THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 24'3,No. 4,Issue of February 25,~~. 1249-1247. 1973

Printed in U.S.A.

Lipid Metabolism in Cultured Cells

XI. UTILIZATION OF SERUM TRIGLYCERIDES*

(Received for publication, August 22, 1972)

J. MARTYN BAILEY, BARBARA V. HOWARD,~ AND SUSAN F. TILLMAN

From the Biochemistry Department, The George WashingtorI University Xchool of Medicine, Washington, ~3.6. moo5

SUMMARY

Utilization of serum triglycerides by cells in tissue culture has been studied in order to determine their importance as a source of cell lipids and to obtain information on the cellular mechanisms of triglyceride uptake. L strain mouse fibro- blasts incorporated serum triglycerides from the growth me- dium, but the rates of uptake were up to IO-fold less than that of free fatty acids or monoglycerides under the same culture conditions. When cells were grown under condi- tions of limited supply of serum lipids, however, only about 7% of the cell lipid came from serum free fatty acids and up to 28% was provided by the serum triglycerides. The major portion of the cell lipid under these conditions was derived by de novo synthesis from glucose.

Hydrolysis of exogenous triglyceride by lipases in the serum-supplemented tissue culture medium was measured and found to be inadequate to account for the rate of triglyc- eride utilization observed in these studies. In addition, heat treatment of the serum to inactivate lipase did not signifi- cantly affect triglyceride uptake. No excretion of lipase ac- tivity by the cells was detected.

Incorporation of triglyceride labeled in the glycerol portion with aH and the fatty acid portion with 14C was measured from untreated, heat-inactivated, or delipidized serum using both monolayers and growing cultures. The 3H:14C ratio in the isolated cellular lipids averaged over 70% of that in the original triglyceride under all conditions of culture. Further- more, incorporation of the [3H]glycerol portion of the molecule was not diluted out by addition to the growth medium of a large excess of nonradioactive glycerol or a nonhydrolyzable monoglyceride analog. In short term uptake experiments most of the radioactivity in cell lipids was found in the tri- glyceride fraction, with only minor amounts in the free fatty acids. In long term growth experiments the incorporated triglyceride was converted extensively (70 to 80%) to cellular phospholipid with maintenance of the 3H:14C ratio. The results indicate that L cells in tissue culture can utilize serum triglycerides and suggest that the predominant mechanism of uptake involves the intact molecule and does not require prior hydrolysis.

* This work was supported by United States Public Health Service Grant HE-05062.

$ Present address, Clinical Research Center, Philadelphia General Hospital, Philadelphia, Pennsylvania 19104.

Several investigators have observed that cells cultured in medium containing animal sera can take up free fatty acids from the serum and convert them to cellular glycerolipids (l-4). These workers reported an accumulation of intracellular tri- glyceride droplets which correlated with free fatty acid concen- tration in the medium. It has also been shown that de ?u)vo cell lipid biosynthesis is inhibited when cells are cultured in medium containing serum or added lipid emulsions (5). The origin of cellular lipid has been evaluated by isotopic dilution experiments by Howard and Kritchevsky (6), using cells cultivated in the presence of calf serum, and by Mackenzie et aZ. (2), using cells cultivated in medium supplemented with horse serum. They found in confirmation of earlier observations (5) that very little cellular lipid was derived from biosynthesis when serum lipids were available. Moreover, both of these studies indicated that the main source of cellular non-sterol lipid was serum free fatty acid. The origin of the glycerol moiety of cellular glycerolipids has also been investigated by both laboratories (7, 8). It was found that the glycerol portion was derived almost exclusively from glucose, further confirming that the cells under these condi- tions were utilizing mainly free fatty acids and esterifying it intracellularly to synthesize glycerolipids.

Other studies have indicated, however, that serum triglycerides may also be utilized by cell cultures (9, 10). MB111 strain mouse lymphoblasts can attain population densities up to 10 times that of most cell lines. In experiments using these high population densities, serum triglycerides were completely de- pleted in 4 days (10). The observation that serum triglyceride could be hydrolyzed by enzymes present in serum suggested that triglyceride might be hydrolyzed before utilization by cells (6). This possibi1it.y was indirectly supported by initial studies on serum lipid utilization using dense cultures of L cells (7), be- cause the specific activity of the free fatty acid in the medium decreased under conditions in which serum triglycerides were being utilized. The extent and mechanism of utilization of serum triglyceride by cultured cells has not, however, been studied directly.

Triglyceride utilization in ciao, on the other hand, has been studied in a number of different tissues. Many studies support the view that the triglyceride is usually hydrolyzed before up- take. In the small intestine, for example, triglycerides are hydrolyzed to monoglyceride and free fatty acids before absorp- tion by the epithelial cells (11, 12). When utilization of tri- glyceride by fat cells and adipose tissue was investigated using

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doubly labeled triglyceride (13, 14), hydrolysis was observed Lipids were extracted from harvested cells and medium using prior to absorption. This hydrolysis is mediated by the enzyme lipoprotein-lipase which is released by the adipose cells. Prior hydrolysis has also been reported to occur before uptake of tri- glyceride by muscle, heart cells (15), and mammary gland (16). Moreover, recent experiments with isolated liver cells indicate that these cells also require prior hydrolysis of chylomicron tri- glyceride before it is utilized (17).

