Fluorocitrate and fluoroacetate effects on astrocyte metabolism in vitro

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BRAIN RESEARCH

ELSEVIER

Brain Research 664 (1994) 94 100

Research report

Fluorocitrate and fluoroacetate effects on astrocyte metabolism in vitro R a y m o n d A. Swanson a,., Steven H. G r a h a m ~ Department of Neurology, University of California and Veterans Affairs Medical Center, San Francisco, CA, USA Accepted 30 August 1994

Abstract

The Krebs cycle inhibitor fluorocitrate (FC) and its precursor fluoroacetate (FA) are taken up in brain preferentially by glia. These compounds are used experimentally to inhibit glial metabolism in situ. The actions of these agents have been attributed to both the disruption of carbon flux through the Krebs cycle and to impairment of ATP production. We used primary astrocytc cultures to evaluate these two possible modes of action. Astrocyte ATP levels exhibited little or no reduction during incubation with 0.5 mM FC or 25 mM FA. Correspondingly, FC and FA caused less than 30% reductions in glutamate uptake (P > 0.05), an important energy-dependent astrocyte function. Carbon flux through the Krebs cycle was assessed by measuring astrocyte glutamine production in the absence of exogenous glutamate or aspartate. Under these conditions, glutamine production was reduced 65 _+5% by 0.5 mM FC and 61 + 3% by 25 mM FA (P < 0.01). In contrast, FC and FA had no effect on glutamine production when 50 #M glutamate was provided in the media. These findings suggest that the metabolic effects of FC and FA on astrocytes in vivo result from impairment of carbon flux through the Krebs cycle, and not from impairment of oxidative ATP production.

Keywords: Astrocyte; ATP; Azide; Fluoroacetate; Fluorocitrate; Glia; Glutamine; Glutamate uptake

I. Introduction

Fonnum and colleagues in 1987 introduced the the use of fluorocitrate (FC) as a means of selectively inhibiting glial metabolism in situ [29]. FC and its precursor fluroacetate (FA) have since become widely used to study the functional roles of glia in brain [4,11,19,30-32,34,39,40,45-47,49]. The relative selectivity of these agents for glia stems from the more rapid uptake of acetate and citrate by glia than by neurons [11,27,38]. FC is a suicide inhibitor of the Krebs cycle enzyme aconitase (aconitate hydratase, EC 4.2.1.3) [7,33], and may also inhibit mitochondrial transport of citrate [12]. FA is thought to act only after metabolic conversion to FC [3,7]. Despite the wide use of FC and FA, it remains unsettled whether the effects of these compounds result from impairment of glial energy metabolism or from reduced carbon flux through the Krebs cycle. FC and FA reduce glutamine synthesis and content in

* Corresponding author. Neurology (127), V.A.M.C., 4150 Clement St., San Francisco, CA 94121, USA. Fax: (1) (415) 751-1343. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0006-8993(94)01 057-9

brain [3,4,6,8,11,28,29,32,49] and reduce the K+-stimu lated release of glutamine, glutamate, and G A B A [30,31,45]. Since glutamine synthesis requires ATP, and oxidative A T P production is fueled primarily by reducing equivalents generated in Krebs cycle reactions [22], it has frequently been suggested that the metabolic effects of FC and FA might result from glial energy failure [4,8,11,29,34,39,45-47]. However, this concept may warrant reconsideration in view of several studies suggesting that astrocytes, unlike neurons, can largely maintain A T P levels and function by glycolytic metabolism alone [1,9,18,23,41-43]. In addition to its role in energy metabolism, the astrocyte Krebs cycle is an integral part of the brain glutamine-glutamate cycle. Krebs cycle intermediates serve as precursors for astrocyte glutamine synthesis, and glutamine released by astrocytes is taken up by neurons and used to form neurotransmitter pools of glutamate and G A B A [15,38]. Some Krebs cycle intermediates may in addition be directly released by astrocytes and taken up by neurons [36,37]. The intermediates that are released or used to form glutamine can be replenished from glucose through the action of pyruvate carboxylase, which is present in astrocytes but not

