help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brubaker, P. L.
Right arrow Articles by Greenberg, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brubaker, P. L.
Right arrow Articles by Greenberg, G. R.
Endocrinology Vol. 139, No. 1 148-155
Copyright © 1998 by The Endocrine Society

ARTICLES

Nutrient and Peptide Regulation of Somatostatin-28 Secretion from Intestinal Cultures1

Patricia L. Brubaker, Karen A. Gronau, Sylvia L. Asa and Gordon R. Greenberg

Departments of Physiology (P.L.B., G.R.G.), Medicine (P.L.B., G.R.G.), and Pathology (S.L.A.), University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Dr. P. Brubaker, Room 3366, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: p.brubaker{at}utoronto.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Of the two known forms of intestinal somatostatin, somatostatin-28 (S28) and S14, S28 predominates in the distal mucosa, whereas S14 is localized in the foregut. Although S14 release has been well studied, little is known about the factors regulating secretion of S28 from the intestine. Therefore, fetal rat intestinal cultures, which have been previously demonstrated to synthesize and secrete predominantly S28, were treated with potential nutrient, neuromodulator/transmitter, and peptide secretagogues (n = 4–6/experiment). Oleic acid dose dependently stimulated the release of somatostatin-like immunoreactivity (SLI) to 272 ± 81% of the control value at 1.5 x 10-4 M (P < 0.01). Gel permeation analysis (n = 3) demonstrated that this increment was accounted for not only by an increase in the release of S28, but also by an increase in that of S14, such that the secretion of both peptides was increased in parallel. Of the neuromodulators tested, only the enteric peptide gastrin-releasing peptide stimulated intestinal SLI secretion, to 386 ± 60% of the control value at 10-6 M (P < 0.001); similar to oleic acid, the effects on S28 and S14 were equivalent. Galanin, vasoactive intestinal peptide, bethanechol, and epinephrine did not affect SLI release. The duodenal hormone secretin also stimulated SLI release to 310 ± 78% of the control value at 10-6 M (P < 0.001); however, secretin caused a preferential release of S14 over that of S28 (S14, 7.8 ± 2.8-fold; S28, 1.5 ± 0.1-fold). In contrast, gastrin, cholecystokinin, glucose-dependent insulinotropic peptide, neurotensin, peptide YY, epidermal growth factor, and transforming growth factor-{alpha} had no effect on intestinal SLI release. Thus, luminal nutrients and neuro/endocrine peptides exert differential effects on S28 release from the rat intestine compared with those on S14. These findings implicate S28 as a distinct regulatory peptide in the physiological setting.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH widely distributed in the brain, pancreas, and peripheral autonomic nervous system, the gastrointestinal tract provides the largest source of somatostatin in the periphery (1, 2, 3). Within the gastrointestinal tract, differential posttranslational processing of prosomatostatin generates two major biologically active peptides, somatostatin-14 (S14) and S28, the distribution of which forms a gradient along the aboral axis. In the mucosa of the stomach and duodenum, S14 is the major product of processing, whereas S28 predominates in the D cells of the distal jejunum and ileum (1, 2, 3, 4). S14 is also distributed throughout the length of the small intestine within the myenteric plexi. Both peptides have potent inhibitory effects on digestive functions, including gastric and pancreatic exocrine secretion, neuro/endocrine peptide release, and intestinal growth (5). Of particular note are studies suggesting that S28, rather than S14, may be the physiological regulator of postprandial insulin secretion in humans (6, 7). Although regulation of S14 secretion from the stomach has been studied extensively using in vivo (8, 9, 10, 11, 12) and in vitro (13, 14, 15) models, there has been a paucity of studies directed toward understanding the regulation of S28 secretion. Such studies have largely been conducted using in vivo models and have relied heavily upon measurement of the circulating levels of somatostatin-like immunoreactivity (SLI) (9, 10, 11, 12, 16, 17). Independent mechanisms modulating the release of the two forms of somatostatin have been suggested by studies demonstrating differential neural regulation of S28 and S14 in the dog (9, 11). However, data derived from in vivo models preclude definitive assessment of those factors (nutrients, peptide secretagogues, and neurotransmitters) that have direct effects at the cellular level. To overcome this difficulty, we developed an intestinal culture system to examine S28 release from intestinal D cells (18, 19, 20). Our initial investigations demonstrated that both S28 and S14 are synthesized and secreted by the intestinal cells in culture, but S28 is the predominant form of somatostatin in this model. In the present study, we used this model to examine the cellular regulation of S28 secretion by nutrients as well as by neuro/endocrine regulatory peptides that have been suggested to have actions on intestinal somatostatin release in vivo. Use of this model has also permitted determination of the differential effects of secretagogues on the release of S28 and S14 from the intestine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental protocol
Fetal rat intestinal cells (FRIC) were placed into monolayer culture, as described in detail previously (18, 19, 20). Animal protocols were approved by the University of Toronto animal care committee according to Canadian Council on Animal Care standards. In brief, 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 Co., St. Louis, MO), hyaluronidase (type II), and deoxyribonuclease I (Sigma Chemical Co.) 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/liter glucose, 20 µU/ml insulin, 50 IU/ml penicillin, and 50 µg/ml streptomycin. All test peptides were obtained from Bachem (Torrance CA), sodium oleate was obtained from Sigma, bethanechol was obtained from Merck Frosst Canada (Dorval, Canada), epinephrine was obtained from Allerex Laboratory (Kanata, Canada), and tetrodotoxin was obtained from Sigma. Groups of two dishes were used for all experiments except those for gel permeation chromatography, in which groups of 10 dishes were used. Each litter of fetal rats was processed independently to make n = 1. All secretion experiments were repeated four to six times, whereas separate gel permeation studies were carried out 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), as described previously (18, 19, 20). We have demonstrated that this methodology affords a greater than 95% recovery of intact, exogenously added S14 and S28 (18). Aliquots of each extract were dried in vacuo for RIA or chromatography, and the samples were stored at -20 C.

