Peptides 23 (2002) 127–133 Albutensin A and complement C3a decrease food intake in mice Kousaku Ohinataa, Akio Inuib, Akihiro Asakawab, Keiji Wadac, Etsuko Wadac, Masaaki Yoshikawaa,* a Division of Food Bioscience and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan b Second Department of Internal Medicine, Kobe University School of Medicine, 7–5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan c Department of Degenerative Neurological Diseases, National Institute of Neuroscience, NCNP 4 –1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan Received 26 April 2001; accepted 17 August 2001 Abstract Albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg) derived from serum albumin dose-dependently decreased food intake after intracerebroventricular (10 –50 nmol/mouse) or peripheral (0.3–1.0 ␮mol/mouse) administration in fasted conscious ddY mice. Albutensin A delayed gastric emptying and elevated blood glucose levels. Although albutensin A showed low affinity for bombesin receptor, it decreased food intake in bombesin receptor knockout mice, indicating that its inhibitory effect on feeding was not mediated through bombesin receptor. Then, we investigated whether the albutensin A-induced decrease in food intake was mediated by complement C3a and C5a receptors, because albutensin A had affinities for these receptors. Des-Arg-albutensin A, lacking affinity for C3a and C5a receptors, did not inhibit food intake. We found for the first time that centrally administered C3a (10 –100 pmol/mouse) by itself decreased food intake in fasted mice. In contrast, C5a increased food intake after central injection. Based on these results, we conclude that the inhibitory effect of albutensin A on food intake is mediated through the C3a receptor. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Albutensin A; Food intake; Blood glucose; Gastric emptying; Complement C3a; Bombesin 1. Introduction Many bioactive peptides had been found in the enzymatic digests of proteins [13,31,36,38]. Among them, albutensin A was isolated from the tryptic digest of serum albumin as an ileum-contracting peptide in our previous studies [29,30,37]. In the present study, we found that centrally or peripherally administered albutensin A decreased food intake in fasted conscious mice. We also investigated gastric emptying [3,19,32] and blood glucose levels [12,20], which are tightly associated with feeding. Next, we investigated the receptor mediating the inhibitory effect of albutensin A on food intake. Bombesin (BN) receptor was initially examined as a candidate mediating the action of albutensin A. In our previous study, proadrenomedullin N-terminal 20 peptide had 4 homologous residues with BN and showed affinity for BN Abbreviations: i.c.v., intra-third cerebroventricular; i.p., intra-peritoneal. * Corresponding author. Tel.: ⫹1-81-774-38-3725, fax: ⫹1-81-77438-3774. E-mail address: yosikawa@food2.food.kyoto-u.ac.jp (M. Yosikawa). receptor [24], which was suggested to mediate an anorectic action [12]. In the present study, we observed that albutensin A also had 3 homologous residues with BN (Fig. 1) and that albutensin A had affinity for BN receptor. BN is an amidated tetradecapeptide originally isolated from amphibian skin [2]. BN-like peptides such as gastrin-releasing peptide [14], neuromedin B [15] and neuromedin C [16] have been demonstrated to exist throughout the mammalian gastrointestinal and nervous system. These peptides were postulated to play roles in thermoregulation, metabolism and satiety [12]. Then, we investigated whether the suppression of food intake after administration of albutensin A was mediated through BN receptor, using BN receptor knockout mice [8,33]. As other candidates mediating inhibitory effect of albutensin A on food intake, complement C3a or C5a receptors were investigated, because albutensin A showed affinities for both receptors and induced ileum-contraction in guinea pigs through them [30]. However, according to previous reports, it has been thought that C3a and C5a rather have facilitatory actions on food intake after central administration in sated rats [12,27]. C3a (77 amino acids) and C5a (74 amino acids) are generally thought to be important inflam- 0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 