Somatostatin-28 regulates GLP-1 secretion via somatostatin
receptor subtype 5 in rat intestinal cultures
Connie
Chisholm and
Gordon R.
Greenberg
Department of Medicine and Physiology, University of
Toronto, Toronto, Ontario, Canada M5G 1X5
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ABSTRACT |
Five somatostatin receptors (SSTRs)
bind somatostatin-14 (S-14) and somatostatin-28 (S-28), but SSTR5 has
the highest affinity for S-28. To determine whether S-28 acting through
SSTR5 mediates inhibition of glucagon-like peptide-1 (GLP-1), fetal rat
intestinal cell cultures were treated with somatostatin analogs with
relatively high specificity for SSTRs 2-5. S-28 dose-dependently
inhibited GLP-1 secretion stimulated by gastrin-releasing peptide more
potently than S-14 (EC50 0.01 vs. 5.8 nM). GLP-1 secretion
was inhibited by an SSTR5 analog, BIM-23268, more potently than S-14
and nearly as effectively as S-28. The SSTR5 analog L-372,588 also
suppressed GLP-1 secretion equivalent to S-28, but a structurally
similar peptide, L-362,855 (Tyr to Phe at position 7), was ineffective. An SSTR2-selective analog was less effective than S-28, and an SSTR3
analog was inactive. Separate treatment with
GLP-1-(7-36)-NH2 increased S-28 and S-14 secretion by
three- and fivefold; BIM-23268 abolished S-28 without altering S-14,
whereas the SSTR2 analog was inactive. The results indicate that
somatostatin regulation of GLP-1 secretion occurs via S-28 through
activation of SSTR5. GLP-1-stimulated S-28 secretion is also
autoregulated by SSTR5 activation, suggesting a feedback loop between
GLP-1 and S-28 modulated by SSTR5.
gastrin-releasing peptide; somatostatin analogs; phorbol
12-myristate 13-acetate; protein kinase C; protein kinase A
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INTRODUCTION |
GLUCAGON-LIKE
PEPTIDE-1 (GLP-1) is an intestinal hormone released after
nutrients from L cells located in the ileum and colon (24). GLP-1 participates in the ileal brake mechanism
causing inhibition of gastric emptying and proximal intestinal motility (41), inhibits gastric acid secretion (10,
36), and is one of the potent incretin hormones enhancing
glucose-dependent insulin secretion (7, 18). Thus GLP-1
plays an important role in the absorption and assimilation of
nutrients. Several factors have been implicated in the stimulation of
GLP-1 secretion including direct effects of nutrients, predominantly
glucose and fat (15, 32); neuropeptides, including
gastrin-releasing peptide (GRP) (8); and in rodents,
circulating hormones, such as glucose-dependent insulinotropic
peptide (29). Much less is known about the
mechanisms involved in counterregulation of GLP-1 secretion.
Somatostatin has been reported to inhibit GLP-1 secretion in certain
species (14, 20, 21). However, within the gastrointestinal
tract, differential posttranslational processing leads to two principle bioactive molecular forms of somatostatin: somatostatin-14 (S-14) and
somatostatin-28 (S-28) (37). S-14 is localized to the
foregut and enteric nervous system, whereas S-28 is found
predominantly in the mucosa of the ileum and colon (28),
similar to GLP-1. The mechanisms regulating S-28 secretion (2,
12, 13) and certain of its inhibitory actions (9,
42) are distinct from S-14, suggesting that the physiological
roles of the two peptides are different. The biological effects of S-14
and S-28 are mediated by five G protein-coupled receptors
[somatostatin receptor (SSTR) subtype 1 to SSTR5], expressed in most
tissues including the gastrointestinal tract (17).
Although S-28 and S-14 bind with similar affinity to SSTR1 to SSTR4,
SSTR5 is characterized by its preferential affinity for S-28 (22,
23, 27). The recent availability of agonists showing relatively
selective affinities for the SSTRs have provided new insights into the
somatostatin receptor subtypes regulating the secretion of certain
hormones (11, 38, 39) and digestive functions
(5, 11, 35, 43).
The proximity of D cells producing S-28 and L cells containing GLP-1 in
the ileum suggests that S-28 acting through SSTR5 may participate in
the direct regulation of GLP-1 secretion. Furthermore, because GLP-1
stimulates S-28 and S-14 release (1), a negative feedback
may be present whereby GLP-1 regulates its own secretion by activating
S-28 release. In the present study, a rat intestinal cell culture
system that secretes proglucagon-derived peptides (16) was
used to examine the roles of S-28, S-14, and SSTRs in the regulation of
GLP-1 secretion.
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MATERIALS AND METHODS |
Cell isolation and culture.
