From the Department of Cell Biology, Lerner Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, September 25, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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The selective uptake of high density lipoprotein
(HDL) cholesteryl ester (CE) by the scavenger receptor class B type I
(SR-BI) is well documented. However, the effect of altered HDL
composition, such as occurs in hyperlipidemia, on this important
process is not known. This study investigated the impact of variable CE
and triglyceride (TG) content on selective uptake. CE selective uptake by Y1 and HepG2 cells was strongly affected by modification of either
the CE or TG content of HDL. Importantly, TG, like CE, was selectively
taken up by a dose-dependent, saturable process in these
cells. As shown by ACTH up-regulation and receptor overexpression experiments, SR-BI mediated the selective uptake of both CE and TG.
With in vitro modified HDLs of varying CE and TG
composition, the selective uptake of CE and TG was dependent on the
abundance of each lipid within the HDL particle. Furthermore, total
selective uptake (CE + TG) remained constant, indicating that these
lipids competed for cellular uptake. These data support a novel
mechanism whereby SR-BI binds HDL and mediates the incorporation of a
nonspecific portion of the HDL lipid core. In this way, TG directly
affects the ability of HDL to donate CE to cells. Processes that raise the TG/CE ratio of HDL will impair the delivery of CE to cells via this
receptor and may compromise the efficiency of sterol balancing pathways
such as reverse cholesterol transport.
High density lipoprotein
(HDL)1 exists in plasma as
several distinct species defined by their size and density properties. Although synthesized initially as CE- and TG-deficient discs, HDL
rapidly becomes spherical as it acquires these core lipids (1, 2). The
transition to spherical particles and the repetitive cycling of
individual HDL species among the various size/density subfractions are
mediated in plasma by the actions of LCAT, CETP, phospholipid transfer
protein, and hepatic lipase (2-6). Together these factors control the
levels of HDL CE and TG, which directly influence the subfraction
content of the HDL pool.
HDL plays an important role in sterol metabolism. In nonsteroidogenic
tissues, cholesterol homeostasis is maintained through the capacity of
HDL to promote the net removal of free cholesterol from cells.
HDL-associated free cholesterol is converted to CE through the action
of LCAT (3). CE is in turn delivered to the liver for excretion. The
essential role of HDL in this process, known as reverse cholesterol
transport, is thought to explain, at least in part, the epidemiologic
evidence that HDL is anti-atherogenic (7, 8). In steroidogenic tissues,
the delivery of HDL cholesterol to cells is essential to the
maintenance of normal hormone biosynthesis (9).
The uptake of HDL-associated cholesterol in both hepatic and
steroidogenic tissues is mediated by the SR-BI receptor (10). SR-BI,
most highly expressed in the liver, adrenal gland, and ovary (10, 11),
is the only known receptor for HDL (10, 12, 13). The importance of
SR-BI in cholesterol homeostasis is readily observed in genetically
altered animals. Mice deficient in SR-BI display marked
hypercholesterolemia characterized by enlarged, cholesterol-rich HDL
particles, impaired HDL cholesterol clearance, and dramatically reduced
sterol content of adrenal tissue (14, 15). Conversely, hepatic
overexpression of SR-BI results in decreased HDL cholesterol content
and increased delivery of HDL-associated lipid to hepatocytes and the bile.
Unlike the well characterized uptake of lipoproteins by
receptor-mediated endocytosis (16), SR-BI facilitates a nonendocytic process known as selective uptake. In selective uptake, HDL binds to
SR-BI on the cell surface, a portion of its CE is transferred to the
cell without concomitant whole particle uptake, and then the
CE-depleted HDL is released into the extracellular fluid (10, 17, 18).
Little is known about the properties of HDL that provide optimum
interaction and cholesterol delivery through SR-BI. Because binding to
SR-BI does not require a specific apolipoprotein (19, 20), the
variations in apoprotein content of HDL subfractions are not likely to
influence markedly their substrate potential. Particle size has been
suggested to be an important determinant of selective uptake, but two
studies with synthetic HDL particles containing CE as their only core
lipid have given contradictory results (18, 19). However, unlike these
artificial lipoproteins, physiologic and pathophysiologic changes in
human HDL particle size are accompanied by alterations in both the
absolute level of CE within the particle and the relative amounts of CE
compared with the other major core lipid, TG. Although TG has been
reported to be a major regulator of cholesterol esterification and
reverse cholesterol transport (21), the influence of HDL TG content on
the capacity of HDL to donate CE via SR-BI is unknown. In this study we
have examined the influence of HDL core lipid (CE and TG) composition
on CE selective uptake in cultured hepatocytes and adrenal cells. The
findings shed light on the selective uptake mechanism and suggest that
the beneficial functions of HDL may be compromised under conditions of
excess HDL TG.
