(Received for publication, July 17, 1995; and in revised form, November 30, 1995)
From the
Using the Chinese hamster ovary cell line, 25-RA, we have
demonstrated that lipoprotein-derived cholesterol and endogenously
synthesized cholesterol are selectively differentiated with respect to
their cellular locations. These cells lack sterol-mediated regulation,
spontaneously storing large amounts of esterified cholesterol, which
turns over with a half-time of 7.5 h. When
[H]cholesterol was provided to the cells in serum
to trace cellular cholesterol, the specific activities of cellular free
and esterified cholesterol (6238 ± 273 and 5128 ± 277
cpm/µg, respectively) failed to equilibrate, indicating that bulk
cellular free cholesterol is isolated from that participating in the
cholesteryl ester cycle. Using [
H]acetate to
trace the fate of endogenously synthesized cholesterol, a failure of
equilibration was also observed (specific activities of free and
esterified cholesterol = 280 ± 37 and 458 ± 8
cpm/µg, respectively). The lower specific activity of the precursor
indicates that endogenously synthesized cholesterol is preferentially
esterified. When cells radiolabeled with
[
H]acetate were post-incubated in the absence of
radiolabel, the specific activity of the esterified cholesterol pool
remained significantly higher than that of the free cholesterol,
suggesting that cholesterol derived from hydrolysis of esterified
cholesterol is preferentially re-esterified.
Many investigators have used cell culture models for studying
cholesterol metabolism to examine the pathology of atherosclerosis at
the cellular level (for review see (1) ). Chinese hamster ovary
(CHO) ()cells are fibroblast-like cells that have been used
extensively to examine the somatic cell genetics of cholesterol
metabolism(2) . They are particularly useful because of their
haploid phenotype, which allows one to obtain cells deficient in gene
products of interest in order to elucidate specific pathways involved
in cell processes(3) . One such mutant CHO cell line (CHO
25-RA), selected for resistance to 25-hydroxycholesterol, spontaneously
stores large amounts of cholesteryl ester, making it similar in
appearance to the foam cells found in the atherosclerotic plaque. When
exposed to 25-hydroxycholesterol, normal cells are subject to
down-regulation of endogenous cholesterol biosynthesis and the low
density lipoprotein (LDL) receptor pathway, and thus the cells normally
maintain low cholesterol levels. The 25-RA cells, which are resistant
to the actions of 25-hydroxycholesterol, continue to take in
cholesterol via LDL receptors and to synthesize cholesterol via
biosynthetic pathways(4, 5) . In characterizing the
cell line, Chang and Limanek (4) documented that the expression
of the first four cholesterogenic enzymes, as well as the activity of
the LDL receptor, is unregulated. Although the precise molecular defect
in 25-RA cells remains to be established, the phenotype is similar to
another CHO line that has been shown to have a mutant sterol regulatory
element binding protein 2(6) . The lack of sterol regulation
proved useful for the studies presented here on intracellular
cholesterol trafficking.
Intracellular movement of cholesterol involves a complex set of pathways (for reviews see (7, 8, 9) ). In a mammalian cell, cholesterol, existing in either the unesterified or stored (esterified) form, can be derived from two distinct sources, either de novo synthesis or exogenous, lipoprotein-derived cholesterol. The LDL receptor-mediated internalization of cholesterol has been well characterized. LDL binds to a cell surface-associated receptor and is internalized, and the lipoprotein cholesteryl ester is degraded to unesterified cholesterol in the lysosomes(10) . At this point, the LDL-derived cholesterol is thought to enter a pool that can either incorporate into the plasma membrane or move to intracellular membranes to be esterified by acyl CoA:cholesterol acyltransferase (ACAT) for storage as cholesteryl esters. Xu and Tabas (11) concluded that trafficking of LDL-derived cholesterol to the plasma membrane precedes the utilization of cholesterol for storage in cholesteryl esters and have demonstrated that in macrophages the esterification of cholesterol occurs only after the free cholesterol pool expands 25% above basal levels. Similar trafficking patterns have been proposed by other investigators(7, 12) .
