Am J Physiol Endocrinol Metab 322: E494–E507, 2022.

First published April 11, 2022; doi:10.1152/ajpendo.00432.2021

RESEARCH ARTICLE

Deciphering the Contribution of the Gastrointestinal Tract on Glucose, Lipid, and Energy
Metabolism

Ghrelin deficiency sex-dependently affects food intake, locomotor activity, and

adipose and hepatic gene expression in a binge-eating mouse model
Karina Prins, Martin Huisman, Anke McLuskey, Rosinda Mies, Bas Karels,† Patric J. D. Delhanty, and
Jenny A. Visser
Department of Internal Medicine, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

Abstract
Binge-eating disorder is the most prevalent eating disorder diagnosed, affecting three times more women than men. Ghrelin
stimulates appetite and reward signaling, and loss of its receptor reduces binge-eating behavior in male mice. Here, we exam-
ined the influence of ghrelin itself on binge-eating behavior in both male and female mice. Five-wk-old wild-type (WT) and ghre-
lin-deficient (Ghrl / ) mice were housed individually in indirect calorimetry cages for 9 wks. Binge-like eating was induced by
giving mice ad libitum chow, but time-restricted access to a Western-style diet (WD; 2 h access, 3 days/wk) in the light phase
(BE); control groups received ad libitum chow (CO), or ad libitum access to both diets (CW). All groups of BE mice showed
binge-eating behavior, eating up to 60% of their 24-h intake during the WD access period. Subsequent dark phase chow intake
was decreased in Ghrl / mice and remained decreased in Ghrl / females on nonbinge days. Also, nonbinge day locomotor
activity was lower in Ghrl / than in WT BE females. Upon euthanasia, Ghrl / BE mice weighed less and had a lower lean body
mass percentage than WT BE mice. In BE and CW groups, ghrelin and sex altered the expression of genes involved in lipid
processing, thermogenesis, and aging in white adipose tissue and livers. We conclude that, although ghrelin deficiency does not
hamper the development of binge-like eating, it sex-dependently alters food intake timing, locomotor activity, and metabolism.
These results add to the growing body of evidence that ghrelin signaling is sexually dimorphic.
NEW & NOTEWORTHY Ghrelin, a peptide hormone secreted from the gut, is involved in hunger and reward signaling, which
are altered in binge-eating disorder. Although sex differences have been described in both binge-eating and ghrelin signaling,
this interaction has not been fully elucidated. Here, we show that ghrelin deficiency affects the behavior and metabolism of mice
in a binge-like eating paradigm, and that the sex of the mice impacts the magnitude and direction of these effects.

binge eating; food intake; ghrelin; locomotor activity; sex differences

INTRODUCTION times more likely to be diagnosed with BED in their life-

Binge-eating disorder (BED) is currently the most diag-
nosed eating disorder (1, 2). It is characterized by binge-
eating episodes: excessive food intake in a short time
span, in which patients experience a loss of control over
their eating (3). During these episodes, patients often
consume palatable, high-sugar/high-fat foods. Besides
the emotional distress this causes, patients do not com-
pensate for the surplus calories they eat during episodes.
This often leads to weight gain and resulting metabolic
comorbidities, such as obesity and metabolic syndrome
(4, 5). Like in other eating disorders, the prevalence of
BED is greater in women than in men: women are three
time (1, 6).
These sex differences in BED have also been researched
using laboratory animals. Several models of binge-like eating
have been developed for rodents (7–9). The model of limited
access to a highly palatable, energy-rich food without food
deprivation, developed for rats and adapted for mice, has
been well characterized and used in many studies. In this
model, animals are provided sporadic, time-limited access to
a high-fat diet or to a Western-style diet (WD) and eat more
calories on access days than on nonaccess days. Although
this model does not cause overt obesity in the short term,
metabolic effects have been observed, such as impaired glu-
cose homeostasis and increased fat mass (9). Importantly,

P. J. D. Delhanty and J. A. Visser contributed equally to this work.

†Deceased 15 December 2021.
Correspondence: J. A. Visser (j.visser@erasmusmc.nl).
Submitted 14 December 2021 / Revised 25 March 2022 / Accepted 4 April 2022

