In Vivo Evidence for Inverse Agonism of Agouti-Related
Peptide in the Central Nervous System of
Virginie Tolle1,2and Malcolm J. Low1,2,3
OBJECTIVE—Melanocyte-stimulating hormone (MSH) pep-
tides processed from proopiomelanocortin (POMC) regulate
energy homeostasis by activating neuronal melanocortin recep-
tor (MC-R) signaling. Agouti-related peptide (AgRP) is a naturally
occurring MC-R antagonist but also displays inverse agonism at
constitutively active melanocortin-4 receptor (MC4-R) expressed
on transfected cells. We investigated whether AgRP functions
similarly in vivo using mouse models that lack all neuronal MSH,
thereby precluding competitive antagonism of MC-R by AgRP.
RESEARCH DESIGN AND METHODS—Feeding and meta-
bolic effects of the MC-R agonist melanotan II (MTII), AgRP, and
ghrelin were investigated after intracerebroventricular injection
in neural-specific POMC-deficient (Pomc?/?Tg/?) and global
POMC-deficient (Pomc?/?) mice. Gene expression was quanti-
fied by RT-PCR.
RESULTS—Hyperphagic POMC-deficient mice were more sen-
sitive than wild-type mice to the anorectic effects of MTII.
Hypothalamic melanocortin-3 (MC3)/4-R mRNAs in POMC-defi-
cient mice were unchanged, suggesting increased receptor sen-
sitivity as a possible mechanism for the heightened anorexia.
AgRP reversed MTII-induced anorexia in both mutant strains,
demonstrating its ability to antagonize MSH agonists at central
MC3/4-R, but did not produce an acute orexigenic response by
itself. The action of ghrelin was attenuated in Pomc?/?Tg/? mice,
suggesting decreased sensitivity to additional orexigenic signals.
However, AgRP induced delayed and long-lasting modifications of
energy balance in Pomc?/?Tg/?, but not glucocorticoid-deficient
Pomc?/?mice, by decreasing oxygen consumption, increasing the
respiratory exchange ratio, and increasing food intake.
CONCLUSIONS—These data demonstrate that AgRP can mod-
ulate energy balance via a mechanism independent of MSH and
MC3/4-R competitive antagonism, consistent with either inverse
agonist activity at MC-R or interaction with a distinct receptor.
Diabetes 57:86–94, 2008
POMC is processed posttranslationally into multiple pep-
tides, including the opioid ?-endorphin and the melano-
cortins ACTH, ?-melanocyte-stimulating hormone (?MSH),
?MSH, and ?MSH. POMC peptides in the central nervous
system (CNS) are essential in the regulation of energy
intake and expenditure as demonstrated in studies using
compound mutant mice (Pomc?/?Tg/?) expressing a
Pomc transgene that selectively restored pituitary POMC
in Pomc?/?mice to produce a neural-selective POMC
deficiency (4). Lack of ?MSH is likely the principal cause
of obesity (3,5) due to the loss of agonist signaling at
central melanocortin receptors (MC-R), melanocortin-3
receptor (MC3-R), and melanocortin-4 receptor (MC4-R),
each of which plays a distinct role in the regulation of
energy homeostasis (6–8).
The anorectic actions of centrally administered ?MSH
or the synthetic MC3/4-R agonist melanotan II (MTII)
(9–11) are blocked by Agouti-related peptide (AgRP), an
endogenous MC3/4-R antagonist (12,13), released from
terminals of neuropeptide Y (NPY)/AgRP arcuate neurons.
In addition to their localization to the same brain regions
as POMC fibers (14), AgRP nerve terminals send projec-
tions to neurons that possess MC4-R (15) but are not
innervated by ?MSH terminals (14,16). These neuroana-
tomic findings indicate that AgRP may modulate MC4-R
activity in the absence of endogenous ?MSH.
In vitro data strongly support the ability of AgRP to
modulate MC4-R by an inverse agonist mode of action
(17–20); however, the physiological significance is unre-
solved. Modulation of MC4-R constitutive activity may be
important to maintain long-term energy homeostasis in
humans (21). In rodents, the concept of inverse agonism
has been buttressed by demonstrations that a single injec-
tion of AgRP induces hyperphagia over several days
(22–24), whereas this long-lasting effect cannot be repro-
duced by synthetic MC4-R antagonists like HS014 or
In the present study, we analyzed the feeding and meta-
bolic responses to intracerebroventricular injections of MTII
and AgRP in mice deficient in all central MSH peptides.
