Alcohol and Alcoholism Advance Access originally published online on June 2, 2006
Alcohol and Alcoholism 2006 41(5):494-504; doi:10.1093/alcalc/agl044
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GLYCOSYLATION IS ALTERED BY ETHANOL IN RAT HIPPOCAMPAL CULTURED NEURONS
1 Section of Cell Biology and Pathology, Center for Investigation, Hospital La Fe, Valencia, Spain and 2 Department of Human Anatomy and Embryology. University of Granada, Spain
* Author to whom correspondence should be addressed at: Sec. Biología y Patología Celular, Centro de Investigación, Hospital Universitario ‘La Fe’, Avda. Campanar 21, E-46009 Valencia, Spain. Tel: +34 96 386 27 00 (ext. 50411); Fax: +34 96 197 30 18; E-mail: renau_jai{at}gva.es
(Received 6 March 2006; first review notified 4 April 2006; in revised form 28 April 2006; accepted 4 May 2006)
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ABSTRACT |
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Aims: Glycoproteins, such as adhesion molecules and growth factors, participate in the regulation of nervous system development. Ethanol affects the synthesis, intracellular transport, distribution, and secretion of N-glycoproteins in different cell types, including astrocytes and hepatocytes, suggesting alterations in the glycosylation process. We analysed the effect of exposure to low doses of ethanol (30 mm, 7 days) on glycosylation in cultured hippocampal neurons. Methods: Neurons were incubated for short (5 min) and long (90 min) periods with the radioactively labelled carbohydrate precursors 2-deoxy-glucose, N-acetyl-D-mannosamine and mannose. The uptake and metabolism of these precursors, as well as the radioactivity distribution in protein gels, were analysed. The levels of the glucose transporters GLUT1 and GLUT3 were also determined. Results: Ethanol exposure reduces the synthesis of proteins, DNA and RNA and decreased the uptake of mannose, but not of 2-deoxy-glucose and N-acetyl-D-mannosamine, and it increased the protein levels of both glucose transporters. Moreover, it altered the carbohydrate moiety of several proteins. Finally, alcohol treatment results in an increment of cell surface glycoconjugates containing terminal non-reduced mannose. Conclusions: Alcohol-induced alterations in glycosylation of proteins in neurons could be a key mechanism involved in the teratogenic effects of alcohol exposure on brain development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Asparagine (N)-linked oligosaccharides, or N-glycans, are found at the cell surface and in the extracellular matrix, and they play pivotal roles in nervous system development (Edelman and Jones, 1998
; Haltiwanger and Lowe, 2004; Kleene and Schachner, 2004; Ye and Marth, 2004). N-glycans structures are branched and diversified in the Golgi complex after being processed in the endoplasmic reticulum. Thus, N-linked glycans are classified into three subgroups termed as complex, high-mannose, and hybrid (Roth, 2002). Results concerning glycans in the mammalian brain indicate the presence of unique structures as well as a greater abundance of high-mannose N-glycans, as compared with other tissues. Furthermore, recent data indicate that hybrid N-glycans are essential for neuronal and postnatal viability in mice, whereas complex N-glycans appear to be dispensable in this cell type (Ye and Marth, 2004).
There is clinical and experimental evidence that alcohol consumption induces a variety of structural anomalies in the CNS during gestation, which include neuronal-glia heterotopias, cerebellar dysplasia, agenesis of the corpus callosum, hydrocephalus, and microcephaly (Miller, 1992; Streissguth et al., 1994; Sokol et al., 2003; Goodlett et al., 2005). These effects are indicative of aberrant migration, decreased proliferation, and of neural cell death (Miller, 1992; Streissguth et al., 1994; Sampson et al., 1997). Recent studies reveal that some of the regional brain shape abnormalities persist in adolescents exposed prenatally to alcohol (Sowell et al., 2002).
