Articles in PresS. Am J Physiol Regul Integr Comp Physiol (February 17, 2005). doi:10.1152/ajpregu.00745.2004
SEROTONIN-TYPE 3 RECEPTORS MEDIATE INTESTINAL POLYCOSE- AND
GLUCOSE- INDUCED SUPPRESSION OF INTAKE
David M. Savastano, Melissa Carelle, and Mihai Covasa
Department, of Nutritional Sciences, College of Health and Human Development, The
Pennsylvania State University, University Park, PA, 16802-6504
Running head: Duodenal glucose and 5-HT3 receptors
Address correspondence to: David M. Savastano
Department of Nutritional Sciences
College of Health and Human Development
The Pennsylvania State University
126 South Henderson
University Park, PA, 16802
Email: [email protected]
Copyright © 2005 by the American Physiological Society.
ABSTRACT
Ondansetron, a selective serotonin-type 3 (5-HT3) receptor antagonist, was used to test
the hypothesis that duodenal infusion of isosmotic solutions of Polycose or its hydrolytic product
glucose, suppressed intake through 5-HT3 receptors. Polycose suppressed sucrose intake across
both concentrations infused (132mM, 7.6 ± 0.6 ml; 263mM, 2.3 ± 0.5 ml), compared to intake
under control conditions (12.6 ± 0.3 ml, P <0.001). Pretreatment with 1.0 mg/kg ondansetron
attenuated reduction of sucrose intake induced only by the highest concentration of Polycose (4.6
± 0.8 ml, P=0.004). Dose response testing revealed that suppression of food intake by 263mM
Polycose was attenuated by ondansetron administered at 1.0, 2.0, and 5.0mg/kg equally, but not
when given at 0.125, 0.25, and 0.5 mg/kg. Acarbose, an alpha-glucosidase inhibitor, attenuated
Polycose-induced suppression of food intake and pretreatment with 1.0 mg/kg ondansetron had
no further effect. Suppression of intake following 990mM glucose, but not mannitol infusion,
was attenuated by pretreatment with 1.0 mg/kg ondansetron. The competitive SGLT1 inhibitor,
phloridzin had no effect on 60 min 990mM glucose-induced suppression of intake or the ability
of ondansetron to attenuate this suppression of intake. Conversely, glucose-induced suppression
of intake was attenuated by phloridzin at earlier time points, and further attenuated when rats
were pretreated with 1.0 mg/kg ondansetron. Ondansetron administration alone had no effect on
intake at any dose tested. We conclude that 5-HT3 receptors participate in the inhibition of food
intake by intraduodenal infusion of carbohydrate solutions through a post-hydrolytic,
preabsorptive mechanism.
Keywords: nutrient absorption, food intake, satiation, serotonin
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INTRODUCTION
The presence of nutrients in the intestine elicits signals essential to feedback control of
ingestion. Considerable advances have been made in understanding the mechanisms by which
nutrients in the intestine suppress intake in a number of species (30, 33), including rats (42, 51,
53), and humans (74). For example, it is well documented that intestinal carbohydrates reduce
food intake in a dose-responsive manner (53, 76) by pre-absorptive (63, 78), as well as postabsorptive (71) factors, largely through vagal afferent pathways (78, 79). However, the complete
mechanism(s) by which intestinal oligosaccharides or monosaccharides are perceived to elicit
negative feedback control of intake is not known. Administration of the alpha-glucosidase
inhibitor, acarbose, attenuates reduction of food intake and c-Fos-immunoreactivity by
maltotriose (62, 63). Moreover, inhibition of sodium-dependent glucose transporter 1 (SGLT1)
by intestinal phloridzin infusion, fails to attenuate suppression of food intake and vagal afferent
activation by maltotriose or glucose, even though the transport of glucose to the blood is almost
entirely blocked (62, 63). These findings suggest that reduction of food intake by intestinally
infused carbohydrates requires hydrolysis to glucose, but that transport of glucose to the blood is
not necessary. Since vagal afferent terminals do not penetrate between epithelial cells or protrude
into the lumen (3), the receptive mechanism by which intestinal glucose elicits a reduction of
intake seems to be localized on the luminal side of the intestinal mucosa, outside of the direct
neural afferent activation locale. Therefore, a paracrine or endocrine product of intestinal origin
must interfere in vagal afferent signaling. The identity of such a product or products is not
certain, however, neuroactive modulators released in response to intestinal stimulation are the
most obvious candidates (60). Duodenal carbohydrate solutions stimulate the release of several
putative gastrointestinal (GI) hormones and neuropeptides that contribute to the negative
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feedback control of food intake (62), one of which is the brain-gut indoleamine serotonin (5hydroxytryptamine, 5-HT) (21, 46, 49, 52, 55, 80).
The serotonergic system has been well studied for its important role in the control of food
intake. For example, administration of agonists activating post-synaptic 5-HT receptors produces
marked reductions in food intake (4, 64, 68). Likewise, administration of 5-HT receptor
antagonists has been shown to elicit food intake in rats (20, 26). Systemic serotonergic activity
has been shown to induce an anorectic response through activation of a number of receptors,
including the excitatory, ligand-gated, cation channel 5-HT3 receptor (1, 37, 80). 5-HT3 receptors
are predominantly located on terminals of vagal afferent fibers (10, 28, 34, 61) in addition to
distinct subpopulations in the brain (9, 56, 58). This receptor class has been shown to be
involved in mediating GI functions such as gastric emptying, pancreatic secretion, and colonic
transit (29, 61, 80), and have also been shown to be involved in the control of intake under a
variety of feeding paradigms. For instance, 5-HT3 receptors mediate anorectic responses to
dietary amino acid deficiency (1, 2, 19), as well as peripheral CCK administration (17, 38, 39).
