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Effects of homocysteine on metabolic pathways in cultured astrocytes
Neurochemistry International 52 (2008) 1410–1415
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Effects of homocysteine on metabolic pathways in cultured astrocytes Ying Jin, Lorraine Brennan * UCD School of Agriculture, Food Science and Veterinary Medicine, UCD Conway Institute, UCD Dublin, Belfield, Dublin 4, Ireland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 February 2008 Received in revised form 2 March 2008 Accepted 6 March 2008 Available online 13 March 2008
Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 mM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-13C]glucose in the presence or absence of homocysteine and analysed using 13C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using 1H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. ß 2008 Elsevier Ltd All rights reserved.
Keywords: Homocysteine Astrocytes Metabolism
1. Introduction Elevated levels of the sulphur containing non-essential amino acid homocysteine have been reported as an independent risk factor for cognitive dysfunction (Bell et al., 1992; Allen et al., 1998; Miller, 2000). A significant correlation has been reported between risk of Alzheimer’s disease and Parkinson’s disease and high-plasma levels of homocysteine (Clarke et al., 1998; Ravaglia et al., 2005). In recent years, it has become clear that exposure to homocysteine induced neuronal cell death in a variety of neuronal types including rat hippocampal and cortical neurons (Langmeier et al., 2003). A recent study on the Purkinje cells showed that exposure to homocysteine inhibited cell survival and the outgrowth of neurites (Oldreive and Doherty,
* Corresponding author. Tel.: +353 1 716 6759; fax: +353 1 283 7211. E-mail address: [email protected] (L. Brennan).
Abbreviations: PDK-2, pyruvate dehydrogenase kinase isoform 2; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; ALAT, alanine aminotransferase; AST, aspartate aminotransferase; GSH, glutathione. 0197-0186/$ – see front matter ß 2008 Elsevier Ltd All rights reserved. doi:10.1016/j.neuint.2008.03.001
2007). Several mechanisms of toxicity have been reported including NMDA receptor and group I metabotropic glutamate receptor (mGluR) mediated neurotoxicity (Ho et al., 2002; Zieminska et al., 2003). In addition homocysteine can induce caspase-dependent apoptosis in human dopaminergic cells, rat hippocampal and mouse cortical neurons (Kruman et al., 2000, 2002; Duan et al., 2002). However, capase-independent cell death has been reported in murine cerebellar granule cells in the presence of high levels of homocysteine (Foister et al., 2005). In addition to the toxic effects homocysteine has also been reported to alter hippocampal plasticity and synaptic transmission (Christie et al., 2005). This non-protein amino acid has to date three known metabolic fates in the cell. In the first instance homocysteine can undergo reversible methylation to form methionine in the methionine cycle. In the second case it can undergo trans-sulfuration to form cystathionine and cysteine and the importance of this pathway in the brain has recently been reported (Vitvitsky et al., 2006). The cysteine formed has two metabolic fates: entry into glutathione (GSH) synthesis or entry into taurine synthesis pathway. Alternatively homocysteine can be used in the production of homocysteine thiolactone which can then react with reactive nitrogen
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species such as nitric oxide (Jakubowski, 2004). Although the neurotoxic effects of homocysteine are well documented there has been very little study on the effect of homocysteine in astrocytes. Maler et al. (2003) reported that homocysteine showed a dose dependent cytotoxic effect at doses at 2 mM and above in cortical astrocytes. The mechanism of these effects are unknown. Astrocytes are an important cell in the central nervous system and are critical in the glial–vascular interface as part of the blood– brain barrier (Verkhratsky and Toescu, 2006). Over the last decade the importance of astrocytes in the regulation of brain metabolism and in particular brain energy metabolism has been realised (Escartin et al., 2006). It is now accepted that brain energy metabolism is regulated by cross-talk between astrocytes and neurons. As a result, any modulation of astrocyte function could have potential detrimental effects to neuronal function. While a large body of literature exists relating the deleterious role of neuronal metabolic impairment in the diseased brain, the role of astrocytes has been largely ignored until recently (Seifert et al., 2006). Considering the increasing profile of astrocytes the present study was undertaken to probe the effects of sub-lethal concentrations of homocysteine on metabolic processes in astrocytes. 2. Experimental procedures 2.1. Reagents All chemicals were obtained from Sigma–Aldrich Chemical Company (Poole, Dorset, UK) except [U-13C]glucose which was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Culture media and foetal bovine serum were obtained from Gibco (Glasgow, UK). 2.2. Cell culture Rat astrocytes were prepared from 2-day-old Wistar rats as previously described (Richter-Landsberg and Besser, 1994; Alves et al., 1996). All procedures were performed in accordance with Irish Government ethical guidelines. The cerebral hemispheres were isolated and the meninges was carefully removed. The cerebral hemispheres were then mechanically disrupted. Cells were grown in DMEM containing 10% (v/v) FBS for 7–10 days. The nonastrocytic cells were removed by vigorous shaking and the astrocytes were subcultured for 20–23 days during which time the media was changed twice a week. Cells were maintained in a Forma Scientific incubator at 37 8C in a humidified atmosphere of 5% CO2 and 95% air. To assess cell viability the LDH assay was performed using a coupled enzymatic reaction where tetrazolium salt was converted into formazan with an absorbance maximum at 490 nm (Wroblewski and Ladue, 1955). 2.3. Extraction procedures To investigate the effects of homocysteine on astrocytic metabolism the cells were pre-incubated for a period of 20 h in the presence of 400 mM homocysteine. The cells were then washed with phosphate-buffered saline (PBS) and were incubated for 4 h in minimal medium supplemented with 5.5 mM [U-13C]glucose in the presence 400 mM homocysteine. Control experiments were run in parallel. As a D,L-mixture of homocysteine was used the effective concentration used in the present studies was 200 mM. A previous study showed that homocysteine concentrations above 2 mM were needed to induce cell death (Maler et al., 2003). The concentration chosen here is well below this toxic concentration and is within the range used in other in vitro studies (20 mM to 25 mM). Following the incubation period, the media was removed and stored at 20 8C and analysed later for glucose concentration and lactate dehydrogenase (LDH) activity. The cells were washed with ice-cold phosphate-buffered saline, placed in liquid N2 and the metabolites were extracted using 6% perchloric acid. The extracts of six culture dishes were pooled and centrifuged at 3000 g for 12 min. The resulting supernatant was neutralised with KOH (5 and 0.1 M solutions) and the pellet was soaked overnight in 0.1 M NaOH. The protein concentration was determined using the Lowry method (Lowry et al., 1951). The neutralised supernatant was centrifuged at 3000 g for 5 min and then lyophilised. Each experiment was carried out on at least three independent cultures. The lyophilised cell extracts were dissolved in 1.2 ml D2O and then centrifuged at 350 g for 5 min. The supernatant was carefully removed and the pH was checked and adjusted when necessary to 7.0 0.1 with 0.1 M NaOH and 0.1 M HCl. An insert containing 1% (v/v) dioxane in water was used as an external signal intensity reference for quantification of the NMR spectra.
