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Intestinal inflammation
Original article
Glucagon-like peptide-1 as a treatment for chemotherapy-induced mucositis
  1. Hannelouise Kissow,
  2. Bolette Hartmann,
  3. Jens Juul Holst,
  4. Steen Seier Poulsen
  1. Department of Biomedical Sciences and the Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark
  1. Correspondence to Dr Hannelouise Kissow, Department of Biomedical Sciences and the Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health Science, University of Copenhagen, Blegdamsvej 3, Copenhagen DK-2200, Denmark; kissow{at}sund.ku.dk

Abstract

Background Glucagon-like peptide-2 (GLP-2) has been suggested for the treatment of mucositis, but the peptide has also been shown to accentuate colonic dysplasia in carcinogen-treated mice. Recently, an effect on intestinal growth was discovered for glucagon-like peptide-1 (GLP-1),

Objective To determine whether endogenous GLP-1 contributes to the healing processes and if exogenous GLP-1 has a potential role in treating mucositis.

Methods Mice were injected with 5-fluorouracil (5-FU) or saline to induce mucositis and were then treated with GLP-1, GLP-2, GLP-2 (3-33), exendin (9-39) or vehicle. The mice were sacrificed 48 or 96 h after the 5-FU injections. The end points were intestinal weight, villus height, proliferation and histological scoring of mucositis severity. Rats were injected with 5-FU or saline, and after 48 h, blood was drawn and analysed for GLP-1 and GLP-2 concentration.

Results GLP-1 and GLP-2 significantly prevented the loss of mucosal mass and villus height and significantly decreased the mucositis severity score in the duodenum and jejunum 48 h after chemotherapy. The effect was equivalent. Exendin (9-39) reduced the intestinal weight 96 h after chemotherapy. The GLP-1 levels in blood were increased more than 10-fold, and GLP-2 levels were increased sevenfold.

Conclusions GLP-1 and GLP-2 were secreted after intestinal injury, and recovery was delayed after treatment with exendin (9-39), indicating an important role for the peptides in the protection of the intestine from injury. GLP-1 treatment ameliorated mucositis, which suggests that mucositis and other acute intestinal disorders might benefit from treatment with GLP-1 analogues.

  • Gut Hormones
  • Mucosal Repair
  • Mucosal Injury
  • Gastrointestinal Peptides
  • Chemotherapy

https://doi.org/10.1136/gutjnl-2012-303280

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    Significance of this study

    What is already known about the subject?

    • Glucagon-like peptide-1 (GLP-1) is an incretin hormone, and receptor agonists (RAs) have been developed for the treatment of type 2 diabetes mellitus.

    • Gastrointestinal mucositis is a severe injury to the intestine with a significant affect on the health, economic outcome and quality of life of patients with cancer receiving chemotherapy.

    • No treatment is available for this condition.

    • The intestinal growth-factor glucagon-like peptide-2 (GLP-2) has previously been suggested as a treatment for mucositis.

    What are the new findings?

    • The GLP-1 RA liraglutide can prevent severe mucositis in an experimental animal model.

    • The treatment effect is comparable to the effect of GLP-2.

    • GLP-1 and GLP-2 are both secreted in large amounts as a result of mucositis induction.

    • The GLP-1 receptor antagonist exendin (9-39) prevented spontaneous healing after mucositis, suggesting the involvement of endogenous GLP-1 in its pathogenesis.

    How might it impact on clinical practice in the foreseeable future?

    • These new findings may result in the further exploration of the possible role of GLP-1 in the pathogenesis of intestinal disorders and of GLP-1 RAs for the prevention of mucositis caused by chemotherapy in patients with cancer.

