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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1128-1134.)
© 1997 American Heart Association, Inc.

Articles

Effects of Short-term Hormone Replacement Therapies on Low-Density Lipoprotein Metabolism in Cynomolgus Monkeys

Janice D. Wagner; Dawn C. Schwenke; Li Zhang; Deborah Applebaum-Bowden; John D. Bagdade; ; Michael R. Adams

From the Comparative Medicine Clinical Research Center (J.D.W., L.Z., M.R.A.) and the Departments of Pathology (D.C.S.) and Internal Medicine (D.A.-B.), Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC; and Rush-Presbyterian–St Luke's Medical Center (J.D.B.), Chicago, Ill.

	Abstract

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Abstract Estrogen replacement therapy reduces the risk of coronary heart disease in women and decreases the extent of atherosclerosis in monkeys. In our previous studies, estrogen treatment decreased arterial LDL degradation and accumulation, thus indicating one mechanism by which estrogen inhibits the progression of atherosclerosis. The influence of progestins on these processes remains unclear. The objective of this study was to determine the effects of oral estrogen (conjugated equine estrogens) and progestin (medroxyprogesterone acetate) alone or in combination on arterial LDL metabolism after 12 weeks of atherogenic stimulus. This relatively short period of treatment was chosen to determine effects on arterial LDL metabolism before substantial subendothelial macrophage accumulation. In contrast to previous studies (16 to 18 weeks of treatment), when macrophages were present in the intima, neither estrogen nor progestin (nor their combination) had any effect on any index of arterial LDL metabolism. These results suggest that estrogen may preferentially reduce LDL metabolism in macrophages with little effect on cells of the normal artery. In contrast to arterial LDL metabolism, hepatic LDL uptake was significantly increased in animals treated with estrogen or estrogen plus progestin. Despite the increased LDL uptake by the liver, hepatic lipid content was significantly decreased by 50% in both estrogen and estrogen-plus-progestin treatment compared with control and progestin-treated animals. The decrease in hepatic cholesterol content is hypothesized to be due to increased biliary secretion of cholesterol.

Key Words: cholesterol • cynomolgus monkeys • liver • estrogen • LDL metabolism • progestin

	Introduction

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Coronary heart disease is the leading cause of death in women in Western societies. Although there is extensive evidence that estrogen replacement therapy reduces the risk of CHD in women^{1 2} and decreases the extent of atherosclerosis in monkeys,^{3 4} the mechanisms for these beneficial effects remain unclear. Because only a small portion of the beneficial effect can be explained by changes in plasma lipid and lipoprotein concentrations,^{2 3} we have proposed that estrogens exert beneficial effects at the level of the arterial wall. In support of this hypothesis, we found that estrogens decrease the arterial rate of LDL degradation and arterial accumulation of nondegraded LDL^{5 6 7 8} as well as improve coronary artery vascular reactivity.⁹ However, in contrast, the effects of added progestins on CHD are less clear. The consequences of the addition of a progestational steroid may depend on the type of progestin, route of administration, and dose.

The purpose of the present study was to assess the short-term (12 weeks) effects of an estrogen (CEE), a progestin (MPA), and their combination (CEE+MPA) on both arterial and hepatic LDL metabolism in ovariectomized cynomolgus monkeys fed atherogenic diets. In past studies of arterial LDL metabolism,^{5 6 7 8} we used experimental periods of 16 to 18 weeks. This duration of atherogenic stimulus caused minimal changes in arterial morphological characteristics (eg, small accumulations of subendothelial foam cells with no treatment differences in intimal thickness) yet resulted in inhibitory effects of estrogen on arterial degradation and accumulation of LDL. However, because the 16 to 18 weeks of atherogenic stimulus did result in recruitment of intimal macrophages, we selected a shorter (12-week) period for the present study in an attempt to investigate further the effect of the hormone therapies on even earlier events in the atherogenic process (eg, hypercholesterolemia, but with the recruitment of few intimal macrophages). In addition, we determined the effects of hormone therapies on plasma lipoprotein patterns and hepatic LDL and cholesterol metabolism.