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 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE
this model emulates aspects of the disturbed eating behav-
iors observed in humans with BED, particularly those related
to hedonic rather than homeostatic control of eating. In this
model, female rats escalated their intake of palatable food
faster than males (10) and were more likely to be classified as
binge-eating prone or high responders (11, 12). The increased
sensitivity of females to food-related reward signaling has
been implicated to play a role in this process (13). Similarly,
in humans, it appears that altered food cues and reward
processing, and sex differences herein, play a role in the eti-
ology of BED (14, 15).
Ghrelin is a peptide hormone that is secreted by the
stomach. It is present in the blood in two forms: acylated
ghrelin (AG) and unacylated ghrelin (UAG). Acylated ghre-
lin is octanoylated by ghrelin-O-acyltransferase (GOAT),
and is the endogenous ligand of the growth hormone se-
cretagogue receptor 1a (GHSR1a) (16). Through this recep-
tor, AG stimulates growth hormone secretion, enhances
appetite, and plays a role in stress and reward signaling
(17, 18). UAG, however, appears to be antidiabetogenic and
its levels correlate with fasting insulin, BMI, waist circum-
ference, and weight loss, although its mechanism of action
is not known (19, 20). Altogether, these functions make
ghrelin an interesting potential modulator of binge-like
eating behavior and resulting metabolic comorbidities.
Data on both postprandial and fasted plasma ghrelin lev-
els in patients with BED are conflicting, however. Although
most studies show that plasma ghrelin concentrations are
lower in patients with BED than in weight-matched controls
(21–24), others find no significant differences between these
groups (25). A recent meta-analysis of genome-wide associa-
tion studies in BED identified ghrelin as a potential gene of
interest: Leu72Met ghrelin is more commonly expressed in
patients with BED than in healthy controls (9, 26). Moreover,
fasting plasma AG levels were observed to correlate posi-
tively with the risk of developing eating disorders in women
with obesity (27). Interestingly, total plasma ghrelin levels
positively correlate with both estrogens in women and tes-
tosterone in men (28–30). In contrast, high androgen levels
in women decrease circulating total ghrelin (31), and anti-
androgen treatment in women increases plasma total ghrelin
(32). Altogether, these studies imply that ghrelin levels are
altered in BED and that ghrelin levels are sex dependent.
In laboratory animals, components of the ghrelin signal-
ing pathway have proven an interesting target in BED
research. Results in Ghsr knockout (Ghsr / ) mice, however,
strongly depend on the frequency of access to the palatable
diet that is used to evoke binge-eating behavior. That is,
when mice are given daily time-limited access to a palatable
diet, Ghsr / mice develop binge-like eating behavior simi-
larly to wild-type (WT) mice (33, 34). In contrast, when the
access to the palatable food is only given once every 2 days,
Ghsr / mice eat less of the palatable diet than WT mice
(34). In a shorter experiment, Valdivia et al. (35) showed that
Ghsr / mice did not escalate the intake of a high-fat diet
over 4 days of restricted access to it, whereas WT mice did.
Despite these findings, Ghrl / and Goat / mice have not
been investigated using these models.
Furthermore, there are very few basic studies so far that
have examined sex-dependent differences in the physiologi-
cal response to ghrelin (36). Intriguingly, one of these studies
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found that male rats are more sensitive to ghrelin treatment,
assessed by acute food intake, than female rats (37). However
in mice, when ghrelin is administered just before the dark pe-
riod, females are not only more sensitive in terms of its orexi-
genic effects, but also the effects of treatment lasted longer
than in males (38). Likewise, the GH secretagogue activity of
ghrelin appears to be more potent in females than in males
(38), and it has been shown that there is estrogen dependency
on ghrelin levels and the orexigenic efficacy of ghrelin. For
example, the orexigenic effects of ghrelin in male and ovariec-
tomized female rats are reduced after estrogen treatment.
Furthermore, the inductive effects of ovariectomy on food
intake and body weight are lost in GHSR-null mice (37). This
could be related to the direct modulation of neuronal sensitiv-
ity to ghrelin by estrogens (39, 40). However, these studies
only give an idea of the acute physiological response to the
administration of pharmacological doses of ghrelin.
Given the sex differences in BED prevalence and in ghre-
lin signaling, it is surprising that most of the ghrelin-related
studies on BED have only been performed in male mice.
This study aims to find out whether sex impacts the effects
of ghrelin on the development and metabolic consequences
of binge-eating behavior. We hypothesized that ghrelin defi-
ciency would attenuate the binge-like pattern of eating that
occurs in WT mice, using an intermittent access model, like
that of King et al. (34) in their study in Ghsr / mice. We also
hypothesized that the sex of the mice would modulate this
binge-like eating behavior, perhaps in interaction with ghre-
lin deficiency. Therefore, to assess these hypotheses, we
exposed male and female mice, with or without ghrelin defi-
ciency, to a limited access model (2 h access to WD/day 3
times a week, relative to mice given either free access to both
WD and regular chow or chow-only controls). We ran this
model for 9 wk, a relatively long time, with the aim of accen-
tuating any metabolic effects. In addition to food intake,
using a metabolic cage system, we have also assessed the sex
and genotype effects of the binge-like eating protocol on
energy expenditure, activity, and substrate use. Finally, the
expression of genes relevant to glucose homeostasis and
lipid metabolism in livers and inguinal white adipose tissue
(WAT), and plasma metabolite levels have been determined.
MATERIALS AND METHODS
Animals and Housing
WT and Ghrl / male and female mice with a C57BL/6
background (41, 42) from the same breeding colony were
bred in-house. The mice (48 males, 48 females) were 5 to 6
wk old at the start of the experiment and selected from litters
of 7–10 pups to reduce variability in body weight. The age of
the mice corresponds to adolescence, which is the most com-
mon age of onset of eating disorders in humans (43).
Housing conditions provided a 12-h light:dark cycle with
lights on at 7:00 AM [zeitgeber time (ZT) 0]. Ambient temper-
ature was maintained at 23 C. Throughout the experiment,
all mice had ad libitum access to water. All experimental pro-
cedures were approved as part of license AVD1010020174317
(Central Animal Testing Committee, The Netherlands).
Mice were individually housed in a Promethion metabolic
cage system (Sable Systems, Las Vegas, NV) for 9 wk,
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 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE
including a 5-day habituation period. The cages (Greenline,
Tecniplast, Italy) with woodchip bedding (Lignocel BK 8/15,
J. Rettenmaier & So € hne GmbH, Germany) were fitted with a
water bottle, a nest, and two food containers. Horizontal and
vertical location, food intake, and body mass were detected
at 1 Hz by an infrared beam grid and load cells from which
the food containers and nests were suspended, respectively.
The cage air influx was set to 2 L/min, and respiratory gases
were collected and analyzed at 5-min intervals. Volumes of
consumed O2 (V_ O2), produced CO2 (V_ CO2), and water vapor
(V_ H2O) were measured. The cage system and data acquisi-
tion were controlled with MetaScreen (v. 2.3.15.11) software.
Diets
Throughout the experiment, mice in the chow-only
(CO) group had ad libitum access to pelleted rodent chow,
containing 3.59 kcal/g [22% from protein, 69% from car-
bohydrate (of which 10% sucrose), 9% from fat; 801722
CRM-P, Special Diets Services, UK]. In the binge-eating
(BE) group, binge-like eating was induced by limiting the
access of the mice to a palatable WD [4.69 kcal/g, 17%
from protein, 43% from carbohydrate (of which 70% su-
crose), 41% from fat; D12079B, Research Diets, New
Brunswick, NJ] (7). After a 1-wk habituation period, in
which mice had free access to both chow and WD, BE
mice could only access WD for 2 h during the light phase
(ZT4-6) on Monday, Wednesday, and Friday. WD access
was automatically regulated using software-controlled
shutters. To control for the effects of WD consumption, a
final group of mice (CW) had free access to both chow and
WD throughout the experiment.
Metabolic Assessment
In week 7, glucose tolerance was determined by an intra-
peritoneal glucose tolerance test (IPGTT), as previously
described (44). Briefly, mice were transferred to new cages
where they were fasted for 5 h (ZT2-7). Next, the mice were
weighed, and fasting blood glucose was measured in tail cut
blood. A 2 g/kg glucose bolus was injected intraperitoneally
and blood glucose levels were determined with a glucometer
at 15, 30, 60, and 120 min after injection.
Euthanasia and Tissue Collection
Mice were euthanized in week 9 between ZT2-5 on
Monday, Wednesday, or Friday (access time/days for BE
mice). Mice were weighed manually, and nonfasting
blood glucose concentrations were measured. In addi-
tion, body composition was determined by magnetic res-
onance imaging (EchoMRI-100V, EchoMRI LLC, TX). Fat
and lean mass are presented as percentages of total body
weight. Next, mice were deeply anesthetized with isoflur-
ane (Pharmachemie BV, The Netherlands) and eutha-
