Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 10455–10460, September 1997
Physiology
Synergistic interaction between leptin and cholecystokinin to
reduce short-term food intake in lean mice
MARÍA DOLORES BARRACHINA, VICENTE MARTÍNEZ, LIXIN WANG, JEN YU WEI,
AND
YVETTE TACHÉ*
CURE: Digestive Diseases Research Center, West Los Angeles Veterans Affairs Medical Center, Department of Medicine, and Brain Research Institute,
University of California, Los Angeles, CA 90073
Communicated by Wylie Vale, Salk Institute for Biological Sciences, La Jolla, CA, July 12, 1997 (received for review May 13, 1997)
initiated at the periphery through the activation of capsaicinsensitive vagal afferent fibers that relays the information to
brain sites (20–24). There is also evidence of synergistic
interaction between peripheral CCK and other hormones
released postprandially such as insulin and glucagon in rats (25,
26). Recently, we observed in an in vitro vagus–stomach
preparation that CCK increases responsiveness of vagal afferents to leptin as assessed electrophysiologically (27). Therefore, in the present study we investigate the potential functional synergistic interaction between peripheral leptin and
CCK in the short-term modulation of food intake in lean mice.
ABSTRACT
Leptin is a circulating protein involved in the
long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety
signals. We investigated the interaction between leptin and
CCK-8 in the short-term regulation of food intake induced by
24-hr fasting in lean mice. Leptin, injected intraperitoneally
(i.p.) at low doses (4–120 mgykg), which did not influence feeding
behavior for the first 3 hr postinjection, decreased food intake
dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was
not observed between leptin and bombesin. The food-reducing
effect of leptin injected with CCK was not associated with
alterations in gastric emptying or locomotor behavior. Leptin–
CCK action was blocked by systemic capsaicin at a dose inducing
functional ablation of sensory afferent fibers and by devazepide,
a CCK-A receptor antagonist but not by the CCK-B receptor
antagonist, L-365,260. The decrease in food intake which occurs
5 hr after i.p. injection of leptin alone was also blunted by
devazepide. Coinjection of leptin and CCK enhanced the number
of Fos-positive cells in the hypothalamic paraventricular nucleus
by 60%, whereas leptin or CCK alone did not modify Fos
expression. These results indicate the existence of a functional
synergistic interaction between leptin and CCK leading to early
suppression of food intake which involves CCK-A receptors and
capsaicin-sensitive afferent fibers.
MATERIALS AND METHODS
Animals. Male lean mice (C57BLy6 1y1, 6 to 7 weeks old,
20–25 g) (Harlan Laboratories) were maintained ad libitum on
standard Purina laboratory chow and tap water. They were
housed, five per cage, under conditions of controlled temperature (20 6 1°C), humidity (30–35%), and lighting (06:00–
18:00 h). All experiments were started between 8:00 and 9:00
a.m. and performed according to protocols approved by the
Veterans Administration Animal Care and Use Committee
(Animal Component of Research Protocol number 96-89-8).
Reagents. Aliquots of recombinant murine leptin (Amgen
Biologicals; 1 mgyml in vehicle: 0.1% fetal bovine serum in 1:10
PBS, pH 7.4) or sulfated CCK octapeptide (Peninsula Laboratories; 1 mgyml in saline) were stored at 270°C until used.
Stock solutions were diluted in saline immediately before each
experiment. Bombesin (Clayton Foundation Laboratories,
Salk Institute, La Jolla, CA) was freshly dissolved in saline
immediately before use. Devazepide and L-365,260 (1 mgykg,
Merck Sharpe & Dohme) were dissolved in 50 ml of dimethyl
sulfoxide and 50 ml of Tween 80, and further diluted in saline.
Capsaicin (8-methyl-N-vanillyl-6-nonenamide; Sigma) was dissolved in 10% ethanol, 10% Tween 80, and 80% saline.
