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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
Departments of Physiology (Y.A., T.H., P.L.B.) and Medicine (P.L.B.), University of Toronto, Toronto, Ontario, Canada M5S 1A8
Address all correspondence and requests for reprints to: Dr. Patricia Brubaker, Department of Physiology, University of Toronto, MSB Room 3366, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: . p.brubaker{at}utoronto.ca
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Abstract |
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Introduction |
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Release of GLP-1 from the distal L cells is under complex regulation. Agents that can directly stimulate the secretion of GLP-1 have been analyzed in various models of the intestinal L cells, including a primary cell culture of fetal rat intestinal cells (FRIC) (12, 13, 14), murine intestinal cell lines (15), and the perfused rat ileum (16) and colon (17). The major secretagogues identified using these models include glucose- dependant insulinotropic peptide (GIP) (14, 15, 16, 17), gastrin- releasing peptide (GRP) (14, 16, 17), calcitonin gene-related peptide (14, 16, 17), and agonists of acetylcholine (14, 15, 16, 17). Fatty acids, when applied directly to L cells in culture, also stimulate GLP-1 secretion (13, 15).
In the in vivo setting, GLP-1 is rapidly released from L cells upon the ingestion of a mixed meal (1, 2) or after infusion of fat directly into the duodenum (18, 19, 20). Indeed, plasma levels of GLP-1 peak within 15–30 min of nutrient administration despite the fact that nutrients do not reach the distal ileum or colon within this time frame (21). These observations are not consistent with the idea that nutrients act directly on ileal L cells to stimulate the release of GLP-1, at least during the early phase of secretion. Several studies by our group have demonstrated that administration of nutrients into the duodenum stimulates GIP secretion by local endocrine K cells, which, in turn, stimulates the afferent vagus nerve to the central nervous system, stimulating the efferent vagus (celiac) nerve to the distal gut (18, 19). As the vagus nerve releases acetylcholine, we have hypothesized that this pathway signals through muscarinic receptors expressed at the level of the L cell. Consistent with this hypothesis, several in vitro studies demonstrated the involvement of cholinergic muscarinic receptors in the regulation of GLP-1 secretion (12, 14, 15, 22). Furthermore, Balks et al. (23) used atropine, a nonspecific muscarinic antagonist, to demonstrate that GLP-1 release in humans is under the control of muscarinic receptors. However, additional studies have shown the involvement of cholinergic nicotinic receptors in the regulation of GLP-1 secretion (1), as well as the neuropeptide, GRP (24). The relationships between these different neuromodulatory pathways remain to be determined. The goal of the present study was to investigate the role of cholinergic muscarinic receptors in the control of GLP-1 secretion, using both the anesthetized rat model and FRIC cultures.
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Materials and Methods |
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After median laparotomy, a loose ligation was set up surrounding the pylorus, and 3 ml fat in the form of corn oil (56% polyunsaturated fatty acids, 32% monounsaturated fatty acids, and 12% saturated fatty acids; Sunfresh Ltd., Toronto, Canada) were then infused into the proximal part of the duodenum at time zero. Blood was collected at -10 and 0 min to ascertain basal levels of secretion, and manipulation of the gastrointestinal tract was performed in the interval between these two time points. Blood samples were also collected at 10, 20, 30, and 60 min thereafter. In a previous study (21) we showed that fat does not reach the distal ileum within 60 min after intraduodenal administration in rats, thus precluding any direct effect of the fat on the distal L cell.
Twenty minutes before corn oil administration, groups of 6 rats received one of the following primed infusions: atropine sulfate (Sigma, St. Louis, MO; 500 µg/kg and 500 µg/kg·h, iv), pirenzepine (RBI, Natick, MA; 200 µg/kg and 200 µg/kg·h, iv), gallamine (RBI; 200 µg/kg and 200 µg/kg·h, iv), or 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; RBI; 200 µg/kg and 200 µg/kg·h, iv). Control animals received a solution of 0.9% NaCl, iv The flow rate was 3 ml/h. These doses were selected according to Steinle and Smith (25).
