From the
Modification of dietary fatty acid composition results in
changes in plasma cholesterol levels in man. We examined the effect of
in vitro fatty acid supplementation on low density lipoprotein
(LDL) receptor activity in cultured cells and questioned whether
changes were related to fatty acid-induced alterations in
acyl-CoA:cholesterol acyltransferase (ACAT) activity. Preincubation of
cultured cells ( i.e. human skin fibroblasts, J774 macrophages,
and HepG2 cells) with oleic acid (oleic acid:bovine serum albumin molar
ratio 2:1) at 37 °C for longer than 2 h resulted in a 1.2- to
1.5-fold increase in LDL cell binding at 4 °C and LDL cell
degradation at 37 °C. Scatchard analysis showed that oleic acid
increased LDL receptor number but not LDL affinity
( K
Dietary fatty acid composition has an important influence on
serum cholesterol levels in man
(1, 2) , but the
detailed cellular mechanisms for this effect remain largely
unexplained. Reduction in plasma cholesterol levels associated with
replacement of dietary saturated with unsaturated fatty acids are about
10 to 15% of initial levels and have been linked to increased hepatic
low density lipoprotein (LDL)
In vitro experiments examining effects of fatty acid supplementation on
LDL-cell interactions have yielded conflicting results. Previous work
from this laboratory has shown that free fatty acids (FFA) at high
FFA:bovine serum albumin (BSA) molar ratios (4-6:1) inhibit
binding of LDL to the LDL receptor of human fibroblasts at 4 °C
(7) . A similar effect has been reported in HepG2 cells
(8) and MA-10 Leydig tumor cells at 37 °C
(9) .
However, at more physiologic FFA:BSA molar ratios (1 or 2:1), other
studies on fibroblasts
(10) , mononuclear cells
(11, 12) , and HepG2 cells
(13) have
demonstrated increased LDL degradation following FFA supplementation.
Exogenous cholesterol decreases both LDL receptor expression and
3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) activity
and up-regulates acyl CoA:cholesterol acyltransferase (ACAT) activity
(14) . Of interest, experiments in J774 macrophages showed that
inhibition of ACAT activity with an ACAT inhibitor, 58-035,
down-regulates LDL receptor activity, suggesting that there may also be
a direct regulatory relationship between ACAT and the LDL receptor
(15) . In addition to cholesterol, ACAT activity can also be
regulated by free fatty acids; for example, in vivo (16, 17, 18, 19) and in vitro (9, 20) experiments in different cell types have
demonstrated that unsaturated fatty acids increase liver ACAT activity.
We therefore questioned whether in vitro supplementation of
cultured cells with free fatty acids would increase cellular LDL
receptor activity and, if so, would these changes be correlated with
changes in cellular ACAT activity. Fibroblasts and J774 macrophages
were chosen for most studies because in fibroblasts the LDL receptor
pathway has been very well defined
(14) , whereas J774
macrophages have been widely used to study ACAT activity
(15, 20, 21) and, unlike other macrophage cell
lines, show a high expression of the LDL receptor
(21) . Our
results demonstrate that cellular FFA supplementation in vitro can increase LDL receptor activity, and that this effect is
mediated through modulation of cell ACAT activity and intracellular
cholesterol redistribution.
Sodium
Fibroblasts from frozen stocks (6-12 passages) were plated at
a cell density of 24
Cells were incubated with DMEM containing FFA at 37 °C either in
the absence (preincubation) or presence (co-incubation) of LDL at
various FFA:BSA molar ratios ranging from 0.5:1 to 6:1. When cells were
preincubated with FFA or triglyceride emulsions, cells were washed
twice with phosphate-buffered saline (PBS), 0.2% BSA (w/v) prior to the
addition of radiolabeled LDL to remove non-cell-associated lipid.
Cellular binding, uptake, and degradation of
Cholesterol mass was measured
by injection of 1-µl aliquots into a Hewlett Packard Model 5790A
gas chromatograph with a Flame Ionization Detector equipped with a
Hewlett Packard Model 7673A automatic sampler (Hewlett Packard Co.)
using a 10 m
The effect of oleic
acid on
To determine if the FFA-mediated increase in cell LDL binding
was due to an increase in the cellular recycling of the LDL receptor,
we compared the effect of oleic acid on normal fibroblasts and JD
receptor-defective fibroblasts (Fig. 5). JD cells express LDL
receptors on the cell surface, but fail to internalize receptor-bound
LDL
(14) . Normal and JD fibroblasts were preincubated with
oleic acid for 4 h, the FFA was removed as described above, and
We next questioned whether the effects of different FFA on
the LDL receptor parallel changes in cell cholesterol distribution
between free cholesterol (FC) and cholesteryl ester (CE). J774
macrophages were incubated in parallel experiments with different FFA
(FFA:BSA molar ratio 2:1) in the presence of 100 µg/ml radiolabeled
or non-radiolabeled LDL to measure both LDL degradation and cellular
cholesterol distribution expressed as CE/FC mass ratio (Fig. 6).
In the presence of 100 µg/ml LDL, free cholesterol mass remained
essentially constant while cell total cholesterol and the CE/FC ratio
increased. Levels of FC were similar for all cells regardless of FFA
supplementation, and there was no correlation between the amount of LDL
degraded and cell FC (data not shown). In contrast, a high correlation
was seen between the ability of the FFA to increase LDL catabolism and
either the amount of cholesteryl ester accumulation ( r
We first examined the effect of the ACAT
inhibitor, 58-035
(3-[decyldimethylsilyl]- N-[2-(4-methylphenyl)-1-phenylethyl]-propamamide,
kindly provided by Sandoz) on FFA-mediated increases in LDL cell
metabolism. J774 macrophages were preincubated in the presence or
absence of increasing concentrations of the ACAT inhibitor 58-035, with
oleic acid, eicosapentanoic acid, and arachidonic acid, which had
previously produced marked changes in the cellular CE/FC ratio.
Addition of the ACAT inhibitor resulted in a dose-dependent decrease of
fatty acid-stimulated LDL degradation in control cells and complete
inhibition of the stimulatory effect of all three FFA at a dose of 1
µg/ml (Fig. 7). Similarly, increases in 4 °C LDL cell
surface binding after preincubation of J774 macrophages with oleic acid
were completely abolished by 1 µg/ml 58-035 (data not shown).
