Journol of NeumcIwnii\rr~. 1978. Vol 30. pp 543-548 Perramon Press. Printed in Great Britain
UTILIZATION OF POLYUNSATURATED FATTY ACID SUPPLEMENTS BY CULTURED NEUROBLASTOMA CELLS J. ROBERT.G REBEL*and P. MANDEL Centre de Neurochimie du C N R S and Institut de Chimie Biologique de la Faculte de Medecine. 11. rue Humann, 67085 Strasbourg Cedex, France (Recerrrd 22 March 1977 R e ~ i ~ e7 dJ u l ) 1977 Accepted 9 August 1977)
Abstract-Brain phosphoglycerides are known to contain large amounts of polyunsaturated fatty acids (PUFA) However. neuroblastoma cells contain very low amounts of PUFA Since the serum used for cell culture has been shown to be deficient in PUFA. a supplementation of this serum with various PUFA was undertaken Linoleic. arachidonic and docosahexaenoic acids were incorporated in cell phosphoglycerides. mainly at the expense of oleic acid. while linolenic acid was only poorly incorporated Linoleic. linolenic and arachidonic acids were transformed to their elongation and desaturation products, but the last step of transformation. which involves the action of a A 4 desaturase, was never observed The levels of incorporation and transformation of exogenous PUFA could vary strikingly in different lines of neuroblastoma cells The simultaneous addition of two PUFA (Iinoleic and linolenic acids) was followed by a reduction in the amount of their respective derivatives in cell phosphoglycerides. compared to that obtained when only one PUFA was given to the cells, suggesting d competitive inhibition of the desaturation of each PUFA Such alterations i n membrane lipids may provide a useful model for the study of membrane structure-function relationships
MANYexperiments have been reported recently concerning the relationship between the lipid composition of membranes and their physical and physiological properties Most of this work has been done with microorganisms. especially microbial fatty acid auxo& VAGELOS.1972, MACHTICER & trophs (CRONAY Fox, 1973) The effect of fatty acid alterations on animal membrane properties has recently been incestigated in cultured cells, by adding exogenous fatty et a / , 1973, acids to the culture medium (WISNIESKI WILLIAMS et a / . 1974, FERGUSON et al., 1975) This type of study has rarely been undertaken (HORWITZ et a/., 1974, KITAJIMA et a!.. 1975; WILLIAMS et a / . 1975) on cells which need serum in their culture medium, because serum contains lipid materials which can interfere with the utilization of the fatty acid supplements, and when such cells were used, serum was freed of its lipid components before the fatty acid supplement was added. We have, however. reported recently on the'induction of altered fatty acid patterns in two strains of glial cells in culture, without prior delipidization of the serum (ROBERTet a / , 1977). Cells of nervous origin are of considerable interest tor such investigations because of the particular fatty acid patterns which are found in phospholipids from nerve structures Membrane phospho-
'This work wds supported by a grant from the D G R S T (A C C Membranes biologiques) ' Charge de Recherchc du C N R S Ahhrri iarion u\ed PU FA, polyunsaturated fatty acids 543
lipids from brain (with the exception of myelin) are known to contain larger amounts of polyunsaturated fatty acids (PUFA) than membrane phospholipids from other organs (CLAUSEN, 1969) In particular. the ethanolamine phosphoglycerides from synaptic plasma membranes contain up to 40",, docosahexaenoic acid (BRECKENRIDCE et a/., 1972). Neuroblastoma cells however do not contain large amounts of PUFA, and their phospholipid fatty acid patterns reflect the fatty acid content of the serum. In fact. th fetal calf serum commonly used to grow neuroblastoma cells is largely deficient in PUFA (ROBERT et a/.. 1977) and contains neither short-chain PUFA (I e. 'essential' fatty acids, linoleic acid 18:2w6 and Iinolenic acid 18 30~3).nor long-chain PUFA (the most common of which being arachidonic acid 2 0 . 4 ~ 6in most tissues and docosahexaenoic acid 2 2 . 6 ~ 3 i n brain tissue) It is thus feasible to study PUFA supplementation to these cells. provided that the fatty acids could be introduced in the culture medium without the toxic effects already observed by several authors 1967) The method we (GEYER,1967, MOSKOWITZ. have already used for glial cells was equally applicable to neuroblastoma and without adverse effects on cellular health and integrity We report here alterations to the lipid composition occurring in PUFAsupplemented aeuroblastoma cells Such alterations may provide useful models for studying the lipiddependant properties of cell membranes. and therefore the structureefunction relationship in cell membranes, with special reference to nerre tissue from which our cells originate
J. ROBERT,G. REBELand P. MANDEL
544
MATERIALS AND METHODS Cell culture. Two clonal lines from the C1300 mouse neuroblastoma were used: the NlE 115 clone from Nirenberg's laboratory (AMANOet a/., 1972) and the M1 clone isolated in our laboratory by CIESIELSKI-TRESKA et a[. (1975). The cells were grown in 75cm' Falcon dishes with Eagle-Dulbecco synthetic medium (Gibco) supplemented with 10% fetal calf serum (Gibco) at 37°C in a humidified atmosphere containing 5"/, C 0 2 . The serum was used either unaltered (for controls) or supplemented with a PUFA. This PUFA, as free form, was added directly to the serum by means of a sterile microsyringe (Hamilton). After magnetic stirring for 3 h, the mixture was homogeneous and was used throughout. A control of the new fatty acid profile of the serum was always performed before its use, to ensure that the amount of the added PUFA was constant and approximately equal to the amount of oleic acid present in the serum. In fact, we had to add about 10-12 mg of PUFA in 100 ml serum to reach this concentration. The final molar concentration of exogenous PUFA in contact with the cells was then 3 0 - 4 0 ~ ~In. some experiments we added two PUFA together; the total exogenous PUFA concentration was then doubled. At the beginning of each experiment, the cells were replicated either with unaltered serum and synthetic medium, o r with supplemented serum and synthetic medium. Unless otherwise specified the cells were grown for 9 days under identical conditions, with a replication on the 5th day and two changes of medium o n the third and 7th days. The cells were harvested by scraping with a rubber policeman, washed 3 times in 0.9% NaCl and pelleted. Lipid analysis. Lipids were extracted according to FOLCH et a / . (1957). Fatty acids were prepared by alkaline methanolysis (DAWSON,1960) either from total saponifiable lipids, or from total phosphoglycerides isolated by column chromatography (HIRSCH& AHRENS,1958). or from indiviet al., dual phosphoglycerides isolated by T L C (NUSSBAUM 1970). G L C of fatty acid methyl esters was performed at 180'C using a glass column packed with 100,; EGSS-X on chromosorb W-HP. Chromatographic separation was conducted using a Varian-Aerograph gas-chromatograph using a flame ionization detector. The peaks were identified by comparison with known standards or by semi-logarithmic plotting. When we needed a discrimination between the different kinds of PUFA. fatty acid methyl esters were fractionated on silver-nitrate impregnated plates (DUDLEY & ANDERSON, 1975) and the different fractions were eluted and analysed by GLC. Lipid phosphorus was assayed according t o ' MACHEBOBUF & DELSAL(1943). Cholesterol was assayed with the Liebermann-Burchard reagent, according to IDLER& BAUMANN (1953). Distribution of phospholipids was obtained by phosphorus assay (BARTLETT, 1959) of the individual et spots obtained after TLC in the system of NUSSBAUM al. (1970). Ethanolamine plasmalogens were separated from other ethanolamine phosphoglycerides by an acidic hydrolysis performed directly on the plate between the two migrations (see HORROCKS,1968).