There is also some preliminary data demonstrating that 5-HT3 receptors mediate suppression of
intake following intestinal preloads of a nutritionally complete meal (7). While these findings
suggest that endogenous 5-HT, in response to intraintestinal nutrients, participates in the control
of food intake via 5-HT3 receptor activation, it is not clear whether specific nutrient classes or
their digestive by-products activate the 5-HT3 receptor to bring forth short-term reductions in
food intake.
Conversely, the products of carbohydrate digestion, in particular, have been shown to
mediate feeding related GI functions via 5-HT3 receptor activation. Functional evidence indicates
that 5-HT3 receptors are involved in initiation of intestinal feedback inhibition of gastric empting
(61) and stimulation of pancreatic secretion (46) in response to intestinal perfusion with glucose,
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and maltose solutions, respectively. Furthermore, electrophysiological studies have shown that
activation of vagal afferent fibers by the products of carbohydrate digestion is abolished by the
administration of a 5-HT3 receptor antagonist (80). Given this evidence, it is conceivable that 5HT3 receptors are functionally important in mediating intestinal feedback inhibition of food
intake in response to carbohydrates. However, there are no reports that directly demonstrate
whether a highly selective 5-HT3 receptor antagonist can attenuate the reduction of intake
resulting from complex carbohydrates (glucose polymers) or monosaccharides (glucose)
delivered into the proximal intestine.
Therefore, our present studies were designed to investigate participation of 5-HT3
receptors in carbohydrate-induced suppression of food intake. Specifically, we examined
whether the reduction of intake following an intraintestinal infusion of Polycose (glucose
polymers) could be attenuated via blockade of 5-HT3 receptors. Additionally, we tested the
contribution of luminal Polycose hydrolysis and free glucose on 5-HT3 receptor mediated
suppression of intake. Finally, we explored whether glucose absorption was required to suppress
food intake through 5-HT3 receptor activation.
METHODS
Animals and Surgical Preparation
Adult (250-350 g) male Sprague Dawley rats (Harlan, Indianapolis, IN) were housed
individually in hanging wire bottom cages and adapted to a 12:12 light-dark cycle (lights on at
06:00) in a temperature-controlled vivarium. Rats had ad libitum access to pelleted rodent chow
(Purina, 5001) and water, except as indicated in the experimental procedure when they were
deprived of food but not water overnight (17 h). Once acclimated to laboratory conditions, rats
were fitted with chronic duodenal catheters, consisting of a 22-cm length of silicone rubber
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tubing [0.025 in (ID), 0.047 in (OD), Dow Corning, Midland, MI] as described previously by
Yox and Ritter (78). Catheters were inserted 2-cm distal to the pylorus and advanced 6-cm
within the duodenal lumen in an aborad direction. The exposed end of the catheter exited through
a skin incision over the dorsal aspect of the cranial portion of the neck and was occluded with a
stainless steel wire (obturator), which was removed only for flushing of the catheter and
infusions. A minimum of seven days was allowed for recovery during which rats attained their
pre-operative body weights before the experiments began. This protocol was approved by The
Pennsylvania State University Institutional Animal Care and Use Committee.
Treatments
5-HT3 receptor blockade. The selective 5-HT3 receptor antagonist Ondansetron HCl (a
gift from GlaxoSmithKline, Barnard Castle, U.K.) was diluted in physiological saline to 0.125,
0.25, 0.5, 1.0, 2.0 and 5.0 mg/ml dilutions. In all experiments, ondansetron was administered
intraperitoneally (i.p.; 1.0 ml/kg), while control treatment consisted of an i.p. injection of
physiological saline.
Intestinal Infusions. Polycose (Ross Laboratories, Columbus, OH) was diluted with
distilled water to 132, and 263mM concentrations. Acarbose, an alpha-glucosidase inhibitor was
isolated via centrifugation of dissolved Precose tablets (100mg, Bayer; Burns Veterinary) and
delivered intra-intestinally in 0.2 % (w/v) concentrations, as described previously (53, 63). In
experiments investigating intestinal glucose transport inhibition, phloridzin (Sigma Chemical, St.
Louis, MO) a competitive SGLT1 inhibitor, was infused at 0.39% (w/v) concentrations, as
previously described (63). Sodium chloride was added to each infusion solution as needed to
yield an isosmotic solution (~300 mOsmol). Glucose (Sigma) and mannitol (Sigma) solutions
were infused in 308, 694, and 990mM concentrations. Intestinal nutrient concentrations were
selected based on their efficacy to suppress 60-minute intake in food-deprived rats, as
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determined in pilot studies by our laboratory. All infusates were prepared immediately prior to
testing and made to a pH of 7.35-7.40.
General Procedure
Overnight food-deprived (17h) rats were given an i.p. injection of either saline or
ondansetron five minutes before the initiation of the intraintestinal infusion. Infusates were
delivered at a rate of 0.4ml/min for 20 minutes. This infusion rate has been shown to be within
the physiological range of gastric emptying (42). After infusion ceased, rats were returned to
their homecages, immediately presented with 15% sucrose in a graduated drinking burette and
intake was measured to the nearest 0.1 ml over the subsequent 60 min. Treatments (ondansetron
injection and/or nutrient infusion) were separated by saline injection combined with an isotonic
(150mM) saline infusion (saline/saline), and each experimental trial was separated by 48 hours.
A minimum of two repetitions of each injection/infusion combination were conducted for each
experiment. Thus, each experimental datum represents the mean sucrose intake (± SE) from at
least two tests separated by saline/saline control tests. In the studies presented here, the effect of
ondansetron on carbohydrate-induced suppression was significant only during the latter half of
the sixty minute period (Figure 6). Therefore, with the exception of experiment four, only 60 min
data are presented.