Materials
[9,10-3H]Triolein (15 Ci/mmol) and
carrier-free 125I in NaOH were purchased from PerkinElmer
Life Sciences. [1,2-3H]Cholesteryl oleyl ether
(3H-COE, 49 Ci/mmol) was purchased from Amersham Pharmacia
Biotech. 3H-Labeled trialkylglycerol ether
(3H-TGE), a triether analogue of TG, was synthesized from
3H-glycerol ([1(3)-3H]glycerol, 3 Ci/mmol,
Amersham Pharmacia Biotech) and >100-fold mole excess oleyl methane
sulfonate (Nu-Chek Prep, Elysian, MN) in the presence of potassium
metal, as previously described (22). Trypsin-EDTA,
penicillin/streptomycin, DME/F12 media, and fetal bovine serum
were from Life Technologies, Inc. Lipoprotein lipase (Pseudomonas) was from ICN (Costa Mesa, CA), and cholesterol
was from Nu-Chek Prep. BSA (fraction V), ACTH, egg
phosphatidylcholine, sodium cholate, trichloroacetic acid,
diethyl p-nitrophenyl phosphate (paraoxon), and all reagents
for salt solutions were obtained from Sigma. Paraoxon was prepared as a
100× stock (100 mM) in 50% methanol, 50 mM
Tris-HCl, pH 6.0, and stored at Isolation of Lipoproteins and Modification of HDL
Lipoproteins were isolated by sequential ultracentrifugation of
fresh human plasma from the Blood Bank of the Cleveland Clinic Foundation (23). Isolated VLDL (d < 1.006 g/ml)
was filtered (0.45 µm) after the addition of 1/100 volume of 0.4 M EDTA, 4% NaN3, pH 7.6. HDL (1.063 < d < 1.21 g/ml fraction) was dialyzed extensively
against 0.9% NaCl, 0.01% EDTA, pH 7.4. HDL and VLDL (500 µg of
cholesterol each) were combined with CETP (200 µl), 3.5% BSA (100 µl/ml), and paraoxon (1 mM final concentration) to block
residual LCAT activity. Samples were incubated at 37 °C from 0 to
24 h to permit CETP to modify the TG and CE content of HDL. Lipid
transfer inhibitor protein, which has been shown to stimulate the
transfer from VLDL to HDL (24), was added in certain cases to augment
the HDL modification. After re-isolation of modified HDLs within their
original density limits, lipoproteins were dialyzed as described above
and assayed for protein, total cholesterol, free cholesterol, TG, and
phospholipid content as described below. CE was calculated as the
difference between total and free cholesterol times 1.69 to correct for
its fatty acid content.
In addition to CETP modification of HDL composition, the levels of TG
and CE were altered independently. To enhance CE content, fresh human
plasma was incubated for 18 h at 37 °C, allowing LCAT to act on
the lipoproteins and thus increasing their CE concentration. Control
plasma was incubated similarly but received paraoxon to block the
action of LCAT. HDL was then isolated by ultracentrifugation and its
lipid constituents assayed as described below. To reduce the TG content
of HDL, doubly labeled HDL (3H-COE/125I-HDL, 1 mg protein), prepared as described below, was treated with 30 units of
lipoprotein lipase (Pseudomonas, from ICN) for 2 h at
37 °C. Lipolytic activity was stopped by the addition of paraoxon (1 mM); control HDL received paraoxon but no lipase. The
extent of triglyceride hydrolysis was determined from the degradation
of identically treated 3H-TG-labeled HDL. Treatment with
this bacterial lipase did not affect the phospholipid content of the
HDL.
The particle size of native and modified HDL was determined using
nondenaturing polyacrylamide gel electrophoresis on 4-30% gradient
gels (Isolab, Inc., Akron, OH) as previously described (25). Gels were
stained with colloidal Coomassie Blue G-250 (Gradipore LTD, Sydney,
NSW, Australia). Images were captured using a ScanMaker III
scanner (Microtek Lab Inc., Redondo Beach, CA), and bands were
quantitated by analysis with NIH Image software (version 1.6).
