belled trichloroacetic acid-soluble noniodide material in the conditioned medium [24].
2
.
8
. Competiti6e enzyme immunoassayfor LDL receptors
Immunoreactive LDL receptors were measured using a competitive enzyme immunoassay. The LDL receptor
was partially purified from bovine adrenals by DEAE cellulose chromatography of octyl-b-
D
-glucoside solubi- lized membrane extracts as described [28,29]. LDL re-
ceptor containing column fractions were identified by SDS polyacrylamide gradient gel electrophoresis T =
5 – 12.5 and immunoblotting with the LDL receptor specific monoclonal antibody C7 [30,31]. The receptor
containing DEAE cellulose fractions were adjusted to a protein concentration of 7.5 mgl and supplemented
with octyl-b-
D
-glucoside and N-hydroxysuccinimide to yield final concentrations of 80 and 0.5 mM, respec-
tively. To immobilize the receptors, 50 ml of this solu- tion were pipetted into the wells of CovaLink
microplates. To each well, 50 ml of 1.0 mM 1-ethyl-3-3- dimethylaminopropyl-carbodiimide were added and
the plates were incubated overnight at room tempera- ture. The plates were washed twice with 200 ml PBS
0.14 M NaCl, 2.7 mM KCl, 10 mM Na
2
HPO
4
,1.5 mM KH
2
PO
4
, pH 7.4 and blocked with 5 gl casein in PBS for at least 2 h. To prepare sample membrane fractions,
the cells were washed twice with ice-cold 0.15 M NaCl. The cells were then overlayered with 4 ml ice-cold PBS
containing, in addition, 1 mM phenylmethylsulfonylfiu- oride PMSF, 2.5 mM leupeptin, and 200.000 KIEI
aprotinin, and scraped with a rubber policeman. The cell suspension was centrifuged for 5 min at 1000 × g.
The resulting pellet was resuspended in 0.5 ml of 50 mM
Tris-hydroxy-methyl-amino-methane-maleate buffer, pH 6.0, 1 mM PMSF, 2.5 mM leupeptin and
200.000 KIEl aprotinin. Another 0.5 ml aliquot of the Tris-maleate buffer and octyl-b-
D
-glucoside to yield a final concentration of 40 mM were added. The suspen-
sion was incubated for 45 min on ice and then ultracen- trifuged at 100 000 × g. Protein in the supernate
membrane fraction was determined according to Lowry [23] as modified by Beisiegel [30].
The membrane specimens were pre-incubated with the LDL receptor specific antibody C7 diluted 1:2000 in
PBS containing either 0.05 volvol Tween 20 or 40 mM octyl-b-
D
-glucoside plus 0.5 gl casein overnight at different concentrations of total protein. One hundred
microlitres of these mixtures were loaded in duplicates to the LDL receptor-coated microplate wells and incu-
bated for 2 h. The plates were washed three times with PBS-Tween and incubated for 1 h with biotinylated
goat anti-mouse IgG diluted 1:2000 in PBS-Tween 20 plus 0.5 g1 casein. The wells were washed three times
and incubated for 1 h with the ABC Peroxidase Elite reagent. After three additional washing steps, colour
was developed with o-phenylenediamine 9.25 mM in 100 mM sodium citrate, pH 5.0 and H
2 2
1.8 mM for 15 to 30 min. The reaction was stopped by adding 0.5
M H
2
SO
4
and absorbance was read at 450 mn Titertek MCC 340, Flow Laboratories. Standard curves were
obtained by running dilutions of a reference prepara- tion of partially purified LDL receptor. This prepara-
tion was arbitrarily assumed to contain 100 units of LDL receptors per mg total protein.
2
.
9
. Other methods Reaction rates were fitted to one-ligand Michaelis
and Menten type equations using P.FIT, a non-linear curve fitting program from FIG.P Software Durham,
NC.
3. Results
3
.