On the other hand, chronic alcohol consumption and prenatal alcohol exposure alter the glycosylation process in hepatocytes and astroglial cells (Renau-Piqueras et al., 1987, 1989a, 1997; Vallés et al., 1994; Kim and Druse, 1996; Miñana et al., 2000; Tomás et al., 2002, 2003; Garige et al., 2005; Lakshman et al., 2005). However, while carbohydrate-deficient transferrin (CDT, transferrin lacking the terminal sialic acid) (Arndt, 2001) is a known biomarker for alcohol exposure synthesized in the liver, the production of proteins such as CDT and how that might affect the CNS is an area worthy of study. In addition, alcohol may inhibit or delay the intracellular transport of nascent glycoproteins and glycolipids in the cell, or else alter the expression of membrane proteins, including NCAM, L1, and growth factors (Vallés et al., 1994; Kim and Druse, 1996; Torok et al., 1997; Bearer et al., 1999; Miñana et al., 2000; Azorín et al., 2004; Goodlett et al., 2005; Tomás et al., 2005). Moreover, other studies have demonstrated that alcohol affects ganglioside sialylation in both the rat brain and in cultured neurons, which could be the result of decreased sialyltransferase activity and increased sialydase activity (Rosenberg and Noble, 1994; Ghosh et al., 1998).
However, the mechanisms behind these alcohol-induced glycoconjugate alterations in neuronal cells remain unresolved. As the biosynthesis of glycans is a complex process that occurs along the secretory pathway, the deleterious effect of alcohol may have several targets. Although it remains unclear whether alcohol-induced alterations of the glycosylation process hinder protein trafficking in neuronal cells, modifications of the protein glycosylation state sometimes affect the folding of a nascent protein, and thus compromise its subsequent transport through the secretory pathway (Rasmunssen, 1992). Therefore, whether the reported alterations in the intracellular storage, trafficking, and constitutive secretion of glycoconjugates are directly attributable to alcohol exposure, or whether they are merely the consequence of the disruption caused by alcohol at some stage of the glycosylation process remains unclear.
In this study, we have used isotopic methods, Western blotting and lectin-probed Western blotting, immunofluorescence, and electron microscopy to explore the effects on in vitro low ethanol concentrations (30 mM, 7 days) on glycoprotein biosynthesis, and in particular on monosaccharide uptake, as well as on the metabolism, composition, and subcellular distribution of glycoconjugates in primary cultures of rat hippocampal neurons.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
The radiochemicals used were:
(i) Acetyl-D-mannosamine (ManNAc), N-[mannosamine-6-3H] (specific activity 5–15 Ci/mmol, American Radiolabeled Chemicals Inc.); (ii) 2-Deoxy-D-[1-3H]Glucose (2-DGlc) (specific activity 13.0 Ci/mmol, Amersham Pharmacia); (iii) D-[2,6-3H]Mannose (Man) (specific activity 20–40 Ci/mmol, American Radiolabeled Chemicals Inc.); (iv) L-[35S]Methionine (Met) (specific activity 40–500 Ci/mmol, Amersham Pharmacia).
Antibodies: anti-MAP2 mouse monoclonal antibody (Sigma-Aldrich, Spain); anti-neurofilament (NF) 200 rabbit polyclonal antibody (Sigma-Aldrich); anti-GLUT1 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc. CA, USA), anti-GLUT3 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.), anti-digoxigenin (DIG) mouse monoclonal antibody (Roche Diagnostics, S.L. Spain) and anti-GFAP rabbit polyclonal antibody (Sigma-Aldrich). Secondary antibodies Alexa-488 (1:50) or Alexa-546 (1:200) F(ab')2 fragments were obtained from Molecular Probes (Invitrogen, Invitrogen S.A., Spain). Alkaline phosphatase-conjugated anti-rabbit IgG (1:20000) was procured from Sigma. Anti-mouse IgG gold conjugate (15 nm) was from Jackson ImmunoResearch (Jackson ImmunoResearch Europe, Inc.) and anti-rabbit IgG alkaline phosphatase conjugate was from Sigma-Aldrich.
A labelling kit from Roche was used (DIG Glycan Differentiation Kit, Cat No. 1210 238) for the characterization of carbohydrate chains. Tissue culture reagents were acquired from Invitrogen (Paisley, UK). Chemicals for electron microscopy were from Electron Microscopy Science (EMS, PA, USA), and all remaining chemicals, including lectin inhibitors, were from Sigma-Aldrich.