Experiments
Four experiments were performed. The first experiment tested the hypothesis that the
reduction of feeding in response to intraintestinal Polycose infusion occurs via a 5-HT3 receptor
mediated mechanism. One group of rats (n=6) received an injection of saline or ondansetron (1.0
mg/kg) followed by intraintestinal infusion of either saline, 132mM, or 263mM Polycose
administered in ascending order of osmotic concentration. In a separate group of rats (n=10), the
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effects of ondansetron across a range of doses (0.125, 0.25, 0.5, 1.0, 2.0 and 5.0 mg/kg) on
263mM Polycose-induced suppression of sucrose intake were examined.
The second experiment examined whether the products of luminal glucose polymer
hydrolysis were necessary to reduce food intake via 5-HT3 receptor activation. Rats previously
used in infusion experiments (n=12) received an injection of saline or ondansetron (1.0 mg/kg)
followed by infusion of solutions that included the alpha-glucosidase inhibitor acarbose (0.2%)
either alone or in combination with 263mM Polycose.
To further assess 5-HT3 receptor participation in the reduction of food intake in response
to products of glucose polymer hydrolysis, the next experiment was performed to test the
hypothesis that free luminal glucose suppresses intake through 5-HT3 receptor. Infusates up to
this point have been delivered in isosmotic concentrations given that high luminal osmolarity
alone has been shown to release 5-HT (52), and elicit 5-HT3 receptor activation (46, 61). Since
glucose has a molecular weight approximately five times less than that of Polycose, it is not
possible to infuse glucose across increasing satiating concentrations while maintaining
isotonicity. Therefore, to control for the osmotic properties and establish whether the satiating
effect was specific to glucose, the same osmotic concentrations of mannitol were also tested. In
the third experiment, experimentally naive rats (n=6), were infused with glucose or mannitol in
0, 308, 694, or 990mM concentrations following an injection of saline or ondansetron.
The final experiment explored whether 5-HT3 receptors participate in the suppression of
intake when active, Na+-dependent absorption of duodenal glucose is inhibited using phloridzin,
the competitive SGLT1 inhibitor. Phloridzin is a useful tool in the study of intestinal glucose
transport because it competitively binds SGLT1 and inhibits its activity (44). In this experiment,
rats (n=12) were injected with either saline or ondansetron (1.0 mg/kg) prior to duodenal
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infusion of either saline, 990mM glucose, phloridzin (0.39%) or glucose combined with
phloridzin.
To determine phloridzin’s efficacy in intestinal glucose transport inhibition, blood
glucose levels were measured in response to infusions of saline, 990mM glucose, phloridzin
(0.39%) or glucose combined with phloridzin. Blood glucose was measured in a drop of tailblood obtained serially from each rat at 0, 20, 30, 45, 60, and 120 minutes relative to the start of
the infusion, using a handheld glucometer (LifeScan, Inc. Milpitas, CA).
Analysis of Results
Data for each respective experiment were analyzed separately. Sucrose intake (ml) for
each rat was subjected to a two-way repeated-measures analysis of variance (rmANOVA), with
drug injection and intestinal infusate as independent variables, using PC-SAS (version 8.02, SAS
Institute, Carey, NC) mixed procedure. Blood glucose data (mg/dL) for each rat was subjected to
a two-way rmANOVA with infusion condition and time as independent variables. Significant
differences among treatment means (adjusted) were analyzed by pairwise t-test for planned
comparisons with P< 0.05 considered statistically significant.
RESULTS
In all experiments, when rats received ondansetron prior to intestinal saline infusion,
intake was not significantly different from control (saline injection/saline infusion) at any time
(see Figures 1, 3-5 and Table 1).
Effects of 5-HT3 receptor blockade on Polycose-induced inhibition of food intake
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Repeated–measures ANOVA demonstrated a significant main effect of injection
[F(1,121)=5.32, P=0.02], and a significant main effect of infusion [F(2,121)=124.36, P<0.0001]
but not a significant interaction between injection and infusion [F(2,121)=1.54 P=0.22]. As
illustrated in Figure 1, infusion of 132mM Polycose significantly suppressed 60 min intake (7.6
± 0.6 ml), compared to intake under control conditions (12.6 ± 0.3 ml, P <0.001). Pretreatment
with ondansetron (1.0 mg/kg) did not significantly alter this suppression (8.3 ± 0.4 ml, P =0.45).
Infusion with 263mM Polycose significantly suppressed intake (2.3 ± 0.5 ml, P<0.001)
compared to saline. This suppression was significantly attenuated following pretreatment with
ondansetron (4.6 ± 0.8 ml, P=0.004).
Table 1 shows the effects of treatment with ondansetron (0.125 - 5.0 mg/kg) on 263mM
Polycose-induced inhibition of 60 min sucrose intake. Infusion with 263mM Polycose produced
a significant reduction of sucrose intake [F(1,195)=976.74, P<0.001]. Repeated–measures
ANOVA also demonstrated an overall effect of injection [F(6,195)=3.85, P=0.0012] as well as a
significant interaction between injection and infusion [F(6,195)=4.42, P<0.001]. The degree to
which suppression of intake was attenuated by each dose of ondansetron is illustrated in Figure 2
via mean difference scores. The difference score for each rat was calculated as intake following
treatment with ondansetron and Polycose minus intake following treatment with saline and
Polycose. One way rmANOVA revealed no significant differences between the degree by which
ondansetron attenuated Polycose-induced suppression of food intake across all doses tested
[F(5,44)=0.286, P=0.91]. However, only 1.0, 2.0, and 5.0 mg/kg ondansetron significantly
attenuated Polycose-induced suppression of intake (Table 1).
Inhibition of luminal glucose polymer hydrolysis accompanied by 5-HT3 receptor blockade
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There was a significant main effect of infusion on intake [F(3,147)=105.52, P<0.0001].
