Glucagon-like peptide-1 inhibits gastropancreatic function by
inhibiting central parasympathetic outflow
André
Wettergren1,
Morten
Wøjdemann1, and
Jens
Juul
Holst2
1 Department of
Gastrointestinal Surgery C, Rigshospitalet, and
2 Department of Medical Physiology
C, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen,
Denmark
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ABSTRACT |
Glucagon-like peptide (GLP)-1 inhibits acid
secretion and gastric emptying in humans, but the effect on acid
secretion is lost after vagotomy. To elucidate the mechanism involved,
we studied its effect on vagally stimulated gastropancreatic secretion
and motility in urethan-anesthetized pigs with cut splanchnic nerves, in which insulin-induced hypoglycemia elicited a marked stimulation of
gastropancreatic secretion and antral motility. In addition, we studied
vagally stimulated motility and pancreatic secretion in isolated
perfused preparations of the porcine antrum and pancreas. GLP-1
infusion (2 pmol · kg
1 · min
1)
strongly and significantly inhibited hypoglycemia-induced antral motility, gastric acid secretion, pancreatic bicarbonate and protein secretion, and pancreatic polypeptide (PP) secretion. GLP-1 (at 10
10-10
8
mol/l) did not inhibit vagally induced antral motility, pancreatic exocrine secretion, or gastrin and PP secretion in isolated perfused antrum and pancreas. We conclude that the inhibitory effect of peripheral GLP-1 on upper gastrointestinal secretion and motility is
exerted via interaction with centers in the brain or afferent neural
pathways relaying to the vagal motor nuclei.
acid secretion; gastrin; pancreatic polypeptide; hypoglycemia; neuroglucopenia; enterogastrone; ileal brake; gastrointestinal effects
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INTRODUCTION |
GLUCAGON-LIKE PEPTIDE (GLP)-1 is an intestinal hormone
(13) arising as the result of tissue-specific posttranslational
processing of proglucagon expressed in open-type endocrine cells (L
cells) in the small intestine (2, 28, 32), from which it is released in
response to meal ingestion (4, 35). GLP-1 has attracted considerable
interest because of its potent actions on carbohydrate metabolism and
potential applicability in the treatment of type 2 diabetes (8, 36). In
addition to its glucoregulatory effects, GLP-1 strongly inhibits
gastric acid secretion and gastric motility in humans (30, 37, 46) and
is thought to represent one of the enterogastrones of the "ileal
brake" mechanism (13). Regarding the mechanism of action with
respect to inhibition of acid secretion in humans, a recent study (45)
from our laboratory has shown that GLP-1 almost abolished sham
feeding-induced acid secretion, whereas its inhibitory effect on
pentagastrin-induced secretion (37) was lost after vagotomy (47),
suggesting that the inhibitory effect of GLP-1 on at least acid
secretion is mediated via neural pathways.
To investigate further the mechanism of GLP-1 inhibition of upper
gastrointestinal (GI) functions, we developed an experimental model
allowing studies of centrally (insulin hypoglycemia) induced stimulation of antral motility and gastric and pancreatic secretion in
anesthetized pigs. The effect of GLP-1 in this model was compared with
its effects in isolated perfused preparations of the porcine pancreas
and antrum with intact vagal innervation.
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MATERIALS AND METHODS |
In Vivo Experiments
Animal preparation. Twenty-three pigs,
strain LYY, weighing 24-38 kg, were used. They were fasted for 24 h but had free access to water. Anesthesia was induced briefly with
2.5% halothane and maintained with intravenous infusion of urethan
(0.8 g · kg
1 · h
1)
after intubation and start of artificial ventilation. The abdomen was
opened through a midline incision. To reduce sympathetic inhibitory effects on pancreatic secretion (18) (in this study elicited by
insulin-induced hypoglycemia), the splanchnic nerves were cut bilaterally at the level of the diaphragm. An orogastric tube was
placed in the fundic region of the stomach. A baby-feeding tube (no. 8, Argyll; Sherwood Medical Industries, St. Louis, MO) was fixed to the
gastric tube allowing intragastric infusion of 57CoCl (Amersham); 370 kBq were
dissolved in 1,000 ml 0.9% saline with 20 ml 5% human albumin (Novo
Nordisk A/S Plasma Product Unit PSU) to avoid adhesion to tubes and
infused at a rate of 2 ml/min. A large-bore draining tube was passed
through the pylorus via a duodenotomy and kept in place by a string
tied around the pylorus without damaging the antropyloric nerves and
blood vessels. The pancreatic duct was isolated and catheterized with a
baby-feeding tube (no. 6), and both the duodenal and pancreatic tubes
were exteriorized through separate skin incisions in the abdominal wall. Two force transducers (RB Products, Madison, WI) were sutured onto the serosa of the antrum, ~4-5 cm from the pylorus, one in a longitudinal direction, the other perpendicular to the first, to
detect circular and longitudinal muscle contractions. The transducers were calibrated using weights. The femoral veins and a femoral artery
were isolated and catheterized (baby-feeding tube no. 8) for blood
sampling, infusion of peptide, and intra-arterial measurements of blood
pressure, respectively. Electrodes were placed on the thorax, and
electrocardiogram and heart rate were monitored continuously. In four
pigs prepared as described above, a cervical bilateral vagotomy was
carried out.
Peptide.
GLP-1-(7
36) amide (porcine, human) was purchased from Peninsula, St.
Helens, UK. The peptide was dissolved in 0.9% saline containing 1%
human serum albumin (pure, dry; Behringwerke, Marburg, Germany) and kept at
20°C until use.
