Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradiol-treated rats

The regulation of hepatic cholesterol and lipoprotein metabolism was studied in the ethinyl estradiol-treated rat in which low density lipoprotein (LDL) receptors are increased many fold. Cholesterol synthesis was reduced at both its diurnal peak and trough by ethinyl estradiol. The diurnal variation in 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was abolished, whereas that for acyl coenzyme A: cholesterol acyltransferase (ACAT) was retained. LDL receptor number did not vary diurnally. Feeding these animals a cholesterol-rich diet for 48 h suppressed cholesterol synthesis and reductase activities to levels similar to those found in cholesterol-fed control animals, but ACAT activity was unaffected. LDL receptors were reduced about 50%. Intravenously administered cholesterol-rich lipoproteins suppressed HMG-CoA reductase and LDL receptors in 2 h but had a variable effect on ACAT activity. Intragastric administration of mevalonolactone reduced reductase and increased acyltransferase activity but had little effect on LDL receptors when given 2 or 4 h before death. Although animals fed a cholesterol-rich diet before and during ethinyl estradiol treatment became hypocholesterolemic, free and esterified cholesterol concentrations in liver were high as was ACAT activity. HMG-CoA reductase was inhibited to levels found in control animals fed the cholesterol-rich diet. LDL receptors were increased to a level about 50% of that reached in animals receiving a control diet and ethinyl estradiol. These data demonstrate that key enzymes of hepatic cholesterol metabolism and hepatic LDL receptors respond rapidly to cholesterol in the ethinyl estradiol-treated rat. Furthermore, estradiol increases LDL receptor activity several fold in cholesterol-loaded livers.     

Diurnal rhythm of rat liver mRNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase. Correlation of functional and total mRNA levels with enzyme activity and protein

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase exhibits a diurnal rhythm of activity which coincides with a diurnal rhythm of reductase protein and reductase mRNA levels. This diurnal rhythm of reductase activity, polypeptide mass, and mRNA exists in rats fed a normal diet (unsupplemented rat chow) and in rats fed a diet supplemented with cholestyramine plus or minus mevinolin. Levels of reductase protein were determined by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Reductase mRNA was measured by in vitro translation or blot hybridization of liver RNA. Functional reductase mRNA levels in rats fed a normal diet were approximately 10-fold higher during the middle of the dark cycle than during the middle of the light cycle. Maximum induction of functional reductase mRNA was observed in rats fed cholestyramine and mevinolin. This latter level was 157-fold higher than the level measured at the diurnal low point in rats fed a normal diet. Blot hybridization of liver RNA showed two predominant mRNAs of 4.6 and 4.2 kilobase pairs and a minor species at 6.9 kilobase pairs. These mRNAs exhibited a diurnal rhythm for rats on all three diets and reached peak levels during the 12-h dark period. These data indicate that the diurnal rhythm of reductase mass and activity is closely paralleled by the level of its mRNA.     

Two distinct poly(A) polymerases in yeast nuclei

β-Hydroxy-β-methylglutaryl coenzyme A reductase activity in rat liver increased 2 to 7-fold after subcutaneous administration of insulin into normal or diabetic animals. Reductase activity began increasing after one hour, rose to a maximum in two to three hours, and then declined to the control level after six hours. This response was elicited during the time of day when the normal diurnal variation in reductase activity approached a minimum. It was also elicited when animals did not have access to food. This stimulation of reductase activity was completely blocked when glucagon was administered in conjunction with insulin. The increase in reductase activity after insulin administration was accompanied by a proportionate increase in activity for the conversion of acetate to cholesterol.     

