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British Journal of DOI:10.1111/j.1476-5381.2011.01484.

BJP Pharmacology
www.brjpharmacol.org

RESEARCH PAPER bph_1484 704..718


Correspondence
Professor RR Ugale, Division of
Neuroscience, Department of

Agmatine in the Pharmacology, Shrimati


Kishoritai Bhoyar College of
Pharmacy, New Kamptee, Nagpur

hypothalamic (Maharashtra) 441 002, India.


E-mail: ugale.rajesh@gmail.com
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paraventricular nucleus Keywords


agmatine; food intake;
neuropeptide Y; a2-adrenoceptors;

stimulates feeding in rats: paraventricular nucleus of


hypothalamus
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involvement of Received
15 November 2010
Revised
neuropeptide Y 2 April 2011
Accepted
2 May 2011
1 1 2 1 2
BG Taksande , NR Kotagale , KT Nakhate , PD Mali , DM Kokare ,
K Hirani3, NK Subhedar4, CT Chopde1 and RR Ugale1
1
Division of Neuroscience, Department of Pharmacology, Shrimati Kishoritai Bhoyar College of
Pharmacy, New Kamptee, Nagpur, Maharashtra, India, 2Department of Pharmaceutical Sciences,
Rashtrasant Tukadoji Maharaj Nagpur University Campus, Nagpur, Maharashtra, India,
3
Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami,
FL, USA and 4Indian Institute of Science Education and Research (IISER), Sutarwadi, Pashan,
Pune, India

BACKGROUND AND PURPOSE


Agmatine, a multifaceted neurotransmitter, is abundantly expressed in the hypothalamic paraventricular nucleus (PVN). Our
aim was to assess (i) the effect of agmatine on feeding behaviour and (ii) its association, if any, with neuropeptide Y (NPY).
EXPERIMENTAL APPROACH
Satiated rats fitted with intra-PVN cannulae were administered agmatine, alone or jointly with (i) a2-adrenoceptor agonist,
clonidine, or antagonist, yohimbine; (ii) NPY, NPY Y1 receptor agonist, [Leu31, Pro34]-NPY, or antagonist, BIBP3226; or (iii)
yohimbine and NPY. Cumulative food intake was monitored at different post-injection time points. Furthermore, the
expression of hypothalamic NPY following i.p. treatment with agmatine, alone or in combination with yohimbine (i.p.), was
evaluated by immunocytochemistry.

KEY RESULTS
Agmatine robustly increased feeding in a dose-dependent manner. While pretreatment with clonidine augmented, yohimbine
attenuated the orexigenic response to agmatine. Similarly, NPY and [Leu31, Pro34]-NPY potentiated the agmatine-induced
hyperphagia, whereas BIBP3226 inhibited it. Moreover, yohimbine attenuated the synergistic orexigenic effect induced by the
combination of NPY and agmatine. Agmatine increased NPY immunoreactivity in the PVN fibres and in the cells of the
hypothalamic arcuate nucleus (ARC) and this effect was prevented by pretreatment with yohimbine. NPY immunoreactivity in
the fibres of the ARC, dorsomedial, ventromedial and lateral nuclei of the hypothalamus was not affected by any of the above
treatments.

CONCLUSIONS AND IMPLICATIONS


The orexigenic effect of agmatine is coupled to increased NPY activity mediated by stimulation of a2-adrenoceptors within the
PVN. This signifies the importance of agmatine or a2-adrenoceptor modulators in the development of novel therapeutic
agents to treat feeding-related disorders.

Abbreviations
3V, third ventricle; aCSF, artificial cerebrospinal fluid; AGM, agmatine; AHC, central part of the anterior hypothalamus;
ARC, hypothalamic arcuate nucleus; BIBP3226, N2-diphenylacetyl)-N-[(4-hydroxy-phenyl)-methyl]-D-arginine amide;
DMH, hypothalamic dorsomedial nucleus; F, fornix; ir, immunoreactive; LH, lateral hypothalamus; LHRH, leutinizing
hormone-releasing hormone; NPY, neuropeptide Y; ME, median eminence; PVN, hypothalamic paraventricular nucleus;
Rch, retrochiasmatic nucleus; SAL, saline; VMH, hypothalamic ventromedial nucleus; YOH, yohimbine

