Pharmacology Laboratories, Central Research
Laboratories, The Green Cross Corporation, 2-25-1, Shodai-Ohtani,
Hirakata, Osaka 573, Japan
We evaluated the effect of AE0047, a dihydropyridine-type calcium
antagonist, on the plasma lipid levels of obese Zucker rats. In rats
treated orally with 3 to 10 mg/kg/day AE0047 for 7 days, plasma
triglyceride (TG) and TG-rich lipoprotein levels dose-dependently decreased, whereas those of high-density lipoprotein cholesterol increased. Total cholesterol and low-density lipoprotein levels did not
change. To elucidate the mechanism by which AE0047 decreases plasma TG
levels, we examined the effect of AE0047 on the synthesis and secretion
of TG-rich lipoproteins in human intestinal cell line Caco-2, as well
as on the association and degradation of very low density lipoprotein
(VLDL) in human hepatoblastoma cells HepG2. When Caco-2 cells were
grown on a membrane filter and 14C-oleic acid was added to
the apical side, 10
5 and 106 M AE0047
inhibited basolateral secretion of 14C-TG. AE0047 also
suppressed the basolateral secretion of apolipoprotein B. In HepG2
cells, AE0047 increased the cellular uptake of 125I-VLDL.
These results suggested that AE0047 decreased plasma TG level by the
inhibition of intestinal chylomicron secretion and the enhancement of
hepatic uptake of VLDL. AE0047 may be beneficial for the treatment of
hypertensive patients with hypertriglyceridemia to reduce the risk
factors of coronary heart disease.
 |
Introduction |
High
blood pressure is a major risk factor in the development of CHD (Kannel
et al., 1978
; Newman et al., 1986).
Epidemiological studies have indicated that essential hypertension
tends to be associated with other risk factors of CHD, such as
hyperlipidemia, diabetes and obesity (Ferrannini et al.,
1987). One goal of antihypertensive therapy should be to reduce CHD by
lowering blood pressure; therefore, an antihypertensive drug that can
reduce the above risk factors is preferred for the management of
hypertension.
Recent clinical studies, however, have revealed that some
antihypertensive drugs have unfavorable effects on plasma lipid profiles (Giles, 1992; Kasiske et al., 1995). For example,
some beta adrenergic receptor blockers increase plasma TG
levels and decrease HDL levels. Thiazide-type diuretics in general
increase TG and LDL levels. Although most of the clinical and
experimental data indicate that calcium antagonists generally do not
influence plasma lipid profiles (Giles, 1992; Kasiske et
al., 1995), several exceptions indicated that they can decrease
plasma TG and increase HDL levels (Canale et al., 1991;
Kazumi et al., 1990; Kihara, 1991; Morris et al.,
1993). Because high TG and low HDL levels are often associated with
hypertension and thought to accelerate atherosclerosis, it is important
to evaluate the effect of calcium antagonists on these lipid profiles.
AE0047 [(±)-2-[4-(4-Benzhydrylpiperazin-1-yl) phenyl] ethyl methyl
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate dihydrochloride; fig. 1] is a DHP-type calcium antagonist. It inhibited the K+-induced constriction of rat aortic strip
and the binding of [3H]PN 200-110 to bovine aorta
microsomes (Yamanaga et al., in press). Oral administration
of AE0047 showed a potent hypotensive effect with slow onset and
long-lasting actions in stroke-prone spontaneously hypertensive rats
(Shinyama et al., 1995). In the present study, we evaluated
the effect of AE0047 on the plasma lipid profiles of Zucker rats. The
obese Zucker fatty (fa/fa) rat is a model of
hypertriglyceridemia that exhibits chronic hyperinsulinemia and insulin
resistance (Bray, 1977; Wang et al., 1984). We describe the
remarkably decreased plasma contents of TG and TG-rich lipoproteins caused by AE0047 in Zucker rats.
To
clarify the mechanism by which AE0047 decreases plasma TG, we examined
each of the rate-limiting steps of TG metabolism: (1) intestinal
absorption of exogenous lipid and secretion of chylomicrons, (2)
synthesis and secretion of VLDL by the liver, (3) catabolism of
lipoproteins by lipases and (4) resorption of the lipoproteins into the
liver. Because TG-rich lipoproteins (chylomicrons and VLDL) are
exclusively generated by the liver and intestine, we used human hepatic
(HepG2) and intestinal (Caco-2) cell lines in which the synthesis,
secretion and clearance of lipoproteins have been widely studied
(Javit, 1990; Levy et al., 1995). We identified two possible
mechanisms from in vitro studies using these cell lines.
