Atherosclerosis 153 2000 69 – 80
The effects of lifibrol K12.148 on the cholesterol metabolism of cultured cells: evidence for sterol independent stimulation of the
LDL receptor pathway
Hubert Scharnagl
a,
, Michael Schliack
b
, Roland Lo¨ser
b
, Markus Nauck
a
, Hedi Gierens
a
, Nikola Jeck
c
, Heinrich Wieland
a
, Werner Groß
c
, Winfried Ma¨rz
a
a
Department of Medicine, Di6ison of Clinical Chemistry, Albert Ludwigs-Uni6ersity, Hugstetter Strasse
55
,
79106
Freiburg, Germany
b
Department of Biochemistry, Klinge Pharma GmbH, Munich, Germany
c
Gusta6 Embden-Center of Biological Chemistry, Johann Wolfgang Goethe-Uni6ersity, Frankfurt, Germany Received 23 September 1999; accepted 25 January 2000
Abstract
Lifibrol 4-4-tert. butylphenyl-1-4-carboxyphenoxy-2-butanol is a new hypocholesterolemic compound; it effectively lowers low density lipoprotein LDL cholesterol. We studied the effects of lifibrol on the cholesterol metabolism of cultured cells. In the
hepatoma cell line HepG2, Lifibrol decreased the formation of sterols from [
14
C]-acetic acid by approximately 25. Similar to lovastatin, lifibrol had no effect on the synthesis of sterols from [
14
C]-mevalonic acid. Lifibrol did not inhibit 3-hydroxy-3-methyl- glutaryl-coenzyme A HMG-CoA reductase. Instead, cholesterol synthesis inhibition by lifibrol was entirely accounted for by
competitive inhibition of HMG-CoA synthase. Lifibrol enhanced the cellular binding, uptake, and degradation of LDL in cultured cells in a dose dependent fashion. The stimulation of LDL receptors was significantly stronger than expected from the
effect of lifibrol on sterol synthesis. In parallel, lifibrol increased the amount of immunologically detectable receptor protein. Stimulation of LDL receptor mediated endocytosis was observed both in the presence and in the absence of cholesterol-containing
lipoproteins. In the absence of an extracellular source of cholesterol, both lifibrol and lovastatin induced microsomal HMG-CoA reductase. Co-incubation with LDL was sufficient to suppress the lifibrol mediated increase in reductase activity, indicating that
lifibrol does not affect the production of the non-sterol derivatives which are thought to regulate HMG-CoA reductase activity at the post-transcriptional level. Considered together, the data suggest that the hypolipidemic action of lifibrol may, at least in
part, be mediated by sterol-independent stimulation of the LDL receptor pathway. A potential advantage of lifibrol is that therapeutic concentrations do not interfere with the production of mevalonate which is required not only to synthesize sterols but
also as a precursor of electron transport moieties, glycoproteins and farnesylated proteins. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Hypercholesterolemia; Cholesterol biosynthesis; 3-Hydroxy-3-methylglutaryl coenzyme A synthase; 3-Hydroxy-3-methylglutaryl coen- zyme A reductase; Low density lipoprotein receptor; Lifibrol K12.148; Atherosclerosis
www.elsevier.comlocateatherosclerosis
1. Introduction
There is now abundant evidence that hypercholes- terolemia due to high concentrations of apolipoprotein
B containing lipoproteins is one of the major risk factors of atherosclerosis and that the reduction of
LDL cholesterol
lowers the
cardiovascular risk
[1 – 5].
Abbre6iations
:
Ac-CoA, acetyl-CoA; AcAc-CoA, acetoacetyl-CoA; cpm, counts per minute; DMSO, dimethyl sulfoxide; EDTA, ethylenedi- aminetetraacetate; FBS, fetal bovine serum; HMG, 3-hydroxy-3-methylglutaric acid; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; K
m
, Michaelis constant; Lifibrol, 4-4-tert. butylphenyl-1-4-carboxyphenoxy-2-butanol; LDL, low density lipoproteins; LPDS, human lipoprotein
deficient serum; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; Tris, trishydroxy- methylaminomethane; V
max
, maximum reaction rate. Corresponding author. Tel.: + 49-761-2707306; fax: + 49-761-2703444.
E-mail address
:
hubschmedl.ukl.uni-freiburg.de H. Scharnagl. 0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 2 1 - 9 1 5 0 0 0 0 0 4 0 5 - 6
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