RIA and chromatography
Total SLI was determined in dried sample aliquots by RIA, as described previously (8, 9, 10, 11, 12, 18, 19, 20), using an antiserum that detects both S14 and S28 with equal affinity. The detection limit of the assay is 0.3 fmol/tube, and the sensitivity (IC50) is 9.5 fmol/tube. Gel permeation chromatography of dried sample aliquots (100 fmol/sample) was performed using a 9 x 1000-mm Sephadex G-50 superfine column, as described previously (8, 9, 10, 11, 12, 18, 19, 20). Columns were calibrated with dextran blue (void volume), cytochrome c (mol wt, 12,384), synthetic S28 (Bachem CA), synthetic S14 (Peninsula Laboratories, Belmont CA), 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 S28 and S14 elute at 53 (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% (n = 24). In control medium, S28 and S14 levels per 10 dishes were 69 ± 25 and 32 ± 6 fmol, respectively; in control cells, S28 and S14 levels per 10 dishes were 975 ± 204 and 983 ± 196 fmol, respectively (n = 7).

Immunohistochemistry
Fetal rat intestines or cells cultured on Lab-Tek tissue culture chamber/slides (Miles Scientific Co., Naperville, IL) were fixed in 10% (vol/vol) or 1% (vol/vol) formalin in PBS, respectively, and intestines were sectioned (5-µm thickness) after paraffin embedding. The avidin-biotin-peroxidase technique was used as described previously (21, 22). A prediluted monoclonal antibody against the neural marker neurofilament (Zymed Laboratories, South San Francisco, CA) was incubated for 2 h at room temperature, and the immunological reaction was visualized by detection of peroxidase activity, using H2O2 and 3,3'-diaminobenzidine tetrahydrochloride. Slides were counterstained with hematoxylin. The specificity of staining was verified by replacing the primary antiserum with normal mouse ascites.

Data and statistical analyses
Secretion of SLI was determined as a percentage of the total cell content of SLI [100 x medium SLI/(medium plus cellular SLI)] at the end of the incubation period. The total cell content of SLI was determined to be constant under all test conditions used in the present study (330 ± 49 fmol/dish; n = 48), consistent with previous findings (18, 19, 20). After normalization of data to a percentage of the control data, statistical differences were determined by ANOVA using a general linear model with n-1 custom hypotheses tests on a SAS (Cary, NC) program for IBM computers.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of sodium oleate
As shown in Fig. 1Go, treatment of intestinal cultures with 10-5 to 1.5 x 10-4 M sodium oleate stimulated a dose-dependent increase in SLI secretion (F = 3.84; P < 0.05), reaching 272 ± 81% of control levels at the highest dose of fatty acid tested (1.5 x 10-4 M; P < 0.01; n = 4). The minimal effective dose of sodium oleate was 10-4 M (208 ± 58% of the control value; P < 0.05 vs. controls and P < 0.05 vs. 1.5 x 10-4 M sodium oleate), whereas doses ranging from 10-10-10-6 M did not affect SLI release.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 1. Secretion of SLI peptides in response to treatment with different concentrations of sodium oleate (n = 4). Cells were incubated with the test agents for 2 h, after which peptides were extracted separately from cells and cell media. Secretion is expressed as a percentage of the total cell content. *, P < 0.05; **, P < 0.01.

