Báo cáo khoa học: Glucagon-like peptide-2 stimulates the proliferation of cultured rat astrocytes pptx

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 G2-M phases were observed after 24 h incubation with
GLP-2. By contrast, the number of hypodiploid cells was not
affected during the experimental time. Also, GLP-2 pro-
duced a significant increase in the mRNAs of c-fos and c-jun
when gene expression was determined by Northern blotting.
These results suggest that GLP-2 directly stimulates the
proliferation of rat astrocytes; this may open new insights in
the physiological role of this novel neuropeptide.
Keywords: GLP-2; astrocytes; proliferation; cell cycle; gene
expression.
Glucagon-like peptide-2 (GLP-2) consists of 33 amino
acids, corresponding to the proglucagon (126–158) carboxy
terminal end, and belongs to the family of so-called
glucagon and related peptides [1]. The structure of the
proglucagon gene, with six exons and five introns [2], is
similar in humans, rat, hamster and guinea pig. This gene
gives rise to an mRNA transcript that is identical in the
pancreas, intestine and brain, although post-translational
processing of the precursor yields different products in these
organ [3]. In the A cells of the pancreas, the products are
glicentin-related pancreatic peptide (GRPP), glucagon, and
a large peptide containing the glucagon-like peptide-1
(GLP-1) and GLP-2 sequences. In the
L
cells of the
intestine, proglucagon is predominantly processed to gli-
centin, oxyntomodulin, GLP-1 and GLP-2. In the brain the
proglucagon is processed in a similar manner to that
observed in the gut [4].
GLP-2 is cosecreted with GLP-1 from intestinal L cells

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

to be involved in the central regulation of food intake, as
occurred with GLP-1 [23,24]. Thus, intracerebroventricular
injection of GLP-2 inhibited food intake in rats [25] and in
both normal [9] and obese [26] mice, although the anorexi-
genic effects of GLP-2 are transient and modulated by the
presence or absence of GLP-1 receptor signaling in vivo [9].
Correspondence to J. M. Ruiz-Albusac, Departamento de Bioquı
´
mica
y Biologı
´
a Molecular III, Facultad de Medicina, Universidad
Complutense, 28040-Madrid, Spain.
Fax: + 34 91 3941691, Tel.: + 34 91 3941446,
E-mail:
Abbreviations: GLP, Glucagon-like peptide; GRPP, glicentin-related
pancreatic peptide; IBMX, isobutyl-1-methylxanthine; PCNA,
proliferating cell nuclear antigen.
(Received 31 October 2002, revised 22 April 2003,
accepted 19 May 2003)
Eur. J. Biochem. 270, 3001–3009 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03677.x
Because activation of gut cell growth is one of the most
relevant functions of GLP-2, and as GLP-2 receptors seem
to be expressed in the brain, in the present study, we used
cultured rat astroglial cells as an in vitro physiological model
to investigate whether or not GLP-2 has a direct effect on
cellular proliferation in the CNS. Here we report for the first
time the expression of GLP-2R mRNA in astrocytes, and
describe that these cells respond to GLP-2 by increasing
cAMP formation and their proliferation activities. This may

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

the DIG DNA labeling Kit were from Roche Molecular
Biochemicals (Barcelona, Spain). DNA polymerase from
Thermus thermophilus was from Biotools (Madrid, Spain).
PGEM
Ò
-T Easy vector was from Promega (Madison, WI,
USA). All other chemicals were reagent grade or molecular
biology grade.
Rat astrocyte cultures
Primary rat astroglial cell cultures were prepared from
cerebral hemispheres of 1-day-old Wistar rats according to
the method of McCarthy and de Vellis [27], with some
modifications. All procedures involving animals were
approved by the appropriate institutional review committee
and met the guidelines for the care of animals specified by
the European Community. Mechanically dissociated cells
were plated onto 75-cm
2
tissue culture flasks at low-density.
Cells were grown in DMEM/F-12 medium, 15 m
M
Hepes
supplemented with 10% (v/v) fetal bovine serum. After
21–28 days, astroglial cells were dispersed by treatment with
trypsin/EDTA for 1–2 min at 37 °C and replated at
approximately 2.3–3 · 10
4
cellsÆcm
)2
in 24-well (for

