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Central administration of ghrelin preferentially enhances fat ingestion
Neuroscience Letters 369 (2004) 75–79
Central administration of ghrelin preferentially enhances fat ingestion Takuya Shimbara, Muhtashan S. Mondal, Takashi Kawagoe, Koji Toshinai, Shuichi Koda, Hideki Yamaguchi, Yukari Date, Masamitsu Nakazato∗ Third Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan Received 10 May 2004; received in revised form 15 July 2004; accepted 28 July 2004
Abstract Ghrelin, a brain-gut peptide discovered from the stomach, stimulates growth hormone release, food intake, adiposity, and weight gain. Circulating ghrelin levels are modulated under conditions of positive and negative energy balance, however its effect on macronutrient selection is not known. The present experiment investigates the effect of ghrelin on single and two-diet feeding paradigms in high-carbohydrate (HC) and high-fat (HF) preferring rats. In the macronutrient selection test in which rats were given free access to either high-carbohydrate or high-fat diet, an intracerebroventricular (i.c.v.) administration of ghrelin potently enhanced fat intake over carbohydrate intake in both HC- and HF-preferring rats. In the diet preference test in which rats were given free access to both high-carbohydrate and high-fat diets simultaneously, an i.c.v. administration of ghrelin also preferentially enhanced fat consumption over carbohydrate in both HF- and HCpreferring rats. Intracerebroventricular administrations of galanin and neuropeptide Y enhanced fat and carbohydrate ingestion, respectively. Centrally administered ghrelin enhanced fat ingestion. These results provide further insights for the role of ghrelin in feeding behavior and the development of obesity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Ghrelin; Neuropeptide Y; Galanin; Central administration; Macronutrient selection
Ghrelin, a 28-amino-acid peptide with an n-octanoyl modification indispensable for its activity, was originally discovered in human and rat stomach as an endogenous ligand for the growth hormone (GH) secretagogue receptor [7]. Ghrelin homologues have been identified in fish, amphibians, birds, and many mammals. Ghrelin stimulates GH release when administered in humans and rodents and when applied directly to rat primary pituitary cells [3,7,26]. Additionally, ghrelin administration increases food intake and body weight gain [1,8,16,18,23,25,26]. Ghrelin secretion is upregulated under negative energy balance conditions, including starvation, insulin-induced hypoglycemia, cachexia, and anorexia nervosa [15,17,22,24]. Its secretion is downregulated under conditions of positive energy balance such as feeding, hyperglycemia, and obesity [15,17,22,24]. Ghrelin’s signals for starvation and GH release from the stomach ∗
Corresponding author. Tel.: +81 985 85 7972; fax: +81 985 85 7902. E-mail address: [email protected] (M. Nakazato).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.07.060
are relayed to the hindbrain via the gastric vagus nerve [4]. Until recently, most studies of the neuropharmacology of eating behavior have utilized a single nutritionally complete diet, measuring total food intake rather than intake of specific macronutrients. Certain orexigenic agents have been reported to induce the selection of specific macronutrients. Neuropeptide Y (NPY), the most potent orexigenic peptide as yet identified in the hypothalamus, preferentially stimulates selection of carbohydrate over fat and protein [14,20]. An intracerebroventricular (i.c.v.) injection of galanin, an orexigenic 29amino acid peptide produced in the central and peripheral nervous systems, causes a strong and selective enhancement of fat intake, with a significantly smaller increase in carbohydrate intake and no change in protein ingestion [21]. However, the effect of ghrelin on dietary carbohydrate and fat has yet to be studied. These might be of primary importance as dietary fat is an important factor involved in the development of obesity. Using a macronutrient self-administration paradigm, in
