nature reviews endocrinology https://doi.org/10.

1038/s41574-023-00927-z

Review article Check for updates

The intersection between ghrelin,

metabolism and circadian rhythms
Soumya S. Kulkarni1,2, Omprakash Singh 2
& Jeffrey M. Zigman 2,3,4

Abstract Sections

Despite the growing popular interest in sleep and diet, many gaps exist Introduction

in our scientific understanding of the interaction between circadian Circadian biology and
rhythms and metabolism. In this Review, we explore a promising, metabolism

bidirectional role for ghrelin in mediating this interaction. Ghrelin Regulation and metabolic
actions of ghrelin and LEAP2
both influences and is influenced by central and peripheral circadian
systems. Specifically, we focus on how ghrelin impacts outputs of Ghrelin action in the brain

circadian rhythm, including neuronal activity, circulating growth Effects of ghrelin on circadian
rhythm
hormone levels, locomotor activity and eating behaviour. We also
consider the effects of circadian rhythms on ghrelin expression and Influence of circadian
rhythms on ghrelin and
the consequences of disrupted circadian patterns, such as shift work GHSR expression
and jet lag, on ghrelin secretion. Our Review is aimed at both the casual
Relevance of the work in
reader interested in gaining more insight into the scientific context mouse models to human
health and disease
surrounding the trending topics of sleep and metabolism, as well as
experienced scientists in the fields of ghrelin and circadian biology Conclusions

seeking inspiration and a comprehensive overview of how these fields

are related.

Medical Scientist Training Program, UT Southwestern Medical Center, Dallas, TX, USA. 2Center for Hypothalamic
1

Research, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA. 3Division of
Endocrinology, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA. 4Department
of Psychiatry, UT Southwestern Medical Center, Dallas, TX, USA. e-mail: jeffrey.zigman@utsouthwestern.edu

Nature Reviews Endocrinology

Review article
Key points
•• Disrupted circadian and metabolic systems are a growing cause of
societal health burden.
•• The ghrelin system regulates many key metabolic functions
including food intake, body weight, blood glucose, food anticipatory
activity, growth hormone secretion and body temperature.
•• Ghrelin and GHSR (growth hormone secretagogue receptor, which
serves as the receptor for ghrelin) mRNA and protein expression are
regulated by the circadian system.
•• Ghrelin affects neuronal firing, food anticipatory activity and
thermoregulation in a circadian-dependent manner.
•• Given the expansion in scientific technology in the past 25 years,
many more tools now exist to better understand the role of the ghrelin
system in metabolism and circadian biology.
•• Future studies are needed to elucidate further the connections
between the ghrelin system, metabolism and circadian systems,
and to provide novel avenues for therapeutic approaches.
Introduction
Ghrelin is an acylated peptide hormone secreted mainly from an elec-
tron microscopically distinct type of enteroendocrine cell that is now
known as a ghrelin cell. Calorie restriction, stress and poor sleep all
increase ghrelin secretion. By contrast, food intake and obesity lower
plasma ghrelin levels. In adults, ghrelin cells are localized primarily
to the lining of the stomach and duodenum (Fig. 1). About one out of
every 100–300 gastric mucosal cells is a ghrelin cell. The receptor to
which ghrelin binds and through which ghrelin acts is the growth hor-
mone secretagogue receptor (GHSR). GHSRs are localized to several
tissues, but have perhaps been best characterized within the brain,
pituitary and pancreatic islets. Ghrelin binding to GHSRs depends on
ghrelin’s acyl group — most usually, an octanoyl group — which is added
as a post-translational modification catalysed by the enzyme ghrelin
O-acyltransferase (GOAT). Liver-expressed antimicrobial peptide 2
(LEAP2), a peptide hormone that is secreted mainly from hepatocytes
and jejunal enterocytes, also binds to GHSR. Unlike ghrelin, LEAP2
serves as an antagonist and inverse agonist at GHSRs, powerfully block-
ing ghrelin action while also decreasing GHSR constitutive activity.
When first reported in 1999 (refs. 1,2), ghrelin’s actions as an agonist
for GHSR and a potent growth hormone secretagogue were the focus.
Yet, ghrelin has since been implicated in several well-known functions
including meal initiation3–5, appetite stimulation (reviewed elsewhere6),
reducing energy expenditure2, raising blood glucose and preventing
life-threatening falls in blood glucose7–9, stimulating gastrointestinal
motility2, and inducing reward behaviours related to food, alcohol and
addictive drugs10–13, among others.
A topic that is not as well studied is the interaction between the
ghrelin system and circadian biology. This interaction is of great
importance because of the links between ghrelin and metabolism
and between metabolism and circadian biology that are already
well established. Moreover, a large and growing population of indi-
viduals are facing the synergistic health burdens of dysregulated
Nature Reviews Endocrinology
circadian and metabolic systems. A growing area of concern is the
effects of working the nightshift on human health. A study of over 300
health-care workers found that those who worked the nightshift had
significantly elevated waist circumference, fasting blood glucose and
high-sensitivity C-reactive protein (a marker for the risk of developing
coronary artery disease)14.
Another growing area of concern is abnormal eating patterns, such
as eating during resting periods15. Healthy young women who engaged
in night-time snacking for just 13 days exhibited decreases in whole
body fat oxidation and increases in LDL cholesterol, both of which
are risk factors for obesity and metabolic disease16. Late-night snack-
ing is associated with increased hunger, decreased waketime energy
expenditure and 24-h core body temperature, altered lipid metabolism
pathways, and, of relevance to this Review, an increased ratio of plasma
ghrelin to the anorexigenic adipose tissue-derived hormone leptin17.
Although not all individuals who engage in night-time snacking
have an eating disorder, some individuals who frequently engage in
night-time snacking meet criteria for night-eating syndrome. These
criteria include morning anorexia, consumption of over 50% of daily
energy intake after the last evening meal, waking up at least once a
night to eat, exclusion of bulimia nervosa or binge-eating disorder,
and persistence of symptoms for at least 3 months18. Individuals with
night-eating syndrome display altered circadian physiology, including
phase delays in timing of meals and in leptin and insulin rhythms19. By
contrast, circulating ghrelin levels in individuals with night-eating
syndrome show a phase advance of 5.2 h19. Given the severity and preva-
lence of the effects of aberrant circadian and metabolic regulation
on society, more steps must be taken to better understand how the
circadian and metabolic systems interact. Exploring whether and how
the ghrelin system may be involved in this relationship is a rational
place to begin.
In this Review, we touch on some salient evidence linking circadian
biology and metabolism. We briefly provide an overview of the ghrelin
system and its key players — ghrelin, GHSR, LEAP2 and GOAT — and
discuss their physiological functions and effects on the brain. We then
take a more specific look at the effects of ghrelin on circadian biology,
including its roles in regulating neuronal activity, locomotor and food
anticipatory behaviours and body temperature. Similarly, we consider
the effects of circadian rhythms on ghrelin secretion and activity,
including what is known regarding this topic in humans, peripheral and
central effects of circadian rhythmicity on ghrelin, and time-restricted
feeding. Finally, we explore some clinically relevant models such as
chronic jetlag, provide an overview of open research questions, and
give our suggestions on how to best begin to tackle them.
Circadian biology and metabolism
An overview of some basic circadian terminology is shown (Box 1).
The interaction between circadian biology and metabolism has been
extensively discussed previously20–22. Here, we discuss only the key
findings that lay the foundation for the subsequent discussion. Dys-
regulation of circadian clock genes or zeitgebers can severely impact
metabolism. Both Turek et al.23 and Oishi et al.24 noted increased appe-
tite in homozygous Clock-mutant mice but had contradictory findings
regarding body weight and liver enzyme levels; these discrepancies
were attributed to differences in the strains of mice used by the two
groups. Mutations in BMAL and CRY are also associated with metabolic
abnormalities25, including impaired glucose and adipocyte regulation.
Thus, most, if not all, of the core clock genes are involved in metabolic
regulation.
Review article