The bulk of cholesterol synthesis is thought to occur in the endoplasmic reticulum (ER) because the biosynthetic enzymes co-fractionate with ER markers(13, 14) . In mammalian cells, cholesterol is important for new membrane synthesis and is found mainly in the plasma membrane(15) . Alternatively, especially when cellular cholesterol levels rise, cholesterol may become a substrate for ACAT, another enzyme found in the endoplasmic reticulum. In a recent review, Liscum and Faust (8) suggest that the majority of cholesterol esterified by ACAT is endogenously synthesized; however, definitive data demonstrating this are lacking. The concept is complementary to the data of Xu and Tabas (11) suggesting that exogenous cholesterol is channeled away from ACAT and appears to reside initially in the plasma membrane. Although these studies suggested that there is selective trafficking of cholesterol derived from either exogenous or endogenous sources, the intact regulation of cholesterol synthesis in these cell systems has precluded rigorous examination.
The 25-RA cell line, having lost its regulatory sensitivity to increasing cholesterol levels, seemed an ideal system to use to examine this compartmentalization, because synthesis continues at high levels regardless of the influx of lipoproteins. Using radiolabeled compounds to trace exogenous and endogenous cholesterol, we observed differences in specific activities that can best be explained by a specific compartmentalization of the pools of cholesterol in the cell. The data presented here demonstrate that not only are these pathways distinct but that a compartmentalization of endogenous cholesterol exists within the cholesteryl ester cycle. These studies using 25-RA cells as a foam cell model provide new information on the preferential handling of endogenous and exogenous cellular cholesterol that may contribute to the process of foam cell formation in atherosclerosis.
CHO 25-RA cells grown
on 10% FBS or 1 mg/ml delipidized calf serum protein were labeled with
[H]acetate to trace endogenously synthesized
cholesterol. The cells were plated on 100-mm culture dishes and allowed
to grow for 24 h. Fresh media containing
[
H]acetate (5 µCi/plate) were added, and the
cells were incubated for 3 days. The media were removed, and the
monolayers were washed three times with phosphate-buffered saline.
Isopropanol was added to the monolayers overnight to extract the
lipids. Dried isopropanol extracts were partitioned using the method of
Bligh and Dyer(27) . Individual lipid classes were separated by
thin layer chromatography on glass-backed Anasil-G 250-µm plates
developed in petroleum ether:diethyl ether:glacial acetic acid
(90:10:1, v/v/v). The areas of the plate corresponding to the major
lipid subclasses, as identified by the migration of standards and by
scanning for radioactivity using a Radiomatic Imaging TLC Scanner, were
scraped into test tubes. The lipids were extracted from the silica gel
by the procedure of Folch et al.(28) as modified by
Bamberger et al.(29) . The individual lipids then were
saponified in 10% alcoholic potassium hydroxide for 1 h at 60 °C
and then extracted three times with petroleum ether and twice with
diethyl ether. The collected extractions were dried under nitrogen and
washed twice with deionized water. The final sample was analyzed for
radioactivity and cholesterol mass. Specific activities of the free and
esterified cholesterol pools were calculated as the number of
cpm/µg of cholesterol.
CHO 25-RA cells, maintained in 10% fetal bovine serum, accumulate extremely high levels of cellular cholesterol. Over a 3-day period, cells maintain fairly constant free cholesterol levels of about 20 µg/mg cell protein while storing approximately 120-130 µg of esterified cholesterol/mg of cell protein (data not shown). Even in the absence of an exogenous source of cholesterol, cells continue to maintain excess cholesterol. As shown in Fig. 1, when 25-RA cells were incubated in lipid-free medium for 3 days, cellular esterified cholesterol decreased, but the cells were by no means depleted of cholesteryl esters, because approximately 60% of the cellular cholesterol remained in the esterified form. The 25-RA stock cultures used for some of the experiments described below, which were maintained on delipidized serum protein for several weeks, continued to contain 40-50% of total cholesterol in the esterified form. The decline in cellular cholesterol upon incubation with lipid-free medium was not due to efflux of cholesterol to the medium but rather was a function of the normalization of the data for cholesterol content as a function of the monolayer protein content, which increased over time (Fig. 1, inset).