704 British Journal of Pharmacology (2011) 164 704–718 © 2011 The Authors
British Journal of Pharmacology © 2011 The British Pharmacological Society
Agmatine stimulates feeding via NPY
BJP
Introduction NPY in the PVN pertaining to the regulation of energy
homeostasis are still largely unexplored.
Agmatine is a biogenic amine synthesized through decar- Based upon the above findings, it appears necessary to
boxylation of L-arginine by arginine decarboxylase in the define the precise role of hypothalamic agmatine system in
mammalian brain. It is considered to be a central neurotrans- feeding behaviour in the satiated rats. Therefore, we exam-
mitter as it is stored in synaptic vesicles, accumulated by ined the effect of agmatine on spontaneous food intake and
uptake, released by depolarization and inactivated by agma- its modulation by a2-adrenoceptors. Subsequently, the influ-
tinase (Raasch et al., 1995; Reis and Regunathan, 2000). It ence of NPY, NPY Y1 receptor agents on the agmatine-induced
activates a2-adrenoceptors and imidazoline receptors (Reis changes in food consumption was assessed. Furthermore, the
and Regunathan, 2000; Halaris and Plietz, 2007), antagonizes effect of agmatine on endogenous NPY-containing elements
NMDA receptors (Yang and Reis, 1999) and inhibits NO syn- in the PVN, ARC, dorsomedial (DMH) and ventromedial
thase (Auguet et al., 1995). Moreover, agmatine augments the (VMH) nuclei of hypothalamus and the lateral hypothalamus
release of catecholamines from the adrenal chromaffin cells (LH) was analysed employing immunocytochemistry.
(Li et al., 1994), insulin from the pancreatic b-cells (Sener
et al., 1989) and leutinizing hormone-releasing hormone
(LHRH) from the hypothalamus (Kalra et al., 1995). Agmatine
exerts several pharmacological effects including anti-
Methods
depressive (Zomkowski et al., 2002; Taksande et al., 2009),
anti-nociceptive (Onal et al., 2004), anxiolytic (Lavinsky Subjects
et al., 2003), anticonvulsive (Bence et al., 2003), anti- Adult male Sprague–Dawley rats (240–260 g) were group
proliferative (Isome et al., 2007), neuroprotective (Olmos housed in acrylic cages (24 ¥ 17 ¥ 12 cm) under ambient
et al., 1999), spatial memory facilitation (Liu and Bergin, room temperature (25 ⫾ 2°C) and relative humidity (50 ⫾
2009) and is also implicated in drug addiction (Kotagale et al., 5%), maintained at 12:12 h dark–light cycle (lights on at
2010; Taksande et al., 2010). Moreover, agmatine also 07:00 h). Food and water were available ad libitum. All pro-
increases caloric intake and dietary carbohydrate preference cedures employed in the present study were approved by
in satiated rats (Prasad and Prasad, 1996). Institutional Animal Ethics Committee and carried out under
Central regulation of feeding behaviour involves complex strict compliance with Committee for the Purpose of Control
interactions between neuropeptides, monoamines and other and Supervision of Experiments on Animals, Ministry of
brain messengers at the level of the hypothalamus (Kalra Environment and Forests, Government of India.
et al., 1999; Konturek et al., 2005). Neuropeptide Y (NPY) has
emerged as a major orexigenic signalling molecule in the Intra-PVN cannulation and injections
hypothalamus (Pedrazzini et al., 2003). Neurons in the hypo- The detailed procedure of intra-PVN cannulation is described
thalamic arcuate nucleus (ARC) abundantly synthesize NPY in our earlier study (Kamdi et al., 2009). Briefly, the rats were
and innervate the paraventricular (PVN) and other hypotha- anaesthetized by an i.p. injection of thiopentone sodium
lamic nuclei (Beck, 2006). The PVN is considered to be a key (45 mg·kg-1; Abbott Pharmaceuticals, Mumbai, India), and
area in the control of appetite, and notably, release of NPY is stainless steel guide cannulae (C316G/Spc; Plastics One,
augmented in this nucleus during fasting or before initiation Roanoke, VA, USA) were implanted bilaterally into the PVN
of feeding (Kalra et al., 1991; Dube et al., 1992). Moreover, using stereotaxic co-ordinates, -1.8 mm posterior, ⫾0.4 mm
exogenous injection of NPY directly into the PVN stimulates bilateral to midline and 7.6 mm ventral with respect to
feeding (Kask et al., 1998). Several neurotransmitter or neu- bregma (Paxinos and Watson, 1998). After surgical implanta-
romodulatory systems interact in the PVN to regulate energy tion and appropriate wound closure, animals were housed
homeostasis (Kalra et al., 1999). Among these, the noradren- individually and allowed to recover for 7 days. During this
ergic system has been widely investigated in relation to period, rats were injected s.c. with cefotaxime sodium
feeding behaviour (Goldman et al., 1985; Morien et al., 1999). (50 mg·kg-1; Cefantral®, Lupin Pharmaceuticals, Ankhalesh-
Contextually, a close anatomical and physiological associa- war, Gujarat, India) immediately prior to, and 2 days after,
tion has been found between a2-adrenoceptors and NPY cannulation to prevent any systemic or intracranial infection.
(Everitt et al., 1984; Sawchenko et al., 1985; Harfstrand et al., Moreover, s.c. buprenorphine (0.05 mg·kg-1; Tidigesic®, Sun
1987; Huguet et al., 1993). While the a2-adrenoceptor Pharmaceuticals, Vadodara, Gujarat, India) was administered
agonist, clonidine, raises NPY immunoreactivity in the dif- to alleviate pain following surgery. Antibiotic ointment
ferent brain regions and peripheral organs (Nagata et al., (Neosporin-H®, GlaxoSmithKline Pharmaceuticals, Nashik,
1986; Franco-Cereceda et al., 1987; Smiałowska et al., 1997), Maharashtra, India) was applied locally twice daily to prevent
its antagonist, yohimbine, attenuates the orexigenic, behav- any infection at the site of the surgery. The bedding in the
ioural and endocrinological effects of NPY (Allen et al., 1987; cages was changed every alternate day. Those rats losing more
Clark et al., 1988; Heilig et al., 1988). than 10% of their body weights during the recovery period
The presence of agmatine in the PVN and its ability to were excluded from the study (Kask et al., 1998; Kamdi et al.,
enhance caloric intake suggests that, agmatine may be an 2009; Dandekar et al., 2011).
additional regulator of feeding behaviour. It is noteworthy Nuclei-specific injections into the brain require a small
that, both, agmatine and NPY are widely distributed through- fluid volume to assure regional specificity. In the present
out the brain including the PVN (Otake et al., 1998; Gor- study, 0.25 mL of fluid was injected in the vicinity of PVN. A
batyuk et al., 2001; Pedrazzini et al., 2003; Beck, 2006). similar volume range has been used previously to inject drugs
However, the pharmacological interaction of agmatine and into the PVN (Kask et al., 1998; Currie et al., 2001). Intra-PVN

British Journal of Pharmacology (2011) 164 704–718 705


BG Taksande et al.
BJP
Table 1
Effect of a2-adrenoceptor ligands and NPY agents on food intake

Food intake (g) Cumulative food


Treatment Dose 0–2 h 2–4 h 4–6 h intake (g) in 24 h

aCSF (n = 6) 3.10 ⫾ 0.35 1.38 ⫾ 0.37 0.94 ⫾ 0.53 17.76 ⫾ 1.37


Clonidine (nmol)
5 (n = 6) 1.6 ⫾ 0.21 1.14 ⫾ 0.30 0.93 ⫾ 0.20 17.05 ⫾ 1.13
10 (n = 6) 4.98 ⫾ 0.28*** 1.21 ⫾ 0.21 0.57 ⫾ 0.16 20.72 ⫾ 1.08*
20 (n = 8) 4.57 ⫾ 0.34*** 1.24 ⫾ 0.29 0.8 ⫾ 0.42 21.43 ⫾ 1.04**
Yohimbine (nmol)
2.5 (n = 8) 2.65 ⫾ 0.24 1.75 ⫾ 0.67 1.36 ⫾ 0.38 16.97 ⫾ 1.12
5 (n = 6) 3.12 ⫾ 0.43 0.76 ⫾ 0.27 1.62 ⫾ 0.47 20.72 ⫾ 1.15
10 (n = 8) 3.20 ⫾ 0.35 0.84 ⫾ 0.26 1.86 ⫾ 0.29 19.17 ⫾ 1.23
NPY (nmol)
0.1 (n = 7) 3.65 ⫾ 0.55 1.35 ⫾ 0.16 0.76 ⫾ 0.18 19.99 ⫾ 1.15
0.5 (n = 8) 7.43 ⫾ 0.59*** 1.05 ⫾ 0.38 0.72 ⫾ 0.31 21.18 ⫾ 0.93**
1 (n = 7) 7.32 ⫾ 0.46*** 0.90 ⫾ 0.31 1.15 ⫾ 0.23 21.19 ⫾ 0.73**
[Leu31, Pro34]-NPY (pmol)
10 (n = 8) 3.67 ⫾ 0.31 1.32 ⫾ 0.41 1.31 ⫾ 0.29 20.27 ⫾ 1.34
20 (n = 8) 6.96 ⫾ 0.32** 1.03 ⫾ 0.35 0.9 ⫾ 0.21 21.33 ⫾ 0.79**
40 (n = 7) 7.39 ⫾ 0.47*** 0.89 ⫾ 0.31 1.36 ⫾ 0.34 21.35 ⫾ 1.44**
BIBP3226 (nmol)
0.5 (n = 8) 3.54 ⫾ 0.29 0.95 ⫾ 0.22 1.3 ⫾ 0.33 19.43 ⫾ 1.49
1 (n = 8) 1.21 ⫾ 0.02 1.78 ⫾ 0.66 2.5 ⫾ 0.28** 18.39 ⫾ 1.4
2 (n = 8) 1.41 ⫾ 0.13 1.18 ⫾ 0.23 3.32 ⫾ 0.45*** 19.17 ⫾ 1.23