Fetal rat intestinal cells were placed into monolayer cultures as
described in detail previously (2). In brief, small and large intestines from 19- to 21-day-old fetal Wistar rats were dissected free of gastric and pancreatic tissues, and the cells were
dispersed by incubation with collagenase (blend type H; Sigma Chemical,
St. Louis, MO), hyaluronidase (type II), and deoxyribonuclease-1 (Sigma Chemical) and placed into monolayer culture for 24 h at a density of 0.625 fetal rat intestines/60-mm dish. Cells were then
washed with Hanks' balanced salt solution and incubated with test
agents for 2 h in DMEM containing 0.5% (vol/vol) FBS, 1 g/l glucose, 20 µU/ml insulin, 50 IU/ml penicillin, 50 µg/ml
streptomycin, 10 µM diprotinin A, 10 µM amastatin, and 1 µM
phosphoramidon. Groups of two dishes were used for all experiments
except for the gel permeation chromatography studies in which groups of
10 dishes were used. The secretion experiments were repeated four to
six times, and separate gel chromatography studies were performed in
triplicate. After the incubation period, medium samples were centrifuged to remove any floating cells and made to 0.1% (vol/vol) trifluoroacetic acid. Cells were homogenized in 1 N HCl containing 5%
(vol/vol) HCOOH, 1% (vol/vol) trifluoroacetic acid, and 1% (wt/vol)
NaCl. The peptides contained in the media and cell samples were then
collected separately by passage through a cartridge of C18
silica (Sep-Pak; Waters Associates, Milford, MA), which affords a
>95% recovery of exogenously added peptides, as reported previously
(2). Aliquots of each extract were dried in vacuo, and the
samples were stored at
20°C for subsequent RIA determinations. Animal protocols were approved by the University of Toronto animal care
committee according to Canadian Counsel on Animal Care standards.
Somatostatin analogs and test agents.
The analogs NC8-12, BIM-23268, and BIM-23058 were gifts from Dr.
J. E. Taylor (Biomeasure, Milford, MA). The analog L-362,855 and
two structurally related compounds L-372,587 and L-372,588 were gifts
from Dr. R. M. Freidinger (Merck Research Laboratories, West
Point, PA). The chemical and pharmacological characteristics of the
analogs are shown in Table 1. The analogs
were dissolved in distilled water at 1 mM, lyophilized, and stored at
20°C until they were used. Gastrin-releasing peptide (GRP),
GLP-1-(7-36)-NH2, and S-28 were obtained from Bachem
(Torrance, CA), and S-14 was obtained from Peninsula Laboratories
(Belmont, CA). Phorbol 12-myristate 13-acetate (PMA), forskolin, IBMX,
diprotinin A, amastatin, and phosphoramidon were obtained from Sigma
Chemical.
RIA and chromatography.
Immunoreactive GLP-1 was measured by RIA using antiserum RA 7168 (Peninsula), as described in detail previously (10). The reactivity of the antiserum was 100% for GLP-1-(7-36)-amide, 42% for
GLP-(1-36)-amide, 0.4% for GLP-1-(7-37), and <0.2% for
GLP-1-(1-37), GLP-2, and other members of the glucagon-secretin group
of peptides. Gel permeation chromatography of sample aliquots has
previously indicated that the RIA detects a single peak corresponding
to the position of synthetic GLP-1-(7-36)-amide (10). The
detection limit of the assay is 0.4 fmol/tube or 2.0 fmol/ml, and the
sensitivity (IC50) is 10.0 fmol/tube or 50.0 fmol/ml.
Somatostatin-like immunoreactivity (SLI) was determined by RIA,
as described previously (12), using an antiserum that
detects both S-14 and S-28 with equal affinity. The detection limit of
the assay is 0.3 fmol/tube or 1.5 fmol/ml, and the sensitivity
(IC50) is 9.5 fmol/tube or 47.5 fmol/ml. Gel permeation
chromatography of dried sample aliquots (100 fmol/sample) was performed
using a 9 × 1,000-mm Sephadex G-50 superfine column, as described
previously (12). Columns were calibrated with dextran blue
(void volume), cytochrome c (mol wt 12,384), synthetic S-28, synthetic S-14, and Na125I (total volume). Elution was
carried out at 6 ml/h and 4°C with 125 mM
NH4HCO3, pH 9.0, containing 100 mM NaCl and
0.1% (wt/vol) BSA. Synthetic S-28 and S-14 elute at 53 ml
[coefficient of distribution (Kav) = 0.68)] and 67 ml (Kav = 1.02),
respectively, under these conditions, and recovery exceeds 95%. The
recovery of experimental SLI added to the column was 94 ± 2%. In
the present study in control medium, S-28 and S-14 levels per 10 dishes
were 26 ± 5 and 21 ± 4 fmol, respectively; in control
cells, S-28 and S-14 levels per 10 dishes were 549 ± 112 and
508 ± 103 fmol, respectively (n = 6).
Data and statistical analysis.