 
To determine the effects of oleic acid treatment on the release of S28 specifically, media and cell extracts were further examined by gel permeation chromatography after incubation under control conditions or with 10-4 M sodium oleate (Fig. 2Go). Control media and cells contained two peaks of SLI that coeluted with synthetic S28 (Kav = 0.68) and S14 (Kav = 1.02), and a third peak (Kav = 0.15) with a molecular mass of approximately 13,000 daltons. In the media, the three peaks corresponded to 60 ± 8%, 27 ± 3%, and 13 ± 5% of the total SLI, respectively. Similarly, the SLI peaks in control cells accounted for 45 ± 1%, 46 ± 1%, and 9 ± 2% of the total SLI, respectively. Thus, together, the two major forms of SLI, S28 and S14, accounted for approximately 90% of the total intestinal SLI, with a predominance of S28. Treatment of paired culture dishes with 10-4 M sodium oleate increased total SLI release by 2.0 ± 0.4-fold, and this increment was equally distributed across the three SLI molecular forms. The proportions of S28 and S14 in media (63 ± 6 and 30 ± 4 in media, respectively) and cells (49 ± 2 and 44 ± 4, respectively) were not altered from those in control media and cells.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 2. Characterization by gel permeation chromatography of the SLI peptides contained in cells and cell media from intestinal cultures treated for 2 h under control conditions (A; n = 3) or with 10-4 M sodium oleate (B; n = 3). Each experiment used a pool of 10 dishes within a single culture, and all experiments were paired.

 
Effects of neuromodulators/neurotransmitters
Analysis of the effects of peptidergic modulators on SLI release from the cultured cells demonstrated that gastrin-releasing peptide (GRP) stimulated secretion (F = 29.63; P < 0.001) to a maximum value of 386 ± 60% of the control level at 10-6 M (P < 0.001; n = 6; Fig. 3Go). However, neurotransmitters, including bethanechol and epinephrine, as well as the neuropeptides, galanin and VIP, demonstrated insignificant or minor effects on intestinal SLI secretion (Table 1Go).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Secretion of SLI peptides in response to treatment with different concentrations of GRP (n = 6). Cells were incubated with the test agents for 2 h, after which peptides were extracted separately from cells and cell media. Secretion is expressed as a percentage of the total cell content. ***, P < 0.001.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Secretion of SLI by FRIC cultures in response to treatment with different concentrations of neuromodulators/neurotransmitters

 
Gel permeation analysis of media and cells from 10-6 M GRP-treated cultures indicated a 3.8 ± 0.6-fold stimulation of total SLI release (Fig. 4Go). This SLI increment was accounted for by parallel increases in S28 and S14, and the proportions of S28 and S14 were not altered in either the media (56 ± 3% and 32 ± 2%, respectively) or cells (47 ± 2% and 42 ± 1%, respectively) of GRP-treated cultures compared with control values. Chromatographic analyses of media and cells from vasoactive intestinal peptide- and bethanechol-treated cultures further demonstrated that neither S28 nor S14 was altered by the treatment. Thus, the proportions of S28 and S14 were comparable to those in controls (data not shown), thereby precluding an increase in one bioactive form with a parallel decrease in the other form as a factor accounting for the lack of effect of these test agents on total SLI.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. Characterization by gel permeation chromatography of the SLI peptides contained in cells and cell media from intestinal cultures treated for 2 h under control conditions (A; n = 3) or with 10-6 M GRP (B; n = 3). Each experiment used a pool of 10 dishes within a single culture, and all experiments were paired.