R.A. Swanson, S.H. Graham/Brain Research 664 (1994) 94-100

neurons [15,20,35]. Consequently, a potential result of astrocyte Krebs cycle blockade, independent of effects on energy metabolism, is a reduction in astrocyte export of glutamine and Krebs cycle intermediates, with a resultant depletion of neurotransmitter glutamate and GABA. In the present study we have employed primary astrocyte cultures to directly assess FC and FA effects on both energy metabolism and glutamine production. The advantage of using astrocyte cultures for these studies is that the potentially confounding effects (direct or indirect) of FC and FA on neuronal metabolism are eliminated. The absence of neurons also ensures that changes in astrocyte metabolites are not masked by potentially larger neuronal pools. These studies suggest that the metabolic effects of FC and FA on astrocytes in vivo result from impairment of carbon flux through the Krebs cycle, and not from impairment of oxidative ATP production. The findings comport with previous studies suggesting a high capacity of astrocytes for non-oxidative energy metabolism and a predominately synthetic role for the astrocyte Krebs cycle.

2. Materials and methods Reagents were obtained from Sigma, St. Louis MO, except where otherwise noted.

2.1. Astrocyte cultures Primary cortical astrocyte cultures were prepared as described by Hertz [13], with minor modifications [41]. O n e day old S p r a g u e Dawley rats (Simonsen, Gilroy, CA) were anesthetized with isoflurane and decapitated. The cortices were dissected free of meninges, minced, and placed in Ca2+/Mg2+-free Hank's balanced salt solution containing 20 U / m l papain and 0.5 m g / m l D N A a s e I. After 30 min incubation, the cells were centrifuged and re-suspended in Eagle's minimal essential medium (MEM) containing 10% fetal bovine serum (Hyclone; Ogden, UT) and 2 m M glutamine. Cell dissociation was completed by trituration through a fire-polished glass pipette. The cells were plated into Falcon 24-well tissue culture plates (Becton-Dickenson, Oxnard, CA) at a density of 0.15 brains per 24-well plate (approximately 1 × 104 cells per each 2 cm 2 well) and incubated at 37°C in a humidified 5% CO 2 atmosphere. The cultures reached confluency at 12-15 days in vitro (DIV), at which time 2 0 / ~ M cytosine arabinoside was added to arrest the proliferation of other cell types. This medium was exchanged after 48 h with fresh medium containing 2.5% fetal bovine serum and 2 m M glutamine plus 0.15 m M dibutyryl cyclic A M P to induce differentiation [12]. Cells prepared in this m a n n e r displayed stellate morphology and were uniformly positive for glial fibrillary acidic protein (ICN, Costa Mesa, CA) and negative for neurofilament protein (Boehringer-Mannheim, Indianapolis, IN). Each study was repeated on cells from at least three different batches of astrocyte cultures at 25-34 DIV.

2.2. Metabolic inhibitors All reagents were prepared as 1 0 x or 5 × s t o c k solutions that were buffered to p H 7.2 with 5 m M N a H C O 3 and adjusted to 300

95

m O s m with NaCI. Fluorocitrate was obtained as its barium salt, and most experiments were conducted after NaSO 4 precipitation of barium as described by Paulsen et al. [29], with NaSO 4 added in slight excess of the total barium concentration. For some experiments the barium precipitation step was omitted. Barium citrate (prepared as barium chloride plus sodium citrate) was carried through the same barium precipitation procedure to produce a control for the fluorocitrate vehicle. The effects of FC and FA were compared in each study to the effects of chemical hypoxia induced by 5 m M azide and to glycolyic blockade induced by 1 m M iodoacetate in glucose-free media. Glucose deprivation alone is not sufficient to block glycolysis in astrocytes because of the substantial glycogen stores in these cells [44]. The highest FC and F A concentrations employed were at least as high as previously used in either brain slices [3,4,6,8,30,34,40,45,46] or brain in situ [10,11,19,28,29,31,32].

2.3. A TP measurements Astrocyte cultures were washed and incubated with the metabolic inhibitors for 180 min in Hank's balanced salt solution (HBSS) at 37°C. The incubations were terminated by lysing the cells in ice-cold 0.1 M N a O H containing 0.01% lauryl sulfate and 1 m M ETDA. Aliquots were removed for protein determination by the Lowry method [25], using ovalbumin standards. The reminder of the samples were treated with perchloric acid and centrifugation to remove proteins. The supernatants were neutralized with K O H / K H z P O 4 and again centrifuged. A T P in the resulting supernatants was determined fluorometrically with the method of Lowry and Passoneau [24]. A T P standards were carried through the protein precipitation steps in parallel with the samples for each assay.

