The steady-state concentration of low density lipo- proteins LDL is mainly regulated by LDL receptors
on the surface of liver cells, which are responsible for approximately 75 of the catabolism of LDL in the
body [6]. In patients lacking functional LDL receptors, not only is the fractional catabolic rate of LDL de-
creased, but also the production of LDL is increased. This is due to the fact that LDL receptors normally
bind and internalize remnants of triglyceride-rich lipo- proteins which serve as precursors for the formation of
LDL [7]. The expression of LDL receptors is finely tuned according to the cell’s demand for cholesterol. In
the absence of extracellular sources of cholesterol, cells enhance the production of LDL receptors, together
with coordinate increases in HMG-CoA reductase and HMG-CoA synthase, the two rate-limiting enzymes of
the sterol biosynthesis pathway [6,8]. Similar responses are elicited in cells in which the endogenous production
of sterols is inhibited, for instance by competitive HMG-CoA reductase inhibitors.
The most successful strategies to reduce the concen- tration of LDL in the circulation have in common that
they involve the up-regulation of the LDL receptor activity by depleting the regulatory pool of cholesterol
in the liver. Anion exchanging resins reduce the reab- sorption of bile salts from the intestine and stimulate
the conversion of cholesterol to bile acids, plant sterols interfere with the absorption of cholesterol from the
intestine, and the HMG-CoA reductase inhibitors re- duce the de no6o production of sterols [9,10].
Lifibrol is a novel, highly effective lipid-lowering agent. The hypolipidemic properties of Lifibrol have
been demonstrated in rats, marmosets, WHHL-rabbits and pigs [11,12]. Lifibrol strongly reduced serum choles-
terol and triglycerides in these studies. In hyperlipi- demic humans, lifibrol lowered LDL cholesterol and
triglycerides by approximately 40 and 25, respectively [13 – 15]. In all trials reported so far, the maximum
hypocholesterolemic effect occurred earlier than with 3-hydroxy-3-methylglutaryl-coenzyme A HMG-CoA
reductase inhibitors, nicotinic acid or bile acid seques- trants [16 – 18] and was sustained during the entire
administration period. The rapid lipid lowering effect of lifibrol strongly suggests that its mechanism of action is
distinct from that of the HMG-CoA reductase in- hibitors. Another interesting feature of lifibrol is that it
brought about substantial decreases of Lpa and fibrinogen [15]. There has been evidence that lifibrol
acts by inhibiting cholesterol biosynthesis [19]. Recently published studies examined the effect of lifibrol on the
in vivo metabolism of apolipoprotein B. These data suggest that lifibrol lowers cholesterol by increasing the
receptor-mediated catabolism of LDL [20].
The purpose of this study was to examine in detail the effects of lifibrol on the cholesterol metabolism of
cultured cells. The results provide evidence that lifibrol enhances the expression of membrane LDL receptors
independent from the cellular regulatory cholesterol pool and suggest that lifibrol sets up a new class of
hypolipidemics with a mode of action clearly distinct from the fibrates and from competitive inhibitors of
HMG-CoA reductase.
2. Materials and methods
2
.
1
. Materials Lifibrol
4-4-tert. butylphenyl-1-4-carboxyphe-
noxy-2-butanol was synthesized at Klinge Pharma GmbH Munich, Germany. Lovastatin was from
Merck Sharp and Dohme. The lactone form of lova- statin was converted into the sodium salt according to
the procedure of Cutts et al. [21]. Stock solutions containing 10 mM of lifibrol in DMSO or lovastatin
in ethanolNaOH were stored at − 20°C until use. The final concentration of DMSO did not exceed 0.1
volvol. [2-
14
C]-acetic acid, sodium salt 56 mCi mmol, [1-
14
C]-Ac-CoA specific activity 50 – 62 mCi mmol,
[1,2-
3
H]-cholesterol 52
Cimmol, DL-3
-hydroxy-3-methyl-[3-
14
C]-glutaryl coenzyme
A 58
mCimmol,
DL
-[2-
3
H]-mevalonic acid lactone 1.26 mCimmol, [2-
14
C]-mevalonic acid lactone 58 mCi mmol and Na[
125
I] carrier free, 17 Cimg were from Du Pont New England Nuclear Bad Homburg, Ger-
many. Silica gel thin layer chromatography plates 20 × 20 cm on aluminium sheet support were from E.
Merck Darmstadt, Germany. ITLC-SA polysilic glass fiber sheets 20 × 20 cm were from Gelman Sciences
Ann Arbor, MI. Scintillation fluid Quicksafe A was from
Zinsser Analytik
Frankfurt, Germany.
Polystyrene tissue culture flasks and CovaLink multi- well plates were from Nunc Roskilde, Denmark. Cell
culture medium, additives glutamine, penicillin G, streptomycin and fetal bovine serum FBS were from
GibcoBRL, Eggenstein, Germany. Glucose-6-phos- phate dehydrogenase EC 1.1.1.49, 350 Umg was from
Boehringer Mannheim Mannheim, Germany. The LDL receptor specific monoclonal antibody C7 was
from Amersham Braunschweig, Germany. Biotiny- lated goat anti-mouse antibody and avidin:biotinylated
horseradish peroxidase reagent ABC Peroxidase Elite were provided by Vector Laboratories Burlingame,
CA.
2
.
