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β-Lactotensin derived from bovine β-lactoglobulin suppresses food intake via the CRF system followed by the CGRP system in mice
Peptides 30 (2009) 2228–2232
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b-Lactotensin derived from bovine b-lactoglobulin suppresses food intake via the CRF system followed by the CGRP system in mice I-Ching Hou a, Masaaki Yoshikawa a,b, Kousaku Ohinata a,* a b
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan Frontier Research Center, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 July 2009 Received in revised form 24 August 2009 Accepted 24 August 2009 Available online 29 August 2009
We found that b-lactotensin (His-Ile-Arg-Leu), which has been isolated as an ileum-contracting peptide from chymotrypsin digest of bovine b-lactoglobulin, dose-dependently suppresses food intake after intracerebroventricular (i.c.v.) or intraperitoneal administration at a dose of 40 nmol/mouse or 100 mg/ kg, respectively, in fasted mice. Orally administered b-lactotensin also suppressed food intake at 500 mg/kg. We previously reported that b-lactotensin acts as an agonist for neurotensin receptors; however, the anorexigenic activity of b-lactotensin was not inhibited by i.c.v. co-administration with SR48692 or levocabastine, an antagonist for neurotensin NT1 or NT2 receptor, respectively. On the other hand, the anorexigenic effect of b-lactotensin was blocked by i.c.v. co-administration with astressin or calcitonin gene-related peptide (CGRP)(8–37), an antagonist for corticotropin releasing factor (CRF) or CGRP, respectively. b-Lactotensin had affinity for neither CRF nor CGRP receptor. In addition, CRFinduced anorexigenic activity after i.c.v. administration was completely blocked by CGRP(8–37), while CGRP-induced anorexigenic activity was not inhibited by astressin. These results suggest that the CGRP system is activated downstream of the CRF system in food intake regulation. Taken together, blactotensin may suppress food intake by activating the CRF system followed by the CGRP system, independently of the neurotensin system. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Food intake b-Lactotensin Neurotensin Calcitonin gene-related peptide Corticotropin releasing factor
1. Introduction It is known that a number of bioactive peptides isolated from enzymatic digests of food proteins show a variety of physiological actions on blood pressure, lipid metabolism, pain response, and hair growth in animals [6,25,27–29]. In addition to these actions, we previously reported that low molecular weight peptides often have actions on the central nervous system (CNS) such as anorexigenic and anxiolytic-like and memory-enhancing activities after oral administration [7,8,12–15,30,31]. In this study, we found that b-lactotensin (His-Ile-Arg-Leu), an ileum-contracting tetrapeptide isolated from chymotryptic digest of bovine b-lactoglobulin [27], suppresses food intake after oral administration, and investigated the mechanism underlying the anorexigenic activity of b-lactotensin. We have reported that b-lactotensin has a variety of actions including antinociceptive, cholesterol-lowering, anti-stress and memory-enhancing activities [12,28–30]. b-Lactotensin has homology with neurotensin, an endogenous anorexigenic tridecapeptide widely distributed in the gastrointestinal tract and the CNS [2,5,22]. NT1 and NT2 receptors for neurotensin are G-protein
coupled receptors with a seven-transmembrane domain [2,5,22]. It is known that the anorexigenic activity of neurotensin itself is mediated through the NT1 receptor [20]. b-Lactotensin has affinity for NT1 and NT2 receptors; however, its affinity for the NT2 receptor is 50-fold higher than that of the NT1 receptor [27]. We demonstrated that the antinociceptive, cholesterol-lowering and anti-stress activities induced by b-lactotensin were mediated via the NT2 receptor [28–30]. The ileum-contracting activity of blactotensin was mediated through the NT1 receptor [27]. Interestingly, the anorexigenic effect of b-lactotensin was not blocked by SR48692 or levocabastine, antagonists for NT1 and NT2 receptors, respectively, suggesting that b-lactotensin suppressed food intake independently of the neurotensin system. A number of endogenous peptides regulating food intake are known [9]. We also investigated whether the peptidergic regulating system is involved in the anorexigenic activity of b-lactotensin using antagonists of receptors for anorexigenic peptides. 2. Materials and methods 2.1. Materials
* Corresponding author. Tel.: +81 774 38 3733; fax: +81 774 38 3774. E-mail address: [email protected] (K. Ohinata). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.08.018
b-Lactotensin was synthesized by the Fmoc strategy and purified using reverse phase-high performance liquid chromatography.
