Regulation of Fatty Acid Biosynthesis in Ehrlich Cells by Ascites Tumor Plasma Lipoproteins 1 RICHARD McGEE, JR. 2, DOUGLAS E. BRENNEMAN, and ARTHUR A. SPECTOR, Departments of Biochemistry and Medicine, University of Iowa, Iowa City, Iowa 52242

ABSTRACT

Fatty acid biosynthesis in Ehrlich cells in vitro was reduced when very low den- sity lipoproteins (VLDL) isolated from the ascites tumor plasma were added to the incubation medium. The degree of inhibition was dependent on the VLDL concentration. At the VLDL concentra- t i o n s usually present in the ascites plasma, there was a 30% decrease in bio- synthesis as measured by 3H20 incor- poration into fatty acids. Analysis of the labeled fatty acids by gas liquid chroma- tography indicated that this decrease was due to a reduction in fatty acid de novo biosynthesis and that chain elongation actually was increased when VLDL were present. Although ascites plasma low- and high density lipoproteins also produced a concentration-dependent inhibition of fatty acid biosynthesis, their effects were much smaller than those of the VLDL. Studies employing VLDL and radioactive free fatty acids indicated that the cells took up and utilized fatty acids derived from these lipoproteins. When VLDL were present, labeled free fatty acid incorporation into cell phospholipids, cholesteryl esters, and CO 2 decreased, whereas its incorporation into the cell free fatty acid pool increased. By con- trast, the cells incorporated only very small amounts of fatty acid from either low- or high density lipoproteins. This suggests that the VLDL exert their inhib- itory effect on fatty acid synthesis by supplying exogenous fatty acids to the cells.

INTRODUCTION

Fatty acid synthesis in mammalian cells is decreased when free fatty acids (FFA) are added to the culture medium (1,2). A similar reduction occurs when Ehrlich ascites tumor cells are exposed to fatty acids complexed with

1presented in part at the AOCS Spring Meeting, Dallas, April 1975.

2 Present address: Laboratory of Biochemical Genetics, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014.

plasma albumin during in vitro incubations (3). FFA are contained in the ascites tumor plasma and are readily taken up and utilized by the Ehrlich cells (4,5). Therefore, fatty acid syn- thesis in the Ehrlich cell probably is regulated to some extent by the FFA concentration of the ascites tumor plasma (6).

In addition to FFA, the Ehrlich ascites plasma contains high concentrations of lipo- proteins when the tumor is grown in CBA mice (7). Very low density lipoproteins (VLDL) that are rich in triglycerides and cholesterol contain most of the lipids present in the ascites plasma (7). A hypertriglyceridemia is produced when CBA mice are inoculated with the Ehrlich ascites tumor, and the triglyceride-rich VLDL that accumulate in the ascites plasma resemble those present in the blood plasma of the host (7,8). Therefore, the ascites plasma VLDL probably are derived from the tumor bearing host. In vitro studies with ascites plasma VLDL containing tracer amounts of radioactive tri- glycerides suggest that these lipoproteins might serve as a source of fatty acids for the Ehrlich cells (9). Therefore, it was of interest to deter- mine whether they, like FFA, also exert a regu- latory effect on lipid synthesis in the Ehrlich cell.

MATERIALS AND METHODS

Lipoproteins and Ceils

The Ehrlich ascites tumor was harvested from male CBA mice from 12 to 14 days fol- lowing transplantation (6). Ascites plasma lipo- protein fractions were isolated by preparative ultracentrifugation at the following densities: VLDL, < 1 . 0 0 6 ; low density lipoproteins (LDL), 1.006-1.063; and high density lipopro- teins (HDL), 1.063-1.21 (7). Prior to these ultracentrifugations, the ascites plasma was centrifuged at 30,000 g for 30 min to remove any trace quantities of chylomicrons that were present (9). Each of the isolated lipoprotein fractions was washed twice by ultracentrifuga- tion through a solution of appropriate maxi- mum density, and the washed fractions were dialyzed for 24 hr at 4 C against 4 1 of Krebs- Ringer phosphate buffer, pH 7.4. The buffer was changed at least twice during the 24 hr period. Each of these washed and dialyzed lipo- protein fractions migrated as a single band on

66

LIPOPROTEINS AND METABOLISM 67

electrophoresis (7). The triglyceride and choles- terol content of the isolated l ipoprotein frac- tions was measured by an automated method (10,ll).