Experimental procedure.
After surgery the animals were left undisturbed for 30 min, allowing
equilibration of the gastric tracer. After a basal period of 20 min, a
bolus of insulin (1.0 IU/kg; Actrapid, Novo Nordisk, Denmark) was
injected intravenously. When the motor response had reached a constant
stimulated activity (~80 min after the injection), intravenous
infusions of either porcine GLP-1 (2 pmol · kg
1 · min
1)
(GLP-1 experiment, n = 8) or NaCl
(control experiment, n = 8), were
given for a 30-min period. The experiment was stopped 140 min after the
insulin injection. Gastric and pancreatic juice were collected on ice
for 10-min intervals throughout the study, and blood samples were drawn
every 10 min. Antral motility was recorded continuously from the
transducers, which were connected to a multichannel pen-writing
recorder via an amplifier. The contractile response to insulin-induced
hypoglycemia was quantified manually and expressed as the mean
frequency and amplitude (force) in 10-min periods.
Laboratory analysis.
ACID SECRETION. The concentration of
titratable acid in the gastric samples was determined by titration with
0.1 mol/l NaOH to pH 7.0 using an automatic titrator (Autoburette
Radiometer, Copenhagen, Denmark). The radioactive concentration of
57Co in each gastric sample was
measured in duplicate in a gamma-spectrometer and employed to calculate
the volume of gastric juice according to the recovery of marker (11).
Gastric output was expressed as milliequivalents of acid per 10 min.
PANCREATIC SECRETION. Pancreatic
samples were collected in chilled gastight tubes, which were capped
immediately after collection and stored frozen until assayed for volume
and bicarbonate and protein content as previously described (20).
BLOOD GLUCOSE. Blood glucose
concentration was measured every 10 min using a glucometer (One Touch
II, Lifescan).
RADIOIMMUNOASSAYS. Blood samples were
collected in chilled tubes containing EDTA (3.9 mmol/l) and aprotinin
(500 KIU/ml) and centrifuged at 4°C. The plasma was separated and
stored at
20°C until analyzed. GLP-1 immunoreactivity was
measured as previously described (35), using synthetic GLP-1-(7
36)
amide as standard (Peninsula),
125I-labeled GLP-1-(7
36) amide
and antiserum 89390, directed against the COOH terminus of
GLP-1-(7
36) amide (35). Plasma concentrations of gastrin and
pancreatic polypeptide (PP) were measured as previously described (39,
43), using synthetic porcine peptides for standards and tracers,
antisera code nos. 2609 (gastrin) and 146-5 (PP), and
plasma-coated charcoal for separation. For all assays, sensitivity was
<5 pmol/l, intra-assay coefficients of variation were <6%, and
recovery of added standards was within ±15% of expected values, whether performed in perfusate (in vitro studies) or in porcine plasma.
In Vitro Experiments
Isolated perfused antrum and pancreas.
The direct effect of GLP-1 on vagally induced antral motility and
pancreatic secretion was investigated using isolated perfused
preparations of the porcine antrum and pancreas with intact vagal
innervation (15). In short, seven pigs weighing 16-19 kg were
anesthetized with chloralose, and the pancreas and the gastric antrum
were isolated together with a segment of the aorta comprising both the
celiac and the anterior mesenteric outlets. The venous effluent from
the preparation was drained from the portal vein. In the perfusion
chamber, the antrum was suspended with rubber bands sutured to the cut
edges of the antral wall. The antral part of this preparation shows annular propulsive contractions with regular intervals. To record these, a suture was tied to the serosal surface of the antrum and
connected to a force-displacement transducer, which was connected to a
Mingograph recorder. The transducer was calibrated by attaching weights
to the suture. Motility was recorded continuously. The vagal
innervation was preserved by including the lesser omentum and the
gastrosplenic ligament in the preparation. The vagal trunks (isolated
at the level of the cardia) were threaded through a tunnel electrode
and stimulated electrically with square-wave impulses of 4 ms and 10 mA
at 8 Hz, previously shown to elicit submaximal effects (18). Pancreatic
secretion was collected for intervals of 5 or 10 min by means of a
catheter inserted into the pancreatic duct and analyzed for volume and
bicarbonate and protein concentrations as described above. The
preparation was perfused in a single-pass system (20), with a
Krebs-Ringer bicarbonate solution containing in addition 0.1% human
serum albumin, 5% dextran T70, 7 mmol/l glucose, and a mixture of
amino acids (Vamin, final concentration of amino acids 5 mmol/l;
Pharmacia, Uppsala, Sweden). The medium was supplemented with 20%
fresh, washed bovine erythrocytes and perfused at a rate of 0.4 ml · g
1 · min
1,
whereby an oxygen consumption of 9-12
µl · g
1 · min
1
was ensured. In four perfusion experiments, 1-min effluent fractions were collected on ice and centrifuged and the supernatants were analyzed for gastrin and PP concentration as described above.
Protocol.
After a 30-min equilibration period, electrical vagal stimulation was
carried out for 5 min, followed by a 10-min "rest." Then GLP-1
was infused for 15 min at a rate resulting in a final perfusate
concentration of 1 nmol/l (7 perfusion experiments). After a 5-min
infusion, vagus stimulation was repeated. In three perfusion
experiments, a third vagus stimulation was carried out 15 min after the
termination of the GLP-1 infusion. For these experiments, the control
values in Table 2 are mean values of results from the first and third
vagus stimulation. In two of the perfusion experiments, additional
vagus stimulations were carried out during infusion of GLP-1 to 10 nmol/l and in three experiments during infusion to 0.1 nmol/l.
