Mice with hyperghrelinemia are hyperphagic and glucose intolerant and have reduced leptin sensitivity.
ABSTRACT Ghrelin is the only known peripheral hormone to increase ingestive behavior. However, its role in the physiological regulation of energy homeostasis is unclear because deletion of ghrelin or its receptor does not alter food intake or body weight in mice fed a normal chow diet. We hypothesized that overexpression of ghrelin in its physiological tissues would increase food intake and body weight.
We used bacterial artificial chromosome transgenesis to generate a mouse model with increased ghrelin expression and production in the stomach and brain. We investigated the effect of ghrelin overexpression on food intake and body weight. We also measured energy expenditure and determined glucose tolerance, glucose stimulated insulin release, and peripheral insulin sensitivity.
Ghrelin transgenic (Tg) mice exhibited increased circulating bioactive ghrelin, which was associated with hyperphagia, increased energy expenditure, glucose intolerance, decreased glucose stimulated insulin secretion, and reduced leptin sensitivity.
This is the first report of a Tg approach suggesting that ghrelin regulates appetite under normal feeding conditions and provides evidence that ghrelin plays a fundamental role in regulating beta-cell function.
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ABSTRACT: Small synthetic molecules called growth-hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through GHS-R, a G-protein-coupled receptor for which the ligand is unknown. Recent cloning of GHS-R strongly suggests that an endogenous ligand for the receptor does exist and that there is a mechanism for regulating GH release that is distinct from its regulation by hypothalamic growth-hormone-releasing hormone (GHRH). We now report the purification and identification in rat stomach of an endogenous ligand specific for GHS-R. The purified ligand is a peptide of 28 amino acids, in which the serine 3 residue is n-octanoylated. The acylated peptide specifically releases GH both in vivo and in vitro, and O-n-octanoylation at serine 3 is essential for the activity. We designate the GH-releasing peptide 'ghrelin' (ghre is the Proto-Indo-European root of the word 'grow'). Human ghrelin is homologous to rat ghrelin apart from two amino acids. The occurrence of ghrelin in both rat and human indicates that GH release from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelin.Nature 01/2000; 402(6762):656-60. · 38.60 Impact Factor
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ABSTRACT: The discovery of the peptide hormone ghrelin, an endogenous ligand for the growth hormone secretagogue (GHS) receptor, yielded the surprising result that the principal site of ghrelin synthesis is the stomach and not the hypothalamus. Although ghrelin is likely to regulate pituitary growth hormone (GH) secretion along with GH-releasing hormone and somatostatin, GHS receptors have also been identified on hypothalamic neurons and in the brainstem. Apart from potential paracrine effects, ghrelin may thus offer an endocrine link between stomach, hypothalamus and pituitary, suggesting an involvement in regulation of energy balance. Here we show that peripheral daily administration of ghrelin caused weight gain by reducing fat utilization in mice and rats. Intracerebroventricular administration of ghrelin generated a dose-dependent increase in food intake and body weight. Rat serum ghrelin concentrations were increased by fasting and were reduced by re-feeding or oral glucose administration, but not by water ingestion. We propose that ghrelin, in addition to its role in regulating GH secretion, signals the hypothalamus when an increase in metabolic efficiency is necessary.Nature 11/2000; 407(6806):908-13. · 38.60 Impact Factor
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ABSTRACT: : Ghrelin, an endogenous ligand for growth hormone secretagogue receptor, was recently identified in the rat stomach. We examined the effects of the gastric peptide ghrelin on energy balance in association with leptin and vagal nerve activity. : Food intake, oxygen consumption, gastric emptying, and hypothalamic neuropeptide Y (NPY) messenger RNA expression were measured after intra-third cerebroventricular or intraperitoneal injections of ghrelin in mice. The gastric vagal nerve activity was recorded after intravenous administration in rats. Gastric ghrelin gene expression was assessed by Northern blot analysis. Repeated coadministration of ghrelin and interleukin (IL)-1 beta was continued for 