© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Biphasic Effects of the Natural Estrogen 17ß-Estradiol on Hepatic Cholesterol Metabolism in Intact Female Rats
From the Metabolism Unit (P.P., B.A., B.F., M.R.), Center for Metabolism and Endocrinology, Department of Medicine, and the Molecular Nutrition Unit, Center for Nutrition and Toxicology Novum, Karolinska Institute at Huddinge University Hospital, Stockholm, Sweden; and the Division of Reproductive Endocrinology (A.S-E., B.F., H.E.), Department of Woman and Child Health, Karolinska Institute at Karolinska Hospital, Stockholm, Sweden.
Correspondence to Paolo Parini, MD, PhD, CME, M63, Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail paolo.parini{at}cnt.ki.se
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Abstract |
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Abstract—The protective influence of estrogens in cardiovascular disease is believed to be partly due to beneficial effects on cholesterol metabolism. Much of the experimental data are based on models in which synthetic estrogens have been used in pharmacological doses, and therefore, the physiological role of estrogens in cholesterol metabolism is uncertain. To evaluate this important issue, we performed experiments in intact female rats with use of the natural estrogen 17ß-estradiol (E2) administered either subcutaneously or orally. After physiological doses of E2 (
0.04 mg ·
kg-1 · d-1) were
administered, plasma levels of high density lipoprotein (HDL)
cholesterol and apolipoprotein (apo) A-I were increased. In
the liver, 3-hydroxy-3-methylglutaryl coenzyme A reductase and
cholesterol 7
-hydroxylase activities were increased, as
well as cholesterol 7-hydroxylase mRNA levels. These
effects were abolished during treatment with higher doses of E2,
whereas apo A-I mRNA increased in a dose-dependent way. After treatment
with pharmacological doses of E2 (
0.2 mg ·
kg-1 · d-1), the
number of hepatic low density lipoprotein receptors increased and
plasma cholesterol was reduced. These effects were similar
after both oral and subcutaneous administration of E2. Our results show
that the responses to E2 are biphasic: plasma HDL, apo A-I, and hepatic
enzyme activities governing bile acid and cholesterol
synthesis increased only at physiological doses of
E2. At pharmacological doses of E2, hepatic low density lipoprotein
receptors are stimulated and plasma cholesterol is reduced.
Therefore, under physiological conditions, E2
exerts its major effects on hepatic cholesterol
metabolism through mechanisms other than stimulation of low
density lipoprotein receptor expression.
Key Words: apolipoprotein A-I • bile acids • lipoproteins • LDL receptors • estrogen receptors
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Introduction |
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Endogenous sex steroids are believed to protect premenopausal women against the development of coronary heart disease.1 2 Estrogens have been reported to have a number of potentially beneficial effects on lipoprotein metabolism, resulting in reduced LDL and increased HDL cholesterol in plasma.1 2 3 4 5 Attempts to identify the mechanisms by which endogenous estrogens modulate their effects in vivo are hampered by the difficulty of interpreting data from experimental models. Thus, the varying effects observed may be related to species, sex, and initial hormonal status (eg, postmenopausal or ovariectomized versus fertile). Differences in dose and efficiency between estrogen preparations, as well as the mode of administration (eg, oral versus parenteral), may also be important.
Much of our knowledge of estrogen’s effects on lipoprotein metabolism is based on studies in rats. In this species, the plasma total and HDL cholesterol levels are higher in females than in males.6 7 High doses of estrogens (1 to 5 mg · kg-1 · d-1 of ethynyl estradiol) reduce plasma cholesterol levels in male rats.8 This effect is in part caused by increased elimination of LDL from the plasma9 10 due to an increased hepatic LDL receptor (LDLR) expression.11 12 These responses to pharmacological doses of estrogens can be quenched by simultaneous administration of antiestrogens,13 indicating that the effects are estrogen receptor (ER) mediated. However, owing to the high doses used, it is still unclear whether these effects of estrogens are physiologically relevant.
