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Departments of Medical Physiology (B.H., J.J.H.) and Anatomy (J.T., H.K., S.T., C.O., C.R., S.S.P.), The Panum Institute, University of Copenhagen, Copenhagen N, DK-2200 Denmark
Address all correspondence and requests for reprints to: Prof. Jens Juul Holst, Department of Medical Physiology, University of Copenhagen, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. E-mail: holst{at}mfi.ku.dk
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Introduction |
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Materials and Methods |
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Peptides
Human recombinant GLP-2 was a generous gift from L. Thim
(Novo Nordisk A/S, Bagsværd, Denmark) and synthetic human
GLP-2 (3–33) was from PolyPeptides Laboratories (Wolfenbüttel,
Germany). Purity (>95%) and correctness of structure were confirmed
by mass, sequence, and HPLC analysis. For DPP-IV inhibition, we used
valine-pyrrolidide (VP; a generous gift from Dr. T. E. Hughes,
Novartis Institute for Biomedical Research, Summit, NJ).
For injections, GLP-2, GLP-2 (3–33), and VP were dissolved in saline containing 3.5 mg/ml Hemaccel (Behringwerke AG, Marburg, Germany), which was also used for control injections. For rats, each injection vol was 400 µl; and for mice, the vol was 100 µl. In effect studies, injections were given sc into separate flanks, twice daily, every 12 h.
Degradation of GLP-2
Forty-eight rats were randomly divided into two groups. Animals
in group 1 received two sc injections, one containing 40 µg GLP-2 and
one containing 15 mg VP, each being given into separate flanks. Animals
in group 2 received also two sc injections, one containing 40 µg
GLP-2 and one control injection. At six different time points (5, 15,
and 60 min and 4, 8, and 12 h; n = 4 at each time point),
animals were anesthetized with barbiturate (Brietal, Methohexital;
Eli Lilly & Co., Indianapolis, IN; 50 mg/kg, ip), and
blood samples were obtained from the inferior vena cava. Samples were
collected into chilled tubes containing, in final concentrations EDTA
(3.9 mM) and VP (0.01 mM), kept on ice, and
centrifuged within 0.5 h. Plasma samples were stored at -20 C
until assay.
RIA
GLP-2 was measured with an N-terminal specific antiserum, code
no. 92160, measuring only GLP-2 with an intact N terminus, as described
in Ref. 6 . For standards, we used recombinant human GLP-2,
and the tracer was bovine GLP-2 with
Thr12
Tyr12 substitution,
125I-labeled using the standard stoichiometric
chloramine T method, as described elsewhere (7). This
assay cross-reacts 5.6 ± 1.8% with synthetic human GLP-2
(3–33).
Total GLP-2, comprising intact GLP-2 and elongated or truncated
molecular forms, was measured using a midregion specific antiserum,
cat. no. RAS 7167 (Peninsula Laboratories, Inc. Europe,
St. Helens, UK; reacting with a midsequence of GLP-2), using rat GLP-2
with an Asp33Tyr33
substitution for iodination and recombinant human GLP-2 for standards.
All plasma samples were extracted in a final concentration of 75%
ethanol before GLP-2 measurements.
For both assays, the experimental detection limit is 5 pM, and the intraassay coefficient of variation is 5% at a concentration of 40 pM.
Intestinotrophic effect of GLP-2 and GLP-2 (3–33) in rats
Thirty animals were randomly divided into the following groups
(six animals in each group): 1) control group 1 (saline); 2) control
group 2 (15 mg VP); 3) 40 µg GLP-2; 4) 40 µg GLP-2+15 mg VP; and 5)
40 µg GLP-2 (3–33). Animals were treated for 2 weeks, and body
weight was measured on day 1 of treatment and before being killed on
day 14. At the time of death, the weight and the length of the small
intestine, the cecum, and the large intestine were measured, after the
luminal contents and the mesenteric fat had been removed. All segments
were vertically suspended with a 10-g weight to provide uniform tension
during the measurement of length.
Intestinotrophic effect of GLP-2 and GLP-2 (3–33) in mice
Animals were weighed and randomly allocated to the following
groups of six: 1) saline; 2) 5 µg GLP-2; 3) 5 µg GLP-2+1.5 mg VP;
4) 25 µg GLP-2; and 5) 25 µg GLP-2 (3–33). Animals were treated
for 10 days, whereupon they were killed, and the weight and the length
of the small intestine, the cecum and the large intestine were measured
as above. All segments were vertically suspended with a 1.5 g
weight to provide uniform tension during the measurement of the
length.
