DTD 5

ARTICLE IN PRESS

Regulatory Peptides xx (2004) xxx – xxx

www.elsevier.com/locate/regpep

Review

Reverse pharmacology of orexin: from an orphan GPCR

to integrative physiology
Takeshi Sakurai*
Department of Pharmacology, Institute of Basic Medical Science, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
ERATO Yanagisawa Orphan Receptor Project, Japan Science and Technology Corporation, Tokyo 135-0064, Japan

Abstract

Orexins, which were initially identified as endogenous peptide ligands for two orphan G-protein coupled receptors (GPCRs), have been
shown to have an important role in the regulation of energy homeostasis. Furthermore, the discovery of orexin deficiency in narcolepsy
patients indicated that orexins are highly important factors for the sleep/wakefulness regulation. The efferent and afferent systems of orexin-
producing neurons suggest interactions between these cells and arousal centers in the brainstem as well as important feeding centers in the
hypothalamus. Electrophysiological studies have shown that orexin neurons are regulated by humoral factors, including leptin, glucose, and
ghrelin as well as monoamines and acetylcholin. Thus, orexin neurons have functional interactions with hypothalamic feeding pathways and
monoaminergic/cholinergic centers to provide a link between peripheral energy balance and the CNS mechanisms that coordinate sleep/
wakefulness states and motivated behavior such as food seeking.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Hypothalamus; Orexin; Arousal; Sleep; Cholinergic; Monoaminergic

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. Identification of orexins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Orexin receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. Orexin-producing neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5. Orexin defficiency in narcolepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
6. Mechanisms of regulation of behavioral states by orexins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
7. Regulation of orexin neuronal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
8. Roles of orexins in the regulation of energy homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
9. Orexin: a link between energy homeostasis and arousal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

bReverse pharmacologyQ, i.e., endogenous ligand screen-

ing using cell lines which express orphan G-protein coupled
receptors (GPCRs), combined with genetic engineering
* Tel.: +81 29 853 3277; fax: +81 298 853 3276. techniques, has increased our understanding of novel
E-mail address: stakeshi@md.tsukuba.ac.jp. signaling systems in the body [1]. Perhaps the most
0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.regpep.2004.08.006

