Somatostatin restrains the secretion of glucagon-like
peptide-1 and -2 from isolated perfused porcine ileum
Lene
Hansen1,
Bolette
Hartmann1,
Thue
Bisgaard1,
Hitoshi
Mineo1,
Peer N.
Jørgensen2, and
Jens J.
Holst1
1 Department of Medical Physiology, the Panum
Institute, University of Copenhagen, DK-2200 Copenhagen; and
2 Novo-Nordisk, DK-2880 Bagsværd,
Denmark
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ABSTRACT |
Suspecting that paracrine inhibition might influence
neuronal regulation of the endocrine L cells, we studied the role of somatostatin (SS) in the regulation of the secretion of the
proglucagon-derived hormones glucagon-like peptide-1 and -2 (GLP-1 and
GLP-2). This was examined using the isolated perfused porcine ileum
stimulated with acetylcholine (ACh, 10
6
M), neuromedin C (NC, 10
8 M), and
electrical nerve stimulation (NS) with or without
-adrenergic blockade (phentolamine 10
5 M), and
perfusion with a high-affinity monoclonal antibody against SS. ACh and
NC significantly increased GLP secretion, whereas NS had little effect.
SS immunoneutralization increased GLP secretion eight- to ninefold but
had little influence on the GLP responses to ACh, NC, and NS. Basal SS
secretion (mainly SS28) was unaffected by NS alone. Phentolamine + NS
and NC abstract strongly stimulated release mainly of SS14, whereas ACh
had little effect. Infused intravascularly, SS14 weakly and SS28
strongly inhibited GLP secretion. We conclude that GLP secretion is
tonically inhibited by a local release of SS28 from epithelial
paracrine cells, whereas SS14, supposedly derived from enteric neurons,
only weakly influences GLP secretion.
paracrine activity; L cells; bombesin; enteric nervous system; gastrointestinal hormones; immunoneutralization
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INTRODUCTION |
GLUCAGON-LIKE PEPTIDE (GLP)-1 is a peptide hormone
secreted from the distal part of the small intestine and the colon in
response to meal ingestion (18). It plays an important role as an
incretin hormone, stimulating insulin secretion (40), and also
functions as one of the hormones of the "ileal brake," the
endocrine inhibition of upper gastrointestinal motility and secretion
elicited by the presence of unabsorbed nutrients in the ileum (22, 28,
42). GLP-2 is a peptide hormone derived from the same precursor (1). Much less is known about GLP-2, but recently it has been shown to act
as an intestinal growth factor (10). Relatively little is known about
the physiological mechanisms that regulate the secretion of GLP-1 and
GLP-2, but because of their common precursor, they are supposed to (and
also are shown to) be secreted synchronously and in equimolar amounts
(29). The presence in the lumen of unabsorbed carbohydrates or lipids
provides an important stimulus for GLP-1 secretion (11, 15, 22).
However, the GLP-1 response to a meal is usually rapid, with increases
occurring within a few minutes after the start of the meal (11, 14,
31), i.e., before the bulk of the meal has reached the lower small
intestine. This suggests that neural and/or endocrine factors may also
play a role. In humans and pigs, none of the duodenal hormones (gastric inhibitory polypeptide, motilin, cholecystokinin, or secretin) seems to
stimulate GLP-1 secretion (27, 28). Furthermore, in humans, neurally
induced responses cannot be provoked by modified sham feeding or a
cephalic vagal stimulus (32), and, in pigs, electrical stimulation of
the mixed intestinal nerves had little effect on GLP-1 secretion in the
absence as well as in the presence of
-adrenergic blockers (13).
However, somatostatin has been reported to inhibit GLP-1 secretion in
sheep (24), as might have been expected in view of the previously
demonstrated inhibitory effect of somatostatin on glicentin (or
enteroglucagon) secretion (4, 6, 7, 23, 35, 39). Like GLP-1 and GLP-2,
glicentin is a product of glucagon gene expression in the gut, and the
three peptides are secreted in parallel (29). We therefore hypothesized that a stimulatory effect of a given stimulus on the secretion of GLP-1
and GLP-2 might be concealed if the stimulus also caused a local
release of somatostatin. If so, the inhibitory effect of somatostatin
might outweigh the stimulatory effect of the primary stimulus on GLP-1
and GLP-2 secretion. To study this, we employed somatostatin
immunoneutralization (20) in the present series of studies of the
neural regulation of GLP-1 and GLP-2 secretion from isolated perfused
porcine ileum.
