Orexins/hypocretins: waking up the scientific world.
Orexins/hypocretins are newly discovered neuropeptides synthesized by neurones located mainly in the lateral hypothalamus. They were originally believed to have an important role in the regulation of appetite. However, nerve fibres from orexin/hypocretin neurones are widely distributed in the brain suggesting that orexins/hypocretins have multiple functions. In particular, these peptides have now been implicated as important regulators of sleep, arousal and locomotor activity. Animal studies suggest these peptides are also involved in the regulation of pituitary hormone secretion and autonomic nervous system activity. The aim of this review is to describe how these fascinating peptides were discovered, what their structures are, the available evidence on their putative actions and their possible relevance to clinical medicine. Orexins (A and B), or hypocretins (1 and 2), have recently been discovered as a result of two independent modern approaches to the identification of neuropeptides. The hypocretins were discovered, using directional tag PCR subtraction, in an effort to determine abundant messenger RNAs that are selectively expressed in the rat hypothalamus (de Lecea et al., 1998). In this study, one clone (clone 35) was found to be particularly abundant in the dorsolateral hypothalamus and resulted in the identification of the prepro-hypocretin and its possible peptide products (hypocretin 1 and 2). These peptides were named 'hypocretins' (hypothalamic incretins) because of some amino acid sequence homology to the peptide hormone secretin. The orexins were discovered through the reverse pharmacology approach in search of endogenous ligands for 'orphan' G protein coupled receptors (GPCRs; Sakurai et al., 1998). Transfectant cell lines, expressing the orphan GCPR HFGAN72 (now orexin-A/hypocretin-1 receptor, OX1R/Hcrt1R) cDNA, were challenged with chromatographic fractions derived from rat brain extracts (Fig. 1). The fractions that elicited an increase in cytoplasmic Ca2+ levels in cells were further purified and analysed to identify and isolate the peptide orexin A. A smaller activity peak was identified as orexin B and led to a search for a different orphan receptor now known as the orexin-2/hypocretin-2 receptor (OX2R/Hcrt2R). The orexins ('stimulators of appetite') were named because orexin neuronal cell bodies were found to be largely confined to the lateral hypothalamus, an area classically described as the 'feeding' centre, and based on observations that when administered into the cerebral ventricles (intracerebroventricular; ICV), orexins potently increased food intake in rats (Sakurai et al., 1998). The 'reverse' pharmacology approach to identification of ligands for orphan G protein-coupled receptors. In the discovery of orexins, transfectant cells (HEK-293) expressing the orphan receptor (HFGAN72) were challenged with chromatographic fractions derived from rat brain extracts. The observed response was a rise in cytoplasmic calcium. Hypocretin-1 and -2 are essentially the same peptides as orexin A and B, respectively (Fig. 2) and the prepro-orexin and prepro-hypocretin genes are the same. Much confusion has occurred because the structure of biologically active hypocretin-1 was not fully determined when the hypocretins were first described. Thus, hypocretin-1, as originally reported (Fig. 2), is longer than orexin A, lacks the intrachain disulphide bonds and carboxy-terminal amidation, and is inactive at the orexin/hypocretin receptors (Smart et al. 2000). It is not surprising that in the original description (de Lecea et al., 1998) only hypocretin-2 was reported to have neuroexcitatory activity since its structure (only different from orexin B by one amino acid), unlike hypocretin-1, was the biologically active form. Whether we should use the name 'hypocretins' or 'orexins' for these neuropeptides is a matter of debate. These peptides have only weak homology to secretin and it is unlikely that they belong to the secretin family of peptides; therefore the name 'hypocretins' may be inappropriate. On the other hand, these peptides are now known to have multiple functions beside the regulation of appetite (Fig. 3) thus making the name 'orexins' confusing. In this review, we will refer to the biologically active peptide products of prepro-orexin/hypocretin as orexin A/Hcrt-1 