Original Article Obesity

OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Effects of Liraglutide in Hypothalamic Arcuate Nucleus

of Obese Mice
Andre R.C. Barreto-Vianna, Marcia B. Aguila, and Carlos A. Mandarim-de-Lacerda

Objective: The neuroprotective effects of liraglutide (200 lg/kg, twice daily, subcutaneous administration)
in the hypothalamic arcuate nucleus (ARC) of diet-induced obese mice were investigated.
Methods: C57BL/6 mice were separated into groups: standard chow treated with vehicle or liraglutide
and the respective liraglutide pair-fed group; high-fat diet treated with vehicle or liraglutide and the
respective pair-fed group. Body mass (BM) evolution, carbohydrate metabolism, leptin resistance, pro-
teins involved in energetic balance, apoptosis, and microglia in the ARC were studied.
Results: Obese animals showed glucose intolerance, resistance to insulin and to anorexigenic effect of
leptin, and microgliosis accompanied by elevated Bax/Bcl2 ratio in the ARC. Liraglutide improved the
carbohydrate metabolism, BM loss, and the activation of pro-opiomelanocortin (POMC) and cocaine and
amphetamine-regulated transcript (CART) in the ARC. The liraglutide enhanced leptin sensitivity and
diminished the microgliosis with decrease in Bax/Bcl2 ratio.
Conclusions: Liraglutide activates central anorexigenic pathways, thereby diminishing the energy intake
of obese mice and improving the metabolic parameters related to obesity. Liraglutide is a relevant neuro-
protective agent, which can decrease the microgliosis and stimulate the anti-apoptotic pathway, a signifi-
cant effect in the treatment of obesity and its comorbidities. Some benefits of liraglutide are independent
of the BM loss, which usually accompanies the drug administration.
Obesity (2016) 24, 626–633. doi:10.1002/oby.21387

Introduction
Liraglutide (an analog of glucagon-like peptide GLP-1) decreases
the appetite with consequent reduction in the body mass (BM) (1).
The suppressive effects of GLP1 on food intake are mediated, in
part, through direct central action (2).
The activation of the GLP1 receptor (GLP1r) is associated with the
central control of food intake in the central nervous system (CNS)
areas involved in the control of energy metabolism (3), including
two subpopulations of neurons localized in the hypothalamus arcuate
(ARC) and paraventricular nuclei (4). The first subpopulation con-
tains neuropeptide Y (NPY) and agouti-related peptide neurons,
which are both orexigenic, and the second subpopulation contains
pro-opiomelanocortin (POMC) neurons, which co-express the ano-
rexigenic neuropeptide cocaine and amphetamine-regulated tran-
script (CART). These neuron subpopulations co-express neurotrans-
mitters to control food intake (5,6).
The small neuroglial cells in the CNS, the microglia, may become
phagocytic in the areas of neural damage or inflammation (7). How-
ever, although microglia are critical to maintaining the homeostasis of
the neuronal environment, in large quantity known as microgliosis,
microglia has a pro-inflammatory state, resulting in a vicious cycle of
inflammation, stimulated by an increased release of inflammatory
cytokines and that microglial dysfunction exacerbates some neuro-
pathological conditions (8,9). Recently, a large quantity of the micro-
glia marker ionized calcium-binding adapter molecule 1 (IBA1) was
reported in the ARC nucleus of obese animals (10), probably due to
increased apoptosis in the neurons of the ARC nucleus (11).
In the present study, we were interested in investigating the effect of
liraglutide on the ARC nucleus neurons involved in appetite control
(POMC, CART, and NPY), leptin resistance, microglia density,
and activation of pro/anti-apoptotic pathway in a model of obesity
induced by diet.

Laboratory of Morphometry, Metabolism, and Cardiovascular Disease, Biomedical Center, Institute of Biology, State University of Rio De Janeiro, Rio De
Janeiro, Brazil. Correspondence: Carlos A. Mandarim-de-Lacerda (mandarim@uerj.br)

Funding agencies: This study was financially supported by FAPERJ (Rio de Janeiro State Foundation for Scientific Research, www.faperj.br, grant number E-26/201.186/
2014) and CNPq (Brazilian Council of Science and Technology, grant numbers 302154/2011-6 and 442673/2014-0). These funding agencies had no role in the study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Disclosure: The authors declared no conflict of interest.
Author contributions: ABV, MBA, and CAML conceived the experiments and analyzed data. ABV drafted the manuscript. All authors were involved in writing the paper
and CAML gives final approval of the submitted and published versions.
Received: 9 June 2015; Accepted: 6 October 2015; Published online 25 February 2016. doi:10.1002/oby.21387