5 days. : Ghrelin exhibited gastroprokinetic activity with structural resemblance to motilin and potent orexigenic activity through action on the hypothalamic neuropeptide Y (NPY) and Y(1) receptor, which was lost after vagotomy. Ghrelin decreased gastric vagal afferent discharge in contrast to other anorexigenic peptides that increased the activity. Ghrelin gene expression in the stomach was increased by fasting and in ob/ob mice, and was decreased by administration of leptin and IL-1 beta. Peripherally administered ghrelin blocked IL-1 beta-induced anorexia and produced positive energy balance by promoting food intake and decreasing energy expenditure. : Ghrelin, which is negatively regulated by leptin and IL-1 beta, is secreted by the stomach and increases arcuate NPY expression, which in turn acts through Y(1) receptors to increase food intake and decrease energy expenditure. Gastric peptide ghrelin may thus function as part of the orexigenic pathway downstream from leptin and is a potential therapeutic target not only for obesity but also for anorexia and cachexia.Gastroenterology 02/2001; 120(2):337-45. · 12.82 Impact Factor
Mice With Hyperghrelinemia Are Hyperphagic and
Glucose Intolerant and Have Reduced Leptin Sensitivity
Gavin A. Bewick, Aysha Kent, Daniel Campbell, Michael Patterson, Mohammed A. Ghatei,
Stephen R. Bloom, and James V. Gardiner
OBJECTIVE—Ghrelin is the only known peripheral hormone to
increase ingestive behavior. However, its role in the physiologi-
cal regulation of energy homeostasis is unclear because deletion
of ghrelin or its receptor does not alter food intake or body
weight in mice fed a normal chow diet. We hypothesized that
overexpression of ghrelin in its physiological tissues would
increase food intake and body weight.
RESEARCH DESIGN AND METHODS—We used bacterial
artificial chromosome transgenesis to generate a mouse model
with increased ghrelin expression and production in the stomach
and brain. We investigated the effect of ghrelin overexpression
on food intake and body weight. We also measured energy
expenditure and determined glucose tolerance, glucose stimu-
lated insulin release, and peripheral insulin sensitivity.
RESULTS—Ghrelin transgenic (Tg) mice exhibited increased
circulating bioactive ghrelin, which was associated with hyperpha-
gia, increased energy expenditure, glucose intolerance, decreased
glucose stimulated insulin secretion, and reduced leptin sensitivity.
CONCLUSIONS—This is the first report of a Tg approach
suggesting that ghrelin regulates appetite under normal feeding
conditions and provides evidence that ghrelin plays a fundamen-
tal role in regulating ?-cell function. Diabetes 58:840–846,
tion from the pituitary (1). Ghrelin also increases food
intake and adiposity, suggesting a role in the control of
energy homeostasis (2). Consistent with this, plasma gh-
relin levels have been shown to increase before a meal and
during fasting (3). Ghrelin circulates in two forms: the
biologically active octanoylated form and the des-octanoyl
form, which is thought to be biologically inactive (4).
Recent data show that ghrelin a-acyltransferase (GOAT), a
membrane-bound enzyme, is responsible for octanoylation
of the serine 3 residue of ghrelin and confers biological
Despite unequivocal pharmacological data, the evidence
for a physiological role for ghrelin in the control of
hrelin is a 28-aa peptide that is expressed at
high levels in the stomach. It is the endogenous
ligand for the growth hormone secretagogue
receptor and increases growth hormone secre-
appetite is much less clear. Mice with targeted deletion of
either ghrelin or the growth hormone secretagogue recep-
tor exhibit an essentially normal metabolic phenotype
when fed a regular chow diet, suggesting that ghrelin may
have a redundant role in the regulation of food intake
(7,8). When fed a high-fat diet, these mice are resistant to
diet-induced obesity, exhibiting reduced adiposity and
increased energy expenditure (9,10). More recent data
suggest that these knockout models are not resistant to
diet-induced obesity when backcrossed to a pure C57BLK6
genetic background. Despite this, calorie restriction in the
pure-bred mice resulted in lower blood glucose in both
knockout models (11). The conflicting food intake and
body weight data from Tg models has made defining a
key role for endogenous ghrelin in the control of
appetite difficult. However, the data do consistently
suggest that ghrelin may be important in the control of
Ghrelin gain-of-function models have not produced the
expected hyperphagic and obese phenotype (12–14).
