Journal of Neurochemistry Raven Press, Ltd., New York 0 1991 International Society for Neurochernistry
Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:60-3) and Arachidonic Acid (20:40-6) *Steven A. Moore, *Elizabeth Yoder, ?Sean Murphy, ?Gary R. Dutton, and $Arthur A. Spector Departments of *Pathology, tPharmacology, and $Biochemistry, University of Iowa, Iowa City, Iowa, U.S.A.
Abstract: Elongated, highly polyunsaturated derivatives of linoleic acid ( I 8:2w-6) and linolenic acid ( I 8:30-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [ 1-'4C]18:2w-6, [ 1-14C]20:4~-6,[ 1-14C]18:3w-3, or [1-14C]20: 5w-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of A4desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2w-6 was 20:4w-6, whereas the primary products
from 1k30-3 were 20:5w-3, 22:5w-3, and 22:6w-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4w-6 and 22:6w-3, was released into the extracellular fluid. Although incapable of producing 20:4w6 and 22:6w-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating w-6 and w3 essential fatty acid precursors to 20:40-6 and 22:6w-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons. Key Words: Astrocyte-Neuron-Arachidonic acid-Docosahexaenoic acid-Fatty acid elongation-Fatty acid desaturation. Moore S. A. et al. Astrocytes, not neurons, produce docosahexaenoic acid (22:6w-3) and arachidonic acid (20:40-6). J. Neurochem. 56, 518-524 (1991).
The brain is more highly enriched than most other tissues in long-chain polyunsaturated fatty acids, particularly docosahexaenoic acid (22:6w-3; Salem et al., 1986; Neuringer et al., 1988). 22:6w-3 is a major component of excitable membranes and may exert considerable influence on membrane fluidity and the functional properties of integral proteins (Stubbs and Smith, 1984; Salem et al., 1986), possibly by promoting optimal acyl chain packing or interactions with transmembrane domains (Applegate and Glomset, 1986). Arachidonic acid (20:4w-6) is the substrate for the production of many biologically active compounds, including prostaglandins, hydroxyeicosatetraenoic acids, leukotrienes, and lipoxins (Wolfe, 1982; Samuelsson et al., 1987). Similarly, eicosapentaenoic acid (20:5w3) and 22:6w-3 are substrates for the production of prostaglandins, hydroxy acids, and leukotrienes (Needleman et al., 1979; Salem et al., 1986; Sprecher, 1986). Although several studies have attempted to explain
this enrichment (Salem et al., 1986; Neunnger et al., 1988), many questions remain about the source of essential fatty acids in the brain and the roles of various cell types in the uptake and retention of these iongchain polyunsaturated fatty acids. Studies of essential fatty acid elongation and desaturation by the brain (Dhopeshwarkar et al., 1971b; Yavin and Menkes, 1974; Dhopeshwarkar and Subramanian, 1976; Cohen and Bersohn, 1978; Clandinin et al., 1985; Anderson and Connor, 1988) have failed to clarify which cell types within this tissue are capable of producing 22: 6w-3. Moreover, recent findings indicating that 22:6w3 is produced in the liver and then circulated to the CNS have led to the view that the brain may not be an important site of 22:6w-3 formation (Scott and Bazan, 1989). We previously have demonstrated that cells of the blood-brain barrier, the cerebromicrovascular endothelium, can elongate and desaturate essential fatty acid
Received May I I , 1990: revised manuscript received July 26. 1990; accepted July 26, 1990. Address correspondence and reprint requests to Dr. S. A. Moore at The University of Iowa, Department of Pathology, Room I19 Medical Laboratories, Iowa City, IA 52242, U.S.A.
Abbreviafions used: FBS, fetal bovine serum; GC-MS, gas chromatography-mass spectrometry; MEM, minimum essential medium.
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ASTROCYTES PRODUCE 20:40-6 AND 22:6~-3 precursors of both the w-3 and w-6 families (Moore et al., 1990). However, these cells are not able to perform the final desaturation step, mediated by A4-desaturase, to produce 22:6w-3 or 22:5w-6 (Moore et al., 1990). This suggested that brain A4-desaturase activity is located in another cell type. To determine whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon glial cells, we have studied the processing of w-3 and w-6 fatty acid precursors in primary cultures of neurons and astrocytes obtained from neonatal rat cerebrum and cerebellum.
MATERIALS AND METHODS Materials Methyl ester standards were purchased from NuChek Prep (Elysian, MN, U.S.A.), and [ I-'4C]linoleic acid ([ I-I4C]18: 2w-6), [ 1-'4C]20:4w-6, [ I-'4C]linolenic acid ([ 1-I4C]18:3w-3), and [ l-'4C]20:50-3 (approximately 55 mCi/mmol) were purchased from New England Nuclear (DuPont, Boston, MA, U.S.A.) and Amersham (Arlington Heights, IL, U.S.A.).
Cell culture Primary cultures of astrocytes and neurons were prepared from 2-7-day-old rat pups by a trypsinization/trituration technique which disaggregates brain perikarya [for methodology details, see Murphy (1990) and Dutton (199O)l. Cerebral cultures were obtained from forebrain gray matter after dissecting away white matter and leptomeninges. Cerebellar cultures were obtained from whole cerebellum. Highly enriched astrocyte and neuronal cultures were obtained from the same starting cell suspension in each anatomical site. Astrocyte cultures were seeded into poly-D-lysine-coated multiwell plates at a density of lo4 cells/cm2and maintained in Eagle's minimum essential medium (MEM) with Earle's salts supplemented with 33 mM glucose, 2 mM glutamine, 180 pMgentamicin, and 10%fetal bovine serum (FBS). Every 3 days in culture, half the medium was replaced until the cultures were confluent (approximately 14 days). Neuronal cultures were seeded into poly-D-lysine-coated multiwell plates at a density of 1.5 X lo6 cells/cm2 and maintained in Eagle's MEM with Earle's salts supplemented with 33 mM glucose, 1 M g l u t a m i n e , I80 pMgentamicin, 20 m M KCI, 80 pM 5-fluoro-2'-deoxyuridine, 2.5% chick embryo extract, and 10%FBS. Neuronal cultures were used 10-12 days after plating without a medium change. Astrocyte cultures were routinely 295% type I astrocytes by immunohistochemical characterization. Immunohistochemical characterization of neuronal cultures revealed those from cerebrum to contain 60-709'0 neurons, with the majority of contaminating cells being type I astrocytes, and those from cerebellum to contain approximately 90% glutamatergic granule neurons and 57% GABAergic inhibitory interneurons, with the majority of contaminating cells being type I astrocytes.
Experimental protocol The uptake, elongation, and desaturation of essential fatty acids were studied in 10-14-day cultures of astrocytes or neurons grown in multiwell tissue culture plates. Incubations were routinely carried out in culture media containing 110% FBS and 10-20 pM I4C-labeled fatty acids. Cells were maintained at 37°C in a 5% C 0 2 incubator during all of the incubations.
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Lipid extractions Lipids were extracted from the incubation medium using a modification of the procedure of Folch et al. (1957). The medium was centrifuged to remove cellular debris, then extracted with a 2: 1 (vol/vol) mixture of chloroform/methanol containing I % acetic acid. The chloroform phase was dried under nitrogen and resuspended in acetonitrile. To isolate cell lipids, the incubation medium was removed and the cellswere washed with serum-free medium containing 10 pM fatty acid-free bovine serum albumin, scraped into methanol, and transferred to a siliconized screw-top glass test tube (Dudley and Spector, 1986).One volume of chloroform was added, and after mixing with a Vortex and adding one volume of 0.88% KCI, each sample was centrifuged at 1,300 g for 10 min to separate the phases. After the aqueous phase was washed once with one volume of chloroform, the two chloroform layers were combined, dried under nitrogen, and resuspended in chloroform/methanol (2: I , vol/vol).
