The regulation of hydroxymethylglutaryl-CoA reductase in cultured cells

Growth-stimulated synchronized cells exhibit a rapid increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.88) activity prior to the onset of DNA synthesis. Under normal culture conditions, HMG-CoA reductase activity exhibits wide variations among experiments. To determine whether this phenomenon is dependent on cell replication, we used J774 macrophage-like cells to compare changes in reductase activity in cells synchronized by serum deprivation and then growth-stimulated by fresh media containing serum to unsynchronized cells treated with fresh media and serum. Under these conditions, no increase in [3H]thymidine incorporation into cell DNA was seen in unsynchronized cells, but a large increase was observed in synchronized cells 10-12 h after media change. Although the growth characteristics differed between the cells, reductase activity was low at the time of media change and increased 10 to 20-fold 5-10 h after media change, returning to basal levels by 24 h in both synchronized and unsynchronized cells. This pattern of reductase activity was observed in unsynchronized cells from a variety of cell lineages, although the magnitude of the changes varied. Fluctuations of [14C]acetate incorporation into cholesterol were observed in parallel to alterations in reductase activity. LDL receptor expression also paralleled the changes in reductase activity, but scavenger receptor expression was not affected. Addition of lipoproteins at the time of media change inhibited the rise in reductase activity by 80-90%. The increase in reductase activity was not due to a stimulation of cholesterol efflux into the medium, but evidence for the secretion into the media of an inhibitory factor was obtained. These results suggest that cell requirements for cholesterol are not always directly related to replication, and that standard culture conditions induce transient fluctuations in reductase activity and lipoprotein receptor expression.     

Effects of mevinolin in rat liver: evidence for a lack of coupling between synthesis of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase activity

The effect of treatment of rats with the hydroxymethylglutaryl-CoA reductase inhibitor, mevinolin, on 7 alpha-hydroxylation of cholesterol was studied. Treatment with 0.1% mevinolin in diet for 3 days was found to have an inhibitory effect on 7 alpha-hydroxylation of cholesterol (about 35%). Treatment with cholestyramine increased 7 alpha-hydroxylation of both exogenously added and endogenous microsomal cholesterol 3-4-fold. Combined treatment with both cholestyramine and mevinolin decreased this stimulation to 2-2.5-fold. Treatment with 2% cholesterol in diet increased 7 alpha-hydroxylation of exogenous cholesterol about 2-fold and 7 alpha-hydroxylation of endogenous cholesterol about 3.5-fold. The stimulatory effect of cholesterol was reduced or abolished when 0.1% mevinolin was added to the cholesterol-containing diet. With the exception of the experiments with cholesterol in the diet, all experiments including mevinolin gave a marked stimulation (up to 60-fold) of the hydroxymethylglutaryl-CoA reductase activity under the in vitro conditions employed. The concentration of free cholesterol in the liver microsomes was not significantly changed in any of these experiments. It is concluded that there is no coupling between induction of synthesis of hydroxymethylglutaryl-CoA reductase protein and cholesterol 7 alpha-hydroxylase activity. The inhibitory effect of mevinolin on cholesterol 7 alpha-hydroxylase activity under experimental conditions where most of the effect of mevinolin on hydroxymethylglutaryl-CoA reductase was abolished by treatment with cholesterol suggest that the effect of mevinolin on the cholesterol 7 alpha-hydroxylase may be independent of its effect on cholesterol synthesis. The over-all results do not favour the hypothesis that cholesterol synthesis and cholesterol availability are the most important determinants for the regulation of the cholesterol 7 alpha-hydroxylase.     

Effects of internal biliary bypass on the regulation of hepatic cholesterol 7alpha-hydroxylase activity in rats.