Statistical analysis.
Data are expressed as means ± SE. For evaluation of the effect of
GLP-1 in vivo, the data for the basal period and the two 10-min periods
immediately before, during, and after GLP-1 infusion were pooled. The
resulting values are shown in Table 1. The
data from each of these four periods were analyzed by two-factor ANOVA for repeated measures followed by contrasting the data at specific time
intervals (typically the GLP-1- or saline-infusion period) using the
software Statistica (Statsoft, Tulsa, OK). In the in vitro experiments,
vagus stimulations were carried out both with and without GLP-1 in all
perfusions, and the vagus stimulation results were, therefore, compared
using a t-test for paired data. Changes as a function of time were evaluated by repeated-measures ANOVA
followed by Bonferroni's test. Further comparisons were made using
t-tests as specified in the text.
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Table 1.
Effect of GLP-1 on centrally (insulin hypoglycemia) induced antral
motility and gastric acid and pancreatic secretion in anesthetized
pigs
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RESULTS |
In Vivo Experiments
Antral motility. The motor responses
recorded by the circular and longitudinally orientated electrodes were
similar; the circular responses have been used for statistical
analysis. Insulin-induced hypoglycemia resulted in a prolonged
stimulation of antral motility lasting 120 ± 21 min and 122 ± 9 min
in the control and GLP-1 experiments, respectively (not significant,
t-test). The motility responses to
hypoglycemia are summarized in Table 1. In the control experiments, the
frequency of contractions increased almost twofold and the amplitude
increased more than eightfold corresponding to a contractile force of
63 ± 10 g. The response showed little variation with time with
respect to amplitude and frequency and no changes were seen during
saline infusion. In the GLP-1 experiments, hypoglycemia increased the
frequency twofold, and the amplitude increased about sevenfold.
Infusion of GLP-1 caused a pronounced inhibition of motility, with
frequency being reduced by 43 ± 6% of the increase
(P = 0.003) and the amplitude to
almost basal values (P = 0.0002).
After termination of the GLP-1 infusion, the frequency and the
amplitude "recovered" to preinfusion levels. A typical GLP-1
experiment is shown in Fig. 1.

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Fig. 1.
Effect of glucagon-like peptide (GLP)-1 on hypoglycemia-induced antral
motility. Recording is from a circularly orientated transducer attached
to serosal surface of the antrum in a single, typical experiment.
Amplitude is in g (contractile force). Figure should be read from
right to
left.
Bottom trace is direct continuation of
top trace. Insulin and GLP-1 were
given intravenously as indicated.
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Acid secretion.
Mean results for both groups are shown in Fig.
2. Recoveries of tracer in the control and
GLP-1 experiments were 94 ± 5% and 103 ± 7%, respectively (NS,
t-test). Insulin-induced hypoglycemia resulted in an increase in gastric acid secretion, reaching a plateau
~10-fold above basal secretion from 80 to 140 min after insulin
injection. In the GLP-1 experiments, a similar response was obtained
initially [P = 0.004 for the
effect of hypoglycemia (=time) in the two groups], but the GLP-1
infusion (started at 80 min) inhibited acid secretion by 40 ± 14% of
the 70-min value (and by 56 ± 12% of the 80-min value) at 120 min.
The P value for the inhibition in the
GLP-1 infusion period was 0.07 (Table 1). The effect of GLP-1, however,
was, as expected, somewhat delayed. The
P value for a comparison of the
secretion in the period of 90-120 min was thus 0.019 [degrees of
freedom (df ) = 14, F = 7.008].
The acid secretion hereafter recovered to a level that was
not significantly different from that observed in the control
experiment.

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Fig. 2.
Effect of GLP-1 on hypoglycemia-induced gastric acid secretion. Means
and SE of 8 control experiments ( ) and 8 GLP-1 experiments ( ) are
shown. See text and Table 1 for statistical evaluation.
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Pancreatic secretion.
Insulin-induced hypoglycemia resulted in an eightfold increase in the
output of juice (Fig. 3). A similar
response was seen at the beginning of the GLP-1 experiments
(P = 0.001 for the combined groups),
but infusion of GLP-1 almost abolished the increase
(P = 0.0016). After termination of the
infusion, the output of juice recovered to the same level as in the
control experiment (Fig. 3). Protein and bicarbonate outputs also
increased significantly after hypoglycemia, and both decreased
significantly in response to the subsequent GLP-1 administration (Table
1), whereas in the control experiments a plateau (protein) or a steady
increase (bicarbonate) was observed. Neither the concentration of
protein nor the concentration of bicarbonate in the juice differed
significantly between saline and GLP-1 experiments (not shown).

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Fig. 3.
Effect of GLP-1 on hypoglycemia-induced pancreatic secretion
[flow of juice (A), protein
output (B), bicarbonate output
(C)]. Means and SE of 8 control experiments ( ) and 8 GLP-1 experiments ( ) are shown.
GLP-1 infusion period was from 80-110 min as indicated by vertical
lines. See text and Table 1 for statistical evaluation.
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Plasma peptide and blood glucose concentrations.
The plasma concentrations of GLP-1, gastrin, and PP are shown in Fig.
