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Departments of Physiology (I.I., A.M., L.D., P.L.B.) and Medicine (J.L., B.Y., P.L.B., D.J.D.), Toronto General Hospital, Banting and Best Diabetes Center, University of Toronto, Toronto, Ontario, Canada M5G 2C4
Address all correspondence and requests for reprints to: Dr. Daniel J. Drucker, Toronto General Hospital, 101 College Street, CCRW3–838, Toronto, Ontario, Canada M5G2C4. E-mail: d.drucker{at}utoronto.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GLP-2, located carboxyl-terminal to GLP-1 in the proglucagon molecule, appears to function as a regulator of intestinal mucosal homeostasis (5). Administration of GLP-2 to normal rodents stimulates crypt cell proliferation and inhibits enterocyte apoptosis, resulting in expansion of the small bowel mucosal epithelium (6, 7). The observations that intestinal injury is associated with increased production of the intestinal PGDPs (8) taken together with the finding of increased circulating levels of bioactive GLP-2 in human patients with inflammatory bowel disease (9) provide indirect evidence supporting a role for GLP-2 as an endogenous trophic regulator of intestinal mucosal repair in vivo. Consistent with this hypothesis, administration of GLP-2 to rodents with experimental intestinal injury reduces weight loss and disease activity scores, enhances mucosal regeneration, and reduces mortality in the setting of both small and large bowel inflammation (10, 11, 12, 46).
Although intestinal proglucagon gene expression is detectable during fetal gut development (13, 14), the biological actions of the intestinal PGDPs during fetal and neonatal life remain unclear. As GLP-2 exhibits intestinotropic activity in adult rodents, it seems possible that GLP-2 may play a role in fetal gut development and/or possibly in the complex transition from the neonatal to the adult gut. The actions of GLP-2 are mediated by the GLP-2 receptor (GLP-2R), a recently identified novel member of the G protein-coupled receptor superfamily (15). Although GLP-2R RNA transcripts have been identified in the adult rodent gastrointestinal tract (15), the developmental onset of GLP-2R expression has not yet been reported. Furthermore, whether the fetal or neonatal gut is capable of processing proglucagon to bioactive GLP21–33 remains unknown. To determine whether the GLP-2/GLP-2R axis is expressed and functional during rat development, we have now analyzed the coordinate expression of GLP-2 and the GLP-2R in fetal and neonatal rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue preparation for HPLC analysis
Fetal rat intestinal cells (19–20 days gestation) were
dispersed with collagenase, hyaluronidase, and deoxyribonuclease (40,
50 and 5 mg/dl, respectively; Blend Type H, Sigma, St.
Louis, MO), and placed into 60-mm culture dishes in DMEM containing 5%
FBS and 4.5 g/liter glucose for 24 h. Cells were then washed and
treated with medium containing 0.5% FBS, 1 g/liter glucose, and 20
µU/ml insulin for 2 h. Cells and cell media were collected
separately at the end of the incubation period (16).
Tissues [whole intestine from fetal (19–20 days gestation) and
postnatal day 1 (PD1) rats, 5 cm of ileum from all other rats (PD12,
PD21, PD42, and adult), and fetal rat intestinal cells in culture;
n = 3–6 each] were homogenized in 1 N HCl containing
5% HCOOH, 1% trifluoroacetic acid (TFA), and 1% NaCl, followed by
extraction of peptides and small proteins by passage through a
cartridge of C18 silica (Sep-Pak, Waters Corp., Milford, MA). Plasma was collected into a 10% volume of
Trasylol (5000 KIU/ml)-EDTA(12 mg/ml)-diprotin A (34
µg/ml). Two volumes of 1% TFA (pH 2.5; with diethylamine) were added
to plasma, and 1 vol 10% TFA was added to the medium before extraction
of the peptides on C18 silica (n = 3–5
each) (2, 17).
HPLC analysis
GLP-2-related peptides were separated by HPLC using a
C18 µBonadapak column (Waters Corp.) with a 45-min linear gradient of 30–60% solvent B
(solvent A = 0.1% TFA in water; solvent B = 0.1% TFA in
acetonitrile), followed by a 10-min purge with 99% solvent B. The flow
rate was 1.5 ml/min, and 0.3-min fractions were collected (2, 17).
RIA
RIAs for PGDPs were conducted on dried samples using a panel of
site-specific antisera. The N-terminal PGDPs (glicentin, oxyntomodulin,
and glucagon) were detected using antiserum K4023 [glucagon-like
immunoreactivity (GLI); Biospacific, Emeryville, CA], whereas
antiserum 04A was used to detect glucagon alone [immunoreactive
glucagon (IRG); Dr. R. Unger, Dallas, TX].