Separate groups of rats were administered, by the intra-PVN route, aCSF (control; 0.25 mL·per rat), clonidine (5, 10, 20 nmol·per rat),
yohimbine (2.5, 5, 10 nmol·per rat), NPY (0.1, 0.5, 1 nmol·per rat), [Leu31, Pro34]-NPY (10, 20, 40 pmol·per rat) or BIBP3226 (0.5, 1,
2 nmol·per rat) 10 min prior to the onset of the dark phase. Each value represents food consumption (g) ⫾ SEM (n = 6–8 per group) at the
specified time point. Data were analysed by one-way ANOVA followed by post hoc Dunnett’s test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus
aCSF-treated control animals.

injections were given over a period of 1 min, through a route in normal saline solution. The doses of agmatine,
31-gauge internal cannula connected to the 10 mL syringe a2-adrenoceptor ligands or NPY agents were selected on the
(Hamilton, NV, USA) and E73-02, -05 programmable infusion basis of a preliminary dose-response study (Table 1) and were
pump (Coulbourn Instruments, Flushing, NY, USA) with found comparable with those used earlier (Clark et al., 1988;
PE10 tubing. Since microinjections were given bilaterally into Kask et al., 1998; Morgan et al., 1998; Nakhate et al., 2009;
the PVN; each microinjection contained one-half of the total Mansur et al., 2010).
stated dose. Before administration, the syringe and the tubing
were filled with double distilled water, and a small air bubble Food intake studies
was introduced to separate the infused solution from the After a recovery period from surgery, animals were acclima-
double distilled water. The movement of an air bubble inside tized to the testing environment for 7 days as described in our
the tubing confirmed the precise flow of the solution during previous reports (Kamdi et al., 2009; Meena et al., 2009;
the injection. Nakhate et al., 2009). Those animals showing stable baseline
Agmatine sulphate, clonidine hydrochloride, yohimbine intake were selected and assigned to different treatment
hydrochloride, NPY, [Leu31, Pro34]-NPY and BIBP3226 (N2- groups (n = 6–8). Rats usually show a peak feeding activity
diphenylacetyl) - N - [(4 - hydroxy - phenyl) -methyl]-D-arginine during the dark phase (Kimura et al., 1970), which coincides
amide) were purchased from Sigma-Aldrich Co. (Saint Louis, with a2-adrenoceptors and NPY levels circadian periodicity
MO, USA). All drugs were dissolved in artificial cerebrospinal (Jhanwar-Uniyal et al., 1986; McKibbin et al., 1991). Hence,
fluid (aCSF: 140 mM NaCl, 3.35 mM KCl, 1.15 mM MgCl2, drug treatments were offered at the onset of the dark phase
1.26 mM CaCl2, 1.2 mM Na2HPO4 and 0.3 mM NaH2PO4, and feeding was monitored periodically.
pH 7.4) containing 0.1% BSA (Bhisikar et al., 2009) and On the day of study, animals were injected with either
injected directly into the PVN. As well as the intra-PVN route, vehicle or different drugs 10 min prior to the onset of the
agmatine and yohimbine were also given by the i.p. dark phase, and an individual rat was housed in a cage with

706 British Journal of Pharmacology (2011) 164 704–718


Agmatine stimulates feeding via NPY
BJP
an arrangement of grid floor. A pre-weighed quantity of food intra-PVN). Yohimbine was given 30 min prior to the injec-
pellets (30 g) was placed in the cage hopper, and food intake tion of NPY; 10 min after that, agmatine was administered.
was monitored (g) manually by weighing the leftover food at Immediately thereafter, the rats were returned to their home
the respective time points (2, 4, 6 and 24 h). Food spillage cages, and pre-weighed food pellets were offered. Food con-
collected from the tray positioned beneath the grid floor was sumption was measured (g) at 2, 4, 6 and 24 h post-injection
subtracted from the total food consumed (Kask et al., 1998; time points. We used the doses of agmatine and NPY that were
Kokare et al., 2006). The spillage by individual rats, across all ineffective by themselves if administered alone, but their
the treatment groups, was found to be negligible and mea- combination resulted in a synergistic response.
sured to the nearest 0.3 g after the 24 h time point.
Cannula placement verification
Effect of agmatine, a2-adrenoceptor ligands At the end of each experiment, the injection sites targeted at
and NPYergic agents on the spontaneous the PVN were confirmed by histological examination as
food intake described in our previous study (Kamdi et al., 2009). Briefly,
Different groups of rats (n = 6–8) were injected with either the rats were killed, and their brains were removed, sectioned,
agmatine (5, 10, 20 nmol·per rat) or aCSF (0.25 mL·per rat) stained with cresyl violet, and the injection sites targeted at the
directly into PVN. Other groups (n = 6–8) received the PVN were verified under a light microscope. The sections were
a2-adrenoceptor agonist clonidine (5, 10, 20 nmol·per rat) or also examined for the anatomical integrity of the PVN; no
antagonist, yohimbine (2.5, 5, 10 nmol·per rat), NPY (0.1, structural disruption was noticed. The data of animals with
0.5, 1 nmol·per rat), and the NPY Y1 receptors agonist, [Leu31, incorrect cannula placement were excluded from the study.
Pro34]-NPY (10, 20, 40 pmol·per rat) or antagonist, BIBP3226
(0.5, 1, 2 nmol·per rat) by the intra-PVN route. To select the Immunocytochemistry
dose for the immunocytochemistry experiments, agmatine Different groups of rats were injected with saline (1 mL·kg-1,
(20, 40, 80 mg·kg-1) or saline (1 mL·kg-1) was injected by the i.p.), agmatine (40 mg·kg-1, i.p.) or yohimbine (0.15 mg·kg-1,
i.p. route. All the injections were given 10 min prior to the i.p.) + agmatine (40 mg·kg-1, i.p.) (n = 6 per group). Two hours
onset of the dark phase. The rats were immediately returned following drug injections, rats were deeply anaesthetized
to their home cages that contained pre-weighed quantities of with thiopentone sodium (60 mg·kg-1, i.p.), perfused tran-
food pellets. Food intake was measured (g) at 2, 4, 6 and 24 h scardially with heparin-containing PBS followed by 4%
post-injection time points. paraformaldehyde in 0.1 M phosphate buffer. The brains
were removed, post-fixed, cryoprotected in 30% sucrose,
Effect of a2-adrenoceptor ligands and NPY embedded and serially sectioned in the coronal plane at
agents on agmatine-induced food intake 30 mm thickness using a cryostat and collected in PBS. The
In light of the high affinity of agmatine for a2-adrenoceptors sections were processed for the immunocytochemical label-
and the close association between a2-adrenoceptors and the ling with NPY-antibody using streptavidin-biotin-peroxidase
NPY system, the effect of their agonists and antagonists on method (Nakhate et al., 2010, 2011). Briefly, the sections were
feeding elicited by agmatine was tested. incubated in polyclonal primary antibodies against NPY
The sub-effective and effective doses were determined fol- diluted in PBS (1:5000) containing 2% normal horse serum,
lowing dose-dependent studies, and the same were employed 0.3% Triton X-100, 0.2% Kodak PhotoFlo solution and 0.08%
in the combination studies. Different groups (n = 6–8) of rats sodium azide for 48 h at 4°C. After being rinsed in PBS,
were administered, via the intra-PVN route, (i) clonidine sections were incubated in biotinylated goat anti-rabbit IgG
(5 nmol·per rat) + agmatine (5 nmol·per rat); (ii) yohimbine (1:100) for 2 h followed by in ExtrAvidin-peroxidase conju-
(2.5 nmol·per rat) + agmatine (10 nmol·per rat); (iii) NPY gate (1:100) for 45 min at room temperature. For visualiza-
(0.1 nmol·per rat) + agmatine (5 nmol·per rat); (iv) [Leu31, tion of immunoreaction product, the sections were incubated
Pro34]-NPY (10 pmol·per rat) + agmatine (5 nmol·per rat) and for 3 min in a solution containing 0.03% hydrogen peroxide
(v) BIBP3226 (0.5 nmol·per rat) + agmatine (10 nmol·per rat). and 3-amino-9-ethyl-carbazole. Reddish-brown precipitate
In all cases, agonists or antagonists of a2-adrenoceptors indicated the presence of antigen in the sections. The sec-
and NPY Y1 receptors were injected 10 min prior to agmatine tions were washed and mounted in glycerol-gelatin.
administration. Immediately thereafter, the rats were
returned to their home cages, and pre-weighed food pellets Relative quantitative analysis of
were offered. Food consumption was measured (g) at the 2, 4, NPY immunoreactivity
6 and 24 h post-injection time points. Brain sections showing NPY immunoreactivity in the PVN,
ARC, DMH, VMH and LH of the rats treated with either saline
Influence of yohimbine on synergistic (1 mL·kg-1, i.p.), agmatine (40 mg·kg-1, i.p.) or yohimbine
orexigenic effect of NPY and agmatine (0.15 mg·kg-1, i.p.) + agmatine (40 mg·kg-1, i.p.) were sub-
In order to substantiate the involvement of a2-adrenoceptors jected to the morphometric analysis (Nakhate et al., 2010,
in the orexigenic effect of agmatine and NPY, rats were treated 2011). The images (480¥) were captured using a Leica Leitz-
with (i) saline + aCSF + aCSF; (ii) yohimbine (0.15 mg·kg-1, LaborLux S microscope and analysed with Leica QWin Stan-
i.p.) + aCSF + aCSF; (iii) saline + NPY (0.1 nmol·per rat, intra- dard software (version 3.1.0) (Leica Microsystems, Wetzlar,
PVN) + aCSF; (iv) saline + aCSF + agmatine (5 nmol·per rat, Germany). The image of brain section was digitized, and the
intra-PVN) and (v) yohimbine (0.15 mg·kg-1, i.p.) + NPY background of non-immunoreactive area was considered as
(0.1 nmol·per rat, intra-PVN) + agmatine (5 nmol·per rat, threshold. The area occupied by immunostained cells and

