A2B receptor activation promotes glycogen synthesis in astrocytes through modulation of gene expression

Igor Allaman, Sylvain Lengacher, Pierre J. Magistretti, and Luc Pellerin

Institut de Physiologie, Faculté de Médecine, Université de Lausanne, 1005 Lausanne, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adenosine has been proposed as a key factor regulating the metabolic balance between energy supply and demand in the central nervous system. Because astrocytes represent an important cellular element in the control of brain energy metabolism, we investigated whether adenosine could induce long-term changes of glycogen levels in primary cultures of mouse cortical astrocytes. We observed that adenosine increased glycogen content, up to 300%, in a time- (maximum at 8 h) and concentration-dependent manner with an EC50 of 9.69 µM. Pharmacological experiments using the broad-spectrum agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and specific agonists for the A1, A2A, and A3 receptors [N6-cyclopentyladenosine (CPA), CGS-21680, and IB-MECA, respectively] suggest that the effect of adenosine is mediated through activation of the low-affinity A2B adenosine receptor subtype. Interestingly, adenosine induces in parallel the expression of the protein targeting to glycogen (PTG), one of the protein phosphatase-1 glycogen-targeting subunits that has been implicated in the control of glycogen levels in various tissues. These results indicate that adenosine can exert long-term control over glycogen levels in astrocytes and might therefore play a significant role in physiological and/or pathological processes involving long-term modulation of brain energy metabolism.

energy metabolism; protein targeting to glycogen; CCAAT/enhancer-binding protein; purinergic receptor; glia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADENOSINE HAS BEEN RECOGNIZED as an important neuromodulator in the central nervous system (7, 9). Its effect on neurotransmission represents a balance between inhibitory and facilitory influences that are mediated via four subtypes of receptors classified as A1, A2A, A2B, and A3 (13). Activation of A1 receptors leads to an overall inhibition of neuronal activity through both presynaptic and postsynaptic mechanisms. Thus adenosine prevents neurotransmitter release by inhibiting the opening of voltage-dependent Ca2+ channels while causing a postsynaptic hyperpolarization via activation of inwardly rectifying K+ channels (Ref. 7 and references therein). In contrast, activation of the other adenosine receptors, and particularly the A2A subtype, is associated with a facilitation of neurotransmission (43). In addition to its role as a neuromodulator, adenosine has been proposed to act as an homeostatic regulator in many tissues including the brain (4, 7). Adenosine has been shown to accumulate in the extracellular space after increased neuronal activity as a consequence of high-energy utilization (25, 28). In such circumstances, it is postulated that adenosine would act to couple energy supply to demand (4). Thus adenosine is a potent vasodilatory agent known to participate in the enhancement of cerebral blood flow triggered by neuronal activity (34). By locally increasing blood flow, adenosine would contribute to bringing more oxygen and glucose to the activated region. At the same time, by acting via A1 receptors to decrease neuronal activity, it would reduce subsequent energy needs. In relation to the energy homeostasis concept, adenosine was also reported to have a glycogenolytic effect in brain slices as well as in cultured cortical astrocytes (22, 37, 44).

Glycogen is the major energy reserve of the brain, and it is almost exclusively localized in astrocytes (6). Although the exact role of cerebral glycogen is not known, strong evidence suggests that glycogen mobilization is tightly coupled to neuronal activity (46). Indeed, glycogen accumulation is observed in conditions of decreased neuronal activity such as phenobarbital anesthesia (33), hibernation (46), and slow-wave sleep (17). In contrast, cerebral glycogen breakdown is observed after sensory stimulation (47). Moreover, in addition to adenosine, various neuroactive substances possess glycogenolytic properties in vitro (Ref. 24 and references therein). In particular, a glycogenolytic effect of vasoactive intestinal peptide (VIP) and norepinephrine (NE) has been demonstrated in mouse cerebral cortical slices as well as in primary cultures of cortical astrocytes (23, 37, 44). It was shown that, in addition to their rapid glycogenolytic effect, both NE and VIP induce, through the activation of the cAMP-dependent signal transduction pathway, a massive glycogen resynthesis in cortical astrocytes that takes place over several hours (45). Interestingly, this effect apparently requires activation of gene expression and synthesis of new proteins.