Primary culture of hippocampal neurons
Hippocampal neuron cultures were prepared from rat fetuses from female rats on day 14 of gestation (Brewer et al., 1993). The hippocampus were dissected from brains and dissociated by passage through a needle 10 times in 1 ml of Hanks Balanced Salt Solution (HBSS) per brain without Ca++ and Mg++ and then supplemented with 10 mM HEPES, pH 7.4 and 1.0 mM sodium piruvate. Then, divalent cations were restored by dilution with 2 ml of HBSS per brain, and supplemented as above. After allowing non-dispersed tissues to settle for 3 min, the supernatant was transferred to a conical bottom tube and centrifuged for 1 min at 200 g. The pellet was resuspended in 10 ml of Neurobasal Medium containing 2 mM Glutamax I and 25 µM glutamate, B27 supplement (2 ml of B27 50x/100 ml) and antibiotics (penicillin and streptomycin) at 50 U/ml and 50 µg/ml, respectively. Cells were plated on polylysine-treated dishes (12.5 µg/ml) at a dilution of 6 ml/fetus. Cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The medium was changed every 2 days. Some cells were grown in the presence of ethanol (Renau-Piqueras et al., 1989b), which was added to the culture medium on the day of plating. Cells were then maintained in a medium containing alcohol until day 7. The ethanol concentration in the medium was checked daily and adjusted to a final concentration of 30 mM (138 mg/dl, ethanol evaporation after 24 h was 10–20%). This is similar to the blood levels reported in pregnant chronic drinkers (Eckardt et al., 1998). The purity of neurons cultures was assessed by immunofluorescence using the anti-MAP2 and anti-NF antibodies, and any possible contamination by astroglial cells was determined using an anti-glial fibrillary acidic protein antibody (Renau-Piqueras et al., 1989b). Cell viability was determined by the trypan blue exclusion test. All experiments using rats were approved by the appropriate institutional review committee, and were performed in strict compliance with the European Community Guide for the Care and Use of Laboratory Animals.
Protein, DNA, and RNA synthesis
In order to assess the effect of ethanol on neuronal proliferation under the above-mentioned culture conditions, cells were washed twice with culture medium, and incubated for 1 and 24 h in a medium containing L-[methyl-3H]Methionine (1.0 µCi/ml), [methyl-3H]Thymidine (1.0 µCi/ml), or [5,6-3H]Uridine (1.0 µCi/ml). The amount of radioactivity incorporated was measured in an automatic scintillation counter (Renau-Piqueras et al., 1988). The results were expressed as dpm/mg of protein, as determined by Lowry et al. (1951).
In some experiments, the proliferation rate of cells was also determined using a colorimetric immunoassay based on the BrdU incorporation measurement during DNA synthesis. For these experiments a Kit form Roche (cell Proliferation ELISA BrdU, cat No 1647229) was used following the manufacturer's instructions.
Immunofluorescence
Neurons growing on 16 mm polylysine-treated glass coverslips were used for MAP2, NF, GLUT1, and GLUT3 immunolocalization, as previously described in detail (Tomás et al., 2002).
Uptake and metabolism of carbohydrates
In order to study the effect of alcohol on the uptake and metabolism of carbohydrates, neurons were incubated with 1 ml of medium containing one of the following radioactive precursors (1.5 µCi/ml): 3H-Man, 3H-2DGlc, or 3H-ManNAc, the direct obligate metabolic precursor for NeuAc (Pellerin and Magistretti, 1994; Rosenberg and Noble, 1994; Ghosh et al., 1998; Tomás et al., 2002). The cells were maintained in this medium for 5 min, and the incorporation was stopped by washing the cultures three times in cold phosphate-buffered saline (PBS), which were then collected for radioactive counting. Cells were incubated overnight at 37°C in the presence of 0.5 M NaOH, and the amount of radioactivity incorporated was measured in an automatic scintillation counter. The dpm were computed to obtain the net dpm corresponding to each isotope. The results where expressed as dpm/mg of protein, as determined by Lowry et al. (1951).