Therefore, blocking the activity of ACAT with 58-035 eliminated the
ability of FFA to increase cell LDL binding and degradation.
Since changes in LDL
receptor activity due to oleic acid do not appear in J774 macrophages
until after 2 to 3 h of preincubation (see Fig. 3), we examined
whether FFA stimulation of ACAT activity would precede the increase in
LDL receptor activity, in the absence of LDL. J774 macrophages were
incubated with [
In vivo, fatty acids
are delivered to cells either in the free form, or as triglyceride in
lipoprotein particles. We questioned whether fatty acids, delivered as
triglyceride, have the same stimulating effect on the LDL receptor
pathway that was observed with free fatty acids. Studies in our
laboratory have demonstrated that J774 macrophages take up and
hydrolyze triglyceride and cholesteryl ester from model
triglyceride-rich emulsion particles within 2-4 h of incubation
(25) . Therefore, J774 cells were incubated with varying
concentrations of model IDL-size apoE emulsion particles containing
either 100% triolein or almost equal amounts of triolein and
cholesteryl ester (Fig. 9). A dose-dependent increase in LDL
degradation up to 70% was observed after preincubation with pure
triolein particles. However, the addition of cholesteryl ester to the
emulsion particle blocked the ability of triglyceride to enhance LDL
metabolism. Other experiments have shown, under similar conditions,
that over 50% of emulsion particle cholesteryl ester is hydrolyzed to
free cholesterol
(25) . Therefore, delivery of oleic acid in a
triglyceride molecule using emulsion particles produced a similar if
not greater increase in LDL degradation as did oleic acid alone, but
the availability of additional cholesterol contained in the same
particle prevented the triglyceride induced increase in LDL receptor
activity.
Previous work has shown close association between LDL
receptor and cellular ACAT activities
(14, 15) , and
separate studies reported certain FFA stimulate free cholesterol
esterification via ACAT activity
(9, 16, 17, 18, 19, 20) .
Our data provide original in vitro evidence demonstrating a
close and concurrent inter-relationship between all three, i.e. ACAT activity, FFA, and the LDL receptor. We show that
supplementation of FFA enhance LDL receptor activity and LDL catabolism
by stimulating cell ACAT activity and consequently altering cell
cholesterol distribution. Increases in LDL receptor activity observed
in vitro (1.2- to 1.5-fold) are likely sufficient to account
for the decreases in plasma and LDL-cholesterol levels seen in man
(about 15% of initial levels) when changing from a diet high in
saturated fat to one rich in unsaturated fat
(1, 2) .
Several experimental observations indicate that the effect of oleic
acid and other FFA on cell LDL metabolism is mediated by changes in LDL
receptor activity. First, cell preincubation with oleic acid increased
LDL receptor number, but not receptor affinity. Second, LDL bound after
oleic acid preincubation was completely metabolized within the same
time frame as LDL bound without oleic acid preincubation. Third, JD
fibroblasts, which express but do not internalize LDL receptors, showed
increased LDL binding after oleic acid preincubation similar to normal
fibroblasts, but did not internalize or degrade this additionally bound
LDL. Fourth, experiments with receptor-negative fibroblasts
demonstrated no increases in LDL binding or degradation after
preincubation with oleic acid. Fifth, fatty acid did not increase the
binding or degradation of acetylated LDL, which is metabolized through
the scavenger receptor pathway.
LDL receptor activity is intimately
linked with cellular cholesterol requirements. Exogenous cholesterol
markedly stimulates the cholesterol esterifying enzyme, ACAT, and
down-regulates the LDL receptor
(14) . Inhibition of cell ACAT
activity, which presumably increases cell-free cholesterol, also
down-regulates the LDL receptor
(15) . In our studies, in the
absence of exogenous cholesterol, three lines of evidence suggest that
activation of ACAT through FFA also results in changes in specific
intracellular cholesterol pools which modulate LDL receptor activity.
Firstly, incubating macrophages with FFA in the absence of an exogenous
source of cholesterol did not produce changes in total cell
cholesterol, but increased cholesteryl ester formation presumably by
redistributing intracellular free cholesterol between the pool
regulating the LDL receptor and the pool accessible for ACAT activity.
Secondly, increases in ACAT activity with FFA preceded by 1-2 h
the appearance of increases in LDL receptor activity. Thirdly, the
effect of three different FFA (oleic acid, EPA, and arachidonic acid),
which markedly increased LDL binding and catabolism, was completely
blocked by inhibition of ACAT activity. A similar mechanism has been
proposed for the effects of sphingomyelinase on cultured cells, which
after a rapid (within 10 min) redistribution of intracellular free
cholesterol, increased ACAT activity, decreased HMG-CoA reductase
activity
(33) , and increased LDL receptor activity
(34, 35) .
Although liver cells metabolize most of
LDL in vivo, we examined the effects of FFA primarily in
fibroblasts and J774 macrophages, since experiments in liver cells can
add a number of potential confounders; e.g. liver cells
synthesize and secrete apoE and apoB lipoproteins, as well as hepatic
lipase and bile acids. Nevertheless, in HepG2 cells, after oleic acid
preincubation, we observed marked increases in LDL binding, which were
then abolished by addition of 58-035. Our results suggest that effects
of oleic acid on LDL receptor activity may be common to a number of
cell types, including those which modulate LDL levels in vivo.
In the presence of different sources of exogenous cholesterol, the
fatty acids showed two different responses. When cells were
co-incubated with fatty acids and LDL, the increase in LDL receptor
binding persisted even after 24 h of incubation, suggesting that,
despite the increase in total cholesterol mass (2-3-fold,
typically), no down-regulation of the LDL receptor activity was seen.
In contrast, the delivery of excess cholesterol by cholesteryl
ester-containing emulsions abolished the effect of triolein-released
FFA on increasing LDL receptor activity. It is unclear why cholesteryl
ester delivered within LDL particles fails to similarly override the
stimulatory effect of FFA supplementation. A possible explanation is
that LDL- and triglyceride-rich particles, which are routed to
different compartments within the cell after internalization
(36) , have a different rate of CE hydrolysis, which could
determine a different final targeting of the free cholesterol generated
by CE breakdown. In addition, concentrations of cholesteryl ester in
experiments with LDL were much lower than those provided by the lipid
emulsions. Thus, the effect of FFA on LDL receptor activity may also be
affected by the source and intracellular distribution of cholesteryl
ester.