British Journal of Pharmacology (2011) 164 704–718 707


BG Taksande et al.
BJP
aCSF (0.25 μL) Agmatine (10 nmol) Saline (1 mL kg–1) Agmatine (40 mg kg–1)
Agmatine (5 nmol) Agmatine (20 nmol) Agmatine (20 mg kg–1) Agmatine (80 mg kg–1)
A 8 B 30 A 8 B 30

Cumulative food intake (g)

Cumulative food intake (g)


25 25
6 6
Food intake (g)

Food intake (g)


20 20

4 15 4 15

10 10
2 2
5 5

0 0 0 0
0-2 2-4 4-6 24 0-2 2-4 4-6 24
Time (h) Time (h)

Figure 1 Figure 2
Effect of intra-PVN injections of agmatine on food intake. Different Effect of i.p. injections of agmatine on food intake. Different group of
group of rats were administered agmatine or aCSF just prior to lights rats were administered agmatine or saline just prior to lights out, and
out, and food intake (g) was measured at 2, 4, 6 and 24 h post- food intake (g) was measured at 2, 4, 6 and 24 h post-injections. (A)
injections. (A) Each column and bar represents the food consump- Each column and bar represents the food consumption (g) ⫾ SEM
tion (g) ⫾ SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 per group). (B)
per group). (B) Each column and bar represents the cumulative food Each column and bar represents the cumulative food consumption
consumption (g) ⫾ SEM over 24 h post injection (n = 6–8 per group). (g) ⫾ SEM over 24 h post injection (n = 6–8 per group). Data were
Data were analysed by one-way ANOVA followed by post hoc Dun- analysed by one-way ANOVA followed by post hoc Dunnett’s test.
nett’s test. *P < 0.01 and **P < 0.001 versus aCSF-treated controls. *P < 0.05 and **P < 0.001 versus saline-treated controls.

fibres, above the threshold, was filled with overlaid colour. mean comparisons by Dunnett’s test showed that, adminis-
The area (per 50 000 mm2) of the colour overlay within the tration of 10 nmol agmatine increased food intake by 263%
evaluated field was automatically obtained using Leica-QWin (P < 0.001) as compared with the control group, while at
Standard software. While evaluating the area of the NPY- 20 nmol dose, it was increased by 233% (P < 0.01) at 0–2 h
immunoreactive cells in the ARC, the area covered by the post-injection time point [F(3, 27) = 10.41, P < 0.001]. However,
immunoreactive fibres was erased and therefore not consid- lower doses of agmatine (5 nmol·per rat, intra-PVN) failed to
ered for the measurement. Similarly, during measurement of show any effect at any of the time points compared with the
immunoreactivity in the NPY-containing fibres, the immu- control group (P > 0.05). Administration of 10 and 20 nmol
noreactive cells area was not taken. The data from all animals induced a 54% (P < 0.001) and 45% (P < 0.01) increase in the
in each group were pooled separately for each brain region, cumulative food intake over a period of 24 h (Figure 1B).
and the mean ⫾ SEM was calculated. Similarly, i.p. injections of agmatine (Figure 2A) resulted
in a dose- and time-dependent increase in food intake com-
Data analyses pared with saline administration. Agmatine (40–80 mg·kg-1)
The data are presented as mean ⫾ SEM. The results were significantly increased the food intake during first 2 h
analysed using one-way ANOVA followed by post hoc analysis [F(3, 23) = 41.03, P < 0.001]. Post hoc Dunnett’s test revealed
of significance with Dunnett’s test (for dose-dependent that, agmatine 40 mg·kg-1 increased food intake by 320% (P <
studies) or Newman–Keuls multiple comparison test (for 0.001) and 44% (P < 0.05), while 80 mg·kg-1 increased by
combination and immunocytochemical studies). Differences 355% (P < 0.001) and 49% (P < 0.05) at the 2 and 24 h
were considered significant at P < 0.05. post-injection time points, respectively, as compared with
control rats. As shown in Figure 2B, the cumulative food
intake over a period of 24 h was significantly higher in the
agmatine 40 (P < 0.05) and 80 mg·kg-1 (P < 0.05) treated rats
Results as compared with control animals [F(3, 23) = 4.95, P < 0.01].
However, 20 mg·kg-1 agmatine i.p. failed to alter the basal
Agmatine stimulated food intake in level of food intake in satiated rats at any of the time points
satiated rats (P > 0.05). Agmatine at all the doses used here failed to alter
Intra-PVN administration of agmatine (10 and 20 nmol·per the food consumption during the 2–4 and 4–6 h time point
rat; Figure 1A) resulted in a significant increase in food intake after injection. This effect of agmatine on feeding was nor-
in the first two hours (0–2 h) [F(3, 27) = 9.28, P < 0.001] com- malized until 48 h (data not shown).
pared with aCSF-treated rats. However, with the 10 nmol dose
of agmatine, no significant difference in the food consump-
tion was observed during 2–4 and 4–6 h time points. More- Influence of clonidine and yohimbine on the
over, at the dose of 20 nmol, agmatine enhanced food intake orexigenic effect of agmatine
(P < 0.01) during the time interval of 2–4 h [F(3, 27) = 8.09, P < Rats injected (intra-PVN) with 10–20 nmol clonidine
0.001] but not at 4–6 h post injection (P > 0.05). Post hoc (Table 1) significantly increased food intake at all time points

