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Central administration of leucine, but not isoleucine and valine, stimulates feeding behavior in neonatal chicks
Neuroscience Letters 354 (2004) 166–168 www.elsevier.com/locate/neulet
Central administration of leucine, but not isoleucine and valine, stimulates feeding behavior in neonatal chicks Tomofumi Izumi, Kazuya Kawamura, Hiroshi Ueda, Takashi Bungo* Laboratory of Animal Science, Department of Agrobiological Science, Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan Received 14 July 2003; received in revised form 16 September 2003; accepted 26 September 2003
Abstract Branched-chain amino acids (BCAAs) are essential amino acids that play a major role in brain energy metabolism. This study was done to elucidate whether central injection of BCAAs influences feeding behavior in chicks. We found that the intracerebroventricular injection of leucine (200 mg) significantly stimulated food intake in neonatal chicks during 30 min postinjection. Additionally, the starting time of feeding and pecking rhythm after injection were significantly accelerated by leucine. In contrast, isoleucine and valine had no effect on ingestive response during experiment periods. Moreover, a metabolite of leucine (a-ketoisocaproic acid) at an equimolar concentration of leucine also did not increase food intake in chicks. These results suggest that leucine induces hyperphagia of neonatal chicks and it may be due to the synthesized glutamate by exogenous leucine. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Feeding behavior; Leucine; Isoleucine; Valine; a-Ketoisocaproic acid; Central nervous system; Chick
The central nervous system plays an important role in regulating the feeding behavior of animals. This behavior is known to be controlled by multiple neurotransmitters including amino acids such as glutamate and g-aminobutyric acid [5]. In addition, neuromodulators are also important factors in the regulation of feeding behavior. Some amino acids have been shown to act as the neuromodulator and affect ingestive behavior. Injection of an amino acid mixture into the hypothalamus inhibits subsequent feeding response [7] and shows that the lateral hypothalamus region is highly sensitive to iontophoresis of essential amino acids [10]. Branched-chain amino acids (BCAAs) modulate various cellular functions, and easily cross the blood – brain barrier [6], with the influx of leucine exceeding that of other amino acids [8]. Moreover, the BCAAs in brain slices are much more rapidly metabolized than incorporated into protein [2]. Therefore, it appears that BCAAs play a major role in brain energy metabolism, and hold an important position in brain nitrogen metabolism. However, their function in controlling any behavior of animals, especially feeding behavior, is unknown. The aim of this study is to elucidate whether * Corresponding author. Tel./fax: þ81-89-946-9820. E-mail address: [email protected] (T. Bungo).
central injection of BCAAs affects the feeding behavior of the neonatal chick. A further experiment was undertaken to explore the influence of a metabolite of leucine (aketoisocaproic acid). Day-old male Leghorn chicks were purchased from a local hatchery (Kudoh-sha, Ehime, Japan). Birds were maintained in a room with 24 h lighting and at a temperature of 30 8C. They were given free access to a commercial starter diet (Nihon Nosan Kogyo Co. Ltd., Yokohama, Japan) and water during the pre-experimental period. Chicks were maintained in accordance with the recommendations of the National Research Council [4]. They were distributed into experimental groups based on their body weight so that the average body weight was as uniform as possible for each treatment. The birds were reared individually in experimental cages and had ad libitum access to food up to the time of the experiments. The birds were given intracerebroventricular (ICV) injections with the solutions (10 ml) using a microsyringe according to the methods used by Davis et al. [3]. L Leucine, L -isoleucine and L -valine were donated by Kyowa Hakko Kogyo Co. Ltd. (Tokyo, Japan). a-Ketoisocaproic acid was purchased from Sigma (St. Louis, MO). The drugs were dissolved in 0.1% Evans Blue solution, which was prepared in 0.85% saline. Saline containing Evans Blue was
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.071