During treatment with low doses of estrogens, HDL
cholesterol is increased in the rat.14 15
Effects of low doses of 17ß-estradiol (E2) on the activity of the
rate-limiting enzyme in the conversion of cholesterol to
bile acids, cholesterol 7-hydroxylase (C7OH), have
also been described.16 The effects of pharmacological
doses of E2 on this enzyme are less clear.17
The current investigation was performed to study the physiological relevance of the effects of estrogens on hepatic cholesterol metabolism in the intact female rat. Our results clearly show that physiological doses of E2 increase plasma HDL, apo A-I, and the enzymatic activities regulating the synthesis of bile acids and cholesterol. At pharmacological doses of E2, these responses are absent, whereas hepatic LDLRs are induced together with reduced plasma cholesterol.
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Methods |
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Materials
E2 (17ß-estradiol; catalog No. E-4) was from Sigma Chemical Co, and Alzet osmotic minipumps (model 2001) were from Alza Corp. All other reagents and chemicals were from previously described sources.18 19
Animals and Experimental Procedure
Altogether, 110 female Sprague-Dawley rats (250 to 300 g, 8
to 9 weeks old; B&K Universal, Stockholm, Sweden) were used in 3
separate experiments, for which each group consisted of 5 or 6 rats.
Animals were kept under standardized conditions with free access to
water and chow. The light cycle hours were between 6 AM and
6 PM. All studies were approved by the institutional Animal
Care and Use Committee.
The drugs were dissolved in propylene glycol and administered
subcutaneously under light ether anesthesia at 9
AM for 7 days. In the experiment in which E2 was infused
subcutaneously, osmotic minipumps were implanted in the dorsal region.
A small (5- to 10-mm) incision was made, and a subcutaneous tunnel was
created with a pair of forceps. Minipumps were put in place and the
skin was sutured. Controls were sham-operated with the same surgical
procedure. The control groups in all experiments received vehicle. When
the experiments were terminated (10 to 11 AM), the rats
were anesthetized with ether. Blood was drawn by cardiac
puncture and the animals killed by cervical dislocation. Livers were
perfused with ice-cold PBS (140 mmol/L NaCl, 2.7 mmol/L KCl,
and 9.5 mmol/L phosphate buffer, pH 7.4), removed, and immediately
frozen in LN2. In the experiment in which the
activities of C7OH and 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase were determined, a piece of fresh liver (
1 g)
was taken immediately after the perfusion for preparation of microsomes
as described below.
Cholesterol Assays
Cholesterol in the plasma and fast protein liquid
chromatography (FPLC) fractions was assayed with the
Boehringer Mannheim cholesterol assay kit (MPR 2 1
442 350) and a 5.2 mmol/L cholesterol standard from
Merck. Size fractionation of lipoproteins by FPLC was performed on a
Superose 6B column and using a previously described
system.19 Equal volumes of plasma from individual animals
were pooled (2.4 mL), and the density was adjusted to 1.21 g/mL with
KBr. After ultracentrifugation at
100x103g for 48 hours, the supernatant was
adjusted to 1.6 mL with 0.15 mol/L NaCl, 0.01% EDTA, and 0.02%
NaN3, pH 7.3. One milliliter of this solution,
corresponding to 1.5 mL of plasma, was injected onto a
540x18-mm Superose 6B column after filtration through a Millipore
0.45x10-3-mm mixed cellulose ester filter. FPLC
fractions of 2 mL were collected at a flow rate of 1 mL/min.
SDS–Polyacrylamide Gel Electrophoresis (PAGE) Separation
of Apolipoproteins
From each group, 7.5 or 15 µL of
ultracentrifuged plasma was added to loading buffer
(final volume, 95 µL) and boiled for 5 minutes in the presence of 5%
(vol/vol) 2-mercaptoethanol. Aliquots of 70 µL were loaded onto a 4%
to 20% gradient SDS/polyacrylamide gel and separated for 4
hours at 45 mA. Gels were stained with Coomassie Blue. For reference,
wide molecular-mass standards (Bio-Rad Laboratories) as well as human
LDL and HDL were used. The bands corresponding to apo A-I were
quantified by densitometry.
