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Department of Psychiatry and Behavioral Science, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We investigated whether dorsal
hindbrain and/or peripheral cocaine- and amphetamine-regulated
transcript peptide (CARTp) acts to suppress gastric emptying of a
caloric stimulus. Furthermore, effects of dorsal hindbrain CARTp on
sucrose consumption and licking microstructure was studied, as well as
the possible contribution of corticotropin-releasing factor (CRF)
receptors to mediate effects of CARTp downstream on emptying and
sucrose intake. Rats bearing gastric fistulas received intragastric
infusions (1.0 ml/min) of 12 ml 12.5% glucose. Gastric samples were
withdrawn immediately after the intragastric infusion to reflect
emptying during gastric fill. CARTp injected in the fourth ventricle
intracerebroventricularly (0.5 and 1.0 µg) suppressed gastric
emptying. CARTp reduced sucrose intake at similar doses and altered a
variety of lick microstructure variables (no. of licks, bursts,
clusters, licks/burst, licks/clusters, interlick interval, first meal
size, and first meal duration). Pretreatment with the CRF antagonist
-helical CRF-(9-41) blocked the effect of 1.0 µg
CARTp on gastric emptying but not on sucrose consumed or on any of the
licking microstructure parameters. These data demonstrate differential
mediation of the feeding and gastric inhibitory effects of CARTp and
suggest that CARTp-induced inhibition of gastric emptying does not
contribute to this peptide's ability to inhibit food intake.
brain stem; corticotropin-releasing factor; ingestive behavior; licking microstructure
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PEPTIDES ENCODED FROM cocaine- and amphetamine-regulated transcript (CART; see Refs. 9 and 33) have been shown to inhibit food intake when administered in the brain ventricles (17, 19) or in the paraventricular nucleus (PVN) of rodents (28), and a role for CART-derived peptides (CARTp) as endogenous brain satiety factors has been proposed (17). CART and CARTp-like immunoreactivity (LI) have been detected in several key areas of the hypothalamus that are involved in the control of ingestive behavior, including the arcuate nucleus, the PVN, and the lateral and dorsomedial hypothalamic nuclei (4, 9, 16). CART and CARTp-LI are also found in the brain stem where they are present in several nuclei that are involved in the control of gastrointestinal functions and food intake. These sites include the area postrema, the nucleus of the solitary tract (NTS), and the parabrachial nucleus (PBN; see Refs. 9 and 16). CARTp-LI has been detected in fibers of the vagus nerve (2, 16), and CART mRNA is expressed in the nodose ganglion of the rat (2). Intracerebroventricular injection of CARTp-(42-89) has been shown to induce c-fos expression in both hypothalamic and brain stem nuclei associated with gastric function and/or food intake control, including the PVN, lateral PBN, and the NTS (35). CARTp-LI is also found in the periphery. For example, CARTp-LI is present in cholinergic neurons of the myenteric plexus (5) and the islets of Langerhans (13), suggesting the possibility that CARTp may also affect gastrointestinal function through peripheral receptor targets.
Recently, CARTp injected intracisternally was shown to inhibit both
gastric acid secretion and the gastric emptying of a small, noncaloric
load in rats (25). Although the distribution and structure
of putative CARTp receptors are not yet known, it is possible that the
targets for the CARTp-induced suppression of gastric emptying obtained
by intracisternal injection (25) are substrates in the
hindbrain. However, given that agents administered in the cisterna
magna first must pass the narrow foramina of Magendi and Luschka to
access the fourth ventricle and relevant brain stem nuclei and that the
cerebrospinal fluid normally circulates in the opposite direction,
intracisternal administration does not allow for a determination of a
hindbrain site of action to be clearly made. In the present study, we
directly assess a dorsal hindbrain site of action for CARTp by
application of recombinant CARTp-(55-102) in the
fourth ventricle (4th icv) of rats and further examine whether an
effect of CARTp in the hindbrain may be dependent on the participation
of corticotropin-releasing factor (CRF) pathways. CRF, a peptide well
known for its key role in the regulation of the
hypothalamic-pituitary-adrenal (HPA) axis, has actions that, to some
extent, resemble those induced by CARTp, including suppression of food
intake (15, 18, 24) and the inhibition of gastric emptying
(20, 27, 32) and gastric acid secretion (20, 29,
31). CART mRNA and CARTp-LI are present at all levels of the HPA
axis (4, 9). Moreover, evidence suggests that CRF and
CARTp may be anatomically and functionally connected in areas related
to gastrointestinal and food intake controls, i.e., CARTp-containing
fibers closely oppose CRF-immunoreactive PVN neurons that, in addition,
respond to intracerebroventricular CARTp with induction of
c-fos (35). In the brain stem, CARTp is present
in the NTS, and receptors for CRF are known to be present in the dorsal
motor nucleus of the vagus (DMX)/NTS complex (26). Importantly, CARTp effects on gastric acid secretion have been demonstrated to be blocked by the CRF antagonist -helical
CRF-(9-41), suggesting that CRF may be a downstream
mediator of CARTp's gastrointestinal actions (25).