626 Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016 www.obesityjournal.org

Original Article
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
Methods
Animals and diets
The procedures were in accordance with conventional guidelines for
animal experimentation (NIH Publication No.85-23, revised 2011),
and the experimental protocols were approved by the animal ethics
committee of the State University of Rio de Janeiro (Protocol
CEUA/009/2012).
a. A group fed standard chow for rodent (SC, 10% energy from
lipids, 14% energy from proteins, and 76% energy from carbohy-
drates—3.8 kcal/kg);
b. A diet-induce obese (DIO) group fed a high-fat diet (HF, 50%
energy from lipids, 14% energy from proteins and 26% energy
from carbohydrates—5.0 kcal/kg).
Sixty male C57BL/6 mice were maintained under controlled condi-
tions in the NexGen system (Allentown Inc., PA, 20 6 28C and
12 h/12 h dark/light cycle). At 3 months of age, the animals were
randomly distributed into two groups (n 5 30 each group) and were
followed for 8 weeks.
SC group (n 5 10), standard chow 1 vehicle
SC/LI group (n 5 10), SC 1 liraglutide
SC/PF group (n 5 10), SC/LI pair feeding 1 vehicle
HF group (n 5 10), high-fat diet 1 vehicle
HF/LI group (n 5 10), HF diet 1 liraglutide
HF/PF group (n 5 10), HF/LI pair feeding 1 vehicle
The diets were manufactured at PragSolucoes (Jau, Sao Paulo, Brazil),
consistent with the recommendations of the American Institute of
Nutrition (AIN 93M) (12). After 8 weeks, each group was separated
into three new groups (n 5 10 each new group), and the diets were
maintained throughout the experiment. However, one new group from
each primary group received a subcutaneous injection of liraglutide
twice daily (1,000 h and 1,700 h) for 6 weeks (200 lg/kg; Novo Nor-
disk, Bagsvaerd, Denmark), and other groups received vehicle (sterile
saline).
The animals were housed singly, were provided free access to food,
except for the pair feeding animals (SC/PF and HF/PF), which
received the same amount of food ingested by their counterparts
(SC/LI and HF/LI, respectively). Fresh chow was provided daily,
and daily food intake was measured by weighing the amount of
food supplied and the amount of food left in the cage. Energy intake
was estimated as the product of food consumption and the energy
content of the diet. The BM of the animals was measured weekly
(Friday, 1,000 h).
Oral glucose tolerance test and intraperitoneal
insulin tolerance test
The oral glucose tolerance test (OGTT) was analyzed before and
after the treatment in the animals 6 h fasted. The animal received
1 g glucose/kg by oral gavage (25% in sterile saline, 0.9% NaCl) to
glucose overload. The intraperitoneal insulin tolerance test (IPITT)
was performed in the animals 4 h fasted. Insulin (0.5 U/kg, Huma-
log Insulin Lispro, Lilly, IN) was administered intraperitoneally.
In both tests, blood was sampled from the tail vein at 0, 15, 30, 60,
and 120 min. The blood glucose was measured using a glucometer
(Accu-Chek, Roche, Sao Paulo, SP, Brazil). The area under the curve
www.obesityjournal.org
Obesity
(AUC) in arbitrary units (a.u.) was determined (Prism version 6.05
for Windows, GraphPad Software, La Jolla, CA).
Intraperitoneal leptin resistance test
In order to determine the anorectic effect of exogenous leptin, ani-
mals were singly housed in monitoring chambers Compulse v 2.7.13
(Harvard/Panlab, Barcelona, Spain) for two days to acclimatize and
then they were intraperitoneally injected with leptin (2.5lg/g, Pepro-
Tech, Eocky Hill, NJ) or saline, and food intake was evaluated in
the following 24 h (13).
Tissue sampling
On the last day of the treatment, the animals were 6 h fasted and
deeply anesthetized (sodium pentobarbital, 150 mg/kg intraperitoneal).
The brain (n 5 5) was rapidly frozen in liquid nitrogen and stored at
2808C for microdissection by a punch of the ARC nucleus, according
to its coordinates (14).
Immunofluorescence and confocal laser
microscopy
For immunofluorescence (n 5 5), animals were deeply anesthetized
and perfused transcardially with 0.9% saline solution and then with
4% paraformaldehyde (PFA, 0.1 M phosphate buffer, PBS, pH 7.4).
The isolated brains were rapidly immersed in a 4% PFA solution
(4 h at 48C), and then cryoprotected with sucrose 30% in PBS (over-
night at 48C). The brains were frozen (Optimal Cutting Temperature
medium, Tissue-Tek, Sakura Finetek Europe, The Netherlands) and
sectioned in a cryostat at a nominal thickness of 20 lm containing
the hypothalamus (14). For immunofluorescence, sections were
treated with 0.3% PBS-Triton solution and blocked with 5% bovine
serum albumin (BSA) for 1 h and immunolabelled with the primary
antibody IBA-1 (#019-19741, Wako Chemicals, VA) overnight. In
addition, sections were exposed to appropriate secondary antibodies
and then counterstained with 4,6-diamidino-2-phenylindole, dihydro-
chloride (DAPI; Sigma-Aldrich, Saint Louis, MO). The slides were
mounted in ProLong Gold Antifade (Invitrogen, Molecular Probes,
Carlsbad, CA). Control procedures were performed with the omis-
sion of primary antibodies and with the inclusion of the secondary
antibody. Fluorescence images were obtained with the laser confocal
microscope Model C2 (Nikon Inc., Tokyo).
The numerical density per area of IBA-1 positive cells in the ARC
nucleus was evaluated by counting the number of immunoreactive
cells in a frame of known area when they did not hit two consecutive
lines of the system (the forbidden lines) (15), in both sides of ARC,
by counting four slides per animal (n 5 5/group). The IBA-1 positive
cells were counted from matched sections ranging from bregma
21.43 mm to 22.03 mm (14).
Western blots
The total proteins of the ARC nucleus were extracted in homogenizing
buffer, centrifuged, and the supernatants were collected. Equal quanti-
ties of total protein were resuspended in SDS-containing sample
buffer, heated for 5 min at 1008C and separated by SDS-PAGE. The
proteins were electroblotted onto a polyvinyl difluoride transfer mem-
brane (Amersham Biosciences, Piscataway, NJ). We used antibodies
against POMC (SC-20148), NPY (SC-28943), and beta-actin (SC-
81178) (purchased from Santa Cruz Biotechnology, Santa Cruz, CA),
Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016 627
Obesity Beneficial Central Effects of Liraglutide Barreto-Vianna et al.