The suprachiasmatic nucleus (SCN) is the body’s master circadian

regulator. It is a bilateral region of the brain located ventromedially in GHSR
Brain Ghrelin
the anterior hypothalamus, rostral to the arcuate nucleus (ARC). The LEAP2
Pituitary gland
SCN regulates many metabolic processes including energy homeo-
stasis and insulin release26. Indeed, organisms with disrupted SCNs
develop dysregulated metabolism23. Bilateral electrolytic SCN lesion-
ing experiments show an increase in body weight, loss of circadian
rhythms of oxygen consumption and food intake, and development of
hepatic insulin resistance26. In addition, SCN-neuron-specific Bmal1-
knockout animals under constant darkness (DD) conditions display
loss of circadian rhythmicity in feeding, oxygen consumption and
thermoregulation, increased body weight attributable to increased
adiposity, and impaired glucose tolerance27,28. Behavioural and molecu-
lar circadian rhythms are restored following 1 week of time-restricted
feeding27. Furthermore, tracing studies indicate an anatomical connec-
tion between the ventromedial ARC (ARCvm) and the SCN29. These ana-
tomical connections might help relay peripheral metabolic information
Liver
to the SCN, although functional evidence is needed to support this
Stomach
hypothesis. In a later section of the Review, we consider the potential
Pancreas
involvement of ghrelin in these maladaptive changes.
Duodenum

Regulation and metabolic actions of ghrelin Jejunum

and LEAP2
As already mentioned, ghrelin is an agonist for GHSR, which is a mem-
ber of the seven-transmembrane G protein-coupled receptor family
(Fig. 2). In adults, ghrelin is primarily secreted from the stomach and
duodenum2,30. As noted, plasma ghrelin level increases upon fasting,
with stress and in settings of poor sleep, while it declines in obesity31–33.
Plasma ghrelin levels are also dynamically affected by feeding status,
rising preprandially and falling after a meal4,34. In the case of calorie
restriction and stress, the increase in plasma ghrelin level is thought to
occur from activation of the sympathetic nervous system and engage-
ment of β1-adrenergic receptors localized to ghrelin cells32,35–38. By con-
trast, meal-driven and obesity-driven increases in insulin, the ensuing
increased engagement of ghrelin cell-expressed insulin receptors, and
in turn reduced ghrelin secretion, are mainly responsible for the des­
cribed postprandial and obesity-related reductions in plasma ghrelin
level39. Also contributing to meal and/or obesity-associated ghre-
lin reductions are lowered sensitivity of ghrelin cells to the usual
potent ghrelin secretagogue noradrenaline, activation of ghrelin
cell-expressed fatty acid receptors, and/or changed numbers of ghre-
lin cells36,39,40. Regarding effects on metabolism, administering ghrelin
or ghrelin mimetics increases food intake, body weight gain, adiposity,
hepatic steatosis and blood glucose level2,33,41–44, lowers energy expendi-
ture, upregulates expression of fat storage-promoting enzymes, and
leads to hedonic eating behaviours including operant responding
for food rewards, sucrose self-administration and cue-potentiated
feeding2,45–50. By contrast, neutralizing ghrelin or treatment with GHSR
antagonists lowers body weight, food intake, various food reward
behaviours and hepatic fat44,47,49,51–55. Although neither ghrelin nor GHSR
loss-of-function (knockout) mice exhibit obvious phenotypes under
normal conditions, clear phenotypes emerge once challenged: when
exposed to a high-fat diet early in life, these mice eat less, gain less
weight and develop less adiposity than wild-type mice56,57. Exposing
these mice to an acute-on-chronic calorie restriction regimen results
in marked hypoglycaemia and increased mortality32,58,59. Following
forced high-intensity interval exercise, these mice eat much less than
exercised wild-type mice60. Further, these loss-of-function models
show reduced stress-induced conditioned place preference for food
Pancreatic islets
Fig. 1 | Major sites of expression of the hormones ghrelin and LEAP2 and
their receptor, GHSR. In adults, ghrelin (red) is secreted mainly by a distinct
type of enteroendocrine cells (ghrelin cells) in the mucosa of the stomach and
duodenum. Growth hormone secretagogue receptor (GHSR; blue) serves as the
receptor for ghrelin. GHSR is expressed in several regions of the brain, including
the suprachiasmatic nucleus of the hypothalamus. GHSR is also expressed
highly in growth hormone-secreting somatotrophs within the pituitary, as
well as in several endocrine cell types within pancreatic islets. Liver-expressed
antimicrobial peptide 2 (LEAP2; yellow), is another endogenous GHSR ligand.
In contrast to ghrelin (which is an endogenous GHSR agonist), LEAP2 is an
endogenous antagonist and inverse agonist of GHSR. LEAP2 is primarily
produced by the liver and the jejunal region of the small intestine.
rewards, reduced binge-like high-fat diet intake and reductions in other
hedonic ingestive behaviours48,61–63.
LEAP2 represents another endogenous GHSR ligand (Fig. 2). In
contrast to ghrelin, which is a GHSR agonist, LEAP2 is a GHSR antagonist
and inverse agonist that potently blocks ghrelin action and decreases
constitutive GHSR activity; it is primarily secreted from the liver and
intestine58,64. Although LEAP2 is not as well characterized as ghrelin,
plasma LEAP2 is known to change inversely to ghrelin in many settings.
For instance, in both mice and humans, LEAP2 falls during fasting, rises
in obesity and rises after a meal64,65. In contrast to ghrelin, LEAP2 and/or
LEAP2 analogues reduce food intake, binge eating and body weight, and
block ghrelin-induced food intake in mice63,66,67. LEAP2 also has been
shown to reduce ad libitum food intake and postprandial blood glucose

