Glucagon-like peptide-2 stimulates the proliferation of cultured
rat astrocytes
Esther Vela
´
zquez, Juan M. Ruiz-Albusac and Enrique Bla
´
zquez
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Complutense University, Madrid, Spain
Glucagon-like peptide-2 (GLP-2) is a potent intestino-
trophic/satiety hormone that acts through a G protein-
coupled receptor. To determine whether or not GLP-2
has any effect on cellular proliferation on neural cells, we
examined the effects of this peptide on cultured astrocytes
from rat cerebral cortex. The expression of the GLP-2
receptor gene in both cerebral cortex and astrocytes was
determined by RT-PCR and Southern blotting. Also, cells
responded to GLP-2, producing cAMP in a dose-dependent
manner (EC
50
¼ 0.86 n
M
). GLP-2 also stimulated the DNA
synthesis rate in rat astrocytes. When proliferation was
assessed by measuring [
3
H]thymidine incorporation into
DNA or staining cells with crystal violet, GLP-2 produced a
dose-dependent increase in both parameters. Similarly, when
the numbers of cells in different phases of the cell cycle were
measured by flow cytometry, a dose-dependent decrease in
those in the G0-G1 phase and an increase in those in the S

and acts as an intestinal growth factor. The actions of GLP-2
are transduced through a recently cloned GLP-2-specific G
protein-coupled receptor (GLP-2R), which is linked to the
activation of the adenylate cyclase pathway [5,6]. The
GLP-2R gene is expressed in a tissue-specific manner,
mainly on subsets of enteric nerves [7] and in gut endocrine
cells [5,8], and in several regions of the central nervous
system, including the murine thalamus, hippocampus,
cerebral cortex and hindbrain [9] and in the compact part
of dorsomedial hypothalamic nucleus [10] of the rat.
It has been widely documented that GLP-2 affects several
functions in the gastrointestinal tract, in this sense it inhibits
gastric acid secretion and motility, reduces intestinal per-
meability and enhances intestinal hexose transport [11].
However, the most striking role of GLP-2 in the intestine
consists in promoting the expansion of small bowel mucosal
epithelium by stimulating crypt cell proliferation, increasing
villus height and crypt depth, and decreasing apoptosis in
both the crypt and enterocyte compartments [6,12–14]. On
the basis to studies carried out in experimental models of
intestinal disease, a potential therapeutic role for GLP-2 has
been proposed that might prevent or ameliorate the effects of
intestinal injury[15–20]. Furthermore, aphysiological growth
factor function of GLP-2 has been proposed in diabetic rats,
whose elevated levels of endogenous GLP-2 were associated
with marked intestinal growth [21], which was significantly
reduced by blockade of endogenous active peptide [22].
In contrast to the increasing number of studies describing
the CNSactions ofGLP-1, the potential effect(s) ofGLP-2 on
the brain are scarcely known. GLP-2 has recently been found

possibly open new insights into the actions of this novel
neuropeptide.
Experimental procedures
Materials
Rat GLP-2 was from Peninsula Laboratories (St. Helens,
UK). The c-AMP enzyme immunoassay (EIA) system was
from Amersham Pharmacia Biotech (Little Chalfont Bucks,
England). [Methyl-
3
H]-thymidine (60–80 CiÆmmol
)1
)and
[a
32
P]-deoxy-CTP (3000 CiÆmmol
)1
) were from NEN Life
Science Products, Inc. (Boston, MA, USA). Whatman GF/C
glass microfiber filters were from Whatman International
Ltd. (Maidstone, England). Mini-Quick Spin DNA col-
umns were from Roche Diagnostics (Bromma, Sweden).
Nylon membranes were from Boerhinger Mannheim
GmbH (Mannheim, Germany). The DNA labeling system
was from Amersham-Pharmacia-Biotech (Uppsala,
Sweden). Ribonuclease A (Ribonucleate 3¢-pyrimidino-
oligonucleotidohydrolase, EC 3.1.27.5) from bovine pan-
creas was from Roche Molecular Biochemicals (Hvidovre,
Germany). The Titan
TM
One Tube RT-PCR System and

