The Journal of Neuroscience, October 15, 2002, 22(20):9048–9052

The Catabolic Action of Insulin in the Brain Is Mediated by

Melanocortins

Stephen C. Benoit,1 Ellen L. Air,1 Lique M. Coolen,2 Richelle Strauss,1 Alana Jackman,3 Deborah J. Clegg,1
Randy J. Seeley,1 and Stephen C. Woods1
Departments of 1Psychiatry and 2Cell Biology, Neurobiology, and Anatomy, University of Cincinnati, Cincinnati, Ohio
45267, and 3Procter & Gamble Pharmaceuticals, Mason, Ohio 45040

Like leptin, the pancreatic hormone insulin is an important Finally, a subthreshold dose of the melanocortin antagonist
adiposity signal to the brain. We report that the hypothalamic SHU-9119 prevented the reduction in food intake caused by
melanocortin system is an important target of the actions of third-ventricular insulin administration. These data suggest that
insulin to regulate food intake and body weight. Hypothalamic the hypothalamic melanocortin system mediates the anorexic
neurons expressing insulin receptors were found to coexpress effects of central insulin, as well as of leptin.
the melanocortin precursor molecule pro-opiomelanocortin
(POMC), and administration of insulin into the third cerebral Key words: insulin; melanocortins; POMC; food intake; obe-
ventricle of fasted rats increased expression of POMC mRNA. sity; leptin

Body weight is regulated by a complex interaction of peripheral signal to the CNS that is proportional to peripheral energy stores
signals with central pathways that influence food intake and and is linked to CNS systems that control food intake and body
energy expenditure. Understanding the high degree of accuracy weight.
displayed by most mammals to maintain energy balance is fun- Like insulin, the more recently characterized adipocyte hor-
damental to our knowledge of how homeostatic systems function, mone leptin also acts to reduce food intake and body weight. The
as well as to the etiology and treatment of clinical syndromes, central mechanisms of this hormone, however, have been well
including obesity and eating disorders. One afferent signal to the characterized during the last several years. Considerable evi-
brain that provides information about the amount of stored dence now implicates the hypothalamic melanocortin system in
calories in the form of adipose tissue is the pancreatic hormone the mediation of the anorexic effects of leptin. We hypothesized
insulin. The secretion of insulin from the pancreas and its level in that the hypothalamic melanocortin system also mediates the
the blood are directly proportional to adipose mass (Bagdade et central effects of insulin to reduce food intake and body weight.
al., 1967), such that plasma insulin increases during periods of This hypothesis makes several predictions that we tested. First,
positive energy balance and decreases during periods of negative insulin receptors exist on ␣-melanocyte-stimulating hormone (␣-
energy balance (Bagdade et al., 1967; Woods et al., 1974). More- MSH) producing neurons within the arcuate nucleus (ARC) of
over, insulin passes through the blood–brain barrier via a satura- the hypothalamus. Second, insulin stimulates expression of the
ble, receptor-mediated process that yields insulin levels in the ␣-MSH precursor pro-opiomelanocortin (POMC). Third, the
CNS that are proportional to plasma insulin (Baura et al., 1993). ability of insulin to reduce food intake depends on melanocortin
The administration of exogenous insulin in small amounts into receptor activation.
either the neuropil of the ventral hypothalamus or the adjacent
third ventricle results in dose-dependent decreases in food intake
MATERIALS AND METHODS
(Woods et al., 1979; Schwartz et al., 1992) and sustained weight
Subjects
loss that is not attributable to incapacitation or illness (Chavez et
Subjects for all experiments were male, L ong–Evans rats (250 –300 gm)
al., 1995), and this has been observed in several species. Con- individually housed in plastic tub cages. All animals had ad libitum access
versely, the central administration of insulin antibodies results in to water and Purina 5001 rat chow unless otherwise noted.
increased food intake and body weight (McGowan et al., 1992).
Consistent with this, the selective removal of insulin receptors Experiment 1: dual-labeling immunohistochemistry
from neurons or else the selective absence of key insulin receptor E xperiment 1 assessed the hypothesis that insulin receptors are located
signaling molecules in the brain results in increased body weight on hypothalamic arcuate neurons that express the melanocortin precur-
sor POMC. To assess this hypothesis, we used qualitative dual-labeling
and susceptibility to diet-induced obesity (Brüning et al., 2000; immunohistochemistry for insulin receptors and POMC.
Stubdal et al., 2000). Hence, insulin provides a negative feedback Procedures. Five rats were given an overdose of pentobarbital (60
mg / kg) and perf used transcardially with 450 ml of 4% paraformaldehyde
Received March 8, 2002; revised July 12, 2002; accepted July 12, 2002. in 0.1 M sodium phosphate buffer (PB). Brains were removed, postfixed
(1.5 hr at room temperature), and stored in 20% sucrose in PB. Coronal
This work was supported by grants from the National Institutes of Health and the
American Diabetes Association (Physician Scientist Training Award to E.L.A.) and sections were cut (40 ␮m) with a cryostat (Leica, Nussloch, Germany)
funds from Procter & Gamble Pharmaceuticals. and stored in cryoprotectant (30% sucrose and 30% ethylene glycol in 0.1
Correspondence should be addressed to Stephen C. Benoit, Department of M PB) (Watson et al., 1986) at ⫺20°C until f urther processing for POMC
Psychiatry, University of Cincinnati, College of Medicine, Box 670559, Cincinnati, and insulin receptor-␤. Available antibodies recognizing these antigens
OH 45267-0559. E-mail: [email protected]. were all raised in rabbit. To eliminate the possibility of cross-reactivity,
Copyright © 2002 Society for Neuroscience 0270-6474/02/229048-05$15.00/0 a modification of a recently described protocol was used (Hunyady et al.,
Benoit et al. • MC Mediation of Insulin Action J. Neurosci., October 15, 2002, 22(20):9048–9052 9049