These models did not, however, exhibit increases in
plasma bioactive ghrelin. Reed et al. developed a model in
which ghrelin was overexpressed in the brain but not the
stomach (15). In one Tg line, circulating bioactive ghrelin
was found to be increased, but this was not associated
with hyperphagia. The lack of an obese phenotype in these
mice was attributed to developmental compensation, al-
terations in peripheral versus central nervous system
ghrelin concentrations, and/or alterations in diurnal pat-
terns of ghrelin release.
The production of bioactive ghrelin critically depends
on its octanoylation by GOAT. To physiologically overex-
press bioactive ghrelin, the ghrelin transgene must be
expressed in tissues that also produce GOAT: the stomach
and small intestines. We used the ghrelin promoter to
drive ghrelin overexpression and generated mice with
increased circulating levels of bioactive ghrelin. We then
investigated the phenotype of these mice.
RESEARCH DESIGN AND METHODS
Generation of ghrelin Tg mice. We identified a bacterial artificial chromo-
some (BAC) containing the ghrelin gene RP23-441K11 (Invitrogen, Huntsville,
AL). Tg mice were created using standard pronuclear injection techniques. F0
mice were mated with CBA/C57Bl6 mice, and Tg lines were maintained
separately. Mice were maintained in cages under controlled temperature
(21–23°C) and light (11 h light/13 h dark) with ad libitum access to food (RM1
diet; SDS UK Ltd) and water. Animal procedures performed were approved
under the British Home Office Animals Scientific Procedures Act 1986.
Body weight, food intake, indirect calorimetry, and body composition.
Mice were singly housed from weaning, and food intake and body weight were
measured. Body composition of 16-week-old mice was calculated using the
method of Salmon and Flatt (16). Metabolic parameters were obtained using
the open-circuit Oxymax comprehensive lab animal monitoring system (Co-
lumbus Instruments, Columbus, OH) as previously described (17).
Glucose tolerance test and insulin tolerance test. An intraperitoneal
glucose tolerance test (IP-GTT) was performed in conscious 16-week-old
From the Department of Investigative Medicine, Hammersmith Campus,
Imperial College London, London, U.K.
Corresponding author: Stephen R. Bloom, email@example.com.
Received 16 October 2008 and accepted 8 January 2009.
Published ahead of print at http://diabetes.diabetesjournals.org on 16 January
2009. DOI: 10.2337/db08-1428.
© 2009 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
840DIABETES, VOL. 58, APRIL 2009
mice. After an 18-h fast, D-glucose (2 g/kg) was administered intraperitoneally
and blood glucose was measured by tail bleeds at 0, 15, 30, 60, 120, and 150
min postglucose administration. For glucose-stimulated insulin release, blood
glucose and insulin levels were measured at 0, 15, 30 and 60 min postglucose.
The insulin tolerance test was performed similarly except that mice were
fasted for 4 h before intraperitoneal administration of Humulin (1.5 units/kg)
(Eli Lillly, Basingstoke, U.K.). Plasma glucose was measured using the Acensia
Contour blood glucose monitoring system (Bayer HealthCare, Newbury, U.K.).
Measurement of circulating hormones. Whole blood was collected by
cardiac puncture from fasted 16-week-old mice. Mouse plasma insulin and
leptin concentrations were determined using reagents and methods from
Crystal Chem (Downers Grove, IL). Plasma corticosterone was measured
using a radioimmunoassay kit from MP Biomedicals (Orangeburg, NY).