In the present study, we report that adenosine enhances glycogen levels in primary cultures of mouse cortical astrocytes over a period of several hours. This effect is apparently mediated by A2B receptor subtypes and involves the activation of genes encoding proteins well known for their role in the regulation of energy metabolism. These results highlight the possibility that, in addition to its rapid glycogenolytic effect, adenosine exerts a long-term control over glycogen levels, an action consistent with its putative role as a homeostatic modulator.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Fetal calf serum was purchased from Fakola (Basel, Switzerland). Amyloglucosidase, hexokinase/glucose-6-phosphate dehydrogenase, ATP, and NADP were from Boehringer Mannheim (Rotkreutz, Switzerland). Basic fuchsin was from Fluka (Buchs, Switzerland). Rolipram was from RBI (Natick, MA). The radioreceptor assay used to mesure cAMP was obtained from Amersham Pharmacia (Duebendorf, Switzerland). Adenosine, N6-cyclopentyladenosine (CPA), 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-beta -D-ribofuranuronamide (IB-MECA), 5'-(N-ethylcarboxamido)adenosine (NECA), 8-[4-(((((2-aminoethyl)amino)carbonyl)methyl)oxy)-phenyl]-1,3-dipropylxanthine (xanthine amine congener, XAC), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89), 1-(6-[([17beta ]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U-73122), Dulbecco's modified Eagle's medium (DMEM), actinomycin D (actD), cycloheximide (CHX), as well as all other chemicals were obtained from Sigma (Buchs, Switzerland).

Cell cultures. All experiments were carried out in accordance with the European Communities Council Directive regarding care and use of animals for experimental procedures. Primary cultures of cerebral cortical astrocytes were prepared from 1- to 2-day-old Swiss Albino mice (OF1; Iffa Credo, Lyon, France) as previously described (45). Briefly, brains were removed aseptically from skulls, meninges were excised carefully, and neocortices were dissected. Cells were dissociated by passage through needles of decreasing gauges (1.2 × 40, 0.8 × 40, and 0.5 × 16 mm) with a 10-ml syringe. No trypsin was used for dissociation. Cells were seeded on polyornithine-coated dishes in DMEM (catalog no. D7777, Sigma) containing 10% fetal calf serum and incubated at 37°C in an atmosphere containing 5% CO2-95% air. Cells were plated on 60 × 15 mm (Northern blots) or 35 × 10 mm (glycogen assay, cAMP assay, and histochemical reaction for glycogen) culture dishes. The culture medium was renewed 5 days after seeding and subsequently twice a week. This procedure yields cultures in which >90% of cells are immunoreactive for glial fibrillary acidic protein (GFAP) (45). Twenty-one-day-old cultures were used for all experiments.

Exposure to pharmacological agents. During all incubations, primary cultures of astrocytes were maintained at 37°C in an atmosphere containing 5% CO2-95% air. Twenty-four hours before treatment, the culture medium was removed and astrocytes were incubated in serum-free DMEM (catalog no. D5030, Sigma) supplemented with 5 mM glucose, 44 mM NaHCO3, 10 ml/l of an antibiotic-antimycotic solution (catalog no. A7292, Sigma), and 0.045 mM phenol red (DMEM5). Without changing the medium, the cells were then exposed to 10 nM-300 µM adenosine, 10 nM-100 µM NECA, 100 nM-1 µM CPA, 10 µM CGS-21680, or 10-100 nM IB-MECA. Adenosine or other pharmacological agents were maintained during the entire incubation period except as shown in Fig. 1B. In this case, astrocytes were exposed for either 15 or 30 min to adenosine, the medium was replaced by fresh medium without adenosine, the incubation was pursued for a total period of 8 h, and then glycogen determination was performed. When indicated, cells were exposed for 30 min to 10 µM CHX, 5 µM actD, 2 µM XAC, 1-5 µM H-89, or 10 µM U-73122 before exposure to adenosine and maintained during the entire incubation period.


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Fig. 1.   Induction of glycogen synthesis by adenosine in primary cultures of cortical astrocytes. A: time course analysis. Cultures were stimulated for various periods of time with 100 µM adenosine, and glycogen levels were determined as described in MATERIALS AND METHODS. Results are means ± SE of triplicate determinations from 1 experiment. Representative experiment repeated twice with similar results. B: glycogen resynthesis after brief adenosine exposure. Astrocytes were exposed for either 15 or 30 min to adenosine (ADE), the medium was replaced by fresh medium without adenosine, and the incubation was pursued for a total period of 8 h, after which glycogen determination was performed. Results are means ± SE of triplicate determinations from 1 experiment. Representative experiment repeated twice with similar results. Data were statistically analyzed with ANOVA followed by Dunnett's test. ** Significantly different from control (P < 0.01). C: histochemical detection. Apparently different levels of glycogen are revealed in subsets of astrocytes after stimulation with adenosine. Histochemical localization of glycogen was performed in control cultures or cultures stimulated with 100 µM adenosine over 8 h. Black and white arrows indicate astrocytes showing strong or moderate labeling for glycogen (in red), respectively.