However, there are reports of work done in vivo which suggest the possibility of cellular incorporation of intact triglyceride. These include studies in adipose tissue where lipoprotein-bound

20 volumes of acidified chloroform-methanol (2: 1, v/v). (Y- Tocopherol (0.1 mg) was added to the extract as an antioxidant. After filtration the solution was washed with >& volume of CaCh (0.05%), according to the procedure of Folch et al. (23). Phos- pholipids were separated from neutral lipids by the acetone- precipitation method of Bloor (24). Free fatty acids were iso- lated by dissolving the lipids in petroleum ether and extracting with ethanolic KOH (0.5%) at 5” (25). Lipid was saponified in 4 N NaOH at 60” for 16 hours. After extraction three times with ethyl ether the saponification mixture was acidified with

triglycerides, as opposed to those in chylomicra, were observed concentrated HCl and fatty acids were extracted with ethyl to enter cell vacuoles intact (18), and experiments using hepatic ether. Glycerol, which remained in the aqueous layer (26), was parenchymal cells where a membrane-bound lipase appeared to determined by the method of Bailey (27). Total lipid was mediate binding and hydrolysis (19). In addition, the experi- quantitated by the method of Bloor (28), fatty acids by the ments by Olivecrona and Belfrage using doubly labeled triglyc- method of Duncombe (29), and triglycerides by the method of eride suggested that spleen and lung could remove intact chylo- van Handel and Zilversmit (30). Removal of solvents from micron triglyceride from the circulation (15). lipid solutions was accomplished by warming under a stream of

We have investigated therefore the utilization of triglyceride nitrogen, and all samples were stored at 5”. by cells in tissue culture to determine its role as a source of lipid When conversion of exogenous triglyceride to other cellular for cells grown in the presence of serum, and to study the mecha- lipids was followed, or when depletion of individual lipid con- nisms of triglyceride uptake in a cell system where interpretation stituents of the medium was monitored, the lipid extracts were is not hampered by the physiological fluxes present in vivo. This fractionated by thin layer chromatography using a solvent sys- paper presents studies in dense cultures of L cells grown in me- tern of petroleum ether-ethyl ether-acetic acid (70 :30 : 1, v/v). dium supplemented with fetal calf serum of low lipid content. For quantitation the plates were sprayed with 70y0 aqueous Balance studies on the source of cellular lipid under these con- sulfuric acid saturated with potassium dichromate, heated at ditions have been performed and the relative contribution of 180” for 25 min, and scanned at a wave length of 560 nm using free fatty acid and triglyceride has been determined. The the densitometer attachment to the Zeiss PM& spectrophotom- mechanism of uptake of triglyceride by these cells has been in- vestigated using doubly labeled triglyceride and attempts have been made to evaluate the role of serum enzymes in this process.

MATERIALS AND METHODS

The L-929 cells used in this study were obtained from the American Type Culture Collection, and were cultured as mono- layers using conventional sterile techniques. The medium consisted of minimal essential medium (Eagle) supplemented with 1 mg per L biotin, 50 pg per ml of aureomycin, and 5 to 10% fetal bovine serum (Microbiological Associates) or delip- idized serum protein. When specified, serum was inactivated by heating for 30 min at 60”. The pH was adjusted to 7.2 to 7.4 by flushing with filtered COz. Cells were routinely subculti- vated using a 0.25% trypsin solution. To harvest the cells for

eter. All solvents were of spectroanalyzed grade or were redistilled

before use. Palmitic acid, tripalmitin, and monopalmitin were obtained from Applied Science Laboratories. [Ui4C]Glucose, [U-14C]glycerol, sodium [14Cl]palmitate, and [carbozy-14C]tripal- mitin were supplied by Amersham-Searle Corporation, and [aH]- glyceryl tripalmitin by Dhom Corporation, Los Angeles. The purity and position of the radioisotope in the [“Cl- and [aH]tri- palmitin were verified by thin layer chromatography and saponi- fication as described above. Radioactivity of aliquots of lipid and medium was assayed by dissolving in Aquasol (New England Nuclear Corporation) and counting in a liquid scintillation spec- trometer which was standardized for dual isotope counting.

