Int J Pept Res Ther (2012) 18:77–88
DOI 10.1007/s10989-011-9281-9
Physiological Low Doses of Leptin and Cholecystokinin Induces
Body Weight-Loss in Juvenile and Lean, but not in Adult-Obese
Rats
Milton Londoño • Luis A. Tellez • Ranier Gutierrez
Accepted: 10 November 2011 / Published online: 19 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Leptin and cholecystokinin (CCK) are two
hormones involved in body weight regulation and feeding
behavior. Leptin, an adiposity-derived signal, and CCK, a
gut-satiety signal, interact in a synergistic manner and their
concomitant treatment causes more weight loss than any
hormone administered alone. The synergistic interaction
between CCK and leptin has been widely characterized in
lean rats, but there are a few studies performed in obese rats.
Herein, in the same individual, we compared the sensitivity
to CCK, leptin and the combined treatment, leptin ? CCK,
on body weight and food intake in two experiments one
performed in juvenile-lean rats, and in a second experiment
comparing adult Chow-fed versus adult-high fat diet (HFD)
rats. We found that only the combined treatment had a long-
term effect on food intake and caused a substantial and
synergistic body weight loss, in juvenile rats. However, the
same combined treatment failed to induce weight loss in
both Chow-fed and HFD rats. We thus conclude that the
sensitivity to leptin ? CCK was age dependent, and at the
doses used ineffective in adult and obese rats.
Keywords Leptin
Cholecystokinin
Synergism