708 British Journal of Pharmacology (2011) 164 704–718


Agmatine stimulates feeding via NPY
BJP
aCSF + aCSF Clonidine (5 nmol) + aCSF aCSF + aCSF Yohimbine (2.5 nmol) + aCSF
aCSF + Agmatine (5 nmol) Clonidine (5 nmol) + Agmatine (5 nmol) aCSF + Agmatine (10 nmol) Yohimbine (2.5 nmol) + Agmatine (10 nmol)
B 30 A 6 B 30
A 6

Cumulative food intake (g)


Cumulative food intake (g)
25
25

Food intake (g)


20
Food intake (g)

20 4
4
15
15
2 10
2 10
5
5
0 0
0 0 0-2 2-4 4-6 24
0-2 2-4 4-6 24
Time (h)
Time (h)

Figure 4
Figure 3
Effect of yohimbine on the orexigenic effect of agmatine. Different
Effect of clonidine on the agmatine-induced feeding. Separate
groups of rats received intra-PVN injections of (1) aCSF + aCSF, (2)
groups of rats were injected by intra-PVN route with (1) aCSF + aCSF,
aCSF + agmatine (10 nmol·per rat), (3) yohimbine (2.5 nmol·per rat)
(2) aCSF + agmatine (5 nmol·per rat), (3) clonidine (5 nmol·per rat)
+ aCSF or (4) yohimbine (2.5 nmol·per rat) + agmatine (10 nmol·per
+ aCSF or (4) clonidine (5 nmol·per rat) + agmatine (5 nmol·per rat)
rat) at the onset of the dark phase. Yohimbine was injected 10 min
at the onset of the dark phase. Clonidine was injected 10 min prior
prior to agmatine. Food intake (g) was measured at 2, 4, 6 and 24 h
to agmatine. Food intake (g) was measured at 2, 4, 6 and 24 h
post-injections. (A) Each column and bar represents the food con-
post-injections. (A) Each column and bar represents the food con-
sumption (g) ⫾ SEM between 0–2, 2–4 and 4–6 h post-injection (n
sumption (g) ⫾ SEM between 0–2, 2–4 and 4–6 h post-injection (n
= 6–8 per group). (B) Each column and bar represents the cumulative
= 6–8 per group). (B) Each column and bar represents the cumulative
food consumption (g) ⫾ SEM over 24 h post injection (n = 6–8 per
food consumption (g) ⫾ SEM over 24 h post injection (n = 6–8 per
group). Data were analysed by one-way ANOVA followed by post hoc
group). Data were analysed by one-way ANOVA followed by post hoc Newman–Keuls mean comparison test. $P < 0.01, $$P < 0.001 versus
Newman–Keuls mean comparison test. *P < 0.001 versus agmatine- saline treatment, *P < 0.01, **P < 0.001 versus agmatine-treated
and $P < 0.001 versus clonidine-treated animals. animals.

compared with control group. However, the lowest dose Influence of NPY, NPY Y1 receptor agonist
(5 nmol·per rat) of clonidine had no effect. As shown in or antagonist on the orexigenic effect
Figure 3A, pretreatment with a low dose (5 nmol·per rat) of of agmatine
clonidine (P < 0.001) significantly potentiated the food intake Injections of NPY (0.5–1 nmol·per rat) or the Y1 receptor
elicited by agmatine (5 nmol·per rat) (P < 0.001) as compared agonist, [Leu31, Pro34]-NPY (20–40 pmol·per rat) into the PVN
with their per se effects at 0–2 h [F(3, 30) = 22.92, P < 0.001]. of satiated rats induced a significant increase in food intake at
Co-administration of agmatine (5 nmol·per rat) and cloni- all time points as compared with aCSF-treated animals
dine (5 nmol·per rat), however, failed to influence the food (Table 1). However, at the lower dose, these peptides (NPY
consumption over the 2–4 and 4–6 h and cumulative food 0.1 nmol; [Leu31, Pro34]-NPY 10 pmol) failed to alter food
intake over the 24 h time point Figure 3A and B). Post hoc intake. Co-administration of a sub-effective dose of NPY
application of Newman–Keuls test showed a significant (0.1 nmol·per rat) or [Leu31, Pro34]-NPY (10 pmol·per rat) with
increase in food intake by 181% (P < 0.001) at the 2 h post- a lower dose of agmatine (5 nmol·per rat) into the PVN pro-
injection time point. The data suggest the participation of duced a synergistic effect on food consumption (Figure 5).
PVN a2-adrenoceptors in the hyperphagic effect of agmatine. Application of post hoc Newman–Keuls test revealed a signifi-
As depicted in Table 1, intra-PVN injections of yohimbine cant difference [158% (P < 0.001)] in food intake during the
(2.5–10 nmol·per rat) per se did not produce any significant 0–2 h [F(3, 32) = 39.29, P < 0.001] (Figure 5A) as well as cumu-
effect on feeding. However, yohimbine (2.5 nmol, intra-PVN) lative food intake during the 24 h [21% (P < 0.05)] [F(3, 32) =
pretreatment inhibited agmatine (10 nmol·per rat, intra- 5.42, P < 0.01] (Figure 5B) time period in rats receiving simul-
PVN)-induced food intake at 0–2 h [F(3, 31) = 18.07, P < taneous treatment of NPY with agmatine. Similarly, admin-
0.001], although it failed to influence the food consumption istration of [Leu31, Pro34]-NPY with agmatine induced an
between 2–6 and 4–6 h after agmatine injection (Figure 4A). increase in food consumption between time point of 0–2 h
Post hoc mean comparison by Newman–Keuls test demon- [142% (P < 0.001)] [F(3, 31) = 26.19, P < 0.001] (Figure 5A) and
strated that yohimbine significantly attenuated agmatine- also cumulative food intake over 24 h [28% (P < 0.001)] [F(3, 31)
induced food intake by 31% (P < 0.001) between the 0 and = 13.16, P < 0.001] post-injection time points (Figure 5B).
2 h post-injection time point. Yohimbine pretreated animals However, co-administration of agmatine with NPY or [Leu31,
also demonstrated significant inhibition of cumulative Pro34]-NPY failed to influence food consumption during
food intake over a period of 24 h [F(3, 31) = 6.44, P < 0.01] 2–4 h and 4–6 h after the treatments. These results suggest
(Figure 4B). These results confirm the involvement of the involvement of the PVN NPYergic system, particularly
a2-adrenoceptors in the PVN in the orexigenic action of NPY Y1 receptors in the food intake stimulating effect of
agmatine. agmatine.