View larger version (54K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of AE0047 and nilvadipine on the plasma lipid
profiles of Zucker rats. Rats were administered the indicated amounts of drugs for 7 days. Animals were deprived of food overnight before the
blood sampling; 2 hr after the final administration, blood was drawn.
There were 11 study animals receiving AE0047 (3 mg/kg), 10 receiving
AE0047 (10 mg/kg) and vehicle and 8 receiving nilvadipine. All values
represent mean ± S.E. Significantly different from vehicle: **,
P < .01; *, P < .05 (Dunnett's method).
|
|
View larger version (49K):
[in this window]
[in a new window]
|
Fig. 3.
Lipase activity of Zucker rats administered AE0047 or
nilvadipine. Rats were treated as described in legend to figure, 2 and postheparin plasma was prepared 2 hr after the last administration. Lipase activity was measured as described in Methods. All values represent mean ± S.E. (n = 7 or 8).
|
|
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of AE0047 on cellular synthesis and
basolateral secretion of TG in Caco-2 cells. Cells were incubated for
24 (n = 5) or 48 (n = 6) hr with
105 M AE0047. Then, 14C-oleic acid was added
to the apical side, and cells were incubated for 18 hr in the presence
of the drug. 14C-TG contents in the cellular lysate
(synthesis) and basolateral medium (secretion) were measured. All
values represent mean ± S.E. Significantly different from
vehicle: **, P < .01 (unpaired t test).
|
|
View larger version (32K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of TG secretion in Caco-2 cells by AE0047.
Caco-2 cells were incubated for 48 hr with 107 to
105 M AE0047 or 105 M nilvadipine, and
14C-TG levels were measured. All values represent mean ± S.E. (n = 6). Significantly different from vehicle:
**, P < .01; *, P < .05 (Dunnett's method).
|
|
View larger version (42K):
[in this window]
[in a new window]
|
Fig. 6.
Apo B antigen contents in the cell and medium of
Caco-2 cells incubated with AE0047. Caco-2 cells were incubated for 48 hr with 105 M AE0047. Thereafter, 14C-oleic
acid was added to the apical side, and cells were incubated for 18 hr
in the presence of the drug. Apo B antigen was measured by
enzyme-linked immunosorbent assay, and 14C-TG contents of
the cellular lysate and basolateral medium were measured as described
in Methods. All values represent mean ± S.E. (n = 5 or 6). Significantly different from vehicle: *, P < .05 (unpaired t test). TG content of the basolateral medium, but
not of the cellular lysate, was significantly lower in the AE0047 group
than in the vehicle group (data not shown).
|
|
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7.
Synthesis of TG, cholesterol and FFA in a sliced
liver from Zucker rats treated with AE0047 or nilvadipine. Rats were
administered with AE0047 or nilvadipine as described in the legend to
figure 2, and liver slices were prepared. Synthesis of
14C-labeled TG, cholesterol and FFA in the slices was
measured as described in Methods. All values represent mean ± S.E. (n = 3-5).
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 8.
Total incorporated lipoprotein in HepG2 cells exposed
to LPDS. Cells were incubated in medium containing 5% LPDS for 24 hr; then, 1 µg/ml 125I-labeled LDL ( ), IDL ( ) or VLDL
( ) was added into the medium. Specific association and degradation
of the lipoproteins in the cell were separately measured, and the sum
is expressed as incorporated lipoproteins. All values represent
mean ± S.E. (n = 4).
|
|
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of AE0047 on the incorporation of LDL (A), IDL
(B) and VLDL (C) into HepG2 cells. Cells were incubated with various concentrations of AE0047 ( ), 107 M;  ,
106 M;  , 105 M) or vehicle ( ) for 48 hr, and 1 µg/ml labeled lipoprotein was added to the medium. All
values represent mean ± S.E. (n = 4).
Significantly different from vehicle at 6 hr: **, P < .01 (Dunnett's method).
|
|
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 10.
Effects of AE0047 and nilvadipine (, vehicle;
, 107 M; 106 M; ,
105 M) on the VLDL incorporated into HepG2 cells. Cells
were incubated with 5% LPDS and drugs for 48 hr, and 1 µg/ml labeled
VLDL was added. All values represent mean ± S.E.
