Lipid Synthesis in Cultured Human Embryonic F ibroblasts MOSELEY WAITE, LOUIS KUCERA, LYNN KING, and SHERYL CROSLAND, The Departments of Biochemistry, Microbiology, and Immunology, The Bowman Gray School of Medicine, Winston-Salem, N.C. 27103 ABSTRACT
This transfer could be similar to that described by Wirtz and Zilversmit (3) or may involve an unknown mechanism. Data reported by BenPorat and Kaplan (1) also showed a decrease in the total amount of labeled lipid in the cytoplasmic fraction after virus infection of host cells. It is not possible from their study to determine whether there had been a net loss of preformed phosphoglyceride from the membrance or if new cytoplasmic membrane synthesis from nonlabeled precursors was inhibited after infection. Asher et al. (4) did not find stimulation of [3HI choline incorporation into phosphoglycerides of cultured monkey kidney cells after HSV infection. From these data, it is reasonable to assume there would be a net loss in the lipids of the endoplasmic reticulum during virus multiplication. To elucidate the metabolic events involved in herpesvirus-host cell interaction, it is imperat i v e t o d e t e r m i n e the existence of key metabolic pathways of lipid synthesis and their interconversion in host cells. Therefore, experiments were designed to investigate in hyman embryonic fibroblasts (HEF) (a) the origin of the glycerol phosphate from both glycolysis and by the action of glycerokinase, fb) the synthesis of fatty acids from acetate and glucose, (c) the incorporation of added fatty acids (both saturated and u n s a t u r a t e d ) a n d endogenously synthesized fatty acid, (d) the synthesis de novo of phosphatidyl choline (PC) and sphingomyelin (Sph) from choline and phosphatidyl ethanolamine (PE) from ethanolamine, and (e) the methylation of PE to form PC. Also, we contrasted the incorporation of labeled ethanolamine into total lipids in grow ing phase and stationary phase cells.
We describe here the pathways by w h i c h h u m a n embryonic fibroblasts synthesize lipids. In these studies, we quantitated the phospholipids by their phosphorus content and by their acyl components. These determinations defined b o t h the chemical composition of the cellular membranes as well as their metabolic turnover. Using radiolabeled precursors, we have shown (a) synthesis of the glycerol moiety via glycolysis and the action of glycerokinase, (b) utilization of both exogenously added and endogenously synthesized fatty acids, (c) synthesis de novo of phosphatidyl choline a n d p h o s p h a t i d y l ethanolamine from their base precursors, and (d) the methyl a t i o n o f phosphatidyl ethanolamine yielding phosphatidyl choline. Dividing cells synthesized phosphoglyceride more rapidly than cells in the stationary phase. However, considerable turnover o f cellular lipid did occur in the stationary phase. INTRODUCTION
During herpesvirus multiplication in hos! ceils, the virus acquires its envelope by budding from the inner nuclear membrane. Ben-Porat and Kaplan reported that the virus envelope was derived from a site of newly-formed lipid in the nuclear membrane (1). Since it is generally accepted that the endoplasmic reticulum is the major, if not exclusive, site for phosphoglyceride synthesis (with the exception o f cardiolipin) (2), it would seem reasonable that herpes simplex virion (HSV) stimulates the transfer of phosphoglyceride from the endoplasmic reticulum to the nuclear membrane.
TABLE I Protein and Phospholipid Content
of HEF
Hour Number o f cells x 10 -5 # g Protein n m o l e s Phospholipid /.~g Protein/i 0 S cells n m o l e s Phospholipid/l 05 ceils
0
4
8
24
28
32
6.3 400 24.1 63 3.8
7.2 400 24.5 56 3.4
7.4 436 33.2 59 4.5
8.3 404 3.117 49 3.8
11.0 516 50.8 47 4.6
12.0 520 47.5 43 3.9
698
LIPID SYNTHESIS IN FIBROBLASTS
/
8,000 Phospholipids -
699
Precursor #4C-glucose Totol Lipids
15,00C
6,000 o PE O PC
/
10,00(3
O DG
2,0OC
mr
1
I
I
0
20
40
I 0
--
I
i
F
I 20
I 40
Hours
FIG. 1. Analysis of continuous uptake of [14C] glucose (1 #Ci/ml); Sp. Act. = 190 mCi/mmole) into phospholipids and total lipids extracted from stationary phase (100% sheeted monolayers) HEF cell monolayers. The cultures were overlaid with 2 ml of growth medium supplemented with [14C] glucose. At various times after incubation at 37 C, some of the cultures were harvested by scraping the cells into the overlay medium and washing each culture dish with 0.5 ml of 0.01 M phosphate buffered saline pH 7.0 (PBS). The wash fluids were pooled with the cell suspension. The cells were pelleted (450 x g, 5 min), and suspended in 1 ml PBS. The cell lipids were extracted and analyzed as described in Experimental Procedures. E X P E R I M E N T A L PROCEDURES Cell Cultures and Media
the fatty acid suspended by ultrasonic irradiation. The protein content of the cells was quantitated by the procedure of Lowry et al. (6) using bovine serum albumin as the standard.
Secondary human embryonic fibroblast cell cultures purchased from Flow Laboratories, Source of Radiolabeled Compounds Rockville, MD, were used between passages 5 1-[ 14C]_acetate, methyl-[ 14C]_methionine, and 17 and propagated as previously described (5). Briefly, the cell growth medium consisted and 1,2-[14C]-choline were purchased from N u c l e a r , B o s t o n , MA. o f Eagle's minimal essential medium with N e w E n g l a n d 1- [ 1 4C] -linoleate, Hank's base salts (Grand Island Biological Com- 9, 1 0-[ 3 H ] - p a l m i t a t e , l - [ 3 H ] - and 1,2-[14C]-ethanolamine were pany, Grand Island, NY) supplemented with 10% heat-inactivated (56 C, 30 rain) fetal calf purchased from Amersham/Searle, Arlington Heights, IL. ICN supplied the uniformly labeled serum (FCS), 10% tryptose phosphate broth, 100 U of penicillin per ml, 100/ag of strepto- glucose and glycerol. mycin per ml, 0.075% NaHCO3, and 2 mM L-glutamine. Cells were serially passaged in 75 Extraction and Separation of the Lipids. cm 2 plastic flasks (BioQuest, Oxnard, CA). For The lipids of the cells were extracted by the experiments, about 1.0 x 105 cells were sus, method of Bligh and Dyer (7). Ahquots of the pended in 2 ml of growth medium with Earle's CHC13 extract were counted to determine the base salts and subcultured to each plastic 35 cm total precursor incorporation into lipid and petri dish (Flow Laboratories, Rockville, MD). chromatographed on Silica Gel H plates in the For lipid quantitation, usually about 1.0 x 107 chloroform-methanol-acetic acid-water system cells were grown in culture bottles. All cell cul- (60:40:5:4, v/v) or the chloroform-methanoltures were incubated at 37 C in a humidified 7N ammonia (60:40:5, v/v) to separate the incubator with a 5% CO2 atmosphere. Experi- phosphoglycerides. The neutral lipids were ments in which radiolabeled fatty acids were separated in the ether-ligroin (bp 63 C-75 C) incorporated, the CHCI3 solution containing formic acid (70:30: 1.5, v/v) system. The lipids the fatty acid was dried, the media added, and were visualized by staining with 12 vapor. The LIPIDS, VOL. 12, NO. 9
M. WAITE, L. KUCERA, L. KING, AND S. CROSLAND
700
Precursor 14C-glycerol )
Phospt~hp~ds
37,000 T~ol
.