In order to re-evaluate the importance of the enterohepatic circulation of bile acid in the regulation of the activities of hepatic cholesterol 7alpha-hydroxylase, bile acid metabolism was examined in internal biliary bypass models of rats. A polyethylene tube was inserted into the common bile duct and another side of the tube was placed in the duodenum (DD), lower jejunum (JD), cecum (CeD), or transverse colon (CoD) as internal biliary bypass models and in the urinary bladder as an external biliary drainage (ED). After bile diversion for 7 days in each group, hepatic cholesterol 7alpha-hydroxylase activities, bile acid concentrations in bile, serum, and portal vein, biliary bile acid compositions, and intestinal absorption rates of infused labeled taurocholic acid were analyzed. Hepatic cholesterol 7alpha-hydroxylase activity was similar in the JD group compared with the DD group, however, it was significantly up-regulated in the CeD (227% of the DD group), CoD (312%), and ED groups (316%). Biliary, serum, and portal bile acid concentrations were not significantly changed in the DD, JD, and CeD groups but those were significantly lower in the CoD and ED groups compared with the DD group. The proportion of the secondary bile acids was significantly increased in the CeD group and was decreased in the CoD and ED groups. The absorption rate of taurocholic acid was almost 100%, 56%, and 23% in the JD, CeD, and the CoD group, respectively. As the cholesterol 7alpha-hydroxylase activity was not significantly changed in the JD group and the predominance of secondary bile acids did not suppress the enzyme activity in the CeD group, the luminal factor, which is absorbed in the presence of bile acids, and the bile acid metabolites are not likely the regulatory factor. The cholesterol 7alpha-hydroxylase activity seems to be primarily regulated by the intestinal absorption of bile acids and partly by the intestinal mucosal factor which is linked to the intestinal bile acid absorption.     

The effect of portacaval transposition of hepatic cholesterol-7alpha-hydroxylase activity in the rat.

Portacaval anastomosis in the rat results in an increase in the activity of the liver microsomal cholesterol-7alpha-hydroxylase enzyme system. The increase in the activity of this oxygenase occurs despite a decrease in the total amount of cytochrome p450 in the liver microsomes after portacaval anastomosis. It is possible to increase further the activity of the cholesterol-7alpha-hydroxylase enzyme in these portacaval shunted animals by feeding them on a diet containing a bile salt sequestering agent. This suggests that one of the factors influencing the activity of the liver microsomal cholesterol-7alpha-hydroxylase enzyme may be the concentration of bile salts reaching the liver from the blood plasma. Portacaval anastomosis in the rat tended to achieve a small decrease in the plasma cholesterol concentration.     

Expression of cholesterol-7alpha-hydroxylase in murine macrophages prevents cholesterol loading by acetyl-LDL

Unlike macrophages, the hepatic parenchymal cells express cholesterol-7 alpha-hydroxylase (CYP7A1) which regulates the conversion of cholesterol into bile acids, the major quantitative pathway maintaining cholesterol homeostasis. We examined if CYP7A1 expression in RAW 264.7 macrophages could prevent the accumulation of cholesterol when they were incubated with acetyl-LDL. A single cell clone (designated as 7 alphaRAW cells) that stably expresses rat CYP7A1 displayed similar rates of growth as non-transfected RAW cells. The CYP7A1 enzymatic activity expressed by microsomes obtained from 7 alphaRAW cells was similar to that expressed by microsomes obtained from mouse liver. Incubating non-transfected RAW with increasing amounts of acetyl-LDL caused a parallel accumulation of cholesterol, whereas 7 alphaRAW cells displayed a complete resistance to cholesterol accumulation. 7 alphaRAW cells displayed increased expression of both ABCA1 mRNA (3.1-fold, P < 0.001) and ABCG1 mRNA (2.2-fold, P < 0.01), whereas the expression of scavenger receptor class A mRNA was unchanged. 7 alphaRAW cells also displayed small but significant increases in the rate of efflux of [(3)H]cholesterol to both delipidated apolipoprotein A1 and to HDL.Thus, CYP7A1 expression in RAW cultured macrophages prevented the accumulation of cholesterol from acetyl-LDL via both increased metabolism and efflux of cholesterol.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

HMGCoA reductase and cholesterol 7 alpha-hydroxylase in human liver

The activities of HMGCoA reductase and cholesterol 7 alpha-hydroxylase were assayed in liver biopsies of patients with or without cholestyramine treatment. The active dephosphorylated form of HMGCoA reductase and the activity of cholesterol 7 alpha-hydroxylase were under the detection limits in untreated subjects. After cholestyramine treatment activities of both enzymes were stimulated and the active form of HMGCoA reductase became detectable in four out of the five tested patients. In two subjects who received cholestyramine, the effect of exogenously added [4-14C] cholesterol on cholesterol 7 alpha-hydroxylase was tested. In the presence of Tween 80, the detergent by which [14C]cholesterol was suspended, the enzyme activity was profoundly inhibited and synthesis of 7 alpha-hydroxycholesterol was extremely low.     