Methyl ester derivatization Aliquots of total cell lipid or medium lipid extracts were combined with an excess of 14%BF3 in methanol and heated to 100°C for 15 min to produce fatty acid methyl esters (Momson and Smith, 1964). Following the addition of water, the methyl esters were extracted in heptane, dried under nitrogen, and resuspended in acetonitrile.
HPLC Radioactive methyl esters prepared from the cell lipids or incubation media were separated by reverse-phase HPLC by a modification of the method of Aveldano et al. (1983). Briefly, a 4.6 X 150-mm C18 reverse-phase Beckman HPLC column with 5-pm spherical packing was used with a mobile phase of water and acetonitrile in a two-step isocratic elution. The more polar fatty acid methyl esters were eluted with 76% acetonitrile, whereas the less polar esters were eluted with 100% acetonitrile. Radioactivity was monitored by mixing the column effluent with scintillator solution at a 1:3 ratio and passing the mixture through an on-line Radiomatic Instruments Flo I@ radioactivity detector (Radioanalytic, Tampa, FL, U.S.A.). The system was standardized with methyl esters of the following I4C-labeledfatty acids: 18:2w6, 18:3~-3,20:4~-6,20:5~-3,and 22:6w-3.
GLC Fatty acid methyl esters also were separated by GLC on a 2 mm X 1.9 m glass column packed with 10% SP2330 on IOO/ 120-mesh Chromosorb W (Supelco, Bellafontane, PA, U.S.A.). A Hewlett-Packard model 5890A gas chromatograph with flame ionization detector was used. The column temperature was raised from 178 to 230°C over 45 min and nitrogen served as the carrier gas at a flow rate of 25 ml/min. Peak areas were determined with a Hewlett-Packard model 3380A integrator-recorder. The methyl ester derivatives of the unknown essential fatty acid metabolites were analyzed with a VG TRIO 1 mass spectrometer containing a 2 mm X 15 m OV-1 column [as described previously, see Moore et al. (1990)J The column temperature was raised from 50 to 250°C at a rate of 20"/min. The energy of the electron beam was 70 eV.
RESULTS Elongation and desaturation of 18:3w-3 During a 24-h incubation, the primary product of I8:3w-3 elongation and desaturation in cerebral astroJ. Neurochem.. Vol. 56. No. 2.1991
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Time (min) FIG. 1. Elongation and desaturationof 18:3w3by primary cultures
of cerebral astrocytes and neurons. Twelve days after plating, primary cultures of cerebral astrocytes (A) or neurons (B) were incubated for 24 h with 18 f l [l -'4C]18:3w-3. Total cell lipids were transmethylated and the resulting fatty acid methyl esters separated by reverse-phase HPLC. The amount of acetonitrile in the mobile phase (acetonitrile/water)was held at 76% during the first 50 min. then raised to 100% over 2 min.
cyte cultures was 22:6w-3 (Fig. 1A). Other major products were 20:5w-3 and docosapentaenoic acid (22:5w-3), the immediate precursors of 22:60-3. Minor metabolites detected by HPLC were 20:3w-3 and 20: 4w-3. The identity of each of these metabolites was confirmed by GLC and/or gas chromatography-mass spectrometry (GC-MS) as previously described (Moore et al., 1990). The isolated 0-3 fatty acid methyl esters of the putative 20:3w-3, 20:4w-3, and 22:5w-3 eluted from the GLC with retention times corresponding to their appropriate standards. Further confirmation of structure was obtained by electron impact GC-MS for the fatty acid methyl ester of 2250-3. A molecular ion was observed at mle 344. Additional ions at m/e 5 5 , 95,135,175, and 2 15 confirmed the w-3 configuration of the double bonds. Cerebral neuronal cultures, on the other hand, produced very little 22:6w-3 (Fig. 1B). The main metabolites of 18:3w-3 in these cultures were 20:3w-3, an elongation product, and 22:5w-3. In mixed cultures containing various ratios of cerebral neurons and astrocytes incubated with [ 1-I4C]18:3w-3, both the relative and absolute amounts of 22:6w-3 formed increased (from 5.8 to 59%, and from 0.96 to 10.43 nmol/well, respectively) as the number of astrocytes in these cultures increased from 37 to 94% (Table l ) . The elongation and desaturation of [ 1-14C]18:3w-3 and [ 1-l4C]20:5w-3also were compared in highly enriched neuronal granule cell and astrocyte cultures from the cerebellum. Like cerebral astrocyte cultures, astrocyte cultures from the cerebellum readily produced 22: 60-3 (Fig. 2A). However, the granule cell neurons were incapable of 2 2 1 6 ~ - production, 3 even after 72 h of
J . Neurochem., Val. 56, No. 2, 1991
incubation with the w-3 fatty acid precursors (Fig. 2B and C). These neuronal cultures have an active elongation system, however, and produced large amounts of 20:3w-3 from 18:3w-3 (Fig. 2B) and 22:5w-3 from 20:5w-3 (Fig. 2C). Small amounts of radioactivity were detected on the HPLC radiochromatograms in peaks eluting between 50 and 60 min (Figs. 1 and 2). These compounds were identified by GLC as 16:O and 18:1 in peak "a" and 18:O and 20: 1 in peak "b". This suggests that some of the 18:3w-3 and 20:5w-3 underwent &oxidation, with reutilization of the released [ I4C]acetate. Figure 2D demonstrates that during continuous incubations with [ 1-I4C]18:30-3, large amounts of radioactive 22:6w-3 were released into the culture medium by astrocytes. A much larger fraction of the 22:6w-3 formed was released into the medium than was retained in the cells (Fig. 3A). Similar production and release of radioactive 22:6w-3 were observed during continuous incubations of astrocytes with [ 1-14C]20:5w-3(data not shown). No desaturation products of either [lI4C]18:30-3 or [ 1-'4C]20:5w-3were observed in the medium of neuronal cultures (data not shown). Elongation and desaturation of 18:2w-6 The ability of brain cells to elongate and desaturate essential fatty acids was further examined in highly enriched neuronal granule cell and astrocyte cultures from the cerebellum incubated with [ 1-I4C]18:2w-6 or [ 1''C]20:4w-6. Astrocyte cultures from the cerebellum readily produced 20:4w-6 (Fig. 4A). Other products TABLE 1. Conversion of 18.3~-3to 2 2 : 6 ~ - 3by mixed glial-neuronal cultures 22:6w-3 production' Astrocyte content of cultures ( % ) b
(% of total label)d
(nmol/well)'
31 k 2.2 48 f 5.4 57 rt 4.5 94 f 3.2
5.8 8.6 25.0 59.0
0.96 1.48 4.45 10.43
a Fluorodeoxyuridine was added to dissociated cerebral cells at various times after plating to establish cultures with various mixtures of neurons and astrocytes. At I2 days after plating, all cultures were incubated for 48 h with I8 pA4 [ 1-I4C]18:3w-3. Following extraction of cell lipids using methanol, immunohistochemistry was performed on all cultures using an antiglial acid fibrillary protein (GFAP) primary antibody and a fluorescein isothiocyanate-labeled secondary antibody. The relative number of astrocytes present in each culture was determined by counting GFAPpositive cells and dividing by the total number of cells obtained by counting nuclei stained with propidium iodide. Values represent the means f SEM for four to seven microscopic fields counted in each culture. Fifty to 100 cells were counted in each field. Culture medium and cell lipids were transmethylated with BFs in methanol and the resulting fatty acid methyl esters separated by HPLC. Values represent the percentage of [ 1-I4C]18:3w-3converted to radioactive 22:6w-3. 'Values represent the amount of radioactive 22:6w-3produced from [I-'4C]18:3w-3.