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MATERIALS AND METHODS |
This study conformed to the Danish legislation governing animal
experimentation (1987) and was carried out after permission was granted
from the National Superintendence for Experimental Animals.
Perfusion experiments.
Young pigs of the YDL-strain weighing 15-20 kg were anesthetized
with ketamine chloride (Ketalar, Parke-Davis, Morris Plains, NJ) for
premedication, pentobarbital for induction, and chloralose (50 mg/kg)
and N2O/O2 for maintenance. After laparotomy,
an 80-cm segment of the central ileum, including its arterial and
venous supply, was isolated, excised, and positioned floating in Ringer solution in a perfusion chamber at 37°C. It was perfused via the artery by use of a pulsatile finger pump (Ole Dich, Copenhagen, Denmark) at a constant rate with a Krebs-Ringer-bicarbonate solution containing, in addition, 0.1% human serum albumin (Behringwerke, Marburg, Germany), 5% dextran T 70 (Pharmacia, Uppsala, Sweden), 7 mmol/l glucose, and a mixture of amino acids (Vamin, 14 g/l, Pharmacia)
in an amount yielding a total amino acid concentration of 5 mmol/l. The
perfusion medium also contained 20% freshly washed bovine erythrocytes
(obtained 2-3 days earlier at a local abattoir), and a
cyclooxygenase inhibitor (indomethacin, Confortid, Dumex, Copenhagen, 5 mg/l) was added to prevent generation of prostaglandins in the
perfusion system. The perfusate was gassed continuously with a mixture
of 5% carbon dioxide in oxygen with a multibulb oxygenator. The
perfusion flow rate was 0.2-0.3
ml · g
1 · min
1,
ensuring an oxygen delivery of 0.75 ml O2/min, which is
about twofold in excess of the oxygen consumption of the preparation. Perfusion pressure was recorded constantly via a sidearm to the arterial catheter by a Statham transducer connected to an amplifier and
a recorder. Motor activity of the gut was estimated visually, but
contractions of the gut were also reliably reflected as short-lasting increments in perfusion pressure (spikes), validated in experiments involving intraluminal manometry (38). The frequency of such increments
(which did not increase the basal perfusion pressure) was recorded. A
bipolar platinum electrode, shaped like a hook, was positioned
carefully around the supplying artery and the network of nerve fibers
surrounding it, without damage to the fibers, and was kept in place by
a loose ligature. Square wave impulses were delivered by a nerve
stimulator. The intestinal lumen was perfused at a rate of 3 ml/min
with preheated perfusate containing 15 mmol/l glucose. The venous
effluent was collected for 1-min periods, chilled in ice, and
centrifuged at 4°C within a few minutes. The supernatants were
frozen immediately pending radioimmunoassays. Arterial and venous
perfusate samples were analyzed at regular intervals for O2
and CO2 using ABL-II [Acid-Base Laboratory II, Radiometer, Copenhagen, Denmark (19)].
Experimental protocol.
Isolated perfused intestines were prepared from a total of 19 pigs. In
six perfusion experiments, sampling of effluent was started after 30 min of equilibration, and after 10 min, a 5-min nerve stimulation (NS;
8 Hz, 10 mA, 4-ms impulse duration) was carried out. A continuous
infusion of phentolamine was then started via the arterial line, giving
a final concentration of 10
5 mol/l.