and Orexin B/Hcrt-2. Schematic representation of the structure of the prepro-orexin/hypocretin gene, mRNA and its prepro-peptide product, and the amino acid structures of orexins (A and B) and hypocretins (1 and 2; as originally described). The amino acids underlined are shared between orexin A and B (and between hypocretin 1 and 2). The main reported actions of orexins/hypocretins. The human prepro-orexin/hypocretin gene is located on chromosome 17q21 (Sakurai et al., 1998). The gene, spanning 1432 bp, consists of 2 exons and 1 intron (Fig. 2; Sakurai et al., 1999). Orexins/hypocretins are derived from a 131 amino acid human (130 amino acids in the rat) precursor prepro-orexin/hypocretin. The first 33 amino acids of prepro-orexin/hypocretin have the characteristics of a signal sequence. Orexin A/Hcrt-1 is a 33 amino acid carboxy-amidated peptide of 3562 Da with an N-terminal pyroglutamyl residue and two intrachain disulphide bonds (Fig. 2). The human orexin A/Hcrt-1 sequence is identical to the mouse, rat, bovine and porcine orexin A/Hcrt-1. Orexin B/Hcrt-2 is also C-terminally amidated, but is a linear peptide of 28 amino acids with a molecular weight of 2937 Da. Human orexin B/Hcrt-2 has two amino acid substitutions compared with rodent orexin B/Hcrt-2 and one substitution compared to porcine orexin B/Hcrt-2. There is 46% amino acid sequence identity between orexin B/Hcrt-2 and orexin A/Hcrt-1 (Fig. 2). Orexin A/Hcrt-1, but not orexin B/Hcrt-2, is lipophilic and crosses the blood–brain barrier (Kastin & Akerstrom, 1999). The OX1R/Hcrt1R receptor has structural homology to several neuropeptide receptors including the NPY Y2 receptor, TRH receptor, CCK-A receptor and NK2 neurokinin receptor. Orexin A/Hcrt-1 has two to three times greater affinity than orexin B/Hcrt-2 for the human OX1R/Hcrt1R receptor. Both orexin A/Hcrt-1 and orexin B/Hcrt-2 have similar affinities for the human OX2R/Hcrt2R receptor. Using oligonucleotide probes, OX1R/Hcrt1R receptor mRNA has been reported to be most abundant in the ventromedial hypothalamus, but has also been detected in the tenia tecta, hippocampus, dorsal raphe and locus coeruleus (Trivedi et al., 1998). OX2R/Hcrt2R receptor mRNA is expressed in the paraventricular hypothalamic nucleus, the subthalamic and thalamic nuclei, the septum, the cerebral cortex, nucleus accumbens, anterior pretectal nucleus and several regions in the medulla oblongata. The subcellular location of these orexin/hypocretin receptors is interesting. Immunohistochemical studies have shown that the location of OX1R/Hcrt1R is mainly cytosolic while that of OX2R/Hcrt2R is mainly nuclear (G. Bewick, unpublished data). OX1R/Hcrt1R and OX2R/Hcrt2R have been detected in the pituitary gland (Date et al. 2000) while OX2R/Hcrt2R mRNA has also been detected in the adrenal medulla (Lopez et al., 1999), where it may alter catecholamine secretion, but the physiological relevance of these observations is at present unclear. Orexins are not found in plasma. However, it is possible that they may be present in nerve endings, in sufficiently low density to be undetectable by radioimmunoassay, in peripheral tissues. Alternatively, peripheral orexin receptors may function as receptors for another ligand. Curiously, orexin/hypocretin neurones appear to be labelled by an antiserum raised against ovine prolactin (Risold et al., 1999). A small proportion of orexin/hypocretin neurones are immunopositive for galanin (Hakansson et al., 1999). Orexin A/Hcrt-1 and Orexin B/Hcrt-2 are found in secretory vesicles at neuronal synapses and are neuroexcitatory (de Lecea et al., 1998; van Den Pol et al., 1998). These peptides cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx in vitro (Smart et al., 1999; Lund et al. 2000). Orexin/hypocretin immunoreactivity and immunoreactive fibres are widely distributed throughout the central nervous system (Peyron et al., 1998; Mondal et al., 1999; Nambu et al., 1999; Taheri et al., 1999), but have only occasionally been reported in peripheral tissues (Kirchgessner & Liu, 1999; Date et al. 2000). Orexin/hypocretin immunoreactive fibres are densely distributed in the hypothalamus, septum, thalamus, brainstem and spinal cord (Peyron et al., 1998; Cutler et al., 1999; Date et al., 1999; Nambu et al., 1999). Orexin fibres innervating neurones in the locus coeruleus appear to represent the greatest peptidergic input