Thyroglobulin was added as an internal standard to each sample. The
lipoprotein particle size was determined as previously reported (25)
with high molecular weight standards (Amersham Pharmacia Biotech). HDLs
were assigned to subfractions based on the criteria reported by Nichols
et al. (25).
Preparation of Liposomes and Synthesis of Radiolabeled HDL
Phospholipid/cholesterol liposomes containing either 50 µCi of
3H-COE or 3H-TG were prepared by a modification
(26) of the cholate dialysis method of Brunner et al. (27).
To radiolabel native or modified HDL, lipoproteins were incubated with
either liposome preparation (4 cpm/ng of protein) in the presence of
excess CETP and 0.5% BSA at 37 °C for 18 h. HDL was
re-isolated within its original density limits and dialyzed as given
above. Final specific activities ranged from 18.9 to 68.7 cpm/ng of CE
for 3H-COE and 17.1 to 57.5 cpm/ng of TG for
3H-TG.
In some instances, 3H-COE or 3H-TG-labeled HDL
was also labeled with the nondegradable conjugate
125I-tyramine-cellobiose (28). The tyramine cellobiose was
first iodinated using the Iodogen reagent (Pierce). The labeled adduct was then conjugated to HDL protein with cyanuric chloride. The specific
activities of the HDL ranged from 4.7 to 24.3 cpm 25I/ng
protein. Alternatively, the proteins of 3H-COE- or
3H-TG-labeled HDL were labeled directly with
125I using the iodine monochloride method of Bilheimer
et al. (29). In this case, specific activities ranged from
147 to 270 cpm 125I/ng of protein.
Selective Uptake of CE and TG by Cultured Cells
Y1 and HepG2 cells were obtained from American Type Culture
Collection (Manassas, VA) and maintained in DME/F12 medium
containing 10% fetal bovine serum and penicillin/streptomycin
(100 units/ml, 100 µg/ml, respectively) at 37 °C in a 5%
CO2 environment. For experiments, cells were seeded in
12-well plates and grown to 80% confluence. Y1 adrenal cells were
pretreated for 18 h with 0.1 µM ACTH in DME/F12
media containing 5% human lipoprotein-deficient plasma to up-regulate
steroidogenesis (30). Two different approaches were taken to assess the
selective uptake of cholesteryl esters and triglycerides from HDL. In
general, these approaches follow the methods described by Pittman
et al. (18) and Rodrigueza et al. (31).
Procedure 1--
Cells were incubated with double-labeled HDL
(3H-COE/125 I-tyramine-cellobiose, 15-60
µg/ml) in DME/F12 medium supplemented with 5 mg/ml BSA and 0.1 µM ACTH. After 5 h at 37 °C, the medium was removed and the cells washed with warm PBS. Following a 30-min chase
period in which cells were incubated in DME/F12 media containing 100 µg/ml unlabeled HDL, cells were washed with PBS and then released from the plate with trypsin. The cells were pelleted, washed twice with
PBS, and finally resuspended in PBS. An aliquot of sonicated cell
lysates was counted to determine 125I content as a measure
of whole HDL particle uptake by cells. The lipids from a second aliquot
were extracted (32), and the hexane phase was dried under
N2. The 3H content was quantitated by
scintillation spectroscopy. No 125I was detectable in the
lipid extracts. Selective uptake was calculated as the difference
between total lipid uptake (3H radioactivity) and the
amount of lipid incorporation that could be accounted for by whole
lipoprotein uptake as determined by the accumulated cellular
125I.
Procedure 2--
Although conceptually similar to Procedure 1, slightly different methods were used to assess the cellular processing
of lipoproteins containing a biodegradable protein marker. Confluent
cultures of Y1 or HepG2 cells were incubated with radiolabeled
lipoproteins as described in Procedure 1 above. To determine the
selective uptake of CE and TG, parallel experiments were performed with 3H-COE-, 3H-TG-, and 125I-labeled
HDLs of identical chemical composition. In this instance, following
incubation and rigorous washing of cells as described above, aliquots
of cell lysate were counted directly to determine lipid
(3H) or protein (125I) uptake. Post-incubation
medium was also collected from 125I-HDL-treated cells, and
an aliquot was precipitated with trichloroacetic acid (33) to determine
the amount of 125I-protein degradation products in the
medium. Total uptake of HDL protein was calculated as the sum of the
125I in the cells plus the noniodide-,
nontrichloroacetic acid-precipitable 125I in the medium.