    Introduction

    Chemotherapy-induced mucositis is a common complication of anticancer treatments, and may reduce the effectiveness of treatment because it often requires a dose reduction and impairs the quality of life of the patient. Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide secreted by enteroendocrine L-cells located in the small and large intestines.1 This peptide has been suggested for the treatment of chemotherapy-induced mucositis,2 ,3 but the peptide has also been shown to accentuate colonic dysplasia in carcinogen-treated mice.4–6

    Glucagon-like peptide-1 (GLP-1) is secreted in parallel with GLP-2 from the L-cells. This secretion is stimulated by food intake, and GLP-1 participates in the maintenance of glucose homoeostasis by stimulating pancreatic β-cells to secrete insulin in a glucose-dependent manner.7 Both GLP-1 and GLP-2 are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4). Because of the insulin-stimulating properties of GLP-1, several GLP-1 receptor agonists (GLP-1 RAs) (resistant to degradation) are being and have been developed for the treatment of type 2 diabetes mellitus. Simonsen et al8 investigated whether exendin-4, a GLP-1 RA, also has intestinal effects, and they unexpectedly found a trophic effect of GLP-1 in the rat small intestine. We reproduced this effect in mice9 and also found increased GLP-1 secretion after intestinal injury in rats (unpublished data). To our knowledge, GLP-1 has not been proposed as a treatment for intestinal disorders. We hypothesised that endogenous GLP-1 is as important as GLP-2 for recovery after chemotherapy. To investigate this hypothesis, we examined the epithelial proliferation after chemotherapy with or without GLP-1 and GLP-2 receptor antagonists and the effects of GLP-1 RAs for the treatment of chemotherapy-induced intestinal damage in mice to compare the protective effects of GLP-1 and GLP-2.

    Materials and methods

    Animals

    The animal studies were approved by the Danish National Committee for Animal Studies. Female CD1 mice (Charles River, Germany) weighing between 20 and 30 g and male Wistar rats weighing approximately 300 g (Taconic, Denmark) were housed in the animal facilities at the Panum Institute, Copenhagen, Denmark. They were kept in temperature (21°C)- and humidity (55%)-controlled rooms with light/dark cycles of 12 h in individually ventilated cages. They were maintained throughout the study on water and chow (no. 1314, Altromin, Germany) ad libitum. Because our test system requires more than the limited amount of blood that can be acquired from a mouse, rats were used to establish whether the plasma levels of GLP-1 and GLP-2 were increased after chemotherapy. The mice were used in the antagonist and treatment studies.

    All animals were monitored every day for signs of severe mucositis, such as cachexia, diarrhoea, bloody stools or piloerection.

    Test solutions

    We used a DPP-4-resistant GLP-2 analogue, NNC-103-0066 (Novo Nordisk, Denmark), referred to as NNC-GLP-2, for these studies,10 and liraglutide (Victoza, Novo Nordisk, Denmark) in a ready-to-use solution purchased from a pharmacy. The dosage of NNC-GLP-2 was 25 µg given subcutaneously twice daily. The concentration of the ready-to-use liraglutide solution was 6 mg/ml, and the initial dose was 0.1 ml twice a day by subcutaneous injection, equivalent to 600 μg×2. With this dose, the animals lost weight (2–5 g) and stopped eating for the first 24 h of treatment. The dosage was then adjusted to 0.05 ml (300 μg) twice a day from day −1. To antagonise the GLP-2 receptor, we used human GLP-2 (3-33) (Schafer-N, Denmark), a partial agonist that acts as a competitive antagonist at the GLP-2 receptor, at a dose of 50 ng twice a day11–13; to antagonise the GLP-1 receptor, we used exendin (9-39) (Bachem, Switzerland) at a dose of 25 µg twice a day.14 All peptides except liraglutide were dissolved in 100 μl phosphate-buffered saline containing 3.5 mg/ml Haemaccel (Behringwerke AG, Germany). Phosphate-buffered saline with Haemaccel served as control solution in all studies and is referred to as vehicle.

