(Received for publication, August 8, 1995)
From the
Previous studies suggest that during sterol synthesis in cells,
cholesterol and precusor sterols are transported to the plasma membrane
and that this transport is stimulated by the binding of high density
lipoprotein (HDL) to its putative cell surface receptor, leading to
enhanced sterol efflux. Little is known about the identities of
synthesized sterols subject to efflux or whether efflux of cholesterol
and precursor sterols are stimulated equally by HDL. To address these
issues, cells were incubated with [H]acetate or
[
H]mevalonate and sterol acceptors, and then the
labeled sterols in cells and efflux media were analyzed by high
pressure liquid chromatography methods that resolved cholesterol and
precursor sterols. In non-hepatic cells (Chinese hamster ovary (CHO),
fibroblasts, and smooth muscle), cholesterol and multiple precursor
sterols accumulated. In CHO cells, the major products were cholesterol
and desmosterol, which together constituted 50% of labeled
nonsaponifiable lipids. When media contained human HDL
(1
mg of protein/ml), the molar efflux of synthesized desmosterol was four
times that of cholesterol, and the 8-h efflux of these sterols, each
normalized to its own production, averaged 48 and 16%, respectively.
When media contained egg phosphatidylcholine vesicles (1 mg/ml), the
efflux of these sterols averaged 18 and 2.4%, respectively. Thus, with
both acceptors, desmosterol was the major synthesized sterol released
from cells, and its efflux was substantially greater than that of
synthesized cholesterol. High relative efflux of desmosterol (or a
desmosterol-like sterol) occurred in all cell types and in both
cholesterol-enriched and unenriched cells. These results demonstrated
qualitatively similar efflux of synthesized sterols in the presence of
HDL
and phospholipid vesicles, arguing against an absolute
requirement for acceptors that interact with the HDL receptor. To probe
for possible quantitative differences in the capabilities of these two
acceptors, the ratios of (efflux to HDL
)/(efflux to
phosphatidylcholine vesicles) were calculated for synthesized
cholesterol and desmosterol, plasma membrane cholesterol, and lysosomal
cholesterol. In comparison to plasma membrane cholesterol, there was
little or no HDL selectivity for lysosomal cholesterol or synthesized
desmosterol, whereas there was a 2-3-fold selectivity for
synthesized cholesterol, suggesting that the ability of HDL to enhance
the efflux of synthesized sterols is a modest quantitative effect and
confined to cholesterol.
In mammalian cells, the efflux of newly synthesized sterols
involves translocation from the intracellular site of synthesis (the
endoplasmic reticulum) to the plasma membrane, followed by sterol
desorption from the plasma membrane to extracellular carriers, such as
high density lipoprotein (HDL) ()(reviewed in Johnson et
al., 1991). In cells that contain normal basal levels of
cholesterol, the translocation step within cells is mediated by
vesicular carriers, but bypasses the Golgi apparatus and thus appears
to be independent of the protein secretory pathway (Lange and Steck,
1985; Urbani and Simoni, 1990). Extensive work by Lange and colleagues
(Lange and Muraski, 1987; Echevarria et al., 1990) has shown
that this transport process results in the delivery of both cholesterol
and biosynthetic sterol intermediates, such as lanosterol and
zymosterol, to the cell surface. In work by Oram and colleagues (Mendez et al., 1991), the efflux of newly synthesized sterol has been
used as a marker for the removal of internal pools of cholesterol that
may contribute to cholesterol and cholesteryl ester deposition in
atherosclerotic foam cells. These experiments suggest that when
cellular cholesterol is elevated above normal levels, the delivery of
newly synthesized sterol to the plasma membrane (and consequently the
efflux of this sterol) becomes a regulated process that is stimulated
when media contain acceptors that bind to the putative HDL receptor.
The most important ligand for this receptor is apolipoprotein AI
(apoAI), the major protein of HDL. The intracellular signaling
mechanism is thought to involve diacyglycerol and protein kinase C
(Mendez et al., 1991). Cyclic AMP also stimulates sterol
translocation in cells, but this appears to be unrelated to HDL binding
(Hokland et al., 1993). Recently, it was reported that in
cholesterol-enriched cells, the delivery of synthesized sterols to the
plasma membrane is sensitive to brefeldin A and proton ionophores,
suggesting participation of the Golgi apparatus in HDL-regulated sterol
translocation (Mendez, 1995).
