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Endocrinology Vol. 144, No. 7 3244-3250
Copyright © 2003 by The Endocrine Society

Muscarinic Receptors Control Glucagon-Like Peptide 1 Secretion by Human Endocrine L Cells

Younes Anini and Patricia L. Brubaker

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

Address all correspondence and requests for reprints to: Dr. Patricia L. Brubaker, Department of Physiology, Medical Sciences Building Room 3366, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: p.brubaker{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucagon-like peptide 1 (GLP-1) released from distal intestinal endocrine L cells after food intake is a potent glucose-dependent stimulant of insulin secretion. Plasma levels of GLP-1 rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve. Our previous studies showed the involvement of M1 muscarinic receptors expressed by the L cells in the regulation of postprandial GLP-1 secretion in rats. The goal of this study was to explore the involvement of muscarinic receptors in the regulation of GLP-1 secretion by human L cells using a newly described human L cell line (NCI-H716). Phorbol 12-myristate 13-acetate (positive control) stimulated GLP-1 secretion to 252 ± 38% of the control (P < 0.001). Bethanechol, a nonselective muscarinic agonist, significantly stimulated GLP-1 secretion to 187 ± 20% of the control (P < 0.01, n = 8). Pirenzepine (M1 antagonist; 10–1000 µM) and gallamine (M2 antagonist; 10–1000 µM) completely inhibited bethanechol-induced GLP-1 secretion, whereas 4-diphenylacetoxy-N-methylpiperidine (M3 antagonist) had no effect on bethanechol-stimulated GLP-1 secretion. McN-A-343 (M1 muscarinic agonist) dose dependently stimulated GLP-1 secretion (to 252 ± 50% of control at 1000 µM; P < 0.01), whereas oxotremorine (M3 agonist) had no effect. M1, M2, and M3 muscarinic receptors were shown to be expressed in NCI-H716 cells by Western blot, immunohystochemistry, and RT-PCR. Expression of the M1, M2, and M3 muscarinic receptor subtypes was also confirmed in paraffin-embedded human small intestine sections by double immunofluorescent staining. These results demonstrate the role of M1 and M2 muscarinic receptors expressed by human L cells in the control of GLP-1 secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HORMONE GLUCAGON-like peptide 1 (GLP-1) is secreted from enteroendocrine L cells localized in the distal ileum and colon (1). GLP-1 acts through specific G protein-coupled receptors to potently stimulate glucose-dependent insulin secretion (2, 3, 4), as well as to inhibit glucagon secretion (5), gastric emptyings (6), and food intake (7). Furthermore, recent finding have demonstrated that chronic administration of GLP-1 also stimulates pancreatic ß-cell proliferation and neogenesis (8, 9). These pleiotropic actions of GLP-1 therefore offer great potential for the treatment of hyperglycemia in patients with type 2 diabetes mellitus (1, 10).

GLP-1 is rapidly released from L cells upon nutrient ingestion, especially fat and carbohydrates (1, 11, 12, 13, 14, 15, 16, 17). The profile of GLP-1 secretion after food intake is biphasic, with the first peak occurring within 15–30 min of nutrient ingestion, and another peak occurring approximately 1 h later (13). The early peak of GLP-1 secretion occurs before the nutrients reach the distal ileum and colon (15), excluding a direct effect of nutrients on the L cells. Several studies by our group have demonstrated that administration of nutrients into the duodenum stimulates GLP-1 secretion indirectly, through pathway involving the glucose-dependent insulinotropic polypeptide (GIP) secretion by endocrine K cells which in turn, stimulates the afferent vagus nerve to the central nervous system, thereby activating the efferent vagus (celiac) to the distal gut (14, 15, 16, 18). Recently, we demonstrated that muscarinic receptors expressed by the L cells, especially the M1 subtype, regulate postprandial GLP-1 secretion in adult rats, whereas both M1 and M2 muscarinic receptor subtypes control GLP-1 secretion from fetal rat L cells (12). Furthermore, atropine, a nonselective muscarinic receptor antagonist, reduces the integrated GLP-1 response to an oral glucose load in humans (17), suggesting a role for the vagus in mediating the early phase of GLP-1 secretion from the human L cell. In the present study, we investigated the role of muscarinic receptors in the control of GLP-1 secretion from human L cells, using both a newly described human endocrine L cell line, NCI-H716, that secretes GLP-1 in a regulated manner (19, 20) and human small intestinal sections. Elucidation of the receptors involved in muscarinic regulation of GLP-1 secretion by human L cells may facilitate development of novel strategies for enhancing GLP-1 secretion in patients with type 2 diabetes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Human NCI-H716 cells were obtained from the American Type Culture Collection (Manassas, VA). The NCI-H716 cell line was developed from ascites fluid of a 33-yr-old Caucasian male with poorly differentiated adenocarcinoma of the colon (21). NCI-H716 cells have been recently described to secrete GLP-1 in a regulated manner (19, 20). For proliferation maintenance, cells were grown in suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cell adhesion was initiated by plating the cells on Matrigel matrix (Becton Dickinson and Co., Bedford, MA) in high-glucose DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin, as described (19, 20). No difference was observed in GLP-1 content of NCI-H716 cells plated on Matrigel matrix or growing in suspension, in accordance with (22) (data not shown).