Phentolamine addition was considered essential to eliminate the
inhibitory effect of the sympathetic innervation on GLP and other
hormone secretions of the mucosa. Electrical stimulation of the mixed
intestinal nerve (as we use here) invariably activates the sympathetic
fibers, and removal of their inhibitory effect may be necessary to
observe the effect of stimulatory neurons (25). After 10 min, the NS
was repeated. After a further 10-min rest, 5-min infusions of
acetylcholine (ACh; Sigma, St. Louis, MO, via preweighed vials and at a
final concentration of 10
6 mol/l) or
neuromedin C [NC, the COOH-terminal decapeptide of gastrin-releasing polypeptide (GRP), or "mammalian bombesin," Peninsula Europe, Merseyside, St. Helens, UK;
10
8 mol/l] were given via the
arterial line in randomized order. After another 10-min rest period, an
infusion of high-affinity (affinity constant, or
Ka, = 1011 l/mol) monoclonal
somatostatin (SS) antibodies (20), which had been dialyzed overnight
(against saline), was started, in an amount yielding an SS binding
capacity of 6 nmol/ml. After 25 min, 5-min periods of NS or ACh and NC
infusions were carried out as before. In six additional perfusion
experiments, a 5-min infusion of SS14 at
10
8 mol/l (final perfusate
concentration) was carried out after the initial equilibration period,
and in four perfusion experiments, stepwise increasing dose-response
experiments were carried out for both SS14 and SS28 (at
10
10 to
10
8 mol/l). At the end of the
experiments, NC was infused as before, without or with SS14 and SS28,
both at 10
8 mol/l. In three perfusion
experiments, monoclonal antibodies against 2,4,6,-trinitrophenyl (3)
were added to yield
-globulin concentrations similar to the anti-SS
experiments. In an earlier study, when the same experimental model was
used, it was demonstrated that vascular and secretory responses
remained unchanged for up to 4 repeated instances of NS (25).
Analyses of the perfusion effluents.
All effluent fractions were analyzed for SS and GLP-1 by use of
previously described radioimmunoassays (17, 31). The SS assay
recognizes equally well SS14 and SS28. The GLP-1 assay was carried out
using the antibody 89390, which has an absolute requirement for the
amidated COOH terminus of the GLP-1 molecule and therefore recognizes
GLP-1-(7
36)amide and the inactive metabolite, GLP-1-(9
36)amide. The
effluent fractions from three of the immunoneutralization experiments
(the effluents from the other experiments were not available for
analysis) and the effluent fractions from the four dose-response
experiments with SS14 and SS28 were analyzed for GLP-2 content by use
of the antibody 92160, which recognizes the NH2 terminus of
GLP-2 and, therefore, only fully processed biologically active GLP-2.
Details of the assay are described in Ref. 43. The detection limits of
the assays were below 5 pmol/l.
Chromatography.
Pools of effluent samples, collected during different experimental
conditions (in the basal state, during NS + phentolamine perfusion,
during NC + phentolamine perfusion, and during SS antibody perfusion),
were applied to Sep-Pak C18 cartridges (Water-Millipore, Milford, MA) and eluted with 70% ethanol + 0.1% trifluoroacetic acid.
The effluent was dried, redissolved in albumin-containing (0.1%)
phosphate buffer (0.05 mM, pH 7.5), and applied to a Sephadex G50 fine
K16/100 column (Pharmacia, Uppsala, Sweden) eluted with the same buffer
at 4°C.
The eluted fractions resulting from gel filtration of the pools of
venous effluent obtained in the basal state, during NS + phentolamine, and during NC + phentolamine perfusion were analyzed for immunoreactive SS to estimate the relative contributions of SS14
and SS28 to the SS response.
The eluted fractions from gel filtration of the pools obtained during
SS antibody perfusion (n = 4) were analyzed for immunoreactive glicentin, GLP-1, and GLP-2 by use of the following antisera: 4304 (recognizes the glucagon part of glicentin, previously described in
Ref. 21), 2135 (a so-called side-viewing antibody, recognizing a
midregion and, therefore, all forms of GLP-1, previously described in
Ref. 30), 89390 (described above and in Ref. 31), 92071 [a
COOH-terminally directed antibody, recognizing specifically glycine-extended GLP-1-(7
37), previously described in Ref. 31], 92160 (described above), and another side-viewing GLP-2 antiserum that
recognizes a midregion of GLP-2 (Peninsula Laboratories, cat. no. RAS
7167, Merseyside, St Helens, UK). For the side-viewing GLP-2 assay, we
employed monoiodinated rat GLP-2, with an Asp33
Tyr33 substitution as tracer but otherwise similar assay
conditions (43).
