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Chronic intracerebroventricular administration of orexin-A to rats increases food intake in daytime, but has no effect on body weight
Brain Research 849 Ž1999. 248–252 www.elsevier.comrlocaterbres
Interactive report
Chronic intracerebroventricular administration of orexin-A to rats increases food intake in daytime, but has no effect on body weight 1 Akihiro Yamanaka a , Takeshi Sakurai a, ) , Takuo Katsumoto a , Masashi Yanagisawa b , Katsutoshi Goto a b
a Department of Pharmacology, Institute of Basic Medical Sciences, UniÕersity of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan Howard Hughes Medical Institute, Department of Molecular Genetics, UniÕersity of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-9050, USA
Accepted 29 July 1999
Abstract Orexins are recently identified neuropeptides, and have been shown to increase food intake when administered intracerebroventricularly. We examined the effects of chronic administration of orexin in rats by continuous intracerebroventricular administration by means of an osmotic minipump. Continuous administration of orexin-A Ž0.5 nmolrh. for 7 days in rats resulted in a significant increase in food intake in the daytime. Daytime food intake increased to 180% of the control value. However, it resulted in a slight decrease nighttime food intake as compared with vehicle-treated rats. The total amount of food intake per day was almost comparable with that of vehicle-administered rats. The gain of body weight and blood glucose, total cholesterol and free fatty acid levels were normal. Chronic orexin-A treatment did not cause obesity in rats. We observed abnormal behavior during the daytime after starting administration of orexin-A; these rats kept awake during the daytime. Our present observation showed that continuous administration of orexin-A could not overcome the regulation of energy homeostasis and body weight. However, orexin-A might be implicated in short-term, immediate regulation of feeding behavior. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Feeding behavior; Feeding circadian rhythm; Chronic treatment; Intracerebroventricular infusion; Blood glucose
1. Introduction Orexin-A and -B are neuropeptides that were identified as endogenous ligands for an orphan G-protein-coupled receptor, which was originally found as an expressed sequence tag in human brain w18x. Orexin-A and -B are derived from the same precursor peptide Žprepro-orexin. by proteolytic processing. They bind and activate two closely related G protein-coupled receptors, termed OX 1 and OX 2 receptors. OX 1 receptor is selective for orexin-A, while OX 2 receptor is a non-selective receptor for both orexin-A and -B. Orexin-containing neurons are specifically localized in the lateral hypothalamic area ŽLHA. and adjacent regions w4,12x. The LHA has been thought to be implicated in feeding behavior, because lesions of LHA induce an anorexic response, while electrical stimulation of LHA causes feeding behavior in rats w1x.
) Corresponding author. Tel.: q81-298-53-3276; Fax: q81-298-533039; E-mail: [email protected] 1 Published on the World Wide Web on 4 August 1999.
Food deprivation for 48 h results in an increase in prepro-orexin mRNA level in the rat hypothalamus. Orexin-A and -B increase food intake when administered centrally w18x. The orexin neurons diffusely project to various regions of brain. In the hypothalamus, orexin neurons project to the ARC, PVN, DMH Ždorsomedial hypothalamus. and VMH Žventromedial hypothalamus. w12x, which has been shown to participate in the regulation of feeding behavior. On the other hand, orexin neurons also project to various regions outside the hypothalamus, including the cerebral cortex, limbic system and brain stem. These observations suggest that orexins may be involved in not only the fundamental regulation of energy homeostasis, but also emotional, cognitive and motivational aspects of feeding behavior. Neuropeptide Y ŽNPY. has been demonstrated to be involved in the regulation of food intake and energy homeostasis as well as in the secretion of various hormones such as insulin and glucocorticoids w3,17x. The chronic administration of exogenous NPY to specific nuclei or into the cerebral ventricles of normal rats results in many of the abnormalities associated with obesity, includ-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 0 5 - 8
A. Yamanaka et al.r Brain Research 849 (1999) 248–252
ing a marked increase in food intake, accelerated body weight gain, hypercorticosteronemia, hyperinsulinemia, muscle insulin resistance, and increased triglyceride accumulation within white adipose tissue w20,23,24x. These results suggest that NPY might be involved in the fundamental regulation of energy homeostasis. However, NPYdeficient mice and NPY Y1 or Y5 receptor-deficient mice feed and grow normally, as well as exhibit a normal response to starvation w5x. One explanation for this is that there are other neuromodulators that compensate for the deficiency of NPY. Alternatively, NPY does not have roles in regulating physiological feeding, though it might be implicated in abnormal feeding in pathological states. Since orexins are thought to be involved in the regulation of feeding behavior, it could be very important to examine the effect of chronic administration of orexins, to further clarify the physiological roles of orexins in the central regulation of energy homeostasis. Therefore, we investigated the effects of chronic orexin administration to normal rats.
