©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Oleate and Other Long Chain Fatty Acids Stimulate Low Density Lipoprotein Receptor Activity by Enhancing Acyl Coenzyme A:Cholesterol Acyltransferase Activity and Altering Intracellular Regulatory Cholesterol Pools in Cultured Cells (*)

Steven C. Rumsey (1), Narmer F. Galeano (2), Barry Lipschitz (2), Richard J. Deckelbaum (1) (2)(§)

From the (1) Institute of Human Nutrition and (2) Department of Pediatrics, Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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). 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.


INTRODUCTION

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)() receptor number (3, 4, 5, 6) .

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.


EXPERIMENTAL PROCEDURES

Sodium 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.

Fibroblasts from frozen stocks (6-12 passages) were plated at a cell density of 24 10cells 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 10cells/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.

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.

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.

Cellular binding, uptake, and degradation of 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.

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 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 Ncarrier 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.


RESULTS

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.

The effect of oleic acid on 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.

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 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.

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= 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.

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.


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).

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 [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.

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.


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.




DISCUSSION

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.() 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.

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 Caionophores (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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 40404 and HL 21006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Pediatrics, Columbia University, 630 W 168th St., New York, NY 10032.

The abbreviations used are: LDL, low density lipoprotein; IDL, intermediate density lipoprotein; FC, free cholesterol; CE, cholesteryl ester; FFA, free fatty acid(s); PBS, phosphate-buffered saline; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; LPDS, lipoprotein-deficient serum; EPA, eicosapentanoic acid; ACAT, acyl-CoA:cholesterol acyltransferase; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA reductase; apoE, apolipoprotein E.