Secretion studies
Two days before each experiment, cells were seeded in 12-well culture plates coated with Matrigel to induce cell adhesion. On the day of the experiment, cells were washed with HBSS, and incubated with FBS-free DMEM alone (negative control), 1 µM phorbol 12-myristate 13-acetate (PMA; positive control), bethanechol (nonspecific muscarinic agonist, Sigma, St. Louis, MO; 100-1000 µM), McN-A-343 (M1 agonist, RBI, Natick, MA; 100-5000 µM) or Oxotremorine-M (M3 agonist, RBI; 100-5000 µM) (23). Some cells were pretreated for 30 min with medium alone, pirenzepine (M1 antagonist, RBI; 100-1000 µM), gallamine (M2 antagonist, RBI; 100-1000 µM) or 4-diphenylacetoxy-N-methylpiperidine (M3 antagonist, RBI, 4-DAMP; 100-1000 µM), before incubation for 2 h with added bethanechol (1000 µM). We have previously demonstrated that no changes in total GLP-1 content of the L cell can be detected during this time period (18). After the incubation period, cells were checked by microscopy and no major difference between controls and treated cells was observed. Cell and medium peptides and small proteins were collected by reverse phase adsorption (C18 Sep-Pak, Waters Corp., Milford, MA) as described previously (12, 20). Extracts were stored at –20 until assay.

Peptides extracted from NCI-H716 cells were assayed for the presence of GLP-1 using an antiserum directed against the carboxyl terminus of GLP-1 (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) amide (total immunoreactive GLP-1; Affinity Research Products, Nottingham, UK). The assay sensitivity is 3–800 pg/tube and the interassay and intraassay variations are 15.9% and 4.9% respectively, as described previously (12, 20). Secretion was calculated as the total amount of GLP-1 in the medium, normalized for the total content of GLP-1 (i.e. medium plus cells).

Immunohistochemistry
NCI-H716 cells were grown for 48 h in eight-well chamber slides (Nalge Nunc International, Naperville, IL) coated with Matrigel. The medium was removed and the cells were washed with PBS and fixed in methanol at –10 C for 5 min. Sections of human small intestine were obtained from Dr. S. Asa (University Hospital Network, Toronto, Ontario, Canada). After deparaffinization and hydration, fixed cells and tissue sections were incubated with 5% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min. Slides were then incubated with primary antisera for GLP-1 (rabbit polyclonal antibody, used at 1:1250 dilution (12, 20), and for M1 (goat polyclonal antibody C-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; used at a 1:50 dilution), M2 (goat polyclonal antibody C-20, 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 (12) for 1 h at room temperature. After three serial washes with PBS, sections were incubated with secondary antisera, Cy3-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.; used at 1:250 dilution) or Cy2-conjugated donkey antigoat IgG (Jackson ImmunoResearch Laboratories, Inc.; used at 1:50 dilution), for 1 h at room temperature. After rinsing with PBS, the sections were mounted and visualized using a fluorescent microscope. Human tissue was collected as part of a therapeutic procedure at the time of surgery and was anonomized. The use of this anonomized tissue was approved by the institutional Research Ethic Board.