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T. Shimbara et al. / Neuroscience Letters 369 (2004) 75–79
Table 1 Time line of diet paradigm study (days)
Table 2 Composition of diets used in experiments Diet (% by weight) Standard Casein Cysteine Choline chloride Cornstarch Sucrose Soybean oil Fiber AIN vitamin mixture 93 AIN mineral mixture 93 Total (kcal)
which animals have access to two diets, we have examined the impact of i.c.v. injection of ghrelin on high-fat and highcarbohydrate diet intake and compared its effects with NPY and galanin. In this study, we demonstrate that ghrelin preferentially enhances fat ingestion. Male Wistar rats weighing 300–350 g (Charles River, Japan Inc., Shiga, Japan) were used in all experiments. Rats were housed individually in plastic cages under controlled temperature (21–23 ◦ C) and a 12-h light:12-h dark cycle (08:00–20:00 h) with ad libitum access to food and water. Intracerebroventricular cannulae were implanted into the lateral cerebral ventricle under anesthesia by intraperitoneal injection of sodium pentobarbital (Abbot Laboratories, Chicago, IL), and proper placement of the cannulae was verified at the end of the experiments by dye administration [6]. Rats were sham injected before the study, and weighed and handled daily. Only animals that showed progressive weight gain after the surgery were used in subsequent experiments. All experiments were repeated two or three times. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Miyazaki Medical College and the Japanese Physiological Society’s guidelines for animal care. Table 1 shows the time line of experimental protocol. Rats were fed standard laboratory diets for 7 days until they were placed on one of two diets (high-carbohydrate or high-fat diet; KBT Oriental Co., Ltd., Saga, Japan). In all experiments, rats were presented with two nutritionally complete diets that were high in either carbohydrate (63.7% of kilocalories derived from carbohydrates and 9.6% of kilocalories derived from fat) or fat (40.1% of kilocalories derived from carbohydrate and 34.7% of kilocalories derived from fat). The dietary compositions are shown in Table 2. Placement of food was changed daily to avoid placement preference and intake measurements were corrected for spillage. Next, rats were given free access to both high-carbohydrate and high-fat diets simultaneously and food intake was measured daily for 7 days. They were divided into two groups based on amounts of fat and carbohydrate consumed. These two subgroups were labeled as “high-carbohydrate (HC) preferring” in which rats consumed more than 50% carbohydrate and less than 25%
Carbohydrate Fat Protein
27.0 0.5 0.3 42.0 10.0 5.0 2.25 5.0 7.0 360.1 60.6% 13.4% 26.0%
High carbohydrate 27.0 0.5 0.3 43.6 10.0 3.4 2.25 5.0 7.0 352.1 63.7% 9.6% 26.6%
High fat 27.0 0.5 0.3 22.0 10.0 14.0 13.25 5.0 7.0 360.7 40.1% 34.7% 25.2%
fat in their diets, and “high-fat (HF) preferring” in which rats consumed more than 30% fat and less than 45% carbohydrate in their diets. The fat/carbohydrate preference ratio was based on the actual percentage of intake that is fat or carbohydrate. In the macronutrient selection test, rats were given free access to both high-carbohydrate and high-fat diets, and the daily food intake was measured for 5 days. Rat ghrelin (Peptide Institute, Inc., Osaka, Japan; 500 pmol/10 l saline), rat neuropeptide Y (Peptide Institute, Inc.; 1 nmol/10 l saline), rat galanin (Peptide Institute, Inc.; 1 nmol/10 l saline), or saline was administered intracerebroventricularly to freefeeding rats (n = 10 per group) in the early light phase (10:00 h). Same rats were given administrations of these peptides in a crossover experiment on separate days. Rats