Previous in vitro studies have demonstrated that FFA
increase cellular ACAT activity
(9, 20) and that the
ability of fatty acid-CoA to stimulate liver microsomal ACAT activity
varied, with a rank order of oleyl > palmityl > stearyl >
linoleyl-CoA
(37) . Other in vitro studies suggest that
one unsaturated fatty acid, linoleic acid, may inhibit ACAT activity
(38, 39) . In vivo studies in animal models
have also shown that, compared with saturated fat diets, diets high in
unsaturated fatty acids increase liver ACAT activity
(16, 17, 18, 19) , probably by mediating
changes in the microsomal fatty acid content and the physical
properties of the ACAT membrane environment
(17, 18, 38) . A recent in vivo study
on hamsters showed that oleic acid, in contrast to myristic acid,
increased both liver cholesteryl ester levels and LDL receptor activity
(6) , probably causing a redistribution of cholesterol from
intracellular regulatory compartments to cholesteryl ester storage
pools. Our results describe a clear in vitro link between FFA,
LDL receptor, and ACAT activity and, thus, provide direct evidence for
this hypothesis.
Besides an increase in LDL receptor activity,
redistribution of intracellular cholesterol toward cholesteryl ester
might also increase cellular HMG-CoA reductase activity. In preliminary
experiments, we found that oleic acid increased HMG-CoA reductase
activity from 20-60% in both fibroblasts and J774
macrophages.
In vivo dietary
studies have shown that the saturated fatty acids, myristate and
palmitate, raise serum cholesterol, whereas linoleic and oleic acids
lower serum cholesterol levels
(40, 41, 42, 43, 44, 45) .
In our in vitro studies, although saturated fatty acids
generally caused little change in receptor activity, data were
variable. Co-incubations of macrophages with palmitic acid stimulated
receptor activity and cholesterol ester accumulation. Interestingly,
palmitic acid has been described as a good substrate for the ACAT
reaction
(37) . Linoleic acid in our system caused little
effect. Of interest, diets containing exclusively linoleic acid do not
increase LDL receptor activity in animals.
In our
studies, the effect of the longer chain FFA, EPA, and arachidonic acid,
which markedly increased LDL metabolism, was somewhat surprising.
Although EPA has been shown to be poorly esterified in HepG2 cells
(30) , we measured similar incorporation of radiolabeled
supplemental EPA, oleic acid, and arachidonic acid into cellular
cholesteryl ester in both fibroblasts and macrophages, and this
cholesteryl ester formation was blocked by the ACAT inhibitor, 58-035
(data not shown).
The in vitro FFA-induced up-regulation of
LDL receptor number might be mediated by increases in LDL receptor mRNA
or by post-transcriptional mechanisms; however, the pathway(s)
responsible for this effect remain to be elucidated. We performed
preliminary investigations to test if increases in LDL binding mediated
by FFA was due to increased synthesis of LDL receptor mRNA, and no
differences due to FFA could be detected. However, since increased LDL
receptor activity on the cell surface was relatively small,
25-40%, it is unlikely that alterations in mRNA of a similar
magnitude if they exist would have been detected. Other investigators
have demonstrated that rapid up-regulation of LDL receptor mRNA
synthesis (within 1 h) is followed by increases in LDL receptor surface
expression within 2-4 h in response to platelet-derived growth
factor
(46) , tumor necrosis factor
(47) , and
Ca
Unsaturated fatty acids may
also alter LDL receptor recycling, cellular distribution, or affinity
for the LDL particle by increasing membrane fluidity
(11, 54) . We observed similar FFA-mediated increases in
LDL cell binding in normal fibroblasts and fibroblasts unable to
internalize the LDL receptor (JD cells), suggesting that the effect of
fatty acids on LDL cell binding is not due to an increase in LDL
receptor recycling. Although not measured herein, our results do
suggest that overall cell membrane fluidity may not be as important as
other mechanisms cited above, since arachidonic acid and EPA increased
cell LDL receptor activity, while linoleic acid, linolenic acid, and
docosahexanoic acid, which also increase fluidity, had much less
effect. In addition, FFA-induced increase in LDL receptor activity was
blocked by an ACAT inhibitor, which would not affect the intracellular
membrane fatty acid profile. Nevertheless, it is possible that by
redistributing cholesterol among specific intracellular pools,
selective changes in fluidity might occur in certain cell membranes.
Hypotheses have been suggested for two distinct control mechanisms
for cellular LDL receptor activity
(6, 55) . The first
relates to mechanisms changing net sterol balance across cells, while
the second involves a redistribution of cholesterol between regulatory
and cholesteryl ester pools, under circumstances where external sterol
balance is kept constant. In our incubations with FFA in the absence of
LDL, no measurable changes in cell cholesterol mass were observed.
However, after FFA preincubations, when LDL was added to cells at 4
°C ( e.g. Figs. 3, 4, and 5), the increase in LDL receptor
binding was immediately apparent. This, together with our experiments
on the blocking effects of ACAT inhibition and cholesterol
supplementation suggests that, in our in vitro cell system,
the major mechanism controlling increased LDL receptor activity was a
shift in intracellular regulatory cholesterol pools, associated with
the effects of FFA on increasing ACAT activity. Wang et al. (56) have recently suggested that regulation of sterol
regulatory element binding protein (SREPB) processing (with subsequent
changes in LDL receptor activity) will be sensitive to localized
endoplasmic reticulum alterations in sterol content.
In summary, a
number of different mechanisms likely exist to explain the modulation
of LDL receptor activity in vivo by different dietary fatty
acids. Our results provide direct evidence for one such pathway,
i.e. unsaturated fatty acids increase cellular LDL receptor
activity by an initial, direct stimulation of cellular ACAT activity.