nized by exsanguination. Cardiac blood was collected
using EDTA-coated syringes (Na3EDTA, Sigma Aldrich,
The Netherlands), aliquoted immediately, and centri-
fuged for 5 min at 1,300 g to obtain plasma. The resulting
plasma samples were snap-frozen in liquid nitrogen and
stored at 80 C until further use. Liver and brown (BAT)
and white adipose tissues (WAT) were dissected, weighed,
and snap-frozen in liquid nitrogen for later RNA isolation.
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Hormone Assessment
The blood aliquot for measuring AG and UAG was stabi-
lized with 4-(2-aminoethyl)benzenesulfonyl fluoride hydro-
chloride (AEBSF, Roche, Germany; final concentration 0.2
mM) before centrifugation. Plasma AG and UAG were meas-
ured by mouse/rat-specific ELISA (RRID:AB_2619624, Bertin
Pharma, France; RRID:AB_2619623, Bertin Pharma, France)
(45). Plasma levels of leptin were determined by mouse/rat
leptin ELISA (RRID:AB_2801467, Alpco Diagnostics, NH),
and total adiponectin concentrations were measured with a
mouse/rat HMW and total adiponectin ELISA kit (RRID:
AB_2801466, Alpco Diagnostics, NH) (46). Corticosterone
was extracted from feces from the cage bedding of weeks 5–7
of the experiment as previously described (47), and meas-
ured by ELISA (RRID:AB_2307314, Enzo Life Sciences,
Switzerland).
Metabolite Analysis
Plasma triglycerides (A11A01640, Horiba Group, France),
non-esterified fatty acids (NEFA) (3055, Instruchemie, The
Netherlands), and cholesterol concentrations (A11A01634,
Horiba Group, France) were measured colorimetrically,
according to the manufacturer’s protocol. Liver glycerol
was determined by colorimetric measurement, according
to the manufacturer’s protocol (2913, Instruchemie, The
Netherlands). Hepatic glycogen content was determined
as previously described by subtracting free glucose from
glycogen, measured using a colorimetric assay (2319,
Instruchemie, The Netherlands) (48, 49).
Gene Expression Analysis
RNA was isolated from snap-frozen livers and inguinal
WAT (TriPure Isolation Reagent, Roche Diagnostics), and
genomic DNA contamination was removed (RQ1 RNase-
Free DNase, Promega Corporation, WI). cDNA was synthe-
sized (Transcriptor high-fidelity cDNA synthesis kit,
Roche Diagnostics), and quantitative PCR was performed
on a QuantSTudio 7 flex real-time PCR system (Applied
Biosystems, Life Technologies, CA), using FastStart Universal
SYBR Green Master (Rox) (Roche Diagnostics). Target gene
expression was normalized to the mean of two housekeeping
genes (b-actin and 18S for liver, b2-microglobulin and 18S for
inguinal WAT) by the 2 DDCT method, as described previously
(46). Amplicon specificity was assessed by melting curve anal-
ysis. Primer sequences are listed in Supplemental Table S1
(https://doi.org/10.6084/m9.figshare.17197274).
Data Processing
Data from Monday to Saturday in week 8, the last full week
of the experiment, were processed with MacroInterpreter
(v2.40, Sable Systems, TX) using One-Click Macro 1 (v2.35.0) to
obtain body mass, energy expenditure, respiratory exchange
ratio (RER), and locomotor activity variables. Energy expendi-
ture was calculated using the Weir equation (50), and the RER
was calculated as V_ CO2/ V_ O2.
Food intake data were analyzed in R (v4.0.2, R Core
Team). Briefly, food container mass was imported using the
SableBase package (51) and median filtered to reduce sensor
noise. The start and end of feeding events were defined as
 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE
differential mass values outside the means ± 2SD range.
Events were defined as lasting more than 10 s and less than
15 min, with two events being separated by at least 2 min.
The position of the mouse in the cage was determined using
the XY infrared beam breaks, and an event was recorded
if the mouse was within reach of the food containers for at
least 10% of the event duration. Intake was determined by
the difference in container mass before and after an event,
and events with an intake over 1.2 g were considered unreal-
istic and excluded.
The resulting data were binned in 2 h blocks to inspect
daily profiles, in which the light phase and subsequent dark
phase were considered for one experimental day. Cumulative
data were binned for the light phase, dark phase, and full 24-h
period. To reduce between-day variation, data for each mouse
were averaged over all days (CO and CW mice), or for access
and nonaccess days separately (BE mice).
Statistical Analysis
Statistical analyses were performed in R using the RStatix
package (52). Data were analyzed by two-way repeated-meas-
ures ANOVA (2-h binned data) or by two-way ANOVA and
considered significant at P < 0.05 (abbreviations for P values:
PGT for genotype effect, PS for sex effect, PT for time effect).
When a significant interaction between factors (genotype and
sex) was detected, the Tukey HSD post hoc test was used to
compare groups. If no significant interaction effect was
observed, t tests with Bonferroni multiple comparison correc-
tions were applied. For the IPGTT data, the area under the
curve (AUC) was normalized to the fasting glucose level (53).
Data are visualized in GraphPad Prism (v. 8, GraphPad
Software, La Jolla, CA). All data are represented as means ± SE.
RESULTS
Food Intake
In the CO group, 2-h binned food intake patterns largely
overlapped between the groups (Fig. 1A). Although the geno-
type effect reached significance (PGT = 0.02), the magnitude
of the difference between WT and Ghrl / was small.
Likewise, over the full 24-h period, we only observed a trend
toward an interaction effect of the ghrelin genotype and sex
(PGT:S = 0.059; Supplemental Fig. S1A, https://doi.org/10.
6084/m9.figshare.19384001).
On access days, BE mice consumed a large proportion of
their total caloric intake from WD during the 2-h access pe-
riod (Fig. 1B). Interestingly, in the subsequent dark phase,
Ghrl / mice, and especially Ghrl / males, ate less
chow than WT mice (PGT = 0.005, Supplemental Fig. S1B).
Consequently, on access days, Ghrl / mice consumed a sig-
nificantly lower percentage of their total food intake from
chow (47.8 ± 3.7%) than WT mice (60.3 ± 3.8%, PGT = 0.020).
On nonaccess days, Ghrl / mice continued to have a lower
chow intake than WT mice (PGT = 0.009; Fig. 1C). Moreover,
particularly during the dark phase, females had a lower
chow intake than males (PS:ZT = 0.019). The resulting total
food intake on the nonaccess day trended toward a geno-
type-sex effect (PGT:S = 0.065; Supplemental Fig. S1C), which
appeared to be mainly driven by the Ghrl / females (P =
0.016 vs. Ghrl / male; P = 0.020 vs. WT female).
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In the CW group, the 2-h binned food intake data showed
a significantly higher food intake in females than in males
(PS = 0.002; Fig. 1D). This sex effect originated from the WD
intake (PS < 0.001; Supplemental Fig. 1D), which made up
over 90% of the total food intake. Notably, as in the BE
group, Ghrl / mice in the CW group showed an even more
decreased preference for chow, consuming 3.5 ± 1.1% of their
calories from chow, compared with 8.8 ± 1.6% in WT mice
(PGT = 0.029).
Energy Expenditure
During the transition from light to dark phase, EE profiles
in the CO group showed a genotype-sex interaction effect
(P < 0.05; Fig. 1E), which affected total light phase energy ex-
penditure (PGT:S = 0.019; Supplemental Fig. S1E). However,
this effect did not last into the dark phase, thus not signifi-
cantly changing the 24-h EE.
In BE mice, EE was increased during the access period on
access days (PZT < 0.001, Fig. 1F). During the light phase,
Ghrl / mice had a higher EE than WT mice (PGT = 0.003,
Supplemental Fig. S1F). In contrast, on nonaccess days, light
phase EE was lower in Ghrl / mice than in WT mice (PGT =
0.001, Fig. 1G and Supplemental Fig. S1G).
Regardless of access, female BE mice expended less
energy than male BE mice (PS < 0.05). Similarly, EE was
increased with ghrelin deficiency and female sex in the CW
group (PGT = 0.027, PS = 0.002, Fig. 1H). Ghrl / females, in
particular, had a higher EE, resulting in a significantly
increased 24-h EE compared with WT females (P = 0.027)
and Ghrl / males (P = 0.014).
Respiratory Exchange Ratio
In the CO group, RER was low during the light phase
and increased upon the transition to the dark phase, corre-
sponding with fat oxidation during the inactive (light)
phase, and increased carbohydrate use during the active
(dark) phase (Fig. 1I). Ghrelin deficiency altered RER in a
sex-dependent way in this group (PGT:S < 0.001). That is,
ghrelin deficiency increased RER in males, whereas the op-
posite occurred in females. In males, this effect occurred
mainly during the dark phase, whereas the RER of Ghrl /
females also remained lower during the light phase, albeit
not significantly.
On access days, the RER of BE mice increased upon WD
access and remained elevated throughout the remainder of
the light phase and subsequent dark phase (Fig. 1J). In the
early dark phase, Ghrl / mice had a lower RER (PGT:ZT =
0.024), corresponding to the lower food intake around that
time. In the late dark phase, females had a lower RER than
males (PS:ZT = 0.011). On nonaccess days, RER is lower in
Ghrl / mice (PGT = 0.027, Fig. 1K). Moreover, females have a
lower RER than males (PS = 0.049).
The RER of CW mice also showed a circadian pattern, but
its amplitude was lower than that of the other groups (PZT <
0.001, Fig. 1L). This indicates reduced variation in the meta-
bolic fuel that these animals use (predominantly fat).
Locomotor Activity
In CO mice, females moved a greater distance than
males, especially during the dark phase (PS:ZT < 0.001,
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 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE

A B C D

E F G H

I J K L

Figure 1. Food intake, energy expenditure, and respiratory exchange ratio. Food intake for CO mice (A), BE mice on access days (B) and nonaccess
days (C), and CW mice (D). Dotted lines represent chow intake, and solid lines represent WD intake. Energy expenditure in kcal of CO mice (E), BE mice
on access days (F) and nonaccess days (G), and CW mice (H). Respiratory exchange ratio of CO mice (I), BE mice on access days (J) and nonaccess days
(K), and CW mice (L). Experimental groups: WT M, wild-type male; Ghrl / M, ghrelin-deficient male; WT F, wild-type female; Ghrl / F, ghrelin-deficient
female. Group size n = 6–8 mice. The access period is marked in gray. Data are presented as means ± SE, and 2-h binned total food intake, energy ex-
penditure, and respiratory exchange ratio were analyzed by two-way repeated-measures ANOVA. Main effects were considered significant if P < 0.05.
g
P < 0.05, (g)P < 0.10 for genotype differences; sP < 0.05, (s)P < 0.10 for sex differences, and P < 0.05, ()P < 0.10 for interaction effects, determined
by post hoc analysis. BE, binge eating; CO, chow only; CW, chow and WD; WD, Western-style diet.

Fig. 2A). Moreover, in both sexes, Ghrl / mice had lower
locomotor activity than WT mice (PGT = 0.029, Supple-
mental Fig. 2F). Although this effect was larger in
females, the interaction did not reach statistical signifi-
cance (PGT:S = 0.080).
During the access period, BE mice increased their loco-
motor activity, although there were no significant differ-
ences between the sexes or genotypes (Fig. 2B). Like CO
mice, BE females walked more than males, especially dur-
ing the dark phase (PS:ZT = 0.045). Interestingly, this sex
effect was ablated in Ghrl / females on nonaccess days
(PGT:S:ZT = 0.027): the activity of Ghrl / females was simi-
lar to that of males during the dark phase.
In contrast to the other groups, ghrelin deficiency has no
effect on locomotor activity in CW mice (Fig. 2D). However,
like in CO mice, females walk more than males, especially
during the dark phase (PS:ZT < 0.001).
Body Weight and Composition
From week 2 of the experiment, CO mice gained weight
significantly, increasing to 23.1 ± 1.0% over baseline at the 8-
wk timepoint (PWK < 0.001, Fig. 3A). The resulting body
mass upon euthanasia was lower in females than in males
(PS < 0.001, Fig. 3B). Interestingly, ghrelin deficiency
sex-dependently altered the body composition of the mice
(PGT:S < 0.005, Fig. 3C): Ghrl / females had a higher fat per-
centage (P = 0.031) and lower lean mass percentage (P =
0.034) than WT females. Notably, the effect of ghrelin defi-
ciency was the opposite in males: Ghrl / males trended to-
ward a higher lean body mass percentage than WT males
(P = 0.067). Differences in body-weight-corrected WAT mass
corresponded with the body fat percentage (PGT:S < 0.05),
whereas the body-weight-corrected BAT mass was similar
across all groups (Fig. 4A).

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A B C D

Figure 2. Directed locomotor activity. Distance walked by CO mice (A), BE mice on access days (B) and non-access days (C), and CW mice (D).
Experimental groups: WT M, wild-type male; Ghrl / M, ghrelin-deficient male; WT F, wild-type female; Ghrl / F, ghrelin-deficient female. Group size n =
6–8 mice. The access period is marked in gray. Data are presented as means ± SE and were analyzed by two-way repeated-measures ANOVA. Main
effects were considered significant if P < 0.05. sP < 0.05, (s)P < 0.10 for sex differences, and P < 0.05, ()P < 0.10 for interaction effects, determined
by post hoc analysis. BE, binge eating; CO, chow only; CW, chow and WD; WD, Western-style diet.