[
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

, in the presence of 10 l
M
3-isobutyl-1-methylxanthine (IBMX). All measurements
were carried out after 30-min drug treatments. Five
micromolar forskolin was used as a positive control. The
half-maximal effective concentration (EC
50
)wascalcula-
ted using the
GRAPHPAD PRISM APPLICATION
(GraphPad
software, Inc.).
[
3
H]-Thymidine incorporation into rat astrocytes
The astrocyte DNA synthesis rate was estimated by
quantitating the amount of tritium incorporated into the
acid-precipitable form after [
3
H]-thymidine incubation,
according to the method of Pietras and Szego [28] with
some modifications. Two different treatments were carried
out 48 h after subconfluent astrocyte cultures had been
shifted to serum-free medium, studying the time-course
effect of 1 l
M
GLP-2 between 10 and 48 h, and the dose-
dependent effect of GLP-2 (10
)12
)10

)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

)12
)10
)6
M
). Reactions were stopped as indicated
above, and then 2 gÆL
)1
crystal violet in 2% (v/v) ethanol
were added to each dish. After incubation at room
temperature, culture dishes were rinsed with distilled water
and air dried. Then, cells were eluted in 1% (w/v) sodium
dodecyl sulphate and optical density at 560 nm (A
560
)
was determined. Results were expressed as the absorbance
per well.
3002 E. Vela
´
zquez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Flow cytometry analysis
The DNA content and the phases of the cell cycle were
analyzed by flow cytometry as described by Vindelov et al.
[30], using propidium iodide as DNA marker. Treatments
were carried out 48 h after confluent astrocyte cultures had
been shifted to serum-free medium. Cells were incubated
with 1 l
M
GLP-2 for 24 h and with GLP-2 (10
)10
)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

both the different phases of the cell cycle and apoptosis by
separating cells with a diploid or higher DNA content from
those with a hypodiploid DNA content. Apoptosis was
expressed as the percentage of hypodiploid cells with respect
to total cells [31] and the stage of the cell cycle as the
percentage of cells in G0/G1 (diploid cells), G2/M (tetra-
ploid cells), and S (intermediate DNA content) phases,
respectively. To avoid mistaken results, a program that
recognized a single cell with a tetraploid DNA content of an
aggregate of two diploid cells was used.
Northern blot analysis
Treatments were carried out 48 h after confluent astrocyte
cultures had been shifted to serum-free medium. Astrocytes
were stimulated with GLP-2 (10
)9
)10
)6
M
) for 30 and
60 min. Total cellular RNA was isolated using a modified
guanidinium thiocyanate procedure [32]. Ten micrograms
of total RNA were electrophoresed on 0.4
M
formaldehyde,
1.2% (w/v) agarose in 1 · Mops at 125 V for 2 h,
transferred to membranes and then immobilized by ultra-
violet light irradiation [33]. Integrity was assessed by
methylene blue staining of membranes [34]. The different
labeled probes (1–2 · 10
9

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

AAGGTAAGTATG-3¢) corresponding to nucleotides
bases 358–379 and the sense priming oligonucleotide
(5¢-GGATCC-ATGAGGCCCCAACCAAGC-3¢) corres-
ponding to nucleotide bases 1–18 [6] were used. According
to the mouse GLP-2R gene structure, our upstream primer
is complementary to exon 1 and the downstream one is
complementary to exons 2 and 3, consequently the PCR-
product amplified from genomic DNA is about 8.5 kb.
Bands with this size have never been visualized on UV
transilluminator using ethidium bromide staining. RT-PCR
amplification was carried out using the Titan
TM
One Tube
RT-PCR System. First strand cDNA was synthesized at
52 °C for 1 h, and amplification of GLP-2R cDNA was
performed at an annealing temperature of 65 °Candan
extension temperature of 72 °C for 30 cycles. RT reactions
were analyzed to control for genomic DNA and template
contamination (PCR amplification was carried out using a
DNA polymerase from T. thermophilus). To control for
nonspecific amplification, PCR reactions were also carried
out in the absence of first-strand cDNA. RT-PCR ampli-
fication products were electrophoresed in 1% agarose and
transferred to a nylon membrane. Blots were probed under
high stringency conditions with a fragment corresponding
to nucleotides 1–848 of GLP-2 receptor from rat hypotha-
lamus labeled with digoxigenin by random priming using
the DIG DNA Labeling kit. This fragment was also ligated
into pGEMÒ-T Easy vector, and the sequence was
confirmed using an automated LKB ALF DNA sequencer