were presented with either a high-carbohydrate or a high-fat diet, and food-intake measurements were made at 1, 2 and 4 h postinjection. All the experiments were repeated twice in all groups of rats. In the diet preference test, rats were given free access to both high-carbohydrate and high-fat diets simultaneously, and the daily food intake was measured for 5 days. Ghrelin (500 pmol), neuropeptide Y (1 nmol), galanin (1 nmol), or saline was administered intracerebroventricularly to freefeeding rats (n = 10 per group) at 10:00 h. Rats were presented with both high-carbohydrate and high-fat diets, and foodintake measurements of both diets were made at 1, 2 and 4 h postinjection. All the experiments were repeated twice. Data are expressed as means ± S.E.M. Statistical analysis was determined by ANOVA with post hoc differences determined by Fisher’s protected least significant difference test. Differences were considered significant at P values less than 0.05. In the macronutrient selection test, i.c.v. administrations of ghrelin to both HC- and HF-preferring rats receiving either the high-carbohydrate or high-fat diet predominantly selected the high-fat over high-carbohydrate diet 2 h postinjection. In HC-preferring rats (Fig. 1A), ghrelin-treated rats selected the high-fat over high-carbohydrate diet (6.30 ± 0.23 g versus 4.46 ± 0.31 g, P < 0.001). In contrast,
T. Shimbara et al. / Neuroscience Letters 369 (2004) 75–79
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Fig. 1. Effects of i.c.v. administration during the light phase of ghrelin (500 pmol), NPY (1 nmol), and galanin (1 nmol) on macronutrient selection in (A) HC-preferring and (B) HF-preferring rats 2 h postinjection. Values are means ± S.E.M. for 10 rats. ∗ P < 0.05, ∗∗ P < 0.001, ∗∗∗ P < 0.0001 vs. high-carbohydrate diet. High-carbohydrate diet (white column) and high-fat diet (black column).
NPY-treated rats predominantly chose the high-carbohydrate over high-fat diet (8.03 ± 0.47 g versus 6.19 ± 0.52 g, P < 0.05) and galanin-treated rats selected both the highcarbohydrate and high-fat diets (3.85 ± 0.38 g versus 2.96 ± 0.43 g, not significant) in these rats. In HF-preferring rats (Fig. 1B), both ghrelin- and galanin-treated rats selected the high-fat over high-carbohydrate diet (ghrelin, 5.94 ± 0.27 g versus 3.28 ± 0.42 g, P < 0.0001; galanin, 3.90 ± 0.36 g versus 2.79 ± 0.33 g, P < 0.05). In contrast, NPY-treated rats selected both the high-carbohydrate and high-fat diets (7.63 ± 1.00 g versus 6.44 ± 1.01 g, not significant). In the diet preference test, i.c.v. administrations of ghrelin to both HC- and HF-preferring rats receiving both the highcarbohydrate and high-fat diets, predominantly selected the high-fat over high-carbohydrate diet 2 h postinjection. In HCpreferring rats (Fig. 2A), both ghrelin- and galanin-treated rats selected high-fat over high-carbohydrate diet (ghrelin, 6.26 ± 0.53 g versus 0.70 ± 0.35 g, P < 0.0001; galanin, 3.30 ± 0.18 g versus 0.11 ± 0.07 g, P < 0.0001) 2 h postinjection. In contrast, NPY-treated rats preferentially consumed the high-carbohydrate over high-fat diet (8.32 ± 1.05 g versus 0.87 ± 0.40 g; P < 0.0001). In HF-preferring rats (Fig. 2B), both ghrelin- and galanin-treated rats preferred the high-fat over high-carbohydrate diet (ghrelin, 6.30 ± 0.35 g versus 0.00 ± 0.00 g, P < 0.0001; galanin, 3.36 ± 1.02 g versus 0.00 ± 0.00 g, P < 0.0001). NPY-treated rats also preferred the high-fat diet, but consumed a comparatively large amount of high-carbohydrate food (6.70 ± 0.64 g versus 2.50 ± 0.26 g, P < 0.0001).
Fig. 2. Effects of i.c.v. administration during the light phase of ghrelin (500 pmol), NPY (1 nmol), and galanin (1 nmol) on diet preference in (A) HC-preferring and (B) HF-preferring rats 2 h postinjection. Values are means ± S.E.M. for 10 rats. ∗ P < 0.0001 vs. high-carbohydrate diet. Highcarbohydrate diet (white column) and high-fat diet (black column).