Preparation of Hepatic Membranes and Ligand Blot Assay of
LDLRs
Liver membranes were prepared from pools of liver as described
previously.18 Gels (6% SDS/polyacrylamide) were
loaded with the indicated amount of membrane protein prepared from
pooled samples of liver. Size markers were reduced with mercaptoethanol
and boiled. Filters were incubated with
125I-labeled rabbit ß-migrating VLDL, as
described previously.18 Filters were exposed on Dupont
Cronex film. LDLR expression was quantified by using a Fujix
Bio-imaging analyzer (BAS 2000, Fuji Photo Film Co). The values
of the 120-kDa bands were expressed in arbitrary units after
subtraction of filter background.
Total Nucleic Acid Preparation
Frozen liver specimens (0.2 g) were homogenized in 4
mL of SET buffer (1% [wt/vol] SDS, 10 mmol/L EDTA, and 20
mmol/L Tris-HCl, pH 7.5) with a Polytron (Kinematica, type PT 10/35,
Kriens). The samples were subsequently sonicated on ice by 10 pulses in
a Branson B 15 sonifier and digested with proteinase K (200 µg/mL)
for 45 minutes at 45°C. Total nucleic acid was precipitated with
ethanol after phenol-chloroform extraction, and the pellet was
suspended in 300 µL of 0.2x SET buffer. The concentration of total
nucleic acid in the samples was measured at 260 nm and assuming
that 1 optical density unit was equivalent to 40 µg of total nucleic
acid per milliliter. The DNA concentration was measured
fluorometrically at 458 nm.
Quantification of mRNA
The mRNA levels for the LDLR, HMG-CoA reductase, and C7OH
were quantified by a solution hybridization titration assay with the
use of mouse cRNA probes.20 The mRNA levels for apo A-I
were quantified by using a rat cRNA probe corresponding to
nucleotides 16 to 419 in the rat apo A-I cDNA. The probe
was developed from a plasmid containing the full cDNA sequence of rat
apo A-I, kindly provided by Dr Bart Staels, INSERM, Lille Cedex,
France. Hybridization specificity was tested on total nucleic acid
extracts from different rat organs (data not shown). The probe used for
ER mRNA corresponds to nucleotides 1470 to 2062 of the
mouse ER cDNA sequence, which encode the C-terminal half of
the steroid-binding domain E and all of domain F.21
The slopes of the linear hybridization signals were calculated by the
method of least squares and compared with the slope generated by the
respective synthetic mRNA standard. Data are expressed as
attomoles (10-18 moles) of mRNA per
microgram of total nucleic acid.
Preparation of Cytosol and ER Determination by Enzymatic
Immunoassay
All procedures were performed at 0°C to 4°C. Liver samples
(0.5 to 1 g) were freeze-dried in glass centrifuge tubes
for 92 hours and cut with a scalpel. Two milliliters of 0.4 mol/L KCl
in TEM-SH buffer (10 mmol/L Tris-HCl, 1.5 mmol/L EDTA,
10 mmol/L sodium molybdate, and 1 mmol/L monothioglycerol, pH
7.4) was added to the tubes. The samples were extracted for 30 minutes,
vortexed every 5 minutes, and then centrifuged at
7700g for 20 minutes. The supernatant was collected and the
incubation and centrifugation procedures repeated with
1 mL of KCl in TEM-SH buffer. The supernatants were centrifuged
again in a swing-out rotor at 230 000g for 65 minutes.
Protein determination was performed according to Lowry et
al.22 The protein concentration in samples was
adjusted to 1 mg/mL, and an enzymatic immunoassay was performed by
using a commercial kit and following the manufacturer’s instructions
(Abbott Scandinavia AB).