2. Materials and methods 2.1. Chemicals Orexin-A and NPY were purchased from Peptide Institute ŽOsaka, Japan.. 2.2. Animals and surgery Male Wistar rats Ž250–300 g on receipt; Charles-River, Kanagawa, Japan. were housed under controlled lighting Ž12 h light–dark cycle; light-on 8:00 am–8:00 pm. and temperature Ž228C. conditions. Food and water were available ad libitum. Rats were anesthetized with pentobarbital Ž50 mgrkg, i.p.., positioned in a sterotaxic frame ŽDavid Kopf Instruments, CA, USA., and a guide cannula was implanted into the lateral ventricle using sterile technique. Rats were then housed singly for a recovery period of at least 7 days. The position of the cannula was verified by central administration of 0.3 nmol human neuropeptide Y to test for a positive response. Rats that ate at least 4 g of food over a one-hour period postinjection were used for experiments. After the recovery period, a mini-osmotic pump ŽAlzet 1007D, ALZA, CA, USA. was implanted subcutaneously in the inter-scapular region under anesthesia. This osmotic minipump releases the contents at a rate of 0.5 mlrh for 7 days under physiological conditions. The infusion rate of orexin-A was 0.5 nmolrh. The mini-
Fig. 1. Effect of orexin-A on ŽA. daytime, ŽB. nighttime, and ŽC. total food intake Žmean"S.E.M... Orexin-A Žhatched bars. or vehicle Žsolid bars. was continuously injected into the lateral ventricle using an osmotic minipump for 7 days. q, P - 0.05 vs. saline Ž ns 4 ratsrgroup..
249
osmotic pump was connected to the inner cannula via a polyethylene tube ŽSP10, Natsume, Tokyo, Japan.. The
250
A. Yamanaka et al.r Brain Research 849 (1999) 248–252
inner cannula was inserted into the guide cannula and fixed using an adhesive agent. Orexin-A was dissolved in sterile water, which was used alone as the vehicle control. During administration, ad libitum food intake and body weight were measured. The food intake in the daytime and nighttime were measured separately; the decrease of food was measured twice a day Žat 8:00 am and 8:00 pm. using a digital scale ŽHL-200, A and D, Tokyo, Japan.. After the experiments, a blood sample was collected from the carotid artery into an EDTA-coated tube. Plasma was used for biochemical analysis Žblood glucose, triglycerides and total-cholesterol.. Data were analyzed by two-way ANOVA followed by post-hoc analysis of significance by the Bonferroni–Dunn statistic, using the Stat view 4.5 software package ŽAbacus Concepts, Berkeley, CA, USA.. Probability Ž P . values - 0.05 were considered statistically significant.
Fig. 3. Effect of orexin-A on plasma levels of glucose, triglycerides, total cholesterol and free fatty acids ŽFFA.. Mean and S.E.M. are presented. Orexin-A Žhatched bars., saline Žsolid bars., ns 4 per group.
3. Results 3.1. Food intake An increase of food intake during the daytime was observed after the start of administration of orexin-A ŽFig. 1.. Daytime food intake was increased 2-fold at 2 days after starting orexin-A infusion. Two-way ANOVA showed a significant overall difference Ž p - 0.05.. On the other hand, orexin-A-administered rats showed almost same or slightly decreased food intake during the nighttime compared with vehicle control rats ŽFig. 1.. 3.2. Body weight The body weight of rats treated with orexin-A for 7 days was not significantly different at the end of treatment
from that of vehicle control rats ŽFig. 2.. The gain of body weight was normal in orexin-A-administered rats after starting infusion. There was no significant difference between vehicle control and orexin-A-administered rats. Inguinal white adipose tissue weight was also normal Ždata not shown.. 3.3. Blood analysis Blood glucose, total cholesterol and free fatty acids in plasma were analyzed at the end of the experiment Žsee Fig. 3.. There were no significant differences between orexin-A-treated and control rats. Blood glucose level of orexin-A-treated and control rats was 183.4 " 4.4 and 173.7 " 2.1 Ž n s 4., respectively. Total cholesterol levels of orexin-A-treated and control rats was 52.0 " 4.7 and 59.7 " 9.3 Ž n s 4., respectively. Free fatty acid level of orexin-A-treated and control rats was 0.13 " 0.02 and 0.11 " 0.003 Ž n s 4., respectively. Triglyceride level of orexin-A-treated and control rats was 52.4 " 5.4 and 46.0 " 2.4 Ž n s 4., respectively.