    The concentrations of the peptides in the test solutions were verified with a radioimmunoassay, as previously described15 using a side-viewing GLP-2 antiserum (code HYB 312-01, in-house) for the detection of GLP-2 (3-33),16 an N-terminal GLP-2 antiserum (code 92160, in-house) for the detection of NNC-GLP-215 and exendin-4 antiserum (code 3145-3, in-house) for the detection of exendin (9-39).17

    Experimental protocol for the mouse study

    Mucositis was induced with a single intraperitoneal (IP) injection of 5-fluorouracil (5-FU) 400 mg/kg (Hospira Nordic AB, Sweden).

    We performed five experiments in total, each including 32 CD1 mice. In each experiment, the mice were divided into four groups: two groups had 5-FU injected at day 0, and two groups had saline. In each experiment, one chemotherapy-injected group (chemo) and one saline-injected group (no chemo) were treated with the test solution, and one chemotherapy-injected group and one saline-injected group were treated with vehicle. The test solutions were exendin (9-39) and GLP-2 (3-33) in the two experiments performed in study 1, NNC-GLP-2 and liraglutide in the two experiments performed in study 2 and exendin (9-39) in the one experiment performed in study 3.

    Study 1: The effect of the GLP-1 and GLP-2 receptor antagonists on the compensatory epithelial proliferation after chemotherapy

    The mice were treated with the antagonists (exendin (9–39) and GLP-2 (3–33)) from the morning of day 0 until day 4, when the compensating hyperproliferation was established in pilot studies to be at its maximum. 5-FU was injected on day 0 at noon. Food intake was monitored, and body weight (BW) was recorded on days 0 and 4. On the day of sacrifice the mice received an IP injection of bromodeoxyuridine (BrdU) 50 mg/kg (Sigma-Aldrich, Germany) 2.5 h before they were anaesthetised. After anaesthesia with ketamine/xylazine 100/10 mg/kg IP (Pharmacy Service, Denmark), the animals were weighed and the abdomen was opened. Blood was drawn from the vena cava inferior. The intestines were moved and gently flushed intraluminally with saline, and the intestinal weight was recorded. The tissue samples from the duodenum, jejunum, ileum and mid-colon were fixed in ice-cold buffered 4% paraformaldehyde for 24 h and thereafter stored in 70% ethanol.

    Study 2: The effect of the GLP-1 and GLP-2 RAs on chemotherapy-induced mucositis

    The mice were treated with the agonists (NNC-GLP-2 and liraglutide) from day −2 until sacrifice on day 2, when intestinal injury was expected to be at its maximum. The 5-FU injection was given on day 0 at noon. BW and food intake were recorded each day. Sacrifice was performed as described above.

    Plasma insulin

    The concentrations of insulin in the plasma from study 2 with liraglutide were measured with a mouse insulin ELISA kit (Mercodia 10-1247-01, Sweden). The manufacturer's protocol was followed.

    Study 3: The effect of the GLP-1 receptor antagonist on chemotherapy-induced mucositis

    The mice were treated with the antagonist exendin (9-39) from day −2 until sacrifice at day 2. The 5-FU injection was given on day 0 at noon. BW and food intake were recorded each day. Sacrifice was performed as described above.

    Experimental protocol for the rat study (alterations in GLP-1 and GLP-2 plasma concentrations)

    In total, 16 rats received an IP injection with either 5-FU 400 mg/kg or saline. After 48 h, the rats were anaesthetised with ketamine/xylazine 100/10 mg/kg IP and blood was drawn from the vena cava inferior (distal from the vena portae) and the vena portae simultaneously. Before euthanisation, a small piece of the ileum was removed and fixed in 4% buffered paraformaldehyde for histological examination to confirm the presence of mucositis. Blood was collected in chilled tubes containing EDTA and valine-pyrrolidide (a DPP-4 inhibitor, Novo Nordisk, Denmark) at final concentrations of 3.9 mmol/l and 0.01 mmol/l, respectively. The samples were centrifuged for 10 min at 2400×g and 4°C, and the plasma was kept at −20°C until analysis. For the determination of GLP-1, the plasma was extracted with 70% ethanol before analysis. For the determination of GLP-2, the plasma was extracted with 75% ethanol before analysis. Hormone concentrations were determined by radioimmunoassay as previously described, using an N-terminal specific antiserum (code 92160, in-house) to measure active GLP-2 only15 and a side-viewing antiserum (code 2135, in-house) to measure all molecules that contain the mid-sequence of GLP-1.18