1996). Free-floating sections were incubated overnight at 4°C with poly- singleplex GAPDH reactions. All reactions completed 40 replication
clonal antiserum recognizing insulin receptor-␤ (Santa Cruz Biotechnol- cycles.
ogy, Santa Cruz, CA) diluted 1:1000 in incubation solution (PBS con- Sequence amplification and fluorescence detection were done using
taining 4% normal donkey serum and 0.1% Triton X-100). Sections were the ABI PRISM 7700 Sequence Detection System (PE Applied Biosys-
subsequently exposed to biotin-conjugated donkey anti-rabbit IgG (1:400 tems). Baseline fluorescence was determined by the system and defined
in incubation solution for 60 min; Jackson ImmunoResearch, West as the average fluorescence detected during cycles 3–15. The threshold
Grove, PA), avidin –biotin –horseradish peroxidase (ABC Elite, 1:1500 in fluorescence was then calculated by the system to be the level of fluo-
PBS for 60 min; Vector Laboratories, Burlingame, CA), biotinylated rescence that was statistically different from baseline (i.e., 10 times the
tyramide (1:250 for 10 min; T yramide Signal Amplification; N EN, Bos- mean SD of fluorescence in all wells over baseline cycles). Relative
ton, M A), and C Y3-conjugated streptavidin (1:400 in PBS for 30 min; expression was calculated based on the cycle number at which the
Jackson ImmunoResearch). Sections were then incubated overnight at fluorescence exceeded the threshold of detection (C T). Specifically, the
room temperature in polyclonal antiserum recognizing POMC (1:1000 in C T for GAPDH was subtracted from that of POMC for each well (⌬C T).
incubation solution; Phoenix Peptides, Belmont, CA) and in Alexa488- The average ⌬C T for each experimental group was derived from the
conjugated goat anti-rabbit (1:400 for 30 min; Molecular Probes, Eugene, average ⌬C T of each rat in that group. The percentage of change in
OR). Sections were mounted on glass slides and coverslipped with POMC expression, relative to the reference fed saline group, was defined
Gelvatol, containing an anti-fading agent [1,4-diazabicyclo (2,2) octane]. as 100 ⫻ 2 ⫺⌬⌬C T, where ⌬⌬C T equals the group ⌬C T minus the ⌬C T of
Immunocytochemical controls included omission of primary antibody the fed saline group. Percentage of change data were analyzed with
or preabsorption of diluted antiserum with nanomolar concentrations of one-way ANOVA, which yielded a significant main effect of treatment
appropriate purified peptides at 4°C for 24 hr. In addition, controls (F(2,19) ⫽ 7.44; p ⬍ 0.05).
included omission of second primary antibody and application of
Alexa488-conjugated secondary antibody. Fluorescent-stained sections Experiment 3: food intake
were examined with a Z eiss (Oberkochen, Germany) laser-scanning E xperiment 3 determined whether a subthreshold for feeding dose of a
confocal microscope system (Z eiss L SM510). Alexa 488 fluorescence was melanocortin antagonist would block the ability of central insulin to
imaged with a 505 nm emission filter and an argon laser (488 nm) and reduce food intake.
visualized as red signal; C Y3 fluorescence was imaged with a 567 nm Surgeries. Intracerebroventricular cannulations were performed as de-
emission filter and a HeNe laser (544 nm) and visualized as green signal. scribed for experiment 2.
Images were imported into Adobe Systems (San Jose, CA) PhotoShop Procedures. The dose of SHU-9119 (Phoenix Peptides) was 0.1 nmol,
6.0 and Microsoft (Seattle, WA) Word to comprise Figure 1. Images were which has been found previously to be subthreshold for augmenting food
not adjusted or altered in any way, except for occasional adjustment of intake but nonetheless blocks the anorexic effects of involuntary over-
brightness. feeding (Hagan et al., 1999). Food was removed 4 hr before the dark
phase, and all inf usions were administered 1 hr before lights-off. Food
Experiment 2: POMC expression was returned at the onset of dark, and intake was measured after 1, 2, 4,
E xperiment 2 determined whether administration of central insulin and 24 hr. Body weights were recorded at 24 hr. This design included four
increases expression of POMC mRNA. within-subjects tests: saline 3 saline, SHU-9119 3 saline, saline 3
Surgeries. With the aid of a stereotaxic device, a stainless steel 21 gauge insulin, and SHU-9119 3 insulin. Order of test inf usions was counter-
cannula was implanted 2.2 mm posterior to bregma, 7.5 mm below the balanced via Latin-square design, with 2 d between treatments.