The diversity of biosynthetic sterols
that find their way to the plasma membrane has led to concern about the
identities of these sterols and whether they are released from cells
with equal efficiency. The data addressing these issues are limited and
somewhat contradictory. In mouse peritoneal macrophages, analysis by
reverse-phase thin layer chromatography indicated that the major sterol
produced from [H]mevalonate was desmosterol and
that this sterol and cholesterol were released to HDL with similar
efficiency (Aviram et al., 1989). In the recent studies of
Hokland et al.(1993), an analysis of biosynthetic products in
fibroblasts by reverse-phase high pressure liquid chromatography
suggested a different pattern, in that the major product in cells was
cholesterol, whereas the major products released to HDL were polar
sterols with retention times somewhat different from that of
desmosterol. In this case, there were significant differences in the
efficiency of release of different sterols. In other work with
fibroblasts, Echevarria et al.(1990) reported that zymosterol
was a major product of sterol synthesis and that its efflux from
glutaraldehye-fixed cells to diluted blood plasma was about two times
more efficient than that of newly synthesized cholesterol.
The objective of the present studies was to provide a more complete quantitative understanding of the efflux of newly synthesized cholesterol in comparison to the major sterol intermediates that had been reported to accumulate in cells during typical labeling procedures. The critical technical aspect of the experiments was to analyze samples using a chromatographic method capable of separating cholesterol and the major intermediates of sterol synthesis. To satisfy this need, all samples were analyzed by a reverse-phase high pressure liquid chromatograph (HPLC), which was connected in series with a flow-through liquid scintillation counter to provide a sensitive, high resolution profile of the distribution of radiolabeled sterols in each experimental sample. The results confirm that in a variety of non-hepatic cells (CHO, fibroblasts, and smooth muscle cells) several sterol intermediates more polar than cholesterol accumulate and are available for efflux. A product comigrating with desmosterol (and conclusively identified as this sterol in CHO cells) was the major biosynthetic sterol released from non-hepatic cells. Additional experiments addressed the dependence of cholesterol and desmosterol efflux on the type of acceptor provided in the medium and on whether cells are pre-enriched with exogenous cholesterol.
To enrich cells with free (unesterified)
cholesterol, the incubation with DLP was followed by a 1-day incubation
in medium containing 50 µg of protein/ml of LDL, 100 µg of
cholesterol/ml of cholesterol-rich egg-PC liposomes (Arbogast et
al., 1976), 1% fetal bovine serum, 0.2% bovine serum albumin
(BSA), and 1 µg/ml Sandoz compound 58035. All subsequent media
contained compound 58035 to prevent sterol esterification. After the
1-day enrichment period, cells were rinsed, incubated 1 h with 0.2% BSA
to allow desorption of any loosely adherent lipids, and then incubated
with labeled [H]acetate with or without sterol
acceptor. Control (unenriched) cells were treated identically except
that during the enrichment period the medium did not contain LDL and
the liposomes were cholesterol-free.
For HPLC, the column dimensions
were 4.6 250 mm (diameter
length), the packing was
5-µm C-18 Spherisorb (Isco), the solvent was acetonitrile-isopropyl
alcohol (3:1, v/v) flowing at 1 ml/min, and the capacity of the
injection loop was 100 µl. The column effluent went directly to a
UV absorbance cell where A
(due to carbon-carbon
double bonds) was monitored. From the UV cell, the effluent was
directed to a Radiomatic Flo-One
counter, which continuously
mixed the effluent with 3 volumes of liquid scintillant and then
monitored the elution of radioactivity by means of a flow-through
liquid scintillation counter. The cell volume for liquid scintillation
counting was 0.5 ml and the data-sampling interval was 6 s. Software
provided by Radiomatic was used for peak integration and the
preparation of side-by-side plots of UV absorbance and
H
radioactivity. There was a 1-min delay between the UV and
H
signals, which was compensated for by the software. The retention times
reflect the times of appearance of peaks at the UV cell. The retention
time of cholesterol was 16-19 min, varying somewhat from day to
day. The retention times of commercial standards relative to
cholesterol were as follows: 25-hydroxycholesterol, 0.35;
4-cholesten-3-one, 0.69; desmosterol, 0.72; 7-dehydrocholesterol, 0.80;
squalene, 0.84; lanosterol (two peaks), 0.82 and 1.14. The presence of
two components was observed in several commercial lanosterol
preparations. This may be due to the presence of both authentic
lanosterol (relative retention time 0.82) and dihydrolanosterol (1.14).