4. Discussion
Fig. 2. Daily body weight Žmean"S.E.M.. of rats before and during continuous injection of either orexin-A Žv . or vehicle Ž`. into the lateral ventricle using an osmotic minipump for 7 days Ž ns 4 ratsrgroup..
An intricate balance between energy intake and expenditure achieves maintenance of body weight. This energy homeostasis is governed by the hypothalamus, where a large array of neurotransmitters, especially neuropeptides, modulate signals though complex neural circuits. Identification of the leptin signaling pathway and recent molecular genetic studies of several hypothalamic neuropeptides start to provide insights on the molecular mechanisms involved in feeding behavior and energy homeostasis. Leptin has been shown by many reports to be an essential component
A. Yamanaka et al.r Brain Research 849 (1999) 248–252
of body weight regulation w6x. The site of leptin action has been thought to exist in the medial part of the hypothalamus. Many neuropeptides involved in the regulation of energy homeostasis are known to exist in the medialrperiventricular part of the hypothalamus and are regulated by leptin w16x. Especially, the arcuate nucleus, located in the mediobasal hypothalamus, is critical for regulation of body weight and metabolism, because it contains neurons that express the leptin receptor as well as neuropeptides implicated in feeding behavior including a-melanocyte stimulating hormone Ž a-MSH., aguti-related protein ŽAgRP. and NPY. NPY infusion in normal rats resulted in hyperphagia, increased body weight gain, hyperinsulinemia, hypercorticosteronemia, and hypertriglyceridemia w23x. In our present study, orexin-A-administered rats did not show such characteristics of obesity. Similar to orexin, chronic intracerebroventricular administration of an orexigenic agent, such as galanin or melanin concentrating hormone ŽMCH., did not induce obesity in normal rats w15,19x. These observations show that these orexigenic peptides do not overcome the regulatory mechanisms of body weight, unlike NPY. Rather, these factors might be implicated in short-term regulation of feeding behavior. A decline of blood glucose level can signal the initiation of food intake. It has been reported that glucose receptor neurons are located in VMH and glucose-sensitive neurons are located in LHA w13,14x. Moriguchi et al. w11x showed that approximately 30% of orexin-containing neurons express Fos-like immunoreactivity under an insulininduced hypoglycemic state in rats. These observations suggest that some of the orexin-containing neurons are glucose-sensitive. The activity of glucose-sensitive neurons was suppressed and that of glucose sensing neurons was activated under hyperglycemia w13x. These observations suggest that orexin-containing neurons is affected by blood glucose. These observations further support our data suggesting that orexin might be involved in the short-term regulation of feeding. We observed that rats treated with orexin-A showed abnormal behavior in the daytime. Normal rats maintained this usual nocturnal habit after the start of infusion; these rats slept during the light-on period, while they behaved actively and consumed 85–90% of their total food intake during the light-off period. However, rats administered orexin-A were awake during the daytime and often showed abnormal behavior, including rearing, grooming, face washing and wet dog shaking. These behaviors were previously reported after bolus administration of orexin-A or -B w9x. Orexin might be involved in the maintenance of arousal and therefore, these rats stay awake during the daytime. This effect may possibly be a mechanism by which orexin increases food intake in the daytime. In rats, the photoperiodic-dependent circadian pattern of feeding is linked to information transmitted from the suprachiasmatic nucleus ŽSCN. to the hypothalamus. Dis-
251
crete lesions in the SCN result in loss of regulated feeding w22x. These results suggest that orexins are involved in the circadian pattern. The LHA has also been shown to be involved in maintenance of the awake state w1,7,10x. Orexin immunoreactive nerve fibers were seen in brain regions involved in the sleep–wake cycle, such as the locus coeruleus, tuberomammillary nucleus, pontine reticular formation, raphe nucleus, preoptic area and dorsal tegmental nucleus w2,4,8,12x, suggesting that orexin might have effects on vigilance and arousal. The effect of orexin-A became weak with increasing number of days of administration. Chronic orexin-A treatment might cause accumulation of peptide in the cerebrospinal fluid ŽCSF. which results in desensitization of orexin receptors. The same phenomenon has been reported with repeated icv administration of galanin w19x or MCH w15x. We showed that chronic icv administration of orexin-A using an osmotic minipump did not produce a significant increase in body weight or blood sugar in rats. An experiment with chronic injection of orexins into specific nuclei in the brain is needed to clarify the role of orexins, because it has been reported that orexins induce potent feeding behavior when injected into specific nuclei, e.g. perifornical hypothalamus or lateral hypothalamus w21x. However, our present observations suggest that orexin is involved in the short-term, immediate regulation of food intake, rather than the long-term maintenance of body weight. Especially, orexins might have a role in maintaining arousal and vigilance for normal feeding.
