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The effect of two isomeric octadecenoic acids on the lipid metabolism and growth of Novikoff hepatoma cells
469
Biochimica et Biophysics Acta, 431 (1976) 469-480 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56786
THE EFFECT OF TWO ISOMERIC OCTADECENOIC ACIDS ON THE LIPID METABOLISM AND GROWTH OF NOVIKOFF HEPATOMA CELLS
DAVID
E. WENNERSTROM
* and HOWARD
M. JENKIN
The Hormel Institute, University of Minnesota, Austin, Minn. 55912 (Received
November
(U.S.A.)
lOth, 1975)
Summary
The origin and metabolism of octadecenoic acid (18 : 1) was examined in intact Novikoff rat hepatoma cells by using labeled precursors and two isomeric octadecenoic acids which differed in their abilities to stimulate cell growth in a serum-free medium. The isomers (cis-6-18 : 1 and cis-9-18 : 1) were measured in the cellular lipid by ozonolysis and reduction of the ozonides. The results indicate that the 18 : 1 fatty acid accumulated in the cell lipid by uptake of the preformed acid from the medium. The ~5-6-18 : 1 was more extensively metabolized than the cis-9-18 : 1 to 16 : 1 and 20 : 1 fatty acids by chain shortening and chain elongation. Both isomers inhibited de novo fatty acid synthesis from acetate by cells suspended in a serum-free medium. The isomers did not exert coordinate control of both fatty acid and cholesterol biosynthesis in the Novikoff cells.
Introduction
Analysis of the fatty acid composition of a number of normal and tumorigenic cells has shown that the cellular content of octadecenoic acid (18 : 1) is increased in transformed cells [l--3], and this increase appears to be related to transformation. For instance, the fatty acid composition of the Novikoff rat hepatoma contains a markedly increased level of 18 : 1 compared to both normal and host liver [4]. Nevertheless, little work has been done in elucidating the importance of the increased level of 18 : 1 in tumorigenic cells. In addition, hepatomas have been reported to lack the ability of normal liver to regulate lipogenesis in response to changes in lipid supply even though the isolated en* Present address: Department of Microbiology. Little Rock. Ark. 72201. U.S.A.
The University of Arkansas for Medical Sciences.
470
zymes are identical to those of normal liver with respect to cofactor requirements and inhibition by long chain fatty acids [ 51. The purpose of this study was to investigate the origin and metabolism of 18 : 1 in intact tumorigenic cells and to evaluate its physiological significance as measured by its effects on cell growth. Novikoff hepatoma cells in suspension culture were exposed to two positional isomers of 18 : 1 which differ in their metabolism and their effects of cell growth. The ability of these fatty acids to regulate de novo synthesis of fatty acids in the intact cell was also observed. Experimental Materials. Swim’s 67-G powdered medium and meat peptone were obtained from Grand Island Biological Co., Grand Island, N.Y.; bovine serum albumin (fatty acid-free bovine serum albumin) was purchased from Miles Laboratories, Kanakakee, Ill.; fatty acids and authentic lipid standards were purchased from NuCheck Prep, Inc., Elysian, Minn.; glycerol ether diester was a gift from Dr. W.J. Baumann, The Hormel Institute; Silica Gel H was obtained from E. Merck AG, Darmstad, Germany; sodium[ l-14C]acetate (62 Ci/mol) and [ l-14C]oleic acid (cis-9-18 : 1, 58 Ci/mol) were purchased from Amersham-Searle Inc., Arlington Heights, Ill.; Permablend scintillator was purchased from Packard Instrument Co., Downers Grove, Ill. Cell culture. Novikoff hepatoma cells (subline NISI-67) were obtained from Dr. P.G.W. Plagemann, University of Minnesota, and grown in suspension in Swim’s 67-G medium containing 5% newborn calf serum [ 61, except that the pancreatic autolysate was replaced by 2.0 g/l of meat peptone. The cells were enumerated with a Coulter counter. In each experiment, cells were incubated in duplicate flasks containing medium supplemented with 2 mg/ml fatty acidfree bovine serum albumin or bovine serum albumin complexed to the sodium salt of an octadecenoic acid (cis-6-18 : 1 or cis-9-18 : 1). The sodium salts of the fatty acids were prepared [6] and were complexed to the bovine serum albumin according to Spector et al. [7]. A starting density of 2 - 10’ hepatoma cells was incubated for 45-48 h at 36°C. Lipid analysis. Following incubation, the cells were sedimented by centrifugation at 300 X g at 4°C and washed twice with a cold serum-free and peptonefree salt solution which was identical in composition to 67-G medium except containing 5 mM glucose, 0.1 mM sodium acetate, and 20 mM N-2-hydroxyethyl-piperazine-l\r’S-ethanesulfonic acid, pH 7.2. This solution was designated 67SS. The cells were resuspended in water and the lipid extracted by the method of Bligh and Dyer as modified by Kates [ 81. The chloroform layer was evaporated to dryness under a stream of Nz and the lipid immediately dissolved in a measured amount of chloroform. Aliquot samples of total lipid were removed for gravimetric measurement of total lipid weight. The remaining lipid was transesterified in 5% HCl in methanol at 95°C for 2 h after the addition to all samples of a constant amount of heneicosanoic acid methyl ester (21 : 0) as an internal standard. Total fatty acid methyl esters were extracted three times by the addition of light petroleum and water, separated by thin-layer chromatography in a solvent system of light
471
petroleum/diethyl ether/glacial acetic acid (90 : 15 : 2, v/v) and analyzed by gas-liquid chromatography. The gas-liquid chromatography was performed on a Victoreen 4000 series instrument equipped with a flame ionization detector and an 8 ft X l/S inch aluminum column packed with 15% ethylene glycol succinate (EGSS-X) on Gas Chrom P 100/120. Helium was the carrier gas, and the column temperature was 170°C. To determine the isomeric composition of monounsaturated fatty acids present in the total lipid of Novikoff cells, the 16 : 1, 18 : 1, and 20 : 1 methyl esters were collected by preparative gas-liquid chromato~aphy using a Beckmen GC-2A instrument equipped with a thermal conductivity detector and the same type of column as the Victoreen instrument. Ozonolysis was performed on the isolated methyl esters as described by Beroza and Bier1 [9] using redistilled pentane as the solvent. Ozonolysis products were detected as described for the methyl esters except that the column temperature was pro~ammed from 100 to 155°C at 3”C/min. The aldehydes and aldesters were identified by comparison with chromatograms of ozonized cis-6-18 : 1; cis-9-18 : 1, and cis11-18 : 1 methyl esters as standards. Incorporation of radioactivity in Novikoff cell lipids. Novikoff cells in the exponental phase of growth were sedimented at 300 Xg for 10 min, washed twice in 67-SS, and resuspended at 1 - lo6 cells/ml in replicate ffasks cont~ning 67-SS with the appropriate fatty acid. After the addition of [1-‘4C]acetate (0.6 &i/ml in all experiments) or l-*4C-labeled cis-9-18 : 1, and incubation at 36”C, the cell suspensions were rapidly cooled in ice, a loo-fold excess of unlabeled sodium acetate was added when [l-*4C]acetate was the labeled precursor, and the cells were sedimented at 300 X g at 4°C. The cells were washed once with 67-SS, resuspended in water, and lipids were extracted. Replicate samples of labeled lipid were applied to 500 E.tm thick Silica Gel H plates together with authentic neutral lipid standards and the lipids were separated by thin-layer chromatography using a solvent system of light petroleum/diethyl ether/glacial acetic acid (90 : 15 : 2, v/v). The lipids were located by staining with iodine vapor. The silica gel bands of the separated lipids were scraped into scintillation vials and the radioactivity was determined by liquid scintillation spectroscopy (Packard Model 2405) using 10 ml of toluene-based scintillation fluid. Methanol (0.5 ml) was added to each vial to help solubilize the lipids. The counting efficiency was 85%. Rest&s The effect of octadecenoic acid isomers on cellgrowth
The Novikoff hepatoma cells grew exponentailly for 40-48 h when they were suspended in the complete growth medium at an initial concentration of 2 - lo5 cells/ml. The population density at 48 h was 8-lo-fold that of the inoculum, Table I shows that cell growth was only about 3.5 times the inoclum after 40 h of growth in medium without serum compared to a 7-fold in the complete medium. The addition of lo-40 Erg/ml cis-9-18 : 1 to the medium stimulated cell growth to approx. 80% of that observed in medium containing serum. The fatty acid was effective in stimulation of growth at the lowest con~ntration tested. ~though the response to increasing doses was minimal, 20
472 TABLE
I
THE DIFFERENTIAL EFFECT OF OCTADECENOIC ACID ISOMERS ON THE GROWTH OF NOVIKOFF HEPATOMA CELLS IN SERUM-FREE
cis-6-18 : MEDIUM
1
AND
cis-9-18
: 1
Supplement added to the growth medium which was serum free. All flasks contained 2 mgjml of fatty acid-free bovine serum albumin. Growth was measured as the increase in the number of cells over the inoculum during an incubation at 36’C for 40 h. Each value is the average of duplicate samples. Supplement
Fatty acid concentration
Growth (-fold the mean
increase in number
of cells ? difference
from
(pg/mI) Experiment
__None
1
Experiment
0
3.45 i 0.04
3.79 ? 0.45
cis-6-18
:1
10 20 40
N.D. 3.54 + 0.63 2.90 * 0.05
3.86 I 0.11 3.58 i 0.21 3.20 + 0.36
cis-9-18
:1
10 20 40
N.D. 5.34 + 0.04 5.01 * 0.17
5.76 ? 0.56 6.56 i 0.48 5.61 f 0.47
20 N.D.
N.D. N.D.