Selective uptake was calculated as the difference between total lipid
uptake, as determined from cellular 3H radioactivity
content, and the amount of lipid incorporation because of whole
particle uptake, as determined from cell-associated 125I
plus 125I degradation products in the media. To minimize
the contribution of low levels of radiolabeled degradation products
present initially in lipoprotein preparations on calculated uptake
values, cellular lipid and protein uptake were determined at both 0.5 and 5 h. Reported uptake values are equal to the difference
between these two values, which reflects cellular uptake over a
4.5-hour time span. Cell protein levels were quantitated (34)
and all uptake values normalized for cellular protein mass.
SR-BI Expression in COS7 Cells
Recombinant adenovirus expressing mouse SR-BI, or a recombinant
virus with analogous adenoviral sequences but containing no transgene
(Adnull), was prepared as previously described (35). These reagents and
a protocol for their use were generously provided by Dr. Deneys van der
Westhuyzen (University of Kentucky Medical Center). COS7 cells were
grown in 12-well plates containing DME medium with 10% fetal bovine
serum and penicillin/streptomycin (100 units/ml, 100 µg/ml,
respectively) until ~80% confluent (~0.8 × 106
cells/well). Adenovirus, stored at Analytical Methods
Protein was quantitated by the method of Lowry et al.
(36) as modified by Peterson (34) with BSA as standard. Lipoprotein cholesterol was assayed by a colorimetric, enzymatic method using a
Cholesterol 100 reagent kit (Sigma). Free cholesterol was determined by
a free cholesterol kit from Wako Diagnostics (Richmond, VA). Triglyceride was assayed using the GPO-Trinder reagent (Sigma) with
glycerol as standard. Lipid phosphorus was determined by the method of
Bartlett (37); phospholipid mass was calculated assuming an average
molecular weight of 800.
HDL Modification--
Plasma lipoproteins undergo continual
remodeling of their composition during their circulatory lifetime. It
is widely recognized that CETP remodels HDL in vivo through
a heteroexchange reaction in which TG from triglyceride-rich
lipoproteins is exchanged for HDL CE. The effect of this modification
on the ability of HDL to donate CE to the SR-BI receptor was studied
herein. When incubated with CETP and VLDL as a source of TG, HDL was
enriched in TG and depleted of CE in a time-dependent
manner (Table I). HDL, initially containing predominately CE in its core, became a TG-rich lipoprotein with the mole ratio of TG to CE exceeding 1 at longer incubation times.
This remodeling of HDL composition was virtually limited to
modification of CE and TG content. Free cholesterol (FC) and phospholipid (PL) increased only modestly, whereas the FC/PL ratio of
modified HDL remained nearly constant (Table I). The equimolar heteroexchange of CE for the larger TG molecule (38) also modified HDL
size. As shown by nondenaturing gradient gel electrophoresis, native
HDL was composed of approximately equal populations of HDL3, HDL2a, and HDL2b (34.1, 37.4, and 28.5%, respectively) (Fig. 1). With TG enrichment this distribution
shifted. A 2-fold decrease in the content of smaller HDL3
particles and a marked rise in the largest HDL subfraction,
HDL2b, were observed. The mean HDL particle size increased
from ~8.9 to ~9.7 nm after extensive CETP modification. Thus, CETP
activity resulted in larger HDL particles that were CE-depleted and
TG-enriched.
CE Selective Uptake from Modified HDL--
The
dose-dependent uptake of CE was measured with COE, a
nondegradable analogue of CE, from native and modified HDL (15-60 µg
of HDL protein) into Y1 adrenal cells (Fig.