Together, these data raise the possibility that effects of CARTp in the
caudal brain stem on gastric emptying and/or on food intake may also be
CRF dependent.
In the following experiments, the ability of fourth intracerebroventricular CARTp to inhibit gastric emptying was examined, and a role for CRF in the mediation of such an inhibition was assessed. We studied gastric emptying in an experimental paradigm that resembles the physiological conditions during consumption of a meal. Differential controls of gastric emptying during and after a period of ingestion have been identified (14, 21, 22). Whereas emptying of a nutritive fluid is regulated by caloric feedback control in the postfill period (21), emptying during ongoing gastric fill is significantly more rapid and appears to be dependent on volumetric, rather than caloric, controls (14, 22). In the present study, animals equipped with chronic gastric fistulas were given intragastric infusions of 12.5% glucose. The volume and rate of administration were chosen to correspond to what rats typically ingest in fluid intake tests. Gastric samples were collected immediately after the termination of the intragastric stimulus delivery to directly assess the effects of CARTp on gastric emptying during gastric fill.
A ligand's effects on gastric emptying have sometimes been proposed to
be an underlying mechanism for its effects on food intake. For example,
CCK inhibits food intake and gastric emptying, and the gastric
inhibitory actions have been proposed to contribute to its satiety
actions (23). Conversely, the increase in gastric emptying
caused by glutamate-receptor activation has been shown to directly
contribute to its orexigenic effect (6). In an effort to
assess a potential role for CARTp's inhibition of gastric emptying in
CARTp-induced reductions of food intake, we addressed whether
pretreatment with -CRF would affect CARTp-induced alterations of
food intake and gastric emptying in similar manners. Because CARTp has
been demonstrated to produce changes in the structure of meals and to
produce an overall inhibition of food intake (1), we used
a lickometry paradigm to assess the potential CRF mediation of CARTp's
feeding inhibitory action.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Male Sprague-Dawley rats weighing 300 g at the time of surgery (Charles River Laboratories, Wilmington, MA) were housed singly under conditions of controlled temperature, humidity, and illumination (lights on 7:00 AM-7:00 PM). The animals had free access to drinking water and chow (Prolab RMH 1000) until 1 h before testing in experiments 1-3. In experiments 4 and 5, animals were allowed free access to drinking water at all times, but food was removed 2 h before testing. Different groups of rats were used for each experiment. The experimental protocols were approved by the Animal Care and Use Committee at the Johns Hopkins University School of Medicine.Surgery
For surgical procedures, rats were anesthetized with a 4:3 mixture of ketamine (100 mg/ml; Phoenix Pharmaceutical, St. Joseph, MO) and xylazine (20 mg/ml; Phoenix Pharmaceutical), injected intramuscularly at a dose of 1.0 ml/kg body wt. All surgeries were performed under aseptic conditions. For gastric emptying experiments, rats were equipped with chronic gastric fistulas. Before all gastric surgeries, the animals were food deprived overnight. After laparotomy, two concentric purse-string sutures were sewn in the ventral forestomach, along the major curvature. A small opening was made in the center of the purse, where a stainless steel gastric fistula was inserted. The purse was closed around the fistula and secured with ligatures. Finally, the distal end of the fistula was exteriorized through a paramidline puncture of the abdominal wall and skin and secured with a purse-string suture. The wound was closed, and the animals were allowed 9 days of recovery before implantation of fourth intracerebroventricular guide cannulas. In experiments 1, 3, 4, and 5, the rats were implanted with chronic guide cannulas aimed at the fourth brain ventricle, as described previously (27). The coordinates were 2.6 mm anterior to the occipital crest in the midline. The animals were allowed 6-7 days of recovery after guide cannula surgeries before the beginning of habituation training sessions.Experimental Design