Results
Body mass
When the study started, the animals were not different in BM (Figure
1). After 2 weeks of consuming their respective diets, BM increased
more in the HF group than in the SC group (8%; P < 0.001). The
difference in BM continued to rise in the HF group compared with
the SC group. After 14 weeks, the BM was greater in the HF group
than in the SC group (19%; P < 0.0001).

After 6 weeks of liraglutide administration, the SC/LI group had

a significant decrease in BM in comparison with the SC group
(7%; P 5 0.04), and the HF/LI group had a decrease in BM
compared with the HF group (13%; P < 0.0001). As expected, the
BM was diminished in the pair-fed groups and was similar between
the groups SC/PF and SC/LI, and between the groups HF/PF
and HF/LI.

Figure 1 Body mass. Weeks 1-8 correspond to the period of obesity induction, and
weeks 9-14 correspond to the period of liraglutide administration. The values are
presented as the mean 6 SD, n 5 10. Significant differences (P < 0.05) were deter-
mined using t-test or one-way ANOVA with the post hoc test of Holm-Sidak:
* 6¼ SC, § 6¼ SC/PF, r 6¼ SC/LI, † 6¼ HF/LI, ‡ 6¼ HF/PF.
Bax (NBP1-28566) and Bcl2 (NBP2-22170) (purchased from Novus
Biologicals, Littleton, CO), and CART (#14437) (purchased from Cell
Signaling Technology, Beverly, MA). The membrane was made using
ECL, and the images of the blots were obtained using the Molecular
Imaging ChemiDoc XRS System (Bio-Rad, Hercules, CA). The intensity
of the chemiluminescent bands was quantified using ImageJ software,
version 1.48s (NIH, imagej.nih.gov/ij, USA). The bound antibodies in
the membranes were stripped to reblotted with another antibody.
Statistical analysis
The values are presented as the mean and the standard deviation
(SD). The differences between the groups were tested using t-test
(the first part of the study and the IPITT) or one-way analysis of
variance (ANOVA, the second part of the study) followed by the
post hoc test of Holm-Sidak. A P-value < 0.05 was considered stat-
istically significant (Prism version 6.05 for Windows, GraphPad
Software, La Jolla, CA).
Food intake
The animals SC and HF consumed the same amount of food (Table
1). The pair feeding groups consumed the same amount of food as
the liraglutide-treated animals. The SC/LI group consumed 20.6%
less food compared with the SC group (P < 0.0001), and the HF/LI
group consumed 20.5% less food compared with the HF group (P <
0.0001). Together with the BM effects, the data indicate that liraglu-
tide has an anorexigenic effect, with a consequent decrease in BM.
The decline in BM is mainly due to a reduction in appetite since the
animals in the pair-fed group showed a similar mass to its respective
pair treated with liraglutide.
Carbohydrate metabolism
We analyzed the area under the curve (AUC) of the OGTT and
IPITT (Table 1, Figure 2). The OGTT AUC of the HF group was
greater than that of the SC group (67%; P < 0.0001). In the HF/LI
group, the AUC was smaller than that of the HF group (34%; P <
0.0001).
The IPITT AUC of the HF group was greater than that of the SC
group (72%; P < 0.0001). The AUC of the HF/LI group was smaller
than the AUC of the HF group (30%; P 5 0.0001).