RESULTS

extraction of cell lipids the monolayer was washed three times Source of Cellular Lipid under Con&tons of Limited Supply of with buffered balanced salt solution (20) and disrupted by freeze- Serum Lipids-Previous studies, establishing that free fatty acid thawing in a small volume of distilled water or by scraping with was the source of cellular lipid, had been conducted under con- a rubber policeman. ditions where free fatty acid was present in the medium in abun-

Delipidized serum protein was prepared by the cold ether-ex- dant quantity. In this series of experiments, therefore, culture traction method of Albutt (21). The protein was redissolved conditions were established such that free fatty acid was present in an equivalent volume of balanced salt solution and sterilized in limited amount. This was accomplished by growing the by filtration. Protein was assayed by the method of Lowry L-929 strain, which can form dense monolayers, in culture flasks et al. (22). of 75-cm2 surface area containing only 20 ml of medium supple-

Radioactive and nonradioactive lipids for inclusion in the mented with 5y0 fetal bovine serum, which is low in lipid con- medium were sterilized by filtration and redissolved in a small tent. At the time of harvesting the flasks contained about 10 amount of sterile ethanol. They were added to the serum or million cells corresponding to 2 to 3 mg of cell protein. Under delipidized serum protein solution and allowed to equilibrate for these culture conditions the total cell lipid isolated from one 30 min at 37” before mixing with medium. At no time was the culture flask averaged 0.91 mg, and the total lipid in the medium ethanol concentration in the final medium more than 0.2%. available to the cells averaged 1.3 mg. Of this only 3.2%, or When cells were cultured in the presence of isotopically labeled about 40 pg, was free fatty acid, which is far less than necessary lipids, these were included in the medium at the time of sub- for cellular requirements over a 7-day growth period. cultivation. For short term assay of lipid incorporation the Utilization of the serum free fatty acid and triglyceride by medium containing radioactive lipids was added directly to these cultures was then compared by labeling the serum lipid washed monolayers of cells. with 14C-fatty acid or “C-triglyceride. The results of a typical

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experiment are presented in Fig. 1. Free fatty acid was rapidly depleted, and by the 4th day was essentially absent from the medium. Triglyceride was also taken up, but the relative rate of depletion was much lower. There was some variation among individual experiments in amount of triglyceride utilized, but

20

Free Fatty Acid

\A A I

A I Ia I I 4

2 4 6 8 10 12 14 TIME (DAYS)

FIG. 1. Depletion of W-triglyceride and ‘“C-fatty acid by dense cultures of L cells. Replicate cultures of L strain mouse fibro- blasts subcultivated at 1:lO ratios were grown in medium supple- mented with 50/, fetal calf serum labeled with either sodium [Wl]palmitate or [carbozy-Wltripalmitin. Aliquots of medium were removed daily and radioactivity remaining in triglycerides and fatty acids was measured as described under “Materials and Methods.” Total lipid content of the medium initially averaged 0.065 mg per ml, of which about 0.04 mg remained at the end of the experiment.

TABLE I

Depletion of serum lipids by L cell cultures L-929 strain mouse fibroblasts were grown in minimal essential

medium (Eagle) supplemented with 5yc calf serum having 134 mg per 100 ml total lipid. Cells were grown for 7 days until they had reached confluency and lipid was extracted from the growth

medium, Cell population was about 10 million per flask corre- sponding to about 2.5 mg of protein as measured on the cell residue after extraction of lipids. Lipid fractions were separated

by thin layer chromatography and quantitated by the method of Duncombe (29). Although some free fatty acid was often found at the end of the cultivation period when non-heat-inactivated

serum was used, independent experiments using W-fatty acid indicate rapid turnover of the free fatty acid pool with complete utilization of that originally present. Of the total of 1.34 mg of lipid per culture present initially, 0.45 mg or approximately 34% was utilized.

Lipid class

Phospholipid. ..........

Cholesterol. ............ Free fatty acid ......... Triglyceride ............

Cholesterol ester. ......

Lipid content of growth medium

At 0 time

Amount Amount Per Total Per

culturea culturea -__

/a %

195 15

273 21 42 3

203 16

625 48

I. __

a Average of five experiments.

After 7 days

Total

fig % P&T

200 23 0

183 21 90

0 0 42

83 6.1 125

426 49 200

Amount utilized

rarely was more than 50% removed from the medium during an 8- to la-day growth period.

In relation to the lipid available to the cells under these con- ditions the low rate of triglyceride incorporation observed sug- gested that cell lipid requirements under these culture conditions might not be completely satisfied by either free fatty acid or triglyceride. We therefore monitored depletion of all of the major lipid components of serum by thin layer chromatography. The data, presented in Table I, indicate that on the average only about one-third of the serum lipid is depleted during the culture period. A proportion of the cholesterol and cholesterol ester are taken up; these amounts are sufficient to supply the cellular sterol content, which is only about 10% of the total cell lipid. The depletion of the other serum lipids, however, is not sufficient to account for the intracellular non-sterol lipid. Al- though all of the free fatty acid is utilized, only about one-half of the triglyceride is depleted and no appreciable depletion of phospholipid is observed.