ASTROCYTES PRODUCE 20:4~-6AND 2216~-3
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FIG. 2. Elongation and desaturationof w-3 fatty acids by primary cultures of cerebellar astrocytes and neurons. Twelve days after plating, primary culturesof cerebellar astrocytes(A and D)or neurons (B) were incubated for 72 h with 18 pM [l -14C]18:3w-3. Cerebellar neuronal cultures were also incubated for 72 h with 18 p.M [1-14C]20:5~-3 (C). Total cell lipids (A, B, and C) or medium lipids (D) were transmethylated and the resultingfatty acid methyl esters separated by reverse-phase HPLC. The amount of acetonitrile in the mobile phase (acetonitrilefwater)was held at 76O%during the first 50 min, then raised to 100% over 2 min.
FIG. 4. Elongation and desaturation of w-6 fatty acids by primary cultures of cerebellar astrocytes and neurons. Twelve days after plating, primary cultures of cerebellar astrocytes (A and D)or neurons (B) were incubated for 72 h with 18 ~ J V [1-14C]18:2w-6.Cerebellar neuronal cultures were also incubated for 72 h with 18 phf [1-14C]20:4w-6 (C). Total cell lipids (A, B, and C) or total medium lipids (D) were transmethylated and the resulting fatty acid methyl esters separated by reverse-phase HPLC. The amount of acetonitrile in the mobile phase (acetonitrilelwater) was held at 76% during the first 70 min. then raised to 100% over 2 min.
were 20:2w-6, 20:3w-6, 22:4w-6, and 22:5w-6. The identity of each of these metabolites was confirmed by GLC and/or GC-MS, as previously described (Moore et al., 1990). The isolated w-6 fatty acid methyl esters of the putative 20:2w-6, 20:3w-6, 22:4w-6, and 2215~6 all eluted from the GLC with retention times corresponding to their appropriate standards. Additional confirmation of structure was obtained by electron impact GC-MS. For 20:2w-6,20:30-6, and 22:4w-6, molecular ions at m/e 322, 320, and 346, respectively, were observed, and, in addition, several major ions confirmed the w-6 configuration of the
double bonds. These included ions at m/e 137, 15 1, and 165 for 20:2w-6, at m/e 177 and 191 for 20:3w-6, and at m/e 217 and 231 for 22:40-6. Cerebellar neuronal cultures, on the other hand, produced no detectable 20:4w-6 (Fig. 4B). Similarly, the granule cell neurons did not desaturate 20:4w-6, even after prolonged incubation (Fig. 4C). These neuronal cultures have an active elongation system, however, and produced large amounts of 20:20-6 from 18: 2w-6 (Fig. 5B) and 22:40-6 from 20:4w-6 (Fig. 4C). Other minor radioactive products detected in the HPLC radiochromatograms, designated as peaks “a” and “b” eluting between 75 and 90 min, were 16-, 18-, and 20-carbon saturated and monounsaturated fatty acids. Analysis by GLC revealed that peak “a” contained a mixture of 16:O and 18:1, whereas peak “b” contained 18:O and 20: 1. This suggests that some of the 18:2w-6 and 20:4w-6 underwent @-oxidation, with incorporation of the released [14C]acetate into newly synthesized fatty acids. Figure 4 D demonstrates that during continuous incubations with [ 1-I4C]18:2w-6, radioactive 20:4w-6 was released into the culture medium by astrocytes. As was observed with 22:6w-3, a larger fraction of the radioactive 20:4w-6 formed was released into the medium than was retained in the cells (Fig. 3B). No desaturation products of either [ 1-l4C]18:20-6 or [ 1-14C]20:4w-6 were observed in the medium of neuronal cultures (data not shown).
B
0 medium’ E4 cells
22:6
m4
a 4
a 5
FIG. 3. Distribution of polyunsaturated fatty acids produced by
cerebellar astrocyte cultures. The distribution of radiolabeled W-3 (A) or w-6 (B) fatty acids was calculated from the HPLC percentage distribution and the total radioactivity in extracted cell or media lipids. The data presented are the means SEM for three separate experiments.
*
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FIG. 5. Uptake of w-3 and W-6fatty acids by neuronal cultures.
Cultures of cerebral neurons were incubated with 18 p M [ l -"C]labeled fatty acid (182w6.20:4&, 18303, OT 22:603) in medium containing 10% FBS. Uptake was determined by measuring the total radioactivity in chloroform/methanol-extracted cell lipids after incubations of 15, 30, 60, 120, or 240 min. The amount of fatty acid taken up has been corrected for micrograms of cell protein. Data represent means k SEM or three separate cultures.