	Methods

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Animals
Forty adult female cynomolgus macaques (Macaca fascicularis) were imported directly from Indonesia (CV Primates; Bogor, Indonesia). During a 3-month quarantine period, they were fed monkey chow (High Protein Monkey Chow, Ralston Purina Co). The monkeys were then fed a moderately atherogenic diet containing 0.28 mg of cholesterol per kilocalorie with 40% of calories from fat³ for a 2-month dietary challenge. TPC and HDL-C concentrations were used in a stratified randomization scheme to divide animals into four groups, with equivalent responses in TPC and HDL-C concentrations to the atherogenic stimulus. After the challenge period, the animals were again fed monkey chow for at least 6 months to allow plasma lipoproteins to return to preatherogenic diet levels.

Before treatment was begun, the monkeys were ovariectomized. The treatment period was 12 weeks, during which monkeys were fed the same atherogenic diet as during the challenge period either with no treatment (postmenopausal controls) or with the addition of the following hormones to the diet at amounts equivalent to the prescribed human dose per 1800 calories: CEE (Premarin, Wyeth-Ayerst) at a dose equivalent to 0.625 mg/d for women, 2.5 mg MPA (Cycrin, Wyeth-Ayerst), or combined CEE+MPA. One monkey from each treatment group entered the study at the same time. Monkeys were housed in social groups of four until 1 week before the study began, after which they were housed singly. Forty-eight hours before the end of the study, radiolabeled LDLs were injected intravenously as described below to determine the whole-body LDL FCR and both arterial and hepatic LDL metabolism.

All procedures involving animals were conducted in compliance with state and federal laws, standards of the US Department of Health and Human Services, and guidelines established by the institutional animal care and use committee. Ovariectomies and femoral catheterizations (for LDL metabolism studies) were done while the animals were anesthetized with ketamine hydrochloride (15 mg/kg) and butorphanol (0.05 mg/kg), and blood samples were obtained while animals were sedated with ketamine hydrochloride (10 mg/kg). One animal from the CEE-treated group died during the treatment period of causes unrelated to the experiment.

Plasma Lipid, Lipoprotein, and Hormone Measurements
During the study, blood samples were collected into tubes containing EDTA (final concentration, 1 mg/mL) after the animals were fasted overnight. TPC,¹⁰ HDL-C,¹¹ and triglycerides¹² were determined when the monkeys were consuming monkey chow and during the diet-challenge period (no hormone treatment) and after 4, 7, and 11 weeks of the experimental period. Analyses for TPC, HDL-C, and triglycerides were in full standardization with the Centers for Disease Control–National Heart, Lung, and Blood Institute Lipid Standardization Program. In addition, after 9 weeks of study, lipoproteins were fractionated by ultracentrifugation and high-performance liquid chromatography.¹³ Cholesterol distributions among plasma lipoproteins were determined by enzymatic methods,¹⁴ and LDL molecular weight was determined by column chromatography.¹⁵

Plasma concentrations of estradiol, estrone, and MPA were measured by radioimmunoassay in samples collected after an overnight fast after 4 and 7 weeks of treatment, as described previously.¹⁶ Samples were assayed at the Yerkes Regional Primate Center (Atlanta, Ga) in the laboratory of Dr Mark Wilson.

Hepatic Lipase and CET Activity
Hepatic lipase activity was measured in seven monkeys per group before treatment and after 12 weeks of treatment. Heparin (100 U/kg) was injected intravenously, and blood samples were collected 20 minutes later. Postheparin plasma hepatic lipase levels were measured with glyceryl tri[³H]oleate in the presence of 1 mol/L NaCl as described previously.¹⁷

CET activity was measured in plasma samples collected at necropsy and stored at -70°C until analysis. CET activity was determined by use of an isotopic assay that measures the movement of labeled cholesteryl ester from donor HDL particles to recipient VLDL and LDL particles.¹⁸ The transfer of cholesteryl ester radioactivity was calculated as the slope of the line that describes the amount of radioactivity in the precipitated VLDL+LDL during a 1-hour period.