The reproducibility of relative retention times was ±0.01.
Thin layer chromatography (TLC) of NSL was performed with
glass-backed Silica Gel G plates using a development solvent of
hexane-ethyl acetate (70:30, v/v). Mass amounts of lipid were
visualized by staining with iodine vapor. Distributions of radiolabel
were imaged using a Radiomatic TLC-600 gas-ionization thin layer plate
scanner. R values of standards can be calculated
from data in Fig. 1.
Figure 1:
HPLC analysis of NSL and cholesterol
TLC fraction from [H]acetate-labeled CHO-K1
cells. CHO-K1 cells were incubated 24 h in medium containing BSA
(0.2%), compound 58035 (1 µg/ml), and
[
H]acetate (200 mCi/ml), and then NSL were
prepared from the cells. A portion of the NSL along with unlabeled
standards was analyzed by thin layer chromatography (stationary phase:
Silica Gel G; mobile phase: hexane-ethyl acetate, 70/30, v/v). Panel A shows the migration of the standards (as determined by
staining with iodine vapor) and the distribution of
H
radioactivity (as determined by scanning with a gas-ionization
counter). Abbreviations: orig., TLC origin; 25HC,
25-hydroxycholesterol; chol., cholesterol; lanost.,
lanosterol; squal., squalene; front, TLC solvent
front. The major peak of radioactivity from the TLC plate (co-migrating
with cholesterol standard) was extracted with chloroform-methanol (1:1,
v/v). Panels B and C show the HPLC analysis of the
total NSL and the cholesterol TLC fraction,
respectively.
Digitonin precipitation was performed as described by Sperry and Webb (1950) using 1 mg of unlabeled cholesterol as a ``carrier'' and 2.5 ml of 0.5% (w/v) digitonin to effect precipitation of sterols. After rinsing, the precipitated sterols were dissociated from the digitonide complex by treatment with hot dimethyl sulfoxide and extracted into petroleum ether (Issidorides et al., 1962) for analysis by HPLC.
Combined gas chromatography (GC)/electron-impact mass spectrometry (MS) was performed without derivatization of sterols using a Hewlett-Packard model 59970 GC-MS, as described previously (Fischer et al., 1989). The sterols were partitioned using a DB17 capillary column (J & W Scientific, Folsom, CA) with a column oven temperature program (235-265 °C, 5 °C/min) to improve resolution. For each sterol, the mass spectrum is that of the major peak eluting from the GC column.
In-vial liquid scintillation counting and assays for protein, cholesterol, and phospholipid were as described previously (Johnson et al., 1990, 1991).
In a given experiment, all incubations were performed in at least triplicate. Values are the means of these replicate determinations. Uncertainties are 1 S.D. Statistical significance was assessed by Student's t test, with p < 0.05 as the criterion of significance.
To establish methods for the analysis of newly synthesized
sterols, NSL were prepared from CHO cells that had been incubated with
[H]acetate for 24 h, and then TLC and
reverse-phase HPLC were compared as methods of detecting and
quantifying
H-labeled cholesterol and other products in the
NSL. By TLC, most of the nonsaponifiable
H co-migrated with
cholesterol (Fig. 1A). However, by HPLC the labeled NSL
were seen to consist of about 20% cholesterol plus three other major
products that were somewhat more polar than cholesterol (Fig. 1B). When the ``cholesterol'' TLC
fraction was isolated and analyzed by HPLC, a profile very similar to
that of the total NSL was obtained (Fig. 1C). Thus, the
``cholesterol'' TLC fraction was a mixture of products, and
most of the radioactivity in this TLC fraction was not associated with
cholesterol when analyzed by HPLC. These results are consistent with
previous work by Echevarria et al.(1990), Burki et
al.(1987), and Hokland et al.(1993), who have also
reported that the incubation of mammalian cells with nonsterol
precursors of cholesterol leads to the accumulation of a diversity of
products, some of which are not resolved from cholesterol using typical
TLC methods. Thus, in studies of this type it appears to be essential
that a method with high resolving ability, such as reverse-phase HPLC,
be used to obtain an accurate assessment of the synthesis and efflux of
individual sterols. The use of TLC alone could yield misleading
results. The data reported in this paper are based entirely on the
reverse-phase HPLC analysis of total NSL, as illustrated in Fig. 1B.