1 ) 0 0 5 8 8 - 5 128 K. Ohinata et al. / Peptides 23 (2002) 127–133 2.3. Food intake Fig. 1. Homology of albutensin A with bombesin, complement C3a and C5a. Identical residues are shown in boxes. Non-identical but similar residues are shown in dotted boxes. matory mediators in host defense [22]. These peptides are enzymatically processed from complement C3 and C5 during activation of the complement system [11]. C3a and C5a receptors were recently reported to exist in the central nervous system (CNS) [1,22]. To investigate the involvement of C3a and/or C5a receptors in the albutensin A-induced reduction of feeding, we used des-Arg-albutensin A derivatives lacking affinity for both receptors. We also examined the effects of C3a or C5a by themselves on food intake after central administration in conscious mice. 2. Materials and methods 2.1. Peptides Albutensin A and des-Arg-albutensin A amide were purchased from American Peptide Company (Sunnyvale, CA). Des-Arg-albutensin A was synthesized by the Fmoc method using a PS3 peptide synthesizer (Protein Technologies, USA). The peptide was purified by reverse-phase HPLC on an octadecyl silica column. Complement C3a was from CALBIOCHEM (Darmstadt, Germany). Complement C5a was obtained from Sigma Chemical (St. Louis, MO). Mice were deprived of food for 18 h with free access to water. After i.c.v. or intraperitoneal (i.p.) injection of peptide in 4 ␮l artificial cerebrospinal fluid (ACSF) or in 100 ␮l saline, respectively, the mice had free access to preweighed food pellets. We precisely measured food intake using the unused food pellets and checking the food spillage. Food intake was determined by sequentially weighing the food pellets after i.c.v. or i.p. treatments. 2.4. Gastric emptying Gastric emptying was measured as described previously [3,25,31]. Prior to this experiment, mice were deprived of food for 18 h with free access to water. The fasted mice had free access to preweighed food pellets for 1 h. Food pellets were reweighed to estimate the weight of food intake after 1 h feeding, and then peptide or ACSF were i.c.v. administered. The mice were deprived of food again for 2 h and killed by cervical dislocation 2 h after peptide injection. The stomach of each mouse was immediately exposed by laparotomy, quickly ligated at both pylorus and cardia, and then removed, and its contents weighed. Gastric emptying (%) was calculated according to the following formula: Gastric emptying (%) ⫽ {1- (weight of contents in stomach/weight of food intake)} ⫻ 100. 2.5. Blood glucose Similarly, mice were deprived of food pellets for 18 h. Blood samples were obtained from the orbital sinus under ether anesthesia 15 min after i.c.v. injection of peptide, and blood glucose was determined by the glucose oxidase method [24]. 2.6. Binding assay 2.2. Cannula implantation Male ddY mice (SLC, Shizuoka, Japan) at 7 weeks old were used. Each mouse was individually housed under regulated conditions (22°C on a 12 h light-dark cycle). Food and water were available ad libitum, except as otherwise indicated. All experiments were approved by our university animal committee. Intra-third cerebroventricular (i.c.v.) injection was performed as described previously [21,24,25]. Mice were anesthetized with sodium pentobarbital (80 – 85 mg/kg i.p.) and placed in a stereotaxic instrument. Using a needle, a hole was made through the skull at 0.9 mm posterior to the bregma and 0.9 mm lateral to the suture. A 24-gauge cannula beveled at one end over a distance of 3 mm (SafeletCas, Nipro, Osaka, Japan) was implanted into the third cerebral ventricle. Animals were tested one week or more after implantation. The radioreceptor assay was performed according to the method of Moody et al. [18,24], with slight modification. Adult male Wistar rats (175–200 g) were decapitated and the brains were dissected. The brain was homogenized in 100 vol of 50 mM Tris-HCl buffer (pH 7.4) in ice cold with Biomixer (Nihonseiki Kaisha Ltd.) for 30 sec and centrifuged at 30,000 g for 15 min. The resulting pellet was resuspended in 100 vol of 50 mM Tris-HCl buffer containing 100 mM NaCl (pH7.4), incubated at 4°C for 60 min and centrifuged at 30,000 g for 20 min. The resulting pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.4) at 