2A) and HepG2 hepatocytes (Fig. 2C). Compared with that for native HDL (CE/TG = 9.1), total CE uptake from CE-depleted particles was markedly reduced
and directly related to the residual CE content of the modified HDL (CE/TG = 4.2 and 3.3) in both cell types. Over this range of CE/TG compositions, although the dose dependence of CE uptake was similar regardless of the HDL substrate, CE uptake declined by 73-76% in both
Y1 and HepG2 cells. The portion of total CE uptake attributed to whole
HDL uptake (Fig. 2, A and C, open symbols)
was small in all cases and accounted for little of the total CE
incorporated by cells. Thus, the extent of CE selective uptake (Fig. 2,
B and D), the difference between total uptake and
whole-HDL uptake by Y1 and HepG2 cells, known to be mediated by the
SR-BI receptor, was related directly to the CE content of the HDL
substrate.
To further investigate the relationship between the CE content of HDL
and its uptake, HDL was modified by an alternate strategy whereby HDL
CE was increased through the action of the LCAT enzyme. Compared with
HDL isolated in the absence of LCAT modification, LCAT-modified
HDL was CE-enriched (CE/protein = 0.66 versus 0.47) but
had similar TG content (TG/protein = 0.10 versus 0.09).
CE-enriched HDL was more effective donors of CE via the selective
uptake mechanism than control HDL (Fig.
3). In both Y1 adrenal cells (panel
A) and HepG2 hepatocytes (panel B), LCAT-modified HDL
was a 2-fold better donor of CE than control.
The foregoing data indicated that the capacity of HDL to donate CE to
the selective uptake pathway was dependent on the CE content of HDL.
However, because both CETP and LCAT modification altered the level of
CE in HDL, it remained undetermined whether the relationship between CE
selective uptake and HDL composition reflected a dependence on the
absolute concentration of CE in HDL or on its relative abundance
compared with TG. To investigate these possibilities, HDL was treated
with a TG-specific bacterial lipase to selectively reduce its TG
content. Despite a constant CE content, TG-deficient HDL was
significantly more effective at donating CE to both Y1 and HepG2 cells
than control HDL (Fig. 3, panels C and D,
respectively). Hydrolysis of ~50% of the TG in HDL led to a 34 (Y1)
and 73% (HepG2) increase in CE selective uptake.
TG as a Selective Uptake Substrate--
The above detailed studies
demonstrate the importance of the relative CE and TG content of HDL as
a determinant of the capacity of various HDLs to donate CE to the
selective uptake process. The importance of TG content, as specifically
demonstrated with lipase-modified HDL, suggests one of two possible
roles for HDL TG. First, HDL TG may serve as an inert diluent for CE,
thus decreasing CE selective uptake simply by controlling the apparent
concentration of CE. Alternatively, HDL TG may also be a substrate for
selective uptake such that CE and TG compete for uptake via a common pathway.
To delineate these possibilities, the substrate potential of TG was
investigated in cultured Y1 cells. Like CE, TG in native HDL was taken
up in a dose-dependent fashion (Fig.
4A). The portion of TG uptake
attributable to the uptake of the whole HDL particle was small,
representing <10-15% of total TG uptake, indicating that most TG
uptake occurred by a selective process. Thin layer chromatographic
analysis of cell lysates showed that cell-associated TG was degraded
extensively (>64%) to fatty acids and intermediate acylglycerols by
both Y1 and HepG2 cells. The possibility that extracellular degradation
products were the source of apparent TG uptake was examined using a
nondegradable ether analogue of TG (TGE). Although performed with
different HDL preparations, when compared with the uptake of CE from
the same HDL, the kinetics of TG ether uptake (Fig. 4B) was
similar to that of the ester (Fig. 4A). This finding
suggests that TG was incorporated selectively into cells prior to
degradation. In addition, within this time frame it does not appear
that significant degradation products were lost from cells because the
apparent uptake of TG was not increased when the extracellular fatty
acid acceptor, BSA, was omitted from the culture medium (78.8%
of control in Y1 cells and 95.9% of control in HepG2 cells). CE
selective uptake was similarly affected by the deletion of BSA. Thus,
SR-BI-expressing cells selectively incorporated both CE and TG from
HDL.
The dependence of TG selective uptake on HDL concentration was similar
to that for CE. The presence of a common pathway for the selective
uptake of these two lipids was further supported by the finding that
the addition of a 10-fold excess of unlabeled HDL to the media
suppressed the selective uptake of CE and TG to similar extents (52.7 and 52.8%, respectively, for Y1 and 54.8 and 58.6%, respectively, for
HepG2). That this pathway was mediated by the SR-BI receptor was
supported by several observations. First, up-regulation of SR-BI on Y1
adrenal cells by pretreatment with the steroidogenic hormone ACTH
equally stimulated the selective uptake of both CE and TG, as
well as TGE, from HDL (Table II). Additionally, the overexpression of murine SR-BI in COS7 cells, as
confirmed by Western analysis (Fig. 5,
inset), enhanced the selective uptake of both CE and TG to
similar extents compared with control cells (Fig. 5). Collectively
these data show that TG is a selective uptake substrate like CE and
that the uptake of these lipids proceeds through SR-BI.