I.-C. Hou et al. / Peptides 30 (2009) 2228–2232
SR48692, a NT1 receptor antagonist, was kindly provided by SanofiAventis (Paris, France). Levocabastine hydrochloride, a NT2 receptor antagonist, was obtained from BIOMOL International (Plymouth Meeting, PA). Corticotropine-releasing factor (CRF), astressin (CRF antagonist), calcitonin gene-related peptide (CGRP), and CGRP(8– 37) (CGRP antagonist) were purchased from the American Peptide Company, Inc. (Sunnyvale, CA). 2.2. Animals Male ddY mice at 7 weeks of age were obtained from Japan SLC (Shizuoka, Japan) [14–19]. Each mouse was individually housed under regulated conditions (22 8C on a 12 h light–dark cycle with lights on at 7 am). Food and water were available ad libitum unless otherwise indicated. All experiments were approved by Kyoto University Ethics Committee for Animal Research Use. 2.3. Cannula implantation Intracerebroventricular (i.c.v.) administration was performed as described previously [14–19]. Briefly, mice were anesthetized with sodium pentobarbital (80–85 mg/kg i.p.) and placed in a stereotaxic instrument. A 24-gauge cannula beveled at one end over a distance of 3 mm (Safelet-Cas; Nipro, Osaka, Japan) was implanted 0.9 mm posterior to the bregma and 0.9 mm lateral to the suture in the third cerebral ventricle. Animals were tested 1 week or more after implantation. After food intake experiments, cannula placement was confirmed by i.c.v. administration of dye.
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2.4. Food intake Each mouse was deprived of food pellets for 18 h with free access to water. The food intake experiment was started at 11 am. For i.c.v. administration, b-lactotensin was dissolved in 4 ml artificial cerebrospinal fluid (ACSF, 138.9 mM NaCl, 3.4 mM KCl, 1.3 mM CaCl2, 4.0 mM NaHCO3, 0.6 mM NaH2PO4, 5.6 mM glucose, pH 7.4). b-Lactotensin in ACSF or the vehicle alone was i.c.v. administered to conscious mice, that were fed pre-weighed food pellets 10 min after administration. Thereafter, the weight of the food pellets was measured, and the cumulative food intake was calculated. Similarly, b-lactotensin in saline was intraperitoneally (i.p.) or orally administered, and food intake was measured. To investigate the mechanism of the anorexigenic activity of b-lactotensin, antagonists including SR48692 (2.5 mg/mouse), levocabastine (2.5 mg/mouse), astressin (6 nmol/mouse) and CGRP(8–37) (30 nmol/mouse) were i.c.v. co-administered with b-lactotensin (40 nmol/mouse). Doses of these antagonists were determined based on previous reports [3,10,24], and we confirmed that their administration did not affect food intake under our experimental conditions (data not shown). CRF (0.03 nmol/mouse) or CGRP (0.3 nmol/mouse) [4] was coadministered with CGRP(8–37) (30 nmol/mouse) [24] or astressin (6 nmol/mouse) [3], respectively, after i.c.v. administration in mice fasted for 18 h with free access to water, and food intake was measured. 2.5. Statistical analysis Values are expressed as the means SEM. Statistical comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by Fisher’s test or the unpaired Student’s t-test. P values less than 0.05 were considered significant.
3. Results 3.1. b-Lactotensin suppresses food intake after oral administration
b-Lactotensin dose-dependently suppressed food intake after i.c.v. (40 nmol/mouse) and i.p. (100 mg/kg) administration in male mice fasted for 18 h (Fig. 1). Orally administered b-lactotensin also suppressed food intake at a dose of 500 mg/kg, and the anorexigenic effect of b-lactotensin lasted for 1 h (Fig. 2). Thus, we found that orally administered b-lactotensin suppresses food intake in mice.
Fig. 1. Effect of i.c.v. or i.p. administration of b-lactotensin on food intake. bLactotensin at a dose of 20–40 nmol/mouse i.c.v. (a) or 30–100 mg/kg i.p. (b) suppressed food intake in male mice fasted for 18 h. Each column represents the mean SEM ((a) n = 4–5, (b) n = 8). *P < 0.05, *P < 0.01 compared with the ACSF- or saline-control group.
Fig. 2. Effect of oral administration of b-lactotensin on food intake. Orally administered b-lactotensin at a dose of 500 mg/kg suppressed food intake in male mice fasted for 18 h. Each column represents the mean SEM (n = 7). *P < 0.05 compared with the control group.
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Fig. 3. Effects of neurotensin receptor antagonists on the anorexigenic activity of blactotensin. SR48692 or levocabastine (2.5 mg/mouse, i.c.v.), an antagonist for NT1 or NT2 receptor, respectively, were co-administered with b-lactotensin (40 nmol/ mouse, i.c.v.). The anorexigenic effect was not blocked by SR48692 or levocabastine. Each column represents the mean SEM. *P < 0.05; **P < 0.01 compared with the ACSF-treated control group.