The depletion of free cholesterol from an, as yet, unidentified
intracellular regulatory pool (to form cholesteryl ester) would then
signal the cell for a need of increased exogenous cholesterol and a
subsequent increase in LDL receptors. This may be one mechanism whereby
certain unsaturated fatty acids increase LDL receptor activity in
vivo, resulting in decreased plasma total and LDL cholesterol and
reducing the risk of atherosclerosis.
We appreciate the helpful critique and suggestions of
Dr. John M. Dietschy after his review of a draft of this manuscript and
thank Dr. Ira Tabas for his repeated helpful advice during the time
period of these experiments. We also thank Dr. Laura Liscum, Tufts
University, for her analysis of cellular HMG-CoA reductase activity,
Dr. Xian Cheng Jiang for his analysis of LDL receptor mRNA levels, Dr.
Bettina Schwiegelshohn for emulsion preparation, Dr. Steve Sturley for
his comments on the manuscript, and Inge Hansen and Anne Gleeson for
their assistance with cell culture.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
). Fatty acid supplementation of J774
macrophages increased both LDL receptor activity and cholesteryl ester
accumulation. The ACAT inhibitor, 58-035, eliminated both effects, and
increased ACAT activity preceded stimulation of LDL receptor activity
by 1-2 h. Supplementation of macrophages with triolein emulsion
particles also increased LDL cell binding and degradation, and addition
of cholesterol to the emulsions abolished this effect. Among fatty
acids tested, oleate (18:1), arachidonate (20:4), and eicosapentanoate
(20:5) demonstrated the greatest effects. We hypothesize that certain
fatty acids delivered to cells either in free form, or as triglyceride,
first increase cellular ACAT activity, which then causes a decrease in
an intracellular free cholesterol pool, signaling a need for increased
LDL receptor activity. This mechanism may play a role in the effect of
certain dietary fatty acids on LDL metabolism in vivo.
(
)
receptor number
(3, 4, 5, 6) .
I was purchased from Amersham. Fatty acids,
bovine serum albumin (Lot A-2153) with a fatty acid content of 0.11 mol
of fatty acid/mol of albumin, disodium EDTA, trichloroacetic acid, and
potassium iodide were obtained from Sigma. 10,000 mg/ml streptomycin,
200 mML-glutamine, and 100,000 units/ml penicillin
were purchased from Hazelton.
Cells
Normal human skin fibroblasts (HS68) and HepG2
cells were bought from American Tissue Culture Collection. Fibroblasts
from familial hypercholesterolemia subjects lacking (GM 2000) or having
a defect in the internalization of the LDL receptor (GM 2408, JD cells)
were purchased from Coriell (Camden, NJ). J774 (A2) murine macrophages
were kindly provided by Dr. Ira Tabas of Columbia University.
10
cells per well in 12-well
plates (22 mm) in medium containing Dulbecco's modified
Eagle's medium, 1% glutamine (v/v), 1% penicillin/streptomycin
(v/v), and 10% fetal bovine serum (v/v), which was replaced after 3
days. By the 5th day, fresh medium containing 10% (v/v)
lipoprotein-deficient serum ( d < 1.21 g/ml) was added to
monolayers. Cells were used for experiments at day 7. Monolayer
cultures of J774 cells were grown and maintained in the medium
described above and plated at a cell density of 10
10
. Twenty-four hours before experiments, the medium was
changed to 10% (v/v) lipoprotein-deficient serum-containing medium.
HepG2 cells were grown in collagen-coated tissue dishes in complete
medium containing minimum essential medium with 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate, 1%
penicillin/streptomycin (v/v), and 10% fetal bovine serum. Cells were
plated onto collagen-coated plates 1 day prior to experiments at a
density of 1-1.5
10
cells/35-mm well in
medium containing 10% (v/v) lipoprotein-deficient serum.
Low Density Lipoproteins
Blood from normolipidemic
subjects was collected with EDTA, and plasma was separated by low speed
centrifugation (3000 rpm). Disodium EDTA (1.2 g/liter), NaN(0.1 g/liter), and aprotinin (100,000 kallikrein inhibiting
units/liter) were added to the plasma, and LDL was isolated by
sequential ultracentrifugation (1.019 g/ml < d < 1.063
g/ml)
(22) . LDL protein was measured using the method of Lowry
et al. (23) . LDL was radioiodinated as described by
Bilheimer et al. (24) . The specific activity of
I-LDL ranged between 100 and 400 cpm/ng of LDL protein,
and over 99% of the radioactivity was precipitable after a 60-min
incubation with 10% (v/v) trichloroacetic acid at 4 °C. LDL was
dialyzed in 150 mM NaCl, 0.24 mM EDTA, pH 7.4, and
then filtered with a 45-µm filter (Gelman Sciences). All isolated
LDL were kept under argon at 4 °C and used within 7 days.
Lipid Emulsions
50 mg of triolein or 25 mg of
triolein and 25 mg of cholesterol oleate (Nu-Check) was added to 50 mg
of egg yolk phosphatidylcholine (Avanti), and the organic solvents were
completely evaporated under N. Homogeneous IDL-size lipid
emulsions were then prepared in 150 mM NaCl, 0.24 mM
EDTA, 10% sucrose (w/v), pH 7.4 at 60 °C, by sonification and
ultracentrifugation, as previously detailed
(25) . Emulsions
were analyzed for triglycerides and total and free cholesterol (FC), as
well as for phospholipid content. Triglycerides and total and free
cholesterol were analyzed using Boehringer Mannheim Enzymatic Kits
877557, 290319, and 310378, respectively. Phospholipids were analyzed
using the Bartlett assay
(26) . ApoE emulsion complexes were
prepared by adding 0.1 µg of recombinant apoE3 (Biotechnology
General, Rehovot, Israel) to 1 µg of neutral lipid in the lipid
emulsion.
Fatty Acid Addition
FFA were dissolved in
2-propanol (Fisher) to produce a concentrated solution (30.3-60.6
mM) which was added slowly to Dulbecco's modified
Eagle's medium (DMEM) containing BSA (usually 1% w/v), at 37
°C, below the surface-air interface to prevent surface dispersion
of FFA. The total volume of propanol did not exceed 1% of the volume of
DMEM, and propanol had no effect on any of the parameters measured. In
some experiments, fatty acid was provided to cells in the form of
triglyceride- and cholesteryl ester-containing emulsion particles.