Plasma was stored at -20 C before RIA for bioactive GLP-1 [GLP-1-(7–36) amide]. Plasma was extracted in ethanol and assayed using an RIA kit from Linco Research, Inc. (St. Charles, MO). The assay uses an antibody specific for the N-terminal end of GLP-1 and therefore does not cross-react with the circulating degradation product, GLP-1-(9–36) amide. The detection limit of the assay is 3 pM.
FRIC cultures
FRIC cultures were prepared as described in detail previously (12, 13, 14). In brief, intestines from a litter of 19- to 21-d gestation fetal Wistar rats were pooled, and the cells were dispersed by two sequential 15-min incubations with collagenase (45 mg/dl), hyaluronidase (50 mg/dl), and deoxyribonuclease I (5 mg/dl; Sigma). The dispersed cells were washed and placed into monolayer culture on 60 x 15-mm dishes in culture medium [DMEM containing 5% (vol/vol) FBS, 4.5 g/liter glucose, and penicillin (50 IU/ml)-streptomycin (50 µg/ml)] at a final concentration of 0.6 fetal rat intestines/2 ml·dish. The cells were then incubated at 37 C with 10% (vol/vol) CO2 and constant humidity for 16–24 h. Before each experiment, cultures were washed twice with HBSS to remove dead or floating cells, and groups of two dishes were then incubated for 2 h with experimental agents in 2 ml/dish experimental medium [DMEM containing 0.5% (vol/vol) FBS, 1 g/liter glucose, 20 µU/ml insulin, and penicillin (50 IU/ml)-streptomycin (50 µg/ml)].
Some cells were pretreated for 30 min with medium alone (control) or with pirenzepine (1 mM), gallamine (1 mM), or 4-DAMP (1 mM), followed by 2-h treatment with medium alone (control), phorbol myristate acetate (PMA; 1 µM; positive control), pirenzepine (1 mM), gallamine (1 mM), 4-DAMP (1 mM), bethanechol (1 mM; Sigma), McN-A-343 (RBI; 10–5000 µM), or oxotremorine-M (RBI; 10–5000 µM). Each treatment was tested in 6–10 independent experiments.
After the incubation period, cell viability was checked visually by phase-contrast microscopy, and then cell and medium peptides were collected as described previously (12, 13, 14). In brief, medium was centrifuged to remove any floating cells, and trifluoroacetic acid was added to the collected medium to a final concentration of 0.1% (vol/vol). Cells were collected by scraping and homogenization in 2 ml/dish 1 N HCl containing 5% (vol/vol) HCOOH, 1% (vol/vol) trifluoroacetic acid, and 1% (vol/vol) NaCl at 4 C. Peptides and small proteins in acidified cell medium or cell extracts were then collected by reverse phase adsorption to C18 silica (C18 Sep-Pak, Waters Corp., Milford, MA) as previously reported (12, 13, 14). We have demonstrated that this methodology permits greater than 88% recovery of intact proglucagon-derived peptides, including GLP-1, from tissues and cell cultures (12). Extracts were stored at -20 C until assay.
Peptides extracted from the intestinal cultures were assayed for the presence of GLP-1 using an antiserum directed against the carboxyl-terminus of GLP-1-(7–36) amide (Affinity Research Products, Nottingham, UK) as described previously (18, 25). Previous studies have demonstrated that FRIC cultures synthesize and secrete GLP-1-(7–36)NH2 (26). Secretion of GLP-1 was calculated as the total amount of GLP-1 released into the medium over the 2-h incubation period, normalized for the total content of GLP-1 in each dish (i.e. medium plus cells).
Immunohistochemistry
Rats were anesthetized with halothane. Pieces of distal ileum were removed, fixed with formalin, embedded in paraffin, and 5-µm sections were prepared. After deparaffinization and hydration, sections were incubated with blocking solution (5% normal donkey serum, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min. Sections were then incubated with primary antiserum for GLP-1 (rabbit polyclonal antibody, used at a 1:1250 dilution) (11) or for M1 (goat polyclonal antibody C-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; used at a1:50 dilution), M2 (goat polyclonal antibody C-18, Santa Cruz Biotechnology, Inc.; used at a 1:50 dilution), or M3 (goat polyclonal antibody C-20, Santa Cruz Biotechnology, Inc.; used at a 1:50 dilution) muscarinic receptors for 1 h at room temperature. After three serial washes with PBS, sections were incubated with secondary antibodies, Cy3-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.; used at a 1:250 dilution) or Cy2-conjugated donkey antigoat IgG (Jackson ImmunoResearch Laboratories, Inc.; used at a 1:50 dilution), for 1 h at room temperature. After rinsing with PBS, the sections were mounted and visualized using a fluorescent microscope.
