© 1996 American Heart Association, Inc.
Articles |
Effect of Estrogen and Progesterone on the Expression of Hepatic and Extrahepatic Sterol 27-Hydroxylase in Baboons (Papio sp)
the Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, Tex.
Correspondence to Rampratap S. Kushwaha, PhD, Southwest Foundation for Biomedical Research, Department of Physiology and Medicine, PO Box 760549, San Antonio, TX 78245-0549.
Abstract
Sterol 27-hydroxylase plays an important role in cholesterol metabolism in hepatic and extrahepatic tissues. To determine whether female sex steroid hormones influence its expression, we measured plasma and hepatic 27-hydroxycholesterol, hepatic mRNA levels, activity of sterol 27-hydroxylase, and adrenal mRNA levels of this enzyme in baboons (n=6 per group) treated with placebo, estrogen, estrogen + progesterone, and progesterone. We also measured hepatic cholesterol concentration and hepatic acyl coenzyme A:cholesterol acyltransferase (ACAT) activity to determine their relationship with hepatic sterol 27-hydroxylase activity. Plasma 27-hydroxycholesterol concentration was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (P=.090) and LDL (P=.026) cholesterol concentrations. Similarly, hepatic sterol 27-hydroxylase activity was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (P=.056) and LDL (P=.052) cholesterol concentrations but was positively correlated with hepatic and plasma 27-hydroxycholesterol concentrations (P<.001). Hepatic ACAT activity was increased by progesterone (P<.004) and was positively correlated with plasma (P=.002) and LDL (P=.009) cholesterol concentrations but was negatively correlated with hepatic sterol 27-hydroxylase activity (P=.035). Hepatic and adrenal gland mRNA levels for sterol 27-hydroxylase were increased by estrogen alone or in combination with progesterone (P<.05). Hepatic sterol 27-hydroxylase activity was positively correlated with hepatic mRNA levels (P<.001), an observation suggesting that estrogen increases the activity of sterol 27-hydroxylase by increasing its synthesis. Hepatic cholesterol concentration was not influenced by the hormone treatment. These observations suggest that estrogen alone or in combination with progesterone increases the synthesis of sterol 27-hydroxylase in hepatic and extrahepatic tissues, and the increased activity of hepatic sterol 27-hydroxylase resulting from the increased synthesis is associated with a hypolipidemic effect on plasma LDL levels. Furthermore, progesterone alone increases the hepatic ACAT activity, but given in combination with estrogen progesterone does not have the same effect on hepatic ACAT activity. The effect of estrogen on hepatic ACAT activity may be mediated by sterol 27-hydroxylase and its effect on cholesterol metabolism (decreased cholesterol synthesis and increased output of cholesterol in the bile) in liver.
Key Words: dietary cholesterol • dietary saturated fat • female sex steroid hormones • 27-hydroxycholesterol • acyl coenzyme A:cholesterol acyltransferase
Sterol 27-hydroxylase is a mitochondrial enzyme that plays an important role in cholesterol metabolism in hepatic and extrahepatic tissues.1 2 In the liver sterol 27-hydroxylase initiates modification of the side chain of cholesterol in both bile acid synthesis pathways2 3 —the pathway initiated by 7
-hydroxylation of cholesterol and that initiated by 27-hydroxylation of cholesterol.4 5
The product of sterol 27-hydroxylation (27-hydroxycholesterol) is the major oxysterol in human plasma6 7 and in the plasma and liver of baboons.8 9 Recently, Björkhem et al10 have reported that 27-hydroxycholesterol is the major oxysterol in human atheromas and that sterol 27-hydroxylase is expressed in several extrahepatic tissues, including macrophages, which can convert cholesterol into 27-hydroxycholesterol and 3-hydroxy-5-cholestenoic acid. Cultured macrophages not only can produce these oxysterols but also are able to transport them from the cell into the medium.10 The 27-hydroxycholesterol that is transported from extrahepatic cells is carried via the bloodstream to the liver, where it is converted to bile acids and then excreted. Thus, sterol 27-hydroxylase may retard arterial lesion development.