LDL Metabolism Studies
LDL particles for labeling and reinjection were isolated from pooled blood obtained from a group of 23 ovariectomized female monkeys consuming the same atherogenic diet as the experimental monkeys. Blood pools of 230 mL were collected in tubes containing aprotinin and D-phenylalanyl-L-prolylarginine chloromethyl ketone at final concentrations of 25 kallikrein inhibitory units/mL and 1 µmol/L, respectively, to limit degradation of apoB by proteolysis and 2 mmol/L Na₂ EDTA to prevent oxidation. The serine protease inhibitor phenylmethylsulfonyl fluoride and the antioxidant butylated hydroxytoluene were added to isolated plasma at a final concentration of 0.5 and 0.5 mmol/L, respectively.

LDLs (1.020 to 1.063 g/mL) were isolated by differential ultracentrifugation followed by exhaustive dialysis against buffer (0.9% NaCl, 0.01% EDTA, pH 7.4). LDL protein was determined¹⁹ with bovine serum albumin used as a standard. Each LDL preparation (30 mg of protein) was first labeled with ¹³¹I using 1,3,4,6-tetrachloro-3,6-diphenylglycouril and then coupled to ¹²⁵I-TC.^{8 20 21} Specific activities for the doubly labeled LDL averaged 454±59 and 150±64 cpm/ng protein (mean±SEM) for ¹²⁵I-TC and ¹³¹I, respectively, for the 10 preparations. Trichloroacetic acid–soluble radioactivities (10% final concentration of trichloroacetic acid) averaged 3.6±0.3% for ¹²⁵I-TC and 0.7±0.2% for ¹³¹I, and radioactivities extractable in chloroform/methanol²² averaged 3.6±0.2% and 3.5±1.0%, respectively. Before injection, LDL preparations were sterilized by filtration (0.45-µm Millipore filter).

After the monkeys had consumed the atherogenic diet for 12 weeks, doubly labeled LDLs (2.79±0.26x10⁹ cpm ¹²⁵I and 5.02±0.60x10⁸ cpm ¹³¹I) were injected through an indwelling femoral venous catheter 48 hours before necropsy.⁵ Subsequent blood samples were collected from the arterial catheter into tubes containing EDTA (1 mg/mL final concentration) at 3, 10, 20, 40, and 60 minutes and 2, 4, 6, 20, 24, and 48 hours after injection to determine the plasma decay of labeled LDL. The FCR of LDL by the whole body was calculated from coefficients and exponents determined by the biexponential equation fitted to the decay of protein-bound radioactivity in the plasma.^{5 23}

After collecting the final (48-hour) blood sample, the animals were anesthetized with sodium pentobarbital (80 mg/kg IV). The cardiovascular system was flushed via the left ventricle with 1 L of lactated Ringer's solution with efflux through the vena cava. Samples of liver and arteries (thoracic and abdominal aortas, right common iliac artery, common carotid arteries and carotid bifurcations, and left anterior descending and left circumflex coronary arteries) were removed and placed in modified Karnovsky solution for 24 hours to provide adequate fixation for radiolabeled LDL.²⁴

Analysis of LDL Metabolism
Fixation of tissue samples in modified Karnovsky solution preserves products of ¹²⁵I-TC–LDL degradation as well as nondegraded ¹²⁵I-TC LDL and ¹³¹I-LDL.^{20 24} The tissue ¹²⁵I-TC radioactivity (cpm/g), which represents both nondegraded LDL and products of LDL degradation, was normalized by the area under the curve of protein-bound ¹²⁵I-TC radioactivity in plasma during the metabolic experiment ([cpm/µL]xh) to express the ¹²⁵I-TC radioactivity in a form (µL·g^-1·h^-1) independent of the plasma LDL concentration and amount of labeled LDL injected.⁶