Secretion of GLP-1 was determined as a percentage of the total cell
content (TCC) of GLP-1 (100× medium GLP-1/medium plus cellular
peptide) at the end of the incubation period. Synthesis of GLP-1,
determined as a function of the total GLP-1 content of media and cells,
did not change under all test conditions used in the present study,
consistent with previous findings (16). GLP-1 suppression
by each analog was calculated as a percentage of the control GLP-1
secretion (GRP, PMA, or forskolin alone) above basal, in the same
experiment. In the gel chromatography studies, the release of S-28 and
S-14 was determined as the sum of SLI in each fraction under the
respective peaks. Statistical comparisons of means within a group were
evaluated by Student's paired t-test, and differences
between groups were tested by ANOVA followed by a multiple comparisons
test using Sigma-Stat (Jandel Scientific, San Rafael, CA). Results are
expressed as means ± SE; P
0.05 was considered significant.
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RESULTS |
Effects of S-14, S-28, and analogs on GLP-1 secretion.
As shown in Fig. 1, treatment of
intestinal cell cultures for 2 h with GRP (100 nM) stimulated
GLP-1 to 225 ± 4% of paired basal control values
(P < 0.001). S-28 and S-14 caused
concentration-dependent inhibition of GRP-stimulated GLP-1 secretion;
however, S-28 was more potent compared with S-14 (P < 0.001). The half-maximal effective concentrations (EC50)
for S-28 and S-14 were 0.01 and 5.8 nM, respectively, and maximal
inhibition to basal values occurred at 1 nM S-28 and 1 µM S-14.

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Fig. 1.
Secretion of glucagon-like peptide-1 (GLP-1) in the basal
state and in response to treatment with 100 nM gastrin-releasing
peptide (GRP) alone and with different concentrations of
somatostatin-14 (S-14) or somatostatin-28 (S-28). Cells were incubated
with test agents for 2 h, after which peptides were extracted
separately from cells and cell media. Secretion is expressed as a
percentage of total cell content. Results are means ± SE of 6 experiments. * P < 0.05, ** P < 0.001 compared with GRP alone.
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To determine whether S-28 inhibition of GLP-1 secretion was
preferentially mediated by SSTR5, the effects of two relatively specific SSTR5-preferring analogs BIM-23268 and L-362,855 were examined. The octapeptide BIM-23268 caused concentration-dependent inhibition of GRP-stimulated GLP-1 secretion with maximal inhibition to
basal values occurring at 10 nM (Fig. 2).
BIM-23268 with an EC50 of 0.09 nM was marginally less
potent than S-28 (P < 0.05) but more effective
compared with S-14 (P < 0.001). In contrast to the
effect of the octapeptide analog, the hexapeptide SSTR5-preferring analog L-362,855 did not alter GLP-1 secretion (Fig. 2). However, the
structurally related peptide L-372,588 (conversion of phenylalanine to
tyrosine at position 7) caused concentration-dependent inhibition of
GLP-1 secretion (EC50 0.03 nM) that was equipotent to S-28, whereas L-372,587 (conversion of phenylalanine to tyrosine at position
2) was inactive at concentrations up to 10 nM (Fig. 2).

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Fig. 2.
GLP-1 response to treatment with 100 nM GRP alone and
with different concentrations of S-14, S-28, and the analogs BIM-23268,
L-372,588, L-362,855, and L-372,587. GLP-1 inhibition to each analog is
expressed as a percentage of the GLP-1 response to GRP alone above
basal and is the mean ± SE of 4-6 experiments.
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To determine the specificity of SSTR5 in the regulation of GLP-1
secretion, the effects of SSTR2- and SSTR3-preferring analogs were
studied. The SSTR2 analog NC8-12 caused concentration-dependent inhibition of GRP-stimulated GLP-1 secretion (EC50 3.1 nM)
more potently than S-14 (P < 0.05) but was less
effective than S-28 (P < 0.001) (Fig.
3). The SSTR3 analog BIM-23058 was
relatively ineffective at concentrations up to 10 nM (Fig. 3).

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Fig. 3.
GLP-1 response to treatment with 100 nM GRP alone and
with different concentrations of S-14, S-28, and the analogs
NC8-12 and BIM-23058. GLP-1 inhibition to each analog is expressed
as a percentage of the GLP-1 response to GRP alone above basal and is
the mean ± SE of 4-6 experiments.
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Because the biological effects of GRP include regulation through
protein kinase C-dependent pathways (19), GLP-1 secretion in response to the phorbol ester PMA was studied. PMA (1 µM)
increased GLP-1 secretion to 15.5 ± 0.9% TCC at 2 h
or by 274 ± 22% of paired basal values (P < 0.001). S-28 (10 nM) inhibited PMA-stimulated GLP-1 to baseline,
whereas S-14 (10 nM) suppressed the GLP-1 response by 72 ± 8%
and was less effective compared with S-28 (P < 0.05) (Fig. 4). Similarly, BIM-23268 (10 nM)
and L-372,588 (10 nM) inhibited PMA-stimulated GLP-1 release to
baseline values, whereas NC8-12 (10 nM) suppressed the GLP-1
response by 76 ± 9% (Fig. 4). NC8-12 was less potent
compared with S-28 (P < 0.05) and the SSTR-5 analogs (both P < 0.05).

