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Cholesterol homoeostasis is the result of the fine tuning between intake and disposal of this molecule. High levels of cholesterol in the blood are detrimental as they may lead to excessive accumulation in vessel walls, a condition predisposing to the development of atherosclerotic lesions. Cholesterol is removed from the vessel wall and transported to the liver through a process called reverse cholesterol transport. Nuclear receptors are among the most important transcription factors regulating genes involved in different steps of reverse cholesterol transport. Here, we discuss the role of the nuclear receptors LXR (liver X receptor) and HNF-4alpha (hepatocyte nuclear factor-4alpha) in different steps of reverse cholesterol transport. LXR controls the transcription of crucial genes in cholesterol efflux from macrophages and its transport to the liver, such as ABCA1 (ATP binding cassette A1), CYP27A1 (sterol 27-hydroxylase), CLA-1 (scavenger receptor type B1) and apolipoprotein E. Some oxysterols present in oxidized low-density lipoproteins and proinflammatory cytokines modulate the activity of LXR by antagonizing the effect of activators of this receptor, thus contributing to cholesterol accumulation in macrophages. Bile acid synthesis, which represents the final step of reverse cholesterol transport, is transcriptionally regulated by several nuclear receptors at the level of the liver-specific cytochrome P450 cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme of this metabolic pathway. Bile acids returning to the liver through the enterohepatic circulation down-regulate CYP7A1 transcription via the bile acid sensors farnesoid X receptor and HNF-4alpha. Based on this evidence, these nuclear receptors are candidate targets of new drugs for the treatment and prevention of atherosclerotic disease.

Bile acid metabolism plays an essential role in cholesterol homeostasis and is critical for the initiation of atherosclerotic disease. However, despite the recent advances, the molecular mechanisms whereby bile acids regulate gene transcription and cholesterol homeostasis in mammals still need further investigations. Here, we show that bile acids suppress transcription of the gene (CYP7A1) encoding cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, also through an unusual mechanism not involving the bile acid nuclear receptor, farnesoid X receptor. By performing cell-based reporter assays, protein/protein interaction, and chromatin immunoprecipitation assays, we demonstrate that bile acids impair the recruitment of peroxisome proliferator-activated receptor-gamma coactivator-1alpha and cAMP response element-binding protein-binding protein by hepatocyte nuclear factor-4alpha, a master regulator of CYP7A1. We also show for the first time that bile acids inhibit transcription of the gene (PEPCK) encoding phosphoenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, through the same farnesoid X receptor-independent mechanism. Chromatin immunoprecipitation assay revealed that bile acid-induced dissociation of coactivators from hepatocyte nuclear factor-4alpha decreased the recruitment of RNA polymerase II to the core promoter and downstream in the 3'-untranslated regions of these two genes, reflecting the reduction of gene transcription. Finally, we found that Cyp7a1 expression was stimulated in fasted mice in parallel to Pepck, whereas the same genes were repressed by bile acids. Collectively, these results reveal a novel regulatory mechanism that controls gene transcription in response to extracellular stimuli and argue that the transcription regulation by bile acids of genes central to cholesterol and glucose metabolism should be viewed dynamically in the context of the fasted-to-fed cycle.

Oxidized LDL exhibits cytotoxic activity towards endothelial cells, which is thought to be mediated by the products derived also from cholesterol oxidation, mainly involving the B-ring. By LC-PB-EI-MS and EI-MS/MS analysis, we have identified 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide among the cholesterol oxidation products in LDL incubated with micromolar concentrations of copper ions.

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).

