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BRIEF COMMUNICATION |
Department of Nutritional Sciences (D.W.N., D.M.N.) and Department of Comparative Biosciences (M.S.B.), School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706; and Department of Anatomy, Physiology, and Cell Biology (J.W.S., H.E.R.), University of California-Davis, School of Veterinary Medicine, Davis, California 95616
Address all correspondence and requests for reprints to: Denise M. Ney, University of Wisconsin-Madison, Department of Nutritional Sciences, 1415 Linden Drive, Madison, Wisconsin 53706. E-mail: ney{at}nutrisci.wisc.edu.
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Introduction |
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GLP-2 is a potent intestinal growth factor, although the pathway and cellular mechanisms responsible for GLP-2 action are largely unknown (2, 3, 4). The trophic actions of GLP-2 are thought to be mediated indirectly by paracrine and/or neural pathways because the GLP-2 receptor (GLP-2R) is not localized to the known target cells of GLP-2 trophic action, the intestinal epithelium (4). The GLP-2R has been localized to murine (12) and porcine (13) enteric neurons, subepithelial myofibroblasts (14), and human enteroendocrine cells (15). Peptide growth factors produced by intestinal subepithelial myofibroblasts (16) and enteroendocrine cells may act alone or integrate with neural pathways to induce GLP-2 action. However, the mechanisms underlying paracrine and/or neural regulation of GLP-2 action remain unknown.
The vagus nerve, a cranial nerve that contains both afferent and efferent fibers, innervates most of the GI tract including afferent sensory nerve endings within the GI mucosa and submucosa (17). The vagal pathway regulates digestive capacity by controlling gastric emptying, gastric acid secretion, and pancreatic secretion in response to nutrient load (17). Vagal afferents express receptors for many GI regulatory peptides and respond to signals from hormones, luminal nutrients, and mechanical stimuli in the intestinal wall. Centrally, vagal afferents synapse with efferents via interneurons in the nucleus of the solitary tract (NTS) of the brainstem and via projections to the dorsal vagal nucleus, the site of vagal efferent cell bodies. Several studies illustrate the role of the vagal pathway in integrating signals from intestinal hormones to stimulate central nervous system (CNS) activity. Vagal afferents express the cholecystokinin (CCK) type 1 receptor (CCK1R) and, in response to intestinal lipid, mediate CCK-dependant activation of the CNS to increase pancreatic secretion (18) and lipid-induced inhibition of gastric acid secretion, gastric emptying (18), and food intake (19). Additionally, vagal afferents express receptors for several other regulatory peptides released in response to luminal nutrients that alter GI function and food intake including CCK, leptin, cannabinoids, orexin, ghrelin, peptide YY, and GLP-1 (20, 21, 22).
The vagal pathway may modulate GLP-2 action, similar to other intestinal hormones such as CCK (18, 23). GLP-2 is a key hormonal mediator of intestinal adaptive growth, and we previously reported that vagal afferents are essential for maximal resection-induced adaptive growth in orally fed rats (24). Thus, the current study was designed to test the hypothesis that the vagal afferents express a functional GLP-2R. The specific aims were to determine whether 1) the GLP-2R is expressed on vagal afferents by localizing its expression to the nodose ganglia, 2) exogenous GLP-2 stimulates the vagal afferent pathway by measuring c-fos protein immunoreactivity in the NTS of rats, and 3) functional ablation of vagal afferents inhibits the intestinal growth induced by exogenous GLP-2 in rats maintained with total parenteral nutrition (TPN).
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Materials and Methods |
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GLP-2R IHC and Western blot
GLP-2R antibody.