 
Effects of hormones and growth factors
To establish the effect of hormones known to circulate in the physiological setting on SLI release, intestinal cultures were treated on separate occasions with peptides arising predominantly from the duodenum/jejunum, at concentrations ranging from 10-12–10-6 M. Secretin stimulated SLI release (F = 8.84; P < 0.001) to 310 ± 78% of the control level at 10-6 M (P < 0.001; n = 6; Fig. 5Go). In contrast, gastrin, cholecystokinin-octapeptide (CCK) and glucose-dependent insulinotropic peptide (GIP) increased SLI release only slightly or not at all (Table 2Go). Similarly, administration of the ileal peptide, neurotensin, or the intestinal growth factors, epidermal growth factor and transforming growth factor-{alpha}, did not influence SLI secretion, whereas the ileal peptide YY (PYY) caused a slight, albeit significant, increase at the highest dose tested (Table 2Go).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Secretion of SLI peptides in response to treatment with different concentrations of secretin (n = 6). Cells were incubated with the test agents for 2 h, after which peptides were extracted separately from cells and cell media. Secretion is expressed as a percentage of the total cell content. ***, P < 0.001.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Secretion of SLI by FRIC cultures in response to treatment with different concentrations of intestinal circulating hormones or growth factors

 
In separate experiments, gel permeation analysis demonstrated that the 3.7 ± 0.7-fold increment in SLI after treatment with 10-6 M secretin was not equally distributed across the three SLI molecular forms. Although S28 was increased only modestly (1.5 ± 0.1-fold), S14 secretion was increased by 7.8 ± 2.3-fold compared with that in paired controls (Fig. 6Go). The proportions of S28 and S14 in the medium after secretin treatment were, therefore, 22 ± 3% and 72 ± 2% of the total SLI, respectively. In contrast, the distribution of these peptides inside the cells was not significantly affected by secretin treatment (45 ± 2% and 47 ± 2%, respectively).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Characterization by gel permeation chromatography of the SLI peptides contained in cells and cell media from intestinal cultures treated for 2 h under control conditions (A; n = 3) or with 10-6 M secretin (B; n = 3). Each experiment used a pool of 10 dishes within a single culture, and all experiments were paired.

 
Confirmation of nonneural origin of intestinal somatostatin
To establish that the S14 produced by the FRIC cultures originated from intestinal endocrine cells and was not neural in origin, the cultures were first examined for the presence of neurons by immunohistochemical staining for neurofilament. This neural marker was undetectable in the cell cultures, whereas parallel analyses of whole fetal rat intestines demonstrated strong staining in the myenteric plexi (Fig. 7Go). Moreover, in separate experiments, SLI secretion stimulated by neuromodulators [GRP and calcitonin gene-related peptide-1 (CGRP-1)], endocrine peptides (secretin and glucagon-like peptide-1 (GLP-1)], and an activator of protein kinase A [(Bu)2cAMP] was not different in the absence (399 ± 156% of controls) or presence (429 ± 118% of controls) of the neural blocker tetrodotoxin (3 x 10-6 M; n = 5).



View larger version (136K):
[in this window]
[in a new window]
  		Figure 7. Immunostaining of cultured intestinal cells (A) or intact fetal rat intestine (B) for neurofilament. The arrowheadsin B indicate staining in neural components of the gut wall. Magnification, x350.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies on the factors regulating secretion of somatostatin from the intestinal D cell have been complicated by the existence of contralateral gradients of S28 and S14 along the aboral axis, as well as by the presence of somatostatin in other, nonmucosal gastrointestinal tissues. To date, limited studies have been carried out using in vivo models to determine the factors that regulate the major bioactive form of somatostatin in the intestine, S28. The development of a rat intestinal culture model (18, 19, 20) now permits characterization of nutrient and peptide secretagogues that directly regulate intestinal somatostatin secretion. Demonstration of the morphological and functional absence of neurons in these cells coupled with previous results indicating the presence of an epithelial cell marker (23) confirm that the different molecular forms of somatostatin produced by our culture system arise from mucosal epithelial D cells. Thus, the present studies were carried out to determine those factors stimulating S28 secretion as well as to assess any differential regulation of S28 and S14 release from the intestine.

Our results have demonstrated that the long chain monounsaturated fatty acid, oleic acid, stimulates secretion of SLI from the intestinal cultures at concentrations that are likely to be physiologically relevant (24). These findings are in accord with in vivo data showing that fat is the most potent nutrient secretagogue of intestinal SLI (10, 16, 25). Of particular interest was the observation that oleic acid stimulated S28 secretion from D cells. Data obtained from humans after mixed fat ingestion (6, 17) and from dogs after mixed fat administration into the duodenum (10) have demonstrated a temporally delayed requirement for the release of S28 that accords with the predominant distribution of this peptide in the distal intestine (1, 2, 3, 4). Thus, our findings indicate that ingested fats may require transit to the distal D cell to induce S28 release. In contrast to S28, but consistent with its proximal distribution (1, 2, 3, 4), in vivo release of S14 in response to intraluminal fat occurs rapidly (within 30 min of administration) (6, 10, 17). Our finding that oleic acid also facilitates S14 release in vitro is consistent with these observations. When taken together, these observations suggest that movement of fat from the duodenum toward the ileum may invoke direct release of S14 initially, followed in a temporal fashion by S28 secretion from the ileum. The suggestion that S28 may be important in the inhibition of postprandial insulin secretion is consistent with the late release of this peptide in response to fat ingestion (6), permitting S28 to serve a physiological role as a decretin and thereby preventing postprandial hyperinsulinemia (7).