Weight loss
Obesity
Introduction
In most societies the prevalence of obesity has reached
epidemic proportions (Kissane 2011). Obesity is now con-
sidered to be a neurobiological disease that reduces life
M. Londoño
L. A. Tellez
R. Gutierrez (&)
Department of Pharmacology, CINVESTAV-IPN, Av. I.P.N.
#2508 México D.F., 07360 Mexico City, DF, Mexico
e-mail: ranier@cinvestav.mx
expectancy and is a risk factor for cardiovascular disease,
certain types of cancers and for the development of type 2
diabetes (Overweight, obesity, and health risk. National
Task Force on the Prevention and Treatment of Obesity
2000). Ideally obesity must be treated with exercise and
dieting, but few people can withstand strict dietary regimes
for too long (Rossner et al. 2008; Jakicic 2009). The relative
lack of successful diets to control weight for the long term
has led to the development of several pharmacological
treatments. However, and despite the discovery of multiple
peptides that regulate appetite, little is known about how
naturally-occurring hormones involved in body weight
regulation interact (Roth et al. 2008), and the use of any
individual hormone has failed to control the obesity pan-
demic (Cannon and Kumar 2009; Wang and Beydoun 2007).
Leptin is an adiposity-derived hormone that is the product of
the ob gene and regulates food intake and energy balance.
Leptin circulates at levels proportional to body fat (Camp-
field et al. 1995), and although leptin is a circulating signal
that reduces appetite mainly by central action (Schwartz
et al. 1996), in general, obese subjects have an unusually
high concentration of circulating leptin, and thus they
developed leptin resistance (Friedman and Halaas 1998). On
the other hand, cholecystokinin (CCK) was the first dis-
covered and perhaps the most studied gut-derived peptide
that signals satiety to the brain (Weller et al. 1990).
Endogenous CCK is normally released by endocrine I cell
type from the upper intestine during meal initiation and
rapidly returns to near baseline levels during meal termina-
tion (Gibbs et al. 1973; Moran and Ladenheim 2011; Dourish
et al. 1989; Weller et al. 1990). In rodents, exogenous
administration of CCK induces satiety at concentrations that
does not induce taste aversion (West et al. 1984).
Leptin and CCK can interact in a synergistic manner
either at the periphery or centrally. The main site of action of
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CCK is within the periphery, activating vagal terminal
nerves that in turn activates the Nucleus of the Tract of the
Solitary (NTS) (Wang et al. 1997). In contrast, leptin has a
central site of action, modulating both anorexigenic neurons
(POMC-expressing cells) and orexigenic cells (AGRP/
NPY-expressing cells) located in the arcuate nucleus of the
hypothalamus (Friedman and Halaas 1998; Horvath et al.
2010; Aponte and Sternson 2011); In this regard, intracra-
nial ventricular (icv) administration of leptin is more potent
to suppress appetite in comparison to peripheral adminis-
trations of the same hormone (Patel and Ebenezer 2008;
Harris et al. 2003). Likewise icv leptin and i.p. CCK co-
administration induces a stronger weight loss than the effect
achieved by peripheral administration of both hormones
together (Matson et al. 2000, 2002; Matson and Ritter 1999;
Emond et al. 1999). Moreover, leptin and CCK can also
interact at the peripheral level in two main ways. (1)
Although the adipocyte is the main source of leptin, recent
studies have shown that the stomach can also secrete leptin
(Sobhani et al. 2000). In the intestine, luminal leptin
can enhance duodenal CCK release to levels comparable
to feeding (Guilmeau et al. 2003). (2) Exogenous lep-
tin ? CCK can act synergistically by potentiating activation
of vagal afferents. E.g., a relatively high local concentration
of leptin in the stomach can enhance CCK activation of a
subpopulation of gastric vagal nerves (Wang et al. 1997).
Thus stomach-derived leptin may have a significant physi-
ological role in controlling vagally mediated responses,
such as satiation, gastrointestinal motility and secretion
(Peters et al. 2004). In summary, all these data highlight the
synergistic interaction between peripheral leptin and CCK
as a potential and useful therapeutic treatment to regulate
both appetite and obesity (Barrachina et al. 1997; Cano et al.
2003; Matson et al. 1997, 2000, 2002); nevertheless, a few
studies have evaluated the synergistic action of these pep-
tides in obese subjects (Trevaskis et al. 2010).
In this study, we used a multi-phase design in order to
evaluate and compare, in the same individual, the periph-
eral action of leptin and CCK in both lean (‘‘Experiment 1:
Juvenile-Lean Rats’’ section) and obese (‘‘Experiment 2:
Adult Rats’’ section) rodents. In regards to CCKs sensi-
tivity, we found that juvenile-lean rats did not developed
tolerance to CCKs satiating effect (Duncan et al. 2005),
whereas Chow-fed adult rats did. Unexpectedly high fat
diet (HFD) rats were paradoxically hypersensitive to the
satiating effect of CCK relative to adult Chow-fed rats. In
regards to CCK/leptin combination, we found that
peripheral leptin and CCK synergistically enhanced body
weight loss in juvenile-lean rats. However, neither Chow-
fed adults nor HFD rats lose weight after the combined
leptin ? CCK treatment. Therefore, we concluded that
unlike juvenile rats, adult rats were more insensitive to
physiological doses of leptin ? CCK combination.
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Int J Pept Res Ther (2012) 18:77–88
Materials and Methods
Animals
Male Sprague–Dawley rats were housed in a communal
house cage of 7–10 rats under standard laboratory condi-
tions with tap water and regular chow food provided
ad libitum, unless otherwise mentioned, in a 12/12 h light/
dark cycle (lights on at 6:00 am) at a constant room tem-
perature of 21–22°C. Body weight and total overnight food
intake were measured daily at 9:00 am. All behavioral
procedures were performed in the same vivarium room and
all protocols were approved by the CINVESTAV-IPN
Institutional Animal Care and Use Committee.
Drugs
Human recombinant leptin (Peprotech, Mexico) and (Tyr
[SO3H] 27) CCK fragment 26–33 amide or CCK-8 (Sigma-
Aldrich, Mexico) were dissolved in 0.9% sterile saline
solution.
Diets
Chow food diet contains 3.38 kcal/g (Purina Mexico; 15%
fat, 23% protein, 62% carbohydrate).
We used the liquid diet EnsureÒ chocolate-flavored that
contains 1.01 kcal/g (Abbott Laboratories, Mexico). The
composition of EnsureÒ was 32% fat, 14% protein, and
54% carbohydrate. We used Ensure, instead of sucrose, in
order to better emulate a real meal, since it is well known
that foods rich in protein and fat releases CCK from duo-
denal mucosal cells (Feltrin et al. 2004; Little et al. 2007).
Furthermore, Ensure has been widely used to characterize
the synergism between CCK and leptin (Matson et al.
1997; Emond et al. 1999).
30-Min Ensure Intake Procedure
Rats were habituated to the following daily schedule. Solid
food (chow and/or HFD) and water (or sucrose) was
removed at 9:00 am every day. After 6 h of mild food and
water deprivation (from 9:00 am to 15:00 pm), rats were
placed in an individual cage where EnsureÒ was available
for 30-min. Ensure intake was carried out at the same time
each day (15:00 pm) in a rack of 8 standard home cages
adapted with a drinking compartment (ENV 250, Med
Associates, Georgia, USA). At the end of 30-min Ensure
intake, total volume consumed was measured at the nearest
0.5 ml and the rats were immediately returned to their
communal house cage where daily chow food ration was
provided from 5:00 pm to 9:00 am. The amount of over-
night food intake per group (communal home cage) was
Int J Pept Res Ther (2012) 18:77–88
recorded to the nearest 0.1 g and corrected by spillage
every day at 9:00 am. All protocols were approved by the
CINVESTAV Mexico, Institutional Animal Care and Use
Committee.
Data Analysis
Unless otherwise mention data is presented as mean ±
SEM. Statistical differences between groups were assessed
by a Student’s t test or repeated-measures ANOVA, with a
Bonferroni post hoc correct.
Methods for Experiment 1: Juvenile-Lean Rats
Animals
Thirteen juvenile (*2 months old) male Sprague–Dawley
rats weighing 250–260 g were randomly split into Saline and
Treatment groups. Saline group (n = 7) served as a control
and received the same number of i.p. injections than the
treated group, but their injections were always of 0.9% saline
solution (1 ml/kg; see below), whereas the second group, the
‘‘Treatment group’’ received CCK and/or leptin following
the next schedule (see Fig. 1a; right panel). Briefly, the
multi-phase design was as follow: the first baseline (BL1)
phase occurred between days 1 and 3: each day, all rats were
transferred to an individual drinking box and were habitu-
ated to drink Ensure for 30-min. CCK phase (days 4–8):
10 min before access to EnsureÒ, ‘‘Treatment group’’
received an i.p. injection of CCK (2 lg kg body wt-1).
Baseline 2 (BL2, days 9–11), all rats had 30-min access to
Ensure and no-injection occurred in this phase. Low leptin
phase (days 12–16): Two hours before giving access to
Ensure, the Treatment group received a single daily i.p. lepin
administration, at 10 lg kg body wt-1 dose. High leptin
phase (days 17–21): same conventions as in the previous
phase, but the experimental treated group received a daily
i.p. leptin injection at a dose of 30 lg kg body wt-1. We
chose this higher, but still physiological dose because it
raises plasma leptin to similar levels than those observed
after feeding (Jaworek et al. 2003) and it delays gastric
emptying (Cakir et al. 2007) (see below). Leptin/CCK phase
(days 22–26): The ‘‘Treatment group’’ received leptin (i.p.,
30 lg kg body wt-1), and 2 h later received an injection of
CCK (i.p. 2 lg kg body wt-1), and 10 min after CCK
injection both ‘‘Treatment’’ and Saline groups received
accesses to EnsureÒ during 30 min. Finally, a third baseline
(BL3) phase was done between days 27 and 31.
Dose Selection
Low and High Physiological Leptin Doses The rationale
for choosing the doses of leptin was based on the fact that
79
exogenous leptin, at low doses (1–10 lg/kg), produced
significant inhibition of pancreatic enzyme secretion;
meanwhile at high dose (30 lg/kg) but still physiological
dose, raises plasma leptin to similar levels than those
observed after feeding (Jaworek et al. 2003). Indeed, the
administration of exogenous leptin (0.1, 1, 5, 10, 20 or
50 lg/kg), results in the dose dependent rise of plasma
leptin activity, reaching the highest level with the dose of
50 lg/kg of leptin, that was about 1.6 ± 0.15 ng/ml, and
was similar to the value obtained from fed rats (Jaworek
et al. 2003). Moreover, 30 lg/kg dose is effective in the
inhibition of gastric emptying rate, whereas other higher
leptin doses do not affect emptying of either caloric or non-
caloric gastric contents (Cakir et al. 2007).
CCK Although 2 lg/kg of CCK is relatively a low dose, it
induces a strong satiating effect in a short-term food intake
(Maggio et al. 1988), and does not induce postprandial sleep
(de Saint Hilaire-Kafi and Nicolaı̈dis 1989). This low dose
of CCK also reduced the probability of inducing condi-
tioned taste aversion (which is observed at higher
doses [1.1 mg/kg (Ervin et al. 1995). It has also demon-
strated that consecutive administrations, during 10 days, of
a high dose 6 lg/kg produced a learned tolerance to the
satiating CCKs effect (Goodison and Siegel 1995; Duncan
et al. 2005), thus we hypothesized that our low dose of CCK
(2 lg/kg) will reduce the occurrence of CCKs tolerance.
Methods for Experiment 2: Adult Rats
Animals
Nineteen male Sprague–Dawley rats weighing 300 g at the
beginning of the experiment were randomly split into two
groups: Chow group (n = 9) served as control (chow food
was its only source of calories) and HFD group (n = 10).
HF group had both ad libitum chow food, and a HFD: and
ad libitum water and 20% sucrose solution throughout the
experiment (HFD-Suc).
High Fat Diet (HFD)
We used a basal purified diet with 60% calories from fat
(energy = 5.21 kcal/g; Test Diet 58G9 Richmond, IN) that
contains 60% fat, 18.6% protein, and 21.4% carbohydrate.
Rats had ad libitum intake of 20% sucrose solution
(HFD ? 20% of sucrose = 6.01 kcal/g).
The pharmacological treatment began, after 12 weeks of
exposure to HFD-Suc diet (Fig. 2a), but both groups con-
tinued receiving their corresponding diets throughout the
experiment. At this stage rats were *6 months old. Pro-
cedures were similar as in Experiment 1 (Fig. 2a), but
Chow and HFD groups received identical pharmacological
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80 Int J Pept Res Ther (2012) 18:77–88