Gastric emptying experiments. The experiments were carried out every 3rd day to avoid possible carryover effects of the peptides or antagonist. Gastric emptying was assessed between 2:00 and 4:30 PM, with each animal tested at approximately the same time point each test day. The animals were habituated to the experimental procedure during four once-daily training sessions in which they underwent the entire protocol, but no intracerebroventricular injections were given (experiments 1 and 3). During the habituation period preceding experiment 2, a subcutaneous saline injection (1.0 ml/kg) was given 20 min before the onset of the intragastric infusions. Before testing (1 h), the gastric fistulas were opened, and intragastric contents were evacuated carefully with water per lavage. After gastric lavage, the animals were placed in Plexiglas test cages with wire mesh floors. Just before testing, the fistulas were opened and connected to a Harvard Pump 22 (Harvard Instruments, South Natick, MA) via a silicone tube. The animals received an intragastric infusion (1.0 ml/min) of 12.5% (0.694 mol/l) glucose for 12 min. After infusion offset, the silicone tube was clamped and cut closely to the opening of the fistula. The remaining intragastric glucose solute was quickly aspirated immediately after the termination of the intragastric infusion, and the volume of the collected sample was measured. The stomachs were then rinsed with 5.0 ml distilled water to detect any solute remaining in the stomach after the initial aspiration. The fistulas were finally closed, and the animals were returned to their respective home cages.
The glucose concentrations of the collected samples and of the rinse returns were determined with a glucose oxidase kit (Trinder; Sigma-Aldrich). Samples were analyzed in duplicate, and absorbance was determined at a wavelength of 505 nm on a Bausch & Lomb Spectronic 20 spectrophotometer. The volumes recovered, the gastric glucose concentrations of the infusate, the primary sample, and the rinse returns were then used to calculate the amount solute emptied, the volumes retained, and the gastric secretion volumes.Sucrose lickometry experiments. The animals were habituated to the lickometry testing protocol during a series of 10-12 once-daily training sessions. The experiments began when their sucrose (10.27% or 0.3 mol/l) intake volumes had reached plateau levels and were unchanged for at least four consecutive days. Before lights off (2 h), the food hoppers were removed from the respective home cages, and 15 min before lights off, the rats were placed in the lickometer testing cages. At the onset of the dark period, the rats were given access to a sucrose solution for 30 min. The experiments were run every 3rd day to avoid possible drug carryover effects. On days between testing sessions, the animals were placed in the test cages and given access to the sucrose; however, no injections were given. Each lickometry testing cage consisted of drinking bottles with stainless steel drinking spouts attached to an interface. Each time an animal's tongue touched the drinking spout, an electrical circuit was closed, and a 60-nA current passed through the rat. The current, well below what the animal can detect (36), was amplified and recorded with an IBM AT computer. The time (ms) for each tongue contact was thereby detected. The collected data files were then analyzed with the Tongue Twister software (12). The data collected during the entire 30-min test period were analyzed according to Davis and Smith (8), and the number of licks, bursts, clusters, licks/burst, and licks/cluster and the average interlick interval length were quantified. We chose an interlick interval 230-550 ms as the criterion for the end of a cluster. Licks that were of a nonburst character were filtered. The sucrose volumes consumed for the entire observation period (30 min) were also recorded. To specifically analyze the rat's behavior during the first meal, a meal pattern analysis was also performed. Three licks separated by interlick intervals <250 ms followed by a period of at least 5 min without licking were defined as a meal. The size of the first meal (no. of licks) and the duration of the first meal (min) were identified.
