Regulation of hepatic apolipoprotein synthesis in the 17 alpha-ethinyl estradiol-treated rat

Regulatory mechanisms of hepatic apolipoprotein synthesis were studied in groups of male Sprague-Dawley rats made severely hypolipidemic by treatment with pharmacological doses of 17 alpha-ethinyl estradiol. Treatment resulted in a marked reduction of plasma cholesterol and apolipoproteins B, A-I, and A-IV. Hepatic apoA-I mRNA and apoA-I synthesis were increased in the ethinyl estradiol-treated animals. Hepatic apoA-IV protein synthesis rates were unaltered; however, a reduction of the apoA-IV mRNA level was observed. Diet-control studies suggested the effects of 17 alpha-ethinyl estradiol on apoA-I, unlike those on apoA-IV, appeared to be related to the steroid and not to reduced caloric intake. Livers of control and ethinyl estradiol-treated rats synthesized both apoBH and apoBL. Total hepatic apoB (apoBL plus apoBH) synthesis and apoB mRNA levels in the ethinyl estradiol-treated rats were similar to ad libitum fed or diet-controls. In ad libitum fed and diet-control rats, 21% and 32%, respectively, of newly synthesized hepatic apoB was apoBH. In contrast, 47% of the newly synthesized apoB in the ethinyl estradiol-treated animal was apoBH. Nucleotide sequence analysis of hepatic apoB mRNA confirmed a marked decrease in the proportion of the apoBL mRNA in ethinyl estradiol-treated animals. After cessation of 17 alpha-ethinyl estradiol treatment, the hepatic apolipoprotein A-I synthesis rate, apolipoprotein A-I and A-IV mRNA levels, and the apoBH and apoBL synthesis rates, as well as plasma apolipoprotein and cholesterol levels, returned to normal. A major finding of the present study is that pharmacological doses of ethinyl estradiol do not affect total hepatic apoB synthesis, but increase the relative amount of apoBH synthesized.     

The influence of estrogen on hepatic cholesterol metabolism and biliary lipid secretion in rats fed fish oil

Both estrogen and dietary n-3 polyunsaturated fatty acids are known to be hypocholesterolemic, but appear to exert their effects by different mechanisms. In this study, the interaction between dietary fish oil (rich in n-3 polyunsaturated fatty acids) and estrogen in the regulation of hepatic cholesterol metabolism and biliary lipid secretion in rats was studied. Rats fed a low fat or a fish oil-supplemented diet for 21 days were injected with 17alpha-ethinyl estradiol (5 mg/kg body weight) or the vehicle only (control rats) once per day for 3 consecutive days. Estrogen-treatment led to a marked reduction in plasma cholesterol levels in fish oil-fed rats, which was greater than that observed with either estrogen or dietary fish oil alone. The expression of mRNA for cholesterol 7alpha-hydroxylase was decreased by estrogen in rats fed a low fat or a fish oil-supplemented diet, while the output of cholesterol (micromol/h/kg b.wt.) in the bile was unchanged in both groups. Cholesterol levels in the liver were increased by estrogen in rats given either diet, but there was a significant shift from cholesterol esterification to cholesteryl ester hydrolysis only in the fish oil-fed animals. Estrogen increased the concentration of cholesterol (micromol/ml) in the bile in rats fed the fish oil, but not the low fat diet. However, the cholesterol saturation index was unaffected. The output and concentration of total bile acid was also unaffected, but changes in the distribution of the individual bile acids were observed with estrogen treatment in both low fat and fish oil-fed groups. These results show that interaction between estrogen-treatment and dietary n-3 polyunsaturated fatty acids causes changes in hepatic cholesterol metabolism and biliary lipid secretion in rats, but does not increase the excretion of cholesterol from the body.     