Nature Reviews Endocrinology

Review article
Box 1
Key circadian biology definitions
Circadian rhythm: fluctuation pattern of any behavioural,
physiological or molecular process over the course of
approximately 24 h.
DD: experimental condition exposing subjects to constant darkness
conditions.
LD: experimental condition exposing subjects to 12-h light–12-h dark.
Peripheral clocks: tissue-specific circadian clocks, influenced by
but independent from the central clock, which drive the circadian
expression of a variety of genes involved in various physiological
processes115.
Phase shift: a measurable change in the phase of a rhythm,
observed by comparing a reference point in the phase (for example,
onset of locomotor activity) with external time. Phase shifts can
be either advances or delays; a phase advance means that the
reference point occurs earlier than normal, whereas a phase delay
means it occurs later than normal116.
Transcription–translation feedback loop (TTFL): first described
by Bass and Takahashi, mammalian cells possess a cell
autonomous circadian clock generated by a transcriptional
autoregulatory feedback loop. Transcriptional activators, CLOCK
and BMAL1, act on their target genes, Period and Cryptochrome.
These products progressively accumulate to form a repressor
complex which, in turn, inhibits transcription of CLOCK and
BMAL1 (ref. 117).
Zeitgeber: external time cues, such as light, access to food and
environmental temperature, which synchronize the circadian
system with external (that is, geophysical) time118.
Zeitgeber time (ZT): standard notation in the circadian field where
ZT0 indicates the beginning of the light phase and ZT12 indicates
the beginning of the dark phase116.
excursions in healthy men68. In lean people, postprandial increases in
LEAP2 correlate positively with postprandial decreases in appetite
ratings and with postprandial decreases in food cue brain reactivity
(measured using a food picture evaluation functional MRI task)69. LEAP2
deletion augments ghrelin-induced food intake and cFos induction in
both the ARC and the olfactory bulb65. Female LEAP2-knockout mice
fed a high-fat diet have higher body weight than wild-type mice, due
to lower energy expenditure and locomotor activity and higher food
intake65. Thus, lowering plasma LEAP2 levels and lowering the plasma
LEAP2 to ghrelin ratio reduces the phenomenon of ghrelin resistance —
or rather, it enhances sensitivity to the actions of endogenous ghrelin.
As LEAP2 was only recently identified as a GHSR ligand, only limited
specific interactions it might have with circadian biology have so far
been reported.
Ghrelin action in the brain
One of the main targets of ghrelin is the brain, which makes sense
given the high expression of GHSR in the brain and the many brain
regions expressing GHSR70–72. This Review highlights some specific
roles of ghrelin within the hypothalamus, although ghrelin also directly
Nature Reviews Endocrinology
engages GHSR-expressing neurons in other brain regions including
the hippocampus, caudal brainstem and olfactory bulb65,73,74. Vari-
ous hypothalamic nuclei express GHSR, including the ventromedial
hypothalamus (VMH), which is involved in feeding cessation, and the
ARC, which helps balance hunger and satiety signals. Previous research
extensively supports the direct interaction of ghrelin with these two
mediobasal hypothalamic nuclei2,75.
In the ARC, ghrelin stimulates AgRP (agouti-related peptide)/NPY
(neuropeptide Y) neurons. Selective GHSR expression in those neurons
enables ghrelin to induce food intake and prevents marked hypogly-
caemia upon caloric restriction7. Conversely, selective GHSR deletion
from ARC AgRP neurons or ablation of ARC AgRP neurons abolishes the
acute orexigenic effects of ghrelin24,44. In the VMH, ghrelin suppresses
the activity of glucose-sensing neurons23, and inhibiting AMPK robustly
impairs the central orexigenic effect of ghrelin23. GHSR-expressing
neurons in the paraventricular nucleus of the hypothalamus, the lateral
hypothalamic nucleus and dorsomedial hypothalamus (DMH) help
mediate the actions of ghrelin on the neuroendocrine axis, food intake
and food reward24–26. However, the effects of ghrelin on the SCN and
vice versa are not yet thoroughly characterized.
As mentioned previously, the SCN is the master circadian clock
and is of critical importance for metabolic regulation. Notably, ghrelin
secretion fluctuates in a circadian pattern, suggesting a role for the
SCN in regulating ghrelin secretion. Evidence also exists suggesting
that ghrelin has direct actions on the SCN, as GHSR mRNA is expressed
in the SCN41,70,71 (Fig. 3). Thus, there is reason to suspect an interaction
between ghrelin and the circadian system.
Could the ghrelin/GHSR system be a bridge between circadian
rhythms and metabolism? So far, indications exist towards this pos-
sibility, but there are no definitive answers. The following two sections
explore the relationship between the ghrelin system and circadian
rhythm in further detail.
Effects of ghrelin on circadian rhythm
Effects of ghrelin or GHSR on SCN neuronal activity
Ghrelin modulates the activity of neurons in the SCN. In one study by
Zhou et al., 5–6-week-old mice were individually housed and initially
entrained to a 2-week 12-h light–12-h dark (LD) cycle, and were then
placed under DD conditions76. Maximal neuronal activity responses
were observed when the mice were given a bolus injection of the ghrelin
mimetic GHRP-6 at the beginning of the subjective night (CT12; which
is the same as the beginning of the active phase for mice). Specifically,
GHRP-6 injected at CT12 gradually increased SCN neuronal firing fre-
quency as measured by in vivo extracellular electrode recordings. Also,
Ca2+ mobilization in SCN neurons, as determined by flow cytometry,
was increased twofold76. Although Zhou et al. switched the animals
from LD to DD after 2 weeks, another study by Tokizawa et al.77 kept