Fourth Intracerebroventricular Injections
For fourth intracerebroventricular injections, a Gilmont microinjector, attached to a 32-G injection needle via a PE-20 tube, was used. The animals were restrained gently by hand, the injection needle was inserted through the guide and into the fourth ventricle, and drug or vehicle (3 µl) was injected into the fourth ventricle intracerebroventricularly over 1 min. The injector was left in place for another 30 s to reduce the risk of backflush. After the injection needle was removed, a new obturator was inserted in the guide. Before the habituation training sessions, functional assessments of the fourth intracerebroventricular cannula placements were performed by fourth ventricle intracerebroventricular injection of 210 µg 5-thio-D-glucose. A doubling in blood glucose over 1 h was taken as a correct placement (10). After the last experimental testing session, the animals were anesthetized, and 3 µl India ink were injected in the fourth ventricle. The animals were killed, and the brains were removed, frozen, and sectioned. The site of injection was confirmed by inspection of the dye location in the fourth ventricle. One animal in experiment 1 and one animal in experiment 3 were excluded from the study since in both cases the dye was found in the cerebellum and not in the fourth ventricle.Drugs
Synthetic CART-(55-102) peptide (rat; American Peptide, Sunnyvale, CA) and-helical CRF-(9-41)
(Sigma-Aldrich) were dissolved in saline and distilled water,
respectively, then separated into aliquots and frozen (
20°C). Fresh
aliquots were thawed on each experimental day before injections, and
any excess was discarded.
Design
Experiment 1. In experiment 1, dose-response effects of CARTp in the caudal brain stem on gastric emptying of glucose during gastric fill and gastric secretion volume were examined. CARTp (0.1, 0.5, and 1.0 µg), or saline as a vehicle, was administered in the fourth ventricle in a randomized crossover design 15 min before the onset of a 12-min intragastric infusion (1 ml/min) of glucose (12.5%). Gastric samples were withdrawn immediately after infusion offset to reflect emptying during gastric fill.
Experiment 2. This experiment examined the possible peripheral effects of CARTp on gastric emptying and gastric secretion volume during gastric fill. The following two questions were addressed: 1) whether centrally administered CARTp could be affecting gastric emptying by a peripheral mechanism, that is, whether the CARTp administered in the fourth ventricle had passed in the peripheral circulation and caused the effects on emptying that were observed in experiment 1, and 2) whether CARTp in a higher peripheral dose may act to control emptying or gastric secretion. For these purposes, the low dose of CARTp administered peripherally was 2.5 µg/kg body wt or ~1.0 µg, analogous to the effective fourth ventricle intracerebroventricular dose; the high dose of CARTp was 25 µg/kg body wt, or 10 µg. Each CARTp dose or vehicle was administered once in a randomized crossover design. The subcutaneous injections of CARTp or vehicle (1.0 ml/kg) were administered 20 min before the onset of a 12-min intragastric infusion of glucose (1.0 ml/min). The remaining stomach solute was collected immediately after termination of the infusion so that gastric emptying during gastric fill could be determined.
Experiment 3.
The hypothesis that CARTp acts at a caudal brain stem site to suppress
gastric emptying during gastric fill via a CRF-dependent mechanism was
addressed. The 10-nmol dose of -CRF has previously been shown to
block fourth intracerebroventricular effects of 1,000 pmol CRF on
emptying suppression (27) and a number of other CRF- and
stress-related physiological and behavioral paradigms (18,
20). The animals were pretreated with 3 µl of 10 nmol of the
CRF antagonist -helical CRF-(9-41) (-CRF)
injected fourth intracerebroventricularly, or vehicle (distilled
water), 10 min before fourth intracerebroventricular CARTp (1.0 µg; 3 µl), or vehicle (saline), injections. The CARTp or vehicle
combination was given 15 min before the intragastric glucose infusion.
Each combination was administered one time in a randomized crossover design. Glucose was infused in the stomach (1.0 ml/min) over 12 min,
and gastric samples were collected immediately after the offset of the infusion.