Visualization of the hepatic low-density lipoprotein receptor in rats by sequential scintiscans

The usefulness of a gamma camera system for external imaging of the degradation sites of low-density lipoprotein (LDL) is demonstrated in rats. [131I]LDL was injected intravenously in normal rats and rats pretreated with 17 alpha-ethinyl estradiol for LDL receptor induction. Distribution of the radioligand was followed for 20 min, for perfusion control [131I]albumin was administered. Rats pretreated with 17 alpha-ethinyl estradiol showed a marked increase in the LDL uptake by the liver, which was not competed by the preinjection of physiological amounts of unlabelled LDL. It is concluded that the injection of small amounts of [131I]LDL is sufficient to image the hepatic LDL receptor and that the endogenous LDL does not compete effectively at physiological LDL levels.     

The effect of hypocholesterolemic drug treatment on adrenocortical cell function

The role of exogenous lipoprotein cholesterol versus endogenous cholesteryl esters as substrates in adrenal steroidogenesis was studied in isolated rat adrenal cells. Hypocholesterolemic drugs were used in rats to depress the plasma cholesterol concentration and the adrenal cholesterol concentration. Adrenal cortical cells were prepared in the usual way. The steroidogenic response to ACTH in normal adrenal cells and in cells which have been cholesterol-depleted was studied. Normal adrenal cells responded specifically over a 6 h incubation period to low doses of ACTH (half-maximal response equivalent to 40 microunits ACTH). These normal cells exhibited no altered response over a 3 h period to ACTH in the presence of serum or serum lipoproteins. The hypocholesterolemic drugs, 4-aminopyrazolo-[3,4-d]-pyrimidine, hexestrol and 17 alpha-ethinyl estradiol were used to lower plasma cholesterol, and after 1 day of 4-aminopyrazolo-[3,4-d]-pyrimidine and 5 days of hexestrol or 17 alpha-ethinyl estradiol treatment the plasma total cholesterol concentrations were similar. After 3 days of 4-aminopyrazolo-[3,4-d]-pyrimidine treatment the adrenal total cholesterol content was lower than after 1 day of this treatment, or 5 days of hexestrol treatment or 5 days of 17 alpha-ethinyl extradiol treatment. Lipoproteins had no significant effect on ACTH-stimulated steroidogenesis in cells isolated from rats treated for 1 day with 4-aminopyrazolo-[3,4-d]-pyrimidine, or for 5 days with hexestrol or 17 alpha-ethinyl estradiol. However, lipoproteins did stimulate steroidogenesis in cells from rats treated for 3 days with 4-aminopyrazolo-[3,4-d]-pyrimidine. The results show that normal adrenal cells contain a reserve of intracellular cholesterol so that the supply of endogenous cholesterol for steroidogenesis does not limit the response to ACTH and exogenous lipoproteins have no effect on steroidogenesis. However, if the cells are severely depleted of cholesterol then exogenous lipoproteins must be added for maximal steroidogenesis to occur.     

The uptake of chylomicron remnants and very low density lipoprotein remnants by the perfused rat liver

The regulation of the hepatic uptake of chylomicron remnants and very-low-density lipoprotein (VLDL) remnants was studied in the rat using a nonrecirculating liver perfusion system. The hepatic removal of remnant lipoproteins was shown to be by receptor-mediated processes since the concentration-dependent uptake was saturable and reductive methylation of the particles reduced the uptake of each lipoprotein by two-thirds. Treatment of liver donor rats with 17 alpha-ethinyl estradiol resulted in a 2-fold increase in the hepatic uptake of VLDL remnants, while cholesterol feeding of liver donor rats caused complete suppression of the receptor-mediated uptake of VLDL remnants. Chylomicron remnant removal was unaffected by estradiol administration and only slightly diminished by cholesterol feeding. The results of competition studies also indicated that a specific chylomicron remnant receptor exists in the liver. Apoprotein E was shown to be required for the receptor-mediated uptake of both remnant lipoproteins. Chylomicron remnants which contained no apoprotein E and VLDL remnants which contained reductively methylated apoprotein E were removed by the liver to about one-third of the extent of native particles. Thus the hepatic uptake of remnant lipoproteins occurs by receptor-mediated processes and the specific removal of both particles is mediated by apoprotein E. In addition, the uptake of VLDL remnants is regulated by the same factors that control hepatic low-density lipoprotein removal, while chylomicron remnant removal is unaffected by these factors.     