dural surface and directly along the midline, with bregma and lambda at
the same vertical coordinate. The cannula was anchored to the skull with RESULTS
screws and dental acrylic. All rats were allowed to recover for 1 week.
C annula patency was then assessed by injection of 10 ng of angiotensin-II Experiment 1: dual-labeling immunohistochemistry
(1 ␮l injection). C annulas were considered patent if rats consumed 5 ml We assessed the hypothesized overlap in distribution of POMC
of water within 1 hr of injection. and the insulin receptor (␤-subunit) by qualitative dual-labeling
Procedures. Beginning at lights-on, each rat received an intrathird
ventricular (i3vt) injection (1 ␮l) of either saline or 4 mU of insulin (n ⫽ immunohistochemistry. First, immunoreactivity for insulin
7 per group). The injections were repeated every 12 hr over a 72 hr receptor-␤ was present in brain areas found previously to contain
period (seven injections total), during which time the rats were food mRNA or binding sites (Baskin et al., 1983), including hippocam-
deprived. An additional group of rats (n ⫽ 7) also received i3vt saline but pus and arcuate nucleus (Fig. 1). No insulin receptor-␤ immuno-
remained fed ad libitum.
POMC mR NA quantification. T wo hours after the final injection, the
reactivity was observed in areas of the brain that do not express
rats were killed, and the brains were collected. RNA was isolated from insulin receptor mRNA. Second, in all sections observed, arcuate
whole hypothalami using Tri-Reagent (Medical Research Council, C in- neurons with POMC immunoreactivity also had immunoreactiv-
cinnati, OH) according to the instructions of the manufacturer. DNA ity for the insulin receptor-␤. Figure 1 depicts a representative
contamination was eliminated using a removable DNase system section with colocalization of POMC and insulin receptor-␤ im-
(DNAfree; Ambion, Austin, TX). The absence of DNA contamination
was confirmed by amplification of glyceraldehyde-3-phosphate dehydro- munoreactivity in the arcuate nucleus at bregma ⫺2.4 mm. Ex-
genase (GAPDH) (250 ng of RNA per well) with and without a preced- tensive colocalization of POMC and insulin receptor-␤ was ob-
ing reverse transcriptase (RT) step (2 min at 50°C and 30 min at 60°C). served throughout the nucleus in both medial and lateral arcuate
Completion of 40 amplification cycles (i.e., 5 min at 95°C for one time; 20 neurons. Most (⬃90%) of the cells with POMC immunoreactivity
sec at 94°C and 60 sec at 62°C for 40 times) without detecting a product
in the non-RT wells indicated that the RNA samples were not
also had insulin receptor-␤ immunoreactivity. However, not all
contaminated. insulin receptor-␤-immunopositive neurons coexpressed POMC
POMC expression was analyzed using the Taqman real-time PCR immunoreactivity. Cells positive only for insulin receptor-␤ were
system (PE Applied Biosystems, Foster C ity, CA) (Medhurst et al., distributed throughout the arcuate but primarily observed in the
2000). POMC was amplified, as above, in triplicate samples of RNA from most rostral sections. Furthermore, immunocytochemical con-
each rat (10 ng /20 ␮l reaction) using forward and reverse primers
(5⬘-CGCCCGTGTTTCCA-3⬘ and 5⬘-TGACCCATGACGTAC TTCC - trols demonstrated specificity of the primary antibody. Both omis-
3⬘, respectively; 300 nM each) coupled with a fluorescent-labeled Taqman sion of the primary antibody and preabsorption in diluted anti-
probe (6-FAM-ACGGAGATGAACAGCCC TTGAC T-TAMR A; 150 serum blocked insulin receptor-␤ immunoreactivity.
nM). GAPDH served as the reference gene in each multiplexed reaction
(forward primer, 5⬘-TGCACCACCAAC TGC TTAG-3⬘; reverse primer, Experiment 2: POMC expression
5⬘-GGATGCAGGGATGATGTTC -3⬘, 80 nM each; probe V IC - To test the hypothesis that insulin upregulates POMC expression
CAGAAGAC TGTGGATGGCCCC TC -TAMR A, 100 nM). Reactions
were run using the Taqman EZ RT-PCR core reagent kit at the recom-
in the ARC, insulin was administered into the third ventricle of
mended concentrations, although reaction volume was reduced to 20 ␮l. fasted rats, and POMC expression compared with fasted, saline-
Standard curves consisted of pooled RNA from each treatment group in treated, and ad libitum fed rats was measured in whole hypotha-
9050 J. Neurosci., October 15, 2002, 22(20):9048–9052 Benoit et al. • MC Mediation of Insulin Action

