The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 1 182-187
Copyright © 1997 by The Endocrine Society
Transfer of Dehydroepiandrosterone- and Pregnenolone-Fatty Acid Esters between Human Lipoproteins1
Pierre R. Provost2,
Bernard Lavallée3 and
Alain Bélanger
Medical Research Council Group in Molecular Endocrinology, CHUL
Research Center and Laval University, Quebec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Dr. Alain Bélanger, Medical Research Council Group in Molecular Endocrinology, CHUL Research Center, 2705 Laurier Boulevard, Quebec, Canada G1V 4G2.
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Abstract
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Dehydroepiandrosterone (DHEA)- and pregnenolone (PREG)-fatty acid
esters (FA) are formed in plasma high density lipoproteins (HDL),
whereas they accumulate in very low density lipoproteins (VLDL), low
density lipoproteins (LDL), and HDL. We have hypothesized that these
lipoidal steroids could be transferred from HDL to VLDL and LDL by the
cholesteryl ester (CE) transfer protein (CETP), which mediates CE
transfer activity in human plasma. In this study, we further
investigated this hypothesis. Lipoproteins and lipoprotein-deficient
plasma (LPDP) were purified and analyzed by Western blots. LPDP was
rich in CETP, in contrast to lipoprotein preparations, which contained
very low amounts. Using these preparations in in vitro
transfer assays, CE transfer from radiolabeled steroid ester-HDL to
VLDL or LDL was only observed in the presence of LPDP. In contrast,
time- and temperature-dependent transfer of DHEA-FA and PREG-FA were
observed in the absence of LPDP. The addition of LPDP had no effect on
the DHEA-FA transfer rate, whereas the PREG-FA transfer rate was
increased. Moreover, in the absence of LPDP, no decrease in the
transfer levels of DHEA-FA and PREG-FA was observed after the removal
of CETP from lipoprotein preparations by immunoaffinity column
chromatography. The PREG-FA transfer activity of LPDP was studied using
the anti-CETP monoclonal antibody TP-2, which is known to block the CE
transfer activity of CETP. In the presence of LPDP, this antibody led
to a dose-dependent decrease in CE transfer activity, whereas PREG-FA
transfer activity was unaffected.
In conclusion, we have shown that DHEA-FA and PREG-FA are transferred
from HDL to VLDL and LDL by a CETP-independent mechanism. There is a
major difference in the transport of lipoidal steroids by human
lipoproteins compared to that of CE.
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Introduction
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THE PRESENCE of steroidal fatty acid esters
(-FA) was characterized many years ago, and almost every family of
steroid is known to occur in esterified form (1, 2). For example,
3ß-hydroxy-5-ene-steroids such as pregnenolone (PREG),
dehydroepiandrosterone (DHEA), and androst-5-ene-3ß,17ß-diol can be
converted to FA counterparts by the lecithin:cholesterol
acyltransferase (LCAT) enzyme present in high density lipoproteins
(HDL) (3, 4, 5, 6, 7, 8). Analysis of the distribution of these compounds in
lipoproteins has revealed the presence of lipoidal steroids in all
major lipoprotein classes, but predominantly in low density
lipoproteins (LDL) (6, 7, 9). This suggests that nonpolar steroids are
transferred to LDL and very low density lipoproteins (VLDL) after their
formation by HDL (10, 11). Recent work by our group has demonstrated
that PREG-FA incorporated into LDL may be used as substrate for adrenal
and ovarian steroid synthesis (9, 12, 13). Moreover, internalization of
[3H]DHEA-FA from LDL carriers was observed after
incubation with breast cancer cells (6), suggesting that peripheral
tissues may also use circulating DHEA-FA as a substrate for androgen or
estrogen synthesis (14). Taken together, these observations suggest a
role for DHEA-FA and PREG-FA, and the mechanism by which these
compounds are transferred between lipoproteins is important to
investigate.
It is well established that cholesteryl esters (CE) are transferred
between human lipoproteins by CE transfer protein (CETP) (15, 16). We,
therefore, postulated that the transfer of PREG-FA and DHEA-FA between
lipoproteins could also depend on the activity of CETP. The present
study demonstrated that tritiated DHEA-FA and PREG-FA incorporated in
HDL are transferred to VLDL and LDL, and that these transfer activities
are not dependent of CETP.