T. Izumi et al. / Neuroscience Letters 354 (2004) 166–168
used as a control. Each chick was injected only once with either one of several doses of drugs or saline. At the end of the experiments, the birds were sacrificed by decapitation after which the location of the injection site was confirmed. Data from individuals having injections that were not verified by the presence of Evans Blue dye in the lateral ventricle were deleted. The number of birds used for data analysis is shown in each figure and table. In Experiments 1, 2 and 3, birds (4-day-old) given free access to food were injected with doses of 50, 100 and 200 mg of each BCAA. In Experiment 4, birds (3-day-old) were injected by the ICV route with four levels (0, 119, 238 and 475 mg) of a-ketoisocaproic acid based on equimolar concentrations of Experiment 1. Food intake was determined 30 min after each injection. In Experiment 5, the birds (2-day-old) were divided into two groups that received either saline or leucine (200 mg). Immediately after the ICV injection, the birds were placed individually in the monitoring cage and the behavior of each bird was recorded for 10 min using a video recording set. Data were collected for the starting time of feeding after injection, food pecking frequency and feeding time (the nearest second for the duration of time during which the chick stayed to peck at the feeder). In addition, pecking rhythm was calculated as food pecking frequency divided by feeding time. In Experiments 1, 2, 3 and 4, ANOVA was used to determine overall statistical significance due to treatment. When a treatment effect was significant, Fisher’s protected LSD test was used to compare the significance among means. The significance of dose was set at P , 0:05. In Experiment 5, data were analyzed using the Mann – Whitney U-test. Results are presented as means ^ SEM. The food intake of chicks was significantly increased by L -leucine (200 mg) when compared with control at 30 min postinjection (Fig. 1A; Fð3; 56Þ ¼ 3:341, P , 0:05). In addition, all parameters for feeding behavior in the leucine group implied hyperphagia (Table 1), and this result is in good agreement with the result of food intake. In particular, starting time of feeding and pecking rhythm were significantly accelerated by leucine (Table 1; P , 0:05). On the other hand, neither L -isoleucine nor L -valine had any effect on food intake compared with saline control during the experimental period (Fig. 1B,C; P . 0:05). We do not have information from the present study as to how leucine alone modulates feeding regulation in the central nervous system of chicks, but some hypotheses may be proposed. First, because the catabolic route of leucine is different from both
167
Fig. 1. Food intake of chicks injected ICV with saline or one of three doses (50, 100 or 200 mg) of (A) L -leucine, (B) L -isoleucine or (C) L -valine. Values are means ^ SEM. The number of chicks used is shown in parentheses. *P , 0:05, compared with saline control. (A) Food intake (g/30 minÞ ¼ 0:29ðSE 0.08) þ 0.02(SE 0.01)X, R2 ¼ 0:148, P , 0:01.
isoleucine and valine, we hypothesize that a metabolite of leucine increases food intake in chicks. Leucine is transaminated to produce glutamate and a-ketoisocaproic acid, and a-ketoisocaproic acid is converted to acetoacetylCoA in the brain [11]. On the other hand, the ketoacids of isoleucine and valine are converted to succinyl-CoA [11]. However, ICV injection of a-ketoisocaproate at an equimolar concentration to the L -leucine used here had no effect on food intake compared with the saline control during the experiment period (Fig. 2; P . 0:05). Therefore, it is unlikely that this metabolite of leucine increases food intake in chicks. Secondly, it can be assumed that glutamate resulting from the exogenous leucine stimulates feeding behavior. There are some reports that BCAAs, particularly leucine,
Table 1 Influence of central administration of leucine on feeding behavior of chicks
Control Leucine P value
Feeding time (s)
Pecking frequency for food
Pecking rhythm
Starting time of feeding (s)
140.6 ^ 62.4 267.0 ^ 66.1 0.175
99.6 ^ 42.2 314.6 ^ 87.8 0.076
0.78 ^ 0.10 1.17 ^ 0.15 0.028
310.0 ^ 48.5 151.8 ^ 23.2 0.047
Pecking rhythm was calculated as food pecking frequency divided by feeding time. Values are means ^ SEM of five birds.