Figure 2. Hypothalamic POMC expression. POMC expression after ad

libitum feeding (left bar), a 72 hr fast (middle bar), and a 72 hr fast with
i3vt infusions of 4 mU of insulin every 12 hr (right bar). Central admin-
istration of insulin increased expression of POMC relative to fasting, and
the resultant levels were not different from those that occurred during ad
libitum feeding. *p ⬍ 0.05.

lamic extracts by real-time PCR (Medhurst et al., 2000). As

depicted in Figure 2, POMC expression in the fasted saline-
treated rats was decreased by 50% (ANOVA; Tukey’s post hoc
test; p ⬍ 0.01) relative to those in a group of fed rats, an effect
similar to that seen in previous reports (Schwartz et al., 1997;
Thornton et al., 1997; Mizuno et al., 1998). In contrast, POMC
expression in insulin-treated fasted rats was significantly higher
than that of the level in the fasted saline rats (Tukey’s post hoc
test; p ⬍ 0.05) and not significantly different from the level in the
fed controls. Hence, the local administration of insulin greatly
attenuated the reduction of ARC POMC caused by fasting. Per-
centage of change data were analyzed with one-way ANOVA,
which yielded a significant main effect of treatment (F(2,19) ⫽
7.44; p ⬍ 0.05).
Experiment 3: food intake
SHU-9119 in the absence of insulin had no effect on food intake
at this dose (Fig. 3), nor did it block the anorexic action of
glucagon-like peptide-1 (Seeley et al., 1997). Insulin by itself
caused a significant reduction of food intake (Tukey’s post hoc
test; p ⬍ 0.05), and this was completely reversed by the presence
of SHU-9119. Food intake data were analyzed with a repeated-
measures ANOVA, yielding a significant main effect of treat-
ments (F(3,12) ⫽ 3.52; p ⬍ 0.05).

DISCUSSION
Although the importance of insulin as an adiposity signal has
been well established, the mechanisms by which it regulates
intake and body weight have been ill defined. The central action
of insulin to reduce food intake is similar in many ways to what is
known about the central action of the adipocyte hormone leptin.
Like insulin, leptin secretion and levels in the circulation are
directly proportional to adiposity; like insulin, leptin penetrates

4
Figure 1. Dual-label immunohistochemistry for insulin receptor-␤ and
POMC. The top two panels are confocal images (5 ␮m optical section) of
ARC neurons. Positive immunoreactivity for the insulin receptor-␤ is
depicted in green ( A), whereas POMC-positive immunoreactivity is de-
picted in red ( B). C, An overlay of the above images. Green arrows point
to single-labeled neurons; yellow arrows indicate dual-labeled neurons.
Scale bar, 50 ␮m.
Benoit et al. • MC Mediation of Insulin Action J. Neurosci., October 15, 2002, 22(20):9048–9052 9051