TABLE 1 Food behavior and carbohydrate metabolism

Data SC SC/LI SC/LI/PF HF HF/LI HF/LI/PF

Food behavior
Food intake (g/day/mouse) 2.6 6 0.2 2.0 6 0.2[a] 2.1 6 0.2[a] 2.5 6 0.2[b][c] 2.0 6 0.2[a][d] 2.1 6 0.2[a][d]
Energy intake (kJ/day/mouse) 40.9 6 2.5 32.4 6 3.5[a] 33.1 6 3.0[a] 52.9 6 4.0[a][b][c] 42.0 6 4.8[b][c][d] 43.3 6 4.4[b][c][d]
Carbohydrate metabolism
IPITT (AUC, a.u.) 8.5 6 1.3 8.2 6 1.4 8.5 6 0.7 14.7 6 2.5[a][b][c] 10.2 6 0.6[b][d] 12.3 6 1.2[a][b][c][d][e]
OGTT (AUC, a.u.) 12.9 6 0.9 11.2 6 1.4 12.7 6 1.2 21.5 6 2.7[a][b][c] 14.1 6 1.7[b][d] 19.4 6 1.5[a][b][c][e]
Glucose (mmol/L) 6.1 6 0.6 4.9 6 0.9[a] 5.6 6 1.0 7.4 6 0.4[a][b][c] 5.5 6 0.4[d] 6.5 6 0.4[b][c][e]

Values are presented as the mean 6 SD (n 5 8).

Significant differences are indicated (P < 0.05), as determined by one-way ANOVA and post hoc test of Holm Sidak: [a] 6¼ SC, [b] 6¼ SC/LI, [c] 6¼ SC/LI/PF, [d] 6¼ HF,
[e] 6¼ HF/LI.
Abbreviations: a.u., arbitrary units; AUC, area under the curve; IPITT, intraperitoneal insulin tolerance test; OGTT, oral glucose tolerance test.

628 Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016 www.obesityjournal.org

Original Article Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Figure 2 Oral glucose tolerance test (OGTT) (left) and intraperitoneal insulin tolerance test (IIPITT) (right). The values are pre-
sented as the mean 6 SD, n 5 8.

Moreover, the HF group had a higher fasting plasma glucose level
compared with the SC group (21%; P 5 0.0001), whereas the
HF/LI group had a lower fasting plasma glucose level compared
with the HF group (25%; P 5 0.0001).
Both OGTT and IPITT indicated a significant difference between
the groups HF/PF and HF, i.e., an improvement in glucose tolerance
and a decrease in insulin resistance effects exclusive by the adminis-
tration of liraglutide and not by a BM reduction.
Leptin resistance
The administration of leptin stimulates the anorexigenic hypothala-
mus pathways, with a consequent reduction in food intake (Figure
3). The HF group showed resistance to leptin, with a reduction of
only 17% of the food intake after leptin administration, while the
SC group showed a decrease of 45% of the food intake (P 5 0.001).
The liraglutide administration resulted in increased sensitivity to
leptin, thereby both groups SC/LI and HF/LI showed a reduction in
food intake after leptin administration (SC/LI, 69%; HF/LI, 56%;

Figure 3 Intraperitoneal leptin resistance test (IPITT). (A) Food intake (grams) and (B) energy intake (kJ). Mice were injected with leptin
(2.5 mg/kg), and 24 h food intake was measured. The values are presented as the mean 6 SD, n 5 5.

www.obesityjournal.org Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016 629