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For the global metabolic profiling the cells were grown and treated as described as above. For the final 4 h incubation period the cells were incubated with unlabelled glucose in the presence or absence of 400 mM homocysteine. Following the incubation period, the media was removed and the cells were washed with icecold PBS. The metabolites were extracted with ice-cold 70% ethanol. The extracts of two culture flasks were pooled and centrifuged at 4000 g for 15 min. The resulting supernatant was stored at 20 8C and the protein concentrations were determined in the pellets. The samples were lyophilised and redissolved in 700-ml D2O. For chemical shift reference 10 ml TSP (sodium 3-(trimethylsilylpropionate-2,2,3,3-d4) was added to each and the pH was adjusted to pH 7.0 0.1 with 1 M HCl. 2.4. NMR spectroscopy Proton decoupled 13C spectra were acquired on a Bruker DRX 500 spectrometer using a QNP 5-mm probe. Typically, spectra were acquired with 32k data points using a 908 pulse angle, 260 ppm spectral width, 2.5 s relaxation delay and 12,000 scans. Spectra were recorded at 25 8C. Chemical shifts in aqueous media were referenced to tetramethylsilane at 0 ppm. Exponential multiplications with 2 Hz line broadening were performed using Bruker WINNMR software. The assignments of the intermediate metabolites were made by comparison with chemical shift tables in the literature (Fan, 1996) or by addition of 100 mM of unlabelled amino acid. 1 H NMR spectra were typically acquired with 32k data points and 256 scans over a spectral width of 8 kHz. Water suppression was achieved during the relaxation delay (2.5 s) and the mixing time (100 ms). All 1H NMR spectra were referenced to TSP at 0.0 ppm and processed manually with the Bruker software using a line broadening of 0.2 Hz. All spectra were baseline corrected. The spectra were then reduced by integrating into bins across spectral regions of 0.005 ppm, using AMIX (Bruker Biospin, Germany). The water region (4.2–5.6 ppm) was excluded and the data was normalised to the sum of the spectral integral. In the case of 13C NMR spectra the amount of 13C in each resonance was evaluated by integration of the extract peaks and the corresponding peaks in the standard sample relative to the dioxane signal. The standard sample contained a solution of Lalanine, L-glutamate, lactate and D-glucose at known concentrations. Corrections for the natural abundance signal were made. The fluxes reported were obtained by analysis of the isotopomers of glutamine C2 and C4. The ratio between flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) was calculated as follows: ([2,3-13C2] + [1,2,3-13C3])/[4,5-13C2] + [3,4,5-13C3] (Lapidot and Gopher, 1994). The fraction of acetyl-CoA labelled from [U-13C]glucose was calculated using the following equation for the C4 peak: (3,4,5-13C) C4/C3 (Malloy et al., 1990). 2.5. NAD(P)H and mitochondrial membrane potential measurements For the NAD(P)H experiments the cells were grown in 96-well plates and preincubated in the presence or absence of 400 mM homocysteine for 20 h. Cells were washed with PBS and incubated in Krebs–Ringer bicarbonate (KRB) buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO47H2O and 0.25% BSA, 10 mM NaHCO3 pH 7.4) at 37 8C for 1 h. Following this incubation period the cells were placed in a temperature-controlled platereader (FLEXstation, Molecule Devices) and the baseline fluorescence was measured at an excitation wavelength of 340 nm and an emission wavelength of 460 nm. After 150 s the cells were stimulated by addition of glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Fluorescence readings were recorded every 2 s over the total duration of the experiment (455 s). For the mitochondrial membrane potential cells were grown in 96-well plates and pre-incubated in the presence or absence of 400 mM homocysteine for 20 h. Cells were incubated in KRB buffer at 37 8C for 15 min, washed and incubated in 5 mg/ml rhodamine-123 for 15 min. Following this incubation period the cells were washed and incubated in KRB buffer in the temperature-controlled platereader (FLEXstation, Molecule Devices). The baseline fluorescence was measured at an excitation wavelength of 505 nm and an emission wavelength of 515 nm. After 50 s the cells were stimulated by addition of glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Fluorescence readings were recorded every 4 s over the total duration of the experiment (150 s). 2.6. Western blot analysis For Western blot analysis cells were grown in six well plates and treated for 20 h in the presence or absence of 400 mM homocysteine. Cells were washed twice with ice-cold PBS and lysed with RIPA buffer. Samples were centrifuged at 14,000 g for 15 min at 4 8C. The supernatant was collected and total protein concentration was determined using a BCA assay. Samples containing 15 mg protein were then subjected to SDS-PAGE electrophoresis using a 10% resolving gel. Resolved proteins were transferred to nitrocellulose membranes and blocked for 1 h at room temperature with Tris-buffered saline supplemented with 5% non-fat powdered milk. Nitrocellulose blots were incubated overnight at 4 8C with the pyruvate dehdyrogenase kinase-2 (PDK-2) polyclonal antibody (Abgent), washed and incubated with horseradish peroxidase (Santa Cruz Biotechnology). Bound
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antibody was visualised by using ECL according to the manufacturers instructions (Pierce, Rockford, IL, USA). Equal loading was verified by analysis of total glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The blots were then exposed to Hyperfilm and bands quantified by scanning densitometry using GeneTools (Syngene).
Table 2 Glucose uptake and lactate production in control cells and homocysteine-treated cells
2.7. Enzyme activities
Control Hcy
For the determination of enzyme activity, homogenates of astrocytes were prepared as previously described (Almeida and Medina, 1998). Briefly, the cells were trypsinised, centrifuged and resuspended in an isotonic sucrose buffer. The cells were homogenised using a Potter S homogeniser at a speed of 550 rpm. The homogenate was frozen and stored at 20 8C for subsequent analysis of enzyme activities. The activity of alanine aminotransferase (ALAT) and aspartate aminotransferase (AST) were determined using kits supplied by Randox Laboratories Co. (Antrim, UK). All assays were carried out according to the manufacturer’s recommendations. 2.8. Total metabolite levels Total glutamate, glucose, lactate and glutamine concentrations were measured in cellular extracts and incubation medium using a YSI 7100 amino acid analyser (YSI Incorporated). For determination of glucose uptake the amount of glucose remaining in the media following the incubation period was determined. GSH levels were measured using the monochlorobimane method (Kamencic et al., 2000). Briefly cells were grown and treated as described above. The cells were washed with PBS and incubated with KRB buffer supplemented with monochlorbimane (100 mM) and glutathione-S-transferase (1 U/ml) for 30 min at 37 8C. Fluorescence was measured with an excitation of 380 nm and emission of 520 nm. Following the experiment the cells were lysed and the protein concentration determined. 2.9. Statistical analysis The results are presented as the mean S.D. for n separate determinations. Groups of data were compared using Student’s unpaired t-test. Differences were considered significant at p < 0.05. For analysis of the metabolic profiles the data was loaded into SIMCA (Umetrics) and mean centred and Pareto scaled (each variable was weighted according to pffiffiffiffiffiffiffiffiffi 1= S:D:, where S.D. is the standard deviation of the variable). Principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) were carried out to analyse the data. The data was visualised by construction of scores plots and corresponding loadings plots. The discriminating metabolites were identified by inspection of the loadings plot and comparison to in-house metabolite libraries and published chemical shift tables. The goodness of the model was assessed using R2 and Q2 values. The R2-value gives an indication of how much of the variation in the data is explained by the principal components of the model. Q2 is an indication of the predictive ability of the model.
3. Results 3.1. Effects of prolonged culture with homocysteine on glucose metabolism and metabolite generation
Condition
Glucose uptake (mmol/mg protein)
Lactate production (mmol/mg protein)
7.28 2.94 11.27 1.40*
7.4 4.6 9.1 5.2
The glucose uptake is reported as the glucose uptake during a 4-h incubation period. Control conditions represent 4 h incubation with 5.5 mM [U-13C]glucose and Hcy conditions represent 20 h incubation with homocysteine followed by a 4-h incubation with 5.5 mM [U-13C]glucose and 400 mM homocysteine. Data are presented as mmol/mg protein and n = 3. * p < 0.05.
However, there was a significant increase in glucose uptake from the media following culture in the presence of homocysteine (see Table 2). The production of lactate did not significantly change. The levels of GSH did not change following incubation with homocysteine (63.4 12.2 nmol/mg protein for control cells and 65.6 8.8 nmol/mg protein for treated cells). Calculation of the ratio of flux through PC and PDH showed significant changes following homocysteine treatment: there was a significant decrease in the PC to PDH ratio (see Fig. 1). The % acetyl-CoA labelled with 13C was significantly increased from 34.2 to 45.8% in the presence of homocysteine ( p < 0.05). As acetyl-CoA can only be labelled via conversion of pyruvate to acetyl-CoA through the PDH complex a change in the percentage acetyl-CoA labelled indicates a change in flux through PDH. As a result the change in the ratio of PC to PDH is dominated by a change in flux through PDH. The PDH complex is highly regulated by a reversible phosphorylation/dephosphorylation cycle. Phosphorylation is achieved by pyruvate dehydrogenase kinase (PDK) which causes an inactivation of the complex. Following culture with homocysteine for 20 h there was a significant decrease in PDK-2 isoform protein levels (Fig. 2). PDK-2 has been reported to be the most abundant isoform in the brain (Nakai et al., 2000). 3.2. Effect of homocysteine on NAD(P)H levels and mitochondrial membrane potential Addition of glucose induced a rapid increase in NAD(P)H levels. Following culture with homocysteine for 20 h this increase was significantly increased ( p < 0.05, Fig. 3). These results agree with the increased flux through PDH and the
Cells were pre-cultured with homocysteine for a period of 20 h followed by a 4-h incubation period in the presence of [U-13C]glucose and homocysteine. There was no indication of cell death during the incubation periods as assessed by measuring LDH activity; the total LDH release was 5.1 1.3% and 5.0 1.4% under control and treatment conditions, respectively. There was no significant change in the amount of glutamine labelled at position C3 and C4 and glutamate labelled at C4 following culture with homocysteine (see Table 1). Table 1 The amount of glutamate and glutamine labelled at specific carbon positions Condition
Glu C4
Gln C3
Gln C4
Control Hcy
3.53 0.04 4.67 0.77
11.76 3.78 10.92 1.71
8.53 2.82 8.68 0.22
Amount of 13C labelled metabolites at specific carbon positions expressed as nmol/ mg protein (n = 3). Control conditions represent astrocytes incubated for 4 h with 5.5 mM [U-13C]glucose. Hcy experiments represent culture with 400 mM homocysteine for 20 h followed by a 4-h incubation period with 400 mM homocysteine and [U-13C]glucose.
