Hepatic cholesterol metabolism in estrogen-treated men

GASTROENTEROLOGY

1992;103:1657-1663

Hepatic Cholesterol Metabolism in EstrogenTreated Men BO ANGELIN, HANS DAGNY STAHLBERG, PETER HENRIKSSON, Metabolism Unit, Departments Hospital, Huddinge, Sweden

OLIVECRONA, EVA REIHNER, MATS RUDLING, MATS ERIKSSON, STAFFAN EWERTH,

and KURT EINARSSON of Medicine

and Surgery, Karolinska

Operative liver biopsies were obtained from two male patients who developed gallstone disease during estrogen treatment of metastatic prostatic carcinoma. The heparin-sensitive binding of ‘251-lowdensity lipoprotein (LDL) to liver homogenates (reflecting the expression of the LDL receptor) was determined, together with the activities of the ratelimiting enzymes in cholesterol synthesis [3-hydroxy+methylglutaryl-coenzyme A (HMG-CoA) reductase], bile acid production (cholesterol 7a-hydroxylase), and cholesterol esterification (acyl CoA:cholesterol acyl transferase). The results were related to data available in 18 patients (5 male, 13 female) who underwent cholecystectomy because of gallstone disease. The hepatic ‘251-LDL-binding activity was increased threefold compared with five controls, and the activity of HMG-CoA reductase was increased twofold. There was no major difference in the activities of cholesterol %+hydroxylase or acyl CoA:cholesterol acyl transferase. The concentration of free and total cholesterol in liver microsomes was -30% lower in the estrogentreated men than in 11 controls. The results indicate that estrogen at pharmacological doses stimulates hepatic LDL-receptor expression and HMG-CoA reductase activity in men. The increased LDL-receptor expression could in part explain the enhanced plasma clearance of injected ‘251-LDLand hence the reduction in plasma LDL cholesterol previously shown to occur in estrogen-treated men. he liver is a key organ in the regulation of body cholesterol, being the major site of both synthesis and net excretion of cholesterol as such or after conversion into bile acids.‘*’ Very low-density lipoproteins (VLDL) emanate predominantly from the liver,3 and more than half of the receptor-mediated elimination of plasma low-density lipoproteins (LDL) occurs in this organa4s5The expression of hepatic LDL receptors has been shown to be regulated by hor-

T

Institute

at Huddinge

University

monal and metabolic stimuli in several animal species.“’ Of particular interest is the observation that pharmacological doses of estrogen increase LDL-receptor expression and LDL uptake in rat liver.g*‘o Previous studies have shown that serum LDL cholesterol levels are reduced in response to estrogen therapy in men’1~12and that an enhanced clearance of LDL from the circulation may explain this phenomenon.” We have hypothesized that the latter change is the result of induced LDL-receptor activity in the liver during pharmacological estrogen therI1 similar to that observed in the rat.g*‘ OA possiapy, bility of directly evaluating the role of estrogen treatment in the regulation of hepatic LDL receptors in humans emerged when two male patients, on chronic estrogen therapy for prostatic cancer, developed gallstone disease and were scheduled for cholecystectomy. After informed consent, we were able to obtain operative liver biopsy specimens at cholecystectomy and could specifically seek the answer to the question of whether pharmacological therapy with estrogen induces LDL-receptor activity in the human liver. The results show a powerful stimulation of LDL-receptor activity by estrogen and further suggest a stimulated activity of the rate-limiting enzyme step in cholesterol biosynthesis, $hydroxy-3methylglutaryl-coenzyme A (HMG-CoA) reductase. Materials and Methods Patients Two male patients in whom estrogen therapy had been given for more than z years because of metastatic cancer of the prostate’l,” were studied. In both subjects, symptoms of gallstone disease occurred during treatment, and they were advised by an independent surgeon to Flave cholecystectomy performed. The clinical data of these two patients are shown in Table 1, together with data on 18 0 1992 by the American

Gastroenterological 0016-5065/92/$3.00

Association

1658

ANGELIN ET AL.