Treatments and Measurement of Food Intake. Mice, deprived of food for 24 hr with free access to water, were injected
i.p. (10 mlykg) with either vehicle, leptin (4, 12, 80, or 120
mgykg), CCK (3.5 mgykg), bombesin (5 mgykg), leptin (4, 12,
80, or 120 mgykg) plus CCK (3.5 mgykg) or leptin (120 mgykg)
plus bombesin (5 mgykg). Thereafter, preweighed Purina chow
was given every hour and food intake was determined by
measuring the difference between the preweighed standard
chow and the weight of chow and spill at the end of each hour
for a 7-hr period. Mice were weighed before and after 24-hr
fasting and again at the end of the 7-hr feeding period. Similar
studies were performed in mice injected i.p. with devazepide
(1 mgykg) or L-365,260 (1 mgykg) and 10 min after with leptin
(120 mgykg) plus CCK (3.5 mgykg) or vehicle. In a series of
experiments, mice were anesthetized with halothane (3%
vapor in O2) and injected with a single subcutaneous injection
Leptin is an adipose tissue-derived circulating protein, originally identified as a key element in the long-term regulation of
food intake and body weight homeostasis in obyob mice (1–6).
Although obyob mice are more sensitive to leptin effects,
reduction of food intake and weight loss can also be elicited by
chronic hyperleptinemia achieved by repeated peripheral injections of leptin or by adenovirus-mediated leptin gene
therapy in lean mice and rats (1–3, 5, 7). By contrast, less is
known about the short-term alteration of feeding behavior
induced by leptin administered peripherally (3, 8, 9). Kinetic
studies indicate that upon a single intravenous or intraperitoneal (i.p.) injection, leptin decreases food intake only after
several hours in obyob or lean mice (3, 8, 9). The deferred onset
may be related to the delayed bioavailability of leptin to reach
or influence its target sites of action in the brain (9–12).
Alternatively, leptin may require the presence of food-related
gastric or postgastric signals. Several peptides released postprandially cause reduction in meal size (13). Cholecystokinin
(CCK) is secreted from small intestinal cells in response to
food ingestion and functions as a postprandial satiety signal
(13–16). Numerous studies using CCK receptor agonists and
antagonists have extended these findings in several species,
including mice and humans (14, 17–19). Convergent reports
indicate that one component of the satiety effect of CCK is
Abbreviations: CCK, cholecystokinin; PVN, paraventricular nucleus
of the hypothalamus.
*To whom reprint requests should be addressed at: CURE: Digestive
Diseases Research Center, Veterans Affairs Medical Center, Building 115, Room 203, 11301 Wilshire Boulevard, Los Angeles, CA
90073. e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424y97y9410455-6$2.00y0
PNAS is available online at http:yywww.pnas.org.
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Physiology: Barrachina et al.
(0.1 ml) of capsaicin (50 mgykg) or vehicle (10% ethanoly10%
Tween 80y80% saline). One week later, the effect of a single
i.p. injection of leptin (120 mgykg) plus CCK (3.5 mgykg) on
food intake was evaluated as described above. Efficiency of
capsaicin pretreatment was verified immediately before euthanasia by the corneal chemosensory test which consists of
monitoring the wiping reflex to ocular instillation of a drop of
0.1% NH4OH solution. None of the capsaicin-pretreated mice
showed a wiping response, indicating an effective ablation of
primary sensory afferents as previously reported in adult mice
with a similar treatment (28), whereas wiping reflex was
present in vehicle-pretreated mice.
In the last group of experiments, mice, fasted for a 20-hr
period, received a single i.p. injection of leptin (120 mgykg) or
vehicle (5 mlykg) and the access to food was restricted to the
4th to the 7th hr after leptin injection. Ten minutes before food
access, mice were injected i.p. with devazepide (1 mgykg) or
vehicle (5 mlykg). Food intake was monitored hourly, as
described above, from the fifth to seventh hour after leptin
administration.
Measurement of Gastric Emptying. Mice fasted for 24 hr
had free access to preweighed Purina chow for 3 hr and
thereafter received a single i.p. injection of either leptin (120
mgykg), CCK (3.5 mgykg), combined treatment (leptin plus
CCK), or vehicles (10 mlykg). Mice were killed by cervical
dislocation 4 hr after i.p. treatment. The stomach was exposed
by laparotomy, quickly ligated at both the pylorus and cardia,
then removed, and the wet content was weighed. Gastric
emptying (% in 4 hr) was calculated according to the following
formula: gastric emptying (%) 5 [1 2 (wet weight of food
recovered from the stomachyweight of food intake)] 3 100.