REGPEP-03271; No of Pages 8
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2 T. Sakurai / Regulatory Peptides xx (2004) xxx–xxx
successful example of such approach is discovery of orexin.
Many works clearly suggested that orexins are highly
important molecules in the regulation of sleep/wakefulness
states as well as feeding behavior. This review summarizes
recent relevant findings on orexins, and discusses physio-
logical roles of these peptides.
2. Identification of orexins
Most neuropeptides work though G protein–coupled
receptor (GPCRs). There are numerous (approximately
100–150) borphanQ GPCR genes in the human genome;
the cognate ligands for these receptor molecules have not
been identified yet. We performed a so-called breverse
pharmacologyQ that aims to identify ligands for orphan
GPCRs. We expressed orphan GPCR genes in transfected
cells and used them as a reporter system to detect
endogenous ligand substances in tissue extracts that can
activate signal transduction pathways in GPCR-expressing
cell lines. In this process, we identified orexin-A and orexin-
B as endogenous ligands for two orphan GPCRs found as
human expressed sequence tags [2]. Orexins constitute a
novel peptide family, with no homology with any previously
described peptides.
Fig. 1. (A) Schematic representation of the orexin system. Orexin-A and -B
are derived from a common precursor peptide, prepro-orexin. The actions
of orexins are mediated via two G protein-coupled receptors named orexin-
1 (OX1R) and orexin-2 (OX2R) receptors. OX1R is selective for orexin-A,
whereas OX2R is a nonselective receptor for both orexin-A and orexin-B.
OX1R is coupled exclusively to the Gq subclass of heterotrimeric G
proteins, whereas OX2R may couple to Gi/o, and/or Gq. (B) Structures of
mature orexin-A and -B peptides. The topology of the two intrachain
disulfide bonds in orexin-A is indicated above the sequence. Amino acid
identities are indicated by boxes. *Mammalian orexin-A sequences thus far
identified (human, rat, mouse, pig, dog, sheep, cow) are all identical.
Mammalian orexin-A is a 33-amino-acid peptide with
two intrachain disulfide bonds, while mammalian orexin-B
is a 28-amino-acid, C-terminally amidated linear peptide
(Fig. 1B). Strong homology between orexin-A and orexin-B
is found in their C terminal halves. Orexin-A and orexin-B
are both derived from a common precursor (prepro-orexin),
which is encoded by a gene composed of two exons and an
intervening intron located at 17q21 in human [2,3]. Prepro-
orexin is a 130–131-residue (depending on the species)
polypeptide, which has a typical secretory signal sequence
at its N-terminus, and is proteolytically cleaved to form
mature orexin-A and -B (Fig. 1A). An mRNA encoding the
same precursor peptide was independently isolated by de
Lecea et al. as a hypothalamus-specific transcript [4]. They
predicted that this transcript encodes two neuropeptides,
named hypocretin-1 and -2. The names bhypocretinQ and
borexinQ are currently used as synonyms.
3. Orexin receptors
Two orexin receptor subtypes, which have 64% amino-
acid identity with each other, named orexin-1 receptor
(OX1R) and orexin-2 receptor (OX2R), have been identified
in mammals [2]. We initially identified orexin peptides
using cells expressing OX1R, which has a 1-order-of-
magnitude greater affinity for orexin-A compared with
orexin-B. BLAST search for EST data bases with OX1R
sequence as a query led us identification of another subtype
of orexin receptor, OX2R, to which both orexin-A and
orexin-B bind with similar affinity (Fig. 1A). OX1R is
coupled exclusively to the Gq subclass of heterotrimeric G
proteins, whereas OX2R is coupled to both Gi/o and Gq
when expressed in cell lines [5].
In situ hybridization studies have demonstrated that
orexin receptors are expressed in regions in which dense
orexin immunoreactive fibers are observed (discussed
below). OX1R and OX2R show a markedly different and
complementary distribution [6]. For instance, within the
hypothalamus, a low level of OX1R mRNA expression is
observed in the dorsomedial hypothalamus (DMH), while
high level of OX2R mRNA expression is observed in this
region. Other areas of OX2R expression in the hypothal-
amus include the arcuate nucleus, paraventricular nucleus
(PVN), LHA, and most significantly, the tuberomammillary
nucleus (TMN) [6]. In these regions, there is little or no
OX1R signal. In the hypothalamus, OX1R mRNA is
abundant in the anterior hypothalamic area and ventrome-
dial hypothalamus (VMH). Outside the hypothalamus, high
levels of OX1R mRNA expression are detected in the tenia
tecta, hippocampal formation, dorsal raphe nucleus, and
most prominently, the locus coeruleus (LC). OX2R mRNA
is abundantly expressed in the cerebral cortex, nucleus
accumbens, subthalamic nucleus, paraventricular thalamic
nuclei, anterior pretectal nucleus, and the raphe nuclei.
Within the brain, OX1R is most abundantly expressed in the
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LC, while OX2R is most abundantly expressed in the TMN,
regions highly important for maintenance of arousal.
4. Orexin-producing neurons
Orexin-producing neurons (orexin neurons) are exclu-
sively localized to the LHA [7–9]. These cells diffusely
project to the entire neuroaxis, excluding the cerebellum [7–
9] (Fig. 2). The densest staining of orexin-immunoreactive
nerve endings in the brain was found in the arcuate nucleus
of the hypothalamus, raphe nuclei, TMN and LC. Together
with the tissue distribution of both orexin receptors, these
observations suggest that these regions are major effector
sites of orexin.
Melanin-concentrating hormone (MCH) neurons show
very similar localization with orexin neurons in the LHA
[10]. However, orexin and MCH neurons are distinct and
independent neuronal populations [11,12]. Orexin does not
colocalize with cocaine and amphetamine-regulated tran-
script (CART) or nitric oxide synthase, either [13]. How-
ever, orexin colocalizes with dynorphin [14], galanin [15],
and glutamate [16]. Recently, Eriksson et al. [17] reported
that dynorphin suppressed GABAergic input and thus
disinhibited histaminergic neurons in the TMN. Therefore,
co-localized dynorphin and orexin might synergistically
activate TMN histaminergic neurons [17].
5. Orexin defficiency in narcolepsy
The importance of orexin in the regulation of sleep/
wakefulness is highlighted by the discovery of orexin
deficiency in human narcolepsy patients [18,19]. Approx-
imately 90% of patients with narcolepsy show decreased
orexin-A levels in the cerebrospinal fluid [20]. Narcolepsy
is a common sleep disorder characterized by a primary
disorganization of behavioral states. This disorder affects
approximately 1 in 2000 individuals in the United States.
Most cases of human narcolepsy usually start during
adolescence. A cardinal symptom of the disorder is
excessive daytime sleepiness (an insurmountable urge to
sleep), manifested particularly as attacks of somnolence at
inappropriate times. Nocturnal sleep is also sometimes
disturbed by hypnagogic hallucinations, vivid dreaming,
and sleep paralysis. Narcolepsy patients often suffer from
cataplexy, which is an attack characterized by sudden
muscle weakness, which can range from jaw dropping and
speech slurring to a complete bilateral collapse of the
postural muscles. These attacks of muscle weakness are
most often triggered by strong emotional stimuli. Con-
sciousness is preserved during cataplexy. The latency for
REM sleep is notably reduced in narcolepsy patients, and
the presence of sleep-onset REM period is a diagnostic
criterion for narcolepsy. It is generally accepted that the
symptoms of narcolepsy can be considered as a pathological
intrusion of factors of sleep, and especially REM sleep-
related phenomena, into the state of wakefulness. Thus,
narcolepsy can be viewed as a behavioral state boundary
disorder [21].
The clues suggesting the possible involvement of orexin
in narcolepsy initially came from animal models; mice
lacking either the orexin gene (prepro-orexin knockout
mice) or orexin neurons (orexin/ataxin-3 transgenic mice),
as well as mice and dogs with null mutations in the OX 2 R
gene, all have phenotypes remarkably similar to narcolepsy
[22–25] (Table 1). Prepro-orexin knockout mice and orexin/
ataxin-3 mice showed a similar phenotype, which is