Calculations.
Because of the constant perfusion flow, the hormone effluent
concentrations parallel secretion rates. The data are presented as
means ± SE. Changes in hormone secretion as a function of time were
evaluated by ANOVA for repeated measures, or, alternatively, mean
hormone outputs for the immediate prestimulatory 5-min periods were
compared with mean 5-min stimulated plateau responses (calculated for
the 3- to 7-min period after start of NS) by use of a t-test for paired data. Hormone outputs after NS without and with phentolamine and SS antibodies were compared using ANOVA, whereas hormone outputs after ACh and NC stimulation with and without SS antibodies were compared using a t-test for paired data. Spike frequencies
(reflecting motor activity) were recorded for 5-min periods.
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RESULTS |
The results concerning perfusion pressure and motor activity responses
to infusions and nerve stimulations are summarized in Table
1. NS significantly increased perfusion
pressure, and phentolamine strongly reduced this response, illustrating
successful stimulation of sympathetic efferent fibers (eliciting
vasoconstriction and therefore increasing perfusion
pressure) and the expected effect of
-adrenergic blockade on this
response. As expected, NS had little effect on motor activity alone but
markedly enhanced motor activity after
-adrenergic blockade,
illustrating effective stimulation of nonadrenergic motor neurons under
these circumstances (38). Both ACh and NC significantly stimulated
motor activity, demonstrating adequate activation of cholinergic and
bombesin receptors.
The main finding of the present study is the dramatic increase in GLP-1
and GLP-2 secretion after SS immunoneutralization (Fig.
1). After 4-5 min, both GLP-1 and
GLP-2 secretion increased, reaching plateau levels of 813 ± 141 and
995 ± 385% of basal secretion after 15-20 min. In preliminary
experiments in which immunoneutralization was carried out without
phentolamine, similar increases in GLP-1 secretion were observed (4- to
8-fold increases, n = 3, not shown). Thus the effect of
immunoneutralization was independent of the presence of absence of
noradrenergic blockade. For GLP-1, plateau values averaged ~400
pmol/l, whereas the GLP-2 plateau amounted to ~800 pmol/l. Because of
this unexpected difference, we investigated the molecular nature of the
secreted peptides by chromatographic analysis of pooled, concentrated
perfusion effluents from four perfusion experiments. The results are
shown in Fig. 2. Glucagon (glicentin)
immunoreactivity eluted at a distribution coefficient (Kd) of 0.35, corresponding to the elution position
of synthetic and natural glicentin. GLP-2 immunoreactivity eluted at
Kd 0.55-0.60, regardless of the assay employed
(side-viewing or NH2 terminal). The side-viewing GLP-1
assay (code no. 2135) identified a peak at Kd 0.65, and a peak with a similar Kd was obtained with the assay for glycine-extended GLP-1 (code no 92071). The assay for amidated GLP-1 revealed a peak around Kd 0.60, slightly shifted to the left compared with that of the 92071 assay.
Synthetic amidated GLP-1 and glycine-extended GLP-1 both eluted at
0.60-0.65. Measured as total eluted amounts and with the amount
identified with the side-viewing GLP-1 assay (code no 2135) set to
100%, both the assay for the amidated and that for the
glycine-extended COOH terminus measured significantly less than the
side-viewing assay (27.6 ± 4.3%, P < 0.001 for
89390 and 30.6 ± 11.5%, P < 0.010 for 92071; Fig.
2C). However, the sum of the results of these assays (89390 + 92071) did not differ significantly from the amount obtained with the
side-viewing assay (P = 0.076). Thus approximately one-half of
the total output of GLP-1 was accounted for by glycine-extended GLP-1,
explaining the difference between the measured GLP-1 (using antiserum
89390) and GLP-2 plateau values, and possibly also some of the other
minor discrepancies between the results of the two assays. There was no
significant difference between amounts measured using the side-viewing
GLP-1 assay and the assays for GLP-2 or glicentin (Fig. 2C).