brain, we hypothesized that insulin likewise interacts with the

central melanocortin system to exert its effects on energy balance.
Consistent with this hypothesis, insulin receptors would be ex-
pected to be located on POMC neurons. This was found to be the
case (Fig. 1). Moreover, some insulin receptor-containing cells
did not contain POMC, indicating that these cells may contain
other important ARC systems, such as neuropeptide Y and
agouti-related protein AgRP. These data provide the first dem-
onstration of a direct anatomical link between central insulin
receptors and a specific neuropeptide system that regulates en-
ergy homeostasis.
One potential way in which insulin may influence the melano-
cortin system is through regulation of POMC expression. Insulin,
at doses that do not influence peripheral glucose levels, was able
to attenuate significantly the reduction in POMC expression that
accompanies a prolonged fast (Fig. 2). These data are consistent
with a previous report that, when circulating insulin is reduced
after streptozotocin administration, POMC gene expression in
Figure 3. Effect of insulin and SHU-9119 on food intake. Mean 4 hr food the ARC is reduced, and the administration of insulin systemi-
intake (in grams) after i3vt administration of saline (Sal ), insulin (8 mU) cally restores POMC expression (Kim et al., 1999). However, it
(Ins), SHU-9119 (0.1 nmol) (Shu), and SHU-9119 followed by insulin could not be concluded from that report whether insulin or leptin
(S⫹I ). SHU-9119, by itself, had no effect on food intake, whereas insulin
significantly reduced intake relative to saline. Administration of SHU-
was the key factor because streptozotocin treatment also results in
9119 before administration of insulin blocked the effect of insulin to reduced circulating leptin, and systemic insulin administration
reduce food intake (*p ⬍ 0.05). leads to an immediate increase in plasma leptin. The current
results indicate that the local administration of a small amount of
insulin directly into the CNS stimulates POMC gene expression,
the blood–brain barrier via a receptor-mediated process, and, as presumably through the insulin receptors found on POMC
occurs with insulin, experimentally or genetically induced in- neurons.
creases in central leptin activity cause a net catabolic response, The observations that insulin receptors are expressed by
whereas decreases cause a net anabolic response (Woods et al., POMC neurons and that insulin stimulates POMC gene expres-
1998; Schwartz et al., 2000). Hence, insulin and leptin share many sion suggest that insulin may reduce food intake and body weight
common activities regarding the control of energy homeostasis, by acting through the central melanocortin system. However,
and recent evidence suggests that they also share common intra- because the POMC precursor encodes a number of important
cellular signaling pathways (Bjorbaek et al., 1998; Barsh et al., ligands, the data from the first two experiments do not unambig-
2000; Emanuelli et al., 2000; Niswender et al., 2001). Based on all uously relate insulin signaling to increased activity at melanocor-
of this evidence, we hypothesized that insulin and leptin act in tin receptors. To this end, we determined the ability of melano-
concert in the CNS to regulate caloric intake and expenditure. cortin receptor antagonists to block the anorexic actions of
Considerable evidence suggests that specific neuronal popula- centrally administered insulin. As illustrated in Figure 3, a non-
tions in the ARC of the hypothalamus mediate the catabolic specific melanocortin 3/4 receptor antagonist, SHU-9119, was
action of leptin (Cheung et al., 1997; Fan et al., 1997; Seeley et al., able to significantly attenuate the reduction in food intake elicited
1997; Thornton et al., 1997; Mizuno et al., 1998). In particular, by insulin administration. Hence, like leptin, insulin appears to
one population of ARC neurons expresses POMC, the precursor exert its central catabolic action by acting through melanocortin
molecule of ␣-MSH. In the CNS, ␣-MSH interacts with melano- receptors.
cortin 3 and 4 (MC3 and MC4) receptors, and administration of Collectively, these experiments provide the first demonstration
␣-MSH or other MC3/4 agonists reduces food intake and body that the CNS melanocortin system is an important downstream
weight (Fan et al., 1997; Thiele et al., 1998). Consistent with this, target for the effects of insulin to regulate food intake and body
overexpression or pharmacological administration of MC3/4 an- weight. Therefore, they provide a significant step in delineating
tagonists increases food intake and body weight (Fan et al., 1997; the critical pathways that allow for the maintenance of energy
Ollmann et al., 1997; Hagan et al., 2000), whereas genetic disrup- balance. The present data also indicate that one important role
tion of either POMC or the MC4 receptor results in a phenotype for the CNS melanocortin system is to integrate the message
that includes increased food intake and obesity (Huszar et al., conveyed by insulin and leptin and likely other important signals,
1997; Yaswen et al., 1999). as well (Woods et al., 1998; Schwartz et al., 2000; Cowley et al.,
Leptin receptors are expressed by POMC neurons in the ARC 2001).
(Cheung et al., 1997), and leptin administration stimulates
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