Fig. 1. PC to PDH ratio’s and % acetyl-CoA labelled from [U-13C]glucose. Data are represented as % control and control conditions (white bars) represent astrocytes incubated for 4 h with 5.5 mM [U-13C]glucose. Treated experiments (black bars) represent culture with 400 mM homocysteine for 20 h followed by a 4-h incubation period with 400 mM homocysteine and [U-13C]glucose. The data are presented as mean W S.D. *p < 0.05.
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Fig. 3. Effects of homocysteine on NAD(P)H levels in astrocytes. Cells were cultured in the presence or absence of 400 mM homocysteine for 20 h and stimulated at the indicated time (arrow) with glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Values are mean of four independent experiments. Control conditions are represented by the black line and homocysteine-treated cells are represented by the grey line. Following the homocysteine treatment the increase in NAD(P)H fluorescence upon stimulation was 15,940 W 4462, which was significantly higher than the control value 9254 W 1201 ( p < 0.05).
Fig. 2. Effects of culture with homocysteine on PDK-2 protein levels in primary cultures of astrocytes. Cells were cultured in the presence or absence of 400 mM homocysteine for 20 h. Proteins extrats were performed and expression levels analysed using Western blot. (a) PDK-2 expression levels are reported as the PDK2:GAPDH ratios. Results are expressed as mean W S.D. (n = 5), *p = 0.01. (b) A representative Western blot showing PDK-2 and GAPDH protein levels in control cells and in homocysteine-treated cells.
significant decrease in PDK-2 expression, the enzyme which controls activity of PDH. As a means to probe the effects of homocysteine on mitochondrial function we investigated the effects on mitochondrial membrane potential. Addition of glucose to control cells resulted in a reduction of the rhodamine-123 signal by 19.2% reflecting hyperpolarisation of the mitochondrial membrane. Exposure to homocysteine for 20 h resulted in an impaired glucose-induced mitochondrial hyperpolarisation (Fig. 4). 3.3. Enzyme activities in the presence of homocysteine The exposure to homocysteine did not alter the enzyme activities of ALAT and AST in the cultured astrocytes. Control values were 14.7 1.8 and 5.0 0.7 mU/mg protein for AST and ALAT, respectively. Following exposure to homocysteine the values for AST and ALAT activity were 13.2 1.1 and 5.2 1.6 mU/mg protein, respectively. The lack of effect on the activity of the amino transferases indicates a more specific action of homocytseine on
Fig. 4. Effects of homocysteine on mitochondrial membrane potential (Dcm) levels in astrocytes. Cells were cultured in the presence or absence of 400 mM homocysteine for 20 h and stimulated at the indicated time (arrow) with glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Values are represented as mean values (n = 4). Control conditions are represented by closed black squares and treated condition by closed grey triangles. Following the homocysteine treatment the decrease in Dcm fluorescence upon stimulation was (2.13 T 107) W (0.29 T 107), which was significantly lower than the control value (2.70 T 107) W (0.22 T 107) ( p < 0.05).
oxidative metabolism compared to that previously reported for gliotoxins (Brennan et al., 2006). 3.4. Effects of culture in the presence of homocysteine on the metabolic profiles 1
H NMR spectra of the metabolite extractions following treatment with homocysteine separated from control extractions (Fig. 5). The PLS-DA model comprised of three components and had an R2X and Q2Y of 0.57 and 0.63, respectively. Inspection of the corresponding loadings plot showed that the following NMR regions were responsible for the separation of the two groups: 3.368, 2.653, 3.383, 2.658, 2.668, 3.228, 3.268, and 3.223 ppm. These regions correspond to hypotaurine (3.368, 2.653, 3.383, 2.658, and 2.668 ppm), choline (3.228 and 2.223 ppm) and an unknown (3.268 ppm).
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Fig. 5. PLS-DA scores plot (a) and corresponding loadings plot (b) of 1H NMR spectra from control cells and cells treated with homocysteine. Cells were incubated for 20 h in the presence or absence of 400 mM homocysteine and then subsequently incubated in the presence of unlabelled glucose (5.5 mM) in the presence or absence of 400 mM homocysteine. The control experiments separated from the homocysteine-treated experiments with a model of R2 = 0.574 and Q2 = 0.627. Control conditions are represented by closed triangles and treated condition by closed squares.
4. Discussion The detrimental effects of homocysteine on neurons is well documented in the literature. However, very little has been reported on the effects of homocysteine on astrocytes and astrocytic function. Maler et al. (2003) reported that concentrations of 2 mM of homocytseine and above induced cell death in astrocytes. However, the mode of action remains unknown and relatively higher concentrations are needed to induce cell death compared to neurons where, for example, concentrations of 50– 500 mM significantly reduced the number of Purkinje neurons (Oldreive and Doherty, 2007). The present paper investigated the effects of sub-lethal concentrations of homocysteine (effective concentration 200 mM) on astrocytic glucose metabolism. Significant alterations in oxidative glucose metabolism and mitochondrial function were found. The PDH complex catalyses the oxidative decarboxylation of pyruvate leading to NADH and acetyl-CoA production. This complex is tightly regulated by a reversible phosphorylation/ dephosphorylation cycle. Inactivation is caused by phosphorylation at three serine residues on the E1a subunit and is mediated by PDK. To date there are four isoforms of PDK and the current study showed that culture with homocysteine at sub-lethal concentrations resulted in a decrease in protein expression of PDK-2. A decrease in the amount of PDK-2 will result in increased PDH activity agreeing with the increased flux through PDH calculated from the 13C NMR data. The increase in glucose consumption agrees with an increased flux through oxidative metabolism. Increased flux through PDH will result in increased oxidative metabolism which in turn should increase NADH production. Indeed there was a significant increase in NADH production following the treatment with homocysteine. To probe further the effects of homocysteine on mitochondrial function experiments were performed to investigate the effects on mitochondrial membrane potential. Following treatment with homocysteine there was impaired glucose-induced mitochondrial hyperpolarisation which indicates impaired mitochondrial ATP production. It is well established that astrocytes produce substantial amounts of mitochondrial ATP which is necessary for astrocytic functions such as glutamate uptake (Hertz et al., 2007). As a result alterations to mitochondrial function, such as that observed here following homocysteine exposure, can impair astrocytic neuroprotective function. In a recent study, Voloboueva et al. (2007) showed using an inhibitor of astrocytic
mitochondria that mitochondrial dysfunction compromised their ability to protect against excitotoxicity. In the current study using sub-lethal concentrations of homocysteine considerable alterations to mitochondrial metabolism was shown highlighting the possibility of a role for altered astrocytic function in neurodegenerative diseases associated with elevated homocysteine levels. Analysis of the metabolic profiles showed that the major change following homocysteine culture at sub-lethal concentrations was the increase in hypotaurine and a decrease in choline. Choline has been reported to be involved in the methylation of homocysteine to methionine through a betaine-dependent pathway (Bidulescu et al., 2007) and the observed decrease in choline suggests that this pathway is active in the astrocytes. The increase in hypotaurine is significant for a number of reasons. Firstly, the conversion of homocysteine to cysteine involves a trans-sulfuration pathway which until recently was considered not to be functional in the brain (Vitvitsky et al., 2006). The current study, which identifies hypotaurine as the major metabolic change following homocysteine culture, adds further evidence to the existence of a functional trans-sulfuration pathway in the brain. A previous study using addition of labelled cysteine showed that the major product of cysteine metabolism in astrocytes was hypotaurine and taurine (Brand et al., 1998). Again the current study corroborates this hypothesis. The conversion of hypotaurine to taurine in astrocytes has been reported to be slow (Brand et al., 1998) and previous studies have reported higher concentrations of hypotaurine compared to taurine in astrocytes. The cysteine produced by the trans-sulfuration pathway has another metabolic fate, namely the entry into the GSH synthesis pathway. Following homocysteine treatment there was no significant increase in total GSH levels. The preferential entry into the taurine synthesis pathway over the glutathione pathway is noteworthy considering that reciprocal levels of GSH and homocysteine have been reported in some neurodegerative diseases. In the long-term under conditions of elevated homocysteine concentrations it is possible to speculate that the increased conversion of cysteine to hypotaurine will have detrimental effects on the production of GSH. Further experiments on the effects of long-term exposure of astrocytes to homocysteine are warranted. In summary, culture with sub-lethal concentrations of homocysteine resulted in altered glucose metabolism and mitochondrial function in primary cultures of astrocytes. In addition, the presence of homocysteine increased the production of hypotaurine which may be of use as an osmolyte or an osmolyte precursor. Overall, the
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observed homocysteine-induced changes may have important implications for neuronal-astrocyte cross-talk and as a result will be important for the understanding of the pathogenesis of neurodegenerative diseases linked to raised homocysteine levels. Acknowledgments We would like to acknowledge the NMR Facility at the School of Biomolecular and Biomedical Sciences. We would like to acknowledge Dr. Brian Mion for technical help. YJ was supported by a UCD Ad Astra Scholarship.