GLP-1-containing peptides were analyzed using an antiserum
for the free C-terminal, Gly-extended form of GLP-1
(antiserum b5; Dr. S. Mojsov, New York, NY) as well as an antiserum
that recognizes the amidated C-terminal end of the molecule
(GLP-17–36NH2;
Affinity Research Products Ltd., Mamhead, UK). GLP-2-related peptides
were detected using antiserum UTTH7, which detects the midsequence
amino acids 25–30 and. thus, both GLP-21–33 and
the degraded form, GLP-23–33 (2, 17). Tissue protein levels were determined by the Lowry protein
assay (18).
Tissue isolation for RT-PCR analysis
Rat gastrointestinal tissues were extracted, cleaned of debris,
immediately snap-frozen in liquid nitrogen, and stored at -80 C until
RNA was isolated. Fetal (19–20 days gestation) stomachs and the entire
lengths of the fetal small and large intestines were pooled separately
(n = 5 each). For neonatal rats, stomachs from PD1 pups were
removed and pooled, and the intestines were divided into small and
large bowels and pooled (n = 4 each). For PD12 rats, stomachs were
removed, and RNA was prepared from each sample. Intestinal samples from
PD12 rats consisted of 5 cm of jejunum (10–15 cm from stomach), 5 cm
of ileum (5 cm proximal to cecum), and the entire length of the colon.
For all other age groups, separate gastrointestinal RNA preparations
were obtained from rats (PD21, PD42, and adults rats) consisting of the
entire stomach alone or 5-cm segments of jejunum (10–15 cm distal to
the duodenum), ileum (5-cm proximal to the cecum), or colon (5–10 cm
distal to cecum).
RNA isolation and semiquantitative RT-PCR analysis
Total RNA was isolated using a modified guanidinium
isothiocyanate protocol (19), and RNA integrity was
assessed by agarose gel electrophoresis. For first strand complementary
DNA (cDNA) synthesis, samples were treated with deoxyribonuclease I
(Life Technologies, Inc., Toronto, Canada), primed with
random hexamers (Life Technologies, Inc.), and reverse
transcribed with Superscript II reverse transcriptase (Life Technologies, Inc.). RT- reactions were
analyzed to control for genomic DNA and template contamination. PCR
amplification was carried out using Taq DNA polymerase (MBI
Fermentas). Oligonucleotide primer pairs for PCR amplification were as
follows: rat GLP-2R, 5'-TTGTGAACGGGCGCCAGGAGA-3' and
5'-GATCTCACTCTCTTCCAGAATCTC-3'; rat proglucagon,
5'-GTTTACATCGTGGCTGGATTG-3' and 5'-TGAATTCCTTTGCTGCCTGGC-3'; and
for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
5'-TCCACCACCCTGTTGCTGTAG-3' and 5'-GACCACAGTCCATGACATCACT-3'.
Semiquantitative RT-PCR analysis of rat GLP-2R messenger RNA (mRNA)
transcripts was achieved by amplifying 2 µl dilute cDNA for 24 cycles
using the indicated primer pairs. The expected PCR product for the rat
GLP-2R is 1672 bp, which corresponds to the full-length rat GLP-2R
cDNA. For proglucagon, 2 µl dilute cDNA was amplified for 22 cycles,
and the expected PCR product is 323 bp. For PCR amplification of GAPDH
cDNA, 1 µl dilute cDNA was amplified for 18 cycles with an expected
PCR product of 452 bp. To control for nonspecific amplification, PCR
reactions were also carried out in the absence of first strand cDNA.
The conditions for linear PCR amplification of GLP-2R, proglucagon, and
GAPDH PCR products were determined by carrying out multiple PCR
reactions at varying cycle numbers (no. 8–28) and different cDNA input
concentrations (0.01–2 µl) as indicated in Fig. 3
. The linear range
for PCR amplification was determined by plotting the PCR product yield
against either the cycle number or cDNA input amount.
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GLP-2 administration to neonatal rats
h[Gly2]-GLP-2 was a gift from NPS Allelix Corp. (Mississauaga,
Canada). Pregnant Wistar rats were acclimatized to the animal
facilities and delivered pups 1 week after arrival. Within 24 h of
birth, all pups in one litter (n = 14 pups) were injected sc with
h[Gly2]-GLP-2 (5 µg in a total volume of 100 µl dissolved in
PBS), and all pups in a second litter (n = 11) were injected sc
with saline (100 µl) alone. Pups in both litters were injected once a
day at approximately 1700 h for a total of 10 days. On day 11,
approximately 20 h after the last injection, the neonatal rats
were killed by CO2 anesthesia. Gastrointestinal
tissues (stomach, jejunum, ileum, and colon) were removed, cleaned, and
weighed as previously described (6, 7, 22, 23). Tissue
samples collected for histological examination were fixed overnight in
10% neutral-buffered formalin, paraffin embedded, and counterstained
in hematoxylin and eosin.
