Full text of DOI:10.1038/sj.bjp.0704696

British Journal of Pharmacology (2002) 136, 341 ± 346
ã
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Blockade of body weight gain and plasma corticosterone levels in
Zucker fatty rats using an orally active neuropeptide Y Y1
antagonist
1
1
1
1
*,1Akane Ishihara, Akio Kanatani, Megumu Okada, Masayasu Hidaka, Takeshi Tanaka,
1
1
1
1
1Satoshi Mashiko, Akira Gomori, Tetsuya Kanno, Mikiko Hata, Maki Kanesaka,
1Yushin Tominaga, Naga-aki Sato, Masahiko Kobayashi, Takashi Murai, Keiko Watanabe,
1
1
1
1
1Yasuyuki Ishii, Takahiro Fukuroda, Takehiro Fukami & Masaki Ihara
1
1
1
1Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
1 An experiment was conducted to examine whether a potent, orally active and highly selective
neuropeptide Y Y1 receptor antagonist attenuates hyperphagia and obesity in genetically obese
Zucker fatty rats.
Oral administration of the Y1 antagonist (30 and 100 mg kg71, once daily for 2 weeks)
2
signi®cantly suppressed the daily food intake and body weight gain in Zucker fatty rats accompanied
with a reduction of fat cell size and plasma corticosterone levels.
3
Despite the fact that food intake was gradually returned to near the control level, the body
weight of the treated animals remained signi®cantly less when compared to that of the controls for
the duration of the treatment.
4
These results suggest that the Y1 receptor, at least in part, participate in pathophysiological
feeding and/or fat accumulation observed in Zucker fatty rats. Y1 antagonists might be useful for
the treatment of obesity.
British Journal of Pharmacology (2002) 136, 341 ± 346
Keywords: Neuropeptide Y, a Y1 antagonist; body weight gain; plasma corticosterone levels; Zucker fatty rats; obesity;
feeding behaviour
Abbreviations: ADX, adrenalectomy; ANOVA, analysis of valiance; Cort., corticosterone; ELISA, enzyme-linked immuno-
sorbent assay; FFA, free fatty acid; HPA, hypothalamic-pituitary-adrenal; ICV, intracerebroventricular; a-
MSH, a-melanocyte-stimulating hormone; NPY, neuropeptide Y; PFA, paraformaldehyde; PP, pancreatic
polypeptide; PYY, peptide YY; RIA, radioimmunoassay; TG, triglyceride; total CH, total cholesterol
Introduction
Neuropeptide Y (NPY) is a highly conserved 36 amino acid
peptide that has been shown to have potent, centrally-
mediated orexigenic e€ects (Tatemoto et al., 1982; Clark et
al., 1984; Stanley & Leibowitz, 1984). It is a member of a
peptide family which also includes peptide YY (PYY), and
Five distinct subtypes of NPY receptors (Y1, Y2, Y4, Y5
and Y6) have been cloned so far, and more than one receptor
subtype seems to be involved in the NPY-mediated feeding
(Blomqvist & Herzog, 1997). Among them, the Y1 receptor is
considered to be one of the major feeding receptors, since Y1
antagonists with distinct chemical structures are reported to
suppress feeding. This is also supported by a ®ndings that
mice lacking Y1 receptor showed a signi®cantly diminished
response to exogenously administered NPY (Kanatani et al.,
2000b).
Genetically obese Zucker fatty rat (fa/fa) is extensively
used for the study of obesity. Hyperphagia, decreased energy
expenditure, hyperinsulinaemia, and hypercorticosteronaemia
based on a disfunction of leptin long-form receptors are
characteristics of Zucker fatty rats (Bray, 1977). As a
pancreatic polypeptide (PP) (Tatemoto
& Mutt, 1980;
Tatemoto et al., 1982). Chronic administration of NPY into
the brain results in hyperphagia and body weight gain,
reduces energy expenditure, and increases lipogenic activity in
the liver and adipose tissue (Stanley et al., 1986; Zarjevski et
al., 1993). It has been also reported that concentrations of
NPY and its mRNA in the hypothalamus are markedly
increased during food deprivation and in some genetic
models of obesity in rodents (Sanacora et al., 1990; White
et al., 1990; Kesterson et al., 1997; Guan et al., 1998).