Acknowledgements This study was supported by a grand-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, the University of Tsukuba Research project, and the Uehara Memorial Foundation. We would like to thank Dr. W.A. Gray for reading our manuscript.
References w1x L.L. Bernardis, L.L. Bellinger, The lateral hypothalamic area revisited: neuroanatomy, body weight regulation, neuroendocrinology and metabolism, Neurosci. Biobehav. Rev. 17 Ž2. Ž1993. 141–193. w2x C. Broberger, L. De Lecea, J.G. Sutcliffe, T. Hokfelt, Hypocretinr ¨ orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems, J. Comp. Neurol. 402 Ž4. Ž1998. 460–474. w3x J.T. Clark, P.S. Kalra, W.R. Crowley, S.P. Kalra, Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats, Endocrinology 115 Ž1. Ž1984. 427–429. w4x Y. Date, Y. Ueta, H. Yamashita, H. Yamaguchi, S. Matsukura, K. Kangawa, T. Sakurai, M. Yanagisawa, M. Nakazato, Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems, Proc. Natl. Acad. Sci. USA 96 Ž2. Ž1999. 748–753.
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w5x J.C. Erickson, K.E. Clegg, R.D. Palmiter, Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y, Nature 381 Ž1996. 415–421. w6x J.M. Friedman, J.L. Halaas, Leptin and the regulation of body weight in mammals, Nature 395 Ž6704. Ž1998. 763–770. w7x N. Goodless-Sanchez, R.Y. Moore, L.P. Morin, Lateral hypothalamic regulation of circadian rhythm phase, Physiol. Behav. 49 Ž3. Ž1991. 533–537. w8x T.L. Horvath, S. Diano, A.N. van den Pol, Synaptic interaction between hypocretin Žorexin. and neuropeptide Y cells in the rodent and primate hypothalamus: a novel circuit implicated in metabolic and endocrine regulations, J. Neurosci. 19 Ž3. Ž1999. 1072–1087. w9x T. Ida, K. Nakahara, T. Katayama, N. Murakami, M. Nakazato, Effect of lateral cerebroventricular injection of the appetite-stimulating neuropeptide, orexin and neuropeptide Y, on the various behavioral activities of rats, Brain Res. 821 Ž2. Ž1999. 526–529. w10x K. Koizumi, H. Nishino, Circadian and other rhythmic activity of neurones in the ventromedial nuclei and lateral hypothalamic area, J. Physiol. 263 Ž3. Ž1976. 331–356. w11x T. Moriguchi, T. Sakurai, T. Nambu, M. Yanagisawa, K. Goto, Neurons containing orexin in the lateral hypothalamic area of the adult rat brain are activated by insulin-induced acute hypoglycemia, Neurosci. Lett. 264 Ž1. Ž1999. 101–104. w12x T. Nambu, T. Sakurai, K. Mizukami, Y. Hosoya, M. Yanagisawa, K. Goto, Distribution of orexin neurons in the adult rat brain, Brain Res. 827 Ž1. Ž1999. 243–260. w13x Y. Oomura, T. Ono, H. Ooyama, M.J. Wayner, Glucose and osmosensitive neurones of the rat hypothalamus, Nature 222 Ž190. Ž1969. 282–284. w14x Y. Oomura, H. Ooyama, M. Sugimori, T. Nakamura, Y. Yamada, Glucose inhibition of the glucose-sensitive neurons in the rat lateral hypothalamus, Nature 247 Ž439. Ž1974. 284–286. w15x M. Rossi, S.J. Choi, D. O’shea, T. Miyoshi, M.A. Ghatei, S.A. Bloom, Melanin-concentrating hormone acutely stimulates feeding, but chronic administration has no effect on body weight, Endocrinology 138 Ž1997. 351–355.