into this pontine region (Horvath et al., 1999a). The locus coeruleus is a major monoaminergic site within the central nervous system involved in the regulation of arousal. Elevated c-fos (a measure of neuronal activation) immunoreactivity in response to orexins (Date et al., 1999; Edwards et al., 1999; Mullett et al. 2000) has been detected in hypothalamic areas involved in food intake, neuroendocrine regulation and sleep and circadian rhythms (the lateral hypothalamus, the posterior and dorsomedial hypothalamus, anterior hypothalamus, the perifornical, arcuate and paraventricular nuclei), in the lateral septal area, the central nucleus of the amygdala, the shell of the nucleus accumbens, the bed nucleus of the stria terminalis, and the nucleus of the solitary tract (involved in autonomic and visceral regulation). Orexin neurones in the lateral hypothalamus have reciprocal connections with neuropeptide Y (NPY) and agouti-related peptide (AgRP) neurones in the arcuate nucleus (Broberger et al., 1998; Horvath et al., 1999b). The lateral hypothalamus has classically been considered to be the 'feeding' centre with lesions of this area being associated with a marked decrease in food intake. The ventromedial hypothalamus is known as the 'satiety' centre since lesions of this area result in increased food intake and obesity. These early experiments have been clarified by the discovery of neuropeptide mediators of food intake (Table 1; Kalra et al., 1999). Several neuropeptides synthesized in the arcuate nucleus of the mediobasal hypothalamus are mediators of food intake including NPY, the pro-opiomelanocortin derivative α-melanocyte-stimulating hormone (α-MSH), AgRP and the peptide product of cocaine and amphetamine-regulated transcript (CART). When administered ICV to rats, NPY and AgRP stimulate food intake, while α-MSH and CART reduce food intake. These neuropeptides are regulated by leptin, a cytokine that signals the extent of fat stores to the central nervous system (Meister 2000). The lateral hypothalamus contains neurones, distinct from orexin/hypocretin neurones, expressing melanin-concentrating hormone (MCH), a central stimulator of food intake (Elias et al., 1998). The anatomical location of neurones expressing orexins/hypocretins, led to the hypothesis that they are important regulators of food intake. When originally administered into the lateral ventricle of rats, both orexin A/Hcrt-1 and orexin B/Hcrt-2 dose-dependently and potently stimulated food intake in rats and prepro-orexin/hypocretin mRNA was shown to be upregulated with fasting (Sakurai et al., 1998). The stimulation of food intake by orexins/hypocretins has been difficult to consistently repeat in several laboratories, particularly for orexin B/Hcrt-2 (Edwards et al., 1999). While orexin A/Hcrt-1 may increase food intake in the first 4 hours after ICV administration, it actually decreases food intake in the subsequent 20 hours (Taheri et al. 2000a). Furthermore, chronic administration of orexin A/Hcrt-1 does not result in obesity (Haynes et al., 1999; Yamanaka et al., 1999). It may be that orexins/hypocretins are not as important in food intake as originally believed, or that they may be important in food intake only in particular circumstances (e.g. in response to hypoglycaemia and/or in the regulation of circadian food intake). Orexin/hypocretin neurones have leptin receptors and are immunoreactive for STAT3, a transcription factor activated by leptin (Hakansson et al., 1999). However, the interaction of leptin with orexins/hypocretins is unlike the interaction observed with the potently orexigenic peptide NPY. NPY mRNA is upregulated in leptin-deficient ob/ob and leptin receptor deficient db/db mice, while prepro-orexin/hypocretin mRNA is downregulated, but increases with starvation in these animals (Yamamoto et al., 1999; Yamamoto et al. 2000). Although one group has recently reported moderately lower prepro-orexin/hypocretin mRNA in obese Zucker rats (Cai et al. 2000), which have defective leptin receptor action compared to their lean controls, we have noted no differences in prepro-orexin/hypocretin mRNA or orexin A/Hcrt-1 immunoreactivity in these strains (Taheri et al., in press). It is therefore unlikely that orexins/hypocretins are directly involved or are altered in the obesity of Zucker rats. These findings do not mean that orexins/hypocretins are not