GASTROENTEROLOGY Vol. 103,No. 5

Table Z. Basal Data on Patients

and Controls Plasma lipidsb

Estrogen treatmeW Patient 1 Patient 2 Controls (n = 18) Mean + SEM Range

Age lyr)

Relative body weight” (%)

Cholesterol [mmol/L (mg/dL1]

68 69

107 103

5.4(209) 6.2(240)

52 + 3 37-71

92 + 3 64-111

5.5-t0.2(213f 8) 3.8-7.3 (147-283)

Triglyceride [mmol/L (mg/dLJ] 2.5(221) 2.4(212) 1.3+ 0.1(1152 9) 0.4-2.2 (35-195)

“Calculated as Body Weight (kg)/[Height (cm] - 1001X 100%. bDetermined in fasting blood samples obtained at the time of operation. “Polyestradiol phosphate, 80 mg intramuscularly per month, and oral ethinyl estradiol, 150 pg daily, for >2 years.

otherwise healthy patients (5 male, 13 female) with uncomplicated gallstone disease who served as untreated controls. Informed consent was obtained from each subject, and the ethical aspects of the study were approved by the Ethical Committee of Karolinska Institutet (January 9, 1989).

Experimental

Procedure

The patients were admitted to the hospital on the day before operation and were given the regular hospital diet. Operations were performed between 8 and 9 AM after a l&hour fast. Standardized anesthesia was given in all 20 patients with tiopentothal induction and continuous treatment with nitrous oxide, diazepam, and phentanyl.13 Immediately after the abdomen was opened, a liver biopsy (2-4 g) was taken from the left lobe and placed in ice-cold homogenizing medium; the preparation of microsomes was started at the laboratory within 10 minutes. A small specimen of the liver was sent for histological examination. The cystic duct was clamped and gallbladder bile was obtained by aspiration. Cholecystectomy was performed without any complications. From analysis by light microscopy, there were no abnormalities of hepatic structure in the two estrogen-treated subjects compared with the untreated controls; there was evidence of slight fatty infiltration in patient 1, whereas the pattern was normal in patient 2. Fatty infiltration was also observed in 5 of the untreated gallstone patients. Analysis of Plasma Lipid and Lipoprotein Levels

and Analyses

The liver biopsy specimens mogenized in a Potter-Elvehjem

Assays

of Liver Microsomes were minced homogenizer

and howith a

of Microsomal

Enzyme

Activities

Microsomal HMG-CoA reductase activity was determined by measuring the conversion of 14C-HMG-CoA to mevalonate.13 This assay measures the (total) activity of the enzyme after activation of inactive enzyme during the preparation of microsomes.‘3 The activity of cholesterol 7a-hydroxylase was determined using a method based on isotope dilution-mass spectrometry.17 The assay of ACAT activity was performed by addition of “C-oleoyl CoA and determination of its conversion to cholesteryl oleate.” The coefficient of variation of the three enzyme assays are 7%, lo%, and 7%, respectively. Assay

Plasma cholesterol and triglycerides were analyzed by enzymatic methods. The different lipoproteins were analyzed by a combination of ultracentrifugation and precipitation.14 One of the two estrogen-treated patients (patient 1) previously participated in a study on “‘1-LDL turnover”; the data from that study are also included here for comparison. Preparation

loosely fitting Teflon pestle in nine volumes of 50 mmol/L Tris-HCl buffer, pH 7.4, containing 0.3 mol/L sucrose and 50 mmol/L NaCl (HMG-CoA reductase assay), 10 mmol/L dithiothreitol (DTT), 10 mmol/L ethylenediaminetetraacetic acid (EDTA) (cholesterol 7a-hydroxylase and HMGCoA reductase assays) or 1 mmol/L EDTA [acyl CoA: cholesterol acyl transferase (ACAT) assay]. DTT was excluded in the ACAT assay. The homogenate was centrifuged at 20,OOOgfor 15 minutes at 4’C. The supernatant was centrifuged at 100,OOOg for 60 minutes and recentrifuged at 100,OOOg (without DTT in the cholesterol 7a-hydroxylase assay) for another 60 minutes (except in the assay of ACAT). The microsomal content of protein was determined by the method of Lowry et a1.15Free and total cholesterol concentrations were determined by isotope dilutionmass spectrometry as previously described.16