References Allen, R.H., Stabler, S.P., Lindenbaum, J., 1998. Relevance of vitamins, homocysteine and other metabolites in neuropsychiatric disorders. Eur. J. Pediatr. 157 (Suppl. 2), S122–S126. Almeida, A., Medina, J.M., 1998. A rapid method for the isolation of metabolically active mitochondria from rat neurons and astrocytes in primary culture. Brain Res. Brain Res. Protoc. 2, 209–214. Alves, P.M., Flogel, U., Brand, A., Leibfritz, D., Carrondo, M.J., Santos, H., Sonnewald, U., 1996. Immobilization of primary astrocytes and neurons for online monitoring of biochemical processes by NMR. Dev. Neurosci. 18, 478–483. Bell, I.R., Edman, J.S., Selhub, J., Morrow, F.D., Marby, D.W., Kayne, H.L., Cole, J.O., 1992. Plasma homocysteine in vascular disease and in nonvascular dementia of depressed elderly people. Acta Psychiatr. Scand. 86, 386–390. Bidulescu, A., Chambless, L.E., Siega-Riz, A.M., Zeisel, S.H., Heiss, G., 2007. Usual choline and betaine dietary intake and incident coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) study. BMC Cardiovasc. Disord. 7, 20. Brand, A., Leibfritz, D., Hamprecht, B., Dringen, R., 1998. Metabolism of cysteine in astroglial cells: synthesis of hypotaurine and taurine. J. Neurochem. 71, 827–832. Brennan, L., Alves, P.M., Hewage, C., Malthouse, J.P., McBean, G.J., 2006. Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes. Neurochem. Int. 48, 739–745. Christie, L.A., Riedel, G., Algaidi, S.A., Whalley, L.J., Platt, B., 2005. Enhanced hippocampal long-term potentiation in rats after chronic exposure to homocysteine. Neurosci. Lett. 373, 119–124. Clarke, R., Smith, A.D., Jobst, K.A., Refsum, H., Sutton, L., Ueland, P.M., 1998. Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch. Neurol. 55, 1449–1455. Duan, W., Ladenheim, B., Cutler, R.G., Kruman, I.I., Cadet, J.L., Mattson, M.P., 2002. Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson’s disease. J. Neurochem. 80, 101–110. Escartin, C., Valette, J., Lebon, V., Bonvento, G., 2006. Neuron-astrocyte interactions in the regulation of brain energy metabolism: a focus on NMR spectroscopy. J. Neurochem. 99, 393–401. Fan, T.W.M., 1996. Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Prog. Nucl. Magn. Reson. Spectrosc. 28, 161–219. Foister, N.S., Oldreive, C.E., Mackie, J.B., Doherty, G.H., 2005. Embryonic cerebellar granule cells are resistant to necrosis induced by homocysteine. Brain Res. Dev. Brain Res. 160, 85–89. Hertz, L., Peng, L., Dienel, G.A., 2007. Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. J. Cereb. Blood Flow Metab. 27, 219–249.
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Ho, P.I., Ortiz, D., Rogers, E., Shea, T.B., 2002. Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J. Neurosci. Res. 70, 694–702. Jakubowski, H., 2004. Molecular basis of homocysteine toxicity in humans. Cell. Mol. Life Sci. 61, 470–487. Kamencic, H., Lyon, A., Paterson, P.G., Juurlink, B.H., 2000. Monochlorobimane fluorometric method to measure tissue glutathione. Anal. Biochem. 286, 35–37. Kruman, I.I., Culmsee, C., Chan, S.L., Kruman, Y., Guo, Z., Penix, L., Mattson, M.P., 2000. Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J. Neurosci. 20, 6920– 6926. Kruman, I.I., Kumaravel, T.S., Lohani, A., Pedersen, W.A., Cutler, R.G., Kruman, Y., Haughey, N., Lee, J., Evans, M., Mattson, M.P., 2002. Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer’s disease. J. Neurosci. 22, 1752–1762. Langmeier, M., Folbergrova, J., Haugvicova, R., Pokorny, J., Mares, P., 2003. Neuronal cell death in hippocampus induced by homocysteic acid in immature rats. Epilepsia 44, 299–304. Lapidot, A., Gopher, A., 1994. Cerebral metabolic compartmentation. Estimation of glucose flux via pyruvate carboxylase/pyruvate dehydrogenase by 13C NMR isotopomer analysis of D-[U-13C]glucose metabolites. J. Biol. Chem. 269, 27198–27208. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Maler, J.M., Seifert, W., Huther, G., Wiltfang, J., Ruther, E., Kornhuber, J., Bleich, S., 2003. Homocysteine induces cell death of rat astrocytes in vitro. Neurosci. Lett. 347, 85–88. Malloy, C.R., Sherry, A.D., Jeffrey, F.M., 1990. Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers. Am. J. Physiol. 259, H987–H995. Miller, J.W., 2000. Homocysteine, Alzheimer’s disease, and cognitive function. Nutrition 16, 675–677. Nakai, N., Obayashi, M., Nagasaki, M., Sato, Y., Fujitsuka, N., Yoshimura, A., Miyazaki, Y., Sugiyama, S., Shimomura, Y., 2000. The abundance of mRNAs for pyruvate dehydrogenase kinase isoenzymes in brain regions of young and aged rats. Life Sci. 68, 497–503. Oldreive, C.E., Doherty, G.H., 2007. Neurotoxic effects of homocysteine on cerebellar Purkinje neurons in vitro. Neurosci. Lett. 413, 52–57. Ravaglia, G., Forti, P., Maioli, F., Martelli, M., Servadei, L., Brunetti, N., Porcellini, E., Licastro, F., 2005. Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am. J. Clin. Nutr. 82, 636–643. Richter-Landsberg, C., Besser, A., 1994. Effects of organotins on rat brain astrocytes in culture. J. Neurochem. 63, 2202–2209. Seifert, G., Schilling, K., Steinhauser, C., 2006. Astrocyte dysfunction in neurological disorders: a molecular perspective. Nat. Rev. Neurosci. 7, 194–206. Verkhratsky, A., Toescu, E.C., 2006. Neuronal-glial networks as substrate for CNS integration. J. Cell. Mol. Med. 10, 826–836. Vitvitsky, V., Thomas, M., Ghorpade, A., Gendelman, H.E., Banerjee, R., 2006. A functional transsulfuration pathway in the brain links to glutathione homeostasis. J. Biol. Chem. 281, 35785–35793. Voloboueva, L.A., Suh, S.W., Swanson, R.A., Giffard, R.G., 2007. Inhibition of mitochondrial function in astrocytes: implications for neuroprotection. J. Neurochem. 102, 1383–1394. Wroblewski, F., Ladue, J.S., 1955. Lactic dehydrogenase activity in blood. Proc. Soc. Exp. Biol. Med. 90, 210–213. Zieminska, E., Stafiej, A., Lazarewicz, J.W., 2003. Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones. Neurochem. Int. 43, 481–492.