A Leptin
30 µg/kg

2h

CCK Leptin Leptin CCK

2 µg/kg i.p. 10 µg/kg 30 µg/kg 2 µg/kg

10 min 2 h 2 h 10 min
30-min
Ensure Ensure Ensure Ensure Ensure Ensure Ensure
intake intake intake intake intake intake intake

BL1 CCK BL2 Low High Leptin/ BL 3

1 3 4 8 12 Leptin 16 17 Leptin 21 22 CCK 26 27 31
Days
B D
Saline group
Treatment group Saline group Treatment group
115 115
30-min Ensure intake (mL)

20
18 110 110

%Body Weight
(Normalized)
16 105
105
14
100 100
12
10 95 95
8 90 90
6
85 85
4 22 23 24 25 26 27 28 29 30 31 22 23 24 25 26 27 28 29 30 31
2
Saline BL 3 Leptin/CCK BL 3
0
0 5 10 15 20 25 30 Days Days
C E
360
450
340
Body weight (g)

400
Overnight Chow intake

320 350
CCK
300 BL 2
300
250 Low Lept in
(Kcal)

280 200 High Lept in

150 Lepti n/ CCK
260 BL 3
100
240 50
0 5 10 15 20 25 30
0
BL1 CCK BL2 Low High Leptin/CCK BL3 Saline group Treatment group
Leptin Leptin
Days

Fig. 1 Effect of the administrations of leptin and/or CCK on throughout the experiment. Despite that chow food was given
juvenile-lean rats. a Schematic representation of the experimental throughout all nights of the experiment, we started measuring
design. b 30-Min Ensure intake. Background rectangles indicate overnight chow intake during the last day of CCK phase, and thus
treatment phases (CCK, low leptin, high leptin and leptin/CCK). no SEM was computed for that phase.*Significant difference relative
c Average body weight and d normalized body weight to high leptin to Saline group (P \ 0.05)
phase for each experimental subject. e Overnight chow intake

treatment (CCK, high leptin and high leptin/CCK). This Results

experimental design was done to determine whether
obesity altered the sensitivity to leptin and/or CCK. The Experiment 1: Juvenile-Lean Rats
multiphasic experimental design was similar to that used in
Experiment 1. Briefly, the BL1 phase occurred between Exogenous CCK decreased 30-min Ensure Intake
days 1 and 3: followed by a CCK phase (days 4–8; 2 lg/kg
i.p.), and BL2 (days 9–11). We decided not to test for a low Figure 1b shows 30-min Ensure intake throughout all pha-
leptin dose due to the lack of anorectic effect observed in ses of Experiment 1. During phase BL1 Ensure intake
juvenile rats (Fig. 1b), hence only the high leptin dose was gradually increased in both groups, indicating that naı̈ve
employed. High Leptin phase (days 12–16): Both groups rats required at least 1–2 days to familiarize with our
received a daily i.p. leptin injection at a dose of 30 lg/kg. drinking box and to learned to drink as much as possible in a
Leptin (30 lg/kg)/CCK (2 lg/kg) phase (days 17–23). 30-min session (at this stage no significant differences
Finally, a BL3 phase was done between days 24 and 27. between groups were observed).