British Journal of Pharmacology (2011) 164 704–718 709


BG Taksande et al.
BJP
aCSF + aCSF [Leu31 Pro34]-NPY (10 pmol) + aCSF aCSF + aCSF BIBP3226 (0.5 nmol) + aCSF
aCSF + Agmatine (5 nmol) NPY (0.1nmol) + Agmatine (5 nmol) aCSF + Agmatine (10 nmol) BIBP3226 (0.5 nmol) + Agmatine (10 nmol)
NPY (0.1 nmol) + aCSF [Leu31 Pro34]-NPY (10 pmol) + A 6 B 30
Agmatine (5 nmol)

Cumulative food intake (g)


A 8 B 30 25

Food intake (g)


Cumulative food intake (g)
25 4 20
6
Food intake (g)

20 15

4 15 2 10
10 5
2
5
0 0
0-2 2-4 4-6 24
0 0 Time (h)
0-2 2-4 4-6 24
Time (h)

Figure 6
Figure 5 Effect of BIBP3226 on the agmatine-induced hyperphagia. Different
Effect of NPY and [Leu31, Pro34]-NPY on the hyperphagic action of groups of rats received intra-PVN injections of (1) aCSF + aCSF,
agmatine. Separate groups of rats were administered, by the intra- (2) BIBP3226 (0.5 nmol·per rat) + aCSF, (3) aCSF + agmatine
PVN route, (1) aCSF + aCSF, (2) aCSF + agmatine (5 nmol·per rat), (10 nmol·per rat) or (4) BIBP3226 (0.5 nmol·per rat) + agmatine
(3) NPY (0.1 nmol·per rat) or [Leu31, Pro34]-NPY (10 pmol·per rat) + (10 nmol·per rat) at the onset of the dark phase. BIBP3226 was
aCSF or (4) NPY (0.1 nmol·per rat) or [Leu31, Pro34]-NPY injected 10 min prior to agmatine. Food intake (g) was measured at
(10 pmol·per rat) + agmatine (5 nmol·per rat) at the onset of the 2, 4, 6 and 24 h post-injections. (A) Each column and bar represents
dark phase. NPY or [Leu31, Pro34]-NPY were injected 10 min prior to the food consumption (g) ⫾ SEM between 0–2, 2–4 and 4–6 h
agmatine. Food intake (g) was measured at 2, 4, 6 and 24 h post- post-injection (n = 6–8 per group). (B) Each column and bar repre-
injections. (A) Each column and bar represents the food consump- sents the cumulative food consumption (g) ⫾ SEM over 24 h post
tion (g) ⫾ SEM between 0–2, 2–4 and 4–6 h post-injection (n = 6–8 injection (n = 6–8 per group). Data were analysed by one-way ANOVA
per group). (B) Each column and bar represents the cumulative food followed by post hoc Newman–Keuls test. $P < 0.001 versus aCSF-
consumption (g) ⫾ SEM over 24 h post injection (n = 6–8 per group). and *P < 0.05, **P < 0.001 versus agmatine-treated animals.
Data were analysed by one-way ANOVA followed by post hoc
Newman–Keuls mean comparison test. *P < 0.01, **P < 0.001 versus
induced increase in food intake by 30% (P < 0.001) between
agmatine treatment, $P < 0.01, $$P < 0.001 versus NPY treatment,
0 and 2 h post-injection time interval, whereas the orexigenic
#P < 0.001 versus [Leu31, Pro34]-NPY-treated animals.
effect of the co-administered agmatine and NPY between 2
and 4 h interval remained unaffected. A compensatory
BIBP3226 (1–2 nmol·per rat, intra-PVN) significantly increase in food intake was observed in the yohimbine/NPY/
inhibited the food intake at 0–2 and 2–4 h time points, which agmatine treated group at the 4–6 h post-injection time point
returned to basal level following an interval of 4–6 h. On the [F(5, 38) = 10.98, P < 0.001]. No significant difference in the
other hand, at an ineffective dose (0.5 nmol·per rat, intra- cumulative food intake at the 24 h time point was noted. The
PVN) (Table 1), BIBP3226, attenuated the hyperphagic effect administration of yohimbine, agmatine or NPY alone at
of agmatine (10 nmol·per rat, intra-PVN) at 0–2 h [F(3, 28) = the doses used here had no effect on food intake as compared
41.70, P < 0.001] (Figure 6A) and cumulative food consump- with saline- or aCSF-treated rats.
tion over a period of 24 h [F(3, 28) = 9.15, P < 0.001] (Figure 6B).
Post hoc application of Newman–Keuls test showed a signifi- Effect of agmatine, alone or in combination
cant attenuation of agmatine-induced food intake by
with yohimbine on the hypothalamic
BIBP3226 by 56% (P < 0.001) between 0 and 2 h and 21% (P
< 0.05) over the 24 h post-injection time points. Agmatine or
NPY immunoreactivity
The paraventricular nucleus of hypothalamus Changes in the
BIBP3226, alone or in combination, failed to alter the food
immunocytochemical profile of the NPY-immunoreactive
consumption during the 2–4 h and 4–6 h post treatment time
fibres in the PVN of saline (1 mL·kg-1, i.p.), agmatine
intervals. These data confirm that NPY Y1 receptors are
(40 mg·kg-1, i.p.) and yohimbine (0.15 mg·kg-1, i.p.) + agma-
involved in the hyperphagic response elicited by agmatine in
tine (40 mg·kg-1, i.p.) treated rats are summarized in
the framework of PVN.
Figure 8A–C, and the data from morphometric analysis of the
fibres are presented in Figure 9A. A significant increase was
Yohimbine attenuated the synergistic noticed in the NPY-immunoreactive fibres (by 50%, P < 0.01)
orexigenic effect induced by NPY in the PVN of agmatine-treated rats as compared with those
and agmatine in the saline-treated animals [F(2, 179) = 12.19, P < 0.01].
As shown in Figure 7, pretreatment with yohimbine However, prior administration of yohimbine with agmatine
(0.15 mg·kg-1, i.p.) significantly blocked the synergistic orexi- did not induce any noticeable change (P > 0.05) in the NPY
genic effect induced by the combination of agmatine immunoreactivity in the PVN fibres as compared with that in
(5 nmol·per rat, intra-PVN) and NPY (0.1 nmol·per rat, intra- saline-treated rats.
PVN) at 0–2 h [F(5, 38) = 17.26, P < 0.001]. Post hoc mean
comparison by Newman–Keuls test demonstrated that The arcuate nucleus of hypothalamus The immunocytochemi-
yohimbine significantly attenuated the NPY plus agmatine- cal profile of the NPY-immunoreactive cells and fibres in the

710 British Journal of Pharmacology (2011) 164 704–718


Agmatine stimulates feeding via NPY
BJP
Saline + aCSF + aCSF Saline + aCSF + Agmatine amine occurs within the neuroanatomical framework of the
Yohimbine + aCSF + aCSF Saline + NPY + Agmatine PVN. Rats injected with agmatine via the i.p. or intra-PVN
Saline + NPY + aCSF Yohimbine + NPY + Agmatine
A 8 B 30 route showed a drastic increase in food intake within 2 h,
which was evident up to 24 h. However, the data from actual

Cumulative food intake (g)


25
6
food intake studies (not cumulative) at 4, 6 and 24 h suggests
Food intake (g)