of LDL Binding

to Hepatic

Receptors

The heparin-sensitive binding of LDL to liver tissue homogenates was determined using a filter assay as described.” Liver homogenates were stored at -20°C and analyzed at the same occasion. The results represent specific heparin-sensitive binding (nanograms of “‘1-LDL per milligram of protein) at an “‘1-LDL protein concentration of 50 pg/mL; the coefficient of variation of the assay is 9%. Analysis

of BiJiary

Lipids

Gallbladder bile was extracted with chloroformmethanol 2:1 (vol/vol), and the chloroform phase was ana-

November

ESTROGEN AND HEPATIC CHOLESTEROL METABOLISM

1992

lyzed with respect to cholesterolzO and phospholipids.zl Total bile acid concentration was determined using an enzymatic method22 in another portion of bile. The relative concentrations of cholesterol, bile acids, and phospholipids were expressed as molar percentage of total biliary lipids. The cholesterol saturation of bile (%) was calculated according to the method of Carey.23 Bile acid composition was determined using gas-liquid chromatography.24 Statistical

Analysis

Data observed in the patients are contrasted to the 95% confidence intervals for the control group. Results Long-term treatment with estrogen significantly influenced the hormonal status in the two men to a therapeutically desired extent (Table 2). The clinical effect on the malignant disease was also satisfactory, and the subjects had no symptoms or evidence of active disease at routine follow-up. Apart from symptoms of gallbladder disease, the subjects did not have any evidence of hepatic dysfunction and, similar to the untreated controls, they did not display any laboratory evidence of cholestasis (Table 2). Although total cholesterol levels in the two estrogen-treated men were within the normal range (Table l), a detailed analysis of lipoprotein distribution (Table 3) demonstrated a tendency for higher highdensity lipoprotein (HDL) and lower LDL cholesterol

Table 2. Hormonal Levels and Serum Enzyme Tests in the Two Patients on Long-Term Estrogen Therapy

Analysis Testosterone (nmol/L) Prolactin &g/L) Follicle-stimulating hormone (U/L) Luteinizing hormone (U/L)

Patient 1

Patient 2

0.7

0.6 5

10

Reference values (men >55 yr old) 6.0-18.5
4

4

4-15

<2

12

4-18

15

22

Acid phosphatase C&G) Aspartate aminotransferase @kot/L) Alanine aminotransferase @kot/L) Alkaline phosphatase Iukat/L1 NOTE. Estrogen therapy included intramuscularly per month, and daily, for 22 years. Analyses were tion; all analyses were performed terone measurement (plasma).

0.29

<0.70

0.31

0.19

<0.70

2.7

2.1

<4.2

polyestradiol oral ethinyl performed at using serum,

Table 3. Changes in Lipoprotein Long-Term

phosphate 80 mg estradiol, 150 pg the time of operaexcept for testos-

Cancer

Basal [mmoI/L (mg/dL)l

Long-term estrogen therapy ImmoI/L (mg/dL)l

Change (%)

2

6.6 (255) 7.8 (302)

5.4 (209) 6.2 (240)

-18 -21

1 2

0.53 (21) 0.54 (21)

0.35 (14) 1.2 (46)

-34 +118

1 2

5.5 (213) 5.8 (225)

3.7 (143) 2.7 (105)

-32 -53

1 2

0.61 (24) 1.5 (58)

1.4 (54) 2.4 (93)

+121 +60

1 2

2.0 (177) 1.2 (106)

2.5 (221) 2.4 (212)

+25 +100

Analysis Cholesterol Total Patient Patient VLDL Patient Patient LDL Patient Patient HDL Patient Patient Triglycerides Total Patient Patient

Levels in Response to Therapy in Two Patients

Estrogen

With Prostatic

1

NOTE. Analyses were performed in fasting blood samples obtained before the initiation of estrogen treatment and at the time of operation.