w16x A. Sahu, Evidence suggesting that galanin ŽGAL., melanin-concentrating hormone ŽMCH., neurotensin ŽNT., proopiomelanocortin ŽPOMC. and neuropeptide Y ŽNPY. are targets of leptin signaling in the hypothalamus, Endocrinology 139 Ž2. Ž1998. 795–798. w17x A. Sainsbury, F. Rohner-Jeanrenaud, E. Grouzmann, B. Jeanrenaud, Acute intracerebroventricular administration of neuropeptide Y stimulates corticosterone output and feeding but not insulin output in normal rats, Neuroendocrinology 63 Ž4. Ž1996. 318–326. w18x T. Sakurai, A. Amemiya, M. Ishii, I. Matsuzaki, R.M. Chemelli, H. Tanaka, S.C. Williams, J.A. Richardson, G.P. Kozlowski, S. Wilson, J.R. Arch, R.E. Buckingham, A.C. Haynes, S.A. Carr, R.S. Annan, D.E. McNulty, W.S. Liu, J.A. Terrett, N.A. Elshourbagy, D.J. Bergsma, M. Yanagisawa, Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior, Cell 92 Ž4. Ž1998. 573–585. w19x B.K. Smith, D.A. York, G.A. Bray, Chronic cerebroventricular galanin does not induce sustained hyperphagia or obesity, Peptides 15 Ž1994. 1267–1272. w20x B.G. Stanley, S.E. Kyrkouli, S. Lampert, S.F. Leibowitz, Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity, Peptides 7 Ž6. Ž1986. 1189–1192. w21x D.C. Sweet, A.S. Levine, C.J. Billington, C.M. Kotz, Feeding response to central orexins, Brain Res. 821 Ž2. Ž1999. 535–538. w22x A.N. van den Pol, X.B. Gao, K. Obrietan, T.S. Kilduff, A.B. Belousov, Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretinrorexin, J. Neurosci. 18 Ž19. Ž1998. 7962–7971. w23x R. Vettor, N. Zarjevski, I. Cusin, F. Rohner-Jeanrenaud, B. Jeanrenaud, Induction and reversibility of an obesity syndrome by intracerebroventricular neuropeptide Y administration to normal rats, Diabetologia 37 Ž12. Ž1994. 1202–1208. w24x N. Zarjevski, I. Cusin, R. Vettor, F. Rohner-Jeanrenaud, B. Jeanrenaud, Chronic intracerebroventricular neuropeptide-Y administration to normal rats mimics hormonal and metabolic changes of obesity, Endocrinology 133 Ž4. Ž1993. 1753–1758.