the mice housed under LD conditions and then administered ghrelin
intraperitoneally either 1 h after lights on or 1 h after lights off. Neuro­
nal activity was measured using immunohistochemistry for cFos.
In the group that received ghrelin during the light phase, the number
of cFos-positive neurons in the SCN was significantly greater in the
ghrelin group than in the vehicle group. In addition, the number of cFos
and NPY double-labelled neurons in the SCN was greater in the ghrelin
group than in the vehicle group77. No difference in neuronal activity
was observed between the vehicle and ghrelin groups when ghrelin was
administered during the dark phase. The results from the two studies
are challenging to compare, since the mice were housed under dif-
ferent circadian conditions. Still, it seems that although both groups
Review article
showed that ghrelin increases neuronal activity in the SCN, the study by
Zhou et al. showed this effect predominantly during the traditional
active phase for mice, while the study by Tokizawa et al. showed this
effect during the traditional resting phase for mice.
In similar experiments, rats were housed under DD conditions
and exposed to a light stimulus 1 h into their subjective night78. Neuro­
nal activity was again measured using immunohistochemistry for
cFos. The photic stimulation resulted in an increase in cFos-positive
neurons in the SCN; however, pretreatment with GHRP-6 attenuated
the light-induced cFos expression by 50%. These results are contrary
to the results of Zhou et al.76, but also conflict with the investigators’
own results when the same paradigm was applied to mice, suggesting
that species differences might exist in the effects of ghrelin on SCN
neuronal activity.
The effects of ghrelin on SCN neuronal activity have also been
studied using an in vitro approach. Coronal sections of mouse brains
containing the SCN region were obtained from mice that had been
housed under LD conditions and killed during the light phase79. Ghrelin
was applied to the sections on the first day ex vivo, at what would have
been the middle of the light phase for the mice. Spontaneous neuronal
activity was recorded on the second day ex vivo. Sections that had been
exposed to ghrelin showed a 3-h advance in the rhythm of spontane-
ous firing when compared with the firing pattern of neurons in brain
sections from control mice that had received artificial cerebrospinal
fluid. These effects were not observed when ghrelin was applied to
the sections at what would have been 2 h into the dark phase for the
mice. Taken together, the results of the studies highlighted in this
section suggest that the effects of ghrelin on SCN neuronal activity
vary depending on phase and housing conditions (LD versus DD).
Specifically, ghrelin has a maximal impact on SCN neuronal activity
during the resting phase when rodents are housed under LD condi-
tions and during the active phase when rodents are housed under
DD conditions.
• Fasting
• Stress
• Poor sleep
Sympathetic Insulin acting
nervous system via insulin
acting via β1ARs receptors on
on ghrelin cells ghrelin cells
Ghrelin
GHSR
Fig. 2 | Overview of the ghrelin system and its physiological functions.
Growth hormone secretagogue receptor (GHSR) is a seven-transmembrane
G protein-coupled receptor expressed by CNS neurons, pituitary somatotrophs
and pancreatic islet endocrine cells. Ghrelin and liver-expressed antimicrobial
peptide 2 (LEAP2) represent two endogenous GHSR ligands — one that activates
GHSR (ghrelin) and the other that blocks ghrelin-dependent and constitutive GHSR
activity (LEAP2). Fasting and stress stimulate ghrelin secretion, in large part
via activation of the sympathetic nervous system and engagement of
β1-adrenergic receptors (β1ARs) expressed by ghrelin cells. Poor sleep also
Nature Reviews Endocrinology
In addition to affecting neuronal activity in the SCN, ghrelin also
impacts neuronal activity in the ARC, specifically the ARCvm. The
ARCvm, as described previously, is thought to have a role in relaying
peripheral metabolic signals to the SCN29. Ghrelin administration
induces greater cFos expression in the mouse ARC in the dark phase
than in the light phase77. The majority of the cFos-positive ARC neurons
in the dark phase are also NPY-positive. The ARC AgRP/NPY neuronal
population is perhaps best known for its effects on feeding. Func-
tionally, these features of the ARC are relevant because they provide
mechanistic insight into circadian patterns of eating — these find-
ings are consistent with a role for ghrelin in promoting food-seeking
behaviours during an organism’s active phase. Not only does ghrelin
administration globally increase cFos expression in the ARC, it also
specifically increases cFos expression in the ARCvm. Similarly, GHRP-6
activated AgRP neurons exclusively in the ARCvm in rats78. Altogether,
these findings suggest that ghrelin has both direct and indirect effects
on the SCN and, therefore, on circadian biology.
Effects of the ghrelin system on circadian patterns of
locomotor activity
Locomotor activity is tightly regulated by circadian rhythm and is an
easily observable behavioural output. Ghrelin has numerous effects
on circadian patterns of locomotor activity. When mice housed under
DD conditions were given GHRP-6 at the start of their subjective night,
a clear phase delay occurred in the rhythm of locomotor activity76.
This effect was abolished when mice were pretreated with a GHSR
antagonist76. By contrast, in mice housed under LD conditions, no
effects on locomotor activity rhythm were observed when the mice
were given ghrelin at the start of their dark phase78. Surprisingly, light
stimulation 1 h into the dark phase produced a phase delay in loco-
motor activity that was reduced when the animals received ghrelin
pretreatment78. In a GHSR-knockout mouse model, when animals were
switched from LD to constant light (LL), knockout animals had longer
• Obesity
• Meals
LEAP2
The
circadian • Food intake