regulated by leptin since the effects of the other neuroendocrine abnormalities present in these animals may mask the effects of leptin deficiency on orexin/hypocretin neurones. Orexins/hypocretins appear to have an important role in the regulation of metabolic rate. Using indirect calorimetry, orexin A/Hcrt-1, but not orexin B/Hcrt-2, increased metabolic rate (Lubkin & Stricker-Krongrad, 1998). Mice overexpressing prepro-orexin/hypocretin have reduced body weight despite increased food intake since they also have a concomitant increase in metabolic rate (Inui 2000). Mice with targeted disruption of the prepro-orexin/hypocretin are hypophagic with reduced metabolic rate, but are prone to diet induced obesity, presumably due to their lower metabolic rate (Willie et al., in press). Orexin/hypocretin fibres innervate hypothalamic regions involved in the regulation of pituitary hormone release. Orexin A/Hcrt-1 releases several neuropeptides and releasing factors from mediobasal hypothalamic explants in vitro (Russell et al. 2000). Centrally administered orexin A/Hcrt-1 alters plasma prolactin, growth hormone and corticosterone. In the studies that originally reported these endocrine responses, orexin A/Hcrt-1 was shown to increase arousal and to activate neurones in the locus coeruleus (Hagan et al., 1999). The profound inhibition of plasma prolactin by ICV orexin A/Hcrt-1 is partially attenuated by the administration of the dopamine receptor antagonist domperidone suggesting that while in the presence of domperidone centrally administered TRH, neurotensin and vasopressin lose their prolactin lowering effect, the effect of orexin A/Hcrt-1 is partly independent of dopamine (Russell et al. 2000). Orexin A/Hcrt-1 increases plasma ACTH and corticosterone and there is increased c-fos mRNA in the paraventricular nucleus (PVN) of the hypothalamus where corticotrophin-releasing factor (CRF) neurones are located (Hagan et al., 1999; Ida et al. 2000; Kuru et al. 2000). It is therefore likely that orexin A/Hcrt-1 is an important regulator of the hypothalamo-pituitary-adrenal axis, which is consistent with its effect on increasing arousal. The mechanism through which orexin A/Hcrt-1 inhibits GH secretion remains to be determined, but may involve the release of somatostatin. Orexins/hypocretins may regulate LH secretion via GnRH neurones in the hypothalamus. In one study, the effects of orexin A/Hcrt-1 and orexin B/Hcrt-2 on LH secretion were evaluated in ovariectomized (ovx) and ovarian steroid-treated ovx rats (Pu et al., 1998). Central injection of orexin A/Hcrt-1 or orexin B/Hcrt-2 rapidly stimulated LH secretion in ovx animals pretreated with oestradiol and progesterone. However, these peptides inhibited LH release in unprimed ovx rats. Interestingly, we have observed changes in orexin A/Hcrt-1 immunoreactivity in the rat CNS with the oestrous cycle (Taheri & Russell, unpublished data). The available evidence for the importance of orexins/hypocretins in the hypothalamic regulation of pituitary hormone release is derived from animal experiments in which these peptides have been administered into the cerebral ventricles. This experimental approach, however, will result in the activation of several circuits in which orexins/hypocretins are involved and any hormonal changes in the plasma will represent the net effect of the circuits activated. It is therefore not surprising that intracerebroventricular administration of orexins/hypocretins stimulates the HPA axis, while paradoxically inhibiting prolactin release. The neuronal circuitry through which orexins/hypocretins may regulate pituitary hormone release remain to be defined. Further, the physiological circumstances in which orexins/hypocretins regulate the neuroendocrine system and the existence of any negative or positive feedback loops remain to be determined. The distribution of orexin/hypocretin immunoreactive fibres within the CNS raised the possibility that they may be involved in the regulation of sleep, arousal and activity. Targeted disruption of the prepro-orexin gene in mice results in a phenotype with features similar to the human narcolepsy syndrome (Chemelli et al., 1999). In the same report, modafinil, a drug used in the treatment of narcolepsy through an unknown mechanism, was shown to activate orexin/hypocretin neurones. Narcolepsy is a debilitating sleep disorder characterized