Our studies in baboons show that estrogen, either alone or combined with progesterone, reduces LDL cholesterol levels and retards the development of arterial lesions.11 The antiatherogenic effects of estrogen include an independent or direct effect of estrogen on the arterial wall that cannot be explained by lower levels of plasma lipoproteins.11 This direct effect of estrogen may be related to its ability to increase the expression of sterol 27-hydroxylase in extrahepatic tissues, as has been suggested by Björkhem et al.10 Thus, we hypothesize that (1) estrogen induces the expression of sterol 27-hydroxylase in hepatic and extrahepatic tissues and (2) hepatic sterol 27-hydroxylase activity is negatively correlated with LDL cholesterol concentration. Since hepatic acyl coenzyme A:cholesterol acyltransferase (ACAT) is related to VLDL secretion12 and progesterone increases VLDL secretion,13 we hypothesized that progesterone increases hepatic ACAT activity. However, because progesterone when combined with estrogen does not increase VLDL secretion, we further hypothesized that estrogen reverses the effect of progesterone on hepatic ACAT activity. We tested these hypotheses by studying frozen plasma, liver, and adrenal glands from ovariectomized and hysterectomized baboons that had been obtained for a previous study.
Methods
Animals
Frozen plasma and liver samples from baboons used in a previous study were used for this study.11 We selected 24 adult female baboons (Papio sp), ranging from 4 to 9 years of age, with similar plasma and lipoprotein cholesterol levels on a basal chow diet as well as on a high cholesterol (1.7 mg/kcal) and high fat (40% from lard) (HCHF) diet fed for 7 weeks. The composition of the diet and characteristics of these animals have been described previously.11 Baboons were fed the HCHF diet for 8 weeks, ovariectomized, hysterectomized, and treated with hormones as described below while being maintained on the HCHF diet. The protocol of this experiment was approved by the Animal Research Committee of the Southwest Foundation for Biomedical Research (SFBR). The SFBR is accredited by the American Association for Accreditation of Laboratory Animal Care and is registered with the US Department of Agriculture.
Treatments
The experiment was conducted in two blocks with 12 baboons in each block as described previously.11 Baboons in each block were randomly divided into four treatment groups: estrogen, progesterone, estrogen + progesterone, and untreated control. The estrogen group received 100 µg/kg per week estradiol cypionate in cotton seed oil injected intramuscularly. They were also given a fig bar daily. The progesterone group was given progesterone at 3 mg/kg per day in a fig bar plus an injection of cotton seed oil. The estrogen + progesterone group was injected with estradiol cypionate and given progesterone in a fig bar. The control group was injected with cotton seed oil and given a fig bar. These treatments were continued for 18 months while the baboons were fed the HCHF diet. Plasma, livers, and adrenal glands were collected at necropsy, immediately frozen with liquid nitrogen, and stored at -80°C until used.
Measurements of 27-Hydroxycholesterol in Plasma and Liver and Mitochondrial Sterol 27-Hydroxylase Activity in Liver
27-Hydroxycholesterol was separated by high-performance liquid chromatography as described previously.8 9 Hepatic mitochondrial sterol 27-hydroxylase activity was measured by the method described by Petrack and Latario.14 We used 0.5 mg of mitochondrial protein in a total volume of 0.5 mL containing 100 mmol/L phosphate, 1 mmol/L DTT, 0.2 mmol/L EDTA, 1.2 mmol/L NADPH, 5.0 mmol/L D,L-trisodium isocitrate, and 0.2 units of isocitrate dehydrogenase (pH 7.5). Exogenous cholesterol (100 mmol) dissolved in 10 µL of 45% 2-hydroxypropyl-ß-cyclodextrin was added in each assay. The assays were conducted at 37°C for 15 minutes. In simultaneous control experiments, reaction mixtures were incubated at 4°C for 15 minutes and reactions stopped by adding 50 µL of 40% sodium cholate.14 At this stage, internal standard (7ß-hydroxy-cholesterol, Sigma Chemical Co) was added to the reaction mixture. The final reaction was started by adding 2 U of cholesterol oxidase (Calbiochem-Novabiochem Corp) in 10 mmol/L potassium phosphate buffer (pH 7.5) containing 1 mmol/L DTT and 20% glycerol.15 Both control and experimental mixtures were incubated for 20 minutes at 37°C to generate the ,ß-unsaturated ketones.16 The final reaction was terminated by adding 1.5 mL methanol followed by 0.5 mL saturated KCl. The mixture was extracted three times with 3.0 mL hexane. The combined extracts were evaporated to dryness under nitrogen and redissolved in 200 µL of 5% isopropanol in dodecane (Aldrich Chemical Co). Oxysterols (ketone derivatives) were separated by high-performance liquid chromatography (Waters) with the use of a silica column (4.6 mmx25 cm, 5 µm; Alltech Associates, Inc). The mobile phase was hexane:isopropanol (95:5), the flow rate was kept at 1 mL/min, and the absorbance was monitored at 240 nm. All assays were carried out in duplicate. Hepatic sterol 27-hydroxylase activity was quantified as the difference between 27-hydroxycholesterol concentrations in reaction mixtures incubated at 37°C and those incubated at 4°C.