/
27,000
Li~ds /
Totol p ~ i p O aPE o Pc
ZS~X)O
s~h
9 TG 1FFA ODG
ZO,O00
5,ooo 0
5,OOOo I
20
I
40
I
HOurs
o
I
20
I
40
FIG. 2. Analysis of continuous uptake of I I4C] glycerol (1 taCi/ml; Sp. Act. = 16 mCi/mmole) into phospholipids and total lipids extracted from stationary phase (100% sheeted monolayers) HEF cells. See legend under Figure 1 for Experimental Procedures. silica gel which contained the lipid was scraped into a scintillation vial and counted using a toluene-Triton x 100-H20 (2:0.2:1, v/v) scintillation cocktail. The lipids were eluted from the silica gel by a mixture of CHCI 3-methanoi-H20 (2:1:0.3, v/v) for phosphorus analysis (8). Those samples taken for analysis by gas liquid chromatography were treated in the same way except that they were detected by rhodamine spray rather than by 12 vapor. The methylation and gas chromatography are described elsewhere (9). An internal standard of behenic acid was used for quantitation. To measure glucose incorporation into the fatty acids of the total glycerides, the CHCI 3 cell extract was dried and redissolved in 1.0 ml of 0.1 M NaOH in methanol. The sample was incubated for I hr at 37 C, 1 ml of 0.2 M HCI was added and the fatty acid was extracted in pentane. Aliquots of each phase were counted by scintillation spectrometry to measure the amount of label in the glycerol moiety (watermethanol phase) and acyl moiety (pentane phase). Chromatography of the initial CHCI3 extract indicated that less than 3% of the label was free fatty acid. LIPIDS, VOL. 12, NO. 9
RESULTS AND DISCUSSION
We first determined the relationship between the number of cells in culture and their protein and phospholipid content. Table ! shows that over a 32 hr period of culture there was a doubling in the number of cells. Since the ratio of protein and phosphoand phospholipid content to cell number remains constant, we concluded that the cellular membranes are not changing appreciably during culture. This is also found when the composition of the membrane lipids is analyzed, in the experiments reported here, there was some variabilit~ in the number of cells used; therefore, the results presented are from a representative experiment rather than an average of all. With the exception of the cell counting, all determinations were done in at least three experiments. Both uniformly labeled [14C] glucose and [14C] glycerol were incorporated into the cellular lipids of growing phase HEF (Fig. 1 and 2). The major class of products was phospholipid and the major phospholipid was PC. Considerable amounts of labeled precursor appeared in Sph. Since this phospholipid does not
LIPID SYNTHESIS 1N FIBROBLASTS
701
Precursors[3HI Palmdote - 114C]Acetote - - I00'000 1 Phospholipids
I0,000
~ I ~..-0
1,00
,ooi
f~//
V
O
I 20
Sph
" J'"~ I/~r !~l
I 40
Ii
x TOIOIphospholipld
:TGA
I
I ODG
I
20
40
Hours FIG. 3. Analysis of continuous uptake of [3H I palmitate (5 #Ci/ml; Sp. Act. = 477 mCi/mmole) and [ 14C] sodium acetate (I #Ci/ml; Sp. Act. = 60 mCi/mmole) into phospholipids extracted from stationary phase (100% sheeted monolayers) HEF cells and total lipids. See legend under Figure 1 for Experimental Procedures. contain glycerol, the labeled precursor would most likely be the acyl chain or the sphingosine derived from palmitic acid. Saponification of the total lipids demonstrated that 20% of the labeled precursor was in the acyl groups of the lipids and 80% in water soluble products, presumably glycerol. Small amounts of free fatty acid accumulated with either precursor which suggests that once synthesized, they are rapidly incorporated into lipids (cf. Fi&s. 3 and 4). An appreciable amount of triglyceride (TG) was also synthesized; relatively more labeled glycerol was incorporated into TG than labeled glucose (about 30% vs. 15%). These data suggest that a pool of diglyceride (DG) arising from glycolysis was used primarily for phospholipid synthesis whereas DG drived from the action of glycerol kinase was utilized to somewhat a greater extent for neutral lipid synthesis. Since the amounts of glycerol and glucose present in the media differed so greatly, no attempt was made to quantitate the relative incorporation of the two. To demonstrate that the cells could utilize both exogenous as well as synthesized fatty acids, we contrasted the incorporation of [3H] palmitate and [14C] acetate (Fig. 3). Results showed a very rapid incorporation of [3H] palrnitate between 0 and 10 h.r; after 24 hr
TOTAL
CPM
RECOVERED Cells
2 - ~,
- -
Flu,OS -- - --
\
\,., 2~ _
OF 0
l
l
4
8
_
I
l
24
30
HOurS
FIG. 4. Analysis of continuous uptake of [3H] palmitate (10 vCi/ml; Sp. Act. = 477 mCi/mmole) and [14C] linoleate (2 vCi/ml; Sp Act. = 61 mCi/mmole) into phospholipids and total lipids extracted from HEF cells and supematant fluids (initial 1.1 x 107 cells). These data are of aliquots of each sample counted directly. The designations are (o), [ 14C] ; (O), [3HI. LIPIDS, VOL. 12, NO. 9
M. WAITE, L. KUCERA, L. KING, AND S. CROSLAND
702
there was only a slight increase in palmitate ~aa '0 ggg
'0
~ ex
incorporation9 At 8 hr, little free [ 3 H ] palmi'0
g
+ ~.