    Histological sections and morphometric estimates

    The fixed tissue samples were dehydrated, embedded in paraffin and cut into 5 μm sections using a microtome. The sections from the mice from studies 2 and 3 that had received chemotherapy were evaluated for the degree of mucositis using a method described previously by Howarth et al.19 A total score for each region of the intestine (duodenum, jejunum and ileum) was obtained by rating 10 histological characteristics of mucositis from 0 (normal) to 3 (severe). The criteria used were villus stunting/atrophy, villus fusion, disruption of enterocytes, crypt loss, architectural disruption of crypts, disruption or distortion of crypt cells, infiltration of inflammatory cells, dilatation of lymphatic vessels and capillaries and thickening or oedema of the submucosa or lamina muscularis. Sections from the rat study were only evaluated to confirm the presence of mucositis.

    To measure the area of the mucosal layer, the crypt depth and villus height, transverse sections from the duodenum, jejunum, ileum and colon from each animal in all mouse studies were stained with haematoxylin/eosin and examined with a light microscope connected to a camera (Zeiss Axioscope 2 plus, Brock & Michelsen, Denmark). Photomicrographs were analysed using Image Pro 7.0 software (Media Cybernetics, USA).

    To determine the proliferative activity, BrdU immunohistochemistry was performed on sections from the duodenum, jejunum, ileum and colon from each animal. Antigen retrieval was performed by heating the sections for 15 min in a microwave oven (750 W) in citrate buffer pH 6.0. The sections were then preincubated for 10 min in 2% bovine serum albumin, followed by overnight incubation with a monoclonal mouse anti-BrdU antibody Ab-4(Bu20a) (Thermo Scientific, USA) diluted 1 : 200.

    To visualise the immunoreactions, the sections were incubated for 40 min with biotinylated horse anti-mouse IgG (Vector BA-2000 Vector Laboratories, USA) diluted 1:200, followed by StreptABComplex/horseradish peroxidase (Vectastain ABC kit, Vector Laboratories, USA), and finally developed with 3, 3’-diaminobenzidine containing nickel ammonium sulphate (Sigma-Aldrich, Denmark) for 15 min. Proliferation was quantified as the area of BrdU immunoreactive cells per crypt, measured in 10 crypts for each section.

    Statistics

    All results are shown as means and SE of the mean (SEM). The comparison between groups was performed with analysis of variance, followed by post hoc comparisons between the two chemo groups and the two no chemo groups with Bonferroni's test to evaluate the influence of the test solutions in either the chemotherapy or saline-injected mice. Dunnett's multiple comparison test was used to compare all groups in each experiment with the no chemo vehicle-treated group, using the latter as a healthy untreated control. Student's t test was used to compare the plasma levels between two groups in the rat study. All analyses were performed using GraphPad Prism V5.01, and probability values p<0.05 were considered significant.

    Results

    Study 1

    The mice given chemotherapy showed, as expected, a decrease in food intake and BW compared with the saline-injected mice. No differences in food intake were seen between the controls and mice treated with exendin (9-39) or GLP-2 (3-33) (figure 1A–D).

    Figure 1
    Figure 1

    Study 1: Food intake and body weight (BW) loss in mice after chemotherapy or saline injection, treated with the GLP-1 R antagonist exendin (9-39) (A,B) or the GLP-2 R antagonist GLP-2 (3-33) (C,D) for 4 days. Intestinal weight relative to BW after chemotherapy or saline injection treated with exendin (9-39) (E,F) or GLP-2 (3-33) (G,H) for 4 days. *p<0.05 compared with vehicle (analysis of variance (ANOVA) followed by Bonferroni's test). a=p<0.05 compared with no chemo vehicle (ANOVA followed by Dunnett's multiple comparison test).