However, rmANOVA did not reveal a significant main effect of injection [F(1,147)=2.57, P=
0.11], or an overall interaction between injection and infusion [F(3,147)=2.10, P= 0.103]. Rats
infused with 263mM Polycose significantly suppressed 60 min sucrose intake (2.1 ± 0.6 ml)
compared to intake under control conditions (12.8 ± 0.3 ml, P <0.0001). This suppression was
significantly attenuated following pretreatment with 1.0 mg/kg ondansetron (4.4 ± 0.9 ml, P
=0.019; Figure 3). A combined infusion of 0.2% acarbose and 263mM Polycose resulted in a
significant suppression of sucrose intake (10.4 ± 0.8 ml, P =0.0015) compared to control.
Pretreatment with ondansetron did not significantly alter this suppression (10.0 ± 0.9 ml, P
=0.65). Infusion of acarbose alone resulted in sucrose intake which was not significantly
different from control when rats were injected with saline (12.2 ± 0.9 ml, P =0.44), or
ondansetron (13.3 ± 0.6 ml, P =0.22).
Effects of 5-HT3 receptor blockade on glucose-induced inhibition of food intake
Repeated–measures ANOVA failed to demonstrate a significant main effect of injection
[F(1, 293)=0.9, P =0.34], but demonstrated a significant main effect of infusion [F(6,
293)=50.66, P <0.0001] as well as an overall interaction between injection and infusion [F(6,
293)=4.63, P =0.0002]. As illustrated in Figure 4, infusion of 308mM glucose resulted in 60 min
sucrose intake (12.5 ±0.5 ml) that was less than intake under control conditions (13.4 ±0.2 ml),
but this difference was not significant (P =0.052). Pretreatment with 1.0 mg/kg ondansetron did
not significantly change this intake (12.3 ±0.5 ml, P =0.84). Similarly, sucrose intake was not
suppressed when rats were infused with 308mM mannitol (14.7 ± 0.6 ml, P =0.09). Pretreatment
with ondansetron did not alter this intake (13.0 ± 0.8 ml, P =0.15). Infusion of 694mM glucose
significantly suppressed intake (10.0 ±0.7 ml, P <0.0001) compared to control, and pretreatment
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with ondansetron did not significantly alter this suppression (10.4 ± 1.3 ml, P =0.69). Likewise,
infusion of 694mM mannitol suppressed intake (11.3 ± 1.1 ml, P =0.001) compared to control.
This suppression was not changed by pretreatment with ondansetron (12.2 ± 0.7 ml, P =0.43).
The 990mM glucose infusion markedly suppressed sucrose intake (0.4 ± 0.1 ml, P <0.001),
which was significantly attenuated by pretreatment with ondansetron (4.4 ± 0.7 ml, P =0.005).
Infusion with 990mM mannitol also significantly suppressed sucrose intake (5.0 ± 0.8 ml, P
<0.001), but this suppression was not altered following pretreatment with ondansetron (4.8 ± 1.2
ml, P =0.89).
Effects of 5-HT3 receptor blockade on glucose-induced reduction of food intake while inhibiting
active intestinal glucose transport
A significant main effect of injection [F(1,109)=4.47, P=0.037], infusion
[F(3,109)=156.47, P<0.0001], as well as a significant interaction between injection and infusion
[F(3,109)=4.53 P= 0.005] on intake were demonstrated by rmANOVA. As illustrated in Figure
5, infusion with the competitive SGLT1 inhibitor phloridzin produced 60 min intake (17.3 ± 1.4
ml) similar to that of control (18.0 ± 0.7 ml, P =0.3). This intake was not altered by pretreatment
with 1.0 mg/kg ondansetron (15.7 ± 1.3 ml, P =0.16). Infusion of 990mM glucose significantly
suppressed sucrose intake (4.3 ± 1.0 ml, P <0.001) compared to intake following control
treatment. The suppression of intake following 990mM glucose infusion was significantly
attenuated when rats were pretreated with ondansetron (7.7 ± 1.0 ml, P =0.002). When rats were
infused with phloridzin in combination with 990mM glucose, sucrose intake was significantly
suppressed (4.9 ± 1.0 ml, P <0.001) compared to control but not different from glucose infusion
alone (P =0.58). The suppression of intake resulting from glucose plus phloridzin infusion was
significantly attenuated when rats were pretreated with ondansetron (7.5 ± 1.3 ml, P =0.018).
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Figure 6 illustrates 60 min feeding period in 5 min intervals for the most relevant
treatments. Infusion of glucose significantly suppressed sucrose intake for the entire 60 min
period (P <0.0001). Suppression of intake following glucose infusion was significantly
attenuated beginning at 40 min when rats were pretreated with ondansetron (P ≤0.01). Duodenal
infusion of phloridzin in combination with glucose significantly attenuated glucose-induced
suppression of intake starting at 5 min, and lasting until 55 min (P ≤0.009), at which time
glucose infused rats began to increase intake. Treatment with ondansetron enhanced this
attenuation of glucose-induced suppression of intake in the presence of phloridzin for the entire
60 min intake was measured (P ≤0.01).
Blood glucose data indicative of phloridzin function are presented in Table 2. Duodenal
infusion (during the first 20 min) of 990mM glucose significantly elevated blood glucose
compared to saline infusion at all time points measured (P <0.05). Significant elevation of blood
glucose, compared to saline infusion, was achieved at 45, 60, and 120 min when glucose was
infused in combination with 0.39% phloridzin (P <0.05). Phloridzin attenuated glucose-induced
elevation in blood glucose from 20 through 45 min (P <0.05).
DISCUSSION
This study provides evidence that 5-HT3 receptors participate in carbohydrate-induced
reduction of intake. More specifically, blockade of 5-HT3 receptors with ondansetron attenuated
the reduction of intake following intraintestinal Polycose infusion. Additionally, by inhibiting
luminal glucose polymer hydrolysis, we demonstrated that the products of hydrolysis are indeed
necessary for the 5-HT3 receptor to mediate Polycose-induced suppression of intake. Finally, we
showed that ondansetron attenuated suppression of food intake in response to intraintestinal
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infusion of free luminal glucose and this response was not altered by blocking active glucose
absorption.