168
T. Izumi et al. / Neuroscience Letters 354 (2004) 166–168
research from the Ministry of Education, Science and Culture, Japan. We wish to thank Kyowa Hakko Kogyo Co. Ltd. for supplying leucine, isoleucine and valine.
References
Fig. 2. Food intake of chicks injected ICV with saline or one of three doses of a-ketoisocaproic acid (119, 238 or 475 mg). Values are means ^ SEM. The number of chicks used is shown in parentheses.
are important as external sources of nitrogen for the synthesis of glutamate and glutamine [1,12]. Moreover, many reports have shown that glutamate injected into the brain elicits a robust feeding response in satiated rats (e.g. Ref. [9]). Thus, it is possible that the orexigenic effect of leucine was due to accelerated synthesis of glutamate. Additionally, because isoleucine and valine are also utilized as sources of nitrogen for the synthesis of glutamate, it is probable that the higher doses of other BCAAs also will stimulate feeding behavior in chicks. The results presented here suggest that L -leucine induces hyperphagia and BCAA(s) may have an important role in the feeding behavior of chicks. However, further experiments will be required to determine the mechanism of leucine’s effect on ingestive behavior in chicks. To our knowledge, this is the first report of a central effect of L leucine on feeding behavior in birds.
Acknowledgements This study was supported by a grant-in-aid for scientific
[1] M.G. Bixel, S.M. Huston, B. Hamprecht, Cellular distribution of branched-chain amino acid aminotransferase isoenzyme among rat brain glial cells in culture, J. Histochem. Cytochem. 45 (1997) 685 –694. [2] E.R. Chaplin, A.L. Goldberg, I. Diamond, Leucine oxidation in brain slices and nerve endings, J. Neurochem. 26 (1976) 710–717. [3] J.L. Davis, D.T. Masuoka, J.F. Gerbrandt, A. Cherkin, Autoradiographic distribution of L -proline in chicks after intracerebral injection, Physiol. Behav. 22 (1979) 693–695. [4] National Research Council, Guide for the Care and Use of Laboratory Animals. NIH Publ. No. 85-23, Department of Health and Human Services, Washington, DC, 1985. [5] F. Nishimura, M. Nishihara, M. Mori, K. Torii, M. Takahashi, Excitability of neurons in the ventromedial nucleus in rat hypothalamic slices: modulation by amino acids at cerebrospinal fluid levels, Brain Res. 691 (1995) 217–222. [6] W.H. Oldendorf, Brain uptake of radiolabeled amino acids, amines and hexoses after arterial injection, Am. J. Physiol. 221 (1971) 1629–1635. [7] J. Panksepp, D.A. Booth, Decreased feeding after injection of amino acids into the hypothalamus, Nature 233 (1971) 341– 342. [8] Q.R. Smith, S. Momma, M. Aoyagi, S.I. Rapport, Kinetics of neutral amino acid transport across the blood-brain barrier, J. Neurochem. 49 (1987) 1651–1658. [9] B.G. Stanley, V.L. Willett III, H.W. Donias, L.H. Ha, L.C. Spears, The lateral hypothalamus: a primary site mediating excitatory amino acid-elicited eating, Brain Res. 63 (1993) 41–49. [10] M.J. Wayner, T. Ono, A. Deyoung, F.C. Barone, Effects of essential amino acids on central neurons, Pharmacol. Biochem. Behav. 3 (Suppl. 1) (1975) 85– 90. [11] M. Yudkoff, Brain metabolism of branched-chain amino acids, Glia 21 (1997) 92–98. [12] M. Yudkoff, Y. Daikihin, L. Glunstein, I. Nissim, J. Stern, D. Pleasure, I. Nissim, Astrocyte leucine metabolism: significance of branched-chain amino acid transamination, J. Neurochem. 66 (1996) 378 –385.
