The activity of the LDL receptor is tightly regulated by changes in cholesterol supply and demand. Hepatic
LDL receptor synthesis and cycling can be markedly suppressed by dietary cholesterol [8,9]. Conversely, ad-
ministration of
both bile
acid binding
resins, which augment hepatocellular bile acid synthesis, and
cholesterol synthesis inhibitors, increase the number of LDL receptors, by increasing the cellular cholesterol
demand [10,11]. The consequent changes in cellular cholesterol
pools modulate
the production
of LDL receptor mRNA and the synthesis of the LDL
receptor. It has previously been shown in vivo, in the golden
Syrian hamster, that chronic feeding of ursodeoxycholic acid UDCA, in contrast to that of its 7a-hydroxyl
epimer, chenodeoxycholic acid CDCA, evoked a sig- nificant increment in hepatic LDL uptake. However,
this occurred in spite of both a 55 – 71 enrichment of the bile acid pool with CDCA and a marked suppres-
sion of bile acid synthesis [12]. This increased LDL receptor-dependent LDL uptake and degradation in
vitro have also been documented, upon acute exposure to UDCA, in hepatocytes isolated from standard ro-
dent chow-fed hamsters [13,14]. Furthermore, recent studies from our laboratory have demonstrated that
UDCA interacts directly with the LDL receptor, in the absence of any effects on either the LDL particle or on
the membrane lipid composition, and independently of any effects on LDL receptor synthesis or cycling
[13,15]. Thus, the ability of UDCA to directly augment LDL receptor-dependent LDL uptake is independent of
changes in cellular cholesterol pools.
Administration of UDCA has been shown to de- crease serum total and LDL cholesterol in hypercholes-
terolemic patients with primary biliary cirrhosis [16]. In light of results of previous studies, it was postulated
that the cholesterol-lowering effect of UDCA may be due, in part, to a direct increment in LDL receptor
binding [13,15]. The present investigation was under- taken in order to determine the ability of UDCA to
enhance hepatocellular LDL receptor recruitment, as determined by its effect both in vivo on LDL uptake,
and in vitro on LDL binding, under conditions of moderately elevated serum cholesterol concentrations
[17]. This was achieved by feeding male golden Syrian hamsters an excess of cholesterol in order to induce
mild hypercholesterolemia. Under these conditions, the effects of UDCA on parameters of hepatic LDL
metabolism were studied both in vivo and in vitro.
2. Materials and methods
2
.
1
. Materials Sodium
125
I specific activity 16 – 20 mCimg and [U-
14
C]sucrose specific activity 380 mCimmol were purchased from Amersham Corporation Arlington
Heights, IL. Gelatin and bovine serum albumin frac- tion V were purchased from Sigma St. Louis, MO.
UDCA was supplied by Tokyo Tanabe Tokyo, Japan, and CDCA by Dr Falk GmbH Freiburg, Germany.
Both UDCA and CDCA were 98 – 99 pure, as judged by gas – liquid chromatography. All other chemicals
used were of analytical grade available from commer- cial sources.
2
.
2
. Animals Male golden Syrian hamsters 115 – 140 g body
weight; Harlan Sprague Dawley, Indianapolis, IN, were divided into four groups, each of which consisted
of 10 – 13 age-matched animals. The first group was fed a standard rodent chow diet Ralston Purina,
St.