In a broader context, the findings of this study corroborate with those from other
laboratories (1, 7, 8, 17 ) as well as our own (38, 39), demonstrating that 5-HT3 receptors are
involved in the negative feedback control of intake. For example, systemic administration of
ondansetron has been shown to reverse suppression of intake resulting from duodenal infusion of
a complete meal (Ensure) (7). Likewise, the inhibitory effect on inter-meal interval following an
intestinal fat infusion was partially mediated by 5-HT3 receptors (8). To our knowledge, this is
the first study to demonstrate participation of 5-HT3 receptors in suppression of food intake by
complex, as well as simple carbohydrates.
Nutrients entering the intestine present a composite of colligative and chemical properties
to the intestinal sensory system which act in concert to generate satiation. Each macronutrient
constituent might influence food intake by distinct mechanisms. It is known that much of the
suppression of food intake following intestinal nutrient infusion is mediated by vagal sensory
neurons (78, 79). However, it is uncertain whether vagal afferents can respond directly to
nutrients in the extracellular space or whether responses to nutrients are mediated by substances
released from the intestinal mucosa or enteric neurons (36). There is support for the hypothesis
that endocrine cells in the gut act as an intermediate between intestinal stimuli and vagal afferent
termini by releasing their contents in response to mechanical and chemical stimulations of the
intestinal wall (59, 60). For example, 5-HT is found in abundance in EC cells of the GI mucosal
epithelia and the enteric neurons (32), and is released in response to luminal disaccharide
solutions (46, 49, 55, 80) as well as glucose, but not mannitol, in a concentration dependent
fashion (21, 43, 52). While there is some evidence that vagotomy either does not change or
enhances the anorectic effect of exogenous 5-HT (22, 27), other data suggest that peripheral 514
HT acts as a signal molecule in carbohydrate-induced inhibition of food intake, most likely
through vagal afferent activation. For example, electrophysiological studies show that
endogenously released 5-HT plays a major role in signal transmission evoked by luminal
carbohydrates to stimulate vagal fibers (47, 80) including nodose neurons (48, 50, 80). Vagal
sensory fibers, which are important in the negative feedback cascade to inhibit food intake (78,
79), are likely involved in 5-HT3 receptor mediated suppression of intake observed in our study.
5-HT3 receptors are amply localized within the gut, specifically on vagal sensory fibers
innervating the duodenum (31, 34, 58, 61), and have been shown to be involved in mediating
glucose-induced inhibition of gastric emptying (61). In addition, electrophysiological evidence
reveals that stimulation of vagal sensory neurons with intestinal maltose is inhibited by
administration of a 5-HT3 antagonist (80). Taken as a whole, these findings suggest that
intestinal carbohydrates elicit 5-HT release and subsequent activation of 5-HT3 receptors present
on vagal sensory pathways as part of a feedback cascade to inhibit food intake.
In the first experiment we did not observe a robust dose response effect of ondansetron
on Polycose-induced suppression of intake. Our results showed that 1.0 mg/kg was the lowest
dose of ondansetron able to significantly attenuate 263mM Polycose-induced suppression of
intake and that higher doses (2.0 and 5.0 mg/kg) did not enhance the effect. The lower doses of
ondansetron tested (0.125 – 0.5 mg/kg) all slightly attenuated Polycose-induced suppression of
intake but did not reach statistical significance. Thus, in the presence of intestinal Polycose,
ondansetron (0.125 – 5.0 mg/kg) exhibited a relatively flat dose-response function, with 1.0
mg/kg dose having the maximal threshold effect on blocking 5-HT3 receptors and that doses five
times higher produced no further effects. Studies examining participation of 5-HT3 receptors in
nutrient-induced satiation are scant. In addition, the few experiments that have investigated the
role of 5-HT3 receptors in negative feedback inhibition of intake or feeding related physiological
15
functions using ondansetron have been single dose studies. To our knowledge, this is the first
study demonstrating the involvement of 5-HT3 receptors in carbohydrate-induced suppression of
food intake. Previous studies examining the effect of ondansetron alone on several functions,
including feeding behavior, have yielded conflicting results. For example, a narrow, inverted-U
dose-response function has been reported in studies attempting to characterize the actions of
ondansetron on anxiolytic activity or benzodiazepine withdrawal (35, 41). In one of these studies
in which food consumption was a secondary outcome measured, 0.01 – 1.0 mg/kg doses of
ondansetron treatment alone produced no effect on food intake (35). Furthermore, studies
examining the effects of much lower doses of ondansetron on food intake are equally
inconsistent. For instance, in one study, administration of ondansetron (30 and 100 µg/kg)
significantly increased sweetened mash intake and decreased 3% sucrose solution intake (10 and
30 µg/kg) in non-food-deprived rats (14) while in a subsequent study, similar doses of
ondansetron (3.0 to 30 µg/kg) had the opposite effect [i.e., significantly reduced sweetened mash
intake (72)]. The reason for such discrepancies is not immediately clear. It is apparent from our
results however, that blockade of 5-HT3 receptors with at least 1.0 mg/kg ondansetron maximally
attenuated the carbohydrate-induced suppression of intake.