2.4. Glutamate uptake Astrocyte cultures were washed and incubated with the metabolic inhibitors for 30-180 min in HBSS at 37°C prior to glutamate uptake determinations. Uptake was measured by the method of Balcar et al. [1], with minor modifications [41]. Each culture well received 0.025 p.Ci/ml L-[2,3-3H]glutamate (American Radiochemicals; St. Louis, MO) plus 5 0 / z M unlabeled glutamate. Uptake was terminated after 7 min incubation at 37°C by 2 washes in ice-cold Hank's balanced salt solution, followed immediately by cell lysis in 0.5 N N a O H / 0 . 0 5 % lauryl sulfate. Aliquots were taken for scintillation counting and for protein determinations. Previous studies have shown uptake to be linear with time through at least 10 min. Correction for glutamate binding was made by performing the uptake assay with osmotically lysed cells. Glutamate binding was typically less than 0.2% of glutamate uptake in control wells. To facilitate comparisons between different batches of astrocytes, uptake in each well was normalized to uptake rates measured in sister wells.

2.5. Glutamine production Glutamine production was assessed as described by Brookes [5]. Two different media were used: Earle's balanced salt solution (EBSS) containing 5 m M glucose and 50 ~ M NHaCI to assess glutamine production from Krebs cycle intermediates, and media that additionally contained 50 /zM glutamate. The metabolic inhibitors were added to aliquots of these media and the aliquots were equillibrated with the 5% CO 2 atmosphere. Cultures were washed twice and pre-incubated in these media for 60 min at 37°C in the 5% CO 2 atmosphere to allow effiux of endogenous intracellular glutamine. Cultures were then placed in 200 p.l of fresh media containing the metabolic inhibitors and incubated for an additional 30 to 180 min. Media aliquots were removed and stored at - 2 0 ° C , and the cells were dissoved in 0.5 N N a O H / 0 . 0 5 % lauryl sulfate for protein determinations. Media amino acid concentrations were determined

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by reverse-phase HPLC of the ortho-phthaldialdehyde deriwttives using electrochemical detection as previously described [43]. Peak areas were determined using Nelson Analytical Software by an observer who was blind to the experimental conditions.

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For all assays, values from each culture well were normalized to the mean of control wells from the same 24-well plate. Significant differences were determined by A N O V A with the Dunnett correction for multiple comparisons.

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The use of FC as its barium salt and FC after barium precipitation gave equivalent results in all of the studies performed, as similarly noted by Stone et al. [40]. For clarity, only results obtained using fluorocitrate after barium precipitation are presented. Studies performed with citrate also produced similar results regardless of whether they were conducted with sodium citrate, barium citrate, or barium citrate after barium precipitation. The mean A T P content of control cultures was 19.3 _+ 3.6 n m o l / m g protein (n = 12). A T P levels were not affected by incubations with FC at concentrations of up to 0.5 mM (Fig. 1) FA at 5 mM also had no significant effect, although 25 mM fluoroacetate produced a 29 + 4% reduction in A T P ( P < 0.05). A modest A T P reduction was also observed after exposure to 5 mM azide (Fig. 1). Azide at 25 mM caused no further effect, consistent with previous observations that azide has a maximal effect at concentrations below 5 mM [41]. In contrast to these modest effects, ATP was reduced by more than 85% after 3 h incubation with the glycolytic inhibitor iodoacetate in glucose free medium (Fig. 1). Citrate and acetate had no significant effect on A T P levels. The effect of the metabolic inhibitors on glutamate

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Fig. 2. Fluorocitrate, fluoroacetate, and azide caused modest reductions in glutamate uptake that did not acheive statistical significance after corrections for multiple comparisons, while iodoacetate (in glucose-free media) caused complete cessation of glutamate uptake by 3 h of incubation, n = 6 9, Abbreviations as in Fig. I

uptake was similar to their effect on ATP levels. Glutamate uptake under control conditions was 9.4_+ 2.4 n m o l / m i n / m g protein (n = 30). FC, FA, and azide each reduced G L U uptake rates by 15-35% after incubation periods of 60 to 180 min (Fig. 2), although these reductions fell short of statistical significance after correction for multiple comparisons. Glycolytic inhibition with iodoacetate (in glucose-free media) had more pronounced effects on glutamate uptake, with reduction to 20 _+ 3% of controls after 1 h and to less than 5% of controls at 3 h. Citrate and acetate did not significantly affect glutamate uptake. Media glutamine increased at a constant rate through at least 3 h in both of the media preparations used for these studies (Fig. 3). Glutamine accumulated at a rate of 2.49 n m o l / m i n / m g protein in medium containing 50 /xM glutamate and 0.95 n m o l / m i n / m g protein in medium without added glutamate. As previously noted by Yu et al. [52], these rates of glutamine production were not significantly different in media with or without 5 0 / z M NH4C1 (results not shown). FC and FA markedly reduced astrocyte glutamine produc-