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Materials and Methods
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Materials
[1,2,6,7-3H]Cholesterol (91.5 Ci/mmol),
[7-3H]PREG (25.0 Ci/mmol), and
[1,2,6,7-3H]DHEA (92.0 Ci/mmol) were purchased from New
England Nuclear-DuPont (Boston, MA). Silica gel thin layer plates were
supplied by E. Merck (Darmstadt, Germany). Sodium heparin and
lyophilized fatty acid-free human serum albumin were purchased from
Sigma Chemical Co. (St. Louis, MO). Cyanogen bromide-activated
Sepharose 4B was obtained from Pharmacia (Uppsala, Sweden).
Human plasma and lipoproteins
Human plasma-ethylenediamine tetraacetate (EDTA) was collected
from fasting men, aged 24–37 yr, who were taking no medication. VLDL
(density, <1.006 g/mL), LDL (1.006< density <1.063 g/mL), HDL
(1.063< density <1.210 g/mL), and lipoprotein-deficient plasma (LPDP;
density, >1.210 g/mL) were isolated by sequential ultracentrifugation
(17) and dialyzed against the buffer used for transfer assays (transfer
assay buffer, 50 mmol/L Tris, pH 7.4; 150 mmol/L NaCl; and 2 mmol/L
EDTA). LPDP was adjusted to the initial plasma volume by the addition
of transfer assay buffer. The protein content of lipoproteins was
measured using a protein assay kit (P5656) from Sigma Chemical Co.
HDL labeling with tritiated PREG or DHEA
HDL were labeled with radioactive steroid esters as previously
described (6, 9, 12). After dialysis against the transfer assay buffer,
radioactivity was determined, and the preparations were analyzed by
thin layer chromatography (TLC). In typical preparations, PREG-FA and
DHEA-FA represented more than 99% of the radioactive counts, whereas
CE-labeled HDL contained 80% and 20% of radiolabeled CE and
unesterified cholesterol, respectively.
In vitro transfer assays
Transfer assays were performed as previously described (18) with
slight modifications. For each determination, two tubes were prepared
and incubated at 0 and 37 C, respectively. Briefly, 23,000–35,000 dpm
of radiolabeled steroid esters-HDL and -VLDL or -LDL, which correspond
to 15 or 20 µg protein, were used. A volume of 12–15 µL LPDP was
added when indicated, and final volumes were adjusted to 100 µL with
the transfer assay buffer. After incubation, the apolipoprotein
B-containing lipoproteins (VLDL or LDL) were precipitated with
heparin-MnCl2, as previously described (18), and removed by
centrifugation at 4 C for 15 min at 3,000 rpm in a Sorvall RC-3
centrifuge (Sorvall, New York, NY). Radioactive counts were determined
in the resulting supernatants using a Wallac liquid scintillation
counter (model 1411). For calculating the percentage of CE transfer,
tubes incubated at 0 C were used as controls without transfer as
described by Tollefson and Albers (19). We have also assumed that the
tubes incubated at 0 C corresponded to controls without transfer for
DHEA-FA and PREG-FA. The percentage of lipoidal steroid transferred
from HDL to VLDL or LDL was thus calculated as follows: [(dpm0
C - dpm37 C) x 100]/dpm0 C.
Immunoaffinity column chromatography
The anti-CETP monoclonal antibody TP-2 (a gift from Dr. Ross
Milne, Ottawa, Canada) was coupled to cyanogen bromide-activated
Sepharose 4B (Pharmacia) using 10 mg IgG/mL Sepharose. Lipoprotein
samples were dialyzed against 10 mmol/L Tris (pH 7.4) and 140 mmol/L
NaCl and then loaded on the column equilibrated in the same buffer.
Unbound fractions were collected. Lipoproteins were dialyzed against
the transfer assay buffer, and the amounts of HDL radioactivity and LDL
proteins were determined.