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Fig. 1.
Effect of infusion of monoclonal somatostatin antibodies [binding
capacity 6 nmol/ml and affinity constant (Ka) of
1011 l/mol] on glucagon-like peptide-1 (GLP-1)
secretion (A) and GLP-2 secretion (B). Values are means ± SE of 6 perfusion experiments (n = 3 for GLP-2).
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Fig. 2.
Gel filtration profiles of immunoreative glicentin and GLP-1 and -2 in
pools of effluent from isolated perfused ileum collected during
somatostatin immunoneutralization. Eluted immunoreactivity is expressed
as a percentage of total eluted amount, plotted as a function of the
coefficient of distribution (Kd). Values are means ± SE of results from gel filtration of 4 effluent pools. A:
results of side-viewing assays for glucagon (×, comprising
glicentin and oxyntomodulin) and GLP-1 ( ) and the
NH2-terminal assay for GLP-2 ( ). B: results of
side-viewing assay for GLP-2 ( ) and COOH-terminal assays for
amidated ( ) and glycine-extended GLP-1 ( ). In C, total
amounts of immunoreactivity eluted as measured in each assay are
compared with amount measured with side-viewing GLP-1 assay (code
2135), set to 100%. Significant differences: ** P < 0.010; *** P < 0.001. In separate experiments, synthetic
glicentin was found to elute at Kd 0.35, oxyntomodulin at 0.75, GLP-2 at 0.55-0.60, and amidated and
glycine-extended GLP-1 at 0.60 and 0.65, respectively.
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Figure 3 shows the results of electrical
mixed NS, either alone or in the presence of phentolamine
(10
5 mol/l) and phentolamine + SS
antibodies. GLP-1 secretion was unaffected by NS regardless of the
perfusion conditions, whereas GLP-2 secretion decreased to a nadir of
52 ± 9% after 3 min of NS. After addition of phentolamine and
phentolamine + SS antibody, GLP-2 secretion increased insignificantly
during NS to maximally 297 ± 129 and 159 ± 30% of basal,
respectively. SS secretion was uninfluenced by NS in the control
experiments but increased greatly (to 470 ± 80% of prestimulatory
levels, P < 0.010) during phentolamine perfusion.
Chromatographic analysis revealed that the SS immunoreactivity released
in the basal state mainly consisted of SS28, whereas during NS + phentolamine, mainly SS14 was released (Fig.
4). SS secretion could not be measured
during antibody perfusion. Figure 5 shows
the NC stimulation experiments during phentolamine alone and during
phentolamine + SS antibody perfusion. GLP-1 secretion, expressed in
percentage of basal, was significantly enhanced by NC during
phentolamine as well as antibody perfusion (to 293 ± 43%,
P < 0.010 and 149 ± 16%, P < 0.050, respectively; P < 0.050 for the difference). The absolute
output of GLP-1 was larger during NC + antibody perfusion (3.4 ± 0.6 vs. 1.1 ± 0.1 pmol/min, P < 0.050). NC, in all three cases,
stimulated GLP-2 secretion under both phentolamine and phentolamine + SS antibody (to 265 ± 42 and 175 ± 52% of prestimulatory levels),
but the difference did not reach significance. SS secretion was greatly
augmented by NC (to 437 ± 73%, P < 0.010), and
chromatographic analysis revealed that SS14 was the predominant
molecular form (Fig. 4).

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Fig. 3.
Effect of nerve stimulation (8 Hz) on secretion of GLP-1 (A),
GLP-2 (B), and somatostatin (C) without additions
(left), during phentolamine administration
(10 5 M; middle), or during
infusion of somatostatin antibodies and phentolamine
(10 5 M) infusion (right). Values
are means ± SE of 6 perfusion experiments (n = 3 for
GLP-2).
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Fig. 4.
Gel filtration profile of immunoreactive somatostatin in a pool of
effluent samples from 6 perfusion experiments, collected in the basal
state (A), during nerve stimulation + phentolamine
administration (B), and during neuromedin C + phentolamine
administration (C). Immunoreactive somatostatin in eluted
fractions, expressed in %total immunoreactivity eluted from column, is
plotted against Kd (a measure of molecular size).