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Int J Pept Res Ther (2012) 18:77–88 81

A
Leptin
30 µg/kg

525 HF group 2h
500 Chow group
475 CCK Leptin CCK
Body Weight (g)

2 µg/kg 30 µg/kg 2 µg/kg

450
425
10 min 2h 10 min
400
375
350
→ 30-min
Ensure Ensure Ensure Ensure Ensure Ensure
intake intake intake intake intake intake
325
300
275 BL 1 CCK BL2 High Leptin/ CCK BL 3
0 1 2 3 4 5 6 7 8 9 10 11 12 1 3 4 Leptin 27
8 12 16 17 23 24
Days
Weeks

B HF group D
** Chow group
Chow group HF group
20
106 106
18 105
105
30-min Ensure intake (Kcal)

16

%Body Weight
104 104

(Normalized)
14 103 103
12 102 102
101 101
10
100 100
8 99 99
6 98 98
4 97 97
17 18 19 20 21 22 23 24 25 26 27 17 18 19 20 21 22 23 24 25 26 27
2 Leptin/CCK BL 3 Leptin/CCK BL 3
0 Days Days
0 5 10 15 20 25
C E
540 1200
520 CCK
Body weight (g)

Overnight intake (Kcal)

1000 BL2
500 High Lept in
800 Lept in/CCK
480
BL 3
460 600

440
400
420
200
400

380 0
0 5 10 15 20 25 chow chow HFD-20% Suc
BL1 CCK BL2 High Leptin Leptin/CCK BL3 Chow group HF group
Days

Fig. 2 Leptin and CCK inhibited 30-min Ensure intake, but did not b 30-Min Ensure’s intake throughout the experiment. c Average body
induce body weight loss in Chow and HF rats. a Schematic representation weight and d Normalized body weight to high leptin phase for each
of experimental design and weight gain after 12 weeks on HFDSuc diet. individual rat. e Overnight calorie intake in each phase

CCK Phase Daily administration of CCK robustly sup- During BL2 phase, consumption of EnsureÒ rapidly
pressed 30-min Ensure intake. The average EnsureÒ intake returned to baseline level, and thus no significant differ-
in this phase was 5.7 ml ± 0.33 (mean ± SEM) for the ences were observed between groups. This result suggests
‘‘Treatment group’’ and 10.2 ml ± 0.47 for the Saline that five contiguous administrations of CCK do not induce
group, the differences between groups were statistically taste aversion to EnsureÒ (see Fig. 1b; BL2 phase), in
significant (ANOVA; F(1, 63) = 56.016, P \ 0.0001). agreement with previous reports (Kulkosky 1985; West
A Repeated Measure ANOVA found a significant main et al. 1987).
effect by group (RM ANOVA; F(1, 11) = 50.669,
P \ 0.0001), but no significant effect by injection day (RM Peripheral Leptin did not Suppress 30-min Ensure Intake
ANOVA; F(1, 4) = 1.794, P = 0.1472, ns) with no sig-
nificant interaction (RM ANOVA; F(1, 44) = 1.881, In the next two phases, we evaluated the effect of leptin at a
P = 0.1306), indicating that, at doses used, juvenile rats do low (10 lg/kg; days 12–16) and a higher (30 lg/kg; days
not developed tolerance to CCKs satiating effect across 17–21) dose on 30-min Ensure intake (Fig. 1b; Low and
five contiguous administrations (Duncan et al. 2005) High Leptin phases). In our experimental conditions, i.p.
(Fig. 1b; CCK phase). leptin administration did not suppress Ensure intake at a