Plasma IGF-I levels were measured using reagents and methods from Immu-
nodiagnostic Systems Ltd (Bolden, UK). Octanoylated ghrelin concentrations
were analyzed using an ELISA kit from LINCO Research (St. Charles, MO).
Northern blot analysis and quantitative PCR. Tissues from 16-week-old
mice were snap-frozen and RNA was extracted using TRI reagent. Northern
blot analysis was used to determine uncoupling protein (UCP)-1 mRNA
expression in brown adipose tissue and ghrelin mRNA expression in stomach.
Real-time quantitative PCR analysis was performed using TaqMan Gene
Expression Assays and TaqMan Universal PCR Master Mix (Applied Biosys-
tems, Foster City, CA) using the ABI Prism 7900 Sequence Detection System
according to the protocols provided by the manufacturer (Applied Biosys-
tems, Melbourne, Australia). The relative mRNA transcript levels were calcu-
lated according to the 2–?CTmethod, with ?CT being the difference in cycle
threshold values between the target mRNA and the 18S internal control.
Peripheral administration of ghrelin and PYY 3–36. At 16 weeks of age, ad
libitum–fed mice were injected intraperitoneally with either saline or ghrelin
(0.3 nmol/g) in a randomized blinded crossover design. A recovery period of
2 days was allowed between each study day. Mice were injected intraperito-
neally in the early light phase at 9:00 A.M. Food intake was measured 1 h
postinjection. At 16 weeks of age, in a randomized blinded crossover design,
mice were injected intraperitoneally with either saline or leptin (3 ?g/g).
Before each study day, mice were fasted for 24 h and leptin was administered
at 9:00 A.M.; food intake was measured at 1 and 4 h postinjection.
Values are means ? SEM unless otherwise stated. Differences in cumula-
tive food intake through time were compared across experimental groups
using generalized estimating equation curve analysis (Stata 9.1; Statacorp,
College Station, TX). For analysis of the effect of peripheral administration of
ghrelin and leptin, a paired Student’s t test with a Bonferroni correction was
used. All other comparisons were made using an unpaired Student’s t test. P
values ?0.05 were considered significant.
Circulating bioactive ghrelin levels are increased in
Tg mice. We identified a BAC, which contained the ghrelin
gene and its promoter. Using this BAC, we generated Tg
mice by standard pronuclear injection. Mice heterozygous
for the ghrelin transgene had significantly increased ghre-
lin expression. Line L91 was chosen for further investiga-
tion. Ghrelin mRNA levels in the stomach were increased
by ?150% in Tg mice compared with wild-type (Wt)
littermates (Wt 2.24 ? 0.23 AU versus Tg 3.37 ? 0.19 AU,
n ? 6, P ? 0.005; Fig. 1A). A similar increase was observed
in stomach ghrelin content for both total and bioactive
octanoylated ghrelin (total ghrelin: Wt 33.1 ? 01.9 ?mol/g
versus Tg 41.2 ? 3.0 ?mol/g, n ? 12, P ? 0.05; octanoy-
lated ghrelin: Wt 4.0 ? 0.4 ?mol/g versus Tg 5.4 ? 0.3
?mol/g, n ? 5, P ? 0.05; Fig. 1B and C). This suggested
that the transgene was increasing ghrelin expression and
that transgene-derived ghrelin was subsequently octanoy-
lated. The increased levels of stomach octanoylated ghre-
lin were reflected by similar increases in fasting plasma
total ghrelin (Wt 907 ? 46 pmol/l versus Tg 1,130 ? 40
pmol/l, n ? 6, P ? 0.01; Fig. 1D) and octanoylated ghrelin
in both males (Wt 25.39 ? 2.38 pmol/l versus Tg 40.07 ?
5.70 pmol/l, n ? 12, P ? 0.05; Fig. 1E) and females (Wt
20.38 ? 2.21 pmol/l versus Tg 33.38 ? 5.20 pmol/l, n ? 12,
P ? 0.05, Fig. 1F). This suggests that the increased
FIG. 1. Total and bioactive ghrelin levels are increased in Tg mice.