The Ehrlich cells were freed of contami- nating erythrocytes (6), and ca. 1 x 108 ceils were incubated with labeled substrates as described in the legends to the tables and figures. The stock preparation of cells used in each experiment was counted with a hemo- cytometer. Aliquots of the suspension then were added by pipet to each incubation flask. In most of the experiments, the number of ceils per flask was 1 x 10 s. After incubation, the contents of the flask were chilled and trans- ferred into 20 ml of cold Krebs-Ringer buffer contained in centrifuge tubes. The cells were sedimented at 600 g for 5 rain at 4 C, washed with 30 ml of fresh buffer and sedimented again (9). Cell lipids were extracted with a chloroform-methanol solution and-the chloro- form phase isolated (12). In some experiments, the lipids were separated by thin layer chroma- tography using a solvent system of hexane: diethyl ether:methanol: acetic acid (90: 20: 2: 3) (13). After visualization of the lipids with 12 vapor and sublimation of the I2, the radio- activity contained in segments of the silica gel was determined (9). Segments of the gel were scraped directly into a scintillator solution con- taining dioxane (14), and the radioactivity was measured in a liquid scintillation spectrometer. Quenching was monitored with the external standard.

The rate of fat ty acid biosynthesis was measured by the incorporation of 3H from 3H20 (2 mCi/flask) into saponifiable lipids (6). In these experiments, the chloroform phase of the chloroform-methanol extract was saponi- fled with ethanolic KOH and the saponifiable fraction isolated after extract ion and subse- quent acidification (6). One aliquot of the saponifiable fraction was taken for liquid scin- tillation counting. Other aliquots were taken for analysis of the amount of 3H in individual f a t t y ac ids . The saponifiable lipids were methylated, and the methyl esters separated and t rapped using a Hewlett-Packard Gas Liquid Chromatograph equipped with a 9:1 effluent stream splitter (3). The radioactive methyl ester fractions were collected in Teflon tubing immersed in 10 ml of a toluene:Tri ton X-100 scintillator solution and counted in the liquid scintillation spectrometer (6).

Collection of 14CO 2 was made using incuba- tion flasks with removable center wells con- taining 0.2 ml of 2 N KOH. Radioactivity con- tained in the KOH solution was measured in 18 ml of a toluene:methanol scintillator solu-

tion (14).

Materials and Analyses

3H20 and 14C-labeled substrates were ob- t a i n e d from either New England Nuclear (Boston, MA) or Amersham/Searle (Arlington Heights, IL). Unlabeled fatty acids and fat ty a c i d m e t h y l esters were purchased from NuChek Prep (Elysian, MN). The labeled fat ty acids were purified by extraction into alkaline ethanol followed by acidification and reextrac- t i o n i n t o n -hep t ane (15). Bovine serum albumin, obtained from Miles Laboratories (Kankakee, IL), was defatted with activated charcoal (16). Fa t ty acids were complexed with albumin by adding dropwise a warm solution of the sodium soap to an albumin solution that was being stirred mechanically (15). The fat ty acid content of these solutions was measured by t i trat ion (17), and the protein content was measured by the Lowry method (18). Gas liquid chromatography columns and packings were obtained from Applied Science Labora- tories (State College, PA).

