroventricular (ICV) administration of QRFP26/P518/26RFa, a constitutive part of QRFP43 (43-amino acid-residue form of QRFP), markedly increases food intake in food-restricted mice over a subsequent 2-h period (4). These results indicate that QRFP may play a role in the central regulation of feeding behavior. However, it remains uncertain whether QRFP is involved specifically in the short-term, immediate regulation of feeding or in the long-term maintenance of energy homeostasis. Here we show that ICV infusion of QRFP43 increases feeding, suppresses energy expenditure, and possibly plays a role in the central regulation of body weight and energy homeostasis.
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Materials and Methods |
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ICV cannulation and injection
Mice were anesthetized with sodium pentobarbital (50 mg/kg, ip, Dinabot, Tokyo, Japan), and a sterile 27-gauge guide cannula was stereotaxically implanted into the right lateral ventricle. The stereotaxic coordinates used were 0.4 mm posterior to the bregma, 0.8 mm lateral to the midline, and 1.0 mm from the surface of the skull using a flat skull position. The cannula was attached to the skull with dental cement, and an antibiotic (Cefamedin 
, 50 mg/kg, Fujisawa Pharmaceutical, Tokyo, Japan) was injected sc. Mice were left for at least 1 wk to recover from the surgical procedure. After this period, mice were given a further week of daily handling to allow acclimation to the experimental procedure and to minimize stress. A positive dipsogenic response to ICV injection of 1 µg/head neuropeptide Y verified cannula placement. Test substances were dissolved in 30% propylene glycol and were injected in a volume of 2 µl using a Hamilton syringe (Hamilton Reno, Reno, NV) attached to polyethylene tubing (inner diameter 0.2 mm, outer diameter 0.5 mm) and a 30-gauge stainless steel injection cannula (Hamilton Reno) extending 1 mm beyond the guide cannula.
Chronic ICV infusion
Mice were anesthetized with sodium pentobarbital (80 mg/kg, ip, Dinabot Tokyo, Japan), and a sterile brain infusion cannula (28 gauge, Alzet; Durect, Cupertino, CA) was stereotaxically implanted into the right lateral ventricle. The stereotaxic coordinates used were 0.4 mm posterior to the bregma, 0.8 mm lateral to the midline, and 2.0 mm from the surface of the skull using a flat skull position. The cannula was attached to the skull with dental cement. The infusion cannula was connected via polyvinylchloride tubing to an osmotic minipump (model 2004; Durect) filled with 10 mM PBS containing 0.05% BSA (PBS). The pump was implanted under the skin of the back, and antibiotic (Cefamedin , 50 mg/kg) was injected sc. Mice were then allowed to recover for at least 2 wk. At the start of the experiment, the infusion pump was replaced with a new pump filled with test substances or PBS.
In vivo protocols
Experiment 1: acute ICV injection of QRFP43 in satiated mice.
Mice (18 wk old; 27.0 g) were randomly divided into five groups (n = 11–13). One day before the experiment, the food was changed to a moderately high-fat (MHF) diet (Oriental Bio Service Kanto, Ibaraki, Japan) to satiate the mice. The MHF diet provides energy as follows: 52.4% as carbohydrate; 15.0% as protein; and 32.6% as fat (4.41 kcal/g). During the early light phase of the experimental day, mice were injected ICV with QRFP43 at 0, 1, 3, 10, or 30 µg/head over a 30-sec period. After ICV treatment, food intake was measured for 2 h.
Experiment 2: chronic ICV infusion of QRFP43 in mice.
Mice (15 wk old; 27.5 g) were divided into six groups matched for average body weight and food intake. This study was performed under two different diet conditions; regular diet and MHF diet. Two groups were fed a regular diet and infused with QRFP43 (30 µg/d; n = 7) or vehicle (PBS; n = 6). In four other groups, the diet was changed to MHF diet at the start of the experiment, and the infusion pump was replaced with a new pump filled with different doses of QRFP43 (3 µg/d, n = 9; 10 µg/d, n = 11; 30 µg/d, n = 11) or vehicle (PBS; n = 9). Food intake and body weight were measured daily. After 2 wk of ICV infusion, mice were fasted for 2 h (0700–0900 h), and blood samples were collected from the infraorbital vein for measurement of plasma glucose, insulin, and leptin levels. Body composition was determined using an nuclear magnetic resonance (NMR) analyzer (Minispec; Bruker Optics, Billerica, MA). Under isoflurane anesthesia, mice were then killed by collecting whole blood from the heart. The mesenteric and epididymal adipose tissues and liver were excised and weighed.
Experiment 3: pair-feeding study.