Biotinylation of low density lipoproteins via free amino groups without loss of receptor binding activity

Low density lipoproteins were biotinylated via free amino groups using carbodiimide-activated biotin or D-biotin-N-hydroxysuccinimide ester. The receptor binding activity of the biotinylated LDL was determined by their ability to displace 125I-labeled LDL from the rat hepatic LDL receptor in the liposome filtration assay. LDL biotinylated with either of the two reagents was able to compete effectively with 125I-labeled LDL providing less than twenty biotin moieties were incorporated per lipoprotein particle. When more than twenty biotins were linked there was a marked loss of activity. The following conditions were adopted as standard for the biotinylation of LDL via free amino groups: 0.3 mumol of D-biotin-N-hydroxysuccinimide ester was incubated with 2 mg of LDL for 1 hr at room temperature. These conditions reproducibly yielded 11.3 +/- 0.6 biotins per LDL particle. With the biotinylated LDL and a performed streptavidin-biotinylated peroxidase complex, the hepatic LDL receptor from rats treated with 17 alpha-ethinyl estradiol was visualized as a single band on electroblots. Finally, the biotinylated LDL was used in an enzyme-linked sorbent assay for the LDL receptor. When solubilized liver membrane proteins from rats treated with 17 alpha-ethinyl estradiol were fixed to the wells with 1.6% paraformaldehyde, a specific binding greater than 0.4 absorbance units was observed which was about ninefold higher than with solubilized proteins from normal rats. We therefore suggest that D-biotin-N-hydroxysuccinimide ester can be used to biotinylate LDL reliably without destroying the lipoprotein's ability to bind specifically to its high affinity receptor.     

A correlated morphological and biochemical study on the rat adrenal steroidogenesis

The ultrastructural and biochemical alterations produced by an hypocholesterolemic drug, 17 alpha-ethinyl estradiol, on the rat adrenal cortex were studied. Male rats aged two months and with approximately 200 g in weight were injected subcutaneously with 10 mg/kg/day of ethinyl estradiol during 9 days; rats injected with 1 ml propylene glycol were used as controls. The animals were sacrificed on the 10th day, and the adrenals from some of them were processed for electron microscopy. The adrenals from the remaining rats were used for measurements of the glands cholesterol and corticosterone, which were also measured in the blood. In estradiol-treated rats the zona fasciculata cells exhibited numerous microvilli, increase in the size of mitochondria and decrease in the number of lipid droplets. The quantitative analysis showed a significant increase of the volumetric density of mitochondria and microvilli and a significant decrease of the lipid droplets in the treated rats, when compared with normal ones. In treated rats, the concentration of cholesterol and corticosterone in the gland and blood were significantly decreased. These data show that hypocholesterolemia produced by estradiol has a remarkable effect on adrenal steroidogenesis, depletes the pool of adrenal cholesteryl esters, and evidences the role of plasma cholesterol in the corticosteroidogenesis.     

Stimulation of hepatic cholesterol 7α-hydroxylase activity by administration of an estrogen (17β-estradiol-3-benzoate) to female rats

Adult female rats were given daily subcutaneous injections of 17β-estradiol-3-benzoate, 25 μg/day for twenty days. Rats were then killed under four different experimental conditions; (1) Fasting, killed in the morning (FM), (2) Non-fasting, killed in the morning (NFM), (3) Fasting, killed at night (FN), and (4) Non-fasting, killed at night (NFN). Estradiol induced a three-fold increase in hepatic cholesterol 7α-hydroxylase activity in group FM and a significant increase in group NFN. Although increases in 7α-hydroxylase activity might be associated with lower serum cholesterol levels (by increasing the rate of conversion of cholesterol to bile acids), serum cholesterol concentrations were, in fact, elevated in all rats given estradiol.     