4°C. 50 pM 125I-[Tyr4]-BN was incubated with the rat membrane at 4°C for 24 min in 50 mM Tris-HCl buffer containing 2 ␮g/ml bacitracin and 0.1% bovine serum albumin (BSA) (W/V) (pH 7.4). Membrane bound 125I-[Tyr4]-BN was separated by filtration through a GF/B filter (Whatman, Maidstone, England). The filter was washed promptly with K. Ohinata et al. / Peptides 23 (2002) 127–133 129 ice-cold 50 mM Tris-HCl buffer containing 0.1% BSA and assayed for radioactivity in a gamma counter. Specific binding was obtained by subtracting nonspecific binding in the presence of excess (100 ␮M) unlabeled BN from total binding. Receptor affinities of each ligands were determined by 50% of specific 125I-BN binding in the competitive inhibition curve. 2.7. Knockout mouse study Three mammalian BN receptors were cloned: neuromedin B receptor [34], gastrin-releasing peptide receptor (GRP-R) [4,28] and bombesin receptor subtype-3 [6,7]. GRP-R is expressed in the gastrointestinal and central nervous system, and mainly mediates BN-induced anorexia in studies using the GRP-R knockout mouse [25,26]. GRP-R deficient mice at 7– 8 months old were subjected to the feeding experiment [33], to confirm whether the inhibitory effect of albutensin A on food intake is mediated through the BN receptor. Food intake was measured after i.p. administration of 1.0 ␮mol/mouse albutensin A in GRP-R deficient mice. 2.8. Statistical analysis Values were expressed as the mean ⫾ SEM. Analysis of variance (ANOVA) followed by Bonferroni’s t test was used to assess differences among groups. P values less than 0.05 were considered significant. 3. Results 3.1. Effects of albutensin A on food intake, blood glucose and gastric emptying Effects of albutensin A on cumulative food intake are shown in Fig. 2. ANOVA indicated significant effects of albutensin A on food intake 20 min [F (4,28) ⫽ 13.433, P ⬍ 0.001] and 60 min [F (4,28) 3.644, P ⬍ 0.05] after central injection and 20 min [F (3,25) 5.181, P ⬍ 0.01] after peripheral injection. The subsequent analysis using Bonferroni’s t test indicated that albutensin A decreased food intake after central (10 –50 nmol/mouse) or peripheral (0.3– 1.0 ␮mol/mouse) administration in fasted conscious ddY mice in a dose-dependent manner. Gastric emptying was decreased after central injection of albutensin A at a dose of 50 nmol/mouse [F (4,27) 9.924, P ⬍ 0.0001] (Fig. 3-A). The blood glucose also increased dose-dependently 15 min after i.c.v. administration of 30 –50 nmol/mouse of albutensin A [F (4,46) 7.729, P ⬍ 0.0001] (Fig. 3-B). In addition, characteristic behaviors of albutensin A were not observed after central and peripheral administration. Fig. 2. Effect of administration of albutensin A on food intake in fasted conscious mice. Male ddY mice were i.c.v. (A) or i.p. (B) injected with albutensin A in 4 ␮l ACSF (artificial cerebrospinal fluid) or in 100 ␮l saline, respectively. Each column represents the mean ⫾ SEM of 5–7 (A) or 7– 8 (B) mice. *P ⬍ 0.05, **P ⬍ 0.01 compared with the ACSF- or saline-treated group by Bonferroni’s t-test. 3.2. Affinities of albutensin A and its derivatives for bombesin receptor As shown in Table 1, albutensin A had affinity for BN receptor (IC50 ⫽ 78 ␮M). The rank order of the affinities of albutensin A-related peptides for BN receptor was des-Argalbutensin A amide ⬎ albutensin A ⬎ des-Arg-albutensin A, indicating that the C-terminal amidation increased the affinity for BN receptor. In addition, albutensin A was reported to show affinities for both receptors for C3a and C5a; the IC50 values were 75 ␮M and 670 ␮M, respectively 130 K. Ohinata et al. / Peptides 23 (2002) 127–133 Table 1 Affinities of albutensin A and its derivatives for bombesin receptor Peptide IC50 (␮M) bombesin albutensin A des-Arg-albutensin A des-Arg-albutensin A amide 0.005 78 360 40 most important role in the regulation of feeding among BN receptor subtypes [21,26,34]. The peripherally administered albutensin A equally decreased food intake in both GRP-R deficient and wild-type mice 20 min after injection [F (3,16) 5.047, P ⬍ 0.05] (Fig. 4), indicating that the inhibitory effect of albutensin A on feeding is not mediated via BN receptor. 