A modi/ icat lon ot the proceduie of Lee and Fritz was used to measure the long chain acyl CoA content of the cells (19). After incubation, the contents of the flasks were transferred to chilled centrifuge tubes containing 0.6 ml of 60% HC104. The material was sonified in an ice bath for 10 sec and the precipitate was col- lected by centrifugation (3). After suspending the precipitate in 3 ml of 15 mM dithiothreitol , the solution was adjusted to pH 11 to 12 with 6 M KOH. The solution was heated at 60 C for 15 rain, acidified with HC104, and neutralized (3). CoA liberated by this hydrolysis was assayed by the method of Mired and Guy (20). The supernatant solution from the original HC104 precipi ta t ion was neutralized, and 1.5 rnl was assayed for citrate content by the spectrophotometr ic method of Moellering and Gruber (21).

Each sample was counted in the liquid scintillation spectrometer for 10 min or, if the radioactivity content was low, until 1 x 104 cpm were recorded. Most of the samples con- tained at least 1000 cpm above background, except in those cases where very short incuba- tion times were employed or the lipid class did not incorporate much radioactivity, i.e., free fatty acids and cholesteryl esters.

RESULTS

Inhibition of Fatty Acid Biosynthesis

As shown in Figure 1, each of the three asc i tes tumor plasma l ipoprotein fractions inhibited fat ty acid synthesis in the Ehrlich

LIPIDS, VOL. 12, NO. i

68 R. McGEE, DoE. BRENNEMAN, AND A.A. SPECTOR

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FIG. 2. The time dependence of 3H20 incorpora- tion into saponifiable lipid in the presence and absence of very low density lipoproteins (VLDL). Incubations were performed as described in Figure 1 for the indi- cated times. VLDL were present, where indicated, at the concentration of 120 #g cholesterol/ml (860 #g tri- glycerides/ml). Each point represents the mean of three separate cell incubations, with S.E. delineated by vertical bars. Points without error bars had S.E. too small to be shown on the figure. All of the data were obtained using the same preparation of cells.

cells as de termined by the incorpora t ion of 3 H 2 0 in to saponifiable lipids during a 90 rain incubat ion. In each case, the inhibi t ion in- creased as the l ipoprote in concent ra t ion was raised. VLDL produced much larger reduct ions

than ei ther LDL or HDL. The t ime course of the VLDL effect is shown in Figure 2. Fa t ty acid synthesis was unaf fec ted during the first 30 rain of incubat ion. Subsequent ly , the degree of inhibi t ion produced by the V L D L increased as the incubat ion progressed. This observation differs f rom that no ted with FFA, where max imum inhibi t ion of fa t ty acid synthesis occurs during the first 30 rain of incubat ion (6).

In order to de termine whether the inhibi tory act ion of VLDL might be due to a nonspecif ic suppression of cellular metabol ism, we exam- ined the effect of VLDL on the ut i l izat ion of labeled glucose by the Ehrlich cells. As shown in Table I, the oxida t ion of bo th 1-14C glucose and 6-14C glucose to 14CO 2 was increased slightly when VLDL were added. Moreover, the incorpora t ion 1 J 4 C glucose into the phospho- lipids and triglycerides of the Ehrl ich cell also was greater when VLDL were present. Similar increases in the incorpora t ion of radioactive glucose into cell lipids occur when fat ty acids are added to the incubat ion medium (22). Previous studies have shown that exposure to VLDL does not cause excessive lactate dehy- drogenase release f rom the Ehrl ich cells (23). Taken together , these findings indicate that VLDL do not have a nonspecif ic toxic effect on the cells. Therefore , like FFA, V L D L appear to specifically inhibit the fat ty acid biosynthet ic process.