Fig. 3. Time course of the effects of ghrelin (500 pmol) on macronutrient selection (A and B) and diet preference (C and D) in HC- and HF-preferring rats 1, 2, and 4 h postinjection. Values are means ± S.E.M. for 10 rats. ∗ P < 0.05, ∗∗ P < 0.001, ∗∗∗ P < 0.0001 vs. high-carbohydrate diet. Highcarbohydrate diet (white column) and high-fat diet (black column).
Fig. 3 shows the time course of ghrelin’s effects on macronutrient selection (A and B) and diet preference (C and D) in both HC- and HF-preferring rats. Intracerebroventricular administration of ghrelin significantly enhanced fat ingestion over carbohydrate in rats at 1 h postinjection and reached peak level at 2 h postinjection (A−D). Intracerebroventricular administrations of galanin and NPY potently enhanced fat and carbohydrate ingestion, respectively, at 1 h postinjection in the same rats that consumed fat in response to ghrelin, and both peptides reached peak level at 2 h postinjection (data not shown). Since orexigenic activities of these three peptides were sustained during 2 h postinjection, we compared the 2-h feeding data in Figs. 1 and 2. Consistent with previous findings, results in this study demonstrate that central administration of ghrelin has a strong stimulatory effect on feeding behavior [1,8,16,18,23,25,26]. This effect is attributable to a preferential enhancement of fat consumption over carbohydrate. This pattern of macronutrient intake after i.c.v. injection of ghrelin is distinctive and comparable to that of galanin, which also strongly enhances fat ingestion [21]. Ghrelin is primarily produced in the mucosal endocrine cells of the stomach [2], but it is also synthesized in the hypothalamic arcuate nucleus [7,13], a region critical for feeding. Ghrelin fibers project to the paraventricular nucleus (PVN), a nucleus that has an important role in modulating nutrient ingestion [12] and a region of high expression of ghrelin receptors [5]. Intracerebroventricular administration of ghrelin induces Fos expression in the PVN [16], suggesting that ghrelin activates neurons in this structure. Galanin-producing neurons are highly expressed in the PVN [21]. Central ad-
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ministration of galanin strongly and preferentially enhanced ingestion of fat over carbohydrate [21]. We here confirmed that galanin enhanced fat ingestion. Ghrelin may function in the PVN to enhance fat ingestion preferentially. There are distinct differences between the neuropeptides that stimulate fat and carbohydrate ingestion. The peptides that stimulate carbohydrate are particularly active during the early hours of the feeding cycle and are dependent on corticosterone and its receptors, which normally peak at this time [10]. NPY’s activity is increased at the start of the natural feeding cycle when carbohydrates are normally preferred [5,19]. This is in contrast to the peptides that stimulate fat intake, which are most potent during the later hours of the cycle and can function independently of corticosterone, which is at low blood levels [9,11]. In animals and humans, appetite for fat progressively rises over the course of the natural feeding cycle, and this appetite shift may be attributed to the increased action of the neuropeptides that stimulate fat ingestion. Galanin enhances fat ingestion through its action in the latter part of the feeding cycle when fat is normally preferred [12,19]. These findings suggest that central ghrelin may stimulate fat ingestion in the latter part of the natural feeding cycle. In summary, we have demonstrated that central administration of ghrelin preferentially enhances fat ingestion, whereas it has a small effect on carbohydrate intake. Dietary fat is a primary contributor to body weight gain and to the development of obesity, producing less satiety and greater total intake, fat deposition, and positive energy balance relative to the other major nutrients. In addition to obesity, fat ingestion and fat accumulation are linked to type II diabetes, cardiovascular disease, and hypertension. The present result provides a foundation for future studies attempting to understand how specific metabolic, endocrine, and environmental signals modulate the activity of ghrelin in the control of appetite for fat.
Acknowledgements This study was supported in part by the 21st Century COE Program and grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Ministry of Health, Labor, and Welfare of Japan; Uehara Memorial Foundation; Ono Medical Research Foundation; Mitsui Life Social Welfare Foundation; Japan Health Foundation for the Prevention of Chronic Diseases and the Improvement of QOL of Patients; Yamanouchi Foundation for Research on Metabolic Disorders to M.N.