Central administration of leucine, but not isoleucine and valine, stimulates feeding behavior in neonatal chicks Tomofumi Izumi, Kazuya Kawamura, Hiroshi Ueda, Takashi Bungo* Laboratory of Animal Science, Department of Agrobiological Science, Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan Received 14 July 2003; received in revised form 16 September 2003; accepted 26 September 2003
Abstract Branched-chain amino acids (BCAAs) are essential amino acids that play a major role in brain energy metabolism. This study was done to elucidate whether central injection of BCAAs influences feeding behavior in chicks. We found that the intracerebroventricular injection of leucine (200 mg) significantly stimulated food intake in neonatal chicks during 30 min postinjection. Additionally, the starting time of feeding and pecking rhythm after injection were significantly accelerated by leucine. In contrast, isoleucine and valine had no effect on ingestive response during experiment periods. Moreover, a metabolite of leucine (a-ketoisocaproic acid) at an equimolar concentration of leucine also did not increase food intake in chicks. These results suggest that leucine induces hyperphagia of neonatal chicks and it may be due to the synthesized glutamate by exogenous leucine. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Feeding behavior; Leucine; Isoleucine; Valine; a-Ketoisocaproic acid; Central nervous system; Chick
The central nervous system plays an important role in regulating the feeding behavior of animals. This behavior is known to be controlled by multiple neurotransmitters including amino acids such as glutamate and g-aminobutyric acid [5]. In addition, neuromodulators are also important factors in the regulation of feeding behavior. Some amino acids have been shown to act as the neuromodulator and affect ingestive behavior. Injection of an amino acid mixture into the hypothalamus inhibits subsequent feeding response [7] and shows that the lateral hypothalamus region is highly sensitive to iontophoresis of essential amino acids [10]. Branched-chain amino acids (BCAAs) modulate various cellular functions, and easily cross the blood – brain barrier [6], with the influx of leucine exceeding that of other amino acids [8]. Moreover, the BCAAs in brain slices are much more rapidly metabolized than incorporated into protein [2]. Therefore, it appears that BCAAs play a major role in brain energy metabolism, and hold an important position in brain nitrogen metabolism. However, their function in controlling any behavior of animals, especially feeding behavior, is unknown. The aim of this study is to elucidate whether * Corresponding author. Tel./fax: þ81-89-946-9820. E-mail address: [email protected] (T. Bungo).
central injection of BCAAs affects the feeding behavior of the neonatal chick. A further experiment was undertaken to explore the influence of a metabolite of leucine (aketoisocaproic acid). Day-old male Leghorn chicks were purchased from a local hatchery (Kudoh-sha, Ehime, Japan). Birds were maintained in a room with 24 h lighting and at a temperature of 30 8C. They were given free access to a commercial starter diet (Nihon Nosan Kogyo Co. Ltd., Yokohama, Japan) and water during the pre-experimental period. Chicks were maintained in accordance with the recommendations of the National Research Council [4]. They were distributed into experimental groups based on their body weight so that the average body weight was as uniform as possible for each treatment. The birds were reared individually in experimental cages and had ad libitum access to food up to the time of the experiments. The birds were given intracerebroventricular (ICV) injections with the solutions (10 ml) using a microsyringe according to the methods used by Davis et al. [3]. L Leucine, L -isoleucine and L -valine were donated by Kyowa Hakko Kogyo Co. Ltd. (Tokyo, Japan). a-Ketoisocaproic acid was purchased from Sigma (St. Louis, MO). The drugs were dissolved in 0.1% Evans Blue solution, which was prepared in 0.85% saline. Saline containing Evans Blue was
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.071
T. Izumi et al. / Neuroscience Letters 354 (2004) 166–168