Experiment 4. The dose-response relationship of CARTp injected in the fourth ventricle on ingestion of a 0.3 mol/l sucrose meal was investigated. CARTp (0.1, 0.5, and 1.0 µg) or saline as a vehicle was injected in the fourth ventricle 15 min before sucrose access. Doses of CARTp were administered in a randomized, crossover design so that each rat received each dose one time. The rats were placed in the lickometer testing cages immediately after the fourth intracerebroventricular injection. The animals were given access to the lickometer spout for 30 min at the onset of the dark period.
Experiment 5.
The hypothesis that CRF may be a downstream messenger for fourth
ventricular CARTp-induced suppression of food intake was tested. The
CRF antagonist -CRF (10 nmol; 3 µl) or vehicle was administered 10 min before the CARTp or vehicle, which in turn was given 15 min before
meal presentation/lights off. The highest CARTp dose from
experiment 4 (1.0 µg) to suppress sucrose intake was
chosen. The animals were given access to the sucrose lickometer spout
for 30 min at lights off.
Data Evaluation
The lickometry raw data were analyzed using Tongue Twister software (12) before statistical evaluation. The data for experiments 1, 2, and 4 were analyzed with repeated-measures ANOVA followed by Dunnett's test for post hoc comparisons and by two-way repeated-measures ANOVA followed by post hoc Tukey's test where appropriate for experiments 3 and 5. In all cases, P < 0.05 was considered significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiment 1
As demonstrated in Fig. 1, repeated-measures ANOVA showed an overall effect of fourth intracerebroventricular CARTp on solute emptied [F(3,18) = 18.66, P < 0.001; n = 7] and on gastric volume retrieved [F(3,18) = 15.62, P < 0.0001; n = 7], but not on gastric secretion volume [F(3,18) = 0.213, not significant (NS); n = 7]. Post hoc comparisons (Dunnett's test) showed that 0.5 and 1 µg CARTp dose-dependently suppressed solute emptying by 35.7% (P < 0.05) and 68.5% (P < 0.01), respectively, compared with vehicle, whereas the 0.1-µg dose was without effect. Reciprocal to the suppression of gastric emptying, gastric volume retained was increased in response to fourth intracerebroventricular injection of 0.05 µg (P < 0.05) and 1.0 µg (P < 0.01; n = 7).
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Experiment 2
As shown in Fig. 2, in contrast to fourth intracerebroventricular CARTp, subcutaneously administered doses of CARTp failed to affect solute emptying [F(2,14) = 0.242, NS; n = 8]. There was no effect on gastric volume retained [F(2,14) = 0.242, NS; n = 8] or gastric secretion volume [F(2,14) = 2.49, NS; n = 8, data not shown].
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Experiment 3
In Fig. 3, the effect of pretreatment with the unselective CRF antagonist-CRF on
CARTp-induced inhibition of gastric emptying during fill is shown.
Two-way repeated-measures ANOVA showed a significant main effect of
CARTp on gastric emptying [F(1,7) = 26.775, P < 0.01] and a significant effect of -CRF
[F(1,7) = 9.781, P < 0.05] but no
significant interaction [F(1,7) = 2.017, P > 0.05]. Post hoc Tukey's test showed a
significant suppression of gastric emptying in response to CARTp
(vehicle/vehicle vs. vehicle/CARTp, P < 0.001),
thereby replicating the results of experiment 1.
Furthermore, although -CRF did not by itself affect gastric emptying
(-CRF/vehicle vs. vehicle/vehicle, NS and -CRF/vehicle vs.
-CRF/CARTp, NS), it blocked the effect of CARTp. Thus the suppression of gastric emptying by CARTp injection was antagonized by
-CRF pretreatment (vehicle/CARTp vs. -CRF/CARTp,
P < 0.05).