(n = 4). Significantly different from vehicle at 6 hr:
**, P < .01 (Dunnett's method).
|
|
| |
Methods |
Materials.
AE0047 was synthesized in our
laboratory. Nilvadipine was obtained from Fujisawa Pharmaceutical Co.
(Tokyo, Japan) and purified in our laboratory. LPL (derived from
Pseudomonas sp.) and lipid assay kits for total cholesterol, TG and HDL
cholesterol were purchased from Wako Chemicals (Osaka, Japan). BSA
(essentially fatty acid free), nonessential amino acid solution and
LPDS were obtained from Sigma Chemical Co. (St. Louis, MA). The protein assay kit was obtained from BioRad Laboratories (Hercules, CA), 14C-oleic acid (50-62 mCi/mmol) was from Amersham
International (Bucks, UK), sodium [125I]iodide was from
DuPont/NEN Research Products (Dreiech, Germany) and FCS was from ICN
Biochemicals Japan (Osaka, Japan).
Cell culture insert (0.4-µm pores, 4.9 cm2) and six-well
tissue culture plates were purchased from Becton Dickinson (Lincoln Park, NJ). Silica gel G TLC plates were obtained from E. Merck (Darmstat, Germany), and collagen-coated tissue culture dishes were
from Iwaki Glass (Chiba, Japan).
Animals.
Female obese Zucker rats (10 or 11 weeks old) were
obtained from Charles River Inc. (Wilmington, MA) and housed for 
1
week before study. Animals were maintained on normal laboratory chow (CE-2) and water ad libitum.
Drug administration and plasma lipid determination.
AE0047
and nilvadipine (40 mg each) were dissolved in 0.4 ml of 99.5% ethanol
and 0.1 ml of Tween 80 and then diluted with water to the desired
concentrations. These solutions were prepared each day immediately
before use. Then, 10 ml/kg concentrations of the drug solutions,
corresponding to 3 or 10 mg/kg AE0047 and 10 mg/kg nilvadipine, were
orally administrated once per day for 7 days to Zucker rats
(n = 8-11). Animals were deprived of food overnight
before blood sampling but were allowed free access to tap water. Two
hours after the final administration on day 7, the animals were
anesthetized with CO2, blood was withdrawn from the heart
and plasma was prepared by centrifugation. Levels of plasma total
cholesterol, TG and HDL cholesterol were measured using commercial
assay kits. Chylomicrons, VLDL and LDL were analyzed by heparin-calcium
precipitation (Matzno et al., 1994).
Determination of lipase activities in postheparin plasma.
AE0047 and nilvadipine were orally adminsitrated for 7 days to Zucker
rats as described above (n = 7 or 8). Two hours after the last administration, 1000 IU/kg heparin was injected intravenously. Postheparin plasma was prepared, and lipase activities were measured as
reported (Matzno et al., 1994).
Synthesis and secretion of TG and apo B in Caco-2 cells.
Caco-2 cells were obtained from American Type Culture Collection
(Rockville, MD) and grown in medium A [Eagle's minimum essential medium (DMEM) supplemented with 1% (v/v) nonessential amino acids, 100 IU/ml penicillin G, 0.1 mg/ml streptomycin sulfate and 20% heat-inactivated FCS] in a humidified atmosphere of 95% air/5% CO2 at 37°C. The cells were subcultured in 75-ml flasks.
Monolayers were studied at passages 23 to 27.
To study TG and apo B, Caco-2 cells (5 × 105
cells/1.5 ml of medium A) were seeded onto filters (cell culture
insert) that were placed in a well of six-well plates containing 2 ml/well of medium A. Cells reached confluence within 2 or 3 days. Media of the upper (apical) and lower (basolateral) sides were exchanged with
fresh media every 2 or 3 days until the transepithelial electrical resistance reached ~300 
cm2. The resistance was
measured using a Millicell-ERS (Millipore, Bedford, MA).
At 7 or 8 days after seeding, media of the both sides of chamber were
changed to medium B (medium A supplemented with 1.5% BSA) containing
AE0047 or nilvadipine. The drugs were dissolved in dimethylsulfoxide,
and the final concentration in the medium was 
0.1%. The cells were
incubated with the drugs for the indicated periods; then, 1 µCi of
14C-oleic acid (400 µM) was added to the apical side.