concentrations. Because baseline determinations of plasma lipoprotein levels were available, rather drastic changes in the lipoprotein classes could be documented in both individuals (Table 3). These findings, with a 30%~50% reduction in LDL cholesterol, and an almost twofold elevation of HDL cholesterol levels are compatible with the results observed in larger series of patients treated similarly.‘2 The reduction in LDL cholesterol has been shown to occur in parallel with an increased fractional catabolic rate (FCR) of LDL,” and the data available from a previous study in patient 1 were in agreement with this concept; patient 1 had a 60% increase in LDL

Table 4. Kinetics Months

of Autologous of Therapy

‘%LDL Before and After 6 with Estrogen in Patient I

Before therapy

<70

0.35

1659

LDL cholesterol [mmol/L (mg/dLI] LDL apoB (mg/dL) LDL FCR (pools/day) LDL catabolic rate (mg . kg-’ . day-‘)

5.5 (213) 146 0.24” 0.24b 14.9

During therapy

Change

3.7 (143) 111 0.39” 0.45b

-32% -24% +63% +88%

18.2

“Calculated from plasma radioactivity decay curve. bCalculated from urine/plasma radioactivity data. Data from Eriksson et al.”

+22%

1660 ANGELIN ET AL.

GASTROENTEROLOGY Vol. 103, No. 5

FCR concomitant with a 20% enhancement of LDL catabolism, which equals synthetic rate, after 6 months of therapy (Table 4)” The possibility of obtaining liver specimens during cholecystectomy in these two estrogen-treated patients provided a unique opportunity to characterize the effects of this hormone on some important parameters involved in hepatic cholesterol and lipoprotein metabolism. When the heparin-sensitive binding of ‘*‘I-LDL to liver homogenates of the two estrogen-treated men was compared with that of samples from untreated gallstone patients (n = 5), it was found that the binding was more than threefold higher in the samples from the estrogen-treated subjects (Table 5). The binding activity was highest in patient 2, who showed the largest reduction in plasma LDL cholesterol levels (Table 3). When the liver biopsy specimens were analyzed for microsomal enzymes related to cholesterol metabolism, the total (maximal) activity of the ratelimiting enzyme in cholesterol biosynthesis, HMGCoA reductase, was increased twofold in the two patients compared with the controls (Table 5). The activities of cholesterol 7a-hydroxylase (catalyzing bile acid production; data available on only one of

Table 5. Measurements of Hepatic LDL-Receptor Binding Activity, Enzyme Activities, and Cholesterol Concentrations in Patients on Estrogen Therapy

LDL-receptor binding (ng/mg protein) HMG-CoA reductase (pmof - min-’ . mg protein-‘) Cholesterol 7a-hydroxylase (pm01 . min-’ . mg protein-‘)

Patient 1

Patient 2

6.0

8.6

231

10.2

153

ND

Controls (95% CI) 1.6-3.2 (n = 5) 76-110 (n = 18) 6.0-11.0 (n = 17)

ACAT (pmol . min-’ . mg protein-‘)

Microsomal cholesterol (nmol/mg protein) Free Esterified Total

4.5

5.7

51.3 7.0 58.3

51.1 12.3 63.4

4.8-6.6 (n = 15)

69.4-84.4

9.6-15.0 80.2-98.4

(n = 11) Total homogenate cholesterol (nmol/mg protein) Free Esterified Total 95% CI, 95% confidence

30.3 4.6 34.9

37.8 1.9 39.7

interval; ND, not determined.