6.97 + 0.05
cis-6-18 : 1 Serum (5%) N.D. Not Determined.
2
-__
5.02 ?: 0.23 -__-
pg/ml appeared to be the most effective in promoting growth. In contrast, all concentrations of the cis-6-18 : 1 were completely without effect in promoting cell growth. The cis-6-18 : 1 did not inhibit cell growth and did not significantly affect the stimulation of the cis-9-18 : 1 when the two isomers were combined in equal amounts (20 pg/ml each). Origin and metabolism of octadecenoic acids in Novikoff cells The ability of the cis-9-18 : 1 to serve as a growth factor suggested
that it
30
20
1
2
4
I
I
6
8
Time (hrs) Fig. 1. The time course of de nova lipid synthesis from acetate and uptake of sodium oleate in Novikoff hepatoma c&s. Duplicate flasks ware inoculated with 1 * lo6 Novikoff hepatoma cells/ml of 67-SS and incubated at 36’C in the presence of [1-14C]acetate (specific activity 2.7 Ci/mol) or sodium [1-14C]oleate (cis-9-18 : 1, specific activity 7.1 Ci/mol). Each point is the average of duplicate samples of 1 . 10’ceIIs.
473
was readily incorporated into the Novikoff cell lipid. The origin of the 18 : 1 in cellular lipid and its metabolism by the Novikoff cellwas investigated by using [1-14C]acetate to measure to de novo synthesis and by employing 1-14C-labeled cis-9-18 : 1 as an exogenous source of fatty acid. The labeled acetate served as a useful indicator for lipogenesis because neither Novikoff cells nor Novikoff tumor homogenates can be demonstrated to oxidize long chain fatty acids (Wennerstrom, D. and Jenkin, H., unpublished data; ref. 10) and because lactate is the principal product of glycolysis [II]. The results in Fig. 1 show that acetate conversion to lipid occurred at a constant rate for 8 h when the cells were suspended in the lipid-free 6’7-5s. In contrast, the uptake and incorporation of the l-14C-labeled c&9-18 : 1 was not linear. The decreasing rate of fatty acid incorporation was not caused by depletion of oleate from the medium because the total activity incorporated at 4 h represented 25% of the activity supplied. The rate change is presumably caused by the differences in the affinity of the bovine serum albumin binding sites for the fatty acid [123. Incorporation of labeled cis-9-18 : 1 into cell lipid at 1 h was 55 times greater than’incorporation of label from acetate after correction for differences in specific activity. The distribution of isotope in the complex lipids labeled from the two sources was also compared (Table II). Phospholipid was the major lipid labeled with 47 and 57% of the label obtained from cis-9-18 : 1 and acetate, respectively. The triacylglycerol (37%) and free fatty acid (4.8%) fractions contained a greater proportion of label from cis-9-18 : 1 whereas the sterol fraction (4.2%) and the sterol ester (10.5%) were more extensively labeled from acetate. Comparison of the labeling pattern with gravimetric analysis of the neutral lipids and phospholipids and the fatty acids of each lipid class by gas-liquid chromatography showed that the labeling of the complex lipid classes occurred in proportion to the amount present in the cells. The distribution of label in the TABLE II THE LABELING PATTERN OF LIPIDS IN NOVIKOFF HEPATOMA CELLS USING [l-*4C]ACETATE AND [1-‘4C]OLEATE Lipid clam
Phospho~pid Monoacylglvcerol+ diaeylgIycerol+ sterol Free fatty acid Triacylglycerol Glycerol ether diester Sterol ester Recovery
Distribution of label (96)
.--
Acetate a
Oleate b
57.2 i 0.5 c 4.2 f 0.2
47.3 z!z0.3 1.6 i 0.0
2.9 19.5 5.8 10.5 75.0
4.8 37.4 7.0 1.5 86.0
+ 0.2 f 0.3 + 0.2 f 0.2 f 1.4
* * t t i
0.1 1.0 1.1 0.2 1.9
a A 2-h incubation in duphcate flasks containing 10 ml of 67-SS with 1.0 . 10’Novikoff hepatoma cells and 6 &tCiof [l-‘Clacetate (specific activity 5.4 Ci/mol). The cpm/106ceBs of total lipids is 3368 f 48. b A lh incubation in duplicate flasks containing 10 ml 67-SS with 1.14 . lo7 Novikoff hepatoma ceils end 20 &g/ml sodium Cl-%]oleate complexed to 2 mg/ml bovine serum albumin (specific activity 7.1 Cilmol). The cpm/106ceila is 118 818 * 1708. ’ Each vafue is the mean percentage f SE. of four determinations.
lipid classes was not changed when the cells were exposed to acetate of lo-fold higher specific activity resulting in 3% of label incorporated. In addition, the uptake of exogenous fatty acid increased the triacylglycerol and free fatty acid content of the cells. Comparison of the fatty acid content of the cells with that of serum in the medium showed that the amount of 18 : 0 was the same (cellular 14%; serum 15.8%) whereas the amount of 18 : 1 was considerably elevated in the cellular fatty acids {cellular 49.6%; serum 28.7%). The amount of 16 : 0 was reduced in cellular fatty acids compared with serum (cellular 14.2%; serum 25.1%). A different pattern was presented by fatty acids labeled from acetate. Saturated fatty acids contained 72% of the label (14 : 0, 3.4%; 16 : 0, 37.1%; and 18 : 0, 31.3%) with the remainder in 16 : 1, 5.8% and 18 : 1,16.3% and longer fatty acids (6.2%). The pattern of fatty acid labeling from acetate is indicative of de novo synthesis. Although 18 : 0 contained more label than would be expected from de novo synthesis alone [13] the amount of endogenous chain elongation appeared to be low. The ability of the Novikoff cells to metabolize cellular complex lipid was investigated by labeling the lipids with acetate followed by further incubation in 67-SS without isotope. Fig. 2 illustra~s that phospholipid increased at the expense of triacylglycerol during the subsequent incubation period of 6 h. During the incubation period, sterol ester lost 46% of its label (10.5 to 5.7%) and the free sterol fraction increased 100% (4.2 to 8.6%). The 15% decrease in the labeled triacylglycerol present in the total lipid represents approx. 80% of label 30
l-
I
I
I
:‘\ !\
20
IO
2.0
4.0
Hours Fig. 2. Changes in the distribution of the 14C label in the lipids of Novikoff hepatoma cells. After a 2-h exposure to fl-14C]acetate, the labeled ceils were washed once in 67SS then suspended at 1 . lo6 cells/ml in fresh 67-SS containing 2 mglml fatty acid-free bovine serum albumin or bovine serum albumin containing 20 &g/ml of sodium oleate. At each sampling time, duplicate samples of 1 * lo7 cells were removed from each of duplicate flasks and the isolated lipids separated by thin-layer chromatography (see ExPefimental). Each bar represents the mean of four lipid extractions and thin-layer separations. All standard errors were less than 1%. BSA represents bovine serum albumin. Fig. 3. The kinetics of redistribution are the same as for Fig. 2.
of r4C label from triacylglycerol
to phospholipid.