3.4. Effect of albutensin A derivatives on food intake The involvement of C3a and/or C5a receptors in the decrease in feeding after administration of albutensin A was also investigated. No suitable C3a antagonist is currently available. To clarify whether the decrease in feeding was mediated through C3a and/or C5a receptors, we measured food intake after central or peripheral injection of des-Argalbutensin A derivatives, lacking affinities for both receptors for C3a and C5a, and compared the relative potencies of these derivatives. Although des-Arg-albutensin A amide showed a more potent affinity for BN receptor than albutensin A, the peptide did not decrease food intake 20 min after i.c.v. or i.p. administration in fasted ddY mice (Fig. 5). These results suggest that albutensin A-induced reduction of feeding is mediated via C3a and/or C5a receptors. Fig. 3. Effect of i.c.v. administration of albutensin A on gastric emptying (A) and blood glucose levels (B) in mice. Gastric emptying was measured 2 h after i.c.v. administration of albutensin A (3–50 nmol/mouse) in ddY mice. Each column represents the mean ⫾ SEM of 6 –7 (A) or 7–15 mice (B). Numbers in parentheses show the number of mice (B). *P ⬍ 0.05, **P ⬍ 0.01 compared with the ACSF-treated group by Bonferroni’s t-test. [30]. On the other hand, des-Arg-albutensin A and des-Argalbutensin A amide have no affinity for these receptor. It was also demonstrated that C-terminal Arg residues of C3a and C5a were indispensable for these biological actions and binding to these receptors [11]. 3.3. GRP-R knockout mouse study To clarify whether the inhibitory effect of albutensin A on feeding was mediated through BN receptor, we used mice lacking GRP-R [26], which was suggested to play the Fig. 4. Effect of administration of albutensin A on food intake in fasted GRP-R deficient mice. Food intake was measured 20 min after i.p. administration of 1.0 ␮mol/mouse albutensin A or saline in GRP-R deficient mice fasted for 18 h. Each column represents the mean ⫾ SEM of 6 (wild-type) or 4 (GRP-R-def) mice. *P ⬍ 0.05, **P ⬍ 0.01 compared with the saline-treated group by Bonferroni’s t-test. K. Ohinata et al. / Peptides 23 (2002) 127–133 131 Fig. 6. Effect of i.c.v. administration of complement C3a on food intake in fasted conscious ddY mice. Complement C3a (0.1–100 pmol/mouse) in ACSF or ACSF alone were i.c.v. injected and food intake was measured in fasted mice. Each column represents the mean ⫾ SEM of 7–14 mice. Numbers in parentheses show the number of mice. *P ⬍ 0.05, **P ⬍ 0.01 compared with the ACSF-treated group by Bonferroni’s t-test. sated mice [F (4,25) 4.319, P ⬍ 0.05] (Fig. 7). In addition, albutensin A has more potent affinity for C3a than C5a receptor [30]. These results indicate that albutensin A-induced reduction of food intake is mediated through C3a not but C5a receptor. Fig. 5. Effects of administration of albutensin A derivatives on food intake in fasted conscious mice. Albutensin A derivatives (■: albutensin A, E: des-Arg-albutensin A amide, 䊐: des-Arg-albutensin A) in 4 ␮l ACSF or in 100 ␮l in saline were i.c.v. (A) or i.p. (B) injected, respectively, and food intake was measured. Each column represents the mean ⫾ SEM of 5–7 (A) or 6 – 8 (B) mice. *P ⬍ 0.05, **P ⬍ 0.01 compared with the ACSF- or saline-treated group by Bonferroni’s t-test. 3.5. Effect of C3a and C5a on food intake To clarify whether C3a or C5a receptors were associated with the inhibitory effect of albutensin A on feeding, we investigated food intake after i.c.v. administration of C3a or C5a by themselves in conscious mice. We found that centrally administered C3a significantly and dose-dependently (10 –100 pmol/mouse) reduced food intake in fasted mice [F (4,49) 2.614, P ⬍ 0.05] (Fig. 6). In contrast, 10 pmol/mouse of C5a increased food intake 2 h after central injection in Fig. 7. Effect of i.c.v. administration of complement C5a on food intake in sated conscious ddY mice. Complement C5a (0.01–10 pmol/mouse) in ACSF or ACSF alone were i.c.v. injected in sated mice and food intake was measured. Each column represents the mean ⫾ SEM of 6 mice. *P ⬍ 0.05, **P ⬍ 0.01 compared with the ACSF-treated group by Bonferroni’s t-test 132 K. Ohinata et al. / Peptides 23 (2002) 127–133 4. Discussion We found that albutensin A derived from serum albumin decreased food intake after central or