1
. Effects of lifibrol on the synthesis of sterols from radioacti6ely labelled precursors
We analysed the effects of lifibrol on the biosynthesis of sterols from [
14
C]-acetate in the human hepatoma cell line HepG2. To up-regulate the mevalonate pathway,
the cells were pre-incubated with medium containing LPDS. As shown in Fig. 1, lifibrol reduced the produc-
tion of sterols in a dose-dependent fashion, regardless whether we considered the production of nonesterified
cholesterol, of esterified cholesterol or of nonsaponifi- able sterols. At concentrations of 10
− 6
and 10
− 5
M, lifibrol reduced the sterol biosynthesis by approximately
20 and 30, respectively. In contrast to lifibrol, lovas- tatin strongly inhibited sterol biosynthesis; less than
2 × 10
− 8
M lovastatin was sufficient to produce a 20 reduction, 50 reduction was obtained at 2.5 × 10
− 8
M, and 10
− 6
M lovastatin decreased the cholesterol synthesis rate by 80. This is in perfect agreement
with data showing that lovastatin inhibited sterol synthesis from acetate with an IC
50
of 2 × 10
− 8
mol1 in mouse L-M cells [32]. Lovastatin is a competitive
inhibitor of HMG-CoA reductase. We reasoned, if lifibrol also acted directly on one of the rate-limiting
steps of the sterol synthesis pathway, then the time kinetics of cholesterol synthesis inhibition by lifibrol
and lovastatin should be similar. To test this hypothesis, HepG2 cells were pre-incubated with LPDS containing
medium and then received lifibrol or lovastatin for different periods of time at concentrations of 10
− 5
or 10
− 6
M, respectively. As predicted from the previous experiment, lifibrol was a less effective inhibitor
of the formation of cholesterol esterified and nones- terified from [
14
C]-acetate than lovastatin. The time kinetics of inhibition, however, were almost perfectly
parallel, suggesting that lifibrol, much as lovastatin, inhibited the cholesterol synthesis pathway directly
rather than by reducing the de novo synthesis or by enhancing the catabolism of a rate-limiting enzyme
Fig. 2.
To identify precisely the step at which lifibrol inhib- ited cholesterol synthesis, we examined its effects on the
formation of nonesterified cholesterol from either [
14
C]- acetate or [
14
C]-mevalonate. As shown in Fig. 3, both lifibrol and lovastatin decreased the synthesis of choles-
terol from acetate but not from mevalonate, indicating that lifibrol inhibited the production of mevalonate
rather than its conversion to sterols.
The production of mevalonate is catalyzed by two sequentially acting enzymes, cytosolic HMG-CoA syn-
thase and microsomal HMG-CoA reductase. Cytosolic HMG-CoA synthase is responsible for the generation
of HMG-CoA from Ac-CoA and AcAc-CoA. HMG- CoA reductase converts HMG-CoA to mevalonate. We
first examined whether lifibrol influenced microsomal HMG-CoA reductase. HepG2 cells were pre-incubated
with medium containing LPDS. The cells were dis- rupted and HMG-CoA reductase in the homogenate
Fig. 2. Effect of lifibrol and lovastatin on the incorporation of acetate into de no6o synthesized cellular sterols. HepG2 cells were grown in
RPMI 1640 medium supplemented with 10 volvol FBS. Forty hours prior to the experiment, the cells were switched to medium
containing 10 volvol human LPDS. The cells were then incubated with lifibrol circles or lovastatin squares at final concentrations of
10
− 5
or 10
− 6
M, respectively, or in the absence of drugs for the time intervals indicated on the abscissa. One hour before the end of each
incubation period, the cells were pulse-labelled with [
14
C]-acetate specific activity 56 mCimmol, final concentration 35 mM and the
incorporation of acetate into esterified left panel and nonesterified right panel cholesterol was determined as described in Section 2.
[
3
H]-cholesterol was used as an internal standard for the recovery of cholesterol. Results are normalized to cellular protein and expressed
in percent of control incubations performed for the respective time periods in the absence of drugs. The data are means from duplicates;
the standard deviations of the replicates were 11 or less of the respective means. The average 100 of control rates of [
14
C]-acetate incorporations into nonesterified cholesterol and into esterified
cholesterol were 6.5 and 0.7 nmolhmg, respectively.