Western blot
NCI-H716 cells were grown for 48 h in six-well plates coated with Matrigel. Cells were then washed with PBS, lysed in 100 µl of sodium dodecyl sulfate sample buffer [62.5 mM Tris base (pH 6.8), 2% wt/vol sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, 0.1% wt/vol bromophenol blue, 2-mercaptoethanol] and placed on ice. Cell lysates were heated to 95 C for 5 min, cooled on ice, microcentifuged for 5 min, and proteins (20–40 µg) were resolved on 7.5% SDS-PAGE gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis.

Membranes were blocked for 3 h with 5% nonfat dry milk in TBS containing 1% Tween 20 (TBST). After washing three times with TBST, the blots were incubated overnight at 4 C with goat anti-M1, M2, or M3 muscarinic receptor antiserum (1:100 dilution). After three washes of 30 min with TBST, blots were incubated for 1 h at room temperature with antigoat HRP-conjugated secondary antibody (1:2000, Cell Signaling Technology, Beverly, MA). The membranes were washed and then probed using the ECL chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). For negative controls, primary antisera were omitted or preadsorbed with the appropriate peptide immunogen and processed as described above.

RNA isolation and RT-PCR
Total RNA was isolated from NCI-H716 cells using an RNeasy Mini kit (QIAGEN, Valencia, CA). Two micrograms of RNA were reverse transcribed and amplified using a OneStep RT-PCR kit (QIAGEN). Cycling parameters were 30 cycles at 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min, followed by a final extension at 72 C for 10 min. The PCR oligonucliotide primers used to amplify cDNA are listed in Table 1Go (24). PCR primers for ß-actin were used to confirm the fidelity of the PCR reaction and to detect genomic DNA contamination. The amplified products were analyzed by electrophoresis on a 1% agarose-TAE [10 mM Tris (pH 7.5), 5.7% glacial acetic acid, and 1 mM EDTA] gel and visualized by ethidium bromide staining.


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  		Table 1. PCR primers

 
Data analysis
GLP-1 secretion is expressed as a percentage of control secretion. 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine the involvement of muscarinic receptors in the regulation of GLP-1 secretion, NCI-H716 cells were incubated for 2 h with medium alone (negative control), PMA [1 µM, a PKC activator known to stimulate GLP-1 secretion by NCI-H716 cells (12, 20)] or graded concentrations of bethanechol (a nonselective muscarinic receptor agonist). Media content of GLP-1 after a 2-h incubation with medium alone was 26.3 ± 4 pg/ml and cell content was 149 ± 9 pg/ml (n = 34). PMA stimulated GLP-1 secretion to 252 ± 38% of control (P < 0.001). Bethanechol (1 mM) also significantly stimulated GLP-1 secretion, to 187 ± 20% of the control (P < 0.001), indicating the involvement of muscarinic receptors in the regulation of GLP-1 secretion by the human L cell line, NCI-H716 (Fig. 1Go).



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  		Figure 1. Release of GLP-1 by NCI-H716 cells in response to a 2-h treatment with medium alone (negative control), PMA (1 µM; positive control) or graded concentrations of bethanechol (a nonselective muscarinic receptor agonist). GLP-1 secretion is expressed as a percentage of the control (n = 6; mean ± SEM). ***, P < 0.001 vs. the control.

 
To determine which subtype of muscarinic receptors is involved in the regulation of GLP-1 secretion by NCI-H716 cells, cells were incubated for 2 h with McN-A-343 (an M1 specific agonist) or oxotremorine-M (an M3 specific agonist). McN-A-343 (500–5000 µM) significantly stimulated GLP-1 secretion in a dose-dependent fashion, to 250 ± 49% of the control (P < 0.001, Fig. 2AGo). Incubating the cells with oxotremorine-M (an M3 muscarinic receptor agonist; 100–5000 µM) had no effect on GLP-1 secretion (Fig. 2BGo).



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  		Figure 2. Release of GLP-1 by NCI-H716 cells in response to a 2-h treatment with medium alone (negative control), PMA (1 µM; positive control) or graded concentrations of McN-A343 (A; an M1 agonist) or oxotremorine (B; an M3 agonist). GLP-1 secretion is expressed as a percentage of the control (n = 6; mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the control.