Louis, MO,
containing 0.027
cholesterol control, CONT. The second group received the stan-
dard diet in which the cholesterol content had been increased to 0.15 cholesterol, CHOL. In the other
two groups, the 0.15 cholesterol-containing diet was supplemented
with 0.1
UDCA CHOL-UDCA
and 0.1 CDCA CHOL-CDCA, respectively. The relative percentage of saturated, monounsaturated,
and polyunsaturated fatty acids in the diet was 1.41, 1.44, and 1.65, respectively, with the predominant
species of 16:0 66, 18:1 92, and 18:2 84 Purina Mills. Each group received the specific
diet for 3 weeks. There were no differences among the treatment groups as far as food intake and weight
gain were concerned. Mean food intake was 6.5 gday, while
mean body
weight ranged
from 120 – 140
g following treatment. The percentage of cholesterol in the
cholesterol-supplemented diets
corresponded to a daily intake of 0.075 gday per kg body
weight. Both bile acids were well tolerated by the animals, and livers appeared macroscopically normal
after 3 weeks of the respective cholesterol and bile acid feeding. All animals received humane care in compli-
ance
with the
George Washington
University guidelines.
2
.
3
. Serum lipid determinations The total serum cholesterol concentration was deter-
mined enzymatically using the cholesterol esterase – cholesterol oxidasephenol-4-amine phenazone reaction
Boehringer Mannheim Diagnostics, Indianapolis, IN. Serum high density lipoprotein HDL cholesterol was
measured as described for total serum cholesterol after precipitation of apo B-containing lipoproteins with dex-
tran sulfate-MgCl
2
, using the method of Warnick et al. [18].
2
.
4
. Hepatocellular lipid determinations Livers were removed from Nembutal-anesthetized
hamsters 3 weeks after feeding a control diet, or a cholesterol-supplemented diet. Liver homogenates and
enriched plasma membrane fractions were prepared by the method of Prpic’ et al. [19], as previously de-
scribed [20]. Cellular and membrane protein content was assessed with a bicinchonic acid BCA protein
assay kit Pierce, Rockford, IL.
Total lipids were extracted from aliquots of liver homogenate and enriched plasma membrane fractions,
respectively, according to the method of Bligh and Dyer [21]. Lipid extracts were kept under nitrogen in
glass tubes to prevent oxidative degradation. Phos- pholipid and cholesterol amounts were determined in
liver homogenate and plasma membrane lipid extracts using HPLC procedures previously described [22].
Quantitation was performed by integration of peak areas in conjunction with internal standards. Total
phospholipid was quantitated by phosphorus mea- surement [23]. Lipid concentrations were expressed
per mg of protein.
2
.
5
. Biliary bile acid composition In order to confirm the enrichment of the respec-
tive bile acid pool by either UDCA or CDCA in the hamsters receiving the bile acid-supplemented diets,
the relative bile acid composition was determined, by gas – liquid chromatography, in gallbladder bile as
previously described [12,15].
2
.
6
. Biliary lipid determinations Total bile acid concentration was measured, using
3a-hydroxysteroid dehydrogenase Worthington Bio- chemicals, Malvern, PA [24]. Biliary phosphatidyl-
choline concentration
was determined
by the
enzymatic measurement of choline content using a choline oxidaseperoxidase reaction following treat-
ment with phospholipase D Nippon Shoji Kaisha, Higashi-Ko, Osaka, Japan [25]. The biliary choles-
terol content was analyzed enzymatically, using a cholesterol
oxidasecatalase reaction
Boehringer Mannheim Biochemicals, Indianapolis, IN [26].
2
.
7
. Separation of plasma LDL Blood was collected from normal human subjects,
as well as from normocholesterolemic hamsters, into EDTA-containing
vacuum collection
tubes. The
plasma lipoproteins
were separated
by density
gradient ultracentrifugation, as previously reported [12 – 14,27]. The total protein concentration of the
LDL fraction was determined by the method of Brad- ford [28], using the Bio-Rad
®
protein assay Bio-Rad Laboratories, Richmond, CA. The purity of the
LDL fraction was confirmed by SDS-page as de- scribed [12 – 14]. In some studies, human LDL was
reductively methylated, as previously described [12 – 14].
2
.