Figure 1:
Effect of
lipid-free medium in CHO 25-RA cells. CHO 25-RA cells were plated in
growth medium (Ham's F-12/10% FBS) and allowed to grow for 3
days. The cells were then rinsed with serum-free medium, and fresh
Ham's F-12 medium containing delipidized serum calf protein at a
concentration of 1 mg/ml was added to the wells. On each day
thereafter, a set of cultures was removed and the lipids were extracted
from the cell monolayers with isopropanol as described under
``Experimental Procedures.'' Each value for esterified
cholesterol () and free cholesterol (
) represents the
average of duplicate cultures. Shown in the inset are the
protein contents of the cell monolayer
(
).
It was of interest to compare the changes in cellular cholesterol contents of 25-RA cells incubated with lipid-free medium with those that occur when the cholesteryl ester cycle was interrupted using the ACAT inhibitor, Sandoz 58035. This compound specifically blocks the synthetic arm of the cholesteryl ester cycle(30) , allowing direct assessment of hydrolase activity through the measurement of the disappearance of cellular esterified cholesterol(31) . As shown in Fig. 2, cellular cholesteryl ester content declined much more rapidly and to a much greater extent in the presence of the ACAT inhibitor than in the absence of exogenous lipoprotein. Because the ACAT inhibitor prevents the reesterification of free cholesterol generated by cholesteryl ester hydrolysis, cellular free cholesterol contents rose to high levels. The differences in the rates of disappearance of esterified cholesterol under the two conditions indicates that the cholesteryl ester cycle remained active in 25-RA cells even in the absence of an exogenous source of cholesterol. Because the decline in cholesteryl ester content of cells incubated with the ACAT inhibitor was so rapid, we performed an additional experiment to examine in more detail the initial kinetics of cholesteryl ester hydrolysis in the presence of the ACAT inhibitor. Using the data shown in Fig. 3, we calculated a half-time of 7.5 h for cholesteryl ester hydrolysis, which is assumed to reflect the turnover of the cholesteryl ester cycle(31) . Additional experiments indicated that this hydrolysis was extra-lysosomal; chloroquine, an inhibitor of lysosomal function(32) , was found to have no inhibitory effect on the cholesteryl ester hydrolysis, whereas umbelliferyl diethyl phosphate, a selective inhibitor of cytoplasmic lipid esterases(33, 34) , blocked hydrolysis (data not shown).
Figure 2:
Effect of inhibiting acyl CoA:cholesterol
acyltransferase. CHO 25-RA cells were plated and harvested as described
in the legend to Fig. 1. Values for esterified cholesterol
() and free cholesterol (
) contents represent the average
of duplicate cultures normalized to cell protein. Control cells (A) were supplemented with 0.25% Me
SO (v/v), a
concentration equal to that used as vehicle for S-58035 in the
experiment shown in B. A final concentration of S-58035
utilized in these studies was 1 µg/ml.
Figure 3:
Kinetics of cholesteryl ester hydrolysis.
CHO 25-RA cells were plated as described in the legend to Fig. 1. After 3 days, the monolayers were rinsed with serum-free
medium, and fresh medium containing 10% FBS, 1 µg/ml S-58035, and
0.25% MeSO (v/v) was added. At indicated intervals,
cultures were harvested and analyzed as described under
``Experimental Procedures.'' The values are the average of
duplicate wells. The dotted lines indicate the time in which
50% of the cellular cholesteryl ester was
hydrolyzed.
Because cholesteryl esters are derived
from free cholesterol and exhibit a turnover with a half-time of
approximately 7.5 h, both the free and esterified cholesterol pools
would be expected to become uniformly radiolabeled quickly in the
presence of radiolabeled cholesterol if the cellular free cholesterol
is in a single freely equilibrating pool. To determine whether this is
the case in CHO 25-RA cells, cells were exposed to
[H]cholesterol, and the percentage of esterified
cholesterol as determined by mass measurements was compared with the
percentage of esterified cholesterol determined after separating
radiolabeled free and esterified cholesterol by TLC. If there were
complete equilibration of all cellular free cholesterol, these values
should converge to a similar level relatively quickly after addition of
exogenous radiolabeled cholesterol. Shown in Fig. 4are data
from an experiment in which [
H]cholesterol was
supplied in growth medium and cells were harvested daily over the
course of 5 days. The percentage of esterified cholesterol by mass
remained higher than that by radiolabel over the entire 5-day period (Fig. 4A). Fig. 4B, which shows data
from the same experiment expressed in terms of specific activities,
confirms that the specific activity of the free cholesterol remained
higher throughout the 5-day period. This implies that the exogenous,
radiolabeled cholesterol is not in rapid equilibrium with the free
cholesterol in the cholesteryl ester cycle.