The composi te effects of V L D L plus F F A and the distr ibut ion o f the 3H incorpora ted into the various fa t ty acid fract ions of the Ehrlich cells are shown in Table II. When VLDL alone were added to the incubat ion medium, there was a 36% reduct ion in 3H incorpora t ion into saponifiable lipids. The extent of the reduct ion was changed very lit t le when laurate, palmitate, or i inoleate was added in addi t ion to the VLDL, as compared with VLDL alone. The effects of VLDL on de novo biosynthesis and chain elongat ion were de termined by collect ing five fat ty acid methy l ester fract ions fol lowing gas l iquid chromatography. When ei ther V L D L or V L D L together with F F A were added, there were 50% or greater reduct ions in 3H incorpora- t ion into the < 1 6 : 0 and 1 6 : 0 + 16:1 methy l ester fractions. Lesser reduct ions (7-29%) were observed in the 18:0 + 18:1 fraction. Except in the case where bo th VLDL and palmita te were added, increases in incorpora t ion were observed in the 18:2-20:4 and > 2 0 : 4 fractions. In the Ehrlich cell, 3H incorpora t ion into the < 1 6 : 0 and 1 6 : 0 + 16:1 fractions occurs primari ly through de n o v o fa t ty acid synthesis, except when laurate is available (3,6). Incorpora t ion of 3H into the 18:0 + 18:1 f rac t ion occurs

LIPIDS, VOL. 12, NO. 1

LIPOPROTEINS AND METABOLISM

TABLE I

Effects of Ascites Plasma VLDL on 1-14C Glucose and 6 -14C Glucose Utilization by Ehrlich Cells a

69

Glucose Incorporat ion c

Metabolite VLDL b 1-14C Glucose 6-14C Glucose

nmoles /108 cells

CO 2 None 559 • 10 119 + 2 Present 608 • 8 d 129 -+ 6

Phospholipids None 149 • 4 ND e Present 175 + 3 d ND

Triglycerides None 22 • 1 ND Present 36 -+ 1 f ND

aThe incubation media contained 1 x 108 cells, 0.4 #mole of a lbumin and 21 #moles o f glucose. Incubation was for 1 hr at 37 C.

bThe very low densi ty l ipoproteins (VLDL) added contained 1.86 #mole triglyceride and 1.32 /~mole cholesterol.

CEach value is the mean -+ SE of 6 separate cell incubations, all done with the same cell preparation.

dp<0 .01 . eNot determined. fP<0.001.

TABLE II

The Composi te Effects of Very Low Density Lipoproteins (VLDL) *tad Fat ty Acids on the Distribution o f ~ among Different

Fat ty Acids Synthesized by Ehrlich Cells a

Incorporation o f 3H into Fat ty Acids

Incubat ion condit ions b Total <16 :0 c 16:0 + 16:1 18:0 + 18:1 18:2-20:4 > 2 0 : 4

nmoles 3H/108 cells

Control 474 90 180 100 66 38 VLDL 300 36 78 69 72 45 VLDL + Laurate 324 45 94 78 71 36 VLDL + Palmitate 288 31 72 94 51 40 VLDL + Linoleate 297 24 80 68 77 48

a lncubat ions were for 90 min at 37 C with the condit ions described in Figures 1 and 2. Saponifiable lipids f rom the exper iments described were methyla ted and analyzed using gas liquid chromatography (3). Each value represents the mean of three GLC analyses o f the pooled lipids f rom three separate incubations.

bThe VLDL concentrat ion was 1.4 mg/ml o f triglyceride. The molar ratio o f fa t ty acid to a lbumin was 1:2. CFatty acid methyl ester fractions eluted f rom gas liquid chromatography are abbreviated as chain length:

number of double bonds. The <16 : 0 fraction contains all fa t ty acid methyl esters with retent ion t imes less than that o f 16:0; the > 2 0 : 4 fractions contain all those wi th re tent ion t imes longer than 20:4.

t h r o u g h b o t h de n o v o s y n t h e s i s a n d c h a i n e l o n g a t i o n , w h e r e a s 3 H i n c o r p o r a t i o n i n t o t h e 1 8 : 2 - 2 0 : 4 a n d > 2 0 : 4 f r a c t i o n s o c c u r s e n t i r e l y t h r o u g h c h a i n e l o n g a t i o n . T h e s e r e s u l t s i n d i c a t e t h a t t h e r e d u c t i o n in 3 H i n c o r p o r a t i o n p r o - d u c e d b y V L D L is d u e p r i m a r i l y t o a d e c r e a s e in de n o v o s y n t h e s i s . M o r e o v e r , t h e y i n d i c a t e t h a t F F A , w h e n a d d e d t o g e t h e r w i t h V L D L , e i t h e r d o e s n o t f u r t h e r s u p p r e s s de n o v o s y n t h e s i s o r s u p p r e s s e s i t b y o n l y a s m a l l add i - t i o n a l a m o u n t .