Activities of C7OH and HMG-CoA Reductase
Microsomes were prepared by differential
ultracentrifugation of individual liver
homogenates in the absence of fluoride as described
previously.23 24 The activity of C7OH was determined as
the formation of 7-hydroxycholesterol (pmol ·
min-1 · mg-1
protein) from endogenous microsomal cholesterol
by using isotope dilution mass spectrometry.24 Microsomal
HMG-CoA reductase activity was assayed by determining the conversion of
[14C]HMG-CoA to mevalonate and expressed as
picomoles formed per minute per milligram protein.23 The
enzyme assays were carried out in duplicate.
Statistics
Data are presented as mean±SEM. The significance
of differences between groups was tested by 1-way ANOVA, followed by
planned comparison or post hoc comparisons of group means according to
least significant difference methods (Statistica software, Stat Soft).
To stabilize the variances, data were logarithmically transformed when
a correlation between means and variances was found.25
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Results |
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In the first experiment, we wanted to determine the effect of increasing doses of E2, given as daily, single subcutaneous injections, on plasma lipoproteins in intact adult female rats. After 1week of treatment with E2 at doses ranging from 0.01 to 4 mg · kg-1 · d-1*, it was found that plasma total cholesterol was reduced in animals treated with high doses (1 and 4 mg · kg-1 · d-1) of E2 (not shown). An
30% reduction was
present in animals receiving 1 mg ·
kg-1 · d-1
(P<0.01), and a 55% reduction was seen in those receiving
4 mg · kg-1 ·
d-1 (P<0.001). Separation of
lipoproteins by FPLC showed that high doses of E2 (1 and 4 mg ·
kg-1 · d-1)
reduced cholesterol within both LDL and HDL fractions
(Figure 1A
). At low doses of E2, HDL
cholesterol increased (Figure 1A). Analysis
of apolipoproteins in ultracentrifuged plasma by SDS-PAGE
separation showed that apo A-I levels were increased among animals
receiving low-dose E2 and that this was most pronounced at a dose of
0.1 mg · kg-1 ·
d-1 (Figure 1B).
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, 0.01 mg ·
kg-1 · d-1 of E2; 
, 0.1 mg ·
kg-1 · d-1 of E2; 
, 1 mg ·
kg-1 · d-1 of E2; and 
, 4 mg
· kg-1 · d-1 of E2. B, Apo A-I
levels in pooled ultracentrifuged plasma. After SDS-PAGE
separation, the bands corresponding to apo A-I were quantified by
densitometry. Changes in lipoprotein profiles or apo A-I levels were
not tested statistically. C, Apo A-I mRNA abundance in hepatic total
nucleic acid extracts from each rat was measured by solution
hybridization. Results are shown as mean±SEM.
Apolipoproteins in animals on the highest dose of E2 (4
mg · kg-1 ·
d-1) could not be measured because of technical
problems. epatic apo A-I mRNA levels increased dose-dependently (Figure 1C
). Analysis of LDLR expression in liver membranes
revealed a 4-fold stimulation at a dose of 1 mg ·
kg-1 · d-1 and an
20-fold stimulation at 4 mg ·
kg-1 · d-1 (Figure 2A). No LDLR stimulation occurred at
lower doses of E2. The LDLR mRNA level was increased 2-fold at a dose
of 1 mg · kg-1 ·
d-1, and no further stimulation was seen at
higher doses (Figure 2B). Assay of hepatic ER expression by
enzyme immunoassay revealed a dose-dependent increase that was maximal
at 1 mg · kg-1 ·
d-1 of E2 (Figure 2C). Quantification of
hepatic ER mRNA showed a dose-dependent reduction that was most
pronounced in animals with the highest abundance of hepatic ERs (Figure 2D).
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We then wanted to clarify whether the route of administration was
important for the level of stimulation of hepatic LDLRs by E2. For this
purpose, increasing doses (0.008 to 1 mg ·
kg-1 · d-1) of E2
were given daily for 5 days to intact female rats by oral or
subcutaneous route. Analysis of total plasma
cholesterol showed reductions at high doses of E2, which
were independent of the route of administration (Figure 3A). Hepatic LDLR expression was
similarly increased in both groups of animals (not shown).
