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: 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) 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. 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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 . Glucagon-like peptide-2 stimulates the proliferation of cultured rat astrocytes Esther Vela ´ zquez, Juan M. Ruiz-Albusac. 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