26.5-38.5 5.4-9.8 32.8-47.4 (n = 11)

Table 6. Biliary Lipid Composition of Gallbladder Bile from Patients on Estrogen Therapy Patient 1 Cholesterol

(mol%)

Bile acids (mol%) Phospholipids

(moI%)

Total biliary lipids (g/dLl Cholesterol

7.4

6.6

83.3

67.2

9.3

26.1

4.2

4.8

Controls (95% CI) 6.3-10.7 (n = 9) 63.7-73.1 (n = 9) 20.3-26.1 (n = 9) 2.6-6.0 (n = 9)

saturation

(%) Cholic acid (%) Chenodeoxycholic (%I Deoxycholic

Patient 2

175

96

69

72

21

20

10

8

108-174 (n = 9) 41-53 (n = 15)

acid

acid (%)

NOTE. Trace amounts of lithocholic acid were observed. 95% CI, 95% confidence interval.

28-36 (n = 15) 13-25 (n = 15)

acid and ursodeoxycholic

the patients) and ACAT (governing the formation of cholesteryl esters) were in the same range as in the controls. Analysis of free, esterified, and total cholesterol levels in the liver homogenates did not show any difference between estrogen-treated and control patients (Table 5). However, the concentrations of free and total microsomal cholesterol were reduced by approximately Xl%-35% in the two hormonetreated men. Analysis of biliary lipid composition did not show any major change in the relative concentration of cholesterol, bile acids, or phospholipids induced by estrogen compared with controls (Table 6). However, the proportion of cholic acid was increased in the estrogen-treated individuals, whereas the proportion of chenodeoxycholic acid tended to decrease (Table 6). This finding is in agreement with observations in gallbladder bile from patients without gallstones on estrogen therapy’2,25,26 and may indicate a redistribution in the composition of the bile acid pool. Discussion To observe maximal metabolic effects of estrogen, we studied a situation in which pharmacological doses of this hormone are administered to men.“vl’ This results in more pronounced changes in cholesterol metabolism than those observed during estrogen treatment in postmenopausal women.26 In

November

1992

estrogen-treated men, there is an increased prevalence of gallstone disease,” and we had the opportunity to obtain liver tissue from two such men who underwent cholecystectomy. This clinical situation permitted a unique opportunity to study the effect of estrogen treatment on several important regulatory steps in cholesterol and lipoprotein metabolism in humans, although it did not allow for complete matching of the controls for age or sex. The estrogentreated patients had no signs of cholestasis, as evaluated on laboratory and histological grounds. The results of the present study represent the first demonstration of an increased expression of hepatic LDL-receptor binding in estrogen-treated humans. This finding is in agreement with previous results obtained in various laboratory animal species under different nutritional conditions.gJ0~27~2g The more than threefold increase in LDL-receptor binding is of major interest for several reasons. First, it represents a larger stimulus of LDL-receptor expression than that induced by hypolipidemic drugs such as cholestyramine or pravastatin.‘g~30~31Second, it shows that expression of the receptor in the liver can be induced considerably in elderly individuals. Third, the stimulation grade of LDL receptors in the liver can be calculated to correspond to a twofold increase in LDL FCR (provided that the liver represents 50% of receptor-mediated catabolism of LDL). This is in good agreement to what has been observed in male patients with prostatic carcinoma.” In these studies (which included one of the patients in this report; Table 4) a mean increase in LDL FCR from 0.27to 0.49 pools/day (81%)was observed; if anything, this probably represents a slight underestimation of the actual receptor stimulation because the affinity for the binding of autologous LDL particles isolated during therapy is somewhat reduced.*l Another interesting finding was that the measured total (maximal] activity of HMG-CoA reductase in human liver microsomes was increased during estrogen therapy. This most probably results in an enhanced cholesterol synthesis; however, there are no data on cholesterol balance in vivo during pharmacological therapy with estrogen as far as we know. The levels of VLDL triglycerides, and most probably the synthetic rate of VLDL, are frequently increased during estrogen therapy.“13’ This is probably relevant for the previously demonstrated” increase in LDL synthetic rate. In the estrogen-treated men, decreases in the concentrations of total and free cholesterol in liver microsomes were observed, despite the presence of increased activities of both pathways for cholesterol delivery to and within the hepatocyte, the LDL receptor, and the HMG-CoA reductase. No conclusions