The rates of LDL degradation and the calculated concentration of nondegraded LDL were determined as described previously.^{6 20} The ¹²⁵I-TC representing LDL degraded by tissues can then be determined by subtracting the tissue ¹³¹I radioactivity from the total ¹²⁵I-TC radioactivity, taking into account the relative activities of these two isotopes in plasma LDL at the time of necropsy. LDL degradation was first calculated in fractional terms and expressed as a fraction of the plasma LDL pool degraded per hour per gram of tissue (FCR_tissue). FCR_tissue was calculated as the product of the whole-body FCR determined from the plasma decay curve and the ratio of LDL degradation products per gram of tissue to the LDL degraded by the whole body (dose injected multiplied by the fraction of the dose irreversibly degraded by the whole body). Degradation of LDL in absolute terms (micrograms of LDL-C per gram of tissue per hour) was calculated as the product of the fractional rate of LDL degradation by the tissue and the total amount of LDL-C in plasma, which was calculated as the product of the plasma LDL-C concentration and the plasma volume.

The arterial concentration of nondegraded LDL (micrograms of LDL-C per gram) was calculated as the ratio of ¹³¹I radioactivity in the tissue (cpm/g) to that in plasma (cpm/mL) at the time of necropsy multiplied by the plasma LDL-C concentration of individual animals.⁶

Tissue Lipid Measurements
Lipid extracts of samples of abdominal and thoracic aortas and hepatic tissue were prepared with chloroform/methanol (2:1, vol/vol) by the method of Folch et al.²⁵ Triglyceride and total and free cholesterol concentrations were then determined enzymatically as described by Carr et al.²⁶ Cholesteryl ester content was determined as the difference between measured total and free cholesterol.

Statistical Analyses
Treatment groups were compared statistically by ANOVA. Post hoc analyses were done by the method of Duncan when significant differences were found for individual treatment differences by ANOVA. Pearson product-moment correlations were used to assess the relationships among variables. In all cases, two-sided tests were used to assess statistical significance. Analyses were performed with the BMDP statistical package (programs 2V and 7D; BMDP Statistical Software). Means presented in the text are reported with the SEM.

	Results

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Plasma estradiol and estrone concentrations were significantly increased in animals receiving CEE and CEE+MPA treatment compared with controls and MPA-treated monkeys (Table 1). In addition, plasma MPA concentrations were increased in those monkeys treated with CEE+MPA and MPA compared with controls and CEE-treated monkeys.

View this table: [in this window] [in a new window]   Table 1. Effect of Hormone Treatment on Plasma Hormone Measurements (Mean±SEM)

Plasma lipid concentrations from two pretreatment phases (while the monkeys were consuming monkey chow and during the atherogenic stimulus) and the means of the values during treatment are shown in Table 2. The atherogenic diet resulted in a twofold to threefold increase in plasma cholesterol values compared with values in monkeys that consumed chow. There were no effects of treatment on TPC or HDL-C; however, plasma triglyceride concentrations were significantly increased in both CEE- and CEE+MPA–treated monkeys.

View this table: [in this window] [in a new window]   Table 2. Effect of Hormone Treatment on Plasma Lipid Measurements (Mean±SEM)

Plasma lipoprotein cholesterol distributions are summarized in Table 3. Although TPC and LDL-C concentrations tended to be slightly lower with CEE treatment and slightly higher with MPA treatment, these differences were not statistically significant. The combined VLDL+IDL fraction was lower with CEE treatment, and this difference was significant compared with MPA-treated monkeys. In addition, LDL size, as determined by the average LDL molecular weight, was significantly reduced in both CEE- and CEE+MPA–treated monkeys compared with control or MPA-treated monkeys. CET activity was not affected by hormone treatment (Table 3). However, as expected,²⁷ hepatic lipase activity was reduced by 40% with CEE or CEE+MPA treatment compared with control or MPA-treated monkeys (P=.07, adjusted for pretreatment values).