Obesity
Figure 4 Western blot analysis in the ARC nucleus. (A) NPY protein levels, (B) POMC protein levels, (C) CART protein levels, (D) Bax/Bcl2 ratio, and (E) representative
bands. Values are presented as the mean 6 SD, n 5 5. Significant differences (P < 0.05) were determined using one-way ANOVA and the Holm-Sidak post hoc test:
[a] 6¼ SC, [b] ¼
6 SC/LI, [c] 6¼ SC/PF, [d] 6¼ HF, [e] 6¼ HF/LI.
P < 0.001). The animals of the groups HF/LI and HF/PF initially
consumed the same amount of energy. After the intraperitoneal
administration of leptin, there was a significant difference in the
quantity of energy ingested between the two groups, indicating that
treatment with liraglutide increased leptin sensitivity. Similar results
were observed for the groups SC/LI and SC/PF. This indicates that
regardless of the condition of obesity liraglutide improved central
anorexigenic response due to leptin administration.
Energy balance-related proteins
The liraglutide administration alters the neuropeptide protein levels
in the ARC nucleus (Figure 4). The POMC protein levels were
higher in the liraglutide-treated groups compared with their
untreated counterparts. The POMC protein levels were 56% higher
in the SC/LI group compared with the SC group (P 5 0.018) and
were higher in the HF/LI group compared with the HF group
(130%; P 5 0.003). Liraglutide administration significantly
increased the CART protein levels in the ARC of both groups SC/LI
(23%; P < 0.0001) and HF/LI (245%; P < 0.0001) in comparison
with their untreated counterparts (Figure 4).
The NPY protein levels were lower in the HF group compared to
the SC group (14%; P < 0.0001) and the administration of liraglu-
tide had no effect on these parameters compared with the untreated
groups. This indicates that liraglutide is able to modulate the hypo-
thalamic control of energetic balance, probably through direct stimu-
lation of anorexigenic neurons.
630 Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016
Beneficial Central Effects of Liraglutide Barreto-Vianna et al.
Apoptosis-related proteins
We examine the pro-apoptotic Bax protein and the anti-apoptotic
Bcl2 protein in the ARC nucleus (Figure 4). The groups that fed a
HF diet have an increase in the Bax protein compared with the
groups that fed a SC, independent of the liraglutide administration.
The Bcl2 protein was increased in the HF/LI group in comparison
with the HF group (64%; P 5 0.003). The Bax/Bcl2 ratio was
greater in the HF group than in the SC group (102%, P 5 0.005)
and was greater in the HF/PF group compared to the SC group
(124%, P 5 0.001). The administration of liraglutide diminished the
Bax/Bcl2 ratio in the HF/LI group in comparison with the HF group
(65%, P<0.0001). Together, these data suggest that the HF diet,
regardless of the amount of food ingested, stimulates the pro-
apoptosis via and liraglutide has an anti-apoptotic effect.
Microglia density
We measured the numerical density per area of the active IBA1
microglia in the ARC nucleus (Figure 5). There was no difference
in microglia density between the groups receiving the SC. However,
both groups HF and HF/PF had an increase in the numerical density
of microglia compared to the SC group (P 5 0.0002), but the HF/LI
group had a similar microglia density as the SC group. The data
indicate that HF diet, regardless of food intake and the BM, led to
microgliosis, and liraglutide was able to control the state of micro-
gliosis in the treated groups.
www.obesityjournal.org
Original Article Obesity
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Figure 5 The density of IBA1 immunoreactivity of microglia in the ARC nucleus. (A) The SC group showed few IBA1 (ionized
calcium-binding adapter molecule 1, green) positive cells in the ARC nucleus. (B) The HF group showed many more IBA1
positive cells than (C) the HF/LI group. The sections were counterstained with DAPI (blue) to show nuclei. (D) Microglia density
in the ARC nucleus is shown. The values are presented as the mean 6 SD, n 5 5. Significant differences (P < 0.05) were
determined using one-way ANOVA with the Holm-Sidak post hoc test: [a] 6¼ SC, [b] 6¼ SC/LI, [c] 6¼ SC/PF, [d] 6¼ HF. [Color figure
can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Discussion
The obesity caused by chronic consumption of a HF diet is accom-
panied by various systemic and central adverse adaptations. In the
current study, obese animals showed glucose intolerance/insulin
resistance, resistance to anorexigenic effect of leptin, elevated
Bax/Bcl2 ratio, and microgliosis, indicating inflammation with
stimulation of the pro-apoptosis in the ARC nucleus. We observed
that liraglutide led to BM loss and carbohydrate metabolism
improvement, confirming previous studies (1). The liraglutide
treatment resulted in a leptin sensitivity enhancement, with reduc-
tion of microgliosis and stimulation of Bcl2, thus reducing the
Bax/Bcl2 ratio. As observed in the pair-fed groups of the current
study, the changes were exclusively due to liraglutide treatment,
not by a decrease in the BM.
We used liraglutide 200 mg/kg twice a day in the study, that is an
antidiabetic dose in mice (16) and considered safe and appropriate
to generate the anorectic effect, the focus of the study (17,18). The
method of administration was chosen according to various experi-
mental studies that reported the peripheral administration of liraglu-
tide as being neuroprotective on the CNS in animal models because
liraglutide crosses the blood–brain barrier and has the potential to
act on the CNS (17,19-21).
The GLP1 agonists stimulate the insulin release from the pancreas,
thereby decreasing glucagon secretion and decreasing the BM (22).
In the present study, the obese animals showed impaired glucose tol-
erance and insulin resistance, which were, as expected, improved
after liraglutide administration. A previous study with liraglutide

www.obesityjournal.org Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016 631