clock • Body weight
• Blood glucose
• Growth hormone secretion
• Locomotor activity;
food anticipatory activity
• Thermoregulation
increases ghrelin levels. Fasting reduces LEAP2 levels. Obesity and meals
decrease ghrelin secretion, in large part via increasing insulin followed by
engagement of insulin receptors expressed by ghrelin cells. By contrast,
obesity and meals increase LEAP2 levels. Ghrelin and LEAP2 regulate food
intake, body weight, blood glucose, growth hormone secretion, locomotor and
food anticipatory activity, and body temperature. Both ghrelin and LEAP2 are
influenced by the circadian clock and have effects on the previously mentioned
outputs (for example, food intake and body weight) that are dependent on the
circadian rhythm.
Review article
GHSR-IRES-Cre: YFP
LV
vhc
PaAP
Pav LH
3V
AHA MPA
BMA LA
opt
SCN
Fig. 3 | GHSR-expressing neurons in SCN of GHSR-IRES-Cre mice.
Photomicrographs demonstrating YFP-immunoreactive cell bodies (green)
in a coronal section of the hypothalamus (located approximately −0.58 mm
from the bregma, at the level of the SCN) of a representative GHSR-IRES-
Cre × ROSA26-YFP reporter mouse. The presence of GHSR-positive neurons
within the SCN provides evidence suggesting a direct effect of the ghrelin
system on the SCN. The methods for visualizing the YFP reporter and acquiring
images are the same as those described by Mani et al.7. Scale bar 100 µm. 3V, third
average periods of locomotor activity and also had delayed phases80.
In addition, under LL conditions, GHSR-knockout animals displayed
increased daily activity when compared with wild-type animals80. A note-
worthy discrepancy in the literature pertains to the effects of ghrelin
on locomotor activity in a food-deprived state: when Yanielli et al.79
administered a high dosage of GHRP-6 in the middle of the subjective
day after 24 h of food deprivation, they noted phase advances of loco-
motor activity. Zhou et al.76 recapitulated this experiment with a slight
variation, but they did not replicate the previous findings. Further,
antisense GHSR shRNA-mediated knockdown of GHSR expression in
the VMH reduces wheel running activity in both ad libitum-fed rats and
rats subjected to a restricted feeding schedule, while also attenuating
body weight loss otherwise induced by the wheel running activity81.
Interestingly, VMH GHSR knockdown in ad libitum-fed rats increases
food intake and body weight gain81.
Less is known regarding the role of LEAP2-mediated effects on
circadian patterns of locomotor activity. However, a study published
in 2021 found that in high-fat diet-fed female mice housed in a stand-
ard LD cycle, LEAP2-knockout mice had a 49% reduction in locomotor
activity during the first 5 h of the dark cycle compared with wild-type
mice, without any differences in the first 5 h of the light cycle65. Thus,
although ghrelin and GHSR have effects on the circadian rhythmicity
of locomotor activity, and although the effects of LEAP2 on locomotor
activity depend on circadian rhythmicity, the wide variation in experi-
mental procedures and a somewhat limited literature make it difficult
to generate any consensus on the nature of the effects of LEAP2.
Effects of the ghrelin system on food anticipatory activity
Besides its effects on locomotor activity in general, ghrelin also impacts
circadian-dependent food intake and food-seeking behaviours. Food
Nature Reviews Endocrinology
DAPI nuclear stain
ventricle; AHA, anterior hypothalamic area, anterior part; BMA, basomedial
amygdaloid nucleus, anterior part; Cre, Cre recombinase; DAPI, 4′,6-diamidino-
2-phenylindole; f, fornix; GHSR, growth hormone secretagogue receptor; IRES,
internal ribosome entry site; LA, lateroanterior hypothalamic nucleus; LH, lateral
hypothalamic area; LV, lateral ventricle; MPA, medial preoptic area; opt, optic
tract; PaAP, paraventricular hypothalamic nucleus, anterior parvicellular part;
Pav, paraventricular hypothalamic nucleus, ventral part; SCN, suprachiasmatic
nucleus; vhc, ventral hippocampal commissure; YFP, yellow fluorescent protein.
anticipatory activity (FAA) is defined as an increase in activity preced-
ing a scheduled meal. In rodents on a restricted meal schedule, FAA is
usually observed beginning several hours prior to the presentation of
the meal with a peak at mealtime82.
Of the many circadian processes reviewed here, the roles of ghre-
lin and GHSR are perhaps the most studied in relation to FAA. Some
groups found that ghrelin produces FAA and that in the absence of
ghrelin signalling, FAA is reduced. LeSauter et al.83 found that periph-
eral administration of ghrelin in the middle of the light phase increased
FAA and food intake. In addition, FAA induced by ghrelin administra-
tion was decreased in GHSR-knockout mice83. Blum et al.84 also found
that FAA was attenuated in GHSR-knockout mice. The decrease in FAA
was associated with decreased cFos expression levels in several hypo-
thalamic areas, including the DMH, but not the SCN. In schedule-fed
goldfish under LL conditions, ghrelin antagonist treatment abolished
the FAA85. However, several groups found no difference in FAA86,87 and
body temperature rise87 between GHSR-knockout mice and wild-type
animals. Several other groups have also noted delays in the onset of
FAA. For instance, Lamont et al.80 noted that GHSR-knockout mice are
less active and develop FAA more slowly than wild-type animals under
DD conditions, although these observations did not reach statistical
significance. Rats with GHSR knockdown in the VMH exhibit a delay
in the onset of FAA81. Therefore, despite these many studies linking
ghrelin and FAA, the effects of ghrelin on FAA are complex and require
further investigation.
Effects of the ghrelin system on thermoregulation
One interaction that is less intuitive than the ones previously mentioned
is the effects of ghrelin on circadian-dependent thermoregulation.
Tokizawa et al.77 investigated this relationship in detail. Mice injected
Review article