Two weeks before the start of infusion, the diet was changed to MHF diet from regular diet. Mice (18 wk old; 30.0 g) were then divided into three groups matched for average food intake, body weight, and diet-induced body weight gain (n = 14–15 per group): 1) vehicle group, which received PBS infusion and were allowed ad libitum access to food; 2) QRFP43 ad libitum-fed (ad lib-fed) group, which received QRFP43 infusion (10 µg/d) and were allowed ad libitum access to food; 3) QRFP43 pair-fed group, which received QRFP43 infusion (10 µg/d) and were provided with the same amount of food as the vehicle group. The infusion pump was replaced with a new pump filled with QRFP43 (10 µg/d) or vehicle (PBS) under isoflurane anesthesia. Daily food intake and body weight were measured for 13 d. At the start of infusion and the on the 12th day after infusion, rectal temperatures were measured. After 13 d of ICV infusion, mice were fasted for 2 h (0700–0900 h), and blood samples were collected from the infraorbital vein for measurement of plasma glucose, insulin, and leptin levels. Body composition was determined using an NMR analyzer (Minispec). Under isoflurane anesthesia, mice were then killed by collecting whole blood from the heart. The mesenteric and brown adipose tissues were excised and weighed.
Experiment 4: measurement of motor activity.
Another set of QRFP43- or vehicle-infused mice (15 wk old; 27.6 g) was prepared for measurement of spontaneous motor activity. QRFP43 (30 µg/d; n = 5) or vehicle (n = 10) was infused for 10 d under regular-diet conditions. On the eighth day after the start of administration, mice were placed into new home cages for 17–18 h for acclimation. Motor activity was measured after 2 d using an activity monitoring system (NS-AS01; Neuroscience, Tokyo, Japan). Briefly, the activity monitor comprised an infrared ray sensor placed over the home cage (21 x 32 x 12.5 cm), a signal amplification circuit, and a control unit. The sensor detected the movement of animals based on the infrared rays associated with their body temperature (5, 6). Data for motor activity were collected at 10-min intervals and analyzed using a computer-associated analyzing system (AB System-24A; Neuroscience).
Measurement of rectal temperature
Rectal temperatures of mice were measured during the early light cycle using a digital thermometer (BAT-12; Physitemp Instruments, Clifton, NJ). The rectal probe (IT-14; Physitemp Instruments) was inserted into a depth of 1.5 cm. Mice were gently wrapped with paper towel and lightly held during insertion. To reduce methodological stress, mice were sufficiently acclimated to the procedure before the test period. All measurements were recorded when rectal temperature reading reached a plateau.
Measurement of plasma and liver parameters
Plasma glucose, triglyceride (TG) and total cholesterol levels were measured using Determiner GL-E, L TGII, L TCII (Kyowa Medex, Tokyo, Japan), respectively. Plasma free fatty acids (FFAs) were determined using NEFA-HA Test Wako (II) (Wako Pure Chemical Industries; Osaka, Japan). Insulin and leptin levels were measured by ELISA (Morinaga, Yokohama, Japan). Hepatic TG contents were measured as described previously (7).
Measurement of uncoupling protein-1 (UCP1) mRNA expression
TaqMan assay using an ABI Prism 7900HT sequence detector (Applied Biosystems, Foster City, CA) was used to determine the level of UCP1 mRNA in brown adipose tissue (BAT). The reaction mixture (10 µl) contained cDNA or non-reverse-transcribed RNA as a negative control (equivalent to 5 ng total RNA). Amounts of UCP1 cDNA were standardized against ß-actin. The relative quantities of transcripts were calculated from the means of quadruplicate reactions using a standard curve method. Detailed conditions, including the sequences of primers and fluorogenic probes were as described previously (7).
Statistical analysis
Data are expressed as means ± SE. Statistical analysis was performed between the vehicle and QRFP43 treatment groups under each diet condition. Data were analyzed by Student’s t test or one-way ANOVA followed by Dunnett’s test. For analysis of body weight changes, repeated-measure one-way ANOVA was performed. P < 0.05 was considered significant.
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Results |
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. ICV injection of QRFP43 increased food intake by 200, 338, 377, and 492% at 1, 3, 10, and 30 µg, respectively, relative to controls. QRFP43-injected mice showed no observable abnormal behaviors, such as sedation or seizure (data not shown).
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). QRFP43 produced a significant increase in daily food intake during the first few days, but intake gradually returned to control levels (Fig. 2B). QRFP43 increased 13-d cumulative food intake by 108% relative to controls, but this change was not significant (Fig. 2C).
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). The MHF diet caused transient hyperphagia. In controls, hyperphagia was observed only on the first and second days, and daily food intake largely returned to basal levels on the third day. After the third day, QRFP43-treated mice showed a significant increase in daily food intake when compared with controls (Fig. 3B). QRFP43 increased cumulative food intake by 113, 121, and 121% at 3, 10, and 30 µg/d, respectively, relative to controls (Fig. 3C). The maximal effects on food intake and body weight gain occurred at 10 µg/d, and higher doses (30 µg/d) did not produce any further increases in food intake or body weight gain.