peripheral administration. The alteration of food intake is known to be associated with changes in gastric emptying, and delayed gastric emptying contributes to the acute satiety effect [19]. In the present study, centrally administered albutensin A also delayed gastric emptying. Furthermore, albutensin A elevated blood glucose levels after central administration, as observed in the case of many other peptides that decrease food intake [12,20]. Next, we investigated what receptor mediated the inhibitory effect of albutensin A on food intake. Albutensin A showed low affinity for BN receptor (Table 1) and had BN-like actions such as the inhibitions of feeding and gastric emptying and elevation of blood glucose. However, albutensin A reduced food intake in GRP-R knockout in addition to in wild-type mice (Fig. 4). This finding suggests that the reduction in food intake is not mediated via BN receptor. Since albutensin A had affinities for complement C3a and C5a receptors and induced ileum-contraction in guinea pigs via both receptors, we investigated the involvement of these receptors in the suppression of food intake after administration of albutensin A. Des-Arg-albutensin A derivatives, lacking affinity for C3a and C5a receptors, did not reduce food intake after central or peripheral administration in mice, suggesting that albutensin A-induced reduction of food intake is mediated through these receptors. Furthermore, C3a alone decreased food intake after central administration in fasted mice and this reduction was not observed after injection of C3a-des-Arg (data not shown), indicating that the effect was specific for C3a. In contrast, centrally administered C5a increased food intake in sated mice (Fig. 7) and this result was consistent with the observation of a previous study [12]. These findings indicate that the albutensin A-induced suppression of food intake was mediated through C3a receptor. The hyperglycemic effect of albutensin A was not affected by GRP-R antagonist, [D-Phe6, Leu-NHEt13, desMet14]-bombesin (6 –14), after i.c.v. administration (data not shown). Although des-Arg-albutensin A showed a more potent affinity for bombesin receptor than albutensin A, des-Arg-albutensin A amide did not elevate blood glucose levels at a dose of 50 nmol/mouse after central injection in fasted mice (data not shown). These findings suggest the hyperglycemic effect of albutensin A is not mediated via BN receptor. Complement C3a is known as one of the most potent inflammatory peptides generated during activation of complement [11]. Until recently, the receptor for complement C3a was thought to be limited mainly to cells of the myeloid line, such as neutrophils, macrophages and mast cells. However, widespread expression of the receptor has been demonstrated throughout many tissues and cells outside the immune system [1,22]. Recent studies have established that astrocytes, microglia and neurons in the CNS possess the C3a receptor under physiological conditions [1]. Central injection of complement C3a has also been reported to exert a psychopharmacological effect on drinking behavior of rats [27]. We recently reported that centrally administered C3a had anti-analgesic and anti-amnesic effects [10]. In the present study, we found the inhibitory effect of C3a on feeding in mice in addition to these actions, indicating that that complement C3a might play various biological roles in the CNS. In the case of anorexia of inflammation or cancer, a number of peptides such as tumor necrosis factor ␣, interleukin-1 and interleukin-6 were postulated to play a role in the inhibition of food intake [9]. C3a, which is considered as a mediator of inflammation, might also be involved in the reduction of food intake during the inflammatory state. In human, albumin is the most abundant plasma protein, accounting for 55– 60% of the measured serum protein [23]. Albumin plays an important role in maintaining osmolarity of blood and is considered to be a transport protein for various endogenous and exogenous hydrophobic compounds [23]. 14 g/day of total albumin is synthesized in a 70 kg adult, and total daily albumin degradation is around 14 g/day or 5% of daily whole-body protein turnover [23]. 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