After a 16-hr incubation, 14C-TG was extracted from the
basolateral medium and cell lysates according to the method of Bligh
and Dyer (1959). TG was separated by TLC using petroleum ether/ethyl
ether/acetic acid (80:20:1, v/v) as the solvent system. Radioactivity
in the TG fraction was measured using a liquid scintillation counter.
Apo B secretion was examined under the same conditions as described
above. After an 18-hr incubation, the apo B contents of the basolateral
medium and cell lysate were measured by enzyme-linked immunosorbent
assay. Briefly, medium or cell suspension was placed into 96-well
plates and incubated for 17 hr at 4°C. After blocking nonspecific
binding with 5% skim milk in PBS, plates were washed three times with
TTBS (10 mM Tris-buffered saline containing 0.01% Tween 80, pH 7.4),
and then 50 µl of anti-apo B antibody (diluted ×5000 with PBS
containing 0.1% BSA) was added and the plates were incubated for 2 hr.
After two washes with TTBS, peroxidase-conjugated anti-goat IgG
(diluted ×5000 with PBS containing 0.1% BSA) was added and incubated
for 2 hr. After washing with TTBS, peroxidase was detected with
o-phenylenediamine, and the amount of antigen was determined
as absorbance at 490 nm.
Synthesis of TG and FFA in sliced liver from Zucker rats.
Zucker rats (n = 3-5) were administered AE0047 and
nilvadipine for 7 days as described above. At 2 hr after the last
administration, rats were bled from the heart, and the livers were
perfused with saline. The livers were removed and sliced. TG and FFA
synthesis was examined according to Yagasaki et al. (1984).
Briefly, liver slices weighing 200 mg were placed in a screwcap tube
(10 ml) containing 2 ml of Krebs-Ringer phosphate buffer, pH 7.4. and 0.8 mCi of 14C-acetate. The tube was gassed with 100%
O2 and incubated at 37°C for 3 hr in a metabolic shaker
at 125 strokes/min. Thereafter, the tube was placed on ice to stop the
reaction, and the liver was homogenized. TG and FFA labeled with
14C were extracted from the homogenate with
chloroform/methanol (2:1, v/v) and separated on TLC, and radioactivity
was measured as described above.
Preparation and 125I-labeling of lipoproteins.
Fresh blood collected from healthy human volunteers was immediately
mixed with 1 mg/ml EDTA and centrifuged (1500 × g, 10 min), and the plasma was collected. VLDL (d < 1.006) and LDL
(1.006 < d < 1.067) were separated by ultracentrifugation
at 110,000 × g for 20 hr and stored at 4°C in the
presence of 5 mM EDTA.
125I-LDL and 125I-VLDL were prepared as
previously described (Yamauchi et al., 1996).
125I-IDL was prepared from 125I-VLDL by lipase
digestion. Briefly, 30 µl of 125I-VLDL (0.1 mg of
protein/ml) was mixed with 30 µl of digestion buffer (0.15 M NaCl,
4% BSA in 0.2 M Tris · HCl, pH 8.6) and LPL (1 IU/ml) and
incubated at 37°C for 1 hr.
Incorporation of lipoproteins by HepG2 cells.
HepG2 cells
were subcultured in 100-cm2 collagen-coated tissue culture
dishes containing Williams' E medium supplemented with penicillin G
(100 IU/ml), 0.1 mg/ml streptomycin sulfate (medium C) and 10%
heat-inactivated FCS.
For the following experiment, 2.5 × 105 cells/well/ml
of HepG2 cells were seeded onto 12-well plates. On the following day, cells were incubated for 48 hr with medium C containing the drugs and
5% LPDS. The media were exchanged with 1 ml of media C containing labeled lipoproteins (1 µg/ml), drugs and 0.1% BSA. After incubation for the indicated periods, 200 µl of the medium was removed, mixed with 100 µl of 20% trichloroacetic acid, stored at 4°C for 30 min
and centrifuged (10,000 × g for 5 min).
Trichloroacetic acid-soluble radioactivity was measured. The remaining
cells were washed with ice-cold PBS and scraped off the dishes with a
rubber policeman. The radioactivity levels were measured, and the data
represent total lipoproteins incorporated by HepG2 (i.e.,
sum of association and degradation).
Statistical analysis.