4. Infusion of GLP-1 (2 pmol · kg
1 · min
1)
resulted in a plateau plasma concentration of 125 ± 27 pmol/l. The
basal levels of GLP-1 did not differ between the experiments, and GLP-1
concentrations were not affected by the hypoglycemia. The plasma
gastrin concentration increased significantly by ~100% during
hypoglycemia in the GLP-1 and control experiments. During GLP-1
infusion, the gastrin concentrations fell compared with preinfusion
values but were not significantly different from the concentrations in
the corresponding period in the control experiments. The plasma levels
of PP also increased significantly during hypoglycemia (P < 0.001 for the combined groups).
In the controls, a steady increase in the plasma concentration was seen
throughout the experiment, whereas during GLP-1 infusion a prolonged
decrease in the PP plasma level was seen, but the concentrations did
not differ significantly (Table 1). However, when the individual
concentrations in the 80- to 110-min period were analyzed by linear
regression, the slopes of the regression lines (
12.2 ± 4.4 and
3.15 ± 4.3) differed significantly between GLP-1 and saline
experiments (P = 0.019, df = 14, F = 7). The basal blood glucose
levels in the two experiments were identical (4.9 ± 0.2 mmol/l in
each series). Insulin decreased the glucose level similarly to 1.7 ± 0.2 mmol/l, a level that remained almost constant throughout the
experiment (Fig. 5).

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Fig. 4.
Effect of GLP-1 on hypoglycemia-induced secretion of pancreatic
polypeptide (PP; A) and gastrin
(B). GLP-1 concentrations are shown
in C. Means and SE of 8 control
experiments ( ) and 8 GLP-1 experiments ( ) are shown. See text and
Table 1 for statistical evaluation.
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Fig. 5.
Plasma glucose levels in response to insulin injection in 8 control
experiments ( ) and 8 experiments with GLP-1 infusion ( ).
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Vagotomy.
In four experiments, bilateral cervical vagotomy was carried out before
induction of hypoglycemia. In these experiments, hypoglycemia had
absolutely no effect on antral motility or gastropancreatic secretion
(not shown). In three experiments involving hypoglycemia-induced stimulation of gastropancreatic motility and secretion, euglycemia was
restored by glucose infusion. This immediately brought motility and
secretion back to basal levels (not shown).
In Vitro Experiments
Electrical stimulation of the vagus nerves in all cases strongly
stimulated antral motor activity and pancreatic secretion (Fig.
6 and Table 2).
Both frequency and amplitude of contractions increased significantly,
reaching a contractile force of 86 ± 13 g in the control experiments.
The flow rate of juice increased 30-fold. GLP-1 at 1 nmol/l had no
effect on antral motility but significantly enhanced the vagally
stimulated rate of pancreatic secretion (by 24%) and bicarbonate and
protein output. The secretion of both gastrin and PP increased in
response to vagus stimulation. GLP-1 had no effect on these responses
(Table 2). Higher (10 nmol/l, n = 2)
and lower (0.1 nmol/l, n = 3)
concentrations of GLP-1 were equally ineffective with respect to
inhibiting vagally induced motility and secretion (not shown).

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Fig. 6.
Motor activity of isolated perfused antrum in response to electrical
stimulation of vagus nerves with
(top) or without
(bottom) addition of GLP-1 to a
concentration of 1 nmol/l in vascular perfusate. Recording is from a
transducer attached to serosal surface of the antrum in a single,
typical experiment. Amplitude is in g (contractile force).
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Table 2.
Effect of GLP-1 on vagally induced antral motility and pancreas
secretion using isolated perfused preparations of porcine antrum and
pancreas with intact vagal innervation
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DISCUSSION |
The distal part of the small intestine participates in the regulation
of upper GI functions (25, 41). Experiments have shown that
intraluminal perfusion of the ileum with fat and
carbohydrate-containing solutions inhibits gastropancreatic secretion
and motility (9, 25, 26, 41, 42). How this endocrine inhibition, the
so-called ileal brake effect, is mediated is not yet clear.
GLP-1 inhibits gastric acid and pancreatic secretion and gastric
emptying when infused in amounts that result in plasma concentrations similar to those observed after meals (30, 37, 46). Moreover, ileal
perfusion with carbohydrates and lipids in physiological amounts
induces increases in GLP-1 plasma levels that reach or exceed those
required to inhibit gastric secretory function (24). These results
suggest that GLP-1 may be at least partly responsible for the ileal
brake effect.
Regarding the mechanism of action of GLP-1, we have previously shown
that GLP-1 infused intravenously almost abolished sham feeding-induced
gastric acid secretion in humans (45). In addition, we have
recently shown that the effect of GLP-1 on pentagastrin-induced acid
secretion is lost in vagotomized humans (47), suggesting that the
gastric effects of GLP-1 in humans are mediated through neural
pathways. GLP-1 may act differently in different species. In the
perfused rat stomach, GLP-1 was found to stimulate somatostatin and
inhibit gastrin release, indicating that GLP-1 in this species could
act directly on the gastrin cells or via local release of paracrine transmitters (3). In contrast, in the pig stomach, GLP-1
affects neither somatostatin nor gastrin secretion (33). In humans,
GLP-1 has no effects on circulating concentrations of gastrin and
somatostatin (45, 46), while still effectively inhibiting gastric acid
secretion stimulated by intragastric meal instillation (46). Part of
the inhibitory effect of GLP-1 on meal-induced pancreatic secretion
observed previously was probably due to its inhibitory effect on
gastric emptying, because the linear relationship between gastric
emptying and pancreatic exocrine secretion was unchanged by GLP-1
infusion (46). However, GLP-1 infused intravenously also effectively
inhibits pancreatic secretion stimulated by intraduodenal perfusion
with amino acids (6). In earlier experiments employing isolated
perfused preparations of the porcine pancreas, GLP-1 had no inhibitory
effect on vagally induced secretion and release of the pancreatic
neurotransmitter vasoactive intestinal polypeptide (VIP) (16). The lack
of effect of GLP-1 on vagally induced pancreatic secretion was
confirmed in the present studies using isolated perfused combined
preparations of the antrum and the pancreas. In fact, the responses to
vagus stimulation were significantly increased by GLP-1. The enhancing effect was observed only at clearly supraphysiological concentrations (1 nmol/l and above) and is unlikely to be of physiological relevance, but presumably reflects the close homology of GLP-1 to the
pancreotropic hormones, secretin, VIP, and pituitary adenylate
cyclase-activating polypeptide. In addition, GLP-1 had absolutely no
effect on antral motility, which was strongly stimulated by electrical
vagus stimulation and/or the secretion of gastrin and PP, both
of which are tightly regulated by efferent vagal activity (17, 38, 39).