~
~
N
=
g
a~ g
"13
el_
~u
.~. o_ 0
F
.g.
t") O
7~7~
~o ~n z.. o= o -r
b tn
T ,nl
>
z
TPPP ~ Q ~ O
o~
ta
LIPIDS, VOL. 12, NO. 9
..i
.l~
t.~
tate was recovered in the cells; most had been incorporated into the lipids. On the other hand, [14C] acetate incorporation into lipid continued to increase between 0 and 48 hr. We did n o t determine whether acetate was incorporated by synthesis de novo or by acyl elongation (10). Similar to the findings with [14C] glycerol and [14C] glucose, PC was the major fipid labeled. More labeled precursor was found in Sph than in PE which indicates that the choline containing lipids are very active metabolically. Considerable label was found in TG as well, although there was a decrease in [3H] TG and [31ti DG after 24 hr. The total recovery of [3H] lipid from the cells and the growth medium decreased from 4.6 x 105 cpm to 3.0 x 105 cpm during the observation period which possibly could be the result of fatty acid oxidation. Since there was a net increase in the total amount of [3H] phospholipid, it is possible that some [3tt] TG and [3HI DG serves as precursor for [ 3H] phospholipid 9 The next experiment was designed to compare the kinetics of incorporation of a saturated ([3H] paimitate) with an unsaturated acid ([14C] linoleate). This comparison required that the specific activities of the precursor fatty acids and product phospholipids be determined. The cells and supernatant fluids were separated and the free fatty acids and phosoholipidsisolated. Aliquots of the purified compounds were counted in a scintillation counter and the methyl esters of the acyl groups formed. Table 1I shows the acyl group composition of the isolated lipids as the average of five time periods of incubation. The only changes that occurred during growth are noted by an asterisk9 A striking feature of these results is the lack of polyunsaturated fatty acid normally found in mammalian cells. The major changes were an increase in the percentage of 18:2 in phosphatidyl serine + phosphatidyl inositol (PS + PI) from 14.4 to 24.9, a decrease in cellular free fatty acid from 22.2 to 10.7, and a decrease in the 18:1 of both pools of free fatty acid from about 24 to about 15. The changes that are concomitant with these did not occur in a single acid and, therefore, did not cause a change of more than a few percent 9 The [3H] palmitate entered the cell more rapidly than did the [14C] linoleate (Fig. 4). When the data of this figure is combined with that of Table I, this means that 4-5 times more palmitate was taken up than 18:2 on a mass basis. Further, there was a greater recovery of [3H] than [14C] as lipid. (The loss presumably
LIPID SYNTHESIS IN F I B R O B L A S T S
|
NEUTRAL
PHOSPHOLIPID
PEo
3-
Sph
TG
703
LIPID
9
FFAm DG
0
6-
~
~Z-
2-
0J
~,
8-
~:) 2-
~ 4-
ol0
4
8
2,4
Hours
30
4
8
24
Hours
30
FIG. 5. The radioactivity of the phospholipids (panel a) and neutral glycerides (panel b) separated by thin layer chromatography. These samples were from the experiment described in Figure 4. Aliquots (0.1 ml) of each lipid extracted (about 3 ml) were counted. SPECIFIC
ACTIVITY OF FATTY ACIDS
Cells - Fluids . . . .
I0-
%
"x
6- ) - " -~
//"
IZ~
,~
0 4 ~o-
\ .~
o~.
8
4
o 0
4
8
Hours
--J 24
50
FIG. 6. The free fatty acids isolated in the experiment- in Figure 5 were quantitated by gas liquid chromatography. The amount of palmitic acid was divided into the total [3HI counts while the amount of linoleic acid was divided into the [ 14C] counts.
o-~"-
0
~
4
,
8
,
Hours
24
I
3O
FIG. 7. The total amount of each phosphoglyceride, quantitated by gas liquid chromatography, was divided by the [3H] counts (top) and [lZ~C] counts (bottom). The data were calculated on the basis of the individual fatty acids as welL Those results are not presented but are discussed in the Results. LIPIDS, VOL. 12, NO. 9
M. WAITE, L. KUCERA, L. KIN(;, AND S. CROSLAND
704
PrecurSOr
14C - chohne
3H- el
Ioo.ooo
15,000
o PE o PC
5O,000
~,00Q
~ IODOO
-
~O00
5Doo
O
oL~ o
I 20
I 413
I o
I
20
I
40
Hours
FIG. 8. Analysis of continuous uptake of [14C] choline (5 uCi/ml; Sp. Act. = 4.2 mCi/mmole) and [3H] ethanolamine (10/aCi/ml; Sp. Act. = 3.8 mCi/mmole) into phospholipids and total lipids extracted from stationary phase (100% sheeted monolayers) HEF cells. See legend under Figure 1 for Experimental Procedures. 60.O00
PE
40,000
~ 20,000
--~ 2,000
" PC p ~,ol4c p
/
u~ l,OO0
J I
Hours
FIG. 9. The specific activities of the phosphatidyl ethanolamine and phosphatidyl choline were determined in an experiment similar to that described in Figure 8 except that each lipid was extracted and the lipid phosphorus quantitatext as described in Experimental Procedures. LIPIDS, VOL. 12, NO. 9
was due to other metabolic fates such as oxidation.) Figure 5 shows that most of both labeled precursors were incorporated into PC (panel a) and TG (panel b). Similar to the findings presented in Figure 3, very little radiolabeled free fatty acid was recovered which indicates a very rapid incorporation of exogenous fatty acid into cellular glycerides. This is further demonstrated by Figure 6 which compares the specific radioactivities of the free fatty acids. At no time did the specific activity of the cellular fatty acid reach that of the extraceUular pool. When compared with individual phosphoglycerides (Fig. 7), we found that, based on the total acyl content, the cellular fatty acid pool had t h e higher specific radioactivity. However, when we consider that only about 20% of the fatty acid was [3H] paimitate and 7-17% was [14C], the specific activities of the individual fatty acid in the phosphoglycerides was actually higher than that of the cellular fatty acid pool. These data suggest that there is a pool of fatty acid derived from exogenous sources that does not mix with the total cellular pool. This exogenous pool is preferentially incorporated into phosphoglycerides which accounts for its high specific radioactivity. Comparison of the three phospholipid classes studied demonstrates that their synthesis (measured by increase in specific
LIPID SYNTHESIS IN FIBROBLASTS
705 [3HI eth0n010mme [14C] ethoflolomine- - - - -
precursors
5I
Precursor 3H ethonolamine
O PE 0 PC 0 Sph 100,000~ c 1 ~ : ] , , _ ~ 3
~
sQ
3 / O s - ' O "~
I0,000~
/
E
// /
d/
i
0
4
8
12
Hours
I 24
,
/
o
o
o
~
//~J 48
~ooO-O.. . . . . .