Figure 4:
Incorporation of exogenous radiolabeled
cholesterol into esterified cholesterol. CHO 25-RA cells were plated at
1 10
cells/ml, 2 ml/35-mm dish in standard growth
medium (Ham's F-12/10% FBS) containing 0.5 µCi/ml
[1,2-
H]cholesterol. On each of 5 days after
plating the cells, a set of cultures was analyzed for esterified lipid
content as described under ``Experimental Procedures.'' Shown
in A are data expressed as the percentage of esterified
cholesterol by mass (
) and by radiolabel (
). The same
data are expressed as specific activity in B where free
cholesterol (
) and esterified cholesterol (
) specific
activities are expressed as cpm/µg cholesterol. The values are
means and standard deviations of triplicate
cultures.
One potential explanation for the differences in specific activities is the continuous presence of exogenous radiolabeled free cholesterol. Because at the outset the cells had a very large unlabeled esterified cholesterol pool, it was possible that equilibration might be observed only when the source of radiolabeled precursor was removed. To examine this, 25-RA cells were plated in the presence of exogenous radiolabeled cholesterol, as in the previous experiment, for 3 days to label the cellular cholesterol pools. Medium containing radiolabel was removed, and the cells were incubated with lipid-free medium containing 1% BSA. Fig. 5shows the specific activities of the free and esterified cholesterol of the cultures harvested at 0, 12, 18, and 24 h. Again, the specific activity of free cholesterol at the beginning of the equilibration period was twice that of esterified cholesterol. Although the specific activity of free cholesterol decreased over time, a significant difference (p < 0.05) was maintained between the two specific activities throughout this time period. Using theoretical concepts involving a two compartmental system (see ``Appendix''), the rate of cholesteryl ester cycle turnover and the masses of the free and esterified cholesterol, we calculated that complete equilibration should have occurred by 12 h. The data presented in Fig. 5, where the cells are placed on label-free, lipid-free medium, show a decline in the specific activity of the free cholesterol, which is expected, because the cells are still synthesizing unlabeled free cholesterol. However, there is no decline in the specific activity of the esterified cholesterol pool, suggesting compartmentalization of the free cholesterol that is part of the cholesteryl ester cycle.
Figure 5:
Specific activity determination during
equilibration with 1% BSA. CHO 25-RA cells were plated in growth medium
containing 0.5 µCi/ml [1,2-H]cholesterol and
allowed to grow for 3 days. Cultures were then rinsed with serum-free
medium and refed with Ham's F-12 medium containing 1% BSA.
Cultures were harvested at the indicated times, and lipids were
extracted and analyzed as described under ``Experimental
Procedures.'' Specific activities, expressed as cpm/µg
cholesterol, for free (
) and esterified (
) cholesterol are
the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test)
between FC SA and EC SA at p < 0.01 (**) and p < 0.005 (***).