LDL Binding, Uptake, and Degradation
Cell binding
of I-LDL to fibroblasts, J774 macrophages, and HepG2
cells at 4 °C was measured in triplicate using established methods
(27) . Cells were incubated for 2.5 h at 4 °C in DMEM
containing
I-LDL, 25 mM HEPES, 1% BSA (w/v), pH
7.4. Afterwards, cells were washed with PBS, 0.2% BSA (w/v) five times,
twice with PBS, solubilized in 0.1 N NaOH, and the total cell
radioactivity was analyzed. Nonspecific binding was measured by the
addition of a 30- to 50-fold excess of non-radiolabeled LDL. Specific
binding was obtained by subtracting nonspecific bound from total bound
I-LDL.
I-LDL at 37 °C was measured after 5 to 24 h of
incubation in DMEM supplemented with 10% lipoprotein-deficient serum
(LPDS)
(27) . The medium was removed and cells were washed three
times with PBS, 0.2% BSA and twice with PBS. At the end of the
incubation, the media were collected and the LDL degradation was
calculated as the difference between the amount of non-iodine,
non-lipid, trichloroacetic acid-soluble radioactivity in the medium and
the amount due to spontaneous degradation measured in parallel
incubations in the absence of cells. Cell surface-bound LDL was
determined as the amount of radioactivity displaced from the cells
after a 60-min incubation at 4 °C in a buffer containing 50
mM NaCl, 10 mM HEPES, 4 mg/ml dextran sulfate, pH
7.4. Internalized LDL was measured as the amount of radioactivity
remaining associated with the cells after incubation with dextran
sulfate buffer. Nonspecific binding uptake and degradation were
determined in the presence of 30- to 50-fold excess non-radiolabeled
LDL.
LDL Receptor mRNA
LDL receptor mRNA of J774
macrophages was quantified by RNase protection assay, as described
(28) .
Cholesterol Mass Analysis
After incubation, cells
were washed five times with PBS, 0.2% BSA, twice with PBS, and then
cellular lipids were extracted for 4 h into hexane:isopropyl alcohol
(3:2 v/v), as described
(26) , using -sitosterol (5
µg/ml) as an internal standard. Samples were split into two
fractions to measure total and free cholesterol separately. To
hydrolyze cellular cholesteryl esters (CE) prior to gas chromatography,
50% KOH (200 µl) and methanol (3 ml) were added to dried aliquots
and heated to 75 °C for 1 h in argon-capped tubes. After cooling,
hexane (5 ml) and water (3 ml) were added. Samples were centrifuged at
1000 rpm for 5 min, and the supernatant containing cholesterol was
removed and dried under N
.
0.53 mm
0.88 µm cross-linked methyl
silicone gum megapore column. Gas chromatography was performed at an
isothermal temperature of 260 °C, injection port temperature of 300
°C, and detector temperature of 265 °C with a N
carrier flow rate of 20 ml/min. Calibration was achieved by
injection of a standard solution of cholesterol (100 µg/ml).
Acyl-CoA:Cholesterol Acyltransferase
Activity
Cellular cholesterol was labeled either by incubating
cells with [H]cholesterol/phospholipid liposomes
(29) or by incubating the cells in the presence of
[
H]mevalonolactone
(30) . In the first
case, cell monolayers were incubated for 1 day with DMEM, 10% LPDS
containing 1 µCi/ml of [
H]cholesterol/egg
lecithin liposomes with concentrations of free cholesterol and
phospholipid of 32 and 62.4 ng/ml, respectively. Following incubation,
cells were washed twice with PBS, 0.2% BSA, prior to treatment with
FFA. In experiments utilizing [
H]mevalonolactone
to label newly synthesized cholesterol, cells were incubated in DMEM,
1% BSA (w/v) containing [
H]mevalonolactone (1
µCi/ml, 0.061 nmol/ml) for 1 h prior to the addition of FFA as well
as during FFA supplementation. In both cases, after cholesterol
labeling, cells were incubated at 37 °C with oleic acid (FFA:BSA
molar ratio 2:1) for up to 4 h, and cellular lipids were extracted with
hexane/isopropyl alcohol (3:2 v/v). Free cholesterol and cholesteryl
ester were then separated by thin layer chromatography in a solvent
mixture of hexane:diethyl ether:acetic acid (70:30:1, v/v/v), and the
radioactivity of each fraction was measured. Squalene was separated
from cholesterol by an initial TLC run in the above solvent system up
to 4 cm from the top of the plate, followed by a second run using
hexane to the top of the plate
(30) .
Statistical Analysis
Differences between group
mean values were compared by two-tailed Student's t test
or analysis of variance, when appropriate.
Free Fatty Acids and LDL Receptor Activity
Human
fibroblasts and J774 macrophages, which had been previously incubated
with lipoprotein-deficient serum (LPDS) to up-regulate the LDL
receptor, were incubated with LDL with or without oleic acid (FFA:BSA
molar ratio 2:1, 0.303 mM) for 5 h or 24 h at 37 °C
(Fig. 1). Compared to control cells at 24 h (Fig. 1,
right), oleic acid supplementation increased LDL receptor
binding by 60% in both cells ( p < 0.001), LDL
internalization by 50% ( p < 0.05) in fibroblasts and 30%
( p < 0.01) in macrophages, and LDL degradation by 20% and
30% ( p < 0.06, p < 0.01) in fibroblasts and
macrophages, respectively. At 5 h, less marked effects of oleic acid
supplementation were observed on specific LDL binding and
internalization via the LDL receptor in fibroblasts ( p <
0.05) and in macrophages ( p < 0.01) but with little early
changes in LDL cell degradation in either cell type (Fig. 1,
left). Similar incubations using octanoic acid resulted in no
alterations in LDL binding, uptake, or degradation at either time
period (data not shown). Nonspecific binding was found to change very
little (less than 10% of net increase in total binding) in response to
oleic acid.
Figure 1:
Effect of oleic acid
supplementation on LDL metabolism in fibroblasts and J774 macrophages.