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Fig. 4.
Secretion of GLP-1 in the basal state and in response to
treatment with 1 µM phorbol 12-myristate 13-acetate (PMA) alone and
PMA plus S-14, S-28, BIM-23268, L-372,588, and NC8-12. Secretion
is expressed as a percentage of total cell content. Results are
means ± SE of 4-6 experiments. * P < 0.05, ** P < 0.001 compared with PMA alone.
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Activation of protein kinase A-dependent pathways is also a known
stimulus for GLP-1 secretion (16), and somatostatin
inhibits adenylyl cyclase and cAMP formation (26).
Treatment with forskolin (10 µM) and IBMX (10 µM) increased GLP-1
secretion to 36.3 ± 3.4% TCC at 2 h (P < 0.001 compared with paired basal values). S-28 (10 nM) inhibited
forskolin-stimulated GLP-1 secretion by 49 ± 5%
(P < 0.01), whereas S-14 was ineffective (Fig.
5). BIM-23268 (10 nM) and NC8-12 (10 nM) caused similar magnitudes of inhibition by 37 ± 4 and 35 ± 3%, respectively (both P < 0.05 compared with forskolin alone), but L-372,588 was inactive (Fig. 5).

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Fig. 5.
Secretion of GLP-1 in the basal state and in response to
treatment with 10 µM each of forskolin (FSK)/IBMX alone and to
FSK/IBMX plus S-14, S-28, BIM-23268, L-372,588, and NC8-12.
Secretion is expressed as a percentage of total cell content. Results
are means ± SE of 4-6 experiments. * P < 0.05, ** P < 0.01 compared with FSK/IBMX alone.
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Effect of analogs on GLP-1-stimulated S-14 and S-28 secretion.
Previous studies with our model have shown that treatment with
GLP-1-(7-36)-NH2 dose-dependently stimulates elevations of S-14 and S-28 secretion, as the culture system is comprised of a
heterogeneous cell population that includes D cells producing somatostatin (2). To determine whether GLP-1 and S-28
secretion are regulated via a feedback loop through SSTR5, gel
permeation chromatography was used to characterize S-28 and S-14
responses after GLP-1-(7-36)-NH2 treatment with and
without the addition of SSTR analogs. Control media and cells contained
two predominant peaks of somatostatin that coeluted with S-28
(Kav = 0.68) and S-14
(Kav = 1.02) (Fig.
6A). Treatment with
GLP-1-(7-36)-NH2 (1 µM) increased secretion of S-28 from
26 ± 5 to 73 ± 7 fmol/10 dishes or threefold above paired
controls (P < 0.001) and secretion of S-14 from
21 ± 4 to 109 ± 10 fmol/10 dishes or fivefold above paired
controls (P < 0.001) (Fig. 6B), similar to
responses reported previously (1). The proportions of S-28
and S-14 in the cells were not altered from controls. The
SSTR5-preferring analog BIM-23268 suppressed to baseline the S-28
response after GLP-1-(7-36)-NH2, without altering S-14
(Fig. 6C). In contrast, the S-28 and S-14 responses to
GLP-1-(7-36)-NH2 after addition of the SSTR2 analog NC8-12 (S-28, 68 ± 9 fmol/10 dishes; S-14, 114 ± 11 fmol/10 dishes; n = 3) were not different from values
observed after GLP-1-(7-36)-NH2 treatment alone.

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Fig. 6.
Characterization by gel chromatography of SLI peptides
contained in cell media from intestinal cultures treated for 2 h
under control conditions (A; n = 3), with 1 µM GLP-1-(7-36)-NH2 (B; n = 3), or with 1 µM GLP-1-(7-36)-NH2 plus BIM-23268
(C; n = 3). The elution positions of dextran
blue [void volume (Vo)], cytochrome c (CC), synthetic
S-28, and synthetic S-14 are indicated.
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 |
DISCUSSION |
The present study shows that regulation of GLP-1 secretion by
somatostatin occurs via S-28 and primarily through activation of SSTR5.
Although both bioactive somatostatin molecular forms caused
dose-dependent inhibition of GLP-1 secretion from rat intestinal cell
cultures when stimulated by the neurotransmitter GRP, S-28 was markedly
more effective than S-14 with an inhibitory action at an
IC50 that is within the range for receptor binding
(30). Our study further shows that GLP-1-stimulated S-28
secretion is autoregulated by activation of SSTR5. Coupled with the
structural localization in the ileum of D cells producing S-28 in
proximity to L cells secreting GLP-1, these findings suggest the
presence of an interrelationship between S-28 and GLP-1 mediated
through SSTR5.