Histological sections and morphometric estimates
Tissue samples [small-bowel segments (proximal, middle, and
distal) and colon segment (middle)] from all animals in the effect
studies were fixed by immersion in ice-cold, freshly prepared buffered
4% paraformaldehyde. The fixed tissue samples were then dehydrated and
embedded in paraffin and were cut perpendicularly to the axis of their
length, into 10-µm sections, using a microtome. The sections were
stained with hematoxylin and eosin and were examined using an Axiophot
microscope (Carl Zeiss, Oberkocken, Germany) connected to
a high-resolution camera (Hamamatsu C2400, Hamamatsu Photonics,
Hamamatsu City, Japan). The cross-sectional area of the mucosa and
muscular layers and villus height and crypt depth were measured using
an image analysis system (NIH Image 1.60). The intestinal lumen and the
boundaries between the mucosal and muscular layers were outlined by the
computer cursor, allowing calculation of the cross-sectional area of
each layer in the wall. Ten well-orientated crypts and villi per
segment were randomly selected and measured for villus height and crypt
depth. The examination and the computer analysis of the histological
sections were performed without knowledge of the origin of tissue
samples.
The effect of DPP-IV inhibition on endogenous GLP-2 concentrations
in rats
Animals were randomly allocated into six groups of six. Groups
1, 2, and 3 were injected with NaCl; and groups 4, 5, and 6 were
injected with 15 mg VP. All animals were fasted for 48 h before
injection. Immediately after injection, animals were either fasted
(groups 1 and 4) or fed ad libitum for 1 h before
death. Groups 2 and 5 were fed with standard rat chow (Altromin 1314,
Altromin, Lage, Germany), and groups 3 and 6 were fed with a palatable
high-energy liquid diet (Complan, Meda, Soeborg, Denmark). At death,
animals were anesthetized, and blood samples were obtained as described
above. Plasma samples were analyzed with the N-terminal specific assay,
to determine the GLP-2 concentrations.
Statistical analysis
The results are shown as mean ± SEM.
Statistical significance of the difference obtained in the GLP-2
degradation study was assessed by an unpaired t test of
results from GLP-2-injected and GLP-2+VP-injected animals. In the GLP-2
effect experiments, comparison between groups was performed by two-way
ANOVA, followed by Fisher’s protected least-significant-difference
post hoc analysis. Probability values of P
< 0.05 were considered significant.
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) and were
significantly higher (P < 0.05) than in rats given
GLP-2 without coinjection with VP. Five and sixty minutes after
injection, the GLP-2 concentrations, measured with the N-terminal
specific assay, were 17.2 ± 1.8 nM and
17.2 ± 2.0 nM, compared with 9.9 ±
1.5 and 6.5 ± 0.7 nM (P <
0.05) for rats injected with GLP-2 alone. There were no longer
significant differences in intact GLP-2 concentrations between the two
groups after 4, 8, and 12 h. There was no significant difference
between the two groups in the concentrations of total GLP-2
immunoreactivity, measured with the sideviewing assay at t = 5 min
and t = 15 min (Table 1), showing that equal doses had been
injected.
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). In the rats, the length
(P < 0.05) and the weight of the small bowel (in
grams, or expressed relative to the body weight) increased
significantly (P < 0.01) in the groups treated with
either GLP-2 alone or with GLP-2 in combination with VP, compared with
the two control groups (NaCl- and VP-treated, respectively) (Fig. 1). Treatment with GLP-2 (3–33) had no
significant effect on length or weight of the small intestine (Fig. 1).
There was no significant difference in any of the measured parameters
of intestinal size or morphometric estimates between the two control
groups (NaCl- and VP-treated) (Fig. 1 and Table 3). The increase in small-bowel weight,
relative to the body weight, corresponded to 19.7% when treated with
GLP-2 alone, and 49.0% when treated with GLP-2 in combination with VP.
There was no significant effect of GLP-2, GLP-2+VP, or GLP-2 (3–33)
treatment on the weight of the cecum or the large intestine (data not
shown). Morphometric estimates of the small bowel revealed that the
area of the mucosa was increased (P < 0.01) in the
proximal (37.1% increase) and the middle (50.4% increase) parts in
normal rats treated with GLP-2+VP, when compared with control treated
animals (Table 3). Treatment with GLP-2 alone had a significant effect
(P < 0.05) on the area of mucosa only in the middle
part of the small intestine, corresponding to a 20.7% increase. The
area of the muscle layer was not increased when normal rats, treated
with GLP-2 alone or in combination with VP, were compared with controls
(Table 3). Villus height was significantly increased (P
< 0.01) in the proximal (25.8% increase) and middle (49.2% increase)
parts of the small intestine in rats treated with GLP-2+VP, compared
with control treated groups (Table 3). Treatment with GLP-2 alone had a
significant effect (P < 0.05) on villus height only in
the distal part, corresponding to a 30.4% increase. In the small
intestine, crypt depth was significantly increased (P
< 0.01) in the proximal (31.3% increase) and middle (19.6% increase)
part in GLP-2+VP-treated rats, compared with controls. Treatment with
GLP-2 alone had significant effect (P < 0.05) on crypt
depth only in the middle part of the small intestine (13.6% increase)
(Table 3).