We developed and characterized an antibody to the rat GLP-2R by targeting a specific peptide fragment within the receptor molecule and used our newly synthesized antibody to localize the GLP-2R to vagal afferents in the nodose ganglia and to enteric neurons and enteroendocrine cells in the small intestine in the rat. Using computer sequence analysis tools, we selected GLP-2R (65–88) extracellular-1 domain as a target for the production of rabbit polyclonal antipeptide antibodies. The full-length sequence was retrieved from the National Center for Biotechnology Information (NCBI) protein database and analyzed using GeneRunner (Hastings Software, Hastings on Hudson, NY), Peptide Structure-GCG Wisconsin Package (Accelrys, San Diego, CA), and NCBI BlastP tools as well as the publication of Yusta et al. (15). The peptide (TGSLLKETQKWANYKEKCLEDLH) selected was based on high indices of antigenicity, surface probability, and surface topology and has 100% homology with rat GLP-2R (1–80) and GLP-2R precursor (1–550). Cysteine was added to the C terminus as a cross-linking site for the preparation of peptide-protein immunogens and affinity media. The peptide was synthesized and its amino acid content verified by HPLC. Rabbits were injected with rat GLP-2R (acetyl-65–88-Cys-amide) conjugated to keyhole limpet hemocyanin once per month for 4 months (Open Biosystems, Huntsville, AL). Development of antisera was followed by ELISA directed against the GLP-2R peptide. Antisera were purified by affinity chromatography using an affinity matrix of Sulfolink gel (Pierce Chemical Co., Rockford, IL) coupled to the GLP-2R (65–88-Cys) peptide immunogen. To purify, crude antisera were diluted 1:1 in 10 mM Tris and applied to the column, and unbound proteins were eluted with the same buffer and then with 10 mM Tris/0.5 M NaCl and discarded. Bound antibody was eluted with 100 mM glycine (pH 2.5), and 1-ml fractions were collected into 1 ml of 1 M Tris (pH 8.0) to neutralize the pH of the fractions. The antibody fractions were extensively dialyzed against 0.02 M PBS and constituted to 1% BSA with 0.02% sodium azide. Affinity-purified antibodies were characterized by IHC and Western blot analysis using baby hamster kidney (BHK) cells transiently transfected with the pcDNA 3.1 expression vector encoding mouse, rat, or human GLP-2R (Fig. 1
). Cells were lysed 24 h after transfection, and protein extracts were resolved by SDS-PAGE under reducing conditions. Blots were probed with a 1:1000 dilution of our GLP-2R antibody in PBS containing 5% milk.
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Sections of the nodose ganglia were incubated in GLP-2R (rabbit polyclonal) primary antibody diluted at 1:1000 and anti-neuronal nuclei (anti-NeuN) antibody (mouse monoclonal MAB377; Chemicon International, Temecula, CA) diluted at 1:200 in GS-PBS (2% GS, 0.2% Triton X-100, and 0.1% BSA in PBS) for 1.5 h at 37 C. Anti-NeuN served as a neuronal marker and labels both in the cytoplasm and in the nucleus, often with predominate labeling in the nucleus. The slides were rinsed in PBS three times for 20 min each. Sections of the nodose ganglia were then incubated with fluorescently conjugated goat antirabbit and goat antimouse antibodies (antirabbit IgG-Alexa Fluor 488 and antimouse IgG-Alexa Fluor 546; Molecular Probes, Eugene, OR) both diluted 1:500 into GS-PBS for 45 min at 37 C. Slides were then washed twice for 15 min each at room temperature and then once at 4 C overnight with PBS and coverglasses mounted with GelMount (BioMeda, Foster City, CA). Control sections were incubated with a nonspecific rabbit IgG or specific antibody absorbed with GLP-2R (65–88) peptide instead of the primary GLP-2R antibody and labeled for the NeuN protein to identify neurons as outlined above. Immunostained sections were viewed and photographed using a Nikon Eclipse E600 microscope equipped with a SpotT digital camera (Diagnostic Instruments, Sterling Heights, MI), or a Bio-Rad (Radiance 2100; Hercules, CA) scanning confocal microscope.
Sections of small intestine were incubated with the GLP-2R primary antibody diluted at 1:1000 and the anti-NeuN antibody diluted at 1:200, with GLP-2R primary antibody diluted at 1:1000 and anti-serotonin (anti-5HT) antibody (Dakocytomation, mouse monoclonal anti-serotonin, MO758; Dako, Carpinteria, CA) diluted at 1:500, or with GLP-2R primary antibody diluted at 1:1000 and anti-Hu antibody diluted at 1:4000 (provided by Miles L. Epstein, Department of Anatomy, University of Wisconsin-Madison) in GS-PBS for 1.5 h at 37 C. The slides were rinsed in PBS three times for 20 min each. Then, sections of the ileum were incubated with fluorescently conjugated goat antirabbit and goat antimouse antibodies (antirabbit IgG-Alexa Fluor 488 and antimouse IgG-Alexa Fluor 546; Molecular Probes) both diluted 1:500 into GS-PBS for 45 min at 37 C. Slides were then washed twice for 15 min each at room temperature then once at 4 C overnight with PBS, and coverglasses were mounted. Control sections were conducted as outlined above. Immunolabeled sections were imaged as above.
