Abstract

Key Words appetite, metabolism, arousal, narcolepsy, lateral hypothalamus s Abstract Orexin-A and orexin-B are neuropeptides originally identified as endogenous ligands for two orphan G-protein-coupled receptors. Orexin neuropeptides (also known as hypocretins) are produced by a small group of neurons in the lateral hypothalamic and perifornical areas, a region classically implicated in the control of mammalian feeding behavior. Orexin neurons project throughout the central nervous system (CNS) to nuclei known to be important in the control of feeding, sleepwakefulness, neuroendocrine homeostasis, and autonomic regulation. orexin mRNA expression is upregulated by fasting and insulin-induced hypoglycemia. C-fos expression in orexin neurons, an indicator of neuronal activation, is positively correlated with wakefulness and negatively correlated with rapid eye movement (REM) and non-REM sleep states. Intracerebroventricular administration of orexins has been shown to significantly increase food consumption, wakefulness, and locomotor activity in rodent models. Conversely, an orexin receptor antagonist inhibits food consumption. Targeted disruption of the orexin gene in mice produces a syndrome remarkably similar to human and canine narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, cataplexy, and other pathological manifestations of the intrusion of REM sleep-related features into wakefulness. Furthermore, orexin knockout mice are hypophagic compared with weight and age-matched littermates, suggesting a role in modulating energy metabolism. These findings suggest that the orexin neuropeptide system plays a significant role in feeding and sleep-wakefulness regulation, possibly by coordinating the complex behavioral and physiologic responses of these complementary homeostatic functions. 0147-006X/01/0301-0429$14.00 429 Annu. Rev. Neurosci. 2001.24:429-458. Downloaded from www.annualreviews.org by SCELC Trial on 11/01/10. For personal use only. WILLIE ET AL INTRODUCTION Feeding behavior is dependent upon the integration of metabolic, autonomic, endocrine, and environmental factors coordinated with an appropriate state of cortical arousal (wakefulness). Historically, the hypothalamus has been recognized to play a critical role in maintaining energy homeostasis by integrating these factors and coordinating the behavioral, metabolic, and neuroendocrine responses Until recently, melanin-concentrating hormone (MCH) was the only neuropeptide implicated in feeding regulation known to be produced solely within the LHA. Annu. Rev. Neurosci. 2001.24:429-458. Downloaded from www.annualreviews.org by SCELC Trial on 11/01/10. For personal use only. ROLE OF OREXIN IN FEEDING AND AROUSAL 431 MCH dose-dependently increases food consumption when administered centrally in rodents. Genetic disruption of the MCH gene in mice results in hypophagia and reduced body weight compared with wild-type littermates OREXIN SIGNALING PATHWAY Orexin Neuropeptides Our group isolated orexin-A and orexin-B while screening high-resolution highperformance liquid chromatography fractions from brain extracts for stimulation of signal transduction in cell lines expressing orphan G-protein-coupled receptors (GPCRs) A single gene composed of two exons and an intervening intron encodes the orexin neuropeptides. This structure is conserved within rodent and human genomes A messenger RNA encoding the same neuropeptide precursor was independently isolated from a hypothalamus-enriched cDNA library by using a differential cloning approach, and the putative encoded peptides were named hypocretins by de . Nucleotide sequence alignment shows that the base-pair sequences of hypocretins-1 and -2 are the same as orexins-A and -B, but that the mature peptides predicted in this report had additional amino acids not found in native orexins. The hypocretin peptides, synthesized according to sequences predicted by 432 WILLIE ET AL (orexin-B) with the gut hormone secretin, but native orexin peptides are, in fact, distantly similar to the bombesin neuropeptide family and not the secretin family ( Orexin Receptors Sakurai et al (1998) identified two orexin receptor subtypes, named orexin-1 receptor (OX 1 R) and orexin-2 receptor (OX 2 R), that are structurally similar to other G-protein-coupled neuropeptide receptors. The OX 1 R is the orphan G-proteincoupled receptor used during ligand hunting to first identify and then purify the orexins. A search of the GenBank dbEST database with the OX 1 R amino acid sequence revealed two candidate ESTs. Using PCR, with primers designed from these ESTs, the OX 2 R receptor was discovered and found to have