Experimental
data
475
in the triacylglycerol pool (Table II). The change in the amount of label in triacylglycerol occurred concomitantly with a 25% increase in total labeled phospholipid which occurred uniformly in all phospholipid classes. The redist~bution of label from triacylglycerol to phospholipid was inhibited after 2 h of incubation by the addition of 20 pi/ml of unlabeled cis-9-18 : 1. The kinetics of the redistribution in the absence of added cis-9-18 : 1 (Fig. 3) shows that the rate of redistribution of label was a first-order process. The apparent half-lives (estimated from Fig. 3) for the decrease in triacylglycerol and increase in phospholipid are 1.0 and 1.25 h, respectively. These results suggest a precursor vs. product relationship. The greater amount of phospholipid reflects the contribution by sterol ester which decreased during incubation, Effect of octadecenoic acids on de nova synthesis of fatty acids
The inability of the cis-6-18 : 1 isomer to serve as a growth factor suggested that it was not incorporated into the cell lipid as the cis-9-18 : 1 (Table If). When both isomers were tested for their ability to modify the distribution of label from acetate in the lipid classes, identical inhibitions of lipogenesis from acetate were observed (Fig. 4). Both fatty acids inhibited incorporation of the label into phospholipid by 74% and triacylglycerol by 45%, but did not alter incorporation into the sterof ester (Table III). These results indicate that inhibition of lipogenesis by the exogenous 18 : 1 isomers was specific for the fatty acids.
60
%
Inhibition
40
20
0
IO
20
I_IQFatty
30
40
Acid/ml
) and cis-6-18 Fig. 4. Inhibition of de nova lipidsynthesis from [l-14CIacetate by cis-S-18 : 1 (00 (*A) in Novikoff hepatoma cells. Each point is the average of duplicate samples of 1 * 10’celh cubated at 36°C for 2 h.
:1 in-
476 TABLE III THE EFFECT OF cis-6-18 : 1 AND &-S-l8 : 1 ON THE DISTRIBUTION OF [1-‘4C]ACETATE INCORPORATION INTO THE LIPID CLASSES OF NOVIKOFF HEPATOMA CELLS CULTIVATED IN SUSPENSION CULTURE The percentage of label in lipid classes separated by thin-layer chromatography on silica gel H using light petroleumidiethyl ether/glacial acetic acid (90 : 15 : 2, v/v) and counted in a liquid scintiRation spectrophotometer. Lipid class
Distribution of label Control
cpm ** Phospholipid
cis-6-18
%
wm
cis-9-18
:1 %
:1 %
cpm
2959 c 34 * 339 I 12
52.3 6.0
760 161
13.4 3.0
780 136
13.8 2.4
stero1 Free fatty acid Triacylglycerol Glycerol ether diester Sterol ester
442 1035 344 540
7.8 18.3 6.1 9.6
184 598 102 564
3.2 10.6 1.8 10.0
135 564 175 524
2.4 10.0 3.1 9.3
Total Recovery f%b)
5660 t 23 75.9 t 1.5
2379 81.8
42.0
2313 83.0
41.0
Monoacylglycerol + diacylglycerol +
f 24 f 66 t 6 I 12
100.0
____-
* Average of duplicate samples. * * cpm /2 3106 cells
The effect of isomeric octudecenoic acids on the fatty acids present in Novikoff cell lipid Although the 18 : 1 isomers were identical in their effect on the labeling of complex lipids by acetate, they had different effects on the amount of saturated and monounsaturated fatty acids present in the cells (Table IV). The cells were grown for 45 h in medium containing serum to obtain a large population which had arisen in the presence of each fatty acid isomer. The five saturated and monoenoic fatty acids in Table IV represent 85% of the fatty acids in the
TABLE IV THE EFFECT OF EXOGENOUS cis-6-18 : 1 AND cis-S-18 : 2 ON SELECTED FATTY ACIDS IN THE TOTAL LIPID OF NOVIKOFF HEPATOMA CELLS The amount of each fatty acid compared to the control (control = 1.00). The peak area of each fatty acid in the chromatogram was normalized to the control by use of an internai standard (21 : 0) and the totaf lipid weight. Values are the mean t S. D. of duplicate samples from each of three independent experlmerits (n = 6). 40 pg of each 18 : 1 isomer was used in aB experiments. Fatty acid
16:0* 16 : 1 18 : 0 18 : 1 20 : 1
Amount of fatty acid Control
cis-9-18
0.97 0.96 1.00 0.98 0.99
1.00 0.64 1.06 1.56 2.35
+ 0.08 +_0.10 + 0.06 f 0.03 + 0.33
:1
i 0.09 t 0.09 ?: 0.20 t 0.09 t 0.68
c&-6-18 0.68 1.27 0.68 1.32 6.09
* Number of carbon atoms in fatty acids: number of double bonds.
f t f f f
:1
0.11 0.07 0.13 0.17 0.47
total lipid of cells which were obtained from complete growth medium containing fatty acid-free bovine serum albumin as the only supplement. The peak area of each fatty acid in each chromatogram was normalized to one sample of the control set by using the peak area of a constant amount of heneicos~oic acid methyl ester (21 : 0) added to each sample as an internal standard and adjusting for the total lipid present (gravimetric analysis) in the sample. Thus, the amount of each fatty acid present in the total lipid was evaluated independently of a change in the qu~tity of any other fatty acid. The addition of either the cis-9 or the cis-6 isomer increased the amount of 18 : 1 by 50 and 32% and increased the amount of 20 : 1 200 and 600%, respectively. The addition of the &s-9-18 : 1 did not affect the amounts of the saturated fatty acids, 16 : 0 and 18 : 0, whereas the cis-6-18 : 1 reduced the amount of these acids by 32%. The c&9-18 : 1 reduced the quantity of 16 : 1 by 35% whereas the cis-6-18 : 1 increased 16 : 1 by 27% compared to the amount of fatty acid present in the lipid from the control cells. No unusual fatty acid of different carbon number or unsaturation appeared when either isomer was added. The isomeric composition of the monoenoic fatty acids The increase in the monounsaturated fatty acids occurring in the lipid of the Novikoff cells exposed to cis-6-18 : 1 prompted an examination of their isomerit composition. The total lipid was isolated from cells grown with bovine serum albumin and cis-6-18 : 1 or cis-9-18 : 1 as a supplement to the complete growth medium, The 16 : 1, 18 : 1 and 20 : 1 methyl esters were isolated by preparative gas-liquid chromatography and their ozonolysis products identified. The data in Table V show that all of the 18 : 1 fatty acid of Novikoff cells was TABLE
v
THE ISOMERIC COMPOSITION OF MONOENOIC FATTY ACIDS CELLS GROWN IN THE PRESENCE OF cis-6-18 : 1 AND cis-9-18 : 1
OF
NOVIKOFF
HEPATOMA
The
Novikoff hepatoma e&s were incubated for 48 h with fatty acid-free bovine serum albumin or bovine serum albumin containing 40 &g/ml of an octadecenoic acid isomer as the addition to the growth medium. The monoenoic fatty acids were isolated from the total cellular lipid and their ozonolysis products identified by gas-Iiguid chromatography (see Experimental). Supplement
Ozoniaed fatty acid
Isomeric
A6
A4 Bovine serum albumin Bovine serum albumin + cis-9-18
:1
Bovine serum albumin + cis-6-18 : 1
18:
1
16 18 20
:1 :1 :1
16 18 20
:1 :1 :1
composition
(WI A’
A8
Ali
Al3
100.0 14.2
62.1
4.8
24.7 35.7 8.5
3.6
5.5
A9 95.4
41.1
37.4
c
b
21.8
3.3 56.0
a The difference between the sum of these values known composition. b Based upon detection of a la-carbon aIdehyde. c Based upon detection of a S-carbon aldehyde.