used as a control. Each chick was injected only once with either one of several doses of drugs or saline. At the end of the experiments, the birds were sacrificed by decapitation after which the location of the injection site was confirmed. Data from individuals having injections that were not verified by the presence of Evans Blue dye in the lateral ventricle were deleted. The number of birds used for data analysis is shown in each figure and table. In Experiments 1, 2 and 3, birds (4-day-old) given free access to food were injected with doses of 50, 100 and 200 mg of each BCAA. In Experiment 4, birds (3-day-old) were injected by the ICV route with four levels (0, 119, 238 and 475 mg) of a-ketoisocaproic acid based on equimolar concentrations of Experiment 1. Food intake was determined 30 min after each injection. In Experiment 5, the birds (2-day-old) were divided into two groups that received either saline or leucine (200 mg). Immediately after the ICV injection, the birds were placed individually in the monitoring cage and the behavior of each bird was recorded for 10 min using a video recording set. Data were collected for the starting time of feeding after injection, food pecking frequency and feeding time (the nearest second for the duration of time during which the chick stayed to peck at the feeder). In addition, pecking rhythm was calculated as food pecking frequency divided by feeding time. In Experiments 1, 2, 3 and 4, ANOVA was used to determine overall statistical significance due to treatment. When a treatment effect was significant, Fisher’s protected LSD test was used to compare the significance among means. The significance of dose was set at P , 0:05. In Experiment 5, data were analyzed using the Mann – Whitney U-test. Results are presented as means ^ SEM. The food intake of chicks was significantly increased by L -leucine (200 mg) when compared with control at 30 min postinjection (Fig. 1A; Fð3; 56Þ ¼ 3:341, P , 0:05). In addition, all parameters for feeding behavior in the leucine group implied hyperphagia (Table 1), and this result is in good agreement with the result of food intake. In particular, starting time of feeding and pecking rhythm were significantly accelerated by leucine (Table 1; P , 0:05). On the other hand, neither L -isoleucine nor L -valine had any effect on food intake compared with saline control during the experimental period (Fig. 1B,C; P . 0:05). We do not have information from the present study as to how leucine alone modulates feeding regulation in the central nervous system of chicks, but some hypotheses may be proposed. First, because the catabolic route of leucine is different from both
167
Fig. 1. Food intake of chicks injected ICV with saline or one of three doses (50, 100 or 200 mg) of (A) L -leucine, (B) L -isoleucine or (C) L -valine. Values are means ^ SEM. The number of chicks used is shown in parentheses. *P , 0:05, compared with saline control. (A) Food intake (g/30 minÞ ¼ 0:29ðSE 0.08) þ 0.02(SE 0.01)X, R2 ¼ 0:148, P , 0:01.
isoleucine and valine, we hypothesize that a metabolite of leucine increases food intake in chicks. Leucine is transaminated to produce glutamate and a-ketoisocaproic acid, and a-ketoisocaproic acid is converted to acetoacetylCoA in the brain [11]. On the other hand, the ketoacids of isoleucine and valine are converted to succinyl-CoA [11]. However, ICV injection of a-ketoisocaproate at an equimolar concentration to the L -leucine used here had no effect on food intake compared with the saline control during the experiment period (Fig. 2; P . 0:05). Therefore, it is unlikely that this metabolite of leucine increases food intake in chicks. Secondly, it can be assumed that glutamate resulting from the exogenous leucine stimulates feeding behavior. There are some reports that BCAAs, particularly leucine,
Table 1 Influence of central administration of leucine on feeding behavior of chicks
Control Leucine P value
Feeding time (s)
Pecking frequency for food
Pecking rhythm
Starting time of feeding (s)
140.6 ^ 62.4 267.0 ^ 66.1 0.175
99.6 ^ 42.2 314.6 ^ 87.8 0.076
0.78 ^ 0.10 1.17 ^ 0.15 0.028
310.0 ^ 48.5 151.8 ^ 23.2 0.047
Pecking rhythm was calculated as food pecking frequency divided by feeding time. Values are means ^ SEM of five birds.