0--0-20
FIG. 10. Analysis of continuous uptake of [3H] ethanolamine (10 #Ci/ml; Sp. Act. = 3.8 mCi/mmole) into growing phase (the cells were about 75% sheeted monolayers) and stationary phase (100% sheeted monolayers) HEF cells. See legend under Figure 1 for Experimental Procedures. radioactivity) paralleled their relative concentrations in the ceils; namely, of the three, PC is the major lipid present, 40%; PE is intermediate, 35%; and PS + PI is least, 25%. It is of interest to note that the incorporation of radiolabeled fatty acid did not continue after the first 8 hr even though only 50% of the fatty acid pool was used. This would be the result of a cessation of phospholipid synthesis or the turnover being at a steady state that cannot be measured by this technique. The data presented in Fig. 8 are consistent with the latter interpretation. The source of the base moiety of the lipid was studied by adding [3H] ethanolamine and [14C] choline (Fig. 8). In this experiment, it is possible to measure both the synthesis de novo of the major phospholipids, PC, PE, and Sph, as well as the methylation of PE which produces PC. We did not attempt to measure any intermediates in the methylation process. Surprisingly, the results with [14C1 choline (Fig. 8) showed an 8 hr delay before labeled Sph was detected. Some lag in the synthesis of [ l s C ] PC was seen, possibly due to the large pool of unlabeled choline in the culture medium. We do not know the pathway which leads to the formation of the small amount of [14C] labeled
0 40
Hours
FIG. 11. Analysis of continuous uptake of ethanolamine into total phospholipids extracted from growing phase (about 75% sheeted monolayers) HEF cells. The cells were suspended in 2 ml growth medium supplemented with [3H] ethanolamine (10 #Ci/ml; Sp. Act. = 3.8 mCi/mmole) and seeded into 35 mm petri dishes. After 24 hr incubation at 37 C, the growth medium was aspirated, the cell monolayers were washed with 0.01 M Tris-saline, pH 7.4, and overlaid with 2 ml fresh growth medium supplemented with [14C] ethanolamine (10 #Ci/ml; Sp. Act. = 58 mCi/mmole). Incubation was continued at 37 C. At various times, some of the cultures were harvested and analyzed for uptake of the labeled precursors into phospholipids extracted from the HEF cells as described in Experimental Procedures. PE (Fig. 8). After 30 hr, there was a marked decrease in [3H] ethanolamine incorporated into PE and in PE which had been converted to PC (Fig. 8, right panel). These results were not seen in all experiments and might reflect some variation in the culture conditions of HEF cells. The incorporation of [3H] ethanolamine into PE did not exhibit any lag whereas the conversion of PE to PC was delayed about 8 hr. These results would be expected of a precursorproduct relationship. In a separate experiment, we determined the specific radioactivity of the PE and PC labeled with [3H] ethanolamine and [14C] choline (Fig. 9). When expressed in this fashion, it can be seen that the amount of lipid newly synthesized, relative to the amount of endogenous lipid, is quite small. This accounts for the initial low specific activity. The specific activity of the [14C] choline in the medium LIPIDS, VOL. 12, NO. 9
706
M. WAITE, L. KUCERA, L. KING, AND S. CROSLAND
initially was 5 x 10s cpm/nmole. Since the maximum spedfic activity of [14C] choline that was found was in the order of 2 x 103, we can conclude that very few of the phospholipid molecules are newly synthesized under these conditions. Similar calculations can be made with the [3HI ethanolamine. In this experiment, there was a 1.25-fold increase in the amount of PE recovered from the cells after 24 hr of culture. The value for PC was 2.0. Results of experiments designed to compare [3HI ethanolamine incorporation into growing phase cells with stationary phase cells showed that maximal rate of incorporation was delayed by several hours (Fig. l 0). Presumably this was the result of difference in the pool size of endogenous ethanolamine in the two types of culture. We also found that the [14C] methyl methione was incorporated into the PC of dividing cells. In this case, there was no lag period in the formation of PC as the preexisting PE served as substrate. To determine the intracellular site and metabolic pathway utilized for HSV envelope phospholipid synthesis, experiments need to be designed to differentiate preexisting membrane lipids from membrane lipids synthesized de novo after virus infection. Toward this end, experiments were done to determine if it is possible to differentiate between preexisting and de novo synthesized membrane lipids by sequentia_l labeling with [3HI ethanolamine and [ 1 4 C ] ethanolamine (Fig. l l ) . We chose labeled ethanolamine as a precursor since two major metabolic events can be measured; first, PE synthesis de novo, and second, methylation of PE. Growing phase ceils were incubated in the presence of [3HI ethanolamine. After 24 hr at 37 C, when the cells had reached a stationary phase of growth (confluent monolayers), the cultures were washed and incubation was continued in the presence of [14C] ethanolamine for various times before extraction of the llpids (Fig. 9). Results showed that during a 48 hr period, there was a net loss of about 50% of the [3H] labeled lipids (1.3 x 105 to 6.5 x 104 cpm). During this same period, there was incorporation of 2.3 x 104 cpm of [14C] ethanolamine in labeled lipids. Based on the relative specific activities of the labeled ethanolamines
LIP1DS, VOL. 12, NO. 9
([3H] specific activity was 65 times greater than [14C]), it appears as if more of the new [14C] PE was formed in the stationary phase cells as compared to PE measured in growth phase cells. In summary, results of this investigation demonstrated the major metabolic pathways of lipid synthesis in HEF cells as well as some of their compositional characteristics. The cells can (a) use both glycerol and glucose for the glycerol backbone of the lipid, (b) use saturated and unsaturated fatty acids and can incorporate acetate into the fatty acids which comprise phosphoglycerides, and (c) synthesize PE and PC de novo and methylate PE to form PC. Further, the cells can synthesize lipids in both the stationary and growing phases although the latter is more rapid. Since wc are able to sequentially label lipids with [3HI and [14C] ethanolamine, we believe this experimental design will be suitable for future studies on the genetic origin of intracellular site of synthesis and metabolic pathway required for the formation of specific lipid components needed for biogenesis of the herpesvirus envelope. ACKNOWLEDGMENTS This work was supported in part by U.S.P.H.S. grants CA 14318 and CA 1219"/. Moseley Waite is the recipient of a Career Development Award AM 17392. REFERENCES I. Ben-Porat, T., and A.S. Kaplan, Virology 45:252 0971). 2. McMurray, W.C., and W.L. Magee, Annu. Rev. Biochem. 41 : ! 29 (I 9"/2).