LDL receptor up-regulated fibroblasts ( solid bars) and J774
macrophages ( hatched bars) were incubated with either DMEM, 1%
BSA with or without oleic acid (FFA:BSA molar ratio 2:1, 0.303
mM FFA) for either 5 or 24 h at 37 °C in the presence of
25 µg/ml I-LDL. LDL cell binding, internalization,
and degradation, as described under ``Experimental
Procedures,'' was performed in triplicate. Results represent the
specific LDL cell binding, internalization, and degradation expressed
as the mean ± S.D. of the percentage values found in control
cells incubated in the absence of oleic acid (*, p < 0.05,
**, p < 0.001 versus control). Control values
expressed as the mean ± S.D. of ng of LDL/mg of cell protein for
cell binding, uptake, and degradation were: 61 ± 6, 538 ±
44, 1764 ± 96 for fibroblasts at 5 h; 31 ± 5, 236
± 80, and 3699 ± 430 for fibroblasts at 24 h; 91 ±
4, 543 ± 26, 4422 ± 60 for macrophages at 5 h; 74
± 8, 546 ± 23, and 11,380 ± 633 for macrophages at
24 h.
To determine whether there was an FFA dose-dependent
effect in LDL cell binding, internalization, and degradation,
up-regulated fibroblasts were incubated at 37 °C with
I-LDL and oleic acid at varying FFA:BSA molar ratios
(1-6:1, 0.15-0.9 mM FFA) for 5 or 24 h
(Fig. 2). After 5 h and 24 h of incubation, maximal increases in
LDL surface binding and internalization of up to 20% ( p <
0.01) occurred at the lower FFA:BSA molar ratios (<2:1), whereas
after 5 h, but not after 24 h, of incubation, higher FFA:BSA molar
ratios (6:1) decreased LDL binding and internalization up to 60%. After
24 h of incubation, a FFA:BSA molar ratio of 6:1 did not inhibit LDL
cell binding or internalization, but degradation was decreased by 20%.
Figure 2:
Dose-dependent effect of oleic acid
supplementation on LDL metabolism in fibroblasts. Fibroblasts were
incubated with DMEM, 1% BSA with or without oleic acid (FFA:BSA molar
ratio 1 to 6:1) for 5 or 24 h at 37 °C in the presence of 25
µg/ml I-LDL. LDL cell binding, internalization, and
degradation was measured as described under ``Experimental
Procedures.'' Results of experiments performed in triplicate are
expressed as a percentage (mean ± S.D.) of the LDL cell binding,
internalization, and degradation of LDL in cells treated with oleic
acid as compared to control cells incubated in the absence of oleic
acid. Control values expressed as the mean ± S.D. of ng of
LDL/mg of cell protein for cell binding, uptake, and degradation were:
107 ± 7, 868 ± 63, and 3491 ± 191, respectively,
at 5 h; and 52 ± 1, 484 ± 24, and 6988 ± 172 at 24
h.
Therefore, depending on the FFA:BSA molar ratio, co-incubation of
fatty acids with LDL may have either a stimulatory or inhibitory effect
on LDL metabolism in vitro. To test whether the effect of
oleic acid on LDL metabolism was dependent on its presence in the media
and perhaps the LDL particle, or was instead dependent on cellular
mechanisms, up-regulated fibroblasts and J774 macrophages were first
preincubated with oleic acid (FFA:BSA molar ratio 2:1) for varying
lengths of time. Then, the cells were washed and specific LDL binding
to the cell surface was assessed at 4 °C, after oleate removal
(Fig. 3). Increases of 20-40% in LDL specifically bound to
the cell surface occurred only after 1 to 2 h of FFA preincubation with
longer preincubation times (up to 24 h) resulting in increases of up to
50%. Fibroblasts appeared to respond to shorter oleic acid
preincubation times than macrophages, but after 4 h of FFA incubation,
the increase in cell surface binding was similar in both cell lines.
Figure 3:
Time
course of effect of FFA preincubation on LDL binding to cells.
Fibroblasts () and J774 macrophages (
) were preincubated
with oleic acid (FFA:BSA molar ratio 2:1) at 37 °C for varying
lengths of time from 10 min to 24 h. Afterwards, cells were washed with
PBS, 0.2% BSA to eliminate non-cell-associated FFA and incubated at 4
°C with 5 µg/ml
I-LDL for 2.5 h. LDL specific
binding was measured by subtracting total bound LDL from LDL bound in
the presence of 50-fold excess non-radiolabeled LDL, as described under
``Experimental Procedures.'' Control values expressed as the
mean ± S.D. of ng of LDL/mg of cell protein were: 84 ± 14
for fibroblasts and 66 ± 3 for J774 macrophages (*, p < 0.05 compared to controls).
Since increased LDL cell binding and degradation can be caused by
increases in LDL receptor number on the cell surface or by increased
affinity of the LDL receptor for its ligand, we next tested the effect
of preincubation with oleic acid on the kinetics of LDL cell binding at
4 °C in both fibroblasts and J774 macrophages (Fig. 4). Cells
preincubated with oleic acid had a 35% and 25% increase in maximal
specific LDL binding in fibroblasts and macrophages, respectively, with
no significant alteration in receptor affinity (Fig. 4,
insets). These data indicate that the increased LDL cell
binding is due to an increase in the number of LDL receptors available
at the cell surface.
Figure 4:
Effect of oleic acid preincubation on
specific LDL receptor binding of LDL in fibroblasts ( A) and
J774 macrophages ( B). Cells previously up-regulated for the
LDL receptor were preincubated for 4 h at 37 °C in DMEM, 1% BSA
with () or without (
) oleic acid (FFA:BSA molar ratio 2:1).
Cells were washed twice with PBS, 0.2% BSA and incubated at 4 °C
for 2.5 h with increasing concentrations of
I-LDL in the
absence or presence of a 50-fold excess of non-radiolabeled LDL, as
described under ``Experimental Procedures.'' The insets in each panel show the Scatchard analysis of the specific LDL
binding. Results are expressed as the mean ± S.D. of ng of
LDL/mg of cell protein of experiments performed in
triplicate.