8
. In-6i6o hepatic uptake of
[
14
C
]
sucrose-labeled LDL Animals were maintained under diethyl ether anes-
thesia for the duration of the surgical procedure as well as the subsequent infusion experiments. For in-
vivo studies, both hamster LDL and methylated hu- man LDL were radiolabeled with [U-
14
C]sucrose [12,27]. The final [
14
C]sucrose-labeled LDL had a spe- cific activity of 1188 9 719 dpmmg protein. Either
[
14
C]sucrose-labeled hamster LDL or methylated hu- man LDL was administered via a jugular vein
catheter Silastic
®
, I.D., 0.012 in.; O.D., 0.025 in., Dow Corning, Midland, MI in a bolus, containing
20 mg of LDL protein, followed by a constant infu- sion for 1 h at a rate of 1 mg70 ml normal salinemin
[12,27]. All LDL infusion experiments were carried out between 09:00 and 12:00 h. Each study on a
given day was carried out in three animals, one of each group CHOL, UDCA-CHOL, CDCA-CHOL,
using the same LDL preparation. After completing the LDL infusion at 60 min, the abdomen was
opened with a midline incision, and a 50 ml sample of blood was withdrawn from the inferior vena cava, the
gallbladder bile was aspirated, the liver was removed, and the animals were exsanguinated. Livers were
combusted in toto in a Packard Oxidizer Packard Instrument, Downers Grove, IL [29]. The radioactiv-
ity in the combusted liver and blood was determined by scintillation counting.
The LDL tissue space in the liver was obtained by subtracting the [
14
C]albumin tissue space, which was previously determined by the infusion of [
14
C]sucrose- labeled hamster albumin Research Plus, Bayonne,
NJ [12,27], from the [
14
C]LDL tissue space. The up- take in the liver was normalized for an animal weigh-
ing 100 g.
2
.
9
. Hepatocyte isolation Hamsters were anesthetized with Nembutal 70 mg
kg body wt, and hepatocytes were isolated by colla- genase perfusion, as previously described [13 – 15].
Cells were suspended in a Krebs – Henseleit bicarbon- ate buffer, pH 7.4, containing 1.5 gelatin, at a con-
centration of approximately 40 – 50 mg cell wet wtml. Prior to each experiment, hepatocytes were incubated
for 20 – 30 min at 37°C under constant agitation and gassing, in order for the cells to reach a steady-state
[13 – 15].
2
.
10
. In-6itro hepatocellular
[
125
I
]
LDL binding Native hamster LDL isolated from normocholes-
terolemic animals was labeled with [
125
I]sodium iodide to a specific radioactivity of 80 – 200 cpmng of protein,
as previously described [13 – 15], using the iodine monochloride technique [30,31].
With hepatocytes isolated in tandem from 1 hamster of each group fed the 0.027 and the 0.15 cholesterol-
containing diet, respectively, LDL binding was studied [13,14]. The cells 150 – 250 mg proteinml were incu-
bated with increasing concentrations of
125
I-labeled hamster LDL 1 – 120 mg proteinml in the presence
and absence of 700 mmoll UDCA. Previous studies have shown that, at this concentration, UDCA is not
toxic to the cell [32], and induces a maximum increase in hepatocellular LDL binding [13,14]. After 1 h incu-
bation at 4°C, the cell-associated radioactivity was mea- sured in a Beckman model 4000 gamma-radiation
counter. Nonspecific binding was determined by incu- bating the cells under the same conditions, but with an
excess of native human LDL 2 – 2.5 mgml [13,15]. Specific binding was determined by subtracting nonspe-
cific binding from total binding. Saturation binding curves were derived, and the maximum number of
binding sites B
max
, ngmg protein and the dissociation constant K
D
, mg LDL proteinml were calculated [13,15].
2
.
11
. Statistical analysis The paired t-test was used to compare the acute
effect of the respective UDCA treatment and dietary cholesterol supplementation with that of the respective
controls. An analysis of variance, and Student – Neu- man – Keuls test were used to compare the differences in
the measured parameters as a result of bile acid feeding.
3. Results