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and 3D and Table 1). In addition, visceral fat (epididymal and mesenteric fat) also increased in the QRFP43 treatment group (Table 1). QRFP43-treated mice tended to show increases in lean mass but the change was not significant (data not shown). QRFP43 at 30 µg/d produced significant increases in liver weight and TG content in MHF diet-fed mice. On the other hand, under regular-diet conditions, QRFP43 tended to increase these parameters but not significantly (Table 1).
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shows the changes in blood chemistry of vehicle- or QRFP43-treated mice after 2 h of fasting in the early light cycle. Under regular-diet conditions, plasma glucose and leptin, but not insulin, were elevated by QRFP43 infusion. Under MHF-diet conditions, QRFP43 increased plasma glucose, insulin, and leptin levels in a dose-dependent manner. Plasma TG levels were elevated by QRFP43 infusion only under regular-diet conditions. QRFP43 increased total cholesterol levels under both diet conditions in a dose-dependent manner.
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). However, NMR analysis showed that the fat content of QRFP43/ad lib-fed and QRFP43/pair-fed were significantly greater than that of controls (Fig. 4C). Mesenteric fat weights were also higher in both QRFP43 treatment groups when compared with controls (vehicle, 0.23 ± 0.02; QRFP43/pair-fed, 0.38 ± 0.03; QRFP43/ad lib-fed, 0.59 ± 0.04 g). In contrast, the lean mass of the QRFP43/pairfed group was slightly reduced when compared with controls, although this change was not statistically significant (vehicle, 22.2 ± 0.3; QRFP43/pair-fed, 21.3 ± 0.3; QRFP43/ad lib-fed, 23.5 ± 0.4 g).
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. After 2 wk of treatment, the rectal temperatures of QRFP43/ad lib-fed and QRFP43/pair-fed mice were significantly lower than those of controls (vehicle, 36.50 ± 0.10; QRFP43/ad lib-fed, 36.10 ± 0.11; QRFP43/pair-fed, 35.89 ± 0.08). In addition, UCP1 mRNA expression levels in BAT significantly decreased in both the QRFP43/ad lib-fed and QRFP43/pair-fed groups when compared with controls at the end of treatment (Fig. 5B).
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Discussion |
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Acute central administration of QRFP43 stimulated food intake in MHF diet-fed mice. The in vitro potency of QRFP43 for GPR103 is comparable with that of QRFP26 (2, 3). QRFP26 is also reported to induce hyperphagia in partially food-deprived mice (4), which agrees with the present observations. Chronic ICV administration of QRFP43 for 13 d also increased food intake under both regular-diet and MHF-diet conditions. Under MHF-diet conditions, QRFP43-induced hyperphagia continued throughout the test period, suggesting that QRFP43 influences both short-term feeding behavior and long-term energy homeostasis.
Chronic QRFP43 administration significantly increased body weight and fat mass, and the QRFP43-infused mice showed typical obese phenotypes, i.e. increases in plasma glucose, insulin, cholesterol, liver weight, and liver triglyceride content. In addition, QRFP43-induced obesity was accelerated in MHF diet-fed mice relative to regular diet-fed mice. These results suggest that QRFP43 contributes more to regulation of energy homeostasis under high-energy diet conditions and plays a pivotal role in the development of obesity. On the other hand, more pronounced orexigenic effects were observed in QRFP43-treated mice on MHF diet, which is a highly palatable diet rich in fat and sucrose. This fact raises the possibility that QRFP43 influences food preference or reward system. To clarify this possibility, further investigation is required.
To clarify whether QRFP43 is able to induce metabolic changes, we performed a pair-feeding study. Although the QRFP43-treated/pair-fed group, which was allowed access to the amount of food at which vehicle-treated mice did not gain body weight, they still exhibited increases in fat mass and plasma levels of leptin and insulin. These results demonstrate that QRFP43 is able to cause metabolic changes independently of increased food intake.
Increased plasma glucose levels by QRFP43 treatment were completely reversed by pair feeding, and thus, hyperglycemia was primarily caused by overfeeding. In contrast with plasma glucose, plasma insulin levels were still elevated in the QRFP43/pair-fed group when compared with the vehicle-treated group. This suggests that QRFP43 could stimulate the secretion of insulin, which is known to be an important hormonal factor in increasing adiposity, via altering vagal activity, and that increased plasma insulin plays an important role in the development of QRFP43-mediated obesity. The question of whether increased plasma insulin in the QRFP43/pair-fed group is due to the increased insulin secretion by QRFP43 through vagal nerve or simply due to the increased insulin resistance by QRFP43-induced adiposity needs to be addressed in future studies. Chronic ICV administration of QRFP43 also increased plasma cholesterol levels in both regular and MHF diet-fed mice. Plasma cholesterol levels were strongly correlated with plasma insulin levels in the present study (r = 0.83, P < 0.001). This suggests that the increased plasma cholesterol levels observed in QRFP43-treated mice are due to increased plasma insulin, which is known to increase the rate of cholesterol biosynthesis.