Some tissue was fresh-frozen and postfixed in methanol, then acetone, at -20 C. Sections (20 µm) were double stained for GLP-1 (as described above) and for neurofilament 68 using a monoclonal antineurofilament 68 antibody (Sigma; used at a 1:100 dilution) for 1 h at room temperature, then incubated with the secondary antibody (donkey antimouse IgG, Jackson ImmunoResearch Laboratories, Inc.; used at a 1:300 dilution). These sections were visualized using 500-nm optical sections on a confocal microscope.
FRIC cultures were grown in eight-well chamber slides (Nalge Nunc International, Naperville, IL) overnight. The medium was removed, and the cells were washed with PBS and fixed in methanol at -10 C for 5 min. Slides were incubated with blocking solution (5% normal donkey serum, Jackson ImmunoResearch Laboratories, Inc.) for 30 min, then incubated with primary antiserum for GLP-1 (rabbit polyclonal antibody, used at a 1:1250 dilution) or for M1 (goat polyclonal antibody C-20, Santa Cruz Biotechnology, Inc.; used at a 1:50 dilution), M2 (goat polyclonal antibody C-18, Santa Cruz Biotechnology, Inc.; used at a 1:50 dilution), or M3 (goat polyclonal antibody C-20, Santa Cruz Biotechnology, Inc.; used at a 1:50 dilution) muscarinic receptors for 1 h at room temperature. After three serial washes with PBS, sections were incubated with secondary antibodies, Cy3-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.; used at a 1:250 dilution) or Cy2-conjugated donkey antigoat IgG (Jackson ImmunoResearch Laboratories, Inc.; used at a 1:50 dilution). After rinsing with PBS, the sections were mounted and visualized using a fluorescent microscope. For the controls, no immunostaining was observed when the primary or secondary antibodies were omitted (not shown).
Data analysis
Peptide secretion is expressed as the change from basal values (in vivo experiments) or as a percentage of control secretion (in vitro experiments). The area under the curve (AUC) for changes in hormone levels was determined using the trapezoidal rule. All data are expressed as the mean ± SEM. Statistical significance between experimental groups was assessed by ANOVA using n-1 post hoc custom hypothesis tests. Significance was determined at the P < 0.05 level in these comparisons.
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Results |
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). The AUC over the basal value after fat infusion was 723 ± 65 pmol/liter·h. Infusion of 3 ml saline into the proximal duodenum had no effect on plasma GLP-1-(7–36) amide during the 1 h of the experiment.
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). The AUC over the basal value during 1 h of atropine infusion was 65 ± 13 pmol/liter·h (i.e. 91% inhibition; P < 0.001), showing the involvement of muscarinic receptors in the control of postprandial GLP-1-(7–36) amide secretion in rats.
To assess which subtype of muscarinic receptor is involved in fat-induced GLP-1 secretion in rats, specific antagonists for the M1, M2, and M3 subtypes of muscarinic receptors were infused into rats receiving intraduodenal corn oil. As shown in Fig. 2, infusion of pirenzepine (200 µg/kg and 200 µg/kg·h; an M1 muscarinic receptor subtype antagonist) significantly inhibited fat-induced GLP-1 secretion by 91 ± 6% (P < 0.01). Infusion of the M2 receptor antagonist, gallamine (200 µg/kg and 200 µg/kg·h) decreased fat- induced GLP-1 secretion by 43 ± 8%, but this did not reach significance. Furthermore, infusion of 4-DAMP (200 µg/kg and 200 µg/kg·h), a specific M3 muscarinic subtype antagonist, had no effect on fat-induced GLP-1 secretion (Fig. 2).
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), M2 (Fig. 3B), and M3 (Fig. 3C) subtypes of muscarinic receptors on all of the ileal L cells. Most of the epithelial cells demonstrated muscarinic receptor positivity. Furthermore, double immunofluorescent staining for GLP-1 (green) and neurofilament-68 (red) confirmed that endocrine L cells are connected to neurons in the enteric nervous system (Fig. 3D).