A collaborative trial for the evaluation of blood cholesterol measurement in Italy was carried out, with the use of two lyophilized controls, whose target values, respectively 4.42 and 6.21 mmol/l, were established by means of "definitive" methodology (isotope dilution/mass spectrometry). Results from 480 participants showed a somewhat broad dispersion (CV 6.1% and 6.3% respectively), and a definite bias (-0.25 mmol/l and -0.61 mmol/l respectively) with respect to the target values. The different analytical systems were characterized by different combinations of inaccuracy and imprecision; however, the bias observed for the higher concentration sample was a constant finding. The behaviour of the control materials, in comparison with that exhibited by patients' sera, was assessed in a manual enzymatic procedure and in the Kodak Ektachem 700 and Technicon Chem 1 systems. The peculiar property of one control material to behave differently from patients' sera in some analytical systems, i.e. the lack of commutability, was found to be partially responsible for the observed bias in the three methods studied. The importance of testing for commutability of the control materials to be used for the control of accuracy is stressed.

Rat hepatocytes isolated by the procedure described here showed 3-hydroxy-3-methylglutaryl-CoA reductase activity in the range of that reported for rat liver at the maximum of the circadian cycle, even if they were taken from rats at the time of the minimum. The enzyme was present in cells as both its active dephosphorylated (20 +/- 8%) and the inactive phosphorylated forms. The enzyme activity and the ratio between the two forms were unaltered during 3 h of cell incubation. 25-Hydroxycholesterol (50 microM) induced about 50% inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase activity during 1 h incubation but the relative amount of the two forms was not modified by the sterol. Cells isolated by the described procedure may therefore be a useful tool in studies on the regulation of cholesterol neogenesis, both through the synthesis of the enzyme, which can be shown by measuring the activity after complete dephosphorylation of the enzyme, and via the rapid reversible shift of the inactive to the active form, resulting from the ratio between the two enzyme forms. The latter mechanism for the modulation of cholesterol synthesis cannot be tested in cell cultures because full activation of the enzyme occurs during hepatocyte plating.

The identification of sterols and steroids formed by incubation of [7alph-3H, 26-14C]cholesterol with mitochondrial enzymes from bovine adrenal cortex is reported. Only 17% of the radioactivity associated with cholesterol metabolites was found in pregnenolone, whereas 15% was reliable to oxygenated sterols and 6% to steroid compounds. The significance of the formation of these compounds is discussed particularly as regards oxygenated cholesterol derivatives.

The effect of a series of beta-adrenoceptor antagonists on cholesterol biosynthesis was studied in vitro in normal human skin fibroblasts. Some, but not all, of the drugs studied stimulated the incorporation of [2-14C]-acetate into cell sterols in a dose-dependent manner. This effect was unrelated to beta-blocking potency, selectivity for beta 1 or beta 2 adrenoceptors and partial agonistic activity of the drugs, thus ruling out a beta-receptor mediated mechanism. A positive, statistically significant correlation was found, however, between the drug lipophilicity and the stimulation of sterol biosynthesis. Propranolol, the most effective agent in increasing [2-14C]-acetate incorporation into cellular sterols, also enhanced the conversion of 3-hydroxy-3-methylglutaryl CoA (HMGCoA) into mevalonic acid, suggesting an interference of lipophilic beta-adrenoceptor antagonists with HMHCoA-reductase, the feed-back regulated rate limiting step of cholesterol biosynthesis.

Scintigraphy of the adrenal gland with 131I-19-iodocholesterol has recently been added to radiological techniques in adrenal imaging and has been used successfully to demonstrate anatomical and functional disorders of the adrenals in a variety of clinical situations. A review of the authors' experience stresses the diagnostic value of this method. Radiological findings and results of scintillation imaging are complementary: their comparison improves and clarifes indications for scintigraphy. Hyperadrenal cortical diseases always gave satisfactory scintigrams, the most interesting results being obtained in adrenal cortical hyperplasia and unilateral hyperfunctioning adenomas. In these cases the evaluation of the response to stimulation or suppression tests was very useful. On the other hand scintigraphy was less valuable in demonstrating malignant and non malignant tumours.