To determine whether the four major
products resolved by HPLC were sterols, labeled NSL were subjected to
digitonin precipitation, and then the precipitable sterols were
recovered from the digitonide complex and compared to total NSL by
HPLC. The results indicated quantitative precipitation of cholesterol
and product 2, but only partial precipitation of products 1 and 3.
Thus, product 2 was identified conclusively as a 3--hydroxysterol
(Haslam and Klyne, 1953). Product peaks 1 and 3 may be poorly
precipitable sterols or may be mixtures of precipitable and
nonprecipitable products.
To examine both the synthesis and efflux
of newly synthesized sterols, CHO cells were incubated simultaneously
with labeled precursor ([H]acetate or
[
H]mevalonate) and an extracellular sterol
acceptor, and then NSL from cells and media were analyzed by HPLC. Over
several experiments, qualitatively similar data were obtained using
either acetate or mevalonate as the precursor. HPLC profiles from an
experiment using [
H]mevalonate are shown in Fig. 2. In this experiment, the medium also contained either BSA
alone or BSA plus HDL
(at a concentration of 1 mg of
protein/ml). Under both conditions (Fig. 2, A and B, respectively), the cells accumulated radiolabel in
cholesterol and in the three polar products noted in Fig. 1(peaks labeled 1-3). When the incubation
medium contained HDL
, there was significant efflux of all
four products (Fig. 2C). Interestingly, it did not
appear that the products were released in proportion to their
accumulation in cells. Product 2 in particular was clearly subject to
disproportionately high efflux.
Figure 2:
HPLC analysis of biosynthetic sterols in
CHO cells and efflux medium. CHO(met18b2) cells were incubated for 8 h
in media containing [H]mevalonate (20 mCi/ml),
compound 58035 (1 µg/ml), and either BSA (0.2%) or BSA plus human
HDL
(1 mg of protein/ml), and then NSL were prepared from
cells and media and analyzed by liquid scintillation counting and
reverse-phase HPLC. For each sample, the HPLC elution profiles of
absorbance at 210 nm (A
) and
H
radioactivity are given. The cholesterol peak and three initially
unidentified (numbered) peaks are indicated on the
H
profiles. The efflux of [
H]NSL to medium
containing just BSA was only 0.75%, making HPLC analysis of this medium
impractical. Panel A, NSL from cells incubated with BSA in the
absence of HDL
. Panel B, NSL from cells incubated
with BSA and HDL
. Panel C, NSL from medium
containing BSA and HDL
.
Because of its interesting efflux
behavior and the strong evidence that it was a sterol, additional
attention was given to the identification of product 2. Its retention
time on HPLC (0.72-0.73 relative to cholesterol) was consistent
with it being a diene sterol (e.g. zymosterol or desmosterol).
As shown by the A profiles in Fig. 2, A and B, small but significant mass amounts of the
sterol were present in CHO cells. This allowed the isolation of
approximately 3 µg of product 2 by preparative HPLC of unlabeled
CHO cell NSL. Electron-impact mass spectral analysis of product 2 in
comparison to zymosterol and desmosterol standards (Table 1)
suggested little similarity to zymosterol, but greater than 90%
correspondence with desmosterol. Thus, with very high probability,
product 2 was desmosterol. In the subsequent data presentation, product
2 isolated from CHO cell incubations is referred to as desmosterol.
Fig. 3illustrates the time courses of synthesis and efflux of
total NSL, cholesterol, and desmosterol in CHO cells incubated with
HDL. The results demonstrated steady production and efflux
of NSL and both sterols over 24 h. The disproportionately high release
of desmosterol in comparison to cholesterol was apparent at all time
points.
Figure 3:
Time courses of efflux of newly
synthesized NSL and sterols from CHO cells to HDL.
Conditions were as described in Fig. 1, except that the
incubation time varied from 0.5 to 25 h. Sterol identifications were
based on HPLC retention times relative to cholesterol. Each incubation
was performed in triplicate. However, at early time points, it
sometimes was necessary to pool triplicate samples of medium NSL in
order to have enough radioactivity for a single useful HPLC profile.