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Experiment 4
The repeated-measures ANOVA for sucrose consumed showed a significant effect of CARTp [F(3,6) = 6.828, P < 0.01], and post hoc Dunnett's test demonstrated that both 0.5 and 1.0 µg significantly (P < 0.01) suppressed intake, but 0.1 µg did not (Fig. 4). Further evaluation of the licking microstructure parameters for the entire 30-min observation period (Table 1) showed overall significant effects of CARTp on number of licks [F(3,6) = 5.276, P < 0.01] and on interlick interval [F(3,6) = 12.220, P < 0.0001]. There was an overall CARTp effect on the number of licks per burst [F(3,6) = 6.419, P < 0.01] and number of licks per cluster [F(3,6) = 6.693, P < 0.01], whereas the number of bursts [F(3,6) = 0.6454, NS] or number of clusters [F(3,6) = 0.6580, NS] was unaffected by CARTp. Post hoc Dunnett's test further showed that 0.5 and 1.0 µg, but not 0.1 µg, CARTp significantly suppressed the number of licks and the number of licks per burst similarly (P < 0.05 and 0.01, respectively). The number of licks per cluster was suppressed in response to 1.0 µg CARTp (P < 0.001). Finally, Dunnett's test showed that the interlick interval was significantly increased in response to 0.5 µg CARTp (P < 0.05) and 1.0 µg CARTp (P < 0.01), but no effect was found in response to 0.1 µg.
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Analysis of the lickometry data for the first meal showed a dose-dependent reduction in meal size (no. of licks) [F(3,18) = 5.882, P < 0.01] in response to CARTp, whereas the duration of the first meal [F(3,18) = 0.1238, NS] was unaffected. Post hoc Dunnett's test showed that all three doses of CARTp suppressed the first meal size (0.1 and 0.5 µg, P < 0.05; 1.0 µg, P < 0.001).
Experiment 5
Two-way repeated-measures ANOVA (2 × 2; CARTp injection ×-CRF pretreatment) showed a main effect of CARTp on the volume of
sucrose consumed [F(1,7) = 49.443, P < 0.001]. There was no effect of -CRF [F(1,7) = 0.0052, P > 0.05] and no significant interaction
between CARTp and -CRF [F(1,7) = 0.147, P > 0.05], indicating that the effect of CARTp was
not dependent on -CRF (Fig. 5).
Statistical evaluation of the lickometry data replicated the results of
CARTp from experiment 4. It showed further that -CRF was
without effect and did not alter any of the effects of CARTp. The
results from the two-way repeated-measures ANOVAs are shown in Table
2. For the licking microstructure
parameters recorded during the entire 30-min test, two-way
repeated-measures ANOVA (2 × 2) showed a significant main effect
of CARTp on the number of licks [F(1,7) = 60.738, P < 0.001] but no main effect of -CRF
[F(1,7) = 1.099, P > 0.05], and
there was no interaction [F(1,7) = 0.377, P > 0.05]. In addition, there was a main effect of
CARTp on the number of licks per burst [F(1,7) = 25.297, P < 0.05] but no main effect of -CRF
[F(1,7) = 1.716, P > 0.05], and
there was no interaction between CARTp and -CRF injection groups
[F(1,7) = 3.588, P > 0.05].
Furthermore, there were no main effects of CARTp or by -CRF on the
number of bursts or on number of clusters, nor were there any main
effects of either CARTp or of -CRF on the number of licks per
cluster. Finally, there was a significant main effect of CARTp on the
interlick interval [F(1,7) = 6.801, P < 0.05] but no main effect of -CRF pretreatment
[F(1,7) = 0.0907, P > 0.05], and the
effect of CARTp was not affected by -CRF, as indicated by the lack
of a significant interaction [F(1,7) = 0.277, P > 0.05].
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The two-way repeated-measures ANOVA for licking parameters from the
first meal showed a significant main effect of CARTp on meal size
[F(1,7) = 130.273, P < 0.001] but
not of -CRF [F(1,7) = 2.156, P > 0.05], and again, there was no significant interaction [F(1,7) = 0.343, P > 0.05]. Finally,
there was a significant main effect of CARTp on duration of the first
meal [F(1,7) = 47.436, P < 0.001]
but no effect of -CRF, and there was no significant interaction
[F(1,7) = 0.544, P > 0.05 and
F(1,7) = 0.858, P > 0.05, respectively].