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low dose (RM ANOVA; F(1, 10) = 0.014, P = 0.9096, ns),
whereas at 30 lg/kg leptin dose, the Treatment group
displayed a slight trend, but not significant over con-
sumption of Ensure in comparison to Saline group
(RM ANOVA; F(1, 11) = 3.213, P = 0.1006, ns; 13.8 ml ±
0.83 mean ± SEM for the ‘‘Treatment group’’, vs. 11.3 ml ±
0.62 for the Saline group). These results indicate that these
doses of leptin were subanorectic and not sufficient to
suppress short-term food intake (Fig. 1b; Low and High
Leptin phases). These results are in agreement with pre-
vious results reporting that only doses well above [
100 lg/kg i.p. of leptin were able to suppressed food intake
(Kinzig et al. 2010).
At Doses Used, Leptin ? CCK did not Induced a Greater
Ensure Intake Suppression than CCK Alone
Direct comparison, of the raw intake values, between CCK
versus Leptin/CCK phases uncovered that the combined
leptin ? CCK treatment failed to produce a greater sup-
pression of EnsureÒ intake, relative to CCK alone. In fact,
we did not observe any significant differences on overall
30-min EnsureÒ intake during the CCK phase versus Leptin/
CCK phase (ANOVA; F(1, 58) = 0.039, P = 0.8437, ns).
The average EnsureÒ intake in the CCK phase was
5.7 ml ± 0.34, whereas in Leptin/CCK phase, it was
5.9 ml ± 0.8. Thus, in our experimental conditions,
peripheral leptin and CCK seems to do not produce a syn-
ergistic greater reduction on short-term food intake. How-
ever, we note that the combined treatment leptin ? CCK,
significantly suppressed 30-min EnsureÒ intake, compared
with saline injection. The Treatment group had 5.9 ml ±
0.77 intake, whereas the Saline group consumed 13.2 ml ±
0.83 (F(1, 63) = 40.554, P \ 0.0001; Fig. 1b, Leptin/CCK
phase). Unexpectedly, Ensure intake gradually decreased
across leptin ? CCK co-administrations. Specifically, a
greater suppression on EnsureÒ intake was achieved in the
4th co-administration (see Fig. 1b; Leptin/CCK phase). For
example, during the 1st co-administration the ‘‘Treatment
group’’ consumed 8.2 ml ± 2.1; meanwhile, in the 4th co-
administration, it consumed 4.3 ml ± 1.4. A Repeated
Measure ANOVA found a significant main effect by group
(RM ANOVA; F(1, 11) = 15.023, P \ 0.003). The experi-
mental group showed a significant effect across injection
days of Leptin ? CCK (RM ANOVA; F(1, 4) = 3.976,
P \ 0.01), and no significant interaction (RM ANOVA;
F(1, 44) = 0.930, P = 0.4552). These results suggest that
CCK and leptin ? CCK not necessarily suppressed Ensure
intake in the same manner. Alternatively, these results also
suggest that previous CCK administrations (CCK phase)
may have interfered with the expected synergism between
leptin ? CCK. However, this explanation is less probable,
since juvenile rats did not develop tolerance to CCKs
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Int J Pept Res Ther (2012) 18:77–88
satiating effect (see ‘‘Exogenous CCK Decreased 30-min
Ensure Intake’’ section).
Finally, during the BL3 phase, although no drug was
administered, the ‘‘Treatment group’’ continued showing a
reduced Ensure intake relative to the Saline group. The average
Ensure intake for treated group was 8.2 ml ± 1.1, whereas for
the Saline group it was 13.8 ml ± 0.73 (ANOVA; F(1,
62) = 19.822, P \ 0.0001).The average Ensure intake gradu-
ally returned to similar levels to those observed by the Saline
group (see Fig. 1b; BL3 phase).
Co-administrations of Both Leptin and CCK
Synergistically Induced Body Weight Loss and Suppressed
Overnight Food Intake
We observed that the combined treatment, leptin ? CCK,
induced a significant body weight loss (F(1, 63) = 19.005,
P \ 0.0001; see Fig. 1c; Leptin/CCK phase). Moreover,
weight loss continued during BL3 phase. Figure 1d shows
the normalized body weight for each rat during both Leptin/
CCK and BL3 phases. The body weight in the Treatment
group gradually decreased, whereas the Saline group con-
tinued gaining weight (Fig. 1d). We note that 2 animals did
not regain body weight after leptin ? CCK withdrawal,
indicating that the combined treatment could most likely
produced pancreatitis, a common side effect of chronic
administration of these peptides (Allman et al. 2009; Wu
et al. 2011; Konturek et al. 2002; Mussa et al. 2008). We did
not experimental confirm the presence of pancreatitis.
We also measured overnight chow food intake (Fig. 1e).
Unexpectedly, the combined treatment leptin ? CCK
significantly reduced overnight chow feeding (ANOVA;
F(1, 7) = 18.086, P \ 0.0005). Accordingly, this reduction
in caloric intake continued during BL3 phase (Fig. 1e).
Although we did not observe a synergistic effect on short-
term food intake (Ensure), our results indicate that leptin
could extended the CCKs satiating action by reducing long-
term (overnight) food intake, and thus it may rationalize the
weight loss observed (Fig. 1c).
In sum, our results indicate that in juvenile rats, and
using a multiphasic experimental design, our physiological
doses of leptin ? CCK resulted in body weight loss and
suppression of overnight Chow intake. We then sought to
evaluate the same doses, but now in adult rats exposed to a
HFD.
Experiment 2: Adult Rats
As expected, consumption of HFD-Suc diet gradually
caused weight gain in the HF group, and body weight was
significantly higher after consumption for 6 weeks of our
HFD (t test(19) = -3.562, P \ 0.005), and this continued
throughout the experiment in comparison to Chow group
Int J Pept Res Ther (2012) 18:77–88
(RM ANOVA; F(1, 17) = 33.765, P \ 0.0001). After 12
weeks, body weight of HFD rats was 496 ± 9.4 g whereas
the Chow group was 407 ± 9.5 g (t test(19) = -6.646,
P \ 0.0001; Fig. 2a). Rats in our dietary regime gai-
ned *90 g of overweight.
HFD Rats were More Sensitive to CCKs Satiety Effect
than Chow-Fed Rats
Figure 2b shows 30-min EnsureÒ intake throughout all
phases of Experiment 2. BL1 phase: During this phase,
both groups gradually increased their EnsureÒ intake until
day 3, and no statistical differences were detected at this
phase. CCK phase: The average EnsureÒ intake during five
daily CCK injection for Chow group was 8.0 ml ± 0.4
(mean ± SEM) versus 6.3 ml ± 0.39 for the HFD group.
Despite that both groups equally suppressed 30-min
EnsureÒ intake over the first three daily CCK administra-
tions, the HF group was slight but significantly more sen-
sitive to CCKs satiating effect (ANOVA; F(1, 93) = 8.290,
P \ 0.005). Specifically during the last 2 days of CCK
administration (4th administration; t test(19) = 2.386,
P \ 0.05; 5th administration; t test(19) = 2.793, P \ 0.02;
Fig. 2b; CCK phase). This result suggest that CCK exerted
a more potent satiety effect in HFD rats than in the Chow
group, and thus, in our experimental conditions, adult rats
in a HFD were more sensitive to CCK (see Fig. 1b, upper
arrow).
Notably, direct comparison against juvenile rats, from
Experiment 1, suggests that adult rats from both Chow and
HF groups developed a gradual insensitivity (or tolerance)
to CCK throughout this phase (compare Fig. 1b CCK
phase vs. Fig. 2b CCK phase). Specifically, a RM ANOVA
comparing the CCK satiety effect in juvenile
(5.7 ml ± 0.34 EnsureÒ intake), versus adults rats of HF
(6.3 ml ± 0.39) and Chow (8.0 ml ± 0.4) groups showed
a near significant effect by group (RM ANOVA; F(2, 22) =
3.418, P = 0.051), significant effect by administration
days (F(2, 4) = 18.905, P \ 0.0001), and significant inter-
action (F(2, 88) = 3.722, P \ 0.001). These results suggest
that in general adult rats were slightly more insensitive to
CCK in comparison to juvenile rats.
During BL2 phase, EnsureÒ intake rapidly returned to
baseline levels, however, there was a significant difference
between groups (ANOVA; F(1, 55) = 3.102, P \ 0.005);
the average Ensure intake was 14.1 ml ± 0.62 for Chow
group, and 10.9 ml ± 0.84 for HF group. The significant
difference could be most likely explained by a lingering
effect of the hypersensitivity to CCK displayed by HFD
rats (Fig. 2b; BL2 phase), or by an overconsumption of