Northern blot. Total RNA from cell cultures was extracted with the Trizol reagent according to the manufacturer's procedure (Life Technologies, Basel, Switzerland). Ten micrograms of RNA from each sample was electrophoresed through denaturing 1.3% agarose gels containing 2 M formaldehyde and transferred onto GeneScreen nylon membranes (NEN Life Sciences, Zaventem, Belgium). Prehybridization was performed for at least 6 h at 65°C in 50% formamide, 5× standard saline citrate (SSC), 50 mM Tris · HCl pH 7.5, 0.1% sodium pyrophosphate, 1% SDS, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 5 mM EDTA, 0.2% bovine serum albumin (BSA), and 150 µg/ml sheared denatured salmon sperm DNA. Hybridization was performed for at least 18 h at 65°C by adding to the prehybridization solution a 32P-labeled antisense riboprobe for CCAAT/enhancer-binding protein (C/EBP)beta , C/EBPdelta , or protein targeting to glycogen (PTG). Membranes were then washed under high-stringency conditions (twice with 2× SSC-0.1% SDS at 65°C for 15 min and twice with 0.1× SSC-0.1% SDS at 65°C for 30 min), and autoradiographed at -70°C with an intensifying screen.

Differences in RNA gel loading and blotting were assessed by stripping and rehybridizing the membranes with a 32P-antisense beta -actin riboprobe. Hybridization and washing conditions for beta -actin were identical to those for the other probes.

Mouse C/EBPbeta and C/EBPdelta 32P-antisense riboprobes were generated as described previously (5). The 32P-antisense riboprobe for PTG was generated from a 768-bp cDNA fragment obtained by reverse transcription (RT) and polymerase chain reaction (PCR) amplification of total RNA from primary cultures of mouse cortical astrocytes with a set of oligonucleotide primers (5'-ATGTCTCAGTGTCAAGCAGG-3' and 5'-GATAGGAGGTCAAGTTCTCC-3') located at 171-190 and 940-921 bp in the coding region of the mouse PTG cDNA sequence (36). The identity of the amplified PTG cDNA fragment was confirmed by sequencing with an automated DNA sequencer (ALF DNA Analysis System; Pharmacia, Uppsala, Sweden). Evaluation of PTG mRNA expression level was performed with quantitative RT-PCR according to the method of Heid et al. (14) with a set of oligonucleotide primers (5'-TCGCAGAGTGAGTGGAAGAGC-3' and 5'-CTTGGAGTCCGCAAACACG-3') located at 199-219 and 264-246 bp in the mouse PTG cDNA sequence (36).

Glycogen assay. After the desired time of incubation, cells were washed three times with ice-cold phosphate-buffered saline (PBS) and sonicated in 30 mM HCl. The suspension was used to measure glycogen as previously described by Sorg and Magistretti (45). Briefly, three 100-µl aliquots were sampled. In the first aliquot, 300 µl of acetate buffer was added. In the second aliquot, 300 µl of a solution containing 1% amyloglucosidase (10 mg/ml) in acetate buffer (0.1 M, pH 4.65) was added and the mixture was incubated at room temperature for 30 min. After incubation with amyloglucosidase, 2 ml of Tris · HCl buffer (0.1 M, pH 8.1) containing 3.3 mM MgCl2, 0.2 mM ATP, 25 µg/ml NADP, 4 µg/ml hexokinase, and 2 µg/ml glucose-6-phosphate dehydrogenase was added and the mixture was incubated at room temperature for 30 min. The first aliquot was treated identically. The fluorescence of the NADPH formed was then read on a fluorometer (excitation 340 nm; emission 450 nm). The first aliquot gives the sum of glucose and glucose-6-phosphate, whereas the second aliquot gives the sum of glycogen, glucose, and glucose-6-phosphate; the amount of glycogen is then determined by the difference between the first two aliquots. The third aliquot was used to measure the protein content by the method of Bradford (3). In this report, one mole of glycogen corresponds to one mole of glycosyl units originating from glycogen.

Histochemical reaction for glycogen. Histochemical localization of glycogen was performed with the method of Rosenberg and Dichter (39). Cell cultures were washed three times with ice-cold PBS, fixed 5 min at room temperature in methanol, and washed three times with 70% (vol/vol) ethanol. After incubation for 30 min at room temperature in 1% (wt/vol) periodic acid dissolved in 70% ethanol, cells were washed three times with 70% ethanol and stained for 60 min at room temperature in 0.5% (wt/vol) basic fuchsin dissolved in acid ethanol (ethanol-water-concentrated HCl, 80:20:1 vol/vol/vol). After staining, cells were rinsed three times in 70% ethanol, dehydrated in absolute ethanol, and counterstained with hematoxylin. Cells were then rinsed in water and dehydrated in absolute ethanol. Stained cells were photographed with an Axioplan2 inverted phase contrast microscope equipped with an AxioCam charge-coupled device (CCD) camera (Zeiss, Feldbach, Switzerland).

cAMP assay. cAMP levels in cultured astrocytes were measured according to the following procedure. Twenty-four hours before treatment, the culture medium was removed and cells were incubated in DMEM5 without phenol red. At the time of the experiment, the extracellular medium was reduced to 1 ml of DMEM5 without phenol red. Ten minutes before exposure to adenosine or NECA, rolipram, an inhibitor of the cAMP-selective (type IV) phosphodiesterase isoenzyme, was added in the medium at a final concentration of 100 µM and maintained over the entire incubation period. Incubation with adenosine and NECA was carried out for 20 min at 37°C. After incubation, cells were briefly sonicated and chilled on ice to terminate the reaction, and two aliquots were sampled. The first aliquot was used to measure the protein content by the method of Bradford (3). Cell lysate from the second aliquot was boiled for 10 min and centrifuged for 2 min at 10,000 g. Aliquots of the supernatant (50 µl) were taken to assess cAMP levels with a radioreceptor assay kit using [3H]cAMP as a tracer.