Insulin also increases the rate of TG biosynthesis. However, QRFP43 increased plasma TG levels under regular-diet conditions but not MHF-diet conditions. It has been reported that the influx of dietary fatty acid inhibits de novo lipogenesis in the liver (8). In the QRFP43/ad lib-fed group, fatty acid derived from increased fat intake may counter the stimulating effects of QRFP43 on plasma TG levels. In agreement with this hypothesis, QRFP43/pair-fed animals showed increased plasma TG levels.
Chronic treatment with QRFP43 caused reductions in rectal temperature in both ad lib-fed and pair-fed animals, suggesting that energy expenditure decreased independently from stimulation of feeding behavior. To support this, QRFP43 infusion also decreased mRNA expression of BAT UCP1, which has an important role in rodent thermogenesis (9, 10), under both feeding conditions. Gene expression of BAT UCP1 is under control of the sympathetic nervous system. In rats, GPR103 is strongly expressed in neurons within the ventromedial hypothalamic nucleus (2), which is known to play an important role in regulating thermogenesis via sympathetic nerves (11, 12). Therefore, QRFP43 may have decreased UCP1 mRNA expression via sympathetic nerve regulation and reduced energy expenditure. To clarify whether QRFP43 really reduces energy expenditure in a whole body, direct measurement of metabolic changes such as oxygen consumption in QRFP43-treated mice remains to be addressed.
In addition to thermogenesis, motor activity is important for regulating energy expenditure. GPR103 is also strongly expressed in the dorsal raphe nucleus and locus coeruleus (2), which are known to be related to sleep and wakefulness (13). To clarify whether QRFP43 modulates activity or circadian rhythms and the involvement of these behavioral changes in QRFP43-induced obesity, we tested the effects of QRFP43 ICV infusion on locomotor activity under regular-diet conditions. QRFP43 did not alter cumulative motor activity during either the light or dark cycle. There were no differences in locomotor circadian rhythms between the QRFP43 treatment group and the vehicle treatment group. We also tested the effect of QRFP43 on locomotor activity under MHF-diet conditions, and no significant change was observed (data not shown). Our results showed that QRFP43-induced obesity is not mediated by changes in motor activity, such as immobility or sedation.
In conclusion, the present study showed that ICV infusion of QRFP43 causes obesity by increasing energy intake and decreasing energy expenditure and suggested that QRFP plays an important role in the central regulation of body weight and energy homeostasis in rodents. Further investigation of the functions of QRFP will help further our understanding of appetite and weight regulation.
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Acknowledgments |
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Footnotes |
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First Published Online March 16, 2006
1 R.M. and H.S. contributed equally to this work. 
Abbreviations: ad lib-fed, ad libitum-fed; BAT, brown adipose tissue; FFA, free fatty acid; ICV, intracerebroventricular; MHF, moderately high-fat; NMR, nuclear magnetic resonance; TG, triglyceride; UCP1, uncoupling protein-1.
Received December 13, 2005.
Accepted for publication March 6, 2006.
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) protein and protein kinase C during the development of sensitization to morphine-induced hyperlocomotion. Neuroscience 111:127–132[CrossRef][Medline]This article has been cited by other articles:
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S. R. Patel, K. G. Murphy, E. L. Thompson, M. Patterson, A. E. Curtis, M. A. Ghatei, and S. R. Bloom Pyroglutamylated RFamide Peptide 43 Stimulates the Hypothalamic-Pituitary-Gonadal Axis via Gonadotropin-Releasing Hormone in Rats Endocrinology, September 1, 2008; 149(9): 4747 - 4754. [Abstract] [Full Text] [PDF]
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 D. A Bechtold and S. M Luckman The role of RFamide peptides in feeding J. Endocrinol., January 1, 2007; 192(1): 3 - 15. [Abstract] [Full Text] [PDF]
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 S. Takayasu, T. Sakurai, S. Iwasaki, H. Teranishi, A. Yamanaka, S. C. Williams, H. Iguchi, Y. I. Kawasawa, Y. Ikeda, I. Sakakibara, et al. A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice PNAS, May 9, 2006; 103(19): 7438 - 7443. [Abstract] [Full Text] [PDF]
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