Locomotor Activity and Stereotyped Behaviors. Locomotor
activity was measured using a similar method to that described
previously (1). Mice were placed in individual Plexiglas cages
(14 3 15 3 25 cm), with the bottom divided into 15 equal squares
of 5 3 5 cm. Locomotor activity (total number of squares crossed
or explored by the animals during the observation time), grooming (washing, licking andyor scratching), and stereotypic activities
(sniffing) were evaluated for 1 min, every 5 min, during the first
hour after a single i.p. injection of either leptin (120 mgykg) plus
CCK (3.5 mgykg) or combined vehicles.
Fos Histochemistry. Immunohistochemistry for Fos was
performed on perfusion-fixed 30-mm-thick frozen brain sections as described (30). Mice, food deprived for 24 hr, were
injected i.p. with either leptin (120 mgykg), CCK (3.5 mgykg),
leptin plus CCK, or combined vehicles, and killed 2 hr later.
Animals were deeply anesthetized with sodium pentobarbital
(5 mg in 0.1 ml, i.p.; Nembutal, Abbott) and transcardially
perfused with 5 ml of 0.9% saline followed by 30 ml of 4%
paraformaldehyde solution and 14% saturated picric acid.
Brains were postfixed for 5 hr and cryoprotected by immersion
in 20% sucrose overnight. An avidin–biotin–peroxidase complex binding method was used with a rabbit antiserum against
human Fos protein N terminal at 1:10,000 dilution (Oncogene
Science) as primary antibody. The staining was abolished by
preabsorbing the antiserum for 24-hr at 4°C with a ratio of
antigenyantibody of 100:1. Cell counting of Fos immunoreactive cells in the paraventricular nucleus of the hypothalamus
(PVN) was made unilaterally in 8–9 sections containing the
biggest volume of parvo- and magnocellular neurons per
animal (bregma: 20.70 to 0.94 mm from Franklin and Paxinos’s mouse brain atlas) (31).
Statistical Analysis. Data are expressed as mean 6 SEM.
Statistical comparisons were done using a nonrepeated measures ANOVA followed by a Newman–Keuls multiple comparisons test. For cell counting, data were analyzed by a
Kruskal–Wallis nonparametric ANOVA followed by Dunn’s
multiple comparisons test. Data were considered statistically
significant when P , 0.05. The ED50 for leptin was determined
by nonlinear regression.
Proc. Natl. Acad. Sci. USA 94 (1997)
RESULTS
Effects of Leptin, Leptin–CCK, or Leptin–Bombesin Coinjection on Food Intake. Leptin (120 mgykg) injected i.p. did not
significantly modify food intake for the first 4 hr after injection,
and CCK (3.5 mgykg) had no effect throughout the 7-hr
experimental period in 24-hr fasted lean mice (Fig. 1). Thereafter, leptin induced a significant reduction of food intake
from the 5th to 7th hr postinjection (Fig. 1). By contrast,
coinjection of leptin with CCK reduced food intake during the
first 3 hr postinjection (Fig. 1). The decrease reached 82.5 6
6.2%, 82.8 6 5.8%, and 75.3 6 8.7% during the first hour
compared with vehicle-, leptin-, or CCK-treated groups, respectively [F(27,203) 5 10.899, P , 0.0001] (Fig. 1, Table 1).
During the fifth to seventh hour after treatments, the hourly
reduction in food intake was no longer different between
groups treated with leptin plus CCK (77.2 6 11.6%, 55.5 6
11.1%, and 61.1 6 7.3%, respectively) and leptin alone (88.1 6
8.4%, 48.0 6 24.1%, and 55.6 6 13.0%, respectively).
In the presence of the subthreshold dose of CCK (3.5
mgykg), leptin (4–120 mgykg)-induced early suppression of
food intake was dose-related in terms of the magnitude
(28–80% reductiony2 hr) (Fig. 2) and duration of the response
(1–3 hr) (Table 1). The ED50, defined as the dose of leptin that
inhibited by 50% the 2-hr cumulative food intake immediately
after coinjection with CCK, was 15.6 mgykg (r2 5 0.988, 95%
interval of confidence: 9.0–26.0 mgykg). By contrast, the
maximal effective dose of leptin (120 mgykg) coinjected with
bombesin (5 mgykg) at a satiating subthreshold dose, equimolar to that of CCK, failed to inhibit the cumulative food intake
throughout the first 2-hr period after injection (Fig. 2).