RT-PCR. Total RNA from cell cultures was extracted with the Trizol reagent according to the manufacturer's procedure (Life Technologies). Four micrograms of RNA was reverse transcribed with oligo(dT)12-18 primer and Superscript II reverse transcriptase (Life Technologies) in a final volume of 20 µl for 1 h at 37°C followed by 30 min at 50°C. cDNAs generated were subjected to PCR amplification with primers (forward primer 5'-CCTCGAGTGCATTACAGACC-3' and reverse primer 5'-TGGAGTGGTCCATCAGTTCC-3') specific for the the A2B receptor subtype mRNA sequence (GenBank accession no. NM007413). Twenty percent of the RT reaction mixture was amplified by PCR in a final volume of 100 µl containing 2.5 U of Taq DNA polymerase (Amersham Pharmacia), 200 µM dNTPs, and 40 pmol of each primer. PCR amplification was performed as follows: 5 min at 95°C and then 30 amplification cycles for 45 s at 94°C, 45 s at 61°C, and 45 s at 72°C, ending with 10 min at 72°C. PCR products were subsequently electrophoresed together with a 0.1-kb DNA ladder marker (Promega, Wallisellen, Switzerland) in a 2% agarose gel in the presence of ethidium bromide. The resulting cDNA fragment from the coding region of the A2B receptor subtype has an expected length of 567 nucleotides, and the correct amplicon identity was confirmed by automated DNA sequencing (ALF DNA analysis System, Pharmacia). A negative control for the RT-PCR experiment was performed with a RT reaction in which reverse transcriptase was omitted.

Statistical analysis. All results are presented as means ± SE. Statistical analysis was performed with either Student's t-test or analysis of variance (ANOVA) followed by Dunnett's or Bonferroni's test with INSTAT software (GraphPad, San Diego, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Regulation of glycogen levels by adenosine was examined in primary cultures of cortical astrocytes. As described previously (44), exposure of cortical astrocytes to adenosine induces glycogenolysis leading to reduced glycogen levels as observed after 30 min of treatment (Student's t-test, P < 0.005 vs. control; Fig. 1A). Interestingly, this short-term action was followed by a marked and delayed induction of glycogen synthesis that occurred several hours after the beginning of adenosine stimulation. As shown in Fig. 1A, glycogen levels increased significantly 2 h after the beginning of adenosine exposure to reach a maximum value, up to three times its initial level, 8 h later. Most importantly, a similar effect was observed when cells were only briefly exposed to adenosine (15 or 30 min) and glycogen levels were assessed 8 h later (Fig. 1B). Histochemical localization of glial glycogen shows that this increase in glycogen is apparent in only a small subset of cortical astrocytes (Fig. 1C). Striking heterogeneity could be observed in adenosine-treated cultures, with some cells presenting either strong or moderate staining for glycogen whereas staining in other cells was not different from control.

Because the glycogenolytic effect of adenosine depends on receptor activation (44), we sought to determine whether this is also the case for glycogen resynthesis and through which adenosine receptor subtype it occurs. Indeed, it was observed that the effect of adenosine on glycogen synthesis was prevented by the general adenosine receptor antagonist XAC (Fig. 2A). The adenosine receptor family is composed of four members, the A1, A2A, A2B, and A3 receptor subtypes. With the exception of the A2B receptor subtype, specific agonists for these receptors are available. Therefore, glycogen levels were determined in cortical astrocytes stimulated with either a nonspecific agonist of all adenosine receptors (NECA) or specific agonists for the A1 (CPA), A2A (CGS-21680), and A3 (IB-MECA) receptors (Fig. 2B). Results show that NECA is able to reproduce the effect of adenosine on glycogen resynthesis whereas CPA, CGS-21680, and IB-MECA were without effect, a pattern consistent with the implication of the A2B receptor subtype. Adenosine and NECA exhibit a low affinity for this receptor compared with the other members of the P1 receptor family. EC50 values determined from concentration-response curves for adenosine and NECA on glycogen resynthesis were 9.69 ± 3.35 and 1.12 ± 0.26 µM (means ± SE of 3 independent experiments), respectively, thus further arguing for the involvement of the A2B receptor subtype in this effect (Fig. 2C). Consistent with these results, the expression of mRNA coding for the A2B receptor was revealed by RT-PCR on cell culture preparations of cortical astrocytes as used in the present study (Fig. 2D).