 
Pirenzepine (an M1 muscarinic receptor antagonist) significantly inhibited bethanechol-induced GLP-1 secretion at 100 and 1000 µM, by 83 ± 24% and 112 ± 19%, respectively (P < 0.05–0.01; Fig. 3AGo). Gallamine (an M2 muscarinic receptor antagonist) also completely inhibited bethanechol-stimulated GLP-1 secretion at 1000 µM (P < 0.001) but had no significant effect at 10 and 100 µM (Fig. 3BGo). In contrast, 4-DAMP (an M3 muscarinic receptor antagonist) had no effect on bethanechol stimulated GLP-1 secretion at any of the doses tested (Fig. 3CGo). None of the antagonists affected basal GLP-1 secretion when tested at a dose of 1000 µM (Fig. 3Go, A–C).



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  		Figure 3. Release of GLP-1 by NCI-H716 cells in response to a 2-h treatment with medium alone (negative control), PMA (1 µM; positive control) or bethanechol (1 mM; a nonselective muscarinic receptor agonist). Cells were pretreated for 30 min with either medium alone or with graded concentrations of pirenzepine (A; an M1 antagonist), gallamine (B; an M2 antagonist), or 4-DAMP (C; an M3 antagonist) before addition of bethanechol. GLP-1 secretion is expressed as a percentage of the control (n = 6; mean ± SEM). ***, P < 0.001 vs. the control. #, P < 0.05; ###, P < 0.001 vs. Bethanechol alone.

 
RT-PCR analysis was performed using primers based on the known human gene sequences encoding the three muscarinic receptor subtypes (Table 1Go; and Ref. 24). Transcripts of the predicted molecular size for the M1, M2, and M3 muscarinic receptors subtypes were identified in the NCI-H716 cells (Fig. 4AGo). The identification of a single RT-PCR product of the predicted size for ß-actin mRNA ruled out contamination by genomic DNA, as the ß-actin primers were designed to span an intron (24).



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  		Figure 4. A, RT-PCR analysis of NCI-H716 cells for mRNA transcripts encoding the muscarinic receptor subtypes M1–M3, or for ß-actin. PCR products of the expected sizes were obtained (Table 1Go). Control, no cDNA present; ladder, molecular mass markers. B, NCI-H716 cells extract were analyzed by Western blot using subtype specific antibodies against the M1, M2, and M3 muscarinic receptors. Immunoreactive proteins with a relative molecular mass of 55 kDa were detected, corresponding to the three receptor subtypes.

 
Protein expression of the muscarinic receptor subtypes was determined by immunoblot analysis using specific polyclonal antibodies directed toward each of the M1, M2, and M3 muscarinic receptor subtypes. Although some nonspecific labeling of other proteins occurred with the M2 and M3 antibodies, one major immunoreactive band was identified that corresponded to the expected mass of the M1, M2, and M3 muscarinic receptor subtypes (~55 kDa; Fig. 4BGo). The major bands were not detected after preincubation of the primary antisera with the respective peptide immunogen (data not shown).

Immunofluorescent labeling was also carried out to localize the different muscarinic receptor subtypes expressed on NCI-H716 cells. Thus, methanol-fixed NCI-H716 cells were double-stained for GLP-1 and the M1, M2, or M3 muscarinic receptor subtypes. All cells demonstrated immunopositive staining for GLP-1 (red), as well as for the M1, M2, and M3 (green) muscarinic receptor subtypes, as indicated by the yellow overlay (Fig. 5AGo). No immunostaining was observed when the primary antisera were omitted (not shown).



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  		Figure 5. Double immunofluorescent staining for GLP-1 (red) and M1, M2, or M3 muscarinic receptor subtypes (green) and overlay of GLP-1 and each muscarinic receptor (Mx) staining (yellow) in methanol-fixed NCI-H716 cells (A) and in human paraffin-embedded 5-µm ileal sections (D). Control indicates omission of both antisera.