Effect of diosgenin on lipid metabolism in rats.

The purpose of this study was to determine whether diosgenin suppresses cholesterol absorption in rats, and to examine relevant changes in cholesterol and bile acid metabolism. Diosgenin fed with the diet for 1 week inhibited cholesterol absorption as determined by the serum isotope ratio technique, as well as by measuring in the feces the amount of unabsorbed radioactivity from orally administered [3H]cholesterol. In addition, diosgenin suppressed the serum and liver uptake of radioactivity from co-administered [3H]cholesterol as well as the accumulation of liver cholesterol in the cholesterol-fed rat; diosgenin was substantially more active than cholestyramine or beta-sitosterol. In vitro, diosgenin had no effect on the activity of rat pancreatic esterase. Diosgenin decreased the elevated cholesterol in serum LDL and elevated cholesterol in the HDL fraction of cholesterol-fed rats; diosgenin had no effect on serum cholesterol in normocholesterolemic rats. In contrast to cholestyramine, diosgenin markedly increased neutral sterol excretion without altering bile acid excretion; in vitro, diosgenin had no effect on bile acid binding. Diosgenin treatment increased hepatic and intestinal cholesterol synthesis as well as the activity of hepatic HMG CoA reductase. This was accompanied by increased biliary concentration of cholesterol, but not of bile acids. Diosgenin had no effect on cholesterol synthesis when added to normal rat liver homogenates. It was concluded that diosgenin interferes with the absorption of cholesterol of both exogenous and endogenous origin; such interference is accompanied by derepressed, i.e., increased, rates of hepatic and intestinal cholesterol synthesis. The increased unabsorbed cholesterol together with enhanced secretion of cholesterol into bile resulted in increased excretion of neutral sterols without affecting the biliary and fecal excretion of bile acids.     

The influence of estradiol on cholesterol processing by the corpus luteum

Recent studies from our laboratory have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular cholesterol metabolism including mobilization of cholesteryl esters, stimulation of lipoprotein receptor activity and induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity. To test the functionality of cholesteryl ester turnover per se, we measured the activities of acyl CoA:cholesterol acyltransferase (ACAT) and cholesteryl esterase, the enzymes involved in cholesteryl ester synthesis and hydrolysis, respectively; we also measured de novo synthesis of cholesterol, cholesteryl esters, and steroids. Pregnant rats, hypophysectomized and hysterectomized on Day 12, were treated for 72 h with either estradiol or testosterone, and luteal microsomal and cytosolic fractions were utilized to measure ACAT and cholesteryl esterase activity, respectively. Intact corpora luteal were employed for [14C]acetate incorporation experiments. Basal ACAT activity (expressed as pmol.min-1.CL-1 increased from a mean of 78 +/- 16 in vehicle-treated rats to 119 +/- 18 and 197 +/- 16 in the estradiol- and testosterone-treated rats, respectively. Similarly, total ACAT activity (measured in the presence of exogenous cholesterol) was also increased in estradiol- and testosterone-treated groups. On the other hand, cholesterol esterase activity (expressed either pmol.min-1.CL-1 or pmol.min-1.mg protein-1) was similar in all three groups and comparable to corpora lutea from intact pregnant rats. Hypophysectomy and hysterectomy caused a 50-60% reduction in [14C]acetate incorporation into sterols when compared with intact pregnant rat. Treatment with either estradiol or testosterone not only restored the cholesterol biosynthetic capacity but also enhanced the overall rate of [14C]acetate incorporation into steroids as compared to intact pregnant rats. The major (-80%), newly synthesized steroid was identified as progesterone. In conclusion, the present studies suggest that the major function of luteal estradiol is to induce de novo cholesterol biosynthesis, regulate ACAT activity, and channel available free cholesterol (derived from both endogenous and exogenous sources) for steroidogenesis.     