3.2. b-Lactotensin suppresses food intake but not as an agonist of neurotensin receptors We investigated the mechanism of the anorexigenic activity of b-lactotensin after central administration using selective antagonists. Initially, we tested the involvement of neurotensin receptors, since b-lactotensin acted as a neurotensin agonist showing a variety of actions on ileum contraction, pain response, cholesterol metabolism and emotional behavior [27–30]; however, the anorexigenic activity of b-lactotensin (40 nmol/mouse, i.c.v.) was blocked by neither SR48692, a NT1 antagonist (2.5 mg/ mouse, i.c.v.) nor levocabastine, a NT2 antagonist (2.5 mg/mouse, i.c.v.) (Fig. 3). The antagonists themselves did not affect food intake after i.c.v. administration under our experimental conditions (data not shown). These results suggest that b-lactotensin suppressed food intake but not as an agonist of neurotensin receptors.
Fig. 4. Effects of CRF or CGRP antagonists on anorexigenic activity of b-lactotensin. The anorexigenic effect of b-lactotensin (40 nmol/mouse, i.c.v.) was abolished by astressin (a, 6 nmol/mouse, i.c.v.) or CGRP(8–37) (b, 30 nmol/mouse, i.c.v.), an antagonist for CRF or CGRP, respectively. Each column represents the mean SEM (n = 6). *P < 0.05, **P < 0.01 compared with the ACSF-treated control group; #P < 0.05 compared with the b-lactotensin-treated group.
3.3. Anorexigenic activity of b-lactotensin was mediated by activating the CRF and CGRP systems
3.4. CRF administration activates the CGRP system in food intake regulation
Next, we examined whether the peptidergic regulating system is involved in the anorexigenic activity of b-lactotensin using antagonists of receptors for several endogenous anorexigenic peptides. CRF is a representative anorexigenic neuropeptide in the hypothalamus of the CNS [9,21,26,32]. We therefore examined whether the anorexigenic effect of b-lactotensin was involved in the CRF system. Food intake suppression of b-lactotesin (40 nmol/ mouse) was blocked by i.c.v. co-administration of astressin (6 nmol/mouse) (Fig. 4a). Astressin alone did not affect food intake (data not shown). The affinity of b-lactotensin (100 mM) for CRF receptor was negligible. These results suggest that blactotensin suppresses food intake by activating the CRF system. CGRP is also an anorexigenic peptide present in the CNS [4]. CGRP(8–37) (30 nmol/mouse, i.c.v.), an antagonist for CGRP blocked the anorexigenic effect of b-lactotensin (40 nmol/mouse, i.c.v.) (Fig. 4b). CGRP(8–37) alone did not affect food intake after i.c.v. administration (data not shown). Furthermore, b-lactotensin also had no affinity for the CGRP receptor (data not shown). These results suggest that b-lactotensin suppresses food intake by activating both CRF and CGRP systems.
We examined the relationship between CRF and CGRP systems in food intake regulation using their agonists and antagonists. CRF (0.03 nmol/mouse)-induced food suppression was completely blocked by CGRP(8–37) (30 nmol/mouse) (Fig. 5a). On the other hand, anorexigenic activity of CGRP (0.3 nmol/mouse) was not blocked by astressin (6 nmol/mouse) (Fig. 5b). These results suggest that central administration of CRF activates the CGRP system in food intake regulation. This is a novel anorexigenic pathway in the CNS. Taken together, b-lactotensin may activate the CRF system followed by the CGRP system.
In contrast, the anorexigenic activity of b-lactotensin was not blocked by lorglumide, an antagonist of cholecystokinin (CCK)1 receptor for CCK, which is known as a satiety factor released postprandially [23] (data not shown). SHU9119, an antagonist of melanocortin (MC)4 receptor for a-melanocyte-stimulating hormone (a-MSH) also did not inhibit food intake suppression of blactotensin (data not shown), suggesting that anorexigenic activity of b-lactotensin was independent of both CCK1 and MC4 receptors.
4. Discussion We found that orally or centrally administered b-lactotensin suppresses food intake in fasted mice. To the best of our knowledge, this is first example of an orally active anorexigenic peptide derived from milk whey protein. b-Lactotensin had cholesterol-lowering and learning-enhancing activities after oral administration in mice [12,30]. Ile-Arg-Leu or His-Ile-Arg, tripetide deleted N-terminal or C-terminal amino acids, respectively, of blactotensin (His-Ile-Arg-Leu) that did not affect food intake after
I.-C. Hou et al. / Peptides 30 (2009) 2228–2232
Fig. 5. Relationship between the CRF and CGRP system in food intake regulation. (a) CRF (0.03 nmol/mouse, i.c.v.)-induced anorexigenic effect was blocked by CGRP(8– 37)(30 nmol/mouse i.c.v.). (b) CGRP (0.3 nmol/mouse i.c.v.)-induced anorexigenic effect was not blocked by astressin (6 nmol/mouse i.c.v.). Each column represents the mean SEM (n = 6). *P < 0.05; **P < 0.01 compared with the ACSF-treated control group; #P < 0.05 compared with the CRF-treated group.