Because responses to melanocortin antagonists appar-
ently require the presence of circulating glucocorticoids
(26), we compared Pomc?/?mice with a global deficiency
of POMC and adrenal insufficiency to Pomc?/?Tg/? mice
with a neural-specific deficiency of POMC but restored
glucocorticoids (4). Feeding effects of the orexigenic gut
peptide ghrelin (27) were also tested.
enetic disruption of either mouse or human
proopiomelanocortin (POMC) causes early-on-
set obesity (1–3), highlighting a major role of
POMC in the regulation of energy homeostasis.
From the1Center for the Study of Weight Regulation and Associated Disor-
ders, Oregon Health and Science University, Portland, Oregon; the2Vollum
Institute, Oregon Health and Science University, Portland, Oregon; and the
3Department of Behavioral Neuroscience, Oregon Health and Science Univer-
sity, Portland, Oregon.
Address correspondence and reprint requests to Virginie Tolle, PhD,
UMR549 Inserm, IFR Broca-Ste Anne, 2 ter rue d’Alesia, 75014 Paris, France.
Received for publication 29 May 2007 and accepted in revised form 26
Published ahead of print at http://diabetes.diabetesjournals.org on 1 Octo-
ber 2007. DOI: 10.2337/db07-0733.
Additional information for this article can be found in an online appendix at
AgRP, Agouti-related peptide; CART, cocaine and amphetamine–related
transcript; CNS, central nervous system; CRH, corticotropin-releasing hor-
mone; DIO, diet-induced obesity; MC-R, melanocortin receptor; MC3-R, mela-
nocortin-3 receptor; MC4-R, melanocortin-4 receptor; MSH, melanocyte-
stimulating hormone; MTII, melanotan II; NPY, neuropeptide Y; PLSD,
protected least squares difference; POMC, proopiomelanocortin; RER, respi-
ratory exchange ratio.
© 2008 by the American Diabetes Association.
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.
86 DIABETES, VOL. 57, JANUARY 2008
RESEARCH DESIGN AND METHODS
A colony of Pomc mutant mice on a hybrid B6;D2;129X1;129S6 genetic
background with independently segregating Pomc?/?and pHalEx2* Tg alleles
was established as described previously (4). Mice were maintained under
controlled temperature and photoperiod (12-h light, 12-h dark; lights on at 7:00
A.M.) with free access to water and chow (4.5% fat, 20% protein, 6% fiber, and
3.4 kcal/g; PicoLab Rodent Diet 20; PMI Nutrition International, St. Louis, MO).
Experimental procedures were approved by the Institutional Animal Care and
Use Committee and followed Public Health Service guidelines.
Peptides. MTII, hAgRP (83–132), mAgRP (82–131), and rGhrelin were pur-
chased from Phoenix Pharmaceuticals (Mountain View, CA) and dissolved in
Intracerebroventricular cannulation. Mice were anesthetized with 2%
Avertin. Twenty-six–gauge stainless steel guide cannulae cut 2.5 mm below
the pedestal (Plastics One, Roanoke, VA) were implanted stereotaxically into
the right lateral ventricle (posterior ?0.4 mm, lateral ?1.0 mm, relative to
bregma), secured to the skull using cap screws (Small Parts) and dental
cement, and occluded with stainless steel dummy obturators. Mice were then
housed individually for 7–10 days of recovery without specific treatment,
except for the Pomc?/?mice that required injection with dexamethasone (0.15
?g i.p. in 1 ml saline) for 3 days.
Feeding and basal metabolic rate. Peptides were injected intracerebrov-
entricularly in a volume of 1 ?l over 1 min using a 33-gauge stainless steel
injection cannula extending 0.5 mm below the guide cannula and connected to
a 1-?l Hamilton syringe with polyethylene tubing. Mice were returned to their
home cage, and the remaining food in containers on the cage floor was
weighed at different time intervals. Basal metabolic rate was determined by
indirect open-circuit calorimetry (Oxymax; Columbus Instruments) as previ-
ously described (4). After 3 days of chamber habituation, six measurements
were recorded daily from each mouse at 60-min intervals. Individual basal
oxygen consumption (Vo2) levels were established by averaging the two
lowest Vo2measurements. Respiratory exchange ratios (RERs) were recorded
as the molar ratio of Vco2to Vo2, and a daily average value was calculated.
