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Energy expenditure by intracerebroventricular administration of orexin to anesthetized rats
Neuroscience Letters 315 (2001) 49–52 www.elsevier.com/locate/neulet
Energy expenditure by intracerebroventricular administration of orexin to anesthetized rats Jian Wang, Toshimasa Osaka*, Shuji Inoue National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan Received 16 August 2001; received in revised form 19 September 2001; accepted 20 September 2001
Abstract Orexin-A and -B are hypothalamic neuropeptides that have been implicated in stimulating food intake and maintaining arousal. Because food intake is closely related to the control of energy homeostasis, we examined the effects of intracerebroventricular administration of orexins on O2 consumption (VO2), an index of energy expenditure, body temperature, skin temperature and heart rate (HR) in urethane-anesthetized rats. VO2 increased significantly after an orexin-A injection, and this increase was accompanied by a significant tachycardiac response. Orexin-B also increased VO2 and HR, although orexin-A was ~30 times more potent in eliciting these responses than orexin-B. The effects of orexin-A were dose dependent over the range of 1 pmol–1 nmol, whereas an injection of the saline vehicle had no effect. These findings suggest that centrally acting orexin-A functions to increase energy expenditure. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Orexin; Energy expenditure; Intracerebroventricular; O2 consumption; Heart rate; Temperature
Orexin-A and -B are neuropeptides synthesized specifically by neurons in the lateral hypothalamus [10,13], which is a region classically established to be involved in the central regulation of feeding behavior and energy homeostasis [12]. Central administration of orexins stimulated food intake [2,13], increased mean arterial pressure and heart rate (HR) [14], and facilitated wakefulness [11]. Neuropeptides implicated in the control of energy homeostasis usually have coordinated effects on both food intake and energy expenditure. For example, orexigenic peptides, such as neuropeptide Y and ghrelin, increase food intake and decrease energy expenditure, facilitating fat storage [1,3,16]. On the other hand, anorexigenic peptides, such as leptin, corticotropin-releasing factor, and a -melanocyte-stimulating hormone, decrease food intake and increase energy expenditure, facilitating fat loss [4,6,7]. Although it has been clearly demonstrated that orexins stimulate food intake, their effects on energy expenditure are unknown. In the present study, we measured whole-body O2 consumption (VO2), which is an index of energy expenditure, HR, colonic temperature (Tco), and tail skin temperature (Tsk) following intracerebroventricular (i.c.v.) administration of orexin-A or -B. * Corresponding author. Tel.: 181-3-3203-5723; fax: 181-33205-9536. E-mail address: [email protected] (T. Osaka).
Male Wistar rats, weighing 280–410 g, were housed in a room maintained at 24 ^ 28C with a 12:12 h light/dark cycle. All experiments were carried out within the range of normal housing temperatures. The care of the animals and surgical procedures followed our institutional guidelines. The rats were anesthetized with urethane (1.2 g kg -1 intraperitoneally), and placed in a stereotaxic apparatus (SR-6N, Narishige, Japan) with the head fixed according to the coordinates of Paxinos and Watson. Their body temperature was maintained at 35.5–378C with a heating pad during the experiments. VO2 was measured with an open-circuit indirect calorimetry system as previously described [8]. Briefly, the head of each rat was covered with a hood, which was continuously ventilated at a constant rate of 1 l min -1. The difference in O2 concentrations between inflow and outflow air was measured with a differential O2 analyzer. VO2 was recorded at 15-s intervals, and the result was expressed in terms of metabolic mass (kg 0.75). We also measured Tsk with a small thermistor attached to the dorsal base of the tail and Tco with a thermistor probe inserted about 60 mm beyond the anus. The electrocardiogram was recorded with needle electrodes inserted into the limbs and monitored on an oscilloscope. HR was calculated every 15 s. After the experiments, data were averaged over 5-min intervals. A 30-gauge stainless steel cannula connected to a 10-ml
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 32 2- 9
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J. Wang et al. / Neuroscience Letters 315 (2001) 49–52
Fig. 1. Effects of i.c.v. administration of 0.1 nmol orexin-A (X, n ¼ 4) or orexin-B (O, n ¼ 4) on VO2 (A), HR (B), and Tco (C). Open circles show lack of effects of the saline vehicle (n ¼ 2). Arrow shows the time of administration. Values are means ^ SEM. Asterisks (*) indicate a significant difference from the baseline level in experiments of orexin-A, and double asterisks (**) indicate a significant difference from the level for orexin-B (P , 0:05).