Interactive report
Chronic intracerebroventricular administration of orexin-A to rats increases food intake in daytime, but has no effect on body weight 1 Akihiro Yamanaka a , Takeshi Sakurai a, ) , Takuo Katsumoto a , Masashi Yanagisawa b , Katsutoshi Goto a b
a Department of Pharmacology, Institute of Basic Medical Sciences, UniÕersity of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan Howard Hughes Medical Institute, Department of Molecular Genetics, UniÕersity of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-9050, USA
Accepted 29 July 1999
Abstract Orexins are recently identified neuropeptides, and have been shown to increase food intake when administered intracerebroventricularly. We examined the effects of chronic administration of orexin in rats by continuous intracerebroventricular administration by means of an osmotic minipump. Continuous administration of orexin-A Ž0.5 nmolrh. for 7 days in rats resulted in a significant increase in food intake in the daytime. Daytime food intake increased to 180% of the control value. However, it resulted in a slight decrease nighttime food intake as compared with vehicle-treated rats. The total amount of food intake per day was almost comparable with that of vehicle-administered rats. The gain of body weight and blood glucose, total cholesterol and free fatty acid levels were normal. Chronic orexin-A treatment did not cause obesity in rats. We observed abnormal behavior during the daytime after starting administration of orexin-A; these rats kept awake during the daytime. Our present observation showed that continuous administration of orexin-A could not overcome the regulation of energy homeostasis and body weight. However, orexin-A might be implicated in short-term, immediate regulation of feeding behavior. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Feeding behavior; Feeding circadian rhythm; Chronic treatment; Intracerebroventricular infusion; Blood glucose
1. Introduction Orexin-A and -B are neuropeptides that were identified as endogenous ligands for an orphan G-protein-coupled receptor, which was originally found as an expressed sequence tag in human brain w18x. Orexin-A and -B are derived from the same precursor peptide Žprepro-orexin. by proteolytic processing. They bind and activate two closely related G protein-coupled receptors, termed OX 1 and OX 2 receptors. OX 1 receptor is selective for orexin-A, while OX 2 receptor is a non-selective receptor for both orexin-A and -B. Orexin-containing neurons are specifically localized in the lateral hypothalamic area ŽLHA. and adjacent regions w4,12x. The LHA has been thought to be implicated in feeding behavior, because lesions of LHA induce an anorexic response, while electrical stimulation of LHA causes feeding behavior in rats w1x.
) Corresponding author. Tel.: q81-298-53-3276; Fax: q81-298-533039; E-mail: [email protected] 1 Published on the World Wide Web on 4 August 1999.
Food deprivation for 48 h results in an increase in prepro-orexin mRNA level in the rat hypothalamus. Orexin-A and -B increase food intake when administered centrally w18x. The orexin neurons diffusely project to various regions of brain. In the hypothalamus, orexin neurons project to the ARC, PVN, DMH Ždorsomedial hypothalamus. and VMH Žventromedial hypothalamus. w12x, which has been shown to participate in the regulation of feeding behavior. On the other hand, orexin neurons also project to various regions outside the hypothalamus, including the cerebral cortex, limbic system and brain stem. These observations suggest that orexins may be involved in not only the fundamental regulation of energy homeostasis, but also emotional, cognitive and motivational aspects of feeding behavior. Neuropeptide Y ŽNPY. has been demonstrated to be involved in the regulation of food intake and energy homeostasis as well as in the secretion of various hormones such as insulin and glucocorticoids w3,17x. The chronic administration of exogenous NPY to specific nuclei or into the cerebral ventricles of normal rats results in many of the abnormalities associated with obesity, includ-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 0 5 - 8
A. Yamanaka et al.r Brain Research 849 (1999) 248–252
ing a marked increase in food intake, accelerated body weight gain, hypercorticosteronemia, hyperinsulinemia, muscle insulin resistance, and increased triglyceride accumulation within white adipose tissue w20,23,24x. These results suggest that NPY might be involved in the fundamental regulation of energy homeostasis. However, NPYdeficient mice and NPY Y1 or Y5 receptor-deficient mice feed and grow normally, as well as exhibit a normal response to starvation w5x. One explanation for this is that there are other neuromodulators that compensate for the deficiency of NPY. Alternatively, NPY does not have roles in regulating physiological feeding, though it might be implicated in abnormal feeding in pathological states. Since orexins are thought to be involved in the regulation of feeding behavior, it could be very important to examine the effect of chronic administration of orexins, to further clarify the physiological roles of orexins in the central regulation of energy homeostasis. Therefore, we investigated the effects of chronic orexin administration to normal rats.
2. Materials and methods 2.1. Chemicals Orexin-A and NPY were purchased from Peptide Institute ŽOsaka, Japan.. 2.2. Animals and surgery Male Wistar rats Ž250–300 g on receipt; Charles-River, Kanagawa, Japan. were housed under controlled lighting Ž12 h light–dark cycle; light-on 8:00 am–8:00 pm. and temperature Ž228C. conditions. Food and water were available ad libitum. Rats were anesthetized with pentobarbital Ž50 mgrkg, i.p.., positioned in a sterotaxic frame ŽDavid Kopf Instruments, CA, USA., and a guide cannula was implanted into the lateral ventricle using sterile technique. Rats were then housed singly for a recovery period of at least 7 days. The position of the cannula was verified by central administration of 0.3 nmol human neuropeptide Y to test for a positive response. Rats that ate at least 4 g of food over a one-hour period postinjection were used for experiments. After the recovery period, a mini-osmotic pump ŽAlzet 1007D, ALZA, CA, USA. was implanted subcutaneously in the inter-scapular region under anesthesia. This osmotic minipump releases the contents at a rate of 0.5 mlrh for 7 days under physiological conditions. The infusion rate of orexin-A was 0.5 nmolrh. The mini-
Fig. 1. Effect of orexin-A on ŽA. daytime, ŽB. nighttime, and ŽC. total food intake Žmean"S.E.M... Orexin-A Žhatched bars. or vehicle Žsolid bars. was continuously injected into the lateral ventricle using an osmotic minipump for 7 days. q, P - 0.05 vs. saline Ž ns 4 ratsrgroup..
