Influence of a maternal cholesterol-enriched diet on [1-14C]-linoleic acid and L-[4, 5-3H]-leucine entry in plasma of rabbit offspring

Fetal development requires an important entry of essential free fatty acids (EFFA) and essential amino acids (EAA) into the fetal circulation. We have reported that a 0.2% enriched-cholesterol diet (ECD) during rabbit gestation significantly reduces fetus weight compared to control diet. It is known that dietary linoleic acid deficiency, an EFFA, during the fetal development induces an important impair to the somatic development. Moreover, intrauterine growth retardation induced a reduction of the flux of leucine, an EAA, from maternal to fetal circulation. Therefore, we hypothesized that the administration of an ECD induces modifications of placental lipid composition concomitant alterations of the transfer of linoleic acid and leucine in fetal circulation. Quantification of placental lipids revealed that in the ECD group a reduction of total-cholesterol (TC) and free-cholesterol (FC) is observed, however an increased in FFA and phospholipids is noticed when compared to the control group. In placenta from the ECD group, the FC/ TC ratio is significantly reduced compared to the control group. In the ECD group, the liver shows an increase of TC, FC and FFA compared to the control group. However, the quantity of triacylglycerol present in the liver from the ECD is significantly reduced compared to the control group. To evaluate the placental transfer of some essential nutrients, intravenous injection of [1-14C]-linoleic acid or L-[4, 5-3H]-leucine to term rabbit (control and ECD group) were done. Two hours later, rabbits were euthanized and we collected placenta, livers and blood from dams and offspring. The concentrations of both radiolabeled molecules (linoleic acid and its esterified form or leucine) were higher in the plasma of ECD offspring than those found in offspring from control diet. Despite such alteration of placental lipid composition, linoleic acid and leucine transfer by the placenta was not compromised but rather increased.     

Age-related changes in cholesterol metabolism in macrosomic offspring of rats with streptozotocin-induced diabetes.

The aim of this study was to determine the impact of diabetic macrosomia on cholesterol and lipoprotein metabolism. Age-related changes in the activities of serum LCAT, hepatic HMG-CoA reductase, cholesterol 7alpha-hydroxylase, and ACAT, the major enzymes involved in cholesterol metabolism, were determined in macrosomic offspring of streptozotocin-induced diabetic rats. Hepatic, serum, and lipoprotein cholesterol contents were also examined. Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin (40 mg/kg body weight) on day 5 of gestation. Control pregnant rats were injected with citrate buffer. At birth, macrosomic pups had higher serum, LDL-HDL(1), and HDL(2-3) cholesterol levels (P < 0.05) associated with increased LCAT activity (+57%) compared with control values. At 1 and 2 months of life, serum and lipoprotein cholesterol concentrations in macrosomic rats were similar to those of controls, whereas LCAT activity remained elevated about 1.5-fold. In addition, there was no change in hepatic cholesterol contents but hepatic HMG-CoA reductase, cholesterol 7alpha-hydroxylase, and ACAT activities were higher in both macrosomic males and females than in their respective controls (P < 0.01). By 3 months, macrosomic rats had developed hypercholesterolemia with a rise in all lipoproteins. Enzyme activities were still increased in these mature macrosomic rats, and hepatic cholesteryl esters were higher only in macrosomic females.These data demonstrate an overproduction, combined with overutilization, of cholesterol during the phase of rapid growth in macrosomic rats. However, cholesterol oversynthesis exceeded its removal and was a major contributor to hypercholesterolemia in adult macrosomic rats. In conclusion, macrosomia was associated with alterations in cholesterol metabolism through adulthood.     

Effect of an enriched cholesterol diet during gestation on fatty acid synthase, HMG-CoA reductase and SREBP-1/2 expressions in rabbits

Pregnancy is associated with hyperlipidemia and hypercholesterolemia in humans. These changes take place to support fetal growth and development, and modifications of these maternal concentrations may influence lipids and cholesterol synthesis in the dam, fetus and placenta. Administration of a 0.2% enriched cholesterol diet (ECD) during rabbit gestation significantly increased cholesterol and triglyceride (TG) levels in maternal livers and decreased fetal weight by 15%. Here we used Western blot analysis to examine the impact of gestation and 0.2% ECD on the expression levels of fatty acid synthase (FAS), HMGR and SREBP-1/2, which are involved in either lipid or cholesterol synthesis. We confirmed that gestation modifies the hepatic and circulating lipid profile in the mother. Our data also suggest that the maternal liver mainly supports lipogenesis, while the placenta plays a key role in cholesterol synthesis. Thus, our data demonstrate a decrease in HMGR protein levels in dam livers by feeding an ECD. In the placenta, SREBPs are highly expressed, and the ECD supplementation increased nuclear SREBP-1/2 protein levels. In addition, our results show a decrease in FAS protein levels in non-pregnant liver and in the liver of offspring from ECD-treated animals. Finally, our data suggest that the placenta does not modify its own cholesterol synthesis in response to an increase in circulating cholesterol. However, the dam liver compensates for this increase by essentially decreasing the level of HMGR expression. Because HMGR and FAS expressions do not correlate with the circulating lipid profile, it would be interesting to find which genes are then targeted by SREBP-1/2 during gestation.     

Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis

The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7alpha-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7alpha-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human liver cells has not been examined. The specific aim of this study was to determine the effects of overexpression of CYP7A1 on key regulatory steps involved in hepatocellular cholesterol homeostasis, using primary human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in HepG2 cells and PHH was accomplished by using a recombinant adenovirus encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted in a marked activation of the classic pathway of bile acid biosynthesis in both PHH and HepG2 cells. In response, there was decreased HMG-CoA-reductase (HMGR) activity, decreased acyl CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl ester hydrolase (CEH) activity, and increased low-density lipoprotein receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and CEH mRNA levels paralleled changes in enzyme specific activities. More specifically, LDLR expression, ACAT activity, and CEH activity appeared responsive to an increase in cholesterol degradation after increased CYP7A1 expression. Conversely, accumulation of the oxysterol 7alpha-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.     

Placental transfer of cholesterol-4-14C into rabbit and guinea pig fetus

A tracer dose of cholesterol-4-(14)C was given daily in the diet of six pregnant guinea pigs to establish an isotopic steady state. At the time of parturition, maternal and fetal blood and fetal tissues were collected and analyzed for cholesterol content and cholesterol specific activity. A comparison of these specific activities in neonatal and maternal serum indicated that about 22% of the fetal serum cholesterol was transferred from maternal blood. In the newborn, tissues generally had the same cholesterol specific activity as serum. Brain tissue was an exception in having a specific activity only 8.4% of that of serum. Dietary cholesterol did not increase serum cholesterol levels in the newborn but did increase the percentage of fetal cholesterol derived from the maternal circulation. The rapid transfer of cholesterol-4-(14)C across the placenta was indicated by the appearance of this isotope in the newborn 2 days after its administration to pregnant rabbits. A considerable amount of the cholesterol content of newborn guinea pigs and rabbits originated from the maternal blood.     

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.     

On the structural specificity in the regulation of the hydroxymethylglutaryl-CoA reductase and the cholesterol-7 alpha-hydroxylase in rats. Effects of cholestanol feeding

The effect of feeding 2% cholestanol or cholesterol on cholesterol-7 alpha-hydroxylase activity and hydroxymethylglutaryl (HMG)-CoA reductase activity was studied in rats. The rate of 7 alpha-hydroxylation of a trace amount of labelled cholesterol increased by about 80% after the cholestanol feeding, whereas the 7 alpha-hydroxylation of endogenous microsomal cholesterol increased by about 40%. The latter conversion was measured with an accurate technique based on isotope dilution-mass spectrometry. After cholesterol feeding, the corresponding figures were about 50 and 60%, respectively. The cholestanol feeding had no significant effect on the HMG-CoA reductase activity, whereas the cholesterol feeding decreased the activity by about 80%. From the results obtained, it is concluded that the increased 7 alpha-hydroxylation observed after cholesterol feeding can not be explained only by a simple expansion of the substrate pool. The similar effect of both cholesterol and cholestanol on the cholesterol 7 alpha-hydroxylase activity and the diverging effect on the HMG-CoA reductase activity show that there is no coupling between cholesterol synthesis and degradation under the conditions employed. The lack of effect of cholestanol on the HMG-CoA reductase activity indicates a high structural specificity of the receptor involved in regulation of the enzyme. If a receptor mechanism is involved in the stimulation of the cholesterol-7 alpha-hydroxylase by cholesterol and cholestanol, these receptor(s) must be different from those involved in the regulation of the HMG-CoA reductase.     

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.     

Sexual Dimorphism of the Feto-Placental Phenotype in Response to a High Fat and Control Maternal Diets in a Rabbit Model

Maternal environment during early developmental stages plays a seminal role in the establishment of adult phenotype. Using a rabbit model, we previously showed that feeding dams with a diet supplemented with 8% fat and 0.2% cholesterol (HH diet) from the prepubertal period and throughout gestation induced metabolic syndrome in adult offspring. Here, we examined the effects of the HH diet on feto-placental phenotype at 28 days post-coïtum (term = 31days) in relation to earlier effects in the blastocyst (Day 6). At 28 days, both male and female HH fetuses were intrauterine growth retarded and dyslipidemic, with males more affected than females. Lipid droplets accumulated in the HH placentas’ trophoblast, consistent with the increased concentrations in cholesteryl esters (3.2-fold), triacylglycerol (2.5-fold) and stored FA (2.12-fold). Stored FA concentrations were significantly higher in female compared to male HH placentas (2.18-fold, p<0.01), whereas triacylglycerol was increased only in HH males. Trophoblastic lipid droplet accumulation was also observed at the blastocyst stage. The expression of numerous genes involved in lipid pathways differed significantly according to diet both in term placenta and at the blastocyst stage. Among them, the expression of LXR-α in HH placentas was reduced in HH males but not females. These data demonstrate that maternal HH diet affects the blastocyst and induces sex-dependent metabolic adaptations in the placenta, which appears to protect female fetuses from developing severe dyslipidemia.     