For this reason, data are plotted without error bars. When three
independent determinations of a value were obtained, S.D. values
typically were 5-15%. Data points missing at the 0.5-h time point
indicate that the medium sample was deemed to have too little
radioactivity to justify HPLC analysis of even a pooled sample. Each
panel shows the recovery of the indicated component in cells (circles), in medium (triangles), and in cells +
medium (squares).
Table 2and Table 3provide a compilation of
data from several experiments in which the synthesis and efflux of
sterols in CHO cells under three acceptor conditions (BSA alone, BSA
plus HDL, and egg phosphatidylcholine vesicles) were
examined after an 8-h incubation period. The data in Table 2demonstrate that under all three conditions, cholesterol
and desmosterol together accounted for approximately half of newly
synthesized NSL in cells, and that the two sterols were present in
roughly equal amounts, although in this regard there was considerable
variability from experiment to experiment. The data in Table 2also show that cholesterol and desmosterol were available
for efflux to both HDL
and PC vesicles and that in both
cases the contribution of desmosterol to the newly sythesized sterol
efflux was three to four times the contribution of cholesterol (i.e. cholesterol was only about 10% of the biosynthetic NSL
in the efflux medium, whereas desmosterol was 30-42% of the
biosynthetic NSL in the medium). In Table 3, the efflux data are
re-expressed in fractional units (i.e. efflux of a given
component normalized to its accumulation in cells + medium during
the 8-h incubations). These data show that with HDL
, the
efflux of desmosterol was about three times more efficient than the
efflux of cholesterol. The difference in efflux efficiencies appeared
to be even greater with PC vesicles (6-9-fold).
To determine
whether the results on synthesis and efflux of sterols obtained with
CHO cells were representative of other cells, an experiment comparing
several different cell types (CHO, human fibroblasts, rabbit smooth
muscle cells, human HepG2 hepatoma cells) was performed. When the
different cell types were incubated for 8 h with
[H]acetate and no sterol acceptor in the medium (Fig. 4, left panels), similar complex sterol profiles
were obtained in the four non-hepatic cell types (CHO-met18b2, CHO-K1,
GM3468A fibroblasts, and rabbit smooth muscle cells), consisting mostly
of cholesterol and two or three other major products with retention
times less than that of cholesterol. In all cases, sterol 2
(co-migrating with desmosterol) was prominent. In contrast, HepG2 cells
accumulated cholesterol and little of any other sterol. When media
contained HDL
(Fig. 4, right panels),
substantial efflux of newly synthesized sterol was obtained and in all
cases the fractional release of sterol 2 was approximately three times
that of cholesterol. These results suggest that the accumulation of
both cholesterol and more polar sterol intermediates is characteristic
of a variety of non-hepatic cells, and that in a wide range of cell
types, newly synthesized desmosterol (or a desmosterol-like sterol) is
much more readily available for efflux than is newly synthesized
cholesterol.
Figure 4:
Production and efflux of newly synthesized
sterols in a variety of cell types. The five indicated cell types were
compared in a single experiment. The base medium for CHO cells was
Ham's F-12, and for the other cells minimum essential medium.
Otherwise, the conditions for all cells were identical. Cells were
incubated for 8 h in media containing [H]acetate
(50 µCi/ml) and either BSA (0.2%) or BSA plus HDL
(1 mg
of protein/ml), and then NSL were prepared from cells and media and
analyzed by HPLC. The panels on the left show HPLC
profiles of NSL from cells incubated with BSA in the absence of
HDL
. The panels on the right show
fractional efflux of
H-labeled NSL, cholesterol, and sterol
2 to media containing HDL
. Sterol 2 in CHO cells was
identified as desmosterol (see Table 2) but was not identified in
the other cell types. The HPLC retention times of cholesterol and the
other major sterols varied somewhat from day to day, although the
retention times of the sterols relative to each other were constant. To
compensate for the variability in absolute retention times, the
retention time of cholesterol was determined each day using labeled and
unlabeled standards, and this determination provided the basis for
identifying cholesterol and sterol 2 (relative retention time
0.72-0.73) in the experimental
profiles.