The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase.

The microsomal activities of the hepatic enzymes hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase exhibit a diurnal rhythm with maximum activities observed during the dark period and minimum activities around noon (12:00h). This diurnal rhythm was maintained for both enzymes after adrenalectomy, but the amplitude of variation for the activity of both enzymes was greatly decreased. A single injection of cortisol administered to adrenalectomized rats 3h before the expected maximum in enzyme activity resulted in a twofold increase in the activity of both enzymes 3h later, at values similar to those observed for control rats killed at the same time. This response appeared to require protein synthesis, since it was blocked by actinomycin D. However, the administration of cortisol to adrenalectomized rats 3 h before the expected minimum did not result in significant change in the activity of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase 3 h later. Kinetic studies of cholic acid metabolism in vivo demonstrated that adrenalectomy results in a significant decrease in the rate of synthesis of cholic acid and a considerable decrease in the pool size of cholic acid and its metabolic products. Treatment of adrenalectomized rats with cortisol increased the rate oonsistent with the effects of adrenalectomy and cortisol treatment on the activity of cholesterol 7alpha-hydroxylase.     

Effects of cholestanol feeding on corneal dystrophy in mice

A cholestanol-enriched diet administered for 8 months to BALB/c mice produced in 20% two kinds of corneal opacities resembling calcific band keratopathy and Schnyder's crystalline dystrophy in humans. The concentrations of cholestanol in serum, liver and cornea of the corneal opacity bearing mice were 30-40-times higher than those of normal mice. On the other hand, brain cholestanol level increased only 7-times in the opacity group as compared with that of control group. There was no significant difference in the cholesterol concentrations of serum and several tissues among opacity, non-opacity and the control group. The crystal particles were observed between epithelial basement membrane and superficial stroma by the electron microscopy. Energy dispersive analysis of the particles revealed that the deposits were composed principally of calcium and phosphorus with other crystalline materials, which was presumed to be cholestanol. These results suggest that the cholestanol may deposit in the cornea from elevated serum levels. Deposition of cholestanol in cornea and related area may be a cause of corneal dystrophy in CTX.     

Circadian rhythm of cholesterol-7alpha-hydroxylase and cortisol in the African green monkey (Cercopithecus aethiops).

Cholesterol-7alpha-hydroxylase in African green monkey liver had an apparent Km of 1.65-10(-4) M cholesterol and a pH optimum of 7.4. The amplitudes of the circadian maxima of enzyme activity and serum cortisol levels were significantly greater in vervets than in grivets. Fluctuations in enzyme activity and cortisol levels during the circadian cycle were positively correlated (r = 0.89). Enzyme activities and hormone levels were 2.7-fold lower over a 24-h period in the grivet than in the vervet. Cholesterol feeding reduced the enzyme activity by 40% and serum cortisol was reduced to 38% of control levels at the diurnal peak. Serum glucocorticoids may be important physiological regulators of cholesterol-7alpha-hydroxylase in non-human primates. The concentration of cortisol and its time of release appear to be factors in the hyperresponsive trait of grivets. Genetic differences between vervet and grivet races may account for differences in the amplitude and timing of the circadian rhythm of cholesterol-7alpha-hydroxylase activity possibly influenced by cortisol.     