Under the conditions used, no changes were detected in the morphology of the cells, nor in their growth characteristics in the presence of any added PUFA. No lipid inclusions were seen in the cellular cytoplasm. The phospholipid and cholesterol content of the cells remained constant, and the relative proportions of each phospholipid were not significantly modified (Table 1). The fatty acid compositions of total phosphoglycerides (Table 2) or of individual phosphoglycerides (Table 3) showed great variation when an exogenous PUFA was added to the serum: 1. Each added PUFA was incorporated at different levels in cell phosphoglycerides, except for linolenic acid which was very poorly incorporated in comparison with other PUFA. 2. The added PUFA were transformed t o their elongation and desaturation products, the percentage of which increased in the cell phosphoglycerides. 18:2w6 was transformed to 20:2w6, 20:4w6 and 22:4w6, 18:303 was transformed to 20:303, 20:403, 20: 5w3 and 22:5w3, 20:4w6 was transformed to 22:4w6 and 24:4w6. N o transformation products could be detected when 22:6w3 was added to the serum. Ethanolamine plasmalogens were the phospholipid fraction into which most of the newly formed PUFA were incorporated, mainly for the a 3 series. 3. The increase observed in the PUFA content of the cell phosphoglycerides were obtained almost exclusively at the expense of the monounsaturated series, the percentage of the saturated fatty acids remaining relatively constant or even increased in the case of P E in the presence of 18:2w6. 4. The added PUFA underwent transformation up until the final step; 22:5w6 was only very slightly increased when 18:2w6 or 20:4w6 were added to the serum; 22:6w3 was not significantly increased when 18:3w3 was added to the serum. The last step of the
TABLE1. LIPIDCOMPOSITION OF MI CELLS (LINE a) 9 days IN THE PRESENCE OF P U F A
GROWN
Control Cells treated with cells 18:2 18:3 20:4 22:6 Cholesterol (Pg/mg protein)
Lipid phosphorus (pglmg protein) PC PE PS PI EPl SM
17.6
18.7
15.7
16.0
16.6
3.88
4.72
3.90
3.69
4.16
54.1 12.6 4.2 . 10.7 15.2 3.2
53.2 12.9 5.2 8.8 17.0 2.9
50.5 13.6 5.2 10.4 16.3 3.9
54.6 12.2 3.5 9.8 16.1 3.8
52.4 11.5 4.9 10.8 16.2 4.2
Each phospholipid is expressed as per cent of total phosphorus. Abbreviations: PC, phosphatidylcholine; PE, phosphaThe fatty acid profiles of unaltered and supple- tidylethanolamine; PS, phosphatidylserine: P1, phosphamented serum have already been published (ROBERT tidylinositol; Epl, ethanolamine plasmogens; SM. sphingomyelin. e t a/., 1977). RESULTS
Fatty acid supplements in neuroblastoma cells TABLE 2. FATTY ACID
COMPOSITION OF TOTAL PHOSPHOGLYCERIDES OF OF PUFA
545
M1 CELLS (LINE a) GROWN 9 days
Control
18:2
18:3
20:4
22:6
23.6 2.5 15.4 17.2 12.2
24.63.2 15.4 20.4 2.4 3.6
23.1 1.9 13.4 17.3 1.3
23: 1 4.8 12.4 33.0
0.8
20: 3w9
24.6 6.6 12.6 38.8 2.0 2.6 0.5 0.4
0.3 0.3
1.5 0.3 0.3
20: 306
0.7
20:406* 20:403 20: 5w3 22 :406 22:5w6 -t 22:4tu3 22: 5w3 22: 6w3 24 :4w6
4.5
3.3 14.0
-
-
-
-
0.7 6.0 1.2 9.6
0.5 0.4 0.4 16.1
0.9
3.4 -
16:O 16: 1 18:O 18: 1 18 :2to6 18:3w3 20: 1
+
20:2
2.6 3.1 -
1.3 2.3 -
2.7 2, I
&.
2.2
IN THE PRESENCE
18:2
+ 18:3
22.2 2.9 14.8 17.9 13.0 4.1 2.4
-
0.8
2.2
4.6
9.5
-
__
0.2
-
0.8
16.9
0.6
0.4 8.2
-
-
1.1 1.2 -
3.1 2.3 0.7
2.2
5.8 2.4
3.0 __
14.’ -
__
* May contain some 20:3w3. Results are expressed as weight percentages. fatty acid desaturation involves the action of a A 4 desaturase the activity of which was not exhibited by the cells. We have observed these findings in two different sublines of the M1 clone, called lines a and a’. Similar results were obtained with another neuroblastoma clone, the NIE 115. However, a third subline of MI clone, that we have called line b, presented striking differences to the other ones (Table 4). The exogenous short-chain PUFA were incorporated in cell phosphoglycerides at higher amounts than in other lines; these PUFA were mostly elongated and very little desaturated to other PUFA. We have studied the effect of the simultaneous addition of both linoleic and linolenic acids in serum to the MI clone (line a) (Table 2) and to NIE 115 clone. In both clones. the incorporation of each PUFA was unchanged, but the proportion of their transformation products was highly reduced. It seemed therefore that each of the two ‘essential’ PUFA could inhibit the desaturation of the other one. The kinetics of the increase of the linoleic and arachidonic acids percentage in cell phosphoglycerides was followed during a period of 9 days in linoleic acid treated cells (Fig. 1). The proportion of the exogenous PUFA increased rapidly and reached its maximum level within 24 h. The percentage of the derived PUFA in contrast increased much more slowly, and was still markedly increasing between day 5 and day 9.
DISCUSSION Several studies have shown that exogenous fatty acids can be taken up by cells in culture and incorporated into phosphoglycerides (WILLIAMSer a/.. 1974; FERGUsOh e f a/.. 1975; HORWITZet a/., 1974; MACKENZIE et al., 1964; SPFCTOR& SOBOROFF, 1971;
BIERMAN & ALBERS, 1975; MENKES,1972; HOWARD & KRITCHEVSKY, 1970; YAWN & MENKES. 1974; & SPECTOR,1974; YAWNet a/., 1975; BRENNEMAN 1965; FILLERUP et a/., 1958; SPECTOR& STEINBERG, RITTENHOUSE et al., 19743. The consequence was sometimes an alteration in cell morphology, consisting frequently of the presence of numerous fatty acid inclusions in the cytoplasm of the cells (GEYER,1967; MOSKOWITZ, 1967). Some authors (WISNIESKI er a/., 1973; WILLIAMS et a/., 1974 & 1975) have stated that exogenous free fatty acids may be toxic for cultured cells growing on serum-free medium. Consequently they used the fatty acids as their tween esters. However we have not seen any alteration to the morphology and growth rate of neuroblastoma cells when serum was supplemented with exogenous non-esterified PUFA. Several authors have used lipid vesicles to induce alterations in animal cell membrane lipid et a/.. 1974; PAGANO er patterns (PAPAHADJOPOULOS a/.. 1974). Such methods d o not seem to be always et a/., compatible with cellular survival (PAGANO 1974). Furthermore, the various mechanisms for the penetration of lipid vesicles into cells have not yet been completely elucidated (PAPAHADJOPOULOS et a/.. 1974) and the modifications which occur concern, simultaneously. various lipid components of the cell membrane. We have preferred. therefore, to add the lipid supplements directly to the culture medium. Another problem was the possible interference between the uptake of fatty acids by the cells from the serum and the de nouo synthesis of fatty acids via the cellular acyl synthetase enzyme complex. In et a/. (1972). VOLPE this respect, BAILEY (1966), BAILEY & MARASA (1975) and ELSBACH (196%. h) showed that cultured cells grown with serum preferentially utilize serum lipids and did not synthesize de noco fatty acids. However. in the present experiments. this interference of de nor0 synthesis has not been checked.