Hamilton microsyringe via a polyethylene tube was unilaterally inserted into the lateral ventricle (LV) by means of a micromanipulator. Rat orexin-A or -B (Peptide Institute, Minoh, Japan), dissolved in physiological saline, was injected at a dose of 1 pmol-1 nmol in a volume of 0.07-2 ml over a period of 1–2 min. The injection cannula was kept in place for 10 min after the end of infusion and was then removed. As a control experiment, 2 ml of vehicle saline was injected into the LV. Values were expressed as the means ^ SEM. A one-way repeated measures ANOVA followed by Dunnett’s multiple comparison test was used to determine significance of differences. Statistical significance was defined as P , 0:05. The time-integrated increase in VO2 reflected the total area of increase in VO2 for 2 h over the resting value, which was determined as an average during a 5-min period before the injection of a solution. VO2 rose significantly from the baseline level at 10 min and reached a peak level of 10.36 ^ 0.08 ml min -1 kg -0.75
(n ¼ 4) at 45 min after an orexin-A (0.1 nmol, 2 ml) injection (Fig. 1A, X). The significant increase in VO2 lasted about 50 min and returned to the baseline level within 2 h. HR also increased after the orexin-A injection to a peak of 389 ^ 4 beats min -1 (n ¼ 4) at 40 min after the 0.1 nmol orexin-A injection (Fig. 1B, X). The elevated HR then slowly dropped, but still remained high at 2 h. The increase in VO2 (Fig. 2) or HR induced by orexin-A was dose dependent: A smaller dose of orexin-A (10 pmol) increased VO2 and HR by 0.34 ^ 0.06 ml min -1 kg -0.75and 47 ^ 9 beats min -1 (n ¼ 4), respectively. A higher dose of orexin-A (1 nmol) induced a greater increase in VO2 and HR, by 1.05 ^ 0.14 ml min -1 kg -0.75 and 95 ^ 8 beats min -1 (n ¼ 4), respectively. After the 0.1 nmol orexin-A injection into the LV, Tco increased significantly after 40 min and reached a peak of 36.88 ^ 0.288C (n ¼ 4) at 115 min (Fig. 1C, X). The increased Tco lasted more than 2 h. A higher dose of orexin-A showed a stronger effect on Tco. On the other hand, Tsk did not change significantly after any dose of orexin-A injected. The administration of orexin-B slightly increased VO2 and HR, although the responses were considerably smaller than those to orexin-A. After an injection of 0.1 nmol orexin-B, VO2 increased to a peak of 9.63 ^ 0.17 ml min 1 kg -0.75 (n ¼ 4) at 15 min and it returned to the baseline within 40 min (Fig. 1A, O). The magnitude of integrated increase in VO2 induced by 1 nmol orexin-B was comparable to that induced by 10-30 pmol orexin-A (Fig. 2). The injection of 0.1 nmol orexin-B also increased HR to 342 ^ 12 beats min -1 (Fig. 1B, O, n ¼ 4). The increased HR lasted more than 2 h. No significant change in Tco was observed after i.c.v. orexin-B (Fig. 1C, O). The threshold dose of orexin-B to elicit changes in VO2 or HR was 10-30
Fig. 2. Dose-dependent effects of orexin-A (X, n ¼ 22) and orexinB (W, n ¼ 15) on VO2. VO2 values are the integrated increase over the baseline levels within 2 h after the orexin injection. Arrows indicate overlapping data taken from different rats. Lines are linear regressions of VO2 versus dose of orexin-A (r ¼ 0:58) and versus that of orexin-B (r ¼ 0:345). The increase in VO2 was significantly correlated with the dose of orexin-A or -B.