To examine the effects of enriching cells with
cholesterol mass on the efflux of newly sythesized sterols, control and
free cholesterol-enriched CHO-K1 cells and fibroblasts were prepared as
described under ``Materials and Methods,'' and then after
rinsing and a brief equilibration period, were incubated for 24 h in
medium containing [H]acetate and either HDL
or egg PC vesicle acceptor particles. Values for the fractional
efflux of total NSL, cholesterol, and sterol 2 (desmosterol in CHO
cells) are given in Table 4. In all cases, the enrichment with
cholesterol either had no significant effect or suppressed the
fractional efflux of sterols. In CHO cells, the enrichment suppressed
the efflux of newly synthesized cholesterol by about one-third using
either HDL
or PC vesicles as the extracellular acceptor,
whereas the suppression of desmosterol efflux was about twice as great
with the vesicle acceptor as with HDL
. In fibroblasts, the
enrichment had no significant effect on the fractional efflux of newly
synthesized cholesterol and suppressed the efflux of sterol 2 by about
one-fourth using either of the acceptors. Under all conditions, the
fractional efflux of sterol 2 remained substantially greater than that
of cholesterol.
To determine whether HDL was
particularly efficient in comparison to PC-SUV at promoting efflux from
specific cellular compartments, fibroblast and CHO cell data on sterol
efflux from different cellular pools were compiled and compared. Data
were available for synthesized sterols from the present work and for
the plasma membrane and lysosomal cholesterol pools from previous
studies performed under similar conditions (see Table 5references). For each sterol pool, the data were expressed
as the ratio of [efflux to HDL
]/[efflux to
PC-SUV], using acceptor concentrations of 1 mg of protein/ml and 1
mg/ml, respectively (mol/mol efflux ratios, Table 5). Then, to
probe for HDL-specific influences on the delivery of internal sterol to
the plasma membrane, the ratio for plasma membrane cholesterol was set
equal to 1, and the other ratios were re-expressed accordingly
(normalized efflux ratios, Table 5). These calculations show that
in both CHO cells and fibroblasts, the efflux ratio for lysosomal
cholesterol was identical to that for plasma membrane cholesterol. In
CHO cells, the ratios for synthesized desmosterol and cholesterol were
greater than plasma membrane cholesterol by factors of 1.2 and 2.9,
respectively. In GM3468A fibroblasts, the corresponding values were 1.3
and 2.2, respectively. These results indicate that synthesized sterol 2
(desmosterol) was released from cells with a low acceptor specificity,
similar to that governing the efflux of lysosomal cholesterol (Johnson et al., 1990, 1991). In contrast, the efflux of newly
synthesized cholesterol, although much slower than that of sterol 2
under all conditions tested (Table 2Table 3Table 4),
was enhanced by HDL
, suggesting that the delivery of
synthesized cholesterol to the plasma membrane may be more efficient in
the presence of HDL
. Using data from Table 4, it can
also be calculated that the enrichment of cells with cholesterol had
little effect on the efflux ratios for synthesized sterols. Thus, the
enhanced efflux of synthesized cholesterol in the presence of HDL
did not require prior elevation of cellular cholesterol content.
To provide an additional test of the selective enhancement of cholesterol efflux by HDL, we compared the abilities of PC-SUV and a reconstituted PC-apoHDL discoidal complex to deplete cholesterol and desmosterol mass from CHO-K1 cells. In this case, the cells were grown in lipid-free medium (Fig. 5) so that they contained only synthesized sterols. In addition, to stabilize the levels of cholesterol and desmosterol in the system during efflux, the cells were treated with a combination of compound 58035, mevinolin, and triparanol (inhibitors of acyl-CoA:cholesterol acyltransferase, hydroxymethylglutaryl-CoA reductase, and sterol 24-reductase, respectively). The mass data indicated that both PC-SUV and PC-apoHDL were able to cause net depletion of cellular desmosterol, with the apoHDL acceptor being two to three times more efficient than SUV (Fig. 5B). For cellular cholesterol (Fig. 5A), the fold difference in acceptor efficiency was much greater, with PC-apoHDL removing 60% of cholesterol after 24 h and SUV removing 10% or less (a change that was not statistically significant but does not contradict the more precise radio-tracer data in Table 3and Table 4). Thus, the mass data were consistent with the efflux patterns described in Table 5and provide independent confirmation of the selective enhancement of endogenous cholesterol release by HDL.