Regulation of hydroxymethylglutaryl-CoA reductase in rat leukocytes

Methods were developed for the assay of hydroxymethylglutaryl-CoA reductase (NADPH) activity in microsomes from rat leukocytes. The activity in freshly isolated leukocytes is low compared to rat liver but can be assayed reliably. The patterns of response of leukocyte reductase in the assay to variation in substrate concentration, protein concentration, and time mimic those of rat liver reductase. Reductase activity in leukocyte microsomes, as in liver microsomes, is depressed by dietary cholesterol and by fasting and is elevated by dietary cholestyramine. Unlike liver reductase, leukocyte reductase activity does not exhibit a detectable diurnal rhythm. We conclude that the assay of reductase in freshly isolated leukocytes holds promise as a technique for detecting the effects of various factors on cholesterol synthesis in vivo.     

Effect of estrogens on β-hydroxy-β-methylglutaryl coenzyme a reductase activity and cholesterol levels

The effect of estrogens on hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity and cholesterol in serum and liver of ovarietcomized rats on normal diet, 2% cholestyramine diet or 2% cholesterol diet was investigated. Estrogen administration to ovariectomized rats on normal diet resulted in increased reductase activity and was correlated with decreased serum cholesterol and increased liver cholesterol levels wlth mestranol (ME), ethinyl estradiol (EE) and estradiol benzoate (EB, 250 μg) but increased serum and liver cholesterol levels with 25 μg and 100 μg EB administration. The increased stimulation of reductase activity by estrogen administration was absolished when rats were fed a 2% cholesterol diet. Cholestyramine feeding markedly increased reductase activity in livers of ovariectomized rats. These studies show that estrogens are not absolutely required for the stimulation of reductase activity and therefore is consistent with the model in which cholesterol functions as a feedback repressor of reductase activity.     

Diurnal variation of HMG CoA reductase activity in rat intestine

HMG CoA reductase activity of rat intestinal mucosa has a diurnal rhythm which coincides with the diurnal variation of the hepatic HMG CoA reductase but has a lower amplitude. The rhythmic variation of the intestinal reductase was present in both jejunal and ileal crypt cell microsomes and was not abolished by cholestyramine administration.     

Regulation of the diurnal rhythm of rat liver beta-hydroxy-beta-methylglutaryl coenzmye A reductase activity by insulin, glucagon, cyclic AMP and hydrocortisone

Rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and the amplitude of the diurnal variation of this enzyme are progressively reduced to very low levels within 1 week after the onset of diabetes induced by streptozotocin. Daily insulin therapy to 7-day diabetic rats restores the activity and the amplitude of this diurnal variation in enzyme activity to near-normal levels within 4 days. Insulin also produces a rapid 2-hr stimulation of the reductase activity in diabetic rats to the level found in normal animals at that time of day regardless of the duration of diabetes. Hence, insulin is required for the diurnal rise of reductase activity in rat liver. Glucagon, dibutyryl cyclic AMP, and hydrocortisone, in contrast, markedly inhibit the diurnal rise of reductase activity in normal rats. Therefore, the relative concentrations of insulin, glucagon, and glucocorticoids are important in the regulation of the diurnal variation of hepatic reductase activity.     

Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity comigrated with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200,000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52,000 +/- 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11,303 and 19,584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy-3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment.     

Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in foetal rats by maternal cholestyramine feeding

Late gestation foetus from rats fed a non-absorbable bile acid binding resin (cholestyramine) have increased hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. This was due to increased unphosphorylated (active) as well as total reductase and was accompanied by higher fatty acid synthetase activity. No increase in foetal hepatic cystathionase or tyrosine aminotransferase activity, or changes in plasma insulin, corticosterone or thyroxine were found. The studies demonstrate that foetal hepatic cholesterol metabolism is sensitive to drug-induced perturbation of maternal lipoprotein metabolism. The data suggest induction of foetal cholesterol and fatty acid synthesis by a specific mechanism not involving generalized hormone-induction of hepatic enzyme systems. Cholestyramine appears to have application for in vivo study of the regulation of foetal cholesterologenesis and its coordination to maternal and foetal steroid requirements.     

Treatment of familial hypercholesterolaemia. United Kingdom lipid clinics study of pravastatin and cholestyramine.