a
68.5
and
100
represents
ozonolysis
products
of un-
478
the A’ isomer when the cells were grown for 48 h in complete medium supplemented with fatty acid-free bovine serum albumin or bovine serum albumin confining 40 pg/ml of c&-9-18 : 1. In contrast, 56% of the A6 isomer was present when the medium was supplemented with 40 @g/ml of cis-6-18 : 1. The 16 : I fatty acid contained 24-27% A9 isomer whether the supplement was cis-6-18 : 3. or cis-9-18 : 1. However, a major difference occurred in the isomerit composition of the remainder of the 16 : 1 which depended on the isomerit composition of the 18 : 1 supplement. When the supplement was c&-918 : 1, 41% of the 16 : 1 was the A7 isomer. When the supplement was cd-8 18 : 1, 37% of the 16 : 1 was the A4 isomer. Similarly, the composition of the 20 : 1 was determined by the structure of the 18 : 1 supplement. The 20 : 1 was 14% A9, 62% Al’ and 4.8% Al3 when the supplement was cis-9-18 : 1, whereas 68% of the 20 : 1 fatty acid was the A8 isomer in cells exposed to cis-6-18 : 1. The A’ isomer replaced the A” in cells c~tivated in medium supplemented with c&6-18 : 1. These results show that both isomers undergo both chain elongation and chain shortening. Discussion The elevation of octadecenoic acid in the lipids of tumors and cultured transformed cells has been noted in several investigations [ 1-4 1. However, little is known of the relationship of the increased 18 : 1 to the altered properties of tumorigenic cells. This investigation has employed the advantages of cells cultivated in a lipid-deficient medium to study the origin, metabolism and physiological effect of 18 : 1 in intact Novikoff hepatoma cells. The amount of 18 : 1 in the cellular lipid was proportionately greater than the amount present in serum. In addition, the accumulation of the fatty acid does not appear to result from de novo synthesis because the products of synthesis from acetate were primarily 16 : 0 and 18 : 0 and because de novo synthesis was inhibited by the exogenous 18 : 1. Thus, the data indicate that cellular 18 : 1 was obtained preformed from the environment under the usual conditions of culture. This conclusion on the uptake and incorporation of exogenous fatty acids in general has previously been reached by other investigators using a variety of other cell types and conditions [ 141. Inhibition of de novo synthesis of fatty acids from acetate was unexpected because lipogenesis in hepatomas has not been known to be subject to regulation by exogenous fatty acids. The absence of feedback inhibition of fatty acid synthesis has been observed for 10 transplantable rat and mouse hepatomas [ 151. This finding has been the basis for the view that lack of regulation of fatty acid synthesis is a characteristic feature of hepatomas. The present data clearly indicate that short-term fatty acid synthesis is subject to regulation by exogenous fatty acids in the intact Novikoff hepatoma cell. The inhibition is probably mediated by fatty acyl-CoA because Sumper [16] has shown that inhibition of acetyl-CoA carboxylase is mediated by the fatty acyl-CoA derivative and because the 18 : 1 isomers were both chain elongated and chain shortened by the Novikoff cells. The inhibition by long chain acids does not appear to be the consequence of a limiting supply of available cofactors such as CoA because the amount of label appearing in the sterol ester remained unaffected
479
(Table III). Recently, McGee and Spector [17] have shown that fatty acid synthesis in Ehrlich ascites tumor cells is also subject to short-term regulation. The obse~ation that unlabeled long chain fatty acid inhibits incorporation of label into all lipid classes except sterol ester is not consistent with coordinate control of both fatty acid and cholesterol biosynthesis by exogenous lipid which has been observed in cultured L cells [ 183. However, evidence has been presented which shows that the pathways of fatty acid and cholesterol synthesis in rat liver diverge at acetyl-CoA [19]. The differential inhibition of de novo lipogenesis from acetate in Novikoff cells is compatible with that evidence and agrees with results obtained with freshly isolated rat hepatocytes [ 201. Most of the label present in lipids from [l-‘4C]acetate or 1-14C-labeled &s-918 : 1 appeared in the phospholipid fraction which is probably a reflection of the growth capacity and membrane formation by these cells. The utilization of triacylglycerol to synthesize phospholipid supports similar findings for L cells [ 213 and is compatible with the synthesis of phospholipid by the direct pathway observed previously with Novikoff cells [22]. Synthesis of phospholipid by the direct pathway shows that the Novikoff cells can rapidly use endogenous lipid which probably contributed to the 3-fold growth of the cells in lipiddeficient medium. However, the pathway would not be expected to operate under the usual conditions of culture since redistribution of the label was inhibited by exogenous fatty acid. This observation suggests that the predominant pathway of phospholipid synthesis is the de novo pathway through phosphatidic acid [ 141. The cis-9-18 : 1 served as a definite requirement for the optimal growth of the Novikoff cells in serum-free medium because it can subst~tially replace serum as a growth factor. In the present study, the bosh-promoting capacity of 18 : 1 was specific for the h-9 isomer. This does not preclude growthpromoting effects by untested 18 : 1 isomers. For instance, the elevation of 18 : 1 in hepatoma tissue culture cells has been shown by Wood et al. [23] to result from accumulation of the cis-11-18 : 1 isomer. The present results are not comparable to those reported by Wood et al. f23] because no cis-11-18 : 1. was detected in the Novikoff cells, although it is possible that the small amount of cis-13-20 : 1 (5%, Table V) originated by chain elongation of cis-11-18 : 1. The cis-9-18 : 1 has also been observed to promote the growth of monkey kidney cells [24] and mouse L [25] cells in culture. The basis for the inability of the c&-6-18 : 1 to stimulate the growth of Novikoff cells is not known. Isomeric specificity does not reside in the inability of the cells to metabolize the cis-6-18 : 1 isomer to other fatty acids. Preliminary data suggest that the extensive conversion of the cis-6 to other fatty acids (16 : 1 and 20 : 1) may result in the incorporation of these fatty acids into neutral lipid and phospholipid classes replacing the saturated fatty acids as observed for the total lipid (Table IV). This evidence supports the findings of Wood and Falch [26] for the lack of class specificity in the fatty acid composition of the lipid of hepatoma tissue culture cells. Acknowledgments The authors express their appreciation to Mr. TX. Yang, Mr. P. Brown, Ms. E. McMeans, and Ms. P. Horvat for excellent technical assistance, and to
480
Dr. H, Brockman for helpful discussion. This work was supported by Grant CA 14003 of the National Cancer Institute, PHS research grant No. HL-08214 from the Program Project Branch extramural program, National Heart and Lung Institute, and by The Hormel foundation. References 1 Buggieri, S. and FaIIani, A. (1973) Tumor Lipids, Biochemistry and Metabolism (Wood, pp. 89-109, American Oil Chemists’s Society, Champaign, III. 2 Wood, R. and Falch. J. (1973) Lipids 8. 702-710 3 Araki, E., PhiUps, F. and Privet& O.S. (1974) Lipids 9.707-712 4 Chiappe, L., Mercuri, 0. and Detomk, M.E. (1974) Lipids 9, 360-362 5 Majerus, P.W.. Jacobs, R. and Smith, M.B. (1968) J. Biol. Chem. 243,35SS-3595 6 Steele, W. and Jenkin, H.M. (1972) Lipids 7, 556-559 7 Spector, A.A., Steinberg, D. and Tanaka, A. (1965) J. Biol. Chem. 240. 1032-1041 8 Kates, M. (1972) Laboratory Technique in Biochemistry and Molecular Biology (Work, Work, E., eds.), Vol. 3, Part II, p. 351, American Elsevier Publishing Co., Inc., New York 9 Beroza, M. and Bier& B.A. (1967) AnaI. Chem. 39,1131-1135 10 Bioch-Frankenthsl, L.. Langan, J., Morris, H.P. and Weinhouse, S. (1965) Cancer Res. 25, 11 Renner, E.D.. Plagemann. P.G.W. and Berniohr. R.W. (1972) J. Biol. Chem. 247. 5765-5776 12 Spector, A.A., John, K. and Fletcher, J.E. (1969) J. Lipid Res. 10, 56-67 13 Bressler. R. and WakiI, S.J. (1961) J. Biol. Chem. 236.1643-1651 14 Spector, A.A. (1972) Growth, Nutrition, and Metabolism of Cells in Culture (Rothblat, Cristofaio, V.J., eds.). Vol. I, pp. 257-288, Academic Press, New York 15 Sabine. J.R. (1975) Progress in Biochemical Pharmacology (Carroll, K.K., ed.), Vol. 10, PP. S. Karger, Base1 16 Sumper, M. (1974) Eur. J. Biochem. 49.469475 17 McGee. R. and Spector, A.A. (1974) Cancer Res. 34, 335!%3362 18 Howard, B.V., Howard, W.J. and Bailey, J.M. (1974) J. Biol. Chem. 249,7912-7921 19 Barth, C.A., Hackenschmidt. H.J., Weis, E.E. and Decker, K.F.A. (1973) J. Biol. Chem. 248, 20 Nilsson. A., SundIer. R. and Akesson, B. (1973) Eur. J. Biochem. 39,613-620 21 Bailey, J.M.. Howard, B.V. and TiIiman, SF. (1973) J. Biol. Chem. 248,1240-1247 22 Plagemann, P.G.W. (19711 J. Lipid Res. 12, 715--724 23 Wood, R., Falch, J. and Wiegand, R.D. (1974) Lipids 9, 987-992 24 Jenkin, H.M. and Anderson, L.E. (1970) Exp. Cell Res. 59, 6-10 25 Morrison, S.J. and Jenkin, H.M. (1972) In Vitro 8,94-100 26 Wood, R. and Falch, J. (1974) Lipids 9,979-986
R., ed.1,
T.S.