Figure 5: Depletion of CHO cell cholesterol and desmosterol mass by PC-SUV and PC-apoHDL acceptors. CHO-K1 cells were grown to approximately 50% confluence in 35-mm tissue culture wells with medium containing DLP (5 mg/ml) and Sandoz compound 58035 (1 µg/ml), and then incubated 2 days with the same medium also supplemented with triparanol (1 µM), an inhibitor of sterol 24-reductase, to induce accumulation of easily measurable amounts of desmosterol. At this time the cells were confluent and were incubated overnight with 1.5 ml/well of medium containing DLP, compound 58035, triparanol, and mevinolin (1 µg/ml), to stabilize the levels of desmosterol and cholesterol in the cells. After this overnight incubation, depletion of cellular sterol was examined under three efflux conditions: 1) BSA (0.2%), 2) BSA + PC-apoHDL (500 µg of PC/ml), and 3) PC-SUV (1 mg/ml). Exposure to the three drugs was continued under all three efflux conditions. Cells were analyzed for content of cholesterol and desmosterol by gas-liquid chromatography after 0, 8, and 24 h of efflux. Results are plotted as sterol content/mg cell protein versus incubation time under the different conditions.
The results of these studies suggest that sterol synthesis in
non-hepatic cells is accompanied by the accumulation of both
cholesterol and several polar sterol intermediates. As implied by
efflux to extracellular acceptors, the intermediates do not remain
confined to the endoplasmic reticulum or other internal organelles, but
rather are delivered along with cholesterol to the outer leaflet of the
plasma membrane. During efflux, cholesterol and the intermediates are
not released from cells with equal efficiency. In particular, a sterol
with the chromatographic properties of desmosterol (and conclusively
identified as this sterol in CHO cells) is released from cells several
times more efficiently than is newly synthesized cholesterol. As a
result of the substantial production of this sterol and its tendency to
undergo rapid efflux, the major synthesized sterol released from
non-hepatic cells appears to be desmosterol (or a desmosterol-like
sterol) rather than cholesterol. This finding holds for both
cholesterol-depleted cells (in which cholesterol synthetic activity is
high) and for cholesterol-enriched cells (in which synthetic activity
is low), and for both lipoprotein (HDL) and non-lipoprotein
(PC-SUV) acceptors. However, the results indicate a significant degree
of enhancement of the efflux of newly synthesized cholesterol in
response to incubation with HDL. This enhancement is not seen for newly
synthesized desmosterol or for the lysosomal pool of cholesterol. The
enhancement suggests the possibility of regulation of the efflux of
newly synthesized cholesterol by HDL and other biological acceptors
that are thought to participate in reverse cholesterol transport.
Previous studies exploring the
acceptor-specificity of biosynthetic sterol efflux suggested that in
cholesterol-enriched cells the efflux was stimulated by the presence of
acceptors that bind to the putative HDL receptor (Oram et al.,
1983; Aviram et al., 1989). Thus, HDL was reported
to promote efflux of newly synthesized sterols, whereas acceptors such
as PC vesicles and nitroslyated HDL, which cannot bind to the receptor,
were much less effective (Slotte et al., 1987). Limited data
have been provided on the composition of sterols released from cells.
Tabacik et al.(1991) used thin layer chromatography methods to
characterize the synthesis and efflux of biosynthetic sterols in smooth
muscle cell cultures and reported the preferential release of sterol
precursors in comparison to cholesterol during the incubation of cells
in diluted calf serum. The desorbed presursors appeared to consist
predominantly of late intermediates (i.e. C-27 dienes), but
precise identification of the sterols was not performed. Aviram et
al.(1989) reported that in human monocyte-derived macrophages,
most biosynthetic sterol that accumulated and was released from cells
co-migrated with desmosterol on reverse-phase thin layer
chromatography. The efflux of this sterol to HDL
was
reported to be similar to that of biosynthetic cholesterol. Hokland et al.(1993) reported that in human fibroblasts the
biosynthetic sterols consisted of cholesterol and multiple unidentified
components with HPLC retention times somewhat different from that of
desmosterol. The fractional efflux of the intermediates to HDL
was greater than that of cholesterol, although the efflux of both
cholesterol and the other sterols was stimulated by cAMP. These
previous results indicate that polar intermediates are the predominant
biosynthetic sterols released from cells and suggest that the efflux
behavior of these sterols resembles that of biosynthetic cholesterol.