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Central administration of ghrelin preferentially enhances fat ingestion Takuya Shimbara, Muhtashan S. Mondal, Takashi Kawagoe, Koji Toshinai, Shuichi Koda, Hideki Yamaguchi, Yukari Date, Masamitsu Nakazato∗ Third Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan Received 10 May 2004; received in revised form 15 July 2004; accepted 28 July 2004
Abstract Ghrelin, a brain-gut peptide discovered from the stomach, stimulates growth hormone release, food intake, adiposity, and weight gain. Circulating ghrelin levels are modulated under conditions of positive and negative energy balance, however its effect on macronutrient selection is not known. The present experiment investigates the effect of ghrelin on single and two-diet feeding paradigms in high-carbohydrate (HC) and high-fat (HF) preferring rats. In the macronutrient selection test in which rats were given free access to either high-carbohydrate or high-fat diet, an intracerebroventricular (i.c.v.) administration of ghrelin potently enhanced fat intake over carbohydrate intake in both HC- and HF-preferring rats. In the diet preference test in which rats were given free access to both high-carbohydrate and high-fat diets simultaneously, an i.c.v. administration of ghrelin also preferentially enhanced fat consumption over carbohydrate in both HF- and HCpreferring rats. Intracerebroventricular administrations of galanin and neuropeptide Y enhanced fat and carbohydrate ingestion, respectively. Centrally administered ghrelin enhanced fat ingestion. These results provide further insights for the role of ghrelin in feeding behavior and the development of obesity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Ghrelin; Neuropeptide Y; Galanin; Central administration; Macronutrient selection
Ghrelin, a 28-amino-acid peptide with an n-octanoyl modification indispensable for its activity, was originally discovered in human and rat stomach as an endogenous ligand for the growth hormone (GH) secretagogue receptor [7]. Ghrelin homologues have been identified in fish, amphibians, birds, and many mammals. Ghrelin stimulates GH release when administered in humans and rodents and when applied directly to rat primary pituitary cells [3,7,26]. Additionally, ghrelin administration increases food intake and body weight gain [1,8,16,18,23,25,26]. Ghrelin secretion is upregulated under negative energy balance conditions, including starvation, insulin-induced hypoglycemia, cachexia, and anorexia nervosa [15,17,22,24]. Its secretion is downregulated under conditions of positive energy balance such as feeding, hyperglycemia, and obesity [15,17,22,24]. Ghrelin’s signals for starvation and GH release from the stomach ∗
Corresponding author. Tel.: +81 985 85 7972; fax: +81 985 85 7902. E-mail address: [email protected] (M. Nakazato).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.07.060
are relayed to the hindbrain via the gastric vagus nerve [4]. Until recently, most studies of the neuropharmacology of eating behavior have utilized a single nutritionally complete diet, measuring total food intake rather than intake of specific macronutrients. Certain orexigenic agents have been reported to induce the selection of specific macronutrients. Neuropeptide Y (NPY), the most potent orexigenic peptide as yet identified in the hypothalamus, preferentially stimulates selection of carbohydrate over fat and protein [14,20]. An intracerebroventricular (i.c.v.) injection of galanin, an orexigenic 29amino acid peptide produced in the central and peripheral nervous systems, causes a strong and selective enhancement of fat intake, with a significantly smaller increase in carbohydrate intake and no change in protein ingestion [21]. However, the effect of ghrelin on dietary carbohydrate and fat has yet to be studied. These might be of primary importance as dietary fat is an important factor involved in the development of obesity. Using a macronutrient self-administration paradigm, in
76
T. Shimbara et al. / Neuroscience Letters 369 (2004) 75–79
Table 1 Time line of diet paradigm study (days)
Table 2 Composition of diets used in experiments Diet (% by weight) Standard Casein Cysteine Choline chloride Cornstarch Sucrose Soybean oil Fiber AIN vitamin mixture 93 AIN mineral mixture 93 Total (kcal)