(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

of the cAMP response to GLP-2 in these cells was
0.86 ± 0.12 n
M
. Forskolin (5 l
M
), used as a positive
control, produced about a 20-fold induction (data not
shown).
Effect of GLP-2 on [
3
H]thymidine incorporation
into DNA in rat astrocyte cultures
We chose incubation times between 10 and 48 h to analyze
thetimecourseoftheeffectofGLP-2(1l
M
) on astrocyte
DNA synthesis. Figure 3 shows that GLP-2 significantly
stimulated [
3
H]thymidine incorporation into DNA through-
out the interval studied, the maximum response occurring at
24 h after exposure to GLP-2.
The dose–response experiments of GLP-2 on astrocyte
DNA synthesis are shown in Fig. 4. Cells were incubated
for24hwithGLP-2(10
)12
)10
)6
M
) and, as shown, GLP-2

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. [

3
H]Thymidine
(5 lCiÆmL
)1
) was present during the last 4 h of treatment. The amount
of tritium incorporated into an acid-precipitable form was measured
by scintillation counting. Results are expressed as percentage with
respect to their respective bovine serum albumin controls. Each point
represents the means ± SEM between three and nine independent
experiments carried out six times. **P < 0.01, ***P < 0.001.
Fig. 4. Dose-dependent effect of GLP-2 on [
3
H]thymidine incorporation
into DNA from rat astrocytes. Cells kept in serum-free medium were
incubated for 24 h with GLP-2 as indicated in Fig. 1. [
3
H]Thymidine
was present during the last 4 h of treatment. Results are expressed as
percentages (left) with respect to the control sample (zero dose of
GLP-2) and as disintegrations per min per well (right). EC
50
was
0.45 n
M
. Each point represents the means ± SEM of four independ-
ent experiments carried out six times. The control value was
3884 ± 301 d.p.m. *P < 0.025, **P < 0.01, ***P < 0.001.
3004 E. Vela
´
zquez et al. (Eur. J. Biochem. 270) Ó FEBS 2003

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

GLP-2 produced a significant increase in cell numbers at all
concentration assayed, the maximum effect (145% with
respect to the control) being observed at the highest dose
used. The EC
50
of the cellular density response to GLP-2
was 6 ± 0.4 p
M
.
Effect of GLP-2 on DNA content in rat astrocytes
in culture
To assess whether or not cell proliferation parameters were
associated with an increased programmed cellular death in
control cells with respect to the GLP-2-treated cells, we
chose incubation times between 8 and 30 h to analyze the
time-course of the effect of 1 l
M
GLP-2 and 10% fetal
bovine serum (positive control) on astrocyte DNA contents
when measured by flow cytometry, using propidium iodide
as a marker. Table 1 shows that the number of hypodiploid
cells was very small in all three experimental situations and,
significant differences were only observed in 10% FBS-
treated (1.4%) as compared with control cells (3.1%) at
24 h. However, there were no significant differences in the
number of hypodiploid cells between control astrocytes and
those incubated with GLP-2 at any time. After the trypan
blue exclusion test, more than 96% of cells were viable in the
three experimental groups studied (data not shown).
The different cell cycle phases were also analyzed in the

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

in rat astrocyte cultures
Because immediate/early gene expression is frequently
associated with cell activation, we examined whether or
not GLP-2 induced c-fos and c-jun mRNA synthesis.
Figure 6 shows representative autoradiographs indicating
the dose-dependent effect of GLP-2 on c-fos (Fig. 6A) and
c-jun (Fig. 6B) mRNAs expression after 30 and 60 min of
incubation, respectively. Also shown are the autoradio-
graphs for 28S rRNA used as a loading control at the same
times. When effects of GLP-2 on c-fos and c-jun expression
were normalized according to the 28S content, a significant
induction of expression was observed at all concentrations
studied, the maximum effects being at 10
)6
M
(2.8-fold
induction, Fig. 6C) for c-fos, and at 10
)8
M
(3.5-fold
induction, Fig. 6D) for c-jun. After 1 h of incubation,
c-fos mRNA expression was not detected at any the doses of
GLP-2 assayed, whereas significant c-jun mRNA expression
was also detected at all doses of GLP-2 assayed after 2 h
of incubation (data not shown).
Discussion
The proglucagon gene seems to be processed in a similar
manner both in gut and brain [4], giving rise to GLP-1 and
GLP-2, which act through specific receptors [5,39]. Some
biological actions of these peptides have been reported on