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Fig. 2.   Pharmacological characterization of the effect of adenosine on glycogen synthesis. A: cultures of cortical astrocytes were stimulated with 100 µM adenosine (ADE) in the absence or presence of 2 µM xanthine amine congener (XAC) for 8 h, and glycogen levels were determined. XAC was added 30 min before adenosine. Results are means ± SE of triplicate determinations from 1 experiment. Representative experiment repeated twice with similar results. Data were statistically analyzed with ANOVA followed by Dunnett's test. ** Significantly different from control (Ctrl) (P < 0.01). B: cultures of cortical astrocytes were stimulated with 100 µM adenosine, 10 µM 5'-(N-ethylcarboxamido)adenosine (NECA), 100 nM N6-cyclopentyladenosine (CPA), 10 µM CGS-21680, or 10 nM IB-MECA for 8 h, and glycogen levels were determined. Similar results were obtained with concentrations up to 1 µM for CPA and 100 nM for IB-MECA. Results are means ± SE of triplicate determinations from 1 experiment. Representative experiment repeated twice with similar results. Data were statistically analyzed with ANOVA followed by Dunnett's test. ** Significantly different from control (P < 0.01). C: concentration-response relationship. Cultures of cortical astrocytes were stimulated with various concentrations of adenosine or NECA for 8 h, and glycogen levels were determined. Results are expressed as % of control values (121 ± 5 and 105 ± 3 nmol/mg protein for adenosine and NECA, respectively) and are means ± SE of 3 determinations from 1 experiment. Representative experiment repeated twice with similar results. D: RT-PCR analysis of A2B receptor mRNA expression. Total RNA from primary cultures of mouse astrocytes was reverse transcribed in the presence (+RT) or absence (-RT) of reverse transcriptase and then amplified by PCR with specific primers for the A2B receptor subtype as described in MATERIALS AND METHODS. Amplification products were electrophoresed on a 2% agarose gel stained with ethidium bromide. The expected fragment size for the A2B receptor was 567 bp. The negative control performed without reverse transcriptase (-RT) showed no amplification product, indicating that the amplified fragment in the +RT condition comes from reverse-transcribed mRNA and not from genomic DNA. Lane M, 0.1-kB DNA ladder marker. A2BR, A2B receptor.

The A2B receptor is positively coupled with the cAMP signaling pathway (10). As shown in Fig. 3A, both adenosine and NECA effectively caused a twofold increase in cAMP levels in cortical astrocytes within 20 min of treatment (control 80.0 ± 7.0, adenosine 163.2 ± 8.8, and NECA 164.2 ± 14.5 pmol/mg protein). Moreover, the effect of adenosine on glycogen synthesis was strongly reduced by H-89, an inhibitor of protein kinase A (PKA), whereas U-73122, an inhibitor of the phosphatidylinositol-specific phospholipase C (PI-PLC), did not have a significant effect (Fig. 3B).


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Fig. 3.   Signal transduction mechanism involved in the effect of adenosine on glycogen synthesis. A: effect of adenosine and NECA on cAMP levels. Cultures of cortical astrocytes were stimulated with 100 µM adenosine or 10 µM NECA for 20 min, and cAMP levels were determined as described in MATERIALS AND METHODS. Results are means ± SE of 9 determinations from 3 independent experiments for control (Ctrl) and adenosine and of 5 determinations from 2 independent experiments for NECA. Data were statistically analyzed with ANOVA followed by Dunnett's test. ** Significantly different from control (P < 0.01). B: effect of phospholipase C (PLC) and protein kinase A (PKA) inhibitors. Cultures of cortical astrocytes were stimulated with 100 µM adenosine for 8 h in the absence or presence of 10 µM U-73122, 1 µM H-89, or 5 µM H-89, and glycogen levels were determined. Inhibitors were added 30 min before adenosine. Results are means ± SE of 3 determinations from 1 experiment. Representative experiment repeated twice with similar results. Data were statistically analyzed with ANOVA followed by Bonferroni's test. *** Significantly different from control (P < 0.001); +++significantly different from adenosine (P < 0.001); ns, not significant vs. adenosine (P > 0.05).

Because glycogen resynthesis occurs several hours after the beginning of adenosine exposure, this suggests that it might depend on stimulation of gene expression. To test this hypothesis, cortical astrocytes were stimulated with adenosine in the presence of the transcription inhibitor actinomycin D (actD). As shown in Fig. 4, actD did not affect basal glycogen content but totally abolished the increase in glycogen levels induced by adenosine, therefore indicating that adenosine acts by stimulating gene transcription. Among the different proteins implicated in the regulation of glycogen, PTG and the transcription factors C/EBPbeta and C/EBPdelta were suggested previously to play a crucial role in astrocytes (1, 5). Therefore, the effect of adenosine on PTG, C/EBPbeta , and C/EBPdelta mRNA expression was analyzed by Northern blot. Results show that PTG as well as C/EBPbeta and C/EBPdelta mRNA levels were increased by adenosine in cortical astrocytes in a time-dependent manner (Fig. 5). PTG mRNA levels increased significantly 2 h after exposure to adenosine, reached their maximal values after 4 h (3- to 5-fold increase), and then decreased to return to their initial values 12 h after the beginning of the stimulation. Interestingly, induction of mRNA levels for the transcription factors C/EBPbeta and C/EBPdelta preceeded the induction of PTG mRNA because it could be observed as early as 30 min after stimulation by adenosine. Moreover, in contrast to C/EBPbeta and PTG, C/EBPdelta presented a biphasic pattern of induction: its expression rose rapidly after exposure to adenosine up to 2 h and then decreased, to increase again after 4 h.