~
~~
~
~
~
~
,
-
~~
0.4 0.8 0.5 0.7 0.2 0.6 0.6 0.6 2.9 0.4 12.0 18.2 8.8 9.1 3.7 9.1 8.1 12.5 16.9 10.1 1.2 4.6 4.9 2.7 1.2 2.7 2.9 2.9 5.3 1.5 24.5 21.3 35.0 42.4 43.5 39.0 42.6 26.3 24.8 36.4 14.2 33.4 34.1 18.7 18.1 19.1 27.3 31.3 14.2 9.8 0.5 1.1 0.9 7.3 0.9 0.6 0.9 0.8 5.9 0.6 0.4 1.4 1.6 i.8 1.5 0.7 1.5 1.7 0.7 0.7 0.1 0.5 1.4 0.5 0.2 4.3 0.4 0.2 1.2 - 4.2 1.0 1.6 0.4 0.2 0.4 - -1.0 1.2 2.1 0.2 0.3 1.6 4.3 2.2 1.1 15.4 2.9 0.7 5.5 1.7 3.2 0.4 9.7 19.4 12.7 -~ -~ -. 7.0 0.7 - 2.6 1.4 12.7 18.0 0.2 1.1 2.2 0.8 13.4 0.3 1.0 5.1 0.3 0.8 4.7 -- 4.1 -4.3 2.0 5.2 2.5 17.9 3.5 2.3 1.3 0.8 4.3 3.5 12.8 3.7 1.5 4.4 2.2 12.0 0.9 0.6 0.7 0.3 .~- - -~ _.
contain some 20:3to3. Results are expressed as weight percentages. The phospholipids are abbreviated as in Table I . The number at the head of each column indicates the PUFA added: ( I ) control; (2) linoleic acid; (3) linolenic acid: (4) arachidonic acid; (5) docosahexaenoic acid.
* May
.
14:O 2.6 2.9 0.9 2.3 2.4 0.8 0.3 0.2 16:O 30.1 32.4 31.2 37.0 33.6 17.1 12.6 9.2 16: I 9.6 5.3 4.0 3.4 7.5 5.1 1.5 1.3 18.0 6.7 9.1 10.8 9.6 7.7 16.3 32.8 28.5 18:l 44.4 19.2 27.5 .22.2 41.0 40.1 18.7 16.2 18:2w6 1.2 12.4 1.9 1.2 1.2 0.9 5.8 0.8 2.5 1.1 3.2 0.8 1.7 2.4 1.0 1.7 18:3tu3 + 20: I -. 0.6 5.9 0.3 20:2 0.4 1.7 0.3 0.3 20: 3to9 0.2 - 0.1 0.3 0.1 1.0 0.3 20: 3016 0.4 2.1 0.4 0.3 0.4 0.7 1.5 0.7 0.8 7.4 3.6 13.6 1.1 5.9 13.1 8.0 20:4~6* - _. 0.9 - - 0.6 20:4tu3 0.2 - 7.8 0.3 1.8 12.4 20: 5013 0.2 4.3 0.4 5.5 0.3 0.2 2.1 0.3 22:4to6 2 2 ~ 5 ~+ 62 2 ~ 4 ~ 0 3 --0.5 0.9 --0.5 0.5 1.1 4.5 1.3 0.4 3.3 2.4 12.7 22:503 0.4 1.0 1.8 0.8 2.3 3.8 2.3 6.2 22 :6013 __ 0.5 __ 24 :4t06
T
-
-
-.
0.4 0.4 . 0.7 10.3 13.3 5.8 1.9 2.0 5.1 38.0 30.6 2.6 10.4 25.8 30.2 0.5 0.5 1.4 0.3 0.9 4.9 -2.1 1.5 0.8 2.4 2.4 0.5 1.3 1.8 28.3 12.8 14.3 - 1.4 5.6 3.4 0.4 1.0 2.3 - 1.8 1.5 11.1 1.1 4.6 11.5
.
0.6 0 9 0.8 1.0 10.1 9.2 0.3 1.7 3.5 3.6 3.9 7.2 3.8 7.8 25.0 0.8 0.6 1.0 1.6 0.4 3.8 0.2 -0.4 -. 0.3 0.7 0.6 - 0.5 1.3 0.3 25.9 8.6 25.7 5.8 - 1.0 - -. - 20.8 1.7 15.3 1.1 29.5 0.5 - 2.3 5.2 -14.1 40.0 7.6 4.7 11.8 13.9 4.9 35.3 1.0 -
0.4 4.8 1.8 7.9 8.2 2.3 4.3 1.2
r
m
Fz
-P
c e
P
W
P
0
4 .
9
8
ZJ
+
Fatty acid supplements in neuroblastoma cells TABLE 4 FATTY ACID CtKIDES OF
MI
COMPOSlTiON OF TOTAL PHOSPHOGLYCELLS (LINF b) GROWN 9 days I N THE PRESENCE OF PUFA
14:O 16:O 16: 1
18:O 18:l 18 : 2106 18:3tu3 20: 1 20:2 20: 3tu9 20: 3 ~ 6 20:3w3 20:4to6 20:4t03 20: 5013 22 :4tu6 22:5tu6 22:4~3 22: 5a,3 22:6tu3
+
Control
18:2
18:3
1.6 21.6 4.6 14.5 37.0 2.4 3.4
1.4 16.7 2.1 15.3
0.7
1.4 20.6 1.8 15.1 16.6 22.5 1.5 5.0
0.5 1.5 5.2
3.6 6.0
+
+
-
-
-
0.7 0.8 0.1 2.4 2.9
-.
15.0 2.2
22.0 0.2 0.1 1.1 10.5
2.0 3.7
1.7
0.8
0.I 2.2 2.0
0.8 3.7 2.2
Results are expressed as weight percentages.