and
732-736
GH.
and
269-307,
738-739
Biochimica et Biophysics Acta, 431 (1976) 469-480 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56786
THE EFFECT OF TWO ISOMERIC OCTADECENOIC ACIDS ON THE LIPID METABOLISM AND GROWTH OF NOVIKOFF HEPATOMA CELLS
DAVID
E. WENNERSTROM
* and HOWARD
M. JENKIN
The Hormel Institute, University of Minnesota, Austin, Minn. 55912 (Received
November
(U.S.A.)
lOth, 1975)
Summary
The origin and metabolism of octadecenoic acid (18 : 1) was examined in intact Novikoff rat hepatoma cells by using labeled precursors and two isomeric octadecenoic acids which differed in their abilities to stimulate cell growth in a serum-free medium. The isomers (cis-6-18 : 1 and cis-9-18 : 1) were measured in the cellular lipid by ozonolysis and reduction of the ozonides. The results indicate that the 18 : 1 fatty acid accumulated in the cell lipid by uptake of the preformed acid from the medium. The ~5-6-18 : 1 was more extensively metabolized than the cis-9-18 : 1 to 16 : 1 and 20 : 1 fatty acids by chain shortening and chain elongation. Both isomers inhibited de novo fatty acid synthesis from acetate by cells suspended in a serum-free medium. The isomers did not exert coordinate control of both fatty acid and cholesterol biosynthesis in the Novikoff cells.
Introduction
Analysis of the fatty acid composition of a number of normal and tumorigenic cells has shown that the cellular content of octadecenoic acid (18 : 1) is increased in transformed cells [l--3], and this increase appears to be related to transformation. For instance, the fatty acid composition of the Novikoff rat hepatoma contains a markedly increased level of 18 : 1 compared to both normal and host liver [4]. Nevertheless, little work has been done in elucidating the importance of the increased level of 18 : 1 in tumorigenic cells. In addition, hepatomas have been reported to lack the ability of normal liver to regulate lipogenesis in response to changes in lipid supply even though the isolated en* Present address: Department of Microbiology. Little Rock. Ark. 72201. U.S.A.
The University of Arkansas for Medical Sciences.
470
zymes are identical to those of normal liver with respect to cofactor requirements and inhibition by long chain fatty acids [ 51. The purpose of this study was to investigate the origin and metabolism of 18 : 1 in intact tumorigenic cells and to evaluate its physiological significance as measured by its effects on cell growth. Novikoff hepatoma cells in suspension culture were exposed to two positional isomers of 18 : 1 which differ in their metabolism and their effects of cell growth. The ability of these fatty acids to regulate de novo synthesis of fatty acids in the intact cell was also observed. Experimental Materials. Swim’s 67-G powdered medium and meat peptone were obtained from Grand Island Biological Co., Grand Island, N.Y.; bovine serum albumin (fatty acid-free bovine serum albumin) was purchased from Miles Laboratories, Kanakakee, Ill.; fatty acids and authentic lipid standards were purchased from NuCheck Prep, Inc., Elysian, Minn.; glycerol ether diester was a gift from Dr. W.J. Baumann, The Hormel Institute; Silica Gel H was obtained from E. Merck AG, Darmstad, Germany; sodium[ l-14C]acetate (62 Ci/mol) and [ l-14C]oleic acid (cis-9-18 : 1, 58 Ci/mol) were purchased from Amersham-Searle Inc., Arlington Heights, Ill.; Permablend scintillator was purchased from Packard Instrument Co., Downers Grove, Ill. Cell culture. Novikoff hepatoma cells (subline NISI-67) were obtained from Dr. P.G.W. Plagemann, University of Minnesota, and grown in suspension in Swim’s 67-G medium containing 5% newborn calf serum [ 61, except that the pancreatic autolysate was replaced by 2.0 g/l of meat peptone. The cells were enumerated with a Coulter counter. In each experiment, cells were incubated in duplicate flasks containing medium supplemented with 2 mg/ml fatty acidfree bovine serum albumin or bovine serum albumin complexed to the sodium salt of an octadecenoic acid (cis-6-18 : 1 or cis-9-18 : 1). The sodium salts of the fatty acids were prepared [6] and were complexed to the bovine serum albumin according to Spector et al. [7]. A starting density of 2 - 10’ hepatoma cells was incubated for 45-48 h at 36°C. Lipid analysis. Following incubation, the cells were sedimented by centrifugation at 300 X g at 4°C and washed twice with a cold serum-free and peptonefree salt solution which was identical in composition to 67-G medium except containing 5 mM glucose, 0.1 mM sodium acetate, and 20 mM N-2-hydroxyethyl-piperazine-l\r’S-ethanesulfonic acid, pH 7.2. This solution was designated 67SS. The cells were resuspended in water and the lipid extracted by the method of Bligh and Dyer as modified by Kates [ 81. The chloroform layer was evaporated to dryness under a stream of Nz and the lipid immediately dissolved in a measured amount of chloroform. Aliquot samples of total lipid were removed for gravimetric measurement of total lipid weight. The remaining lipid was transesterified in 5% HCl in methanol at 95°C for 2 h after the addition to all samples of a constant amount of heneicosanoic acid methyl ester (21 : 0) as an internal standard. Total fatty acid methyl esters were extracted three times by the addition of light petroleum and water, separated by thin-layer chromatography in a solvent system of light
471
petroleum/diethyl ether/glacial acetic acid (90 : 15 : 2, v/v) and analyzed by gas-liquid chromatography. The gas-liquid chromatography was performed on a Victoreen 4000 series instrument equipped with a flame ionization detector and an 8 ft X l/S inch aluminum column packed with 15% ethylene glycol succinate (EGSS-X) on Gas Chrom P 100/120. Helium was the carrier gas, and the column temperature was 170°C. To determine the isomeric composition of monounsaturated fatty acids present in the total lipid of Novikoff cells, the 16 : 1, 18 : 1, and 20 : 1 methyl esters were collected by preparative gas-liquid chromato~aphy using a Beckmen GC-2A instrument equipped with a thermal conductivity detector and the same type of column as the Victoreen instrument. Ozonolysis was performed on the isolated methyl esters as described by Beroza and Bier1 [9] using redistilled pentane as the solvent. Ozonolysis products were detected as described for the methyl esters except that the column temperature was pro~ammed from 100 to 155°C at 3”C/min. The aldehydes and aldesters were identified by comparison with chromatograms of ozonized cis-6-18 : 1; cis-9-18 : 1, and cis11-18 : 1 methyl esters as standards. Incorporation of radioactivity in Novikoff cell lipids. Novikoff cells in the exponental phase of growth were sedimented at 300 Xg for 10 min, washed twice in 67-SS, and resuspended at 1 - lo6 cells/ml in replicate ffasks cont~ning 67-SS with the appropriate fatty acid. After the addition of [1-‘4C]acetate (0.6 &i/ml in all experiments) or l-*4C-labeled cis-9-18 : 1, and incubation at 36”C, the cell suspensions were rapidly cooled in ice, a loo-fold excess of unlabeled sodium acetate was added when [l-*4C]acetate was the labeled precursor, and the cells were sedimented at 300 X g at 4°C. The cells were washed once with 67-SS, resuspended in water, and lipids were extracted. Replicate samples of labeled lipid were applied to 500 E.tm thick Silica Gel H plates together with authentic neutral lipid standards and the lipids were separated by thin-layer chromatography using a solvent system of light petroleum/diethyl ether/glacial acetic acid (90 : 15 : 2, v/v). The lipids were located by staining with iodine vapor. The silica gel bands of the separated lipids were scraped into scintillation vials and the radioactivity was determined by liquid scintillation spectroscopy (Packard Model 2405) using 10 ml of toluene-based scintillation fluid. Methanol (0.5 ml) was added to each vial to help solubilize the lipids. The counting efficiency was 85%. Rest&s The effect of octadecenoic acid isomers on cellgrowth
The Novikoff hepatoma cells grew exponentailly for 40-48 h when they were suspended in the complete growth medium at an initial concentration of 2 - lo5 cells/ml. The population density at 48 h was 8-lo-fold that of the inoculum, Table I shows that cell growth was only about 3.5 times the inoclum after 40 h of growth in medium without serum compared to a 7-fold in the complete medium. The addition of lo-40 Erg/ml cis-9-18 : 1 to the medium stimulated cell growth to approx. 80% of that observed in medium containing serum. The fatty acid was effective in stimulation of growth at the lowest con~ntration tested. ~though the response to increasing doses was minimal, 20
472 TABLE
I
THE DIFFERENTIAL EFFECT OF OCTADECENOIC ACID ISOMERS ON THE GROWTH OF NOVIKOFF HEPATOMA CELLS IN SERUM-FREE
cis-6-18 : MEDIUM
1
AND
cis-9-18
: 1
Supplement added to the growth medium which was serum free. All flasks contained 2 mgjml of fatty acid-free bovine serum albumin. Growth was measured as the increase in the number of cells over the inoculum during an incubation at 36’C for 40 h. Each value is the average of duplicate samples. Supplement
Fatty acid concentration
Growth (-fold the mean
increase in number
of cells ? difference
from
(pg/mI) Experiment
__None
1
Experiment
0
3.45 i 0.04
3.79 ? 0.45
cis-6-18
:1
10 20 40
N.D. 3.54 + 0.63 2.90 * 0.05
3.86 I 0.11 3.58 i 0.21 3.20 + 0.36
cis-9-18
:1
10 20 40
N.D. 5.34 + 0.04 5.01 * 0.17
5.76 ? 0.56 6.56 i 0.48 5.61 f 0.47
20 N.D.
N.D. N.D.