Fig. 2. Schematic drawing of para-agittal section through the rat brain, summarizing the organization of the orexin neuronal system. Orexin neurons are found
only in the lateral hypothalamic area and project to the entire central nervous system. Abbreviations: 3V, third ventricle; 4V, fourth ventricle; Amyg, amygdala;
VLPO, ventrolateral preoptic area; SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; VMH, ventromedial hypothalamus; ARC, arcuate nucleus;
DMH, dorsomedial hypothalamus; LH, lateral hypothalamus; TMN, tuberomamillary nucleus; PPN, pedunculopontine nucleus; LC, locus coeruleus.
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Table 1
Rodent narcolepsy models produced by genetic engineering
Phenotype Reference
Prepro-orexin knockout Cataplexy (+), sleep attack (+) [23]
Sleep/wake fragmentation (severe)
OX1R knockout Cataplexy ( ), sleep attack (+) [43]
Sleep/wake fragmentation (mild)
OX2R knockout Cataplexy ( ), sleep attack (+) [25]
Sleep/wake fragmentation (severe)
Orexin/ataxin-3 mouse Cataplexy (+), sleep attack (+) [24]
Sleep/wake fragmentation (severe)
Orexin/ataxin-3 rat Cataplexy (+), sleep attack (+) [56]
Sleep/wake fragmentation (severe)

characterized by behavioral arrest similar to cataplexy,
occasional direct transition to REM sleep from wakefulness,
and highly fragmented behavioral states, resulted from
behavioral instability [23,24] (Fig. 3).
Consistently, a postmortem study in narcoleptic subjects
showed undetectable levels of orexin peptides in projection
sites such as the cortex and pons and an 80–100% reduction
in the number of orexin-containing neurons in the hypo-
thalamus [18,19], supporting an earlier report showing
undetectable CSF orexin-A peptide levels in most narco-
lepsy patients [26]. These results suggest either a loss of
orexin-containing neurons or a lack of orexin production in
these neurons if still present. One of the predisposing factors
is a specific class II HLA haplotype on human chromosome
6, with HLA DQB1*0602 and DQA1*0102 alleles, which
were found in more than 85% of all narcoleptic patients