Ghrelin stomach mRNA expression was significantly increased in Tg
mice compared with Wt littermates (A). In addition, both total ghrelin
(B) and octanoylated ghrelin (C) were significantly increased in
stomach extracts. Plasma total ghrelin concentrations were increased
in male mice (D). Fasting plasma octanoylated ghrelin concentrations
were increased in both female (E) and male (F) mice. Ghrelin expres-
sion was found exclusively in the hypothalamus and stomach of Tg and
Wt mice (G). The results are means ? SEM; n ? 6. *P < 0.05, **P < 0.01
Tg and WT controls at 16 weeks of age. int, intestine; RQ, relative
quantification compared with control stomach.
G.A. BEWICK AND ASSOCIATES
DIABETES, VOL. 58, APRIL 2009 841
bioactive ghrelin in the stomach was released into the
circulation. To determine if the transgene expression
matched the endogenous expression pattern for ghrelin,
we measured ghrelin mRNA levels in various tissues.
Ghrelin mRNA was found exclusively in the hypothalamus
and stomach of both Wt and Tg mice (Fig. 1G). The
increased stomach ghrelin expression was associated with
a nonsignificant increase in stomach GOAT expression
(Wt 1.16 ? 0.26 versus Tg 1.42 ? 0.7, n ? 6). Two
additional independent lines were produced that had
smaller increases in circulating ghrelin levels and exhib-
ited a phenotype similar to that of L91 (data not shown).
Phenotypic characterization of ghrelin overexpress-
ing mice. Overexpression of ghrelin does not affect the
growth hormone axis but inhibits the hypothalmic-adrenal-
pituitary (HPA) axis. Administration of a single dose of
ghrelin is known to stimulate growth hormone release
(18). To study the possible effects of ghrelin overexpres-
sion on the growth hormone axis, we measured IGF-1 as a
surrogate marker for growth hormone and longitudinal
growth. At 16 weeks of age, there were no differences in
circulating IGF-1 levels between Tg and Wt littermates (Wt
774 ? 49 ng/ml versus Tg 799 ? 80 ng/ml). There was also
no difference in nose-to-anus length between genotypes
(Wt 7.52 ? 0.09 cm versus Tg 7.57 ? 0.10 cm, n ? 10).
These data suggest that overexpression of bioactive ghre-
lin did not affect the growth hormone axis. Ghrelin is
known to acutely affect the HPA axis; central administra-
tion of ghrelin increases corticosterone release, whereas
intravenous administration inhibits corticosterone release
in rats with high basal corticosterone levels (19,20). We
observed a significant reduction in corticosterone levels in
Tg mice (Wt 77.06 ? 8.05 ng/ml versus Tg 47.3 ? 6.34
ng/ml, n ? 12–15, P ? 0.05).
Bioactive ghrelin overexpression increases both food
intake and energy expenditure. Chronic pharmacolog-
ical administration of ghrelin significantly increases food
intake and body weight. However, previous studies of
genetic overexpression of ghrelin have not produced the
expected hyperphagic and obese phenotype. To determine
the effects of overexpression of bioactive ghrelin on
energy homeostasis, we monitored food intake and body
weight of mice fed regular chow. Tg mice exhibited
hyperphagia compared with Wt controls. Daily food intake
was increased in both male and female mice from 6 weeks
of age (males: Wt 98.8 ? 2.2 kj/day versus Tg 107.0 ? 3.3
kj/day, n ? 6, P ? 0.05; females: Wt 51.25 ? 1.25 kj versus
Tg 56.13 ? 1.9 kj, n ? 6, P ? 0.05) until the end of the study
at 16 weeks of age (males: Wt 99.5 ? 1.2 kj/day versus Tg
114.3 ? 5.0 kj/day, n ? 6, P ? 0.05; females: 54.25 ? 1.75
kj/day versus 61.63 ? 2.13 kj/day, n ? 6, P ? 0.05; Fig. 2A).