Ensure intake of Chow-fed rats. We note that for Chow-fed
rats, Ensure was its only source of palatable food, whereas
for the HFD group they had access to both HFD and 20%
83
sucrose, hence it is possible that Ensure could be less
motivating for HFD rats.
Peripheral Leptin did not Suppress 30-min EnsureÒ Intake
Next we evaluated leptin at a 30 lg/kg dose (days 12–16)
in the 30-min Ensure intake. The Ensure intake in High
Leptin phase for Chow group was 15.4 ml ± 0.5, versus
12.2 ml ± 0.7 for the HF group (F(1, 93) = 13.897,
P \ 0.0005). Leptin treatment did not suppress 30-min
Ensure intake in comparison to BL2 intakes (Chow;
ANOVA; F(1, 70) = 2.53, P = 0.1162, ns; HF; ANOVA;
F(1, 78) = 1.359, P = 0.2473, ns). Hence, these results
indicate that 30 lg/kg of leptin was not sufficient to inhibit
short-term food intake in neither Chow-fed nor HFD rats
(Fig. 2b; High Leptin phase).
Leptin ? CCK Gradually Decreased Short-Term Food
Intake in Adult Rats
We analyzed the temporal dynamics of leptin/CCKs satiety
action across administration days. During the first three
daily administrations of leptin and CCK, the average
EnsureÒ intake was 12.1 ml ± 0.6, and 9.7 ml ± 0.68, for
Chow and HFD group, respectively. Moreover, during the
last fourth leptin/CCK co-administration it was 7.9 ml ±
0.6 and 5.6 ml ± 0.6 for the Chow and HFD groups,
respectively. A RM ANOVA confirmed the significant
and decreasing tendency between days (RM ANOVA;
F(1, 6) = 18.405, P \ 0.0001), but the main effect of group
and the interaction were not significant (RM ANOVA;
F(1, 4) = 3.622, P = 0.0741; RM ANOVA; F(1, 102) = 1.881,
P = 0.5076, ns, respectively). These results indicate that both
groups gradually decreased Ensure intake upon lep-
tin ? CCK treatment (Fig. 2b; Leptin/CCK phase).
Finally, we compared the satiety effect induced by CCK
alone with the one observed when CCK was coadminis-
tered with leptin (CCK phase vs. Leptin/CCK phase). As
we noted, rats in CCK phase initially suppressed Ensure
intake, but then they gradually increased their intake
(Fig. 2b; CCK phase). In contrast, during Leptin/CCK
phase, the opposite pattern was observed, i.e. Ensure intake
was initially high and then gradually decreased. In fact, the
average intake during the first 3 single daily administration
of CCK was 5.9 ml ± 0.5 versus 10.8 ml ± 0.5 for the
first 3 injections of leptin ? CCK, and the differences
were statistically significant (ANOVA; F(1, 112) = 68.423,
P \ 0.0001). In contrast to the expected synergistic inter-
action, using a multiphasic design, our results suggested
that in adult rats co-administration of leptin initially seems
to antagonize and thus overshadow the satiating action of
CCK. However, this effect rapidly disappeared and was
123
84
followed by strong intake suppression in the following days
(see Fig. 2b).
During the first 2 days of the BL3 phase, we found a
significant difference between groups (ANOVA; F(1, 36) =
10.537, P \ 0.005). However, in the final 2 days of BL3,
we found that Ensure intake returned to baseline levels and
was similar for both groups (ANOVA; F(1, 36) = 3.713,
P = 0.0619, ns).
Effect of Daily Co-administration of Leptin and CCK
on Body Weight in Adult Rats
We observed that combined leptin ? CCK did not alter
body weight in the HFD group (F(6, 63) = 0.028, P [ 0.99)
or the Chow group (F(6, 56) = 0.025, P [ 0.99; Fig. 2c).
Unlike juvenile rats from Experiment 1, the body weight in
adult rats was remarkably constant throughout the experi-
ment. In fact, normalized body weight in adult rats was
unaltered during leptin ? CCK administrations (Fig. 2d),
and moderately increased during BL3 phase (Fig. 2d). For
the doses employed in this study, our results suggest that
weight loss induced by leptin ? CCK was age dependent
and ineffective in adult and obese rats.
Finally, we analyzed the overnight intake for each group
as a function of experimental phase (Fig. 2e). Throughout
the experiment, we found that Chow group did not sig-
nificantly modify the total amount of calories consumed
from chow food (Fig. 2e; Chow group). As expected for
the HFD group, the HFD-Suc diet was the largest caloric
source, and thus HFD rats drastically reduced chow food
intake. However, no significant differences on overnight
food intake were observed during any phase of the exper-
iment (Fig. 2e). These results suggest that leptin/CCK
synergy on overnight intake, observed in juvenile-lean rats
(Fig. 1e) was completely absent in adult rats, rationalizing
the failure to induce weight loss.
Individual Sensitivity to Leptin and CCK on
Short-Term Intake does not Explain Weight Loss
We finally addressed the question whether the individual
sensitivity to leptin and/or CCK on short-term intake can
predict weight loss, i.e. if the rats that displayed the
stronger synergistic Ensure intake suppression corre-
sponded to animals with the largest body weight loss. In
order to address this hypothesis, we performed a simple
linear regression of normalized body weights during both
Leptin/CCK and BL3 phases (Fig. 2d) and computed the
slope or ‘‘b weight’’ for each subject. Positive values
indicate weight gain, whereas negative b weight indicates
weight loss. Finally we calculated the ‘‘D Intake index’’ by
subtracting the average Ensure intake during Leptin/CCK
phase minus CCK phase for each rat. Negative values
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Int J Pept Res Ther (2012) 18:77–88
indicate a stronger Ensure intake suppression, whereas
positive ‘‘D Intakes’’ indicate no suppression on short-term
Ensure intake (Fig. 3). In contrast to our hypothesis, we
found no significant correlation between ‘‘b weights’’ and
‘‘D Intake’’ values in any group. These results indicate that
rats that were more sensitive to daily co-administration of
leptin ? CCK were not necessarily the rats that lost more
weight. Therefore, our data suggest that leptin ? CCK
inhibited short-term food intake using a different mecha-
nism than that which induces weight loss. We acknowledge
that our total ‘‘n’’ of subjects that experience weight loss
was relatively small and thus further studies are necessary
to enhance our statistical power.
Discussion
In this study, we evaluated in the same rat the therapeutic
efficacy of the synergism between leptin and CCK as a
potential treatment to induced weight loss. We first evaluated
the effectiveness of daily leptin ? CCK co-administration
on body weight loss in juvenile and lean rats (2 months old;
250–350 g). As expected, we found that the combined
treatment caused a reliable body weight loss, associated with
a strong suppression on long-term overnight food intake, in
juvenile rats (Fig. 1). Based on these results, we then eval-
uated the responsiveness of adult Chow and HFD rats
(6 months old; 400–500 g) to the same combined treatment
(Experiment 2). In contrast to juvenile rats, we found that
co-administration of leptin and CCK failed to induce weight
loss in both Chow and HFD group (Fig. 2). This result
strongly suggests that the synergistic interaction between
leptin and CCK decreased in aged rats and was also inef-
fective under a HFD.
As noted, in comparison with lean rats there is a scarcity
of studies characterizing the synergy leptin ? CCK in rats
under a HF context (Barrachina et al. 1997); (Emond et al.
1999); (Matson et al. 1997). To our knowledge the only
report has been publish by Trevaskis et al. (2010) and
similar to us, they also showed that leptin ? CCK co-
administration was unable to reduce body weight in HFD
rats, even upon administration of higher leptin doses
([200 lg/kg), and using independent groups (Trevaskis
et al. 2010). Herein using a multiphasic design, our results
provide further evidence that adult rats (400–500 g) appear
to developed a gradual unresponsiveness to the synergy
induced by leptin ? CCK on body weight loss (compare
Figs. 1c vs. 2c).
CCKs Sensitivity
We found that juvenile rats, in comparison to adult rats
were the most sensitive to CCK as measured by the lack of
Int J Pept Res Ther (2012) 18:77–88 85