Of the neuropeptides tested in the present study, only GRP dose dependently increased the release of S28. GRP is localized in enteric neurons throughout the small intestine (26, 27) and is known to regulate the release of a number of other ileal peptides, including PYY (22, 28) and glucagon-like peptide-1 (28, 29). Thus, GRP appears to be a major determinant of ileal peptide secretion. GRP exerts its effects through a protein kinase C-dependent pathway (30), which is consistent with our previous report that activation of this pathway with phorbol esters stimulates the release of intestinal SLI (18). Interestingly, we also observed release of S14 in parallel with that of S28 in response to GRP treatment. These findings are consistent with previous studies showing GRP to be a potent stimulator of SLI release from the perfused rat stomach (31, 32) and suggest that proximal and distal rat intestinal D cells may be similar to the gastric D cell in their sensitivity to GRP. It must be noted, however, that GRP/bombesin was ineffective in modulating SLI release from isolated canine fundic and human antral D cells (14, 33), suggesting that there may be species-dependent differences in the expression of GRP receptors by gastrointestinal D cells.

Studies in vivo have elucidated an important role for the vagus nerves in regulating intestinal SLI release (8, 9, 11, 34). Moreover, differential effects of muscarinic pathways on S14 and S28 release have been reported in the dog, as acetylcholine stimulates S28 secretion, but causes inhibition of S14 (9, 11). Similarly, muscarinic effects on SLI secretion from the isolated rat stomach are predominantly inhibitory (35, 36). However, in the present study, bethanechol had no effect on intestinal SLI release. Our chromatographic studies further confirmed an absence of either stimulation of S28 or suppression of S14 by bethanechol in the intestinal cultures. There is no clear explanation for the absence of a cholinergic effect on intestinal D cell function, although we have previously shown in the same intestinal culture model system that bethanechol is biologically active, stimulating the release of proglucagon-derived peptides (29).

In contrast to the effects of fat and GRP on intestinal S28 and S14 release, the endocrine peptide secretin released mainly S14 from the intestinal cultures. These findings are interesting given the proximal distribution of both secretin (37) and S14 (1, 2, 3, 4) in the intestine. Similarly, we have previously reported that CGRP-1, a sensory neuropeptide found only in the proximal gut (38), also shows specificity for the release of S14 from intestinal cultures (19). Both of these peptides are known to exert their biological effects through protein kinase A-dependent pathways (39, 40). These findings suggest that there may exist two distinct populations of D cells in the intestinal mucosa, the more proximal of which releases S14 and expresses the receptors for secretin and CGRP-1. Our use of the entire fetal rat small intestine for the preparation of FRIC cultures would be consistent with the presence of both cell types in our model system. Interestingly, in vitro and in vivo studies on the human, canine, and rat gastric D cell have also demonstrated stimulatory effects of secretin on total SLI release (15, 31, 41, 42). Furthermore, the enterogastrone effect of secretin in vivo is abolished by administration of an anti-somatostatin antiserum (42). As S14 potently inhibits acid secretion by the parietal cell (43), the results of the present study lend support to the concept of a feedback loop between the proximal intestine and the parietal cell through secretin-mediated S14 release. The effects of secretin we observed occurred at relatively high concentrations, raising the possibility that in vivo, the actions of secretin on proximal D cells are a local rather than an endocrine effect.

In contrast to the effects of secretin, neither CCK nor GIP affected intestinal somatostatin release in the present study, whereas the influence of gastrin was marginal. These results differ from those of previous studies on human, rat, and canine antral and canine fundic D cell cultures (13, 14, 15, 44), in which CCK potently stimulated the release of somatostatin. Furthermore, GIP has been shown to stimulate SLI release from isolated sections of rat antral mucosa (45). In addition to the possibility of species-specific responses, tissue-specific differences may provide an explanation for these differential effects, as suggested previously (46), possibly through preferential distribution of GIP, CCK-A, and CCK-B receptors on D cells in the stomach. It also remains possible that CCK binding is not fully developed in the term fetal rat ileum, as has been demonstrated for the newborn rat pancreas (47).