In accordance with increased daily food intake, cumula-
tive food intake was also increased by 13% between 5 and
16 weeks of age in Tg mice (males: Wt 7,440 ? 68 kj versus
Tg 8,480 ? 379 kj; females: Wt 4,087 ? 115 kj versus Tg
4,544 ? 148 kj, n ? 6, P ? 0.05; Fig. 2C). This increase in
food intake would be expected to significantly increase
both weight gain and adiposity. However, the increased
energy intake did not result in increased weight gain in
either male or female Tg mice (Fig. 2B and D). There was
also no change in adiposity or lean mass between the two
groups (Fig. 2E). Consistent with this finding, circulating
leptin levels were unaltered between Tg and Wt mice (Wt
2.08 ? 0.68 ng/ml versus Tg 2.66 ? 0.63 ng/ml; Fig. 2F). The
hyperphagia without increased body weight was indicative
of increased energy expenditure. To determine the mech-
anism by which Tg mice remained lean in the face of
increased food intake, we measured UCP-1 expression
levels in brown adipose tissue as a surrogate marker of
energy expenditure. UCP-1 expression levels were found
to be significantly increased in Tg mice compared with Wt
controls (Wt 4.42 ? 0.45 AU versus Tg 5.62 ? 0.5 AU; Fig.
3A), suggesting that energy expenditure was increased in
Tg mice. This conclusion was supported by indirect calo-
rimetry, which indicated that oxygen consumption was
increased during both the light and dark cycles (Fig. 3B).
Energy expenditure was found to be increased by 15% in
Tg mice compared with Wt controls (hourly average VO2
wt 5,458 ? 114 ml/h/kg versus Tg 6,207 ? 367 ml/h/kg, n ?
6–8, females, P ? 0.01; Fig. 3C). In addition, we measured
locomotor activity and found no differences in either dark
or light cycle locomotor activity between genotypes (Fig.
3D). This suggested that the normal body weight observed
in the Tg mice was a consequence of increased basal
metabolic rate rather than an increase in locomotor activ-
ity. Exogenous ghrelin administration has been shown to
decrease fat use, whereas mice with targeted deletion of
ghrelin have increased fat use when fed a high-fat diet. Our
ghrelin overexpressing mice did not show an altered
respiratory exchange ratio suggesting chronic exposure to
ghrelin does not alter nutrient partitioning in our model
Bioactive ghrelin overexpression decreases glucose-
stimulated insulin release. Pharmacological administra-
tion of ghrelin inhibits glucose stimulated insulin release,
whereas both knockout and overexpression models of the
ghrelin system exhibit changes in glucose homeostasis
(15,21,22). Our Tg mice had similar fasting plasma glucose
concentrations as Wt littermates (Wt 5.1 ? 0.4 mmol/l
versus Tg 5.5 ? 0.4 mmol/l, mean ? SEM, n ? 6). Despite
similar plasma glucose levels, Tg mice had elevated fasting
plasma insulin levels, although this was not statistically
significant (Wt 0.37 ? 0.1 ng/ml, n ? 9; Tg 0.61 ? 0.17
ng/ml, n ? 5; P ? 0.2). We carried out IP-GTT to further
explore the effects of ghrelin on glucose homeostasis. Tg
mice were glucose intolerant with significantly increased
plasma glucose concentrations at 30 and 60 min (Fig. 4A)
after glucose (2 g/kg) injection compared with Wt controls
(area under the curve [AUC]: Wt 1,253 ? 58 versus 1,769 ?
158 Tg, n ? 6, P ? 0.01; Fig. 4B). This was indicative of
either increased insulin resistance or impaired glucose-
stimulated insulin release. To determine the mechanism,
we measured glucose-stimulated insulin release and per-
formed insulin tolerance tests. After insulin administra-
tion, there were no differences in plasma glucose
concentrations between genotypes (AUC: Wt 5,301 ? 388
versus Tg 5,740 ? 438, n ? 6–10; Fig. 4C and D).