Fig. 3. Summary of vigilance state parameters recorded from orexin/ataxin-3 hemizygous transgenic and wild-type littermate control (WT) mice. Upper panels:
Total time spent in each state (minutes, meanFS.E.M.), itemized separately for light (left panel) and dark (right panel) periods. Lower pannels: Mean episode
duration of each vigilance state observed in light (left panel) and dark (right panel) periods. Significant differences ( Pb0.05; repeated measurements ANOVA)
between Tg and WT mice are indicated with an asterisk.
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[27]. This suggests a possibility that narcolepsy may result
from selective autoimmune degeneration of orexin neurons.
The decreased CSF orexin-A peptide level in most
narcolepsy patients also suggests that measuring CSF
orexin-A might be a definitive diagnostic test [28].
Since narcolepsy is a disorder of sleep–wake cycle
organization resulting from absence of orexin, replacement
therapy using orexin receptor agonists may provide treat-
ment of narcolepsy. Our recent study showed that chronic
over-production of both orexin-A and orexin-B peptides
from an ectopically expressed transgene prevented the
development of narcolepsy syndrome in orexin neuron-
ablated (orexin/ataxin-3) mice [29]. Similarly, acute admin-
istration of orexin-A maintained wakefulness, suppressed
sleep, and inhibited cataplectic attacks in narcoleptic mice
[29]. Together, these findings provide strong evidence for a
specific relationship between absence of orexin peptides in
the brain and the development of narcolepsy syndrome.
6. Mechanisms of regulation of behavioral states by
orexins
The finding of orexin deficiency in narcoleptic patients
suggests that orexin has an important role in the normal
regulation of sleep/wakefulness. Orexin neurons might be
especially important for stabilization of behavioral states,
because the major symptom in narcolepsy is inability to
maintain each behavioral state, which results in sleep/
wakefulness fragmentation. When orexin-A was injected
intracerebroventricularly into rats during the light period, it
caused increased wakefulness time and decreased REM and
non-REM sleep time [30]. In rats, Fos expression of orexin
neurons is increased during the dark active period [31], and
orexin level in cerebrospinal fluid also peaks during the dark
period and decreases during the light rest period [32]. These
observations suggest that orexin neurons are active during
the active period and support wakefulness, and are inactive
during the sleep period. The activities of the monoaminergic
neurons in the brain stem are also synchronized and strongly
associated with behavioral states: they fire tonically during
wakefulness, less during non-REM sleep, and not at all
during REM sleep [33]. This regulation might be at least in
part by orexin neurons, which are also wake-active, because
orexin neurons project to and excite histaminergic neurons
in the TMN, noradrenergic neurons in the LC and
serotonergic neurons in the dorsal raphe (DR) [7–9], and
the presence of OX1R in the LC and OX2R in TMN and
both receptors in the DR has been confirmed [6]. Consistent
with this hypothesis, isolated cells from these nuclei are all
activated by orexins in vitro [30,34,35]. Orexins also have a
strong direct excitatory effect on cholinergic neurons of the
basal forebrain [36], which is hypothesized to play an
important role in behavioral and electrocortical arousal [37].
These observations suggest that orexin neurons are active
during the wake period, and exert an excitatory influence on
5
the basal forebrain cholinergic neurons and monoaminergic
neurons in the brain stem to maintain arousal. In addition,
orexin neurons also appear to act on LDT/PPT cholinergic
neurons, because orexin neurons project directly to the PPT/
LDT nuclei and direct injection of orexin-A into the LDT of
cats results in an increase in wakefulness and a decrease in
REM sleep [38]. In addition, several reports showed that
orexin induces long-lasting excitation of cholinergic neu-
rons in the LDT [39]. However, a group of cholinergic
neurons in the LDT/PPT, which are silent during wakeful-
ness and active in the REM period, constitute a system to
induce and maintain REM sleep [40]. Therefore, orexin
neurons might not exert a direct excitatory influence on
these cells during wakefulness. Orexin neurons might
activate another type of cholinergic neurons in the PPT
and LDT, which are active in wakefulness as well as the
REM-sleep period. Recent work also shows that orexin
inhibits cholinergic neurons in the PPT via activation of
GABAergic local interneurons and GABAergic neurons in
the substantia nigra pars reticulata [41]. These results
suggest that hypothalamic orexin neurons affect the activity
of LDT/PPT cholinergic neurons directly and/or indirectly
to appropriately regulate the activity of these cells to control
behavioral states.
Several reports showed that the effect of orexin on