    The mice receiving chemotherapy corresponded to a normal healthy mouse (no chemo vehicle) when we measured the relative small intestinal weight 4 days after chemotherapy (figure 1E–H). Treatment with GLP-2 (3-33) had no influence on this outcome, but treatment with exendin (9-39) prevented the intestine from returning to its normal weight after the injury (3.1% of BW (±0.15) vs 3.6% (±0.2) of BW p<0.05). Surprisingly, exendin (9-39) treatment of the no chemo mice increased the small intestinal weight (4.0% of BW (±0.1) vs 3.6% of BW (±0.1) p<0.05), resulting in a large gap between the chemo and no chemo exendin (9-39)-treated mice. The relative colon weight increased after chemotherapy, but GLP-2 (3-33) and exendin (9-39) had no influence on this outcome (figure 1E–H).

    The morphometric analysis of the cross-sectional area, villus height and crypt depth of the duodenum, jejunum, ileum and colon confirmed that there were no differences between GLP-2 (3-33)-treated mice and vehicle-treated mice with or without chemotherapy (data not shown). The cross-sectional area was still decreased in the duodenum and jejunum after chemotherapy, and this result was significantly more pronounced in the jejunum in the exendin (9-39) group (table 1). Villus height was decreased and crypt depth was increased in all parts of the small intestine after chemotherapy, and thus, the crypt:villus ratio was increased twofold. Treatment with either GLP-2 (3-33) or exendin (9-39) had no influence on these outcomes.

    View this table:
    Table 1

    Morphometric estimates of the intestine 4 days after a single injection of 5-fluorouracil (400 mg/kg) or saline, treated with either exendin (9-39) 25 μg twice a day or vehicle (cross-sectional area of the mucosa in mm2, villus height and crypt depth in μm)

    Morphometric analysis showed no difference in colonic tissue samples.

    The estimation of the area of BrdU-immunoreactive cells in the small and large intestines showed a large increase in proliferation in all investigated parts of the intestine 4 days after chemotherapy. The proliferation was increased approximately fourfold in the small intestine and sixfold in the colon. Treatment with the antagonists GLP-2 (3-33) and exendin (9-39) did not influence compensatory hyperproliferation after chemotherapy (data not shown).

    Study 2

    Treatment with NNC-GLP-2 did not influence the food intake, but treatment with NNC-GLP-2 protected the mice from BW loss after chemotherapy (1.6 g (±0.5) vs 0.4 g (±0.3) p<0.05) (figure 2D). The liraglutide-treated mice showed a markedly reduced food intake and a reduced BW during the first 24 h, but decreasing the liraglutide dosage to 0.05 ml completely normalised both parameters (figure 2A,C). Treatment with either of the peptides abolished the considerable decrease in intestinal weight relative to BW observed after chemotherapy. This result was highly significant for both liraglutide (5.8% of BW (±0.1) vs 4.0% of BW (±0.1) p<0.001) and NNC-GLP-2 (4.4% of BW (±0.1) vs 3.6% of BW (±0.1) p<0.01) (figure 2E–H). Insulin was measured in the liraglutide study, and there was no difference between groups, either in the chemo (0.17 μg/l (±0.02) vs 0.18 μg/l (±0.02)) or the no-chemo groups (0.17 μg/l (±0.03) vs 0.22 μg/l (±0.01)).

    Figure 2
    Figure 2

    Study 2: Food intake and body weight (BW) loss in mice after chemotherapy or saline injection, treated with the GLP-1 R agonist liraglutide (A,B) or the GLP-2 R agonist NNC-GLP-2 (C,D) from two days before until two days after chemotherapy. Intestinal weight relative to BW after chemotherapy or saline injection treated with liraglutide (E,F) or NNC-GLP-2 (G,H) from 2 days before until end of experiment 2 days after chemotherapy. **p<0.01 and ***p<0.001 compared with vehicle (analysis of variance (ANOVA) followed by Bonferroni's test). a=p<0.05 compared with no chemo vehicle (ANOVA followed by Dunnett's multiple comparison test).