Competition of CE and TG for Selective Uptake--
Given the
observed effects of variable HDL TG/CE composition on CE selective
uptake and our finding that TG is also a substrate for this process, we
investigated the relationship between TG and CE selective uptake as a
function of varied HDL core lipid composition. CETP-modified HDL
particles were altered in their capacity to support CE and TG selective
uptake. Continuous TG enrichment and CE depletion by CETP resulted in
particles that were progressively more efficient donors of TG and less
effective donors of CE. Over a 4-fold increase in HDL TG/CE content, TG selective uptake increased nearly 2-fold (Fig.
6A). Concurrently, as the CE
content of HDL decreased, CE selective uptake decreased by almost 50%.
Overall, the selective uptake of TG or CE was proportional to the mole
fraction of that lipid in the HDL core (i.e. TG/CE + TG or
CE/CE + TG, respectively). This resulted in a near linear relationship
between the TG/CE content of the donor HDL particle and the ratio of
these lipids selectively taken up by cells (Fig. 6B). The
slope of this line (panel B) was near a value of 1, suggesting that CE and TG were equivalent substrates for selective
uptake. Notably, the total amount of lipid selectively imported from
HDL (CE + TG) remained rather constant. Together, these data illustrate that TG and CE compete for selective uptake, with the relative rate of
uptake for each lipid being defined by the relative abundance of TG and
CE in the HDL core.
Few studies have investigated the properties of HDL that influence
its capacity to donate CE to cells through the selective uptake
pathway. Early work with synthetic HDL suggests that the rate of
selective uptake is a function of particle size and density, with
small, dense HDL particles being more effective donors of CE (19).
Additionally, selective uptake has been reported to be more extensive
with particles containing apoA-I (19, 39). Consistent with this
finding, selective uptake of CE from endogenous lipoproteins is
nearly absent in knockout mice lacking apoA-I but not in apoA-II or
apoE knockout animals (40). An interpretation of this finding is
complex, however, because apoA-I-deficient mice fail to synthesize HDL.
In contrast, binding studies show that multiple apoproteins can mediate
particle interaction with SR-BI (41). These different results may be
explained in part by the observation that particle electronegativity is
a key determinant of SR-BI binding (42, 43). Nevertheless, it is
important to note that although particle binding is essential, fruitful
selective uptake also requires subsequent, distinct interactions
between SR-BI and the bound ligand to facilitate lipid incorporation
(44). Features of HDL that influence the availability of CE for
selective uptake during this second interaction are addressed in this study.
Physiologically, CETP is an important determinant of HDL core lipid
composition. Its remodeling of HDL composition alters the balance of
HDL subclasses and directly influences the functionality of the HDL
fraction (4, 45, 46). In this study we have used CETP as a tool to
investigate the importance of core lipid composition on the selective
uptake of CE from HDL. Our data demonstrate that the neutral lipid
composition of the HDL core has a direct effect on the ability of HDL
to donate CE to the selective uptake pathway. As HDL is made
progressively CE-deficient and TG-enriched, CE selective uptake
diminishes. Although these modified HDL are increased in size,
this appears to contribute little to the decline in CE selective
uptake because enlarged, CE-enriched HDL, produced by LCAT
modification, are also more effective donors of CE. CE selective uptake
was also found to increase when HDL TG is hydrolyzed by lipase even
though CE content is not affected. Taken together, these data
demonstrate that HDL TG levels influence CE selective uptake.
Our observation that HDL TG and its nonhydrolyzable analogue, TGE, are
selectively taken up by Y1 and HepG2 cells supports previous studies
with Y1-BS1 cells in which TG was selectively taken up from
reconstituted HDL (22). Like CE, TG selective uptake is mediated by
SR-BI, as demonstrated by the costimulation of CE and TG selective
uptake in ACTH-treated cells and by overexpression of the murine SR-BI
receptor in COS7 cells. Although selectively incorporated TG is rapidly
degraded by cells, the similarity between TG and TGE uptake kinetics
suggests that TG degradation products are quantitatively retained,
perhaps through their efficient incorporation into cellular lipids as
reported by Hilaire et al. (47).