i.c.v. administration (data not shown). Thus, b-lactotensin might be absorbed from the gastrointestinal tract and across the blood– brain barrier (BBB) to act in the CNS, though the possibility that blactotensin might transmit signals from nerves such as the vagus to the CNS could not be ruled out. We previously reported that b-lactotensin has affinity for neurotensin receptors, and acts as an agonist for neurotensin NT1 or NT2 receptors [27–30]; however, the anorexigenic activity of blactotensin was blocked by antagonists for neither NT1 or NT2 receptor (Fig. 3). These results suggest that food intake suppression of b-lactotensin was independent of the neurotensin system. Recently, b-lactotensin was also reported to have affinity for the adrenergic a2B receptor [1]; however, the anorexigenic activity of b-lactotensin was not blocked by imiloxan, an antagonist for the a2B receptor (data not shown), suggesting that b-lactotensin suppresses food intake independently of the a2B receptor. bLactotensin has also homology with His-Phe-Arg-Trp, a pharmacophore for melanocortin receptor activation, and marginal affinity for MC4 receptor (Ki = 830 mM); however, MC4 receptor is ruled out from the candidate for b-lactotensin receptor, since its anorexigenic activity was not blocked by an antagonist of MC4 receptor. The anorexigenic effect of b-lactotensin was blocked by antagonists for CRF and CGRP receptors; however, b-lactotensin had no affinity for them. We also found a novel anorexigenic pathway that CRF activates the CGRP system in food intake regulation in the CNS. Taken together, b-lactotensin may suppress food intake by activating the CRF system followed by the CGRP system downstream of unidentified receptor for b-lactotensin. CRF and its receptors are widely distributed throughout the brain, and are particularly present in the PVN of the hypothalamus [9,21,32]. A number of endogenous agonist peptides for the CRF receptor have been identified: urocortins I, II and III, stresscopin, and
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stresscopin-related peptide as well as CRF itself [32]. Astressin has affinity for both receptor subtypes for CRF (CRF1 and CRF2 receptors) [32]. On the other hand, CGRP has been also reported to decrease food intake after central and peripheral administration [4]. CGRP(8–37) has affinity not only for the CGRP1 receptor but also for adrenomedullin (AM)1 and AM2 receptors [11]. CGRP and AM 1 and 2 are known as endogenous agonist peptides for these receptors [11]. It is known that AM 1 and 2 as well as CGRP decrease food intake after central administration [24], and are present in the CNS, including the hypothalamus. Further investigation will clarify which of these endogenous agonist peptides for CRF or CGRP receptors are activated in response to b-lactotensin administration. As for the interaction between CGRP and CRF systems, it has been reported that CGRP stimulated release of CRF from hypothalamic explants [4]; however, it remained unknown whether CRF activates the CGRP system in food intake regulation or not. We found for the first time that the anorexigenic activity of CRF was inhibited by a CGRP antagonist, while that of CGRP was not blocked by a CRF antagonist. Thus, the CRF system may be upstream of the CGRP system in food intake regulation in the CNS. Among the endogenous peptides regulating food intake, few orally active peptides are known; however, we previously reported that orally active Arg-Ile-Tyr, a hypotensive peptide derived from rapeseed protein, suppresses food intake via CCK release and CCK1 receptor activation [7]. Furthermore, we found that a complement C3a agonist peptide, [Trp5]-oryzatensin(5–9), and an angiotensin AT2 agonist peptide, novokinin, which were designed based on the primary structure of isolated peptides from food proteins, also decreased food intake after oral administration by activating prostaglandin (PG) E2 and EP4 receptor among four receptor subtypes for PGE2 [14–18]. We then tested whether the anorexigenic activity of b-lactotensin was coupled to the activation of the CCK and/or PG system. Food intake suppression of b-lactotensin was blocked by neither lorglumide, a CCK1 receptor antagonist, nor a cyclooxygenase (COX) inhibitor, indomethacin (data not shown), suggesting that b-lactotensin suppressed food intake independently of activating CCK and PG systems. In conclusion, we found that b-lactotensin suppresses food intake after i.c.v. and i.p. administration. Orally administered blactotensin was also active. The anorexigenic effect of blactotensin is mediated by CRF and CGRP systems. We also found a novel anorexigenic pathway that the CGRP system is activated downstream of the CRF system in the CNS.
