Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase.

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.     

Acetoacetyl-CoA ligase activity in the isolated rat hepatocyte: Effects of 25-hydroxycholesterol and high density lipoprotein

The activity of acetoacetyl-CoA (AcAc-CoA) ligase (E.C.6.2.1.16) in hepatocytes from rats was shown to be the same as the activity in homogenates of their livers. In hepatocytes treated with 25-hydroxycholesterol, AcAc-CoA ligase, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and rates of sterol synthesis were substantially decreased. Hepatocytes treated with high density lipoprotein (HDL) exhibited a 2 to 4 fold induction of HMG-CoA reductase activity; however an accompanying increase in AcAc-CoA ligase activity and the rate of cholesterol synthesis was not observed. We conclude (a) that increases in the activity of HMG-CoA reductase when mediated by HDL in hepatocytes do not result in a corresponding change in the capacity for sterol synthesis and (b) that changes in the activity state of HMG-CoA reductase can be dissociated from that of AcAc-CoA ligase.     

Regulation of early enzymes of ergosterol biosynthesis in Saccharomyces cerevisiae.

In order to determine the regulation mechanisms of ergosterol biosynthesis in yeast, we developed growth conditions leading to high or limiting ergosterol levels in wild type and sterol-auxotrophic mutant strains. An excess of sterol is obtained in anaerobic sterol-supplemented cultures of mutant and wild type strains. A low sterol level is obtained in aerobic growth conditions in mutant strains cultured with optimal sterol supplementation and in wild type strain deprived of pantothenic acid, as well as in anaerobic cultures without sterol supplementation. Measurements of the specific activities of acetoacetyl-CoA thiolase, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase (the first three enzymes of the pathway), show that in cells deprived of ergosterol, acetoacetyl-CoA thiolase and HMG-CoA synthase are generally increased. In an excess of ergosterol, in anaerobiosis, the same enzymes are strongly decreased. A 5-10-fold decrease is observed for acetoacetyl-CoA thiolase and HMG-CoA synthase. In contrast, HMG-CoA reductase is only slightly affected by these conditions. These results show that ergosterol could regulate its own synthesis, at least partially, by repression of the first two enzymes of the pathway. Our results also show that exogenous sterols, even if strongly incorporated by auxotrophic mutant cells, cannot suppress enzyme activities in aerobic growth conditions. Measurement of specific enzyme activities in mutant cells also revealed that farnesyl pyrophosphate thwarts the enhancement of the activities of the two first enzymes.     

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.     

Inhibitors of sterol synthesis. Effects of dietary 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on early enzymes in hepatic cholesterol biosynthesis

The effects of dietary administration (0.1% in diet for 8 days) of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on the levels of activity of cytosolic acetoacetyl coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, and microsomal HMG-CoA reductase in liver have been studied in male Sprague-Dawley rats. Significant increases in the levels of activity of acetoacetyl-CoA thiolase and of HMG-CoA synthase were observed. The levels of microsomal HMG-CoA reductase activity were increased, relative to pair-fed control animals, in three experiments and increased, relative to ad libitum control animals, in one of three experiments. When compared with other agents for which the primary mode of action is an inhibition of the intestinal absorption of cholesterol, the magnitude of the increases in the levels of hepatic microsomal HMG-CoA reductase activity in the 15-ketosterol-fed rats was considerably smaller. In view of the previously described marked activity of the 15-ketosterol in the inhibition of the intestinal absorption of cholesterol, as well as its known effects in lowering HMG-CoA reductase activity in mammalian cells in culture, it is proposed that the 15-ketosterol may suppress the elevated levels of hepatic microsomal HMG-CoA reductase activity induced by the reduced delivery of cholesterol to liver as a consequence of the inhibition of the intestinal absorption of cholesterol.     

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.     

Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.     

Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells

Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.     

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631)

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.     

Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase in a tobacco mutant that overproduces sterols.