YAWNel ul. (1975) found that when neuroblastorna cells were grown in the presence of [‘“CJinoleic acid. half of the cell fatty acid radioactivity was recovered in linoleic acid. and the other half in 20:3w6 and 20:4w6. When these cells were grown in the presence of [i4C]linolenic acid, however, a little radioactivity was found in the cell linolenic acid. while 20: 5w3 and 22:5m3 were highly labeled and 22:6u3 unlabeled. Our results are in notable agreement with the above report in three respects: 1. The discrepancy existing between the incorporations of exogenous linoleic and linolenic acids; 2. The fact that ethanolarnine plasmalogens are the principal acceptors for PUFA of the 0 3 series:
547
3. The absence of transformation of linoleic and linolenic acid to 22: 5 0 6 and 2 2 . 6 ~ 3respectively. GEYER (1967) and FERGUSON er al. (1975) observed that cultured cell’s lost their capacity to synthesize PUFA DUNBAR& BAILEY (1975) reported that all the transformed cells they analyzed had lost A6 desaturase activity, but had retained A 5 desaturase activity and the ability to elongate PUFA. N o enzyme deletion was found in their normal diploid cells. Our data and that of YAWNel al. (1975) show that neuroblastoma cells, as well as the glial cell lines we have already studied, keep their A 6 and A 5 desaturase activities and the elongation procedures, but have lost the A 4 desaturase activity. In contrast, primary cell cultures converted [i4C]linolenic acid to labeled docosahexaenoic acid (YAWNer al., 1975). It must be underlined however that manipulation such as subcloning can lead to the non-specific loss of desaturase activities, or at least to striking changes in the regulation of elongation and desaturation procedures. When comparing the results obtained by the simultaneous addition of both Iinoleic and linolenic acids to the serum with those obtained when only one of them was added, we can conclude that the presence of linolenic acid inhibited the transformation of Iinoleic acid to other PUFA; and that the presence of linoleic acid inhibited the transformation of linolenic acid to other PUFA. Such findings were already observed in cultured glial cells (ROBERT ei al., 1977). These results were surprising since MOHRHAUER et a/. (1967) and BRENNER & PELUFFO(1966) have reported in rat liver that the affinity of the A 6 desaturase is highe? for linolenic acid than for linoleic acid. Linolenic acid must. therefore, strongly inhibit the desaturation of linoleic a c ~ dI t seemed in contrast that .in our cells both linoleic and linolenic acids could inhibit the desaturation of the other
% I
( CLONE M I , line a
2
0: %
1
5
1
days 9
FIG 1 Kinetics of the incorporation dnd trdnsformation of linoicic acid i n neurObld\tomd cells (clone M I line d ) The percentage of linoleic and ardchidonic acids in cell total phosphoglyceridcs 15 plotted as ‘I function of time The values obtained at 0 and 9 days differ alightly from thosc presented i n Table 2 because of the use of d different batch of fetal calf serum i n this experiment
548
J. ROBERT,G. REBELand P. MANDEL
Our results suggest a rather fast turnover of phosphoglyceride fatty acids, since the linoleic acid percentage reached a plateau within 24 h. Such a rapid turnover of phosphoglycerides in cultured cells was also observed by GALLAHER & BLOUCH(1975). We were, therefore, able to increase markedly the PUFA content of phosphoglycerides in neuroblastoma cells. Since no modifications occurred to the gross lipid composition (in particular no modification of the cholesterol/phospholipid molar ratio), we can expect this procedure may increase the fluidity of :he cell membrane. Such alterations may provide a useful model for studying the influence of membrane physical state on its physiological properties. This is presently under investigation.
GALLAHER W. R. & BLOUCHH. A. (1975) Archs Biochem. Biophys. 168, 104114. GEYERR. P. (1967) in Lipid Metabolism in Tissue Culture G. H. & KRITCHEVSKY D., eds) pp. Cells (ROTHBLAT 3 3 4 7 . Wistar Institute Press, Philadelphia. HIRSCHJ. & AHRENSE. H. (1958) J. biol. Chem. 233, 31 1-320. HORROCKS L. A. (1968) J. Lipid. Res. 9, 469-472. HORWITZA. F.. HATTENM. E. & BURGERM. M. (1974) Proc. natn. Acad. Sci. U.S.A. 71, 3115-3119. D. (1969) Biochim. biophys. HOWARDB. V. & KRITCHEVSKY Acta 187. 293-301. D. (1970) Lipids 5, 49-55. HOWARDB. V. & KRITCHEVSKY C. A. (1953) J. biol. Chem. 203, IDLERD. R. & BAUMANN 389-396. KITAJIMAK., TASHIROS. & NUMAS. (1975) Eur. J . Biochem. 54, 373-383. MACHEBOEUF M. & DELSALJ. (1943) Bull. SOC. Chim. Biol. Acknowledgements-We thank Mr N. BECKERfor his excel25, 116120. lent technical assistance. MACHTICER N. A. & Fox C. F. (1973) Ann. Rev. Biochem. 42, 575-600. MACKENZIE C . G., MACKENZIE J. B. & REISS0. K. (1964) Exptl Cell Res. 36, 533-547. REFERENCES MENKESJ . H. (1972) Lipids 7, 135-141. H., CHRISTIANSEN K., G A N M. V., DEUBIG E. & NIRENRERG M. (1972) Proc. MOHRHAUER AMANOT., RICHELSON M. & HOLMANR. T. (1967) J . biol. Chem. 242, natn. Acad. Sci., U.S.A. 69, 258-263. 4507-4514. BAILEYJ. M. (1966) Biochim. biophys. Acta 125, 226-236. M. S. (1967) in Lipid Metabolism in Tissue BAILEYJ. M., HOWARDB. V., DUNBARL. M. & TILLMAN MOSKOWITZ G . H. & KRITCHEVSKY D., eds.) Culture Cells (RQTHBLAT S. F. (1972) Lipids 7, 125-134. pp. 49-62. Wistar Institute Press, Philadelphia. BARTLETTG. R. (1959) J. biol. Chem. 234, 466-468. J. L., NESKOVIC N. & KOSTICD. (1970) in Les BIERMAN E. L. & ALBERSJ. J. (1975) Biochim. biophys. Acta NUSSBAUM M., ed.) Mutants Pathologiques chez /'Animal (SABOURDY 388, 198-202. pp. 33-55. Editions du C.N.R.S., Paris. BRECKENRIDCE W. C., GOMBOS G. & MORGAN 1. G. (1972) PAGANOR., HUANGL. & WEY C. (1974) Nature 252. Biochim. biophys. Acta 266, 695-707. 166-167. BRENNEMAN D. E. & SPECTORA. A. (1974) J . Lipid. Res. PAPAHADJOPOULOS D., MAYHEWE., POSTEG . , SMITHS. 15, 309-316. & VAILW. J. (1974) Nature 252, 163-165. BRENNERR. R. & PELUFFOR. 0. (1966) J. biol. Chem. H. G.. WILLIAMS R. E., WISNIESKI B. & Fox RITTENHOUSE 241, 5213-5219. C. F. (1974) Biochem. biophys. Res. Commun. 58, 222-228. CIESIELSKI-TRESKA J., WARTERS. & MANDELP. (1975) ROBERTJ., REBEL G. & MANDELP. (1977) Biochimie 59, Neurobiology 5, 382-392. J. (1969) in Handbook of Neurochemistry (LAJTHA 417-423. CLAUSEN J. M. (1971) J . Lipid Res. 12, A., ed.) Vol. 1, pp. 273-300. Plenum Press, New York. SPECTORA. A. & SOBOROFF 545-552. CRONAN J. E.. JR. & VAGELOS P. R. (1972) Biochim. biophys. SPECTORA. A. & STEINBERG D. (1965) J. biol. Chem. 240, act^ 265, 25-60. 3747-3753. DAWSONR. M. C. (1960) Biochem. J. 75, 45-53. VOLPEJ. J. & MARASAJ. C. (1975) J. Neurochem. 25, P. A. &ANDERSON R. E. (1975) Lipids 10, 113-115. DUDLEY 333-340. J. M. (1975) J . biol. Chem. 250, DUNBAR L. M. & BAILEY WILLIAMS R. E., WISNIESKI B. J.. RITTENHOUSE H. G. & 1152-1153. FOXC. F. (1974) Biochemistry 13. 1969-1977. ELSBACHP. (1965a) Biochim. biophys. Acta 98, 402419. WILLIAMS R. E., LI J. K. K. & Fox C. F. (1975) Biochem. ELSBACHP. (1965b) Biochim. biophys. Acta 98, 420-431. FERGUSON K. A., GLASERM., BAYERW. H. & VAGELOS biophys. Res. Commun. 62, 462469. WISNIESKI B. J., WILLIAMS R. E. & FOXC. F. (1973) Proc. P. R. (1975) Biochemistry 14, 146-151. natn. Acad. Sci., U.S.A. 10, 3669-3673. FILLERUP D., MIGLIORE J. & MEADJ. F. (1958) 1. biol. YAWN E. & MENKESJ. H. (1974) Lipids 9. 248-253. Chem. 233, 98-101. J. H. (1975) J. Neurochem. FOLCHJ., LEESM. & SLOANE-STANLEY G. H. (1957) J. biol. YAVINE.. YAWNZ. & MENKES 24. 71-77. Chem. 226, 497-509.