Metabolism of radioactive 17alpha-ethynylestradiol by women

Data on the urinary excretion and subsequent fractionation of radioactivity derived from 6,7-tritiated-17alpha-ethinyl estradiol (tritiated EE) are presented. Tritiated EE was administered orally to 9 women. 17alpha-ethinyl estradiol 2-methoxy-17alpha-ethinyl estradiol, 2 -hydroxy-17alpha-ethinyl estradiol 3-methyl ether, and D-homoestradiol-1 7abeta were identified as urinary metabolites by reverse isotope dilution. The extent of D-homoannulation was much less than that reported previously with rabbits.     

Regulation of hepatic cholesterol ester hydrolase and acyl-coenzyme A:cholesterol acyltransferase in the rat

Cholesterol exists within the hepatocyte as free cholesterol and cholesteryl ester. The proportion of intrahepatic cholesterol in the free or ester forms is governed in part by the rate of cholesteryl ester formation by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolysis by neutral cholesterol ester (CE) hydrolase. In other cell types both ACAT and CE hydrolase activities are regulated in response to changes in the need for cellular free cholesterol. In rats, we performed a variety of experimental manipulations in order to vary the need for hepatic free cholesterol and to examine what effect, if any, this had on the enzymes that govern cholesteryl ester metabolism. Administration of a 20-mg bolus of lipoprotein cholesterol or a diet supplemented with 2% cholesterol resulted in an increase in microsomal cholesteryl ester content with little change in microsomal free cholesterol. This was accomplished by an increase in cholesteryl esterification as measured by ACAT but no change in CE hydrolase activity. An increased need for hepatic free cholesterol was experimentally induced by intravenous bile salt infusion or cholestyramine (3%) added to the diet. ACAT activity was decreased with both experimental manipulations compared to controls, while CE hydrolase activity did not change. Microsomal cholesteryl ester content decreased significantly with little change in microsomal free cholesterol content. Addition of exogenous liposomal cholesterol to liver microsomes from cholestyramine-fed and control rats resulted in a 784 +/- 38% increase in ACAT activity. Nevertheless, the decrease in ACAT activity with cholestyramine feeding was maintained. These studies allowed us to conclude that changes in hepatic free cholesterol needs are met in part by regulation of the rate of cholesterol esterification by ACAT without a change in the rate of cholesteryl ester hydrolysis by CE hydrolase.     

Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver

Pharmacological doses of 17 alpha-ethinyl estradiol induce a low density lipoprotein (LDL) receptor in the liver of male rats. Our aim was to solubilize this receptor. Isolated liver membranes (8,000-100,000 g fraction) from male rats treated with 17 alpha-ethinyl estradiol and from control rats were solubilized in 1% (w/v) Triton X-100. Using Amberlite XAD-2, more than 90% of the detergent was then removed. Liposomes were prepared by precipitating the solubilized proteins with acetone in the presence of phosphatidylcholine. The receptor activity of these liposomes was assayed using human 125I-labeled LDL. Filtration was used to separate bound from free 125I-labeled LDL. The assay was optimized; 0.25 mM CaCl2, 25 mM NaCl, pH 8.0, were chosen as the standard conditions. Binding of 125I-labeled LDL was dependent on Ca2+. Liposomes containing solubilized membrane proteins from treated rats displayed Ca2+-dependent binding which was 11 times higher than for control rats. The specific binding of 125I-labeled LDL was saturable with a Kd = 18 micrograms/ml. 125I-Labeled LDL was displaced by unlabeled lipoproteins containing apolipoproteins B and E and by dimyristoylphosphatidylcholine liposomes containing purified apolipoprotein E, but not by HDL3. The binding was abolished by pronase and was inhibited by suramin. Ligand blotting with 125I-labeled LDL revealed one band of protein with an apparent molecular weight of 133,000 daltons. These properties are characteristic of the low density lipoprotein receptor.     