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

free medium for 48 h and then incubated for the times indicated with 1.4 gÆL
)1
of bovine serum albumin alone (control) or in presence of 1 l
M
GLP-2 or 10% fetal bovine serum. DNA contents were assessed by flow cytometry using propidium iodide as DNA marker.
Time (h) Control (%)
a
GLP-2 (%)
a
Fetal bovine serum (%)
a
8 6.3 ± 0.6 7.4 ± 0.3 6.7 ± 1.4
15 2.4 ± 1.5 1.9 ± 0.6 1.1 ± 0.3
18 1.2 ± 0.1 1.4 ± 0.3 1.0 ± 0.2
21 1.0 ± 0.3 1.1 ± 0.3 0.8 ± 0.2
24 3.1 ± 0.7 2.3 ± 0.7 1.4 ± 0.1
b
30 5.2 ± 2.4 3.1 ± 1.0 2.4 ± 0.5
a
Percentage of cells with a hypodiploid DNA content, means ± SD (n ¼ 3, carried out in quadruplicate).
b
P < 0.05 vs. control.
Table 2. Time course of the effects of GLP-2 and fetal bovine serum 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.
Time (h) Treatment
a
% G0-G1
b
%S
b

% 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

39.8 ± 1.6
d
13.2 ± 0.7
d
24 Control 75.3 ± 0.5 17.4 ± 2.1 7.3 ± 1.0
GLP-2 55.5 ± 2.0
d
33.5 ± 2.3
d
11.0 ± 0.3
d
Fetal bovine serum 53.9 ± 1.3
d
33.2 ± 0.7
d
12.9 ± 0.4
d
30 Control 83.9 ± 1.3 8.6 ± 1.2 7.5 ± 0.6
GLP-2 73.1 ± 1.2
d
24.0 ± 1.0
d
2.9 ± 0.2
d
Fetal bovine serum 86.6 ± 2.0 6.5 ± 0.2
c
6.9 ± 0.1
a
Treatments were with 1.4 gÆL
)1

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

23.5 ± 2.0
d
8.0 ± 1.0
10 66.5 ± 1.4
e
24.8 ± 1.2
e
8.7 ± 0.2
c
100 62.0 ± 3.4
e
28.4 ± 3.5
e
9.6 ± 0.1
d
1000 55.5 ± 2.0
e
33.5 ± 2.3
e
11.0 ± 0.3
e
a
Astrocytes were incubated with GLP-2 for 24 h.
b
Means ± SD (n ¼ 3, carried out in quadruplicate).
c
P < 0.05;
d
P < 0.01;
e

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

into DNA. Our results clearly demonstrate that GLP-2
produces a dose-dependent increase in [
3
H]thymidine
incorporation along a prolonged period of time. This might
represent the synthesis of new DNA, because in parallel an
increase in the relative cell number, as measured by staining
with crystal violet, was observed. These effects of GLP-2 on
rat astrocytes are similar to those already reported in gut
from experiments using antisera directed against bromo-
deoxyuridine or proliferating cell nuclear antigen (PCNA)
[12,14,17,18] or by measuring total mucosal DNA content
[20,45,46]. In addition, GLP-2 increases the cell numbers in
GLP-2R-transfected BHK cells [6] and [
3
H]thymidine
incorporation in the Caco-2 human epithelial cell line [47].
Further information about the effect of GLP-2 on
astrocyte proliferation was obtained upon analyzing its
action on the progression of the cell cycle. The percentage of
astrocytes in the different phases of the cell cycle was
determined as cell DNA contents, using propidium iodide
[30]. Addition of GLP-2 to the incubation medium induced
a dose-dependent astrocyte proliferation, as reflected by the
percentage of cells progressing from a diploid DNA content
to a stage where DNA content was higher. Thus, 10 n
M
GLP-2 produced a 1.4-fold increase in the percentage of
cells in S + G2-M phases, similar to what was obtained
when the relative cell number was measured by staining with