    To investigate the effect of the different treatments on the intestinal mucosa, we measured the cross-sectional area of the mucosa in different parts of the intestine. Both liraglutide and NNC-GLP-2 significantly prevented the chemotherapy-induced reduction in the mucosal area in the duodenum (liraglutide 3.7 mm2 (±0.2) vs vehicle 2.7 mm2 (±0.2), p<0.01 and NNC-GLP-2 3.3 mm2 (±0.3) vs vehicle 2.4 mm2 (±0.2), p<0.01) and in the jejunum (liraglutide 2.6 mm2 (±0.2) vs vehicle 1.9 mm2 (±0.2), p<0.05 and NNC-GLP-2 2.5 mm2 (±0.1) vs vehicle 1.7 mm2 (±0.1), p<0.001). For both peptides, the treatment resulted in a mucosal area comparable with that of the healthy mice (figure 3).

    Figure 3
    Figure 3

    Study 2: Cross-sectional area of the mucosa layer and villus height in duodenum, jejunum and ileum in mice after chemotherapy or saline injection. Mice were treated with the GLP-1 R agonist liraglutide or the GLP-2 R agonist NNC-GLP-2 from 2 days before until end of experiment 2 days after chemotherapy. *p<0.05, **p<0.01 and ***p<0.001 compared with vehicle (analysis of variance followed by Bonferroni's test).

    The villus height was significantly decreased in all chemo mice treated with vehicle, but after treatment with NNC-GLP-2 or liraglutide, the villus height remained comparable to that of the healthy control mice in all parts of the intestine (figure 3).

    Regarding the disease activity in the chemo mice treated with NNC-GLP-2, the mucositis scores were significantly lower in the duodenum (8.71 (±0.68) vs 11.40 (±1.02), p<0.05) and in the jejunum (10.88 (±0.72) vs 13.50 (±0.95), p<0.05). Similar results were seen in the liraglutide-treated group (10.63 (±0.94) vs 13.50 (±0.87), p<0.05 in the duodenum and 14.00 (±0.95) vs 17.25 (±2.55), p<0.05 in the jejunum) (figure 4A).

    Figure 4
    Figure 4

    Study 2: Mucositis severity score in duodenum, jejunum and ileum in mice after chemotherapy injection. Mice were treated with the GLP-1 R agonist liraglutide or the GLP-2 R agonist NNC-GLP-2 from 2 days before until 2 days after chemotherapy (A). Area of bromodeoxyuridine (BrdU) immunoreactive cells per crypt as a measure of proliferation rate in mice after chemotherapy or saline injection treated with liraglutide or NNC-GLP-2 from 2 days before until end of experiment 2 days after chemotherapy. *p<0.05 and ***p<0.001 compared with vehicle (analysis of variance followed by Bonferroni's test).

    The proliferative activity almost stopped in all chemo mice, and neither NNC-GLP-2 nor liraglutide prevented this outcome. However, we found that NNC-GLP-2 treatment in the no chemo mice significantly increased the natural proliferation rate (figure 4B).

    Study 3

    The chemo mice showed reductions in BW, food intake, small intestinal weight, cross-sectional area of the small intestine and villus height compared with the no chemo mice, as seen in study 2. Treatment with the GLP-1 R antagonist exendin (9-39) did not influence any of these parameters (data not shown).