Obesity
administration have demonstrated an increase in insulin sensitivity,
improving beta-cell function in both diabetic and normoglycemic
animals (23).
In the ARC nucleus, leptin activates the anorexigenic pathways and
promotes a negative energy balance, but obese animals have this neg-
ative feedback system interrupted, resulting in central leptin resistance
(13). One of the mechanisms underlying central leptin resistance is
hyperleptinemia, which occurs in obese animals (24,25), with a con-
sequent reduction of anorectic response to exogenous leptin (13). As
seen in the current study, liraglutide reversed the resistance to exoge-
nous leptin. In addition, we detected increased POMC and CART
protein levels in animals treated with liraglutide, indicating that lira-
glutide stimulates the activity of the melanocortin system, which leads
to hypophagia. POMC is the precursor of melanocyte-stimulating hor-
mone (a-MSH), which activates the melanocortin-4 receptor, thus
leading to a reduction in food intake.
A series of sophisticated experiments made by Secher et al. showed
that the GLP1r is necessary for liraglutide uptake in the brain (the
GLP1r was expressed in POMC/CART neurons). CART-positive
cells in the ARC nucleus were positive for liraglutide after the
peripheral administration of fluorescently labeled liraglutide, indicat-
ing that the decrease in BM caused by peripheral administration of
liraglutide appears to be consistent with the direct activation of
POMC/CART neurons in the ARC, with increased gene expression
of CART, and no change in POMC gene expression (26). In the cur-
rent study, we observed an increase in the POMC protein level and
an even greater increase in CART protein level. Secher et al. used
100 mg/kg daily (26), while we have used 200 mg/kg twice a day in
the current study, which may explain the different finding relative to
POMC in neurons in the ARC. Similarly, previous studies have
shown that exendin-4 improved the gene expression of POMC
(27,28), but little is known about the activities of neuropeptide
CART relative to GLP-1 administration.
In contrast to the activation of the melanocortin system, which indu-
ces BM loss, the release of NPY in the ARC nucleus stimulates
food intake (29). In the present study, we observed a reduction of
the NPY levels in the HF group compared to the SC group. How-
ever, this subject is controversial because studies have shown that a
HF diet diminish the NPY levels (30-32), while other studies have
demonstrated that a HF diet was associated with increased levels of
NPY (33,34). In the present study, the liraglutide administration did
not alter the NPY protein levels, similar to that observed by Secher
et al. (26). In addition, the groups treated with liraglutide and their
pair-fed counterparts have their BM associated with the food intake.
The microgliosis occurs in the ARC nucleus induced by the HF diet,
thus indicating hypothalamus inflammation and consequent disrup-
tion of the neuronal network involved in energy balance control and
even death of POMC neurons (9,35). A recent study demonstrated
that, in obese animals, the ARC nucleus microgliosis is not related
to BM, but with hormones and diet (10). Likewise, we observed an
increased density of microglia in the HF and in the HF/PF (the pair
feeding group associated with the HF/LI group, which loses BM),
but not in the HF/LI group, where the liraglutide administration was
efficient in reducing the microgliosis, corroborating that the decrease
in BM is not relevant to change the number of microglia in the
ARC nucleus. A similar result was described previously using
exendin-4 treatment, indicating that this effect of decreasing micro-
632 Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016
Beneficial Central Effects of Liraglutide Barreto-Vianna et al.
gliosis is not exclusive to exendin-4, but probably for all GLP1r
agonists (10).
The hypothalamus inflammation induced by the HF diet causes apo-
ptosis of the neurons in the ARC nucleus that is stimulated by an
activation of the toll-like receptor (TLR)24 (11,36). In the present
study, the Bax levels, a pro-apoptotic protein, increased with the HF
diet, but did not reduce in the HF/PF groups (the pair feeding group
associated with the HF/LI group that had a decreased food intake)