2
. Lipoproteins and serum Human LDL 1.030 – 1.050 kg1 were isolated from
plasma by preparative ultracentrifugation. LDL were iodinated using iodine-monochloride as oxidizing agent
[22]. Protein was determined as described [23]. Specific activities ranged from 180 – 250 cpmng protein. Human
lipoprotein-deficient serum LPDS was prepared by ultracentrifugation as described [24] and stored at −
25°C.
2
.
3
. Cell culture HepG2 were obtained from the American Type Cul-
ture Collection Rockville, MD. Human skin fibro- blasts were from skin biopsies of normolipidemic
individuals. The cells were grown in 80 cm
2
flasks containing RPMI 1640 medium supplemented with
penicillin G 100 000 unitsl, streptomycin 100 mgl and 10 volvol FBS in a humidified incubator at 5
volvol CO
2
and 37°C.
2
.
4
. Incorporation of acetate or me6alonate into cellular sterols
HepG2 cells were seeded in 25 cm
2
flasks, each well containing 5 ml medium with FBS and grown to 70
confluence. Forty hours before the experiments, the cells were washed with PBS and switched to medium
supplemented with 10 volvol human LPDS. The monolayers were then incubated for 6 h with lifibrol or
lovastatin at the indicated concentrations. Two hours before the end of the incubation period, the cells were
pulse-labelled with [
14
C]-acetate or [
14
C]-mevalonate both at final concentrations of 35 mM 2 mCil
medium. After incubation, the cells were washed three times with 3 ml of 150 mM NaCl and suspended in 3
ml n-hexane:isopropanol 3:2 by volume. After adding 0.25 mCi of [1,2
3
-H]-cholesterol, each monolayer was extracted for 30 min. The resulting suspension was
transferred to a glass tube and centrifuged at 3300 × g for 20 min. The lipid phase was removed, evaporated to
dryness under a stream of nitrogen and resuspended in 1 ml chloroform:methanol 2:1 by volume. The cell
pellet was dissolved in 1 ml of 1 M NaOH and used for the determination of protein. The lipid extracts were
subjected to thin layer chromatography on aluminium sheet supported silica gel plates. The plates were devel-
oped with a solvent of hexane:isopropanol:formic acid 80:30:2 by volume. The nonesterified i.e. ‘free’ and
the esterified cholesterol spots were visualized by iodine vapour, cut out and counted in a scintillation counter.
The data were expressed as nmol of [
14
C]-acetate incor- porated per h and per mg of total cell protein. To
analyze the incorporation of acetate into nonsaponifi- able sterols, 0.5 ml of the n-hexane:isopropanol extract
were evaporated to dryness and saponified with 3 ml of 15 wtvol KOH in 70 volvol ethanol at 70°C for
5 h. After adding 6 ml of bidistilled water, the nonsa- ponifiable lipids were extracted with diethyl ether three
times with 6 ml each. The lipid extracts were com- bined, evaporated to dryness and suspended in 5 ml
aceton:ethanol 1:1 by volume. One hundred mi- crolitres of a 1 gl cholesterol solution in aceton and
50 ml of 10 volvol acetic acid were added and the sterols were precipitated overnight with 2 ml of 0.5
wtvol digitonin, dissolved in 50 volvol ethanol. After centrifugation 3000 × g, 20 min, the precipitate
was washed once with 6 ml of aceton:diethylether 1:2 by volume and once with diethylether alone, dissolved
in 1 ml of chloroforin:methanol 2:1 by volume and subjected to thin layer chromatography as described
above. The cholesterol spot was then again counted in a liquid scintillation counter.
2
.
5
. Microsomal HMG-CoA reduclase acti6ity HMG-CoA reductase activity was determined essen-
tially as described [24]. The cells were washed twice with 150 mM NaCl, 50 mM Tris – HCl, pH 7.4, scraped
with a rubber policeman and pelleted by centrifugation 900 × g, 5 min, 4°C. The cell pellet was kept in liquid
nitrogen until the time of assay. Cell extracts were prepared by incubating the pellets at 37°C for 10 min
with 5 0 mM K
2
HPO
4
, pH 7.4, 5 mM dithiothreitol, 5 mM EDTA · Na
2
, 0.2 mM KCl, 1 volvol Triton X-100. The detergent solubilized extract was cen-
trifuged at 1000 × g for 5 min. Aliquots of the super- nate were assayed for protein [25] and for HMG-CoA
reductase activity using [
14
C]-HMG-CoA as substrate [24].
2
.
6
. HMG CoA synthase acti6ity Cytosolic HMG-CoA synthase was partially purified
from chicken liver using the method of Gil and cowork- ers [26] with the modifications described previously [19].
The enzyme activity was measured by following the formation HMG-CoA from radioactively labelled ace-
tyl-CoA Ac-CoA and acetoacetyl-CoA AcAc-CoA. HMG-CoA was isolated from the reaction mixture by
reversed-phase ion-pair chromatography [19].
2
.
7
. Uptake and degradation of
[
125
I
]
-labelled lipoproteins
We used the procedures described by Goldstein et al [24] with slight modifications [27]. Cells were grown to
70 confluence in RPMI 1640 medium with 10 vol vol FBS in 24-well tissue culture plates. Cells were
pre-incubated for 40 h in medium containing 10 volvol LPDS to up-regulate LDL receptors. To de-
termine cellular uptake surface binding plus internal- ization and degradation, cells were incubated for 4 h at
37°C with [
125
I]-labelled LDL in RPMI 1640 pH 7.4 in the absence of serum. The amount of [
125
I]-labelled material associated with the cells binding plus internal-
ization was determined after lysis in 0.3 M NaOH. Lysosomal degradation was determined as [
125
I]-la-
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