a 64% aminoacid identity with OX 1 R. Competitive radio-ligand binding assays reveal that the orexin receptors have different binding profiles for the respective orexin peptides. The OX 1 R has a 1-order-of-magnitude greater affinity for orexin-A [50% Inhibitory Concentration (IC 50 = 20 nM)] compared with orexin-B (IC 50 = 250 nM). In contrast, orexins-A and -B bind the OX 2 R with equal affinity (IC 50 = 20 nM). Therefore, it appears that the OX 1 R is moderately selective for orexin-A, whereas OX 2 R is a nonselective receptor for both neuropeptide agonists. Evidence from receptor-transfected cell lines and isolated receptor-expressing hypothalamic neurons suggest that the OX 1 R is coupled exclusively to the G q subclass of heterotrimeric G proteins, whereas OX 2 R may couple to G i/o , and/or G q (Sakurai et al 1998; van den Pol et al 1998; T Yada, S Muroya, H Funahashi, S Shioda, A Yamanaka, et al, submitted for publication). Neuroanatomy of the Orexin System In the rodent CNS, orexin-producing cells ( In situ hybridization studies with orexin receptor riboprobes demonstrate that orexin receptors are expressed in a pattern consistent with orexin projections, but that they have a marked differential distribution 434 WILLIE ET AL interpreted cautiously since nuclei in close proximity to the ventricular space would be preferentially activated from ventricular delivery, and no conclusions regarding orexin-mediated inhibition of neuronal pathways were obtained. The potential importance of orexin-mediated inhibition is confirmed by the work of van den . These investigators found that orexins increase the release of the inhibitory neurotransmitter γ -aminobutyric acid (GABA), as well as the excitatory neurotransmitter glutamate, by acting directly on axon terminals of neuroendocrine cells in the ARC nucleus. Orexin neurons also express mRNAs for the orexigenic opioid dynorphin and the secretory marker secretogranin II , and the biosynthesis of these three peptides may be similarly regulated . Immunoreactivity for the appetite stimulating neuropeptide galanin has also been identified in orexin neurons OREXINS MAINTAIN WAKEFULNESS The regulation of sleep-wakefulness cycling within the context of circadian and environmental influences is critical for the efficient maintenance of energy homeostasis. In mammals, the sleep-wakefulness cycle can be divided into periods of waking, non-rapid-eye-movement (non-REM), and rapid-eye-movement (REM) sleep on the basis of distinct behavioral and electroencephalographic (EEG) criteria. A role for LHA in sleep regulation was suggested by early animal experiments that showed electrical stimulation of the LHA promoted wakefulness while destruction of the LHA caused somnolence and inattentiveness 435 is also associated with behavioral changes indicative of an aroused state including increased locomotor activity, rearing, grooming, burrowing, searching behaviors, and food consumption (discussed below) Several recent studies confirm the contribution of disrupted orexin signaling to the etiology of human narcolepsy. Nishino et al Based on behavioral and polysomnographic criteria, orexin knockout mice exhibit a phenotype strikingly similar to human narcoleptics ROLE OF OREXIN IN FEEDING AND AROUSAL 437 and consequent production of non-functional OX 2 R receptors. Our lab has also created mice with targeted disruptions of the ox1r and ox2r receptor genes. Preliminary studies in the receptor knockouts show that, as expected, the ox2r knockout mice have characteristics of narcolepsy. However, the behavioral and electroencephalographic phenotype of the ox2r-null mice is less severe than that found in the orexin neuropeptide knockout mice The ascending cortical activating system (ACAS) is a diffuse collection of brainstem nuclei grouped together based on their ability to stimulate cortical arousal and wakefulness. Dense projections from orexin neurons to the ACAS including the histaminergic TMN, noradrenergic LC, serotonergic dorsal raphe, and cholinergic pedunculopontine nucleus provide further anatomic evidence of orexin's important role in sleep-wakefulness regulation OREXINS INFLUENCE INGESTIVE BEHAVIORS AND METABOLISM Feeding Early lesioning experiments of the LHA consistently caused a syndrome of decreased food and water intake that lowered body weight set-point to about 75%-80% that of sham-operated controls in several species ROLE OF OREXIN IN FEEDING AND AROUSAL 439 suppresses spontaneous feeding in fasted rats Circadian processes have a marked influence on feeding behavior and disruptions of normal circadian feeding patterns are well-described effects of LHA