249
osmotic pump was connected to the inner cannula via a polyethylene tube ŽSP10, Natsume, Tokyo, Japan.. The
250
A. Yamanaka et al.r Brain Research 849 (1999) 248–252
inner cannula was inserted into the guide cannula and fixed using an adhesive agent. Orexin-A was dissolved in sterile water, which was used alone as the vehicle control. During administration, ad libitum food intake and body weight were measured. The food intake in the daytime and nighttime were measured separately; the decrease of food was measured twice a day Žat 8:00 am and 8:00 pm. using a digital scale ŽHL-200, A and D, Tokyo, Japan.. After the experiments, a blood sample was collected from the carotid artery into an EDTA-coated tube. Plasma was used for biochemical analysis Žblood glucose, triglycerides and total-cholesterol.. Data were analyzed by two-way ANOVA followed by post-hoc analysis of significance by the Bonferroni–Dunn statistic, using the Stat view 4.5 software package ŽAbacus Concepts, Berkeley, CA, USA.. Probability Ž P . values - 0.05 were considered statistically significant.
Fig. 3. Effect of orexin-A on plasma levels of glucose, triglycerides, total cholesterol and free fatty acids ŽFFA.. Mean and S.E.M. are presented. Orexin-A Žhatched bars., saline Žsolid bars., ns 4 per group.
3. Results 3.1. Food intake An increase of food intake during the daytime was observed after the start of administration of orexin-A ŽFig. 1.. Daytime food intake was increased 2-fold at 2 days after starting orexin-A infusion. Two-way ANOVA showed a significant overall difference Ž p - 0.05.. On the other hand, orexin-A-administered rats showed almost same or slightly decreased food intake during the nighttime compared with vehicle control rats ŽFig. 1.. 3.2. Body weight The body weight of rats treated with orexin-A for 7 days was not significantly different at the end of treatment
from that of vehicle control rats ŽFig. 2.. The gain of body weight was normal in orexin-A-administered rats after starting infusion. There was no significant difference between vehicle control and orexin-A-administered rats. Inguinal white adipose tissue weight was also normal Ždata not shown.. 3.3. Blood analysis Blood glucose, total cholesterol and free fatty acids in plasma were analyzed at the end of the experiment Žsee Fig. 3.. There were no significant differences between orexin-A-treated and control rats. Blood glucose level of orexin-A-treated and control rats was 183.4 " 4.4 and 173.7 " 2.1 Ž n s 4., respectively. Total cholesterol levels of orexin-A-treated and control rats was 52.0 " 4.7 and 59.7 " 9.3 Ž n s 4., respectively. Free fatty acid level of orexin-A-treated and control rats was 0.13 " 0.02 and 0.11 " 0.003 Ž n s 4., respectively. Triglyceride level of orexin-A-treated and control rats was 52.4 " 5.4 and 46.0 " 2.4 Ž n s 4., respectively.
4. Discussion
Fig. 2. Daily body weight Žmean"S.E.M.. of rats before and during continuous injection of either orexin-A Žv . or vehicle Ž`. into the lateral ventricle using an osmotic minipump for 7 days Ž ns 4 ratsrgroup..