or in the HF/LI group (the HF group treated with liraglutide). How-
ever, we observed an increase in the anti-apoptotic Bcl-2 protein
levels in liraglutide-treated animals, with consequent reduction in
Bax/Bcl2 ratio, indicating that, despite the fact that liraglutide did
not decrease the pro-apoptotic signals, it was able to increase the
anti-apoptotic signals. This effect is crucial since obesity increases
the susceptibility to neurodegenerative diseases. Although these ben-
eficial protective effects of liraglutide were reported in other experi-
mental models of disease, like cerebral ischemia, Alzheimer’s dis-
ease, Parkinson’s disease, and other neurodegenerative diseases (37-
39), an original finding of the present study is the stimulation of an
anti-apoptotic pathway in the ARC nucleus of diet-induced obesity.
The reduced amount of tissue from the ARC nucleus, which did not
allow the analysis of proteins related to the apoptosis pathway, is a
limitation of the study.
Conclusion
In conclusion, liraglutide administration activates central anorexi-
genic pathways, thereby diminishing the energy intake of the diet-
induced obese mice and improving the metabolic parameters related
to obesity. The findings also indicate that liraglutide is a relevant
neuroprotective agent, which can decrease the microgliosis and stim-
ulate the anti-apoptotic pathway, a very significant effect in the
treatment of obesity and its comorbidities. Some benefits of liraglu-
tide are independent of the BM loss, which usually accompanies the
drug administration.O
C 2016 The Obesity Society
V
References
1. Ando T, Haraguchi A, Matsunaga T, et al. Liraglutide as a potentially useful agent
for regulating appetite in diabetic patients with hypothalamic hyperphagia and
obesity. Int Med 2014;53:1791-1795.
2. Alhadeff AL, Rupprecht LE, Hayes MR. GLP-1 neurons in the nucleus of the
solitary tract project directly to the ventral tegmental area and nucleus accumbens
to control for food intake. Endocrinology 2012;153:647-658.
3. Hayes MR, Leichner TM, Zhao S, et al. Intracellular signals mediating the food
intake-suppressive effects of hindbrain glucagon-like peptide-1 receptor activation.
Cell Metab 2011;13:320-330.
4. Kanoski SE, Fortin SM, Arnold M, Grill HJ, Hayes MR. Peripheral and central
GLP-1 receptor populations mediate the anorectic effects of peripherally
administered GLP-1 receptor agonists, liraglutide and exendin-4. Endocrinology
2011;152:3103-3112.
5. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous
system control of food intake and body weight. Nature 2006;443:289-295.
6. Schwartz MW, Woods SC, Porte D Jr., Seeley RJ, Baskin DG. Central nervous
system control of food intake. Nature 2000;404:661-671.
7. Neumann H, Kotter MR, Franklin RJ. Debris clearance by microglia: an essential
link between degeneration and regeneration. Brain 2009;132:288-295.
8. Fernandes A, Miller-Fleming L, Pais TF. Microglia and inflammation: conspiracy,
controversy or control? Cell Mol Life Sci 2014;71:3969-3985.
9. Smith JA, Das A, Ray SK, Banik NL. Role of pro-inflammatory cytokines released
from microglia in neurodegenerative diseases. Brain Res Bull 2012;87:10-20.
www.obesityjournal.org
Original Article
OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
10. Gao Y, Ottaway N, Schriever SC, et al. Hormones and diet, but not body weight,
control hypothalamic microglial activity. Glia 2014;62:17-25.
11. Moraes JC, Coope A, Morari J, et al. High-fat diet induces apoptosis of
hypothalamic neurons. PloS One 2009;4:e5045.
12. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents:
final report of the American Institute of Nutrition ad hoc writing committee on the
reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939-1951.
13. Olofsson LE, Unger EK, Cheung CC, Xu AW. Modulation of AgRP-neuronal
function by SOCS3 as an initiating event in diet-induced hypothalamic leptin
resistance. Proc Natl Acad Sci USA 2013;110:E697-E706.
14. Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates, 4th ed.
Academic Press: Oxford, 2013.
15. Gundersen HJG. Notes on the estimation of the numerical density of arbitrary
profiles: the edge effect. J Microsc 1977;111:219-227.
16. Knudsen LB. Liraglutide: the therapeutic promise from animal models. Int J Clin
Pract Suppl 2010;64 (Suppl 167):4-11.