wakefulness is largely mediated by activation of the
histaminergic system mediated through OX2R. In rats,
i.c.v. injection of orexin during the light period potently
increases the wake period, and this effect is markedly
attenuated by the H1 antagonist, pyrilamine [35]. Further-
more, the effect of orexin-A on wakefulness in mice is
almost completely absent in H1-receptor deficient mice [42].
Furthermore, OX 2 R knockout mice exhibit a narcoleptic
phenotype, while OX 1 R knockout mice show only mild
fragmentation of behavioral states [43]. Because OX2R is
strongly expressed in the TMN, while OX1R is strongly
expressed in the LC, the TMN seems to be an important
effector site of orexin for sleep/wakefulness regulation.
Orexin neurons also contain an additional neurotransmitter,
dynorphin [14]. Dynorphin in orexin neurons might inhibit
GABAergic input to TMN neurons, and thus act in concert
with orexin to increase the excitability of these neurons [17].
However, several findings indicate that signaling through
OX1R is also important for the regulation of wakefulness.
As mentioned before, OX 2 R knockout mice exhibit charac-
teristics of narcolepsy [25]. Interestingly, OX 1 R knockout
mice do not have any overt behavioral abnormalities and
exhibit only mild fragmentation of behavioral states [43].
However, the behavioral and electroencephalographic phe-
notype of OX 2 R knockout mice is less severe than that
found in prepro-orexin knockout mice and double receptor
knockout (OX 1 R- and OX 2 R-null) mice, which appear to
have the same phenotype as prepro-orexin knockout mice.
These observations suggest that OX 1 R also has additional
effects on sleep/wakefulness regulation. These findings
suggest that despite the lack of an overt OX 1 R phenotype,
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loss of signaling through both receptor pathways is
necessary for severe narcoleptic phenotype.
Willie et al. [25] classified, using behavioral, electro-
physiological, and pharmacological criteria, two distinct
classes of behavioral arrest exhibited by mice deficient in
orexin-mediated signaling. Both OX 2 R and prepro-orexin
knockout mice are similarly affected with behaviorally
abnormal attacks of non-REM sleep (bsleep attacksQ) and
show similar degrees of disrupted wakefulness. In contrast,
OX 2 R knockout mice are only mildly affected with
cataplexy-like attacks of REM sleep, whereas orexin
knockout mice are severely affected. Absence of OX 2 R
eliminates orexin-evoked excitation of histaminergic neu-
rons in the hypothalamus, which gate non-REM sleep/
wake transition. While normal regulation of wake/non-
REM sleep transition depends critically upon OX2R
activation, the profound dysregulation of REM sleep
control unique to the narcolepsy syndrome emerges from
loss of signaling through both OX2R-dependent and
OX1R-dependent pathways in mice.
Thus, orexin neurons may be active during wakefulness,
helping sustain activity in monoaminergic arousal regions
[31], which in turn send inhibitory input to the ventro-
lateral preoptic area (VLPO) sleep-active neurons, and
thereby further maintain wakefulness [44]. In the absence
of orexin, these arousal regions may have reduced or non-
synchronized activity, resulting in an inappropriately low
threshold for transition into non-REM sleep. Narcoleptic
mice also have fragmented non-REM sleep; orexin-
deficient mice have more frequent transitions between all
states, as has been noted in human narcolepsy. Therefore,
orexin neurons might also be necessary for maintenance of
non-REM sleep.
7. Regulation of orexin neuronal activity
Until recently, little was known about the factors that
influence the activity of orexin neurons. Recent electro-
physiological studies have identified several activators and
inhibitors of orexin neurons. By recording from hypothala-
mic slices of transgenic mice expressing green fluorescent
protein (GFP) only in orexin neurons, it was shown that
agonists of ionotropic glutamate receptors (AMPA and
NMDA) excite orexin neurons, whereas glutamate antag-
onists (AP-5, CNQX or NBQX) reduce their activity [45].
These results indicate that orexin neurons are tonically
activated by glutamate. Although orexin has little direct
effect on the activity of orexin neurons, it increases this
glutamate signaling by acting on presynaptic terminals
[45]. This mechanism may reinforce and coordinate the
activity of orexin neurons in the LHA.
Several researchers have hypothesized that monoamines
excite orexin neurons, forming positive feedback loops that
would maintain wakefulness [21], but our electrophysio-
logical studies showed just the opposite; both noradrena-
line and serotonin hyperpolarize and inhibit GFP-
expressing orexin neurons [45,46]. Histamine has no effect
on orexin neurons. It seems strange that wake-active
monoaminergic areas would inhibit wake-active orexin
neurons. Therefore, we hypothesize that orexin neurons
might be innervated by monoaminergic cells in regions