To ascertain if the effect of FFA on LDL cell
binding was exclusively mediated via the LDL receptor or if other cell
pathways might also contribute, experiments were performed in LDL
receptor-negative fibroblasts (data not shown). Preincubation of these
cells with oleic acid at different FFA:BSA molar ratios (0.5:1 to 4:1)
resulted in no changes in total LDL cell surface binding at 4 °C at
ratios of <2:1 and up to a 25% decrease in LDL 4 °C binding and
degradation at 37 °C at higher ratios. These results further
suggested that the effects of FFA on increasing LDL binding to the cell
surface were mediated through the LDL receptor.
I-acetylated LDL metabolism in J774 macrophages
was also studied, and no alterations in cell binding or degradation of
modified LDL were found (data not shown), suggesting that oleic acid
has no effect on the activity of the scavenger receptor in this cell
line.
I-LDL was bound to the cell surface at 4 °C. The
cells were then washed to eliminate excess
I-LDL and
warmed to 37 °C to follow the fate of the surface-bound LDL. At 4
°C, compared with unsupplemented control cells, cells preincubated
with oleic acid had 38% and 37% more LDL bound to the cell surface in
normal and JD fibroblasts, respectively (Fig. 5 A). After
warming at 37 °C for 2 h, normal cells treated with oleic acid
degraded 40% more LDL than untreated cells. JD cells, however, despite
the initially increased cell surface-bound LDL after oleic acid
treatment, degraded 13% less LDL than control JD cells, not treated
with oleic acid (Fig. 5 B). Therefore, receptor
internalization is not required for oleic acid to increase LDL binding
at the cell surface, but is required for additionally bound LDL to be
metabolized.
Figure 5:
Comparison of effect of oleic acid on LDL
cell binding and degradation in normal and JD fibroblasts. Normal
( solid bars) and LDL receptor-defective JD fibroblasts
( hatched bars) were preincubated with oleic acid (FFA:BSA
molar ratio 2:1) for 4 h at 37 °C, after which, cells were washed
with PBS, 0.2% BSA, cooled to 4 °C, and incubated with 5 µg/ml
I-LDL for 2.5 h to bind LDL to the cell surface.
Afterwards, cells were either solubilized in 0.1 N NaOH to
measure initial cell surface total binding ( A), or parallel
plates were washed and warmed to 37 °C for 2 h to measure the
degradation of surface-bound LDL ( B), as described under
``Experimental Procedures.'' Data are expressed as a
percentage (mean ± S.D.) of the results obtained in control
cells (both normal and JD fibroblasts) not pretreated with oleic acid.
Control values for LDL cell binding and degradation expressed as the
mean ± S.D. of ng of LDL/mg of cell protein were: 65 ± 4
and 152 ± 7 for normal fibroblasts and 194 ± 28 and 40
± 6 for JD fibroblasts, respectively.
To investigate whether the increase in maximal binding
was due to increased synthesis of the LDL receptor mRNA, mRNA was
quantified using an RNase protection assay, and differences due to FFA
were not detected (data not shown). Experiments in both fibroblasts and
macrophages demonstrated that the addition of a protein synthesis
inhibitor, cycloheximide (10 µg/ml), 30 min prior and throughout
oleic acid supplementation, abolished the increase in cell surface LDL
binding at 4 °C. Similar incubations with the mRNA synthesis
inhibitor, actinomycin D, showed no effect (data not shown), suggesting
that FFA affected the LDL receptor by a post-translation pathway.
Effects of Fatty Acids on Cell Cholesterol Distribution
and LDL Receptor Activity
To probe further whether FFA-mediated
increases in LDL receptor activity were associated with changes in
cellular cholesterol homeostasis, we compared the effects of oleic acid
on LDL cell binding at 4 °C in non-up-regulated and up-regulated
fibroblasts. Cells were either grown continuously in medium containing
10% calf serum to suppress the LDL receptor (non-up-regulated) or in
medium containing 10% lipoprotein-deficient serum for 2 days to
up-regulate the LDL receptor (up-regulated). Cells were then
preincubated for 4 h with oleic acid and washed, and, then,
I-LDL binding to the cell surface was measured at 4
°C. In both cell preparations, oleic acid increased LDL binding to
the cell surface, but, compared to cells incubated in absence of oleic
acid, up-regulated cells demonstrated a greater increase in LDL binding
than non-up-regulated cells (30.7 ± 3.2 versus 19.3
± 4.7 ng of LDL/mg of cell protein, respectively). This suggests
that relative depletion of cellular cholesterol by incubation in
lipoprotein-deficient serum makes the cell more sensitive to the
effects of FFA, and that the effect of FFA may still occur despite the
LDL receptor down-regulation induced by 10% calf serum on cultured
cells.
= 0.85) or the CE/FC ratio ( r
= 0.78) after the 18-h incubation. For example, as
compared to control, cholesteryl ester mass and the CE/FC ratio both
increased by over 1.5-fold in the presence of oleic acid. Incubation of
cells with fatty acids without LDL resulted in no changes in total
cellular cholesterol mass. Unsaturated fatty acids had the most marked
effect on LDL degradation and on changing cell cholesterol distribution
between free cholesterol and cholesteryl ester. Saturated fatty acids
had less effect, and short chain fatty acids had no effects. Myristic
acid was not included in Fig. 6because in different experiments
it produced variable and inconsistent results.
Figure 6:
Effect of different FFA on LDL
degradation and CE/FC mass ratio in J774 macrophages. In parallel
experiments, J774 macrophages were incubated with DMEM, 1% BSA with
different fatty acids (FFA:BSA molar ratio 2:1) for 18 h in the
presence of either 100 µg/ml non-radiolabeled LDL to determine
cellular free and total cholesterol mass accumulation or 100 µg/ml
I-LDL to determine cellular degraded LDL. Cholesterol
mass was determined by extracting cellular lipids into hexane:isopropyl
alcohol (3:2 v/v). The samples were split into two aliquots, and total
and free cholesterol were measured using gas liquid chromatography as
described under ``Experimental Procedures.'' Cholesteryl
ester was calculated as ((TC - FC)
1.67). Degraded LDL
represent the differences between the amount of non-iodine,
non-trichloroacetic acid-precipitable radioactivity remaining in the
media and the amount due to spontaneous LDL degradation in the absence
of cells, after 18 h of incubation ( r
=
0.78).