A d d i t i o n a l e x p e r i m e n t s i n d i c a t e d t h a t n e i t h e r t h e l o n g c h a i n f a t t y a cy l C o A n o r t h e c i t r a t e c o n t e n t o f t h e cel ls c h a n g e d a p p r e c i a b l y

as a r e s u l t o f i n c u b a t i o n in m e d i a c o n t a i n i n g V L D L . T h e s e f i n d i n g s a re s i m i l a r t o t h o s e m a d e w i t h p a l m i t a t e a n d s t e a r a t e c o m p l e x e d w i t h b o v i n e s e r u m a l b u m i n (3) . A l t h o u g h t h e s e f ree f a t t y a c i d s r e d u c e d f a t t y ac id de n o v o b i o s y n - t h e s i s b y 5 0 - 6 0 % , t h e y a l so p r o d u c e d v e r y l i t t l e c h a n g e in t h e t o t a l l o n g c h a i n f a t t y a c y l C o A or c i t r a t e c o n t e n t s o f t h e E h r l i c h cel ls .

Lipoprotein Effects on FFA Utilization

W h e n V L D L c o n t a i n i n g t r a c e a m o u n t s o f r a d i o a c t i v e t r i g l y e e r i d e s were i n c u b a t e d w i t h E h r l i c h cells , t h e cel ls t o o k u p a n d u t i l i z e d s o m e o f t h e r a d i o a c t i v i t y . T h e s e r e s u l t s sug-

LIPIDS, VOL. 12, NO. 1

70 R. McGEE, D.E. BRENNEMAN, AND A.A. SPECTOR

TABLE III

Effects of Ascites Plasma VLDL, LDL, and HDL on Palmitate Utilization by Ehrlich Cells a

Lipoprotein fraction 1-14C Palmitate IncorporationC

added b CO 2 Phospholipids Triglycerides Cholesteryl esters Free fatty acids

nEq/lO 8 cells

None 82.0 + - 1.3 186 + - 0.7 1 0 8 - + 0.6 16.4-+ 0.4 9.1 -+ 0.5 VLDL 40.3 -+ 0.3 f 143 -+ 0.5 f 97 -+ 1.0 f 5.7 + 0.1 f 19.0 +-0.6 f LDL 79.0 -+ 0.8 175 • 1.5 f 103 -+ 1.1 e 17.1 +- 1.2 7.1 -+ 1.2 d HDL 73.7 +- 0.2 173 +- 2.5 e 105 -+ 1.6 13.3 -+ 0.5 e 8.8 -+ 0.2

aThe incubation media contained 0.84 #Eq of 1-14C palmitate, 0.4 pmole of albumin, 21 #moles of glucose and 1.1 x 108 cells in a total volume of 4 mL Incubation was for 1 hr at 37 C with air as the gas phase. VLDL = very low density lipoproteins, LDL = low density lipoproteins, HDL = high density lipoproteins.

bVLDL, LDL, and HDL added contained 1570, 124, and 55/~g/ml of triglycerides and 235, 46, and 65/~g/ml of cholesterol, respectively.

CEach value is the mean -+ SE of four separate cell incubations. dp<0.05. Comparisons were made against the values obtained from flasks which contained no added lipopro-

tein. ep<.01. fP<.o01.