with either vehicle or ghrelin at the start of the light phase (ZT0) had
an immediate increase in body temperature, probably partially as an
effect of animal handling. However, the increase in body tempera-
ture was less in the ghrelin group than in the vehicle group. Over the
next 2 h, body temperature decreased in both groups, with greater
decreases in the ghrelin group. At ZT2 (2 h after the start of the light
phase), mice were either exposed to 10°C or stayed under 27°C condi-
tions. At both temperatures, no change in body temperature occurred
in the vehicle-treated animals. Body temperature of the ghrelin-treated
mice that remained under 27°C conditions increased until it was not
significantly different from the body temperature in the vehicle-treated
group. However, the ghrelin-treated group exposed to 10°C condi-
tions showed a significant decrease in body temperature compared
with all the other groups. No differences were observed when injec-
tions of vehicle or ghrelin were given during the dark phase. Related
to this finding, the increase in oxygen consumption in the vehicle
group exposed to cold conditions was significantly greater than the
increase in oxygen consumption in the cold-exposed ghrelin group.
Interestingly, in a study of a cohort of female mice fed a high-fat diet,
heat production was reduced (by 9.5%) in LEAP2-knockout mice com-
pared with wild-type mice during the first 5 h of the dark cycle65. No
differences were observed between groups during the first 5 h of the
light cycle65. These findings suggest that both activating GHSRs with
ghrelin and knocking out the endogenous GHSR antagonist, LEAP2,
impair an organism’s ability to generate heat, resulting in hypothermia,
but only during the dark phase. Many unanswered questions remain
regarding the intersection of thermogenesis, circadian rhythm and
the ghrelin system.
Therefore, although ghrelin clearly influences circadian-driven
processes such as neuronal activity, locomotor activity, food antici-
patory behaviour and thermoregulation, much debate remains in the
field regarding the exact role of the ghrelin system. More targeted
experiments with standardized protocols for light–dark conditions
and fed versus fasted states, should be conducted to better define the
effects of ghrelin on circadian rhythms.
Influence of circadian rhythms on ghrelin and
GHSR expression
Circadian rhythmicity of ghrelin in humans
Circadian rhythms affect the expression of ghrelin and GHSR and are
relevant to humans. The human ghrelin gene contains an Ebox ele-
ment, a sequence of DNA that allows protein binding and regulates
gene expression88. Ebox elements are strongly associated with circadian
rhythm89. Functionally, plasma ghrelin has been measured in humans
over 24-h periods. In humans with access to three meals a day on a fixed
schedule, ghrelin fluctuates across 24 h both as a function of mealtime
as well as in a diurnal rhythm4 (Fig. 4). Plasma ghrelin levels rise approxi-
mately twofold immediately before every meal and fall to trough levels
1 h after eating4. These preprandial and postprandial plasma ghrelin
patterns are influenced by the types of nutrients within meals: both
carbohydrate-containing beverages and protein-containing bever-
ages suppress post-meal nadirs in plasma ghrelin more robustly than
lipid-containing beverages90. Over the course of the day, ghrelin levels
exhibit a diurnal rhythm, reaching a peak at 0100 and falling overnight
to their lowest point at 0900 (ref. 4).
Peripheral effects of circadian rhythmicity of ghrelin
The effects of circadian rhythms on ghrelin and GHSR have been studied
in further detail in animal models, both in the periphery and in the cen-
tral nervous system (CNS). In the periphery in mice, ghrelin-secreting
cells of the stomach express the circadian clock proteins Per1 and Per283.
Ghrelin expression is rhythmic, antiphase to Per1 and Per2 expression,
and does not require light input83. In ad libitum-fed rats, Sánchez et al.3
found that ghrelin mRNA expression in the gastric mucosa remains
high throughout the light phase (Fig. 4). At the onset of the dark phase,
ghrelin mRNA expression in the gastric mucosa decreases and reaches
its lowest point in the middle of the dark phase3. By contrast, plasma
ghrelin levels reach a peak at the start of the dark phase, drop to a
nadir shortly after, then gradually increase for the remainder of the
dark phase3. Notably, Bodosi et al.91 observed a slightly earlier peak in
plasma ghrelin levels in ad libitum-fed mice than did Sánchez et al.3;