OBJECTIVE--To compare the efficacy and safety of cholestyramine, an anion exchange resin, and pravastatin, a new hydrophilic specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, in the treatment of heterozygous familial hypercholesterolaemia. DESIGN--Double blind, double dummy, placebo controlled study with three parallel groups. SETTING--Six specialist lipid clinics in the United Kingdom. PATIENTS--128 patients aged 18-70 with heterozygous familial hypercholesterolaemia diagnosed on strict biochemical and clinical findings. MAIN OUTCOME MEASURES--Total plasma cholesterol, triglyceride, and lipoprotein subfractions and biochemical and haematological safety parameters. RESULTS--Pravastatin (40 mg/day) led to a 25% reduction in total plasma cholesterol concentration and a reduction in low density lipoprotein cholesterol concentration of 30%. Cholestyramine (24 g/day) led to similar reductions in concentrations of total cholesterol (23%) and low density lipoprotein cholesterol (31%). No consistent changes occurred in high density lipoprotein cholesterol values with either compound. Plasma triglyceride concentrations showed a small rise (18%) on resin therapy. No serious adverse drug reactions occurred during the study. CONCLUSIONS--Pravastatin seems to be a highly effective, well tolerated drug for severe hypercholesterolaemia. Patients chosen for this study were recruited on the basis that they could tolerate a full dose of cholestyramine, and in this situation cholestyramine was also highly effective in lowering plasma low density lipoprotein cholesterol concentrations.     

Lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat.

In the adrenal gland of the rat, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol synthesis, is shown to be regulated by cholesteerol carried in plasma lipoproteins. When plasma cholesterol levels were lowered 90% by administration of the drug 4-aminopyrazolopyrimidine, the cholesteryl ester content of the adrenal gland declined by more than 90% and this was associated with a 150- to 200-fold increase in the activity of adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase and a 30-fold increase in cholesterol synthesis from [14C]acetate. The subsequent intravenous infusion of cholesterol contained in either rat or human high density or low density lipoproteins restored the adrenal content of cholesteryl esters and reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase to basal levels. The depletion of adrenal cholesteryl esters and the enhancement in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that occurred in the 4-aminopyrazolopyrimidine-treated rat required the action of adrenocorticotropic hormone (ACTH) since neither was observed when ACTH secretion was blocked by administration of dexamethasone. The current data indicate that the low rate of cholesterol synthesis normally observed in the rat adrenal gland is due to a suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that is mediated by plasma lipoproteins.     

The interaction of various control mechanisms in determining the rate of hepatic cholesterogenesis in the rat.

In these studies rats were subjected to diurnal light-cycling, stress, fasting and the feeding of cholestyramine, beta-sitosterol and cholesterol in various combinations. In control animals exposed to light cycling for 2 weeks the rate of hepatic cholesterogenesis was 3.7 fold higher in the mid-dark than in the mid-light phase of the light cycle. The magnitude of this difference varied with the duration of light cycling and the size of the animals. Similarly, enhanced rates of cholesterol synthesis were seen in the mid-dark phase relative to the mid-light phase of the light cycle in rats where the base-line level of hepatic cholesterogenesis was increased by feeding cholestyramine (1.6-fold) or beta-sitosterol (2.9-fold) or was depressed by fasting (19-fold) or cholesterol feeding (2.1-fold). Restraining animals for 48 h also increased the rate of cholesterol synthesis in the liver; in control animals, this stress enhanced the level of cholesterogenesis seen at both the mid-light and mid-dark phases of the light cycle. In addition, both the effects of stress and of diurnal light cycling could be identified in groups of animals where base-line cholesterogenic activity was varied by fasting or by feeding cholestyramine, beta-sitosterol or cholesterol. These studies illustrate the complexity of the control of hepatic cholesterol synthesis and suggest that the final rate of cholesterogenesis may be the result of several different effectors modifying by different mechanisms the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase localization in rat liver peroxisomes and microsomes of control and cholestyramine-treated animals: quantitative biochemical and immunoelectron microscopical analyses