Rodrigueza et al. (31) reported that CE selective uptake is
directly dependent on the concentration of CE within the HDL core,
suggesting a mechanism whereby CE moves down a concentration gradient
from HDL particles docked on SR-BI into the cell plasma membrane.
Although this mechanism can explain some of our findings with
CETP-modified HDLs, it does not appear to explain the higher transfer
rates mediated by HDL that have been modified by bacterial lipase
(reported here), hepatic lipase (48-50), or a combination of CETP and
hepatic lipase (51), since CE in these particles is either unchanged or
reduced. Our observation that TG is also a selective uptake substrate
suggests that CE selective uptake may be modulated by the uptake of TG.
This possibility was investigated directly with a series of HDL
particles of varying TG/CE composition. Our data demonstrated that the
rates of CE and TG selective uptake were directly related to the
content of these lipids in the donor HDL. Even though the selective
uptake of TG or CE differed by several-fold over the range of modified
HDL studied, the sum of these two lipids transferred to cells remained
essentially constant. This strongly suggests that TG and CE compete for
transfer by SR-BI. In general, the extent of TG and CE selective uptake
was the same when the mole ratio of these two lipids in HDL was near 1, indicating little preference for CE or TG in selective uptake. Also,
because total lipid uptake (TG + CE) remained constant, these data
further suggest that modest changes in HDL size, which resulted from TG
enrichment, did not influence selective uptake rates.
In conclusion, the data reported here demonstrate that TG, like CE, is
a substrate for selective uptake by the SR-BI receptor. The selection
of CE or TG for transfer is related directly to the mole ratio of these
lipids. This relationship was observed with HDL of widely varying TG/CE
content, including particles more CE-rich and particles more TG-rich
than native HDL. Based on the results obtained here, we propose a
modification of the nonaqueous channel model described for SR-BI (31).
Our data suggest a mechanism in which HDL binds to SR-BI, creating a
channel through which a nonspecific portion of core lipid, defined by its TG/CE ratio, diffuses to the cell plasma membrane and is
incorporated into the cell (Fig. 7).
Although this channel does not appear to discriminate between molecules
of similar size, such as CE and TG, it apparently cannot accommodate
very large hydrophobic molecules (18). HDL processed by such a
mechanism is depleted of both CE and TG, with the resulting HDL product
retaining the same TG/CE ratio of the parent molecule (Fig. 7). Such a
mechanism provides a plausible explanation for the higher CE selective
uptake from HDL depleted of TG by hepatic lipase (48-51). In this way, the ability of HDL to donate cholesterol for steroidogenesis or to
promote reverse cholesterol transport may be augmented when HDL contain
a high CE/TG ratio, such as that promoted by the combined actions of
CETP and hepatic lipase (51) and enhanced by lipid transfer inhibitor
protein (24, 52). Conversely, these beneficial functions of HDL may be
impaired when these particles contain elevated TG, such as occurs in
hypertriglyceridemia and noninsulin-dependent diabetes
mellitus. In this regard our data suggest a novel mechanism by
which elevated TG may promote atherosclerosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
80 °C, was thawed on ice, diluted in DME medium to 8 × 106 plaque-forming
units/ml, and filtered (0.45 µm), and 1 ml was added per well.
After 24 h, the treatment medium was removed and cells were
incubated for 0.5 or 5 h with radiolabeled HDL as described above.
TG and CE uptake was determined as detailed above, and SR-BI protein
levels were determined by Western blot using rabbit anti-mouse SR-BI
antiserum (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Modification of HDL composition by CETP
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Fig. 1.
Separation of HDL subclasses by nondenaturing
polyacrylamide gradient gel electrophoresis. HDL subpopulations
were separated on 4-30% gradient gels as described under
"Experimental Procedures." Numbers represent the
relative percentage of each subpopulation contained within the HDL
fraction. Subpopulations are noted by vertical dashed lines.
A, native HDL. B, HDL modified with VLDL and CETP
for 24 h.
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Fig. 2.