17. Hamilton A, Patterson S, Porter D, Gault VA, Holscher C. Novel GLP-1 mimetics
developed to treat type 2 diabetes promote progenitor cell proliferation in the brain.
J Neurosci Res 2011;89:481-489.
18. Cummings BP, Stanhope KL, Graham JL, et al. Chronic administration of the
glucagon-like peptide-1 analog, liraglutide, delays the onset of diabetes and lowers
triglycerides in UCD-T2DM rats. Diabetes 2010;59:2653-2661.
19. McClean PL, Parthsarathy V, Faivre E, Holscher C. The diabetes drug liraglutide
prevents degenerative processes in a mouse model of Alzheimer’s disease.
J Neurosci 2011;31:6587-6594.
20. Hunter K, Holscher C. Drugs developed to treat diabetes, liraglutide and
lixisenatide, cross the blood brain barrier and enhance neurogenesis. BMC Neurosci
2012;13:33.
21. Holscher C. Central effects of GLP-1: new opportunities for treatments of
neurodegenerative diseases. J Endocrinol 2014;221:T31-T41.
22. Meloni AR, DeYoung MB, Lowe C, Parkes DG. GLP-1 receptor activated insulin
secretion from pancreatic beta-cells: mechanism and glucose dependence. Diab
Obes Metabol 2013;15:15-27.
23. Ellenbroek JH, Tons HA, Westerouen van Meeteren MJ, et al. Glucagon-like
peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic
mice. Diabetologia 2013;56:1980-1986.
24. Friedman JM. Obesity: causes and control of excess body fat. Nature 2009;459:
340-342.
25. Volpato AM, Schultz A, Magalhaes-da-Costa E, Correia ML, Aguila MB,
Mandarim-de-Lacerda CA. Maternal high-fat diet programs for metabolic
disturbances in offspring despite leptin sensitivity. Neuroendocrinology 2012;96:
272-284.
www.obesityjournal.org
Obesity
26. Secher A, Jelsing J, Baquero AF, et al. The arcuate nucleus mediates GLP-1
receptor agonist liraglutide-dependent weight loss. J Clin Invest 2014;124:4473-
4488.
27. Yang Y, Moghadam AA, Cordner ZA, Liang NC, Moran TH. Long term exendin-4
treatment reduces food intake and body weight and alters expression of brain
homeostatic and reward markers. Endocrinology 2014;155:3473-3483.
28. Shirazi R, Palsdottir V, Collander J, et al. Glucagon-like peptide 1 receptor induced
suppression of food intake, and body weight is mediated by central IL-1 and IL-6.
Proc Natl Acad Sci USA 2013;110:16199-16204.
29. Sanchez-Lasheras C, Konner AC, Bruning JC. Integrative neurobiology of energy
homeostasis-neurocircuits, signals and mediators. Front Neuroendocrinol 2010;31:
4-15.
30. Briggs DI, Enriori PJ, Lemus MB, Cowley MA, Andrews ZB. Diet-induced obesity
causes ghrelin resistance in arcuate NPY/AgRP neurons. Endocrinology 2010;151:
4745-4755.
31. Kohsaka A, Laposky AD, Ramsey KM, et al. High-fat diet disrupts behavioral and
molecular circadian rhythms in mice. Cell Metab 2007;6:414-421.
32. Hansen MJ, Jovanovska V, Morris MJ. Adaptive responses in hypothalamic
neuropeptide Y in the face of prolonged high-fat feeding in the rat. J Neurochem
2004;88:909-916.
33. Gao J, Ghibaudi L, van Heek M, Hwa JJ. Characterization of diet-induced obese
rats that develop persistent obesity after 6 months of high-fat followed by 1 month
of low-fat diet. Brain Res 2002;936:87-90.
34. Ziotopoulou M, Mantzoros CS, Hileman SM, Flier JS. Differential expression of
hypothalamic neuropeptides in the early phase of diet-induced obesity in mice.
Amer J Physiol Endocrinol Metabol 2000;279:E838-E845.
35. Thaler JP, Yi CX, Schur EA, et al. Obesity is associated with hypothalamic injury
in rodents and humans. J Clin Invest 2012;122:153-162.
36. Milanski M, Degasperi G, Coope A, et al. Saturated fatty acids produce an
inflammatory response predominantly through the activation of TLR4 signaling in
hypothalamus: implications for the pathogenesis of obesity. J Neurosci 2009;29:
359-370.
37. Han WN, Holscher C, Yuan L, et al. Liraglutide protects against amyloid-beta
protein-induced impairment of spatial learning and memory in rats. Neurobiol
Aging 2013;34:576-588.
38. Kelly P, McClean PL, Ackermann M, Konerding MA, Holscher C, Mitchell CA.
Restoration of cerebral and systemic microvascular architecture in app/ps1
transgenic mice following treatment with liraglutide. Microcirculation 2015;22:
133-145.
39. Briyal S, Shah S, Gulati A. Neuroprotective and anti-apoptotic effects of liraglutide
in the rat brain following focal cerebral ischemia. Neuroscience 2014;281C:
269-281.
Obesity | VOLUME 24 | NUMBER 3 | MARCH 2016 633