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Effects of homocysteine on metabolic pathways in cultured astrocytes Ying Jin, Lorraine Brennan * UCD School of Agriculture, Food Science and Veterinary Medicine, UCD Conway Institute, UCD Dublin, Belfield, Dublin 4, Ireland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 February 2008 Received in revised form 2 March 2008 Accepted 6 March 2008 Available online 13 March 2008
Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 mM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-13C]glucose in the presence or absence of homocysteine and analysed using 13C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using 1H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes. ß 2008 Elsevier Ltd All rights reserved.
Keywords: Homocysteine Astrocytes Metabolism
1. Introduction Elevated levels of the sulphur containing non-essential amino acid homocysteine have been reported as an independent risk factor for cognitive dysfunction (Bell et al., 1992; Allen et al., 1998; Miller, 2000). A significant correlation has been reported between risk of Alzheimer’s disease and Parkinson’s disease and high-plasma levels of homocysteine (Clarke et al., 1998; Ravaglia et al., 2005). In recent years, it has become clear that exposure to homocysteine induced neuronal cell death in a variety of neuronal types including rat hippocampal and cortical neurons (Langmeier et al., 2003). A recent study on the Purkinje cells showed that exposure to homocysteine inhibited cell survival and the outgrowth of neurites (Oldreive and Doherty,
* Corresponding author. Tel.: +353 1 716 6759; fax: +353 1 283 7211. E-mail address: [email protected] (L. Brennan).
Abbreviations: PDK-2, pyruvate dehydrogenase kinase isoform 2; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; ALAT, alanine aminotransferase; AST, aspartate aminotransferase; GSH, glutathione. 0197-0186/$ – see front matter ß 2008 Elsevier Ltd All rights reserved. doi:10.1016/j.neuint.2008.03.001
2007). Several mechanisms of toxicity have been reported including NMDA receptor and group I metabotropic glutamate receptor (mGluR) mediated neurotoxicity (Ho et al., 2002; Zieminska et al., 2003). In addition homocysteine can induce caspase-dependent apoptosis in human dopaminergic cells, rat hippocampal and mouse cortical neurons (Kruman et al., 2000, 2002; Duan et al., 2002). However, capase-independent cell death has been reported in murine cerebellar granule cells in the presence of high levels of homocysteine (Foister et al., 2005). In addition to the toxic effects homocysteine has also been reported to alter hippocampal plasticity and synaptic transmission (Christie et al., 2005). This non-protein amino acid has to date three known metabolic fates in the cell. In the first instance homocysteine can undergo reversible methylation to form methionine in the methionine cycle. In the second case it can undergo trans-sulfuration to form cystathionine and cysteine and the importance of this pathway in the brain has recently been reported (Vitvitsky et al., 2006). The cysteine formed has two metabolic fates: entry into glutathione (GSH) synthesis or entry into taurine synthesis pathway. Alternatively homocysteine can be used in the production of homocysteine thiolactone which can then react with reactive nitrogen
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species such as nitric oxide (Jakubowski, 2004). Although the neurotoxic effects of homocysteine are well documented there has been very little study on the effect of homocysteine in astrocytes. Maler et al. (2003) reported that homocysteine showed a dose dependent cytotoxic effect at doses at 2 mM and above in cortical astrocytes. The mechanism of these effects are unknown. Astrocytes are an important cell in the central nervous system and are critical in the glial–vascular interface as part of the blood– brain barrier (Verkhratsky and Toescu, 2006). Over the last decade the importance of astrocytes in the regulation of brain metabolism and in particular brain energy metabolism has been realised (Escartin et al., 2006). It is now accepted that brain energy metabolism is regulated by cross-talk between astrocytes and neurons. As a result, any modulation of astrocyte function could have potential detrimental effects to neuronal function. While a large body of literature exists relating the deleterious role of neuronal metabolic impairment in the diseased brain, the role of astrocytes has been largely ignored until recently (Seifert et al., 2006). Considering the increasing profile of astrocytes the present study was undertaken to probe the effects of sub-lethal concentrations of homocysteine on metabolic processes in astrocytes. 2. Experimental procedures 2.1. Reagents All chemicals were obtained from Sigma–Aldrich Chemical Company (Poole, Dorset, UK) except [U-13C]glucose which was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Culture media and foetal bovine serum were obtained from Gibco (Glasgow, UK). 2.2. Cell culture Rat astrocytes were prepared from 2-day-old Wistar rats as previously described (Richter-Landsberg and Besser, 1994; Alves et al., 1996). All procedures were performed in accordance with Irish Government ethical guidelines. The cerebral hemispheres were isolated and the meninges was carefully removed. The cerebral hemispheres were then mechanically disrupted. Cells were grown in DMEM containing 10% (v/v) FBS for 7–10 days. The nonastrocytic cells were removed by vigorous shaking and the astrocytes were subcultured for 20–23 days during which time the media was changed twice a week. Cells were maintained in a Forma Scientific incubator at 37 8C in a humidified atmosphere of 5% CO2 and 95% air. To assess cell viability the LDH assay was performed using a coupled enzymatic reaction where tetrazolium salt was converted into formazan with an absorbance maximum at 490 nm (Wroblewski and Ladue, 1955). 2.3. Extraction procedures To investigate the effects of homocysteine on astrocytic metabolism the cells were pre-incubated for a period of 20 h in the presence of 400 mM homocysteine. The cells were then washed with phosphate-buffered saline (PBS) and were incubated for 4 h in minimal medium supplemented with 5.5 mM [U-13C]glucose in the presence 400 mM homocysteine. Control experiments were run in parallel. As a D,L-mixture of homocysteine was used the effective concentration used in the present studies was 200 mM. A previous study showed that homocysteine concentrations above 2 mM were needed to induce cell death (Maler et al., 2003). The concentration chosen here is well below this toxic concentration and is within the range used in other in vitro studies (20 mM to 25 mM). Following the incubation period, the media was removed and stored at 20 8C and analysed later for glucose concentration and lactate dehydrogenase (LDH) activity. The cells were washed with ice-cold phosphate-buffered saline, placed in liquid N2 and the metabolites were extracted using 6% perchloric acid. The extracts of six culture dishes were pooled and centrifuged at 3000 g for 12 min. The resulting supernatant was neutralised with KOH (5 and 0.1 M solutions) and the pellet was soaked overnight in 0.1 M NaOH. The protein concentration was determined using the Lowry method (Lowry et al., 1951). The neutralised supernatant was centrifuged at 3000 g for 5 min and then lyophilised. Each experiment was carried out on at least three independent cultures. The lyophilised cell extracts were dissolved in 1.2 ml D2O and then centrifuged at 350 g for 5 min. The supernatant was carefully removed and the pH was checked and adjusted when necessary to 7.0 0.1 with 0.1 M NaOH and 0.1 M HCl. An insert containing 1% (v/v) dioxane in water was used as an external signal intensity reference for quantification of the NMR spectra.