Analysis of hepatic LDLR mRNA abundance revealed that oral
administration of E2 increased the LDLR mRNA levels significantly by
2-fold at 1 mg · kg-1 ·
d-1 only (Figure 3B). Subcutaneous
injection of E2 resulted in a 2-fold increase of LDLR mRNA, not only
at 1 mg · kg-1 ·
d-1 but also at the lower dose of 0.2 mg
· kg-1 · d-1
(Figure 3B). Thus, there was no evidence of a more potent effect
of oral compared with subcutaneous administration of E2.
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Because single, daily bolus injections of E2 may result in
nonphysiological oscillating plasma E2 levels, we
then proceeded to determine whether a more
physiological administration, viz, by continuous
infusion with osmotic minipumps, could alter hepatic
cholesterol metabolism at lower doses of E2.
After 1 week of subcutaneous infusion with E2 into intact female rats,
the animals were killed. Analysis of plasma total
cholesterol showed an 35% reduction among rats
receiving 0.2 mg · kg-1 ·
d-1 of E2 and an 60% reduction in animals
receiving 1 mg · kg-1 ·
d-1. There were no reductions in plasma
cholesterol in animals receiving lower doses of E2 (Figure 4A). When hepatic LDLR expression was
analyzed by ligand blotting, a 2-fold increase was already
found at a dose of 0.04 mg · kg-1
· d-1 (Figure 4B). A 5-fold increase
was observed in animals infused with 0.2 mg ·
kg-1 · d-1 of E2,
whereas a 4-fold increase was seen in animals receiving 1 mg ·
kg-1 · d-1 (Figure 4B). Analysis of LDLR mRNA abundance showed increased
levels only in animals receiving 1 mg ·
kg-1 · d-1 of E2
(Figure 4C).
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We then determined the activities of the rate-limiting enzymes
in bile acid and cholesterol synthesis, viz, C7OH and
HMG-CoA reductase, in hepatic microsomes. The C7OH activity was
stimulated at low doses of E2: a 65% stimulation was present in
animals infused with 0.008 mg · kg-1
· d-1 of E2, and rats infused with 0.04
mg · kg-1 ·
d-1 showed a 45% increase (Figure 5A). No stimulation of C7OH activity
occurred at higher doses of E2. The C7OH mRNA levels were increased
by 60% in animals receiving the lowest dose of E2 (Figure 5B).
At higher doses of E2, there were no significant increases in C7OH
mRNA. The activity of HMG-CoA reductase in hepatic microsomes was
increased by 75% in animals infused with a dose of 0.008 mg
· kg-1 · d-1 E2
(Figure 5C). The induction of HMG-CoA reductase activity by E2
was maximal (90% increase) at a dose of 0.04 mg ·
kg-1 · d-1 (Figure 5C). In animals infused with 0.2 mg ·
kg-1 · d-1 of E2,
the microsomal activity of HMG-CoA reductase was increased by 45%
(Figure 5C). There were no significant changes in HMG-CoA
reductase mRNA levels at any dose (not shown).