Like the CO mice, body weights of BE mice increased sig-
nificantly from week 2 of the experiment, up to 16 ± 1.03% in
week 8 (PWK < 0.001, Fig. 3D). Upon euthanasia, females in
this group also weighed less than males (PS < 0.001, Fig. 3E).
Body weights were also lower in Ghrl / mice versus WT
mice in both sexes (PGT = 0.001). Although body fat percent-
age was not significantly affected, lean body mass percent-
age was lower in Ghrl / mice and in females (PGT = 0.014,
PS = 0.002, Fig. 3F). Consistent with the fat percentages, the
relative weights of most adipose tissues in BE mice were not
affected by sex or genotype (Fig. 4B). Only inguinal WAT rel-
ative weight was increased in Ghrl / mice compared with
WT mice (PGT = 0.022).
Remarkably, female CW mice gained 15% more weight
over baseline than male CW mice (PS:WK < 0.001, Fig. 3G).
Although body weight upon euthanasia remained lower in
females than in males (PS = 0.047, Fig. 3H), females of both
genotypes had a higher fat percentage (PS < 0.001), at the
expense of a lower lean body mass percentage (PS < 0.001,
Fig. 3I). Accordingly, relative subcutaneous WAT and ingui-
nal WAT weights were higher in females (PS < 0.05, Fig. 4C).
In addition, inguinal WAT was relatively heavier in Ghrl /
animals (PGT = 0.036). Interestingly, ghrelin deficiency pre-
vented the increase in relative retroperitoneal WAT weight
in CW females (PGT:S = 0.018). Moreover, relative BAT weight
was increased in CW Ghrl / males compared with WT
males (PGT:S = 0.035).
Glucose Tolerance
In the IPGTT, CO females cleared the glucose bolus faster
than CO males (PS:T = .015, Fig. 5A). Ghrl / animals were
more glucose tolerant than WT mice (PGT:T = .025), especially
in males (P = 0.037, Fig. 5B).
In contrast, all BE groups cleared glucose at a similar rate,
not showing any effects of genotype or sex (Fig. 5, C and D).
Conversely, the genotype and sex-dependent effects
seen in the CO group were exaggerated in the CW diet
(PGT:T = 0.007, PS:T = 0.002, Fig. 5, E and F). Although WT
females cleared glucose faster than WT males (P = 0.014),
this sex effect was lost in Ghrl / animals. Instead, the
AUC of Ghrl / males was similar to that of the females of
both genotypes, being significantly lower than that of WT
males (P = 0.035).
Metabolic Gene Expression in Livers and Inguinal WAT
As the inguinal WAT mass was affected by genotype and/
or sex in all diets, we decided to analyze gene expression pro-
files in this depot. In CO mice, both sex and genotype
affected gene expression in the inguinal WAT depot (Fig. 6A;
Supplemental Table S2). Mainly genes involved in lipid stor-
age and mobilization were expressed less in females (Plin1,
Hsl). However, macrophage markers (Cd206, Cd11c) and
browning-related genes (Fgf21, Pgc1a) were expressed differ-
entially in Ghrl / and WT animals. In livers, gene expres-
sion was mainly affected by sex (Fig. 6B; Supplemental Table
S3): females showed higher expression of genes involved in
fatty acid uptake and metabolism (Cd36, Ppara, Fasn) and of
secreted proteins (Lcn2, Leap2).
Likewise, in BE mice, the expression of genes involved in
lipid storage and mobilization in inguinal WAT was lower in
females (Plin1, Atgl, Hsl). Expression of uncoupling proteins,
however, was affected by genotype: Ghrl / females
expressed significantly more Ucp1 than any other group,
whereas Ghrl / mice of both sexes expressed less Ucp2. In
addition, there was a suppressive effect of ghrelin deficiency
on the expression of the proinflammatory M1 macrophage
marker Cd11c (Fig. 6A; Supplemental Table S2). In contrast to
CO mice, hepatic gene expression in the BE group was
mainly affected by genotype. Ghrl / mice showed a lower
expression of genes involved in triglyceride and fatty acid
metabolism (Dgat1, Gpat1, Ppara, Elovl3, Fasn, Scd1,
Srebp1c), and in the storage and transport of glucose (Gck1,
Gys2, Slc2a2) (Fig. 6B; Supplemental Table S3).
Finally, in CW mice, inguinal WAT gene expression only
showed minor effects of sex (Plin1, Ucp1), whereas genotype
affected the expression of macrophage polarization and
uncoupling proteins. As in the BE dietary group, but more
pronounced, inguinal WAT of Ghrl / mice of both sexes
had lower expression of Cd11c, as well as a trend to lower
expression of Cd68, a general macrophage marker. Ucp1
expression was increased, but Ucp2 expression was decreased
in iWAT of Ghrl / mice compared with their WT

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 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE

A B C

Figure 3. Body weight and composition.

Weight gain over baseline for CO mice (A),
BE mice (D), and CW mice (G). Body
weight upon euthanasia for CO mice (B),
BE mice (E), and CW mice (H). Percentage
fat and lean body mass of CO mice (C), BE
mice (F), and CW mice (I). Experimental
D E F
groups: WT M, wild-type male; Ghrl / M,
ghrelin-deficient male; WT F, wild-type
female; Ghrl / F, ghrelin-deficient female.
Group size n = 6–8 mice. Data are pre-
sented as means ± SE and were analyzed
by two-way repeated-measures ANOVA
for weight gain over baseline and two-
way ANOVA for the other panels. Main
effects were considered significant if P <
0.05. (g)P < 0.10 for genotype differences;
s
P < 0.05, (s)P < 0.10 for sex differences,
and ()P < 0.10 for interaction effects,
determined by post hoc analysis. BE,
binge eating; CO, chow only; CW, chow
and WD; WD, Western-style diet. G H I

counterparts (Fig. 6A; Supplemental Table S2). In the livers of
CW mice, gene expression effects were more pronounced
compared with those of BE mice, being mainly affected by ge-
notype and not sex (Fig. 6B; Supplemental Table S3).
Circulating and Hepatic Metabolites
Upon euthanasia, Ghrl / CO mice had lower nonfasted
blood glucose concentrations (PGT = 0.003), but higher
NEFA (PGT = 0.006) and cholesterol plasma levels (PGT =
0.021, Table 1). Both glucose (PS = 0.004) and cholesterol
concentrations were lower in females (PS = 0.002).
Triglyceride and free glycerol plasma levels, and liver glyc-
erol content were comparable across groups, similar to
previous reports (54). Liver glycogen content, however,
showed a trend toward a genotype-sex interaction effect
(PGT:S = 0.054), with ghrelin deficiency increasing glyco-
gen content in males, but decreasing it in females.
In BE animals, NEFA (PGT:S = 0.030), triglyceride (PGT:S =
0.020), and free glycerol levels (PGT:S = 0.044) showed inter-
action effects of genotype and sex: although WT females
tended to have lower levels than WT males, this sex differ-
ence was reversed in Ghrl / animals. In addition, plasma
cholesterol levels were lower in females than in males (PS <
0.001). Liver glycerol content was higher in females (PS =
0.003), and liver glycogen content trended toward being
lower in Ghrl / mice (PGT = 0.060).
In contrast to the other diets, plasma metabolite concen-
trations were neither affected by genotype nor affected by
sex in the CW group. However, like in the BE group, liver
glycerol content was higher in females (PS < 0.001), and liver
glycogen content showed a trend toward being lower in
Ghrl / mice than in WT mice (PGT = 0.092) and higher in
females than in males (PS = 0.077).
Circulating and Fecal Hormones
Although plasma ghrelin and leptin levels were affected
by the dietary interventions (P < 0.001), for none of the diets
these levels were affected by genotype or sex (Table 2). In
contrast, fecal corticosterone levels were higher in females
than in males in the CO (PS = 0.002) and CW (PS = 0.027)

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 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE

were able to develop binge-like eating similar to WTs in lon-

ger-term daily access paradigms (33, 34). It was based on
A these previous findings that we chose an intermittent model
to assess the longer-term effects of bingeing in our mice. The
reason for the difference in response to daily as opposed to
intermittent access models may be related to entrainment
effects, although the mechanisms for this remain to be

Figure 4. Fat distribution. Fat depot weights as a percentage of body

weight for CO mice (A), BE mice (B), and CW mice (C). Experimental
groups: WT M, wild-type male; Ghrl / M, ghrelin-deficient male; WT F,
wild-type female; Ghrl / F, ghrelin-deficient female. Group size n = 6–8
mice. Data are presented as means ± SE and were analyzed by two-way
ANOVA. Main effects were considered significant if P < 0.05. gP < 0.05,
(g)
P < 0.10 for genotype differences; sP < 0.05, (s)P < 0.10 for sex differen-
ces, determined by post hoc analysis. BE, binge eating; CO, chow only;
CW, chow and WD; WD, Western-style diet.

groups. In the BE group, however, only a trend toward a ge-

notype-sex interaction was observed (PGT:S = 0.086): Ghrl /
males had lower fecal corticosterone levels than all other
groups, albeit not significantly.