    Results of the rat study (alterations in GLP-1 and GLP-2 plasma concentrations)

    The concentrations of GLP-1 and GLP-2 were measured in blood from the vena cava and vena portae 48 h after the chemotherapy or saline injections. In the blood from the vena cava, the concentrations of both peptides were significantly increased (GLP-1 109 pmol/l (±11) vs 10 pmol/l (±2), p<0.001 and GLP-2 70 pmol/l (±8) vs 11 pmol/l (±1), p<0.001) (figure 5A). Similar results were seen in the blood from the vena portae (GLP-1 195 pmol/l (±25) vs 38 pmol/l (±2), p<0.001 and GLP-2 149 pmol/l (±11) vs 19 pmol/l (±2), p<0.001). There was a significant correlation between the GLP-1 and GLP-2 concentrations in both the vena cava and vena portae. There was a significant correlation between GLP-2 concentrations in the vena portae and GLP-2 concentrations in the vena cava, but with approximately half of the amount in the vena cava. This result was seen in both the chemo and no chemo rats, and the slope was almost the same for the two groups (figure 5C,D). There was no correlation between the GLP-1 concentrations in the vena cava and in the vena portae (data not shown).

    Figure 5
    Figure 5

    Rat study: Plasma concentrations of GLP-1 and GLP-2 obtained from vena cava and vena portae in rats 48 h after chemotherapy or saline injection (A). Correlations between plasma concentrations of GLP-1 and GLP-2 obtained from vena cava and vena portae (B). Correlation between plasma concentrations of GLP-2 obtained from vena cava or vena portae 48 h after saline injection (C) or chemotherapy injection (D). ***p<0.001 compared with no chemo (Student's t test).

    Discussion

    Mucositis does not solely involve direct damage to the intestinal surface epithelium, leading to cell death, atrophy and ulceration, but also involves connective tissue elements in the lamina propria.20 The process can be divided into five phases, starting with the initiation phase, where the generation of reactive oxygen species appears to be a primary event, leading to the primary damage response with the upregulation and generation of messenger signals. Reactive oxygen species and chemotherapy cause DNA damage and cell death directly, but at the same time, transcription factors activated by chemotherapy with NF-κB have been suggested to play a key role in the genesis of mucositis. As a consequence, proinflammatory cytokine genes are upregulated, leading to the third phase in which the signalling and amplification of a number of pathways lead to apoptosis. In this phase, the tissue may still have a normal appearance. The next phase, the ulceration phase, involves epithelial defects or erosions and carries a risk of bacterial infiltration. Finally, the tissue enters a healing phase with epithelial proliferation and differentiation.21 We found that both GLP-1 and GLP-2 are secreted in increased amounts after chemotherapy. We measured the peptide levels 48 h after 5-FU injection, and from simultaneous histological analyses, we determined the intestinal tissue to be somewhere between the signalling/amplification phase and the ulceration phase, which coincides with the increase in proinflammatory cytokines observed before the severe histological damage appears.22 The increased secretion of GLP-1 and GLP-2 could be the endogenous response to mucosal damage, leading to the initiation of the healing phase. The increased GLP-2 levels are probably also responsible for the small increase in the colon weight after chemotherapy.11

    The correlation between the concentrations in the plasma of the two peptides was expected because the peptides are secreted in equimolar amounts from the L-cells. There also was a correlation between the GLP-2 concentrations in the vena portae and in the vena cava, with approximately half of the amount in the vena cava. The slope was almost the same for the chemo and no chemo rats, suggesting that the degradation rate was similar in the two groups. We found no correlation between the GLP-1 concentrations in the vena portae and in the vena cava and a relatively higher concentration of GLP-1 than of GLP-2. We used a processing-independent antibody to measure GLP-1, which may explain the difference and lack of correlation.

    A number of studies have investigated the restorative and protective effects of GLP-2 following damage to the intestinal mucosa. Exogenous GLP-2 has been shown to improve the barrier function of the intestinal epithelium in healthy mice,23 have a protective effect on the murine small intestinal mucosa after gamma-irradiation,24 protect the rat intestine from ischaemia and reperfusion-induced injury25 ,26 and ameliorate experimentally induced inflammatory bowel diseases.27 ,28 To our knowledge, these effects have never been examined for GLP-1, but given that GLP-1 has an intestinotrophic effect, it is tempting to suggest that GLP-1 might also be involved in the intestinal adaptation in a manner similar to that of GLP-2 and that it possesses restorative and protective effects in the intestine.