Uptake of fatty acids by neuronal cultures Although the neuronal cultures utilized in these studies were incapable of producing their own 22:6w3 or 20:4w-6, they readily took up [ 1-I4C]22:6w-3and [1-'4C]20:4w-6 from the culture medium (Fig. 5). In fact, they incorporated progressively more 204w-6 than 1k2w-6 and more 22:6w-3 than 18:3w-3 into glycerolipids as the incubation continued. DISCUSSION
The data presented here strongly suggest that a major site in the brain for the processing of long-chain w-3 and w-6 essential fatty acids is the astrocyte. Primary cultures of rat type I astrocytes from either cerebrum or cerebellum actively elongated and desaturated 18and 20-carbon precursor essential fatty acids to form 22:6w-3 and 20:4w-6 (Figs. 1, 2, and 4). Astrocyte cultures released the majority of the 22:60-3 and 20:4w6 they formed into the extracellular fluid (Fig. 3). These polyunsaturated fatty acids are enriched in brain lipids (Salem et al., 1986; Neuringer et al., 1988) where they are major components of excitable membranes (Stubbs and Smith 1984; Applegate and Glomset 1986; Salem et al., 1986)and precursors of many biologically active compounds, including prostaglandins, hydroxyeicosatetraenoic acids, leukotrienes, and lipoxins (Wolfe, 1982; Samuelsson et al., 1987; Murphy et al., 1988). In contrast to the astrocytes, primary cultures of rat cerebral and cerebellar neurons did not perform the fatty acid desaturation steps necessary to produce 22: 6w-3 or 20:4w-6 (Figs. 2 and 4). Instead, they only elongated 18- and 20-carbon precursor fatty acids. Many earlier studies of essential fatty acid metabolism in the brain may be interpreted in a different light as a result of these findings. Some investigators, delivering radiolabeled precursors to the brain through direct or intravenous injection, or by incubating these pre-
J. Neurochem., Val. 56, No. 2,1991
cursors with brain homogenates, have provided evidence that 20:4w-6 and 22:6w-3 formation can occur in some brain cells (Yavin and Menkes, 1974; Dhopeshwarkar and Subramanian, 1976;Cohen and Bernsohn, 1978;Clandinin et al., 1985; Anderson and Connor, 1988). Others, introducing 18:20-6 or 1k30-3 through the gut or peritoneal cavity, have observed substantial fatty acid elongation and desaturation in the liver (Sinclair and Crawford, 1972;Nouvelot et al., 1986; Scott and Bazan, 1989). These latter publications have led to the widely held view that the main source of brain 20:4w-6 and 22:6w-3 is the liver via the circulation. Our present studies with astrocytes, together with our previous work with cerebral endothelium (Moore et al., 1990) indicate that, independent of the liver, the brain is indeed capable of producing 20:4w6 and 22:60-3 from precursor fatty acids. Some of the earliest studies with isolated brain cells suggested that both glial and neuronal elements could desaturate and chain-elongate 18:2w-6 or 18:3w-3 (Yavin and Menkes, 1974; Cohen and Bernsohn, 1978). Later, oligodendroglial cells and C6 glioma cells (neoplastic astrocytes) were shown to be incapable of 20: 4w-6 and 22:6w-3 formation (Fewster et al., 1975; Robert et al., 1977), whereas human retinoblastoma cell cultures were found to produce 20:4w-6 and 22: 6w-3 from precursors (Hyman and Spector, 1981; Yorek et al., 1985). Because the latter cell lines were considered to be neuronal in origin, the perception arose that neurons, not glia, are responsible for long-chain polyunsaturated fatty acid formation in brain. Contrary to this widely held view, we find that neither cerebral nor cerebellar neurons are capable of significant desaturation of 0-3 or w-6 fatty acids (Table 1 and Figs. 2 and 4). This instead appears to be a function of astrocytes, a conclusion supported by the present data (Table 1 and Figs. 1, 2, and 4) and earlier work by Robert et al. (1983). The desaturation and elongation of 1k2w-6 and 18: 3w-3 in astrocytes appear to follow the pathways described in other tissues (Naughton, 1981; Holman, 1986).Of note is the presence of A4-desaturaseactivity in these cells. The A4-desaturase activity often is lost during cell culture (Spector et al., 1981) and is replaced by an alternative, dead-end pathway that forms 20:2w6 from 18:2w-6,and 20:3w-3 from 1k3w-3. This pathway appears to be more active in neurons than in astrocytes (Figs. 1, 2, and 4). Apparent substrate preference for w-3 fatty acids (Figs. 2, 3, and 4) also is consistent with findings reported in other tissues (Naughton, 1981; Holman, 1986), including the cerebral microvessel endothelium (Moore et al., 1990). This substrate specificity may take on added significance in astrocytes and in the cerebromicrovascular endothelium in light of the 22:6w-3 enrichment found in the brain (Salem et al., 1986;Neuringer et al., 1988). Another difference between the desaturation and elongation of 18:2w-6 and lk3w-3 by astrocytes is that w-
ASTROCYTES PRODUCE 2 0 : 4 ~ - 6AND 22.6~-3 6 metabolism does not continue appreciably beyond 20:40-6 (Figs. 3 and 4), whereas w-3 metabolism extends to 22:6w-3 (Figs. 2 and 3). Thus, in each polyunsaturated fatty acid family, astrocytes produce primarily the long-chain derivative that accumulates in neural tissues. Astrocytes are abundant glial cells that perform numerous supportive functions in the CNS, e.g., processing metabolites such as glucose and amino acids, deactivating neurotransmitters, and producing neurotrophic factors (Levi, 1990). They also influence blood-brain barrier function (Stewart and Wiley, 198 1 ; Tao-Cheng and Brightman, 1988; Maxwell et al., 1989). Because of their strategic anatomical location between the cerebromicrovascularendothelialcells and neurons, astrocytes have initial access to all of the incoming fatty acid precursors. These anatomical relationships, coupled with their demonstrated ability to produce and release both 20:4w-6 and 22:6w-3, indicate that astrocytes may have an additional supportive role in providing long-chain essential fatty acids to neurons. In doing so, astrocytes take up essential fatty acid precursors as they cross the blood-brain bamer, convert them into 20:4w-6 or 22:6w-3, and release these elongation/desaturation products into the interstitium of the brain for uptake and sequestration in neurons. Further, astrocytes can supply their own needs for 20: 4w-6 and 22:6w-3 for the production of eicosanoids that may play key roles in the regulation of cerebromicrovascular, glial, or neuronal function (Murphy et al., 1988). These findings suggest that astrocyte function must be intact for normal essential fatty acid metabolism to occur in the CNS. Acknowledgment: This work was supported by National Institutes of Health Grants NS-01096, NS-20632, NS-2462 1, and HL-39308. Joel Carl assisted in preparation of the figures. MaryAnn Barry and Greg Welk assisted in preparation of the cell cultures.
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Dutton G. R. (1990) Isolation, culture, and use of viable central nervous system perikarya, in Methods in Neuroscience, Vol. 2 (Conn P. M., ed), pp. 87-102. Academic Press, New York. Dyerberg J., Bang H. O., Stofferson E., Moncada S., and Vane J. R. (1 978) Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis. Lancet ii, I 17- 1 19. Fewster M. E., Ihrig T., and Mead J. F. (1975) Biosynthesis of long chain fatty acids by oligodendroglia isolated from bovine white matter. J. Neurochem. 25,207-2 13. Folch J., Lees M., and Sloane-StanleyG. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509. Holman R. T. (1986) Nutritional and biochemical evidence of acyl interaction with respect to essential polyunsaturated fatty acids. Prog. Lipid Res. 25, 29-39. Hyman B. T. and Spector A. A. (1981) Accumulation of N-3 polyunsaturated fatty acids by cultured human Y79 retinoblastoma cells. J. Neurochem. 37, 60-69. Levi G., ed (1990) Diflerentiafionand Functions of Glial Cells. WileyLiss, New York. Maxwell K., Berliner J. A,, and Cancilla P. A. (1989) Stimulation of glucose analogue uptake by cerebral microvessel endothelial cells by a product released by astrocytes. J. Neuropathol. Exp. Neurol. 48,6940.
Moore S. A., Yoder L., and Spector A. A. (1990) Role of the bloodbrain bamer in the formation of long-chain w-3 and w-6 fatty acids from essential fatty acid precursors. J. Neurochem. 55, 39 1-402.