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory enzyme involved in cholesterol biosynthesis, has recently been reported to be present in rat liver peroxisomes (Keller, G.A., M.C. Barton, D.J. Shapiro, and S.J. Singer, 1985, Proc. Natl. Acad. Sci. USA, 82:770-774). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is present in the matrix of peroxisomes. This study is a quantitative biochemical and immunoelectron microscopical analysis of HMG-CoA reductase in rat liver peroxisomes and microsomes of normal and cholestyramine-treated animals. Cholestyramine treatment produced a six- to sevenfold increase in the specific activity of peroxisomal HMG-CoA reductase, whereas the microsomal HMG-CoA reductase specific activity increased by about twofold. Using a computer program that calculates optimal linear combinations of marker enzymes, it was determined that between 20 and 30% of the total reductase activity was located in the peroxisomes of cholestyramine-treated animals. Less than 5% of the reductase activity was present in peroxisomes under control conditions. Quantitation of the immunoelectron microscopical data was in excellent agreement with the biochemical results. After cholestyramine treatment there was an eightfold increase in the density of gold particles per peroxisome, and we estimate about a threefold increase in the labeling of the ER.     

Regulation of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in the rat adrenal gland.

The activities of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in rat adrenal gland were measured at various time intervals over 24 h. The activity of cholesterol esterase displayed diurnal rhythm, with a major peak at the onset of darkness coinciding with the peak in the diurnal rhythm of plasma corticosterone concentration. The activity of acyl-CoA : cholesterol acyltransferase also exhibited a characteristic diurnal rhythm, with the minimum activity occurring 3 h after the onset of darkness. The profile of the rhythm exhibited by the activity of the esterifying enzyme was similar to the mirror image of the pattern of diurnal rhythm in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Microsomal non-esterified cholesterol showed a gradual decline with a significant decrease in concentration at the onset of darkness, thus suggesting that diurnal removal of cholesterol in the environment of the esterifying enzyme and hydroxymethylglutaryl-CoA reductase leads to such diurnal decrease or increase in the activities of these two enzymes. Acute administration of corticotropin led to a 3-fold increase in the activity of cholesterol esterase, a 50% decrease in the activity of acyl-CoA : cholesterol acyltransferase and a 2-fold increase in the activity of hydroxymethylglutaryl-CoA reductase. Corticotropin administration also resulted in a significant decrease in microsomal non-esterified cholesterol and increase in plasma corticosterone concentration. These observations suggest that corticotropin plays an important part in generating the diurnal rhythm in the activities of the three enzymes.     

Variations of hepatic cholesterol precursors during altered flows of endogenous and exogenous squalene in the rat

Hepatic and serum levels of cholesterol precursors were analyzed in rats under basal (control) conditions and when cholesterol synthesis was activated by feeding 1% squalene or 5% cholestyramine. Exogenous squalene stimulated the activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT) but strongly inhibited the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase; cholestyramine did not affect ACAT but increased HMG-CoA reductase several-fold, indicating enhanced production of endogenous squalene. Activation of cholesterol synthesis by the two methods markedly increased the hepatic and serum contents of cholesterol precursor sterols. However, the sterol profiles were clearly different. Thus, exogenous squalene raised most significantly (up to 109-fold) free and esterified methyl sterols, and less so (up to 2-fold) demethylated C27 sterols (desmosterol and cholestenols) and also esterified cholesterol. Activation of endogenous squalene production by cholestyramine was associated with a depletion of esterified cholesterol and by a marked, up to 8-fold, increase of the free demethylated sterol precursor levels, whereas the increase of methyl sterols, up to 5-fold, was less conspicuous than during the squalene feeding. The changes were mostly insignificant for esterified sterols. The altered serum sterol profiles were quite similar to those in liver. Serum cholestenols and especially their portion of total serum precursor sterols were closely correlated with the hepatic activity of HMG-CoA reductase.     