which animals have access to two diets, we have examined the impact of i.c.v. injection of ghrelin on high-fat and highcarbohydrate diet intake and compared its effects with NPY and galanin. In this study, we demonstrate that ghrelin preferentially enhances fat ingestion. Male Wistar rats weighing 300–350 g (Charles River, Japan Inc., Shiga, Japan) were used in all experiments. Rats were housed individually in plastic cages under controlled temperature (21–23 ◦ C) and a 12-h light:12-h dark cycle (08:00–20:00 h) with ad libitum access to food and water. Intracerebroventricular cannulae were implanted into the lateral cerebral ventricle under anesthesia by intraperitoneal injection of sodium pentobarbital (Abbot Laboratories, Chicago, IL), and proper placement of the cannulae was verified at the end of the experiments by dye administration [6]. Rats were sham injected before the study, and weighed and handled daily. Only animals that showed progressive weight gain after the surgery were used in subsequent experiments. All experiments were repeated two or three times. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Miyazaki Medical College and the Japanese Physiological Society’s guidelines for animal care. Table 1 shows the time line of experimental protocol. Rats were fed standard laboratory diets for 7 days until they were placed on one of two diets (high-carbohydrate or high-fat diet; KBT Oriental Co., Ltd., Saga, Japan). In all experiments, rats were presented with two nutritionally complete diets that were high in either carbohydrate (63.7% of kilocalories derived from carbohydrates and 9.6% of kilocalories derived from fat) or fat (40.1% of kilocalories derived from carbohydrate and 34.7% of kilocalories derived from fat). The dietary compositions are shown in Table 2. Placement of food was changed daily to avoid placement preference and intake measurements were corrected for spillage. Next, rats were given free access to both high-carbohydrate and high-fat diets simultaneously and food intake was measured daily for 7 days. They were divided into two groups based on amounts of fat and carbohydrate consumed. These two subgroups were labeled as “high-carbohydrate (HC) preferring” in which rats consumed more than 50% carbohydrate and less than 25%
Carbohydrate Fat Protein
27.0 0.5 0.3 42.0 10.0 5.0 2.25 5.0 7.0 360.1 60.6% 13.4% 26.0%
High carbohydrate 27.0 0.5 0.3 43.6 10.0 3.4 2.25 5.0 7.0 352.1 63.7% 9.6% 26.6%
High fat 27.0 0.5 0.3 22.0 10.0 14.0 13.25 5.0 7.0 360.7 40.1% 34.7% 25.2%
fat in their diets, and “high-fat (HF) preferring” in which rats consumed more than 30% fat and less than 45% carbohydrate in their diets. The fat/carbohydrate preference ratio was based on the actual percentage of intake that is fat or carbohydrate. In the macronutrient selection test, rats were given free access to both high-carbohydrate and high-fat diets, and the daily food intake was measured for 5 days. Rat ghrelin (Peptide Institute, Inc., Osaka, Japan; 500 pmol/10 l saline), rat neuropeptide Y (Peptide Institute, Inc.; 1 nmol/10 l saline), rat galanin (Peptide Institute, Inc.; 1 nmol/10 l saline), or saline was administered intracerebroventricularly to freefeeding rats (n = 10 per group) in the early light phase (10:00 h). Same rats were given administrations of these peptides in a crossover experiment on separate days. Rats were presented with either a high-carbohydrate or a high-fat diet, and food-intake measurements were made at 1, 2 and 4 h postinjection. All the experiments were repeated twice in all groups of rats. In the diet preference test, rats were given free access to both high-carbohydrate and high-fat diets simultaneously, and the daily food intake was measured for 5 days. Ghrelin (500 pmol), neuropeptide Y (1 nmol), galanin (1 nmol), or saline was administered intracerebroventricularly to freefeeding rats (n = 10 per group) at 10:00 h. Rats were presented with both high-carbohydrate and high-fat diets, and foodintake measurements of both diets were made at 1, 2 and 4 h postinjection. All the experiments were repeated twice. Data are expressed as means ± S.E.M. Statistical analysis was determined by ANOVA with post hoc differences determined by Fisher’s protected least significant difference test. Differences were considered significant at P values less than 0.05. In the macronutrient selection test, i.c.v. administrations of ghrelin to both HC- and HF-preferring rats receiving either the high-carbohydrate or high-fat diet predominantly selected the high-fat over high-carbohydrate diet 2 h postinjection. In HC-preferring rats (Fig. 1A), ghrelin-treated rats selected the high-fat over high-carbohydrate diet (6.30 ± 0.23 g versus 4.46 ± 0.31 g, P < 0.001). In contrast,
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Fig. 1. Effects of i.c.v. administration during the light phase of ghrelin (500 pmol), NPY (1 nmol), and galanin (1 nmol) on macronutrient selection in (A) HC-preferring and (B) HF-preferring rats 2 h postinjection. Values are means ± S.E.M. for 10 rats. ∗ P < 0.05, ∗∗ P < 0.001, ∗∗∗ P < 0.0001 vs. high-carbohydrate diet. High-carbohydrate diet (white column) and high-fat diet (black column).