TABLE IV

Effects of Ascites Plasma Very Low Density Lipoprotein (VLDL) Concentration on Palmitate Utilization by Ehrlich Cells a

1-14C Palmitate Utilization

VLDL added b CO 2 Phospholipids Triglycerides Cholesteryl esters Free fatty acids

#g/ml nEq/108 cells

0 44 122 41 5.5 2.5 89 42 120 40 5.0 2.6

199 38 117 42 5.0 2.9 487 36 113 48 5.0 4.5 974 30 106 53 3.5 5.5

1460 27 102 55 3.0 10.0 1950 25 96 58 2.5 15.0

aEach flask contained 0.87 ,uEq of 1-14C palmitate, 0.5 gmole of albumin, 21 #moles of glucose, and 0.9 x 108 cells in a total volume of 4 ml. The incubation was for l hr at 37 C with air as the gas phase.

bExpressed as pg/ml of triglycerides in the VLDL. CEach value is the mean of two closely agreeing determinations.

ges ted t h a t V L D L t r ig lycer ides were able to

s u p p l y f a t t y acids to the cells (9). Because F F A also s u p p r e s s e s f a t t y acid s y n t h e s i s in these cells (3 ,6) , it was r e a s o n a b l e to a:ssume tha t the i n h i b i t i o n p r o d u c e d by the V L D L m i g h t be due to the i r s u p p l y i n g f a t t y acids to t he cells. T h e r e f o r e , we wished to d e t e r m i n e w h e t h e r the i n h e r e n t l ipids c o n t a i n e d in the V L D L ac tua l ly can p rov ide f a t t y acids to t he cells du r i ng in v i t ro i n c u b a t i o n s . This was t e s t ed ind i r ec t ly by e x a m i n i n g the e f fec t o f u n l a b e l e d asci tes p l a sma l i p o p r o t e i n s o n the u t i l i za t ion o f 1-14C f a t t y acids also p r e s e n t in the i n c u b a t i o n m e d i u m . I f the ceils o b t a i n e d u n l a b e l e d f a t t y acids f r o m the V L D L lipids, one w o u l d e x p e c t s o m e r e d u c t i o n in the ra te o f u t i l i za t ion o f the rad ioac t ive F F A . As s h o w n in Table I I I , addi-

t ion o f asci tes p l a sma V L D L r e d u c e d the utili- za t ion o f 1-14C p a l m i t a t e b y the Ehr l ich cells, p r i m a r i l y by decreas ing its i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s and its o x i d a t i o n to CO 2. By c o n t r a s t , 1-14C p a l m i t a t e i n c o r p o r a t i o n i n to

the cell F F A p o o l inc reased w h e n VLDL were added . Since the V L D L did n o t s u p p r e s s g lucose u t i l i za t ion (Tab le I) or cause lac ta te d e h y d r o g e - nase release, these e f fec t s are n o t due to a n o n - specif ic i n h i b i t i o n o f cel lular m e t a b o l i s m or tox ic i ty . N e i t h e r L D L n o r H D L had any apprec i ab le e f fec t on 1-14C p a l m i t a t e incor - p o r a t i o n as c o m p a r e d wi th the l i p o p r o t e i n - f r e e m e d i u m .

Table IV d e m o n s t r a t e s t h a t the e f fec t o f V L D L on 1-14C p a l m i t a t e u t i l i za t ion was con- c e n t r a t i o n d e p e n d e n t . As the V L D L c o n c e n t r a -

LIPIDS, VOL. 12, NO. 1

L I P O P R O T E I N S A N D M E T A B O L I S M

tion was raised, there was a progressive reduc- tion in 1-14C palmitate oxidation and incor- poration into phospholipids and cholesteryl esters and a progressive increase in palmitate radioactivity present in the cell FFA pool. Surprisingly, 1-~4C palmitate incorporation into cell triglycerides also increased as the VLDL concentration was raised.