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For the global metabolic profiling the cells were grown and treated as described as above. For the final 4 h incubation period the cells were incubated with unlabelled glucose in the presence or absence of 400 mM homocysteine. Following the incubation period, the media was removed and the cells were washed with icecold PBS. The metabolites were extracted with ice-cold 70% ethanol. The extracts of two culture flasks were pooled and centrifuged at 4000 g for 15 min. The resulting supernatant was stored at 20 8C and the protein concentrations were determined in the pellets. The samples were lyophilised and redissolved in 700-ml D2O. For chemical shift reference 10 ml TSP (sodium 3-(trimethylsilylpropionate-2,2,3,3-d4) was added to each and the pH was adjusted to pH 7.0 0.1 with 1 M HCl. 2.4. NMR spectroscopy Proton decoupled 13C spectra were acquired on a Bruker DRX 500 spectrometer using a QNP 5-mm probe. Typically, spectra were acquired with 32k data points using a 908 pulse angle, 260 ppm spectral width, 2.5 s relaxation delay and 12,000 scans. Spectra were recorded at 25 8C. Chemical shifts in aqueous media were referenced to tetramethylsilane at 0 ppm. Exponential multiplications with 2 Hz line broadening were performed using Bruker WINNMR software. The assignments of the intermediate metabolites were made by comparison with chemical shift tables in the literature (Fan, 1996) or by addition of 100 mM of unlabelled amino acid. 1 H NMR spectra were typically acquired with 32k data points and 256 scans over a spectral width of 8 kHz. Water suppression was achieved during the relaxation delay (2.5 s) and the mixing time (100 ms). All 1H NMR spectra were referenced to TSP at 0.0 ppm and processed manually with the Bruker software using a line broadening of 0.2 Hz. All spectra were baseline corrected. The spectra were then reduced by integrating into bins across spectral regions of 0.005 ppm, using AMIX (Bruker Biospin, Germany). The water region (4.2–5.6 ppm) was excluded and the data was normalised to the sum of the spectral integral. In the case of 13C NMR spectra the amount of 13C in each resonance was evaluated by integration of the extract peaks and the corresponding peaks in the standard sample relative to the dioxane signal. The standard sample contained a solution of Lalanine, L-glutamate, lactate and D-glucose at known concentrations. Corrections for the natural abundance signal were made. The fluxes reported were obtained by analysis of the isotopomers of glutamine C2 and C4. The ratio between flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) was calculated as follows: ([2,3-13C2] + [1,2,3-13C3])/[4,5-13C2] + [3,4,5-13C3] (Lapidot and Gopher, 1994). The fraction of acetyl-CoA labelled from [U-13C]glucose was calculated using the following equation for the C4 peak: (3,4,5-13C) C4/C3 (Malloy et al., 1990). 2.5. NAD(P)H and mitochondrial membrane potential measurements For the NAD(P)H experiments the cells were grown in 96-well plates and preincubated in the presence or absence of 400 mM homocysteine for 20 h. Cells were washed with PBS and incubated in Krebs–Ringer bicarbonate (KRB) buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO47H2O and 0.25% BSA, 10 mM NaHCO3 pH 7.4) at 37 8C for 1 h. Following this incubation period the cells were placed in a temperature-controlled platereader (FLEXstation, Molecule Devices) and the baseline fluorescence was measured at an excitation wavelength of 340 nm and an emission wavelength of 460 nm. After 150 s the cells were stimulated by addition of glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Fluorescence readings were recorded every 2 s over the total duration of the experiment (455 s). For the mitochondrial membrane potential cells were grown in 96-well plates and pre-incubated in the presence or absence of 400 mM homocysteine for 20 h. Cells were incubated in KRB buffer at 37 8C for 15 min, washed and incubated in 5 mg/ml rhodamine-123 for 15 min. Following this incubation period the cells were washed and incubated in KRB buffer in the temperature-controlled platereader (FLEXstation, Molecule Devices). The baseline fluorescence was measured at an excitation wavelength of 505 nm and an emission wavelength of 515 nm. After 50 s the cells were stimulated by addition of glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Fluorescence readings were recorded every 4 s over the total duration of the experiment (150 s). 2.6. Western blot analysis For Western blot analysis cells were grown in six well plates and treated for 20 h in the presence or absence of 400 mM homocysteine. Cells were washed twice with ice-cold PBS and lysed with RIPA buffer. Samples were centrifuged at 14,000 g for 15 min at 4 8C. The supernatant was collected and total protein concentration was determined using a BCA assay. Samples containing 15 mg protein were then subjected to SDS-PAGE electrophoresis using a 10% resolving gel. Resolved proteins were transferred to nitrocellulose membranes and blocked for 1 h at room temperature with Tris-buffered saline supplemented with 5% non-fat powdered milk. Nitrocellulose blots were incubated overnight at 4 8C with the pyruvate dehdyrogenase kinase-2 (PDK-2) polyclonal antibody (Abgent), washed and incubated with horseradish peroxidase (Santa Cruz Biotechnology). Bound
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antibody was visualised by using ECL according to the manufacturers instructions (Pierce, Rockford, IL, USA). Equal loading was verified by analysis of total glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The blots were then exposed to Hyperfilm and bands quantified by scanning densitometry using GeneTools (Syngene).
Table 2 Glucose uptake and lactate production in control cells and homocysteine-treated cells
2.7. Enzyme activities
Control Hcy
For the determination of enzyme activity, homogenates of astrocytes were prepared as previously described (Almeida and Medina, 1998). Briefly, the cells were trypsinised, centrifuged and resuspended in an isotonic sucrose buffer. The cells were homogenised using a Potter S homogeniser at a speed of 550 rpm. The homogenate was frozen and stored at 20 8C for subsequent analysis of enzyme activities. The activity of alanine aminotransferase (ALAT) and aspartate aminotransferase (AST) were determined using kits supplied by Randox Laboratories Co. (Antrim, UK). All assays were carried out according to the manufacturer’s recommendations. 2.8. Total metabolite levels Total glutamate, glucose, lactate and glutamine concentrations were measured in cellular extracts and incubation medium using a YSI 7100 amino acid analyser (YSI Incorporated). For determination of glucose uptake the amount of glucose remaining in the media following the incubation period was determined. GSH levels were measured using the monochlorobimane method (Kamencic et al., 2000). Briefly cells were grown and treated as described above. The cells were washed with PBS and incubated with KRB buffer supplemented with monochlorbimane (100 mM) and glutathione-S-transferase (1 U/ml) for 30 min at 37 8C. Fluorescence was measured with an excitation of 380 nm and emission of 520 nm. Following the experiment the cells were lysed and the protein concentration determined. 2.9. Statistical analysis The results are presented as the mean S.D. for n separate determinations. Groups of data were compared using Student’s unpaired t-test. Differences were considered significant at p < 0.05. For analysis of the metabolic profiles the data was loaded into SIMCA (Umetrics) and mean centred and Pareto scaled (each variable was weighted according to pffiffiffiffiffiffiffiffiffi 1= S:D:, where S.D. is the standard deviation of the variable). Principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) were carried out to analyse the data. The data was visualised by construction of scores plots and corresponding loadings plots. The discriminating metabolites were identified by inspection of the loadings plot and comparison to in-house metabolite libraries and published chemical shift tables. The goodness of the model was assessed using R2 and Q2 values. The R2-value gives an indication of how much of the variation in the data is explained by the principal components of the model. Q2 is an indication of the predictive ability of the model.
3. Results 3.1. Effects of prolonged culture with homocysteine on glucose metabolism and metabolite generation
Condition
Glucose uptake (mmol/mg protein)
Lactate production (mmol/mg protein)
7.28 2.94 11.27 1.40*
7.4 4.6 9.1 5.2
The glucose uptake is reported as the glucose uptake during a 4-h incubation period. Control conditions represent 4 h incubation with 5.5 mM [U-13C]glucose and Hcy conditions represent 20 h incubation with homocysteine followed by a 4-h incubation with 5.5 mM [U-13C]glucose and 400 mM homocysteine. Data are presented as mmol/mg protein and n = 3. * p < 0.05.
However, there was a significant increase in glucose uptake from the media following culture in the presence of homocysteine (see Table 2). The production of lactate did not significantly change. The levels of GSH did not change following incubation with homocysteine (63.4 12.2 nmol/mg protein for control cells and 65.6 8.8 nmol/mg protein for treated cells). Calculation of the ratio of flux through PC and PDH showed significant changes following homocysteine treatment: there was a significant decrease in the PC to PDH ratio (see Fig. 1). The % acetyl-CoA labelled with 13C was significantly increased from 34.2 to 45.8% in the presence of homocysteine ( p < 0.05). As acetyl-CoA can only be labelled via conversion of pyruvate to acetyl-CoA through the PDH complex a change in the percentage acetyl-CoA labelled indicates a change in flux through PDH. As a result the change in the ratio of PC to PDH is dominated by a change in flux through PDH. The PDH complex is highly regulated by a reversible phosphorylation/dephosphorylation cycle. Phosphorylation is achieved by pyruvate dehydrogenase kinase (PDK) which causes an inactivation of the complex. Following culture with homocysteine for 20 h there was a significant decrease in PDK-2 isoform protein levels (Fig. 2). PDK-2 has been reported to be the most abundant isoform in the brain (Nakai et al., 2000). 3.2. Effect of homocysteine on NAD(P)H levels and mitochondrial membrane potential Addition of glucose induced a rapid increase in NAD(P)H levels. Following culture with homocysteine for 20 h this increase was significantly increased ( p < 0.05, Fig. 3). These results agree with the increased flux through PDH and the
Cells were pre-cultured with homocysteine for a period of 20 h followed by a 4-h incubation period in the presence of [U-13C]glucose and homocysteine. There was no indication of cell death during the incubation periods as assessed by measuring LDH activity; the total LDH release was 5.1 1.3% and 5.0 1.4% under control and treatment conditions, respectively. There was no significant change in the amount of glutamine labelled at position C3 and C4 and glutamate labelled at C4 following culture with homocysteine (see Table 1). Table 1 The amount of glutamate and glutamine labelled at specific carbon positions Condition
Glu C4
Gln C3
Gln C4
Control Hcy
3.53 0.04 4.67 0.77
11.76 3.78 10.92 1.71
8.53 2.82 8.68 0.22
Amount of 13C labelled metabolites at specific carbon positions expressed as nmol/ mg protein (n = 3). Control conditions represent astrocytes incubated for 4 h with 5.5 mM [U-13C]glucose. Hcy experiments represent culture with 400 mM homocysteine for 20 h followed by a 4-h incubation period with 400 mM homocysteine and [U-13C]glucose.