It may be argued that electrical vagal stimulation is unphysiological and cannot be compared with central activation by meals or
hypoglycemia. An inhibitory effect of GLP-1 might therefore be
concealed by unphysiological activation of stimulatory mechanisms
(e.g., antidromic stimulation of afferent fibers). However, we have
previously shown that in this preparation, administration of the
nicotinic cholinergic blocker hexamethonium abolishes all effects of
vagus stimulation, proving that ganglionic transmission is involved in
all effects (15, 18). In addition, somatostatin, which inhibits
cholinergic and peptidergic neurons, powerfully inhibits the effects of
electrical vagus stimulation in this model (16). Thus, if GLP-1 had
similar effects, they should not escape notice. In addition, GLP-1 has no direct effect on the exocrine secretion of the isolated perfused pig
pancreas (16) and does not inhibit secretin- and
cholecystokinin-induced secretion (unpublished studies). Furthermore,
in preparatory experiments in anesthetized pigs, infusions of GLP-1
(same doses as described here) had no effect on secretin-stimulated
(0.5 clinical units/kg) pancreatic secretion or secretion brought about
by electrical stimulation (18) of the peripheral cut ends of the vagus
nerves (same parameters as in the present in vitro studies). We
conclude, therefore, that the GLP-1 inhibitory mechanism, which may be
similar in humans and pigs, involves the vagus nerves but neither
peripheral transmission of vagal impulses to the ganglia of the stomach
and the pancreas nor the function of their intrinsic excitatory
neurons.
Further studies of the mechanism of the inhibitory effects of GLP-1
necessitated a new experimental approach. For the experimental animal
we chose the pig because of its similarity to humans with respect to GI
physiology (27) and because of the similar apparent lack of direct
effects of GLP-1 on the stomach and the exocrine pancreas. The
combination of urethan anesthesia, cutting of the splanchnic nerves,
and insulin-induced hypoglycemia turned out to provide a reproducible
and robust model for centrally induced stimulation of antral motility
as well as gastric and pancreatic secretion. The stimulated pancreatic
secretion amounts to about one-half of the secretion elicited by
maximal electrical stimulation of the vagus nerves (9), and the gastric
secretory response is similar to that obtained with maximal
pentagastrin stimulation in conscious pigs of similar size (49). The
frequency and strength of the insulin-induced antral contractions are
very similar to those observed in response to maximal electrical
stimulation of the vagus nerves in vitro (14). Arterial gastrin and PP
concentrations increased to levels similar to those observed after
maximum vagus stimulation in anesthetized pigs (31, 38). All effects
were completely abolished by truncal vagotomy (and intravenous
glucose), proving their central origin. In a recent study in conscious
pigs with permanent pancreatic duct cannulas, Karlsson et al. (22) failed to demonstrate cephalic stimulation of the exocrine pancreatic secretion when neuroglycopenia was induced with
2-deoxy-D-glucose, a finding
that contrasts with the present results and with the profuse pancreatic
secretion that may be elicited by electrical stimulation of the vagus
nerves (10, 18). The sympathectomy included in our preparation probably
provides the explanation. We have shown previously that concomitant
stimulation of the splanchnic nerves almost abolished the pancreatic
response to electrical vagal stimulation, and, in addition, cutting of
the splanchnic nerves increased the vagus response (18). In pigs,
therefore, activation of the sympathetic nervous system induced by
central hypoglycemia may counteract or obscure its stimulatory effect on pancreatic secretion. In the intact pig, the exocrine pancreatic response to electrical vagus stimulation is greatly potentiated by
simultaneous acidification of the duodenal bulb (18). In the present
study, pyloric ligation prevented acidification of the duodenum, and
the pancreatic response to central hypoglycemia therefore represents a
"pure" pancreatic response. However, if the stomach was allowed
to drain to the duodenum, as occurs under natural circumstances, the
pancreatic response to central stimulation would probably be markedly
enhanced. Thus our results support a prominent role for central
regulation of pancreatic secretion (12, 23).