NPY-treated rats predominantly chose the high-carbohydrate over high-fat diet (8.03 ± 0.47 g versus 6.19 ± 0.52 g, P < 0.05) and galanin-treated rats selected both the highcarbohydrate and high-fat diets (3.85 ± 0.38 g versus 2.96 ± 0.43 g, not significant) in these rats. In HF-preferring rats (Fig. 1B), both ghrelin- and galanin-treated rats selected the high-fat over high-carbohydrate diet (ghrelin, 5.94 ± 0.27 g versus 3.28 ± 0.42 g, P < 0.0001; galanin, 3.90 ± 0.36 g versus 2.79 ± 0.33 g, P < 0.05). In contrast, NPY-treated rats selected both the high-carbohydrate and high-fat diets (7.63 ± 1.00 g versus 6.44 ± 1.01 g, not significant). In the diet preference test, i.c.v. administrations of ghrelin to both HC- and HF-preferring rats receiving both the highcarbohydrate and high-fat diets, predominantly selected the high-fat over high-carbohydrate diet 2 h postinjection. In HCpreferring rats (Fig. 2A), both ghrelin- and galanin-treated rats selected high-fat over high-carbohydrate diet (ghrelin, 6.26 ± 0.53 g versus 0.70 ± 0.35 g, P < 0.0001; galanin, 3.30 ± 0.18 g versus 0.11 ± 0.07 g, P < 0.0001) 2 h postinjection. In contrast, NPY-treated rats preferentially consumed the high-carbohydrate over high-fat diet (8.32 ± 1.05 g versus 0.87 ± 0.40 g; P < 0.0001). In HF-preferring rats (Fig. 2B), both ghrelin- and galanin-treated rats preferred the high-fat over high-carbohydrate diet (ghrelin, 6.30 ± 0.35 g versus 0.00 ± 0.00 g, P < 0.0001; galanin, 3.36 ± 1.02 g versus 0.00 ± 0.00 g, P < 0.0001). NPY-treated rats also preferred the high-fat diet, but consumed a comparatively large amount of high-carbohydrate food (6.70 ± 0.64 g versus 2.50 ± 0.26 g, P < 0.0001).
Fig. 2. Effects of i.c.v. administration during the light phase of ghrelin (500 pmol), NPY (1 nmol), and galanin (1 nmol) on diet preference in (A) HC-preferring and (B) HF-preferring rats 2 h postinjection. Values are means ± S.E.M. for 10 rats. ∗ P < 0.0001 vs. high-carbohydrate diet. Highcarbohydrate diet (white column) and high-fat diet (black column).
Fig. 3. Time course of the effects of ghrelin (500 pmol) on macronutrient selection (A and B) and diet preference (C and D) in HC- and HF-preferring rats 1, 2, and 4 h postinjection. Values are means ± S.E.M. for 10 rats. ∗ P < 0.05, ∗∗ P < 0.001, ∗∗∗ P < 0.0001 vs. high-carbohydrate diet. Highcarbohydrate diet (white column) and high-fat diet (black column).