The next set of experiments examined the
fate of synthesized cholesterol. CHO 25-RA cells maintained on 10% FBS
were plated in growth medium and incubated for 24 h. The cells were
then exposed to [H]acetate in fresh growth medium
for 3 days, followed by a 24-h chase period using fresh, unlabeled
medium. At various time intervals, cellular lipids were extracted with
isopropanol and separated on TLC plates, and the separate lipid classes
were isolated. Cholesteryl esters were subjected to an additional
saponification step to remove the fatty acyl moiety and ensure that we
were examining only the radiolabeled sterol derived from the
cholesteryl ester. Specific activities of free and esterified
cholesterol at various time points are shown in Fig. 6. As in
prior experiments, esterified cholesterol mass levels in the cells on
10% serum comprised approximately 75% of the total cholesterol. At the
end of the 3-day labeling period, the specific activity of esterified
cholesterol was 1.6 times greater that of free cholesterol (EC SA
= 1550 ± 41; FC SA = 945 ± 18 cpm/µg
cholesterol). Because esterified cholesterol is a product derived from
free cholesterol, the higher specific activity of esterified
cholesterol demonstrates that endogenously synthesized cholesterol is
preferentially esterified. At the first determination after changing
the medium (4 h), a small increase was seen in the specific activities
of free and esterified cholesterol that can probably be attributed to
continued incorporation of radiolabeled precursor into cholesterol
after removal of the radiolabeled medium. Beyond 4 h, the specific
activities of free and esterified cholesterol appeared to drop in
parallel. However, the decrease was significant only for free
cholesterol (p < 0.05), and the specific activity of
esterified cholesterol remained high throughout the chase period, even
though the source of radioisotope was removed. The differences seen in
specific activity during chase period again suggest that there is a
specific compartmentalization of the cholesterol that is part of the
cholesteryl ester cycle; once cholesterol becomes part of the cycle, it
tends to remain there.
Figure 6:
Endogenous labeling of endogenously
synthesized cholesterol: effect of a chase period. CHO 25-RA cells
maintained on 10% serum were plated on 100-mm culture dishes (10
ml/plate) and allowed to grow for 24 h. The media were then changed to
fresh Ham's F-12/10% FBS containing
[H]acetate (1 µCi/ml), and the cells were
incubated for 3 days. To begin the chase period, cultures were rinsed
with phosphate-buffered saline, and fresh growth medium (Ham's
F-12/10% FBS) was added to the plates. At each time point, cultures
were harvested, and cellular lipids were extracted and saponified as
described under ``Experimental Procedures.'' Specific
activities are calculated to be cpm/µg cholesterol. Final SA shown
for esterified cholesterol (
) and free cholesterol (
) are
the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test)
between FC SA and EC SA at p < 0.005
(***).
In the previous experiment, cells had large
stores of cholesteryl ester because they had been previously maintained
on 10% FBS. It was possible that such cells might not exhibit
equilibration of cholesterol pools within the time frame examined due
to the large amount of unlabeled cholesterol initially present in the
cells. Therefore, a similar experiment was conducted wherein cells
maintained on delipidized serum protein for several weeks to lower
their cholesteryl ester stores were exposed to
[H]acetate in lipid-free medium. For this
experiment, cells were exposed to the radiolabel for 3 days to produce
significantly different specific activities in free and esterified
cholesterol. The cells were then exposed to medium containing 1% BSA,
and cultures were harvested at intervals over the next 24 h. As shown
in Fig. 7, whereas the specific activities of both free and
esterified cholesterol decreased with time, that of esterified
cholesterol remained higher at all points, again supporting our
conclusion that endogenously synthesized cholesterol is preferentially
esterified and cholesterol derived from hydrolysis of cholesteryl ester
is recaptured and recycled in the cholesteryl ester cycle. If the
failure to achieve equal specific activities in free and esterified
cholesterol is indeed a consequence of the elevated, unregulated
synthesis of cholesterol, one would predict that inhibition of
cholesterol synthesis would result in equilibration of radiolabel
between the two cholesterol pools. Shown in Fig. 8are the
specific activities of free and esterified cholesterol from 25-RA cells
cultured in the presence of radiolabeled cholesterol, mevinolin, and
mevalonate for a period of 5 days. As predicted, an equilibration of
specific activities was achieved but not until 5 days of culture. The
extended delay in equilibration supports our conclusion about the
relative compartmentalization of cholesteryl ester and is likely due to
the large store of unlabeled cholesteryl ester present at the beginning
of the experiment.
Figure 7:
Endogenous labeling of endogenously
synthesized cholesterol in CHO 25-RA lipid-depleted cells: effect of a
chase in cholesterol-free medium. CHO 25-RA cells maintained on
lipid-free medium (Ham's F-12/1 mg/ml delipidized serum calf
protein) were plated and treated as described in the legend to Fig. 6. Specific activities are calculated to be cpm/µg
cholesterol. Final SA shown for esterified cholesterol () and
free cholesterol (
) are the mean and standard deviations of
triplicate cultures. The asterisks indicate significant
differences (t test) between FC SA and EC SA at p < 0.05 (*) and p < 0.005
(***).