J. Wang et al. / Neuroscience Letters 315 (2001) 49–52
pmol, whereas that of orexin-A was 1-3 pmol. The saline injection had no effect on VO2, HR or Tco (Fig. 1A–C, W). In the present study, i.c.v. administration of orexins especially orexin-A induced dose-dependent energy expenditure in urethane-anesthetized rats. The effective dose (10 pmol) of orexin-A for eliciting the thermogenic response was much smaller than those doses (0.3–10 nmol) in the previous studies that demonstrated stimulation of the food intake, as well as sympathetic and cardiovascular actions [2,14,18]. The extremely low effective dose of orexin-A in the present study suggests that orexin-A has more powerful potential to enhance energy expenditure than to stimulate feeding behavior. As shown in the present study, the orexininduced energy expenditure was always accompanied by long-lasting tachycardia. These results suggest that orexininduced thermogenesis was mediated by activation of the sympathetic nervous system, although the duration of thermogenesis was shorter than that of tachycardia. The nonshivering thermogenesis is mediated by the sympathetic nervous system and occurs mainly in the brown adipose tissue (BAT). Thus, the BAT could be the site of orexininduced thermogenesis. It was also demonstrated that central orexin-A stimulated gastric acid secretion through activation of the vagus nerves [15]. Accordingly, it might be possible that thermogenesis was caused by secretory or motor of functions of the gastrointestinal tract. Further investigations are, however, necessary to elucidate the precise mechanism. Two kinds of orexin receptor (OX1R and OX2R) have been reported [13,17], and the affinity of orexin-A for OX1R is 100 times higher than that of orexin-B, whereas both orexins have similar affinity for OX2R [13]. In the present study, the effect of orexin-A on VO2 was ~30 times more potent than that of orexin-B. Similarly, it was reported that orexin-A was more potent than orexin-B in stimulating cardiovascular parameters and sympathetic nerve activity [14]. Accordingly, OX1R plays an important role on the regulation of energy expenditure. On the other hand, orexin-B is likely to be more important in regulating sleep-wakefulness than modulating energy expenditure because a mutation of the orexin-B gene results in narcolepsy [9]. Continuous i.c.v. administration of orexin-A resulted in a significant increase in food intake but had no effect on body weight in rats [18]. Moreover, transgenic mice whose orexin-sensitive neurons had been ablated genetically showed late-onset obesity despite eating less than non-transgenic mice [5]. The results of present study can explain the results of these findings: orexin-A stimulates the food intake but prevents obesity by simultaneously increasing energy expenditure. Usually a neuropeptide that stimulates food intake also reduces energy expenditure, whereas a neuropeptide that reduces food intake also increases energy expenditure [1,3,4,6,7,16]. However, orexins stimulate both food intake and energy expenditure. In conclusion, the present study demonstrated that orexins, mainly orexin-A, enhanced energy expenditure, probably through
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activation of sympathetic nervous system, and that orexins are exceptional neuropeptides that simultaneously exert opposite actions with respect to energy storage; i.e. they stimulates energy expenditure as well as food intake. This study was supported in part by a grant (KH51059) from the Japan Health Science Foundation. [1] Billington, C.J., Briggs, J.E., Grace, M. and Levine, A.S., Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism, Am. J. Physiol., 260 (1991) R321– R327. [2] Dube, M.G., Kalra, S.P. and Kalra, P.S., Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action, Brain Res., 842 (1999) 473–477. [3] Egawa, M., Yoshimatsu, H. and Bray, G.A., Neuropeptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats, Am. J. Physiol., 260 (1991) R328– R334. [4] Egawa, M., Yoshimatsu, H. and Bray, G.A., Preoptic area injection of corticotropin-releasing hormone stimulates sympathetic activity, Am. J. Physiol., 259 (1990) R799–R806. [5] Hara, J., Beuckmann, C.T., Nambu, T., Willie, J.T., Chemelli, R.M., Sinton, C.M., Sugiyama, F., Yagami, K., Goto, K., Yanagisawa, M. and Sakarai, T., Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity, Neuron, 30 (2001) 345–354. [6] Haynes, W.G., Morgan, D.A., Djalali, A., Sivitz, W.I. and Mark, A.L., Interactions between the melanocortin system and leptin in control of sympathetic nerve traffic, Hypertension, 33 (1999) 542–547. [7] Haynes, W.G., Morgan, D.A., Walsh, S.A., Mark, A.L. and Sivitz, W.I., Receptor-mediated regional sympathetic nerve activation by leptin, J. Clin. Invest., 100 (1997) 270– 278. [8] Kobayashi, A., Osaka, T., Namba, Y., Inoue, S. and Kimura, S., CGRP microinjection into the ventromedial or dorsomedial