Fig. 1. Effect of lifibrol and lovastatin on the incorporation of acetate into de no6o synthesized cellular sterols. HepG2 cells were grown in
RPMI 1640 medium supplemented with 10 volvol FBS. Forty hours prior to the experiment, the cells were switched to medium
containing 10 volvol human LPDS. The cells then received lifibrol left panel or lovastatin right panel at the concentrations
indicated on the abscissa for 8 h. Two hours before the end of the incubation period, the cells were pulse-labelled with [
14
C]-acetic acid, sodium salt specific activity 56 mCimmol, final concentration 35
mM. The incorporation of acetate into esterified circles and nones- terified squares cholesterol or into the digitonin precipitable sterols
triangles were determined as described in Section 2. [
3
H]-cholesterol was used as an internal standard for the recovery of cholesterol.
Results were normalized to the amount of cellular protein and expressed in percent of the respective control incubations. The data
are means from duplicates, except that the controls are average values of four replicate incubations. The standard deviations of the repli-
cates were 9 or less of the respective means. The average 100 of control rates of [
14
C]-acetate incorporations into esterified circles, nonesterified squares cholesterol, and into digitonin precipitable
sterols triangles incorporations were 0.76, 4.11, and 3.78 nmolh mg, respectively.
was measured in the presence of increasing concentra- tions of lifibrol and lovastatin. As shown in Fig. 4,
lovastatin dose-dependently inhibited HMG-CoA re- ductase, the IC
50
being around 3 × 10
− 8
M. In con- trast, lifibrol did not affect HMG-CoA reductase. We
then examined the effect of lifibrol on HMG-CoA synthase using partially purified enzyme from chicken
liver and a reversed-phase ion-pair chromatography based method to recover HMG-CoA, the product of
the reaction, from the incubation mixture [19]. Lifibrol 10
− 5
M inhibited HMG-CoA synthase Fig. 4. When the concentration of AcAc-CoA was kept constant at
60 mM, the inhibitory effect of lifibrol was greatest between 400 and 1200 mM Ac-CoA. Further increases
of the Ac-CoA substrate concentration reversed the inhibitory effect of lifibrol; at 4000 mM Ac-CoA,
HMG-CoA synthase activity was inhibited by 10 only. To determine the type of inhibition, we estimated
V
max
, the maximum reaction rate, and K
m
, the Michaelis constant, from the data shown in Fig. 4. V
max
was 44.7 and 47.1 nmolminmg and K
m
was 1408 and 2708 mM in the absence and in the presence of 10
− 4
M lifibrol, respectively. Formally, the results were thus
compatible with the assumption that lifibrol was a competitive inhibitor, the inhibitor constant K
i
being approximately 108 mM.
3
.
2
. Effects of lifibrol on the acti6ity of the LDL receptor
To examine the effect of lifibrol on the activity of LDL receptors, we measured receptor mediated uptake
and degradation of [
125
I]-1abelled LDL in HepG2 cells and normal human skin fibroblasts. The cells were
incubated for 40 h with medium containing LPDS alone or with the same medium containing, in addition,
lifibrol at concentrations of 10
− 7
– 10
− 5
M. As shown in Fig. 5a, lifibrol increased receptor mediated endocy-
tosis of LDL in both cell types in a dose-dependent fashion. At 10
− 5
M, lifibrol increased receptor medi- ated binding, uptake, and degradation by approxi-
mately 50 in human skin fibroblasts Fig. 5b. The effect of lifibrol was even greater in HepG2 cells, where
lifibrol enhanced receptor mediated catabolism of LDL by : 80. We wished to compare the effects of lifibrol
and lovastatin on the receptor mediated uptake of LDL. In these studies, lifibrol and lovastatin were used
at 10
− 5
and 10
− 6
M, respectively. At these concentra- tions, the two compounds inhibited sterol synthesis by
30 and 80, respectively. However, despite the smaller inhibition of sterol synthesis, 10
− 5
M lifibrol was more effective in enhancing receptor mediated binding, up-
take, and degradation of LDL than lovastatin in HepG2 cells Fig. 6a. This was confirmed in normal
Fig. 4. Effects of lifibrol and lovastatin on the activity of microsomal HMG-CoA reductase and HMG-CoA synthase. Left panel
:
HepG2 cells were grown in 80 cm
2
polystyrene flasks containing RPMI 1640 medium supplemented with 10 volvol FBS. To upregulate HMG-
CoA reductase, the cells were incubated with medium containing 10 volvol human LPDS for 48 h before the experiment. Cell extracts
were prepared by lysis with 50 mM potassium phosphate, pH 7.4, 5 mM dithiothreitol, 5 mM EDTA, 200 mM KCl, 1 volvol Triton
X-100, and pre-incubated at 38°C for 10 min with lifibrol circles or lovastatin squares at the indicated concentrations. The extracts were
then assayed for HMG-CoA reductase activity as described in Section 2 using [
3
H]-mevalonate as an internal standard. Two independent experiments were performed with Lifibrol open and closed circles,
respectively. The results are means from duplicates, normalized to extract protein. The 100 of control values for HMG-CoA reductase
activity were: 158 lifibrol, closed circles, 184 lifibrol, open circles, and 106 lovastatin, squares pmolhmg, respectively. Right panel:
cytosolic HMG-CoA synthase was purified from chicken liver as described [19,26]. The activity of the partially purified enzyme was
determined using 60 mM AcAc-CoA and the indicated concentrations of [1-
14
C]-Ac-CoA as substrates, either in the absence circles or in the presence of Lifibrol 10
− 5
M, squares. HMG-CoA synthase activity was measured using an assay in which the HMG-CoA formed
during the reaction is isolated by reversed phase-ion pair chromatog- raphy [19].