lesions Chronic infusion of orexin-A over several days disrupts the normal circadian feeding pattern in rats by increasing daytime and decreasing nighttime food intake Nuclei-specific studies performed by ROLE OF OREXIN IN FEEDING AND AROUSAL 441 Drinking Early electrical stimulation studies also suggested that the LHA is a "primary drinking center" Metabolic, Autonomic, and Endocrine Effects The LHA is also important in controlling metabolic rate. Animal models with lesions in the LHA consistently become hypercatabolic and remain so, even after they reach a lower body weight set-point (Bernardis & Bellinger 1993 INTERACTION WITH HYPOTHALAMIC FEEDING PATHWAYS Leptin is an anorectic protein produced and secreted in proportion to adipocyte fat stores and therefore it is commonly referred to as the "adipostat." Exogenously administered leptin can inhibit the increased feeding stimulated by several orexigenic neuropeptides. In experiments to test whether orexin-mediated feeding is sensitive to leptin, Yamanaka et al (A Yamanaka, K Kunii, N Tsujino, I Matsuzaki, K Goto, T Sakurai, submitted for publication) pre-treated rats with leptin at doses that completely inhibit NPY-and galanin-stimulated feeding. They found that leptin only partially inhibited orexin-mediated feeding. The effect of anorectic peptides, thought to mediate leptin effects on food intake, were then examined. Pre-treatment with either CART (cocaine-and amphetamine-related transcript), αMSH (proopiomelanocortin-derived α-melanocyte-stimulating hormone), or GLP-1 (glucagon-like peptide-1), at doses that abolished NPY-induced feeding, again only partially inhibited orexin-induced feeding. These studies suggest that appetite-stimulating pathways involving NPY and galanin are leptinsensitive, while orexin-mediated feeding involves both leptin-sensitive and -insensitive pathways. Because leptin has recently been shown to promote slow wave (delta) at the expense of REM sleep , the effect of leptin on orexin-mediated arousal should be studied. The ARC nucleus is a major site of leptin-responsive neurons and is regarded as an important "satiety center" on the basis of classic lesioning studies . This nucleus is a complex collection of neurons that express most of the well-described orexigenic and anorectic neuropeptides. Leptin-mediated inhibition of orexigenic NPY/agouti-related protein (AgRP)-coexpressing neurons and excitation of anorectic proopiomelanocortin (POMC)/ CART-coexpressing neurons are thought to underlie the suppression of appetite by Isolated ARC neurons were studied to determine their responses to orexin and leptin using an in vitro fura-2 microfluorimetry system (T Yada, S Muroya, H Funahashi, S Shioda, A Yamanaka, et al, submitted for publication). Application of orexin-A or -B to POMC neurons decreased cytosolic calcium ([Ca 2+ ] i ) and inhibited [Ca 2+ ] i oscillations with a picomolar sensitivity. This effect had an equimolar sensitivity to orexin-A and orexin-B and was inhibited by pertussis toxin suggesting that it is mediated through an OX 2 R pathway that is coupled to G i and/or G o . Interestingly, many of the POMC neurons inhibited by orexin were excited by picomolar concentrations of leptin. In contrast, arcuate NPY neurons are inhibited by leptin and excited by orexin-A and -B, with orexin-B 100-fold less potent than orexin-A. Selective inhibitors of phospholipase C, protein kinase C, and inositol tri-phosphate-dependent Ca 2+ channels block these excitatory effects suggesting the involvement of OX 1 R signaling through a G q -coupled pathway. The VMH is another important component of the "satiety center" and is characterized by a high density of glucose and insulin-responsive neurons (Oomura & Kita Annu. Rev. Neurosci. 2001.24:429-458 REGULATION OF OREXIN NEURONS The LHA receives innervation from much of the neuroaxis and can also be influenced by actively transported peripheral factors, such as leptin, insulin, and other hormones, as well as diffusible factors including glucose, electrolytes, amino acids, and peptides Circadian Influences Orexin neurons are significantly influenced by circadian processes because Fos studies indicate that orexin neurons are primarily "waking active" ROLE OF OREXIN IN FEEDING AND AROUSAL 445 and may influence circadian activity in the LHA (Mikkelsen 1990). Interestingly, Nutritional and Visceral Satiety Signals The LHA reward system is modulated by inhibitory signals from the VMH (Margules & Olds 1962) and it is highly dependent on nutritional state, demonstrating marked potentiation following food deprivation Cai et al (1999) performed a detailed study of orexin mRNA expression in rats after physiologic and pharmacologic manipulations designed to stimulate food Annu. Rev. Neurosci. 