The source of the cellular lipid was then evaluated by isotopic dilution experiments. Tracer amounts of radioactive precursors were added to the medium and the specific activity of the cell lipid at the end of the culture period was compared to that of the original precursor. The data, shown in Table II, indicate that only 6 to 7% of the lipid is derived from fatty acid, and about 9 to 28% from triglyceride. These values agree with those predicted based on the amounts of depletion shown in Table I. The amount of lipid derived from triglyceride varies

TABLE II

Source of cellular lipid in dense cultures of L cells

L strain mouse fibroblasts were grown to confluency in minimal essential medium supplemented with 5% fetal calf serum to which had been added various W-labeled precursors as follows: [l%]glucose, sodium [Wllpalmitate, [carbosy X?]tripalmitin and sodium [14Ci]acetate. The specific activities of the precursors in the medium and in the harvested cells were determined as de- scribed under “Materials and Methods.” Cultures averaged

about 2.5 mg of cell protein per flask. Total lipid content of the medium initially was 1.34 mg in each experiment, about 35y0 of which was utilized during the 4-day growth period.

Lipid precursor

d,%n/patom of carbon %

Free fatty acid Experiment 1. Experiment 2.

3684 263 7.lb

559 36.3 6.5

Triglyceride Experiment 1. Experiment 2.

1406 191

Glucose Experiment 1. 9.97 Experiment 2. 56.5

Acetate................. 219

-r Specific activities

Lipid derived from precursor”

28C

9.4

47.1 52.0

6.3

a Percentage of cell lipid derived from each precursor is equal to specific activity of cell lipid/specific activity of precursor X

100. b The value predicted for lipid derived from free fatty acid on

the basis of data from Table I would be 5yc. c Value predicted from data in Table I and Fig. 1 would be 8 to

20%.

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in the same manner as did the degree of depletion (Fig. 1). The data show that there is a great deal of de nova lipid biosynthesis in these cultures. Approximately one-half of the lipid under these dense culture conditions is derived by de nova synthesis from glucose; and a small proportion is also synthesized from acetate.

Uptake of Serum Triglyceride-The results of the above ex- periments suggested that serum triglycerides are taken up by cultured L cells, but that under the usua.1 conditions of culture the rate of incorporation is too slow to supply all of the cell lipid requirements. In order to investigate the rates of uptake of triglyceride in the absence of other serum lipids, the cells were grown in medium supplemented with delipidized serum protein. 14C-Labeled fatty acid, triglyceride, or monoglyceride was then added to the delipidized serum and the relative cellular incorpo- ration of each of these compounds was measured over a B-day growth period. The results, shown in Fig. 2, indicate that the relative uptake of free fatty acid and monoglyceride uptake from the medium are rapid and are at least 10 times higher than that of triglyceride. This result is also confirmed by the measured relative incorporation of these compounds into the lipids isolated from the harvested cells after the 4-day growth period (Table III), which indicated that much less triglyceride was incorpo- rated than was monoglyceride or free fatty acid.

Our earlier studies had indicated the presence of lipolytic activity in serum-supplemented medium, suggesting that the incorporation of triglyceride may have involved hydrolysis be- fore uptake (6, 7). We therefore quantitated the hydrolysis of triglyceride under the culture conditions employed in order to determine whether it was sufficient to account for the rate of utilization observed in Figs. 1 and 2 and Table III. This was accomplished by labeling the serum triglyceride with tracer amounts of [14C]tripalmitin and measuring the release of radio- activity into the free fatty acid fraction of the serum lipid. The rates of hydrolysis are shown in Fig. 3. The hydrolysis observed in the medium containing unheated serum was about 2 pg per day. This rate varied with different batches of serum, but in no case was it of sufficient magnitude to account for the rate at

2 4 6 TIME (DAYS)

FIG. 2. Uptake of ‘%-labeled precursors by L cells cultured in delipidized serum protein medium. L-929 mouse fibroblasts were grown to confluency in minimal essential medium (Eagle) supple- mented with 5% delipidized fetal calf serum containing either [carbozy-14C]tripalmit&, [carbozy-%]monopalmitin, or sodium P4C11ualmitate. Each livid was added at a concentration of 5 ;g p; ml of medium. Aiiquots (0.2 ml) of medium were sampled at intervals and the amount of radioactive lipid remaining-was determined as described under “Materials and Methods.” Values are expressed in @moles taken up per gram of cell protein.

which triglycerides were taken up by cells, which approximates 10 to 15 pg per day in dense cultures (Fig. 3 and Table I). The hydrolysis can, however, account for the transfer of radioactivity into the free fatty acid pool of the medium which was noted in experiments when triglyceride utilization was monitored using 14C-triglyceride. It is also of sufficient magnitude to account for the dilution of the radioactivity of the serum free fatty acids noted when the medium was supplemented with 14C-labeled free fatty acids (7). As shown in Fig. 3 the hydrolysis of triglyceride does not occur in medium containing serum that has been heat- inactivated, indicating that the cause of hydrolysis is a serum enzyme. In addition, no hydrolysis was observed in “condi- tioned” medium (i.e. medium supplemented with heated serum that had been exposed for 7 days to growing cells). This is an indication that triglyceride utilization by the cells was not the consequence of secretion of a cellular lipase.