ESTROGEN AND HEPATIC CHOLESTEROL METABOLISM

1661

can be drawn from the present study with regard to the effect of estrogen treatment on the activity of the rate-limiting enzymatic steps of bile acid biosynthesis, the cholesterol 7a-hydroxylase, or cholesterol esterification, the ACAT. However, if present, the effects would be of a lesser magnitude. The current findings in humans are not in full agreement with the available data in estrogentreated rats. Thus, there have been reports of both increased33 and decreased2gp34 activities of HMG-CoA reductase, whereas the activity of ACAT appears to be increased.” This species difference could be attributable to the suppressed bile secretion that occurs in estrogen-treated rats.35 There was no evidence of a cholestatic effect in the men investigated in our study, and previous experiments have not shown any decrease in secretion of bile lipids at these doses of estrogen in men.” The increased LDL-receptor expression and HMGCoA reductase activity observed in the estrogentreated men in the present study would indicate an enhanced demand for cholesterol, and a lower concentration of free cholesterol would be in agreement with this concept. The critical question of what mechanism causes the lowered microsomal cholesterol levels in estrogen-treated humans then emerges. Although other mechanisms may well be considered, the most plausible explanation would be that membrane cholesterol is lost because of the increased secretion of free cholesterol into bile.” Thus, in the human situation, where cholestasis is not present, the direct or indirect effects of estrogen treatment may be exerted on biliary canalicular membranes and/or the intracellular distribution of cholesterol available for subsequent biliary excretion The close correlation obtained between the increase in biliary cholesterol output and the decrease in plasma LDL cholesterol levels” tends to support this concept. Finally, it is important to consider that the drastic effects of estrogen treatment on LDL-receptor expression in human liver are obtained in the in vivo situation. That such changes have been difficult to confirm in studies with cultured human hepatoma cells exposed to estrogen in vitro36,37 may indicate that estrogen treatment does not act directly on the hepatocyte but results in alterations of hormonal homeostasis which in turn influence cholesterol metabolism. In this respect, it is interesting that the secretion of growth hormone is stimulated considerably during estrogen therapy.38 In preliminary studies, we recently showed that growth hormone is involved in the stimulation of hepatic LDL receptors that occurs during estrogen treatment of rats3’ In conclusion, we have shown an increased ex-

1662

GASTROENTEROLOGY

ANGELIN ET AL.

pression

of LDL receptor

HMG-CoA tained

reductase

and an enhanced

in liver

from two men treated

biopsy

activity

specimens

with pharmacological

doses of estrogen.

The fact that microsomal

terol

decreased

levels

were

ment may indicate terol

into

bile

during

during

driving

force

response

in humans.