Juvenile Chow HF
.5 .5

Gain
.5

β Weight 0 0 0

-.5 -.5 -.5

-1 -1 -1
Loss

-1.5 -1.5 -1.5

R=-0.53; P >0.05 R=0.25; P >0.05 R=0.074; P >0.05
-2 -2 -2
-6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6

Sensitive Insensitive Δ Intake (Kcal)

Fig. 3 Individual leptin/CCK sensitivity on short-term Ensure does panel Experiment 1), Chow (middle) and HFD rats (right panel). ‘‘R’’
not predict body weight loss. D Intake index (leptin/CCK minus CCK shows the Pearson correlation coefficient and significant P value
phase) versus b weight (body weight change) for juvenile rats (left

tolerance to CCKs satiety action. Paradoxically, we results do not seem to implicate CCK has an important
observed that in comparison to Chow-fed group, HF rats player in hyperphagia and weight gain under a diet-induced
strongly suppressed Ensure intake after 5 days of CCK obesity context (Swartz et al. 2010). Rather, we propose
administration (Fig. 2b), suggesting that HF rats were that HF rats may have been more sensitive to CCK as a
slightly hypersensitive to CCK than Chow-fed rats of the compensatory mechanism to their failure to decrease
same age. One possible explanation to this phenomenon energy intake in the HFD-Suc diet in comparison to their
could be that persistent elevated CCK in plasma, such as Chow-fed group (Fig. 2e). Nevertheless, in comparison to
that observed after long-term HFD consumption (French juvenile rats, our data suggest that adult rats gradually
et al. 1995) may lead to several adaptive changes in became more insensitive to circulating satiety signals such
nutrient and CCK signaling pathways, for example rats fed as leptin (Scarpace and Zhang 2009) and even to CCK
on a HFD exhibit increased pancreatic secretory and (Covasa 2010), and thus it shows that sensitivity to circu-
plasma CCK release to intestinal fat (Spannagel et al. lating signals is dynamic and can change throughout life.
1996). Moreover, it has been recently shown that obese- Moreover, in Experiment 2, we observed that the sup-
prone rats, maintained, on a HFD, have increased expres- pression of Ensure by CCK decreased over contiguous
sion of CCK-1R, in the nodose ganglion (Paulino et al. daily administrations. It is already established that rats can
2009), and a stronger CCKs vagal nerve sensitivity mea- develop tolerance to repeated and contiguous daily CCK
sured by a greater number of C-fos reactive neurons administrations (Duncan et al. 2005; Goodison and Siegel
recruited in the NTS of obese rats, after CCK administra- 1995). These results are consistent with previous findings
tion. Although one previous study has reported that short by Goodison and Siegel that administered CCK 15 min
exposure to an isocaloric HFD decreased CCK sensitivity prior to sucrose access on nine consecutive days, and
(Covasa and Ritter 1998), our data in contrast suggest that whereas sucrose intake was suppressed on Day 1, rats
CCK exerted a more potent satiety action on HF adult rats developed tolerance over 9 days such that CCK no longer
on our 60% HFD. In agreement with our results, Torre- reduced sucrose intake (Goodison and Siegel 1995). Our
grossa and Smith (2003) found that CCK was significantly data extent this observation and since tolerance to CCKs
more potent in rats maintained on a 60% fat diet (Torre- satiety action did not occurred in juvenile rats, then we
grossa and Smith 2003). Accordingly, Chandler et al. suggest that CCKs tolerance can in fact reflect a slight
(2004) reported that DIO-prone rats under a HFD ([800 g insensitivity to CCK in the adult rat.
Body wt) were more sensitive to a low dose of CCK
(0.3 lg/kg) (Chandler et al. 2004). Moreover and more Leptin ? CCK Sensitivity
recently, Swartz and Covasa (2010) found that obese-prone
rats, maintained on a standard rat chow diet, were indeed We did not observe leptin/CCK synergy on short-term
more sensitive to several doses of CCK (1 and 4 lg/kg) Ensure intake using intraperitoneal daily administrations
than obese-resistant rats. Nevertheless both obese-prone (Figs. 1b and 2b). In agreement with previous reports e.g.,
and resistant rats were equally sensitive to 2 lg/kg CCK Matson et al. (1997) used leptin (50 lg/kg) and CCK
dose (Swartz et al. 2010). This is in agreement with our (2–4 lg/kg) and also found no synergistic reduction on
results since HF rats and Chow-fed group in this study were 30-min Ensure intake more than that achieved by CCK
equally sensitive to 2 lg/kg CCK during the first three alone (Matson et al. 1997). Moreover, we also observed a
injection days, but afterward, HF rats displayed a stronger gradual and cumulative suppression of Ensure intake as a
sensitivity to CCK dose (Fig. 2; CCK phase). In sum, our function of leptin ? CCK coadministrations (Fig. 1b), and