Acknowledgements This work was supported in part by a Grant-in-Aid for Young Scientists from the Japanese Society for the Promotion of Science to KO, and by a Grant-in-Aid from the Japan Dairy Association to KO.
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Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
b-Lactotensin derived from bovine b-lactoglobulin suppresses food intake via the CRF system followed by the CGRP system in mice I-Ching Hou a, Masaaki Yoshikawa a,b, Kousaku Ohinata a,* a b
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan Frontier Research Center, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 July 2009 Received in revised form 24 August 2009 Accepted 24 August 2009 Available online 29 August 2009
We found that b-lactotensin (His-Ile-Arg-Leu), which has been isolated as an ileum-contracting peptide from chymotrypsin digest of bovine b-lactoglobulin, dose-dependently suppresses food intake after intracerebroventricular (i.c.v.) or intraperitoneal administration at a dose of 40 nmol/mouse or 100 mg/ kg, respectively, in fasted mice. Orally administered b-lactotensin also suppressed food intake at 500 mg/kg. We previously reported that b-lactotensin acts as an agonist for neurotensin receptors; however, the anorexigenic activity of b-lactotensin was not inhibited by i.c.v. co-administration with SR48692 or levocabastine, an antagonist for neurotensin NT1 or NT2 receptor, respectively. On the other hand, the anorexigenic effect of b-lactotensin was blocked by i.c.v. co-administration with astressin or calcitonin gene-related peptide (CGRP)(8–37), an antagonist for corticotropin releasing factor (CRF) or CGRP, respectively. b-Lactotensin had affinity for neither CRF nor CGRP receptor. In addition, CRFinduced anorexigenic activity after i.c.v. administration was completely blocked by CGRP(8–37), while CGRP-induced anorexigenic activity was not inhibited by astressin. These results suggest that the CGRP system is activated downstream of the CRF system in food intake regulation. Taken together, blactotensin may suppress food intake by activating the CRF system followed by the CGRP system, independently of the neurotensin system. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Food intake b-Lactotensin Neurotensin Calcitonin gene-related peptide Corticotropin releasing factor
1. Introduction It is known that a number of bioactive peptides isolated from enzymatic digests of food proteins show a variety of physiological actions on blood pressure, lipid metabolism, pain response, and hair growth in animals [6,25,27–29]. In addition to these actions, we previously reported that low molecular weight peptides often have actions on the central nervous system (CNS) such as anorexigenic and anxiolytic-like and memory-enhancing activities after oral administration [7,8,12–15,30,31]. In this study, we found that b-lactotensin (His-Ile-Arg-Leu), an ileum-contracting tetrapeptide isolated from chymotryptic digest of bovine b-lactoglobulin [27], suppresses food intake after oral administration, and investigated the mechanism underlying the anorexigenic activity of b-lactotensin. We have reported that b-lactotensin has a variety of actions including antinociceptive, cholesterol-lowering, anti-stress and memory-enhancing activities [12,28–30]. b-Lactotensin has homology with neurotensin, an endogenous anorexigenic tridecapeptide widely distributed in the gastrointestinal tract and the CNS [2,5,22]. NT1 and NT2 receptors for neurotensin are G-protein
coupled receptors with a seven-transmembrane domain [2,5,22]. It is known that the anorexigenic activity of neurotensin itself is mediated through the NT1 receptor [20]. b-Lactotensin has affinity for NT1 and NT2 receptors; however, its affinity for the NT2 receptor is 50-fold higher than that of the NT1 receptor [27]. We demonstrated that the antinociceptive, cholesterol-lowering and anti-stress activities induced by b-lactotensin were mediated via the NT2 receptor [28–30]. The ileum-contracting activity of blactotensin was mediated through the NT1 receptor [27]. Interestingly, the anorexigenic effect of b-lactotensin was not blocked by SR48692 or levocabastine, antagonists for NT1 and NT2 receptors, respectively, suggesting that b-lactotensin suppressed food intake independently of the neurotensin system. A number of endogenous peptides regulating food intake are known [9]. We also investigated whether the peptidergic regulating system is involved in the anorexigenic activity of b-lactotensin using antagonists of receptors for anorexigenic peptides. 2. Materials and methods 2.1. Materials
* Corresponding author. Tel.: +81 774 38 3733; fax: +81 774 38 3774. E-mail address: [email protected] (K. Ohinata). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.08.018
b-Lactotensin was synthesized by the Fmoc strategy and purified using reverse phase-high performance liquid chromatography.