hypothalamic nucleus activates heat production, Brain Res., 827 (1999) 176–184. [9] Lin, L., Faraco, J., Li, R., Kadotani, H., Rogers, W., Lin, X., de Jong, P.J., Nishino, S. and Mignot, E., The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene, Cell, 98 (1999) 365–376. [10] Nambu, T., Sakurai, T., Mizukami, K., Hosoya, Y., Yanagisawa, M. and Goto, K., Distribution of orexin neurons in the adult rat brain, Brain Res., 827 (1999) 243–260. [11] Piper, D.C., Upton, N., Smith, M.I. and Hunter, A.J., The novel brain neuropeptide, orexin-A, modulates the sleepwake cycle of rats , Eur. J. Neurosci., 12 (2000) 726–730. [12] Powley, T.L., Opsahl, C.A., Cox, J.E. and Weingarten, H.P., The role of the hypothalamus in energy homeostasis, In P.J. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalamus, Marcel Dekker, New York, 1980, pp. 211– 298. [13] Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H., Williams, S.C., Richardson, J.A., Kozlowski, G.P., Wilson, S., Arch, J.R.S., Buckingham, R.E., Hayhes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.S., Terrett, J.A., Elshourbagy, N.A., Bergsma, D.J. and Yanagisawa, M., Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior, Cell, 92 (1998) 573–585. [14] Shirasaka, T., Nakazato, M., Matsukura, S., Takasaki, M. and
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Kannan, H., Sympathetic and cardiovascular actions of orexins in conscious rats, Am. J. Physiol., 277 (1999) R1780–R1785. [15] Takahashi, N., Okumura, T., Yamada, H. and Kohgo, Y., Stimulation of gastric acid secretion by centrally administered orexin-A in conscious rats, Biochem. Biophys. Res. Commun., 254 (1999) 623–627. [16] Tscho¨ p, M., Smiley, D.L. and Heiman, M.L., Ghrelin induces adiposity in rodents, Nature, 407 (2000) 908–913.
[17] Trivedi, P., Yu, H., MacNeil, D.J., van der Ploeg, L.H.T. and Guan, X.M., Distribution of orexin receptor mRNA in the rat brain, FEBS Lett., 438 (1998) 71–75. [18] Yamanaka, A., Sakurai, T., Katsumoto, T., Yanagisawa, M. and Goto, K., Chronic intracerebroventricular administration of orexin-A to rats increases food intake in daytime, but has no effect on body weight, Brain Res., 849 (1999) 248–252.
Energy expenditure by intracerebroventricular administration of orexin to anesthetized rats Jian Wang, Toshimasa Osaka*, Shuji Inoue National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan Received 16 August 2001; received in revised form 19 September 2001; accepted 20 September 2001
Abstract Orexin-A and -B are hypothalamic neuropeptides that have been implicated in stimulating food intake and maintaining arousal. Because food intake is closely related to the control of energy homeostasis, we examined the effects of intracerebroventricular administration of orexins on O2 consumption (VO2), an index of energy expenditure, body temperature, skin temperature and heart rate (HR) in urethane-anesthetized rats. VO2 increased significantly after an orexin-A injection, and this increase was accompanied by a significant tachycardiac response. Orexin-B also increased VO2 and HR, although orexin-A was ~30 times more potent in eliciting these responses than orexin-B. The effects of orexin-A were dose dependent over the range of 1 pmol–1 nmol, whereas an injection of the saline vehicle had no effect. These findings suggest that centrally acting orexin-A functions to increase energy expenditure. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Orexin; Energy expenditure; Intracerebroventricular; O2 consumption; Heart rate; Temperature
Orexin-A and -B are neuropeptides synthesized specifically by neurons in the lateral hypothalamus [10,13], which is a region classically established to be involved in the central regulation of feeding behavior and energy homeostasis [12]. Central administration of orexins stimulated food intake [2,13], increased mean arterial pressure and heart rate (HR) [14], and facilitated wakefulness [11]. Neuropeptides implicated in the control of energy homeostasis usually have coordinated effects on both food intake and energy expenditure. For example, orexigenic peptides, such as neuropeptide Y and ghrelin, increase food intake and decrease energy expenditure, facilitating fat storage [1,3,16]. On the other hand, anorexigenic peptides, such as leptin, corticotropin-releasing factor, and a -melanocyte-stimulating hormone, decrease food intake and increase energy expenditure, facilitating fat loss [4,6,7]. Although it has been clearly demonstrated that orexins stimulate food intake, their effects on energy expenditure are unknown. In the present study, we measured whole-body O2 consumption (VO2), which is an index of energy expenditure, HR, colonic temperature (Tco), and tail skin temperature (Tsk) following intracerebroventricular (i.c.v.) administration of orexin-A or -B. * Corresponding author. Tel.: 181-3-3203-5723; fax: 181-33205-9536. E-mail address: [email protected] (T. Osaka).