123
86
this effect was stronger in adult rats (Fig. 2b; Leptin/CCK
phase). Therefore, in our multiphasic design, leptin instead
of synergizing seems to initially antagonized short-term
CCK satiety action (Fig. 2b; compare the first three
injections CCK vs. Leptin/CCK phase). This result is
puzzling because there is a growing evidence indicating
that peripheral leptin can potentiate CCK vagal nerve
activation (Wang et al. 1997), and it is known that leptin
and CCK receptors are co-expressed in a subpopulation of
vagal terminals (Guilmeau et al. 2004). And vagal activity
is the primary target to potentiate CCK satiety action (de
Lartigue et al. 2010). In fact our data suggest that adult rats
under a HFD were hypersensitive to CCKs satiety action.
However, this CCK hypersensitivity appears not to be
sufficient to potentiate leptin ? CCK synergy in HFD rats.
Future experiments are necessary to uncover the physio-
logical mechanism that underlies this apparent leptin
antagonism on short-term CCK satiety action. Alterna-
tively, it is possible that the initial lack of synergism
between leptin ? CCK (Fig. 2b; Leptin/CCK phase) can
be explained by a lingering effect due to the pretreatment
administrations of CCK or leptin (CCK or High Leptin
phases), future studies using independent groups are nec-
essary to rule out this alternative.
We finally evaluated whether within-subject sensitivity
to leptin ? CCK on short-term intake predicted subsequent
weight loss. We found no significant correlation between
Ensure intake suppression and subsequent weight loss
(b weights). We propose that leptin ? CCK caused weight
loss using a different mechanism than that accountable for
suppressing short-term food intake (Matson et al. 2000,
2002).
Biosafety Concerns of Leptin ? CCK Treatment
Despite the strong body weight loss induced by lep-
tin ? CCK, we note in Experiment 1 that two juvenile rats
continued losing weight even after 5 days from withdrawal
of treatment, to our knowledge there is no precedent for
leptin, CCK or the combination to continue suppressing
food intake neither to lose weight for such a long period
post-administration. These results strongly suggest that the
2 animals in the Treatment group might have developed an
enteric illness caused most likely by the combined treat-
ment. We note that it is unlikely that the high number of
i.p. injections itself could cause any enteric tissue damage,
since no animal in the Saline group lose body weight
during the entire experiment. Moreover, it has been shown
that the administration of exogenous leptin (Allman et al.
2009) and CCK (Wu et al. 2011) significantly reduced the
weight of pancreas, and resulted in acute pancreatitis (AP),
an inflammatory disease that can lead to a severe necrotic
condition, renal failure, and eventual death (Konturek et al.
123
Int J Pept Res Ther (2012) 18:77–88
2002). On the one hand AP, in rats and in humans, is
associated with a marked increase in the plasma levels of
leptin, and since leptin promotes hepatic inflammation and
local inflammation on mesenteric adipose tissue (Allman
et al. 2009). Likewise, CCK stimulates pancreatic secretion
and induces growth of the pancreas (Mussa et al. 2008).
Despite that we did not perform any histological analysis;
our present findings suggest that the combination lep-
tin ? CCK could most likely caused pancreatitis as a side
effect of their combined treatment.
Future Perspective for Neurohormonal Synergies
Our main result clearly shows that under DIO context,
leptin ? CCK treatment had an age-limited therapeutic
efficacy. Nevertheless, leptin is a major player regulating
body weight and homeostatic energy and it has been shown
that leptin resistance occurs in HFD rats (Levin and Dunn-
Meynell 2002), thus one therapeutic alternative may be first
to rescue leptin responsiveness and then co-administrate
leptin ? CCK. One possible way to re-sensitize leptin
action is by performing physical exercise (Levin and Dunn-
Meynell 2006). Alternatively, reducing endoplasmic retic-
ulum stress using a chemical chaperones such as 4-phenyl
butyric acid and tauroursodeoxycholic acid has been shown
that acts as a leptin-sensitizing agent that can enhance the
sensitivity to leptin in obese mice (Ozcan et al. 2009).
Leptin can also interact with other peripheral satiety
hormones for example leptin extends the anorectic effect of
PYY3-36 and caused greater weight loss in lean rats (Ha-
latchev and Cone 2005). PYY3-36 is another gut-satiety
signal released by L cells in ileum and colon. Leptin and
PYY3-36 have been explored in a DIO context and have
been shown to be mathematically additive (Roth et al.
2008). However, exogenous PYY3-36 induces taste aversion
(Halatchev and Cone 2005). More recently, amylin phar-
maceutics showed that the combined administration of the
b-cell hormone amylin and leptin induced a marked and
reproducible synergistic interaction on DIO rats. In fact,
the triple combination amylin ? leptin ? CCK achieved
greater weight loss than any other treatment combined
(Trevaskis et al. 2010). This is a promising therapeutic
avenue indicating that treatment combinations are in gen-
eral more effective than monotherapy to control body
weight.
Concluding Remarks
In conclusion, under our experimental conditions, our data
demonstrate that the therapeutic efficacy of leptin/CCK
synergy on weight loss had a limited therapeutic window
Int J Pept Res Ther (2012) 18:77–88
related to age. Therefore, our data indicates that at least for
adult rats it would be necessary first to rescue leptin sen-
sitivity and then administer it in a combined treatment.
Thus so far from all leptin synergies reported in the liter-
ature, the amylin ? leptin combination (Roth et al. 2008)
is by far the most promising leptin synergy for the treat-
ment of obesity.
Acknowledgments We thank to Garcia Osornio E. for technical
help, Daniel Garcia for help with Matlab programming and to Eric E.
Thomson for discussion on this manuscript. This work was supported
by CONACYT Grants 78879, Salud 2010-02-151001, ICYTDF-
PICDS08-59 and Productos Medix 000652 to R.G.
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