Our results clearly show that GLP-1 not only inhibits cephalically
induced acid secretion but also strongly inhibits cephalically induced
antral motility and pancreatic exocrine secretion as well. These
effects were observed at plasma concentrations around 125 pmol/l,
values that are only slightly higher than those observed in response to
ingestion of a meal in normal subjects (35) and similar to those
observed in patients with accelerated gastric emptying (13). Taken
together, our results suggest that the inhibitory effect of GLP-1 on
upper gastric functions involves receptors located either in the
central nervous system or associated with afferent pathways to the
brain stem. Recently, cephalic stimulation of antroduodenal motility
induced by sham feeding in humans was shown to approximate 70% of the
motor response to a standard meal (23). The finding that the cephalic
phase may account for more than two-thirds of the motor response to
meal ingestion combined with the observation in the present study that
GLP-1 nearly abolished the cephalically induced antral motility could
explain the pronounced effect of GLP-1 on gastric emptying in humans,
where infusion of doses similar to those employed here may cause a
complete arrest of gastric emptying for several hours (48). The
inhibition of cephalically induced pancreatic secretion, independent of
gastric emptying, suggests that GLP-1 may also inhibit the cephalic
phase of the pancreatic secretory response to meal ingestion in
addition to the inhibition caused by delayed gastric emptying. The
inhibition by GLP-1 of pancreatic secretion stimulated by duodenal
amino acid perfusion in human volunteers (6) is probably due to a lowering of vagal, cholinergic tone, known to be essential for intraduodenally stimulated secretion (1).
The precise site of the inhibitory action of GLP-1 remains obscure.
However, high-affinity receptors for GLP-1 in the rat brain were
demonstrated previously (7, 21). In addition, we have recently reported
that specific binding sites in various regions around the third
ventricle in rats, in particular the subfornical organ and the area
postrema, were accessible to GLP-1 from the systemic circulation (34).
GLP-1 might interact with these receptors. However, interaction with
afferent pathways to the brain stem, similar to those mediating the
satiating effect of cholecystokinin (40), is another possibility,
supported by recent findings in rats in which intraportal
administration of GLP-1 was demonstrated to evoke a vagal
hepatopancreatic reflex (29). Similarly, vagal deafferentation
abolished the inhibitory effect of peripheral GLP-1 on gastric emptying
[but, surprisingly, also the effects of intracerebroventricularly
administered GLP-1 (19)]. It is of interest that activation of
the cerebral GLP-1 receptors leads to inhibition of food intake in
experimental animals (44). In addition, intravenous infusion of GLP-1
was recently demonstrated to promote satiety and inhibit food intake in
healthy volunteers (5). Possibly, the central actions of GLP-1 are part
of a spectrum of activities whereby the distal gut helps to regulate
food intake.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: J. J. Holst, Dept. of Medical Physiology
C, The Panum Institute, Univ. of Copenhagen, Blegdamsvej 3, DK-2200
Copenhagen, Denmark.
Received 20 January 1998; accepted in final form 20 July 1998.
 |
REFERENCES |
1.
Adler, G.,
C. Beglinger,
U. Braun,
M. Reinshagen,
I. Koop,
B. Göke,
A. Schafmayer,
L. C. Rovati,
and
R. Arnold.
Interaction of the cholinergic system and cholecystokinin in the regulation of endogenous and exogenous stimulation of pancreatic secretion in humans.
Gastroenterology
100:
537-543,
1991[Medline].
2.
Bell, G. I.,
R. Sanchez-Pescador,
P. J. Laybourn,
and
R. C. Najarian.
Exon duplication and divergence in the human preproglucagon gene.
Nature
304:
368-371,
1983[Medline].
3.
Eissele, R.,
H. Koop,
and
R. Arnold.
Effect of glucagon-like peptide-1 on gastric somatostatin and gastrin secretion in rats.
Scand. J. Gastroenterol.
25:
449-454,
1990[Medline].
4.
Elliot, R. M.,
L. M. Morgan,
T. A. Tredger,
S. Deacon,
J. Wright,
and
V. Marks.
Glucagon-like peptide-1-(7
36) amide and glucose-dependent insulinotropic polypeptide secretion in response to nutrient ingestion in man: acute post-prandial and 24-h patterns.
J. Endocrinol.
138:
159-166,
1993[Abstract].
5.
Flint, A.,
A. Raben,
A. Astrup,
and
J. J. Holst.
Glucagon-like peptide-1 promotes satiety and supresses energy intake in humans.
J. Clin. Invest.
101:
515-520,
1998[Abstract/Free Full Text].
6.
Franke, A.,
J. Keller,
J. J. Holst,
D. Grandt,
H. Goebell,
and
P. Layer.
Effects of glucagon-like peptide-1 on human pancreatic enzyme secretion (Abstract).
Digestion
57:
227,
1996.
7.
Göke, R.,
P. J. Larsen,
J. D. Mikkelsen,
and
S. P. Sheikh.
Distribution of GLP-1 binding sites in the rat brain: evidence that exendin-4 is a ligand of brain GLP-1 binding sites.
Eur. J. Neurosci.
7:
2294-2300,
1995[Medline].
8.
Gutniak, M.,
C. Ørskov,
J. J. Holst,
B. Ahrén,
and
S. Efendic.
Anti-diabetogenic effect of glucagon-like peptide-1-(7
36) amide in normal subjects and patients with diabetes mellitus.
N. Engl. J. Med.
326:
1316-1322,
1992[Abstract].
9.
Harper, A. A.,
J. C. A. Hood,
and
J. Mushens.
Pancreatone, an inhibitor of pancreatic secretion in extracts of ileal and colonic mucosa.
J. Physiol. (Lond.)
292:
455-467,
1979[Abstract].
10.
Hickson, J. C. D.
The secretion of pancreatic juice in response to stimulation of the vagus nerves in the pig.
J. Physiol. (Lond.)
206:
275-297,
1970[Medline].
11.
Hobsley, M.,
and
W. Silen.
Use of an inert marker (phenol red) to improve accuracy in gastric acid secretion studies.
Gut
10:
787-795,
1969[Medline].
12.
Holst, J. J.