[
3
H]thymidine incorporation and crystal violet assays),
12-well (for cAMP measurement) or 100 · 20 mm (for
flow cytometry and gene expression analysis) tissue culture
dishes. After 7–10 days, the cultures were 80–90% conflu-
ent. Immunocytochemical analysis of these cultures revealed
that at least 95% of the cells were positive for the astrocyte-
specific marker, glial fibrillary acidic protein. GLP-2 stimu-
lation experiments were carried out in serum-free medium
containing bovine serum albumin (1.4 gÆL
)1
; 0.001% fatty
acid). Then, reactions were stopped by removing the
supernatants and adding ice-cold phosphate buffered saline
(NaCl/P
i
) to cells. Finally, cells were washed twice with
NaCl/P
i
and harvested for the different analyses as
described below.
Intracellular cAMP measurements
Intracellular cAMP was measured using the commercial
protocol for the nonacetylation enzyme immunoassay
(EIA) system with a curve range of 12.5–3200 fmol per
well for cell culture samples. Subconfluent cells were shifted
to serum-free medium for 24 h. GLP-2 was used at a final
concentration of 0.1–50 n
M

)6
M
) for 24 h.
[
3
H]Thymidine at a final concentration of 5 lCiÆmL
)1
was present during the last four hours of treatment.
Reactions were stopped as indicated above. Following this,
cells were incubated with 0.5
M
NaOH for 1 h at room
temperature and then with 20% (v/v) cold trichloroacetic
acid for 1 h at 4 °C. Acid-precipitable material was
collected by filtration on Whatman GF/C glass microfiber
filters washed three times with ice-cold 10% trichloroacetic
acid and once with 70% ethanol at ) 20 °C. The radio-
activity incorporated into DNA was measured by liquid
scintillation counting on the filters. Results are expressed
as disintegrations per min of [
3
H]thymidine incorporated
per well.
Crystal violet staining
The relative number of cells was measured using the method
described by Barna et al. [29] based on the staining of cells
with crystal violet. Briefly, treatments were carried out 48 h
after subconfluent astrocyte cultures had been shifted to
serum-free medium. Cells were incubated for 24 h with
GLP-2 (10

)6
M
)
or 10% fetal bovine serum (positive control) for times
between 8 and 30 h. After stopping the reactions, cells were
dispersed by treatment with trypsin-EDTA and centrifuged
at 420 g for 2 min at 4 °C in presence of freshly prepared
fetal bovine serum. Pellets were washed with NaCl/P
i
and
incubated on ice water with 70% ethanol at ) 20 °C. After
the addition of NaCl/P
i
, lysed cells were sedimented at
1700 g for 4 min at 4 °C and then incubated for 30 min at
37 °Cwith1gÆL
)1
ribonuclease A in 50 m
M
Tris, pH 8.0,
10 m
M
EDTA. Then, propidium iodide (0.05 gÆL
)1
final
concentration) was added and the samples were maintained
in the darkness at 4 °C until used. The intensity of
fuorescence was measured at 560 nm by flow cytometry
(FAC Scan, Becton-Dickinson, San Jose, CA, USA). With
this experimental procedure it was possible to analyze

cpmÆlg
)1
) were generated using
random primers [35] and hybridized with the membranes
for 20 h at 42 °C (in 50% formamide, 5 · Denhart,
3 · NaCl/Cit, 0.2% sodium dodecyl sulfate); the c-fos
probe corresponding to bases 553–853 in the rat c-fos
complementary DNA sequence was a gift of P. Esbrit
(Madrid, Spain) [36]; the c-jun probe corresponding to the
rat c-jun complementary DNA completed sequence [37] and
the nuclear 28S rRNA probe corresponding to bases 4200–
4505 in the rat 28S rRNA complementary DNA sequence
was a gift of A. Santos (Madrid, Spain) [38]. The washing
conditions were 2 · NaCl/Cit/0.5% sodium dodecyl sulfate
at 65 °C, for mild washing, and then 0.2 · NaCl/Cit/0.5%
SDS at 65 °C for stringent washing. A probe labeled for the
nuclear 28S rRNA was used as loading control. For
quantification, autoradiographs were scanned with a Silver
Scanner densitometer and the optical densities of each
specific signal were normalized with the loading control.
Values are expressed as fold-induction with respect to the
control sample.
cDNA synthesis, PCR amplifications, and Southern-blot
analysis
Total RNA from cultured astrocytes, cerebral cortex tissue
from newborn rats, hypothalamus and jejunum intestinal
mucose from adult rats, and Chinese hamster ovary cells
were isolated as described above. Sequence-specific primers
were designed to amplify rat GLP-2R mRNA. The
antisense priming oligonucleotide (5¢-CATTCCACC