S. Rumsey, R. J. Deckelbaum, and L. Liscum, unpublished data.

J. M. Dietschy, personal communication.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Grundy, S. M., and Denke, M. A. (1990) J. Lipid Res. 31, 1149-1171 [Abstract]
  2. Hegsted, D. M., Ausman, L. M., Johnson, J. A., and Dallal, G. E. (1993) Am. J. Clin. Nutr. 57, 875-883 [Abstract]
  3. Spady, D. K., and Woollett, L. A. (1990) J. Lipid Res. 31, 1809-1819 [Abstract]
  4. Woollett, L. A., Spady, D. K., and Dietschy, J. M. (1992) J. Clin. Invest. 89, 1133-1141 [Medline] [Order article via Infotrieve]
  5. Woollett, L. A., Spady, D. K., and Dietschy, J. M. (1992) J. Lipid Res. 33, 77-88 [Abstract]
  6. Daumerie, C. M., Woollett, L. A., and Dietschy, J. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10797-10801 [Abstract]
  7. Bihain, B. E., Deckelbaum, R. J., Yen, F. T., Gleeson, A. M., Carpentier, Y. A., and Witte, L. D. (1989) J. Biol. Chem. 264, 17316-17321 [Abstract/Free Full Text]
  8. Wong, S., and Nestel, P. J. (1987) Atherosclerosis 64, 139-146 [Medline] [Order article via Infotrieve]
  9. Freeman, D. A., and Ontko, J. A. (1992) J. Lipid Res. 33, 1139-1146 [Abstract]
  10. Gavigan, S. J. P., and Knight, B. L. (1981) Biochim. Biophys. Acta 665, 632-635 [Medline] [Order article via Infotrieve]
  11. Loscalzo, J., Freedman, J., Rudd, A. M., Barsky-Vasserman, I., and Vaughan, D. E. (1987) Arteriosclerosis 7, 450-455 [Abstract]
  12. Kuo, P., Weinfeld, M., Rudd, A. M., Amarante, P., and Loscalzo, J. (1990) Arteriosclerosis 10, 111-118 [Abstract]
  13. Kuo, P., Weinfeld, M., and Loscalzo, J. (1990) Biochemistry 29, 6626-6632 [Medline] [Order article via Infotrieve]
  14. Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34-47 [Medline] [Order article via Infotrieve]
  15. Tabas, I., Weiland, D. A., and Tall, A. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 416-420 [Abstract]
  16. Mitropoulos, K. A., Venkatesan, S., and Balasubramaniam, S. (1980) Biochim. Biophys. Acta 619, 247-257 [Medline] [Order article via Infotrieve]
  17. Spector, A. A., Kaduce, T. L., and Dane, R. W. (1980) J. Lipid Res. 21, 169-179 [Abstract]
  18. Johnson, M. R., Mathur, S. N., Coffman, C., and Spector, A. A. (1983) Arteriosclerosis 3, 242-248 [Abstract]
  19. Field, F. J., Albright, E. J., and Mathur, S. N. (1987) J. Lipid Res. 28, 50-58 [Abstract]
  20. McCloskey, H. M., Glick, J. M., Ross, A. C., and Rothblat, G. H. (1988) Biochim. Biophys. Acta 963, 456-467 [Medline] [Order article via Infotrieve]
  21. Tabas, I., Boykow, G. G., and Tall, A. R. (1987) J. Clin. Invest. 79, 418-426 [Medline] [Order article via Infotrieve]
  22. Havel, R. J., Eder, H. A., and Bragdon, J. H. (1955) J. Clin. Invest. 34, 1345-1353 [Medline] [Order article via Infotrieve]
  23. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  24. Bilheimer, D. W., Eisenberg, S., and Levy, R. I. (1972) Biochim. Biophys. Acta 260, 212-218 [Medline] [Order article via Infotrieve]
  25. Schwiegelshohn, B., Presley, J. F., Gorecki, M., Vogel, T., Carpentier, Y. A., Maxfield, F. R., and Deckelbaum, R. J. (1995) J. Biol. Chem. 270, 1761-1769 [Abstract/Free Full Text]
  26. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468 [Free Full Text]
  27. Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260 [Medline] [Order article via Infotrieve]
  28. Rudling, M. (1992) J. Lipid Res. 33, 493-501 [Abstract]
  29. Tabas, I., Rosoff, W. J., and Boykow, G. C. (1988) J. Biol. Chem. 263, 1266-1272 [Abstract/Free Full Text]
  30. Rustan, A. C., Nossen, J. O., Osmundsin, H., and Drevon, C. A. (1988) J. Biol. Chem. 263, 8126-8132 [Abstract/Free Full Text]
  31. Ontko, J. A. (1972) J. Biol. Chem. 247, 1788-1800 [Abstract/Free Full Text]
  32. Spector, A. A., Kiser, R. E., Denning, G. M., Shay-Whey, M. K., and DeBault, L. E. (1979) J. Lipid Res. 20, 536-547 [Abstract]
  33. Slotte, J. P., and Bierman, E. L. (1988) Biochem. J. 250, 653-658 [Medline] [Order article via Infotrieve]
  34. Chatterjee, S. (1993) J. Biol. Chem. 268, 3401-3406 [Abstract/Free Full Text]
  35. Xu, X.-X., and Tabas, I. (1991) J. Biol. Chem. 266, 24849-24858 [Abstract/Free Full Text]
  36. Tabas, I., Myers, J. N., Innerarity, T. L., Xu, X.-X., Arnold, K., Boyles, J., and Maxfield, F. R. (1991) J. Cell Biol. 115, 1547-1560 [Abstract]
  37. Goodman, D. S., Deykin, D., and Shiratori, T. (1964) J. Biol. Chem. 239, 1335-1345 [Free Full Text]
  38. Mathur, S. N., Simon, I., Lokesh, B. R., and Spector, A. A. (1983) Biochim. Biophys. Acta 751, 401-411 [Medline] [Order article via Infotrieve]
  39. Hashimoto, S., and Dayton, S. (1978) Biochim. Biophys. Res. Commun. 82, 1111-1120 [Medline] [Order article via Infotrieve]
  40. Hegsted, D. M., McGandy, R. B., Myers, M. L., and Stare, F. J. (1965) Am. J. Clin. Nutr. 17, 281-295 [Medline] [Order article via Infotrieve]
  41. Keys, A., Anderson, J. T., and Grande, F. (1957) Lancet 1, 66-68
  42. Hayes, K. C., and Khosla, P. (1992) FASEB J. 6, 2600-2607 [Abstract/Free Full Text]
  43. Mattson, F. H., and Grundy, S. M. (1985) J. Lipid Res. 26, 194-202 [Abstract]
  44. Mensink, R. P., and Katan, M. B. (1989) N. Engl. J. Med. 321, 436-441 [Abstract]
  45. Berry, E. M., Eisenberg, S., Haratz, D., Friedlander, Y., Norman, Y., Kaufmann, N. A., and Stein, Y. (1991) Am. J. Clin. Nutr. 53, 899-907 [Abstract]
  46. Roth, M., Emmons, L. R., Perruchoud, A., and Block, L. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1888-1892 [Abstract]
  47. Hamanaka, R., Kohno, K., Seguchi, T., Okamura, K., Morimoto, A., Ono, M., Ogata, J., and Kuwano, M. (1992) J. Biol. Chem. 267, 13160-13165 [Abstract/Free Full Text]
  48. Auwerx, J. H., Chait, A., and Deeb, S. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1133-1137 [Abstract]
  49. Sharkey, M. F, Miyanohara, A., Elam, R. L., Friedmann, T., and Witztum, J. L. (1990) J. Lipid Res. 31, 2167-2178 [Abstract]
  50. Tam, S.-P., Brissette, L., Ramharack, R., and Deeley, R. G. (1991) J. Biol. Chem. 266, 16764-16773 [Abstract/Free Full Text]
  51. Dixon, J. L., Furukawa, S., and Ginsberg, H. N. (1991) J. Biol. Chem. 266, 5080-5086 [Abstract/Free Full Text]
  52. Furukawa, S., Sakata, N., Ginsberg, H. N., and Dixon, J. L. (1992) J. Biol. Chem. 267, 22630-22638 [Abstract/Free Full Text]
  53. Bihain, B. E., and Yen, F. T. (1992) Biochemistry 31, 4628-4636 [Medline] [Order article via Infotrieve]
  54. Kuo, P. C., Rudd, A. M., Nicolosi, R. J., and Loscalzo, J. (1989) Arteriosclerosis 9, 919-927 [Abstract]
  55. Spady, D. K., Woollett, L. A., and Dietschy, J. M. (1993) Annu. Rev. Nutr. 13, 355-381 [CrossRef][Medline] [Order article via Infotrieve]
  56. Wang, X., Sato, R., Brown, M. S., Hua, X., and Goldstein, J. L. (1994) Cell 77, 53-62 [Medline] [Order article via Infotrieve]

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