In some experiments, cultures were incubated for 90 min in the presence of the labelled precursor (3H-Man or 3H-ManNAc, in the presence of 35S-Met) (Tomás et al., 2002). After washing with PBS, cells were stored at –80°C. For the purpose of characterizing proteins by their molecular weights, samples containing 30 µg of protein were solubilized in SDS, and then electrophoresed in 3–8% gradient Tris–acetate gel. After staining with Coomassie blue and drying the gel (Kit DryEasy), the bands were cut and rehydrated for 20 min at 50°C. Then, the bands were incubated with 0.5 ml Solvable (Packard Packard Instrument Company, CT, USA) for 3 h at 50°C. After adding 10 ml of scintillation fluid, the amount of incorporated radioactivity was measured in an automatic scintillation counter, and as the energy distribution spectra of the 3H and 35S were superposed, a two-channel counting programme was used. The dpm obtained in both channels were computed to obtain the net dpm corresponding to each isotope. Results were expressed as dpm per milligram of protein (Tomás et al., 2002). The intensity of each band in the gel was assessed by densitometry using a computer program (Scion Image v. Beta 4.0.2).
Lectin fluorescence
Neurons were fixed as described (Tomás et al., 2002). For the detection of terminal Man of glycoproteins, cells were incubated with GNA (Galanthus nivalis agglutinin), and were conjugated with FITC for 60 min at room temperature. This lectin has a binding specificity for terminal Man, 
(1-3), (1-6), or (1-2) linked to Man (Shibuya et al., 1988). For the detection of terminal sialic acid, cells were first incubated with lectin MAA (Maackia amurensis agglutinin), conjugated with DIG, then with an anti-DIG monoclonal antibody, and finally with an anti-mouse antibody labelled with FITC. In all cases, incubations lasted 60 min at room temperature. MAA is specific for sialic acid (NeuAc) linked (2-3) to Gal (Wang and Cummings 1988; Knibbs et al., 1991).
Electron microscopy and lectin cytochemistry
Neurons were processed for transmission electron microscopy, as previously described (Tomás et al., 2002). Ultrathin sections of cell monolayers embedded in Epon were either mounted on formvar-coated copper grids for ultrastructural examination, or on nickel grids for the cytochemical demonstration of GNA and MAA, as previously described (Megías et al., 2000; Tomás et al., 2002).
Lectin-mediated cytotoxicity assay
Neurons were plated on polylysine-treated 24-well plates, and were cultured both with and without ethanol. The medium was changed every 2 days. On day 6 of culture, cells were incubated for 24 h with several concentrations of GNA and MAA (0, 15, 35, 75 µg/ml). At the end of this period, the medium was removed to eliminate dead cells, and the cell monolayer was rinsed in PBS without Ca2+ and Mg2. Cells were detached and counted, as previously described (Tomás et al., 2002). The results were expressed as the survival percentage of cells for each lectin concentration by comparing each treatment (cells growing in the presence of the lectin) with its respective controls (cells growing in the absence of the lectin) (Tomás et al., 2002).
Lectin-probed Western blots
Lectin blotting using GNA and MAA was used to characterize glycoproteins containing terminal Man or sialic acid (da Silva and Gordon, 1999; Tomás et al., 2002; Azorin et al., 2004). A labelling kit from Roche was used (DIG Glycan Differentiation Kit, Cat. n 1 210 238) following the manufacturer's instructions. Gel quantification was carried out as described above.
Lectin activity inhibitors
The following low molecular weight sugars were used as competitive inhibitors in all the experiments using lectins: D-mannose for GNA (Shibuya et al., 1988) and 2,3-sialyllactose (NeuAc2,3Galß1,4Glc) for MAA (Wang and Cummings 1988; Sata et al., 1989; Knibbs et al., 1991).
Western blotting
Western blotting was carried out as previously described (Tomás et al., 2002, 2005). Neurons were washed with PBS, homogenized in extraction buffer (6 mM Tris–buffer, 10 mM EDTA and 2% SDS, pH 7.0) and incubated on ice for 15 min. Proteins were quantified as previously described (Lowry et al., 1951). Cell lysates (20 µg per lane) were mixed with lithium dodecyl sulphate (LDS) sample buffer, boiled for 3 min, and then separated on SDS polyacrylamide slab gels by loading an equal amount of protein per lane, and by using the discontinuous gel and buffer system described by Laemmli (1970) with different percentages of polyacrylamide (6–10%) in the separating gel. Following electrophoresis, the proteins were transferred to nitrocellulose filters, which were incubated for 60 min with a primary antibody in Tris–buffered saline containing 0.05% Tween-20. Filters were then incubated for 60 min with an alkaline phosphatase-conjugated secondary antibody. After 10–20 min of colour development, filters were washed and photographed. Gel quantification was conducted using the Scion Image program (Beta 4.0.2, Scion Corporation, Maryland, USA, www.scioncorp.com). The results are shown as the mean values of at least 4–6 different experiments per group.