2001.24:429-458. Downloaded from www.annualreviews.org by SCELC Trial on 11/01/10. For personal use only. 446 WILLIE ET AL intake through different mechanisms. These authors fasted rats for 48 hours and found that orexin mRNA levels significantly increased over controls, accompanied by decreases in serum leptin and glucose. No significant difference in orexin mRNA levels were found when rats were restricted to 50% of their usual daily intake for six days. Serum leptin and glucose levels at the end of this experiment were 38% and 83% of control values, respectively. These starvation studies suggest that orexin expression increase markedly in response to acute metabolic challenges, but approach normal after sub-acute stresses when compensatory metabolic processes are likely to intervene (glucose and leptin returning toward baseline values). These authors then studied rats after 6 hours of acute hypoglycemia and after six days of chronic hypoglycemia induced by subcutaneous insulin injections. A significant increase in orexin mRNA expression was found after acute hypoglycemia when rats were food-deprived after insulin injection, but no change was seen when they were allowed to feed freely. Leptin and glucose levels were 228% and 49% vs. 228% and 17% of control values in the freely fed vs. fooddeprived conditions, respectively. An increase in orexin mRNA expression that approached significance was observed after chronic hypoglycemia and freely-fed conditions, with serum leptin and glucose levels 288% and 47% of controls. These authors identified subnormal plasma glucose levels and absence of food intake as key factors associated with increased orexin mRNA expression. They further suggested that orexin neurons may belong to a glucose-sensitive sub-population of LHA neurons that are stimulated by falls in serum glucose and inhibited by vagally-mediated prandial signals, such as gastric distension and/or rising portal venous glucose concentrations Leptin and Hypothalamic Feeding Peptides The LHA receives afferent projections from many CNS areas known to be important in energy homeostasis ROLE OF OREXIN IN FEEDING AND AROUSAL 447 Leptin regulates the expression of many orexigenic and anorectic signals . Genetic mouse models of obesity have been instrumental in identifying and characterizing many hypothalamic feeding pathways. Deficient leptin signaling causes hyperphagia, abnormal glucose utilization, infertility, and early-onset obesity in ob/ob and db/db mice. The leptin gene has a nonsense mutation in the ob/ob mouse whereas the db/db mouse has an inactivating mutation in the leptin receptor gene Studies of Mch, Npy, and orexin gene expression in these mouse models of obesity suggest that orexin is regulated in a manner opposite to that of MCH and NPY. In freely-fed ob/ob, db/db, and A y /a mice, Mch and Npy expression are upregulated compared with normal controls 448 WILLIE ET AL mouse models of obesity. It is intriguing to speculate, however, that decreased orexin expression contributes to the decreased metabolic rate that is believed necessary to explain the extreme obesity of these genetic models since it cannot be explained on grounds of increased food intake alone. Apparently contradictory results have been obtained from studies examining orexin expression when ob/ob and A y /a mice are fasted. DISCUSSION Feeding behavior is critically dependent on appropriate sleep-wakefulness cycling at environmentally advantageous times and in response to homeostatic needs. While feeding and sleep are mutually exclusive behaviors, wakefulness, with increased sensory awareness and motor activity, is required for successful feeding. The relationship among waking, feeding, and environment are highly species dependent. While humans consolidate waking and feeding cycles during the daytime, rodents are awake and feed primarily at night. It can be argued that the balance between food availability and the relative risks of predation were the evolutionary driving forces behind these divergent behavioral patterns. Similarly, during periods of nutritional depletion, central mechanisms capable of augmenting wakefulness and therefore feeding opportunities within the proper circadian phase would confer an advantage. Because of the co-dependency between sleep-wakefulness and feeding, stressors that primarily affect one very often affect the other. Indeed, comorbid disturbances of sleep, appetite, and metabolism are well-described symptoms of obesity, anorexia nervosa, depression, and Cushings disease Substantial