3. Wirtz, K.W.A.,and D.B. Zilversmit, J. Biol. Chem. 243:3596 (1968). 4. Asher, Y., M. lieUer, and Y. Becket, J. Gen. Virol. 4:65 (1969). 5. Melvin, P., and L.S. Kucera, Ibid. 15:534 (1975). 6. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall, J. Biol. Chem. 192:265 (1951). 7. Blight, E.G., and W.J. Dyer, Can. J. Biochem. Physiol. 3"/:911 (1959). 8. Chalvardjian, A., and E. Rudnicki, Anal. Biochem. 36:225 ( ! 9"/0). 9. Waite, M., B. Parce, R. Morton, C. Cunningham, and H.P. Morris, Cancer Res. 37:2092 0977). 10. Wakil, S.J., J. Lipid Res. 2:1 (1961). [ Revision received June !, 1977 ]
This transfer could be similar to that described by Wirtz and Zilversmit (3) or may involve an unknown mechanism. Data reported by BenPorat and Kaplan (1) also showed a decrease in the total amount of labeled lipid in the cytoplasmic fraction after virus infection of host cells. It is not possible from their study to determine whether there had been a net loss of preformed phosphoglyceride from the membrance or if new cytoplasmic membrane synthesis from nonlabeled precursors was inhibited after infection. Asher et al. (4) did not find stimulation of [3HI choline incorporation into phosphoglycerides of cultured monkey kidney cells after HSV infection. From these data, it is reasonable to assume there would be a net loss in the lipids of the endoplasmic reticulum during virus multiplication. To elucidate the metabolic events involved in herpesvirus-host cell interaction, it is imperat i v e t o d e t e r m i n e the existence of key metabolic pathways of lipid synthesis and their interconversion in host cells. Therefore, experiments were designed to investigate in hyman embryonic fibroblasts (HEF) (a) the origin of the glycerol phosphate from both glycolysis and by the action of glycerokinase, fb) the synthesis of fatty acids from acetate and glucose, (c) the incorporation of added fatty acids (both saturated and u n s a t u r a t e d ) a n d endogenously synthesized fatty acid, (d) the synthesis de novo of phosphatidyl choline (PC) and sphingomyelin (Sph) from choline and phosphatidyl ethanolamine (PE) from ethanolamine, and (e) the methylation of PE to form PC. Also, we contrasted the incorporation of labeled ethanolamine into total lipids in grow ing phase and stationary phase cells.
We describe here the pathways by w h i c h h u m a n embryonic fibroblasts synthesize lipids. In these studies, we quantitated the phospholipids by their phosphorus content and by their acyl components. These determinations defined b o t h the chemical composition of the cellular membranes as well as their metabolic turnover. Using radiolabeled precursors, we have shown (a) synthesis of the glycerol moiety via glycolysis and the action of glycerokinase, (b) utilization of both exogenously added and endogenously synthesized fatty acids, (c) synthesis de novo of phosphatidyl choline a n d p h o s p h a t i d y l ethanolamine from their base precursors, and (d) the methyl a t i o n o f phosphatidyl ethanolamine yielding phosphatidyl choline. Dividing cells synthesized phosphoglyceride more rapidly than cells in the stationary phase. However, considerable turnover o f cellular lipid did occur in the stationary phase. INTRODUCTION
During herpesvirus multiplication in hos! ceils, the virus acquires its envelope by budding from the inner nuclear membrane. Ben-Porat and Kaplan reported that the virus envelope was derived from a site of newly-formed lipid in the nuclear membrane (1). Since it is generally accepted that the endoplasmic reticulum is the major, if not exclusive, site for phosphoglyceride synthesis (with the exception o f cardiolipin) (2), it would seem reasonable that herpes simplex virion (HSV) stimulates the transfer of phosphoglyceride from the endoplasmic reticulum to the nuclear membrane.
TABLE I Protein and Phospholipid Content
of HEF
Hour Number o f cells x 10 -5 # g Protein n m o l e s Phospholipid /.~g Protein/i 0 S cells n m o l e s Phospholipid/l 05 ceils
0
4
8
24
28
32
6.3 400 24.1 63 3.8
7.2 400 24.5 56 3.4
7.4 436 33.2 59 4.5
8.3 404 3.117 49 3.8
11.0 516 50.8 47 4.6
12.0 520 47.5 43 3.9
698
LIPID SYNTHESIS IN FIBROBLASTS
/
8,000 Phospholipids -
699
Precursor #4C-glucose Totol Lipids
15,00C
6,000 o PE O PC
/
10,00(3
O DG
2,0OC
mr
1
I
I
0
20
40
I 0
--
I
i
F
I 20
I 40
Hours
FIG. 1. Analysis of continuous uptake of [14C] glucose (1 #Ci/ml); Sp. Act. = 190 mCi/mmole) into phospholipids and total lipids extracted from stationary phase (100% sheeted monolayers) HEF cell monolayers. The cultures were overlaid with 2 ml of growth medium supplemented with [14C] glucose. At various times after incubation at 37 C, some of the cultures were harvested by scraping the cells into the overlay medium and washing each culture dish with 0.5 ml of 0.01 M phosphate buffered saline pH 7.0 (PBS). The wash fluids were pooled with the cell suspension. The cells were pelleted (450 x g, 5 min), and suspended in 1 ml PBS. The cell lipids were extracted and analyzed as described in Experimental Procedures. E X P E R I M E N T A L PROCEDURES Cell Cultures and Media
the fatty acid suspended by ultrasonic irradiation. The protein content of the cells was quantitated by the procedure of Lowry et al. (6) using bovine serum albumin as the standard.