Western blot analysis
SDS-PAGE was performed as previously described (20). Gels were
blotted onto Hybond-C Extra membranes (Amersham Corp., Arlington
Heights, IL) using the method of Towbin et al. (21). CETP
was detected using a mixture of the anti-CETP monoclonal antibodies
TP-2 (15) and TP-14 (22) and with antimouse IgG-F(ab')2
linked to horseradish peroxidase (Sigma Chemical Co.). Signals were
revealed with a chemiluminescence detection kit (ECL, Amersham Corp.)
following exposure on a Reflection autoradiography film (NEF-495,
DuPont Co.) for 1.5 min.
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Results
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Incubation of radiolabeled unconjugated cholesterol, DHEA, and
PREG with human plasma for 24 h led to the production of their FA
counterparts in HDL by endogenous LCAT and the transfer of these newly
formed lipoidal derivatives between lipoproteins. After isolation of
lipoproteins, the relative amounts of newly formed lipoidal steroids
associated with VLDL, LDL, and HDL were determined by TLC analysis.
Figure 1
shows the distribution of newly formed FA of
steroids in lipoproteins at the end of the incubation. Similar
proportions of newly formed CE, DHEA-FA, and PREG-FA were transferred
from HDL to VLDL and LDL after their formation in HDL. The antibody
TP-2 inhibits the transfer of CE by its interaction with the neutral
lipid transfer domain of CETP (23), and therefore, the ability of this
antibody to inhibit the transfer of lipoidal steroids was tested. When
TP-2 was added, the proportion of CE associated with HDL was higher.
However, the presence of the antibody had no effect on the DHEA-FA and
PREG-FA transfer activities (Fig. 1).
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Figure 1. Distribution of newly formed CE, DHEA-FA,
and PREG-FA among human plasma lipoproteins. Fifty microcuries of
[3H]cholesterol, [3H]DHEA, and
[3H]PREG were added to 1 mL human plasma-EDTA, and the
mixtures were incubated for 24 h at 37 C alone or after addition
of 10 µL of the monoclonal antibody TP-2. At the end of the
incubation, lipoproteins were isolated and then analyzed by TLC to
determine the proportion of radioactivity corresponding to lipoidal
derivatives in each fraction compared to the total radioactivity count.
The results are expressed as the percentage of total newly formed CE,
DHEA-FA, and PREG-FA associated with VLDL ( ), LDL ( ), and HDL
( ). Results are the mean of duplicate determinations.
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It is well documented that during isolation of lipoproteins by
sequential ultracentrifugation, CETP dissociate from particles and are
collected in LPDP (16, 24). Therefore, the addition of LPDP or another
source of CETP is essential for the transfer of CE in in
vitro transfer assays (24). As expected, this was confirmed in our
transfer assays, as demonstrated in Fig. 2. The fact
that the transfer of CE in the absence of LPDP did not increase over
time (Fig. 2B) showed that no residual CETP activity was associated
with isolated lipoproteins. As illustrated in Fig. 2, transfer of
PREG-FA increased over time in the absence of LPDP, showing that a
significant part of the PREG-FA transfer activity remained associated
with lipoproteins. The addition of LPDP increased the PREG-FA transfer
rate, indicating that a part of the PREG-FA transfer activity was also
present in LPDP. In contrast, a high rate of DHEA-FA transfer was
observed in the absence of LPDP, and this transfer was not affected by
the addition of LPDP (Fig. 2). Similar results were obtained using LDL
instead of VLDL as a source of acceptor lipoproteins (data not
shown).
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Figure 2. Transfer of lipoidal steroids between
isolated lipoproteins. In vitro transfer assays were
performed using 23,000 dpm (A and B) or 35,000 dpm (C) radiolabeled
steroid ester-HDL (0.7, 0.9, and 1.4 µg HDL proteins for radiolabeled
CE, PREG-FA, and DHEA-FA, respectively) and an excess of acceptor VLDL
(15, 20, and 15 µg VLDL proteins for A, B, and C, respectively).