The time course of these VLDL effects on 1 -~ 4C palmitate utilization is shown in Table V. Changes were apparent after 5 min of incuba- tion, the earliest time point tested. In the case of 1-~ 4C palmitate incorporation into phospho- lipids, the percentage reduction produced by the VLDL after 5 min was almost as large as that occurring after 2 hr of incubation. The maximum percentage reduction in incorpora- tion into phospholipids occurred after only 15 min of incubation with VLDL. In the case of oxidation to C02, however, VLDL produced a progressively larger percentage reduction up to 1 hr of incubation. Likewise, the percent change in 1-14C palmitate incorporated into the cell FFA pool continued to increase during the first 9 0 m i n of incubation. More 1-t4C palmitate also was incorporated into triglyc- erides in this experiment when VLDL were present in the medium.

As shown in Table VI, VLDL produced similar effects on the incorporation of radio- active stearate, oleate and linoleate. In each case, less labeled FFA was utilized by the Ehrlich cells when VLDL were present. Labeled FFA oxidation to CO 2 and incorporation into

phospho l ip ids and cholesteryl esters were reduced in every case. Likewise, the amount of labeled fatty acid recovered in the cell FFA pool was considerably greater in every case when VLDL were present. By contrast, the effect of VLDL on labeled fatty acid incorpora- tion into cell triglycerides was variable. When VLDL were present, the incorporation of radio- activity into triglycerides was less with 1-14C stearate, unchanged with 1-14C oleate and more with 1-~ 4C linoleate and 1-14 C palmitate.

DISCUSSION

Regulatory actions of plasma lipoproteins on mammalian cell lipid metabolism have been reported previously, but they have dealt almost entirely with cholesterol synthesis (24-26). One study has shown that the lipoproteins present in fetal calf serum regulate fatty acid synthesis in cultured hepatoma cells (27). This study was confined to comparisons between intact and lipoprotein-poor serum, and no experiments were done to specify which lipoproteins were predominantly responsible for the control of

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fatty acid biosynthesis. The present results, obtained with Ehrlich cells and the lipoproteins isolated from the ascites tumor plasma, suggest that VLDL exert a regulatory effect on fatty acid synthesis in this system. At a given concen- tration, the VLDL are about four times more effective than ascites plasma HDL and seven times more effective than ascites plasma LDL in r educ ing fatty acid biosynthesis (Fig. 1). Although the relative amounts of the three types of lipoproteins present in the ascites plasma vary with the age of the tumor, VLDL are the main lipid-containing lipoproteins at all times (7,8). For example, on the twelfth day of tumor growth, 94% of the triglyceride and 73% of the cholesterol present in the ascites plasma were recovered in the VLDL fraction (8). Taken together, these data suggest that VLDL may contribute to the control of fatty acid synthesis in the intact Ehrlich ascites tumor�9

Two types of regulation of the fatty acid biosynthesis have been observed in mammalian cells. One is long-term control which involves changes in the cellular content of the lipogenic enzymes (28). The other is short-term control in which the activity of the lipogenic enzymes change without any change in their cellular content (1,2,28). FFA regulate fatty acid synthesis through the short-term mechanism in cultured cells (1,2), including Ehrlich cells (3,6). Since the inhibitory effects of VLDL and FFA are not additive (Table II) and the VLDL appear to supply the cells with fatty acids (Tables IV and VI), it is reasonable to assume that they might also inhibit fatty acid synthesis through a short-term regulatory mechanism�9 On the other hand, FFA produce maximal inhibi- tion of fatty acid synthesis within 30 rain (3,6), whereas the VLDL require about 60 min of incubation before an inhibitory effect is mani- fested (Fig. 2). This may be due to a slower uptake of fatty acids when they are derived from VLDL. For example, the build-up of labeled FFA in the cells continues during the first 90 rain of exposure to VLDL (Table IV) but is completed within 5 min during exposure to a FFA-albumin complex (15). Yet, sufficient fatty acids are transferred from the VLDL to produce other effects on radioactive FFA utilb zation within 5 to 15 min (Table V). Therefore, the possibility that the ascites plasma lipopro- teins act through a long-term control mech- anism must be considered even though it is manifested within I hr of incubation.