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Fig. 4.   Inhibition of transcription prevents glycogen synthesis induced by adenosine. Cultures of cortical astrocytes were stimulated for 8 h with 100 µM adenosine in the absence or presence of 5 µM actinomycin D (actD), and glycogen levels were determined. actD was added 30 min before adenosine. Results are means ± SE of triplicate determinations from 1 experiment. Representative experiment repeated twice with similar results. Data were statistically analyzed with ANOVA followed by Dunnett's test. ** Significantly different from control (P < 0.01).



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Fig. 5.   Induction of protein targeting to glycogen (PTG), CCAAT/enhancer-binding protein (C/EBP)beta , and C/EBPdelta mRNA expression by adenosine. Cultures of cortical astrocytes were stimulated for various periods of time with 100 µM adenosine. Extraction of total RNA and Northern blot analysis were performed as described in MATERIALS AND METHODS. The same blot was sequentially hybridized with 32P-labeled riboprobes complementary to PTG, C/EBPbeta , C/EBPdelta , and beta -actin mRNAs. beta -Actin was used as a loading control. Representative experiment repeated twice with the same results.

Upregulation of PTG mRNA expression by adenosine could result from a stimulation of PTG transcription or from an increase in PTG mRNA stability. As shown in Fig. 6A, the increase in PTG mRNA levels by adenosine was totally abolished by actD, therefore indicating that adenosine acts by stimulating PTG transcription. In addition, stimulation of PTG transcription by adenosine requires de novo protein synthesis because the increase in PTG mRNA expression is prevented by the protein synthesis inhibitor cycloheximide (Fig. 6A).


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Fig. 6.   Pharmacological characterization of the effect of adenosine on PTG expression. A: inhibition of either transcription or protein synthesis prevents PTG mRNA induction by adenosine. Cultures of cortical astrocytes were stimulated for 4 h with 100 µM adenosine in the absence or presence of 5 µM actD or 10 µM cycloheximide (CHX). actD and CHX were added 30 min before adenosine. Extraction of total RNA and Northern blot analysis were performed as described in MATERIALS AND METHODS. beta -Actin was used as a loading control. Representative experiment repeated twice with the same results. B: effect of agonists for different adenosine receptor subtypes on PTG mRNA expression. Cultures of cortical astrocytes were stimulated with 100 µM adenosine, 10 µM NECA, 100 nM CPA, 10 µM CGS-21680, or 10 nM IB-MECA for 4 h. Extraction of total RNA and Northern blot analysis were performed; beta -actin was used as a loading control. Similar results were obtained with concentrations up to 1 µM for CPA and 100 nM for IB-MECA. Representative experiment repeated twice with the same results.

To pharmacologically characterize the effect of adenosine on gene expression, the levels of PTG mRNA expression were determined after exposure to NECA, CPA, CGS-21680, and IB-MECA. As shown by Northern blot analysis, NECA was able to reproduce the effect of adenosine on the induction of PTG mRNA expression whereas CPA, CGS-21680, and IB-MECA were without effect (Fig. 6B).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It was shown previously that adenosine induces glycogenolysis in cultured cortical astrocytes that is evidenced by lower levels of glycogen after 30 min of treatment (44). Here we report that adenosine also exerts long-term control on glycogen levels, i.e., an important glycogen resynthesis occurring after several hours of continuous treatment or even several hours after the end of a brief exposure to adenosine, by a mechanism involving the activation of the cAMP signal transduction cascade. Moreover, data reported here demonstrate that glycogen resynthesis induced by adenosine requires stimulation of gene expression.