Recent investigations with the isolated perfused porcine ileum
similarly showed that S-28 is a potent inhibitor of GLP-1 secretion, whereas S-14 is ineffective (14). The concordance of these
findings in an integrated model (14) with our results in a
cell culture system therefore lends further support to the notion of a
relationship between S-28 and GLP-1. However, five somatostatin
receptor subtypes mediate the biological actions of S-28 and S-14
(26). Although all receptor subtypes are expressed in the
gastrointestinal tract (17), SSTR5 is the only subtype
that preferentially binds S-28 (22, 23, 27). Our results
obtained with agonists that provide relatively selective affinities for
different somatostatin receptor subtypes show that activation of SSTR5
modulates GLP-1 suppression by S-28. BIM-23268, an analog with high
selectivity for SSTR5 (6, 38), caused dose-dependent
inhibition of GLP-1 secretion similar to S-28 and more potently than
S-14. The present data also support and extend previous investigations
(4, 25, 40), indicating the importance of single hydroxyl
groups in ligand binding to SSTR5. L-362,855, with two phenylalanine
residues in its structure, is a peptide with relatively high affinity
for rat and human SSTR5 (6, 23, 30), yet did not influence GLP-1 secretion. However, the structurally related analog L-372,588 with the conversion of Phe to Tyr at position 7 caused inhibition equipotent to S-28, whereas L-372,587 with the conversion of Phe to Tyr
at position 2 was ineffective. Similar differences in the agonist
effects of these compounds were shown on inhibition of L-type
Ca2+ channel current conductance in AtT-20 cells and on
inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster
ovary (CHO-K1) cells expressing rat SSTR5
(40). Thus the presence of a hydroxyl group at position 7 markedly enhances the agonist properties of this group of peptides at
SSTR5. Although the SSTR3 analog showed only a weak effect,
NC8-12, an analog that binds to the two isoforms of the SSTR2
receptor with high affinity (35, 43), also caused
inhibition of GLP-1 secretion marginally more potent than S-14 but less
effective than S-28. These results suggest that the two functional SSTR
receptor subtypes SSTR5 and SSTR2 mediate regulation of GLP-1 secretion
by S-28, although activation of SSTR-5 predominates.
GRP potently stimulated GLP-1 release, consistent with results from in
vivo studies (8). Signal transduction pathways modulating the biological effects of GRP include influx of extracellular Ca2+ through L-type voltage-gated channels, elevations of
intracellular Ca2+, and activation of protein kinase
C-dependent pathways (19). Although nitrendipine does not
influence GLP-1 release in our model, PMA, an activator of
diacylglycerol-sensitive protein kinase C isozymes, stimulated GLP-1
secretion to levels comparable to those observed after GRP. The
patterns of suppression after PMA-stimulated GLP-1 secretion by S-28
and the SSTR5 analogs were consistent with the results obtained after
GRP. These results suggest that SSTR5 and SSTR2 couple to protein
kinase C, similar to findings demonstrated for S-28 inhibition of
smooth muscle cells (5). However, our results differ from
observations obtained with SSTR5-transfected cells where activation of
this receptor subtype stimulated phosphoinositide metabolism
(44). These divergent findings may reflect differences between normal and transfected cells where a higher number of receptors
cause more variable coupling to this effector pathway.
Activation of protein kinase A-dependent pathways is also a potent
stimulus for GLP-1 secretion (16), whereas somatostatin inhibits adenylyl cyclase and cAMP formation (26). The
more effective suppression of forskolin-stimulated GLP-1 secretion by
S-28 compared with S-14 accords with the preferential role for S-28 in
the regulation of GLP-1 secretion. The equipotent inhibition by
BIM-23268 and NC8-12 of GLP-1 stimulated by forskolin is
consistent with suppression of adenylyl cyclase described in SSTR5- and
SSTR2-transfected cells (26) and implicates involvement of
both receptor subtypes in the regulation of protein kinase A-dependent
GLP-1 secretion. In contrast to its potent inhibition of PMA-stimulated
GLP-1 secretion, L-372,588 did not alter forskolin-stimulated GLP-1
secretion, suggesting suppression of protein kinase C-dependent pathways may be a relatively selective action of this SSTR5 analog.
That more than one SSTR subtype is expressed in the same cell type and
individually contributes to the functions of somatostatin is well
recognized (38). However, recent studies indicate that in
certain settings there may be the requirement for the combined action
of two SSTRs, as exemplified by the activation of both SSTR5 and SSTR2
for inhibition of platelet-derived growth factor-induced proliferation
via Ras-dependent pathways (3). That two SSTRs may
undergo heterodimerization to alter various functional properties of the receptors, including agonist regulation, may account for this
finding (33). Although we found SSTR5 and SSTR2 coupled individually to protein kinase C and protein kinase A, additional L-cell functions may be modulated by the combined actions of these two
receptor subtypes and requires further study.
Certain lines of evidence point to the presence of a regulatory
feedback loop between L cells and D cells producing S-28 in the ileum.