Statistical analysis
Results are expressed as mean ± standard errors, and an unpaired two-tailed t-test (GraphPad InStat, GraphPad Software, San Diego, CA, USA) was used for statistical analysis. A P-value of <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Main characteristics of neurons in primary culture
We analysed some characteristics concerning cell growth, cell markers, and cell morphology. As summarized in Figure 1, both control and alcohol-exposed neurons displayed the morphology of mature neurons, and were positive for labelling with MAP2 and NF, two widely used markers for neurons. Moreover, electron microscopy indicated that neurons showed similar ultrastructural characteristics as those previously described (data not shown) (Deitch and Banker, 1993
). Finally, the glial contamination was 1.0%, which agrees with previous studies (Brewer et al., 1993). On the other hand, we found that ethanol exposure reduced the synthesis of proteins, DNA, and RNA (Fig. 2). This effect was more pronounced in 4-day-old cultures (data not shown). However, no significant effect of chronic exposure to ethanol on the cell viability was observed.
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Uptake of carbohydrates
The effect of alcohol exposure on the uptake of carbohydrates was analysed by incubating cells with 3H-2DGlc, 3H-Man, or 3H-ManNAc. As shown in Figure 3, 30 mM ethanol exposure significantly decreased the uptake of 3H-Man, but not of 3H-2DGlc and 3H-ManNAc, after a short 5 min pulse period.
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Effect of ethanol on the protein levels of total GLUT1 and GLUT3
We examined the effect that ethanol had on glucose transporters GLUT1 (the 45 kDa isoform) and GLUT3, which significantly increased both protein levels, as revealed by immunoblotting (Fig. 4). In contrast, no clear differences in the subcellular distribution of both transporters were observed using immunofluorescence.
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Effect of ethanol exposure on the metabolic labelling of glycoproteins
We used metabolic labelling with monosaccharides to probe for structural modifications occurring after alcohol exposure. The metabolic labelling of glycoproteins in control and alcohol-exposed neurons was studied after a 90 min pulse with 3H-Man, 3H-ManNAc, and 35S-Met. After electrophoresis and staining with Coomassie blue, the bands were cut and the radioactivity was counted. Figure 5 shows how alcohol decreased the 3H-Man incorporation in the bands with apparent molecular weights of 90, 62, 59, 55, and 47 kDa. In contrast, the effect of 3H-ManNAc incorporation was heterogeneous. While alcohol provoked an increase in the incorporation in the precursor in some bands (90 and 59 kDa), the incorporation actually decreased in a 55 kDa band (Fig. 5). On the other hand, no changes in the incorporation of 35S-Met in these bands were observed after alcohol exposure (data not shown).
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Lectin-probed Western blot and inmunocytochemistry of terminal man and sialic acid
Labelled sugars may be metabolized by the neurons and incorporated in another form, so we set up experiments in which unlabelled proteins from control and alcohol-exposed neurons were probed with GNA and MAA. Thus, by using SDS–PAGE Western blotting and lectin overlay, we resolved the glycoproteins of control and alcohol-exposed neurons. Figure 6 illustrates how ethanol decreased the amount of several glycoproteins incubated with GNA with apparent molecular weight of 90, 130, and 230 kDa. In contrast, no differences were found between control and alcohol-exposed cells when glycoproteins were incubated with MAA (data not shown). However, lectin fluorescence revealed that exposure to ethanol did not seem to alter either the amount or the distribution of GNA- and MAA-labelling (Fig. 7). The subcellular distribution of GNA- and MAA-binding sites was also analysed using electron microscopy. A qualitative analysis of the binding sites of both lectins showed a similar gold labelling pattern in control and ethanol-treated cells. In both cases, gold particles were visualized mainly at the Golgi complex, endosomes, vacuoles, vesicles, lysosomes, and plasma membrane (Fig. 8).