Moreover, it was well known that NPY-de®cient ob/ob mice
are less obese than ob/ob mice with reduction of food intake
(Erikson et al., 1996). From these data it is inferred that
NPY may be one of the major regulators of energy
homeostasis.
consequence,
a large dys-regulation of leptin-controlled
hypothalamic neuropeptides is observed, and NPY is thought
to be one responsible factor for the abnormality (Beck et al.,
1990; 1993). Levels of NPY (McKibbin et al., 1991) and its
mRNA (Sanacora et al., 1990), and NPY secretion (Dryden
et al., 1995) are increased in the hypothalamic nuclei involved
in the regulation of feeding behaviour. In addition,
hyperphagia, obesity and metabolic changes such as hyper-
insulinaemia, hypercorticosteroidimia and hyperlipidaemia,
*Author for correspondence; E-mail: isihraan@banyu.co.jp

342
A. Ishihara et al
Anti-obesity effect of a Y1 antagonist in Zucker rats
which were seen in Zucker fatty rats, were mimicked by
repeated administration of NPY in normal rats (Stanley et
al., 1986). We previously demonstrated that a potent peptidic
Y1 antagonist, 1229U91 more potently inhibited the
spontaneous feeding in Zucker fatty rats than lean litter
mates after intracerebroventricular (ICV) administration
(Ishihara et al., 1998). Taken together, we hypothesized that
Y1 receptor play a key role in the generation of obesity in
Zucker fatty rats.
Recently we reported that an orally-active and highly
selective Y1 antagonist can suppress NPY-induced feeding in
SD rats (Kanatani et al., 1999). To clarify the physiological
role of the Y1 receptor in the development of obesity in
Zucker fatty rats, here we further examined the chronic e€ect
of the Y1 antagonist in this model animal.
ELISA kit (Morinaga, Japan), and corticosterone level was
measured by RIA kit (Amersham, Biosciences, Japan). Liver
was taken for weighing. Epididymal adipose tissue and liver
were saved for histological investigation. The adipose tissue
was ®xed with 4% paraformaldehyde (PFA, Chiyoda
Junyaku, Japan) for overnight and imbedded in paran
wax. Three mm thick sections were made, stained with
Masson trichrome staining and cell size was measured. Liver
tissues were freshly frozen and 10 mm sections were made.
Slices were ®xed with 4% PFA and stained with oil red O
(Ohroma, Germany).
Zucker lean rats (n=6) did not undergo administration.
After overnight fasting, blood and histological parameters
were collected similarly to the Zucker fatty rats. These values
were used as references, and not included in the statistical
analysis.
Methods
Statistical analysis
Drugs
ANOVA or repeated measures ANOVA followed by
Bonferroni test was used for statistical analysis.
The NPY Y1 antagonist (6-(5-ethyl-1,3-thiazol-2-ylthio-
methyl)-2-[3-methoxy-5-(2-propenyloxycarbonylamino)benzy-
lamino]-4-morpholinopyridine) was synthesized by Banyu
Pharmaceutical Co. Ltd. The structure and in vitro
pharmacological pro®les of the Y1 antagonist are reported
previously (Kanatani et al., 1999). Other chemical substances
were purchased from commercial sources.
Results
Figure 1 shows the daily food (A), water intake (B) and body
weight change (C) in Zucker fatty rats for 2 weeks. Orally
administered Y1 antagonist at
a
dose of 100 mg kg71
signi®cantly suppressed spontaneous feeding (Figure 1A).
The greatest fall in food intake was observed on the 4th day
after starting drug administration. Thereafter, food intake
gradually returned near to control levels, although a small
reduction of food intake was still observed even after 2
weeks. The Y1 antagonist showed a tendency to suppress
water intake at 100 mg kg71, however the e€ect was not
Animals
Male Zucker fatty (fa/fa) and age-matched lean (Fa/Fa) rats
(11 ± 14 weeks old, Charles River Japan) were used. They
were housed in individual cages under controlled temperature
(23+28C), humidity (55+15%) and light-dark cycle (7:00 ±
19:00 h). Water and pellet food (CE-2, CLEA Japan Inc.)
were available ad libitum. All experimental procedures
followed the Japanese Pharmacological Society Guideline
for Animal Use.
Experimental procedure
Zucker fatty rats were divided into three groups to match
average values of basal food intake and body weight (n=6).
Each group was orally administered either vehicle or the Y1
antagonist (suspended in 0.5% methylcellulose (Wako Pure
Chemical Industries, Japan) in distilled water) at doses of 30
or 100 mg kg71 daily for 2 weeks by gavage. Daily food and
water intake and body weight were measured. The adminis-
tration was done during the last hour of the light period
following the measurement.