Studies undertaken with the isolated perfused porcine ileum indicate
that nearly all the somatostatin immunoreactivity secreted in the basal
state is comprised of S-28, and somatostatin immunoneutralization
causes a marked rise of GLP-1 secretion (14). Our results
compliment these findings by showing that GLP-1 potently stimulated
somatostatin release which included elevations of S-28 and S-14, and
coadministration of the SSTR5 agonist BIM-23268 caused preferential
inhibition of S-28. Thus SSTR5 modulates not only inhibition of GLP-1
secretion, but also autoregulates S-28 secretion when stimulated by
GLP-1. Although the culture model employed in this study is comprised
of a heterogeneous cell population that also includes cells secreting
peptide YY and cholecystokinin, neither of these peptides modulates
somatostatin or GLP-1 release when administered to the cultures
(2, 16). Our findings, coupled with in vivo results
(14), therefore suggest the presence of an
interrelationship between ileal L cells and D cells whereby, via
paracrine or endocrine mechanisms, GLP-1 stimulates S-28 secretion, and
both S-28 and GLP-1 release are restrained through activation of SSTR5.
GLP-1 is a potent stimulus of glucose-dependent insulin secretion
(7, 18), and inhibition of insulin secretion by
somatostatin is also mediated by S-28 through SSTR5. Studies of
glucose-stimulated insulin secretion in vitro with pancreatic islets
and in vivo demonstrated that SSTR5-selective analogs decreased insulin
secretion, whereas SSTR2 analogs were ineffective (9, 39).
These observations together with our present results on GLP-1 secretion
lend further support to the idea that S-28 via SSTR5 plays an important
role as a decretin in the counterregulation of the enteroinsular axis.
In conclusion, our studies indicate that somatostatin inhibition of
GLP-1 secretion is mediated by S-28 mainly through activation of SSTR5,
with a lesser effect by SSTR2. Both receptor subtypes modulate GLP-1
responses to activation of protein kinase C- and protein kinase
A-dependent pathways. SSTR5 also autoregulates S-28 secretion when
stimulated by GLP-1-(7-36)-NH2. Single hydroxyl group substitutions modify the agonist properties of SSTR5 analogs. The findings suggest the presence of an interrelationship between L and D cells in the ileum whereby regulation of GLP-1 and S-28 secretion is modulated through SSTR-5.
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ACKNOWLEDGEMENTS |
This work was supported in part by Medical Research Council of
Canada Grant MA-6763.
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FOOTNOTES |
Address for reprint requests and other correspondence:
G. R. Greenberg, Room 445, 600 University Ave., Mt.
Sinai Hospital, Toronto, Ontario, Canada M5G 1X5 (E-mail:
ggreenberg{at}mtsinai.on.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 9, 2002;10.1152/ajpendo.00434.2001
Received 27 September 2001; accepted in final form 4 April 2002.
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REFERENCES |
1.
Brubaker, PL,
Efendic S,
and
Greenberg GR.
Truncated and full-length glucagon-like peptide-1 (GLP-1) differentially stimulate intestinal somatostatin release.
Endocrine
6:
91-95,
1997[ISI][Medline].
2.
Brubaker, PL,
Groneau KA,
Asa SL,
and
Greenberg GR.
Nutrient and peptide regulation of somatostatin-28 secretion from intestinal cultures.
Endocrinology
139:
148-155,
1998[Abstract/Free Full Text].
3.
Cattaneo, MG,
Taylor JE,
Culler MD,
Nisoli E,
and
Vicentini LM.
Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells.
FEBS Lett
481:
271-276,
2000[ISI][Medline].
4.
Chisholm, C,
and
Greenberg GR.
Somatostatin receptor subtype 5 mediates inhibition of peptide YY secretion from rat intestinal cultures.
Am J Physiol Gastrointest Liver Physiol
279:
G983-G989,
2000[Abstract/Free Full Text].
5.
Corletto, VD,
Severi C,
Coy DH,
Delle Fave G,
and
Jensen RT.
Colonic smooth muscle cells possess a different subtype of somatostatin receptor from gastric smooth cells.
Am J Physiol Gastrointest Liver Physiol
272:
G689-G697,
1997[Abstract/Free Full Text].
6.
Coy, DH,
and
Taylor JE.
Receptor-specific somatostatin analogs: correlation with biological activity.
Metabolism
45, Suppl 1:
21-23,
1996[ISI][Medline].
7.
D'Alessio, DA,
Kahn SE,
Leusner CR,
and
Ensinck JW.
Glucagon-like peptide 1 enhances glucose tolerance both by stimulation of insulin release and by increasing insulin dependent glucose disposal.
J Clin Invest
93:
2263-2266,
1994[ISI][Medline].
8.
Dumoulin, V,
Dakka T,
Plaisancie P,
Chayvialle J-A,
and
Cuber J-C.
Regulation of glucagon-like peptide-1-(7
36) amide, peptide YY, and neurotensin secretion by neurotransmitters and gut hormones in the isolated vascularly perfused rat ileum.