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). PMA stimulated GLP-1 secretion to 199 ± 24% to 299 ± 80% of the control level (P < 0.01 to P < 0.001). Bethanechol (1 mM; a nonspecific muscarinic receptor agonist) also stimulated GLP-1 secretion by FRIC cultures to 200 ± 34% to 253 ± 42 of the control level (P < 0.05 to P < 0.001).
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), whereas pirenzepine alone had no effect on GLP-1 secretion by FRIC cultures (102 ± 30% of the control). Gallamine (1 mM) also completely inhibited bethanechol-stimulated GLP-1 secretion (102 ± 8% of the control; P < 0.01 vs. bethanechol alone; Fig. 4B), whereas gallamine alone had no effect on GLP-1 secretion in vitro (92 ± 19% of the control). Unexpectedly, 4-DAMP (1 mM) alone stimulated GLP-1 secretion to 324 ± 52% of the control level (P < 0.01; Fig. 4C). However, 4-DAMP had no significant effect on bethanechol-stimulated GLP-1 secretion by FRIC cultures (343 ± 92% of the control; Fig. 4C).
To further establish the role of muscarinic receptors in regulating GLP-1 secretion by FRIC cultures, cells were incubated for 2 h in the presence of graded concentrations of McN-A-343 (an M1 muscarinic receptor subtype agonist) or oxotremorine-M (an M3 muscarinic receptor subtype agonist). McN-A-343 significantly stimulated GLP-1 secretion to 197 ± 24% of the control level (P < 0.01) when used at a concentration of 5 mM (Fig. 5A). By contrast, oxotremorine-M had no effect on GLP-1 secretion by FRIC cultures when used at concentrations between 10 and 5000 µM (Fig. 5B).
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, E and F), M2 (Fig. 3, G and H), or M3 (Fig. 3, I and J) further showed the expression of all three subtypes of muscarinic receptors on L cells. In contrast to staining for GLP-1, which was found in only a small percentage of FRIC cells, the majority of cells were immunopositive for the muscarinic receptors. |
Discussion |
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Infusion of atropine, a nonspecific muscarinic receptor antagonist, 20 min before intraduodenal administration of corn oil completely blocked nutrient-induced GLP-1 secretion. This finding clearly demonstrates the involvement of cholinergic muscarinic receptors in the control of postprandial GLP-1 secretion in the rat. Furthermore, as atropine does not delay intestinal transit in the anesthetized rat (21), this effect was probably not due to any alterations in nutrient fluxes through the gut. In addition, a previous study has shown that atropine inhibits the stimulatory effect of a cholinergic agonist on GLP-1 secretion in the isolated vascularly perfused rat ileum (27). These findings are also similar to the results of a study in humans showing that atropine reduces the integrated GLP-1 response to an oral glucose load (23). In contrast, D’Alessio et al. (28) reported that nicotinic blockade, but not muscarinic blockade, prevents nutrient-induced GLP-1 secretion in rhesus macaques. We have also shown that hexamethonium (a cholinergic nicotinic receptor antagonist) strongly inhibits GLP-1 secretion induced by intraduodenal fat or a mixed meal in rats (1). Thus, in rat, monkey, and human, GLP-1 secretion is regulated by cholinergic receptors. However, the involvement of muscarinic receptors in the control of this secretion appears to be species specific.
Five different isoforms of muscarinic receptors have been described (M1–M5), although M4 and M5 have been reported to localize mainly in the brain (29). We therefore also performed experiments to establish which of the muscarinic receptor subtypes (M1, M2, or M3) is involved in regulating GLP-1-(7–36 amide) secretion in vivo in rats. As shown in Fig. 1, pirenzepine (an M1 antagonist) completely inhibited corn oil-induced GLP-1 secretion. By contrast, neither gallamine (an M2 antagonist) nor 4-DAMP (an M3 antagonist) had a significant effect on GLP-1 secretion. Furthermore, double immunostaining of the rat ileum for GLP-1 and M1, M2, or M3 muscarinic receptors showed that L cells express all three muscarinic receptor subtypes, suggesting that acetylcholine acts directly on muscarinic receptors localized at the level of the L cell. Finally, a direct role for muscarinic receptors in regulating GLP-1 secretion in the rat was established using FRIC cultures. This study confirmed the results of the in vivo experiments, in that the M1 receptor was found to be the major regulator of GLP-1 secretion from the L cell in vitro. Thus, although both the rat ileal L cell and the fetal rat intestinal L cell express multiple subtypes of muscarinic receptors, only the M1 receptor appears to play a role in GLP-1 secretion in adult rats. These findings are consistent with the mechanism of action of the M1 muscarinic receptor to activate PLC/calcium (30), a pathway that is known to stimulate GLP-1 secretion from the L cell (12, 15).