20 that the differences in cumulative food intake seem to be a


carryover effect from first 2 h time point. We may recall that
4 15
a2-adrenoceptors in the PVN mediate the hyperphagic effect
10 of noradrenaline or other a2-adrenoceptor agonists (Goldman
2 et al., 1985). Since agmatine is ubiquitously expressed in
5
the PVN (Gorbatyuk et al., 2001) and acts primarily via
0 0 a2-adrenoceptors (Li et al., 1994), it is possible that
0-2 2-4 4-6 24
Time (h)
a2-adrenoceptors may mediate the hyperphagia induced by
agmatine.
Agmatine shows a considerable degree of overlap with
Figure 7 NPY, an orexigenic peptide in the hypothalamus, with refer-
Effect of yohimbine on the orexigenic response induced by intra-PVN ence to physiological and pharmacological properties. For
co-administration of NPY and agmatine. Rats received injections of example, both these neuromodulators enhance carbohydrate
yohimbine (0.15 mg·kg-1, i.p.) or saline (1 mL·kg-1, i.p.) 30 min preferences (Prasad and Prasad, 1996; Kask et al., 1998),
before the administration of NPY (0.1 nmol·per rat, intra-PVN) or increase LHRH (Crowley and Kalra, 1987; Kalra et al., 1995)
aCSF and agmatine (5 nmol·per rat, intra-PVN) or aCSF at the onset
and are implicated in the regulation of anxiety (Deo et al.,
of the dark phase. Food intake (g) was determined at 2, 4, 6 and 24 h
2010; Taksande et al., 2010) and depression (Goyal et al.,
post-injections. (A) Each column and bar represents the food con-
sumption (g) ⫾ SEM between 0–2, 2–4 and 4–6 h post-injection (n 2006; Taksande et al., 2009). However, the direct relationship
= 6–8 per group). (B) Each column and bar represents the cumulative between endogenous agmatine and the NPY system is still
food consumption (g) ⫾ SEM over 24 h post injection (n = 6–8 per unclear. It is worth noting that a2-adrenoceptors are localized
group). Data were analysed by one-way ANOVA followed by post hoc on cell bodies in the ARC (Li et al., 1996; Levin et al., 1998),
Newman–Keuls test. $P < 0.001 versus agmatine, #P < 0.001 versus a major site of NPY synthesis (Pedrazzini et al., 2003; Beck,
NPY, *P < 0.001 versus agmatine + NPY. 2006). In the present study, treatment with agmatine signifi-
cantly raised NPY immunoreactivity in the ARC somata and
ARC following treatment with saline (1 mL·kg-1, i.p.), agma- PVN fibres, which correlate with the anatomical evidence for
tine (40 mg·kg-1, i.p.) and yohimbine (0.15 mg·kg-1, i.p.) + biosynthesis and release of NPY (Kalra et al., 1999). Interest-
agmatine (40 mg·kg-1, i.p.) are summarized in Figure 8D–F, ingly, this effect of agmatine was prevented by the pretreat-
and the data from morphometric analysis of the fibres are ment with the a2-adrenoceptor antagonist, yohimbine. These
presented in Figure 9B. While agmatine treatment caused results clearly suggest the involvement of a2-adrenoceptors in
a considerable increase (by 51%, P < 0.01) in NPY- orexigenic effect of agmatine; however, yohimbine also has
immunoreactive cells in the ARC [F(2, 179) = 6.42, P = 0.002], no significant activity at 5-HT1A receptors (Fletcher et al., 2008;
significant change (P > 0.05) was observed in yohimbine + Le et al., 2009). Although yohimbine mimics certain behav-
agmatine-treated rats as compared with that in saline- ioural effects of 5-HT1A receptor agonists (Winter and Rabin,
injected animals. Moreover, NPY-immunoreactive fibres 1992), it is unlikely that the inhibitory action of yohimbine
population in the ARC did not alter significantly (P > 0.05) on the orexigenic effect of agmatine is mediated by 5-HT1A
across all the groups of animals [F(2, 179) = 0.04, P = 0.9]. receptors. Several 5-HT1A agonists, including 8-OH-DPAT, bus-
pirone and gepirone, have been shown to produce hyperph-
The dorsomedial and ventromedial nucleus of hypothalamus and agia in free-fed rats (Dourish et al., 1985; Gilbert and Dourish,
the lateral hypothalamus Immunocytochemical application 1987; Fletcher and Davies, 1990; Ebenezer, 1992; Scopinho
of the antibodies against NPY to the brain sections of saline- et al., 2011), whereas yohimbine, even though it is a 5-HT1A
injected control rats revealed several fibres in the DMH agonist, is anorectic (Currie and Wilson, 1992; Banihashemi
(Figure 10A), while moderate fibre density was detected and Rinaman, 2006) and reported to inhibit the orexigenic
in the VMH (Figure 10E) and the LH (Figure 10I). Agma- effect of clonidine and noradrenaline (Leibowitz et al., 1985;
tine treatment, alone or in combination with yohimbine Clark et al., 1988).
did not produce any significant change (P > 0.05) in the The presynaptic localization of a2-adrenoceptors in the
NPY-immunoreactive fibres population in the DMH PVN is well-documented (Boudaba et al., 2003; Li et al.,
[F(2, 179) = 0.1, P = 0.8] (Figure 10B–D), VMH [F(2, 179) = 0.5, 2005). Therefore, the possibility exists that, in addition to
P = 0.6] (Figure 10F–H) and the LH [F(2, 179) = 3.03, P = 0.05] increasing NPY synthesis in the ARC, agmatine may trigger
(Figure 10J–L) as compared with those in the saline-treated NPY release by its presynaptic action in the PVN. In this
group. connection, we note that rats with hypothalamic 6-OHDA
lesions show reduce noradrenaline levels and an increase in
NPY expression (Kalra et al., 1998). More importantly, agma-
Discussion tine reduces noradrenaline release from presynaptic nerve
terminals, an effect possibly mediated through presynaptic
The results of the present study demonstrate, for the first a2-adrenoceptors (Molderings and Gothert, 1995; Gonzalez
time, that the orexigenic role of agmatine, an endogenous et al., 1996). Based on these findings, we suggest that agma-

British Journal of Pharmacology (2011) 164 704–718 711


BG Taksande et al.
BJP

Figure 8
Photomicrographs showing NPY-immunoreactive fibres (arrows) and/or cells (arrowheads) in the PVN (paraventricular nucleus of hypothalamus)
and ARC (arcuate nucleus of hypothalamus) of rats following administrations of saline (1 mL·kg-1, i.p.; A and D), agmatine (40 mg·kg-1, i.p.; B and
E) and yohimbine (0.15 mg·kg-1, i.p.) + agmatine (40 mg·kg-1, i.p.; C and F). Note a significant increase in NPY immunoreactivity in the PVN fibres
and ARC cells following agmatine treatment. These changes were prevented by pretreatment with yohimbine. 3V, third ventricle. Scale bar =
200 mm.