    In study 1, we aimed to elucidate the importance of endogenous GLP-1 and GLP-2 in the healing phase by antagonising the receptors. The GLP-2 R antagonist GLP-2 (3-33) did not cause any differences in either the intestinal recovery or the proliferation, which might be because endogenous GLP-2 is not necessarily involved in the healing process and that GLP-2 (3-33) is a partial agonist and that the receptor is thus not completely antagonised. When antagonising the GLP-1 receptor with exendin (9-39), a known receptor antagonist,14 we found that the intestinal recovery stagnated and the intestinal weight in the chemo mice treated with exendin (9-39) was significantly lower than that of the vehicle-treated mice, suggesting that the restoration of the damaged intestine was delayed. This possibility was confirmed by morphometric analysis. This result is indirect proof that the GLP-1 RA is involved in the compensatory intestinal proliferation caused by injury.

    Even though GLP-2 R activation has not been directly proved to be important in the pathogenesis of mucositis, several studies have found a therapeutic effect of GLP-2 in experimentally induced mucositis. The protective effect of GLP-2 has been investigated by Boushey et al,2 Yamazaki et al,29 Tavakkolizadeh et al3 and Rasmussen et al,30 and data from their reports and our laboratory have convincingly shown an effect of the GLP-2 analogue NNC-66-103 in the treatment of chemotherapy-induced mucositis in rats.10 Our study demonstrated that the GLP-1 analogue liraglutide and the GLP-2 analogue NNC-103-0066 have comparable effects on chemotherapy-induced mucositis. In both cases, the treatments significantly improved the same parameters—the small intestinal weight loss, reduction of the cross-sectional area of the mucosa and reduction in villus height. Most importantly, there was a significant reduction in the histological scoring of mucositis severity after treatment with both analogues.

    The mechanism of action of both peptides remains unknown, but because GLP-1 potentiates insulin-like growth factor-1 receptor signalling,31 and growth factors have been associated with beneficial effects in experimental mucositis,32 this pathway could be involved in the described effect on mucositis. In clinical settings, only the keratinocyte growth factor analogue palifermin has shown an effect in the treatment of oral mucositis.33

    We also hypothesised that the secretion of GLP-1 plays an important role during the initial phase by targeting the receptor before and during the first days after the injury. We did not find any differences between the chemo mice treated with exendin (9-39) and those treated with vehicle in the acute state of mucositis, indicating that GLP-1 secretion and receptor targeting is important only during the healing phase.

    GLP-2 analogues have been under development for the treatment of chemotherapy-induced mucositis, but Thulesen et al,5 Iakoubov et al4 and Trivedi et al6 have suggested that GLP-2 has tumour-promoting effects in carcinogen-treated animals. Recently, we tested whether liraglutide had the same effect as GLP-2 in the colon and found that it has a potent growth-stimulating effect on the healthy mouse intestine but, despite that result, no stimulatory effect on colonic dysplasia.9 This experiment suggests liraglutide as a new treatment for chemotherapy-induced mucositis and opens up the possibility that GLP-1 analogues might also be beneficial in other acute intestinal disorders.

    Acknowledgments

    The authors thank Heidi Paulsen and Lise Strange for their technical support, and Lars Thim for providing NNC-GLP-2.

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    Footnotes

    • Contributors HK planned and performed the experimental studies and wrote the manuscript. BH participated in the experimental studies and analysed the plasma samples. JJH and SSP supervised the experiment and participated in the writing of the article.

    • Competing interests SSP and BH declare no conflicts of interest. JJH received a fee for speaking or consulting from Novo Nordisk, Merck, Glaxo, Novartis Pharmaceuticals and Roche, and HK is the inventor of ‘GLP-1 RA as treatment for chemotherapy-induced mucositis’ for which Danish patent application no. PA 2012 70142 was filed on 23 March 2012.

    • Provenance and peer review Not commissioned; externally peer reviewed.