such

choles-

estrogen

that the increased

primary

of ob-

treat-

loss of choles-

therapy

for the regulatory

could

be

a

metabolic

References 1. Myant

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acid synthesis in man: assay of hepatic microsomal cholesterol 7a-hydroxylase activity by isotope dilution-mass spectrometry. J Lipid Res 1986;27:82-88. ia. Einarsson K, Benthin L, Ewerth S, Hellers G, Stihlberg D, Angelin B. Studies on acyl-coenzyme A:cholesterol acyltransferase activity in human liver microsomes. J Lipid Res 1989;30: 739-746. 19. Rudling MJ, Reihner E, Einarsson K, Ewerth S, Angelin B. Low density lipoprotein receptor binding activity in human tissues: quantitative importance of hepatic receptors and evidence for regulation of their expression in vivo. Proc Nat1 Acad Sci USA 1990;87:3469-3473. 20. Roda D, Festi D, Sama C, Mazella G, Aldini T, Roda E, Barbara L. Enzymatic determination of cholesterol in bile. Clin Chim Acta 1975$X337-341. 21. Rouser G, Fleischer S, Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1970;5:494-496. 22. Fausa 0, Skllhegg BA. Quantitative determination of bile acids and their conjugates using thin-layer chromatography and a purified 3a-hydroxysteroid dehydrogenase. Stand J Gastroent 1974;9:249-254. 23. Carey M. Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 1978;19:945-955. 24. Angelin B, Einarsson K, Leijd B. Biliary lipid composition during treatment with different hypolipidaemic drugs. Eur J Clin Invest 1979;9:185-190. 25. Everson CT, Fennessy P, Kern F Jr. Contraceptive steroids alter the steady-state kinetics of bile acids. J Lipid Res i988;29:6a-76. 26. Everson GT, McKinley C, Kern F Jr. Mechanisms of gallstone formation in women. Effects of exogenous estrogen (Premarin) and dietary cholesterol on hepatic lipid metabolism. J Clin Invest 1991;87:237-246. 27. Ma PTS, Yamamoto T, Goldstein JL, Brown MS. Increased mRNA for low density lipoprotein receptor in livers of rabbits treated with 17a-ethinyl estradiol. Proc Nat1 Acad Sci USA 1986;83:792-796. 28. Henriksson P, Stamberger M, Eriksson M, Rudling M, Diczfalusy U, Berglund L, Angelin B. Oestrogen-induced changes in lipoprotein metabolism: role in prevention of atherosclerosis in the cholesterol-fed rabbit. Eur J Clin Invest 1989;19:395403. 29. Erickson SK, Jaeckle S, Lear SR, Brady SM, Have1 RJ. Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats. J Lipid Res 1989;30:1763-1771. 30. Reihner E, Rudling M, Stdhlberg D, Berglund L, Ewerth S, Bjorkhem I, Einarsson K, Angelin B. Influence of pravastatin, a specific inhibitor of HMG CoA reductase, on hepatic metabolism of cholesterol. N Engl J Med 1990;323:224-228. 31. Reihner E, Angelin B, Rudling M, Ewerth S, Bjijrkhem I, Einarsson K. Regulation of hepatic cholesterol metabolism in man: stimulatory effects of cholestyramine on HMG-CoA reductase activity and low density lipoprotein receptor expression in gallstone patients. J Lipid Res 1990;31:2219-2226. 32. Schaefer EJ, Foster DM, Zech LA, Lindgren FT, Brewer HB Jr, Levy RI. The effects of estrogen administration on plasma lipoprotein metabolism in premenopausal females. J Clin Endocrinol Metab 1983;57:262-267. 33. Abul-Haji YJ. Effect of estrogens on 6-hydroxy+methylglutaryl coenzyme A reductase activity and cholesterol levels. Steroids 1981;37:601-607. 34. Mukherje S, Bhose A. Studies on estrogen regulation of choles-

November

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37.

38.

1992

terol biosynthesis in rat liver microsomes. Biochim Biophys Acta 1968;164:357-368. Gumucio JJ, Valdivieso VD. Studies on the mechanisms of the ethynyl estradiol impairment of bile flow and bile salt excretion in the rat. Gastroenterology 1971;61:339-344. Semenkovich CF, Ostlund RE Jr. Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2. Biochemistry 1987;26:4987-4992. Wade DP, Knight BL, Soutar AK. Hormonal regulation of lowdensity lipoprotein (LDL) receptor activity in human hepatoma Hep G2 cells. Insulin increases LDL receptor activity and diminishes its suppression by exogenous LDL. Eur J Biothem 1988;174:213-218. Thorner MO. Vance ML. Growth hormone. J Clin Invest 1988;82:745-747.

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39. Rudling M, Norstedt G, Olivecrona H, Reihner E, Gustafsson J-A, Angelin B. Importance of growth hormone for the induction of hepatic low density lipoprotein receptors. Proc Nat1 Acad Sci USA 1992;89:6983-6987. Received October 17, 1991. Accepted June 16, 1992. Address requests for reprints to: Bo Angelin, M.D., Department of Medicine, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Supported by grants from the Swedish Medical Research Council (03X-4793, 03X-7137, and 03K-87223, the King Gustav V and Queen Victoria Foundation, the Hans and Loo Osterman Foundation, and the Karolinska Institute. The skilful technical assistance of Lisbet Benthin, lngela Svensson, and Kristina Soderberg-Reid is gratefully acknowledged. The authors thank Lena Ericsson for editorial assistance.