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Fig. l. Astrocyte A T P levels were reduced by 5 - 3 0 % by 3 h incubation with the Krebs cycle inhibitors FC and FA. Comparable effects were obtained with azide, while the glycolytic inhibitor iodoacetate (in glucose free media) reduced A T P by 88%. CNTRL, control; FC, fluorocitrate; FA, fluoroacetate; AZD, azide; 1A, iodoacetate, n = 6. * P < 0.05, * * P < 0.01.

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Fig. 3. Media glutamine concentrations increased linearly with time (r > 0.99) through 180 min of incubation both in medium without added glutamate and in medium with 50 ~xM added glutamate. Linear regression lines are drawn through the data points.

R.A. Swanson, S.H. Graham/Brain Research 664 (1994) 94-100

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Fig. 4. FC and FA reduced astrocyte glutamine production in media without added glutamate (A), but not in media containing 50 # M added glutamate (B). Acetate and citrate had no significant effects under either condition. FC, flurocitrate; CITR, citrate; FA, fluoroacetate; ACET, acetate; AZD, azide. (n > 9, except azide n = 6) ** P < 0.01

tion (Fig. 4A), unlike the modest effects of these agents on ATP and glutamate uptake. In medium without glutamate, glutamine accumulation was reduced 65.5 _+ 5% by 0.5 mM FC and 61 _+ 3% by 25 mM FA. Citrate, acetate, and azide had no significant effects. The inhibitory effects of FC and FA were negated by the addition of 50 /~M glutamate to the media as an immediate precursor for glutamine synthesis (Fig. 4B).

4. Discussion

This report provides the first assessment of FC and FA effects on astrocytes in primary culture. Use of the culture system ensures that the observed metabolic effects do not reflect changes in neuronal metabolism that could potentially result from either direct actions of these agents on neurons or from secondary effects of their actions on glia. The neuron-free system also prevents masking of changes in astrocyte metabolite concentrations by potentially larger neuronal pools. Our results suggest that the functional effects of FC and FA on astrocyte metabolism result from inhibition of carbon flux through the Krebs cycle, and not from impairment of oxidative energy metabolism. This finding is consistent with previous observations indicating that

ods without oxidative ATP production [1,9,18,23,4143]. Studies in brain and brain slices have repeatedly shown that FC and FA do not alter ATP content [3,4,8,10,28]. However, as proposed by Clarke et al. [8], these observations do not exclude the possibility that ATP levels might be reduced in a small (glial) compartment. Although the glial ATP pool is probably not small relative to the neuronal pool [14], the idea that FC and FA might cause ATP depletion selectively in astrocytes is frequently considered [4,8,11,29,34,39,40, 45-47]. This notion is not borne out by the present studies, as FC and FA did not cause significant reductions in astrocyte ATP except at 25 mM FA, a concentration that is far in excess of levels attained by administration of this agent in situ. Moreover, ATP production should be reduced at least as much by azide blockade of cytochrome c as by FC inhibition of the Krebs cycle. This is because Krebs cycle inhibition would not prevent oxidative production of ATP from glycolytically produced NADH. The observed minimal reduction in ATP content in either condition presumably reflects the high capacity for non-oxidative ATP production in these cells [14,23,42], an interpretation that is further supported by the marked reduction in ATP resulting from glycolytic blockade by iodoacetate. These results also suggest that FC does not produce glycolytic blockade, which could potentially result from FC-induced citrate accumulation [6,7,10,22,28]. The failure of FC and FA to cause large ATP reductions does not necessarily mean that ATP production is unaffected, because ATP levels could be maintained by a compensatory reduction or cessation of normal ATP-consuming functions. Similarly, ATP levels under 'resting' conditions may not reflect the ability of these cells to meet a metabolic challenge. For these reasons, glutamate uptake, which is an important ATP-dependent astrocyte function [2,50], was assessed as a measure of astrocyte functional capacity in these studies. A secondary rationale for examining glutamate uptake in this system was that decreased glutamate uptake has been considered as a possible mechanism for some of the observed effects of F A and FC in vivo [30,31,45,46]. In keeping with their effects on ATP levels, FC and FA caused negligible reductions in glutamate uptake. These small reductions are probably not physiologically significant given the very high capacity of glutamate uptake in brain [2,43]. These resuits are also consistent with the finding by Paulsen et al. that D-aspartate uptake into striatal slices is not impaired by FC [30]. Glutamate uptake occurs primarily by Na+-dependent transport [2], and loss of the transmembrane Na + gradient causes marked reduction in glutamate uptake [48]. Preservation of glutamate uptake in the presence of FC and FA suggests that