 
Finally, as the NCI-H716 cells are transformed L cells, immunofluorescence was performed to explore the expression of the muscarinic receptors by normal human enteroendocrine L cells. Small intestinal sections were therefore double stained for GLP-1 and for the M1, M2, or M3 muscarinic receptor subtypes. Most of the epithelial cells stained positively for the muscarinic receptors. Furthermore, all endocrine L cells (positive for GLP-1; red) were also found to express the M1, M2, and M3 muscarinic receptor subtypes, as indicated by the yellow overlay (Fig. 5BGo). No immunostaining was observed when both primary antisera were omitted (Fig. 5BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The intestinal hormone GLP-1 is a potent insulinotropic and glucagonostatic hormone that also inhibits food intake and reduces body weight following long-term administration (1, 13). We have previously established that M1 muscarinic receptors play an important role in mediating the rapid response of the distal L cell to ingested nutrients in rats (12), whereas the later phase of secretion is stimulated by direct interaction of the nutrients with the L cell (11, 25, 26). Consistent with a role for the vagus in the early response to nutrients, atropine a nonselective muscarinic receptor antagonist inhibits glucose-induced GLP-1 secretion in humans (17). Atropine was also shown to significantly reduce GLP-1 secretion 15–40 min after a standard meal in adult women (27). Interestingly, the cholinergic-dependent cephalic phase induced by sensory stimulation in the oral cavity, does not affect GLP-1 secretion. However, the postabsorptive phase, induced by the entry of the chyme into the stomach stimulates GLP-1 release, and this was shown to be reduced by atropine, although not by trimethaphan, a ganglionic blocker (27). In the present study, we examined the muscarinic regulation of GLP-1 secretion in humans using the newly described endocrine L cell line, NCI-H716. NCI-H716 cells were derived from a human colon adenocarcinoma and have been shown to secrete GLP-1 in a regulated manner (19, 20). Consistent with our hypothesis of an early, vagally mediated L cell response to nutrients (14), NCI-H716 cells are responsive to both carbachol and the neuropeptide gastrin-releasing peptide (19). In addition, nutrients, such as palmitic acid, oleic acid, and meat hydrolysate, also stimulate GLP-1 secretion from these cells (19), suggesting a direct role in the second phase of GLP-1 secretion in humans. Our recent study has also demonstrated a direct effect of the adipocyte satiety hormone leptin, on GLP-1 secretion by the NCI-H716 cells, as well as by rodent L cells (20). Thus, NCI-H716 cells provide a unique model to study the cellular mechanisms underlying GLP-1 secretion from the human L cell.

In the present study, we demonstrated that NCI-H716 cells express the M1, M2, and M3 subtypes of muscarinic receptors, by RT-PCR, Western blot analysis and immunofluorescence. We also investigated the involvement of the muscarinic receptors in controlling GLP-1 secretion by the NCI-H716 cells. Bethanechol, a nonselective muscarinic receptors agonist, stimulated GLP-1 secretion in a dose-dependent fashion, in accord with the results obtained with carbachol by Reimer et al. (19). We therefore performed experiments to establish which of the muscarinic receptor subtypes (M1–M3) is involved in GLP-1 secretion by these cells. Pirenzepine (an M1 muscarinic receptor subtype specific antagonist) and gallamine (an M2 muscarinic receptor subtype specific antagonist) both inhibited bethanechol-induced GLP-1 secretion, indicating the involvement of both the M1 and M2 muscarinic receptor subtypes in controlling GLP-1 secretion by the human L cell. In contrast, only the M1 muscarinic receptor regulates secretion from the adult rat L cell, whereas both the M1 and M2 muscarinic receptors are involved in GLP-1 secretion by fetal rat L cells (12). This may represent a species-specific difference in the regulation of GLP-1 secretion in humans and rats. Finally, 4-DAMP (an M3 muscarinic receptor subtype specific antagonist) had no effect on bethanechol-induced GLP-1 secretion by either the NCI-H716 or rat L cells (12), indicating that M3 muscarinic receptor subtypes are not involved in the control of GLP-1 secretion in either species. Specific agonists for M1 and M3 muscarinic receptor subtype further confirmed the involvement of the M1 but not of the M3 receptors in the regulation of GLP-1 secretion by NCI-H716 cells. As there is no M2-specific selective agonist available, we did not perform such experiments. Finally, as the NCI-H716 model is a cell line and not normal human L cells, we performed double immunofluorescence staining to show colocalization of GLP-1 and the M1, M2, or M3 muscarinic receptor subtypes in human small intestine sections. Our experiments, therefore, show that human L cells express M1, M2, and M3 muscarinic receptor subtypes, but that only the M1 and M2 receptors play a functional role in the regulation of GLP-1 secretion.