Endocrinology
136:
5182-5188,
1995[Abstract].
9.
Ensinck, JW,
Vogel RE,
Laschansky EC,
Koerker DJ,
Prigeon RL,
and
Kahn SE.
Endogenous somatostatin-28 modulates postprandial insulin secretion. Immunoneutralization studies in baboons.
J Clin Invest
100:
2295-2302,
1997[Abstract/Free Full Text].
10.
Fung, LC,
Chisholm C,
and
Greenberg GR.
Glucagon-like peptide-1 (7
36) amide and peptide YY mediate intraduodenal fat-induced inhibition of acid secretion in dogs.
Endocrinology
139:
189-194,
1998[Abstract/Free Full Text].
11.
Fung, LC,
and
Greenberg GR.
Characterization of somatostatin receptor subtypes mediating inhibition of nutrient-stimulated gastric acid and gastrin in dogs.
Regul Pept
68:
197-203,
1997[ISI][Medline].
12.
Greenberg, GR.
Differential neural regulation of circulating somatostatin-14 and somatostatin-28 in conscious dogs.
Am J Physiol Gastrointest Liver Physiol
264:
G902-G909,
1993[Abstract/Free Full Text].
13.
Greenberg, GR,
Fung L,
and
Pokol-Daniel S.
Regulation of somatostatin-14 and -28 secretion by gastric acid in dogs: differential role of cholecystokinin.
Gastroenterology
105:
1387-1395,
1993[ISI][Medline].
14.
Hansen, L,
Hartmann B,
Bisgaard T,
Mineo H,
Jørgensen PN,
and
Holst JJ.
Somatostatin restrains the secretion of glucagon-like peptide-1 and -2 from isolated perfused porcine ileum.
Am J Physiol Endocrinol Metab
278:
E1010-E1018,
2000[Abstract/Free Full Text].
15.
Herrmann, C,
Göke R,
Richter G,
Fehmann H-C,
Arnold R,
and
Göke B.
Glucagon-like peptide-1 and glucose-dependent insulin-releasing polypeptide plasma levels in response to nutrients.
Digestion
56:
117-126,
1995[ISI][Medline].
16.
Huang, THJ,
and
Brubaker PL.
Synthesis and secretion of glucagon-like peptide-1 by fetal rat intestinal cell in culture.
Endocrine
3:
499-503,
1995[ISI].
17.
Kremplels, K,
Hunyady B,
O'Carroll AM,
and
Mezey E.
Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract.
Gastroenterology
112:
1948-1960,
1997[ISI][Medline].
18.
Kreymann, B,
Williams G,
Ghatei MA,
and
Bloom SR.
Glucagon-like peptide-1 7
36: a physiological incretin in man.
Lancet
2:
1300-1304,
1987[ISI][Medline].
19.
Kroong, GS,
Jensen RT,
and
Battey JF.
Mammalian bombesin receptors.
Med Res Rev
15:
389-417,
1995[ISI][Medline].
20.
Marco, J,
Hedo JA,
and
Villaneuva ML.
Inhibition of intestinal glucagon-like immunoreactivity (GLI) secretion by somatostatin in man.
J Clin Endocrinol Metab
44:
695-698,
1977[Abstract].
21.
Martin, PA,
and
Faulkner A.
Effects of somatostatin-28 on circulating concentrations of insulin and gut hormones in sheep.
J Endocrinol
151:
107-112,
1996[Abstract].
22.
O'Carroll, AM,
Lolait S,
Konig M,
and
Mahan L.
Molecular cloning and expression of a pituitary receptor with preferential affinity for somatostatin-28.
Mol Pharmacol
42:
939-946,
1992[Abstract].
23.
O'Carroll, AM,
Raynor K,
Lolait SJ,
and
Reisine T.
Characterization of cloned human somatostatin receptor SSTR 5.
Mol Pharmacol
48:
291-298,
1994.
24.
Ørskov, C,
Holst JJ,
Knuhtsen S,
Baldissera FG,
Poulsen SS,
and
Nielson OV.
Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene are secreted separately from the pig small intestine but not pancreas.
Endocrinology
119:
1467-1475,
1986[Abstract].
25.
Ozenberger, BA,
and
Hadcock JR.
A single amino acid substitution in somatostatin receptor subtype 5 increases affinity for somatostatin-14.
Mol Pharmacol
47:
82-87,
1995[Abstract].
26.
Patel, YC.
Somatostatin and its receptor family.
Front Neuroendocrinol
20:
157-198,
1999[ISI][Medline].
27.
Patel, YC,
and
Srikant CB.
Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hsstr1-5).
Endocrinology
135:
2814-2817,
1994[Abstract].
28.
Penman, E,
Wass JAH,
Butler MG,
Penny ES,
Price J,
Wu P,
and
Rees LH.
Distribution and characterization of immunoreactive somatostatin in human gastrointestinal tract.