None of the other peptides or growth factors tested had any major effect on intestinal SLI release, with the exception of PYY, which did have a modest stimulatory effect at the highest dose tested. The effects of these agents on intestinal somatostatin release have not previously been examined; however, the negative findings are in accord with previous reports by other investigators using gastric D cell models (31, 41, 48). We have previously reported that the ileal peptide GLP-1, cosecreted from ileal L cells with PYY (49), is a potent stimulus for both S28 and S14 release from the intestinal cultures (20). As S28 and, to a lesser extent, S14 are known inhibitors of L cell secretion (29), these findings suggest the existence of a local regulatory feedback loop between the L and D cells in the ileum. It should be noted, however, that although L cells are also present in the FRIC cultures (20, 22), levels of PYY and GLP-1 in the medium are unlikely to reach sufficient concentrations to exert coregulatory effects.

In summary, the results of the present study demonstrate that S28 release from the intestine is subject to regulation by fatty acids and the neuropeptide GRP, but not by a wide variety of other regulatory peptides. Our demonstration of differential regulation of intestinal S28 and S14 secretion lends support to the concept that S28 functions as an independent regulatory peptide in physiological settings.


   Acknowledgments
 
The authors are grateful to C. Chisholm, S. Pokol-Daniel, A. S. Rocca, and K. So for technical assistance.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada (MT-9940 to P.L.B.; MA-6763 to G.R.G.) and the Canadian Diabetes Association (to P.L.B.; Margaret Adele Mollet Grant). Back