by excessive daytime sleepiness, cataplexy (loss of muscle tone in response to emotional stimuli) and disturbance of rapid eye movements (REM) sleep. Canine models of narcolepsy, which inherit narcolepsy in an autosomal recessive fashion with full penetrance through the canarc-1 gene, have been shown to have mutations in the OX2/Hcrt-2 receptor gene (Lin et al., 1999). In rats, ICV administration of orexin A/Hcrt-1 increases arousal while reducing REM sleep and prolonging the latency to the first occurrence of REM sleep (Piper et al. 2000). These studies suggested that orexins have a role in the regulation of arousal and the sleep-wake cycle. Indeed, orexin A/Hcrt-1 immunoreactivity shows diurnal variation in areas of the brain involved in the regulation of sleep, arousal and circadian hormone release (Taheri et al. 2000a). When orexin A/Hcrt-1 immunoreactivity was measured in the cerebrospinal fluid of patients with narcolepsy, it was shown that the majority of the patients examined had undetectable orexin A/Hcrt-1 compared to controls (Nishino et al. 2000; Taheri et al. 2000b). This is now further supported by undetectable prepro-orexin mRNA in autopsy specimens from patients with narcolepsy (Peyron et al. 2000). It is likely that in human narcolepsy, unlike canine narcolepsy, there is an abnormality in orexin production or prepro-orexin processing rather than mutations in the OX2/Hcrt-2 receptor gene. Since orexin A/Hcrt-1 abnormalities have not been detected in all patients diagnosed with narcolepsy suggests that there may be different subsets of patients with narcolepsy. Interestingly, little is known about endocrine abnormalities in both canine and human narcolepsy except a greater propensity towards type 2 diabetes mellitus in human narcolepsy (Honda et al., 1986). It is not surprising that orexins/hypocretins are involved in the regulation of hormones such as prolactin, GH and corticosterone, which are intimately associated with sleep and arousal. Orexins have been implicated in the CNS regulation of gastric acid secretion (vagally mediated), sympathetic activation, cardiovascular function and drinking behaviour. Both orexin A/Hcrt-1 and orexin B/Hcrt-2, when injected ICV increase blood pressure and heart rate in rats (Samson et al., 1999; Shirasaka et al., 1999). ICV orexin A/Hcrt-1 increases water intake, while prepro-orexin/hypocretin mRNA increases with water deprivation (Kunii et al., 1999). In the spinal cord, orexins/hypocretins may influence the sensory and autonomic nervous systems (van Den Pol, 1999). There are several reports on the effect of orexins/hypocretins on peripheral tissues such as the gastrointestinal tract, the pancreas and adrenal gland (Kirchgessner & Liu, 1999; Malendowicz et al., 1999; Nowak et al. 2000). Orexins/hypocretins are good examples of how modern molecular biology techniques have contributed to the discovery and greater understanding of the physiological actions of neuropeptides. It is remarkable how quickly these peptides have made the transition from basic scientific research to clinical medicine. Since the secretion of most pituitary hormones is intimately linked to sleep and arousal, it is likely that under basal conditions, orexins/hypocretins link sleep and arousal to pituitary hormone release. It now remains to study the orexin/hypocretin neuronal circuitry in conditions that disturb this basal state, for example, ageing, sleep loss, night or shift work, jet lag, affective disorders and endocrine diseases (e.g. Cushing's syndrome). Manipulation of the orexin/hypocretin ligand-receptor system may prove therapeutically useful not only in the treatment of sleep disorders such as narcolepsy and insomnia, but also in the treatment of several medical and psychiatric disorders associated with sleep disturbance. However, a greater understanding of the role of the individual peptides and receptors is required combined with an effort to tease out the neuronal circuits involved. Further study of these fascinating peptides may also clarify how hormone release is linked to sleep and arousal. Shahrad Taheri is funded by the Wellcome Trust. We would like to thank members of our laboratory who have contributed to the study of orexins/hypocretins and Dr Mignot, Dr Yanagisawa and Dr Chemelli for providing us with information regarding their work.
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