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Ethanol increases cell surface glycoproteins containing terminal Man
As diverse lectins not only specifically bind to the glycan structures on the cell surface, but can also kill cells in culture, we aimed to test whether ethanol also affects the cell surface glycoproteins containing terminal Man or sialic acid. To this end, we determined the GNA and MAA-mediated cytotoxicity in control and alcohol-exposed neurons. Whereas GNA incubation at low concentrations significantly increased cytotoxicity in alcohol-exposed cells, which reflects an increase in the content of high Man and/or hybrid type sugar chains in the cell membrane of treated cells (Fig. 9), no noticeable effects of MAA on the cells were observed (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ethanol affects neuron growth in primary culture
Suppression of protein and DNA synthesis have been considered as key mechanisms in alcohol teratogenesis (Goddlett et al., 2005
), and our results indicate that chronic exposure of neurons to low ethanol doses during the first 7 days of culture results in a decreased synthesis of DNA, RNA, and protein. Although there are controversial results concerning the effect of alcohol on the synthetic activity of neurons, our data are in accordance with previous studies (Rawat, 1975, 1985; Sharma and Rawat, 1989; Crews and Nixon, 2003; He et al., 2005). Nevertheless, it is important to point out that ‘in vivo’ effects depend on the brain region, in a time-dependent manner (Miller, 1996), and of the neuron population (Lindsley et al., 2002). Decreased synthetic activity after ethanol exposure has been also shown in other cell types, including non-nervous cells (Sharma and Rawat, 1989; Volpi, 2002) as well as nervous cells such as astrocytes (Renau-Piqueras et al., 1988; Snyder et al., 1992), where the effect of ethanol on DNA synthesis and content were more affected during the proliferation period. This effect correlated with an accumulation of cells in the G0/G1 phase of the cell cycle (Guerri et al., 1990; Li et al., 2001). Moreover, it has been reported that alcohol content at concentrations within the range of blood alcohol levels in intoxicated humans can also induce DNA alterations with no apparent effect on cell viability (Lamarche et al., 2004).
Effect of ethanol in the uptake of monosaccharides and on the Glc transporters GLUT1 and GLUT3
Some of the deleterious effects of prenatal exposure to ethanol may be associated with a reduction in the fetal and neonatal brain utilization of the Glc (Goddlett et al., 2005). These effects are of interest, first because Glc provides almost 90% of the energy requirements in the normal brain, and second it serves as a substrate for lipid and nucleic acid synthesis. However, only a few portions of total Glc in the brain are used for the biosynthesis of glycoconjugates. In this study, we have found that alcohol exposure neither affects the uptake of 2-DGlc nor that of ManNAc in neurons rather it significantly decreased the Man incorporation. These results are not only in contrast with studies in a chick embryo model, indicating an alcohol-induced increase of Glc uptake but also with others where the Glc uptake was also measured with 2-DGlc, which resulted in a reduction of the cultured hippocampal neurons as well as in the whole brain (Singh et al., 1989, 1992; Hu et al., 1995). It is interesting to note the disparate effects that ethanol exposure has on the uptake of Glc in astroglial cells with respect to neuronal cells. Thus, we previously showed that an ethanol exposure similar to the one used here, induces alterations in some steps of the glycosylation process in growing astrocytes by increasing the uptake of 2-DGlc, Man, and ManNAc (Tomás et al., 2002, 2003). The discrepancies concerning the effect on cultured neurons and astrocytes could be mainly because of the different experimental conditions and, when whole brain is used, the inability to identify which CNS cell population, glial, endothelial, and/or others have been actually affected by ethanol exposure.