Measurement of ACAT Activity in Liver
Liver microsomes were prepared by the method of Hylemon et al16 and ACAT activity was measured by the method of Carr et al.17 Briefly, 200 µg of microsomal protein was assayed in the presence of 50 nmol of exogenous cholesterol dissolved in tyloxapol (Sigma) and 1.0 mg BSA. Preincubation was carried out for 30 minutes after which the reaction was initiated by the addition of 30 nmol of [14C]-oleoyl-CoA (New England Nuclear) in a final volume of 300 µL. After 2 minutes of incubation, the reaction was stopped by the addition of chloroform and methanol containing 15 µg of cholesteryl oleate as a carrier. The cholesteryl esters were extracted and separated by thin-layer chromatography and counted by scintillation spectrometry (Beckman). ACAT activity was expressed as pmol/mg per minute.
Measurement of Hepatic Free and Esterified Cholesterol Concentrations
Liver samples (200 mg) were homogenized and extracted with chloroform-methanol as described by Folch et al.18 A small amount of [14C]-cholesteryl oleate (New England Nuclear) (0.016 µCi=0.6 kBq) was added during homogenization as a recovery standard for the extraction procedure. The chloroform extract was evaporated to dryness and dissolved in 5 mL chloroform. Aliquots of the extract were measured for liquid scintillation counting and for analysis of triglycerides, free cholesterol, and total cholesterol. For measurement of cholesterol concentration, the hepatic lipid extract was dissolved in isopropanol and total cholesterol and free cholesterol were measured by an enzymatic method using a kit (Wako Pure Chemical Industries, Ltd). Esterified cholesterol was determined by subtracting free cholesterol from total cholesterol.
Measurements of Hepatic and Adrenal Gland mRNA Levels
Total cellular RNA from frozen liver and adrenal gland samples was extracted using the guanidinium thiocyanate-phenol-chloroform extraction technique.19 The total RNA samples (15 µg each) from hormone-treated baboons and a control baboon were fractionated on denaturing agarose gel containing methyl mercuric hydroxide20 and transferred onto a nylon membrane (Genescreen Plus, Du Pont) by the capillary transfer method21 to immobilize the RNA. The 1322-bp SmaI fragment of full-length cDNA of rabbit sterol 27-hydroxylase cloned in pGEM-4 (kindly provided by Dr David Russell, University of Texas Southwestern Medical School, Dallas, Tex) was used as a probe to carry out the northern blotting analysis. The probe was radiolabeled by the random priming method.22 23 Hybridization was carried out with Denhardt's protocol.24 The blots were exposed to the x-ray film, and the autoradiograms were scanned using a laser densitometer (LKB-Ultroscan-XL). All data are expressed as ratios between experimental samples and a control sample. The blots were stripped and reprobed with albumin (for liver) and ß-actin (for adrenal gland) genes (housekeeping genes). A 2000-bp BamHI-EcoRI fragment of pILMALB5–full-length albumin cDNA cloned into pUC19 (ATCC, #61357) and a 1100-bp EcoRI fragment of ß-actin cDNA cloned into pBluescript SK-,HHCI89 (ATCC #65129) were used for northern blotting.
Statistical Analysis
Data in tables are presented as mean±SD. The data for effects of hormone treatment were analyzed by ANOVA. Associations between plasma and hepatic variables were calculated using univariate regression analysis. Significance was set at P<.05, but we also report differences at P<0.1 to balance between type I and type II statistical errors.
Results
Effect of Hormone Treatment on Plasma and Lipoprotein Cholesterol in Ovariectomized Baboons
The effects of treatment with estrogen, progesterone, and estrogen + progesterone on plasma and lipoprotein cholesterol in these ovariectomized baboons were reported earlier.11 The baboons treated with estrogen and estrogen + progesterone had lower VLDL + LDL cholesterol and higher HDL cholesterol concentrations than baboons treated with progesterone or placebo control.11 The baboons treated with progesterone had the highest concentration of VLDL + LDL cholesterol in their plasmas.11
Plasma and Liver Concentrations of 27-Hydroxycholesterol
The concentration of plasma and liver 27-hydroxycholesterol was highest in the estrogen + progesterone group followed by the estrogen, control, and progesterone groups (Table 1
). The concentration of 27-hydroxycholesterol in the plasmas and livers of baboons in the estrogen and estrogen + progesterone groups was higher than that in the plasmas and livers of baboons in the control and progesterone groups (P<.05). Plasma 27-hydroxycholesterol concentration was negatively correlated with plasma (r=.354, P=.090) and LDL (r=-.0454, P=.026, Fig 1A) cholesterol concentrations.