Arrows indicate elution positions of synthetic somatostatin 28 (SS28)
and somatostatin 14 (SS14).
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Fig. 5.
Effect of neuromedin C (NC, 10 8 M)
infusion on secretion of GLP-1 (A), GLP-2 (B), and
somatostatin (C) during phentolamine administration
(10 5 M) (left) or during
infusion of SS antibodies and phentolamine
(10 5 M) infusion (right). Values
are means ± SE of 6 perfusion experiments (n = 3 for
GLP-2).
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Figure 6 shows the ACh stimulation
experiments during phentolamine alone and during phentolamine + SS
antibody perfusion. GLP-1 secretion showed a somewhat delayed increase
in response to ACh, both during phentolamine and SS antibody perfusion
(to 169 ± 12 and 116 ± 4%, in the 5- to 8-min period after start
of ACh, P < 0.010 and 0.050, respectively). The absolute
outputs of GLP-1 did not differ significantly (0.8 ± 0.2 vs. 1.3 ± 0.4 pmol/min, respectively). ACh also tended to increase GLP-2
secretion under both conditions (to 265 ± 50 and 111 ± 18% of
basal secretion, but the changes did not reach statistical
significance). ACh did not affect SS secretion.

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Fig. 6.
Effect of acetylcholine (ACh, 10 6 M)
infusion on secretion of GLP-1 (A), GLP-2 (B), and SS
(C) during phentolamine administration
(10 5 M) (left) or during
infusion of SS antibodies and phentolamine
(10 5 M) infusion (right). Values
are means ± SE of 6 perfusion experiments (n = 3 for
GLP-2).
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In the six experiments in which SS14 was infused to a final
concentration of 10
8 mol/l, GLP-1
secretion decreased to 79 ± 7%, P < 0.050. Because of this
finding, a dose-response study was carried out. Figure 7 shows the effect of SS28 and SS14 infused
to final concentrations of 10
10,
10
9, and
10
8 mol/l on GLP-1 and GLP-2 secretion.
SS28 dose dependently inhibited both GLP-1 and GLP-2 secretion,
reaching 38 ± 10% of basal secretion for GLP-1 and 27 ± 7% for
GLP-2 at 10
8 mol/l (P < 0.01).
SS14 had weak and inconsistent inhibitory effects on GLP secretion.
During co-infusion of SS28 and NC, GLP-1 secretion was inhibited to 66 ± 5% and GLP-2 secretion to 50 ± 9% (P < 0.05), both
significantly different from the stimulated secretion observed with NC
alone (P < 0.01). In contrast, during co-infusion of SS14 and
NC, both GLP-1 and GLP-2 secretion increased (P = 0.08 and 0.09, respectively) to levels not significantly different from those
obtained with NC alone.

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Fig. 7.
Effect of infusions of SS28 and SS14 in final concentrations of 0.1 nM,
1 nM, and 10 nM (left) on GLP-1 secretion (open bars,
A) and GLP-2 secretion (solid bars, B). At
right, effect of co-infusion of SS28 + NC (GRP) and SS14 + NC, and NC alone, on GLP-1 and GLP-2 secretion. All responses are
expressed as %basal secretion. Values are means ± SE of 4 perfusion
experiments. Significant differences from basal secretion:
* P < 0.050; ** P < 0.010; *** P < 0.001.
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Addition of the 2,4,6-trinitrophenyl antibodies had no influence on any
registered parameter (data not shown).
 |
DISCUSSION |
The most important finding of the present study is
the dramatic increase in the secretion of GLP-1 and -2 in response to
somatostatin immunoneutralization. The antibodies were administered as
a highly purified solution of
-globulins, and there was no effect of
infusion of control antibodies, speaking against unspecific effects. In addition, the high concentrations of the glucagon-like peptide measured
during immunoneutralization were verified by chromatographic analysis
of the effluent (see further discussion). Furthermore, the inhibitory
effect of exogenous somatostatin was confirmed. It must be concluded,
therefore, that under the prevailing experimental conditions, local
somatostatin secretion exerts a marked restraint of the secretory
activity of the L cells, the endocrine cells known to be the source of
the glucagon-like peptides. This raises the question of the cellular
source of somatostatin involved. In previous studies, we found that the
density of endocrine epithelial somatostatin-secreting cells is lower
in the ileum compared with the more proximal segments of the gut (41).