UTILIZATION OF POLYUNSATURATED FATTY ACID SUPPLEMENTS BY CULTURED NEUROBLASTOMA CELLS J. ROBERT.G REBEL*and P. MANDEL Centre de Neurochimie du C N R S and Institut de Chimie Biologique de la Faculte de Medecine. 11. rue Humann, 67085 Strasbourg Cedex, France (Recerrrd 22 March 1977 R e ~ i ~ e7 dJ u l ) 1977 Accepted 9 August 1977)
Abstract-Brain phosphoglycerides are known to contain large amounts of polyunsaturated fatty acids (PUFA) However. neuroblastoma cells contain very low amounts of PUFA Since the serum used for cell culture has been shown to be deficient in PUFA. a supplementation of this serum with various PUFA was undertaken Linoleic. arachidonic and docosahexaenoic acids were incorporated in cell phosphoglycerides. mainly at the expense of oleic acid. while linolenic acid was only poorly incorporated Linoleic. linolenic and arachidonic acids were transformed to their elongation and desaturation products, but the last step of transformation. which involves the action of a A 4 desaturase, was never observed The levels of incorporation and transformation of exogenous PUFA could vary strikingly in different lines of neuroblastoma cells The simultaneous addition of two PUFA (Iinoleic and linolenic acids) was followed by a reduction in the amount of their respective derivatives in cell phosphoglycerides. compared to that obtained when only one PUFA was given to the cells, suggesting d competitive inhibition of the desaturation of each PUFA Such alterations i n membrane lipids may provide a useful model for the study of membrane structure-function relationships
MANYexperiments have been reported recently concerning the relationship between the lipid composition of membranes and their physical and physiological properties Most of this work has been done with microorganisms. especially microbial fatty acid auxo& VAGELOS.1972, MACHTICER & trophs (CRONAY Fox, 1973) The effect of fatty acid alterations on animal membrane properties has recently been incestigated in cultured cells, by adding exogenous fatty et a / , 1973, acids to the culture medium (WISNIESKI WILLIAMS et a / . 1974, FERGUSON et al., 1975) This type of study has rarely been undertaken (HORWITZ et a/., 1974, KITAJIMA et a!.. 1975; WILLIAMS et a / . 1975) on cells which need serum in their culture medium, because serum contains lipid materials which can interfere with the utilization of the fatty acid supplements, and when such cells were used, serum was freed of its lipid components before the fatty acid supplement was added. We have, however. reported recently on the'induction of altered fatty acid patterns in two strains of glial cells in culture, without prior delipidization of the serum (ROBERTet a / , 1977). Cells of nervous origin are of considerable interest tor such investigations because of the particular fatty acid patterns which are found in phospholipids from nerve structures Membrane phospho-
'This work wds supported by a grant from the D G R S T (A C C Membranes biologiques) ' Charge de Recherchc du C N R S Ahhrri iarion u\ed PU FA, polyunsaturated fatty acids 543
lipids from brain (with the exception of myelin) are known to contain larger amounts of polyunsaturated fatty acids (PUFA) than membrane phospholipids from other organs (CLAUSEN, 1969) In particular. the ethanolamine phosphoglycerides from synaptic plasma membranes contain up to 40",, docosahexaenoic acid (BRECKENRIDCE et a/., 1972). Neuroblastoma cells however do not contain large amounts of PUFA, and their phospholipid fatty acid patterns reflect the fatty acid content of the serum. In fact. th fetal calf serum commonly used to grow neuroblastoma cells is largely deficient in PUFA (ROBERT et a/.. 1977) and contains neither short-chain PUFA (I e. 'essential' fatty acids, linoleic acid 18:2w6 and Iinolenic acid 18 30~3).nor long-chain PUFA (the most common of which being arachidonic acid 2 0 . 4 ~ 6in most tissues and docosahexaenoic acid 2 2 . 6 ~ 3 i n brain tissue) It is thus feasible to study PUFA supplementation to these cells. provided that the fatty acids could be introduced in the culture medium without the toxic effects already observed by several authors 1967) The method we (GEYER,1967, MOSKOWITZ. have already used for glial cells was equally applicable to neuroblastoma and without adverse effects on cellular health and integrity We report here alterations to the lipid composition occurring in PUFAsupplemented aeuroblastoma cells Such alterations may provide useful models for studying the lipiddependant properties of cell membranes. and therefore the structureefunction relationship in cell membranes, with special reference to nerre tissue from which our cells originate
J. ROBERT,G. REBELand P. MANDEL
544
MATERIALS AND METHODS Cell culture. Two clonal lines from the C1300 mouse neuroblastoma were used: the NlE 115 clone from Nirenberg's laboratory (AMANOet a/., 1972) and the M1 clone isolated in our laboratory by CIESIELSKI-TRESKA et a[. (1975). The cells were grown in 75cm' Falcon dishes with Eagle-Dulbecco synthetic medium (Gibco) supplemented with 10% fetal calf serum (Gibco) at 37°C in a humidified atmosphere containing 5"/, C 0 2 . The serum was used either unaltered (for controls) or supplemented with a PUFA. This PUFA, as free form, was added directly to the serum by means of a sterile microsyringe (Hamilton). After magnetic stirring for 3 h, the mixture was homogeneous and was used throughout. A control of the new fatty acid profile of the serum was always performed before its use, to ensure that the amount of the added PUFA was constant and approximately equal to the amount of oleic acid present in the serum. In fact, we had to add about 10-12 mg of PUFA in 100 ml serum to reach this concentration. The final molar concentration of exogenous PUFA in contact with the cells was then 3 0 - 4 0 ~ ~In. some experiments we added two PUFA together; the total exogenous PUFA concentration was then doubled. At the beginning of each experiment, the cells were replicated either with unaltered serum and synthetic medium, o r with supplemented serum and synthetic medium. Unless otherwise specified the cells were grown for 9 days under identical conditions, with a replication on the 5th day and two changes of medium o n the third and 7th days. The cells were harvested by scraping with a rubber policeman, washed 3 times in 0.9% NaCl and pelleted. Lipid analysis. Lipids were extracted according to FOLCH et a / . (1957). Fatty acids were prepared by alkaline methanolysis (DAWSON,1960) either from total saponifiable lipids, or from total phosphoglycerides isolated by column chromatography (HIRSCH& AHRENS,1958). or from indiviet al., dual phosphoglycerides isolated by T L C (NUSSBAUM 1970). G L C of fatty acid methyl esters was performed at 180'C using a glass column packed with 100,; EGSS-X on chromosorb W-HP. Chromatographic separation was conducted using a Varian-Aerograph gas-chromatograph using a flame ionization detector. The peaks were identified by comparison with known standards or by semi-logarithmic plotting. When we needed a discrimination between the different kinds of PUFA. fatty acid methyl esters were fractionated on silver-nitrate impregnated plates (DUDLEY & ANDERSON, 1975) and the different fractions were eluted and analysed by GLC. Lipid phosphorus was assayed according t o ' MACHEBOBUF & DELSAL(1943). Cholesterol was assayed with the Liebermann-Burchard reagent, according to IDLER& BAUMANN (1953). Distribution of phospholipids was obtained by phosphorus assay (BARTLETT, 1959) of the individual et spots obtained after TLC in the system of NUSSBAUM al. (1970). Ethanolamine plasmalogens were separated from other ethanolamine phosphoglycerides by an acidic hydrolysis performed directly on the plate between the two migrations (see HORROCKS,1968).