Secondary human embryonic fibroblast cell cultures purchased from Flow Laboratories, Source of Radiolabeled Compounds Rockville, MD, were used between passages 5 1-[ 14C]_acetate, methyl-[ 14C]_methionine, and 17 and propagated as previously described (5). Briefly, the cell growth medium consisted and 1,2-[14C]-choline were purchased from N u c l e a r , B o s t o n , MA. o f Eagle's minimal essential medium with N e w E n g l a n d 1- [ 1 4C] -linoleate, Hank's base salts (Grand Island Biological Com- 9, 1 0-[ 3 H ] - p a l m i t a t e , l - [ 3 H ] - and 1,2-[14C]-ethanolamine were pany, Grand Island, NY) supplemented with 10% heat-inactivated (56 C, 30 rain) fetal calf purchased from Amersham/Searle, Arlington Heights, IL. ICN supplied the uniformly labeled serum (FCS), 10% tryptose phosphate broth, 100 U of penicillin per ml, 100/ag of strepto- glucose and glycerol. mycin per ml, 0.075% NaHCO3, and 2 mM L-glutamine. Cells were serially passaged in 75 Extraction and Separation of the Lipids. cm 2 plastic flasks (BioQuest, Oxnard, CA). For The lipids of the cells were extracted by the experiments, about 1.0 x 105 cells were sus, method of Bligh and Dyer (7). Ahquots of the pended in 2 ml of growth medium with Earle's CHC13 extract were counted to determine the base salts and subcultured to each plastic 35 cm total precursor incorporation into lipid and petri dish (Flow Laboratories, Rockville, MD). chromatographed on Silica Gel H plates in the For lipid quantitation, usually about 1.0 x 107 chloroform-methanol-acetic acid-water system cells were grown in culture bottles. All cell cul- (60:40:5:4, v/v) or the chloroform-methanoltures were incubated at 37 C in a humidified 7N ammonia (60:40:5, v/v) to separate the incubator with a 5% CO2 atmosphere. Experi- phosphoglycerides. The neutral lipids were ments in which radiolabeled fatty acids were separated in the ether-ligroin (bp 63 C-75 C) incorporated, the CHCI3 solution containing formic acid (70:30: 1.5, v/v) system. The lipids the fatty acid was dried, the media added, and were visualized by staining with 12 vapor. The LIPIDS, VOL. 12, NO. 9
M. WAITE, L. KUCERA, L. KING, AND S. CROSLAND
700
Precursor 14C-glycerol )
Phospt~hp~ds
37,000 T~ol
.
/
27,000
Li~ds /
Totol p ~ i p O aPE o Pc
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9 TG 1FFA ODG
ZO,O00
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20
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40
I
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I
20
I
40
FIG. 2. Analysis of continuous uptake of I I4C] glycerol (1 taCi/ml; Sp. Act. = 16 mCi/mmole) into phospholipids and total lipids extracted from stationary phase (100% sheeted monolayers) HEF cells. See legend under Figure 1 for Experimental Procedures. silica gel which contained the lipid was scraped into a scintillation vial and counted using a toluene-Triton x 100-H20 (2:0.2:1, v/v) scintillation cocktail. The lipids were eluted from the silica gel by a mixture of CHCI 3-methanoi-H20 (2:1:0.3, v/v) for phosphorus analysis (8). Those samples taken for analysis by gas liquid chromatography were treated in the same way except that they were detected by rhodamine spray rather than by 12 vapor. The methylation and gas chromatography are described elsewhere (9). An internal standard of behenic acid was used for quantitation. To measure glucose incorporation into the fatty acids of the total glycerides, the CHCI 3 cell extract was dried and redissolved in 1.0 ml of 0.1 M NaOH in methanol. The sample was incubated for I hr at 37 C, 1 ml of 0.2 M HCI was added and the fatty acid was extracted in pentane. Aliquots of each phase were counted by scintillation spectrometry to measure the amount of label in the glycerol moiety (watermethanol phase) and acyl moiety (pentane phase). Chromatography of the initial CHCI3 extract indicated that less than 3% of the label was free fatty acid. LIPIDS, VOL. 12, NO. 9
RESULTS AND DISCUSSION
We first determined the relationship between the number of cells in culture and their protein and phospholipid content. Table ! shows that over a 32 hr period of culture there was a doubling in the number of cells. Since the ratio of protein and phosphoand phospholipid content to cell number remains constant, we concluded that the cellular membranes are not changing appreciably during culture. This is also found when the composition of the membrane lipids is analyzed, in the experiments reported here, there was some variabilit~ in the number of cells used; therefore, the results presented are from a representative experiment rather than an average of all. With the exception of the cell counting, all determinations were done in at least three experiments. Both uniformly labeled [14C] glucose and [14C] glycerol were incorporated into the cellular lipids of growing phase HEF (Fig. 1 and 2). The major class of products was phospholipid and the major phospholipid was PC. Considerable amounts of labeled precursor appeared in Sph. Since this phospholipid does not
LIPID SYNTHESIS 1N FIBROBLASTS
701
Precursors[3HI Palmdote - 114C]Acetote - - I00'000 1 Phospholipids
I0,000
~ I ~..-0
1,00
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Sph
" J'"~ I/~r !~l
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Hours FIG. 3. Analysis of continuous uptake of [3H I palmitate (5 #Ci/ml; Sp. Act. = 477 mCi/mmole) and [ 14C] sodium acetate (I #Ci/ml; Sp. Act. = 60 mCi/mmole) into phospholipids extracted from stationary phase (100% sheeted monolayers) HEF cells and total lipids. See legend under Figure 1 for Experimental Procedures. contain glycerol, the labeled precursor would most likely be the acyl chain or the sphingosine derived from palmitic acid. Saponification of the total lipids demonstrated that 20% of the labeled precursor was in the acyl groups of the lipids and 80% in water soluble products, presumably glycerol. Small amounts of free fatty acid accumulated with either precursor which suggests that once synthesized, they are rapidly incorporated into lipids (cf. Fi&s. 3 and 4). An appreciable amount of triglyceride (TG) was also synthesized; relatively more labeled glycerol was incorporated into TG than labeled glucose (about 30% vs. 15%). These data suggest that a pool of diglyceride (DG) arising from glycolysis was used primarily for phospholipid synthesis whereas DG drived from the action of glycerol kinase was utilized to somewhat a greater extent for neutral lipid synthesis. Since the amounts of glycerol and glucose present in the media differed so greatly, no attempt was made to quantitate the relative incorporation of the two. To demonstrate that the cells could utilize both exogenous as well as synthesized fatty acids, we contrasted the incorporation of [3H] palmitate and [14C] acetate (Fig. 3). Results showed a very rapid incorporation of [3H] palrnitate between 0 and 10 h.r; after 24 hr
TOTAL
CPM
RECOVERED Cells
2 - ~,
- -
Flu,OS -- - --
\
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l
l