Previous studies have demonstrated that the Ehrlich cells can utilize the triglycerides con- tained in the ascites plasma VLDL (9). Since l a b e l e d fatty acids from triglycerides are metabolized by these cells in a manner similar

LIPIDS, VOL. 12, NO. 1

LIPOPROTEINS AND METABOLISM 73

to labeled FFA (9), the fatty acids released from VLDL-triglycerides probably enter the same cell FFA pools as fatty acids that are supplied as FFA. Such a mechanism would account for the trapping of more of the incoming labeled FFA in the cell FFA pools when VLDL are present (Tables III-VI). It also would account for the decreased rate of labeled FFA incorporation into phospholipids, choles- teryl esters, and CO2 in the presence of VLDL; for the specific radioactivity of the cell FFA would be decreased by the influx of unlabeled fatty acids from the VLDL. Likewise, the increased 1-14C glucose incorporation into cell lipids in the presence of VLDL (Table I) also is compatible with a utilization of fatty acids derived from the VLDL, the glucose providing the glycerol moieties needed for de novo tri- glyceride and phospholipid synthesis (22).

An observation that is difficult to reconcile with the above explanation is the different effects of VLDL on FFA incorporation into phospholipids and triglycerides, especially the s t imulat ion of triglyceride synthesis under certain conditions. If all of the phospholipids and triglycerides were synthesized by the same de novo pathway, one would expect qualita- tively similar effects of VLDL on radioactive FFA incorporation into both of these ester fractions. This would follow from the observa- tions that diglycerides are used randomly by Ehrlich cells for phospholipid or triglyceride synthesis and that the t-0-alkyl derivatives of glycerophosphatides and acylglycerols also are synthesized from an alkyl acyl intermediate without selectivity (29). Therefore, the dif- ferences in the effects of VLDL on labeled FFA incorporation into phospholipids and triglyc- erides probably reflect additional pathways for synthesis of one or both of these lipid esters. One possibility is that in the presence of VLDL some triglyceride synthesis may occur directly f r o m lower glyceride intermediates. When VLDL containing glycerol-labeled glycerides were incubated with Ehrlich cells, glycerol-

labeled glycerides were recovered in the cells (9). Since free glycerol is utilized very poorly by Ehrlieh cells (9), this findings suggests the existence of a pathway in which monoglyc- e r i d e s are used for triglyceride synthesis (30-32), the monoglycerides being derived from partial hydrolysis of VLDL-triglycerides. This could explain the increase in radioactive FFA incorporation into triglycerides noted in some cases, particularly when the VLDL concentra- tion was raised (Table IV), for the greater availability of monoglyceride substrate may stimulate the incorporation of certain fatty acids into-triglycerides. Another possibility is

that some of the fatty acid incorporation into glycerophosphatides occurs by the acylation of lysophosphatides (33), for an acylation path- way has been demonstrated in the Landschfitz ascites tumor (34).

It remains to be demonstrated whether other forms of VLDL also regulate fatty acid syn- thesis. Although the Ehrlich ascites plasma VLDL resemble VLDL from other species in a number of ways, some differences in their chemical composition and physical properties have been observed (7). For example, the ascites plasma VLDL have a higher free to esterified cholesterol ratio, more phospholipids, increased electrophoretic mobility and a dif- ferent apolipoprotein composition than human VLDL. As compared with the usual lipid con- centrations present in the plasma of many species, the Ehrlich ascites plasma can be characterized as being hypertriglyceridemic (8). In this regard, it should be noted that lipo- proteins obtained from hyperlipidemic rabbit plasma are much more effective in transferring lipids to cultured hepatoma cells than those isolated from normal plasma (35). Since the mechanism of suppression of fatty acid syn- thesis is thought to be transfer of fatty acids from the VLDL to the Ehrlich cells, it is possible that this action is dependent on the fact that these are "abnormal" VLDL and that other forms of VLDL might not produce this effect.

ACKNOWLEDGMENTS

This work was supported by research grants HL 14,781 and HL 14,230 from the National Heart and Lung Institute and 74-689 from the Iowa and Ameri- can Heart Associations.

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[Received May 6, 1976]

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