DISCUSSION
The aim of this study was to characterize the effects of sex
and ghrelin and their interaction on the development of
BED and its metabolic consequences in a model of binge-like
eating in mice. Using an intermittent access to a palatable
diet model, we successfully induced binge-like eating behav-
ior in mice. Unexpectedly, Ghrl / mice of both sexes devel-
oped binge-like eating behavior to a similar extent as their
WT counterparts. Our observations are different from find-
ings in male Ghsr / mice, which were less prone to develop
binge-like eating in both short-term daily access (35) and
long-term intermittent access BED mouse models (34).
However, it is important to note that male Ghsr / mice
Figure 5. Glucose tolerance, as assessed by intraperitoneal glucose toler-
ance test. Blood glucose concentrations after glucose injection at 0 min
and area under the curve of blood glucose levels in CO mice (A, B), BE
mice (C, D), and CW mice (E, F). Group size n = 5–8 mice. Data are pre-
sented as means ± SE and were analyzed by two-way ANOVA. Main
effects were considered significant if P < 0.05. gP < 0.05 for genotype
differences; sP < 0.05, (s)P < 0.10 for sex differences, determined by post
hoc analysis. BE, binge eating; CO, chow only; CW, chow and WD; WD,
Western-style diet.

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 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE
A Ghsr / mice. In contrast, the Ghrl / mice of both sexes in
B other receptors, such as dopaminergic receptors (59, 60),
Figure 6. Gene expression profiles of inguinal WAT and liver. mRNA
expression in inguinal WAT (A) and liver (B), normalized to WT CO males.
Group size n = 6–8 mice. Data are presented as means ± SE and were an-
alyzed by two-way ANOVA. gP < 0.05, (g)P < 0.10 for genotype differen-
ces; sP < 0.05, (s)P < 0.10 for sex differences, determined by post hoc
analysis. WAT: white adipose tissue; WT CO, wild-type chow only.
elucidated. Another notable difference between Ghrl / and
Ghsr / mice occurred in the mice given access to both WD
and chow. When given free access to both rewarding/high-
energy food and standard chow, WT mice spontaneously
consumed most of their calories from the rewarding food.
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However, in the study by Egecioglu et al. (55), it was found
that this preference for rewarding food was lost in female
our study maintained their preference for the rewarding
WD, and even lowered their chow intake, in both the BE and
CW dietary groups. The latter finding is thus more in line
with the unexpected effects of acute AG infusions described
in a binge-like eating rat model: acute ghrelin administration
increased preference for less rewarding, but healthier, chow
(33). It could be speculated that this indicates that ghrelin, as
a hormone, enhances healthy eating, perhaps even inde-
pendent of GHSR.
The differential effects of GHSR deficiency in these
paradigms, as well as our finding that binge-like eating is
not impaired in Ghrl / mice, may point to alternative
functions of GHSR, that are independent of peripheral
ghrelin, and vice versa. Indeed, in vitro evidence has
shown that GHSR has constitutive activity, which may
have physiological relevance (56, 57), although this is pres-
ently poorly understood (58). It is present in brain struc-
tures that are involved in reward signaling and memory
formation. Here, it is thought to form heteromers with
modulating their downstream signaling pathways. It is
still debated, however, whether ghrelin can access and
bind GHSR in these brain regions (61). For example,
although peripheral ghrelin administration has been
shown to strongly affect neuronal activity and connectiv-
ity in areas such as the ventral tegmental area (62), it needs
an intact arcuate nucleus to do so (63). However, others
have suggested that ghrelin might be able to penetrate
deeper into the brain (64), for example, by crossing into
the cerebrospinal fluid (65). Considering the difference
between our results and those obtained in Ghsr / mice, it
is pivotal to gain more clarity on how accessible different
regions of the brain are to ghrelin. Finally, differential
actions between AG and UAG might also contribute to the
differences observed between Ghrl / and Ghsr / mice.
In previous work by our group, we demonstrated that UAG
infusion affects metabolism in mice (45). This form of
ghrelin does not bind GHSR, in contrast to AG. Effects that
are observed in our Ghrl / mice, that express neither AG
nor UAG, might thus be attributable to decreased UAG
action.
An interesting observation in this study was the timing of
periods of reduced chow intake in Ghrl / relative to WT
mice following the binge period. This developed very early
in the study, as it was already observed during the second
week in Ghrl / females (Supplemental Fig. S2). Although
Ghrl / males mainly reduced their chow intake during
the dark phase following WD access, Ghrl / females also
showed a lower chow intake during the following nonaccess
day dark phase. This decreased food intake at the start of the
dark phase in Ghrl / mice after a large meal has, to the best
of our knowledge, not been described before. Perhaps the
homeostatic drive to eat, particularly at the start of the
active—dark—phase, is diminished in Ghrl / mice: either
the balance between orexigenic and anorexigenic signaling
could be disturbed, or ghrelin deficiency could worsen the
disruption of the circadian rhythms of the mice. Ghrelin
counteracts the effects of other gut hormones that regulate
 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE

/
Table 1. WT and ghrelin-deficient (Ghrl ) male (M) and female (F) mice
CO
Metabolite Sig. WT M (n = 5–8) Ghrl /
M (n = 8) WT F (n = 6–8) Ghrl /
F (n = 8)
Plasma, all mmol/L
Glucose GT,S 6.94 ± 0.16 6.13 ± 0.22 6.16 ± 0.27 5.30 ± 0.33
NEFA GT 0.66 ± 0.04 0.74 ± 0.03 0.60 ± 0.04 0.77 ± 0.06(g)
Triglycerides 0.69 ± 0.18 1.34 ± 0.24 1.31 ± 0.28 1.14 ± 0.29
Free glycerol 0.49 ± 0.12 0.83 ± 0.15 0.82 ± 0.15 0.80 ± 0.10
Cholesterol GT,S 2.30 ± 0.11 2.63 ± 0.09 1.72 ± 0.11 2.15 ± 0.25
Liver
Glycogen, mmol glucose/g liver () 85.4 ± 21.1 119.1 ± 29.8 165.6 ± 25.8 99.2 ± 22.0
Glycerol, mmol/g liver 17.7 ± 2.8 14.0 ± 1.1 13.9 ± 2.2 16.9 ± 2.0
BE
Metabolite Sig. WT M (n = 7–8) Ghrl /
M (n = 6) WT F (n = 7–8) Ghrl /
F (n = 8)
Plasma, all mmol/L
Glucose 6.21 ± 0.29 6.00 ± 0.26 6.87 ± 0.58 6.10 ± 0.30
NEFA  0.69 ± 0.04 0.63 ± 0.02 0.56 ± 0.04 0.71 ± 0.07
Triglycerides  1.17 ± 0.18 0.71 ± 0.11 0.52 ± 0.07 0.98 ± 0.28(g)
Free glycerol  0.70 ± 0.08 0.63 ± 0.06 0.44 ± 0.08 0.90 ± 0.22(g)
Cholesterol (GT),S 2.80 ± 0.10 2.87 ± 0.15 1.91 ± 0.08 2.39 ± 0.17
Liver
Glycogen, mmol glucose/g liver (GT) 191.1 ± 28.4 164.5 ± 30.0 183.3 ± 34.9 96.4 ± 14.5
Glycerol, mmol/g liver S 18.3 ± 1.4 16.1 ± 1.2 32.2 ± 9.2 34.4 ± 5.1(s)
CW
Metabolite Sig. WT M (n = 4–8) Ghrl /
M (n = 7) WT F (n = 5–6) Ghrl /
F (n = 7)
Plasma, all mmol/L
Glucose 6.93 ± 0.38 6.48 ± 0.12 6.86 ± 0.57 6.49 ± 0.43
NEFA 0.85 ± 0.09 0.84 ± 0.09 0.88 ± 0.13 0.82 ± 0.05
Triglycerides 2.31 ± 0.72 2.24 ± 0.61 1.91 ± 0.63 1.82 ± 0.37
Free glycerol 1.32 ± 0.32 1.25 ± 0.32 1.31 ± 0.39 1.15 ± 0.18
Cholesterol 3.50 ± 0.42 4.14 ± 0.33 3.34 ± 0.15 3.62 ± 0.18
Liver
Glycogen, mmol glucose/g liver (GT),(S) 137.8 ± 29.5 92.2 ± 31.4 194.3 ± 32.0 140.3 ± 20.1
Glycerol, mmol/g liver S 49.4 ± 5.9 54.0 ± 5.4 81.4 ± 8.1s 82.8 ± 5.3s
Data were analyzed by two-way ANOVA with a Tukey HSD post hoc analysis, or Bonferroni analysis. Sig. lists significant main effects
(P < 0.05): GT, main effect for genotype; S, main effect for sex; , interaction effect. Parenthesized symbols indicate a trend toward sig-
nificance (P < 0.10). gP < 0.05, or (g)P < 0.10 vs. WT mice of the same sex; sP < 0.05, or (s)P < 0.10 vs. males of the same genotype. WT M,
wild-type male; Ghrl / M, ghrelin-deficient male; WT F, wild-type female; Ghrl / F, ghrelin-deficient female. BE, binge eating; CO,
chow only; CW, chow and WD; WD, Western-style diet.

satiety (66, 67). These satiety hormones are released not only
during meals, but also in the subsequent hours (68). Thus
ghrelin deficiency may have resulted in an inadequate orexi-
genic drive to counteract the actions of these hormones fol-
lowing the large meal during the light phase. In addition, the
disruption of ghrelin signaling affects circadian rhythmicity
in mice (69, 70), conceivably decreasing the drive to move
and eat in the early dark phase. However, we did not observe
reductions in locomotor activity at this time, which were
observed in Ghsr / mice (71). The mechanism of the effects
of ghrelin deficiency on postbinge food intake thus remains
to be elucidated.
Besides the differentially timed reduction in food intake,
we also observed a strong interaction effect of sex and geno-
type on locomotor activity in the binge paradigm. Namely,
on nonaccess days, Ghrl / females walked less than WT
females, whereas ghrelin deficiency did not affect locomotor
activity in males. Although in the other interventions, no
such genotype-sex interaction was observed, in CO mice,
females of both genotypes had increased locomotor activity
compared with males and mice of both sexes with ghrelin
deficiency had lower locomotor activity than wild types. In
CW mice, locomotor activity was only significantly increased
in females. The modulation of physical activity by ghrelin
signaling has been described by several authors (41, 72–74).
In male mice, acute AG administration was shown to
enhance locomotor activity (73). Consistently, Mifune et al.
(72) demonstrated that male Ghrl / mice have lower wheel-
running activity than WT mice under time-restricted feed-
ing. However, other authors reported little to no effects of
ghrelin or GHSR deficiency in nonrearing locomotor activity
in male or female mice (41, 74). Combined with these previ-
ous findings, our results suggest that the impact of ghrelin
signaling on physical activity is not only dependent on die-
tary intervention, but also on the sex of the mice.
This sex difference in locomotor activity has been well
described for C57BL/6J mice: especially young females have
increased locomotor activity compared with males of a similar
age (75). It is important to note, though, that locomotor activ-
ity can be affected by social isolation. Although some authors
report increased locomotor activity in socially isolated males
compared with group-housed males (76), others report sex
effects in locomotor activity upon isolation: females were
shown to have higher locomotor activity than males when
housed individually (77). Moreover, Yamada et al. (77)
reported sexually dimorphic alterations in ghrelin signaling

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/
Table 2. WT and ghrelin-deficient (Ghrl ) male (M) and female (F) mice
CO
Hormone Sig. WT M (n = 5–8) Ghrl /
M (n = 8) WT F (n = 6-8) Ghrl /
F (n = 8)
Plasma ghrelin
AG, pg/mL 134.4 ± 22.9 108.9 ± 0.142
UAG, pg/mL 863.0 ± 125.4 778.7 ± 103.5
AG:UAG 0.153 ± 0.032 0.142 ± 0.050
Plasma leptin, ng/mL 1.68 ± 0.29 1.16 ± 0.20 1.21 ± 0.29 1.29 ± 0.26
Plasma adiponectin, mg/mL (GT),S 14.5 ± 0.5 15.9 ± 0.3 20.9 ± 1.1s 23.5 ± 1.6s
Fecal corticosterone (nmol/day) S 0.060 ± 0.007 0.059 ± 0.010 0.098 ± 0.011 0.150 ± 0.045(s)
BE
Hormone Sig. WT M (n = 4–8) Ghrl /
M (n = 7) WT F (n = 5–6) Ghrl /
F (n = 7)
Plasma ghrelin
AG, pg/mL 63.4 ± 6.4 71.2 ± 8.7
UAG, pg/mL 513.2 ± 129.9 743.1 ± 81.5
AG:UAG 0.138 ± 0.033 0.098 ± 0.008
Plasma leptin, ng/mL 2.12 ± 0.51 1.28 ± 0.29 1.30 ± 0.30 0.77 ± 0.07
Plasma adiponectin, mg/mL S 12.3 ± 0.9 12.8 ± 0.7 18.9 ± 1.8s 22.3 ± 1.4s
Fecal corticosterone, nmol/day () 0.222 ± 0.022 0.134 ± 0.019 0.215 ± 0.025 0.235 ± 0.047
CW
Hormone Sig. WT M (n = 7–8) Ghrl /
M (n = 6) WT F (n = 7–8) Ghrl /
F (n = 8)
Plasma ghrelin
AG, pg/mL) 42.9 ± 9.4 49.9 ± 7.0
UAG, pg/mL 408.5 ± 55.1 410.2 ± 53.0
AG:UAG 0.123 ± 0.037 0.122 ± 0.008
Plasma leptin, ng/mL 4.64 ± 0.89 5.02 ± 0.77 4.38 ± 0.82 5.67 ± 0.87
Plasma adiponectin, mg/mL S 15.2 ± 0.9 17.7 ± 1.3 23.6 ± 1.4s 22.2 ± 2.2s
Fecal corticosterone (nmol/day) S 0.021 ± 0.003 0.035 ± 0.020 0.057 ± 0.010 0.045 ± 0.047
Ghrelin data were analyzed by one-way ANOVA, and corticosterone data by two-way ANOVA with a Tukey HSD post hoc analysis, or
Bonferroni analysis. Sig. lists significant main effects (P < 0.05): GT, main effect for genotype; S, main effect for sex; , interaction effect.
Parenthesized symbols indicate a trend toward significance (P < 0.10). sP < 0.05, or (s)P < 0.10 vs. males of the same genotype. WT M,
wild-type male; Ghrl / M, ghrelin-deficient male; WT F, wild-type female; Ghrl / F, ghrelin-deficient female. BE, binge eating; CO,
chow only; CW, chow and WD; WD, Western-style diet.