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), estrogen + progesterone (
), progesterone (
) and placebo control (
). LDL cholesterol concentration is negatively correlated with plasma 27-hydroxycholesterol concentration (r=-.454, P=.026) and hepatic sterol 27-hydroxylase activity (r=-.402, P=.052).
Hepatic Mitochondrial Sterol 27-Hydroxylase Activity in Baboons Treated With Hormones
The activity of sterol 27-hydroxylase was highest in the estrogen + progesterone group followed by the estrogen, control, and progesterone groups (Table 2). The activity of sterol 27-hydroxylase in the livers of the estrogen and estrogen + progesterone groups was significantly higher than that in the control and progesterone groups (P<.015). Hepatic sterol 27-hydroxylase activity was negatively correlated with plasma (r=-.395, P=.056) and LDL (r=.402, P=.052, Fig 1B
) cholesterol but positively correlated with hepatic (r=.733, P=.000, Fig 2A) and plasma (r=.698, P=.000, Fig 2B) 27-hydroxycholesterol.
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Hepatic ACAT Activity
Hepatic ACAT activity was higher (10-fold) in baboons treated with progesterone than in those treated with estrogen, estrogen + progesterone, or placebo control (Table 2). Hepatic ACAT activity was positively correlated with plasma cholesterol (r=.612, P=.002) and LDL cholesterol (r=.526, P=.009, Fig 3A) concentrations. Hepatic ACAT activity was negatively correlated with hepatic sterol 27-hydroxylase activity (r=.440, P=.035, Fig 3B).
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Hepatic Lipid Composition
The hepatic cholesterol concentration by treatment group is given in Table 3. Hepatic concentrations for both esterified and unesterified (free) cholesterol were not different among treatment groups. Unesterified cholesterol was twofold higher than esterified cholesterol in the livers of baboons of each treatment group.
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Hepatic Sterol 27-Hydroxylase mRNA Levels
As shown in Fig 4, the levels of hepatic mRNA for sterol 27-hydroxylase were affected by the hormone treatment (Fig 4A), whereas the levels of hepatic mRNA for albumin were not (Fig 4B). The hepatic mRNA levels of sterol 27-hydroxylase in the estrogen (2.91±0.08, relative units compared with a control sample, n=3) and estrogen + progesterone (2.98±0.03) groups were significantly higher (P<.005) than in the progesterone (0.83±0.25) and control (0.42±0.05) groups. Hepatic sterol 27-hydroxylase activity was positively correlated (r=.869, P<.001) with hepatic mRNA levels.
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Adrenal Sterol 27-Hydroxylase mRNA Levels
As shown in Fig 5, the levels of adrenal gland mRNA for sterol 27-hydroxylase were affected by the hormone treatment (Fig 5A), whereas the levels of mRNA for a control gene (ß-actin) were not (Fig 5B). The adrenal gland mRNA levels of sterol 27-hydroxylase in estrogen (3.33±0.21) (relative units compared with a control sample, n=3) and estrogen + progesterone (3.40±0.10) were significantly higher (P<.008) than those in progesterone (1.30±0.10) and control (0.73±0.27) groups. Adrenal and hepatic sterol 27-hydroxylase mRNA levels were positively correlated (r=.985, P<.001).
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Discussion
The present studies support our hypothesis that estrogen given alone or in association with progesterone increases the expression of sterol 27-hydroxylase in both hepatic and extrahepatic tissues of baboons. Hepatic mRNA levels of sterol 27-hydroxylase were positively correlated with hepatic activity of this enzyme. This association suggests that estrogen and estrogen + progesterone increase the activity of sterol 27-hydroxylase by increasing its synthesis. Hepatic and adrenal mRNA levels were positively correlated, an observation suggesting that estrogen has similar effects on the expression of sterol 27-hydroxylase in both hepatic and extrahepatic tissues. Progesterone treatment did not influence the expression of sterol 27-hydroxylase in either hepatic or extrahepatic tissues. Progesterone, which increased the VLDL apo B secretion in these baboons,13 increased the hepatic ACAT activity. However, progesterone given in combination with estrogen did not produce such an increase in hepatic ACAT activity. Thus, estrogen counterbalances the effect of progesterone on hepatic ACAT activity.