Under the conditions used, no changes were detected in the morphology of the cells, nor in their growth characteristics in the presence of any added PUFA. No lipid inclusions were seen in the cellular cytoplasm. The phospholipid and cholesterol content of the cells remained constant, and the relative proportions of each phospholipid were not significantly modified (Table 1). The fatty acid compositions of total phosphoglycerides (Table 2) or of individual phosphoglycerides (Table 3) showed great variation when an exogenous PUFA was added to the serum: 1. Each added PUFA was incorporated at different levels in cell phosphoglycerides, except for linolenic acid which was very poorly incorporated in comparison with other PUFA. 2. The added PUFA were transformed t o their elongation and desaturation products, the percentage of which increased in the cell phosphoglycerides. 18:2w6 was transformed to 20:2w6, 20:4w6 and 22:4w6, 18:303 was transformed to 20:303, 20:403, 20: 5w3 and 22:5w3, 20:4w6 was transformed to 22:4w6 and 24:4w6. N o transformation products could be detected when 22:6w3 was added to the serum. Ethanolamine plasmalogens were the phospholipid fraction into which most of the newly formed PUFA were incorporated, mainly for the a 3 series. 3. The increase observed in the PUFA content of the cell phosphoglycerides were obtained almost exclusively at the expense of the monounsaturated series, the percentage of the saturated fatty acids remaining relatively constant or even increased in the case of P E in the presence of 18:2w6. 4. The added PUFA underwent transformation up until the final step; 22:5w6 was only very slightly increased when 18:2w6 or 20:4w6 were added to the serum; 22:6w3 was not significantly increased when 18:3w3 was added to the serum. The last step of the
TABLE1. LIPIDCOMPOSITION OF MI CELLS (LINE a) 9 days IN THE PRESENCE OF P U F A
GROWN
Control Cells treated with cells 18:2 18:3 20:4 22:6 Cholesterol (Pg/mg protein)
Lipid phosphorus (pglmg protein) PC PE PS PI EPl SM
17.6
18.7
15.7
16.0
16.6
3.88
4.72
3.90
3.69
4.16
54.1 12.6 4.2 . 10.7 15.2 3.2
53.2 12.9 5.2 8.8 17.0 2.9
50.5 13.6 5.2 10.4 16.3 3.9
54.6 12.2 3.5 9.8 16.1 3.8
52.4 11.5 4.9 10.8 16.2 4.2
Each phospholipid is expressed as per cent of total phosphorus. Abbreviations: PC, phosphatidylcholine; PE, phosphaThe fatty acid profiles of unaltered and supple- tidylethanolamine; PS, phosphatidylserine: P1, phosphamented serum have already been published (ROBERT tidylinositol; Epl, ethanolamine plasmogens; SM. sphingomyelin. e t a/., 1977). RESULTS
Fatty acid supplements in neuroblastoma cells TABLE 2. FATTY ACID
COMPOSITION OF TOTAL PHOSPHOGLYCERIDES OF OF PUFA
545
M1 CELLS (LINE a) GROWN 9 days
Control
18:2
18:3
20:4
22:6
23.6 2.5 15.4 17.2 12.2
24.63.2 15.4 20.4 2.4 3.6
23.1 1.9 13.4 17.3 1.3
23: 1 4.8 12.4 33.0
0.8
20: 3w9
24.6 6.6 12.6 38.8 2.0 2.6 0.5 0.4
0.3 0.3
1.5 0.3 0.3
20: 306
0.7
20:406* 20:403 20: 5w3 22 :406 22:5w6 -t 22:4tu3 22: 5w3 22: 6w3 24 :4w6
4.5
3.3 14.0
-
-
-
-
0.7 6.0 1.2 9.6
0.5 0.4 0.4 16.1
0.9
3.4 -
16:O 16: 1 18:O 18: 1 18 :2to6 18:3w3 20: 1
+
20:2
2.6 3.1 -
1.3 2.3 -
2.7 2, I
&.
2.2
IN THE PRESENCE
18:2
+ 18:3
22.2 2.9 14.8 17.9 13.0 4.1 2.4
-
0.8
2.2
4.6
9.5
-
__
0.2
-
0.8
16.9
0.6
0.4 8.2
-
-
1.1 1.2 -
3.1 2.3 0.7
2.2
5.8 2.4
3.0 __
14.’ -
__
* May contain some 20:3w3. Results are expressed as weight percentages. fatty acid desaturation involves the action of a A 4 desaturase the activity of which was not exhibited by the cells. We have observed these findings in two different sublines of the M1 clone, called lines a and a’. Similar results were obtained with another neuroblastoma clone, the NIE 115. However, a third subline of MI clone, that we have called line b, presented striking differences to the other ones (Table 4). The exogenous short-chain PUFA were incorporated in cell phosphoglycerides at higher amounts than in other lines; these PUFA were mostly elongated and very little desaturated to other PUFA. We have studied the effect of the simultaneous addition of both linoleic and linolenic acids in serum to the MI clone (line a) (Table 2) and to NIE 115 clone. In both clones. the incorporation of each PUFA was unchanged, but the proportion of their transformation products was highly reduced. It seemed therefore that each of the two ‘essential’ PUFA could inhibit the desaturation of the other one. The kinetics of the increase of the linoleic and arachidonic acids percentage in cell phosphoglycerides was followed during a period of 9 days in linoleic acid treated cells (Fig. 1). The proportion of the exogenous PUFA increased rapidly and reached its maximum level within 24 h. The percentage of the derived PUFA in contrast increased much more slowly, and was still markedly increasing between day 5 and day 9.
DISCUSSION Several studies have shown that exogenous fatty acids can be taken up by cells in culture and incorporated into phosphoglycerides (WILLIAMSer a/.. 1974; FERGUsOh e f a/.. 1975; HORWITZet a/., 1974; MACKENZIE et al., 1964; SPFCTOR& SOBOROFF, 1971;
BIERMAN & ALBERS, 1975; MENKES,1972; HOWARD & KRITCHEVSKY, 1970; YAWN & MENKES. 1974; & SPECTOR,1974; YAWNet a/., 1975; BRENNEMAN 1965; FILLERUP et a/., 1958; SPECTOR& STEINBERG, RITTENHOUSE et al., 19743. The consequence was sometimes an alteration in cell morphology, consisting frequently of the presence of numerous fatty acid inclusions in the cytoplasm of the cells (GEYER,1967; MOSKOWITZ, 1967). Some authors (WISNIESKI er a/., 1973; WILLIAMS et a/., 1974 & 1975) have stated that exogenous free fatty acids may be toxic for cultured cells growing on serum-free medium. Consequently they used the fatty acids as their tween esters. However we have not seen any alteration to the morphology and growth rate of neuroblastoma cells when serum was supplemented with exogenous non-esterified PUFA. Several authors have used lipid vesicles to induce alterations in animal cell membrane lipid et a/.. 1974; PAGANO er patterns (PAPAHADJOPOULOS a/.. 1974). Such methods d o not seem to be always et a/., compatible with cellular survival (PAGANO 1974). Furthermore, the various mechanisms for the penetration of lipid vesicles into cells have not yet been completely elucidated (PAPAHADJOPOULOS et a/.. 1974) and the modifications which occur concern, simultaneously. various lipid components of the cell membrane. We have preferred. therefore, to add the lipid supplements directly to the culture medium. Another problem was the possible interference between the uptake of fatty acids by the cells from the serum and the de nouo synthesis of fatty acids via the cellular acyl synthetase enzyme complex. In et a/. (1972). VOLPE this respect, BAILEY (1966), BAILEY & MARASA (1975) and ELSBACH (196%. h) showed that cultured cells grown with serum preferentially utilize serum lipids and did not synthesize de noco fatty acids. However. in the present experiments. this interference of de nor0 synthesis has not been checked.