Figure 8:
Effect of mevinolin on the incorporation
of exogenous radiolabeled cholesterol into esterified cholesterol. CHO
25-RA cells were plated at 1 10
cells/ml, 2
ml/35-mm dish in standard growth medium (Ham's F-12/10% FBS)
containing 0.5 µCi/ml [1,2-
H]cholesterol,
mevinolin (20 µM), and mevalonate (0.5 mM). On
each of 5 days after plating the cells, a set of cultures was analyzed
for esterified lipid content as described under ``Experimental
Procedures.'' Specific activities, expressed as cpm/µg
cholesterol, for free (
) and esterified (
) cholesterol are
the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test)
between FC SA and EC SA at p < 0.05 (*), p <
0.01 (**), and p < 0.005 (***).
Because equilibration of radiolabeled cholesterol
was not achieved in the 25-RA cell system unless cholesterol synthesis
was inhibited, we examined two cell lines we have used extensively as
foam cell models to determine rates of equilibration of the specific
activities of free and esterified cholesterol. Cells, J774 mouse
macrophages and Fu5AH rat hepatomas, were exposed to
cholesterol-enriched media containing
[H]cholesterol as described under
``Experimental Procedures'' to load the cells with
cholesteryl ester. Equilibration was initiated by washing the
monolayers and incubating the cells with 1% BSA for 24 h. Careful
examination of the specific activities of the free and esterified
cholesterol revealed statistically different values at the onset of the
24-h equilibration period (Fig. 9). The specific activities of
free and esterified cholesterol move toward equilibration. However,
with the exception of two analyses of J774 cells, the specific
activities remain significantly distinct at every time point,
suggesting that these cells also sequester cholesterol in the
cholesteryl ester cycle. We conclude that the compartmentalization of
cholesterol derived from exogenous and endogenous sources is a general
cellular phenomenon that is exaggerated in the CHO 25-RA cells because
of their lack of regulation of cholesterol synthesis and uptake.
Figure 9:
Specific activity determination in
loaded Fu5AH hepatoma cells and J774 macrophages after 1% BSA
equilibration. Fu5AH cells and J774 mouse macrophages were plated and
loaded with free cholesterol-rich lipid dispersions as described under
``Experimental Procedures.'' Equilibration was initiated
after the cultures were rinsed three times with phosphate-buffered
saline, and medium containing 1% BSA was added to the wells. Fu5AH FC
SA (), EC SA (
), J774 FC SA (
), and EC SA (
)
are expressed as cpm/µg cholesterol and are the mean and standard
deviations of triplicate cultures. The asterisks indicate
significant differences (t test) between FC SA and EC SA
within the same cell type at p < 0.05 (*), p <
0.01 (**), and p < 0.005 (***).
The formation of foam cells, a key event of the atherosclerotic process, rests on the continual presence of excess cholesterol available for storage within the cell, which is stored as cholesteryl ester in cytoplasmic droplets. Using mouse peritoneal macrophages, it has been demonstrated that this cholesteryl ester is in a dynamic state, undergoing continuous hydrolysis and re-esterification unless an extracellular acceptor of free cholesterol, such as high density lipoprotein, is present(35, 36) . In that case, there is a net efflux of free cholesterol, the only form of cholesterol that can leave the cell, that effectively diverts free cholesterol from the cholesteryl ester cycle. The morphological studies of McGookey and Anderson (37) documented that in the presence of extracellular acceptors, the size of the cholesteryl ester droplets decreases, but the number of droplets was not changed. When progesterone was used to inhibit re-esterification by ACAT in the absence of an extracellular acceptor, the free cholesterol was seen in multilamellar whorls. These experiments suggested that the free cholesterol derived from cholesteryl ester hydrolysis could move freely from the droplets to other locations in the cell and could fully equilibrate with the bulk cellular free cholesterol. The potential for clearance of cholesteryl esters from foam cells thus rests on hydrolysis and subsequent diversion of the free cholesterol away from ACAT.