Neural regulation of pancreatic exocrine function.
In: The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by W. Vay Liang,
M. D. Go,
P. Eugene,
and M. D. Dimagno. New York: Raven, 1993, p. 381-402.
13.
Holst, J. J.
Enteroglucagon.
Annu. Rev. Physiol.
59:
257-272,
1997[Medline].
14.
Holst, J. J.,
H. Harling,
T. Messell,
and
D. Coy.
Identification of the neurotransmitter/neuromodulator functions of the neuropeptide GRP in the porcine antrum using the antagonist (Leu13-psi-CH2NH-Leul4)-bombesin.
Scand. J. Gastroenterol.
25:
89-96,
1989.
15.
Holst, J. J.,
S. L. Jensen,
S. Knuhtsen,
O. V. Nielsen,
and
J. F. Rehfeld.
Effect of vagus, gastric inhibitory polypeptide, and HCl on gastrin and somatostatin release from perfused pig antrum.
Am. J. Physiol.
244 (Gastrointest. Liver Physiol. 7):
G515-G522,
1983[Abstract/Free Full Text].
16.
Holst, J. J.,
T. N. Rasmussen,
H. Harling,
and
P. Schmidt.
Effect of intestinal inhibitory peptides on vagally induced secretion from isolated perfused porcine pancreas.
Pancreas
8:
80-87,
1993[Medline].
17.
Holst, J. J.,
T. N. Rasmussen,
P. Schmidt,
and
S. S. Poulsen.
Transmitters in the control of gastrin and acid secretion in the pig stomach.
In: Gastrin, edited by J. H. Walsh. New York: Raven, 1993, p. 243-258.
18.
Holst, J. J.,
O. B. Schaffalitzky de Muckadell,
and
J. Fahrenkrug.
Nervous control of pancreatic exocrine secretion in pigs.
Acta Physiol. Scand.
105:
33-51,
1979[Medline].
19.
Ineryuz, N.,
B. C. Yegen,
A. Bozkurt,
T. Coskun,
M. L. Villanueva-Penacarillo,
and
N. B. Ulusoy.
Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G920-G927,
1997[Abstract/Free Full Text].
20.
Jensen, S. L.,
J. Fahrenkrug,
and
J. J. Holst.
Secretory effects of secretin on isolated perfused porcine pancreas.
Am. J. Physiol.
235 (Endocrinol. Metab. Gastrointest. Physiol. 4):
E381-E386,
1978.
21.
Kanse, S. M.,
B. Kreymann,
M. A. Ghatei,
and
S. R. Bloom.
Identification and characterization of glucagon-like peptide-1-(7
36) amide binding sites in the rat brain and lung.
FEBS Lett.
241:
209-212,
1988[Medline].
22.
Karlsson, S.,
S. G. Pierzynowski,
B. R. Weström,
M.-J. Thaela,
B. Ahrén,
and
B. W. Karlsson.
Stimulation of endocrine, but not exocrine, pancreatic secretion during 2-deoxy-D-glucose-induced neuroglycopenia in the conscious pig.
Pancreas
11:
271-275,
1995[Medline].
23.
Katschinski, M.,
G. Dahmen,
M. Reinshagen,
C. Beglinger,
H. Koop,
R. Nustede,
and
G. Adler.
Cephalic stimulation of gastrointestenal secretory and motor responses in humans.
Gastroenterology
103:
383-391,
1992[Medline].
24.
Layer, P.,
J. J. Holst,
D. Grandt,
and
H. Goebell.
Ileal release of glucagon-like peptide-1 (GLP-1): association with inhibition of gastric acid in humans.
Dig. Dis. Sci.
40:
1074-1082,
1995[Medline].
25.
Lloyd, K. C. K,
and
H. T. Debas.
Peripheral regulation of gastric secretion.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 1185-1226.
26.
Miller, L. J.,
J. R. Malagelada,
W. F. Tayler,
and
V. L. W. Go.
Intestinal control of human postprandial gastric function: the role of components of jejunoileal chyme in regulating gastric secretion and gastric emptying.
Gastroenterology
80:
763-769,
1981[Medline].
27.
Miller, E. R.,
and
D. E. Ullrey.
The pig as a model for human nutrition.
Annu. Rev. Nutr.
7:
361-382,
1987[Medline].
28.
Mojsov, S.,
G. Heinrich,
I. B. Wilson,
M. Ravazzola,
L. Orci,
and
J. F. Habener.
Preproglucagon gene expression in pancreas and intestine diversifies at the level of posttranslational processing.
J. Biol. Chem.
261:
11880-11889,
1986[Abstract/Free Full Text].
29.
Nakabayshi, H.,
M. Nishizawa,
A. Nakagawa,
R. Takeda,
and
A. Niijima.
Vagal hepatopancreatic reflex effect evoked by intraportal appearance of tGLP-1.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E808-E813,
1996[Abstract/Free Full Text].
30.
O'Halloran, D. J.,
G. C. Nikou,
B. Kreyman,
M. A. Ghatei,
and
S. R. Bloom.
Glucagon-like peptide-1-(7
36)-NH2: a physiological inhibitor of the gastric acid secretion in man.
J. Endocrinol.
126:
169-173,
1990[Abstract].
31.
Olesen, M.,
J. J. Holst,
C. Sottimano,
and
O. V. Nielsen.
Autonomic nervous control of fundic secretion of somatostatin and antral secretion of gastrin and somatostatin in pigs.
Digestion
36:
24-35,
1987[Medline].
32.