Male Wistar rats, weighing 280–410 g, were housed in a room maintained at 24 ^ 28C with a 12:12 h light/dark cycle. All experiments were carried out within the range of normal housing temperatures. The care of the animals and surgical procedures followed our institutional guidelines. The rats were anesthetized with urethane (1.2 g kg -1 intraperitoneally), and placed in a stereotaxic apparatus (SR-6N, Narishige, Japan) with the head fixed according to the coordinates of Paxinos and Watson. Their body temperature was maintained at 35.5–378C with a heating pad during the experiments. VO2 was measured with an open-circuit indirect calorimetry system as previously described [8]. Briefly, the head of each rat was covered with a hood, which was continuously ventilated at a constant rate of 1 l min -1. The difference in O2 concentrations between inflow and outflow air was measured with a differential O2 analyzer. VO2 was recorded at 15-s intervals, and the result was expressed in terms of metabolic mass (kg 0.75). We also measured Tsk with a small thermistor attached to the dorsal base of the tail and Tco with a thermistor probe inserted about 60 mm beyond the anus. The electrocardiogram was recorded with needle electrodes inserted into the limbs and monitored on an oscilloscope. HR was calculated every 15 s. After the experiments, data were averaged over 5-min intervals. A 30-gauge stainless steel cannula connected to a 10-ml
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 32 2- 9
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J. Wang et al. / Neuroscience Letters 315 (2001) 49–52
Fig. 1. Effects of i.c.v. administration of 0.1 nmol orexin-A (X, n ¼ 4) or orexin-B (O, n ¼ 4) on VO2 (A), HR (B), and Tco (C). Open circles show lack of effects of the saline vehicle (n ¼ 2). Arrow shows the time of administration. Values are means ^ SEM. Asterisks (*) indicate a significant difference from the baseline level in experiments of orexin-A, and double asterisks (**) indicate a significant difference from the level for orexin-B (P , 0:05).
Hamilton microsyringe via a polyethylene tube was unilaterally inserted into the lateral ventricle (LV) by means of a micromanipulator. Rat orexin-A or -B (Peptide Institute, Minoh, Japan), dissolved in physiological saline, was injected at a dose of 1 pmol-1 nmol in a volume of 0.07-2 ml over a period of 1–2 min. The injection cannula was kept in place for 10 min after the end of infusion and was then removed. As a control experiment, 2 ml of vehicle saline was injected into the LV. Values were expressed as the means ^ SEM. A one-way repeated measures ANOVA followed by Dunnett’s multiple comparison test was used to determine significance of differences. Statistical significance was defined as P , 0:05. The time-integrated increase in VO2 reflected the total area of increase in VO2 for 2 h over the resting value, which was determined as an average during a 5-min period before the injection of a solution. VO2 rose significantly from the baseline level at 10 min and reached a peak level of 10.36 ^ 0.08 ml min -1 kg -0.75
(n ¼ 4) at 45 min after an orexin-A (0.1 nmol, 2 ml) injection (Fig. 1A, X). The significant increase in VO2 lasted about 50 min and returned to the baseline level within 2 h. HR also increased after the orexin-A injection to a peak of 389 ^ 4 beats min -1 (n ¼ 4) at 40 min after the 0.1 nmol orexin-A injection (Fig. 1B, X). The elevated HR then slowly dropped, but still remained high at 2 h. The increase in VO2 (Fig. 2) or HR induced by orexin-A was dose dependent: A smaller dose of orexin-A (10 pmol) increased VO2 and HR by 0.34 ^ 0.06 ml min -1 kg -0.75and 47 ^ 9 beats min -1 (n ¼ 4), respectively. A higher dose of orexin-A (1 nmol) induced a greater increase in VO2 and HR, by 1.05 ^ 0.14 ml min -1 kg -0.75 and 95 ^ 8 beats min -1 (n ¼ 4), respectively. After the 0.1 nmol orexin-A injection into the LV, Tco increased significantly after 40 min and reached a peak of 36.88 ^ 0.288C (n ¼ 4) at 115 min (Fig. 1C, X). The increased Tco lasted more than 2 h. A higher dose of orexin-A showed a stronger effect on Tco. On the other hand, Tsk did not change significantly after any dose