Morrison W. R. and Smith L. M. (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. 1.Lipid Res. 5, 600-608. Murphy S. (1990) Generation of astrocyte cultures from normal and neoplastic central nervous system, in Methods in Neuroscience, Vol. 2 (Conn P. M., ed), pp. 33-47. Academic Press, New York. Murphy S., Pearce B., Jeremy J., and Dandona P. (1988) Astrocytes as eicosanoid-producing cells. Glia 1, 24 1-245. Naughton J. M. (198 1) Supply of polyenoic fatty acids to the mammalian brain: the ease of conversion of the short-chain essential fatty acids to their longer chain polyunsaturated metabolites in liver, brain, placenta, and blood. Int. J. Biochem. 13, 21-32. Needleman P., Raz A., Minkes M. S., Ferrendelli J. A,, and Sprecher H. ( 1979) Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties. Proc. Natl. Acad. Sci. USA 76,944-948. Neuringer M., Anderson G. J., and Connor W. E. (1988) The essentiality of N-3 fatty acids for the development and function of the retina and brain. Annu. Rev. Nutr. 8, 517-541. Nouvelot A., Delbart C., and Bourre J. M. (1986) Hepatic metabolism of dietary alpha-linolenic acid in suckling rats, and its possible importance in polyunsaturated fatty acid uptake by the brain. Ann. Nutr. Metab. 30, 3 16-323. Robert J., Rebel G., and Mandel P. (1977) Essential fatty acid metabolism in cultured astroblasts. Biochimie 59,417-423. Robert J., Montaudon D., and Hugues P. (1983) Incorporation and metabolism of exogenous fatty acids by cultured normal and tumoral dial cells. Biochim. Biophys. Acta 752, 383-395. Salem N., Jr., Kim H.-Y., and Yergey J. A. (1986) Docosahexaenoic acid: membrane function and metabolism, in Health Effects of
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Polyunsaturated Fatty Acids in S e a f d s (Simopoulos A. P., ed), pp. 263-317. Academic Press, New York. Samuelsson B., Dahlen S.-E.L., Lindgren J. A,, Rouzer C. A., and Serhan C . N. (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 237, 1171- I 175. Scott B. L. and Bazan N. G. (1989) Membrane docosahexaenoate is supplied to the developing brain and retina by the liver. Proc. Natl. Acad. Sci. USA 86, 2903-2907. Sinclair A. J. and Crawford M. A. (1 972) The incorporation of linolenic acid and docosahexaenoic acid into liver and brain lipids of developing rats. FEBS Left.26, 127-129. Spector A. A., Mathur S. N., Kaduce T. L., and Hyman B. T. (198 I ) Lipid nutrition and metabolism of cultured mammalian cells. Prog. LipidRes. 19, 155-186. Sprecher H. (1986) The metabolism of (n-3) and (n-6)fatty acids and their oxygenation by platelet cyclooxygenase and lipoxygenase. Prog. Lipid Res. 25, 19-28. Stewart P. A. and Wiley M. J. (1981) Developing nervous tissue
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induces formation of blood-brain barrier characteristics in invading endothelial cells: a study using quail-chick transplantation chimeras. Do.Biol. 84, 183-192. Stubbs C. D. and Smith A. D. (1984) The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim. Biophys. Acta 779, 89-137. Tao-Cheng J.-H. and Brightman M. W. (1988) Development of membrane interactions between brain endotheliai cells and astrocytes in vitro. Int. J. Dev.Neurosci. 6,26-37. Wolfe L. S. (1982) Eicosanoids: prostaglandins, thromboxanes, leukotrienes, and other derivatives of carbon-20 unsaturated fatty acids. J. Neurochem. 38, 1-14. Yavin E. and Menkes J. H. (1974) Polyenoic acid metabolism in cultured dissociated brain cells. J. Lipid Res. 15, 152-157. Yorek M. A., Figard P. H., Kaduce T. L., and Spector A. A. (1985) A comparison of lipid metabolism in two human retinoblastoma cell lines. Invest. Ophthalmol. Vis. Sci. 26, 1148-1 154.
Astrocytes, Not Neurons, Produce Docosahexaenoic Acid (22:60-3) and Arachidonic Acid (20:40-6) *Steven A. Moore, *Elizabeth Yoder, ?Sean Murphy, ?Gary R. Dutton, and $Arthur A. Spector Departments of *Pathology, tPharmacology, and $Biochemistry, University of Iowa, Iowa City, Iowa, U.S.A.
Abstract: Elongated, highly polyunsaturated derivatives of linoleic acid ( I 8:2w-6) and linolenic acid ( I 8:30-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [ 1-'4C]18:2w-6, [ 1-14C]20:4~-6,[ 1-14C]18:3w-3, or [1-14C]20: 5w-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of A4desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2w-6 was 20:4w-6, whereas the primary products
from 1k30-3 were 20:5w-3, 22:5w-3, and 22:6w-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4w-6 and 22:6w-3, was released into the extracellular fluid. Although incapable of producing 20:4w6 and 22:6w-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating w-6 and w3 essential fatty acid precursors to 20:40-6 and 22:6w-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons. Key Words: Astrocyte-Neuron-Arachidonic acid-Docosahexaenoic acid-Fatty acid elongation-Fatty acid desaturation. Moore S. A. et al. Astrocytes, not neurons, produce docosahexaenoic acid (22:6w-3) and arachidonic acid (20:40-6). J. Neurochem. 56, 518-524 (1991).
The brain is more highly enriched than most other tissues in long-chain polyunsaturated fatty acids, particularly docosahexaenoic acid (22:6w-3; Salem et al., 1986; Neuringer et al., 1988). 22:6w-3 is a major component of excitable membranes and may exert considerable influence on membrane fluidity and the functional properties of integral proteins (Stubbs and Smith, 1984; Salem et al., 1986), possibly by promoting optimal acyl chain packing or interactions with transmembrane domains (Applegate and Glomset, 1986). Arachidonic acid (20:4w-6) is the substrate for the production of many biologically active compounds, including prostaglandins, hydroxyeicosatetraenoic acids, leukotrienes, and lipoxins (Wolfe, 1982; Samuelsson et al., 1987). Similarly, eicosapentaenoic acid (20:5w3) and 22:6w-3 are substrates for the production of prostaglandins, hydroxy acids, and leukotrienes (Needleman et al., 1979; Salem et al., 1986; Sprecher, 1986). Although several studies have attempted to explain
this enrichment (Salem et al., 1986; Neunnger et al., 1988), many questions remain about the source of essential fatty acids in the brain and the roles of various cell types in the uptake and retention of these iongchain polyunsaturated fatty acids. Studies of essential fatty acid elongation and desaturation by the brain (Dhopeshwarkar et al., 1971b; Yavin and Menkes, 1974; Dhopeshwarkar and Subramanian, 1976; Cohen and Bersohn, 1978; Clandinin et al., 1985; Anderson and Connor, 1988) have failed to clarify which cell types within this tissue are capable of producing 22: 6w-3. Moreover, recent findings indicating that 22:6w3 is produced in the liver and then circulated to the CNS have led to the view that the brain may not be an important site of 22:6w-3 formation (Scott and Bazan, 1989). We previously have demonstrated that cells of the blood-brain barrier, the cerebromicrovascular endothelium, can elongate and desaturate essential fatty acid
Received May I I , 1990: revised manuscript received July 26. 1990; accepted July 26, 1990. Address correspondence and reprint requests to Dr. S. A. Moore at The University of Iowa, Department of Pathology, Room I19 Medical Laboratories, Iowa City, IA 52242, U.S.A.
Abbreviafions used: FBS, fetal bovine serum; GC-MS, gas chromatography-mass spectrometry; MEM, minimum essential medium.
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ASTROCYTES PRODUCE 20:40-6 AND 22:6~-3 precursors of both the w-3 and w-6 families (Moore et al., 1990). However, these cells are not able to perform the final desaturation step, mediated by A4-desaturase, to produce 22:6w-3 or 22:5w-6 (Moore et al., 1990). This suggested that brain A4-desaturase activity is located in another cell type. To determine whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon glial cells, we have studied the processing of w-3 and w-6 fatty acid precursors in primary cultures of neurons and astrocytes obtained from neonatal rat cerebrum and cerebellum.
MATERIALS AND METHODS Materials Methyl ester standards were purchased from NuChek Prep (Elysian, MN, U.S.A.), and [ I-'4C]linoleic acid ([ I-I4C]18: 2w-6), [ 1-'4C]20:4w-6, [ I-'4C]linolenic acid ([ 1-I4C]18:3w-3), and [ l-'4C]20:50-3 (approximately 55 mCi/mmol) were purchased from New England Nuclear (DuPont, Boston, MA, U.S.A.) and Amersham (Arlington Heights, IL, U.S.A.).