Statistics
Area under the curve for HPLC peaks was determined as the sum of
the immunoreactivity under the peak. Differences between groups were
determined by Student’s unpaired t test or ANOVA using n-1
post-hoc comparisons, as appropriate, on an SAS system
(SAS Institute, Inc., Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The content of intestinal GLI
increased progressively in the neonatal rat and reached adult levels by
PD21. Consistent with the known profile of proglucagon processing in
the intestine (1, 24), the two principal bioactive forms
of GLP-1,
GLP-17–36NH2
and GLP-17–37, were detected in all
intestinal extracts analyzed (Fig. 1), with a progressive increase in
the levels of both molecular forms of GLP-1 from fetal to
adult life. Similarly, the changes in immunoreactive intestinal GLP-2
paralleled the pattern observed for both GLI and GLP-1,
with relatively lower levels in newborn intestine (P < 0.001
vs. adult) increasing to adult levels by PD21.
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). The small amount of plasma available
from fetal rats precluded assessment of circulating GLP-2 in these
animals. Accordingly, to ascertain whether the fetal rat
enteroendocrine cell exhibits the capacity for processing and secretion
of bioactive GLP-21–33, we assessed whether
immunoreactive forms of GLP-2 were detectable in fetal rat intestinal
cell cultures (27). Both GLP-21–33
and GLP-23–33 were detected in the medium from
fetal rat intestinal cultures (Fig. 2B). Furthermore,
GLP-21–33 and GLP-23–33
were also detected in fetal rat intestine cell extracts (Fig. 2B).
Taken together, these findings clearly demonstrate that fetal and, by
inference from plasma studies, neonatal rat intestines exhibit the
capacity to process proglucagon into bioactive
GLP-21–33, which is then secreted into the
circulation.
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). Similar
results were obtained for analysis of intestinal rat proglucagon and
GAPDH mRNA transcripts in comparable experiments (Fig. 3A).
Furthermore, the relative levels of PCR products for all three
transcripts exhibited a linear relationship between product abundance
and input cDNA (Fig. 3B). A representative PCR analysis from
experiments demonstrating this relationship is shown in Fig. 3C.
As the biological actions of GLP-2 may exhibit developmental and
region-specific differences in distinct gastrointestinal compartments
(5), we studied the ontogeny of GLP-2R expression in the
stomach and both small and large intestines. GLP-2R transcripts were
detected in RNA isolated from fetal, neonatal (PD1 and PD12), weaned
(PD21 and PD42), and adult rat stomachs and in both small and large
intestines at all ages examined (Fig. 4).
In the stomach, the relative levels of GLP-2R mRNA transcripts
decreased slightly from fetal to adult levels, but levels were not
significantly different in animals of different ages (data not shown).
The relative levels of jejunal GLP-2R mRNA were higher in fetal and
neonatal rats than those in adult rats (P < 0.001),
reaching adult levels by PD21. The relative abundance of proglucagon
mRNA transcripts resembled the pattern obtained for GLP-2R RNA in the
jejunum, with comparatively higher levels observed in fetal and
neonatal jejunum (P < 0.01 vs. adult),
followed by a decline to adult levels in PD21 jejunum (Fig. 4). The
developmental expression of the GLP-2R in the ileum was similar to that
observed in the jejunum, with higher levels of GLP-2R mRNA transcripts
detected in fetal gut and PD1 and PD12 ileum (P < 0.05
vs. adult), followed by a decline to lower levels in older
animals. In contrast, proglucagon mRNA transcripts were lower in the
fetus and PD1 ileum, increased markedly by PD12 (P <
0.05 vs. adult), then decreased to lower levels in the ileum
of older animals (Fig. 4). In the colon, the levels of GLP-2R mRNA
transcripts were most abundant at PD12 (P < 0.001
vs. adult), followed by a progressive decrease to adult
levels at PD12. In contrast, proglucagon gene expression in the colon
was not significantly different at various stages of rat
development.