Fig. 3 shows the time course of ghrelin’s effects on macronutrient selection (A and B) and diet preference (C and D) in both HC- and HF-preferring rats. Intracerebroventricular administration of ghrelin significantly enhanced fat ingestion over carbohydrate in rats at 1 h postinjection and reached peak level at 2 h postinjection (A−D). Intracerebroventricular administrations of galanin and NPY potently enhanced fat and carbohydrate ingestion, respectively, at 1 h postinjection in the same rats that consumed fat in response to ghrelin, and both peptides reached peak level at 2 h postinjection (data not shown). Since orexigenic activities of these three peptides were sustained during 2 h postinjection, we compared the 2-h feeding data in Figs. 1 and 2. Consistent with previous findings, results in this study demonstrate that central administration of ghrelin has a strong stimulatory effect on feeding behavior [1,8,16,18,23,25,26]. This effect is attributable to a preferential enhancement of fat consumption over carbohydrate. This pattern of macronutrient intake after i.c.v. injection of ghrelin is distinctive and comparable to that of galanin, which also strongly enhances fat ingestion [21]. Ghrelin is primarily produced in the mucosal endocrine cells of the stomach [2], but it is also synthesized in the hypothalamic arcuate nucleus [7,13], a region critical for feeding. Ghrelin fibers project to the paraventricular nucleus (PVN), a nucleus that has an important role in modulating nutrient ingestion [12] and a region of high expression of ghrelin receptors [5]. Intracerebroventricular administration of ghrelin induces Fos expression in the PVN [16], suggesting that ghrelin activates neurons in this structure. Galanin-producing neurons are highly expressed in the PVN [21]. Central ad-
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ministration of galanin strongly and preferentially enhanced ingestion of fat over carbohydrate [21]. We here confirmed that galanin enhanced fat ingestion. Ghrelin may function in the PVN to enhance fat ingestion preferentially. There are distinct differences between the neuropeptides that stimulate fat and carbohydrate ingestion. The peptides that stimulate carbohydrate are particularly active during the early hours of the feeding cycle and are dependent on corticosterone and its receptors, which normally peak at this time [10]. NPY’s activity is increased at the start of the natural feeding cycle when carbohydrates are normally preferred [5,19]. This is in contrast to the peptides that stimulate fat intake, which are most potent during the later hours of the cycle and can function independently of corticosterone, which is at low blood levels [9,11]. In animals and humans, appetite for fat progressively rises over the course of the natural feeding cycle, and this appetite shift may be attributed to the increased action of the neuropeptides that stimulate fat ingestion. Galanin enhances fat ingestion through its action in the latter part of the feeding cycle when fat is normally preferred [12,19]. These findings suggest that central ghrelin may stimulate fat ingestion in the latter part of the natural feeding cycle. In summary, we have demonstrated that central administration of ghrelin preferentially enhances fat ingestion, whereas it has a small effect on carbohydrate intake. Dietary fat is a primary contributor to body weight gain and to the development of obesity, producing less satiety and greater total intake, fat deposition, and positive energy balance relative to the other major nutrients. In addition to obesity, fat ingestion and fat accumulation are linked to type II diabetes, cardiovascular disease, and hypertension. The present result provides a foundation for future studies attempting to understand how specific metabolic, endocrine, and environmental signals modulate the activity of ghrelin in the control of appetite for fat.
Acknowledgements This study was supported in part by the 21st Century COE Program and grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Ministry of Health, Labor, and Welfare of Japan; Uehara Memorial Foundation; Ono Medical Research Foundation; Mitsui Life Social Welfare Foundation; Japan Health Foundation for the Prevention of Chronic Diseases and the Improvement of QOL of Patients; Yamanouchi Foundation for Research on Metabolic Disorders to M.N.
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