Fig. 4. Roles of orexin in coordination of energy and sleep homeostasis. Orexin neurons stimulate the hypothalamic nuclei involved in feeding behavior and
increase cortical arousal and promote wakefulness through the aminergic nuclei and other sleep-related nuclei. Stimulation of dopaminergic, limbic, and
cholinergic centers by orexins can modulate reward systems, motor activity, and emotional arousal. Peripheral metabolic signals, leptin, ghrelin, and glucose
and circadian rhythms influence orexin neuronal activity to coordinate arousal and energy homeostasis.
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T. Sakurai / Regulatory Peptides xx (2004) xxx–xxx
other than the wake-active regions. To evaluate this
hypothesis, precise retrograde mapping study of afferent
neurons to orexin neurons is required.
8. Roles of orexins in the regulation of energy
homeostasis
The altered energy homeostasis in human narcolepsy
patients suggests roles of orexin in regulation of energy
homeostasis [47,48]. The finding of decreased caloric
intake [49] combined with an increased body mass index
[47] suggests that narcolepsy patients have a feeding
abnormality with reduced energy expenditure or low
metabolic rate, and orexin neurons have a role in the
regulation of energy homeostasis. Consistently, orexin
neuron-ablated mice show hypophagia and late-onset
obesity [24].
Electrophysiological studies on orexin neurons showed
that, in addition to monoamines and acetylcholine,
peripheral humoral factors related to energy metabolism
also influence the activity of orexin neurons; activity of
isolated orexin neurons is inhibited by glucose and leptin,
and stimulated by ghrelin [50]. Consistently, orexin
expression of normal and ob/ob mice correlates nega-
tively with changes in blood glucose, leptin, and food
intake. These findings are consistent with our original
idea that orexins have a role in the regulation of feeding
and energy homeostasis [2].
9. Orexin: a link between energy homeostasis and
arousal
Proper maintenance of arousal during food search and
intake of an animal is essential for its survival. Therefore,
the two vital processes, feeding and sleep/wake behavior,
have to be appropriately coordinated. When faced with a
negative energy balance due to reduced food availability,
mammals respond behaviorally with phases of increased
wakefulness and locomotor activity that support food
seeking [51–55]. The discovery that orexin neurons are
regulated by peripheral metabolic cues suggests that
orexin neurons might have important roles in the
molecular and physiological basis of this phenomenon.
During starvation, orexin neurons might be activated by
low leptin and glucose levels, along with high ghrelin
level. These mechanisms may directly modulate activity
of orexin neurons according to appetite and body energy
stores. Indeed, we found that transgenic mice, in which
orexin neurons are ablated, fail to respond to fasting with
increased wakefulness and activity [50].
These findings indicate that orexin neurons provide a
crucial link between energy balance and arousal (Fig. 4).
These properties might allow the orexin neurons to promote
alertness in a hungry animal and maintain long periods of
7
wakefulness throughout the day. These mechanisms may be
important in maintenance of prolonged wakefulness during
the active period, and in the regulation of energy homeo-
stasis that helps to ensure survival in nature, but may
counteract attempts to treat obesity by food restriction.
Acknowledgements
This study was supported in part by a grant-in-aid for
scientific research from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of
Japan; University of Tsukuba Project Research; The 21st
century COE program; the Uehara Memorial Foundation;
and the ERATO from the Japan Science and Technology
Corporation.
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