Western blot.
Nodose ganglia and jejunum from five to six rats were removed and immediately snap-frozen on dry ice. Frozen tissue samples were homogenized in PBS (pH 7.4) containing protease inhibitors (Protease cocktail I; Upstate, Temecula, CA) and centrifuged at 10,000 x g for 15 min at 4 C. Nodose ganglia (100 µg) and jejunum (200 µg) samples were run on 10% SDS-PAGE. Separated proteins were transferred on a polyvinylidene difluoride membrane at 100 mA overnight at 4 C. After blocking with KPL block solution (KPL, Gaithersburg, MD; catalog no. 71-80-00) for 3 h, blots were incubated in 1:1000 anti-GLP-2R antibody from Alpha Diagnostics (catalog no. GLP2R11-A; San Antonio, TX) or anti GLP-2R antibody raised in our lab overnight at 4 C. Blots were washed in KPL wash solutions and incubated for 1 h in horseradish-peroxidase-conjugated antirabbit IgG (Sigma Chemical Co., St. Louis, MO) at room temperature. After washing, blots were treated with LumiGLO ReserveKPL chemiluminescent substrate (KPL; catalog no. 54-71-01). Bands were visualized by exposing the blots to x-ray film. The image was scanned, and band intensities were quantified using OptiQuant software (Packard Instruments, Meriden, CT).
Neural activity in the NTS measured by c-fos immunoreactivity
We evaluated the ability of exogenous GLP-2 (ip) to activate the vagal afferent pathway by measuring c-fos protein immunoreactivity in the NTS and used perivagal application of capsaicin to determine the role of vagal afferents in mediating brainstem activation. Expression of the immediate-early gene c-fos protein, Fos, can be used as an index of neuronal activity in the CNS (25). NTS activation in rats after ip administration of GLP-2 or saline was evaluated by counting the number of neuronal nuclei immunohistochemically labeled for Fos. Three days before GLP-2 treatment, rats received perivagal application of capsaicin (<1 mg/rat), a specific vagal afferent neurotoxin, or saline as previously described (24). Briefly, a cervical trunk of the vagus nerve was exposed, freed from the carotid artery, and wrapped in a cotton pledget. A drop of 1% capsaicin (10% Tween 80 in olive oil) was applied for 30 min before flushing the area with saline. The procedure was then repeated on the contralateral trunk of the vagus nerve. The dose of capsaicin used in the current study was previously reported to significantly reduce calcitonin gene-related peptide in the trachea by approximately 41%, consistent with partial functional ablation of vagal afferents (24).
A preliminary study was conducted with ip administration of 25, 100, or 200 µg human GLP-2 (California Peptide Research, Napa, CA) to establish the dose of ip GLP-2 sufficient to induce c-fos immunoreactivity. After an overnight fast, rats received an ip injection of 200 µg human GLP-2 or saline (n = 5 per group). At 120 min after injection of GLP-2 or saline, rats were deeply anesthetized and fixed by rapid infusion of 4% paraformaldehyde dissolved in 0.1 M PBS (pH 7.4). The brains were removed and postfixed in the paraformaldehyde solution for 1 h and then stored in PBS overnight at 4 C. Transverse brain sections (100 µm) were cut in ice-cold PBS by a vibratome. Brain sections were washed three times for 10 min each. All antibodies were diluted in a solution of 2% GS (Vector), 0.2% Triton X-100, and 0.1% BSA (Sigma) dissolved in PBS (GS-PBS). Sections were washed three times in PBS and then blocked for 1 h in GS-PBS. The blocking medium was drained, and sections were incubated with the anti-Fos antibody (SC-52, rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 for 3 h at room temperature and then overnight at 4 C. The antibody was drained away, and sections were washed three times for 10 min each with PBS. Sections were then incubated with biotinylated goat antirabbit secondary antibody (Vector) diluted 1:200. The secondary antibody was drained away; sections were washed three times in PBS for 10 min each. Sections were then incubated with the avidin-biotin complex solution (Standard Elite Vectasatin ABC Kit; Vector) for 3 h at room temperature. Sections were again washed three times with PBS for 10 min each. Sections were then incubated with 3,3-diaminobenzidine (DAB) dissolved in PBS (30 mg/100 ml) for 5 min at room temperature. H2O2 (0.3% in ddH2O) was added to the DAB solution (50 µl/ml), and the peroxidase reaction was allowed to continue until a light brown color emerged. Sections were taken through an ethanol-xylene dehydration procedure and mounted on slides with Depex mounting medium.