~
~~
~
~
~
~
,
-
~~
0.4 0.8 0.5 0.7 0.2 0.6 0.6 0.6 2.9 0.4 12.0 18.2 8.8 9.1 3.7 9.1 8.1 12.5 16.9 10.1 1.2 4.6 4.9 2.7 1.2 2.7 2.9 2.9 5.3 1.5 24.5 21.3 35.0 42.4 43.5 39.0 42.6 26.3 24.8 36.4 14.2 33.4 34.1 18.7 18.1 19.1 27.3 31.3 14.2 9.8 0.5 1.1 0.9 7.3 0.9 0.6 0.9 0.8 5.9 0.6 0.4 1.4 1.6 i.8 1.5 0.7 1.5 1.7 0.7 0.7 0.1 0.5 1.4 0.5 0.2 4.3 0.4 0.2 1.2 - 4.2 1.0 1.6 0.4 0.2 0.4 - -1.0 1.2 2.1 0.2 0.3 1.6 4.3 2.2 1.1 15.4 2.9 0.7 5.5 1.7 3.2 0.4 9.7 19.4 12.7 -~ -~ -. 7.0 0.7 - 2.6 1.4 12.7 18.0 0.2 1.1 2.2 0.8 13.4 0.3 1.0 5.1 0.3 0.8 4.7 -- 4.1 -4.3 2.0 5.2 2.5 17.9 3.5 2.3 1.3 0.8 4.3 3.5 12.8 3.7 1.5 4.4 2.2 12.0 0.9 0.6 0.7 0.3 .~- - -~ _.
contain some 20:3to3. Results are expressed as weight percentages. The phospholipids are abbreviated as in Table I . The number at the head of each column indicates the PUFA added: ( I ) control; (2) linoleic acid; (3) linolenic acid: (4) arachidonic acid; (5) docosahexaenoic acid.
* May
.
14:O 2.6 2.9 0.9 2.3 2.4 0.8 0.3 0.2 16:O 30.1 32.4 31.2 37.0 33.6 17.1 12.6 9.2 16: I 9.6 5.3 4.0 3.4 7.5 5.1 1.5 1.3 18.0 6.7 9.1 10.8 9.6 7.7 16.3 32.8 28.5 18:l 44.4 19.2 27.5 .22.2 41.0 40.1 18.7 16.2 18:2w6 1.2 12.4 1.9 1.2 1.2 0.9 5.8 0.8 2.5 1.1 3.2 0.8 1.7 2.4 1.0 1.7 18:3tu3 + 20: I -. 0.6 5.9 0.3 20:2 0.4 1.7 0.3 0.3 20: 3to9 0.2 - 0.1 0.3 0.1 1.0 0.3 20: 3016 0.4 2.1 0.4 0.3 0.4 0.7 1.5 0.7 0.8 7.4 3.6 13.6 1.1 5.9 13.1 8.0 20:4~6* - _. 0.9 - - 0.6 20:4tu3 0.2 - 7.8 0.3 1.8 12.4 20: 5013 0.2 4.3 0.4 5.5 0.3 0.2 2.1 0.3 22:4to6 2 2 ~ 5 ~+ 62 2 ~ 4 ~ 0 3 --0.5 0.9 --0.5 0.5 1.1 4.5 1.3 0.4 3.3 2.4 12.7 22:503 0.4 1.0 1.8 0.8 2.3 3.8 2.3 6.2 22 :6013 __ 0.5 __ 24 :4t06
T
-
-
-.
0.4 0.4 . 0.7 10.3 13.3 5.8 1.9 2.0 5.1 38.0 30.6 2.6 10.4 25.8 30.2 0.5 0.5 1.4 0.3 0.9 4.9 -2.1 1.5 0.8 2.4 2.4 0.5 1.3 1.8 28.3 12.8 14.3 - 1.4 5.6 3.4 0.4 1.0 2.3 - 1.8 1.5 11.1 1.1 4.6 11.5
.
0.6 0 9 0.8 1.0 10.1 9.2 0.3 1.7 3.5 3.6 3.9 7.2 3.8 7.8 25.0 0.8 0.6 1.0 1.6 0.4 3.8 0.2 -0.4 -. 0.3 0.7 0.6 - 0.5 1.3 0.3 25.9 8.6 25.7 5.8 - 1.0 - -. - 20.8 1.7 15.3 1.1 29.5 0.5 - 2.3 5.2 -14.1 40.0 7.6 4.7 11.8 13.9 4.9 35.3 1.0 -
0.4 4.8 1.8 7.9 8.2 2.3 4.3 1.2
r
m
Fz
-P
c e
P
W
P
0
4 .
9
8
ZJ
+
Fatty acid supplements in neuroblastoma cells TABLE 4 FATTY ACID CtKIDES OF
MI
COMPOSlTiON OF TOTAL PHOSPHOGLYCELLS (LINF b) GROWN 9 days I N THE PRESENCE OF PUFA
14:O 16:O 16: 1
18:O 18:l 18 : 2106 18:3tu3 20: 1 20:2 20: 3tu9 20: 3 ~ 6 20:3w3 20:4to6 20:4t03 20: 5013 22 :4tu6 22:5tu6 22:4~3 22: 5a,3 22:6tu3
+
Control
18:2
18:3
1.6 21.6 4.6 14.5 37.0 2.4 3.4
1.4 16.7 2.1 15.3
0.7
1.4 20.6 1.8 15.1 16.6 22.5 1.5 5.0
0.5 1.5 5.2
3.6 6.0
+
+
-
-
-
0.7 0.8 0.1 2.4 2.9
-.
15.0 2.2
22.0 0.2 0.1 1.1 10.5
2.0 3.7
1.7
0.8
0.I 2.2 2.0
0.8 3.7 2.2
Results are expressed as weight percentages.