Cell culture Primary cultures of astrocytes and neurons were prepared from 2-7-day-old rat pups by a trypsinization/trituration technique which disaggregates brain perikarya [for methodology details, see Murphy (1990) and Dutton (199O)l. Cerebral cultures were obtained from forebrain gray matter after dissecting away white matter and leptomeninges. Cerebellar cultures were obtained from whole cerebellum. Highly enriched astrocyte and neuronal cultures were obtained from the same starting cell suspension in each anatomical site. Astrocyte cultures were seeded into poly-D-lysine-coated multiwell plates at a density of lo4 cells/cm2and maintained in Eagle's minimum essential medium (MEM) with Earle's salts supplemented with 33 mM glucose, 2 mM glutamine, 180 pMgentamicin, and 10%fetal bovine serum (FBS). Every 3 days in culture, half the medium was replaced until the cultures were confluent (approximately 14 days). Neuronal cultures were seeded into poly-D-lysine-coated multiwell plates at a density of 1.5 X lo6 cells/cm2 and maintained in Eagle's MEM with Earle's salts supplemented with 33 mM glucose, 1 M g l u t a m i n e , I80 pMgentamicin, 20 m M KCI, 80 pM 5-fluoro-2'-deoxyuridine, 2.5% chick embryo extract, and 10%FBS. Neuronal cultures were used 10-12 days after plating without a medium change. Astrocyte cultures were routinely 295% type I astrocytes by immunohistochemical characterization. Immunohistochemical characterization of neuronal cultures revealed those from cerebrum to contain 60-709'0 neurons, with the majority of contaminating cells being type I astrocytes, and those from cerebellum to contain approximately 90% glutamatergic granule neurons and 57% GABAergic inhibitory interneurons, with the majority of contaminating cells being type I astrocytes.
Experimental protocol The uptake, elongation, and desaturation of essential fatty acids were studied in 10-14-day cultures of astrocytes or neurons grown in multiwell tissue culture plates. Incubations were routinely carried out in culture media containing 110% FBS and 10-20 pM I4C-labeled fatty acids. Cells were maintained at 37°C in a 5% C 0 2 incubator during all of the incubations.
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Lipid extractions Lipids were extracted from the incubation medium using a modification of the procedure of Folch et al. (1957). The medium was centrifuged to remove cellular debris, then extracted with a 2: 1 (vol/vol) mixture of chloroform/methanol containing I % acetic acid. The chloroform phase was dried under nitrogen and resuspended in acetonitrile. To isolate cell lipids, the incubation medium was removed and the cellswere washed with serum-free medium containing 10 pM fatty acid-free bovine serum albumin, scraped into methanol, and transferred to a siliconized screw-top glass test tube (Dudley and Spector, 1986).One volume of chloroform was added, and after mixing with a Vortex and adding one volume of 0.88% KCI, each sample was centrifuged at 1,300 g for 10 min to separate the phases. After the aqueous phase was washed once with one volume of chloroform, the two chloroform layers were combined, dried under nitrogen, and resuspended in chloroform/methanol (2: I , vol/vol).
Methyl ester derivatization Aliquots of total cell lipid or medium lipid extracts were combined with an excess of 14%BF3 in methanol and heated to 100°C for 15 min to produce fatty acid methyl esters (Momson and Smith, 1964). Following the addition of water, the methyl esters were extracted in heptane, dried under nitrogen, and resuspended in acetonitrile.
HPLC Radioactive methyl esters prepared from the cell lipids or incubation media were separated by reverse-phase HPLC by a modification of the method of Aveldano et al. (1983). Briefly, a 4.6 X 150-mm C18 reverse-phase Beckman HPLC column with 5-pm spherical packing was used with a mobile phase of water and acetonitrile in a two-step isocratic elution. The more polar fatty acid methyl esters were eluted with 76% acetonitrile, whereas the less polar esters were eluted with 100% acetonitrile. Radioactivity was monitored by mixing the column effluent with scintillator solution at a 1:3 ratio and passing the mixture through an on-line Radiomatic Instruments Flo I@ radioactivity detector (Radioanalytic, Tampa, FL, U.S.A.). The system was standardized with methyl esters of the following I4C-labeledfatty acids: 18:2w6, 18:3~-3,20:4~-6,20:5~-3,and 22:6w-3.
GLC Fatty acid methyl esters also were separated by GLC on a 2 mm X 1.9 m glass column packed with 10% SP2330 on IOO/ 120-mesh Chromosorb W (Supelco, Bellafontane, PA, U.S.A.). A Hewlett-Packard model 5890A gas chromatograph with flame ionization detector was used. The column temperature was raised from 178 to 230°C over 45 min and nitrogen served as the carrier gas at a flow rate of 25 ml/min. Peak areas were determined with a Hewlett-Packard model 3380A integrator-recorder. The methyl ester derivatives of the unknown essential fatty acid metabolites were analyzed with a VG TRIO 1 mass spectrometer containing a 2 mm X 15 m OV-1 column [as described previously, see Moore et al. (1990)J The column temperature was raised from 50 to 250°C at a rate of 20"/min. The energy of the electron beam was 70 eV.
RESULTS Elongation and desaturation of 18:3w-3 During a 24-h incubation, the primary product of I8:3w-3 elongation and desaturation in cerebral astroJ. Neurochem.. Vol. 56. No. 2.1991
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Time (min) FIG. 1. Elongation and desaturationof 18:3w3by primary cultures
of cerebral astrocytes and neurons. Twelve days after plating, primary cultures of cerebral astrocytes (A) or neurons (B) were incubated for 24 h with 18 f l [l -'4C]18:3w-3. Total cell lipids were transmethylated and the resulting fatty acid methyl esters separated by reverse-phase HPLC. The amount of acetonitrile in the mobile phase (acetonitrile/water)was held at 76% during the first 50 min. then raised to 100% over 2 min.