Fig. 1. PC to PDH ratio’s and % acetyl-CoA labelled from [U-13C]glucose. Data are represented as % control and control conditions (white bars) represent astrocytes incubated for 4 h with 5.5 mM [U-13C]glucose. Treated experiments (black bars) represent culture with 400 mM homocysteine for 20 h followed by a 4-h incubation period with 400 mM homocysteine and [U-13C]glucose. The data are presented as mean W S.D. *p < 0.05.
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Fig. 3. Effects of homocysteine on NAD(P)H levels in astrocytes. Cells were cultured in the presence or absence of 400 mM homocysteine for 20 h and stimulated at the indicated time (arrow) with glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Values are mean of four independent experiments. Control conditions are represented by the black line and homocysteine-treated cells are represented by the grey line. Following the homocysteine treatment the increase in NAD(P)H fluorescence upon stimulation was 15,940 W 4462, which was significantly higher than the control value 9254 W 1201 ( p < 0.05).
Fig. 2. Effects of culture with homocysteine on PDK-2 protein levels in primary cultures of astrocytes. Cells were cultured in the presence or absence of 400 mM homocysteine for 20 h. Proteins extrats were performed and expression levels analysed using Western blot. (a) PDK-2 expression levels are reported as the PDK2:GAPDH ratios. Results are expressed as mean W S.D. (n = 5), *p = 0.01. (b) A representative Western blot showing PDK-2 and GAPDH protein levels in control cells and in homocysteine-treated cells.
significant decrease in PDK-2 expression, the enzyme which controls activity of PDH. As a means to probe the effects of homocysteine on mitochondrial function we investigated the effects on mitochondrial membrane potential. Addition of glucose to control cells resulted in a reduction of the rhodamine-123 signal by 19.2% reflecting hyperpolarisation of the mitochondrial membrane. Exposure to homocysteine for 20 h resulted in an impaired glucose-induced mitochondrial hyperpolarisation (Fig. 4). 3.3. Enzyme activities in the presence of homocysteine The exposure to homocysteine did not alter the enzyme activities of ALAT and AST in the cultured astrocytes. Control values were 14.7 1.8 and 5.0 0.7 mU/mg protein for AST and ALAT, respectively. Following exposure to homocysteine the values for AST and ALAT activity were 13.2 1.1 and 5.2 1.6 mU/mg protein, respectively. The lack of effect on the activity of the amino transferases indicates a more specific action of homocytseine on
Fig. 4. Effects of homocysteine on mitochondrial membrane potential (Dcm) levels in astrocytes. Cells were cultured in the presence or absence of 400 mM homocysteine for 20 h and stimulated at the indicated time (arrow) with glucose (5.5 mM) or glucose (5.5 mM) plus homocysteine (400 mM). Values are represented as mean values (n = 4). Control conditions are represented by closed black squares and treated condition by closed grey triangles. Following the homocysteine treatment the decrease in Dcm fluorescence upon stimulation was (2.13 T 107) W (0.29 T 107), which was significantly lower than the control value (2.70 T 107) W (0.22 T 107) ( p < 0.05).
oxidative metabolism compared to that previously reported for gliotoxins (Brennan et al., 2006). 3.4. Effects of culture in the presence of homocysteine on the metabolic profiles 1
H NMR spectra of the metabolite extractions following treatment with homocysteine separated from control extractions (Fig. 5). The PLS-DA model comprised of three components and had an R2X and Q2Y of 0.57 and 0.63, respectively. Inspection of the corresponding loadings plot showed that the following NMR regions were responsible for the separation of the two groups: 3.368, 2.653, 3.383, 2.658, 2.668, 3.228, 3.268, and 3.223 ppm. These regions correspond to hypotaurine (3.368, 2.653, 3.383, 2.658, and 2.668 ppm), choline (3.228 and 2.223 ppm) and an unknown (3.268 ppm).
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Fig. 5. PLS-DA scores plot (a) and corresponding loadings plot (b) of 1H NMR spectra from control cells and cells treated with homocysteine. Cells were incubated for 20 h in the presence or absence of 400 mM homocysteine and then subsequently incubated in the presence of unlabelled glucose (5.5 mM) in the presence or absence of 400 mM homocysteine. The control experiments separated from the homocysteine-treated experiments with a model of R2 = 0.574 and Q2 = 0.627. Control conditions are represented by closed triangles and treated condition by closed squares.
4. Discussion The detrimental effects of homocysteine on neurons is well documented in the literature. However, very little has been reported on the effects of homocysteine on astrocytes and astrocytic function. Maler et al. (2003) reported that concentrations of 2 mM of homocytseine and above induced cell death in astrocytes. However, the mode of action remains unknown and relatively higher concentrations are needed to induce cell death compared to neurons where, for example, concentrations of 50– 500 mM significantly reduced the number of Purkinje neurons (Oldreive and Doherty, 2007). The present paper investigated the effects of sub-lethal concentrations of homocysteine (effective concentration 200 mM) on astrocytic glucose metabolism. Significant alterations in oxidative glucose metabolism and mitochondrial function were found. The PDH complex catalyses the oxidative decarboxylation of pyruvate leading to NADH and acetyl-CoA production. This complex is tightly regulated by a reversible phosphorylation/ dephosphorylation cycle. Inactivation is caused by phosphorylation at three serine residues on the E1a subunit and is mediated by PDK. To date there are four isoforms of PDK and the current study showed that culture with homocysteine at sub-lethal concentrations resulted in a decrease in protein expression of PDK-2. A decrease in the amount of PDK-2 will result in increased PDH activity agreeing with the increased flux through PDH calculated from the 13C NMR data. The increase in glucose consumption agrees with an increased flux through oxidative metabolism. Increased flux through PDH will result in increased oxidative metabolism which in turn should increase NADH production. Indeed there was a significant increase in NADH production following the treatment with homocysteine. To probe further the effects of homocysteine on mitochondrial function experiments were performed to investigate the effects on mitochondrial membrane potential. Following treatment with homocysteine there was impaired glucose-induced mitochondrial hyperpolarisation which indicates impaired mitochondrial ATP production. It is well established that astrocytes produce substantial amounts of mitochondrial ATP which is necessary for astrocytic functions such as glutamate uptake (Hertz et al., 2007). As a result alterations to mitochondrial function, such as that observed here following homocysteine exposure, can impair astrocytic neuroprotective function. In a recent study, Voloboueva et al. (2007) showed using an inhibitor of astrocytic
mitochondria that mitochondrial dysfunction compromised their ability to protect against excitotoxicity. In the current study using sub-lethal concentrations of homocysteine considerable alterations to mitochondrial metabolism was shown highlighting the possibility of a role for altered astrocytic function in neurodegenerative diseases associated with elevated homocysteine levels. Analysis of the metabolic profiles showed that the major change following homocysteine culture at sub-lethal concentrations was the increase in hypotaurine and a decrease in choline. Choline has been reported to be involved in the methylation of homocysteine to methionine through a betaine-dependent pathway (Bidulescu et al., 2007) and the observed decrease in choline suggests that this pathway is active in the astrocytes. The increase in hypotaurine is significant for a number of reasons. Firstly, the conversion of homocysteine to cysteine involves a trans-sulfuration pathway which until recently was considered not to be functional in the brain (Vitvitsky et al., 2006). The current study, which identifies hypotaurine as the major metabolic change following homocysteine culture, adds further evidence to the existence of a functional trans-sulfuration pathway in the brain. A previous study using addition of labelled cysteine showed that the major product of cysteine metabolism in astrocytes was hypotaurine and taurine (Brand et al., 1998). Again the current study corroborates this hypothesis. The conversion of hypotaurine to taurine in astrocytes has been reported to be slow (Brand et al., 1998) and previous studies have reported higher concentrations of hypotaurine compared to taurine in astrocytes. The cysteine produced by the trans-sulfuration pathway has another metabolic fate, namely the entry into the GSH synthesis pathway. Following homocysteine treatment there was no significant increase in total GSH levels. The preferential entry into the taurine synthesis pathway over the glutathione pathway is noteworthy considering that reciprocal levels of GSH and homocysteine have been reported in some neurodegerative diseases. In the long-term under conditions of elevated homocysteine concentrations it is possible to speculate that the increased conversion of cysteine to hypotaurine will have detrimental effects on the production of GSH. Further experiments on the effects of long-term exposure of astrocytes to homocysteine are warranted. In summary, culture with sub-lethal concentrations of homocysteine resulted in altered glucose metabolism and mitochondrial function in primary cultures of astrocytes. In addition, the presence of homocysteine increased the production of hypotaurine which may be of use as an osmolyte or an osmolyte precursor. Overall, the
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observed homocysteine-induced changes may have important implications for neuronal-astrocyte cross-talk and as a result will be important for the understanding of the pathogenesis of neurodegenerative diseases linked to raised homocysteine levels. Acknowledgments We would like to acknowledge the NMR Facility at the School of Biomolecular and Biomedical Sciences. We would like to acknowledge Dr. Brian Mion for technical help. YJ was supported by a UCD Ad Astra Scholarship.