YAWNel ul. (1975) found that when neuroblastorna cells were grown in the presence of [‘“CJinoleic acid. half of the cell fatty acid radioactivity was recovered in linoleic acid. and the other half in 20:3w6 and 20:4w6. When these cells were grown in the presence of [i4C]linolenic acid, however, a little radioactivity was found in the cell linolenic acid. while 20: 5w3 and 22:5m3 were highly labeled and 22:6u3 unlabeled. Our results are in notable agreement with the above report in three respects: 1. The discrepancy existing between the incorporations of exogenous linoleic and linolenic acids; 2. The fact that ethanolarnine plasmalogens are the principal acceptors for PUFA of the 0 3 series:
547
3. The absence of transformation of linoleic and linolenic acid to 22: 5 0 6 and 2 2 . 6 ~ 3respectively. GEYER (1967) and FERGUSON er al. (1975) observed that cultured cell’s lost their capacity to synthesize PUFA DUNBAR& BAILEY (1975) reported that all the transformed cells they analyzed had lost A6 desaturase activity, but had retained A 5 desaturase activity and the ability to elongate PUFA. N o enzyme deletion was found in their normal diploid cells. Our data and that of YAWNel al. (1975) show that neuroblastoma cells, as well as the glial cell lines we have already studied, keep their A 6 and A 5 desaturase activities and the elongation procedures, but have lost the A 4 desaturase activity. In contrast, primary cell cultures converted [i4C]linolenic acid to labeled docosahexaenoic acid (YAWNer al., 1975). It must be underlined however that manipulation such as subcloning can lead to the non-specific loss of desaturase activities, or at least to striking changes in the regulation of elongation and desaturation procedures. When comparing the results obtained by the simultaneous addition of both Iinoleic and linolenic acids to the serum with those obtained when only one of them was added, we can conclude that the presence of linolenic acid inhibited the transformation of Iinoleic acid to other PUFA; and that the presence of linoleic acid inhibited the transformation of linolenic acid to other PUFA. Such findings were already observed in cultured glial cells (ROBERT ei al., 1977). These results were surprising since MOHRHAUER et a/. (1967) and BRENNER & PELUFFO(1966) have reported in rat liver that the affinity of the A 6 desaturase is highe? for linolenic acid than for linoleic acid. Linolenic acid must. therefore, strongly inhibit the desaturation of linoleic a c ~ dI t seemed in contrast that .in our cells both linoleic and linolenic acids could inhibit the desaturation of the other
% I
( CLONE M I , line a
2
0: %
1
5
1
days 9
FIG 1 Kinetics of the incorporation dnd trdnsformation of linoicic acid i n neurObld\tomd cells (clone M I line d ) The percentage of linoleic and ardchidonic acids in cell total phosphoglyceridcs 15 plotted as ‘I function of time The values obtained at 0 and 9 days differ alightly from thosc presented i n Table 2 because of the use of d different batch of fetal calf serum i n this experiment
548
J. ROBERT,G. REBELand P. MANDEL
Our results suggest a rather fast turnover of phosphoglyceride fatty acids, since the linoleic acid percentage reached a plateau within 24 h. Such a rapid turnover of phosphoglycerides in cultured cells was also observed by GALLAHER & BLOUCH(1975). We were, therefore, able to increase markedly the PUFA content of phosphoglycerides in neuroblastoma cells. Since no modifications occurred to the gross lipid composition (in particular no modification of the cholesterol/phospholipid molar ratio), we can expect this procedure may increase the fluidity of :he cell membrane. Such alterations may provide a useful model for studying the influence of membrane physical state on its physiological properties. This is presently under investigation.
GALLAHER W. R. & BLOUCHH. A. (1975) Archs Biochem. Biophys. 168, 104114. GEYERR. P. (1967) in Lipid Metabolism in Tissue Culture G. H. & KRITCHEVSKY D., eds) pp. Cells (ROTHBLAT 3 3 4 7 . Wistar Institute Press, Philadelphia. HIRSCHJ. & AHRENSE. H. (1958) J. biol. Chem. 233, 31 1-320. HORROCKS L. A. (1968) J. Lipid. Res. 9, 469-472. HORWITZA. F.. HATTENM. E. & BURGERM. M. (1974) Proc. natn. Acad. Sci. U.S.A. 71, 3115-3119. D. (1969) Biochim. biophys. HOWARDB. V. & KRITCHEVSKY Acta 187. 293-301. D. (1970) Lipids 5, 49-55. HOWARDB. V. & KRITCHEVSKY C. A. (1953) J. biol. Chem. 203, IDLERD. R. & BAUMANN 389-396. KITAJIMAK., TASHIROS. & NUMAS. (1975) Eur. J . Biochem. 54, 373-383. MACHEBOEUF M. & DELSALJ. (1943) Bull. SOC. Chim. Biol. Acknowledgements-We thank Mr N. BECKERfor his excel25, 116120. lent technical assistance. MACHTICER N. A. & Fox C. F. (1973) Ann. Rev. Biochem. 42, 575-600. MACKENZIE C . G., MACKENZIE J. B. & REISS0. K. (1964) Exptl Cell Res. 36, 533-547. REFERENCES MENKESJ . H. (1972) Lipids 7, 135-141. H., CHRISTIANSEN K., G A N M. V., DEUBIG E. & NIRENRERG M. (1972) Proc. MOHRHAUER AMANOT., RICHELSON M. & HOLMANR. T. (1967) J . biol. Chem. 242, natn. Acad. Sci., U.S.A. 69, 258-263. 4507-4514. BAILEYJ. M. (1966) Biochim. biophys. Acta 125, 226-236. M. S. (1967) in Lipid Metabolism in Tissue BAILEYJ. M., HOWARDB. V., DUNBARL. M. & TILLMAN MOSKOWITZ G . H. & KRITCHEVSKY D., eds.) Culture Cells (RQTHBLAT S. F. (1972) Lipids 7, 125-134. pp. 49-62. Wistar Institute Press, Philadelphia. BARTLETTG. R. (1959) J. biol. Chem. 234, 466-468. J. L., NESKOVIC N. & KOSTICD. (1970) in Les BIERMAN E. L. & ALBERSJ. J. (1975) Biochim. biophys. Acta NUSSBAUM M., ed.) Mutants Pathologiques chez /'Animal (SABOURDY 388, 198-202. pp. 33-55. Editions du C.N.R.S., Paris. BRECKENRIDCE W. C., GOMBOS G. & MORGAN 1. G. (1972) PAGANOR., HUANGL. & WEY C. (1974) Nature 252. Biochim. biophys. Acta 266, 695-707. 166-167. BRENNEMAN D. E. & SPECTORA. A. (1974) J . Lipid. Res. PAPAHADJOPOULOS D., MAYHEWE., POSTEG . , SMITHS. 15, 309-316. & VAILW. J. (1974) Nature 252, 163-165. BRENNERR. R. & PELUFFOR. 0. (1966) J. biol. Chem. H. G.. WILLIAMS R. E., WISNIESKI B. & Fox RITTENHOUSE 241, 5213-5219. C. F. (1974) Biochem. biophys. Res. Commun. 58, 222-228. CIESIELSKI-TRESKA J., WARTERS. & MANDELP. (1975) ROBERTJ., REBEL G. & MANDELP. (1977) Biochimie 59, Neurobiology 5, 382-392. J. (1969) in Handbook of Neurochemistry (LAJTHA 417-423. CLAUSEN J. M. (1971) J . Lipid Res. 12, A., ed.) Vol. 1, pp. 273-300. Plenum Press, New York. SPECTORA. A. & SOBOROFF 545-552. CRONAN J. E.. JR. & VAGELOS P. R. (1972) Biochim. biophys. SPECTORA. A. & STEINBERG D. (1965) J. biol. Chem. 240, act^ 265, 25-60. 3747-3753. DAWSONR. M. C. (1960) Biochem. J. 75, 45-53. VOLPEJ. J. & MARASAJ. C. (1975) J. Neurochem. 25, P. A. &ANDERSON R. E. (1975) Lipids 10, 113-115. DUDLEY 333-340. J. M. (1975) J . biol. Chem. 250, DUNBAR L. M. & BAILEY WILLIAMS R. E., WISNIESKI B. J.. RITTENHOUSE H. G. & 1152-1153. FOXC. F. (1974) Biochemistry 13. 1969-1977. ELSBACHP. (1965a) Biochim. biophys. Acta 98, 402419. WILLIAMS R. E., LI J. K. K. & Fox C. F. (1975) Biochem. ELSBACHP. (1965b) Biochim. biophys. Acta 98, 420-431. FERGUSON K. A., GLASERM., BAYERW. H. & VAGELOS biophys. Res. Commun. 62, 462469. WISNIESKI B. J., WILLIAMS R. E. & FOXC. F. (1973) Proc. P. R. (1975) Biochemistry 14, 146-151. natn. Acad. Sci., U.S.A. 10, 3669-3673. FILLERUP D., MIGLIORE J. & MEADJ. F. (1958) 1. biol. YAWN E. & MENKESJ. H. (1974) Lipids 9. 248-253. Chem. 233, 98-101. J. H. (1975) J. Neurochem. FOLCHJ., LEESM. & SLOANE-STANLEY G. H. (1957) J. biol. YAVINE.. YAWNZ. & MENKES 24. 71-77. Chem. 226, 497-509.