Interestingly, in FRIC cultures both M1 and M2 receptors seem to be important in regulating GLP-1 secretion, suggesting that there are some differences in the control of the L cell between fetal and adult rats. Furthermore, the M3 receptor antagonist, 4-DAMP, significantly stimulated GLP-1 secretion from FRIC cultures, whereas there was no effect of 4-DAMP on nutrient-induced GLP-1 secretion in the adult rat. As we were most interested in the factors regulating postprandial secretion of GLP-1, we did not determine the effect of any muscarinic antagonist on basal GLP-1 secretion. However, the findings with 4-DAMP suggest that either this antagonist exerts partial agonist activity on the M3 receptor in the fetal L cell or, alternatively, this receptor is inhibitory in the fetal, but not the adult, L cell.
The neuropeptide, GRP, has been shown to potently stimulate GLP-1 secretion in vitro as well as in vivo in humans and several animal species (14, 16, 17, 24, 27, 31). Consistent with a role for GRP in the regulation of postprandial GLP-1 secretion, the administration of a GRP antagonist was found to completely inhibit intraduodenal fat-induced GLP-1 secretion in vivo in rats (24). Furthermore, Persson et al. (31) have shown that mice lacking the GRP receptor have reduced GLP-1 and insulin responses as well as oral glucose intolerance, showing that intact GRP receptors are required for normal GLP-1 release. No study to date has reported on the interaction between the muscarinic and GRP systems in regulating GLP-1 secretion, but as both muscarinic and GRP antagonists completely inhibit fat-induced GLP-1 secretion in vivo in rats, these findings suggest that the two pathways are connected. When taken together with the results of studies of the role of the endocrine hormone GIP (18, 19) as well as of nicotinic receptors (1) in the regulation of GLP-1 secretion in the rat, the results of the present study lead us to propose the following pathway, as summarized in Fig. 6. Arrival of chyme in the duodenum stimulates GIP secretion by local endocrine K cells. This, in turn, stimulates the afferent vagus nerve to the central nervous system, thereby activating the efferent vagus (celiac) nerve to the ileum. Acting via a nicotinic synapse, this stimulates the release of GRP by intrinsic neurons, which, in turn, release acetylcholine from cholinergic neurons in the enteric nervous system to activate muscarinic receptors on the L cell. This complex interplay between nutrients in the duodenum and the ileal L cell, as mediated by neuro/endocrine modulators in the central and enteric nervous systems, thereby results in a highly integrated GLP-1 response to ingested nutrients.
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Acknowledgments |
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Footnotes |
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Abbreviations: AUC, Area under the curve; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine; FRIC, fetal rat intestinal cells; GIP, glucose-dependant insulinotropic peptide; GLP-1, glucagon-like peptide-1; GRP, gastrin-releasing peptide; PMA, phorbol myristate acetate.
Received December 12, 2001.
Accepted for publication February 12, 2002.
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) or saline (
) 20 min before 3 ml fat in the form of corn oil were infused into the proximal duodenum. A control group received 3 ml saline into the proximal duodenum (
). Bioactive GLP-1 secretion is expressed as the change from basal values (n = 6). Values are the mean ± SEM. (P < 0.01 for fat infusion vs. saline at t = 10, 30, and 60 min.)
), M2 (gallamine; 
; 200 µg/kg and 200 µg/kg·h), on corn oil-induced GLP-1 secretion (
). Bioactive GLP-1 secretion is expressed as the change from basal values (A) or as the 1 h integrated response over the basal level (B). Values are the mean ± SEM. **, P < 0.01 [vs. the control (saline) group (n = 6)].