A comparative study of the rate-limiting enzymes of cholesterol synthesis, esterification and catabolism in the alloxan-induced diabetic rat and rabbit

1. Hyperlipidaemia is associated with diabetes mellitus. 2. A comparison of cholesterol synthesis and utilization in alloxan-induced diabetic rats and rabbits revealed that interspecies differences existed only in the response of the key enzymes regulating cholesterol utilization, namely cholesterol 7 alpha-hydroxylase and ACAT in the liver and intestine respectively. 3. The activity of cholesterol 7 alpha-hydroxylase was enhanced in diabetic rats but was markedly reduced in diabetic rabbits. 4. Intestinal ACAT activity, though unchanged in diabetic rats was reduced in diabetic rabbits. 5. Such species differences in cholesterol utilization may underlie the different degree of susceptibility to hypercholesterolaemia that exists between these two species.     

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.     

Lectin histochemistry for detecting cadmium-induced changes in the glycosylation pattern of rat placenta

Cadmium (Cd) is an industrial and environmental pollutant that produces toxic effects on gametogenesis, pre- and post-implantation embryos, and the placenta. Because the effects of acute Cd intoxication on the placenta are not well understood, we investigated changes in its glycosylated components in Cd treated dams at days 4, 7, 10 and 15 of gestation using lectin histochemistry. CdCl₂ was administered to pregnant rats; control animals received sterile normal saline. Placentas were processed for DBA, Con A, SBA, PNA, UEA-I, RCA-I and WGA lectin histochemistry to evaluate changes in the carbohydrate pattern of the placenta that might modify cell interactions and contribute to embryonic alterations. Lectin binding was analyzed in the yolk sac; trophoblast giant cells; trophoblast I, II and III; spongiotrophoblast cells and endovascular trophoblast cells in the chorioallantoic placenta. Our lectin binding patterns showed that Cd caused alteration of SBA and DBA labeling of trophoblast-derived cells, which suggested increased expressions of α and β GalNAc. Cd also caused decreased UEA-1 binding affinity, which indicated fewer α-L-Fuc residues in placentas of Cd treated dams. The nonreactivity in trophoblast I of the control placentas incubated with Con-A contrasted with the labeling in placentas of experimental dams, which indicated increased expression of terminal α-D-Man, and α-D-Glc residues. We found that Cd altered the reactivity of placenta to several lectins, which indicated modification of the glycotype presented by the fetal component of the placenta. We report that Cd exerts a deleterious effect on the glycosylation pattern of the placenta.     

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.     

Cholesterol metabolism in liver at different levels of motor activity

Biosynthesis and decomposition of cholesterol were studied in the rabbit liver under a dosed physical loading. Judging from the intensity of [2-14C]acetate incorporation, the cholesterol biosynthesis intensity rises during loading and decreases under conditions of limited mobility. The cholesterol content in the liver tissue of trained animals is unchanged and in animals, who were under conditions of limited mobility, it rises as compared with the control. Using [7-3N1] cholesterol as a substrate, it established that the activity of cholesterol-7 alpha-hydroxylase, as compared to the initial level, is increased under training and decreased under conditions of low mobility. The enzymic activity was determined by the amount of released 3H2O.     

Purification of cholesterol 7 alpha-hydroxylase from human and rat liver and production of inhibiting polyclonal antibodies

Cholesterol 7 alpha-hydroxylase, the cytochrome P-450-dependent and rate-controlling enzyme of bile acid synthesis, was purified from rat and human liver microsomes. The purified fractions were assayed in a reconstituted system containing [4-14C]cholesterol, and cholesterol 7 alpha-hydroxylase activities in these fractions increased 500-600-fold relative to whole microsomes. Polyacrylamide gel electrophoresis of rat microsomes followed by immunoblotting with polyclonal rabbit antisera raised against purified cholesterol 7 alpha-hydroxylases revealed two peaks at molecular masses of 47,000 and 49,000 daltons for both rat and human fractions. Increasing amounts of rabbit anti-rat and anti-human antibodies progressively inhibited rat microsomal cholesterol 7 alpha-hydroxylase activity up to 80%. In contrast, monospecific antibodies raised against other purified cytochrome P-450 enzymes (P-450f, P-450g, and P-450j) did not inhibit rat or human cholesterol 7 alpha-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7 alpha-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7 alpha-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7 alpha-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7 alpha-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7 alpha-hydroxylase antibodies that can be used to measure the change in cholesterol 7 alpha-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7 alpha-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.     