The increased expression of sterol 27-hydroxylase by estrogen and estrogen + progesterone may have several beneficial effects on lipoprotein metabolism and atherosclerosis. Björkhem et al10 detected 27-hydroxycholesterol in human atherosclerotic arteries and suggested that the conversion of cholesterol into 27-hydroxycholesterol and 3-hydroxy-5-cholestenoic acid is a defense mechanism for macrophages involved in atherosclerotic lesions. The beneficial effect of estrogen and estrogen + progesterone on arterial lesions not explained by their effect on lipoprotein patterns in baboons (as reported earlier11 ) may be mediated by increased expression of sterol 27-hydroxylase in extrahepatic tissues. Since 27-hydroxycholesterol inhibits cholesterol synthesis,25 the 27-hydroxycholesterol produced in extrahepatic tissues would inhibit cholesterol synthesis and would produce a hypolipidemic effect. The plasma 27-hydroxycholesterol concentration and hepatic sterol 27-hydroxylase activity were negatively correlated with LDL cholesterol in the plasma and these correlations support the hypothesis that the increase in hepatic and extrahepatic sterol 27-hydroxylase may be hypolipidemic.
LDL apo B turnover studies in these hormone-treated baboons13 showed that treatment with estrogen alone or in association with progesterone increases LDL apo B catabolism, whereas treatment with progesterone increases VLDL apo B production. Since the hepatic LDL receptor is mainly responsible for the removal of plasma LDL apo B, estrogen-treated baboons may increase their hepatic LDL receptor activity as has been reported in rats26 and rabbits.27 Since the hepatic LDL receptor is regulated by the sterol regulatory pool,28 decreased hepatic cholesterol synthesis mediated by sterol 27-hydroxycholesterol25 may decrease the hepatic sterol regulatory pool. The decrease in hepatic sterol regulatory pool, in turn, would upregulate the hepatic LDL receptor and increase LDL apo B catabolism as reported earlier.13 The present studies are not able to demonstrate whether the increased LDL apo B catabolism is associated with increased expression of hepatic sterol 27-hydroxylase. However, the difference in hepatic sterol 27-hydroxylase activity between estrogen and control groups in the present study is much larger than the difference in LDL apo B catabolism between these groups reported earlier13 and there was no significant difference in hepatic mRNA levels between the treatment groups (data not presented).
Hepatic cholesterol (free or esterified) concentration was not influenced by the hormone treatment in baboons, desspite a considerable difference in hepatic activities of sterol 27-hydroxylase and ACAT. These results are consistent with those reported previously in which low and high LDL responding baboons did not differ in hepatic cholesterol concentration29 even though low LDL responding baboons induced hepatic sterol 27-hydroxylase activity two- to threefold greater than that in high LDL responding baboons when challenged with the HCHF diet.8 As explained in low and high LDL baboons,29 after the hepatic cholesterol pool is saturated by consuming the HCHF diet, hepatic lipoprotein secretion becomes balanced between chylomicron cholesterol delivery plus de novo synthesis and cholesterol excretion in the bile. There is no difference in chylomicron cholesterol delivery to the liver among the treatment groups because the estrogen treatment does not affect cholesterol absorption.30 In estrogen- and estrogen + progesterone–treated baboons as compared with control and progesterone-treated baboons, because of increased hepatic 27-hydroxycholesterol concentration, a lower amount of cholesterol is synthesized; and because of upregulation of cholesterol 7-hydroxylase15 and sterol 27-hydroxylase, a higher amount of cholesterol is excreted in the bile as observed by Everson et al31 and Bennion et al32 in women taking oral contraceptives. Thus, estrogen- and estrogen + progesterone–treated baboons have lower rates of secretion of VLDL apo B and lower LDL cholesterol concentrations in plasma as compared with control and progesterone-treated baboons, as has been reported earlier.13 Since hepatic ACAT activity is regulated by the concentration of the substrate,33 an increased availability of cholesterol in the hepatocytes of progesterone-treated baboons will increase hepatic ACAT activity. However, estrogen when given in combination with progesterone will induce the sterol 27-hydroxylase and cholesterol 7-hydroxylase and, therefore, more cholesterol will be excreted in the bile. An increase in bile secretion will decrease hepatic cholesterol available for VLDL secretion and will downregulate hepatic ACAT activity. Thus, hepatic ACAT activity is regulated by the availability of cholesterol for VLDL secretion and not by a specific effect of progesterone in the liver.
Acknowledgments
This work was supported by NIH grants HL 34982 and HL 41256 and contract HV 53030 from NHLBI. We thank George Lyman for providing technical assistance for these studies. We also thank Dr S.Q. Hasan for the measurement of 27-hydroxycholesterol concentration in plasma and liver.
Received July 17, 1995; revision received February 7, 1996; References
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