Ørskov, C.,
J. J. Holst,
S. Knutsen,
F. G. A. Baldissera,
S. S. Poulsen,
and
O. V. Nielsen.
Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separately from the pig small intestine, but not the pancreas.
Endocrinology
119:
1467-1475,
1986[Abstract].
33.
Ørskov, C.,
J. J. Holst,
and
O. V. Nielsen.
Effect of truncated glucagon-like peptide-1 (proglucagon 78
107 amide) on endocrine secretion from pig pancreas, antrum and stomach.
Endocrinology
123:
2009-2013,
1988[Abstract].
34.
Ørskov, C.,
S. S. Poulsen,
M. Møller,
and
J. J. Holst.
Glucagon-like peptide 1 receptors in the subfornical organ and the area postrema are accessible to circulating glucagon-like peptide 1.
Diabetes
45:
832-835,
1996[Abstract].
35.
Ørskov, C.,
L. Rabenhøj,
H. Kofod,
A. Wettergren,
and
J. J. Holst.
Production and secretion of amidated and glycine-extended glucagon-like peptide-1 (GLP-1) in man.
Diabetes
43:
535-539,
1994[Abstract].
36.
Ørskov, C.,
A. Wettergren,
and
J. J. Holst.
Secretion of the incretin hormones glucagon-like peptide-1 and gastric inhibitory polypeptide correlates with insulin secretion in normal man throughout the day.
Scand. J. Gastroenterol.
31:
665-670,
1996[Medline].
37.
Schjoldager, B. T. G.,
P. E. Mortensen,
J. Christensen,
C. Ørskov,
and
J. J. Holst.
GLP-1 (glucagon-like peptide 1) and truncated GLP-1, fragments of the human proglucagon, inhibit gastric acid secretion in humans.
Dig. Dis. Sci.
34:
703-708,
1989[Medline].
38.
Schwartz, T. W.,
J. J. Holst,
J. Fahrenkrug,
C. Kühl,
S. L. Jensen,
J. F. Rehfeld,
and
O. B. Scffalitzky de Muckadell.
Vagal, cholinergic regulation of the secretion of pancreatic polypeptide.
J. Clin. Invest.
61:
781-789,
1978[Medline].
39.
Schwartz, T. W.,
B. Stenquist,
and
L. Olbe.
Cephalic phase of pancreatic polypeptide secretion studied by sham feeding in man.
Scand. J. Gastroenterol.
14:
313-320,
1979[Medline].
40.
Smith, G. P.,
C. Jerome,
B. J. Cushin,
R. Eterno,
and
K. J. Simansky.
Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat.
Science
213:
1036-1037,
1981[Medline].
41.
Solomon, T. E.
Control of exocrine pancreatic secretion.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 1499-1530.
42.
Spiller, R. C.,
I. F. Trotman,
B. E. Higgins,
M. A. Ghatei,
G. K. Grimble,
Y. C. Lee,
S. R. Bloom,
J. J. Misiewicz,
and
D. B. Silk.
The ileal brake-inhibition of jejunal motility after ileal fat perfusion in man.
Gut
25:
365-374,
1984[Abstract].
43.
Stadil, F.,
and
J. F. Rehfeld.
Determination of gastrin in serum. An evaluation of the reliability of a radioimmunoassay.
Scand. J. Gastroenterol.
8:
101-112,
1973[Medline].
44.
Turton, M. D.,
D. O'Shea,
I. Gunn,
S. A. Beak,
C. M. Edwards,
K. Meeran,
S. J. Choi,
G. M. Taylor,
M. M. Heath,
P. D. Lambert,
J. P. Wilding,
D. M. Smith,
M. A. Ghatei,
J. Herbert,
and
S. R. Bloom.
A role for glucagon-like peptide-1 in the central regulation of feeding.
Nature
379:
69-72,
1996[Medline].
45.
Wettergren, A.,
H. Petersen,
C. Ørskov,
J. Christiansen,
S. P. Sheikh,
and
J. J. Holst.
Glucagon-like peptide-1 (GLP-1)-7
36 amide and peptide YY from the L-cell in the ileal mucosa are potent inhibitors of vagally induced gastric acid secretion in man.
Scand. J. Gastroenterol.
29:
501-505,
1994[Medline].
46.
Wettergren, A.,
B. Schjoldager,
P. E. Mortensen,
J. Myhre,
J. Christiansen,
and
J. J. Holst.
Truncated GLP-1 (proglucagon 72
107 amide) inhibits gastric and pancreatic function in man.
Dig. Dis. Sci.
38:
665-673,
1993[Medline].
47.
Wettergren, A.,
M. Wøjdemann,
S. Meisner,
F. Stadil,
and
J. J. Holst.
The inhibitory effect of glucagon-like peptide-1 (GLP-1) 7
36 amide on gastric acid secretion in humans depends on an intact vagal innervation.
Gut
40:
597-601,
1997[Abstract].
48.
Willms, B.,
J. Werner,
J. J. Holst,
C. Ørskov,
W. Creutzfeldt,
and
M. A. Nauck.
Gastric emptying, glucose responses and insulin secretion after a liquid test meal: effects of exogenous glucagon-like peptide-1 (GLP-1 7
36 amide) in type 2 (non-insulin dependent) diabetic patients.
J. Clin. Endocrinol. Metab.
81:
327-332,
1996[Abstract].
49.
Xu, R.-J.,
and
P. D. Cranwell.
Development of gastric acid secretion in pigs from birth to thirty days of age: the response to pentagastrin.
J. Dev. Physiol. (Eynsham)
13:
315-326,
1990[Medline].
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