Dose-dependent uptake of
cholesterol ester from native HDL or CE-depleted HDL in Y1 and HepG2
cells. HDL were depleted of CE by incubation with CETP and VLDL as
described under "Experimental Procedures." Native and modified HDLs
were doubly labeled with 3H-COE and
125I-tyramine-cellobiose. A and
C, total CE (3H-COE) uptake from native HDL
(CE/Prot = 0.32) (closed squares) and from two
different CETP-treated HDLs (CE/Prot = 0.29 (closed
circles) or CE/Prot = 0.19 (closed triangles)). CE
uptake attributable to whole HDL uptake is shown by the respective
open symbols. B and D, selective CE
uptake calculated as the difference between total CE uptake and whole
HDL uptake. Results are the mean ± S.D. and are representative of
four experiments each for Y1 and HepG2 cells.
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Fig. 3.
CE uptake from enzyme-modified HDL. HDL
was treated with either LCAT or bacterial lipase as described under
"Experimental Procedures." Doubly labeled (3H-COE,
125I-tyramine-cellobiose) native and modified HDL were
incubated with Y1 (A and C) or HepG2
(B and D) cells, and the selective uptake of CE
from HDL (60 µg) was assessed as described under "Experimental
Procedures." Results are the mean ± S.D. and are representative
of three experiments for Y1 and two experiments for HepG2.
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Fig. 4.
Dose-dependent uptake of HDL-TG
in Y1 cells. HDL was labeled with either 3H-TG,
3H-COE, or 125I, and uptake was measured as
described under "Experimental Procedures." A, total TG
uptake (open circles), TG from whole HDL uptake (open
triangles), TG selective uptake (closed circles), and
CE selective uptake (closed squares). Results are
representative of three separate experiments. B, HDL,
distinct from that studied in panel A, was labeled with
either 3H-TGE or 3H-COE, and uptake was
measured as described under "Experimental Procedures."
Response of CE and TG uptake by Y1 cells to ACTH
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Fig. 5.
Effect of murine SR-BI overexpression on
selective uptake. COS7 cells were transiently transfected with
empty adenovirus (Adnull) or adenovirus containing murine
SR-BI (AdmSRBI), and lipid uptake from 3H-TG- or
3H-COE-labeled HDL (60 µg) was measured as described
under "Experimental Procedures." Data (mean ± S.D.) show the
increase in lipid uptake over control COS7 cells not transfected.
Inset, Western analysis of SR-BI levels in control and
transfected COS7 cells detected with anti-mouse SR-BI. Ctl,
control; Ad, Adnull; AdSR, AdmSRBI.
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Fig. 6.
Competition of CE and TG for selective
uptake. Lipid uptake from CETP-modified HDL was measured as
described under "Experimental Procedures" from 3H-TG-
and 3H-COE-labeled HDL. A, CE
(3H-COE, closed squares) and TG (closed
circles) uptake as a function of the mole lipid ratio of these
lipids in the donor HDL. B, ratio of TG/CE uptake,
calculated from the data given in panel A, as a function of
particle composition. Inset, total neutral lipid uptake (TG + CE). Results are the mean ± S.D. and are representative of five
similar experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Model of competitive CE and TG selective
uptake by SR-BI. This model, based on a mechanism proposed by
Rodrigueza et al. (31), proposes that HDL binds to SR-BI
creating a channel through which a portion of the core lipid, defined
by the CE and TG content of the donor particle, diffuses into the cell
plasma membrane and is incorporated into the cell. Because of the
competitive nature, the lipid-depleted HDL that is released contains
the same relative core composition as the original donor HDL.
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FOOTNOTES |
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* This research was supported in part by Grant HL60934 from NHLBI, National Institutes of Health and by Grant-in-aid 0050075N from the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Biology Dept., Mount Union College, Alliance, OH 44607.
§ To whom correspondence should be addressed: Cell Biology, NC10, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-5850; Fax: 216-444-9404; E-mail: mortonr@ccf.org.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M008725200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; CETP, cholesteryl ester transfer protein; LCAT, lecithin:cholesterol acyltransferase; VLDL, very low density lipoprotein; CE, cholesteryl ester; COE, cholesteryl oleyl ether; TG, triglyceride; TGE, trialkylglycerol ether; BSA, bovine serum albumin; DME medium, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; ACTH, adrenocorticotropic hormone; SR-BI, scavenger receptor class B type I.
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