tine reduces noradrenaline activity in the PVN via presynap- et al., 2003; Beck, 2006). Since these nuclei also display
tic a2-adrenoceptors, which in turn may promote the release a2-adrenoceptors (Jhanwar-Uniyal and Leibowitz, 1986;
of NPY and consequentially stimulate eating. Jhanwar-Uniyal et al., 1991; Levin et al., 1998), it is possible
Within the hypothalamus, NPY is synthesized in the ARC that agmatine might influence NPY in the framework of these
and transported to the PVN, DMH, VMH and LH (Pedrazzini nuclear groups. However, in the present study, although NPY

712 British Journal of Pharmacology (2011) 164 704–718


Agmatine stimulates feeding via NPY
BJP

Figure 9
Representation of the semiquantitative morphometric analysis of NPY immunoreactivity in fibres and/or cells of the paraventricular nucleus of
hypothalamus (PVN; A) and arcuate nucleus of hypothalamus (ARC; B) of rats following injections of saline (SAL;1 mL·kg-1, i.p.), agmatine (AGM;
40 mg·kg-1, i.p.) and yohimbine (YOH; 0.15 mg·kg-1, i.p.) + AGM (40 mg·kg-1, i.p.). The outline of the transverse section through the brain
indicates the regions of the PVN and the ARC at the co-ordinates -1.80 mm and -2.56 mm with reference to bregma, respectively (Paxinos and
Watson, 1998), from which the measurements were collated (square, not to scale). AHC, central part of the anterior hypothalamus; DMH,
dorsomedial nucleus of hypothalamus; ME, median eminence; Rch, retrochiasmatic nucleus; VMH, ventromedial nucleus of hypothalamus. The
values in the histograms are shown as the mean ⫾ SEM of five measurements from predetermined fields of the PVN and ARC on both the sides
of each brain (n = 6). Data were analysed by one-way ANOVA followed by post hoc Newman–Keuls multiple comparison test. *P < 0.01 versus SAL
+ SAL-treated animals.

immunoreactivity was increased following agmatine treat- 1985; Matthews et al., 1986). Interestingly, both agmatine
ment in the ARC and PVN, it was not altered in the DMH, and a2-adrenoceptors are particularly enriched in the PVN
VMH and LH. We do not know the underlying reasons for (Gorbatyuk et al., 2001). We found that intra-PVN adminis-
this; however, the available literature suggest a key role for tration of the a2-adrenoceptor agonist clonidine potentiated,
NPY within the ARC–PVN pathway in the regulation of whereas the antagonist yohimbine attenuated, the hyperph-
energy homeostasis. Earlier studies demonstrated the inter- agic effect of agmatine. Moreover, yohimbine not only pre-
relationship of NPY with other systems like cocaine- and vented the augmentation of NPY immunoreactivity in the
amphetamine-regulated transcript (CART) peptide and ARC and PVN associated with the orexigenic effect of agma-
leptin, mainly at the level of ARC and PVN (Satoh et al., 1997; tine but also blocked the synergistic effect of exogenously
Lambert et al., 1998). Anorectic agents like nicotine specifi- administered NPY and agmatine. It is also noteworthy that
cally influence NPY within the ARC and PVN, but not in the NPY administration into the PVN does not stimulate feeding
LH (Nakhate et al., 2009). It is also worthy of note that obesity through the release of endogenous noradrenaline (Stanley
is associated with increased a2-adrenoceptors in the PVN, but and Leibowitz, 1985), whereas the a2-adrenoceptor agonist,
not in other hypothalamic nuclei (Jhanwar-Uniyal et al., clonidine increases the NPY content (Nagata et al., 1986;
1991). Similarly, food deprivation mainly reduces a2- Franco-Cereceda et al., 1987; Smiałowska et al., 1997). Collec-
adrenoceptors binding sites in the ARC and PVN (Jhanwar- tively, these findings raise the possibility that the NPY system
Uniyal and Leibowitz, 1986; Jhanwar-Uniyal et al., 1991). We in the PVN acts downstream to endogenous agmatine and
therefore suggest that NPY within the neuroanatomical noradrenaline to stimulate food intake.
framework of the ARC and PVN may play a key role in The endogenous regulation of feeding behaviour by NPY
mediating the hyperphagic effect of agmatine. Thus, it can be via NPY Y1 receptors is now well established. NPY is known to
argued that NPY in the DMH, VMH and LH is not involved in act via six types of receptors identified as NPY Y1-6, and of
the effects of agmatine, associated with feeding behaviour. these, NPY Y1 and NPY Y5 receptors in the PVN are implicated
Noradrenergic projections into the PVN are crucially in the orexigenic effects of NPY (Pedrazzini et al., 2003; Beck,
involved in the regulation of feeding and energy balance 2006). However, other findings have suggested a predomi-
(Goldman et al., 1985; Peinado and Myers, 1987; Wellman nant role of NPY Y1 receptors in the feeding behaviour (Kana-
et al., 1993). The PVN exhibits a sharp increase in noradrena- tani et al., 2000; Turnbull et al., 2002; Beck, 2006). NPY Y1,
line levels and a2-adrenoceptors at the onset of the dark but not NPY Y5, knockout mice showed significant suppres-
phase, a phase of peak feeding activity in rats (Jhanwar- sion in feeding behaviour (Kanatani et al., 2000). In the
Uniyal et al., 1986; Morien et al., 1999). Moreover, noradrena- present study, intra-PVN injections of NPY as well as the NPY
line injection into the PVN stimulates food intake through Y1 receptor agonist, [Leu31, Pro34]-NPY significantly potenti-
a2-adrenoceptors (Goldman et al., 1985; Leibowitz et al., ated, while the antagonist, BIBP3226, completely reversed

British Journal of Pharmacology (2011) 164 704–718 713


BG Taksande et al.
BJP

Figure 10
Photomicrographs showing NPY-immunoreactive fibres (arrows) in the dorsomedial nucleus of hypothalamus (DMH), ventromedial nucleus of
hypothalamus (VMH) and lateral hypothalamus (LH) of rats following saline (SAL; A, E and I), agmatine (AGM; B, F and J) and yohimbine (YOH)
+ AGM (C, G and K). Moreover, the semiquantitative morphometric analysis of NPY immunoreactivity in fibres of the DMH, VMH and LH is
represented in panels D, H and I respectively. The outlines of the transverse sections through brain indicate the regions of the DMH or LH at the
coordinates -2.56 mm and the VMH at the coordinates -2.80 mm with reference to bregma, respectively (Paxinos and Watson, 1998), from
which the measurements were collated (not to scale). 3V, third ventricle; ARC, arcuate nucleus of hypothalamus; F, fornix; ME, median eminence.
The values in the histograms are shown as the mean ⫾ SEM of five measurements from predetermined fields of the DMH, VMH and LH on both
the sides of each brain (n = 6). Data were analysed by one-way ANOVA followed by post hoc Newman–Keuls multiple comparison test. Scale bar
= 200 mm (DMH) and 100 mm (VMH and LH).

714 British Journal of Pharmacology (2011) 164 704–718


Agmatine stimulates feeding via NPY
BJP
the agmatine-induced hyperphagia in satiated rats. Kask et al.
(1998) proposed that activation of NPY Y1 receptors is the
final common pathway initiating feeding response in
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Feeding behaviour often follows a circadian rhythm, with
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Conflict of interest putative serotonin agonist 8-hydroxy-2-(di-n-propylamino) tetralin
(8-OH-DPAT) elicit feeding in the rat. Psychopharmacology (Berl)
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