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.. Results
Trang 1Glucagon-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 (EC50¼ 0.86 nM) GLP-2 also stimulated the DNA
synthesis rate in rat astrocytes When proliferation was
assessed by measuring [3H]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,a physiologicalgrowth 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 CNS actions of GLP-1,the potential effect(s) of GLP-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: cazorla@med.ucm.es
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)
Trang 2Because 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-3H]-thymidine (60–80 CiÆmmol)1) and
[a32P]-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 TitanTM One Tube RT-PCR System and
the DIG DNA labeling Kit were from Roche Molecular
Biochemicals (Barcelona,Spain) DNA polymerase from
Thermus thermophiluswas from Biotools (Madrid,Spain)
PGEM-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-cm2tissue culture flasks at low-density
Cells were grown in DMEM/F-12 medium,15 mMHepes
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 37C and replated at
approximately 2.3–3· 104 cellsÆcm)2 in 24-well (for
[3H]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/Pi) to cells Finally,cells were washed twice with NaCl/Pi 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 nM,in the presence of 10 lM 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 (EC50) was calcula-ted using the GRAPHPAD PRISM APPLICATION (GraphPad software,Inc.)
[3H]-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 [3H]-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 lMGLP-2 between 10 and 48 h,and the dose-dependent effect of GLP-2 (10)12)10)6
M) for 24 h [3H]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 0.5M NaOH for 1 h at room temperature and then with 20% (v/v) cold trichloroacetic acid for 1 h at 4C 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 [3H]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)6M) Reactions were stopped as indicated above,and then 2 gÆL)1crystal 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 (A560) was determined Results were expressed as the absorbance per well
Trang 3Flow 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 lMGLP-2 for 24 h and with GLP-2 (10)10)10)6M)
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 4C in presence of freshly prepared
fetal bovine serum Pellets were washed with NaCl/Piand
incubated on ice water with 70% ethanol at) 20 C After
the addition of NaCl/Pi,lysed cells were sedimented at
1700 g for 4 min at 4C and then incubated for 30 min at
37C with 1 gÆL)1ribonuclease A in 50 mMTris,pH 8.0,
10 mM EDTA Then,propidium iodide (0.05 gÆL)1 final
concentration) was added and the samples were maintained
in the darkness at 4C 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)6M) 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 0.4Mformaldehyde,
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· 109cpmÆlg)1) were generated using
random primers [35] and hybridized with the membranes
for 20 h at 42C (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 65C,for mild washing,and then 0.2 · NaCl/Cit/0.5%
SDS at 65C 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 TitanTMOne Tube RT-PCR System First strand cDNA was synthesized at
52C for 1 h,and amplification of GLP-2R cDNA was performed at an annealing temperature of 65C and an extension temperature of 72C 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 pGEM-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
Trang 4cortex 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)8M
The EC50of the cAMP response to GLP-2 in these cells was
0.86 ± 0.12 nM Forskolin (5 lM),used as a positive
control,produced about a 20-fold induction (data not
shown)
Effect of GLP-2 on [3H]thymidine incorporation
into DNA in rat astrocyte cultures
We chose incubation times between 10 and 48 h to analyze
the time course of the effect of GLP-2 (1 lM) on astrocyte
DNA synthesis Figure 3 shows that GLP-2 significantly
stimulated [3H]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 for 24 h with GLP-2 (10)12)10)6M) 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 [3H]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)1of bovine serum albumin alone (d) or in 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 [3H]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 [3H]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 experimindepend-ents carried out six times The control value was
3884 ± 301 d.p.m *P < 0.025,**P < 0.01,***P < 0.001.
Trang 5induced a proportional increase in [3H]thymidine
incorpor-ation into DNA,the maximum effect being observed (185%
with respect to the control) at 10)8M The EC50 of the
[3H]-thymidine incorporation response into astrocyte
cultures was 0.45 ± 0.05 nM
Effect of GLP-2 on the cellular density of rat astrocytes
in culture
To assess whether GLP-2 induction of [3H]thymidine
incorporation was associated with increased cell numbers,
astrocytes were incubated for 24 h with GLP-2
(10)12)10)6M) 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 EC50of the cellular density response to GLP-2
was 6 ± 0.4 pM
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 lM 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 lM) 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)10M)
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)6M (2.8-fold induction,Fig 6C) for c-fos,and at 10)8M (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.
Trang 6recently 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)1of 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.
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.
a Treatments were with 1.4 gÆL)1bovine serum albumin (control),1 l M GLP-2 and 10% fetal bovine serum b Means ± SD (n ¼ 3,carried out in quadruplicate).cP < 0.05.dP < 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.
a
Astrocytes were incubated with GLP-2 for 24 h. bMeans ± SD (n ¼ 3,carried out in quadruplicate). cP < 0.05; dP < 0.01;
e
P < 0.001 vs the zero dose.
Trang 7expression 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 EC50 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 EC50
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 [3H]thymidine incorporation
into DNA Our results clearly demonstrate that GLP-2
produces a dose-dependent increase in [3H]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 [3H]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 nM 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
specific32P-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.
Trang 8pathway 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´n Cientı´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.
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