Fig. 3. Effect of lifibrol and lovastatin on the incorporation of acetate or mevalonate into nonesterified cholesterol. HepG2 cells were grown
in RPMI 1640 medium supplemented with 10 volvol FBS. Forty hours prior to the experiment, the cells were switched to medium
containing 10 volvol human LPDS. The cells were then incubated with lifibrol left panel or lovastatin right panel at the indicated
concentrations for 8 h. Two hours before the end of the incubation period, the cells were pulse-labelled with either [
14
C]-acetate solid bars or [
14
C]-mevalonate grey bars, both specific activity 56 mCi mmol, final concentration 3 5 mM. The rates of incorporation of
radioactively labelled precursors into nonesterified cholesterol was determined after separating the cellular lipids by thin layer chro-
matography. [
3
H]-Cholesterol was included as an internal standard to correct for the recovery of cholesterol. The data are means from two
experiments, each performed in duplicate. Results are normalized to cellular protein and expressed in percent of the control incubations. :
P B 0.05 versus the respective control incubations. The average rates of [
14
C]-acetate and [
14
C]-mevalonate incorporations in the control incubations were 3.48 and 0.65 nmolhmg, respectively.
human skin fibroblasts Fig. 6b. No additional effect was obtained when lifibrol and lovastatin were used in
combination. To determine whether upregulation of LDL receptors by lovastatin and lifibrol followed dif-
ferent time kinetics, we incubated human skin fibrob- lasts with medium containing LPDS plus lifibrol or
lovastatin for time intervals between 12 and 48 h. Compared to medium containing LPDS alone, both
drugs enhanced LDL receptor mediated endocytosis Fig. 7a and b. Interestingly, the effect of lifibrol
occurred earlier and was stronger than that of lovastatin.
Cells incubated in the presence of an exogenous source of cholesterol downregulate their LDL recep-
tors. If lifibrol stimulated LDL receptor mediated endo- cytosis by a sterol independent mechanism rather than
by depleting the regulatory sterol pool of the cell, then it should still be effective in medium containing an
extracellular source of cholesterol. We, therefore, incu- bated human skin fibroblasts with lifibrol and lova-
statin in FBS containing medium which provides cholesterol sufficient to suppress LDL receptors in the
absence of drugs. Under these conditions, lifibrol still markedly enhanced receptor mediated uptake and
degradation of radiolabelled LDL at all incubation times considered Fig. 7c and d.