Active phase Inactive phase Scheduled
mealtimes
Human
Daily plasma ghrelin
fluctuations with three
• In both humans and rats with scheduled meals
three scheduled meals, plasma
ghrelin levels peak at
mealtimes.
• During the active phase,
plasma ghrelin levels continue
Rat
to rise between mealtimes.
Daily plasma ghrelin
fluctuations with three
scheduled meals
Fig. 4 | Representative traces of human and
rat plasma ghrelin secretion patterns. Ghrelin
levels in humans and rodents are influenced both
by circadian rhythm and anticipated mealtimes.
Humans are largely a diurnal species whereas
rodents are nocturnal. However, patterns of plasma
ghrelin levels are similar between humans and
rodents when the active and inactive phases for
each species are compared, suggesting that ghrelin
secretion patterns are more heavily dependent on
circadian time than external time. Ghrelin levels also
peak at scheduled mealtimes in both humans and
rodents. The graphs are representations inspired by
multiple sources.

• With ad libitum access to food,

plasma ghrelin levels reach a
peak at the start of the active Rat
phase, then immediately drop Daily plasma ghrelin
down to a nadir. fluctuations with ad libitum
• Subsequently, plasma ghrelin meal access
levels rise until the beginning
of the inactive state.

Nature Reviews Endocrinology

Review article
the plasma ghrelin peak in the study by Bodosi et al. occurred during the
middle of the light phase as opposed to at the start of the dark phase
in the study by Sánchez et al.3. Circadian patterns of peripheral ghrelin
levels are also sensitive to the effects of chronic stress92. Mice exposed to
a chronic social defeat stress model of clinical depression had increased
plasma ghrelin levels in the late light phase immediately preceding lights
off, as compared with controls that did not face the chronic stressor92.
CNS effects of circadian rhythmicity of ghrelin
CNS effects of ghrelin are observed in mice with ad libitum access to
food: administration of GHRP-6, a ghrelin mimetic, downregulates GHSR
expression in the SCN, suggesting a circadian-related feedback loop for
ghrelin and GHSR76. Ghrelin protein expression is significantly reduced
in Clock-mutant mice at all time points of the LD cycle23. The importance
of ghrelin mRNA expression in the brain has been hotly debated, with
Cabral et al.93 concluding that “no irrefutable and reproducible evidence
exists supporting the notion that ghrelin is synthetized, at physiologi-
cally relevant levels, in the central nervous system of adult mammals”;
however, one study showed that ghrelin mRNA levels in the mediobasal
hypothalamus, as detected by PCR with reverse transcription, are mark-
edly decreased in Clock-mutant mice compared with wild-type mice23.
Thus, both ghrelin protein and/or mRNA expression in the periphery
and in the CNS seem to be under circadian control.
Effect of time-restricted feeding on ghrelin secretion
Numerous studies have shown promising results in the improvement
of metabolic health in humans in response to time-restricted feeding
protocols94. Understanding the effects of time-restricted feeding on
ghrelin secretion is, therefore, of interest. Mimicking the traditional
pattern of three meals a day in humans, rats that were restricted to three
meals a day also showed increases in spontaneous activity and ghrelin
levels associated with the scheduled meals95 (Fig. 4). In rats, this inter-
mittent fasting was also associated with adipogenesis95. Restriction of
Ghrelin
• Nighttime snacking
• Night-eating syndrome
• Shift work
• Chronic jetlag
• CLOCK gene SNP
Metabolism Circadian
rhythms
Diet-induced obesity and
other metabolic disorders
Fig. 5 | Intersections of ghrelin, metabolism and circadian rhythms.
The ghrelin system (ghrelin, growth hormone secretagogue receptor (GHSR),
liver-expressed antimicrobial peptide 2 (LEAP2) and ghrelin O-acyltransferase
(GOAT), circadian system (central and peripheral) and metabolism are
interconnected and affect one another. While some aspects of these relationships
are well understood, much remains to be further explained. Regardless of
the mechanisms, what is known is that when any one of these chain links is
disrupted — for instance, by night-time snacking, night-eating syndrome, shift
work, chronic jetlag or certain CLOCK gene single nucleotide polymorphisms
(SNPs) — serious consequences such as diet-induced obesity and other metabolic
disorders ensue. The severity and prevalence of these consequences make it
critical to understand how all three systems interact.
Nature Reviews Endocrinology
food to the light phase in rats resulted in the complete reversal of the
diurnal rhythmicity of plasma ghrelin, with the peak plasma ghrelin
levels occurring towards the end of the dark phase preceding food
presentation91. In mice, restriction of feeding to the light phase led to
increased adiposity, weight gain, hyperleptinaemia and hypothermia96.
Dark phase-restricted feeding (DRF) resulted in an increase in plasma
ghrelin levels prior to the start of the dark phase that was significantly
greater than the increase seen in mice with ad libitum food access97.
DRF led to an increase in expression of GHSR and AgRP 1 h into the
dark phase compared with ad libitum feeding97. DRF also enhanced
the obesogenic properties of ghrelin: a low dose of ghrelin in mice
receiving DRF was sufficient to significantly increase fat preservation
in these mice compared with their counterparts fed ad libitum97.
Several groups have also investigated food anticipatory increases
in plasma ghrelin levels. Drazen et al.98 noted that when rats were
restricted to eating during a 4-h period, an increase in plasma ghrelin
levels occurred in the 2 h preceding the meal, peaking 30 min before the
anticipated mealtime. This rise in plasma ghrelin in time-restricted feed-
ing rats corresponded to an increase in plasma ghrelin in ad libitum-fed
rats immediately before the onset of the dark phase, when rats typi-
cally eat their largest meal98. Frecka and Mattes5 made similar obser-
vations in humans: they placed two groups of participants on feeding
regimens with either small intervals between meals or large intervals
between meals and found that in both groups, the time of peak plasma
ghrelin shifted to prior to the anticipated mealtimes. Interestingly,
mealtime-associated increases in plasma ghrelin and subsequently
FAA are sensitive to perceived food palatability: ad libitum-fed rats that
anticipated chocolate had greater increases in plasma ghrelin and FAA
than chow-fed control animals99. Taken together, these findings suggest
that ghrelin may be involved in priming the body for effective meal
consumption, perhaps as part of the arousal state described by Saper100.
Relevance of the work in mouse models to human
health and disease
Some concern always exists regarding how well an animal model sys-
tem replicates a given disease process in humans, whether because of
inherent differences in baseline physiology or in disease manifestations
between the two species or because of variations in the mechanisms
involved in regulating the underlying physiological processes. As sci-
entists, we do our best to choose models based on some aspects of
similarity to the process we are studying, with the understanding that
not all of our findings will be directly translatable to humans. In the
field of ghrelin biology and metabolism overall, as well as in circadian
biology, animal models have been the cornerstone for furthering our
understanding of basic human physiology. Wang et al.15 offer a detailed
review of the current widely used animal models in circadian research,
the limitations and advantages of each of these models, the similarities
and differences between mouse and human biology overall and specifi-
cally with regard to circadian biology, and the advances in human health
that have been made possible as a result of these models. Wang et al.
conclude that “considering the cost, life cycle, ethical issues, etc., the
advantages of mouse models outweigh the limitations, and the major-