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). Analysis of intestinal tissues
demonstrated a significant increase in stomach and small bowel weight
and small bowel length in the h[Gly2]-GLP-2-treated rats
(P < 0.05; Fig. 5). The crypt and villus compartment
appeared similar in control and h[Gly2]-GLP-2-treated rats (Fig. 5, E
and F). In contrast, no increase in colon weight, but an increase in
colon length, was detected after h[Gly2]-GLP-2 administration. These
findings demonstrate that activation of the GLP-2/GLP-2 receptor axis
is coupled to increased intestinal growth in the neonatal rat
intestine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Developmental analysis of enteroendocrine cell differentiation has established the presence of glucagon-immunoreactive fetal L cells in both small and large intestines (35, 36, 37). Although proglucagon-immunoreactive cells and proglucagon RNA transcripts are first detected by fetal day 14 in the rat gut (13), a major up-regulation in the levels of L cell density and intestinal proglucagon mRNA transcripts occurs between fetal days 17 and 18. Intriguingly, an intestinal profile of glucagon-like immunoreactive peptides is also first detected in the developing rat intestine between fetal days 17–19 (4, 38), suggesting that the molecular machinery required for intestinal processing of proglucagon may be highly regulated at this stage of intestinal development. Consistent with these findings, prohormone convertase 1/3, the enzyme required for liberation of GLP-1 and GLP-2 from proglucagon (2, 39, 40, 41), is developmentally regulated and expressed in the intestine of the fetal rat (42). The finding that bioactive GLP-21–33 is synthesized in and secreted from fetal rat intestinal endocrine cells taken together with the detection of GLP-2R mRNA transcripts in fetal intestine, raise the possibility that the GLP-2/GLP-2R axis may also be functional during the period of rapid intestinal development in utero.
GLP-2 administration to adult rats and mice promotes crypt cell proliferation and inhibits apoptosis, leading to expansion of the mucosal epithelium (7, 23, 25); however, the putative action of GLP-2 in the fetal or neonatal gut has remained unclear. Although we have demonstrated the presence of both bioactive GLP-2 and GLP-2R mRNA transcripts in the rat fetal gut, our data do not allow us to make specific inferences about the biological role, if any, of GLP-2 during fetal intestinal development. We recently determined that pax6 SEYneu mice exhibit a marked deficiency of intestinal GLP-2-producing L cells and a greater than 95% reduction in intestinal proglucagon mRNA transcripts (43). Nevertheless, these mutant proglucagon-deficient mice exhibit apparently normal intestinal development (43), strongly suggesting that normal levels of intestinal GLP-2 are not essential for development of the fetal murine intestine.
The relative levels of GLP-2R expression were comparatively greater in fetal and neonatal rat intestines and declined to lower levels in older animals. Whether these differences reflect changes in GLP-2R transcription and/or RNA stability or developmental differences in the numbers of GLP-2R-positive cells cannot presently be determined, as the cellular localization of GLP-2R expression in the rat intestine has not yet been reported. As the developing intestine undergoes a complex series of molecular changes in the growth, differentiation, and function of the intestinal epithelium in response to enteral nutrition (44), it seems likely that gut peptides such as GLP-2 that are secreted in a nutrient-dependent manner may contribute to the development and maturation process of the neonatal intestinal epithelium in vivo. Although previous experiments demonstrated that GLP-2 is trophic to the mucosal epithelium of 4- to 5-week-old mice (7), the data presented here extend the window of intestinotropic GLP-2 action to the immediate neonatal period in the developing rat gut. Whether GLP-2 is essential for one or more aspects of intestinal adaptation in the transition from the neonatal to adult intestinal epithelium awaits the development of specific GLP-2 antagonists or a GLP-2R knockout mouse.
As the fetal and neonatal intestinal epithelium remains comparatively immature, exhibits defective barrier function, and continues to develop postnatally (44), it remains highly susceptible to external injury. Indeed, premature infants are prone to the development of necrotizing enteritis, a disease characterized by necrotizing infection and destruction of the intestinal mucosa that frequently requires surgical intervention and prolonged hospitalization in the neonatal intensive care unit (45). Although GLP-2 reduces bacterial infection (46) and decreases mucosal epithelial permeability in adult mice, whether GLP-2 exhibits similar actions in the premature developing human gut remains unknown. Nevertheless, our demonstration that the rat intestinal GLP-2/GLP-2R axis is present and functional during the neonatal period suggests that a role for GLP-2 in the prevention or treatment of neonatal intestinal injury be examined in future experiments.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Doctoral Research Award from the Medical Research
Council of Canada.
3 D.J.D. and P.L.B. are equal co-senior authors.
4 Senior Scientist of the Medical Research Council of Canada and
consultant to NPS Allelix, Inc.
Received June 8, 2000.
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) and IRG (
). Middle panel,
GLP-17–36NH2 (