Experimental design. Male and female mice were used in all experiments.
Peptides or saline were administered between 11:00 A.M. and 12:00 P.M. for
daytime experiments and between 6:00 and 7:00 P.M. (lights off at 7:00 P.M.) for
nighttime experiments. Mice were randomized into different groups based on
their 24-h food intake before experiments and received each dose of peptide
and saline in a counterbalanced order. Detailed designs for each experiment
and the number of animals per group are provided in the online appendix
(available at http://dx.doi.org/10.2337/db07-0733).
Real-time RT-PCR. Mice were decapitated between 9:00 and 11:00 A.M., and
hypothalami were dissected on ice, harvested in TRIzol reagent (Invitrogen
Life Technologies, Carlsbad, CA), and extracted according to the manufactur-
er’s directions. PCRs were performed on an ABI Prism 7300 Sequence
Detection System instrument (Perkin-Elmer Applied Biosystems, Foster City,
CA) using TaqMan Gene Expression Assays containing a set of sequence-
specific primers and a 6-FAM dye-labeled TaqMan MGB probe for MC4-R,
MC3-R, AgRP, NPY, or cocaine and amphetamine–related transcript (CART)
and the TaqMan endogenous control 18S. cDNA samples obtained from
reverse transcription of 1 ?g RNA were run in duplicate in total reaction
volumes of 20 ?l containing 5 ?l cDNA, 1? TaqMan Gene Expression Assay,
and 1? TaqMan Universal Master Mix. Thermal cycling conditions included an
initial denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for
15 s and 60°C for 1 min. Real-time PCR data were analyzed with the 2???CT
method as previously described (28).
Statistical analyses. All data presented are means ? SE. Data were analyzed
by repeated-measures ANOVAs or multifactor ANOVAs appropriate for the
design of each experiment with genotype and/or drug as independent vari-
ables using Stat View Power PC for Macintosh version 5.0.1 (SAS Institute).
One-factor ANOVAs were used to follow up significant main effects; post hoc
pairwise comparisons between groups were performed by Fisher’s protected
least squares difference (PLSD) or paired two-tail t tests. P values ?0.05 were
Feeding effects of MTII injected at the onset of the
dark cycle. A single intracerebroventricular injection
of 0.5 nmol MTII at the onset of the dark cycle de-
creased food intake in control Pomc?/?Tg/? and mutant
Pomc?/?Tg/? and Pomc?/?mice compared with vehicle-
treated animals 2 h after the injection (Fig. 1A). In
Pomc?/?Tg/? mice, food intake was inhibited by 67%, and
the effect was completely reversed by 24 h (Fig. 1A and B).
In both Pomc?/?Tg/? and Pomc?/?mice, MTII acutely
decreased food intake by 95%, and in contrast to control
mice, this effect was sustained with 60 and 54% cumulative
reductions at 24 h (P ? 0.01 vs. saline). Long-term an-
orexia induced by 0.5 nmol MTII was also associated with
significant weight loss in Pomc?/?Tg/? and Pomc?/?mice
(P ? 0.01 and 0.05 vs. saline, respectively) (Fig. 1C).
FIG. 1. Effects of intracerebroventricular injection of MTII on food
intake and body weight. Two-hour food intake (A), 24-h food intake
(B), and 24-h body weight change (C) after injection of 0.5 nmol MTII
in Pomc?/?Tg/? control, Pomc?/?Tg/?, and Pomc?/?mice at the onset
of the dark cycle. Repeated-measures ANOVAs showed a significant
effect of 0.5 nmol–MTII treatment on 2-h food intake in all genotypes
(F2,21? 46.6, P < 0.0001) and on 24-h food intake (F2,21? 38.3, P <
0.0001) and nearly a significant effect on body weight change (F2,21?
3.4, P ? 0.08). The effects of 0.5 nmol MTII were prolonged over 24 h
in Pomc?/?Tg/? and Pomc?/?mice but not in Pomc?/?Tg/? controls.