Binding of 17-alpha-ethinyl estradiol and mestranol to uterine and pituitary gland receptors of the rat in vitro

Comparative study, through radiochemical analysis, of the binding of estrogens to the uterine and pituitary receptors in immature rats showed that, for binding to the uterine recpetors, a free OH group in Position 3 is necessary. 17alpha-ethinyl estradiol as well as mestranol (17alpha-ethinyl estradiol-3-methyl ether), showed a stronger affinity for the pituitary than the uterine receptors. Indeed, mestranol, among the 3-methyl ether estrogens, has a special place in respect to its affinity for the pituitary. Since the binding to the receptors is an essential stage in inhibition or stimulation effects, this is a significant point. It may be that mestranol exercises its recognized central effect not only indirectly through release of ethinyl estradiol, but also directly.     

Acute effects of filipin on the plasmic, hepatic, and biliary cholesterol of the rat

Twenty-one male Wistar rats, 13 weeks old, were fed ad libitum hyperlipidic diets (28% fats) loaded with cholesterol (1.2%) for 5 weeks. One group of 11 rats was fed saturated fats (diet group "S") and another group of 10 rats was fed polyunsaturated fats (diet group "PU"). On the day they were sacrificed 10 of the rats were injected intravenously with 1 mg of filipin. Contrary to the rats in diet group "PU," the rats in diet group "S" treated with filipin presented certain characteristics that were not found in the nontreated group: They provided evidence of biliary cholestasis accompanied by a decline in the level of secretion of bile salts and phospholipids into bile. The concentrations of both free and esterified cholesterol in plasma fell and the amount of (esterified) hepatic cholesterol rose, although there was no change due to the filipin in the amounts of hepatic phospholipids. Explanatory hypotheses for these phenomena were considered, first, at the site of plasma membranes where filipin binds selectively to the cholesterol in the membrane, causing a disruption which probably disturbs the absorbance of circulating lipoproteins, especially that of hepatocyte cells, particularly in diet group "PU." Second, the effects of filipin on subcellular membranes seem to disturb the secretion of lipids and lipoproteins into bile and plasma, especially in diet group "S." Last, at the intracellular level, filipin appears to have a blocking effect on the organelles involved in biliary lipid secretion. The activity of certain enzymes such as cholesterol esterase may also be blocked, particularly in diet group "S," which would explain the accumulation of esterified cholesterol in liver.     

Hormonal regulation of acid cholesterol ester hydrolase activity: effects of triiodothyronine and 17 alpha-ethynylestradiol

Cholesterol ester hydrolase activity was measured in isolated rat hepatocytes and adipocytes. Administration of triiodothyronine to rats resulted in a specific and selective increase in lysosomal acid (pH 4.5) cholesterol ester hydrolase activity in hepatocytes. Since the majority of lipoprotein degradation occurs in liver parenchymal cells (hepatocytes), the stimulation of liver (hepatocyte) acid cholesterol ester hydrolase activity by triiodothyronine could contribute to the hypocholesterolemic action of thyroid hormones. Treatment of rats with 17 alpha-ethynylestradiol to increase the hepatic degradation of lipoprotein did not change acid cholesterol ester hydrolase activity in liver, indicating that the thyroid hormone induced stimulation of acid cholesterol ester hydrolase activity in hepatocytes is not a secondary effect owing to the increased hepatic catabolism of low density lipoproteins (LDL). In contrast to the results with hepatocytes, hyperthyroidism did not increase acid cholesterol ester hydrolase activity in rat adipocytes.     