Received July 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Patel YC, Wheatley T, Ning C 1981 Multiple forms of immunoreactive somatostatin: comparison of distribution in neural and nonneural tissues and portal plasma of the rat. Endocrinology 109:1943–1949[Medline]
  2. Baldissera FGA, Holst JJ, Jensen SL, Krarup T 1985 Distribution and molecular forms of peptides containing somatostatin immunodeterminants in extracts from the entire gastrointestinal tract of man and pig. Biochim Biophys Acta 838:132–143[Medline]
  3. Francis BH, Baskin DG, Saunders DR, Ensinck JW 1990 Distribution of somatostatin-14 and somatostatin-28 gastrointestinal-pancreatic cells of rats and humans. Gastroenterology 99:1283–1291[Medline]
  4. Trent DF, Weir GC 1981 Heterogeneity of somatostatin-like peptides in rat brain, pancreas, and gastrointestinal tract. Endocrinology 108:2033–2038[Abstract]
  5. Newman JB, Lluis F, Townsend CM 1987 Somatostatin. In: Thompson JC, Greeley GH, Rayford PL, Townsend CM (eds) Gastrointestinal Endocrinology. McGraw-Hill, New York, pp 286–299
  6. D’Alessio DA, Sieber C, Beglinger C, Ensinck JW 1989 A physiologic role for somatostatin 28 as a regulator of insulin secretion. J Clin Invest 84:857–862
  7. Ensinck JW, Laschansky EC, Vogel RE, D’Alessio DA 1991 Effect of somatostatin-28 on dynamics of insulin secretion in perfused rat pancreas. Diabetes 40:1163:1169[Abstract]
  8. Greenberg GR 1987 Influence of vagal integrity on gastrin and somatostatin release in dogs. Gastroenterology 93:994–1001[Medline]
  9. Greenberg GR, Pokol-Daniel S, Fung L 1992 Influence of gastric acid on circulating somatostatin-14 and -28 released after insulin-induced hypoglycemia in conscious dogs. Endocrinology 131:1527–1533[Abstract]
  10. Greenberg GR, Fung L, Pokol-Daniel S 1993 Regulation of somatostatin-14 and -28 secretion by gastric acid in dogs: differential role of cholecystokinin. Gastroenterology 105:1387–1395[Medline]
  11. Greenberg GR 1993 Differential neural regulation of circulating somatostatin-14 and somatostatin-28 in conscious dog. Am J Physiol 264:G902–G909
  12. Fung L, Pokol-Daniel S, Greenberg GR 1994 Cholecystokinin type A receptors mediate intestinal fat-induced inhibition of acid secretion through somatostatin-14 in dogs. Endocrinology 134:2376–2382[Abstract]
  13. Soll AH, Amirian DA, Park J, Elashoff JD, Yamada T 1985 Cholecystokinin potently releases somatostatin from canine fundic mucosal cells in short-term culture. Am J Physiol 248:G569–G573
  14. Buchan AMJ, Curtis SB, Meloche RM 1990 Release of somatostatin immunoreactivity from human antral D cells in culture. Gastroenterology 99:690–696[Medline]
  15. Buchan AMJ, Meloche RM, Kwok YN, Kofod H 1993 Effect of cholecystokinin and secretin on somatostatin release from cultured antral cells. Gastroenterology 104:1414–1419[Medline]
  16. Ensinck JW, Vogel RE, Laschansky EC, Francis BH 1990 Effect of ingested carbohydrate, fat, and protein on the release of somatostatin-28 in humans. Gastroenterology 98:633–638[Medline]
  17. D’Alessio DA, Ensinck JW 1990 Fasting and postprandial concentrations of somatostatin-28 and somatostatin-14 in type II diabetes in men. Diabetes 39:1198–1202[Abstract]
  18. Brubaker PL, Drucker DJ, Greenberg GR 1990 Synthesis and secretion of somatostatin-28 and -14 by fetal rat intestinal cells in culture. Am J Physiol 258:G974–G981
  19. Brubaker PL, Greenberg GR 1993 Calcitonin gene-related peptide-1 preferentially stimulates secretion of somatostatin from intestinal cultures. Endocrinology 133:2833–2837[Abstract]
  20. Brubaker PL, Efendic S, Greenberg GR 1997 Truncated and full-length glucagon-like peptide-1 (GLP-1) differentially stimulate intestinal somatostatin release. Endocrine 6:91–95[Medline]
  21. Lui EYT, Asa SL, Drucker DJ, Lee YC, Brubaker PL 1990 Glucagon and related peptides in fetal rat hypothalamus in vivo and in vitro. Endocrinology 126:110–117[Abstract]
  22. Brubaker PL, Drucker DJ, Asa SL, Greenberg GR 1991 Regulation of peptide-YY synthesis and secretion in fetal rat intestinal cultures. Endocrinology 129:3351–3358[Abstract]
  23. Brubaker PL, Vranic M 1987 Fetal rat intestinal cells in monolayer culture: a new in vitro system to study the glucagon-related peptides. Endocrinology 120:1976–1985[Abstract]
  24. McNeil NI, Bingham S, Cole TJ, Grant AM, Cummings JH 1982 Diet and health of people with an ileostomy. Br J Nutr 47:407–415[CrossRef][Medline]
  25. Lucey MR, Fairclough PD, Wass JAH, Kwasowski P, Medbak S, Webb J, Rees LH 1984 Response of circulating somatostatin, insulin, gastrin and GIP, to intraduodenal infusion of nutrients in normal man. Clin Endocrinol (Oxf) 21:209–217[Medline]
  26. Moghimzadeh E, Ekman R, Hakanson R, Yanaihara N, Sundler F 1983 Neuronal gastrin-releasing peptide in the mammalian gut and pancreas. Neuroscience 10:553–563[CrossRef][Medline]
  27. Dockray GJ, Vaillant C, Walsh JH 1979 The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract. Neuroscience 4:1561–1568[CrossRef][Medline]
  28. Dumoulin V, Dakka T, Plaisancie P, Chayvialle J-A, Cuber J-C 1995 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[Abstract]