6.97 + 0.05
cis-6-18 : 1 Serum (5%) N.D. Not Determined.
2
-__
5.02 ?: 0.23 -__-
pg/ml appeared to be the most effective in promoting growth. In contrast, all concentrations of the cis-6-18 : 1 were completely without effect in promoting cell growth. The cis-6-18 : 1 did not inhibit cell growth and did not significantly affect the stimulation of the cis-9-18 : 1 when the two isomers were combined in equal amounts (20 pg/ml each). Origin and metabolism of octadecenoic acids in Novikoff cells The ability of the cis-9-18 : 1 to serve as a growth factor suggested
that it
30
20
1
2
4
I
I
6
8
Time (hrs) Fig. 1. The time course of de nova lipid synthesis from acetate and uptake of sodium oleate in Novikoff hepatoma c&s. Duplicate flasks ware inoculated with 1 * lo6 Novikoff hepatoma cells/ml of 67-SS and incubated at 36’C in the presence of [1-14C]acetate (specific activity 2.7 Ci/mol) or sodium [1-14C]oleate (cis-9-18 : 1, specific activity 7.1 Ci/mol). Each point is the average of duplicate samples of 1 . 10’ceIIs.
473
was readily incorporated into the Novikoff cell lipid. The origin of the 18 : 1 in cellular lipid and its metabolism by the Novikoff cellwas investigated by using [1-14C]acetate to measure to de novo synthesis and by employing 1-14C-labeled cis-9-18 : 1 as an exogenous source of fatty acid. The labeled acetate served as a useful indicator for lipogenesis because neither Novikoff cells nor Novikoff tumor homogenates can be demonstrated to oxidize long chain fatty acids (Wennerstrom, D. and Jenkin, H., unpublished data; ref. 10) and because lactate is the principal product of glycolysis [II]. The results in Fig. 1 show that acetate conversion to lipid occurred at a constant rate for 8 h when the cells were suspended in the lipid-free 6’7-5s. In contrast, the uptake and incorporation of the l-14C-labeled c&9-18 : 1 was not linear. The decreasing rate of fatty acid incorporation was not caused by depletion of oleate from the medium because the total activity incorporated at 4 h represented 25% of the activity supplied. The rate change is presumably caused by the differences in the affinity of the bovine serum albumin binding sites for the fatty acid [123. Incorporation of labeled cis-9-18 : 1 into cell lipid at 1 h was 55 times greater than’incorporation of label from acetate after correction for differences in specific activity. The distribution of isotope in the complex lipids labeled from the two sources was also compared (Table II). Phospholipid was the major lipid labeled with 47 and 57% of the label obtained from cis-9-18 : 1 and acetate, respectively. The triacylglycerol (37%) and free fatty acid (4.8%) fractions contained a greater proportion of label from cis-9-18 : 1 whereas the sterol fraction (4.2%) and the sterol ester (10.5%) were more extensively labeled from acetate. Comparison of the labeling pattern with gravimetric analysis of the neutral lipids and phospholipids and the fatty acids of each lipid class by gas-liquid chromatography showed that the labeling of the complex lipid classes occurred in proportion to the amount present in the cells. The distribution of label in the TABLE II THE LABELING PATTERN OF LIPIDS IN NOVIKOFF HEPATOMA CELLS USING [l-*4C]ACETATE AND [1-‘4C]OLEATE Lipid clam
Phospho~pid Monoacylglvcerol+ diaeylgIycerol+ sterol Free fatty acid Triacylglycerol Glycerol ether diester Sterol ester Recovery
Distribution of label (96)
.--
Acetate a
Oleate b
57.2 i 0.5 c 4.2 f 0.2
47.3 z!z0.3 1.6 i 0.0
2.9 19.5 5.8 10.5 75.0
4.8 37.4 7.0 1.5 86.0
+ 0.2 f 0.3 + 0.2 f 0.2 f 1.4
* * t t i
0.1 1.0 1.1 0.2 1.9
a A 2-h incubation in duphcate flasks containing 10 ml of 67-SS with 1.0 . 10’Novikoff hepatoma cells and 6 &tCiof [l-‘Clacetate (specific activity 5.4 Ci/mol). The cpm/106ceBs of total lipids is 3368 f 48. b A lh incubation in duplicate flasks containing 10 ml 67-SS with 1.14 . lo7 Novikoff hepatoma ceils end 20 &g/ml sodium Cl-%]oleate complexed to 2 mg/ml bovine serum albumin (specific activity 7.1 Cilmol). The cpm/106ceila is 118 818 * 1708. ’ Each vafue is the mean percentage f SE. of four determinations.
lipid classes was not changed when the cells were exposed to acetate of lo-fold higher specific activity resulting in 3% of label incorporated. In addition, the uptake of exogenous fatty acid increased the triacylglycerol and free fatty acid content of the cells. Comparison of the fatty acid content of the cells with that of serum in the medium showed that the amount of 18 : 0 was the same (cellular 14%; serum 15.8%) whereas the amount of 18 : 1 was considerably elevated in the cellular fatty acids {cellular 49.6%; serum 28.7%). The amount of 16 : 0 was reduced in cellular fatty acids compared with serum (cellular 14.2%; serum 25.1%). A different pattern was presented by fatty acids labeled from acetate. Saturated fatty acids contained 72% of the label (14 : 0, 3.4%; 16 : 0, 37.1%; and 18 : 0, 31.3%) with the remainder in 16 : 1, 5.8% and 18 : 1,16.3% and longer fatty acids (6.2%). The pattern of fatty acid labeling from acetate is indicative of de novo synthesis. Although 18 : 0 contained more label than would be expected from de novo synthesis alone [13] the amount of endogenous chain elongation appeared to be low. The ability of the Novikoff cells to metabolize cellular complex lipid was investigated by labeling the lipids with acetate followed by further incubation in 67-SS without isotope. Fig. 2 illustra~s that phospholipid increased at the expense of triacylglycerol during the subsequent incubation period of 6 h. During the incubation period, sterol ester lost 46% of its label (10.5 to 5.7%) and the free sterol fraction increased 100% (4.2 to 8.6%). The 15% decrease in the labeled triacylglycerol present in the total lipid represents approx. 80% of label 30
l-
I
I
I
:‘\ !\
20
IO
2.0
4.0
Hours Fig. 2. Changes in the distribution of the 14C label in the lipids of Novikoff hepatoma cells. After a 2-h exposure to fl-14C]acetate, the labeled ceils were washed once in 67SS then suspended at 1 . lo6 cells/ml in fresh 67-SS containing 2 mglml fatty acid-free bovine serum albumin or bovine serum albumin containing 20 &g/ml of sodium oleate. At each sampling time, duplicate samples of 1 * lo7 cells were removed from each of duplicate flasks and the isolated lipids separated by thin-layer chromatography (see ExPefimental). Each bar represents the mean of four lipid extractions and thin-layer separations. All standard errors were less than 1%. BSA represents bovine serum albumin. Fig. 3. The kinetics of redistribution are the same as for Fig. 2.
of r4C label from triacylglycerol
to phospholipid.