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). The weight of the small bowel,
expressed relative to the body weight, was increased significantly
(P < 0.01) in the mice treated with 5 µg GLP-2, 5
µg GLP-2+VP, or 25 µg GLP-2 and 25 µg GLP-2 (3–33)
(P < 0.05), when compared with the control group (Fig. 2). The largest effect was achieved with
5 µg GLP-2+VP and 25 µg GLP-2, corresponding to increases of 43.8%
and 48.9% respectively. Treatment with 5 µg GLP-2 alone induced a
29.0% increase, and treatment with 25 µg GLP-2 (3–33) induced a
small (10.3%), but significant, increase in small-bowel weight. In the
mice, none of the groups treated with GLP-2 alone or in combination
with VP, or treated with GLP-2 (3–33), showed growth of the large
intestine or cecum (data not shown). Morphometric estimates of the
small bowel revealed that the area of the mucosa was increased
(P < 0.01) in the proximal part in mice treated with 5
µg GLP-2+VP (86.6% increase) or 25 µg GLP-2 (121.6% increase),
when compared with the control group (Table 5). In the middle part of the small
bowel, the area of mucosa was increased in mice treated with 5 µg
GLP-2 (69.6% increase; P < 0.01), 5 µg GLP 2+VP
(62.1% increase; P < 0.05), or 25 µg GLP-2 (90.2%
increase; P < 0.01), when compared with controls. The
cross-sectional area of the muscle layer in the proximal part of the
small intestine was significantly increased in mice treated with 25
µg GLP-2 (87.0%; P < 0.01) or 25 µg GLP-2 (3–33)
(39.1%; P < 0.05), when compared with control mice.
In the middle part of the small bowel, the area of the muscle layer
increased only in mice treated with 25 µg GLP-2 (26.5% increase;
P < 0.05), when compared with controls. In the distal
part, the muscle layer increased after 5 µg GLP-2 (43.6%;
P < 0.05) and after 5 µg GLP-2+VP (74.4%), when
compared with controls. The villus height in the proximal part was
increased significantly in all four groups, when compared with controls
(Table 5). The largest effect was achieved with 25 µg GLP-2, causing
a 68% increase, compared with controls. In the middle part, treatment
with 5 µg GLP-2, 5 µg GLP-2+VP, or 25 µg GLP-2 increased villus
height by 83.1%, 100.4%, or 73.1%, compared with controls. There was
no significant difference among the five treatment groups, with
respect to villus height in the distal part of the small bowel. In the
proximal part of the small bowel, the crypt depth increased
significantly in mice treated with 5 µg GLP-2, 5 µg GLP-2+VP, or 25
µg GLP-2, compared with controls (Table 5). The largest effect was
achieved with 25 µg GLP-2, corresponding to a 31.8% increase. There
was no significant difference among the groups, with respect to crypt
depth in the middle part; and, in the distal part, only mice treated
with 5 µg GLP-2+VP had increased crypt depth, corresponding to a
15.2% increase (Table 5).
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). Rats fed a palatable liquid
diet (Complan) had significantly (P < 0.01) elevated
plasma GLP-2 concentrations, and VP caused a further significant
(P < 0.01) increase.
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Discussion |
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In the present study, therefore, we studied the intestinotrophic effect of GLP-2 in both rats and mice and also tested the hypothesis that the degrading activity of DPP-IV would be limiting for the effects of GLP-2. We found GLP-2 to stimulate significantly mucosal growth in the small intestine in both rats and mice. However, treatment with GLP-2 in combination with VP, the specific inhibitor of DPP-IV markedly enhanced the growth effects on intestinal weight in the rats. Moreover, the effects on mucosal surface area, villus height, and crypt depth were also markedly enhanced in the proximal and middle parts of the small intestine, after treatment with both GLP-2 and VP. In addition, we were able to show that the plasma levels of intact GLP-2 increased by at least a factor of 2, after injection of identical doses of the peptide, if the inhibitor was given simultaneously. VP also enhanced the growth effects of GLP-2 in mice, in which almost similar growth effects on the small-bowel weight were observed after 5 µg GLP-2+VP, and 25 µg of GLP-2 alone. Finally, our results confirm that the truncated metabolite, GLP-2 (3–33), has no effect on mucosal growth in rats, thus explaining why, in spite of similar plasma concentrations of total GLP-2, the growth was enhanced in rats after GLP-2+inhibitor administration vs. GLP-2 alone, because VP treatment results in elevated levels of the intact peptide. A very small increase in intestinal weight (in percent of body weight) was noted in mice treated with a relatively high dose of GLP-2 (3–33), 25 µg (increases of 10.3%, compared with 48.9% with GLP-2 25 µg), in agreement with data from Munroe et al. (5), who found binding of GLP-2 (3–33) to the rat GLP-2 receptor but associated with less than a 20% increase in in vivo growth effects. Our results, therefore, suggest that the degrading activity of DPP-IV is indeed limiting for the effects of GLP-2 in rats and mice.