Statistical analysis was made with an
unpaired t test for two groups. For more than three groups,
we used one-way analysis of variance and then Dunnett's
multiple-comparison method (Wallenstein et al., 1980).
Results were considered significant at P < .05.
| |
Results |
In vivo effects of AE0047 on plasma lipid profiles of
obese Zucker rats.
Plasma lipid profiles of hypertriglyceridemic
Zucker rats given AE0047 or nilvadipine for 7 days are shown in figure
2. AE0047 (3 and 10 mg/kg/day) dose-dependently decreased plasma TG
level without altering total cholesterol and LDL levels. AE0047 (10 mg/kg) also decreased plasma TG-rich lipoproteins (chylomicrons and
VLDL), whereas HDL cholesterol significantly increased. The administration of 10 mg/kg nilvadipine, a typical DHP-type calcium antagonist, tended to decrease plasma TG and chylomicron levels.
Effect of AE0047 on plasma LPL activity in Zucker rats.
To
assess the mechanism by which AE0047 decreased the plasma TG level of
Zucker rats, we administered AE0047 or nilvadipine for 7 days and
measured the lipase activity in postheparin plasma, which represents
the sum of the LPL and H-TGL activities. The results showed that
neither drug affected the lipase activity (fig. 3).
Inhibition of the secretion of TG and apo B by AE0047 in Caco-2
cells.
Dietary TG is adsorbed and secreted as chylomicrons from
the intestine. To check this process, we used the human intestinal cell
line (Caco-2). Approximately 80% of the secreted 14C-TG
appeared in the basolateral medium when 14C-oleic acid was
added to the apical side of Caco-2 cells and incubated for 18 hr (data
not shown). The effect of AE0047 on the synthesis and secretion of TG
in Caco-2 cells is shown in figures 4 and 5. AE0047 (105
M) incubated with the cells for 24 hr before the addition of 14C-oleic acid reduced the amount of 14C-TG in
the basolateral medium by ~35% (fig. 4). In contrast, cellular TG
synthesis was not significantly affected. On extending the incubation
with AE0047 to 48 hr, the effect was more pronounced and TG secretion
was suppressed by ~87% without changing the cellular TG synthesis.
TG secretion was depressed in a dose-dependent manner between
107 and 105 M AE0047 (fig. 5). On the
contrary, 105 M nilvadipine affected neither cellular TG
synthesis nor its secretion (fig. 5).
We next examined the effect of AE0047 on the secretion of apo B, a
major apolipoprotein constituent of TG-rich lipoproteins (VLDL and
chylomicrons). As shown in figure 6, 105 M AE0047
inhibited the basolateral secretion of apo B without affecting its
cellular level.
Effect of AE0047 on TG and FFA synthesis in hepatocytes.
We
evaluated the effect of AE0047 and nilvadipine on the hepatic synthesis
of FFA and TG by using liver slices from Zucker rats that had been
treated for 7 days with the drugs. Figure 7 shows that neither AE0047
nor nilvadipine influenced the hepatic synthesis of FFA and TG.
Enhancement of VLDL incorporation by AE0047 in HepG2 cells.
Sequential reactions including binding, incorporation and degradation
by the liver constitute the major pathway of lipoprotein clearance from
the circulation. We evaluated the effect of AE0047 or nilvadipine on
this step using a human hepatic cell line (HepG2). Figure 8 shows the
total incorporation (association and degradation) of
125I-labeled LDL, IDL and VLDL. The total incorporation
efficiency of VLDL was only 45.9% and 70.3% of that of LDL and IDL,
respectively, after a 6-hr incubation. We next studied the
incorporation of lipoproteins in HepG2 cells exposed to AE0047 (fig.
9). Cells were incubated for 48 hr with AE0047 before the addition of
lipoproteins. AE0047 (0.1-10 µM) did not affect the incorporation of
labeled LDL (fig. 9A) or IDL (fig. 9B). In contrast, VLDL incorporation was significantly increased by 10 µM AE0047 (fig. 9C).
We also studied the effect of various concentrations of nilvadipine on
the incorporation of VLDL by HepG2 cells (fig. 10). In contrast to
AE0047, this calcium antagonist did not affect the VLDL incorporation,
even at a concentration of 10 µM.
| |
Discussion |
AE0047 decreases plasma TG levels in Zucker rats.