To analyse the effects of lifibrol on the relative amount of immunoreactive LDL receptors of human
skin fibroblasts, we developed a competitive enzyme immunoassay. Partially purified LDL receptors were
immobilized to microwell plates. Cell membrane sam- ples were incubated with the LDL receptor specific
monoclonal antibody C7, either in the presence of octyl-b-
D
-glucoside 40 mM or Tween 20 0.5 mM as detergent. Antibody not absorbed to LDL receptors in
the sample was then allowed to bind to the immobilized bovine receptors and quantitated. The assay was cali-
brated using a preparation of bovine adrenal LDL receptors. This preparation was arbitrarily considered
to contain 100 units of LDL receptor per mg protein. As expected, we obtained an inverse relationship be-
tween the amount of LDL receptors in the sample and the absorbances Fig. 8. The presence of octyl-b-
D
-glu- coside during incubation of the membrane extracts with
the monoclonal antibody C7 improved the immunore- activity of the LDL receptors compared to Tween 20
Fig. 8. When we estimated LDL receptors in mem- branes from cells pre-incubated with lifibol 10
− 5
M approximately 2-fold increases over control cells was
observed. In contrast, lovastatin 10
− 6
M enhanced the amount of immunoreactive LDL receptors by not
more than 1.5-fold. These data indicate that the en- hanced uptake and degradation caused by lifibrol was
paralleled by an increase of the number of receptor molecules on the cell surface Fig. 8.
3
.
3
. Effect of lifibrol on the expression of HMG-CoA reductase in cultured cells
The expression of HMG-CoA reductase in cultured cells is regulated by sterol and non-sterol mevalonate
derived products [33]. In cells incubated with HMG- CoA reductase inhibitors at a concentration sufficient
to block the production of the nonsterol mevalonate derived effectors of HMG-CoA reductase, extracellular
cholesterol only partly suppresses HMG-CoA reduc- tase. We wondered whether lifibrol, similarly to HMG-
CoA reductase inhibitors, affected the provision of cells with the regulatory mevalonate derived products.
HepG2 cells were incubated in medium containing LPDS alone or, in addition, lifibrol or lovastatin and
increasing concentrations of LDL as a source of extra- cellular cholesterol. After washing the monolayers three
times with 3 ml of 50 mM Tris – HCl, 150 mM NaCl, pH 7.4, to remove the drugs, we determined the activity
of the microsomal HMG-CoA reductase in the cells. In the absence of extracellular LDL, both lifibrol and
lovastatin stimulated HMG-CoA reductase at approxi- mately the same rate over lipoprotein deficient medium
alone Fig. 9. However, the two compounds behaved entirely differently when exogenous LDL was added at
increasing concentrations. Whereas the lifibrol induced increase in HMG-CoA reductase could completely be
repressed to control levels by LDL, mevalonate plus LDL was required to suppress the lovastatin mediated
increase in reductase activity.
Fig. 5. Effect of lifibrol on the uptake and degradation of LDL in cultured cells. HepG2 cells panel 5a and human skin fibroblasts panel 5b were grown in RPM1 1640 medium supplemented with 10 volvol FBS. The cells were incubated for 40 h with medium containing 10
volvol LPDS alone open circles or with Lifibrol at concentrations of 10
− 7
M circles, 10
− 6
M triangles or 10
− 5
M squares, respectively. The cells then received [
125
I]-1abelled LDL 1.030 B d B 1.050 kgl at the indicated concentrations. Binding at CC left panels, uptake center panels, and degradation right panels at 37°C of [
125
I]-labelled LDL were determined as described in Section 2. Each data point represents the average from two independent experiments, each performed in triplicate. Data are adjusted for non-specific binding, uptake, and degradation
determined in the presence of a 50-fold excess of unlabelled LDL. P B 0.05 versus the respective control incubations.
Fig. 6. Effect of lifibrol and lovastatin on the uptake and degradation of LDL in cultured cells. HepG2 cells panel 6a and human skin fibroblasts panel 6b were grown in RPMI 1640 medium supplemented with 10 volvol FBS. The cells were incubated for 40 h with medium containing
10 volvol LPDS alone open circles or, in addition, Lifibrol 10
− 5
M, solid circles, lovastatin 10
− 6
M, squares or the combination of both lifibrol and lovastatin 10
− 5
and 10
− 6
M, respectively, open squares. The cells then received [
125
I]-labelled LDL at the indicated concentrations. Binding at 4°C left panels, uptake center panels and degradation at 37°C right panels of [
125
I]-LDL 1.030 – 1.050 kg l were measured as
described in Section 2. Each data point represents the average from two independent experiments, each performed in triplicate. Data are adjusted for non-specific binding, uptake, and degradation determined in the presence of a 50-fold excess of unlabelled LDL. P B 0.05 versus the
respective control incubations.
4. Discussion