Food deprivation for 24 hr induced a 4.3 6 0.1 g body weight
loss. After a 7-hr refeeding period, the body weight gain in
leptin plus CCK treated mice was significantly lower (0.7 6
0.3 g) to that gained after vehicle, CCK, or leptin treatment
(1.7 6 0.2, 1.8 6 0.2, and 1.5 6 0.2 g, respectively). There was
a good correlation between the body weight gain and the
FIG. 1. Early inhibition of cumulative food intake induced by i.p.
injection of leptin plus CCK in fasted lean mice (1y1). Animals were
exposed to food immediately after a single i.p. injection of control
vehicles (10 mlykg, E; n 5 6), vehicle plus leptin (120 mgykg, F, n 5
8), vehicle plus CCK (3.5 mgykg, Œ, n 5 7) or leptin plus CCK (‚, n 5
8). Data are expressed as mean 6 SEM. p, P , 0.05 vs. the group
treated with combined vehicles [ANOVA, F(27,203) 5 10.899].
Physiology: Barrachina et al.
Proc. Natl. Acad. Sci. USA 94 (1997)
10457
Table 1. Dose-related inhibition of hourly food intake induced by
leptin coinjected with CCK (3.5 mgykg) during the first 3
hr postinjection
Inhibition of food intake per hour,* %
Hour
postinjection
1
2
3
Leptin dose, mgykg, i.p.
4
12
80
120
47.4 6 6.8†
62.4 6 9.7†
46.8 6 11.1†
84.9 6 3.4†
66.4 6 9.3†
66.1 6 8.3†
82.5 6 6.2†
80.4 6 5.7†
79.3 6 4.7†
28.1 6 8.3
12.5 6 8.1
20.1 6 8.0
*Mean 6 SEM of 6–12 animals per group; the percentage of food
intake inhibition was calculated taking values of combined vehiclestreated mice as 0% inhibition.
†P , 0.05 compared with combined vehicles-treated group.
amount of food ingested among the different experimental
groups (r2 5 0.768).
Effects of CCK Receptor Antagonists and Systemic Capsaicin on Leptin–CCK Interaction. Pretreatment with the specific CCK-A receptor antagonist devazepide completely prevented the early inhibition of food intake induced by leptin
(120 mgykg) plus CCK (3.5 mgykg) observed during the 2-hr
period posttreatment, whereas the specific CCK-B receptor
antagonist L-365,260 had no effect (Fig. 3). Capsaicin pretreatment 1 week before the experiments also abolished leptin
(120 mgykg) plus CCK (3.5 mgykg)-induced reduction of food
intake during the 2-hr postpeptide administration period (Fig.
3). Although there was slight food intake increase in mice
treated with devazepide or capsaicin alone during the same
2-hr period, the difference did not reach statistical significance
(Fig. 3).
Effect of Devazepide on Leptin-Induced Reduction of Food
Intake. When mice fasted for 20 hr were injected i.p. with
leptin (120 mgykg) and the food access was withheld up to the
fourth hour after leptin administration, the decrease in food
consumption compared with the vehicle group was not signif-
FIG. 2. Dose-related inhibition of the 2-hr cumulative food intake
induced by leptin in presence of CCK in lean mice (1y1) fasted for
24 hr. Leptin was coinjected i.p. with either vehicle, CCK (3.5 mgykg),
or bombesin (5 mgykg), and mice were immediately exposed to food
for 2 hr. Data are expressed as mean 6 SEM of 6–12 animals per
group. p, P , 0.05 vs. the respective leptin alone-treated group or vs.
the group receiving CCK alone [ANOVA, F(11,94) 5 9.047].
FIG. 3. Blockade of leptin–CCK interaction by the CCK-A receptor antagonist devazepide or by systemic capsaicin in lean mice (1y1).
After 10-min pretreatment with L-365,260 (1 mgykg, i.p.), 10-min
pretreatment with devazepide (1 mgykg, i.p.), 7-day pretreatment with
capsaicin (50 mgykg, s.c.), or pooled vehicles, 24-hr fasted mice
received a single i.p. injection of either vehicle plus vehicle or leptin
(120 mgykg) plus CCK (3.5 mgykg) and were exposed to food for 2 hr.