The finding that blockade of 5-HT3 receptors only attenuated the carbohydrate-induced
suppression of intake to a relatively small degree supports the likelihood that either other
postsynaptic 5-HT receptors are involved in the mediation of this signal and/or that redundant
signaling mechanisms are involved. In fact, Raybould and colleagues (61) found that both
glucose-and mannitol- induced inhibition of gastric emptying is abolished by pretreatment with
tropisetron, a receptor antagonist which exhibits affinity for both 5-HT3 and 5-HT4 receptors
(70). These results suggest the possibility that both receptor types cooperate to mediate intestinal
feedback inhibition mechanisms in response to carbohydrates. Several studies have also shown
16
that inhibition of intake by carbohydrate is mediated by other signals, one of which is
cholecystokinin (CCK). Specifically, selective antagonists for the CCK type-1 (CCK-1)
receptors attenuate the feeding inhibition (5, 57, 77) and dorsal hindbrain expression of c-Fos
(73), resulting from duodenal carbohydrate solutions. Additionally, rats lacking CCK-1 receptors
are insensitive to the suppressive effects of duodenal glucose infusions (16). While both
serotonergic and cholecystokinergic systems work independently to reduce food intake, their
actions impinge on each other at several levels to modify intake in an integrated control of food
intake. Original investigations (11-13, 66) suggested that CCK’s inhibitory action on food intake
is potentiated by 5-HT, and vice versa. More recently, Li and colleagues (48) reported that some,
but not all CCK-responsive vagal neurons are also responsive to intra-luminal 5-HT perfusion. It
is possible that signals such as CCK and 5-HT, arising from the GI tract synergistically produce
a physiologic inhibition of food intake at the level of vagal afferent neurons. Additional evidence
supporting this hypothesis is that CCK-induced expression of c-Fos positive nuclei in select subnuclei of the nucleus tractus solitarius (NTS) and area postrema is attenuated by a blockade of 5HT3 receptors (17), suggesting that 5-HT3 receptors may directly mediate a select subpopulation
of CCK receptive vagal neurons. Indeed, it has repeatedly been established that CCK-induced
inhibition of food intake is attenuated by a blockade of 5-HT3 receptors (17, 38, 39). Finally, we
recently reported that sucrose intake is enhanced following simultaneous blockade of CCK-1 and
5-HT3 receptors (39). Collectively, these findings suggest that 5-HT3 receptors functionally
mediate the suppression of intake generated by intestinal carbohydrates, in part through
cooperation with CCK acting at CCK-1 receptors, and that this interaction likely takes place at
the level of vagal afferent neurons. Additional work is required to delineate the extent to which
5-HT3 receptors interact with CCK-1 or other postsynaptic 5-HT receptors in carbohydrateinduced suppression of intake.
17
The exact anatomical site of ondansetron’s effects on carbohydrate-induced suppression
of intake cannot be determined from the present studies. While pharmacokinetic studies indicate
that ondansetron exhibits limited penetration of the blood brain barrier (65), some reports suggest
that central 5-HT3 receptors are blocked by peripherally administered 5-HT3 receptor antagonists
(15, 75). Conversely, there is no change in food intake when a highly selective 5-HT3 receptor
agonist, m-chlorophenylbiguanide hydrochloride (1-(3-chlorophenyl)biguanide (m-CPBG), is
administered into the third ventricle (9). However, this evidence does not permit us to entirely
discount any central 5-HT3 receptor participation in the current study. Electrophysiological,
immunocytochemical, and autoradiographic studies have revealed that 5-HT3 receptors are
extensively located throughout the rat brain (24, 45) including areas related to control of food
intake, such as the hindbrain and the hypothalamus (58, 69). Furthermore, the NTS expresses
binding sites for 5-HT3 ligands (6, 45) and is densely innervated by serotonergic terminals (67,
69). Given that minute amounts of ondansetron may permeate the blood brain barrier (25),
further investigation is surely warranted to directly examine participation of hindbrain 5-HT3
receptors in the control of food intake, including suppression of intake resulting from intestinal
carbohydrates.
Blockade of 5-HT3 receptors in the absence of intestinal carbohydrate infusion had no
significant effect on sucrose consumption at any dose of ondansetron tested. This is in agreement
with our laboratory’s previous work (38, 39) as well as that of others (7, 17) showing that
administration of ondansetron in equivalent doses did not alter intake compared to control. Since
ondansetron (1.0 mg/kg) does not increase sucrose intake in non-food-deprived rats (39), it is not
likely that an orexigenic effect of ondansetron was masked due to a ceiling effect resulting from
overnight food-deprivation. Furthermore, simultaneous administration of CCK-1 and 5-HT3
receptor antagonists produces intake greater than that of control in food-deprived rats (39)
18
showing that sucrose intake following overnight deprivation is not at its upper limit per se. Thus,
it is not likely that our observations result from independent, additive orexigenic and
anorexigenic effects of ondansetron and nutrient infusion respectively, given that ondansetron
attenuated the suppression of intake only when rats were infused with higher concentrations of
carbohydrate solutions. Instead, our results imply that the feeding effects evoked by 5-HT3
receptor antagonism are due to inhibition of anorectic signals arising from intestinal
carbohydrate-induced activation of 5-HT3 receptors.
Consistent with the findings of others (53, 62, 63), we found that administration of the
competitive alpha-glucosidase inhibitor, acarbose, attenuated reduction of food intake by
intestinal Polycose. This feeding effect of a combined infusion of acarbose and Polycose was
maintained when rats were pretreated with ondansetron. We reason that hydrolysis of glucose
polymers to the monosaccharide, glucose is necessary to elicit reduction of food intake mediated
through 5-HT3 receptors. Supporting this notion is the fact that suppression of intake following
duodenal infusion of free luminal glucose was attenuated by blockade of 5-HT3 receptors.