In contrast to the view that free cholesterol can move freely
between intracellular locations are several pieces of inferential
evidence that cholesterol derived from different sources is
preferentially moved to different locations. These include the studies
of Xu and Tabas (11) from which they concluded that
lipoprotein-derived cholesterol moves preferentially to the plasma
membrane prior to becoming available for esterification by ACAT, and
the conclusions of Liscum and Faust (8) that ACAT
preferentially esterifies endogenously synthesized cholesterol. In
contrast, the studies of Lange et al.(38) showed that
only a small fraction of endogenously synthesized cholesterol was
esterified, whereas the remaining was rapidly moved to the plasma
membrane. To definitively demonstrate this selective trafficking has
been difficult because the intact regulatory mechanisms found in normal
cells minimize de novo synthesis of cholesterol and make
studies tracing the fate of synthesized cholesterol very difficult. The
CHO 25-RA system, which fails to down-regulate cholesterol biosynthesis
or uptake of exogenous cholesterol, has proved to be an informative
model in which to demonstrate the validity of the assumptions drawn
from other systems. These cells have large stores of unlabeled
cholesteryl ester that turn over rapidly (t = 7.5 h). If there were no selective handling of
cholesterol within the cell, provision of exogenous radiolabeled free
cholesterol would lead very quickly to the uniform labeling of both
free and esterified cholesterol pools. As demonstrated here, this is
not the case. As seen in Fig. 5, the specific activity of the
free cholesterol remains far greater than the specific activity of the
esterified cholesterol, even when the radiolabeled precursor
([
H]cholesterol) is removed from the medium.
These data support the idea that cholesteryl ester is derived largely
from de novo synthesis. To demonstrate this, the converse
experiments, using radiolabeled acetate to track endogenous
cholesterol, were done. Again, there were large differences in the
specific activities of the free and esterified cholesterol. However,
with endogenous labeling the specific activity of the esterified
cholesterol exceeded that of the free cholesterol, as would be expected
if the cell preferentially esterifies endogenously synthesized
cholesterol. An interesting result is the maintenance of the higher SA
found in the esterified cholesterol pool, which was seen when the
radiolabeled precursor was removed from the experimental system. These
data imply a recycling and recapturing of the free cholesterol
generated during hydrolysis in the cholesteryl ester cycle, suggesting
that there is a physical compartmentalization of cholesterol that is
part of the cycle from the other pools of free cholesterol within the
cell.
The results presented here demonstrate that in CHO 25-RA foam
cells, there is a specific compartmentalization of cholesterol within
the cholesteryl ester cycle. This conclusion is based on the
observation that equilibration of the specific activity of cholesterol
in free and esterified cholesterol is not attained regardless of
whether the radiolabeled precursor is exogenous cholesterol or is
synthesized from radiolabeled acetate. In addition, the differences in
specific activity are maintained after the radiolabeled precursors are
removed. Fig. 10summarizes our findings in light of current
thinking about intracellular cholesterol trafficking. There are two
distinct pathways of cholesterol transport. First, endogenous
cholesterol synthesized at the ER is preferentially used as a substrate
for ACAT and stored in cholesteryl ester droplets, a reasonable
hypothesis because enzymes of cholesterol synthesis and ACAT both
appear to reside in ER membranes(13, 14) . In our
experiments tracing the fate of endogenously synthesized cholesterol, a
higher specific activity was consistently seen in the product
(esterified cholesterol) rather than in the precursor (free
cholesterol), implying that the cells were preferentially esterifying
endogenously synthesized cholesterol. Our data also suggest that the
cholesteryl ester cycle is isolated. The droplets turn over, generating
free cholesterol, which apparently does not fully equilibrate with
cellular cholesterol prior to re-esterification. Removal of the
radiolabeled precursor ([H]acetate) did not
change the distribution of radiolabel between free and esterified
cholesterol. If there were complete mixing of cholesterol, i.e. that which is part of the cycle leaving and becoming part of bulk
cellular cholesterol pools, the specific activities would have moved
toward equilibrium. Instead, the product (esterified cholesterol)
remained significantly higher. One possible explanation for this
compartmentalization is the following. As the build-up of cholesterol
occurs in the ER, ACAT rapidly esterifies the excess cholesterol. It
has been proposed that the esterification of excess cholesterol in the
membrane by ACAT may in fact be the cellular means of detoxifying the
ER membrane, because abundance of cholesterol may alter protein
function of membranes(39) . When ACAT forms cholesteryl esters,
vesicles bud off and form droplets that contain ER membrane
constituents and cholesteryl esters. Thus, a lipid droplet may contain
all the enzymes of esterification and hydrolysis, explaining the
dynamic nature of the cholesteryl ester cycle and compartmentalization
of cholesterol seen in model foam cells. With the recent cloning of the
cDNA encoding human ACAT(40) , experimental means documenting
the localization of ACAT and whether the intracellular cholesteryl
ester droplets contain this enzyme should be possible in the near
future. A more likely possibility for the compartmentalization is that
cholesterol generated by cholesteryl ester hydrolysis is selectively
moved to the ER, where it again becomes a substrate for ACAT. A recent
publication by Mazzone et al.(41) suggests a similar
trafficking pattern.