Since the results so far suggested, contrary to previous expec- tations, that triglyceride utilization by cells might not be depend-

ent upon hydrolysis, the nature of triglyceride incorporation by

TABLE III

Relative utilization of W-triglyceride, monoglyceride, and free fatty acid by L cells grown in delipidized serum medium

W-Lipids were added to delipidized serum protein as de- scribed under “Materials and Methods” so that the final con-

centration of each lipid was 5 fig per ml of serum. Cells were subcultivated at 1:5 ratios into medium containing the added lipids and harvested after 6 days of growth. Radioactivity in the extracted cell lipids and in ‘the medium was determined as described under “Materials and Methods.”

Lipid Depletion of medium” Relative incorporation

Triglyceride, Free fatty acid. . . . . . Monoglyceride.

% &w/g cell protein

33 zk 5.7 6.7 90 f 2.0 76.0

79.8 f 4.2 37.6

Q Values represent the average of three experiments f standard error of the mean.

MEM plus serum

Conditioned MEM

i 4 DAYS INCUBATED

FIG. 3. Hydrolysis of triglyceride by fetal bovine serum. [carbozy-‘4ClTripalmitin was added to fetal bovine serum, and minimal essential medium supplemented with 5oj, of the labeled serum was added to culture flasks in amounts equivalent to those used in routine culture procedures. “Conditioned medium” had previously been exposed to cells for 7 days. At intervals the medium was sampled, the free fatty acid isolated, and radioac- tivity measured as described under “Materials and Methods.”

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cultured L cells was examined further by using triglyceride labeled with tritium in the glycerol portion and 14C in the fatty acid portion of the molecule ([3H]glyceryl-[14Ci]tripalmitin). Serum triglyceride was labeled using this isotope and 3H:14C ratios in the isolated cell lipids were measured in cultures grown in the presence of the labeled serum for 7 to 14 days. Additional short term experiments were also carried out using monolayers of cells exposed to medium containing the doubly labeled tri- glyceride for only 6 hours. The experiments were carried out both in the presence of untreated serum and serum which had been heated to inactivate lipases. In addition, uptake of the doubly labeled triglyceride was followed in cultures supple- mented only with delipidized serum protein. In none of these different types of conditions was there any large change in the 3H:idC ratios of the cellular lipid as compared to the original triglyceride supplied in the medium (Table IV). This finding, taken in conjunction with the supporting data outlined below, is consistent with the interpretation that the bulk of the tri- glyceride was taken up intact in all these different experimental situations.

To confirm that the tritium and 14C content of the cells did, in fact, represent incorporation of triglyceride into the glycerol and fatty acid portions of the cellular lipids, respectively, the lipid was saponified and the fatty acid and glycerol fractions were iso- lated by extraction. The results given in Table V showed that only 4% of the 3H incorporated was found in the isolated fatty acid fraction, and conversely less than 1 yO of the 14C was present in the glycerol.

It was possible that the observed incorporation of tritium might have resulted from prior hydrolysis of the triglyceride to

TABLE IV Relative utilization of [3H]glycerol and X-fatty acid by cells

exposed to doubly labeled triglycerides

Doubly labeled triglyceride was prepared by adding a mixture of [3H]glyceryl and [14Cl]tripalmitin to either untreated, heat- inactivated or delipidized fetal calf serum as described under “Materials and Methods” and then mixing the labeled serum with medium. The experimental medium consisted of minimal essential medium supplemented with 5% of the labeled serum sample. The total lipid content of the serum or heat-inactivated serum medium was 1.4 mg, of which about 35y0 was utilized in the ‘i-day experiments. When delipidized serum was used the triglyceride content was 100 rg per culture flask and about 30y0 was utilized. When growing cells were exposed to the [3H]glyc- eryl-[14Cl]tripalmitin in whole serum, they were subcultivated at 1:lO ratios in the labeled medium. When delipidized serum was used the cells were subcultivated at 1:3 ratios and grown for only 4 days before harvesting. For 6-hour experiments confluent monolayers were washed and exposed to the labeled medium. The percentages in the last column are included to indicate the probable magnitude of triglyceride incorporated intact.

Experimental conditions [3HIGlycerol to K-fatty acid ratios Uptake

as intact triglyceride

6 Hours. Unheated 7 Days.. Unheated

14 Days. Unheated 6 Hours. Heat-inactivated 7 Days.. Heat-inactivated 4 Days.. Delipidized

2.85 1.75 2.29 1.72 4.2 3.3 1.25 1.06 2.28 1.43 2.24 1.64

% 62 75 79 78 63 73

free fatty acid and either glycerol or monoglyceride accompanied by simultaneous stoichiometric uptake of the hydrolysis products. The incorporation of doubly labeled triglyceride was therefore measured in the presence of an excess of nonradioactive glycerol or glycerol monoether (glycerol-2-octadecyl ether). This was done in order to dilute out any hydrolysis products and thus reduce uptake of the tritium unless it was as intact triglyceride. Glycerol monoether has been shown to be an efficient and com- petitive substitute for monoglyceride during intestinal absorp- tion, and its use avoids complications due to action of mono- glyceride lipases (31, 32). In control experiments using the 14C-labeled compound the monoether was utilized by L strain mouse fibroblasts at about the same rate as monoglyceride. Thin layer chromatography of the extracted cell lipids showed that the isotope was incorporated into triglycerides and phos- pholipids, confirming that the L cell enzyme systems have speci- ficities toward monoether similar to those demonstrated during intestinal absorption of this analog. Uptake of the glycerol moiety of exogenous triglyceride in the presence of the diluents is shown in Table VI. Experiments were carried out with both unheated and heat-inactivated serum, and 3H:14C ratios in the isolated cell lipids were measured. The results show that the incorporation of the [*HIglycerol portion of the triglyceride is not affected either by added excess glycerol or monoglyceride. The amount of glycerol employed was such that greater than a 200-fold excess was present throughout the culture period, and for glycerol monoether there was a 2- to 5-fold excess; however, both the total incorporation and the 3H:14C ratios remain con- stant, confirming that most of the triglyceride is being taken up by the cell without prior hydrolysis. It must be noted that oc- casionally dilution of some of the glycerol label by monoether was noted in untreated serum. The extent of this dilution was most noticeable with non-heat-inactivated serum preparations containing the highest lipolytic activity.

Finally, conversion of the incorporated doubly labeled tri- glyceride into other cell lipids was assayed by thin layer chroma- tography of the cell lipid extract. The percentage of incor- poration of radioactive triglyceride into other cell lipid fractions and the 3H:14C ratios in these fractions are shown in Table VII. The data show that after only 6 hours most of the radioactivity of the triglyceride taken up is present in cellular triglyceride.

TABLE V Distribution of assimilated [~H]glyceryZ-[14C~]tripalmitin in

L cell lipids

As a control to examine the possibility of recycling of isotope and also to validate the separating procedures for glycerol and fatty acids, cells were exposed to minimal essential medium (Eagle) supplemented with 5y0 calf serum and containing doubly labeled triglyceride for 8 to 12 days. The total lipid content of the medium was 1.34 mg initially, of which not more than 50% was utilized during the incubation period. The cell lipids were extracted, and separated into fatty acid and glycerol fractions as described under “Materials and Methods,” and the radio- activity in each extract was determined. Note that the 3H and 14C were recovered almost exclusively in those extracts appropri- ate to glycerol and fatty acids, respectively.

Fraction of cell lipid [~H]Glycerol

Qm %

Fatty acids.. 27,685 4 Glycerola. 584,065 96

a As nonsaponifiable residue.

W-Fatty acid

dpm %

58,919 99 655 1

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TABLE VI TABLE VII

Effect of additional nonradioactive sources of glycerol on the incorporation of the glycerol moiety of exogenous

triglycerides into cell lipid glycerol

These isotopic dilution experiments were carried out to de-

Distribution of exogenous [3H]glyceryZ-[W&ripalmitin info

cell lipids

termine if exogenous triglyceride was hydrolyzed in the medium

before being taken up by cells. Glycerol or glycerol monoether- as a nonhydrolyzable monoglyceride analog (40)-was added to either heated or regular calf serum which had been labeled with

[3H]glyceryl-[14Cl]tripalmitin. The growth medium consisted of

minimal essential medium (Eagle) supplemented with 5yo of the labeled serum. The medium contained initially 208 rg of tri- glyceride in 20 ml and in Experiment 1, 500 rg of glycerol per ml

was added, representing a 24@fold molar excess of free glycerol compared to triglyceride glycerol. At the end of the experiment, about 92yc of the glycerol, or a 220-fold molar excess, remained.

In Experiment 2, 100 pg of octadecyl monoether, representing approximately an initial 2-fold molar excess compared to tri- glyceride, was added. About 50% of the monoether analog was

utilized during the 7-day growth period. In Experiment 3, 500

pg of the monoether, representing a 5-fold molar excess, was added and the medium was incubated with confiuent layers of cells for only 4 hours. The pattern of incorporation of triglycer-

ide in each experiment was followed by measuring the ratio of 3H to 1% in cell lipid for cells grown with diluent or without. If hydrolysis of exogenous triglyceride to glycerol had occurred, an approximately 200-fold decrease in uptake of 3H-triglyceride

would be expected in medium containing glycerol and the 3H:W ratio would be 200-fold less; similarly, if hydrolysis of triglyceride to monoglyceride were the case, up to 2- or 5-fold decreases in

uptake and 3H:W ratios would be predicted.

L-929 strain mouse fibroblasts were grown in minimal essential

medium (Eagle) supplemented with 5% calf serum containing doubly labeled triglyceride for 7 days (long term), or alter- natively, monolayers of cells were exposed to the doubly labeled

medium for 6 hours (short term). The total lipid content of the medium was 1.4 mg per flask, of which about 35yo was utilized in the 7-day experiments. The cells were washed three times with

balanced salt solution and cell lipids were extracted and sepa- rated into the various components by thin layer chromatography. The percentage of the 1% radioactivity and the ratio of aH to 14C in each fraction of lipid is indicated. The 3H appearing in the free fatty acid fraction is an experimental artifact resulting from

trailing of the triglycerides during thin layer chromatography. Note the extensive conversion of triglyceride to phospholipid in the long term experiments with maintenance of the 3H:W ratio.

Lipid Short term (6 hours) Long term (7 days)

Per cent aH:“C Ratio Per cent aH:“C Ratio

Original medium 1.21 1.65 Triglyceride 82 1.1 6.9 1.6 Free fatty acid 8.6 0.28 15.0 0.42 Phospholipid 5.9 0.77 71.6 1.1

down is minor and that the major portion is incorporated and converted into cell lipids without extensive extracellular hy- drolysis. -

EX- peri men NO.

-

1

2

3

Diluent

Glycerol None

(200 Xl” Heat-inactivated Monoether None

(2 X)” Heat-inactivated Monoether None

(5 XY Heat-inactivated

[~HlGlyceryl tripalmitin

taken up aH:“C Ratio

Without With Withou It With diluent diluent diluenl t liluent

M/ml

8.2 7.33 1.72 13.2 10.6 1.43

8.2 5.2 1.72 13.2 10.1 1.43

0.37 0.54 1.32 0.37 0.43 1.34

1.55 1.33 1.64 1.31 1.54

1.63

a Experiments conducted for 7 days in growing cultures. * Experiments conducted for 4 hours using confluent mono-

layers.

In addition, the 3H : 14C ratio in the cell triglyceride is essentially the same as that in the medium, again confirming incorporation intact. A minor proportion of the triglyceride has been hy- drolyzed to free fatty acid and about 6% has already been con- verted to phospholipid. The 3H:14C ratio of this fraction is somewhat lower than that of the triglyceride. Nevertheless, it indicates that a significant amount of the phospholipid is syn- thesized with conservation of the glycerol portion, presumably through diglyceride as an intermediate (33). After 7 days, over 70% of the incorporated triglyceride is present as phospholipid. (A similar result was also obtained at 24 hours.) Again the 3H:14C ratio of the triglyceride resembles the ratio of that sup- plied in the medium, indicating intact incorporation. Retention of tritium-labeled glycerol in the phospholipid is also high, showing that synthesis probably occurs via the diglyceride pathway with conservation of the glycerol and fatty acid portions. In neither the 6-hour nor 7-day samples was there excess of label in the free fatty acid fraction, confirming that triglyceride break-

1245

DISCUSSION

The results of these experiments indicate that the sources of lipids in cultured cells can vary depending upon the culture conditions. In earlier experiments (6), cells were cultured in an environment where there was an excess of free fatty acid avail- able to satisfy cellular lipid requirements. The studies of Mackenzie et al. (2), which also pointed to free fatty acid as the major cellular lipid source, were similarly conducted under culture conditions where free fatty acid was in excess. The data obtained in this study on comparative amounts of lipid uptake suggest that the reason for preferential utilization of free fatty acid under those conditions is simply that this is incorporated into the cell at a much faster rate than other lipids.

Under the present conditions the bulk of the cell lipid is not derived from free fatty acid since this is rapidly depleted. A considerable proportion of the cell lipid is derived from triglyc- eride, but there is also a great deal of de nova biosynthesis. This is probably because the rate of triglyceride uptake is too slow to satisfy cell lipid requirements. Glucose is the primary carbon source for the de 7u)vo synthesis. This is to be expected since earlier observations showed that glucose is the major source of lipid when cells are cultured in a lipid-free medium (5, 7). It should also be noted that glucose is the principal source of the glycerol portion of cell lipids in the presence of excess serum lipids even when fatty acid synthesis from glucose is completely repressed by the lipid-induced negative feedback controls (7).

No utilization of phospholipid was detected in the present studies. Although phospholipid uptake has not been studied directly, Bailey et al. (10) observed some depletion of phos- pholipid by dense cultures of MB111 cells, and phospholipid exchange has been observed in fibroblasts (34). It is possible that the rate of uptake of phospholipid by L cells was too slow to be a significant lipid source under the conditions used here.