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]

significantly reduces the extent of apoptosis in both the crypt
and enterocyte compartments. Also, BHK-GLP-2R cells
have been recently used to study the antiapoptotic pathways
following the activation of GLP-2R signaling [13,50,51].
GLP-2 inhibits cycloheximide- and irinotecan hydrochlo-
ride-induced apoptosis by decreasing activation of caspases-
3, -8 and -9; by reducing caspase-3 and poly(ADP-ribose)
polymerase cleavage, and by decreasing mitochondrial
cytochrome c release in a cAMP-dependent protein kinase
A (PKA), phosphatidylinositol 3-kinase (PI3K)- and
mitogen activated protein kinase (MAPK)-independent
Fig. 6. Northern blot analysis of c-fos and c-jun expression in astrocyte
cultures following GLP-2 treatment. Cells were maintained in serum-
free medium for 48 h. Then, cells were incubated with GLP-2 for 30
(A) and 60 (B) min. Total RNA was isolated, and the c-fos, c-jun and
28S ribosomal mRNAs were determined by Northern blotting using
specific
32
P-cDNA probes. Experiments were carried out in duplicate.
Membranes were exposed at ) 70 °C for 100 h (c-fos), 65 h (c-jun) and
3 h (28S rRNA). (A) and (B) show one representative autoradiogram
of three independent experiments. Autoradiograms of blots probed for
c-fos and c-jun were quantified by densitometric scanning and the
values obtained normalized with those obtained for 28S ribosomal
RNA. Histograms are expressed as fold-induction with respect to the
control sample (zero dose of GLP-2) for c-fos (C) and c-jun (D) and
represent means ± SEM of three independent experiments carried out
in duplicate. *P <0.05,**P < 0.01, ***P <0.001.
Ó FEBS 2003 GLP induces proliferation of astrocytes (Eur. J. Biochem. 270) 3007
pathway. In addition, it has been demonstrated that GLP-

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

(DGICYT), Fondo de Investigacio
´
n Sanitaria de la Seguridad Social
(FIS), Comunidad de Madrid, and from the Instituto de Salud Carlos
III, RGDM (G03/212), Madrid, Spain.
References
1. Hartmann, B., Johnsen, A.H., Orskov, C., Adelhorst, K., Thim,
L. & Holst, J.J. (2000) Structure, measurement, and secretion of
human glucagon-like peptide 2. Peptides 21, 73–80.
2. Heinrich, G., Gros, P. & Habener, J.F. (1984) Glucagon gene
sequence: four of six exons encode separate functional domains of
rat preproglucagon. J. Biol. Chem. 259, 14082–14087.
3. Mojsov, S., Heinrich, G., Wilson, I.B., Ravazzola, M., Orci, L. &
Habener, J.F. (1986) Preproglucagon gene expression in pancreas
and intestine diversifies at the level of post-transcriptional pro-
cessing. J. Biol. Chem. 261, 11880–11889.
4. Larsen, P.J., Tang-Christensen, M., Holst, J.J. & Orskov, C.
(1997) Distribution of glucagon-like peptide-1 and other pre-
proglucagon-derived peptides in the rat hypothalamus and
brainstem. Neuroscience 77, 257–270.
5. Munroe, D.G., Gupta, A.K., Kooushesh, F., Vyas, T.B., Rizkalla,
G., Wang, H., Demchyshyn, L., Yang, Z., Kamboj, R.K.,
Chen, H., McCallum, K., Sumner-Smith, M., Drucker, D.J. &
Crivici, A. (1999) Prototypic G protein-coupled receptor for the
intestinotrophic factor glucagon-like peptide 2. Proc.NatlAcad.
Sci. USA 96, 1569–1573.
6. Yusta, B., Somwar, R., Wang, F., Munroe, D., Grinstein, S., Klip,
A. & Drucker, D.J. (1999) Identification of glucagon-like peptide-2
(GLP-2)-activated signaling pathways in baby hamster kidney
fibroblasts expressing the rat GLP-2 receptor. J. Biol. Chem. 274,