Fifteen microliters of LPDP were added when indicated, and the final
volume was completed to 100 µL. The results are expressed as the
percentage of radiolabeled CE (A and B), PREG-FA (A and B), or DHEA-FA
(A and C) transferred from HDL to VLDL in 5.5 h (A) or the
indicated time (B and C). The values are the mean (±SD) of
duplicates from one selected experiment and correspond to the transfer
at 37 C, calculated using the values obtained at 0 C as controls
without transfer. Similar results were obtained using lipoproteins
isolated from another subject (data not shown). A: , Without LPDP;
, with LPDP. B: , [3H]CE in the absence of LPDP;
, [3H]CE plus LPDP;  , [3H]PREG-FA in
the absence of LPDP; •, [3H]PREG-FA plus LPDP. C: ,
[3H]DHEA-FA in the absence of LPDP; ,
[3H]DHEA-FA plus LPDP.
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In subsequent experiments, we investigated the transfer rates of
DHEA-FA and PREG-FA from HDL donors in the presence of an excess of
VLDL or LDL acceptors. Because PREG-FA and DHEA-FA transfer activities
are mainly associated with lipoproteins, as shown in Fig. 2
, we
expressed the rate of transfer of these two lipoidal steroids in the
absence of LPDP in picomoles of transferred steroid per mg HDL
protein/h of incubation (Table 1). Our results suggest
that the in vitro transfer rate of DHEA-FA is higher than
that of PREG-FA. Table 1 also shows that VLDL are better acceptors of
PREG-FA than LDL, whereas no preferential acceptor was observed for
DHEA-FA. The LPDP-dependent rates of transfer (rate of transfer in the
presence of LPDP - rate of transfer in the absence of LPDP) were
also determined (Table 1).
Our data demonstrate that DHEA-FA and PREG-FA transfer activities are
associated with isolated lipoproteins, in contrast to CE transfer
activity, which is found exclusively in LPDP. The presence of CETP in
preparations of isolated lipoproteins and LPDP was then determined by
Western blot analysis. Figure 3A shows that LPDP
contained a high amount of this protein, in contrast to HDL, VLDL, and
LDL preparations, which retained very low amounts of this protein.
Densitometric analysis revealed that the proportion of CETP contained
in LPDP vs. that contained in lipoprotein preparations was
99:1. This observation agrees with our results showing that the CE
transfer activity was detected only in LPDP, suggesting that CETP has
no DHEA-FA or PREG-FA transfer activities.
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Figure 3. Transfer of DHEA-FA from HDL to LDL using
unbound fractions of lipoproteins eluted from an anti-CETP
immunoaffinity column. LDL and [3H]DHEA-FA-labeled HDL
were purified on an anti-CETP immunoaffinity column using the
monoclonal antibody TP-2. The unbound fractions were tested by Western
blot analysis (A) and used in in vitro transfer assays
(B). A, [3H]DHEA-FA-labeled HDL (lanes 1 and 2; 17,500
dpm) and LDL (lanes 3 and 4; 7.5 µg LDL proteins) collected before
(lanes 1 and 3) or after (lanes 2 and 4) immunoaffinity chromatography,
and typical preparations of VLDL and LPDP used in the present report
(lane 5, 5 µg VLDL proteins; lane 6, 0.5 µL LPDP) were loaded onto
a 10% SDS-PAGE. After transfer, CETP was detected using a mixture of
TP-14 and TP-2 anti-CETP monoclonal antibodies. The positions of mol wt
protein standards are indicated at the left. B,
In vitro transfer assays were performed using 35,000 dpm
[3H]DHEA-FA-labeled HDL (1.4 µg HDL proteins) and an
excess of acceptor LDL (15 µg LDL proteins) in a final volume of 100
µL without LPDP. The results are expressed as a percentage of
radiolabeled DHEA-FA transferred from HDL to LDL in 1 h using
lipoproteins collected before or after immunoaffinity chromatography,
as indicated. The values are the mean (±SD) of triplicates
from one selected experiment and correspond to the transfer at 37 C
calculated using the values obtained at 0 C as controls without
transfer. TP-2, Unbound fractions of lipoproteins eluted from the TP-2
immunoaffinity column; C, lipoproteins collected before immunoaffinity
column chromatography (control).