While the mechanism by which 5-HT is released in response to intestinal glucose remains
unclear, there is some evidence that luminal SGLT1 may be a factor. Specifically, Kim and
colleagues (43), employing an enterochromaffin cell model demonstrated that glucose stimulates
a concentration dependent release of 5-HT, and that this response was inhibited by treatment
with phloridzin. In our final experiment, we sought to examine whether active glucose
absorption by SGLT1 was necessary to elicit 5-HT3 receptors to reduce food intake. Here,
duodenal infusion of phloridzin attenuated glucose-induced suppression of intake. This effect
was immediate while transport of glucose into the blood was markedly inhibited to the level of
blood glucose following saline infusion. It is conceivable that blocking active glucose absorption
with phloridzin attenuated the immediate glucose-induced suppression of intake by impeding
19
SGLT1-mediated release of 5-HT. Moreover, since 5-HT3 receptors function as autoreceptors in
the regulation of 5-HT release from the small intestine such that activation of the 5-HT3
receptors triggers a positive feedback mechanism leading to an increase of 5-HT release (23, 54),
the attenuation of glucose-induced suppression by ondansetron in our study may have resulted
from inhibition of 5-HT release. This notion, along with the inhibitory effect phloridzin has on 5HT release (43), could elucidate why ondansetron plus phloridzin had the greatest effect on
glucose-induced suppression of intake. In other words, some but not all glucose-induced 5-HT
release was independently inhibited by ondansetron or by phloridzin treatments alone, and this
effect was potentiated when these treatments occurred concurrently. While blockade of 5-HT3
receptors did independently produce a significant inhibition of glucose-induced suppression of
intake in the presence or absence of phloridzin, we can only speculate the role of 5-HT release
and its activity in our study. However, on the basis of the evidence presented here we can
conclude that intestinally infused glucose suppresses intake partly through a pre-absorptive
activation of 5-HT3 receptors.
Since 5-HT3 receptors have been shown to be responsive to hyperosmolar liquids to
mediate GI functions (46, 49, 61), whenever possible, we chose to infuse solutions that were
isosmotic to luminal osmolarity. Although we did not directly measure osmoconcentration of our
solutions in the duodenum, it has been shown that infusion of glucose polymer solutions result in
lower luminal osmolarity than infusion of glucose solutions of the same concentration by weight
(18) and that duodenal infusion of hypertonic glucose solutions produce a maximal duodenal
osmoconcentration much lower than that of the solution infused (18, 40). In our study,
ondansetron attenuated the suppression induced by 263mM Polycose as well as 990mM glucose,
but not 990mM mannitol. This suggests that the feeding responses observed when we infused a
solution with a high osmotic concentration, or potentially high luminal concentration, were not
20
due to osmotic activation of the 5-HT3 receptors. Additionally, experiment 3 revealed that 1.0
mg/kg ondansetron attenuated glucose-induced suppression of intake to the level of mannitolinduced suppression of intake, suggesting that glucose itself and not simply the colligative
properties of the infusate are eliciting activation of 5-HT3 receptors to inhibit food intake.
In conclusion, the present studies demonstrate that 5-HT3 receptors participate in food
intake inhibition in response to intraintestinal infusion of both glucose polymer and glucose
solutions. We determined that suppression of feeding due to infusion of Polycose as mediated
through 5-HT3 receptors requires luminal hydrolysis of the glucose polymer. Likewise, blockade
of 5-HT3 receptors resulted in a significant attenuation of suppressed intake when glucose, but
not mannitol, was infused at the highest concentration. Ondansetron independently produced a
significant inhibition of glucose-induced suppression of intake when luminal glucose absorption
was inhibited by phloridzin. Collectively, these findings support the hypothesis that duodenal
carbohydrate solutions suppress intake in part through 5-HT3 receptors. Furthermore, 5-HT3
receptor mediation of intestinal Polycose-induced suppression of intake requires hydrolysis, but
is independent of active glucose transport by luminal SGLT1.
21
ACKNOWLEDGEMENTS
We thank Bart C. De Jonghe, Matthew R. Hayes and Jonathan G. Stine for their technical
assistance and preparation of the manuscript. We acknowledge GlaxoSmithKline for generous
donation of ondansetron.
22
FIGURE LEGENDS
Fig. 1. Duodenal infusion of 132mM and 263mM concentrations of Polycose in food deprived
rats (n = 6), significantly suppressed 60-min 15% sucrose intake compared to saline:saline
treatment. Pretreatment with ondansetron (1.0 mg/kg, i.p.) attenuated 263mM Polycose-induced
suppression of intake but did not significantly alter 132mM Polycose-induced suppression of
intake. * Significantly different from saline:saline treatment (P <0.05).
‡
Significantly different from saline:Polycose treatment (P <0.05).
Fig. 2. Data points are mean ±SE food intake difference scores. Difference scores were
calculated for each rat (n= 10) as 60-min sucrose intake (ml) following treatment with
ondansetron and Polycose minus 60-min sucrose intake following treatment with saline and
Polycose. Ondansetron administered at 1.0, 2.0, and 5.0mg/kg each significantly attenuated the
Polycose-induced suppression of intake (see Table 1). Suppression of intake was not
significantly attenuated by the lower doses of ondansetron tested although difference scores for
all doses were statistically similar (one way rmANOVA, P=0.91).
Fig. 3. Duodenal infusion of 263mM Polycose in food deprived rats (n = 12), significantly
suppressed 60-min 15% sucrose intake with or without presence of an alpha-glucosidase
inhibitor acarbose, compared to saline:saline treatment. Pretreatment with ondansetron (1.0
mg/kg, i.p.) attenuated 263mM Polycose-induced suppression of intake but not suppression
elicited by simultaneous infusion of acarbose and Polycose. Intake following infusion of
acarbose alone was not significantly different from control when rats were pretreated with saline
or ondansetron. * Significantly different from saline:saline treatment (P<0.05).
different from saline:Polycose treatment (P<0.05).
23
‡
Significantly
Fig. 4. Duodenal infusion of glucose or mannitol (694mM and 990mM) both significantly
suppressed 60-min 15% sucrose intake in food deprived rats (n = 6). Pretreatment with
ondansetron (1.0 mg/kg, i.p.), significantly attenuated the 990mM glucose- but not mannitolinduced suppression of intake. * Significantly different from saline:saline treatment (P ≤0.05). ‡
Significantly different from saline:glucose treatment (P <0.05).
Fig. 5. Duodenal infusion of 990mM glucose alone or in combination with the competitive
SGLT1 inhibitor, phloridzin (0.39%) significantly suppressed 60-min 15% sucrose intake in food
deprived rats (n = 12) compared to saline:saline treatment. Pretreatment with ondansetron (1.0
mg/kg, i.p.), significantly attenuated the glucose-, as well as the glucose/phloridzin- induced
suppression of intake. * Significantly different from saline:saline treatment (P ≤0.05). ‡
Significantly different from saline:glucose treatment (P <0.05).