Regul Pept
7:
53-65,
1983[ISI][Medline].
29.
Raynor, K,
Murphy WA,
Coy DH,
Taylor JE,
Moreau J-P,
Yasuda K,
Bell GI,
and
Reisine T.
Cloned somatostatin receptors: identification of subtype-selective peptides and demonstration of high affinity binding of linear peptides.
Mol Pharmacol
43:
838-844,
1993[Abstract].
30.
Raynor, K,
O'Carroll AM,
Kong H,
Yasuda K,
Mahan LC,
Bell GI,
and
Reisine T.
Characterization of cloned somatostatin receptors SSTR4 and SSTR5.
Mol Pharmacol
44:
385-392,
1993[Abstract].
31.
Roberge, JN,
and
Brubaker PL.
Regulation of intestinal proglucagon-derived peptide secretion by glucose-dependent insulinotropic peptide in a novel enteroendocrine loop.
Endocrinology
133:
233-240,
1993[Abstract].
32.
Rocca, AS,
and
Brubaker PL.
Stereospecific effects of fatty acids on proglucagon-derived peptide secretion in fetal rat intestinal cultures.
Endocrinology
136:
5593-5599,
1995[Abstract].
33.
Rocheville, M,
Lange DC,
Kumar U,
Sasi R,
Patel RC,
and
Patel YC.
Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers.
J Biol Chem
275:
7862-7869,
2000[Abstract/Free Full Text].
34.
Rossowski, WJ,
and
Coy DH.
Specific inhibition of rat pancreatic insulin or glucagon release by receptor-selective somatostatin analogs.
Biochem Biophys Res Commun
205:
341-346,
1994[ISI][Medline].
35.
Rossowski, WJ,
Gu ZF,
Akarca US,
Jensen RT,
and
Coy DH.
Characterization of somatostatin receptor subtypes controlling rat gastric acid and pancreatic amylase release.
Peptides
15:
1421-1424,
1994[ISI][Medline].
36.
Schjoldager, BGT,
Mortensen PE,
Christiansen J,
Ørskov C,
and
Holst JJ.
GLP-1 (glucagon-like peptide-1) and truncated GLP-1 fragments of human proglucagon inhibit gastric acid secretion in humans.
Dig Dis Sci
35:
703-708,
1989.
37.
Sevarino, K,
Felix R,
Banks C,
Low M,
Montminy M,
Mandel G,
and
Goodman R.
Cell-specific processing of preprosomatostatin in cultured neuroendocrine cells.
J Biol Chem
262:
4987-4993,
1987[Abstract/Free Full Text].
38.
Shimon, I,
Taylor JE,
Dong JZ,
Bitonte RA,
Kim S,
Morgan B,
Coy DH,
Culler MD,
and
Melmed S.
Somatostatin receptor subtype specificity in human fetal pituatary cultures. Differential role of SSTR2 and SSTR5 for growth hormone, thyroid-stimulating hormone, and prolactin regulation.
J Clin Invest
99:
789-798,
1997[Abstract/Free Full Text].
39.
Strowski, MZ,
Parmer RM,
Blake AD,
and
Schaeffer JM.
Somatostatin inhibits insulin and glucagon secretion via two receptor subtypes: an in vivo study of pancreatic islets from somatostatin receptor 2 knockout mice.
Endocrinology
141:
111-117,
2000[Abstract/Free Full Text].
40.
Tallent, M,
Liapakis G,
O'Carroll AM,
Lolait SJ,
Dichter M,
and
Reisine T.
Somatostatin receptor subtypes SSTR2 and SSTR5 couple negatively to an L-type Ca2+ current in the pituatary cell line AtT-20.
Neuroscience
71:
1073-1081,
1996[ISI][Medline].
41.
Tolessa, T,
Gutniak M,
Holst JJ,
Efendic S,
and
Hellstrom PM.
Inhibitory effect of glucagon-like peptide-1 on small bowel motility. Fasting but not fed motility via nitric oxide independently of insulin and somatostatin.
J Clin Invest
102:
764-774,
1998[Abstract/Free Full Text].
42.
Wang, H,
Bogen C,
Reisine T,
and
Dichter M.
SRIF-14 and SRIF-28 induce opposite effects on potassium currents in rat neocortical neurons.
Proc Natl Acad Sci USA
86:
9616-9620,
1989[Abstract].
43.
Warhurst, G,
Higgs NB,
Fakhoury H,
Warhurst AC,
Giarde J,
and
Coy DH.
Somatostatin receptor subtype 2 mediates somatostatin inhibition of ion secretion in rat distal colon.
Gastroenterology
111:
325-333,
1996[ISI][Medline].
44.
Wilkinson, GF,
Feniuk W,
and
Humphrey PPA
Characterization of human recombinant somatostatin sst5 receptors mediating activation of phosphoinositide metabolism.
Br J Pharmacol
121:
91-96,
1997[Abstract].
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