Data are mean 6 SEM of 6–11 mice per group. p, P , 0.05 vs. all other
groups [ANOVA, F(7,65) 5 24.694].
icantly different during the first hour of food access, corresponding to the fifth hour after leptin injection (Fig. 4).
Thereafter, leptin significantly reduced hourly food intake by
FIG. 4. Blockade of leptin-induced inhibition of food intake by
devazepide. Mice fasted for 20 hr were injected i.p. with either saline
(5 mlykg) or leptin (120 mgykg) and 4 hr later were exposed to food
for 3 hr. Vehicle (5 mlykg) or devazepide (1 mgykg) was injected i.p.
10 min before food exposure. p, P , 0.05 vs. other experimental groups
at the same time period [ANOVA, F(11,102) 5 9.876].
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Physiology: Barrachina et al.
72.2 6 13.9% and 75.3 6 9.8% at the sixth and seventh hour,
respectively (Fig. 4). Reduction of food intake elicited by leptin
during the sixth and seventh hour postadministration was
completely prevented by pretreatment with devazepide, administered 10 min before food access (Fig. 4).
Proc. Natl. Acad. Sci. USA 94 (1997)
Fos Immunoreactivity in the PVN after Leptin–CCK Coinjection. In mice deprived of food for 24 hr, Fos immunoreactivity in the PVN showed a similar number of Fos-positive cells
in untreated animals or 2 hr after i.p. injection of vehicle, CCK
(3.5 mgykg), or leptin (120 mgykg) (Fig. 5 A–C, E, and F). Cells
FIG. 5. Fos immunoreactivity in the PVN after leptin 1 CCK coinjected in lean mice (1y1). Representative microphotographs show Fos protein
immunoreactivity in the PVN 2 hr after a single i.p. injection of (A) combined vehicles (10 mlykg), (B) CCK (3.5 mgykg), (C) leptin (120 mgykg),
or (D) leptin plus CCK, and (E) in fasted untreated animals. Marked increase in Fos immunoreactivity, resulting in dark and well-shaped nuclei,
was observed in the parvo- and magnocellular divisions of the PVN after leptin plus CCK treatment, whereas other groups exhibited only scattered
and lightly labeled cells. (Bar 5 100 mm.) (F) The number of cells per section (unilateral). Data are mean 6 SEM of four animals per group. p,
P , 0.05 vs. all other groups (Kruskal–Wallis ANOVA, KW 5 21308.4).
Physiology: Barrachina et al.
were found mainly in the magnocellular division and in lower
proportion in the ventral and parvocellular divisions. In contrast, CCK plus leptin-treated mice exhibited a 60% significant
increase in the number of Fos-positive cells in the PVN
compared with the other experimental groups (KW 5
21308.4, P , 0.001) (Fig. 5 D and F).
Effect of Leptin–CCK Coinjection on Gastric Emptying.
The 4-hr rate of gastric emptying of a nutrient solid meal was
not modified by leptin (120 mgykg) coinjected with CCK (3.5
mgykg) (87.2 6 3.5%, n 5 7), compared with either vehicles
(92.5 6 0.9%, n 5 6), leptin (91.5 6 0.6%, n 5 7), or CCK
(91.6 6 1.3%, n 5 7) alone.
Behavioral Effects of Leptin–CCK Coinjection. Locomotor
activity during the first hour after i.p. injection of leptin (120
mgykg) plus CCK (3.5 mgykg) resulted in 96.3 6 25.5 squares
crossed or explored (n 5 4). This value was not significantly
different from that observed in mice injected with combined
vehicles (119.3 6 48.4 squares, n 5 4, P 5 0.689). Similarly, no
difference in the total numbers of stereotypic and grooming
activities was observed between the two experimental groups
(leptin plus CCK: 54.5 6 6.1 events; combined vehicles: 50.0 6
10.0 events, P 5 0.715).