30459–30467.
7. Bjerknes, M. & Cheng, H. (2001) Modulation of specific intestinal
epithelial progenitors by enteric neurons. Proc. Natl Acad. Sci.
USA 98, 12497–12502.
8. Yusta, B., Huang, L., Munroe, D., Wolff, G., Fantaske, R.,
Sharma, S., Demchyshyn, L., Asa, S.L. & Drucker, D.J. (2000)
Enterocrine localization of GLP-2 receptor expression. Gastro-
enterology 119, 744–775.
9. Lovshin, J., Estall, J., Yusta, B., Brown, T.J. & Drucker, D.J.
(2001) Glucagon-like peptide (GLP)-2 action in the murine central
nervous system is enhanced by elimination of GLP-1 receptor
signaling. J. Biol. Chem. 276, 21489–21499.
10. Tang-Christensen, M., Vrang, N. & Larsen, P.J. (2001) Glucagon-
like peptide containing pathways in the regulation of feeding
behaviour. Int. J. Obes. Relat. Metab. Disord. 5, S42–S47.
11. Drucker, D.J. (2001) Glucagon-like peptide 2. J. Clin. Endocrinol.
Metab. 86, 1759–1764.
12. Drucker, D.J., Ehrlich, P., Asa, S.L. & Brubaker, P.L. (1996)
Induction of intestinal epithelial proliferation by glucagon-like
peptide 2. Proc. Natl Acad. Sci. USA 93, 7911–7916.
13. Yusta, B., Estall, J. & Drucker, D.J. (2002) Glucagon-like peptide-
2 receptor activation engages bad and glycogen synthase kinase-3
in a protein kinase A-dependent manner and prevents apoptosis
following inhibition of phosphatidylinositol 3-kinase. J. Biol.
Chem. 277, 24896–24906.
14. Tsai, C H., Hill, M., Asa, S.L., Brubaker, P.L. & Drucker, D.J.
(1997) Intestinal growth-promoting properties of glucagon-like
petide 2 in mice. Am. J. Physiol. 273, E77–E84.
15. Chance, W.T., Foley-Nelson, T., Thomas, I. & Balasubraniam, A.
(1997) Prevention of parenteral nutrition induced gut hypoplasya

by coinfusion of glucagon-like peptide 2. Am.J.Physiol.36,
G599–G563.
16. Scott, R.B., Kirk, D., MacNaughton, W.K. & Meddings, J.B.
(1998) GLP-2 augments the adaptive response to massive
intestinal resection in rat. Am.J.Physiol.275, G911–G921.
17. Boushey, R.P., Yusta, B. & Drucker, D.J. (1999) Glucagon like
peptide-2 decreases mortality and reduces the severity of
indomethacin-induced murine enteritis. Am.J.Physiol.277,
E937–E947.
18. Drucker, D.J., Yusta, B., Boushey, R.P., DeForest, L. & Bruba-
ker, P.L. (1999) Human (Gly2) GLP-2 reduces the severity of
colonic injury in a murine model of experimental colitis. Am. J.
Physiol. 276, G79–G91.
19. Alavi, K., Schwartz, M.Z., Palazzo, J.P. & Prasad, R. (2000)
Treatment of inflammatory bowel disease in a rodent model with
the intestinal growth factor glucagon-like peptide 2. J. Pediatr
Surg. 35, 847–851.
20. Prasad, R., Alavi, K. & Schwartz, M.Z. (2000) Glucagon-like
peptide-2 analogue enhances intestinal mucosal mass after ische-
mia and reperfusion. J. Pediatric Surg. 35, 357–359.
21. Fischer, K.D., Dhanvantari, S., Drucker, D. & Brubaker, P.L.
(1997) Intestinal growth is associated with elevated levels of
glucagon-like peptide 2 in diabetic rats. Am.J.Physiol.273,
E815–E880.
22. Hartmann, B., Thulesen, J., Hare, K.J., Kissow, H., Orskov, C.,
Poulsen, S.S. & Holst, J.J. (2002) Immunoneutralization of
endogenous glucagon-like peptide-2 reduces adaptative intestinal
growth in diabetic rats. Regul. Pept. 105, 173–179.
23. Navarro, M., Rodrı
´