Experimental
data
475
in the triacylglycerol pool (Table II). The change in the amount of label in triacylglycerol occurred concomitantly with a 25% increase in total labeled phospholipid which occurred uniformly in all phospholipid classes. The redist~bution of label from triacylglycerol to phospholipid was inhibited after 2 h of incubation by the addition of 20 pi/ml of unlabeled cis-9-18 : 1. The kinetics of the redistribution in the absence of added cis-9-18 : 1 (Fig. 3) shows that the rate of redistribution of label was a first-order process. The apparent half-lives (estimated from Fig. 3) for the decrease in triacylglycerol and increase in phospholipid are 1.0 and 1.25 h, respectively. These results suggest a precursor vs. product relationship. The greater amount of phospholipid reflects the contribution by sterol ester which decreased during incubation, Effect of octadecenoic acids on de nova synthesis of fatty acids
The inability of the cis-6-18 : 1 isomer to serve as a growth factor suggested that it was not incorporated into the cell lipid as the cis-9-18 : 1 (Table If). When both isomers were tested for their ability to modify the distribution of label from acetate in the lipid classes, identical inhibitions of lipogenesis from acetate were observed (Fig. 4). Both fatty acids inhibited incorporation of the label into phospholipid by 74% and triacylglycerol by 45%, but did not alter incorporation into the sterof ester (Table III). These results indicate that inhibition of lipogenesis by the exogenous 18 : 1 isomers was specific for the fatty acids.
60
%
Inhibition
40
20
0
IO
20
I_IQFatty
30
40
Acid/ml
) and cis-6-18 Fig. 4. Inhibition of de nova lipidsynthesis from [l-14CIacetate by cis-S-18 : 1 (00 (*A) in Novikoff hepatoma cells. Each point is the average of duplicate samples of 1 * 10’celh cubated at 36°C for 2 h.
:1 in-
476 TABLE III THE EFFECT OF cis-6-18 : 1 AND &-S-l8 : 1 ON THE DISTRIBUTION OF [1-‘4C]ACETATE INCORPORATION INTO THE LIPID CLASSES OF NOVIKOFF HEPATOMA CELLS CULTIVATED IN SUSPENSION CULTURE The percentage of label in lipid classes separated by thin-layer chromatography on silica gel H using light petroleumidiethyl ether/glacial acetic acid (90 : 15 : 2, v/v) and counted in a liquid scintiRation spectrophotometer. Lipid class
Distribution of label Control
cpm ** Phospholipid
cis-6-18
%
wm
cis-9-18
:1 %
:1 %
cpm
2959 c 34 * 339 I 12
52.3 6.0
760 161
13.4 3.0
780 136
13.8 2.4
stero1 Free fatty acid Triacylglycerol Glycerol ether diester Sterol ester
442 1035 344 540
7.8 18.3 6.1 9.6
184 598 102 564
3.2 10.6 1.8 10.0
135 564 175 524
2.4 10.0 3.1 9.3
Total Recovery f%b)
5660 t 23 75.9 t 1.5
2379 81.8
42.0
2313 83.0
41.0
Monoacylglycerol + diacylglycerol +
f 24 f 66 t 6 I 12
100.0
____-
* Average of duplicate samples. * * cpm /2 3106 cells
The effect of isomeric octudecenoic acids on the fatty acids present in Novikoff cell lipid Although the 18 : 1 isomers were identical in their effect on the labeling of complex lipids by acetate, they had different effects on the amount of saturated and monounsaturated fatty acids present in the cells (Table IV). The cells were grown for 45 h in medium containing serum to obtain a large population which had arisen in the presence of each fatty acid isomer. The five saturated and monoenoic fatty acids in Table IV represent 85% of the fatty acids in the
TABLE IV THE EFFECT OF EXOGENOUS cis-6-18 : 1 AND cis-S-18 : 2 ON SELECTED FATTY ACIDS IN THE TOTAL LIPID OF NOVIKOFF HEPATOMA CELLS The amount of each fatty acid compared to the control (control = 1.00). The peak area of each fatty acid in the chromatogram was normalized to the control by use of an internai standard (21 : 0) and the totaf lipid weight. Values are the mean t S. D. of duplicate samples from each of three independent experlmerits (n = 6). 40 pg of each 18 : 1 isomer was used in aB experiments. Fatty acid
16:0* 16 : 1 18 : 0 18 : 1 20 : 1
Amount of fatty acid Control
cis-9-18
0.97 0.96 1.00 0.98 0.99
1.00 0.64 1.06 1.56 2.35
+ 0.08 +_0.10 + 0.06 f 0.03 + 0.33
:1
i 0.09 t 0.09 ?: 0.20 t 0.09 t 0.68
c&-6-18 0.68 1.27 0.68 1.32 6.09
* Number of carbon atoms in fatty acids: number of double bonds.
f t f f f
:1
0.11 0.07 0.13 0.17 0.47
total lipid of cells which were obtained from complete growth medium containing fatty acid-free bovine serum albumin as the only supplement. The peak area of each fatty acid in each chromatogram was normalized to one sample of the control set by using the peak area of a constant amount of heneicos~oic acid methyl ester (21 : 0) added to each sample as an internal standard and adjusting for the total lipid present (gravimetric analysis) in the sample. Thus, the amount of each fatty acid present in the total lipid was evaluated independently of a change in the qu~tity of any other fatty acid. The addition of either the cis-9 or the cis-6 isomer increased the amount of 18 : 1 by 50 and 32% and increased the amount of 20 : 1 200 and 600%, respectively. The addition of the &s-9-18 : 1 did not affect the amounts of the saturated fatty acids, 16 : 0 and 18 : 0, whereas the cis-6-18 : 1 reduced the amount of these acids by 32%. The c&9-18 : 1 reduced the quantity of 16 : 1 by 35% whereas the cis-6-18 : 1 increased 16 : 1 by 27% compared to the amount of fatty acid present in the lipid from the control cells. No unusual fatty acid of different carbon number or unsaturation appeared when either isomer was added. The isomeric composition of the monoenoic fatty acids The increase in the monounsaturated fatty acids occurring in the lipid of the Novikoff cells exposed to cis-6-18 : 1 prompted an examination of their isomerit composition. The total lipid was isolated from cells grown with bovine serum albumin and cis-6-18 : 1 or cis-9-18 : 1 as a supplement to the complete growth medium, The 16 : 1, 18 : 1 and 20 : 1 methyl esters were isolated by preparative gas-liquid chromatography and their ozonolysis products identified. The data in Table V show that all of the 18 : 1 fatty acid of Novikoff cells was TABLE
v
THE ISOMERIC COMPOSITION OF MONOENOIC FATTY ACIDS CELLS GROWN IN THE PRESENCE OF cis-6-18 : 1 AND cis-9-18 : 1
OF
NOVIKOFF
HEPATOMA
The
Novikoff hepatoma e&s were incubated for 48 h with fatty acid-free bovine serum albumin or bovine serum albumin containing 40 &g/ml of an octadecenoic acid isomer as the addition to the growth medium. The monoenoic fatty acids were isolated from the total cellular lipid and their ozonolysis products identified by gas-Iiguid chromatography (see Experimental). Supplement
Ozoniaed fatty acid
Isomeric
A6
A4 Bovine serum albumin Bovine serum albumin + cis-9-18
:1
Bovine serum albumin + cis-6-18 : 1
18:
1
16 18 20
:1 :1 :1
16 18 20
:1 :1 :1
composition
(WI A’
A8
Ali
Al3
100.0 14.2
62.1
4.8
24.7 35.7 8.5
3.6
5.5
A9 95.4
41.1
37.4
c
b
21.8
3.3 56.0
a The difference between the sum of these values known composition. b Based upon detection of a la-carbon aIdehyde. c Based upon detection of a S-carbon aldehyde.