Brain sections were each assigned a rostral-caudal position relative to the boney skull landmark of bregma according to the anatomical features illustrated by Paxinos (26). Digital images of the NTS were made with a Provis optical imaging system. Counts of the numbers of immunopositive nuclei within the NTS were made with Scion Image (Scion, Frederick, MD) computer software. The total number of Fos-positive neurons was counted in the NTS for at least 10 sections per rat. Regions of the NTS sections were classified as being rostral to the area postrema (13.2–13.5 mm caudal to bregma), in the area postrema (13.6–14.2 mm caudal to bregma), and caudal to the area postrema (14.3–14.7 mm caudal to bregma).
Intestinotrophic action of exogenous GLP-2
We used a model of TPN-induced mucosal atrophy to determine whether functional ablation of vagal afferents by capsaicin would attenuate the ability of exogenous GLP-2 to reverse the mucosal atrophy induced by TPN. Rats were pretreated with capsaicin or vehicle and then fed orally or by TPN for 7 d. The TPN rats were given either TPN alone or TPN coinfused with GLP-2 via a jugular catheter (27). Exogenous human GLP-2 (100 µg/kg body weight·d) was continuously coinfused with TPN solution. This dose of GLP-2 was previously reported by our lab to preserve GI structure in TPN rats and normalize plasma concentration of bioactive GLP-2 compared with orally fed rats (28). To functionally ablate vagal afferents, we treated the rats with systemic capsaicin 8 d before beginning the experiment as previously described (24). Briefly, rats were anesthetized and received three sc injections of capsaicin over 24 h to a total dose of 125 mg/kg body weight. Treatment resulted in a significant 87% reduction in calcitonin gene-related peptide immunoreactivity in the stomach of capsaicin-treated rats (capsaicin, 0.07 ± 0.01 pmol/g; control, 0.56 ± 0.03 pmol/g; P < 0.001), indicating successful functional ablation of vagal afferents. Rats were maintained with nutritionally adequate TPN for 7 d (29) before being anesthetized and killed; intestinal tissue was collected as previously described (28). Briefly, mucosa (small intestine) or intact tissue (colon) was analyzed to determine dry mass and concentrations of protein and DNA (24).
Statistical analyses
Differences between treatment groups in each experiment were determined by one-way ANOVA followed by the protected least significant differences technique. All data are presented as mean ± SE. P < 0.05 was considered statistically significant.
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Results |
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). Results indicate that our GLP-2R antibody recognizes rat and human but not mouse GLP-2R. Specificity of the antibody for IHC was confirmed by peptide competition studies with the antigen to which the antibody was raised. Preabsorption of the GLP-2R antibody with the peptide antigen or secondary antibody completely abolished antibody staining in sections of rat jejunum.
The GLP-2R is differentially glycosylated, similar to many receptors with multiple extracellular domains, as verified in Fig. 1. Whole-cell lysate or immunoprecipitated protein was treated with peptide N-glycosidase F (PNGase F) to remove the carbohydrate residues, and the blots were probed with anti-GLP-2R antisera, which shifted the GLP-2R protein to a single immunoreactive band at approximately 50 kDa, as reported using a different antiserum (31). Taken together, these data support the high specificity of our new GLP-2R antibody to multiple glycosylated forms of the rat GLP-2R for Western assay and IHC.