Glucose-stimulated insulin release was significantly inhib-
ited in Tg mice during an IP-GTT (glucose and insulin
percent baseline, Fig. 4E and F; insulin AUC: Wt 25116 ?
6,182 versus Tg 11202 ? 2,136; Fig. 4G). These results
suggest that ghrelin inhibits glucose-stimulated insulin
release but has no effect on insulin sensitivity.
Bioactive ghrelin overexpressing mice are equally
sensitive to exogenous ghrelin but have reduced
leptin sensitivity. To determine if overexpression of
ghrelin attenuated the response to exogenously adminis-
tered ghrelin, we measured 1-h food intake after intraperi-
toneal injection of ghrelin (0.3 nmol/g). Ghrelin was
equally potent at increasing 1-h food intake in both geno-
types (Wt 0.02 ? 0.01 g versus 0.11 ? 0.03 g saline versus
ghrelin, mean ? SEM, n ? 6, P ? 0.01; Tg 0.03 ? 0.01 g
GHRELIN TRANSGENIC MICE ARE GLUCOSE INTOLERANT
842DIABETES, VOL. 58, APRIL 2009
versus 0.12 ? 0.03 g saline versus ghrelin, mean ? SEM,
n ? 6, P ? 0.01; Fig. 5A).
Within hypothalamic feeding centers, ghrelin and leptin
have been shown to be functional antagonists (23). To
determine if ghrelin overexpression altered leptin sensitiv-
ity, we measured the effect of peripherally administered
leptin (3 ?g/g) on food intake. Tg mice were less sensitive
to the anorexigenic effect of leptin than Wt controls.
Leptin significantly reduced food intake 0 to 1 h postad-
ministration in Wt but not in Tg mice compared with saline
(saline versus leptin: Wt 0.54 ? 0.12 g versus 0.22 ? 0.05 g,
P ? 0.05; Tg 0.69 ? 0.15 g versus 0.62 ? 0.12 g; P ?
nonsignificant, mean ? SEM, n ? 6; Fig. 5B). Four hours
postadministration, leptin significantly reduced food in-
take in both genotypes (saline versus leptin: Wt 1.08 ?
0.15 g versus 0.57 ? 0.12 g, Tg 1.33 ? 0.17 g versus 0.99 ?
0.12 g, mean ? SEM, n ? 6, P ? 0.05; Fig. 5C). The
magnitude of the reduction in food intake, however, was
less in Tg than Wt mice. Leptin reduced 4-h food intake by
approximately half in Wt animals, but only by one-fourth
in Tg animals. These results suggest that Tg mice are less
sensitive to the effects of leptin but equally sensitive to
Bioactivity of ghrelin is conferred by octanoylation of its
serine 3 residue. This reaction is catalyzed by the enzyme
GOAT. Bioactive ghrelin is only produced in GOAT-
expressing tissues. In the mouse, GOAT is expressed
exclusively in the gastrointestinal tract (5,6). To increase
bioactive ghrelin concentrations, the ghrelin transgene
should ideally be expressed in tissues that produce endog-
FIG. 2. Overexpression of bioactive ghrelin increases food intake. Cumulative food intake was measured from 5 to 16 weeks of age in both male
(A) and female (C) mice fed on regular chow. Body growth curves from 5 to 16 weeks of age for male (B) and female (D) mice. Body composition
(E; ?, lean; o, protein; f, fat) and plasma leptin concentrations (F) were measured in 16-week-old male mice. F (solid lines), Wt; ? (broken
lines), Tg. The results are means ? SEM; n ? 6–8. *P < 0.05, **P < 0.01 Tg and Wt controls.
G.A. BEWICK AND ASSOCIATES
DIABETES, VOL. 58, APRIL 2009 843
enous ghrelin and GOAT. To do this, we chose to drive
ghrelin transgene expression using its own promoter. The
approach successfully increased both stomach and plasma
concentrations of bioactive ghrelin. Our data constitute
the first report of the targeted overexpression of bioactive
ghrelin in its physiological sites of production: the stom-
ach and hypothalamus.