Effects of FFA, Triglyceride, and Cholesteryl Ester
Supplementation on ACAT and LDL Receptor Activities
It has been
previously reported that unsaturated FFA increase ACAT activity in J774
macrophages
(20) , and in vivo studies suggest that, in
the liver, FFA may regulate LDL receptor activity by increasing ACAT
activity and modulating an LDL receptor-sensitive regulatory
cholesterol pool
(5, 6) . We therefore investigated
whether FFA-mediated increases in LDL receptor activity and LDL
catabolism were the result of increased ACAT activity in the absence of
exogenous LDL cholesterol.
Figure 7:
Effect
of ACAT inhibitor 58-035 on FFA-mediated stimulation of LDL degradation
in J774 macrophages. Cells were preincubated at 37 °C for 4 h with
DMEM, 1% BSA alone (), with oleic acid (
), EPA (
), or
arachidonic acid (
) (FFA:BSA molar ratio 2:1) in the presence of
varying amounts of ACAT inhibitor, 58-035 (0-1 µg/ml). 58-035
was added 30 min prior to the addition of FFA. Afterwards, cells were
washed to remove excess FFA and 58-035, and fresh medium containing 20
µg/ml
I-LDL was added. After 18 h, media were
removed, and degraded LDL was measured as described under
``Experimental Procedures.'' Control value expressed as the
mean ± S.D. of ng of LDL/mg of cell protein was 8169 ±
37.
Because
in animals and man most of LDL catabolism occurs via LDL receptors in
the liver, we tested whether oleic acid preincubation would increase
LDL binding in cultured HepG2 cells, a hepatocyte-like cell line. Cells
were preincubated with oleic acid (2:1 FFA:BSA ratio) for 18 h and
cooled to 4 °C, and LDL binding to the cell surface was measured.
Oleate resulted in a 45% increase in binding to the cell surface ( p < 0.05, n = 3). Addition of 58-035 (1
µg/ml) during the FFA preincubation eliminated the FFA-induced
increase in LDL binding, but had little effect on control cells. LDL
degradation by HepG2 cells after oleate preincubation was up to 30%
higher than control cells, similar to the the results obtained with
fibroblasts and macrophages (data not shown).
H]mevalonolactone with or without
oleic acid in the presence or absence of 58-035, and cholesteryl ester
formation was measured (Fig. 8). Oleic acid increased cholesteryl
ester formation by 20-80% above control. This increase was seen
as early as 30 min after FFA supplementation, and the ACAT inhibitor,
58-035, completely blocked the increase in cholesteryl ester.
Therefore, ACAT activity is stimulated 1 to 2 h earlier than increases
in cell surface LDL receptor activity ( cf. with results in
Fig. 3
).
Figure 8:
Effect of
FFA on ACAT activity in J774 macrophages. J774 macrophages were
incubated in DMEM, 1% BSA with [H]mevalonolactone
(1 µCi/ml, 0.061 nmol/ml) in the presence or absence of oleic acid
(FFA:BSA molar ratio 2:1) and 58-035 (1 µg/ml).
[
H]Mevalonolactone was added to cells 1 h prior
to the addition of oleic acid (
). At each time point, cells were
washed, lipids were extracted into hexane/isopropyl alcohol (3:2 v/v),
and cellular free cholesterol and cholesteryl ester were separated by
TLC, as described under ``Experimental Procedures.'' ACAT
activity represents the mean ± S.D. of cellular cholesteryl
ester formation at each time point, expressed as a percentage of
control cells not treated with oleic acid or 58-035. Each time period
was performed in six different dishes. 58-035 was added to control
cells (
) or oleate-treated cells (
) to inhibit cholesterol
esterification.
In vitro-supplemented FFA are quickly
converted by cells into triglyceride, presumably to protect the cells
from damage from excess FFA
(31, 32) . When we incubated
both fibroblasts and macrophages with radiolabeled fatty acids (FFA:BSA
molar ratio 2:1), over 80% of the oleic acid taken up by cells was
found as triglyceride after a 4-h incubation. In experiments not shown,
we have observed that, after a 4-h preincubation with FFA, LDL
degradation remained elevated for at least 18 h after exogenous FFA had
been removed, suggesting that FFA released from intracellular
triglyceride formed during FFA preincubations may have a long lasting
effect on the LDL receptor activity.
Figure 9:
Effect of emulsion particles with or
without cholesteryl ester on LDL degradation in J774 macrophages. Cells
were preincubated for 6 h at 37 °C with DMEM, 1% BSA containing
various concentrations (0-800 µg/ml) of emulsion particles
containing 0.1 µg of apoE/µg of neutral lipid, as described
under ``Experimental Procedures.'' Emulsions were either
composed of 100% triolein () or 43:57 (w:w) triolein:cholesteryl
oleate (CE) (
). Afterwards, cells were washed with PBS, 0.2% BSA
and PBS alone, the medium was changed to DMEM, 10% LPDS containing 20
µg/ml
I-LDL, and LDL degraded over an 18-h period was
measured. The mean ± S.D. of control was 5241 ± 101 ng of
LDL/mg of cell protein.
(
)
(
)
The
poor ability of linoleic acid to stimulate ACAT activity in vitro (37, 38, 39) , combined with data which
showed little effects of linoleic acid on cellular cholesterol
distribution, suggest that linoleic acid may modify LDL cholesterol
levels in vivo by mechanisms other than effects on ACAT
activity. Thus, a number of different mechanisms likely account for the
effects of different fatty acids on LDL receptor activity.
ionophores
(48) . This coincides with the
length of time required by oleic acid to increase LDL as demonstrated
herein. Regulation of LDL receptor activity has been shown to occur
post-transcriptionally as well
(49, 50) , e.g. in HepG2 cells; oleic acid supplementation decreases apoprotein B
degradation
(51, 52) , raising the possibility that
oleic acid may also stabilize LDL receptor protein intracellularly by
decreasing its degradation. A recent report has proposed the existence
of a fatty acid-stimulated LDL binding receptor in receptor-deficient
fibroblasts
(53) . However, the investigators used doses of FFA
greater than 6:1 (FFA:BSA molar ratio), which might have stimulated a
phenomenon unrelated to our results.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.