An intricate balance between energy intake and expenditure achieves maintenance of body weight. This energy homeostasis is governed by the hypothalamus, where a large array of neurotransmitters, especially neuropeptides, modulate signals though complex neural circuits. Identification of the leptin signaling pathway and recent molecular genetic studies of several hypothalamic neuropeptides start to provide insights on the molecular mechanisms involved in feeding behavior and energy homeostasis. Leptin has been shown by many reports to be an essential component
A. Yamanaka et al.r Brain Research 849 (1999) 248–252
of body weight regulation w6x. The site of leptin action has been thought to exist in the medial part of the hypothalamus. Many neuropeptides involved in the regulation of energy homeostasis are known to exist in the medialrperiventricular part of the hypothalamus and are regulated by leptin w16x. Especially, the arcuate nucleus, located in the mediobasal hypothalamus, is critical for regulation of body weight and metabolism, because it contains neurons that express the leptin receptor as well as neuropeptides implicated in feeding behavior including a-melanocyte stimulating hormone Ž a-MSH., aguti-related protein ŽAgRP. and NPY. NPY infusion in normal rats resulted in hyperphagia, increased body weight gain, hyperinsulinemia, hypercorticosteronemia, and hypertriglyceridemia w23x. In our present study, orexin-A-administered rats did not show such characteristics of obesity. Similar to orexin, chronic intracerebroventricular administration of an orexigenic agent, such as galanin or melanin concentrating hormone ŽMCH., did not induce obesity in normal rats w15,19x. These observations show that these orexigenic peptides do not overcome the regulatory mechanisms of body weight, unlike NPY. Rather, these factors might be implicated in short-term regulation of feeding behavior. A decline of blood glucose level can signal the initiation of food intake. It has been reported that glucose receptor neurons are located in VMH and glucose-sensitive neurons are located in LHA w13,14x. Moriguchi et al. w11x showed that approximately 30% of orexin-containing neurons express Fos-like immunoreactivity under an insulininduced hypoglycemic state in rats. These observations suggest that some of the orexin-containing neurons are glucose-sensitive. The activity of glucose-sensitive neurons was suppressed and that of glucose sensing neurons was activated under hyperglycemia w13x. These observations suggest that orexin-containing neurons is affected by blood glucose. These observations further support our data suggesting that orexin might be involved in the short-term regulation of feeding. We observed that rats treated with orexin-A showed abnormal behavior in the daytime. Normal rats maintained this usual nocturnal habit after the start of infusion; these rats slept during the light-on period, while they behaved actively and consumed 85–90% of their total food intake during the light-off period. However, rats administered orexin-A were awake during the daytime and often showed abnormal behavior, including rearing, grooming, face washing and wet dog shaking. These behaviors were previously reported after bolus administration of orexin-A or -B w9x. Orexin might be involved in the maintenance of arousal and therefore, these rats stay awake during the daytime. This effect may possibly be a mechanism by which orexin increases food intake in the daytime. In rats, the photoperiodic-dependent circadian pattern of feeding is linked to information transmitted from the suprachiasmatic nucleus ŽSCN. to the hypothalamus. Dis-
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crete lesions in the SCN result in loss of regulated feeding w22x. These results suggest that orexins are involved in the circadian pattern. The LHA has also been shown to be involved in maintenance of the awake state w1,7,10x. Orexin immunoreactive nerve fibers were seen in brain regions involved in the sleep–wake cycle, such as the locus coeruleus, tuberomammillary nucleus, pontine reticular formation, raphe nucleus, preoptic area and dorsal tegmental nucleus w2,4,8,12x, suggesting that orexin might have effects on vigilance and arousal. The effect of orexin-A became weak with increasing number of days of administration. Chronic orexin-A treatment might cause accumulation of peptide in the cerebrospinal fluid ŽCSF. which results in desensitization of orexin receptors. The same phenomenon has been reported with repeated icv administration of galanin w19x or MCH w15x. We showed that chronic icv administration of orexin-A using an osmotic minipump did not produce a significant increase in body weight or blood sugar in rats. An experiment with chronic injection of orexins into specific nuclei in the brain is needed to clarify the role of orexins, because it has been reported that orexins induce potent feeding behavior when injected into specific nuclei, e.g. perifornical hypothalamus or lateral hypothalamus w21x. However, our present observations suggest that orexin is involved in the short-term, immediate regulation of food intake, rather than the long-term maintenance of body weight. Especially, orexins might have a role in maintaining arousal and vigilance for normal feeding.
Acknowledgements This study was supported by a grand-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, the University of Tsukuba Research project, and the Uehara Memorial Foundation. We would like to thank Dr. W.A. Gray for reading our manuscript.
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