The first question to be adressed was whether glycogen resynthesis induced by adenosine in cortical astrocytes was due to the activation of adenosine receptors and in such case which subtype(s) was(were) involved. Four different adenosine receptors have been described and classified as A1, A2A, A2B, and A3 subtypes based on their effect on adenylate cyclase and their selectivity toward specific agonists and antagonists (12, 13). A1 and A3 subtypes are negatively coupled to adenylate cyclase, whereas A2 receptors are positively coupled to adenylate cyclase. All adenosine receptor subtypes have been found in the brain, with higher expression levels for A1 and A2A receptors compared with the A2B and A3 receptor subtypes (8, 13). Inhibition of adenosine response by the nonselective receptor antagonist XAC at a concentration of 2 µM demonstrates that glycogen resynthesis effectively depends on adenosine receptor activation. Consistent with this observation, the effect of adenosine could be mimicked by the adenosine analog NECA, a nonselective adenosine receptor agonist. Pharmacological analysis showed that the effect of adenosine is most likely mediated through the activation of the A2B receptor subtype. In contrast to other members of the adenosine receptor family, no selective agonist or antagonist for the A2B receptor subtype is available. Nevertheless, in light of the inability of specific agonists for the A1 (CPA), A2A (CGS-21680), and A3 (IB-MECA) receptor subtypes to reproduce the effect of adenosine on glycogen resynthesis as well as the EC50 values for the effect of both adenosine and NECA, the implication of the A2B subtype can be suggested. A2B receptors are known to stimulate the formation of cAMP in virtually every cell in which they are expressed, but a mobilization of intracellular calcium has also been observed in certain cases. In astrocytes, activation of both signal transduction pathways by adenosine has been described (29, 32, 35). Activation of the A2B receptor in acutely isolated astrocytes resulted in a phospholipase C-dependent increase in intracellular calcium that could be completely blocked in the presence of the PI-PLC inhibitor U-73122 (35). Because in our hands U-73122 did not significantly inhibit glycogen resynthesis induced by adenosine, this implies that a phospholipase C-dependent mechanism is not essential for the metabolic response caused by adenosine. In contrast, glycogen resynthesis was substantially reduced in the presence of the PKA inhibitor H-89, thus strongly suggesting the involvement of a cAMP-mediated pathway. Moreover, both adenosine and NECA were effective in stimulating cAMP formation (Fig. 3). Finally, another argument in favor of the implication of an A2B-cAMP process comes from the concentration-response curve analyses for the effect of adenosine and NECA on glycogen resynthesis. NECA is a nonselective agonist at all adenosine receptor subtypes but with distinct affinities. Thus, although it is considered the most potent A2B receptor agonist with an EC50 for stimulation of adenylate cyclase in the low micromolar range, it displays greater affinity toward the other adenosine receptors, with EC50 values in the nanomolar range (12). In a similar manner, adenosine stimulates adenylate cyclase with a lower affinity at the A2B receptor (in the micromolar range) compared with activation via other receptors (in the nanomolar range, except for A3 receptors with an EC50 in the low micromolar range) (12). Therefore, such EC50 values for adenosine and NECA on glycogen resynthesis, 9.69 ± 3.35 and 1.12 ± 0.26 µM, respectively, are consistent with the participation of an A2B-cAMP-mediated process. Moreover, these EC50 values are in good agreement with those obtained in other studies on cAMP formation in cultured astrocytes after A2B receptor activation (1-1.3 and 5-9.6 µM for NECA and adenosine, respectively; Refs. 29, 32).

Effects similar to those observed for adenosine on long-term control of glycogen levels in cortical astrocytes have already been described for NE and VIP (45). Interestingly, it has been shown that these effects depend on cAMP-mediated processes and, like adenosine, require stimulation of gene expression (45). We previously identified (1, 5) the transcription factors C/EBPbeta and C/EBPdelta , as well as PTG, as likely key components of the neurotransmitter-induced glycogen synthesis. The present results show that adenosine is able to stimulate expression of C/EBPbeta , C/EBPdelta , and PTG mRNA in a time-dependent manner compatible with a role of these genes in glycogen synthesis. C/EBPbeta and C/EBPdelta are two members of a family of transcription factors involved in the transcriptional regulation of several genes, including genes related to energy metabolism (19, 26). In primary cultures of cortical astrocytes, overexpression of C/EBPbeta and C/EBPdelta enhanced glycogen synthesis elicited by NE (5). It was shown previously that C/EBPbeta and C/EBPdelta are expressed as immediate-early genes in cultured astrocytes after NE or VIP exposure (5). The rapid induction of C/EBPbeta and C/EBPdelta mRNA expression observed in the present study indicates that this is probably also the case after adenosine stimulation. Consistent with our observations, Schwaninger and collaborators (41, 42) observed an increase in the transcriptional activity of C/EBPbeta in cultured astrocytes after activation of the A2B receptor by adenosine. PTG, the other target of adenosine action, is a member of the glycogen-targeting subunits of protein phosphatase-1, which has been shown to play a key role in the regulation of glycogen metabolism in peripheral tissues (30). PTG overexpression in CHO cells (36), rat hepatocytes (2), and human muscle cells (21) leads to glycogen synthesis without the need for other stimuli. Stimulation of PTG mRNA expression by adenosine might therefore represent a key step in glycogen synthesis induced by this modulator. This view is strengthened by the observation that glycogen synthesis and induction of PTG mRNA expression induced by adenosine share the same pharmacological profile. It has been postulated that PTG might be a target gene of C/EBPbeta and C/EBPdelta action (1). Results obtained in this study are consistent with such a hypothesis. Indeed, C/EBPbeta and C/EBPdelta mRNA induction preceeds that of PTG mRNA. Moreover, stimulation of PTG gene expression was blocked in the presence of the protein synthesis inhibitor CHX, thus indicating that synthesis of other proteins (e.g., transcription factors) are required. Together, these observations suggest that adenosine, NE, and VIP exert their long-term effect on glycogen synthesis through similar transcriptional mechanisms.