Conclusive identification of the intermediates has not been provided.
Nor has the efflux of each sterol been quantified separately.
The
present results add to previous findings by clearly identifying
desmosterol as the main biosynthetic sterol released from CHO cells and
by indicating that either desmosterol or a structurally similar sterol
is the main biosynthetic sterol released from other extrahepatic cells,
including fibroblasts. In contrast to the conclusions of previous
studies, we found that the sterols synthesized in cholesterol-enriched
cells are available for efflux to an acceptor (PC-SUV) that does not
bind to the HDL receptor (Table 4). Thus, the efflux of
synthesized sterols does not require acceptors that bind to the
putative HDL receptor. The methodology of reverse-phase HPLC in
combination with continuous liquid-scintillation monitoring of the HPLC
effluent provided a detailed quantitative profile of newly synthesized
sterols undergoing efflux. The ability to quantify individual sterols
using this methodology provided evidence that HDL
selectively enhances the efflux of synthesized cholesterol (Table 5). In contrast to the results of Aviram et
al.(1989), this enhancement appears to be confined to synthesized
cholesterol and is not observed for synthesized desmosterol or for
LDL-derived lysosomal cholesterol (Table 5). The enhancement may
be due to the interaction of HDL with the putative HDL receptor,
although its occurrence in both cholesterol-enriched and unenriched
cells argues against this explanation. Additional experiments will be
needed to determine the basis for this enhancement. These experiments
could involve direct tests of the diacylglycerol and cAMP signaling
mechanisms, which have been reported to stimulate sterol translocation
to the plasma membrane.
The greater efflux of desmosterol in comparison to cholesterol may have been due to greater delivery of desmosterol to the plasma membrane, greater desorption of desmosterol from the plasma membrane to extracellular acceptor particles, or a combination of such differences. Analysis of the sterol profile in isolated plasma membranes and studies on sterol efflux from isolated plasma membranes will be used to resolve this question. Relevant to this issue are studies of Clejan and Bittman (1984) reporting that the efflux of desmosterol from the surface membrane of Mycoplasma gallisepticum to egg phosphatidylcholine vesicles is approximately 30% slower than the efflux of cholesterol. If this finding also applies to the plasma membrane of mammalian cells, it would suggest that the greater fractional efflux of desmosterol relative to cholesterol may be due to more rapid delivery of desmosterol to the plasma membrane. Consistent with this possibility, Lange et al.(1991) reported that the transport of newly synthesized zymosterol to the plasma membrane in fibroblasts was two times faster than that of newly synthesized cholesterol.
The results of these experiments indicated substantial efflux of several nonsaponifiable lipid products other than cholesterol (e.g.Fig. 2C, peaks 1-3). Major attention was focused on desmosterol (peak 2) in this article because of the major contribution of this sterol to the efflux of the newly synthesized sterols and because we were able to provide a conclusive identification of this sterol in CHO cells. It will be of interest in future studies to establish the identities of the other intermediates that accumulate during sterol synthesis and to further explore their availability for efflux.
The presence of low concentrations of sterol intermediates in blood plasma is well documented in the work of Mietennin and colleagues (Bjorkhem et al., 1987; Vanhanen et al., 1993). Previously, the presence of these intermediates in blood has been attributed largely to leakage from the liver (Bjorkhem et al., 1987). However, in the present studies non-hepatic cells appeared to be much better sources of sterol intermediates than either HepG2 human hepatoma cells (Fig. 4) or Fu5AH rat hepatoma cells (data not shown). In these liver-derived cell lines, cholesterol was the major sterol synthesized and released to extracellular acceptors. These findings may indicate that non-hepatic tissues are major sources of the sterol intermediates that are found in blood plasma. This possibility is consistent with the conclusion reached by Dietschy et al. (1993) that non-hepatic tissues contribute substantially to sterol synthesis in mammals, based on the results of in vivo metabolic studies. Another interesting contrast derived from the present results is the large efflux of sterol intermediates from non-hepatic cells in comparison to the very low concentrations of intermediates relative to cholesterol in blood plasma (Bjorkhem et al., 1987). This disparity may imply unusually efficient mechanisms for the clearance of sterol intermediates from plasma. There appears to be very little direct information relating to this question or to the subsequent metabolism of these sterols after uptake into cells. These topics deserve further investigation.