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As PREG-FA and DHEA-FA levels are very low in lipoproteins compared
with levels of unlabeled CE, it is possible that only a low amount of
CETP is sufficient for the transfer of a significant proportion of
radiolabeled PREG-FA and DHEA-FA. To answer this question, CETP from
both HDL donors labeled with radioactive steroid esters and LDL
acceptors were removed by immunoaffinity column chromatography using
the anti-CETP monoclonal antibody TP-2. The resulting lipoprotein
preparations were then tested by Western blot (Figs. 3A and 4A), and the amounts of CETP were compared by
densitometric analysis in samples collected before and after the
treatment. The signals of 66 and 68 kDa correspond to the two isoforms
of CETP (16). For the study of DHEA-FA, the unbound fractions of
lipoproteins eluted from the immunoaffinity column contained
approximately 18% and 10% the amount of CETP associated with
untreated HDL and LDL, respectively. As shown in Fig. 3B, 100% of the
DHEA-FA transfer activity remained associated with lipoproteins after
their elution from the column. For the study of PREG-FA, the amounts of
CETP associated with HDL and LDL after their passage through the
immunoaffinity column compared to that of untreated lipoproteins were
19% and 13%, respectively (Fig. 4A). Again, after their elution from
the column, the lipoproteins did not lose their PREG-FA transfer
activity (Fig. 4B). Moreover, a slight increase in PREG-FA transfer
activity was observed using CETP-depleted HDL.
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Figure 4. Transfer of PREG-FA from HDL to LDL using
unbound fractions of lipoproteins eluted from an anti-CETP
immunoaffinity column. LDL and [3H]PREG-FA-labeled HDL
were purified on an anti-CETP immunoaffinity column using the
monoclonal antibody TP-2. The unbound fractions were tested by Western
blot analysis (A) and used in in vitro transfer assays
(B). A, Aliquots of [3H]PREG-FA-labeled HDL (lanes 1 and
2; 17,500 dpm) and LDL (lanes 3 and 4; 7.5 µg LDL proteins) collected
before (lanes 1 and 3) or after (lanes 2 and 4) immunoaffinity
chromatography were loaded onto a 10% SDS-PAGE. After transfer, CETP
was detected using a mixture of TP-14 and TP-2 anti-CETP monoclonal
antibodies. The positions of mol wt protein standards are indicated at
the left. B, In vitro transfer assays
were performed using 35,000 dpm [3H]PREG-FA-labeled HDL
(1.5 µg HDL proteins) and an excess of acceptor LDL (15 µg LDL
proteins) without LPDP in a final volume of 100 µL. The results are
expressed as a percentage of radiolabeled PREG-FA transferred from HDL
to LDL in 5 h using lipoproteins collected before or after the
immunoaffinity chromatography, as indicated. The values are the mean
(±SD) of duplicates from one selected experiment and
correspond to the transfer at 37 C, calculated using the values
obtained at 0 C as controls without transfer. TP-2, Unbound fractions
of lipoproteins eluted from the TP-2 immunoaffinity column; C,
lipoproteins collected before immunoaffinity column chromatography
(control).
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In contrast to DHEA-FA transfer activity, which was found only in
lipoprotein preparations, PREG-FA transfer activity was detected in
both lipoproteins and LPDP. We next investigated PREG-FA transfer
activity in LPDP for its dependence on the neutral lipid transfer
domain of CETP. For this purpose, the monoclonal antibody TP-2 was
added to in vitro transfer assays to inhibit the transfer
activity of the neutral lipid transfer domain of CETP. In the presence
of LPDP, the increase in TP-2 was accompanied by a significant decrease
in CE transfer activity, whereas the PREG-FA transfer level was
unaffected (Fig. 5). Despite the addition of TP-2, the
level of transfer of PREG-FA remained significantly higher than the
control level with no LPDP added.