The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E

Hepatic catabolism of lipoproteins containing apolipoproteins B or E is enhanced in rats treated with pharmacologic doses of 17 alpha-ethinyl estradiol. Liver membranes prepared from these rats exhibit an increased number of receptor sites that bind 125I-labeled human low density lipoproteins (LDL) in vitro. In the present studies, this estradiol-stimulated hepatic receptor was shown to recognize the following rat lipoproteins: LDL, very low density lipoproteins obtained from liver perfusates (hepatic VLDL), and VLDL-remnants prepared by intravenous injection of hepatic VLDL into functionally eviscerated rats. The receptor also recognized synthetic lamellar complexes of lecithin and rat apoprotein E as well as canine high density lipoproteins containing apoprotein E (apo E-HDLc). It did not recognize human HDL or rat HDL deficient in apoprotein E. Much smaller amounts of this high affinity binding site were also found on liver membranes from untreated rats, the number of such sites increasing more than 10-fold after the animals were treated with estradiol. Each of the rat lipoproteins recognized by this receptor was taken up more rapidly by perfused livers from estrogen-treated rats. In addition, enrichment of hepatic VLDL with C-apoproteins lowered the ability of these lipoproteins to bind to the estradiol-stimulated receptor and diminished their rate of uptake by the perfused liver of estrogen-treated rats, just as it did in normal rats. The current data indicate that under the influence of pharmacologic doses of estradiol the liver of the rat contains increased amounts of a functional lipoprotein receptor that binds lipoproteins containing apoproteins B and E. This hepatic lipoprotein receptor appears to mediate the uptake and degradation of lipoproteins by the normal liver as well as the liver of estradiol-treated rats. The hepatic receptor bears a close functional resemblance to the LDL receptor previously characterized on extrahepatic cells.     

Hypolipidemic principle of the inflorescence stalk of plantain (Musa sapientum)

A pectin present in the juice of the inflorescence stalk of plantain(Musa sapientum) has been isolated. The material contained 32.4% hexoses and 52.5% uronic acid. On administration to rats fed both cholesterol free and cholesterol diet, this material showed significant lowering of cholesterol and triglycerides in the serum, liver and aorta. There was decreased cholesterogenesis in the liver as was evident from decreased activity of hydroxymethylglutaryl coenzyme A reductase and decreased incorporation of labelled acetate into hepatic cholesterol. Hepatic bile acids showed significant increase and there was increased fecal excretion of neutral sterols and bile acids. Release of lipoproteins into the circulation was lower. The material also caused increase in the activity of lipoprotein lipase in the heart and adipose tissue and also of plasma lecithin: cholesterol acyl transferase     

Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Regulation of bile acid synthesis via direct effects on the microsomal membrane

Rats treated with ethinylestradiol (5 mg kg-1 day-1 for 5 days) secrete de novo synthesized bile acids at a markedly reduced rate (-57%). Administration of the nonionic detergent Triton WR-1339 to estradiol-treated rats rapidly restored the rate of secretion of de novo synthesized bile acids to control levels. In contrast, when Triton was administered to control rats, the secretion rate of bile acids was unaffected. The reduction in bile acid synthesis displayed by estradiol-treated rats was similar to the 50% decrease in the activity of hepatic microsomal 7 alpha-hydroxylase. The activity of 7 alpha-hydroxylase was also restored to control levels by the administration of Triton to estradiol-treated rats. We examined the possibility that estradiol acts directly on the hepatic microsomes. Adding increasing amounts of estradiol to microsomes obtained from control rats resulted in decreasing activities of 7 alpha-hydroxylase. The inhibition by estradiol of 7 alpha-hydroxylase obtained in vitro occurred with amounts of estradiol that were found to accumulate in the liver via in vivo treatment. Double-reciprocal analysis showed that at and below 50 micrograms of estradiol/0.5 mg of protein uncompetitive inhibition was displayed. Additional experiments showed that adding Triton to microsomes obtained from estradiol-treated rats increased the activity of 7 alpha-hydroxylase to control levels. In contrast, Triton did not increase the activity of 7 alpha-hydroxylase when it was added to control microsomes. These data show for the first time that the estrogenic steroid estradiol acts directly on the microsomes and inhibits both the activity of 7 alpha-hydroxylase and the rate of bile acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)