*P < 0.05, **P < 0.01 vs. saline, paired t test analyses. Data are
means ? SE, n ? 7–10.
V. TOLLE AND M.J. LOW
DIABETES, VOL. 57, JANUARY 200887
Feeding effects of co-administered MTII and AgRP.
To test the ability of AgRP to antagonize MSH agonists at
MC3/4-R, 0.5 nmol AgRP was injected alone or in combi-
nation with 0.1 nmol MTII at the onset of the dark cycle.
Although this lower dose of MTII was slightly less potent
than 0.5 nmol to reduce feeding 2 h after injection, we
chose it in combination with AgRP because its anorectic
effects had dissipated in all genotypes at 24 h. There was a
significant main effect of the peptide treatments on 2-h
food intake but no significant interaction of treatment ?
genotype (Fig. 2A). Post hoc analyses collapsed across
genotype confirmed that 0.1 nmol MTII alone significantly
decreased acute food intake (P ? 0.0001 vs. saline). In
contrast, there was no significant main effect of treatment
or treatment ? genotype interaction on 24-h food intake
(Fig. 2B). After nighttime injection, when maximum feed-
ing activity is already observed, there was no additive,
short-term orexigenic effect of 0.5 nmol AgRP alone on any
genotype. However, AgRP significantly blocked the ano-
rectic effect of 0.1 nmol MTII in all genotypes 2 h after
co-injection of both peptides (P ? 0.0001, AgRP plus MTII
vs. MTII alone, Fisher PLSD) (Fig. 2A), consistent with a
competitive antagonist action at the MC3/4-R.
Short-term–feeding effects of AgRP and ghrelin in-
jected during the light cycle. Administration of 0.5 or 2
nmol AgRP during the daytime increased 2-h food intake
by 160 and 250%, respectively, in Pomc?/?Tg/? mice (P ?
0.05 vs. saline). In Pomc?/?Tg/? mice, neither dose of
AgRP had short-term orexigenic effects (Fig. 3A; data with
2 nmol not shown). To test the ability of Pomc?/?and
Pomc?/?Tg/? mice to respond to other orexigenic signals,
1 nmol ghrelin was injected during the daytime (Fig. 3B).
Ghrelin stimulated 2-h food intake in Pomc?/?Tg/? and, to
a much lesser extent, in Pomc?/?Tg/? mice (P ? 0.0001
and 0.05 vs. saline, respectively) but had no effect in
Short- and long-term effects of AgRP injected at the
onset of the dark cycle on food intake and body
weight. Nighttime injection of 0.5 nmol AgRP, when
maximum feeding activity occurs naturally, did not further
increase 2-h food intake in any genotype (Fig. 4A, D, G,
and J). However, hyperphagia was observed 24 h after the
injection of AgRP, was sustained up to 72 h, and was
accompanied by body weight gain in Pomc?/?Tg/? mice
(Fig. 4B and C). Pomc?/?mice were more sensitive to the
long-term effects of AgRP with sustained hyperphagia up
to 96 h after injection and more pronounced body weight
gain than Pomc?/?Tg/? mice (Fig. 4K and L). Neither
Pomc?/?Tg/? nor Pomc?/?mice increased their food
consumption after nighttime injection of 0.5 nmol AgRP
(Fig. 4E and H). Furthermore, injection of 0.5 or 2 nmol
AgRP during the light cycle also did not produce any
long-lasting orexigenic effect in the mutant Pomc?/?Tg/?
mice, whereas it was orexigenic in Pomc?/?Tg/? control
mice (data not shown). Similar to Pomc?/?Tg/? controls,
the rate of body weight gain was greater in Pomc?/?Tg/?