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SR48692, a NT1 receptor antagonist, was kindly provided by SanofiAventis (Paris, France). Levocabastine hydrochloride, a NT2 receptor antagonist, was obtained from BIOMOL International (Plymouth Meeting, PA). Corticotropine-releasing factor (CRF), astressin (CRF antagonist), calcitonin gene-related peptide (CGRP), and CGRP(8– 37) (CGRP antagonist) were purchased from the American Peptide Company, Inc. (Sunnyvale, CA). 2.2. Animals Male ddY mice at 7 weeks of age were obtained from Japan SLC (Shizuoka, Japan) [14–19]. Each mouse was individually housed under regulated conditions (22 8C on a 12 h light–dark cycle with lights on at 7 am). Food and water were available ad libitum unless otherwise indicated. All experiments were approved by Kyoto University Ethics Committee for Animal Research Use. 2.3. Cannula implantation Intracerebroventricular (i.c.v.) administration was performed as described previously [14–19]. Briefly, mice were anesthetized with sodium pentobarbital (80–85 mg/kg i.p.) and placed in a stereotaxic instrument. A 24-gauge cannula beveled at one end over a distance of 3 mm (Safelet-Cas; Nipro, Osaka, Japan) was implanted 0.9 mm posterior to the bregma and 0.9 mm lateral to the suture in the third cerebral ventricle. Animals were tested 1 week or more after implantation. After food intake experiments, cannula placement was confirmed by i.c.v. administration of dye.
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2.4. Food intake Each mouse was deprived of food pellets for 18 h with free access to water. The food intake experiment was started at 11 am. For i.c.v. administration, b-lactotensin was dissolved in 4 ml artificial cerebrospinal fluid (ACSF, 138.9 mM NaCl, 3.4 mM KCl, 1.3 mM CaCl2, 4.0 mM NaHCO3, 0.6 mM NaH2PO4, 5.6 mM glucose, pH 7.4). b-Lactotensin in ACSF or the vehicle alone was i.c.v. administered to conscious mice, that were fed pre-weighed food pellets 10 min after administration. Thereafter, the weight of the food pellets was measured, and the cumulative food intake was calculated. Similarly, b-lactotensin in saline was intraperitoneally (i.p.) or orally administered, and food intake was measured. To investigate the mechanism of the anorexigenic activity of b-lactotensin, antagonists including SR48692 (2.5 mg/mouse), levocabastine (2.5 mg/mouse), astressin (6 nmol/mouse) and CGRP(8–37) (30 nmol/mouse) were i.c.v. co-administered with b-lactotensin (40 nmol/mouse). Doses of these antagonists were determined based on previous reports [3,10,24], and we confirmed that their administration did not affect food intake under our experimental conditions (data not shown). CRF (0.03 nmol/mouse) or CGRP (0.3 nmol/mouse) [4] was coadministered with CGRP(8–37) (30 nmol/mouse) [24] or astressin (6 nmol/mouse) [3], respectively, after i.c.v. administration in mice fasted for 18 h with free access to water, and food intake was measured. 2.5. Statistical analysis Values are expressed as the means SEM. Statistical comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by Fisher’s test or the unpaired Student’s t-test. P values less than 0.05 were considered significant.
3. Results 3.1. b-Lactotensin suppresses food intake after oral administration
b-Lactotensin dose-dependently suppressed food intake after i.c.v. (40 nmol/mouse) and i.p. (100 mg/kg) administration in male mice fasted for 18 h (Fig. 1). Orally administered b-lactotensin also suppressed food intake at a dose of 500 mg/kg, and the anorexigenic effect of b-lactotensin lasted for 1 h (Fig. 2). Thus, we found that orally administered b-lactotensin suppresses food intake in mice.
Fig. 1. Effect of i.c.v. or i.p. administration of b-lactotensin on food intake. bLactotensin at a dose of 20–40 nmol/mouse i.c.v. (a) or 30–100 mg/kg i.p. (b) suppressed food intake in male mice fasted for 18 h. Each column represents the mean SEM ((a) n = 4–5, (b) n = 8). *P < 0.05, *P < 0.01 compared with the ACSF- or saline-control group.
Fig. 2. Effect of oral administration of b-lactotensin on food intake. Orally administered b-lactotensin at a dose of 500 mg/kg suppressed food intake in male mice fasted for 18 h. Each column represents the mean SEM (n = 7). *P < 0.05 compared with the control group.
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Fig. 3. Effects of neurotensin receptor antagonists on the anorexigenic activity of blactotensin. SR48692 or levocabastine (2.5 mg/mouse, i.c.v.), an antagonist for NT1 or NT2 receptor, respectively, were co-administered with b-lactotensin (40 nmol/ mouse, i.c.v.). The anorexigenic effect was not blocked by SR48692 or levocabastine. Each column represents the mean SEM. *P < 0.05; **P < 0.01 compared with the ACSF-treated control group.