(Pharmacia, Barcelona, Spain).
Statistical analysis
One-way analysis of variance was carried out using the
GraphPad Prism Application (GraphPad software Inc.).
Values are reported as the means ± SE or SD. P-values
from < 0.05 were considered statistically significant.
Results
GLP-2 receptor expression in rat astrocytes in culture
In an attempt to know whether or not GLP-2 had some
biological effect on rat astrocytes in culture, we first
determined the expression of its receptors in these cells. As
expected, only one band of 379 bp, which corresponded
with the predicted size of the PCR products, was obtained
(Fig. 1). Also, the GLP-2R mRNA was expressed in both
intestine and hypothalamus, and was also found in cerebral
Ó FEBS 2003 GLP induces proliferation of astrocytes (Eur. J. Biochem. 270) 3003
cortex and astrocytes. However, GLP-2R mRNA was not
present in the Chinese hamster ovary cells used as negative
control.
Effect of GLP-2 on cAMP formation by rat astrocytes
As cAMP is considered to be a chemical mediator of the
action of GLP-2, we determined the effect of this peptide on
cAMP formation by rat astrocytes in culture. As shown in
Fig. 2, GLP-2 induced a dose-dependent cAMP produc-
tion, with a maximal effect (twofold induction) at 10
)8
M
.
The EC
50

Fig. 1. RT-PCR analysis of GLP-2R mRNA transcripts in newborn rat
cerebral cortex and in astrocyte cultures. Total RNA from newborn
cerebral cortex (C), subcultured astrocytes (A), hypothalamus (H),
intestine (I) and CHO cells were reverse-transcribed and amplified by
PCR using GLP-2R specific primers and analyzed by Southern
blotting.
Fig. 2. Dose-dependent effect of GLP-2 on intracellular cAMP
production in culture astrocytes. Astrocyte cultures maintained in serum-
free medium for 24 h were incubated for 30 min with GLP-2 in presence
of 10 l
M
IBMX. Then, culture media were aspirated and intracellular
cAMP measured by an enzyme-immunoassay system. EC
50
was
0.86 n
M
. Data represent means ± SEM of three independent experi-
ments carried out in triplicate. **P < 0.01, ***P < 0.001.
Fig. 3. Time course of the GLP-2 induced stimulation of [
3
H]thymidine
incorporation into DNA from rat astrocytes. Astrocyte cultures main-
tained in serum-free medium for 48 h were incubated with fresh
medium containing 1.4 gÆL
)1
of bovine serum albumin alone (d)orin
presence of 1 l
M
GLP-2 (j) for the indicated times. [

induced a proportional increase in [
3
H]thymidine incorpor-
ation into DNA, the maximum effect being observed (185%
with respect to the control) at 10
)8
M
.TheEC
50
of the
[
3
H]-thymidine incorporation response into astrocyte
cultures was 0.45 ± 0.05 n
M
.
Effect of GLP-2 on the cellular density of rat astrocytes
in culture
To assess whether GLP-2 induction of [
3
H]thymidine
incorporation was associated with increased cell numbers,
astrocytes were incubated for 24 h with GLP-2
(10
)12
)10
)6
M
) and variations in cellular density was
determined using a crystal violet assay. Figure 5 shows that