After the ®nal administration, Zucker fatty rats were
fasted overnight and euthanized by collecting whole blood
from the abdominal aorta under ether anaesthesia. Blood
collection was done during early morning. Plasma glucose,
triglyceride, free fatty acid (FFA), total cholesterol, insulin
and corticosterone levels were measured. Glucose, triglycer-
ide, FFA, total cholesterol levels were measured by
enzymatic methods using commercial kits (Determiner GL-
E kit, Determiner L TGII, Determiner L TCII (Kyowa
Medex, Japan) and NEFA-HA testwako (II) (Wako Pure
Chemical Industries, Japan)). Insulin level was measured by
Figure 1 E€ect of the Y1 antagonist on daily food (A) and water
intake (B), and body weight change (C) in Zucker fatty rats. Values
are mean+s.e.mean. *P50.05 vs vehicle-treated group (n=6,
repeated measurement ANOVA followed by Bonferroni test).
British Journal of Pharmacology vol 136 (3)

A. Ishihara et al
Anti-obesity effect of a Y1 antagonist in Zucker rats
343
signi®cant because of large variation (Figure 1B). The 2-week
treatment of the higher dose of the Y1 antagonist
not detect signi®cant change of plasma levels of glucose,
triglyceride, cholesterol and insulin.
(100 mg kg71
) signi®cantly suppressed body weight gain
(Figure 1C). Despite the return of food intake near to
control level, body weight remained signi®cantly decreased
from the control until the end of the experiment period.
Figure 2 shows the adipose cell size of epididymal fat
depot. Data from Zucker lean rats are also shown as a
reference. The Y1 antagonist signi®cantly reduced the fat cell
size at both doses.
Figure 3 shows the liver weight. Liver weight of the Zucker
fatty rats was twice as that of lean rats. The Y1 antagonist
showed no e€ect on liver weight. However, histological
observation showed that the Y1 antagonist tended to
decrease liver lipid accumulation assessed by oil red-O
staining.
Discussion
The present ®ndings show that chronic treatment with a Y1-
selective antagonist can suppress the body weight gain in
Zucker fatty rats. Two-week treatment with the Y1
antagonist at 100 mg kg71 induced a slight, but sustained
reduction of the body weight. The body weight was 3% less
than that of vehicle-treated rats at the end of the experiment.
This change in body weight was accompanied by feeding
suppression, reduction of adipose cell size, and plasma
corticosterone levels. The present results indicate that NPY
Y1 system is, at least in part, involved in the development of
obesity in Zucker fatty rats.
Table 1 shows the fasting plasma biochemical parameters.
Data from lean rats are also shown as a reference. After 2
weeks of treatment, fasting plasma levels of glucocorticoid
were reduced signi®cantly by the treatment, while we could
Previously we showed that ICV administration of the Y1
antagonist inhibited the NPY-induced feeding in SD rats at
a dose of 200 mg, and that acute ICV (200 mg) or PO dosing
Figure 2 Chronic e€ect of Y1 antagonist on epididymal adipose cell size. (A) Microscopic observation of epididymal fat tissues
from lean, vehicle- and 100 mg kg71 of the Y1 antagonist-treated Zucker fatty rats. (B) Fat cell size of the epididymal adipose tissue
from vehicle- and Y1 antagonist-treated Zucker fatty rats. Values are mean+s.e.mean. *P50.05 vs vehicle-treated group (n=6,
ANOVA followed by Bonferroni test). Values of Zucker lean rats are shown as a reference (not included in statistical analysis).
Figure 3 Chronic e€ect of Y1 antagonist on liver weight and fat accumulation. (A) Liver weight of the vehicle- or Y1 antagonist-
treated Zucker fatty rats. Values are mean+s.e.mean. (n=6). Values of Zucker lean rats are shown as a reference. (B) Microscopic
observation of liver from lean, vehicle- or 100 mg kg71 of the Y1 antagonist-treated Zucker fatty rats.
British Journal of Pharmacology vol 136 (3)

344
A. Ishihara et al
Anti-obesity effect of a Y1 antagonist in Zucker rats
Table 1 E€ect of Y1 antagonist on endocrine parameters in plasma of Zucker fatty rats
Treatment
(mg kg71
TG
(mg dl71
Total CH
(mg dl71
Glucose
(mg dl71
Insulin
(mU ml71
Cort.
(ng ml71
)
)
)
)
)
)
Fatty
Lean
Vehicle
30
100
658.7+200.4
540.7+85.0
481.0+61.6
140.0+16.3
154.3+15.3
140.8+6.0
187.4+18.4
160.2+33.2
164.2+19.9
150.6+8.8
139.3+20.7
113.9+17.3
485.9+53.4
412.1+39.5
279.9+36.8*
±
34.8+3.6
57.7+2.9
113.2+3.4
5.4+1.5
279.3+40.4
TG: triglyceride; Total CH: total cholesterol; Cort.: corticosterone. *P50.05 vs vehicle-treated group. Values are mean+s.e.mean
(n=6). Values of Zucker lean rats are shown as a reference (not included in statistical analysis).