of orexin-A injected. The administration of orexin-B slightly increased VO2 and HR, although the responses were considerably smaller than those to orexin-A. After an injection of 0.1 nmol orexin-B, VO2 increased to a peak of 9.63 ^ 0.17 ml min 1 kg -0.75 (n ¼ 4) at 15 min and it returned to the baseline within 40 min (Fig. 1A, O). The magnitude of integrated increase in VO2 induced by 1 nmol orexin-B was comparable to that induced by 10-30 pmol orexin-A (Fig. 2). The injection of 0.1 nmol orexin-B also increased HR to 342 ^ 12 beats min -1 (Fig. 1B, O, n ¼ 4). The increased HR lasted more than 2 h. No significant change in Tco was observed after i.c.v. orexin-B (Fig. 1C, O). The threshold dose of orexin-B to elicit changes in VO2 or HR was 10-30
Fig. 2. Dose-dependent effects of orexin-A (X, n ¼ 22) and orexinB (W, n ¼ 15) on VO2. VO2 values are the integrated increase over the baseline levels within 2 h after the orexin injection. Arrows indicate overlapping data taken from different rats. Lines are linear regressions of VO2 versus dose of orexin-A (r ¼ 0:58) and versus that of orexin-B (r ¼ 0:345). The increase in VO2 was significantly correlated with the dose of orexin-A or -B.
J. Wang et al. / Neuroscience Letters 315 (2001) 49–52
pmol, whereas that of orexin-A was 1-3 pmol. The saline injection had no effect on VO2, HR or Tco (Fig. 1A–C, W). In the present study, i.c.v. administration of orexins especially orexin-A induced dose-dependent energy expenditure in urethane-anesthetized rats. The effective dose (10 pmol) of orexin-A for eliciting the thermogenic response was much smaller than those doses (0.3–10 nmol) in the previous studies that demonstrated stimulation of the food intake, as well as sympathetic and cardiovascular actions [2,14,18]. The extremely low effective dose of orexin-A in the present study suggests that orexin-A has more powerful potential to enhance energy expenditure than to stimulate feeding behavior. As shown in the present study, the orexininduced energy expenditure was always accompanied by long-lasting tachycardia. These results suggest that orexininduced thermogenesis was mediated by activation of the sympathetic nervous system, although the duration of thermogenesis was shorter than that of tachycardia. The nonshivering thermogenesis is mediated by the sympathetic nervous system and occurs mainly in the brown adipose tissue (BAT). Thus, the BAT could be the site of orexininduced thermogenesis. It was also demonstrated that central orexin-A stimulated gastric acid secretion through activation of the vagus nerves [15]. Accordingly, it might be possible that thermogenesis was caused by secretory or motor of functions of the gastrointestinal tract. Further investigations are, however, necessary to elucidate the precise mechanism. Two kinds of orexin receptor (OX1R and OX2R) have been reported [13,17], and the affinity of orexin-A for OX1R is 100 times higher than that of orexin-B, whereas both orexins have similar affinity for OX2R [13]. In the present study, the effect of orexin-A on VO2 was ~30 times more potent than that of orexin-B. Similarly, it was reported that orexin-A was more potent than orexin-B in stimulating cardiovascular parameters and sympathetic nerve activity [14]. Accordingly, OX1R plays an important role on the regulation of energy expenditure. On the other hand, orexin-B is likely to be more important in regulating sleep-wakefulness than modulating energy expenditure because a mutation of the orexin-B gene results in narcolepsy [9]. Continuous i.c.v. administration of orexin-A resulted in a significant increase in food intake but had no effect on body weight in rats [18]. Moreover, transgenic mice whose orexin-sensitive neurons had been ablated genetically showed late-onset obesity despite eating less than non-transgenic mice [5]. The results of present study can explain the results of these findings: orexin-A stimulates the food intake but prevents obesity by simultaneously increasing energy expenditure. Usually a neuropeptide that stimulates food intake also reduces energy expenditure, whereas a neuropeptide that reduces food intake also increases energy expenditure [1,3,4,6,7,16]. However, orexins stimulate both food intake and energy expenditure. In conclusion, the present study demonstrated that orexins, mainly orexin-A, enhanced energy expenditure, probably through