View this table: [in this window] [in a new window]   Table 3. Effect of Hormone Treatment on Plasma Measurements (Mean±SEM)

There was also a 40% increase in the whole-body LDL FCR with CEE treatment compared with controls; however, this did not reach statistical significance (P<.10; Table 4). Consistent with the trend for increased plasma LDL FCR with CEE treatment, there were significant increases the both the fractional degradation rate (P=.02) and total accumulation of ¹²⁵I-TC (¹²⁵I-TC and products of ¹²⁵I-TC–LDL degradation) (P<.01) by the liver in CEE-treated monkeys, with a similar trend in the combined CEE+MPA group (Table 4). The majority (>90%) of the LDL taken up by the liver was present as degradation products, with no effect of treatment on the amount of nondegraded LDL. The plasma LDL FCR correlated positively with the liver FCR (r=.49) and liver ¹²⁵I-TC accumulation (r=.56). The plasma FCR also correlated negatively with TPC (r=-.55), LDL-C (r=-.47), and combined VLDL+IDL cholesterol fraction (r=-.56) as well as the LDL size (r=-.56) in all animals (all P<.01). There was no correlation among measures of liver cholesterol metabolism or plasma FCR with HDL-C (all P>.05).

View this table: [in this window] [in a new window]   Table 4. Effect of Hormone Therapy on Hepatic LDL Metabolism and Lipid Content (Mean±SEM)

Interestingly, whereas there was an increase in hepatic LDL uptake as reflected by hepatic ¹²⁵I-TC accumulation, there was a decrease in total hepatic cholesterol content with both CEE and CEE+MPA treatment compared with control and MPA-treated monkeys (Table 4; P<.001 by ANOVA). The 50% decrease in total cholesterol content was due to a 60% decrease in cholesteryl ester (P<.001 by ANOVA) and an 30% decrease in free cholesterol (P<.01 by ANOVA). In addition, there was a greater than 70% decrease in hepatic triglyceride concentrations with CEE treatment compared with controls (P=.0001 by ANOVA). There was a significant correlation between both hepatic total and esterified cholesterol content and LDL size (r=.57 and .56, respectively; P.001) and the plasma VLDL+IDL fraction (r=.57 and r=.54, respectively; P.001).

There was no effect of treatment on any aspect of arterial LDL metabolism (Table 5). Although there were regional differences in arterial metabolism, with greater LDL degradation rates and ¹²⁵I-TC accumulation in the thoracic aorta and carotid bifurcation, there were no differences with treatment in either the degradation rates, total ¹²⁵I-TC accumulation, or amounts of nondegraded LDL (data not shown) at any site. There was also no difference among groups in total, free, or esterified cholesterol concentrations in the thoracic aorta (Table 5) or abdominal aorta (data not shown). Likewise, after 12 weeks of treatment, there was very little intimal thickening in the coronary arteries and no differences among groups.

View this table: [in this window] [in a new window]   Table 5. Effect of Hormone Treatment on Arterial LDL Metabolism, Cholesterol Content, and Intimal Area (Mean±SEM)

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The purpose of this study was to determine the effects of hormone replacement therapies on early events in atherogenesis. In previous studies, we have found reductions in arterial LDL degradation rate, arterial concentration of nondegraded LDL, and total ¹²⁵I-TC accumulation with various hormone treatments. These included combined estradiol and progesterone delivered subcutaneously^{5 6} and oral esterified estrogens with or without methyltestosterone in ovariectomized monkeys⁸ as well as oral contraceptive steroids given to intact females.⁷ In our previous studies, LDL metabolism was determined after 16 to 18 weeks of treatment, during which time the monkeys consumed an atherogenic diet. In the present study, we chose a shorter period of study (12 weeks) to investigate the effects of hormone treatment in earlier stages of atherogenesis and found no effect of CEE or MPA treatment on arterial LDL metabolism.