GLP-2R localization
We used our antirat GLP-2R antibody to localize GLP-2R immunoreactivity in the nodose ganglia and small intestine of rats. GLP-2R immunoreactivity was demonstrated for the first time on nerve cell bodies in nodose ganglia, where cell bodies of vagal afferent neurons are located, using IHC (Fig. 2). Immunoreactivity was pronounced throughout the soma (perikaryon) and absent in the nucleus of vagal afferents in the nodose ganglia. Colocalization of GLP-2R protein to cells immunoreactive for vertebrate neuron-specific nuclear protein NeuN, predominately present in the nucleus, confirms GLP-2R expression on vagal afferent neurons in nodose ganglia (Fig. 3). In addition to IHC, we confirmed the presence of the GLP-2R in nodose ganglia by Western blot using a commercially available GLP-2R antibody (Alpha Diagnostics no. GLP2R11-A) and our new GLP-2R antibody. An immunoreactive band at approximately 66 kDa was detected based on Western blots of tissue homogenates from rat nodose ganglia using each antibody (Fig. 4).
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) (12, 13, 15). Moreover, colocalization of GLP-2R immunoreactivity with Hu, the neuron-specific RNA-binding protein shown to have extensive colocalization with other neuronal markers (32), establishes that the GLP-2R strongly localizes to enteric neurons within the muscularis (Fig. 5).
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). There were a small number of c-fos-immunoreactive neurons in saline-treated rats (140 ± 39) and a significant 5-fold increase with GLP-2 infusion (853 ± 230; Fig. 6B). Increased activation was more pronounced in the NTS rostral to the area postrema and within the area postrema region (Fig. 6C). To determine whether vagal afferents mediate GLP-2 signaling to the NTS, we applied perivagal capsaicin to ablate vagal afferent function. In capsaicin-treated rats, there were no Fos-positive neurons visible in the NTS after ip injection of saline or GLP-2. Capsaicin treatment was applied locally to the vagus nerve, and at doses used for this study, resulting systemic levels did not affect the behavior of treated rats after they recovered from surgery. Examination of the NTS and surrounding brain tissue showed no observable pathological changes as a result of capsaicin treatment. Thus, we conclude that the absence of c-fos immunoreactivity in capsaicin-treated rats was not due to damage to the NTS.
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). Coinfusion of GLP-2 with TPN solution reversed the TPN-induced atrophy in duodenum and jejunum based on significant increases in mucosal dry mass, protein, and DNA. Ileum showed significant increases in dry mass and protein but not DNA. Pretreatment with capsaicin to functionally ablate vagal afferents did not attenuate the intestinotrophic effects of GLP-2 to reverse the TPN-induced mucosal hypoplasia throughout the small bowel. There was no effect of TPN alone or TPN plus GLP-2 in the colon.
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Discussion |
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Expression of the GLP-2R is primarily restricted to the brain and the GI tract (34, 35), although the precise cellular distribution of GLP-2R expression in the GI tract has been controversial (4). In the absence of a good commercial antibody at the time this research was conducted, we developed our own antibody to localize GLP-2R expression within the small intestine. We have confirmed previous findings colocalizing the receptor to enteric neurons (12, 13) expressing NeuN or Hu and enteroendocrine cells expressing 5HT (15).
To understand the functional significance of the localization of the GLP-2R to vagal afferent neurons, we examined activation of the vagal afferent pathway by measuring the expression of Fos-positive neurons in the NTS, the region of the brainstem where vagal afferents terminate. It is well established that peripheral administration of CCK, for which receptors are also expressed by vagal afferents, activates the brainstem via a capsaicin-sensitive vagal afferent pathway (23). The ip injection of GLP-2 stimulated neural activity in the NTS based on a significant 5-fold increase in the number of neurons expressing c-fos protein immunoreactivity compared with saline. This is in agreement with previous reports indicating that ip injection of CCK-8 and iv injection of ghrelin induce c-fos immunoreactivity in the NTS via CCK1R (23) and ghrelin receptors (21) present on the vagus nerve, respectively. These observations, in combination with localization of the GLP-2R to vagal neurons in the nodose ganglia, suggest that peripheral administration of GLP-2 stimulates vagal sensory input to the brainstem. Moreover, functional ablation of vagal afferents by capsaicin treatment abolished c-fos protein immunoreactivity in the NTS after ip administration of GLP-2, suggesting that activation of the NTS due to GLP-2 is dependent on vagal afferents. Results also demonstrate that normal neuronal activity from the vagus produces low-level background c-fos immunoreactivity in the NTS, which becomes undetectable after capsaicin treatment. This is in agreement with a previous report indicating that perivagal application of capsaicin completely abolishes c-fos immunoreactivity in the NTS and blocks increased food intake after peripheral administration of ghrelin but does not alter the food intake response to centrally administered ghrelin (21). Thus, although centrally administered GLP-2 directly induces CNS activation in the hypothalamus (35), we conclude that peripheral administration of GLP-2 is dependent on vagal afferent input for central activation in the NTS.