Muscarinic receptors subtypes are expressed in all parts of the gastrointestinal tract and are involved in the control of numerous gastrointestinal functions, including motility and exocrine pancreatic, gastric, and intestinal secretions (28). Interestingly, muscarinic receptors have also been found to regulate the secretion of several gastrointestinal hormones. Dumoulin et al. (29) showed that carbachol stimulates neurotensin, GLP-1, and peptide YY release from enteroendocrine cells in the isolated perfused rat ileum, whereas Zhang et al. (30) demonstrated that atropine blocks postprandial peptide YY release in dogs. Similarly, Sandor et al. (31) demonstrated that M1 but not M3 muscarinic receptors stimulate histamine release from gastric enterochromaffin-like cells, and Tanikawa et al. (32) reported that M3 muscarinic receptors mediate pancreatic polypeptide secretion. Thus, there appear to be marked cell-specific differences between the responses of different gastroenteropancreatic cells to muscarinic agonists.

The M1 muscarinic receptor is known to activate the PLC/calcium signaling pathway (33), a pathway that is well established to stimulate GLP-1 secretion from the L cell (25, 34). In contrast, the M2 muscarinic receptor subtype is negatively coupled to adenylyl cyclase through G{alpha}i (33). As the L cell is normally stimulated by activation of the PKA pathway (25, 34), the present finding of enhanced GLP-1 secretion in association with the M2 receptor appears contradictory. One potential explanation of the involvement of M2 muscarinic receptor subtypes in controlling GLP-1 secretion by NCI-H716 cells would be that the M2 receptor antagonizes an inhibitory tonus to the L cells. A similar model has been proposed to explain the predominance of M3 receptor effects in the face of significant M2 receptor expression in the gastrointestinal smooth muscle (35). Such relationships between these receptor subtypes in the regulation of GLP-1 secretion may be further testable in the established models of receptor subtype-specific deficiency, the M1, M2, and M3 receptor null mice (36, 37, 38). Although the M3 receptor knockout mice have been reported to exhibit impaired gastrointestinal contractility (38), none of these models have been investigated for other gastrointestinal abnormalities, nor have they been tested for alterations in GLP-1 secretion.

Interestingly, several studies have now demonstrated aberrant regulation of proglucagon gene expression in the NCI-H716 cells line (19, 22). Nonetheless, the results of both the present report and a previously published study (19) demonstrate that fatty acids stimulate and cholinergic blockers inhibit GLP-1 secretion in the NCI-H716 cells, consistent with in vivo data in the literature for humans (13, 17, 27). Although little is known about the factors that control proglucagon gene expression in the human intestine, these findings suggest that the NCI-H716 cells are a good model with which to investigate GLP-1 secretion from the human L cell.

In conclusion, the results of the present study demonstrate the involvement of M1 and M2 muscarinic receptor subtypes expressed by human L cells in the control of GLP-1 secretion. Because of its pleiotropic actions in nutrient homeostasis, GLP-1 is now under investigation as a potential treatment for patients with type 2 diabetes. Understanding the mechanisms controlling GLP-1 secretion from the enteroendocrine L cells in humans may therefore allow development of treatments to reduce hyperglycemia in patients with type 2 diabetes via enhancement of endogenous GLP-1 secretion.


   Acknowledgments
 
The authors are grateful to Dr. D. Drucker (University of Toronto) for providing the GLP-1 antibody, and to Dr. S. Asa and Ms. B. Terrett (University Hospital Network, Toronto, Ontario, Canada) for providing the human small intestinal sections.


   Footnotes
 
This work was supported by a Flora I. Nichol operating grant from the Canadian Diabetes Association. Y.A. was the recipient of a postdoctoral fellowship from the Canadian Diabetes Association (in the name of Margaret Francis), and P.L.B. is supported by the Canada Research Chair Program.

Abbreviations: 4-DAMP, 4-Diphenylacetoxy-N-methylpiperidine; FBS, fetal bovine serum; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide 1; PMA, phorbol 12-myristate 13-acetate.

Received January 28, 2003.

Accepted for publication April 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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