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FIG. 4. Analysis of continuous uptake of [3H] palmitate (10 vCi/ml; Sp. Act. = 477 mCi/mmole) and [14C] linoleate (2 vCi/ml; Sp Act. = 61 mCi/mmole) into phospholipids and total lipids extracted from HEF cells and supematant fluids (initial 1.1 x 107 cells). These data are of aliquots of each sample counted directly. The designations are (o), [ 14C] ; (O), [3HI. LIPIDS, VOL. 12, NO. 9
M. WAITE, L. KUCERA, L. KING, AND S. CROSLAND
702
there was only a slight increase in palmitate ~aa '0 ggg
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LIPIDS, VOL. 12, NO. 9
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tate was recovered in the cells; most had been incorporated into the lipids. On the other hand, [14C] acetate incorporation into lipid continued to increase between 0 and 48 hr. We did n o t determine whether acetate was incorporated by synthesis de novo or by acyl elongation (10). Similar to the findings with [14C] glycerol and [14C] glucose, PC was the major fipid labeled. More labeled precursor was found in Sph than in PE which indicates that the choline containing lipids are very active metabolically. Considerable label was found in TG as well, although there was a decrease in [3H] TG and [31ti DG after 24 hr. The total recovery of [3H] lipid from the cells and the growth medium decreased from 4.6 x 105 cpm to 3.0 x 105 cpm during the observation period which possibly could be the result of fatty acid oxidation. Since there was a net increase in the total amount of [3H] phospholipid, it is possible that some [3tt] TG and [3HI DG serves as precursor for [ 3H] phospholipid 9 The next experiment was designed to compare the kinetics of incorporation of a saturated ([3H] paimitate) with an unsaturated acid ([14C] linoleate). This comparison required that the specific activities of the precursor fatty acids and product phospholipids be determined. The cells and supernatant fluids were separated and the free fatty acids and phosoholipidsisolated. Aliquots of the purified compounds were counted in a scintillation counter and the methyl esters of the acyl groups formed. Table 1I shows the acyl group composition of the isolated lipids as the average of five time periods of incubation. The only changes that occurred during growth are noted by an asterisk9 A striking feature of these results is the lack of polyunsaturated fatty acid normally found in mammalian cells. The major changes were an increase in the percentage of 18:2 in phosphatidyl serine + phosphatidyl inositol (PS + PI) from 14.4 to 24.9, a decrease in cellular free fatty acid from 22.2 to 10.7, and a decrease in the 18:1 of both pools of free fatty acid from about 24 to about 15. The changes that are concomitant with these did not occur in a single acid and, therefore, did not cause a change of more than a few percent 9 The [3H] palmitate entered the cell more rapidly than did the [14C] linoleate (Fig. 4). When the data of this figure is combined with that of Table I, this means that 4-5 times more palmitate was taken up than 18:2 on a mass basis. Further, there was a greater recovery of [3H] than [14C] as lipid. (The loss presumably
LIPID SYNTHESIS IN F I B R O B L A S T S
|
NEUTRAL
PHOSPHOLIPID
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FIG. 5. The radioactivity of the phospholipids (panel a) and neutral glycerides (panel b) separated by thin layer chromatography. These samples were from the experiment described in Figure 4. Aliquots (0.1 ml) of each lipid extracted (about 3 ml) were counted. SPECIFIC
ACTIVITY OF FATTY ACIDS
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FIG. 6. The free fatty acids isolated in the experiment- in Figure 5 were quantitated by gas liquid chromatography. The amount of palmitic acid was divided into the total [3HI counts while the amount of linoleic acid was divided into the [ 14C] counts.
o-~"-
0
~
4
,
8
,
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FIG. 7. The total amount of each phosphoglyceride, quantitated by gas liquid chromatography, was divided by the [3H] counts (top) and [lZ~C] counts (bottom). The data were calculated on the basis of the individual fatty acids as welL Those results are not presented but are discussed in the Results. LIPIDS, VOL. 12, NO. 9
M. WAITE, L. KUCERA, L. KIN(;, AND S. CROSLAND
704
PrecurSOr
14C - chohne
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FIG. 8. Analysis of continuous uptake of [14C] choline (5 uCi/ml; Sp. Act. = 4.2 mCi/mmole) and [3H] ethanolamine (10/aCi/ml; Sp. Act. = 3.8 mCi/mmole) into phospholipids and total lipids extracted from stationary phase (100% sheeted monolayers) HEF cells. See legend under Figure 1 for Experimental Procedures. 60.O00
PE
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~ 20,000
--~ 2,000
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/
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Hours
FIG. 9. The specific activities of the phosphatidyl ethanolamine and phosphatidyl choline were determined in an experiment similar to that described in Figure 8 except that each lipid was extracted and the lipid phosphorus quantitatext as described in Experimental Procedures. LIPIDS, VOL. 12, NO. 9
was due to other metabolic fates such as oxidation.) Figure 5 shows that most of both labeled precursors were incorporated into PC (panel a) and TG (panel b). Similar to the findings presented in Figure 3, very little radiolabeled free fatty acid was recovered which indicates a very rapid incorporation of exogenous fatty acid into cellular glycerides. This is further demonstrated by Figure 6 which compares the specific radioactivities of the free fatty acids. At no time did the specific activity of the cellular fatty acid reach that of the extraceUular pool. When compared with individual phosphoglycerides (Fig. 7), we found that, based on the total acyl content, the cellular fatty acid pool had t h e higher specific radioactivity. However, when we consider that only about 20% of the fatty acid was [3H] paimitate and 7-17% was [14C], the specific activities of the individual fatty acid in the phosphoglycerides was actually higher than that of the cellular fatty acid pool. These data suggest that there is a pool of fatty acid derived from exogenous sources that does not mix with the total cellular pool. This exogenous pool is preferentially incorporated into phosphoglycerides which accounts for its high specific radioactivity. Comparison of the three phospholipid classes studied demonstrates that their synthesis (measured by increase in specific
LIPID SYNTHESIS IN FIBROBLASTS
705 [3HI eth0n010mme [14C] ethoflolomine- - - - -
precursors
5I
Precursor 3H ethonolamine