3.2. b-Lactotensin suppresses food intake but not as an agonist of neurotensin receptors We investigated the mechanism of the anorexigenic activity of b-lactotensin after central administration using selective antagonists. Initially, we tested the involvement of neurotensin receptors, since b-lactotensin acted as a neurotensin agonist showing a variety of actions on ileum contraction, pain response, cholesterol metabolism and emotional behavior [27–30]; however, the anorexigenic activity of b-lactotensin (40 nmol/mouse, i.c.v.) was blocked by neither SR48692, a NT1 antagonist (2.5 mg/ mouse, i.c.v.) nor levocabastine, a NT2 antagonist (2.5 mg/mouse, i.c.v.) (Fig. 3). The antagonists themselves did not affect food intake after i.c.v. administration under our experimental conditions (data not shown). These results suggest that b-lactotensin suppressed food intake but not as an agonist of neurotensin receptors.
Fig. 4. Effects of CRF or CGRP antagonists on anorexigenic activity of b-lactotensin. The anorexigenic effect of b-lactotensin (40 nmol/mouse, i.c.v.) was abolished by astressin (a, 6 nmol/mouse, i.c.v.) or CGRP(8–37) (b, 30 nmol/mouse, i.c.v.), an antagonist for CRF or CGRP, respectively. Each column represents the mean SEM (n = 6). *P < 0.05, **P < 0.01 compared with the ACSF-treated control group; #P < 0.05 compared with the b-lactotensin-treated group.
3.3. Anorexigenic activity of b-lactotensin was mediated by activating the CRF and CGRP systems
3.4. CRF administration activates the CGRP system in food intake regulation
Next, we examined whether the peptidergic regulating system is involved in the anorexigenic activity of b-lactotensin using antagonists of receptors for several endogenous anorexigenic peptides. CRF is a representative anorexigenic neuropeptide in the hypothalamus of the CNS [9,21,26,32]. We therefore examined whether the anorexigenic effect of b-lactotensin was involved in the CRF system. Food intake suppression of b-lactotesin (40 nmol/ mouse) was blocked by i.c.v. co-administration of astressin (6 nmol/mouse) (Fig. 4a). Astressin alone did not affect food intake (data not shown). The affinity of b-lactotensin (100 mM) for CRF receptor was negligible. These results suggest that blactotensin suppresses food intake by activating the CRF system. CGRP is also an anorexigenic peptide present in the CNS [4]. CGRP(8–37) (30 nmol/mouse, i.c.v.), an antagonist for CGRP blocked the anorexigenic effect of b-lactotensin (40 nmol/mouse, i.c.v.) (Fig. 4b). CGRP(8–37) alone did not affect food intake after i.c.v. administration (data not shown). Furthermore, b-lactotensin also had no affinity for the CGRP receptor (data not shown). These results suggest that b-lactotensin suppresses food intake by activating both CRF and CGRP systems.
We examined the relationship between CRF and CGRP systems in food intake regulation using their agonists and antagonists. CRF (0.03 nmol/mouse)-induced food suppression was completely blocked by CGRP(8–37) (30 nmol/mouse) (Fig. 5a). On the other hand, anorexigenic activity of CGRP (0.3 nmol/mouse) was not blocked by astressin (6 nmol/mouse) (Fig. 5b). These results suggest that central administration of CRF activates the CGRP system in food intake regulation. This is a novel anorexigenic pathway in the CNS. Taken together, b-lactotensin may activate the CRF system followed by the CGRP system.
In contrast, the anorexigenic activity of b-lactotensin was not blocked by lorglumide, an antagonist of cholecystokinin (CCK)1 receptor for CCK, which is known as a satiety factor released postprandially [23] (data not shown). SHU9119, an antagonist of melanocortin (MC)4 receptor for a-melanocyte-stimulating hormone (a-MSH) also did not inhibit food intake suppression of blactotensin (data not shown), suggesting that anorexigenic activity of b-lactotensin was independent of both CCK1 and MC4 receptors.
4. Discussion We found that orally or centrally administered b-lactotensin suppresses food intake in fasted mice. To the best of our knowledge, this is first example of an orally active anorexigenic peptide derived from milk whey protein. b-Lactotensin had cholesterol-lowering and learning-enhancing activities after oral administration in mice [12,30]. Ile-Arg-Leu or His-Ile-Arg, tripetide deleted N-terminal or C-terminal amino acids, respectively, of blactotensin (His-Ile-Arg-Leu) that did not affect food intake after
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Fig. 5. Relationship between the CRF and CGRP system in food intake regulation. (a) CRF (0.03 nmol/mouse, i.c.v.)-induced anorexigenic effect was blocked by CGRP(8– 37)(30 nmol/mouse i.c.v.). (b) CGRP (0.3 nmol/mouse i.c.v.)-induced anorexigenic effect was not blocked by astressin (6 nmol/mouse i.c.v.). Each column represents the mean SEM (n = 6). *P < 0.05; **P < 0.01 compared with the ACSF-treated control group; #P < 0.05 compared with the CRF-treated group.