On the other hand, the ileal mucosa harbors a dense network of
somatostatin-containing nerve fibers, which seem to originate in the
nerve cell bodies of the submucous plexus (41). The predominant
secretory product of the endocrine somatostatin-producing cells is
thought to be SS28 (corresponding to the COOH-terminal 28 amino acids
of prosomatostatin) (2, 12), whereas the main product of the intestinal
neurons is SS14 (the COOH-terminal 14 amino acids of prosomatostatin) (37). In the present study, the chromatographic analysis of ileal
effluent revealed that almost all of the immunoreactivity released in
the basal state corresponded to SS28. Together, these findings indicate
that the source of somatostatin interacting with the L cells in the
basal state is the endocrine cells, whereas somatostatin released
during nerve stimulation or neuromedin C infusion corresponds to SS14,
and therefore presumably is released from the enteric neurons. In
addition, our experiments showed that SS28 was much more efficacious
and potent as an inhibitor of GLP secretion than SS14. We conclude,
therefore, that the local somatostatinergic control of GLP secretion is
exerted by paracrine cells of the mucosa releasing mainly SS28, which
is likely to interact with sstr-5 receptors on the L cells, because
sstr-5 is the only somatostatin receptor with a preference for SS28
(33). Brubaker et al. (5) reported that GLP-1 was capable of releasing both SS14 and SS28 from rat intestinal cultures. In preliminary experiments using the experimental model of the present study, in which
the morphological integrity of the tissue and the paracrine relationships are preserved, we found a dose-related release of somatostatin upon intravascular infusions with GLP-1, with
10
9 mol/l being the most effective
concentration (unpublished studies). This suggests that
GLP-1 and somatostatin secretion may be mutually interdependent in a
paracrine relationship, whereby GLP-1 limits its own secretion by
activating a SS28-mediated paracrine inhibition.
The possible influence of SS14 released from enteric neurons on GLP
secretion was investigated in a series of nerve stimulation experiments. Electrical stimulation of the mixed extrinsic nerves innervating the ileal segment elicited vasoconstriction in the segment,
attesting to the efficiency of stimulation. However, this had little
effect on the secretion of the GLPs or somatostatin, although a
tendency to inhibition was noted. This is in agreement with the results
of yet unpublished studies, in which intra-arterial norepinephrine
inhibited GLP release (Mineo and Holst, unpublished studies). In the
present experiments, addition of the
-adrenergic blocker
phentolamine reduced the hypertensive response as expected (36), but
now nerve stimulation strongly stimulated somatostatin release. This
suggests that the intestinal somatostatinergic neurons are innervated
by inhibitory, noradrenergic fibers as well as strongly stimulatory
fibers. Acetylcholine alone had no effect on somatostatin release and
is therefore unlikely to act as the stimulatory transmitter; vasoactive
intestinal polypeptide, which is released from intrinsic ileal neurons
in the pig under similar conditions (25), might be responsible. GLP
secretion was slightly augmented by nerve stimulation during
-adrenergic blockade. In addition, acetylcholine weakly but
significantly stimulated secretion, in agreement with previous
investigations performed in vivo and in other species, suggesting that
acetylcholine may stimulate L cell secretion (16, 34, 44). Thus it
appears that cholinergic mechanisms may lead to a weak stimulation of
GLP-1 secretion.
We then hypothesized that the dramatic release of somatostatin that was
observed during
-blockade might dampen the secretory response of the
L cells. To test this hypothesis, nerve stimulation was repeated during
somatostatin immunoneutralization, with a technique previously
demonstrated to bring about a lasting and virtually complete
elimination of the actions of exogenous as well as endogenous
somatostatin (20). However, under these conditions (which included
-adrenergic blockade), nerve stimulation had no significant effects.