Identification of radioactive 5 alpha-cholest-8(14)-ene-3 beta,7 alpha-diol in extracts obtained from incubations of 3 beta-hydroxy-5 alpha-[7-3H]cholest-7-ene-14 alpha-carbaldehyde with rat liver microsomes is reported. Levels of this diol in incubations of the 14 alpha-[32-3H]carbaldehyde were measured by multiple selected ion monitoring and were found to be of the same order of those of [3H]formate released from the substrate during the removal of the C-32 atom. The results demonstrate that the diol does not originate from known intermediates of cholesterol biosynthesis, i.e. 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholest-7-en-3 beta-ol and from 5 alpha-cholest-8(14)-en-3 beta-ol. Functionalization at position 7 in the metabolism of 3 beta-hydroxy-5 alpha-cholest-7-ene-14 alpha-carbaldehyde suggests the direct involvement of the double bond in the elimination of the 14 alpha-formyl group in the biosynthetic pathway from lanosterol to cholesterol. 5 alpha-Cholest-8(14)-en-3 beta-ol appears not to be involved in the metabolism of the 14 alpha-carbaldehyde.

Scintigraphy of the suprarenal glands with 131I-iodocholesterol has been performed by means of a linear scanner or gamma-camera in 56 subjects. The results are presented and discussed and some cases are reported in detail.

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.

Results presented here show that when isolated rat hepatocytes are incubated with increasing concentrations of [2-14C]mevalonolactone, incorporation of the substrate into cholesterol is progressively reduced. Correspondingly, an increase of the incorporation of the substrate into precursors of cholesterol (methyl sterols and squalene) occurs. These effects and the observed inhibition of HMGCoA reductase at high mevalonolactone concentration (0.5 mM) are in agreement with those shown by others in cultured hepatocytes. Since pantethine was reported to affect cholesterol biosynthesis from mevalonate in cultured fibroblasts, effects of its addition to hepatocyte incubations at low and high mevalonolactone concentration were studied. Neither the amount of radioactivity incorporated into cholesterol and in its sterol precursors nor sterol levels were modified by pantethine when a mevalonolactone concentration (0.01 mM) that did not alter the levels of intermediates of cholesterol synthesis was used. Pantethine was shown instead to potentiate the decrease of mevalonate incorporation into cholesterol induced by high concentrations of mevalonolactone (0.5 mM). Decrease of 3-hydroxy-3-methylglutaryl CoA reductase activity induced by 1 mM pantethine was twice that caused by mevalonolactone alone. These results may explain the fact that both in laboratory animals and in humans pantethine administration is effective in reducing cholesterol plasma levels in hyperlipidemic conditions.

The aim of this study was to characterize the plasma lipoprotein pattern and some aspects of cholesterol metabolism in a line of hyperlipemic male rats. Plasma cholesterol and triglycerides were increased about 3-fold as compared to control animals (238 vs. 75 and 185 vs. 59 mg/dl respectively). The plasma lipoprotein distribution and the chemical composition of the isolated lipoproteins was unaffected. Plasma triglyceride production rate was increased (40%, P less than 0.01) and post-heparin lipoprotein lipase activity in plasma decreased (-28%, P less than 0.01) in the hyperlipemic rat. The activity of 3 enzymes involved in cholesterol metabolism (HMG-CoA reductase, cholesterol 7 alpha-hydroxylase, and acyl-CoA cholesterol-acyltransferase) did not differ from control values. 3H2O incorporation into digitonin-precipitable sterols, however, was significantly higher than in controls. This finding was due, in part, to an increased liver weight in the hyperlipemic animals. Furthermore kinetic data using 125I-LDL showed that the fractional catabolic rate of lipoprotein was within the normal range, while the synthetic rate of LDL protein was increased (0.67 vs. 0.3 mg/kg/h, P less than 0.01) in the hyperlipemic rat. These observations suggest that multiple metabolic defects underline the hyperlipemia observed in this animal model.

Adult human brain contains cholestanol and two series of cholesterol precursors having 30, 29, 28, and 27 carbon atoms; one has an unsaturated steroid nucleus, and the other is unsaturated in both nucleus and side chain. The ability of preparations of brain to incorporate a specific precursor into cholesterol, as well as into these sterol metabolites in vitro, indicates that cholesterol synthesis continues long after brain maturation ceases.