FIG. 2. Feeding effects of intracerebroventricular injection of AgRP on the anorectic response to MTII. Two-hour food intake (A) and 24-h food
intake (B) after administration of saline, 0.5 nmol AgRP alone, 0.1 nmol MTII alone, or co-administration of 0.1 nmol MTII and 0.5 nmol AgRP
at the onset of the dark cycle in Pomc?/?Tg/? control, Pomc?/?Tg/?, Pomc?/?, and Pomc?/?mice. Repeated-measures ANOVAs showed a
significant effect of the treatments on 2-h food intake in all genotypes (F3,29? 18.5, P < 0.0001) that was not observed after 24 h (F3,29? 1.7,
P ? 0.17). Post hoc analyses showed that MTII treatment decreased 2-h food intake significantly in all genotypes (P < 0.0001 vs. saline) and that
AgRP antagonized the anorectic action of MTII (P < 0.0001 MTII plus AgRP vs. MTII). Data are means ? SE; n ? 7–14, except for Pomc?/?Tg/?
mice (n ? 3).
AgRP ACTIONS IN POMC-DEFICIENT MICE
88DIABETES, VOL. 57, JANUARY 2008
mice injected with 0.5 nmol AgRP than with saline, and
this difference was significant at 72 h (P ? 0.001) (Fig. 4F).
Although a short-lived reduction in food intake and body
weight was sometimes observed after intracerebroventric-
ular injections of saline in Pomc?/?Tg/?, Pomc?/?, and to
a greater degree in Pomc?/?Tg/? mice, food consumption
usually rebounded to baseline values within 48 h. In
contrast, Pomc?/?mice had prolonged anorexia and
weight loss in response to a saline injection (Fig. 4H and
Effect of AgRP injected at the onset of the dark cycle
on Vo2and RER in mice with restricted food access.
To test the hypothesis that AgRP may be a long-term
modulator of energy expenditure in Pomc?/?Tg/? mice,
we measured Vo2and RER during 7 consecutive days
after the injection of saline or AgRP at the onset of the
dark cycle (Fig. 5), conditions that induced weight gain
in both Pomc?/?Tg/? and Pomc?/?Tg/? mice. In most
Pomc?/?Tg/? mice, as depicted in two representative
individuals (Fig. 5A), the effects of AgRP were observed up
to 72 h after the injection. In contrast, the onset of AgRP
effects was delayed by 24 h but subsequently lasted longer
in Pomc?/?Tg/? mice (Fig. 5B). Consequently, changes
were subtler but more prolonged in Pomc?/?Tg/? com-
pared with Pomc?/?Tg/? mice.
Analyses performed for the relevant, genotype-specific
timeframes showed an increased body weight of 12% over
3 days in Pomc?/?Tg/? (P ? 0.01) and 6% over 6 days in
Pomc?/?Tg/? (P ? 0.01) mice after AgRP treatment,
compared with their initial body weights. In Pomc?/?Tg/?
mice, increased daily food consumption, increased aver-
age daily RER, and decreased Vo2were observed within
24 h after the injection (0–24 h) and were sustained up to
72 h after the injection (24–72 h) (Fig. 5C, E, and G). In
contrast, none of the parameters was modified within 24 h
after AgRP injection (0–24 h) in Pomc?/?Tg/? mice, but
the delayed effects on food intake, Vo2, and RER were
significant between 24 and 72 h and persisted up to 168 h
(Fig. 5D, F, and H).
MC3/4-R, AgRP, NPY, and CART gene expression in
the hypothalamus. Expression levels of MC4-R (Fig. 6A)
and MC3-R (Fig. 6B) from the whole hypothalamus were
unchanged in Pomc?/?Tg/? and Pomc?/?mice compared
with control Pomc?/?Tg/? mice. Unlike MC3/4-R, levels of
AgRP (Fig. 6C), NPY (Fig. 6D), and CART (Fig. 6E) mRNA
all differed by genotype. In Pomc?/?mice, AgRP expres-
sion was significantly decreased by 64%, CART expression
was increased by 47%, and NPY expression was unchanged
compared with Pomc?/?Tg/? mice. In Pomc?/?Tg/?
mice, CART expression was increased by 67%, but AgRP
and NPY were unchanged compared with Pomc?/?Tg/?.
Notably, however, in Pomc?/?Tg/? mice, expression of
both AgRP and NPY was significantly increased compared
with that in the glucocorticoid-deficient Pomc?/?mice.