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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These data demonstrate that fourth ventricular, but not peripheral, CARTp potently suppresses glucose gastric emptying during fill and that this gastric inhibitory action of central CARTp depends on a CRF intermediary. In contrast to the CRF mediation of the gastric inhibitory actions of CARTp, CARTp-induced suppression of sucrose ingestion is independent of CRF pathways. Together, these findings suggest that the effects of CART on food intake are independent of its effects on gastric emptying.
Inhibition of gastric emptying by intracisternally administered CARTp has been demonstrated previously (25). Those experiments assessed the effects of CARTp on the emptying of a small, noncaloric load given as a bolus. The present experiments extend these observations to demonstrate that fourth intracerebroventricular CARTp inhibits the emptying of a nutrient solution given in a paradigm that mimics gastric fill during a meal. Thus these data suggest a potential role for CARTp in determining rates of gastric emptying during ingestion of a meal.
The observation that CARTp injected in the fourth ventricle (Fig. 1), but not peripherally administered CARTp (Fig 2), inhibits gastric emptying suggests that there is no peripheral action by CARTp to affect gastric motor function. Instead, it would appear that CARTp acts at a dorsal hindbrain site to cause suppression of gastric emptying. CART and CARTp-LI have not only been detected within the central nervous system but also in the vagus nerve and nodose ganglion, and in the periphery. For example, CARTp is present in the duodenal myenteric plexus (5) and in the islets of Langerhans (13). However, effects of CARTp delivered in the periphery on gastrointestinal function have, as yet, only been reported in one study. Continuous intravenous infusion of CARTp increased amylase output in rats (7), an effect that was blocked by vagotomy and attenuated by pretreatment with atropine or the CCK-A receptor antagonist L-364718. There was no effect by CARTp on amylase secretion in vitro, however. The latter indicates that the intravenously delivered CARTp may act at a central, rather than a peripheral, site to induce a vagal-, cholinergic-, and CCK-dependent increase in amylase in the periphery. Another possibility would be that CARTp acts on vagal afferent fibers to induce pancreatic exocrine secretion. Our experiments begin to address whether peripheral CARTp signaling may play a role in the gastric inhibitory actions of CARTp. Subcutaneous administration of 2.5 µg/kg CARTp, a dose equivalent to the 1.0-µg fourth intracerebroventricular dose, as well as a 10-fold higher dose, failed to affect either gastric emptying or gastric secretion volume (Fig. 2). These data argue against a direct peripheral site of action for either intracerebroventricular, or for subcutaneously, delivered CARTp to induce alterations in gastric function. However, many gastrointestinal peptides (i.e., CCK, somatostatin) are very rapidly degraded in the periphery. Thus the possibility remains that, with higher subcutaneous doses, or continuous intravenous infusions, motoric or secretory actions of peripheral CARTp may be revealed.
The gastric inhibitory actions of CARTp were blocked by pretreatment
with -CRF, indicating that the effect was dependent on CRF pathways.
Therefore, it appears that CARTp may act centrally, using CRF as a
downstream messenger to control inhibition of gastric acid secretion
(25) and, as shown for the first time here, gastric emptying (Fig. 3). The nonselective CRF antagonist -CRF is known to
reverse a variety of stress-induced physiological effects, including
inhibition of gastric acid secretion (20, 29), gastric motor function (20), and food intake (18).
Moreover, while blocking effects of centrally administered exogenous
CRF, -CRF administered centrally exhibits no effects by itself
either on acid secretion, gastric emptying, gastrointestinal transit,
or on food intake in rats that have not been subjected to stress stimulation (18, 20, 27, 29). Because -CRF by itself, in line with previous observations (20, 27), did not
affect emptying while blocking the effects of CARTp (Fig. 3), the
present results suggest that CARTp, at least in part, acted via a CRF intermediary. CRF-containing neurons are present within the DMX (11, 30), the Barrington's nucleus (34), or
within scattered CRF-containing cells within the reticular formation
(34). Both of these latter sites project to the DMX/NTS
complex. A mapping of the receptors for CARTp and the identification of
the neurons expressing CARTp receptors will allow a more complete
determination of the neuronal sites and mechanisms of action of
hindbrain CARTp in the control of gastric emptying.