DISCUSSION
Leptin did not significantly modify food intake for the first 4
hr after a single i.p injection at 120 mgykg in 24-hr fasted lean
mice exposed to food. These results are consistent with
previous time course studies using similar or higher doses of
leptin injected i.p. (8, 9). By contrast, leptin injected simultaneously with CCK results in a dose-dependent reduction of
food intake as it relates to the magnitude and duration of the
response. A dose as low as 12 mgykg of leptin in the presence
of CCK decreased hourly food intake by 62% and 46% during
the first and second hour postinjection, respectively. Such a
response is unlikely to reflect the anorectic effect of CCK. The
peptide was injected at a dose of 3.5 mgykg, subthreshold to
induce a significant decrease in hourly food intake in lean
mice, as reported previously (18, 19, 32). These data provide
clear evidence for a synergistic interaction between leptin and
CCK leading to short-term reduction in food intake upon a
single peripheral injection in lean mice. Such a potentiating
interaction is specific for peripheral administration of CCK as
bombesin, injected i.p. at a subthreshold dose, failed to influence the pattern of leptin-induced change in food intake.
Although bombesin is released postprandially and reduces
food ingestion in a variety of species including mice, its action
is mediated through different mechanisms than CCK, which
may account for the difference observed (18, 33–35).
The reduction of food consumption can be secondary to the
nonspecific behavioral suppressing effects of treatments under
study. However, leptin injected i.p. at doses up to 10 mgyday
did not affect total locomotor or grooming activity in lean mice
(1). Likewise, in the present study, leptin plus CCK-treated
mice were alert and no changes in exploratory locomotion or
induction of any form of stereotyped behaviors were observed
during the first hour, when food intake was reduced by 83%.
Leptin–CCK action is also unlikely to reflect an aversive effect
of treatments. Consistent reports established that the foodreducing effect of i.p. injection of CCK is unrelated to taste
aversion and CCK doses over 300-fold higher than used in the
present study are required to elicit a mild aversive effect in rats
(14, 29). There is also evidence that leptin, injected into the
lateral brain ventricle at a dose reducing short-term food
intake, did not cause aversive side effects as monitored by the
lack of conditioned taste aversion to saccharine (36). Therefore it may be inferred that leptin–CCK action most likely
reflects a specific effect on ingestive behavior.
Gastric satiety signals resulting from faster or slower clearance of nutrients from the stomach andyor stimulation of
gastric stretch receptors by gastric distention may play a role
Proc. Natl. Acad. Sci. USA 94 (1997)
10459
in the short-term regulation of food intake (37–40). In the
present study, leptin injected i.p. at a dose resulting in maximal
suppression of food intake in presence of CCK did not
significantly modify the 4-hr rate of gastric emptying of a
nutrient solid meal ingested before the treatment. Peripheral
injection of leptin alone, as reported previously, did not
influence gastric emptying of a solid nutrient meal in mice (8).
CCK at the dose used in the present study was also subthreshold to inhibit gastric emptying, which occurs at peptide doses
higher than 10 mgykg in mice (41). These data, added to the
rapid onset of food intake suppression induced by leptin-CCK
injection, support the view that gastric satiety signals linked
with alterations of gastric emptying are unlikely to play a role
in the food intake reducing effect of leptin–CCK interaction in
normal lean mice. The lack of change in gastric emptying in
response to leptin–CCK treatment at doses inducing maximal
food intake reducing effect also provides functional evidence
that leptin–CCK action does not result from a nonspecific
stress-related response. Convergent reports showed that delayed gastric emptying is a reliable autonomic response to
activation of brain corticotrophin-releasing factor receptors
induced by stress (42).
The biological actions of CCK are exerted through interaction with two subtypes of CCK receptors, CCK-A and CCK-B,
which are selectively antagonized by devazepide and L-365,260
respectively (42). Leptin coinjected with CCK induced shortterm suppression of food intake was completely prevented by
an i.p. injection of devazepide whereas L-365,260 had no
effect. The reversal by the CCK-A receptor antagonist was
observed under conditions that did not influence basal food
intake as reported previously (18, 32). It is also unlikely to
represent the anxiolytic-like effect of devazepide because
L-365,260, which has established anxiolytic-like properties in
mice (43, 44), did not influence the food-suppressing effect of
leptin–CCK. These results indicate that the CCK-A receptor
subtype is critical for leptin–CCK-induced food intake suppression as established previously for the satiating effect of
peripherally administered CCK alone (14, 18, 45, 46).