Fig. 6. Duodenal infusion of 990mM glucose significantly suppressed 15% sucrose intake in
food deprived rats (n = 12) compared to saline:saline treatment for the entire 60 min period.
Suppression of intake following glucose infusion was significantly attenuated when rats were
pretreated with ondansetron beginning at 40 min. Duodenal infusion of phloridzin (0.39%) in
combination with glucose significantly attenuated glucose-induced suppression of intake starting
at 5 min, and lasting until 55 min. Treatment with ondansetron enhanced attenuation of glucoseinduced suppression of intake in the presence of phloridzin for the entire 60 min intake was
measured. * Significantly different from saline:glucose treatment (P ≤0.009). ‡ Significantly
different from saline:glucose and phloridzin treatment (P ≤0.01).
24
Table 1. Values are means ±SE 60-min 15% sucrose intakes (ml) in overnight food deprived rats
(n= 10). Infusion with 263mM Polycose produced a significant reduction of 15% sucrose intake.
Ondansetron administered at 1.0, 2.0, and 5.0mg/kg each significantly attenuated Polycoseinduced suppression of intake. * Significantly different from saline:saline treatment within a
column (P <0.05). ‡ Significantly different from saline:Polycose treatment within a column (P
<0.05).
Table 2. Values are means ± SE blood glucose in mg/dL. Duodenal infusion (during the course
of 0-20 min) of 990mM glucose significantly elevated blood glucose compared to saline infusion
at all time points. Compared to saline infusion, significant elevation of blood glucose occurred at
45, 60, and 120 min when glucose, in combination with the competitive SGLT1 inhibitor
phloridzin (0.39%), was infused. Phloridzin attenuated the glucose-induced elevation in blood
glucose from 20 through 45 min. * Significantly different from saline infusion for a given time
point (P <0.05). ‡ Significantly different from glucose infusion for a given time point (P <0.05).
25
16
Ondansetron
Saline
60-min Sucrose Intake (ml)
,
14
12
10
*
*
8
*‡
6
4
*
2
0
Saline
132 mM
Polycose
Figure 1
26
263 mM
Polycose
3
Difference Score
2.5
2
1.5
1
0.5
0
0.125
0.25
0.5
1.0
Ondansetron dose (mg/kg)
Figure 2
27
2.0
5.0
16
Saline
Ondansetron
60-min Sucrose Intake (ml),
14
12
*
*
10
8
6
*‡
4
*
2
0
Saline
Acarbose
Polycose
Figure 3
28
Polycose/Acarbose
18
308 mM
60-min Sucrose Intake (ml) , .
16
694 mM
14
*
12
*
*
*
10
990 mM
8
*‡
6
*
*
4
2
0
*
Injection
Sal
Ond
Sal
Ond
Sal
Ond
Sal
Ond
Sal
Ond
Sal
Ond
Sal
Ond
Infusion
Sal
Sal
Glu
Glu
Man
Man
Glu
Glu
Man
Man
Glu
Glu
Man
Man
Figure 4
29
20
Saline
60-min Sucrose Intake (ml) ,
18
Ondansetron
16
14
12
10
*‡
*‡
8
6
*
*
Glucose
Glucose &
Phloridzin
4
2
0
Saline
Phloridzin
Figure 5
30
20
saline/saline
saline/ glucose & phloridzin
ondansetron/ glucose & phloridzin
saline/ glucose
ondansetron/ glucose
18
16
Sucrose Intake (ml)
14
12
10
8
‡
6
4
‡
‡
‡
‡
‡
‡
‡
‡
*
*
*
*
*
*
*
*
*
*
*
2
0
Time (min) 5
*
*
*
*
*
‡
‡
‡
10
15
20
25
30
Figure 6
31
35
40
45
50
55
60
Table 1. Effects of ondansetron on 263mM Polycose-induced inhibition of food intake
Ondansetron Dose (mg/kg)
0.125
0.25
0.5
1.0
2.0
5.0
saline/saline
15.8 ±0.9
13.0 ±0.6
13.0 ±0.7
15.0 ±0.6
13.6 ±0.5
15.0 ±0.6
ondansetron/saline
15.6 ±0.6
13.3 ±0.4
12.8 ±0.7
14.9 ±0.5
13.2 ±0.7
15.3 ±0.8
Injection/Infusion
saline/Polycose
3.3 ±0.8 *
3.4 ±0.5 *
3.6 ±0.5 *
3.5 ±0.7 *
3.5 ±0.6 *
3.5 ±0.7 *
ondansetron/Polycose
4.8 ±0.8 *
5.2 ±0.8 *
5.0 ±0.7 *
5.8 ±0.5 * ‡
5.6 ±0.5 * ‡
5.6 ±0.6 * ‡
32
Table 2. Effect of phloridzin on blood glucose
Time (min)
Saline
Phloridzin
Glucose
Glucose &
Phloridzin
0
64.3 ± 2.3
67.0 ± 2.3
55.8 ± 3.5
56.7 ± 2.7
20
69.3 ± 4.8
57.7 ± 4.8
‡
102.6 ± 4.9 *
68.0 ± 3.5
‡
30
73.3 ± 1.3
61.3 ± 1.9
‡
102.8 ± 4.9 *
80.7 ± 2.9
‡
45
67.7 ± 4.8
60.3 ± 0.3
‡
103.8 ± 2.8 *
86.2 ± 4.5 *
60
63.7 ± 4.4
58.7 ± 0.9
‡
92.8 ± 2.5 *
120
62.7 ± 2.4
53.0 ± 0.6
‡
76 ± 4.7 *
33
‡
93.3 ± 5.5 *
85.0 ± 1.8 *
‡
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