Rate-limiting, diurnal activity of hepatic microsomal cholesterol-7 alpha-hydroxylase in pigeons with high serum cholesterol

Efficiency of regulating serum cholesterol by cholesterol-7 alpha-hydroxylase was studied in pigeon strains hypo-(SR-39) and hypercholesterolemic (SR-37) with respect to dietary cholesterol. Diurnal hydroxylase activity in SR-37 was 10% of that in strain SR-39 adapted to a light-dark cycle and fed a non-cholesterol diet. Acrophase (6 p.m.) activity was 54-fold greater in SR-39 than in SR-37 pigeons. Dietary cholesterol elevated enzyme activity 2.8-fold in SR-37 pigeons. Dietary cholestyramine plus cholesterol increased hydroxylase activity 21-fold in SR-37 and 3-fold in SR-39 strain; yet, activity remained greater in SR-39. Cholestyramine feeding prevented elevated cholesterol levels in both groups. The circadian rhythms of hydroxylase and serum corticosterone were determined. The diurnal activity in SR-37 was 10% of that in SR-39 and acrophase activity was 34-fold greater in SR-39. Hormone levels were comparable. Programmed acrophase was asynchronous between strains. Hydroxylase activity was positively correlated with corticosterone levels and inversely correlated with serum cholesterol. A defect in the up-regulation of cholesterol-7 alpha-hydroxylase is proposed which limits the catabolism of cholesterol in strain SR-37.     

Contribution of cholesterol 7alpha-hydroxylase to the regulation of lipoprotein metabolism.

Clinical studies have clearly established a relationship between bile acid synthesis and plasma LDL-cholesterol concentrations. Interruption of the enterohepatic circulation of bile acids leads to increased bile acid synthesis and a reduction in plasma LDL-cholesterol concentrations. New studies indicate that genetic variation in cholesterol 7alpha-hydroxylase activity accounts for a significant fraction of the inter-individual variation in plasma LDL-cholesterol concentrations in the general population, and a specific CYP7A1 allele associated with increased plasma LDL-cholesterol concentrations has been identified. Studies in which cholesterol 7alpha-hydroxylase was transiently overexpressed in hamsters and mice indicate that direct manipulation of cholesterol 7alpha-hydroxylase leads to changes in plasma LDL-cholesterol concentrations. Interestingly, targeted inactivation of the gene encoding cholesterol 7alpha-hydroxylase does not lead to increased plasma LDL-cholesterol concentrations in mice.     

Deoxycholate 7 alpha-hydroxylase in the hamster: substrate specificity and effect of phenobarbital

In a recent publication, we reported that deoxycholic acid is 7 alpha-hydroxylated to yield glycocholate or taurocholate in vivo in the hamster (1987. Kuroki et al. Hepatology. 7: 229-234). In order to explore the possibility that amidation of free deoxycholic acid precedes the 7 alpha-hydroxylation, we assayed 7 alpha-hydroxylase activities of free and conjugated deoxycholates in vitro. 7 alpha-Hydroxylase activities of glycodeoxycholate and taurodeoxycholate were 720 +/- 132 and 640 +/- 160 pmol/mg.min-1, respectively. Activity of 7 alpha-hydroxylation of free deoxycholate was very low (60 +/- 20 pmol/mg.min-1). After treatment with phenobarbital in a dose of 100 mg/kg per day for 6 days, 7 alpha-hydroxylase activities of conjugated deoxycholates were decreased significantly (40%, P less than 0.01, n = 8), whereas that of free deoxycholate was not significantly changed. In the rat, 7 alpha-hydroxylase activities of conjugated deoxycholates were induced significantly (45% increase, P less than 0.05, n = 5) by phenobarbital treatment in sharp contrast to the hamster. There were significant correlations between the 7 alpha-hydroxylase activity of taurodeoxycholate and that of glycodeoxycholate both in the hamster and in the rat (hamsters: n = 16, r = 0.98, P less than 0.01; rats: n = 10, r = 0.82, P less than 0.01). These studies suggested that deoxycholic acid is 7 alpha-hydroxylated after amidation with glycine or taurine in vivo and that the same enzyme may well catalyze the 7 alpha-hydroxylation of glycodeoxycholate and taurodeoxycholate in the hamster.(ABSTRACT TRUNCATED AT 250 WORDS)