experiments described above. Table 2 shows the time-course
effect of GLP-2 (1 l
M
) and fetal bovine serum (10%) on the
percentage of cells present in each phase of the cell cycle in
comparison with the control cells. The decrease in the
number of cell in the G0-G1 phase and the corresponding
increase in the number of cells in the S and the G2-M phases
began to be significant (P < 0.05) after 18 h of incubation
with GLP-2. Although maximum effects were observed
after 24 h of incubation, a significant (P < 0.001) inducing
effect could still be observed after 30 h of incubation with
this peptide. Similar results were obtained with 10% fetal
bovine serum, except that the maximum effect was observed
after 21 h of incubation, and that after 30 h of incubation
the percentage of cells in each phase of the cell cycle was
very similar to that seen in the control cells. These results
suggest that the cell division cycle of astrocytes lasts about
30 h.
The dose–response experiments of GLP-2 on astrocyte
cell division are shown in Table 3. GLP-2 induced a dose-
dependent effect on the number of cells in different phases
of the cell cycle after 24 h of incubation, with significant
(P < 0.05) differences in the G0-G1 (70.3% vs. 75.3%) and
S (20.7% vs. 17.4%) phases, even at the lowest dose of
GLP-2 assayed (10
)10
M
).
Effect of GLP-2 on c-fos and c-jun mRNAs expression

the CNS, including their effects on feeding behavior [9,23–
26]. It is also known that expression of GLP-1R is increased
in glial cells after mechanical injury [40], supporting the
Fig. 5. Dose-dependent effect of GLP-2 on cellular density in rat astro-
cytes. Astrocyte cultures maintained in serum-free medium for 48 h
were incubated for 24 h with GLP-2 as indicated in the Fig. 1. Then,
cells were stained with crystal violet and the absorbance at 560 nm was
determined in each well. Results are expressed as percentages with
respect to the control sample (zero dose of GLP-2). EC
50
was 6 p
M
.
Each point represents the means ± SEM of three independent
experiments carried out six times. Absorbance in control sample was
0.217 ± 0.002. *P <0.05,**P <0.01.
Ó FEBS 2003 GLP induces proliferation of astrocytes (Eur. J. Biochem. 270) 3005
recently reported neuroprotective/neurotrophic function of
GLP-1 [41,42].
The most striking role of GLP-2 on the intestine consists
in stimulating cell proliferation and decreasing cell apoptosis
[12–14]. However, although GLP-2R are expressed in
several regions of the CNS [9,10], no proliferative effects
of GLP-2 have yet been reported for neural cells. Thus, we
speculated that culture astrocytes from cerebral cortex
could offer a good physiological cellular model to study the
expression of GLP-2R and the effects of GLP-2 on cell
proliferation. As the percentage of astrocytes in our cell
cultures was greater than 95%, as determined by the
Table 1. Effect of GLP-2 and fetal bovine serum on the number of hypodiploid cells in rat astrocytes in culture. Astrocytes were maintained in serum

% G2-M
b
8 Control 83.1 ± 3.3 13.8 ± 2.9 3.1 ± 0.3
GLP-2 87.7 ± 0.8 9.5 ± 0.5 2.8 ± 0.2
Fetal bovine serum 87.8 ± 5.0 9.5 ± 1.3 2.7 ± 0.8
15 Control 84.3 ± 2.0 12.6 ± 2.4 3.2 ± 0.4
GLP-2 87.6 ± 2.3 9.7 ± 1.7 2.7 ± 0.6
Fetal bovine serum 91.0 ± 4.7 7.0 ± 1.1
c
2.0 ± 0.1
c
18 Control 79.2 ± 5.3 16.0 ± 4.3 4.9 ± 1.0
GLP-2 70.1 ± 1.9
c
24.7 ± 1.6
c
5.2 ± 0.3
Fetal bovine serum 72.9 ± 3.7 23.9 ± 3.4
c
3.2 ± 0.3
c
21 Control 75.7 ± 2.1 20.6 ± 2.1 3.7 ± 0.1
GLP-2 62.5 ± 1.0
d
32.1 ± 1.0
d
5.4 ± 0.1
c
Fetal bovine serum 47.0 ± 0.9
d

bovine serum albumin (control), 1 l
M
GLP-2 and 10% fetal bovine serum.
b
Means ± SD (n ¼ 3, carried
out in quadruplicate).
c
P < 0.05.
d
P < 0.001 vs. control.
Table 3. Dose-dependent effect of GLP-2 on the cell cycle in cultured rat astrocytes. The phases of the cell cycle were assessed by flow cytometry using
propidium iodide as DNA marker.
GLP-2 (n
M
)
a
% G0-G1
b
%S
b
% G2-M
b
0 75.3 ± 0.5 17.4 ± 0.5 7.3 ± 1.0
0.1 70.3 ± 3.2
c
20.7 ± 0.8
c
8.7 ± 1.2
1 68.5 ± 3.0
d