i.c.v. administration (data not shown). Thus, b-lactotensin might be absorbed from the gastrointestinal tract and across the blood– brain barrier (BBB) to act in the CNS, though the possibility that blactotensin might transmit signals from nerves such as the vagus to the CNS could not be ruled out. We previously reported that b-lactotensin has affinity for neurotensin receptors, and acts as an agonist for neurotensin NT1 or NT2 receptors [27–30]; however, the anorexigenic activity of blactotensin was blocked by antagonists for neither NT1 or NT2 receptor (Fig. 3). These results suggest that food intake suppression of b-lactotensin was independent of the neurotensin system. Recently, b-lactotensin was also reported to have affinity for the adrenergic a2B receptor [1]; however, the anorexigenic activity of b-lactotensin was not blocked by imiloxan, an antagonist for the a2B receptor (data not shown), suggesting that b-lactotensin suppresses food intake independently of the a2B receptor. bLactotensin has also homology with His-Phe-Arg-Trp, a pharmacophore for melanocortin receptor activation, and marginal affinity for MC4 receptor (Ki = 830 mM); however, MC4 receptor is ruled out from the candidate for b-lactotensin receptor, since its anorexigenic activity was not blocked by an antagonist of MC4 receptor. The anorexigenic effect of b-lactotensin was blocked by antagonists for CRF and CGRP receptors; however, b-lactotensin had no affinity for them. We also found a novel anorexigenic pathway that CRF activates the CGRP system in food intake regulation in the CNS. Taken together, b-lactotensin may suppress food intake by activating the CRF system followed by the CGRP system downstream of unidentified receptor for b-lactotensin. CRF and its receptors are widely distributed throughout the brain, and are particularly present in the PVN of the hypothalamus [9,21,32]. A number of endogenous agonist peptides for the CRF receptor have been identified: urocortins I, II and III, stresscopin, and
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stresscopin-related peptide as well as CRF itself [32]. Astressin has affinity for both receptor subtypes for CRF (CRF1 and CRF2 receptors) [32]. On the other hand, CGRP has been also reported to decrease food intake after central and peripheral administration [4]. CGRP(8–37) has affinity not only for the CGRP1 receptor but also for adrenomedullin (AM)1 and AM2 receptors [11]. CGRP and AM 1 and 2 are known as endogenous agonist peptides for these receptors [11]. It is known that AM 1 and 2 as well as CGRP decrease food intake after central administration [24], and are present in the CNS, including the hypothalamus. Further investigation will clarify which of these endogenous agonist peptides for CRF or CGRP receptors are activated in response to b-lactotensin administration. As for the interaction between CGRP and CRF systems, it has been reported that CGRP stimulated release of CRF from hypothalamic explants [4]; however, it remained unknown whether CRF activates the CGRP system in food intake regulation or not. We found for the first time that the anorexigenic activity of CRF was inhibited by a CGRP antagonist, while that of CGRP was not blocked by a CRF antagonist. Thus, the CRF system may be upstream of the CGRP system in food intake regulation in the CNS. Among the endogenous peptides regulating food intake, few orally active peptides are known; however, we previously reported that orally active Arg-Ile-Tyr, a hypotensive peptide derived from rapeseed protein, suppresses food intake via CCK release and CCK1 receptor activation [7]. Furthermore, we found that a complement C3a agonist peptide, [Trp5]-oryzatensin(5–9), and an angiotensin AT2 agonist peptide, novokinin, which were designed based on the primary structure of isolated peptides from food proteins, also decreased food intake after oral administration by activating prostaglandin (PG) E2 and EP4 receptor among four receptor subtypes for PGE2 [14–18]. We then tested whether the anorexigenic activity of b-lactotensin was coupled to the activation of the CCK and/or PG system. Food intake suppression of b-lactotensin was blocked by neither lorglumide, a CCK1 receptor antagonist, nor a cyclooxygenase (COX) inhibitor, indomethacin (data not shown), suggesting that b-lactotensin suppressed food intake independently of activating CCK and PG systems. In conclusion, we found that b-lactotensin suppresses food intake after i.c.v. and i.p. administration. Orally administered blactotensin was also active. The anorexigenic effect of blactotensin is mediated by CRF and CGRP systems. We also found a novel anorexigenic pathway that the CGRP system is activated downstream of the CRF system in the CNS.
Acknowledgements This work was supported in part by a Grant-in-Aid for Young Scientists from the Japanese Society for the Promotion of Science to KO, and by a Grant-in-Aid from the Japan Dairy Association to KO.
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