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Discussion
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In the present study, we showed that DHEA-FA and PREG-FA are
transferred from HDL to VLDL and LDL by a temperature- and
time-dependent mechanism independent of the presence of CETP. We showed
that high levels of DHEA-FA and PREG-FA transfer activity remain
associated with lipoproteins after ultracentrifugation, in contrast to
both CETP protein and CE transfer activities. When CETP was removed
from lipoprotein preparations by anti-CETP-immunoaffinity column
chromatography, the resulting lipoproteins retained all of their
DHEA-FA and PREG-FA transfer activities. Although CE transfer was
observed only in the presence of LPDP, as shown in time-course studies,
the transfer of DHEA-FA was independent of the addition of LPDP. In
contrast to both CE and DHEA-FA transfer activities, the PREG-FA
transfer activity was found in lipoproteins as well as in LPDP.
Although CETP was present at high concentrations in LPDP, the PREG-FA
transfer activity of LPDP did not depend on the neutral lipid transfer
domain of CETP, as demonstrated by use of the monoclonal antibody TP-2,
which selectively inhibited the transfer of CE, but not that of
PREG-FA. Taken together, our results show that the CETP is not
responsible for the transfer of DHEA-FA and PREG-FA from HDL to VLDL
and LDL.
The observation that newly formed lipoidal steroids were found in VLDL
and LDL suggests that DHEA-FA and PREG-FA are transferred from HDL to
VLDL and LDL. However, it was not excluded that the lipoidal steroids
could be added to VLDL and LDL immediately after their formation by
LCAT. This hypothesis is unlikely because we have clearly shown in the
present report that the transfer of FA steroids can be dissociated from
the LCAT reaction. Moreover, dextran-coated charcoal treatment failed
to remove lipoidal steroids from lipoproteins (13), suggesting that
DHEA-FA and PREG-FA could not have been transferred by simple diffusion
in the aqueous solvent. Therefore, the existence of one or more
transfer proteins, distinct from CETP, responsible for the transfer of
DHEA-FA and PREG-FA between human lipoproteins is postulated.
As mentioned above, PREG-FA transfer activity was detected in both
lipoproteins and LPDP. The relative importance of these two sources of
PREG-FA transfer activity can be deduced from Table 1, which shows the
rates of transfer in vitro. Based on the concentration of
HDL proteins in plasma, we determined that approximately 90–95% of
the total plasma PREG-FA activity remains associated with lipoproteins
after their isolation by ultracentrifugation. One possible explanation
for this observation is that the PREG-FA transfer activity may
partially dissociate from lipoproteins during ultracentrifugation.
Alternatively, it is not excluded that a factor present in LPDP could
stimulate the transfer of PREG-FA or that CETP could have a low
affinity for PREG-FA. However, using the antibody TP-2, we showed that
the neutral lipid transfer domain of CETP is not responsible for the
PREG-FA transfer activity of LPDP. In contrast, DHEA-FA transfer
activity was detected in lipoprotein, but not in LPDP preparations,
whereas CE transfer activity was undetectable in the absence of LPDP.
Because the level of DHEA-FA transfer is relatively high in the absence
of LPDP, we cannot exclude the possibility that CETP may have a low
level of DHEA-FA transfer activity.
A plasma phospholipid transfer protein has been previously described
(25, 26). Although the level of this transfer protein was not measured
in our lipoproteins and LPDP, it is known that, like CETP, it
dissociates from lipoproteins during ultracentrifugation (27). In
addition, because this protein does not participate in the transfer of
neutral lipids such as CE and triglycerides (15, 16), it is an unlikely
candidate for a DHEA-FA and/or a PREG-FA transfer protein.
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Acknowledgments
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We acknowledge Dr. Ross Milne (Heart Institute, Ottawa, Ontario,
Canada) for providing the anti-CETP monoclonal antibodies TP-2 and
TP-14.
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Footnotes
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1 This work was supported by grant from the Medical Research Council
of Canada (to A.B., Medical Research Council Group in Molecular
Endocrinology) and Endorecherche. 
2 Supported by a fellowship from the Heart and Stroke Foundation of
Canada and selected by the Pharmaceutical Roundtable for a Parke-Davis
award.
3 Supported by a studentship from Le Fonds de la Recherche en
Santé du Québec-Fonds de Formation pour les Chercheurs et
l’Aide à la Recherche (FRSQ-FCAR-Santé).
Received July 8, 1996.
Revised September 19, 1996.
Accepted September 24, 1996.
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Abstract
Introduction