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activation of sympathetic nervous system, and that orexins are exceptional neuropeptides that simultaneously exert opposite actions with respect to energy storage; i.e. they stimulates energy expenditure as well as food intake. This study was supported in part by a grant (KH51059) from the Japan Health Science Foundation. [1] Billington, C.J., Briggs, J.E., Grace, M. and Levine, A.S., Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism, Am. J. Physiol., 260 (1991) R321– R327. [2] Dube, M.G., Kalra, S.P. and Kalra, P.S., Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action, Brain Res., 842 (1999) 473–477. [3] Egawa, M., Yoshimatsu, H. and Bray, G.A., Neuropeptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats, Am. J. Physiol., 260 (1991) R328– R334. [4] Egawa, M., Yoshimatsu, H. and Bray, G.A., Preoptic area injection of corticotropin-releasing hormone stimulates sympathetic activity, Am. J. Physiol., 259 (1990) R799–R806. [5] Hara, J., Beuckmann, C.T., Nambu, T., Willie, J.T., Chemelli, R.M., Sinton, C.M., Sugiyama, F., Yagami, K., Goto, K., Yanagisawa, M. and Sakarai, T., Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity, Neuron, 30 (2001) 345–354. [6] Haynes, W.G., Morgan, D.A., Djalali, A., Sivitz, W.I. and Mark, A.L., Interactions between the melanocortin system and leptin in control of sympathetic nerve traffic, Hypertension, 33 (1999) 542–547. [7] Haynes, W.G., Morgan, D.A., Walsh, S.A., Mark, A.L. and Sivitz, W.I., Receptor-mediated regional sympathetic nerve activation by leptin, J. Clin. Invest., 100 (1997) 270– 278. [8] Kobayashi, A., Osaka, T., Namba, Y., Inoue, S. and Kimura, S., CGRP microinjection into the ventromedial or dorsomedial hypothalamic nucleus activates heat production, Brain Res., 827 (1999) 176–184. [9] Lin, L., Faraco, J., Li, R., Kadotani, H., Rogers, W., Lin, X., de Jong, P.J., Nishino, S. and Mignot, E., The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene, Cell, 98 (1999) 365–376. [10] Nambu, T., Sakurai, T., Mizukami, K., Hosoya, Y., Yanagisawa, M. and Goto, K., Distribution of orexin neurons in the adult rat brain, Brain Res., 827 (1999) 243–260. [11] Piper, D.C., Upton, N., Smith, M.I. and Hunter, A.J., The novel brain neuropeptide, orexin-A, modulates the sleepwake cycle of rats , Eur. J. Neurosci., 12 (2000) 726–730. [12] Powley, T.L., Opsahl, C.A., Cox, J.E. and Weingarten, H.P., The role of the hypothalamus in energy homeostasis, In P.J. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalamus, Marcel Dekker, New York, 1980, pp. 211– 298. [13] Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H., Williams, S.C., Richardson, J.A., Kozlowski, G.P., Wilson, S., Arch, J.R.S., Buckingham, R.E., Hayhes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.S., Terrett, J.A., Elshourbagy, N.A., Bergsma, D.J. and Yanagisawa, M., Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior, Cell, 92 (1998) 573–585. [14] Shirasaka, T., Nakazato, M., Matsukura, S., Takasaki, M. and
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J. Wang et al. / Neuroscience Letters 315 (2001) 49–52
Kannan, H., Sympathetic and cardiovascular actions of orexins in conscious rats, Am. J. Physiol., 277 (1999) R1780–R1785. [15] Takahashi, N., Okumura, T., Yamada, H. and Kohgo, Y., Stimulation of gastric acid secretion by centrally administered orexin-A in conscious rats, Biochem. Biophys. Res. Commun., 254 (1999) 623–627. [16] Tscho¨ p, M., Smiley, D.L. and Heiman, M.L., Ghrelin induces adiposity in rodents, Nature, 407 (2000) 908–913.
[17] Trivedi, P., Yu, H., MacNeil, D.J., van der Ploeg, L.H.T. and Guan, X.M., Distribution of orexin receptor mRNA in the rat brain, FEBS Lett., 438 (1998) 71–75. [18] Yamanaka, A., Sakurai, T., Katsumoto, T., Yanagisawa, M. and Goto, K., Chronic intracerebroventricular administration of orexin-A to rats increases food intake in daytime, but has no effect on body weight, Brain Res., 849 (1999) 248–252.