We recently completed a long-term (30-month) study of the same hormone treatments as in the present study and found a 72% reduction in coronary artery atherosclerosis with CEE treatment compared with controls.⁴ Atherosclerosis extent in animals treated with combined CEE+MPA or MPA alone did not differ significantly from controls. Thus, the lack of effect of CEE in the present short-term study does not appear to be due to a lack of effect of CEE in decreasing atherosclerosis progression but suggests that the 12-week period may have been too short to induce arterial changes required for the upregulation of arterial LDL degradation rate and ¹²⁵I-TC accumulation observed in arteries of rabbits^{28 29} and pigeons³⁰ during early atherogenesis and monkeys fed atherogenic diets for longer periods.^{5 6 7 8} In support of this, arterial degradation rates for control postmenopausal monkeys at 12 weeks range from 4% to 14% of those for the corresponding arterial sites of control postmenopausal monkeys studied after 18 weeks,⁶ and the cholesterol concentrations for the thoracic aorta observed in the present study at 12 weeks were 50% of those obtained after 16 weeks (Table 5).⁸ Thus, estrogen may be able to reduce only arterial degradation rates that are elevated by early atherogenic changes and not normal basal values of arterial LDL metabolism. There may be other unidentified reasons why CEE did not decrease arterial LDL accumulation at this stage of atherogenesis.

One postulated mechanism for the beneficial effects of estrogen on CHD risk and atherosclerosis relates to its antioxidant activity. For example, high concentrations of estrogens increase the resistance of LDL to oxidation^{31 32} and inhibit LDL accumulation in macrophages.³³ We have also found that estrogens decrease the amount of thiobarbituric acid–reactive material in arteries.⁸ Thus, if estrogens decrease arterial LDL metabolism by preventing the oxidative modification of LDLs and their subsequent uptake and degradation by macrophages, it is plausible that estrogens would be less effective in decreasing arterial LDL metabolism if few macrophages were present in the intima. This theory is consistent with studies in rabbits in which the antioxidant probucol inhibited lesion progression and decreased rates of arterial LDL degradation but only in lesioned areas that were rich in macrophage-derived foam cells.²⁸ No differences in LDL degradation rate were found in nonlesioned areas in which degradation predominantly occurred in smooth muscle cells. The effect of estrogens on macrophages is also consistent with a recent report by Tomita et al³⁴ in which estrogen was found to inhibit macrophage cholesteryl ester accumulation via increased neutral cholesterol esterase activity.

Despite having no effect on arterial LDL metabolism, CEE treatment had a number of effects on plasma lipoproteins and on hepatic LDL metabolism. In general, CEE treatment resulted in only a slight decrease in total cholesterol concentrations. However, combined and LDL VLDL+IDL cholesterol concentrations were reduced almost 40% compared with controls; in addition, there was a significant reduction in size of LDL particles (Table 3). Contrary to studies in women, the present study and previous studies with this animal model^{3 4} found either no effect or a lowering effect⁴ of estrogen on HDL-C concentrations. However, consistent with studies in postmenopausal women,²⁷ there was a 40% decrease (P=.07) in hepatic lipase activity with CEE and no effect of MPA. Also, the lack of effect of hormone treatment on CET activity is in agreement with studies in women in which CEE had no effect on CET protein.³⁵ Thus, despite similar effects of CEE on enzyme activities, there was no effect of CEE on HDL-C concentrations; yet estrogens still have antiatherogenic properties.^{3 4}