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Discussion |
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The present study has established that the metabolic response to E2 is biphasic in intact female rats. Thus, whereas some effects of estrogen, such as increased plasma apo A-I and HDL cholesterol levels as well as stimulated C7
OH and HMG-CoA reductase activities, were already observed in the
physiological dose range, the well-established
effects of estrogens on hepatic LDLR and plasma
cholesterol8 11 clearly required
pharmacological doses. Ovariectomized animals are commonly used as a model in which to study the effects of estrogens. However, it has been previously shown that ovariectomy results in a 2-fold increase in hepatic ER expression in rats.29 Thus, to avoid possible overinterpretation of the data, we chose to study the effects of different doses of estrogen on cholesterol and lipoprotein metabolism in intact female rats. In fertile females (humans as well as rats), there is a day-to-day variation in serum estrogen levels,30 31 and this variation throughout the cycle may be more important for biological responses than has been generally believed. The levels of serum E2 reached at the lower doses were clearly within this range, however.* Furthermore, at least in rats, ER and ER mRNA levels do not vary in the liver as much as in the uterus throughout the ovulatory cycle.31
In agreement with previous work,18 19 32 the increase in LDLR expression obtained at high doses was also present at the mRNA level. This stimulation was much lower than that of LDLR protein, however. The cause for this discrepancy is unknown,18 19 but it may be due to an estrogen-induced increased efficiency of translation of LDLR mRNA.33 The fact that the hepatic ER number was increased by E2 treatment may also contribute to this phenomenon. In preliminary experiments in intact female rats (P.P. et al, unpublished observations, 2000), we found that the stimulatory effect of high-dose E2 on LDLR expression could be completely blocked by the concomitant administration of a highly selective antiestrogen, ICI-182,780.34 Because there was no effect of the antiestrogen when given alone, this finding indicates that the normal LDLR expression in the liver of female rats is not under control of endogenous estrogens, in consonance with our previous observations in the male rat.13 Lending further support to this contention, we have not been able to demonstrate a reduced LDLR expression in ovariectomized rats (P.P. et al, unpublished observations, 2000).
The finding that there was no major difference in LDLR response after oral and subcutaneous administration of E2 may at first seem unexpected, particularly when regarding previous data from human studies, for example.1 2 3 4 However, from analysis of the plasma E2 concentrations,* it appeared that the steroid-metabolizing capacity was saturated at a dose between 0.01 and 0.1 mg · kg-1 · d-1. If higher doses, much above saturation, are required for stimulation of hepatic LDLRs in the rat, then the mode of administration of E2 would not necessarily be of major importance, in agreement with our findings.
In contrast to the findings for the LDLR, we could identify several
important effects on hepatic lipoprotein and cholesterol
metabolism that occurred at
physiological doses of E2. Thus, the plasma apo A-I
level increased by 3-fold at a low E2 dose (0.01 mg ·
kg-1 · d-1) and
was maximal at the 0.1 mg · kg-1 ·
d-1 dose of E2 (Figure 1B). The
disappearance of apo A-I at the highest dose is probably the
consequence of pronounced stimulation of HDL and LDL clearance due to
the very high expression of hepatic LDLRs at that dose. The discrepancy
of these observations in intact female rats with the established
increase in plasma apo A-I levels, which occurs after estrogen therapy
in human females,35 is probably due to species
differences. The concentration of apo E, an efficient ligand for the
LDLR, is indeed higher in rats than in humans, especially in HDL
particles.36 37 An increase in LDLR expression in the
liver of women treated with estrogen would therefore not affect the
plasma levels of HDL cholesterol and apo A-I as it does in
rats. The hepatic apo A-I mRNA levels were not significantly increased
at the lowest dose but displayed a clear dose-dependent relationship
throughout all E2 concentrations. These findings are in agreement with
previous work in vitro38 and clearly also indicate that
the variation of E2 levels within the physiological
range influences plasma apo A-I and HDL cholesterol. Thus,
the higher HDL cholesterol levels in female compared with
male rats7 are probably determined by their higher
endogenous E2 levels. Accordingly, ovariectomized rats have
lower plasma HDL cholesterol.7 Furthermore,
the fact that HDL cholesterol is decreased in response to
tamoxifen treatment in male rats (P.P. et al, unpublished observations,
2000) may argue in favor of a role for the ER in HDL
cholesterol levels in this sex. Thus, estrogen "tonus"
would seem to be an important factor in the normal regulation of HDL
cholesterol levels in the rat.