Histochemical localization of glycogen revealed that only a small subset of astrocytes undergo glycogen synthesis induced by adenosine. The reason for this heterogeneity in glycogen distribution is unknown, but it is unlikely to derive from differences between astrocyte populations found in distinct cortical areas because this was not the case in vivo. Rather, it probably reflects the presence of distinct populations of astrocytes with different capacities to synthesize glycogen within each cortical area (33). Indeed, previous studies in vitro also showed that not all cells contain glycogen in rodent astrocyte cultures (40). Differences in the ability of astrocytes to synthesize glycogen after adenosine exposure may arise from heterogeneity in expression of the A2B receptors and/or enzymes linked to glycogen metabolism. Jiménez and collaborators (16) showed that not all astrocytes possess A2B receptors in culture. Moreover, immunocytochemical analysis also showed that glycogen phosphorylase is not expressed at the same level by all astrocytes (38).

A2B receptors are widely distributed in the brain and have been found both on neurons and on glial cells in cultures (8, 10), but little is known about their exact function. The affinity of A2B receptors for adenosine (5-20 µM) is largely above the reported range of concentrations (50-200 nM) measured by microdialysis under normal, resting conditions in the brain extracellular space (20). This method, however, has the caveat of being relatively insensitive to very localized increases in concentration and thus largely underestimates real concentrations at the receptor level (20). This is particularly relevant in the case of adenosine because it was demonstrated that ectonucleotidases present at the surface of cells can convert ATP, which is released by synaptic activity, to adenosine at levels sufficient to activate A2B receptors (27, 31). Although activation of these receptors is more likely to occur after events that would cause a substantial increase in extracellular adenosine concentration, there is even evidence that they could be activated tonically under basal conditions (18). Increases in adenosine release from different cell types including neurons have been shown to occur under conditions of large energy requirements or metabolic imbalance (9). In such cases, even a small percentage of ATP breakdown (ATP is found at concentrations of 1-3 mM in cells) will lead to a large increase in intracellular adenosine concentration via the formation and conversion of AMP. Adenosine can then rapidly exit the cell via nonconcentrative, bidirectional transporters and reach receptors on the cell membrane of neighboring cells (20). It has been postulated that in such circumstances adenosine may become a messenger to signal an important metabolic demand (7). Numerous physiological and pathological conditions have been reported to induce adenosine appearance in the extracellular space. Apart from the aforementioned case of ATP conversion by ectonucleotidases, treatments leading to sustained neuronal depolarization such as electrical stimulation, elevated extracellular K+ concentration, and application of glutamate receptor agonists such as NMDA or kainate have all been shown to enhance adenosine release in cultured cells, in tissue slices, or in vivo (Refs. 9 and 20 and references therein). In addition, important elevation of extracellular adenosine levels was detected in pathological conditions leading to major metabolic disturbances such as seizures, hypoxia/anoxia, or ischemia (Refs. 9 and 20 and references therein). It is likely that in many of these conditions, adenosine levels might rise sufficiently to activate A2B receptors on astrocytes and trigger glycogen resynthesis. Interestingly, increased glycogen levels were observed in the rat neocortex after transient forebrain ischemia (11). In that study, glycogen content increased above normal values after a few hours of recirculation. Furthermore, another study showed that glycogen synthesis after transient ischemia occurs in astrocytes (15). Such observations could be accounted for by the mechanism described above involving activation of the A2B receptors on astrocytes by adenosine. Because glycogen forms a readily available energy reserve, this might represent a mechanism to replenish energy stores after a sustained energy demand or metabolic disturbances and help face future requirements. Finally, such a process would be entirely coherent with the purported role of adenosine as an homeostatic regulator, not only on a short-term but also on a long-term basis.


    ACKNOWLEDGEMENTS

We express gratitude to Dr. J.-L. Martin and Dr. J.-M. Petit for helpful discussions and to M. Maillard and M. Marti for expert technical assistance.


    FOOTNOTES

This work was supported by a grant from the Swiss National Science Foundation to P. J. Magistretti and L. Pellerin (no. 56930.99).

Address for reprint requests and other correspondence: L. Pellerin, Institut de Physiologie, 7 Rue du Bugnon, 1005 Lausanne, Switzerland (E-mail: luc.pellerin{at}iphysiol.unil.ch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 6, 2002;10.1152/ajpcell.00202.2002

Received 3 May 2002; accepted in final form 30 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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