upon social isolation. Altogether, future research into the sex-
specific effects of ghrelin deficiency on the locomotor activity
should take the social environment of the mice into account.
Another striking sex-dependent effect of ghrelin defi-
ciency was observed in the CW group: Ghrl / females had a
higher energy expenditure than any of the other groups on
the same diet. Energy expenditure is affected by body size,
and especially fat-free mass, by food intake, and by physical
activity (78). Compared with CW males, food intake and
physical activity were higher, but the fat-free mass was lower
in CW females, regardless of genotype. These factors partly
explain the difference between male and female mice, but
not between Ghrl / and WT female mice. Another explana-
tion could lie in a difference in another component of basal
metabolic rate, such as thermogenesis. In Ghsr / mice,
thermogenesis has been shown to be enhanced in brown and
white adipose tissues upon aging (79). Indeed, microscopic
analysis of the inguinal WAT, a browning-prone depot,
revealed more brown-like areas in Ghrl / than in WT
females (data not shown). Consistently, we showed an up-
regulation of Ucp1 expression in this depot. This upregula-
tion did not reach significance in male Ghrl / mice,
suggesting that ghrelin deficiency not only sex-dependently
alters behaviors as discussed above, but also energy metabo-
lism through enhanced thermogenesis in mice.
Furthermore, in both the CO and CW groups, we demon-
strated that ghrelin deficiency improved glucose tolerance in
males, but it did not significantly affect glucose tolerance in
females. It is well known that female rodents are more glu-
cose tolerant than males (80). Moreover, the improvement in
glucose tolerance upon deletion of genes in the ghrelin sig-
naling pathway has been extensively reported in male mice
(81–85). However, Ghsr deficiency does not improve glucose
tolerance in female mice, which, generally, already have bet-
ter glucose tolerance than males (74). Consistently, we show
that the AUC of Ghrl / males in the IPGTT remains similar
to that of the female mice, regardless of diet. Our findings on
gene expression profiles in WAT, in particular, may provide
one of the mechanisms by which glucose homeostasis is
improved in Ghrl / mice. Cd68 and Cd11c, markers for mac-
rophage abundance and proinflammatory status, respec-
tively, were suppressed in Ghrl / mice. This effect occurred
particularly in the CW, and to a lesser extent in the BE group.
Adipose macrophages play a major role in modulating insu-
lin sensitivity and like our findings in Ghrl-deficient mice,
macrophage-specific deletion of Ghsr leads to an improved
adipose inflammatory profile with aging (86). In addition to
direct markers of macrophage infiltration and phenotype,
the gene expression of Fgf21 was also increased in the ingui-
nal WAT of the Ghrl / mice relative to WTs. FGF21 has been
shown to promote anti-inflammatory M2 macrophage polar-
ization, as well as the insulin sensitivity of subcutaneous fat
(87). Further investigation of the possible modulation of adi-
pose tissue macrophage recruitment and phenotype by ghre-
lin deficiency under conditions of high fat-induced obesity
is thus warranted.

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 SEX-DEPENDENT EFFECTS OF GHRELIN DEFICIENCY IN MICE
As mentioned earlier, a possible drawback of the current
experiment was the prolonged individual housing of the
mice. Individual housing is known to affect several meta-
bolic parameters, such as food intake and visceral fat mass,
when comparing socially housed mice to individually
housed mice (88). The sex of the mice did not appear to mod-
ulate these effects (88). In our study, all groups were exposed
to the same social isolation conditions. Fecal corticosterone
levels, as an assessment of stress, in these mice were lower
than or comparable to levels observed in group-housed ani-
mals from our previous study (46). Importantly, corticoster-
one levels were comparable between WT and Ghrl /
females on the same diet, whereas most of the described
effects of ghrelin deficiency were observed in female mice.
Based on these findings, we conclude that social isolation
stress did not have a major impact on the observed effects.
In conclusion, we found that binge-like eating can be
induced successfully in both male and female ghrelin-defi-
cient mice, as measured by WD intake. We showed that par-
ticularly females were affected by ghrelin deficiency, both in
the BE and CW groups. Overall, this study provides extensive
evidence that both diet and sex should be taken into account
when researching ghrelin signaling.
SUPPLEMENTAL DATA
Supplemental Tables S1–S3: https://doi.org/10.6084/m9.figshare.
17197274 and Figures S1 and S2: https://doi.org/10.6084/m9.
figshare.19384001.
ACKNOWLEDGMENTS
We gratefully acknowledge Prof. Paul Pfluger for the gift of the
Ghrl / mice.
GRANTS
This work was supported by departmental laboratory funds (to
K. Prins, M. Huisman, A. McLuskey, R. Mies, B. Karels, P. J. D.
Delhanty, and J. A. Visser).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by
the authors.
AUTHOR CONTRIBUTIONS
K.P., P.J.D.D., and J.A.V. conceived and designed research;
K.P., M.H., A.M., R.M., B.K., and P.J.D.D. performed experiments;
K.P. and M.H. analyzed data; K.P., P.J.D.D., and J.A.V. interpreted
results of experiments; K.P. prepared figures; K.P., P.J.D.D., and
J.A.V. drafted manuscript; K.P., P.J.D.D., and J.A.V. edited and re-
vised manuscript; K.P., M.H., A.M., R.M., B.K., P.J.D.D., and J.A.V.
approved final version of manuscript.
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