(100 mg kg71) of this compound signi®cantly reduced the
spontaneous feeding in Zucker fatty rats (Kanatani et al.,
1999). The present result is very consistent with the previous
one, and the extent of the feeding suppression was similar in
these two studies. The brain concentration of this compound
was 0.5 mM at 24 h after PO dosing at 100 mg kg71, which
is more than 100 fold higher concentration than the IC50
value in functional assay (3.2 nM, NPY-induced Ca2+ in¯ux
in cells expressing human Y1 receptors, Kanatani et al.,
1999). This compound showed a high speci®city for Y1
receptors (Kanatani et al., 1999). In addition, we identi®ed
that this compound hardly a€ected normal feeding in SD
rats (Ishihara et al., unpublished observations, data not
shown), which is well matched to the results from
structurally diverse Y1 antagonists showing that the anti-
orexigenic e€ects of these compounds were much smaller in
lean animals than those in obese animals (Ishihara et al.,
1998; Kanatani et al., 2001). Deduced from these data, it is
expected that the anti-obese e€ect of this compound is
mediated by the Y1 receptor blockade, and that the dosage
level is enough to block the brain Y1 receptors for 24 h.
However, further con®rmation of brain Y1 receptor
occupancy with this compound using ex vivo binding is
remaining to be addressed.
Although both Y1 and Y5 receptors are reported to be
involved in feeding regulation, it is not clear which subtype
has a physiological role in development of obesity in Zucker
fatty rats. In the present experiment, the reduction of body
weight was accompanied by a signi®cant feeding suppression
induced by the Y1 antagonist treatment. This observation is
in agreement with our previous study that the spontaneous
feeding in Zucker fatty rats is remarkably suppressed by
1229U91, a peptidic NPY Y1 antagonist with weak agonistic
activities for Y4 and Y5 receptors (Ishihara et al., 1998;
Kanatani et al., 1998). In that study, the feeding suppression
by 1229U91 was much more potent in Zucker fatty rats than
that in Zucker lean rats. These ®ndings are in agreement with
the hypothesis that the NPY Y1 system is much stimulated in
the brain of the Zucker fatty rats. On the other hand, an
orally active and selective Y5 antagonist inhibited a Y5
agonist-induced feeding but not the NPY-induced feeding in
SD rats (Kanatani et al., 2000a). In addition, we showed that
structurally-distinct two Y5 antagonists did not a€ect the
spontaneous feeding in Zucker fatty rats (Kanatani et al.,
1997; 2000c). Consistent with this, it is reported that the
density of Y5 or Y2, but not Y1 receptors, is signi®cantly
reduced in a number of hypothalamic and non-hypothalamic
brain regions of Zucker fatty rats as compared to the lean
litter mates (Widdowson, 1997). Together, the present results
indicate that the NPY Y1 receptor system is involved in the
hyperphagia in Zucker fatty rats.
The greatest fall in food intake was observed on the 4th
day after starting drug administration. Thereafter, food
intake gradually returned near to control levels, although a
small reduction of food intake was still observed even after 2
weeks. This result may indicate that some other counter-
regulatory mechanisms are induced by continuous blockade
of the NPY Y1 system. A similar attenuation of feeding
suppression is also seen in other anti-orexigenic substances
such as amphetamine, fen¯uramine and dexfen¯uramine
(Levitsky et al., 1980; Rowland & Carlton, 1986; Rowland,
1994). It is also reported that reducing body weight prior to
drug administration diminished the anorectic e€ectiveness of
amphetamine (Levitsky et al., 1980). In addition, the
decrease in the e€ectiveness of amphetamine is limited to
the anorexia; the behavioural e€ects of the drug such as
increased motor activity and stereotypy do not show
tolerance with repeated administrations (Lewander, 1971).
Thus, the attenuation of feeding suppression may be
common feature of drugs which a€ect feeding, and it may
re¯ect a successful physiological and behavioural adjustment
to an appropriate level of body weight (Levitsky et al., 1980).
Precise mechanisms for these phenomena are still unclear. To
clarify the mechanism, further evaluation of the change of
hypothalamic levels of NPY, NPY receptors and other
factors, such as leptin, a-MSH, orexin, galanin and so on, is
needed.
Although the reduction of food intake diminished at the
end of the experiment, the Y1 antagonist at 100 mg kg71
day71 suppressed body weight gain signi®cantly. The result
suggests that some mechanism(s) other than feeding
suppression is also concerned with the inhibition of body
weight gain. One possible mechanism is suppression of
corticosterone; the Y1 antagonist signi®cantly reduced the
plasma corticosterone levels to near that of lean litter mates.