Figure 10: Hypothesis. For an explanation for lack of equilibration of cholesterol pools see ``Discussion.''
The second major pathway (see Fig. 10) involves lipoprotein-derived cholesterol, which appears to be separately regulated within the cell, as evidenced by specific disorders of cholesterol trafficking(42, 43, 44, 45) , e.g. Niemann Pick type C disease. Cholesteryl esters in exogenous lipoprotein, taken in by receptor mediated endocytosis and degraded to free cholesterol in the lysosomal compartment(34) , appear to preferentially provide cholesterol for new membrane synthesis prior to entering the ACAT substrate pool(10, 11, 18, 46) . These two pathways, designated here as the endogenous and exogenous pathways, although distinct, are not mutually exclusive of one another. As evidenced by all studies presented herein using radiolabel tracers, as well as the work of other investigators, lipoprotein-derived cholesterol is esterified by ACAT, and endogenously synthesized cholesterol is incorporated into the plasma membrane.
Taken together, the data presented here and suggestions made by several other investigators have many implications for foam cell metabolism. First, with regard to cholesterol trafficking, the data presented confirm conclusions of other investigators. The discrepancy in specific activities of the free and esterified cholesterol, seen whether the 25-RA cells were endogenously or exogenously radiolabeled, demonstrate that endogenously synthesized or lipoprotein-derived cholesterol undergoes directed transport along different pathways. Secondly, even if synthesis is minimal in cells with intact regulatory mechanisms, these findings hold significance for the maintenance of foam cells. Unless there is a concerted movement of free cholesterol from the cholesteryl ester cycle to the plasma membrane for efflux, the cholesterol may remain isolated in the cycle, as demonstrated here.
The fundamentals of first-order kinetics can be used to predict how rapidly two cholesterol pools should equilibrate. Assuming the limiting case of a closed system where the entire cellular pool of free cholesterol constitutes that which participates in the cholesteryl ester cycle, a two compartment system can be described by the following general scheme:
where A (substrate) and B (product) represent
the free and esterified cholesterol pools, respectively, and k and k
are the rate
constants describing esterification and hydrolysis, respectively. The
data from the experiment shown in Fig. 3, in which
esterification is inhibited with 58035, demonstrate a half-time for
hydrolysis of approximately 7.5 h. Using the following equation which
describes the relationship between rate constant and half-time,
the rate constant for hydrolysis (k)
was determined to be 0.09 h
. When a system is at
equilibrium, the forward and reverse reactions are equal, and an
equilibrium constant K
, can be defined as
follows:
For the experiment shown in Fig. 5, the esterified
cholesterol mass [B] was 3.3 times greater than the
mass of free cholesterol [A]. Using Equation 3, the
rate constant for esterification (k) is predicted
to be 0.30 h
. For two-pool compartment analysis, the
half-time to reach equilibrium (t
)
depends on the sum of the two rates within the system (47) and
is defined as follows:
Using the measured rate constant for hydrolysis and the
predicted rate constant for esterification, the t is predicted to be 1.75 h. This
would lead to essentially complete equilibration in 12 h (approximately
7 half-times).