  29. Brubaker PL 1991 Regulation of intestinal proglucagon-derived peptide secretion by intestinal regulatory peptides. Endocrinology 128:3175–3182[Abstract]
  30. Battey JF, Way J, Caorjay MH, Shapira H, Kusano K, Harkins R, Wu JM, Slattery T, Mann E, Feldman RI 1991 Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. Proc Natl Acad Sci USA 88:395–399[Abstract/Free Full Text]
  31. Chiba T, Taminato T, Kadowaki S, Inoue Y, Mori K, Seino Y, Abe H, Chihara K, Matsukura S, Fujita T, Goto Y 1980 Effects of various gastrointestinal peptides on gastric somatostatin release. Endocrinology 106:145–149[Abstract]
  32. Guo Y-S, Thompson JC, Singh P 1989 Effect of gamma-aminobutyric acid on bombesin-evoked release of somatostatin and gastrin from isolated rat stomach. Regul Pept 24:179–186[CrossRef][Medline]
  33. Sugano K, Park J, Soll AH, Yamada T 1987 Stimulation of gastrin release by bombesin and canine gastrin-releasing peptides: studies with isolated canine G cells in primary culture. J Clin Invest 79:935–942
  34. Ahrén B, Paquette TL, Taborsky Jr GJ 1986 Effect and mechanism of vagal nerve stimulation on somatostatin secretion in dogs. Am J Physiol 250:E212–E217
  35. Saffouri B, DuVal JW, Makhlouf GM 1984 Stimulation of gastrin secretion in vitro by intraluminal chemicals: regulation by intramural cholinergic and noncholinergic neurons. Gastroenterology 87:557–561[Medline]
  36. Schubert ML, Hightower J 1990 Functionally distinct muscarinic receptors on gastric somatostatin cells. Am J Physiol 258:G982–G987
  37. Bussolati G, Capella C, Solcia E, Vassallo G, Vezzadini P 1971 Ultrastructural and immunofluorescent investigations on the secretin cell in the dog intestinal mucosa. Histochemie 26:218–227[CrossRef][Medline]
  38. Mulderry PK, Ghatei MA, Spokes RA, Jones PM, Pierson AM, Hamid QA, Kanse S, Amara SG, Burrin JM, Legon S, Polak JM, Bloom SR 1988 Differential expression of {alpha}-CGRP and ß-CGRP by primary sensory neurons and enteric autonomic neurons of the rat. Neuroscience 25:195–205[CrossRef][Medline]
  39. Ishihara T, Nakamura S, Kaziro Y, Takahashi T, Takahashi K, Nagata S 1991 Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J 10:1635–1641[Medline]
  40. Seifert H, Sawchenko P, Chenut J, Rivier J, Vale W, Pandol SJ 1985 Receptor for calcitonin gene-related peptide: binding to exocrine pancreas mediates biological actions. Am J Physiol 249:G147–G151
  41. Rouiller D, Schusdziarra V, Harris V, Unger RH 1980 Release of pancreatic and gastric somatostatin-like immunoreactivity in response to octapeptide of cholecystokinin, secretin, gastric inhibitory polypeptide, and gastrin-17 in dogs. Endocrinology 107:524–529[Medline]
  42. Chung I, Li P, Lee K, Chang T, Chey WY 1994 Dual inhibitory mechanism of secretin action on acid secretion in totally isolated, vascularly perfused rat stomach. Gastroenterology 107:1751–1758
  43. Seal A, Yamada T, Debas H, Hollingshead J, Osadchey B, Aponte G, Walsh J 1982 Somatostatin-14 and -28: clearance and potency on gastric function in dogs. Am J Physiol 243:G97–G102
  44. Zeng N, Walsh JH, Kang T, Helander KG, Helander HF, Sachs G 1996 Selective ligand-induced intracellular calcium changes in a population of rat isolated gastric endocrine cells. Gastroenterology 110:1835–1846[CrossRef][Medline]
  45. Wolfe MM, Reel GM 1986 Inhibition of gastrin release by gastric inhibitory peptide mediated by somatostatin. Am J Physiol 250:G331–G335
  46. Sandvik AK, Dimaline R, Forster ER, Evans D, Dockray GJ 1993 Differential control of somatostatin messenger RNA in rat gastric corpus and antrum: role of acid, food, and capsaicin-sensitive afferent neurons. J Clin Invest 91:244–250
  47. Werlin SL, Colton DG, Harb J, Reynolds E, Hoffman RG, Williams JA 1987 Ontogeny of secretory function and cholecystokinin binding capacity in immature rat pancreas. Life Sci 40:2237–2245[CrossRef][Medline]
  48. Greeley Jr GH, Guo Y-S, Gomez G, Lluis F, Singh P, Thompson JC 1988 Inhibition of gastric acid secretion by peptide YY is independent of gastric somatostatin release in the rat. Proc Soc Exp Biol Med 189:325–328[Abstract]
  49. Bottcher G, Sjolund K, Ekblad E, Hakanson R, Schwartz TW, Sundler F 1984 Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut. Regul Pept 8:261–266[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
C. Chisholm and G. R. Greenberg
Somatostatin-28 regulates GLP-1 secretion via somatostatin receptor subtype 5 in rat intestinal cultures
Am J Physiol Endocrinol Metab, August 1, 2002; 283(2): E311 - E317.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
N. M. Sherwood, S. L. Krueckl, and J. E. McRory
The Origin and Function of the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)/Glucagon Superfamily
Endocr. Rev., December 1, 2000; 21(6): 619 - 670.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brubaker, P. L.
Right arrow Articles by Greenberg, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brubaker, P. L.
Right arrow Articles by Greenberg, G. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals