Download the source of lipid accumulation in l cells

Published January 1, 1961
T H E S O U R C E OF L I P I D A C C U M U L A T I O N I N L C E L L S
KLAUS
G. BENSCH,
EDWARD
M.D.,
L. SOCOLOW,
DONALD
W. KING,
M.D.,
and
M.D.
From the Department of Pathology, Yale University School of Medicine, New Haven
ABSTRACT
Strain L cells accumulate lipid, concurrent with cessation of protein synthesis, in the
stationary phase of growth from the extracellular m e d i u m and as a result of de novo synthesis. Cells which have been more severely d a m a g e d with a n a m i n o acid analogue also
accumulate lipid from the extracellular m e d i u m , b u t synthesize very little lipid from labeled
acetate. T h e possible roles which lipid a c c u m u l a t i o n m a y play in the cell are discussed.
INTRODUCTION
MATERIALS
AND METHODS
Tissue Culture: Strain L cells were grown in suspension tissue culture with Eagle's basal medium
supplemcnted with l0 pcr cent horse serum as
previously described (8). Ceil counts werc done using
a standard hemocytometer. Each culture was grown
in a 250 ml. Erlenmeyer flask and consisted of 80 ml.
of medium and free floating cells, initially inoculated
in a concentration of 200,000 to 400,000 cells/ml.
The flasks were agitated on a rotary shaker at
37.5°C.
Isotopes: Isotopes were obtained from the New
England Nuclear Corporation with the following
specific activities: sodium acetate 1-C TM, 24.2 /~c./
mgm.; cholesterol 4-C TM stearate, 10 /zc./mgm.; and
palmitic acid 1-C TM, 6.8 #c./mgm.
Preparation of Isotopes." Cholesterol 4-C TM stearate
was dissolved in hot ethanol and 0.05 ml. containing
200,000 c.P.M, was pipetted directly into the culture.
Acetate 1-C I4 was introduced directly into the culture
after being dissolved in sterile distilled water. Potassium palmitate C TM was prepared by the method of
Fillerup (4). Labeled lipoprotein was synthesized by
the cells from palmitic acid I-C 14 as described below.
The isotope (2 X l0 s C.P.M.) was incubated with
strain L cells in log growth for a period of 3 days.
The cells were then washed 3 times, ruptured by
sonic oscillation, centrifuged at 30,000 times g for 3
hours and the supernatant solution dialyzed against
7,000 cc. Krebs-Ringer phosphate buffer for 2 days.
The labeled lipoprotein was then incubated in a
flask with sterile tissue culture medium on a rotary
shaker at 37.5°C. for 24 hours and the complete
medium centrifuged to insure that no particulate
components were present. Ethanol-ether extraction of
the TCA precipitate of the labeled lipoprotein showed
that over 96 per cent of the total counts were in the
lipid-extractable fraction.
135
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It has been shown previously in this laboratory
t h a t strain L cells in old suspension tissue cultures
accumulate lipid concurrent with cessation of
growth. A similar accumulation of lipid is seen in
cells in which protein synthesis has been inhibited
by the a m i n o acid analogue para-fluorophenylalanine (8). O n e of the major controversies regarding the a p p e a r a n c e of fat in cells centers on
the source of the lipid. A l t h o u g h it is well recognized t h a t most cells are capable of synthesizing
lipid, m a n y reports support the theory t h a t excess
stainable intracellular lipid usually arises from
extracellular lipoproteins. Most of these studies
involve experiments in vivo (13, 16, 14) a l t h o u g h
the incorporation of extracellular fat by cells in
vitro has also been demonstrated (10, 2, 4). O t h e r
investigators believe that excess amounts of lipid
m a n y accumulate intracellularly as a result of de
novo synthesis (11, 3, 15). O n e worker has noted a
distinct species difference in regard to the synthesis
of cholesterol in aortic tissue (1). This study was
initiated to evaluate the c o m p a r a t i v e role of
exogenous a n d endogenous sources in the a c c u m u lation of lipid in strain L cells.
Published January 1, 1961
Preparation of Samples." Five ml. aliquots were removed daily from the culture flasks, the cells washed
3 times by centrifugation in Krebs-Ringer buffer, and
the cell protein precipitated by the addition of 2 ml.
of cold 10 per cent TCA. After being left overnight
at 4°C., the precipitated protein was separated by
centrifugation, and extracted twice at 50°C. for 45
minutes each in a mixed solution of ether ethanol
(2 : 1). The resultant extract was plated on planchetts
as the total lipid-extractable fraction.
Cholesterol was extracted from 10 ml. aliquots of
washed cells according to the method of Sperry and
Webb (12), dried, and plated on planchetts. The
saponified acidic acetone alcohol extract was precipitated with dlgitonin; aliquots of the supernatant
were made alkaline with a few drops of 50 per cent
potassium hydroxide diluted with equal volume of
water and extracted 3 times with equal volumes of
petroleum ether. This extract was dried, plated on
planchetts, and called the non-cholesterol nonsaponifiable fraction. Corrections were made for
self-absorption when necessary.
cells
Ill
4"oot
2
I
I
I
I ooo,
2
I.~.~,. II
t protein
,
lO
I
,'°°°I
I
5
I000
III
0
/
7.0 9.9 4,2 5.9 3.8
r,,,,o-
2
I
2
4.1
[.°,.,,o,, 1
ZOO0
n l
t hill
0
[,.,m,,°,,
I/'ooo
~ I0
o :,ooo[
( X l O 5) per m l .
/ sooo[
III
~2°°°I"
The first group of experiments involved the accumulation of lipid in cultures which were in
the logarithmic and the stationary period of
growth. As shown in Fig. l, cells in the stationary
phase, which had ceased active growth, continued
to incorporate cxogenous palmitate and lipoprotein from the extracellular m e d i u m and also
continued to synthesize lipid de novo from acetate
1-C 14. The acetate label was found in high concentration in the cholesterol, as well as other lipid
fractions, although it is recorded for comparative
purposes in Fig. 1 as the total lipid-extractable
fraction.
In the second group of experiments, protein
synthesis was inhibited by para-fluorophenylalaninc (8 X l0 -4 M) as previously described (8).
3.0 5.5 9.0 3.6 3.6 3.5
/00.,0,, n l oo.'o'' 1
._~ ~ 5oo[
RESULTS
'I II
=°LJAL_LIJ
II III
0
I
2
hme
0
I
2
0
2
0
I
(days)
FIGURE 1
Cells from stock cultures were pooled, divided, and incubated either in fresh tissue culture medium
or in exhausted medium taken fi'om a 5-day-old culture. Labcled acetate (200,000 C.P.M.), palmirate (500,000 C.P.M.), and lipoprotein (100,000 G.P.M,) were introduced into cultures in the logarithmic (white bar) and stationary (black bar) phase of the growth cyclc. Aliquots were removed daily
for determination of cell count, cell protein, and radioactive assay of the total lipid-extractable
fraction by the procedures described under methods. Results were recorded as c.P.M, per rag.
cell protein and c.e.M./106 cells for each sample. The figures at the top of the graphs represent the
cell number (X 10~) per ml. in each culture. Each bar represents the average of 3 flasks.
136
THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 9, 1961
2
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:5.7 8.0 I0.0 5.2 3.6 5.2
Cell protein was determined by Oyama's modi
fication of the Lowry method (9).
Published January 1, 1961
lipoprotein
1200
•_- ~ I 0 0 0
OID
o
•a
-o
o
"-
800
~
dE.
d
600
400
200
.NInl
•~ 300E
-.,,=
l
°2500
o~ 2 0 0 0
~. E 1500
-~
~. ~ o o o
d. 5 0 0
6
.HI
0
3
['] control
6
24
30
0
3
6
24
30
0
3
6
24
30
II p - f - p h e n y l .
olonine
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time ( h o u r s }
FIGURE
Cells from stock c u h u r c s were pooled, divided, a n d i n c u b a t e d in control flasks without a n a l o g u e
or in e x p e r i m e n t a l flasks c o n t a i n i n g p a r a - f l u o r o p h c n y l a l a n i n e (8 X l0 ~ ~). Labeled acetate (100,000
(].P.M.), p a l m i t a t c (100,000 C.P.M.), a n d lipoprotein (100,000 c.P.M.) were introduced into both control a n d e x p e r i m e n t a l flasks a n d aliquots r e m o v e d at the stated period for d e t e r m i n a t i o n of cell
count, cell protein, a n d radioactive assay of the total l i p i d - - e x t r a c t a b l e fraction by the procedures
described u n d e r methods. Results were recorded as C.P.M./mg. cell protein or c.P.i./106 cells
for cach sample. E a c h bar represents t h e average of 3 flasks.
c e l l s ( X l O 5) p e r ml.
3.5
4.5 7.5
5 0 0 1 0 % horse
p-f,-phenyl -
serum
"~ 4 0 0
3.0 3 . 0 2.8
olonlne
2.9
3.0 2.85
proteinfree
~°0 3 0 0
~,
Q.
200
E
o
I00
.,,t
0
I
2
0
iI
I
2
_II
0
I
2
hme (days)
]J'IGURE 3
Cells from stock cultures were pooled, divided, a n d i n c u b a t e d with labeled cholesterol stearate
(200,000 C.P.M.) in one of 3 different m e d i a : basal m c d i u m a n d l0 per cent horse serum, basal m e d i u m a n d l0 per cent horse s e r u m a n d p a r e fluorophcnylalanine (8 X l0 -4 M), or basal m e d i u m
without s e r u m protein. Aliquots were taken daily a n d extracted for total lipids as described u n d e r
methods.
K. G. BENSCH, D. ~V. KING, AND E. L. SOCOLOW Lipid Accumulation
137
Published January 1, 1961
cells
3.5 3.6 9.0
400C
10% horse serum
(XlO5) per
3.6 4.0 3.0
0 . 4 % albumin
ml.
3.6 3.4 3.0
proteinfree
3000
0
--
2000
1000
0
I
2
U total lipid
_Hi
0
I
I cholesterol
2
O-
I
2
time ( d a y s )
FIGURE4
Ceils from stock cultures were pooled, divided, and incubated with labeled cholesterol stearate
(200,000 C.P.M.) in one of 3 different media: basal medium and 10 per cent horse serum, basal medium and 0.4 per cent albumin, and basal medium without serum protein. Aliquots were taken
daily and extracted for cholesterol and total lipids as described under methods.
DISCUSSION
W e have presented evidence in these experiments
to show t h a t old cells in the stationary phase of
the growth cycle a c c u m u l a t e d lipid b o t h as a result of de nova synthesis from labeled acetate a n d
also from extracellular lipoproteins. I t is not
known whether the lack of growth in the stationary phase is the result of a deficient m e d i u m or the
accumulation of toxic metabolites. As a result of
inhibition of protein synthesis the cells m a y be
able to utilize a larger portion of the acetate pool
for lipid synthesis. T h e active acetate could be
]38
derived not only from glycolysis but also from the
ketogenic amino acids. These lipid products may
later, once the conditions again become favorable
for protein synthesis, be degraded both for the
synthesis of high energy molecules a n d for nonessential amino acids. A somewhat analogous
sitation has been shown in E. coll. These cells, in
a nitrogen-deficient medium, synthesized large
quantities of glycogen which was later degraded
w h e n protein synthesis was initiated (7). It is, of
course, possible t h a t some of the acetate incorporated into the cells represented " e x c h a n g e " or
reversible metabolic processes a n d not true de nova
synthesis.
Those cultures, in which protein synthesis was
more drastically inhibited by the amino acid
analogue, accumulated the major portion of their
intracellular lipid from extracellular sources. This
lipid was not reversible a n d the cells eventually
died. It is interesting t h a t an amino acid analogue
appears to inhibit lipid synthesis from labeled
acetate along with protein synthesis. It has been
suggested that lipids might be involved in protein
synthesis (5) a n d present evidence indicates t h a t
the microsomes are the site of lipid as well as
protein synthesis.
T h e r e will always be some controversy concerning the question of whether the labeled lipoprotein
was really incorporated into the cellular lipid
ThE JOI'RNAL OF BIOPHYSICALAND BIOCHEMICALCYTOLOGY • VOLUME9, 1961
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T h e cells were again i n c u b a t e d with labeled acetate as a source of endogenous lipid a n d labeled
palmitate a n d lipoprotein as a source of exogenous
lipid. As shown in Fig. 2, the lipid which acc u m u l a t e d within the cells came principally from
the exogenous sources a n d comparatively little
was synthesized from labeled acetate.
Cholesterol stearate was also used as a source of
exogenous lipid as shown in Figs. 3 a n d 4. It was
incorporated to a m u c h greater degree in the cells
of those cultures in which growth h a d been inhibited, either by para-fluorophenylalanine or
serum free medium, t h a n in the cells of control
cultures containing no analogue a n d supplem e n t e d with 10 per cent horse serum.
Published January 1, 1961
fraction or merely adsorbed on the membrane. We
have tried to take all possible precautions to avoid
the latter possibility. The labeled lipoprotein was
dialysed for 2 days to remove all small loosely attached free acids. The soluble lipoprotein was
preincubated at 37.5°C. on the shaker with complete medium and later centrifuged to insure that
particulate lipoprotein was not removed with the
cell aliquots. After removal of the daily samples,
the cells were washed 3 times in buffered saline
until the final wash was completely free of radioactivity. Cells stained with Sudan I V showed
significant increases of intraeellular lipid droplets.
U p to 50 per cent of the label in the cholesterol
experiments appeared in the non-digitonin precipitate, indicating that the cholesterol was actively metabolized to a further degradation product and not merely adsorbed to the cell membrane.
Finally, the incorporation of labeled lipoprotein
in this system was temperature-dependent and less
than 10 per cent incorporation occurred at 4°C.
In the experience of our laboratory there exists
a definite difference in membrane permeability of
cells during different phases of growth. We have
previously reported the greater incorporation of
acridine orange in L cells during the stationary
period (6). We would now speculate in regard to
the present experiments that during the stationary
phase cells take up more lipoprotein and other
metabolites in an attempt to compensate for
cellular nutritional deficiencies. We believe a
somewhat similar process takes place in the injured cell, perhaps by pinocytosis (2), again in an
attempt to gain essential materials to repair
cellular damage. This compensatory action, of
course, would be of little value to those cells in
which the synthetic mechanism had been irreparably injured.
This work was supported by a grant from the USPHS
C2928 (C3) King.
Receivedfor publication, .4/Iay 19, 1960.
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