guez de Fonseca, F., Alvarez, E., Chowen,
J.A., Zueco, J.A., Gomez, R., Eng, J. & Bla
´
zquez, E. (1996)
Colocalization of glucagon-like peptide-1 (GLP-1) receptors,
glucose transporter GLUT-2, and glucokinase mRNAs in rat
hypothalamic cells: Evidence for a role of GLP-1 receptor agonists
as an inhibitory signal for food and water intake. J. Neurochem.
67, 1982–1991.
24. Rodrı
´
guez de Fonseca, F., Navarro, M., Alvarez, E., Roncero, I.,
Chowen, J.A., Maestre, O., Go
´
mez, R., Mun
˜
oz, R.M., Eng, J. &
Blazquez, E. (2000) Peripheral versus central effects of glucagon-
like peptide-1 receptor agonists on satiety and body weight loss in
Zucker obese rats. Metabolism 49, 709–717.
25. Tang-Christensen, M., Larsen, P.J., Thulesen, J., Romer, J. &
Vrang, N. (2000) The proglucagon-derived peptide, glucagon-like
peptide-2, is a neurotransmitter involved in the regulation of food
intake. Nat. Med. 6, 802–807.
3008 E. Vela
´
zquez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
26. Dhillo, W.S. & Bloom, S.R. (2001) Hypothalamic peptides as drug
targets for obesity. Curr. Opin. Pharmacol. 1, 651–655.
27. MacCarthy, K.D. & de Vellis, J. (1980) Preparation of separate

astroglial and oligodendroglial cell cultures from rat cerebral
tissue. J. Cell Biol. 85, 890–892.
28. Pietras, R.J. & Szego, C.M. (1979) Metabolic and proliferative
responses to estrogen by hepatocytes selected for plasma-mem-
brane binding-sites specific for estradiol-17a. J. Cell Physiol. 98,
145–160.
29. Barna, B.P., Estes, M.L., Jacobs, B., Hudson, S. & Ransohoff,
R.M. (1990) Human astrocytes proliferate in response to tumor
necrosis factor alpha. J. Neuroinmunol. 30, 239–243.
30. Vindelov, L.L., Christensen, I.J., Keiding, N., Spang-Thomson,
M. & Nissen, N.I. (1983) Long-term storage of samples for flow
cytometric DNA analysis. Cytometry 3, 317–322.
31. Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz,
M.A., Lassota, P. & Traganos, F. (1992) Features of apoptotic
cells measured by flow cytometry. Cytometry 13, 795–808.
32. Chomezynsky, P. & Sacchi, N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate–phenol–chloroform
extraction. Anal Biochem. 162, 156–159.
33. Church, G.M. & Gilbert, W. (1984) Genomic sequencing. Proc.
NatlAcad.Sci.USA81, 1991–1995.
34. Herrin, D.L. & Schmidt, G.W. (1988) Rapid reversible staining of
northern blots prior to hybridization. Biotechniques 6, 196–200.
35. Feinberg, A. & Vogelstein, B. (1984) Technique for radiolabeling
DNA restriction endonuclease fragments to high specific activity.
Anal Biochem. 137, 266–273.
36. Valı
´
n, A., Guille
´
n, C. & Esbrit, P. (2001) C-Terminal parathyroid

hormone-related protein (PTHrP) (107–139) stimulates intracel-
lular Ca
2+
through a receptor different from the type 1 PTH/
PTHrP receptor in osteoblastic osteosarcoma UMR 106 cells.
Endocrinology 142, 2752–2759.
37. Angel, P., Allegretto, E.A., Okino, S.T., Hattori, K., Boyle, W.J.,
Hunter, T. & Karin, M. (1988) Oncogene jun encodes a
sequence-specific trans-activator similar to AP-1. Nature 322,
166–170.
38. Martı
´
nez, B., del Hoyo, P., Martı
´
n, M.A., Arenas, J., Pe
´
rez-
Castillo, A. & Santos, A. (2001) Thyroid hormone regulates
oxidative phosphorylation in the cerebral cortex and striatum of
neonatal rats. J. Neurochem. 78, 1054–1063.
39. Thorens, B. (1992) Expression cloning of the pancreatic b cell
receptor for the glucoincretin hormone glucagon-like peptide-1.
Proc.NatlAcad.Sci.US89, 8641–8645.
40. Chowen, J.A., Rodrı
´
guez de Fonseca, F., Alvarez, E., Navarro,
M., Garcı
´
a-Segura, L.M. & Bla
´