cyte cultures was 22:6w-3 (Fig. 1A). Other major products were 20:5w-3 and docosapentaenoic acid (22:5w-3), the immediate precursors of 22:60-3. Minor metabolites detected by HPLC were 20:3w-3 and 20: 4w-3. The identity of each of these metabolites was confirmed by GLC and/or gas chromatography-mass spectrometry (GC-MS) as previously described (Moore et al., 1990). The isolated 0-3 fatty acid methyl esters of the putative 20:3w-3, 20:4w-3, and 22:5w-3 eluted from the GLC with retention times corresponding to their appropriate standards. Further confirmation of structure was obtained by electron impact GC-MS for the fatty acid methyl ester of 2250-3. A molecular ion was observed at mle 344. Additional ions at m/e 5 5 , 95,135,175, and 2 15 confirmed the w-3 configuration of the double bonds. Cerebral neuronal cultures, on the other hand, produced very little 22:6w-3 (Fig. 1B). The main metabolites of 18:3w-3 in these cultures were 20:3w-3, an elongation product, and 22:5w-3. In mixed cultures containing various ratios of cerebral neurons and astrocytes incubated with [ 1-I4C]18:3w-3, both the relative and absolute amounts of 22:6w-3 formed increased (from 5.8 to 59%, and from 0.96 to 10.43 nmol/well, respectively) as the number of astrocytes in these cultures increased from 37 to 94% (Table l ) . The elongation and desaturation of [ 1-14C]18:3w-3 and [ 1-l4C]20:5w-3also were compared in highly enriched neuronal granule cell and astrocyte cultures from the cerebellum. Like cerebral astrocyte cultures, astrocyte cultures from the cerebellum readily produced 22: 60-3 (Fig. 2A). However, the granule cell neurons were incapable of 2 2 1 6 ~ - production, 3 even after 72 h of
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incubation with the w-3 fatty acid precursors (Fig. 2B and C). These neuronal cultures have an active elongation system, however, and produced large amounts of 20:3w-3 from 18:3w-3 (Fig. 2B) and 22:5w-3 from 20:5w-3 (Fig. 2C). Small amounts of radioactivity were detected on the HPLC radiochromatograms in peaks eluting between 50 and 60 min (Figs. 1 and 2). These compounds were identified by GLC as 16:O and 18:1 in peak "a" and 18:O and 20: 1 in peak "b". This suggests that some of the 18:3w-3 and 20:5w-3 underwent &oxidation, with reutilization of the released [ I4C]acetate. Figure 2D demonstrates that during continuous incubations with [ 1-I4C]18:30-3, large amounts of radioactive 22:6w-3 were released into the culture medium by astrocytes. A much larger fraction of the 22:6w-3 formed was released into the medium than was retained in the cells (Fig. 3A). Similar production and release of radioactive 22:6w-3 were observed during continuous incubations of astrocytes with [ 1-14C]20:5w-3(data not shown). No desaturation products of either [lI4C]18:30-3 or [ 1-'4C]20:5w-3were observed in the medium of neuronal cultures (data not shown). Elongation and desaturation of 18:2w-6 The ability of brain cells to elongate and desaturate essential fatty acids was further examined in highly enriched neuronal granule cell and astrocyte cultures from the cerebellum incubated with [ 1-I4C]18:2w-6 or [ 1''C]20:4w-6. Astrocyte cultures from the cerebellum readily produced 20:4w-6 (Fig. 4A). Other products TABLE 1. Conversion of 18.3~-3to 2 2 : 6 ~ - 3by mixed glial-neuronal cultures 22:6w-3 production' Astrocyte content of cultures ( % ) b
(% of total label)d
(nmol/well)'
31 k 2.2 48 f 5.4 57 rt 4.5 94 f 3.2
5.8 8.6 25.0 59.0
0.96 1.48 4.45 10.43
a Fluorodeoxyuridine was added to dissociated cerebral cells at various times after plating to establish cultures with various mixtures of neurons and astrocytes. At I2 days after plating, all cultures were incubated for 48 h with I8 pA4 [ 1-I4C]18:3w-3. Following extraction of cell lipids using methanol, immunohistochemistry was performed on all cultures using an antiglial acid fibrillary protein (GFAP) primary antibody and a fluorescein isothiocyanate-labeled secondary antibody. The relative number of astrocytes present in each culture was determined by counting GFAPpositive cells and dividing by the total number of cells obtained by counting nuclei stained with propidium iodide. Values represent the means f SEM for four to seven microscopic fields counted in each culture. Fifty to 100 cells were counted in each field. Culture medium and cell lipids were transmethylated with BFs in methanol and the resulting fatty acid methyl esters separated by HPLC. Values represent the percentage of [ 1-I4C]18:3w-3converted to radioactive 22:6w-3. 'Values represent the amount of radioactive 22:6w-3produced from [I-'4C]18:3w-3.
ASTROCYTES PRODUCE 20:4~-6AND 2216~-3
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FIG. 2. Elongation and desaturationof w-3 fatty acids by primary cultures of cerebellar astrocytes and neurons. Twelve days after plating, primary culturesof cerebellar astrocytes(A and D)or neurons (B) were incubated for 72 h with 18 pM [l -14C]18:3w-3. Cerebellar neuronal cultures were also incubated for 72 h with 18 p.M [1-14C]20:5~-3 (C). Total cell lipids (A, B, and C) or medium lipids (D) were transmethylated and the resultingfatty acid methyl esters separated by reverse-phase HPLC. The amount of acetonitrile in the mobile phase (acetonitrilefwater)was held at 76O%during the first 50 min, then raised to 100% over 2 min.
FIG. 4. Elongation and desaturation of w-6 fatty acids by primary cultures of cerebellar astrocytes and neurons. Twelve days after plating, primary cultures of cerebellar astrocytes (A and D)or neurons (B) were incubated for 72 h with 18 ~ J V [1-14C]18:2w-6.Cerebellar neuronal cultures were also incubated for 72 h with 18 phf [1-14C]20:4w-6 (C). Total cell lipids (A, B, and C) or total medium lipids (D) were transmethylated and the resulting fatty acid methyl esters separated by reverse-phase HPLC. The amount of acetonitrile in the mobile phase (acetonitrilelwater) was held at 76% during the first 70 min. then raised to 100% over 2 min.
were 20:2w-6, 20:3w-6, 22:4w-6, and 22:5w-6. The identity of each of these metabolites was confirmed by GLC and/or GC-MS, as previously described (Moore et al., 1990). The isolated w-6 fatty acid methyl esters of the putative 20:2w-6, 20:3w-6, 22:4w-6, and 2215~6 all eluted from the GLC with retention times corresponding to their appropriate standards. Additional confirmation of structure was obtained by electron impact GC-MS. For 20:2w-6,20:30-6, and 22:4w-6, molecular ions at m/e 322, 320, and 346, respectively, were observed, and, in addition, several major ions confirmed the w-6 configuration of the
double bonds. These included ions at m/e 137, 15 1, and 165 for 20:2w-6, at m/e 177 and 191 for 20:3w-6, and at m/e 217 and 231 for 22:40-6. Cerebellar neuronal cultures, on the other hand, produced no detectable 20:4w-6 (Fig. 4B). Similarly, the granule cell neurons did not desaturate 20:4w-6, even after prolonged incubation (Fig. 4C). These neuronal cultures have an active elongation system, however, and produced large amounts of 20:20-6 from 18: 2w-6 (Fig. 5B) and 22:40-6 from 20:4w-6 (Fig. 4C). Other minor radioactive products detected in the HPLC radiochromatograms, designated as peaks “a” and “b” eluting between 75 and 90 min, were 16-, 18-, and 20-carbon saturated and monounsaturated fatty acids. Analysis by GLC revealed that peak “a” contained a mixture of 16:O and 18:1, whereas peak “b” contained 18:O and 20: 1. This suggests that some of the 18:2w-6 and 20:4w-6 underwent @-oxidation, with incorporation of the released [14C]acetate into newly synthesized fatty acids. Figure 4 D demonstrates that during continuous incubations with [ 1-I4C]18:2w-6, radioactive 20:4w-6 was released into the culture medium by astrocytes. As was observed with 22:6w-3, a larger fraction of the radioactive 20:4w-6 formed was released into the medium than was retained in the cells (Fig. 3B). No desaturation products of either [ 1-l4C]18:20-6 or [ 1-14C]20:4w-6 were observed in the medium of neuronal cultures (data not shown).
B
0 medium’ E4 cells
22:6
m4
a 4
a 5
FIG. 3. Distribution of polyunsaturated fatty acids produced by
cerebellar astrocyte cultures. The distribution of radiolabeled W-3 (A) or w-6 (B) fatty acids was calculated from the HPLC percentage distribution and the total radioactivity in extracted cell or media lipids. The data presented are the means SEM for three separate experiments.