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It must be noted that in studies of cell lipid utilization there is at present no completely satisfactory way to present individually labeled lipids to a cell with the assurance that they are in the same state as the native lipoproteins in serum. There is evi- dence from the work of Quarfordt and Goodman (35) and Gut- mand and Shafrir (36) that tracer amounts of radioactive lipid equilibrate with the endogenous serum lipoprotein. When delipidized serum protein was used, lipid was added to the pro- tein solution to form an emulsion. There is some indication that lipids added in this way can bind to the serum proteins in a state approximating that of serum lipoproteins (21), although undoubtedly part of the added lipid remains as emulsion or as- sociates in random micellar form with the proteins. It is en- couraging that a similar pattern of uptake and conversion of triglyceride was noted with either method of presenting tri- glyceride.

All of the data obtained using doubly labeled triglyceride in- dicates that L cells in culture can incorporate the triglyceride molecule intact. The 3H : 14C ratio in the lipid of cells exposed to the doubly labeled exogenous triglyceride was, under all experi- mental conditions, about 70% of the original, and this ratio in the cell lipid was not changed, nor was the amount of triglyceride uptake decreased, when excess nonradioactive glycerol or mono- glyceride analog was added to dilute out possible hydrolysis products. This observation is contrary to the generally ob- served uptake of triglyceride in small intestine (11, 12), adipose cells (13, 14), and most somatic tissue (15, 17), where prior hy- drolysis occurs before entry. Uptake of intact triglyceride has been observed in viva under certain conditions (15, 18, 19), and it might be that the abundant lipase activity in intestine and serum masks the ability of cells in vivo to take up the intact tri- glyceride molecule. Lynch and Geyer (31) have observed up- take of the glycerol portion of monoglyceride by cultured cells when horse serum esterase is inactivated, and Spector (37) has recently suggested that intact incorporation of triglyceride oc- curs in Ehrlich ascites cells. It is also possible that the uptake mechanism could depend on the presence of the lipoprotein car- rier. Many of the experiments both in vitro and in vivo have been conducted using lipid emulsions, micellar preparations, and chylomicron triglycerides, whereas in adipose tissue Markscheid and Shafrir (18) observed intact incorporation of lipoprotein- bound triglycerides.

In the cell culture system serum enzyme can hydrolyze some of the triglyceride before incorpoation, and the slight dilution of labeled glyceride glycerol incorporation by monoether observed occasionally in these experiments suggests that the hydrolysis products are free fatty acid and monoglyceride. The obser- vations that triglyceride uptake is not decreased when serum is heat-inactivated or when triglyceride is presented in association with delipidized serum protein indicates that serum hydrolysis is not necessary for entry, and the consistent maintenance of the 3H:14C ratio in the cell lipid under all cases where cells were exposed to doubly labeled exogenous triglyceride confirms that the triglyceride was not hydrolyzed prior to uptake under these experimental conditions. The presence of the lipolytic activity in the serum, however, does account for the transfer of triglyc- eride label into the free fatty acid fraction of the medium which is observed in experiments when radioactive triglyceride was added to non-heat-inactivated serum. This route thus may play a significant role in the entry of triglyceride into the cell in situations when very little is utilized (6, 7). It must also be pointed out that there is a variability in the lipolytic activity of different samples of serum. Boone et al. (38) have noted differ-

ences in the free fatty acid concentration between batches of commercial fetal bovine serum, which could be a reflection of this variable lipase.

The results of these experiments also indicate that the incor- porated triglyceride can be converted to other cellular lipids. The decrease observed in the 3H:14C ratio in the intracellular triglyceride and the appearance of some 14C in the cellular free fatty acid demonstrates that some triglyceride is hydrolyzed intracellularly. The maintenance of high aH:14C ratio in the cell phospholipids, however, suggests that most of the triglyceride is converted to phospholipid with conservation of the glycerol portion, probably via phosphorylation of diglyceride (33).

The mechanism by which the intact triglycerides are taken up will be the subject of further investigation. Some consideration has been given to the possible role of pinocytosis in the uptake of large molecules by cultured cells. It has been shown, however, that the rate of fluid incorporation by pinocytosis is too small by an order of magnitude or more to account for the observed rate of triglyceride uptake (39). Although the washing proce- dure employed in harvesting the cells is efficient in removing loosely bound radioactive material, it is likely that the initial step in uptake is the adsorption of the triglyceride to specific lipoprotein acceptors on the cell surface. This would then be followed by its entry into the metabolic pool without hydrolysis, since conversion of intact triglyceride to phospholipid was ob- served. It seems more likely therefore that some specific up- take by lipoprotein acceptors in the cell membranes will be involved. The uptake of intact triglyceride by such a process would thus be analogous to that which has been found to operate for cholesterol transport by cells in culture (40, 41).

Acknowledgment-We thank Jon D. Finley for technical assistance.

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J. Martyn Bailey, Barbara V. Howard and Susan F. TillmanTRIGLYCERIDES

Lipid Metabolism in Cultured Cells: XI. UTILIZATION OF SERUM

1973, 248:1240-1247.J. Biol. Chem. 

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