In a subset of monkeys from the present study, a detailed analysis of the LDL particles revealed that in addition to reducing the size of LDL particles, CEE treatment resulted in enrichment of LDL with protein and triglyceride and depletion of cholesteryl ester and apoE compared with LDL from control monkeys.³⁶ These changes in the LDL particles were most pronounced in the largest and lightest subfractions (d= 1.015 to 1.025). The decrease in larger, more apoE-enriched LDL with CEE treatment may be due to the greater affinity of these particles for either the apoB/E or scavenger receptor as well as the moderately increased whole-body LDL FCR in the CEE group (Table 4). The selective removal of large LDL could also explain why estrogen treatment results in smaller LDL, as shown in this and other studies in monkeys^{4 5 6 7 8} as well as in women.^{37 38 39 40} The selective decrease in large LDL particles as well as in the VLDL+IDL lipoprotein fraction seen with CEE treatment (Table 3) is consistent with a number of previous studies. In these, estrogen treatment resulted in (1) selective removal of cholesterol-rich and apoE-rich VLDL in perfused rabbit liver,⁴¹ (2) selective removal of apoE-containing ß-VLDL in Watanabe rabbits,⁴² (3) increased removal of large VLDL, which then were not further delipidated to IDL and LDL,⁴³ and (4) increased chylomicron removal and lower IDL cholesterol concentrations.⁴⁴ Our findings of correlations among measures of plasma LDL FCR with plasma LDL and VLDL+IDL concentrations and LDL size are consistent with these previous findings. Although there were no significant changes in LDL-C concentrations, the decrease in VLDL+IDL and the smaller size of the LDL particles are consistent with a selective removal of larger particles. The decrease in larger, apoE-enriched particles with estrogen treatment may result in the decreased plasma apoE concentrations reported in women²⁷ and baboons.⁴⁵

Despite the increased removal of plasma LDL particles by the liver, there was a substantial decrease in hepatic lipid content in both the CEE and the CEE+MPA groups (Table 4). Estrogens have been shown to increase biliary cholesterol secretion in women⁴⁶ and to increase the activity for 7-hydroxylase, the rate-limiting enzyme for bile acid synthesis.⁴⁷ In addition, we found that CEE increased the 7-hydroxylase mRNA abundance in monkeys from the present study.⁴⁸ Thus, increased cholesterol secretion into bile might explain the decrease in hepatic cholesterol content even with increased LDL catabolism. This would be important in preventing a subsequent downregulation of LDL receptor activity, which could decrease LDL uptake by the liver. The decrease in hepatic cholesteryl ester content also may be responsible for smaller LDL particles, as suggested by studies of Parks et al⁴⁹ in which a correlation among hepatic cholesteryl ester content, VLDL cholesterol secretion, and LDL size was found. In support of those findings, we found a correlation in the present study between hepatic total and esterified cholesterol content with LDL size and concentration of plasma VLDL+IDL cholesterol.

In conclusion, short-term hormone treatments in the present study did not affect arterial LDL metabolism in surgically menopausal monkeys. Comparisons with earlier studies^{5 6 7 8} suggest that expression of an inhibitory effect on arterial LDL metabolism by estrogen may require upregulation of arterial LDL metabolism by early atherogenic changes in the intima. However, we did find significant effects of CEE on plasma and hepatic cholesterol metabolism. In our previous studies using parenteral hormone replacement therapy,⁶ we did not see significant effects on hepatic uptake of LDL. In contrast to these earlier studies, the significant effects on hepatic cholesterol metabolism in the present study and in our studies of oral contraceptive steroids⁵⁰ are most likely due to the higher portal concentrations of the steroids after oral treatment.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein CEE = conjugated equine estrogen CET = cholesteryl ester transfer CHD = coronary heart disease FCR = fractional catabolic rate HDL-C = HDL cholesterol LDL-C = LDL cholesterol MPA = medroxyprogesterone acetate 125I-TC = 125I-tyramine cellobiose TPC = total plasma cholesterol

	Acknowledgments

 
This study was supported in part by grants from the National Center for Research Resources (KO1 RR000072 [Dr Wagner]) and the National Heart, Lung, and Blood Institute (HL-PO145666) of the National Institutes of Health, Bethesda, Md. The authors thank Karen Potvin Klein for editorial assistance.

	Footnotes

 
Reprint requests to Janice D. Wagner, DVM, PhD, Department of Comparative Medicine, Bowman Gray School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1040.

Received August 15, 1996; accepted October 30, 1996.

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