Exogenously administered ghrelin has been shown to
have powerful effects on food intake (2,24). Despite the
overwhelming pharmacological evidence, data from Tg
models have not supported the expected role for ghrelin in
the control of appetite, leading to the suggestion that
ghrelin is not a critical regulator of appetite. On the
contrary, our data suggest it is an important regulator of
food intake. Overexpression of bioactive ghrelin in our
model causes hyperphagia, increased energy expenditure,
and glucose intolerance. The increased food intake sug-
gests ghrelin may physiologically regulate appetite under
normal feeding conditions.
The hyperphagia observed in our Tg mice was in con-
trast to the phenotype described for mice with nonspecific
neuronal overexpression of ghrelin (15). These mice are
reported to exhibit an increase in plasma octanoylated
ghrelin without an increase in feeding behavior. A likely
explanation for this difference is the nonspecific nature of
the central nervous system overexpression. Results from
this model are likely to be pharmacological because
physiologically only a few neurones in the hypothalamus
produce ghrelin. The hyperphagic phenotype is also in
contrast to mice with targeted deletion of the ghrelin gene
(ghr?/?) or its receptor. These mice have normal growth
rates and appetites when fed on regular chow (7). A
possible explanation for this is that knockout mice are
susceptible to developmental compensation, which can
mask the true role of the gene in question.
To fully interpret Tg studies, it is important to consider
the genetic background of the model. This is particularly
true for mice with targeted deletion of genes in which it
has been suggested that c57blk/6j genetic traits cosegre-
gate in the null mice, whereas 129Sv traits are more
influential in the Wt controls (7). This can lead to misin-
terpretation of results because c57bl/6j mice are more
obesity prone than 129Sv mice. In classical Tg mice,
however, there is no evidence that the transgene cosegre-
gates with a particular background and, therefore, the Tg
and Wt littermates are likely to be of similar genetic
background. Backcrossing our model to a pure C57bl/6j
background may indeed increase the magnitude of the
Our Tg mice were hyperphagic but did not have an
increase in body weight, which suggested they had in-
creased energy expenditure. Tg mice were found to have
increased oxygen consumption and brown adipose tissue
UCP-1 expression, both of which are surrogate markers of
energy expenditure. This is in contrast to the pharmaco-
logical administration of ghrelin, which decreases energy
expenditure (25). It is possible that the increased meta-
bolic rate in Tg mice is the result of an indirect effect of
ghrelin, which manifests itself after chronic exposure.
Circulating corticosterone levels, for example, were sup-
pressed in our Tg mice. Because corticosterone is known
to suppress energy expenditure, the increased metabolic
rate observed in our Tg mice may be attributed to an
indirect effect of their attenuated corticosterone levels
It is generally accepted that ghrelin activates the HPA
axis, and this is thought to occur at the level of the
hypothalamus (19). In contrast, corticosterone levels were
reduced in our Tg mice. When given intravenously, how-
ever, growth hormone secretagogues (GHSs) reduce cor-
ticosterone in rats with high basal corticosterone levels
(20). It has therefore been suggested that high circulating
levels of glucocorticoids feed back to reduce the ACTH
response to GHS. A similar mechanism could account for
FIG. 3. Overexpression of bioactive ghrelin
increases energy expenditure. Brown adi-
pose tissue UCP-1 mRNA expression in 16-
week-old mice (A) and average oxygen
consumption measured during one 24-h pe-
riod (B) and average hourly oxygen con-
sumption (C) using the comprehensive lab
animal monitoring system in 16-week-old
mice. Average ambulatory activity estimated
as X beam breaks during either the light or
dark period (D). Respiratory exchange ratio
was calculated as VCO2/ VO2(E). The results
are means ? SEM; n ? 6–8. *P < 0.05, **P <
0.01, Tg and Wt controls, respectively.
GHRELIN TRANSGENIC MICE ARE GLUCOSE INTOLERANT
844DIABETES, VOL. 58, APRIL 2009