a
68.5
and
100
represents
ozonolysis
products
of un-
478
the A’ isomer when the cells were grown for 48 h in complete medium supplemented with fatty acid-free bovine serum albumin or bovine serum albumin confining 40 pg/ml of c&-9-18 : 1. In contrast, 56% of the A6 isomer was present when the medium was supplemented with 40 @g/ml of cis-6-18 : 1. The 16 : I fatty acid contained 24-27% A9 isomer whether the supplement was cis-6-18 : 3. or cis-9-18 : 1. However, a major difference occurred in the isomerit composition of the remainder of the 16 : 1 which depended on the isomerit composition of the 18 : 1 supplement. When the supplement was c&-918 : 1, 41% of the 16 : 1 was the A7 isomer. When the supplement was cd-8 18 : 1, 37% of the 16 : 1 was the A4 isomer. Similarly, the composition of the 20 : 1 was determined by the structure of the 18 : 1 supplement. The 20 : 1 was 14% A9, 62% Al’ and 4.8% Al3 when the supplement was cis-9-18 : 1, whereas 68% of the 20 : 1 fatty acid was the A8 isomer in cells exposed to cis-6-18 : 1. The A’ isomer replaced the A” in cells c~tivated in medium supplemented with c&6-18 : 1. These results show that both isomers undergo both chain elongation and chain shortening. Discussion The elevation of octadecenoic acid in the lipids of tumors and cultured transformed cells has been noted in several investigations [ 1-4 1. However, little is known of the relationship of the increased 18 : 1 to the altered properties of tumorigenic cells. This investigation has employed the advantages of cells cultivated in a lipid-deficient medium to study the origin, metabolism and physiological effect of 18 : 1 in intact Novikoff hepatoma cells. The amount of 18 : 1 in the cellular lipid was proportionately greater than the amount present in serum. In addition, the accumulation of the fatty acid does not appear to result from de novo synthesis because the products of synthesis from acetate were primarily 16 : 0 and 18 : 0 and because de novo synthesis was inhibited by the exogenous 18 : 1. Thus, the data indicate that cellular 18 : 1 was obtained preformed from the environment under the usual conditions of culture. This conclusion on the uptake and incorporation of exogenous fatty acids in general has previously been reached by other investigators using a variety of other cell types and conditions [ 141. Inhibition of de novo synthesis of fatty acids from acetate was unexpected because lipogenesis in hepatomas has not been known to be subject to regulation by exogenous fatty acids. The absence of feedback inhibition of fatty acid synthesis has been observed for 10 transplantable rat and mouse hepatomas [ 151. This finding has been the basis for the view that lack of regulation of fatty acid synthesis is a characteristic feature of hepatomas. The present data clearly indicate that short-term fatty acid synthesis is subject to regulation by exogenous fatty acids in the intact Novikoff hepatoma cell. The inhibition is probably mediated by fatty acyl-CoA because Sumper [16] has shown that inhibition of acetyl-CoA carboxylase is mediated by the fatty acyl-CoA derivative and because the 18 : 1 isomers were both chain elongated and chain shortened by the Novikoff cells. The inhibition by long chain acids does not appear to be the consequence of a limiting supply of available cofactors such as CoA because the amount of label appearing in the sterol ester remained unaffected
479
(Table III). Recently, McGee and Spector [17] have shown that fatty acid synthesis in Ehrlich ascites tumor cells is also subject to short-term regulation. The obse~ation that unlabeled long chain fatty acid inhibits incorporation of label into all lipid classes except sterol ester is not consistent with coordinate control of both fatty acid and cholesterol biosynthesis by exogenous lipid which has been observed in cultured L cells [ 183. However, evidence has been presented which shows that the pathways of fatty acid and cholesterol synthesis in rat liver diverge at acetyl-CoA [19]. The differential inhibition of de novo lipogenesis from acetate in Novikoff cells is compatible with that evidence and agrees with results obtained with freshly isolated rat hepatocytes [ 201. Most of the label present in lipids from [l-‘4C]acetate or 1-14C-labeled &s-918 : 1 appeared in the phospholipid fraction which is probably a reflection of the growth capacity and membrane formation by these cells. The utilization of triacylglycerol to synthesize phospholipid supports similar findings for L cells [ 213 and is compatible with the synthesis of phospholipid by the direct pathway observed previously with Novikoff cells [22]. Synthesis of phospholipid by the direct pathway shows that the Novikoff cells can rapidly use endogenous lipid which probably contributed to the 3-fold growth of the cells in lipiddeficient medium. However, the pathway would not be expected to operate under the usual conditions of culture since redistribution of the label was inhibited by exogenous fatty acid. This observation suggests that the predominant pathway of phospholipid synthesis is the de novo pathway through phosphatidic acid [ 141. The cis-9-18 : 1 served as a definite requirement for the optimal growth of the Novikoff cells in serum-free medium because it can subst~tially replace serum as a growth factor. In the present study, the bosh-promoting capacity of 18 : 1 was specific for the h-9 isomer. This does not preclude growthpromoting effects by untested 18 : 1 isomers. For instance, the elevation of 18 : 1 in hepatoma tissue culture cells has been shown by Wood et al. [23] to result from accumulation of the cis-11-18 : 1 isomer. The present results are not comparable to those reported by Wood et al. f23] because no cis-11-18 : 1. was detected in the Novikoff cells, although it is possible that the small amount of cis-13-20 : 1 (5%, Table V) originated by chain elongation of cis-11-18 : 1. The cis-9-18 : 1 has also been observed to promote the growth of monkey kidney cells [24] and mouse L [25] cells in culture. The basis for the inability of the c&-6-18 : 1 to stimulate the growth of Novikoff cells is not known. Isomeric specificity does not reside in the inability of the cells to metabolize the cis-6-18 : 1 isomer to other fatty acids. Preliminary data suggest that the extensive conversion of the cis-6 to other fatty acids (16 : 1 and 20 : 1) may result in the incorporation of these fatty acids into neutral lipid and phospholipid classes replacing the saturated fatty acids as observed for the total lipid (Table IV). This evidence supports the findings of Wood and Falch [26] for the lack of class specificity in the fatty acid composition of the lipid of hepatoma tissue culture cells. Acknowledgments The authors express their appreciation to Mr. TX. Yang, Mr. P. Brown, Ms. E. McMeans, and Ms. P. Horvat for excellent technical assistance, and to
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Dr. H, Brockman for helpful discussion. This work was supported by Grant CA 14003 of the National Cancer Institute, PHS research grant No. HL-08214 from the Program Project Branch extramural program, National Heart and Lung Institute, and by The Hormel foundation. References 1 Buggieri, S. and FaIIani, A. (1973) Tumor Lipids, Biochemistry and Metabolism (Wood, pp. 89-109, American Oil Chemists’s Society, Champaign, III. 2 Wood, R. and Falch. J. (1973) Lipids 8. 702-710 3 Araki, E., PhiUps, F. and Privet& O.S. (1974) Lipids 9.707-712 4 Chiappe, L., Mercuri, 0. and Detomk, M.E. (1974) Lipids 9, 360-362 5 Majerus, P.W.. Jacobs, R. and Smith, M.B. (1968) J. Biol. Chem. 243,35SS-3595 6 Steele, W. and Jenkin, H.M. (1972) Lipids 7, 556-559 7 Spector, A.A., Steinberg, D. and Tanaka, A. (1965) J. Biol. Chem. 240. 1032-1041 8 Kates, M. (1972) Laboratory Technique in Biochemistry and Molecular Biology (Work, Work, E., eds.), Vol. 3, Part II, p. 351, American Elsevier Publishing Co., Inc., New York 9 Beroza, M. and Bier& B.A. (1967) AnaI. Chem. 39,1131-1135 10 Bioch-Frankenthsl, L.. Langan, J., Morris, H.P. and Weinhouse, S. (1965) Cancer Res. 25, 11 Renner, E.D.. Plagemann. P.G.W. and Berniohr. R.W. (1972) J. Biol. Chem. 247. 5765-5776 12 Spector, A.A., John, K. and Fletcher, J.E. (1969) J. Lipid Res. 10, 56-67 13 Bressler. R. and WakiI, S.J. (1961) J. Biol. Chem. 236.1643-1651 14 Spector, A.A. (1972) Growth, Nutrition, and Metabolism of Cells in Culture (Rothblat, Cristofaio, V.J., eds.). Vol. I, pp. 257-288, Academic Press, New York 15 Sabine. J.R. (1975) Progress in Biochemical Pharmacology (Carroll, K.K., ed.), Vol. 10, PP. S. Karger, Base1 16 Sumper, M. (1974) Eur. J. Biochem. 49.469475 17 McGee. R. and Spector, A.A. (1974) Cancer Res. 34, 335!%3362 18 Howard, B.V., Howard, W.J. and Bailey, J.M. (1974) J. Biol. Chem. 249,7912-7921 19 Barth, C.A., Hackenschmidt. H.J., Weis, E.E. and Decker, K.F.A. (1973) J. Biol. Chem. 248, 20 Nilsson. A., SundIer. R. and Akesson, B. (1973) Eur. J. Biochem. 39,613-620 21 Bailey, J.M.. Howard, B.V. and TiIiman, SF. (1973) J. Biol. Chem. 248,1240-1247 22 Plagemann, P.G.W. (19711 J. Lipid Res. 12, 715--724 23 Wood, R., Falch, J. and Wiegand, R.D. (1974) Lipids 9, 987-992 24 Jenkin, H.M. and Anderson, L.E. (1970) Exp. Cell Res. 59, 6-10 25 Morrison, S.J. and Jenkin, H.M. (1972) In Vitro 8,94-100 26 Wood, R. and Falch, J. (1974) Lipids 9,979-986
R., ed.1,
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and
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