Our immediate conclusion would be that somatostatin, released during
nerve stimulation, had no influence on the GLP response to nerve
stimulation. A similar conclusion was reached from the experiments
involving the neurotransmitter neuromedin C. Neuromedin C, the
COOH-terminal active fragment of mammalian bombesin or GRP, has been
shown to provide a strong stimulus for GLP secretion (29), as also
confirmed here. Neuromedin C, however, also stimulated somatostatin
secretion, almost as much as nerve stimulation under phentolamine
infusion. The GLP response to neuromedin C was preserved during
somatostatin immunoneutralization. The fractional increase was much
smaller than before antibody infusion, but the absolute amount
secreted, and also the amount above that elicited by the
immunoneutralization, was larger. Similar results were obtained with
acetylcholine. These results would, again, be interpreted to indicate
that somatostatin released in response to nerve stimulation had only a
minor influence on nerve- or neurotransmitter-stimulated GLP-1
secretion. This notion is supported by the finding of a weak effect of
SS14, the molecular form released during nerve stimulation, and by the
finding that only SS28, but not SS14, could inhibit neuromedin
C-stimulated GLP secretion.
From our experiments, we conclude that paracrine SS28 is a main
regulator of GLP secretion and that the extrinsic innervation of the
gut exerts a weak, presumably cholinergic stimulatory effect on GLP
secretion that is independent of somatostatinergic intestinal neurons.
GRP (or neuromedin C) is much more efficacious than acetylcholine, raising the possibility that GRP-producing neurons could be involved in
a stimulatory secretory control of GLP secretion. However, in nerve
stimulation experiments (Orskov C, Knuhtsen S, and Holst JJ,
unpublished studies), we have never been able to measure a co-release
of GRP and GLPs, which seems to preclude an association between the
two. With respect to somatostatin, we were unable to identify
mechanisms that regulate the paracrine somatostatinergic cells
restraining GLP secretion (except for the potential paracrine actions
of GLP-1 itself). The epithelial somatostatin cells seem to function
independently of the extrinsic innervation. Our results show that
adrenergic mechanisms strongly inhibit somatostatin secretion but also
seem to inhibit GLP secretion, so that diffuse activity in the
extrinsic noradrenergic neurons does not cause a stimulated release of
GLPs, despite lifting of the somatostatin restraint.
We were concerned about the discrepancy between the amounts of GLP-1
and GLP-2 secreted during somatostatin immunoneutralization; therefore,
we performed a chromatographic analysis, which was facilitated by the
large amount of immunoreactive material released. Of the
glucagon-containing peptides, glicentin predominated; amidated GLP-1
[GLP-1-(7
36)amide] and glycine-extended GLP-1
[GLP-1-(7
37)] contributed about equally to the total
amount of GLP-1 secreted, and GLP-2 occurred predominantly as a single
component, identified equally well by side viewing and
NH2-terminal assays. Together the two forms of GLP-1
approximately equaled the amount of GLP-2, in agreement with the
concept that the two are secreted in equimolar amounts. However, the
large contribution of GLP-1-(7
37) was unexpected and distinguishes
pigs from humans, in whom the amidated form predominates (31). Pigs
seem to resemble rats in this respect (26). This observation
illustrates the difficulties involved in determining secretion of GLP-1
in vivo, where side-viewing assays, measuring "total" GLP-1,
cannot be used because of pancreatic secretion of GLP-1-containing
molecules that are picked up by such assays (30). In addition, both
NH2-terminally modified and COOH-terminally modified
molecular forms are circulating (8, 9), precluding the use of single
antibody or sandwich approaches for detection of the total secretory
rate of GLP-1. The rate of GLP-2 secretion, estimated using an
NH2-terminal assay, may thus provide the best estimate of L
cell activity.
 |
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 and other correspondence: J. J. Holst,
Dept. of Medical Physiology, University of Copenhagen, the Panum
Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark (E-mail:
holst{at}mfi.ku.dk).
Received 5 April 1999; accepted in final form 8 December 1999.
 |
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