The activities of C7OH and HMG-CoA reductase were enhanced at low
doses of E2. However, in contrast to the effects on apo A-I, there was
no further stimulation at higher doses of E2. It is clear that the
response patterns for C7OH and LDLR are different. The activity of
HMG-CoA reductase may in part be induced by an increased C7OH
activity at low E2 doses, whereas the reduced activity of the reductase
at high E2 doses is probably related to the massive influx of
lipoprotein cholesterol to the liver via the increased
LDLRs. In a previous study from our group, the total
cholesterol concentration in the livers of rats treated
with high-dose estrogen was indeed shown to be
increased.19 The fact that HMG-CoA reductase mRNA levels
were not altered in these situations of varying enzyme activity may
indicate that posttranscriptional mechanisms of regulation are
predominant.39
Finally, it should be realized that the stimulation of C7OH activity
observed here may not be a direct effect of E2. Low doses of E2 induce
the release of pituitary growth hormone in the intact
rat.40 Growth hormone administration has been shown to
stimulate C7OH activity and mRNA in intact rats.41 42
Therefore, the effects on C7OH after E2 treatment may be secondary
due to an enhanced growth hormone release. Further studies will be
necessary to evaluate this interesting possibility.
In conclusion, our detailed studies on hepatic cholesterol
metabolism in intact female rats treated with a natural
estrogen have established that the responses to this steroid are
biphasic. At low but not at high doses, C7OH and HMG-CoA reductase
activities are increased. At both low and high doses, apo A-I synthesis
is increased in a progressive fashion, resulting in an increase in HDL
cholesterol at low doses. At high (pharmacological) doses,
LDLR expression is stimulated, resulting in drastic reductions of LDL
and HDL cholesterol. The recognition of this response
pattern is most important for understanding the normal regulation of
plasma lipoproteins, because it implies that E2 normally exerts its
major beneficial effects on hepatic cholesterol
metabolism through mechanisms other than the stimulation of
LDLR expression. Although the relevance of these findings to the human
species has not been established, the finding that estrogen at
physiological doses could induce hepatic responses
and affect cholesterol metabolism in the liver
suggests that not only the day-to-day variation in plasma estrogen
levels but also the expression of the ER in the liver may play an
important role in the physiological regulation of
cholesterol metabolism. It will be important to
verify whether the biphasic response observed in this study is similar
for other estrogenic compounds, particularly in relation to their
potential role in the treatment of diseases such as osteoporosis and
atherosclerosis.1 2
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Acknowledgments |
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This investigation was supported by grants from the Swedish Medical Research Council (03X-7137 and 03X-3972); the Swedish Society for Medical Research; the Swedish Foundation for Strategic Research; the Ax:son Johnson, Jeansson, Widengren, Ruth and Richard Julin, and Novo Nordisk AB foundations; the Swedish Heart-Lung Foundation; "Förenade Liv" Mutual Group Life Insurance Co; the foundation of Old Female Servants; and the Karolinska Institute. We thank Ingela Arvidsson, Lisbet Benthin, Ewa Ellis, Lilian Larsson, and Tatiana Egereva for technical assistance; Dr Mats Gåfvels for expert help in developing the probe for apo A-I mRNA determination; and Dr Walter Elger, EnTec, Jena, Germany, for his invaluable help in the plasma estradiol determinations.
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Footnotes |
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1 Physiological levels of serum estradiol in intact female rats vary throughout the cycle. Normal values are considered to be between 2 and 50 pg/mL,26 27 and in pregnant rats, serum estradiol concentrations have previously been shown to be 79±10 pg/mL.28 In the control group, the plasma estradiol concentration of E2 was 25±6.2 pg/mL, and in the group treated with 0.01 mg · kg-1 · d-1, it was 51±14 pg/mL. These plasma estradiol concentrations were thus within the normal physiological range. Treatment with doses of 0.1 mg · kg-1 · d-1 of E2 resulted in a dose-dependent increase in mean plasma estradiol concentration way above physiological levels: 2870±405, 13 900±3470, and 48 400±3595 pg/mL at 0.1, 1, and 4 mg · kg-1 · d-1, respectively. Plasma estradiol concentrations were determined by a commercially available kit (Diagnostic Products Corp), following the manufacturer’s instructions.
Received July 16, 1999; accepted January 20, 2000.
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