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R.A. Swanson, S.H. Graham/Brain Research 664 (1994) 94-100

other astrocyte processes coupled to the transmembrane Na + gradient, such as K + and H + uptake [50], are probably also maintained during FC and FA exposure. FC and FA have consistently been shown to reduce total glutamine content of brain [3,4,6,8,11,28,29,32,49]. Astrocytes, but not neurons, contain glutamine synthetase [26] as well as the anaplerotic enzyme pyruvate carboxylase, that allows replenishment of the Krebs cycle intermediates consumed during de novo synthesis of glutamine and other compounds [20,35]. Since this synthetic pathway requires carbon flux through the Krebs cycle, FC and F A inhibition of the Krebs cycle could reduce glutamine synthesis. The present findings confirm both that FC and FA can significantly reduce astrocyte glutamine production, and that this reduction can occur in the absence of marked ATP depletion. Media glutamine can reasonably be taken to approximate net glutamine production because previous studies with astrocyte cultures have shown intracellular accumulation to be negligible relative to glutamine release over this time period [5]. The observed rate of glutamine release in the presence of media glutamate is lower than the value of 5.8 n m o l / m i n / m g protein reported by Brookes [5], but close to the values of 1.7-2.4 n m o l / m i n / m g protein reported by Yu et al. [51,52], and close to the rate of flux from glutamate to glutamine (2.0-2.2 n m o l / r a i n / mg protein) estimated by Huang et al. [17]. An important aspect of this study is that FC and FA failed to reduce glutamine production when glutamate was added to the media as an immediate precursor for glutamine production. This indicates that FC and FA do not act directly on glutamine synthetase or glutamine transport [8]. These results also support the conclusion that FC and FA do not act by limiting astrocyte ATP production. The dissociation between oxidative ATP production and astrocyte glutamine synthesis is further confirmed by the failure of azide to reduce glutamine production. Extrapolation of these in vitro findings to brain in situ requires caution. It is possible that astrocytes in brain respond differently to FC or FA than do astrocytes in culture. Moreover, astrocytes in vivo may be subject to much higher energy demands, such that modest reductions in A T P synthetic capacity could have greater functional effects. Additionally, other effects of these compounds, such as increased citrate levels [7,28,6,10] and resultant chelation of Ca 2+ or Mg 2+ [16], could have systemic or local actions in brain that might not be apparent in culture. It should also be noted that some of the effects observed after FC or FA administration in situ may reflect actions on neuronal as well as glial metabolism. Indeed Paulsen et al., in their original description of stereotaxic administration of FC as a selective glial toxin [29], stressed that a

selective effect on glial morphology is attained only over a very narrow dose range. The cardinal findings of this study are consistent with those of previous studies perlbrmed in situ: FC and FA reduce glutamine synthesis without reducing ATP. Together, these findings suggest that the metabolic effects of FC and FA in brain result from reduced carbon flux through the astrocyte Krebs cycle and not from reduced ATP production. Based on the metabolism of acetate and other Krebs cycle substrates, Clarke and colleagues [8] proposed that the Krebs cycle in brain is compartmentalized into a large 'energetic' cycle and a distinct, smaller, ~synthetic' cycle. The present in vitro study confirms that the Krebs cycle has an important synthetic function in astrocytes and supports the idea that astrocytes are the site of the the smaller, 'synthetic' Krebs cycle compartment in brain.

Acknowledgements

We thank Florence Cheng for expert technical assistance. This work was supported by Merit Review (S.H.G., R.A.S.) and Career Development (R.A.S.) awards from the US Department of Veterans Affairs.

References

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