References Allen, R.H., Stabler, S.P., Lindenbaum, J., 1998. Relevance of vitamins, homocysteine and other metabolites in neuropsychiatric disorders. Eur. J. Pediatr. 157 (Suppl. 2), S122–S126. Almeida, A., Medina, J.M., 1998. A rapid method for the isolation of metabolically active mitochondria from rat neurons and astrocytes in primary culture. Brain Res. Brain Res. Protoc. 2, 209–214. Alves, P.M., Flogel, U., Brand, A., Leibfritz, D., Carrondo, M.J., Santos, H., Sonnewald, U., 1996. Immobilization of primary astrocytes and neurons for online monitoring of biochemical processes by NMR. Dev. Neurosci. 18, 478–483. Bell, I.R., Edman, J.S., Selhub, J., Morrow, F.D., Marby, D.W., Kayne, H.L., Cole, J.O., 1992. Plasma homocysteine in vascular disease and in nonvascular dementia of depressed elderly people. Acta Psychiatr. Scand. 86, 386–390. Bidulescu, A., Chambless, L.E., Siega-Riz, A.M., Zeisel, S.H., Heiss, G., 2007. Usual choline and betaine dietary intake and incident coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) study. BMC Cardiovasc. Disord. 7, 20. Brand, A., Leibfritz, D., Hamprecht, B., Dringen, R., 1998. Metabolism of cysteine in astroglial cells: synthesis of hypotaurine and taurine. J. Neurochem. 71, 827–832. Brennan, L., Alves, P.M., Hewage, C., Malthouse, J.P., McBean, G.J., 2006. Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes. Neurochem. Int. 48, 739–745. Christie, L.A., Riedel, G., Algaidi, S.A., Whalley, L.J., Platt, B., 2005. Enhanced hippocampal long-term potentiation in rats after chronic exposure to homocysteine. Neurosci. Lett. 373, 119–124. Clarke, R., Smith, A.D., Jobst, K.A., Refsum, H., Sutton, L., Ueland, P.M., 1998. Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch. Neurol. 55, 1449–1455. Duan, W., Ladenheim, B., Cutler, R.G., Kruman, I.I., Cadet, J.L., Mattson, M.P., 2002. Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson’s disease. J. Neurochem. 80, 101–110. Escartin, C., Valette, J., Lebon, V., Bonvento, G., 2006. Neuron-astrocyte interactions in the regulation of brain energy metabolism: a focus on NMR spectroscopy. J. Neurochem. 99, 393–401. Fan, T.W.M., 1996. Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Prog. Nucl. Magn. Reson. Spectrosc. 28, 161–219. Foister, N.S., Oldreive, C.E., Mackie, J.B., Doherty, G.H., 2005. Embryonic cerebellar granule cells are resistant to necrosis induced by homocysteine. Brain Res. Dev. Brain Res. 160, 85–89. Hertz, L., Peng, L., Dienel, G.A., 2007. Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. J. Cereb. Blood Flow Metab. 27, 219–249.
1415
Ho, P.I., Ortiz, D., Rogers, E., Shea, T.B., 2002. Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J. Neurosci. Res. 70, 694–702. Jakubowski, H., 2004. Molecular basis of homocysteine toxicity in humans. Cell. Mol. Life Sci. 61, 470–487. Kamencic, H., Lyon, A., Paterson, P.G., Juurlink, B.H., 2000. Monochlorobimane fluorometric method to measure tissue glutathione. Anal. Biochem. 286, 35–37. Kruman, I.I., Culmsee, C., Chan, S.L., Kruman, Y., Guo, Z., Penix, L., Mattson, M.P., 2000. Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J. Neurosci. 20, 6920– 6926. Kruman, I.I., Kumaravel, T.S., Lohani, A., Pedersen, W.A., Cutler, R.G., Kruman, Y., Haughey, N., Lee, J., Evans, M., Mattson, M.P., 2002. Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer’s disease. J. Neurosci. 22, 1752–1762. Langmeier, M., Folbergrova, J., Haugvicova, R., Pokorny, J., Mares, P., 2003. Neuronal cell death in hippocampus induced by homocysteic acid in immature rats. Epilepsia 44, 299–304. Lapidot, A., Gopher, A., 1994. Cerebral metabolic compartmentation. Estimation of glucose flux via pyruvate carboxylase/pyruvate dehydrogenase by 13C NMR isotopomer analysis of D-[U-13C]glucose metabolites. J. Biol. Chem. 269, 27198–27208. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Maler, J.M., Seifert, W., Huther, G., Wiltfang, J., Ruther, E., Kornhuber, J., Bleich, S., 2003. Homocysteine induces cell death of rat astrocytes in vitro. Neurosci. Lett. 347, 85–88. Malloy, C.R., Sherry, A.D., Jeffrey, F.M., 1990. Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers. Am. J. Physiol. 259, H987–H995. Miller, J.W., 2000. Homocysteine, Alzheimer’s disease, and cognitive function. Nutrition 16, 675–677. Nakai, N., Obayashi, M., Nagasaki, M., Sato, Y., Fujitsuka, N., Yoshimura, A., Miyazaki, Y., Sugiyama, S., Shimomura, Y., 2000. The abundance of mRNAs for pyruvate dehydrogenase kinase isoenzymes in brain regions of young and aged rats. Life Sci. 68, 497–503. Oldreive, C.E., Doherty, G.H., 2007. Neurotoxic effects of homocysteine on cerebellar Purkinje neurons in vitro. Neurosci. Lett. 413, 52–57. Ravaglia, G., Forti, P., Maioli, F., Martelli, M., Servadei, L., Brunetti, N., Porcellini, E., Licastro, F., 2005. Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am. J. Clin. Nutr. 82, 636–643. Richter-Landsberg, C., Besser, A., 1994. Effects of organotins on rat brain astrocytes in culture. J. Neurochem. 63, 2202–2209. Seifert, G., Schilling, K., Steinhauser, C., 2006. Astrocyte dysfunction in neurological disorders: a molecular perspective. Nat. Rev. Neurosci. 7, 194–206. Verkhratsky, A., Toescu, E.C., 2006. Neuronal-glial networks as substrate for CNS integration. J. Cell. Mol. Med. 10, 826–836. Vitvitsky, V., Thomas, M., Ghorpade, A., Gendelman, H.E., Banerjee, R., 2006. A functional transsulfuration pathway in the brain links to glutathione homeostasis. J. Biol. Chem. 281, 35785–35793. Voloboueva, L.A., Suh, S.W., Swanson, R.A., Giffard, R.G., 2007. Inhibition of mitochondrial function in astrocytes: implications for neuroprotection. J. Neurochem. 102, 1383–1394. Wroblewski, F., Ladue, J.S., 1955. Lactic dehydrogenase activity in blood. Proc. Soc. Exp. Biol. Med. 90, 210–213. Zieminska, E., Stafiej, A., Lazarewicz, J.W., 2003. Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones. Neurochem. Int. 43, 481–492.