ity of our knowledge in this field has been conducted in mice”15. Also,
numerous similarities exist in the ghrelin systems between rodents
and humans. The predicted amino acid sequences for GHSR and ghre-
lin in humans and in rodents show high sequence homology101, and
LEAP2 is identical in humans and in rodents58,101,102. The overall effects
of ghrelin on glucose and insulin levels are conserved between rodents
and humans, as are its overall effects103 on growth hormone secretion,
Review article
wakefulness, food intake and weight gain79,103. Differences also exist.
For example, islet ghrelin production, which is predominant in the fetal
period, persists at a low level in adult humans whereas it disappears
in adult mice104. Thus, although it is certainly important to consider
the limitations of animal models, we find no reason to believe that the
data presented in this Review from studies in animal models will not
be relevant to humans.
Conclusions
Given the strong evidence for GHSR in the brain (including the SCN
and ARC), the diurnal rhythmicity of plasma ghrelin levels, and the
bidirectional interactions between ghrelin and the circadian system,
ghrelin seems to be a promising candidate to explain the link between
metabolism and circadian biology (Fig. 5). Several clinically relevant
scenarios exist, in addition to the ones described above, where the role
of ghrelin is less well understood (Box 2).
For instance, Garaulet et al.105 genotyped 1,290 individuals with
overweight and obesity for the CLOCK3111T/C single nucleotide poly-
morphism (SNP). Of the individuals genotyped, 606 had either one or
two copies of the minor allele C, and 684 were non-carriers for the SNP.
Individuals who had either one or two copies of the minor allele C had
higher plasma ghrelin concentrations, lower sleep duration, delayed
breakfast time, subjective preference for evening-time as opposed to
morning-time, and less compliance with a Mediterranean diet pattern.
Mechanistically, how higher plasma ghrelin concentrations could
explain the observed behavioural findings or vice versa is unknown,
although poor sleep is associated with higher plasma ghrelin levels106.
Disrupted circadian rhythms are increasingly becoming part of
day-to-day life. One such lifestyle that frequently produces chrono-
disruption is chronic jetlag due to frequent time zone travelling. In a
mouse model of chronic jetlag, jetlagged mice were housed for 4 days a
week under an LD schedule, were shifted 8 h forward for the remaining
3 days of the week, after which the jetlagged mice were shifted back
to the normal LD schedule, for 4 consecutive weeks107. In the mucosa of
the stomach, ghrelin mRNA expression fluctuated diurnally in control
mice, consistent with previously described findings. By contrast, the
jetlagged mice did not display this rhythm. The study also looked at
GOAT, the enzyme that catalyses the acylation reaction required for
ghrelin to bind to GHSR. Stomach mucosa expression of the Mboat4
gene that encodes GOAT also fluctuated diurnally in control animals,
although this pattern was not significantly different in jetlagged mice.
In addition, plasma ghrelin levels were also rhythmic in both groups107.
More work is needed to understand why chronic jetlag affects only
ghrelin mRNA but not GOAT mRNA in the stomach mucosa and what
the functional significance of this finding could be.
Ghrelin is not alone as a potential link between metabolism and cir-
cadian biology. Roles for several other hormones have been described,
although it is beyond the scope of this Review to discuss them in detail.
As just a few examples, rats with dopaminergic neuron-specific SCN
lesions exhibited signs of metabolic syndrome including increased
weight gain, increased levels of insulin, leptin and noradrenaline, and
altered lipid synthesis, and were hypertensive108. Fibroblast growth
factor 21 (FGF21), a liver molecule that signals starvation, mediates cir-
cadian rhythm via B-Klotho receptors located in the SCN; mice housed
under LD conditions and fed a ketogenic diet showed increases in FGF21
as well as decreases in overall locomotor activity and increases in
locomotor activity in the light phase109. Leptin displays circadian rhyth-
micity opposite to that of ghrelin and also has time-of-day-dependent
effects on feeding and gene expression28.
Nature Reviews Endocrinology
Box 2
Open research questions
•• What would be the effects on phase shift if ghrelin was
systematically administered at multiple timepoints throughout
the day, as opposed to general timeframes such as early
morning or early night, or limited timepoints?
•• Are variations in endogenous levels of ghrelin, such as through
time-restricted feeding or fasting, sufficient to produce changes
in circadian rhythm?
•• Is circadian rhythmicity of ghrelin expression in gastric mucosa
cells a cell-intrinsic pattern or does it depend on external
cues such as nutrient sensing or suprachiasmatic nucleus
(SCN) input?
•• Does ghrelin acting on growth hormone secretagogue receptor
(GHSR) in the SCN have some role in mediating appropriate
mealtimes and meal-seeking behaviour, potentially through the
generation of an arousal state at the start of an organism’s active
phase?
•• Where do GHSR neurons in the SCN project to? Where do they
receive input from?
•• What is the role for liver-enriched antimicrobial peptide 2 (LEAP2)
in circadian regulation of neuronal and locomotor activity?
•• Is LEAP2 expression and secretion influenced by circadian
rhythms?
•• Is there some role for ghrelin in linking phenotypes of poor
metabolic health with disrupted circadian biology, such as that
seen in humans and animal models of chronic jetlag, night-time
snacking and mutations in the CLOCK gene?
To settle some discrepancies and advance our understanding
of the role of the ghrelin system in linking metabolism and circadian
biology, we suggest applying ghrelin/GHSR-specific tools. For exam-
ple, conditional Ghsr-knockout mouse models can help determine
whether the ghrelin system has effects on the circadian system and
metabolism during development or during adulthood. In vivo calcium
imaging of GHSR neurons in the SCN can provide real-time insight into
how neuronal activity is changing over the course of the day. Viral
tracing methods can be employed to better understand and manipu-
late the ghrelin-engaged neuronal circuitry involved, and to better
understand the connections between the DMH or ARCvm and the SCN.
Chemogenetic and optogenetic strategies can be targeted specifically
to GHSR-expressing neurons in the SCN. Additionally, we propose
investigating further the role of ghrelin, a potent growth hormone
secretagogue, in influencing the well-documented circadian regu­
lation of growth hormone1,110–114. Finally, all of these studies should be
done in both male and female animals, as sex differences have not been
well-studied in this field. These strategies are already being used in sev-
eral fields to answer questions that were previously difficult to answer.
Given the clinical potential and strong societal need to find solutions
for dysregulation of circadian and metabolic systems, it seems reason-
able to pursue the question of the role of the ghrelin system in these
processes with increased urgency and vigour.
Published online: xx xx xxxx
Review article

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Acknowledgements
The authors’ works is supported through research grants from the NIH (R01 DK103884,
P01DK119130 and 5T32GM008014).
Author contributions
All authors researched data for the article. S.S.K. and J.M.Z. contributed to the discussion of
content, wrote the article and reviewed/edited it before submission.
Competing interests
J.M.Z. owns stock in Medtronic, Eli Lilly and Novo Nordisk, and received research funding from
Novo Nordisk for another project during the writing of this Review. The other authors declare
no competing interests.
Additional information
Peer review information Nature Reviews Endocrinology thanks Yu Zhou and the other,
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