P < 0.001 vs. the zero dose.
3006 E. Vela
´
zquez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
expression of glial fibrillary acidic protein, we propose that
the findings described refer to astrocytes and not other cells
types, such as oligodendrocytes and O-2 A precursors [27],
which may or may not be targets of GLP-2.
Here we report that the GLP-2 receptor is expressed in rat
cerebral cortex and also in cultured astrocytes. GLP-2R
mRNA transcripts were detected by RT-PCR analysis and
were found to be similar to those detected in the hypotha-
lamus and intestine of adult rats. GLP-2R gene expression
in rat astrocytes lends support to the biological effects of
GLP-2 described here. Thus, GLP-2 elicited an increase in
cAMP formation in astrocytes in culture in a dose-
dependent manner, with an EC
50
similar to that reported
for rat GLP-2R-transfected COS cells [5]. GLP-2-induced
cAMP formation has been also assayed in rat and human-
GLP-2R-transfected BHK cells [6,9,43,44], but the EC
50
values obtained were different as compared to those found
in COS cells or in our cultured rat astrocytes.
Several tests were used to explore the proliferative effect
of GLP-2 on cultured astrocytes, the first ones addressing
the action of this peptide on [
3
H]thymidine incorporation

crystal violet. Because there are no previous studies on the
effects of GLP-2 on the cell cycle, we performed experiments
in parallel in which the effect of a mitogen ) in our case
fetal bovine serum ) was studied; the results obtained in
both experimental groups were very similar.
Because the activation of the AP-1 pathway is frequently
associated with a stimulation of cell proliferation [48], we
studied the effect of GLP-2 on the mRNA expression of the
immediate/early c-fos and c-jun genes. From previous
studies, we knew that in vivo administration of GLP-2
results in a significant increase in the number of c-Fos-
positive cells in the dorsomedial hypothalamic nucleus of
the rat [25] and in the enteric ganglia of the mouse [7], while
h(Gly2)GLP-2 increases the levels of c-fos, c-jun and zif-268
mRNAs in BHK-GLP-2R cells [6]. Our results indicate that
GLP-2 also induces c-fos and c-jun activation in astrocytes
in culture, even at the lowest concentration assayed, lending
further support to the proliferative effect of this peptide on
cells of the CNS.
Recent data suggest that GLP-2 exerts a direct cytopro-
tective effect via inhibition of apoptosis [13]. In enterocytes,
a significant reduction on natural apoptosis was only
observed after 10 days of treatment with GLP-2 [14]. For
the detection of apoptosis in cultured rat astrocytes, we also
used a quantitative technique: the propidium iodide labeling
of DNA followed by flow cytometric analysis [49]. By
contrast, we observed that natural apoptosis in rat astrocytes
was not affected by either GLP-2 or fetal bovine serum
after 30 h of treatment. However, GLP-2 administration to
rodents with experimental intestinal injury [17,18,20,50–52]

2R signaling enhances cell survival via mechanisms that
involve Bad and glycogen synthase kinase-3 phosphoryla-
tion in a PKA-dependent manner and independently of
PI3K/Akt.
The results reported here show for the first time that the
expression of the GLP-2 receptor in isolated rat astrocytes
seems to have functional activity, as judged by the stimula-
ting effect of GLP-2 on cAMP formation. Also, based on
several criteria our findings indicate that, at circulating
concentrations in vivo, GLP-2 exerts proliferative effects on
astrocytes; this may contribute to a better understanding of
the actions of these cells in the central nervous system.
Further studies must be carried out to determine the role of
GLP-2 in glial cells under both normal and pathophysio-
logical situations, especially in response to brain injury.
Acknowledgements
We thank Dr Angel Santos and Dr Elvira Alvarez for their helpful
advice in RNA analysis, and Dr Antonio Santos and Dr Patricia
Va
´
zquez for excellent technical assistance. This study was supported by
grants from Direccio
´
n General de Investigacio
´
nCientı
´
fica y Te
´
cnica

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