Diurnal and dietary-induced changes in cholesterol synthesis correlate with levels of mRNA for HMG-CoA reductase

We determined the extent to which diurnal variation in cholesterol synthesis in liver is controlled by steady-state mRNA levels for the rate-limiting enzyme in the pathway, hydroxymethylglutaryl (HMG)-CoA reductase. Rats 30 days of age and maintained on a low-cholesterol diet since weaning were injected intraperitoneally with (3)H(2)O. The specific radioactivity of the whole-body water pool soon became constant, allowing for expression of values for incorporation of label into cholesterol as absolute rates of cholesterol synthesis. In liver, there was a peak of cholesterol synthesis from 8 pm to midnight, a 4-fold increase over synthesis rates from 8 am to noon. Increases in synthesis were quantitatively in lock step with increases in mRNA levels for HMG-CoA reductase occurring 4 h earlier. In a parallel experiment, rats received 1% cholesterol in the diet from weaning to 30 days of age. Basal levels of hepatic cholesterol synthesis were greatly diminished and there was little diurnal variation of cholesterol synthesis or of levels of mRNA for HMG-CoA reductase. Levels of mRNA for the low density lipoprotein receptor and scavenger receptor-B1 (putative high density lipoprotein receptor) showed little diurnal variation, regardless of diet. This suggests that diurnal variation of hepatic cholesterol synthesis is driven primarily by varying the steady-state mRNA levels for HMG-CoA reductase. Other tissues were also examined. Adrenal gland also showed a 4-fold diurnal increase in accumulation of recently synthesized cholesterol. In contrast to liver, however, there was little corresponding change in mRNA expression for HMG-CoA reductase. Much of this newly synthesized cholesterol may be of hepatic origin, imported into adrenal by SR-B1, whose mRNA was up-regulated 2-fold. In brain, there was no diurnal variation in either cholesterol synthesis or mRNA expression, and no influence of high- or low-cholesterol diets on synthesis rates or HMG-CoA reductase mRNA levels.     

Independent regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and chylomicron remnant receptor activities in rat liver.

Treatment of rats with pharmacological doses of oestrogen resulted in a 3-fold decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and a 4-fold increase in saturable binding of 125I-labelled chylomicron remnants to liver membranes in vitro. Intragastric administration of mevalonolactone to rats did not affect the capacity of the liver membranes to bind to labelled chylomicron remnants even though there was a substantial decrease in the activity of HMG-CoA reductase. Similar results were obtained after cholesterol feeding. Simultaneous treatment of rats with cholestyramine and compactin increased hepatic HMG-CoA reductase activity 6-fold. However, liver membranes derived from these animals showed no change in their capacity to bind to labelled chylomicron remnants in vitro. Administration of mevalonolactone to the cholestyramine/compactin-treated animals also failed to produce a change in remnant-binding capacity. Although administration of mevalonolactone alone produced a significant 3-fold decrease in the activity of hepatic HMG-CoA reductase it was unable to suppress significantly the increase in enzyme activity caused by treatment with cholestyramine and compactin.     

3-Hydroxy-3-methylglutaryl coenzyme a reductase activity in liver of athymic mice with or without an implanted human carcinoma

Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activities and cholesterol content in the liver of athymic mice either bearing or not an implanted human lung mucoepidormoid carcinoma (HLMC) and in the neoplasic tissue, were analyzed. The properties of the HMG-CoA reductase of HLMC grown in nude mice and those ones found in the liver of these animals, sacrificed either at mid-light or mid-dark, were similar. The hepatic reductase activity was found to be four- to five-fold greater at mid-dark than at mid-light (462±141 vs. 123±22 pmol min⁻¹ mg protein⁻¹). Since the K_m value was not modified, the mid-dark activity could be due to an increase in the amount of enzyme. In contrast, HLMC reductase activity and cholesterol content showed similar values at mid-light and mid-dark points. HLMC reductase does not appear to have any diurnal variation and the cholesterol synthesis and content seems to be independent of food intake. HLMC-bearing nude mice undergo several alterations in the biosynthesis and homeostasis of cholesterol. Hypocholesterolemia, lower hepatic cholesterol content and higher HMG-CoA reductase activity are characteristic of host mice.