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FIG. 10. Analysis of continuous uptake of [3H] ethanolamine (10 #Ci/ml; Sp. Act. = 3.8 mCi/mmole) into growing phase (the cells were about 75% sheeted monolayers) and stationary phase (100% sheeted monolayers) HEF cells. See legend under Figure 1 for Experimental Procedures. radioactivity) paralleled their relative concentrations in the ceils; namely, of the three, PC is the major lipid present, 40%; PE is intermediate, 35%; and PS + PI is least, 25%. It is of interest to note that the incorporation of radiolabeled fatty acid did not continue after the first 8 hr even though only 50% of the fatty acid pool was used. This would be the result of a cessation of phospholipid synthesis or the turnover being at a steady state that cannot be measured by this technique. The data presented in Fig. 8 are consistent with the latter interpretation. The source of the base moiety of the lipid was studied by adding [3H] ethanolamine and [14C] choline (Fig. 8). In this experiment, it is possible to measure both the synthesis de novo of the major phospholipids, PC, PE, and Sph, as well as the methylation of PE which produces PC. We did not attempt to measure any intermediates in the methylation process. Surprisingly, the results with [14C1 choline (Fig. 8) showed an 8 hr delay before labeled Sph was detected. Some lag in the synthesis of [ l s C ] PC was seen, possibly due to the large pool of unlabeled choline in the culture medium. We do not know the pathway which leads to the formation of the small amount of [14C] labeled
0 40
Hours
FIG. 11. Analysis of continuous uptake of ethanolamine into total phospholipids extracted from growing phase (about 75% sheeted monolayers) HEF cells. The cells were suspended in 2 ml growth medium supplemented with [3H] ethanolamine (10 #Ci/ml; Sp. Act. = 3.8 mCi/mmole) and seeded into 35 mm petri dishes. After 24 hr incubation at 37 C, the growth medium was aspirated, the cell monolayers were washed with 0.01 M Tris-saline, pH 7.4, and overlaid with 2 ml fresh growth medium supplemented with [14C] ethanolamine (10 #Ci/ml; Sp. Act. = 58 mCi/mmole). Incubation was continued at 37 C. At various times, some of the cultures were harvested and analyzed for uptake of the labeled precursors into phospholipids extracted from the HEF cells as described in Experimental Procedures. PE (Fig. 8). After 30 hr, there was a marked decrease in [3H] ethanolamine incorporated into PE and in PE which had been converted to PC (Fig. 8, right panel). These results were not seen in all experiments and might reflect some variation in the culture conditions of HEF cells. The incorporation of [3H] ethanolamine into PE did not exhibit any lag whereas the conversion of PE to PC was delayed about 8 hr. These results would be expected of a precursorproduct relationship. In a separate experiment, we determined the specific radioactivity of the PE and PC labeled with [3H] ethanolamine and [14C] choline (Fig. 9). When expressed in this fashion, it can be seen that the amount of lipid newly synthesized, relative to the amount of endogenous lipid, is quite small. This accounts for the initial low specific activity. The specific activity of the [14C] choline in the medium LIPIDS, VOL. 12, NO. 9
706
M. WAITE, L. KUCERA, L. KING, AND S. CROSLAND
initially was 5 x 10s cpm/nmole. Since the maximum spedfic activity of [14C] choline that was found was in the order of 2 x 103, we can conclude that very few of the phospholipid molecules are newly synthesized under these conditions. Similar calculations can be made with the [3HI ethanolamine. In this experiment, there was a 1.25-fold increase in the amount of PE recovered from the cells after 24 hr of culture. The value for PC was 2.0. Results of experiments designed to compare [3HI ethanolamine incorporation into growing phase cells with stationary phase cells showed that maximal rate of incorporation was delayed by several hours (Fig. l 0). Presumably this was the result of difference in the pool size of endogenous ethanolamine in the two types of culture. We also found that the [14C] methyl methione was incorporated into the PC of dividing cells. In this case, there was no lag period in the formation of PC as the preexisting PE served as substrate. To determine the intracellular site and metabolic pathway utilized for HSV envelope phospholipid synthesis, experiments need to be designed to differentiate preexisting membrane lipids from membrane lipids synthesized de novo after virus infection. Toward this end, experiments were done to determine if it is possible to differentiate between preexisting and de novo synthesized membrane lipids by sequentia_l labeling with [3HI ethanolamine and [ 1 4 C ] ethanolamine (Fig. l l ) . We chose labeled ethanolamine as a precursor since two major metabolic events can be measured; first, PE synthesis de novo, and second, methylation of PE. Growing phase ceils were incubated in the presence of [3HI ethanolamine. After 24 hr at 37 C, when the cells had reached a stationary phase of growth (confluent monolayers), the cultures were washed and incubation was continued in the presence of [14C] ethanolamine for various times before extraction of the llpids (Fig. 9). Results showed that during a 48 hr period, there was a net loss of about 50% of the [3H] labeled lipids (1.3 x 105 to 6.5 x 104 cpm). During this same period, there was incorporation of 2.3 x 104 cpm of [14C] ethanolamine in labeled lipids. Based on the relative specific activities of the labeled ethanolamines
LIP1DS, VOL. 12, NO. 9
([3H] specific activity was 65 times greater than [14C]), it appears as if more of the new [14C] PE was formed in the stationary phase cells as compared to PE measured in growth phase cells. In summary, results of this investigation demonstrated the major metabolic pathways of lipid synthesis in HEF cells as well as some of their compositional characteristics. The cells can (a) use both glycerol and glucose for the glycerol backbone of the lipid, (b) use saturated and unsaturated fatty acids and can incorporate acetate into the fatty acids which comprise phosphoglycerides, and (c) synthesize PE and PC de novo and methylate PE to form PC. Further, the cells can synthesize lipids in both the stationary and growing phases although the latter is more rapid. Since wc are able to sequentially label lipids with [3HI and [14C] ethanolamine, we believe this experimental design will be suitable for future studies on the genetic origin of intracellular site of synthesis and metabolic pathway required for the formation of specific lipid components needed for biogenesis of the herpesvirus envelope. ACKNOWLEDGMENTS This work was supported in part by U.S.P.H.S. grants CA 14318 and CA 1219"/. Moseley Waite is the recipient of a Career Development Award AM 17392. REFERENCES I. Ben-Porat, T., and A.S. Kaplan, Virology 45:252 0971). 2. McMurray, W.C., and W.L. Magee, Annu. Rev. Biochem. 41 : ! 29 (I 9"/2).
3. Wirtz, K.W.A.,and D.B. Zilversmit, J. Biol. Chem. 243:3596 (1968). 4. Asher, Y., M. lieUer, and Y. Becket, J. Gen. Virol. 4:65 (1969). 5. Melvin, P., and L.S. Kucera, Ibid. 15:534 (1975). 6. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall, J. Biol. Chem. 192:265 (1951). 7. Blight, E.G., and W.J. Dyer, Can. J. Biochem. Physiol. 3"/:911 (1959). 8. Chalvardjian, A., and E. Rudnicki, Anal. Biochem. 36:225 ( ! 9"/0). 9. Waite, M., B. Parce, R. Morton, C. Cunningham, and H.P. Morris, Cancer Res. 37:2092 0977). 10. Wakil, S.J., J. Lipid Res. 2:1 (1961). [ Revision received June !, 1977 ]