In a tobacco mutant callus, containing up to tenfold more sterols than the wild-type genotype, HMG-CoA reductase activity is increased by a factor of approximately three, as is the case in mutant seedlings and plants. The rate of HMG-CoA synthesis from acetyl-CoA by the coupled enzyme system acetoacetyl-CoA thiolase/HMG-CoA synthase, as well as its conversion to acetyl-CoA plus acetoacetate by action of HMG-CoA lyase are not affected. These results confirm the key-regulating role of HMG-CoA reductase in sterol biosynthesis, which seems not to be confined only to the animal kingdom, but can also be extended to plants.     

Differential regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, synthase, and low density lipoprotein receptor genes.

The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.     

Effect of phenylmethylsulfonyl fluoride on sterol biosynthesis in 10,000 x g supernatant fraction of rat liver homogenates

Phenylmethylsulfonyl fluoride (PMSF), a reagent commonly employed for the inhibition of serine proteases, has been found to cause significant inhibition of the incorporation of labeled acetate, but not mevalonate, into nonsaponifiable lipid and digitonin-precipitable sterols in the 10,000 X g supernatant fraction of rat liver homogenate preparations. In two experiments, the extent of inhibition of the synthesis of digitonin-precipitable sterols from acetate by PMSF at 1 mM was 81 and 65%. PMSF inhibited the synthesis of nonsaponifiable lipid from acetate at concentrations as low as 0.1 microM. Preincubation of the 10,000 X g supernatant fraction of rat liver homogenates with PMSF (1 mM) resulted in a significant reduction of the activities of acetate thiokinase and 3-hydroxy-3-methylglutaric acid (HMG)-CoA synthase, but did not affect the activities of acetoacetyl-CoA thiolase. Preincubation of rat liver microsomes with PMSF (1 mM) caused a 50% reduction in the level of HMG-CoA reductase activity. The combined results indicate that major sites of action of PMSF in the inhibition of sterol biosynthesis from labeled acetate appear to be on the activities of acetate thiokinase, HMG-CoA synthase, and HMG-CoA reductase. Another reagent used to inhibit serine proteases, diisopropylfluorophosphate, had (at a concentration of 1 mM) no effect on the activities of cytosolic acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase.     

Localization of 3-hydroxy-3-methylglutaryl CoA reductase and 3-hydroxy-3-methylglutaryl CoA synthase in the rat liver and intestine is affected by cholestyramine and mevinolin

In the normal fed rat, both 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase and HMG-CoA reductase are found in high concentrations in hepatocytes that are localized periportally. The majority of the liver cells show little or no evidence of either enzyme. Addition of cholestyramine and mevinolin to the diet resulted in all liver cells showing strong positive staining for both HMG-CoA reductase and HMG-CoA synthase. These two drugs increased the hepatic HMG-CoA reductase and HMG-CoA synthase activities 92- and 6-fold, respectively, and also increased the HMG-CoA reductase activity in intestine, heart, and kidney 3- to 15-fold. We used immunofluorescence and avidin-biotin labeled antibody to localize HMG-CoA reductase in the rat intestine. In rats fed a normal diet, the most HMG-CoA reductase-positive cells were the villi of the ileum greater than jejunum greater than duodenum. Crypt cells showed no evidence of HMG-CoA reductase. Addition of cholestyramine and mevinolin to the diet led to a dramatic increase in the concentration of HMG-CoA reductase in the apical region of the villi of the ileum and jejunum and in the crypt cells of the duodenum. Hence these two drugs affected both the relative concentration and distribution of intestinal HMG-CoA reductase. Cholestyramine and mevinolin feeding induced in the liver, but not intestine, whorls of smooth endoplasmic reticulum that were proximal to the nucleus and contained high concentrations of HMG-CoA reductase. Administration of mevalonolactone led to the rapid dissolution of the hepatic whorls within 15 min, at a time when there is little or no change in the mass of HMG-CoA reductase. We conclude that the whorls are present in the livers of rats fed cholestyramine and mevinolin because the cells are deprived of a cellular product normally synthesized from mevalonate.     

Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A synthase and the chromosomal localization of the human gene

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was purified to homogeneity from rat liver cytoplasm. The active enzyme is a dimer composed of identical subunits of Mr = 53,000. The amino acid composition and the NH2-terminal sequence are presented. Partial cDNA clones for the enzyme were isolated by screening of a rat liver lambda gt11 expression library with antibodies raised against the purified protein. The identity of the clones was confirmed by hybrid selection and translation. When rats were fed diets supplemented with cholesterol, cholestyramine, or cholestyramine plus mevinolin, the hepatic protein mass of cytoplasmic synthase, as determined by immunoblotting, was 25, 160, and 1100%, respectively, of the mass observed in rats fed normal chow. Comparable changes in enzyme activity were observed. Approximately 9-fold increases in both HMG-CoA synthase mRNA mass and synthase mRNA activity were observed when control diets were supplemented with cholestyramine and mevinolin. When rats were fed these two drugs and then given mevalonolactone by stomach intubation, there was a 5-fold decrease of synthase mRNA within 3 h. These results indicate that cytoplasmic synthase regulation occurs primarily at the level of mRNA. This regulation is rapid and coordinate with that observed for HMG-CoA reductase. The chromosomal localization of human HMG-CoA synthase was determined by examining a panel of human-mouse somatic cell hybrids with the rat cDNA probe. Interestingly, the synthase gene resides on human chromosome 5, which has previously been shown to contain the gene for HMG-CoA reductase. Regional mapping, performed by examination of a series of chromosome 5 deletion mutants and by in situ hybridization to human chromosomes indicates that the two genes are not tightly clustered.     

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA

We here report the isolation and nucleotide sequencing of a full-length 3.3-kilobase cDNA for the cytoplasmic form of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a regulated enzyme in the cholesterol biosynthetic pathway. The cDNA was isolated from UT-1 cells, a compactin-resistant line of Chinese hamster ovary cells. UT-1 cells produce large amounts of mRNA for HMG-CoA synthase and the next enzyme in the pathway, HMG-CoA reductase, as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. The identity of the cDNA for HMG-CoA synthase was confirmed through comparison of the NH2-terminal amino acid sequence predicted from the cDNA with that determined chemically from the purified enzyme. Anti-peptide antibodies directed against the amino acid sequence predicted from the cDNA precipitated HMG-CoA synthase activity from liver cytoplasm. The feeding of cholesterol to hamsters led to a decrease of more than 85% in the amount of mRNA for HMG-CoA synthase and HMG-CoA reductase in hamster liver. These data indicate that the mRNAs for cytoplasmic HMG-CoA synthase and for HMG-CoA reductase, two sequential enzymes in the cholesterol biosynthetic pathway, are coordinately regulated by cholesterol.     

Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl-CoA carboxylase in xenopus laevis

Administration of estradiol-17 beta to male Xenopus laevis evokes the proliferation of the endoplasmic reticulum and the Golgi apparatus and the synthesis and secretion by the liver of massive amounts of the egg yolk precursor phospholipoglycoprotein, vitellogenin. We have investigated the effects of estrogen on three key regulatory enzymes in lipid biosynthesis, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the major regulatory enzyme in cholesterol and isoprenoid synthesis, and acetyl-CoA carboxylase and fatty acid synthetase, which regulate fatty acid biosynthesis. HMG-CoA reductase activity and cholesterol synthesis increase in parallel following estrogen administration. Reductase activity in estrogen stimulated Xenopus liver cells peaks at 40-100 times the activity observed in control liver cells. The increased rate of reduction of HMG-CoA to mevalonic acid is not due to activation of pre-existing HMG-CoA reductase by dephosphorylation, as the fold induction is unchanged when reductase from control and estrogen-stimulated animals is fully activated prior to assay. The estrogen-induced increase of fatty acid synthesis is paralleled by a 16- to 20-fold increase of acetyl-CoA carboxylase activity, indicating that estrogen regulates fatty acid synthesis at the level of acetyl-CoA carboxylase. Fatty acid synthetase activity was unchanged during the induction of fatty acid biosynthesis by estrogen. The induction of HMG-CoA reductase and of acetyl-CoA carboxylase by estradiol-17 beta provides a useful model for regulation of these enzymes by steroid hormones.     

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.