CCK-A receptors are present in vagal afferent fibers and
systemic administration of CCK stimulates gastric vagal afferent discharge in rats (22, 47–51). Convergent findings indicate
that afferent fibers in the vagus nerve represent a major initial
target in peripheral CCK-induced satiety (20, 24, 52). Systemic
capsaicin pretreatment in mice at a dose that causes selective
degeneration of small diameter unmyelinated sensory neurons
(28) prevented the decrease in food intake for the 2-hr period
after i.p. injection of leptin and CCK. These observations are
consistent with leptin plus CCK interaction involving activation of CCK-A receptors located in visceral capsaicin-sensitive
afferents, most likely of vagal origin. This is further supported
by recent electrophysiological recordings in an isolated vagus–
stomach preparation in rats showing an increased responsiveness of gastric vagal afferents to leptin by pretreatment with
CCK (27).
Afferent sensory fibers are the primary neuroanatomical
link between nutrient-related events in the gut and the central
neural substrates that mediate the control of food intake
(53–55). Among them, the PVN is a target site at which visceral
signals are interpreted by limbic structures to regulate homeostatic functions, including ingestive behavior and the satiety
effect of peripheral injection of CCK (56–58). In addition, Fos
expression was observed in the PVN in response to a single i.p.
injection of leptin (1 mgykg) in obyob mice, but not in lean
mice (59) or rats (60). Leptin coinjected with CCK enhanced
the number of Fos-positive cells in the PVN by 60%. Preliminary studies on the immunohistochemical mapping of oxytocin and vasopressin neurons in the PVN in mice suggest that
neurons in both the parvo- and magnocellular parts of the PVN
are activated (unpublished data). By contrast, leptin or CCK
alone did not modify Fos expression above the basal level
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Physiology: Barrachina et al.
which normally occurs in lean mice after a 24-hr fast (61). The
activation of PVN neurons in response to coinjection of leptin
and CCK may be part of the central neural pathways underlying the mechanism of action of leptin–CCK interaction. The
biochemical coding and exact mapping of activated neurons in
the PVN needs to be further characterized. Previous studies in
rats showed that peripheral CCK activates oxytocinergic neurons in the PVN (62, 63).
The synergistic interaction between CCK and leptin may
have physiological relevance. CCK is known to be released
after a meal and to produce postprandial satiety by nonendocrine mechanisms (14, 16, 64). In the present study, the onset
time required to reduce food intake after an i.p. injection of
leptin alone was further delayed when the food was withheld
during the 4-hr period after leptin injection suggesting an
interaction with a food-related signal. In addition, the CCK-A
receptor antagonist devazepide prevented the reduction of
food intake occurring 5–7 hr after i.p. injection of leptin alone.
The interaction between leptin and endogenous CCK was
observed at 5–7 hr after an i.p. injection of leptin, at a time
when circulating leptin levels may have returned to physiological ranges as shown by kinetic studies in lean fasted or fed mice
injected i.p. with 40 mgykg of leptin (65).
Leptin is exclusively produced in the periphery by adipocytes
and has been proposed to be transported from the circulation
to the brain where hypothalamic cells are the target of chronic
leptin treatment (1, 10, 66, 67). In addition to this long-term
action, the present data reveal the existence of a peripheral
neurally mediated synergistic interaction between leptin and
CCK released postprandially to induce short-term modulation
of food intake. Results obtained also show that this process
depends upon CCK-A receptors and activation of capsaicinsensitive afferent neurons sending signals to the PVN. Abnormalities in the release or sensitivity to either factor may be
involved in alterations of food intake. In addition, the potentiation of peripheral leptin action by a subthreshold dose of
CCK may provide a useful concept for the understanding of
multifactorial control of ingestive behavior and open new
avenues for the treatment of obesity and eating disorders.
We thank Amgen Biologicals for providing the mouse recombinant
leptin, and Paul Kirsch for helping in the preparation of the manuscript. This work was supported by Research Scientist Award Grant
MH 00663 (Y.T.), National Institute of Diabetes and Digestive and
Kidney Diseases Grants DK 30110 (Y.T.) and DK 41301 (Animal
Models Core, Y.T.). and DK 48476 (J.Y.W.). M.D.B. is in receipt of
a fellowship from the J. Esplugues Foundation (Valencia, Spain).
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