evidence is accumulating to support the hypothesis that pathologic disruption of orexin signaling causes narcolepsy in humans, mice, and dogs. Several studies suggest that altered energy homeostasis may accompany narcolepsy in these species. ROLE OF OREXIN IN FEEDING AND AROUSAL 449 of non-insulin-dependent diabetes mellitus in an adult Japanese population with narcolepsy. Another study found lower daily caloric intake in narcoleptics compared with matched controls, and that the difference was due primarily to reduced carbohydrate intake No feeding or metabolic abnormalities have been reported in the ox2r-null dog model of narcolepsy. While this may be explained by a lack of surveillance for a subtle phenotype, it may indicate that the OX 1 R-signaling pathway is more important in orexin-mediated energy homeostasis. Several lines of evidence support this hypothesis: There is a marked differential expression pattern of orexin receptors in the CNS, and importantly, a high density of OX 1 Rs are found in the ARC and VMH nuclei of the hypothalamus Researchers have long recognized that fasting alters sleep-wakefulness patterns in several species 450 WILLIE ET AL acids) stimulate increased waking time, thus presumably increasing the chance of feeding opportunities. The magnitude of this effect is highly dependent upon endogenous energy reserves (possibly through leptin as an indicator of fat stores and metabolic state). And, as food deprivation is prolonged, compensatory mechanisms that conserve energy are engaged and energetically costly increases in wakefulness diminish. It is interesting to speculate that the persistent suppression of REM may also increase feeding opportunities by shifting sleep-rest periods into vigilance states with increased sensory arousal and motor tone. The regulation of both sleeping and feeding behaviors in mammals is thought to result from the integration of homeostatic and circadian influences Orexin neurons have the requisite anatomic connections, physiologic effects, interactions with hypothalamic feeding pathways, and regulation by circadian and nutritional factors to suggest that they may be an important cellular and molecular link in the integration of sleep and energy homeostasis ( ROLE OF OREXIN IN FEEDING AND AROUSAL 451 Figure have documented that acute orexin injection dose-dependently increases arousalassociated behavioral and physiologic process including food-intake, waking time, motor activity, and metabolic rate, as well as heart rate and blood pressure. It is unlikely, however, that orexin-signaling plays a significant role in long-term "lipostat" mechanisms since chronic administration fails to increase body weight, total daily food intake, or adiposity. Furthermore, it is likely that orexin-mediated food intake results partly from stimulation of ARC feeding pathways such as those involving NPY. The increased expression of Npy and Mch and decreased expression of orexin found in hyperphagic mouse models of obesity are consistent with this hypothesis. Fasting studies in normal and fat-mutant mice suggest that orexin neurons are more responsive to acute changes in energy substrate availability than endogenous energy stores. The potential role of orexin as a barometer Annu. Rev. Neurosci. 2001.24:429-458. Downloaded from www.annualreviews.org by SCELC Trial on 11/01/10. For personal use only. 452 WILLIE ET AL of acute energy homeostasis is further exemplified by its upregulation when integrating contradictory metabolic signals such as hypoglycemia in the face of hyperleptinemia. The strong "waking active" character of orexin neurons also reinforces the hypothesis that orexin signaling is important in the acute regulation of energy homeostasis: short-term changes in substrate availability likely lead to increased orexin neuronal firing that in turn increases sensory arousal, wakefulness, motor activity, and substrate mobilization from energy stores, while stimulating and reinforcing hypothalamic feeding pathways. The orexin neuropeptide signaling system is an important central pathway that promotes adaptive behavioral and physiologic responses in response to metabolic and environmental signals. During periods of nutritional depletion, orexin-induced increases in arousal and reinforcement of appetite/feeding pathways may be an evolutionarily conserved mechanism that helps to ensure survival. In summary, the orexin neuropeptide system plays a significant role in coordination of feeding and sleep-wakefulness regulation, probably by coordinating the complex behavioral and physiologic responses of these complementary homeostatic functions. ACKNOWLEDGMENTS