Catabolic effects of MTII were accentuated in obese
POMC-deficient mice. Both strains of POMC-deficient
mice were more sensitive to the short-term anorectic
action of MT-II than their control siblings. The mechanism
of increased sensitivity to MTII in POMC-deficient mice or
other animal models of obesity, such as Zucker rats (fa/fa)
(29), diet-induced obesity (DIO) rats (30), and DIO and
ob/ob mice (31) with decreased POMC expression or
decreased melanocortin tone (32–34), may involve in-
creased expression, density, or functional coupling of
MC-R in response to the chronic absence or decrease of
endogenous melanocortin ligands. There were no signifi-
cant differences in the expression of MC4-R or MC3-R
in Pomc?/?Tg/? and Pomc?/?mice compared with
Pomc?/?Tg/? mice, suggesting that either increased MC-R
density or signaling is responsible for the heightened
anorectic effect of MTII. However, our quantification did
not take into account differential regional expression of
the MC4-R in various hypothalamic nuclei or extra-
hypothalamic expression of MC4-R (15), which are impor-
tant in the regulation of energy homeostasis and have been
shown to mediate the effects of melanocortin agonists and
antagonists and long-term orexigenic actions of AgRP (35).
Increased sensitivity to MTII could also be due to
secondary alterations in the expression of other hypotha-
lamic orexigenic/anorectic signals. AgRP mRNA was re-
duced in Pomc?/?mice, consistent with data reported by
Coll et al. (36) and supporting the hypothesis that reduced
levels of AgRP contributed to the increased response to
MTII. However, this cannot be the only explanation be-
cause AgRP expression was almost normalized in glu-
FIG. 3. Short-term feeding effects of intracerebroventricular injection
of AgRP and ghrelin. A: 2-h food intake after injection of AgRP 0.5 nmol
during the light cycle in Pomc?/?Tg/? and Pomc?/?Tg/? mice. B: 2-h
food intake after injection of 1 nmol ghrelin during the light cycle in
Pomc?/?Tg/?, Pomc?/?Tg/?, and Pomc?/?mice. Repeated-measures
ANOVAs showed a significant effect of 0.5 nmol AgRP (F1,13? 7.7, P ?
0.0016) and of 1 nmol ghrelin injection (F2,33? 12.3, P ? 0.0013).
Pomc?/?Tg/? but not Pomc?/?Tg/? mice responded significantly to
AgRP treatment. Pomc?/?Tg/? and Pomc?/?Tg/? but not Pomc?/?mice
responded significantly to ghrelin treatment. *P < 0.05, ***P < 0.0001
vs. saline, paired t test analyses. Data are means ? SE, n ? 6–9 for
AgRP treatment, n ? 6–13 for ghrelin treatment.
V. TOLLE AND M.J. LOW
DIABETES, VOL. 57, JANUARY 200889
cocorticoid-replete Pomc?/?Tg/? mice, which still displayed
an exaggerated anorectic response to MTII.
Differential short- and long-term effects of AgRP in
neural selective POMC-deficient mice. AgRP did not
alter short-term food intake but was able to antagonize the
anorectic effect of MTII in Pomc?/?Tg/? mice, indicating
that the short-term orexigenic effect of AgRP requires the
presence of ?MSH and therefore is due to a competitive
antagonist action at MC3/4-R. Despite reexpression of
POMC in the pituitary gland, Pomc?/?Tg/? mice had
FIG. 4. Short- and long-term effects of AgRP on food intake and body weight. Two-hour food intake (A, D, G, and J), daily 24-h food intake (B,
E, H, and K), and body weight change (C, F, I, and L) over a period of 96 h after a single injection of 0.5 nmol AgRP at the onset of the dark cycle
in Pomc?/?Tg/? control (A–C), Pomc?/?Tg/? (D–F), Pomc?/?(G–I), and Pomc?/?(J–L) mice. Baseline corresponds to 24-h food intake measured
in noninjected animals. Paired t test (saline vs. AgRP) applied on individual genotypes showed a significant effect of 0.5 nmol AgRP on 24-h food
intake in Pomc?/?Tg/? control and Pomc?/?mice but not in Pomc?/?Tg/? and Pomc?/?mice and on body weight gain in Pomc?/?Tg/? controls,
Pomc?/?Tg/?, and Pomc?/?but not Pomc?/?mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline. All data are means ? SE, n ? 5–8.
AgRP ACTIONS IN POMC-DEFICIENT MICE
90DIABETES, VOL. 57, JANUARY 2008