Regulation of cholesterol 7 alpha-hydroxylase by hepatic 7 alpha-hydroxylated bile acid flux and newly synthesized cholesterol supply

We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalytic efficiency. The administration of mevalonolactone, which is converted to cholesterol, modestly increased cholesterol 7 alpha-hydroxylase activity and enzyme mass in the bile acid-depleted rats. However, when taurocholate was infused together with mevalonolactone, cholesterol 7 alpha-hydroxylase activity and catalytic efficiency were markedly depressed while enzyme mass did not change as compared with bile acid-depleted rats. These results show that (a) hepatic bile acid depletion increases bile acid synthesis mainly by activating cholesterol 7 alpha-hydroxylase with only a small rise in enzyme mass, (b) replacement with taurocholate for 24 h decreases both cholesterol 7 alpha-hydroxylase activity and mass proportionally, (c) when cholesterol is available (mevalonolactone supplementation), the infusion of taurocholate results in the formation of a catalytically less active cholesterol 7 alpha-hydroxylase, and (d) tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, does not inhibit cholesterol 7 alpha-hydroxylase. Thus, bile acid synthesis is modulated by the catalytic efficiency and mass of cholesterol 7 alpha-hydroxylase. The enterohepatic flux of 7 alpha-hydroxylated bile acids and the formation of hepatic cholesterol apparently control cholesterol 7 alpha-hydroxylase by different mechanisms.     

Effect of taurine on cholesterol metabolism in hamsters: up-regulation of low density lipoprotein (LDL) receptor by taurine

The effects of taurine on hepatic cholesterol metabolism were investigated in hamsters fed a high-fat diet or normal chow. Two weeks-treatment of taurine at 1% in drinking water prevented high-fat diet-induced increase in cholesterol levels of serum and liver. The decrease in serum cholesterol by taurine was due to decrease in non-HDL cholesterols. A similar tendency was noted in serum and liver cholesterol levels of hamsters fed a normal diet. In hamsters fed a high-fat diet, taurine prevented elevation in hepatic activity of acyl-CoA:cholesterol acyltransferase (ACAT) and increased the activity of cholesterol 7alpha-hydroxylase. Taurine also increased cholesterol 7alpha-hydroxylase activity in hamsters fed normal chow. Studies on liver membranes revealed that taurine increased 125I-labeled LDL binding by 52% and 58% in hamsters fed either a normal chow or high-fat diet, respectively. Furthermore, LDL kinetic analysis showed that taurine intake resulted in significant faster plasma LDL fractional catabolic rates (FCR). These results suggest that taurine elevates hepatic LDL receptor and thereby decreases serum cholesterol levels, an event which may be the result of hepatic cholesterol depletion as a consequence of increased bile acid synthesis via enhancement of cholesterol 7alpha-hydroxylase activity. Thus, up-regulation of the LDL receptor and subsequent increase in receptor- mediated LDL turnover may be a key event in the cholesterol-lowering effects of taurine in hamsters.     

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes

The effect of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), tauroursodeoxycholic (TUDCA), cholic (CA), ursocholic (UCA) acids, analogues of CDCA and UDCA with a cyclopropyl ring at C22, C23 (cypro-CDCA and cypro-UDCA) and 23-methylursodeoxycholic acid (MUDCA) on cholesterol 7 alpha-hydroxylase was studied in rat liver microsomes. Cypro-analogues consisted of a mixture of four diasteroisomers, while MUDCA was the racemic mixture of two enantiomers. Each steroid was added to liver microsomes at concentrations ranging from 10 to 200 microM. With the exception of UCA and CA, all the bile acids inhibited cholesterol 7 alpha-hydroxylase activity. The inhibition shown by cypro-CDCA and cypro-UDCA was stronger than that observed with the corresponding natural compounds. 22S,23S cypro-UDCA exhibited an inhibitory effect which was more pronounced than that of the diasteroisomer mixture. The isomer 22R,23S was less effective and decreased cholesterol 7 alpha-hydroxylase activity in a manner comparable to that of UDCA. The effect of CDCA, UDCA and the cyclopropyl analogues was also tested with respect to HMG-CoA reductase and acylCoA cholesterol acyltransferase (ACAT) activities. ACAT was stimulated by the isomer 22S,23S cypro-UDCA but not affected by the other bile acids. No effect was observed as regards HMG-CoA reductase.