We evaluated
the effect of AE0047 on TG metabolism in the hypertriglyceridemic obese
Zucker rat. This animal model is characterized by primary
hypertriglyceridemia (Bray, 1977) and is widely used to evaluate the
hypolipidemic action of drugs (Kasin et al., 1992). We
demonstrated that AE0047 significantly decreased plasma TG and TG-rich
lipoprotein levels and increased HDL levels in obese Zucker rats (fig.
2).
It is controversial whether hypertriglyceridemia is an independent risk
factor for CHD. However, recent epidemiological studies have confirmed
that it is indeed a risk factor in certain populations (Cambien
et al., 1986; Castelli, 1986; Fontbonne et al.,
1989). The association of hypertriglyceridemia with a low plasma HDL level (Gotto, 1992), high procoagulant activities (Hoffman et al., 1992; Simpson et al., 1983) and high plasminogen
activator inhibitor activities (Raccah et al., 1993) may
facilitate atherosclerosis. Therefore, hypertriglyceridemia must be
treated to prevent CHD, especially in patients with multiple risk
factors for CHD, such as hypertension, obesity and diabetes. The
hypotriglyceridemic action of AE0047, as well as its potent
antihypertensive activity, may help reduce the incidence of CHD. We
previously showed that AE0047 has antiatherogenic activity in rabbits
fed with cholesterol (Yamanaga et al., 1993).
Hypothesis of TG-lowering and HDL-increasing actions.
We first
evaluated the effect of AE0047 on the lipase activities of Zucker rats
because LPL and H-TGL play important roles in TG metabolism by
degrading TG of VLDL, IDL and chylomicrons. However, AE0047
administration did not change the plasma lipase activities (fig. 3).
Moreover, AE0047 did not affect the hepatic synthesis of FFA and TG in
the Zucker rats (fig. 7). In contrast, AE0047 inhibited TG secretion in
Caco-2 cells in a dose- and time-dependent fashion without affecting
cellular TG synthesis (figs. 4 and 5). AE0047 also decreased the
secretion of apo B (fig. 6), a major apolipoprotein in VLDL and
chylomicrons. These results suggested that AE0047 inhibits the
secretion of TG-rich lipoprotein particles into the medium in Caco-2
cells. In vitro data also suggested that one of the
TG-lowering mechanisms of AE0047 is the reduction in chylomicron
secretion from intestine.
The above notion that AE0047 may reduce intestinal chylomicron
secretion is supported by the following. Hughes et al.
(1988) demonstrated that calcium ionophores increase the synthesis and secretion of apo B in Caco-2 cells. Strauss and Jacob (1981) showed that calcium stimulates the secretion of TG in the isolated jejunum of
the hamster. On the contrary, a benzothiazepine-type calcium antagonist
(TA-3090) inhibits TG secretion in jejunal explants (Levy et
al., 1992).
The studies on HepG2 cells indicated another possibility. AE0047
increased only VLDL incorporation in HepG2 cells (fig. 9), suggesting
that AE0047 facilitates the hepatic clearance of VLDL in Zucker rats.
Then, to explain the selectivity of hepatic VLDL incorporation, we
noticed the role of hepatic LRP. In fact, VLDL and IDL include both apo
B and apo E in their particles. However, the portion of these
apolipoproteins in the cellular binding is different. Previous studies
(Connely and Kuksis, 1981; Granot et al., 1994) showed that
large and TG-rich lipid particles caused more rapid binding to apo
E-specific sites (i.e., LRP). On the other hand, Krul
et al. (1985) reported that apo B-specific binding in VLDL
increased with the reduction of diameter and that virtually all of LDL
binding is mediated by apo B. Taken together, these results suggested
that VLDL incorporation is mainly mediated by apo E (through LRP),
whereas IDL is mainly mediated by apo B (through LDL receptor). Thus,
LRP enhancement by AE0047 in HepG2 cells might increase only the VLDL
incorporation.
In addition, it is likely that hepatic LRP enhancement also
increases lipolysis of lipoproteins. The present study showed that
AE0047 treatment did not affect the plasma lipase activity (fig. 3).
Then, we considered the possibility that AE0047 enhanced the
lipase-VLDL association rate. We suggested that AE0047 stimulated hepatic LRP manifestation as mentioned above. LRP also recognizes the
lipase/lipoprotein complex as well as apo E (Krieger and Herz, 1994);
therefore, this multifunctional receptor might help the lipase/lipoprotein interaction, lipolysis and resulting HDL formation. Further investigations remain to clarify these hypotheses.