zquez, E. (1999) Increased gluca-
gon-like peptide-1 receptor expression in glia after mechanical
lesion of the rat brain. Neuropeptides 33, 212–215.
41. Perry,T.A.,Lahiri,D.K.,Chen,D.,Zhou,J.,Shaw,K.T.Y.,
Egan, J.M. & Greig, N.H. (2002) A novel neurotrophic property
of glucagon-like peptide-1: a promoter of nerve growth factor-
mediated differentiation in PC12 cells. J. Pharmacol. Exp Ther
300, 958–966.
42. Perry, T.A., Haughey, N.J., Mattson, M.P., Egan, J.M. & Greig,
N.H. (2002) Protection and reversal of excitotoxic neuronal
damage by glucagon-like peptide-1 and exendin-4. J. Pharmacol.
Exp. Ther. 302, 881–888.
43. DaCambra, M.P., Yusta, B., Sumner-Smith, M., Crivici, A.,
Drucker, D. & Brubaker, P.L. (2000) Structural determinants for
activity of glucagon-like peptide-2. Biochemistry 39, 8888–8894.
44. Thulesen, J., Knudsen, L.B., Hartmann, B., Hastrup, S., Kissow,
H., Jeppesen, P.B., Ørskov, C., Holst, J.J. & Poulsen, S.S. (2002)
The truncated metabolite GLP-2 (3–33) interacts with the GLP-2
receptor as a potential agonist. Reg. Peptides 103, 9–15.
45. Litvak, D.A., Hellmich, M.R., Evers, B.M., Banker, N.A. &
Townsend, C.M. (1998) Glucagon-like peptide 2 is a potent
growth factor for small intestine and colon. J. Gastrointest. Surg.
2, 146–150.
46. Kato, Y., Yu D. & Schwartz, M.Z. (1999) Glucagon-like peptide-2
enhances small intestinal absorptive function and mucosal mass
in vivo. J. Pediatr. Surg. 34, 18–20.
47. Jasleen, J., Shimoda, N., Shen, E.R., Tavakkolizadeh, A., Whang,
E.E., Jacobs, D.O., Zinner, M.J. & Ashley, S.W. (2000) Signaling
mechanisms of glucagon-like peptide 2-induced intestinal epithe-
lial cell proliferation. J. Surg. Res. 90, 13–18.

48. Rauscher, F.J., Voulalas, P.J., Franza, B.R. & Curran, T. (1988)
Fos and Jun bind cooperatively to the AP-1 site: reconstitution
in vitro. Genes Dev. 12B, 1687–1699.
49. Micoud, F., Mandrand, B. & Malcus-Vocanson, C. (2001)
Comparison of several techniques for the detection of apoptotic
astrocytes in vitro. Cell Prolif. 34, 99–113.
50. Boushey, R.P., Yusta, B. & Drucker, D.J. (2001) Glucagon-like
peptide (GLP) -2 reduces chemotherapy-associated mortality and
enhances cell survival in cells expressing a transfected GLP-2
receptor. Cancer Res. 61, 687–693.
51. Yusta, B., Boushey, R.P. & Drucker, D.J. (2000) The glucagon-
like peptide-2 receptor mediates direct inhibition of cellular
apoptosis via a cAMP-dependent protein kinase-independent
pathway. J. Biol. Chem. 275, 35345–35352.
52. Burrin, D.G., Stoll, B., Jiang, R., Petersen, Y., Elnif, J.,
Buddington, R.K., Schmidt, M., Holst, J.J., Hartmann, B. &
Sangild, P.T. (2000) GLP-2 stimulates intestinal growth in pre-
mature TPN-fed pigs by suppressing proteolysis and apoptosis.
Am. J. Physiol. Gastrointest. Liver Physiol. 279, G1249–G1256.
Ó FEBS 2003 GLP induces proliferation of astrocytes (Eur. J. Biochem. 270) 3009