Four hours after the combined administration of GLP-2 and VP, a
significant effect of the inhibitor on the plasma concentrations of
intact GLP-2 was no longer detectable, even though GLP-2 levels were
still elevated, but this could be attributable to metabolisation of the
inhibitor at this time. The inhibitor had no effect on intestinal
growth when given alone, in agreement with our observation of unchanged
concentrations of intact GLP-2 in the fasting state and after 1-h chow
feeding. However, as shown in the experiment with administration of a
particularly palatable liquid diet, whereas VP strongly enhanced GLP-2
levels, DPP-IV also influences the levels of endogenous intact GLP-2.
The lack of effect of VP treatment alone on intestinal growth can,
therefore, possibly be explained partly by too short a duration of
DPP-IV inhibition elicited by VP (<4 h, Table 1
) and by limited or
delayed stimulation of GLP-2 secretion with regular chow (Table 6).
However, GLP-2 may also act as a paracrine growth factor for the
intestinal epithelium. For this, GLP-2 only needs to travel from the
L-cells to the target cells of the villi and, in this way, probably
escapes DPP-IV degradation. If true, this would explain the
ineffectiveness of VP alone.
Presently, the intestinotrophic effect of GLP-2 is attracting considerable interest among clinical gastroenterologists. Thus, a continuous iv administration of GLP-2 to rodents subjected to total parenteral nutrition was found to prevent the mucosal atrophy that otherwise accompanies total parenteral nutrition (15). This raises the possibility that GLP-2 could be used in the treatment of human disease. The extent of DPP-IV-mediated degradation of GLP-2 in humans has been investigated in a few studies (3, 4, 16) showing that GLP-2 is degraded to the metabolite GLP-2 (3–33), which probably will limit its effects, as was the case for the related peptide, GLP-1, which requires continuous infusion or protection against DPP-IV to exert sufficient activity to be of clinical interest. Drucker et al. (14) reported the development of an analog of GLP-2, in which the alanine in position 2 was substituted by glycine, thereby rendering the peptide resistant to the actions of DPP-IV, as was the case for an analog of GLP-1. Indeed, this GLP-2 analog exhibited intestinotrophic effects in rats, and its administration in various mouse models of inflammatory bowel disease has been shown to reduce or prevent the mucosal damage (17, 18). Such analogs may, therefore, represent a clinically useful alternative to the use of natural GLP-2. On the other hand, compared with natural peptides, the use of structurally modified analogs may be associated with unforeseen side effects and antibody formation. An alternative approach could consist of combining GLP-2 with DPP-IV inhibitors. Such inhibitors are currently being investigated as a treatment for type 2 diabetes because of their protective effect on GLP-1; and orally available, widely atoxic compounds, have already been developed. Combined with such inhibitors, one would expect a GLP-2-based therapy to be more effective; or, alternatively, the required doses of GLP-2 could be reduced, thereby reducing the costs of therapy. Furthermore, using this approach, clinical investigations of the effects of GLP-2 in intestinal insufficiency could be carried out without being delayed by the clinical development of GLP-2 analogs.
Compared with other growth factors, GLP-2 is of particular interest in gastroenterology, because its growth effects seem to be confined to the gastrointestinal tract. In agreement with this, the recently cloned receptor for GLP-2 has been demonstrated to be expressed in intestinal tissues, although the exact cellular localization of the receptor still remains to be established (5). Other growth factors, e.g. GH, which have been investigated in clinical trials of treatment of human intestinal insufficiency (19), have growth effects also in other tissues, which greatly increases the risk of untoward and possibly dangerous side effects (20). In the present investigation, the major effect of the treatment was noted in the mucosa of the upper and middle small intestine, with lesser effects in the distal small intestine. This order of sensitivity contrasts with the order of L-cell density, where the highest density is observed in the distal small intestine and in the colon (21). This could indicate, as also supported by experimental studies involving intestinal transposition, that a substantial part of the effect of GLP-2 is endocrine, i.e. that GLP-2 is transported to the target tissues by the blood stream to exert its effects rather than acting locally in a paracrine fashion. GLP-2 receptors were also found in the brain (5); but here, they probably are targets for GLP-2 produced in brain stem neurones projecting to the hypothalamus (22), rather than being accessible to circulating GLP-2.
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Acknowledgments |
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Footnotes |
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Received February 25, 2000.
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