*
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FIG. 5. Uptake of w-3 and W-6fatty acids by neuronal cultures.
Cultures of cerebral neurons were incubated with 18 p M [ l -"C]labeled fatty acid (182w6.20:4&, 18303, OT 22:603) in medium containing 10% FBS. Uptake was determined by measuring the total radioactivity in chloroform/methanol-extracted cell lipids after incubations of 15, 30, 60, 120, or 240 min. The amount of fatty acid taken up has been corrected for micrograms of cell protein. Data represent means k SEM or three separate cultures.
Uptake of fatty acids by neuronal cultures Although the neuronal cultures utilized in these studies were incapable of producing their own 22:6w3 or 20:4w-6, they readily took up [ 1-I4C]22:6w-3and [1-'4C]20:4w-6 from the culture medium (Fig. 5). In fact, they incorporated progressively more 204w-6 than 1k2w-6 and more 22:6w-3 than 18:3w-3 into glycerolipids as the incubation continued. DISCUSSION
The data presented here strongly suggest that a major site in the brain for the processing of long-chain w-3 and w-6 essential fatty acids is the astrocyte. Primary cultures of rat type I astrocytes from either cerebrum or cerebellum actively elongated and desaturated 18and 20-carbon precursor essential fatty acids to form 22:6w-3 and 20:4w-6 (Figs. 1, 2, and 4). Astrocyte cultures released the majority of the 22:60-3 and 20:4w6 they formed into the extracellular fluid (Fig. 3). These polyunsaturated fatty acids are enriched in brain lipids (Salem et al., 1986; Neuringer et al., 1988) where they are major components of excitable membranes (Stubbs and Smith 1984; Applegate and Glomset 1986; Salem et al., 1986)and precursors of many biologically active compounds, including prostaglandins, hydroxyeicosatetraenoic acids, leukotrienes, and lipoxins (Wolfe, 1982; Samuelsson et al., 1987; Murphy et al., 1988). In contrast to the astrocytes, primary cultures of rat cerebral and cerebellar neurons did not perform the fatty acid desaturation steps necessary to produce 22: 6w-3 or 20:4w-6 (Figs. 2 and 4). Instead, they only elongated 18- and 20-carbon precursor fatty acids. Many earlier studies of essential fatty acid metabolism in the brain may be interpreted in a different light as a result of these findings. Some investigators, delivering radiolabeled precursors to the brain through direct or intravenous injection, or by incubating these pre-
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cursors with brain homogenates, have provided evidence that 20:4w-6 and 22:6w-3 formation can occur in some brain cells (Yavin and Menkes, 1974; Dhopeshwarkar and Subramanian, 1976;Cohen and Bernsohn, 1978;Clandinin et al., 1985; Anderson and Connor, 1988). Others, introducing 18:20-6 or 1k30-3 through the gut or peritoneal cavity, have observed substantial fatty acid elongation and desaturation in the liver (Sinclair and Crawford, 1972;Nouvelot et al., 1986; Scott and Bazan, 1989). These latter publications have led to the widely held view that the main source of brain 20:4w-6 and 22:6w-3 is the liver via the circulation. Our present studies with astrocytes, together with our previous work with cerebral endothelium (Moore et al., 1990) indicate that, independent of the liver, the brain is indeed capable of producing 20:4w6 and 22:60-3 from precursor fatty acids. Some of the earliest studies with isolated brain cells suggested that both glial and neuronal elements could desaturate and chain-elongate 18:2w-6 or 18:3w-3 (Yavin and Menkes, 1974; Cohen and Bernsohn, 1978). Later, oligodendroglial cells and C6 glioma cells (neoplastic astrocytes) were shown to be incapable of 20: 4w-6 and 22:6w-3 formation (Fewster et al., 1975; Robert et al., 1977), whereas human retinoblastoma cell cultures were found to produce 20:4w-6 and 22: 6w-3 from precursors (Hyman and Spector, 1981; Yorek et al., 1985). Because the latter cell lines were considered to be neuronal in origin, the perception arose that neurons, not glia, are responsible for long-chain polyunsaturated fatty acid formation in brain. Contrary to this widely held view, we find that neither cerebral nor cerebellar neurons are capable of significant desaturation of 0-3 or w-6 fatty acids (Table 1 and Figs. 2 and 4). This instead appears to be a function of astrocytes, a conclusion supported by the present data (Table 1 and Figs. 1, 2, and 4) and earlier work by Robert et al. (1983). The desaturation and elongation of 1k2w-6 and 18: 3w-3 in astrocytes appear to follow the pathways described in other tissues (Naughton, 1981; Holman, 1986).Of note is the presence of A4-desaturaseactivity in these cells. The A4-desaturase activity often is lost during cell culture (Spector et al., 1981) and is replaced by an alternative, dead-end pathway that forms 20:2w6 from 18:2w-6,and 20:3w-3 from 1k3w-3. This pathway appears to be more active in neurons than in astrocytes (Figs. 1, 2, and 4). Apparent substrate preference for w-3 fatty acids (Figs. 2, 3, and 4) also is consistent with findings reported in other tissues (Naughton, 1981; Holman, 1986), including the cerebral microvessel endothelium (Moore et al., 1990). This substrate specificity may take on added significance in astrocytes and in the cerebromicrovascular endothelium in light of the 22:6w-3 enrichment found in the brain (Salem et al., 1986;Neuringer et al., 1988). Another difference between the desaturation and elongation of 18:2w-6 and lk3w-3 by astrocytes is that w-
ASTROCYTES PRODUCE 2 0 : 4 ~ - 6AND 22.6~-3 6 metabolism does not continue appreciably beyond 20:40-6 (Figs. 3 and 4), whereas w-3 metabolism extends to 22:6w-3 (Figs. 2 and 3). Thus, in each polyunsaturated fatty acid family, astrocytes produce primarily the long-chain derivative that accumulates in neural tissues. Astrocytes are abundant glial cells that perform numerous supportive functions in the CNS, e.g., processing metabolites such as glucose and amino acids, deactivating neurotransmitters, and producing neurotrophic factors (Levi, 1990). They also influence blood-brain barrier function (Stewart and Wiley, 198 1 ; Tao-Cheng and Brightman, 1988; Maxwell et al., 1989). Because of their strategic anatomical location between the cerebromicrovascularendothelialcells and neurons, astrocytes have initial access to all of the incoming fatty acid precursors. These anatomical relationships, coupled with their demonstrated ability to produce and release both 20:4w-6 and 22:6w-3, indicate that astrocytes may have an additional supportive role in providing long-chain essential fatty acids to neurons. In doing so, astrocytes take up essential fatty acid precursors as they cross the blood-brain bamer, convert them into 20:4w-6 or 22:6w-3, and release these elongation/desaturation products into the interstitium of the brain for uptake and sequestration in neurons. Further, astrocytes can supply their own needs for 20: 4w-6 and 22:6w-3 for the production of eicosanoids that may play key roles in the regulation of cerebromicrovascular, glial, or neuronal function (Murphy et al., 1988). These findings suggest that astrocyte function must be intact for normal essential fatty acid metabolism to occur in the CNS. Acknowledgment: This work was supported by National Institutes of Health Grants NS-01096, NS-20632, NS-2462 1, and HL-39308. Joel Carl assisted in preparation of the figures. MaryAnn Barry and Greg Welk assisted in preparation of the cell cultures.
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