TPN is a well characterized model for studying GI atrophy due to the lack of luminal nutrients without the metabolic complications of fasting (29, 36, 37). To further characterize the role of the GLP-2R-activated vagal afferent pathway in the intestinotrophic response to GLP-2, we used exogenous GLP-2 and capsaicin in the TPN model to determine the role of vagal afferents in modulating the intestinotrophic effects of GLP-2 in preventing TPN-induced mucosal atrophy. As in previous studies in rodents (38, 39) and pigs (33, 40), we observed significant mucosal atrophy in the small bowel due to TPN. Furthermore, exogenous GLP-2 prevented TPN-induced mucosal atrophy as determined by increased mucosal dry mass and concentrations of protein and DNA in the small bowel (33, 38, 39, 40). However, functional ablation of vagal afferents with capsaicin did not attenuate GLP-2 action in the TPN model where exogenous luminal nutrients are absent.
The present data indicate that vagal afferents are not essential for the intestinotrophic effects of exogenous GLP-2 during TPN. This finding is in agreement with our previous report (24) where functional ablation of vagal afferents with capsaicin failed to attenuate resection-induced growth in TPN rats. However, in that study, vagal afferents were essential for resection-induced adaptive growth in orally fed rats (24). This suggests that vagal afferents likely work to mediate GLP-2 action through mechanisms involving stimulation by luminal nutrients. Thus, the role of vagal afferents in GLP-2 action is different depending on the presence of luminal nutrients and the experimental model, e.g. in the post-resection hyperproliferative state with elevated endogenous GLP-2 vs. the TPN-induced hypoplastic state with exogenous GLP-2. Taken together with our previous publication and this study, it seems that GLP-2 may act both through vagally mediated pathways and through pathways independent of vagal afferent function to induce GI adaptive growth.
In summary, in the present study, we demonstrate localization of the GLP-2R, for the first time, to vagal afferents in the nodose ganglia and show that peripheral administration of GLP-2 stimulates neural activity in the CNS in a manner dependent on vagal afferents. This research adds to the growing list of intestinal hormones including CCK, ghrelin, 5HT, and GLP-1 that target vagal afferents (17, 21) and emphasizes the important role of the vagal afferents in integrating input from a variety of intestinal hormones. The inability of capsaicin to attenuate the intestinotrophic effects of GLP-2 during TPN, taken together with our previous observation that vagal afferents are essential for maximal resection-induced mucosal adaptive growth in orally fed rats (24), emphasizes potential differences in the role of vagal innervation and GLP-2 action in different physiological states. Additional research is needed to elucidate the cellular mechanisms by which a vagal pathway may mediate the intestinotrophic effects of GLP-2.
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Acknowledgments |
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, along with Sangita Murali for developing the Western blot for the GLP-2R. |
Footnotes |
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Disclosure Statement: D.W.N., D.M.N., J.W.S., and H.E.R. have nothing to declare. M.S.B. consults for Immunostar, Inc. (Stillwater, MN).
First Published Online January 18, 2007
Abbreviations: BHK, Baby hamster kidney; CCK, cholecystokinin; CCK1R, CCK type 1 receptor; CNS, central nervous system; DAB, 3,3-diaminobenzidine; GI, gastrointestinal; GLP-2, glucagon-like peptide-2; GLP-2R, GLP-2 receptor; GS, goat serum; 5HT, serotonin; IHC, immunohistochemistry; NeuN, neuronal nuclear protein; NTS, nucleus of the solitary tract; PNGase F, peptide N-glycosidase F; TPN, total parenteral nutrition.
Received September 8, 2006.
Accepted for publication January 11, 2007.
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