168
T. Izumi et al. / Neuroscience Letters 354 (2004) 166–168
research from the Ministry of Education, Science and Culture, Japan. We wish to thank Kyowa Hakko Kogyo Co. Ltd. for supplying leucine, isoleucine and valine.
References
Fig. 2. Food intake of chicks injected ICV with saline or one of three doses of a-ketoisocaproic acid (119, 238 or 475 mg). Values are means ^ SEM. The number of chicks used is shown in parentheses.
are important as external sources of nitrogen for the synthesis of glutamate and glutamine [1,12]. Moreover, many reports have shown that glutamate injected into the brain elicits a robust feeding response in satiated rats (e.g. Ref. [9]). Thus, it is possible that the orexigenic effect of leucine was due to accelerated synthesis of glutamate. Additionally, because isoleucine and valine are also utilized as sources of nitrogen for the synthesis of glutamate, it is probable that the higher doses of other BCAAs also will stimulate feeding behavior in chicks. The results presented here suggest that L -leucine induces hyperphagia and BCAA(s) may have an important role in the feeding behavior of chicks. However, further experiments will be required to determine the mechanism of leucine’s effect on ingestive behavior in chicks. To our knowledge, this is the first report of a central effect of L leucine on feeding behavior in birds.
Acknowledgements This study was supported by a grant-in-aid for scientific
[1] M.G. Bixel, S.M. Huston, B. Hamprecht, Cellular distribution of branched-chain amino acid aminotransferase isoenzyme among rat brain glial cells in culture, J. Histochem. Cytochem. 45 (1997) 685 –694. [2] E.R. Chaplin, A.L. Goldberg, I. Diamond, Leucine oxidation in brain slices and nerve endings, J. Neurochem. 26 (1976) 710–717. [3] J.L. Davis, D.T. Masuoka, J.F. Gerbrandt, A. Cherkin, Autoradiographic distribution of L -proline in chicks after intracerebral injection, Physiol. Behav. 22 (1979) 693–695. [4] National Research Council, Guide for the Care and Use of Laboratory Animals. NIH Publ. No. 85-23, Department of Health and Human Services, Washington, DC, 1985. [5] F. Nishimura, M. Nishihara, M. Mori, K. Torii, M. Takahashi, Excitability of neurons in the ventromedial nucleus in rat hypothalamic slices: modulation by amino acids at cerebrospinal fluid levels, Brain Res. 691 (1995) 217–222. [6] W.H. Oldendorf, Brain uptake of radiolabeled amino acids, amines and hexoses after arterial injection, Am. J. Physiol. 221 (1971) 1629–1635. [7] J. Panksepp, D.A. Booth, Decreased feeding after injection of amino acids into the hypothalamus, Nature 233 (1971) 341– 342. [8] Q.R. Smith, S. Momma, M. Aoyagi, S.I. Rapport, Kinetics of neutral amino acid transport across the blood-brain barrier, J. Neurochem. 49 (1987) 1651–1658. [9] B.G. Stanley, V.L. Willett III, H.W. Donias, L.H. Ha, L.C. Spears, The lateral hypothalamus: a primary site mediating excitatory amino acid-elicited eating, Brain Res. 63 (1993) 41–49. [10] M.J. Wayner, T. Ono, A. Deyoung, F.C. Barone, Effects of essential amino acids on central neurons, Pharmacol. Biochem. Behav. 3 (Suppl. 1) (1975) 85– 90. [11] M. Yudkoff, Brain metabolism of branched-chain amino acids, Glia 21 (1997) 92–98. [12] M. Yudkoff, Y. Daikihin, L. Glunstein, I. Nissim, J. Stern, D. Pleasure, I. Nissim, Astrocyte leucine metabolism: significance of branched-chain amino acid transamination, J. Neurochem. 66 (1996) 378 –385.