The present results demonstrating that fourth ventricular CARTp significantly reduced short-term sucrose consumption replicate and extend earlier findings (1, 37). CARTp inhibited intake through a dose-dependent reduction in meal size (Table 1). In addition, CARTp caused a decrease in the number of licks per burst, whereas the number of clusters and bursts, and the number of licks per cluster, were unaffected. Moreover, we found no effect on meal duration. These findings are similar to lickometry data from experiments examining the ability of lateral ventricle CART to affect intake during more long-term (6 h) access to an Ensure diet (1). In addition, CARTp specifically increased the interlick interval, supporting the notion that this peptide may modify oral motor function. In contrast to Aja et al. (1), we found a dose-dependent reduction in the number of licks per burst in response to CARTp. Interestingly, although we used the identical CARTp fragment and same route of delivery (4th intracerebroventricular), we also detected significant effects at lower doses than Aja et al. The lowest effective dose to reduce the number of licks was 0.1 µg, and 0.5 µg significantly suppressed licks/burst compared with 1.0 µg in the previous study. It is likely that the higher efficacy is the result of fourth rather than lateral, intracerebroventricular administration. Zheng et al. (37) have demonstrated greater inhibition of sucrose intake after fourth than after lateral intracerebroventricular administration of a single CARTp dose and found significant suppression of sucrose intake at a fourth intracerebroventricular threshold dose of 0.08 nmol (0.408 µg). This higher potency may also be because of differences in stimulus (sucrose vs. Ensure), the differences in feeding regimen (scheduled access followed by access to chow vs. maintenance diet), or that the shorter observation time in our experiment compared with Aja et al. (30 min vs. 6 h; see Ref. 1) simply caught significant effects that otherwise may diminish in the hours after drug administration.
The presence of CART and CARTp in close relation to CRF-containing
hypothalamic and hindbrain structures known to be involved in food
intake controls provides anatomic support for a potential functional
interaction. Kochavi et al. (15) showed that, similar to
CARTp, injection of 2.5 µg CRF in the third ventricle of rats dose
dependently suppressed volume consumed, decreased the number of licks,
and caused a reduction in meal size via a specific reduction in the
number of clusters. In addition, CRF shortened the meal duration,
whereas latency was increased. We found that, although pretreatment
with an unselective CRF antagonist blocked the CARTp-induced effects on
gastric emptying, it failed to antagonize the effects of CARTp on
ingestive behavior. Instead, on average, there appeared to be a
tendency for -CRF to exaggerate the suppressive effect of CARTp on
some of the microstructure variables (Table 2). However, the two-way
repeated-measures ANOVAs failed to verify such a potential interaction,
and, in this case, the use of a post hoc test would not be appropriate.
Together, this would suggest that, in contrast to its effect on gastric
emptying, CARTp acts independently of CRF to reduce meal size or volume consumed.
In summary, acute application of CARTp-(55-102) in the fourth ventricle of rats suppresses gastric emptying during gastric fill. The during-fill suppression of gastric emptying was reversed by pretreatment with a CRF antagonist. These results demonstrate that CARTp acts in the caudal brain stem to reduce gastric emptying via a CRF-dependent mechanism. In contrast, CARTp-induced suppression of sucrose intake is not dependent on the integrity of CRF pathways. This differential mediation suggests that CARTp gastric inhibitory actions are not a necessary component of CARTp ability to inhibit food intake.
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ACKNOWLEDGEMENTS |
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Thomas Houpt is gratefully acknowledged for providing the Tongue Twister software.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-19302. U. Smedh was supported by a Research Fellowship from the Wenner-Gren Foundations, Sweden.
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FOOTNOTES |
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Address for reprint requests and other correspondence: U. Smedh, Dept. of Surgery, Lund University Hospital, SE-22185, Lund, Sweden (E-mail: ulrika.smedh{at}fyfa.ki.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 5, 2002;10.1152/ajpregu.00665.2002
Received 29 October 2002; accepted in final form 3 December 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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