Although the above mechanisms can explain the in vivo action
of AE0047 against the plasma TG level, other mechanisms that were not
tested in the present study might be involved. For example, AE0047
in vivo might alter the composition of apolipoproteins because enrichment of apo E or depletion of apo C in TG-rich
lipoproteins accelerates their uptake by the liver (Kowal et
al., 1990; Takahashi et al., 1995). Moreover, AE0047
might inhibit VLDL secretion, as reported for a benzothiazepine-type
calcium antagonist (Nossen et al., 1987). An indirect
mechanism such as an enhanced hepatic blood flow might also be
involved.
Plasma TG level may be reduced by AE0047 through its calcium
channel-blocking activity.
There are discrepancies regarding the
effect of calcium antagonist on the TG metabolism; therefore, it is
difficult to determine whether the plasma TG level is reduced through
its calcium channel-blocking activity. Clinical data suggest that
calcium antagonists generally do not affect plasma lipid profiles
(Giles, 1992; Kasiske et al., 1995), but results have
differed, presumably due to clinical settings. Nicardipine is a typical
example that may lower (de Cesaris et al., 1991; Kihara,
1991), have no effect on (Soro et al., 1990; Wang et
al., 1993) or increase (Naukkarinen, 1988) plasma TG levels. Similarly, many calcium antagonists, such as amlodipine (Canale et al., 1991), nifedipine (Kazumi et al., 1990;
Maldonado et al., 1992), isradipine (Morris et
al., 1993) and nilvadipine (Berger and Albert, 1992), sometimes
decrease plasma TG levels.
In the present study, AE0047 significantly decreased plasma TG and
chylomicron levels of Zucker rats. In contrast, nilvadipine tended to
decrease them, but the effects were not statistically significant (fig.
2). The discrepancy between AE0047 and nilvadipine on the in
vivo efficacy can be explained by the following. First, we showed
here that AE0047, but not nilvadipine, inhibited the intestinal
chylomicron secretion and enhanced the hepatic uptake of VLDL. Because
intestine and liver are the major organs responsible for the lipid
metabolism, these findings strongly suggest that AE0047 decreased
plasma TG level mainly by the above mechanisms, which were not shared
by nilvadipine. In addition, the discrepancy may be partially due to
the poor bioavailability of nilvadipine (~5% in rats; Tokuma
et al., 1987) compared with that of AE0047 (~20%; Ohkubo
et al., in press) if the calcium channel-blocking activity
of AE0047 caused the reduction of plasma TG in Zucker rats. The
observation that the hypotensive activity of AE0047 in normotensive
rats was twice than that of nilvadipine1
might reflect the difference in bioavailability. As mentioned above,
the relationship between the calcium channel-blocking activity and TG
metabolism remains obscure, however, so more detailed and precise
studies are needed to address the above issue.
In conclusion, AE0047 is a calcium antagonist that decreases plasma TG
and increases plasma HDL levels in Zucker rats. Experiments in
vitro suggest that the plasma TG is reduced through the
suppression of chylomicron secretion from the intestine, as well as by
enhancement of VLDL uptake by the liver. Treating hypertensive patients
who have hypertriglyceridemia with AE0047 might reduce the risk factors of CHD.
Y. Ohtaki, T. Uchida, M. Nishikawa, and K. Yamanaga,
unpublished observations.
DHP, dihydropyridine;
TG, triglyceride;
HDL, high-density lipoprotein;
LDL, low-density lipoprotein;
VLDL, very low
density lipoprotein;
apo, apolipoprotein;
CHD, coronary heart disease;
LPL, lipoprotein lipase;
BSA, bovine serum albumin;
LPDS, lipoprotein-deficient serum;
FCS, fetal calf serum;
TLC, thin-layer
chromatography;
PBS, phosphate-buffered saline;
TTBS, Tris-buffered
saline containing Tween 80;
FFA, free fatty acid;
IDL, intermediate-density lipoprotein;
H-TGL, hepatic triglyceride lipase;
LRP, LDL receptor-related protein.
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
-chlorobenzyloxy)benzyl nicotinate (KCD-232) on triglyceride and fatty acid metabolism in rats.
Biochem. Pharmacol.
33: 3151-3163, 1984[Medline].