Elevated circulating levels of corticosterone is thought to
play a role in the development and maintenance of obesity;
Obesity is often accompanied by hypercorticism, over-
responsiveness of the hypothalamic-pituitary-adrenal (HPA)
axis or increased 24-h urinary corticosterone output
(Cunningham et al., 1986; Guillaume-Gentil et al., 1990).
Treatment with corticosterone can promote the development
of obesity with a regional fat accumulation (Tataranni et al.,
1996). On the other hand, adrenalectomy (ADX) prevents
genetic as well as dietary obesity in animals, and the e€ect
are reversed by corticosterone replacement (Castonguay et
al., 1986; Freedman et al., 1986; Bray et al., 1992). In
addition, ADX is reported to prevents NPY-induced obesity
British Journal of Pharmacology vol 136 (3)

A. Ishihara et al
Anti-obesity effect of a Y1 antagonist in Zucker rats
345
(Sainsbury et al., 1997). It is known that NPY increases
corticosterone levels through the action of HPA axis
(Zarjevski et al., 1993), and that chronic NPY infusion is
reported to induce a sustained increase in corticosterone
(Raposinho et al., 2001). From these ®ndings, the present
results suggest that the stimulation of NPY system produced
hypercorticosteronism in Zucker fatty rats and that the Y1
antagonist may have ameliorated the obesity via suppression
of plasma corticosterone levels.
a precise examination of the anti-obesity mechanism(s) of this
compound such as measurement of oxygen consumption,
hormones and enzyme activities concerning lipid/glucose
metabolism.
In this study, the Y1 antagonist tended to suppress water
intake at a dose of 100 mg kg71, although the di€erence was
not signi®cant because of large variation. NPY is reported to
induce water intake as well as food intake (Stanley et al.,
1986). Food and water intake could change co-relatively, so
the e€ect of the Y1 antagonist on water intake may be the
secondary e€ect of feeding suppression. In agreement with
this, the tendency of reduction in water intake is observed on
the days 4 ± 7 at 100 mg kg71, which is parallel to the change
of food intake. Any abnormal change in gross behaviour or
motor activity was not observed after administration of the
Y1 antagonist. These ®ndings suggest that the suppression of
food and water intake is not due to abnormal changes in
behaviour. Likewise, it seems not likely that the anti-
orexigenic e€ects of Y1 antagonists are due to conditioned
taste aversions or changes of taste sensation, since this
compound hardly a€ected the feeding of lean SD rats
(Ishihara et al., data not shown). However, currently we
have no data which can exclude the possibility that the Y1
antagonist produced a non-speci®c e€ect on taste sensation,
and this is remaining to be addressed.
Cell size of epididymal adipose tissue was signi®cantly
reduced by the Y1 antagonist even at a dose of 30 mg kg71
,
at which dose body weight was not changed. Similar results
was reported that ADX decreases the size of the gonadal and
retroperitoneal fat by reducing cell size, and in short-term
studies, these e€ects were observed before signi®cant changes
in total carcass fat content or food intake (Castonguay et al.,
1986). The Y1 antagonist may have speci®cally a€ected the
adipose tissue cell size, although more precise examination of
total/regional body fat content is necessary by body
composition analysis or a densitometry.
NPY is also reported to be involved in many other
physiological functions. For example, NPY suppresses
thermogenesis in brown adipose tissue via suppression of
sympathetic nervous activities (Egawa et al., 1991; Szreder et
al., 1994). Inhibitory regulation of lipolysis in white
adipocytes is also reported (Castan et al., 1994). It is known
that chronic ICV administration of NPY mimics hormonal
and metabolic changes of obesity in rats even when they are
pair-fed with the control animals, implying that NPY has
diverse e€ects that produce obesity via NPY receptors
(Zarjevski et al., 1993). The Y1 antagonist may have elicited
an anti-obese e€ect in Zucker fatty rats by inhibiting these
e€ects of NPY as well as altering feeding regulation. We need
In conclusion, an orally active and selective Y1 antagonist
signi®cantly suppressed daily food intake and body weight
gain, as well as hypercorticism in Zucker fatty rats. These
results suggest that the Y1 receptor, at least in part,
participates in the development of obesity in Zucker fatty
rats. The Y1 receptor may be a promising target to regulate
energy balance, and a Y1 antagonist is a potential agent for
treatment of obesity.
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(Received December 4, 2001
Revised February 14, 2002
Accepted February 27, 2002)
British Journal of Pharmacology vol 136 (3)