In addition, we also analysed the possible alcohol-induced alterations on Glc transporters, GLUT1, and GLUT3. The 45 kDa GLUT1 isoform is expressed in astrocytes and neurons, whereas GLUT3 is considered as the neuron-specific Glc transporter (Vannucci et al., 1998; Vannucci and Simpson, 2003; McEwen and Reagan, 2004). However, other GLUTs are also expressed in the brain (McEwen and Reagan, 2004). Our results show that alcohol exposure significantly increased the levels of total GLUT1 and GLUT3. In contrast, no clear alcohol-induced differences in the subcellular distribution of both transporters were observed. As already stated, previous studies showed contradictory results concerning the effect of alcohol exposure on Glc transporters in cultured nervous cells and in the brain (Hu et al., 1995; Carver et al., 1999; Fattoretti et al., 2003). The alcohol-induced increase in the total GLUT1 and GLUT3 levels, which occurs without a Glc uptake increment as described in our experiments, could be because of a deregulation of transporters induced by ethanol exposure, as previously suggested (Carver et al., 1999). This in turn could result in an adaptive mechanism of neurons (i.e. an increase in the plasma membrane GLUTs) to provide cells with the required amount of Glc. It is also important to point out that the same alcohol treatment followed here for neurons also induced an increase in the levels of GLUT1 in astrocytes in primary cultures (Tomás et al., 2002, 2003). On the other hand, it is interesting to note that ethanol exposure decreases Man uptake in neurons and that, although Man is efficiently transported by D-mannose-specific Man transporters in several cell types, it is transported by Glc carriers in nervous cells (Wiesinger et al., 1997; Weber et al., 2001; Durán et al., 2004), which however, have a higher affinity for Glc than for Man (Maher et al., 1996). This effect of ethanol on Man uptake remains unclear.
The carbohydrate moiety of N-glycoproteins is modified by ethanol exposure
Our results also suggest that alcohol alters the biosynthesis of glycoproteins in neurons, as ethanol affected the incorporation of these precursors in several newly synthesized glycoproteins after a 90 min pulse period in the presence of 3H-Man and 3H-ManNac. The decrease in the incorporation of 3H-Man together with the heterogeneous incorporation of 3H-ManNAc, strongly supports the concept raised when using other cell models such as astrocytes and hepatocytes (Tomás et al., 2002; Azorin et al., 2004), that is that alcohol has specific effects on the complex-carbohydrate moiety, depending on the glycoproteins and the cell type. This is also supported by the results obtained using lectin blotting, indicating that ethanol decreased the intensity of several bands in GNA-stained gels, whereas it does not affect those glycoproteins labelled with MAA. In contrast, using a GNA-mediated cytotoxicity assay, which reveals only surface glycoproteins containing terminal non-reduced mannose, we have found an increase in this type of N-glycans in the surface of neurons exposed to ethanol. These results agree with previous results observed in astrocytes (Tomás et al., 2002). Although it is currently difficult to explain this increase in metabolic and functional terms, it could constitute a significant effect of alcohol exposure as it has been proposed that cell surface carbohydrates play critical roles in axon guidance and targeting (Lipscomb et al., 2002; Ye and Marth, 2004). In addition, the fact that alcohol mainly affects cell surface glyconjugates containing terminal non-reduced mannose is an interesting result because of the fact that the brain shows a greater abundance of high-mannose N-glycans in comparison with other tissues. Moreover, these glycans are essential in synapse (Martin, 2002) as well as for neuronal and postnatal viability in mice, whereas complex N-glycans appear to be dispensable in this cell type (Ye and Marth, 2004).
In conclusion, our results mainly indicate that moderate ethanol exposure selectively affects the uptake of some monosaccharides, it increases the levels of glucose transporters, and it modifies the carbohydrate moiety of glycoproteins in neurons, indicating that alcohol affects the biosynthesis of glycoconjugates in these cells. These effects could be a key mechanism involved in the teratogenic effects of alcohol exposure on brain development.
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ACKNOWLEDGEMENTS |
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We are very grateful to I. Monserrat and J. Llorens for technical help and to H. Warburton for linguistic assistance. This work was supported by grants from ‘Fundación para Estudio, Prevención y Asistencia a las Drogodependencias’ (FEPAD), ‘Fondo de Investigaciones Sanitarias’ (FIS, PI020073) and ‘Ministerio de Educación y Ciencia’ (MEC, SAF2005-00615). M.T. and M.P.M. are recipients of fellowships from FIS and the CIEN programmes, respectively. Funding to pay the Open Access publication charges for this article was provided by xxxx.
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FOOTNOTES |
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The first two authors contributed equally to this study.
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