Studies on the cholesterol-free mouse: strong activation of LXR-regulated hepatic genes when replacing cholesterol with desmosterol.

OBJECTIVE: Characterization of cholesterol homeostasis in male mice with a genetic inactivation of 3beta-hydroxysteroid-delta24-reductase, causing replacement of almost all cholesterol with desmosterol. METHODS AND RESULTS: There was an increase in hepatic sterol synthesis and markedly increased fecal loss of neutral sterols. Fecal excretion of bile acids was similar in knockout mice and in controls. The composition of bile acids was changed, with reduced formation of cholic acid. It was shown that both Cyp7a1 and Cyp27a1 are active toward desmosterol, consistent with the formation of normal bile acids from this steroid. The levels of plant sterols were markedly reduced. Hepatic mRNA levels of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase, Srebp-1c, Srebp-2, Cyp7a1, Abcg5, Abcg8, and Fas were all significantly increased. CONCLUSIONS: The changes in hepatic mRNA levels in combination with increased biliary and fecal excretion of neutral steroids, reduced tissue levels of plant sterols, increased plasma levels of triglyceride-rich VLDL, are consistent with a strong activation of LXR-targeted genes. The markedly increased fecal loss of neutral sterols may explain the fact that the Dhcr24-/- mice do not accumulate dietary cholesterol. The study illustrates the importance of the integrity of the cholesterol structure--presence of a double bond in the steroid side-chain is compatible with life but is associated with serious disturbances in sterol homeostasis.

Mechanism of accumulation of cholesterol and cholestanol in tendons and the role of sterol 27-hydroxylase (CYP27A1).

OBJECTIVE: Tendon xanthomas are deposits of lipids and connective tissue commonly found in hypercholesterolemic patients. Macrophages are likely to be responsible for the lipid accumulation. Normolipidemic patients with the rare disease cerebrotendinous xanthomatosis, lacking the enzyme sterol 27-hydroxylase (CYP27A1), develop prominent xanthomas in tendons and brain containing both cholestanol and cholesterol, with a cholestanol:cholesterol ratio higher than that in the circulation. Because of its ability to convert cholesterol into polar metabolites that leave the cells faster, CYP27A1 has been suggested to be an antiatherogenic enzyme. The hypothesis was tested that tendons contain CYP27A1 that may be of importance for the normal efflux of both steroids. METHODS AND RESULTS: Western blotting and combined gas chromatography-mass spectrometry showed that human tendons contain significant amounts of CYP27A1 and its product, 27-hydroxycholesterol. Immunohistochemistry showed that CYP27A1 is present in macrophages and tenocytes. The tendons also contained cholestanol, with a cholestanol:cholesterol ratio slightly higher than that in the circulation. Recombinant human CYP27A1, and cultured human macrophages containing this enzyme, had similar activity toward cholesterol and cholestanol. After loading of macrophages with labeled cholesterol and cholestanol, there was an efflux of these steroids in both unmetabolized and 27-oxygenated form, resulting in a significant cellular accumulation of cholestanol compared with cholesterol. CONCLUSION: The results are consistent with the possibility that CYP27A1 is of importance for the efflux of both cholesterol and cholestanol from tendons.

Mutation in the sterol 27-hydroxylase gene associated with fatal cholestasis in infancy.

BACKGROUND: Inborn errors of bile acid synthesis are rare but potentially treatable causes of neonatal cholestasis. We here present a cholestatic infant with an ongoing cytomegalovirus infection who despite intensive treatment died of severe liver disease at 4 months of age. METHODS: The urinary steroids were investigated by electrospray mass spectrometry and gas chromatography mass spectrometry. Oxysterols in plasma were analysed by isotope dilution mass spectrometry. Mutations in the sterol 27-hydroxylase gene were detected by PCR. RESULTS: Glucuronidated bile alcohols, which are known to be excreted by patients with cerebrotendinous xanthomatosis (CTX) were detected in the urine. Analysis of plasma revealed markedly reduced levels of 27-hydroxycholesterol. Mutation analysis showed the presence of a stop codon in exon 7, confirming the diagnosis of CTX, a rare disease not previously diagnosed in Sweden. CONCLUSIONS: Fetal and neonatal deaths among siblings of patients with CTX have been reported previously and the present case supports the contention that reduced activity of the sterol 27-hydroxylase may predispose to the development of neonatal cholestasis. The associated viral infection may have further precipitated the liver disease. Since CTX, like other inborn errors of bile acid synthesis may be treated with bile acids an early diagnosis is essential. Thus, the analysis of urine by electrospray mass spectrometry is highly recommended in the investigation of patients with neonatal cholestasis.

On the substrate specificity of human CYP27A1: implications for bile acid and cholestanol formation.

The mitochondrial sterol 27-hydroxylase (CYP27A1) is required for degradation of the C27-sterol side chain in bile acid biosynthesis. CYP27A1 seems, however, to have roles beyond this, as illustrated by patients with a deficient sterol 27-hydroxylase due to mutations of the CYP27A1 gene [cerebrotendinous xanthomatosis (CTX)]. These subjects have symptoms ranging from accumulation of bile alcohols and cholestanol to accelerated atherosclerosis and progressive neurologic impairment. The present work describes a detailed investigation on the substrate specificity of recombinant human CYP27A1. In accordance with some previous work with rat liver mitochondria, the activity in general increased with the polarity of the substrate. An obvious example was the finding that cholesterol was 27-hydroxylated more efficiently than cholesterol oleate but less efficiently than cholesterol sulfate. The oxysterols 24S-hydroxycholesterol and 25-hydroxycholesterol were 27-hydroxylated less efficiently than cholesterol, possibly due to steric hindrance. Surprisingly, sterols with a 3-oxo-Delta4 structure were found to be hydroxylated at a much higher rate than the corresponding sterols with a 3beta-hydroxy-Delta5 structure. The rates of hydroxylation of the sterols were: 7alpha-hydroxy-4-cholesten-3-one>4-cholesten-3-one>7alpha-hydroxycholesterol>24-hydroxy-4-cholesten-3-one> cholesterol>25-hydroxy-4-cholesten-3-one>24-hydroxycholesterol>or=25-hydroxycholesterol. The possibility is discussed that the findings may have implications for oxysterol-mediated regulation of gene expression. The very high activity of CYP27A1 towards the cholestanol precursor 4-cholesten-3-one may be of importance in connection with the accumulation of cholestanol in patients with CTX.

Studies on the mechanism of accumulation of cholesterol in the gallbladder mucosa. Evidence that sterol 27-hydroxylase is not a pathogenetic factor.

BACKGROUND/AIMS: Cholesterolosis is characterized by accumulation of esterified cholesterol in human gallbladder mucosa. The present study aimed at investigating possible pathogenetic factors for cholesterolosis. The hypothesis was tested that a reduced sterol 27-hydroxylase or an increased amount of ACAT-1 enzyme may be of importance. METHODS: Gall bladder mucosa and bile were obtained from patients with cholesterol gallstones undergoing cholecystectomy (30 with and 43 without cholesterolosis). RESULTS: In cholesterolosis, the gall bladder mucosa was characterized by a several-fold increase in esterified cholesterol and normal content of free cholesterol. The amount of ACAT-1 protein, measured by immunoblotting, was similar in patients with and without cholesterolosis. The level of 27-hydroxycholesterol in gallbladder mucosa was elevated sevenfold as compared with cholesterol in patients with cholesterolosis. Most (87%) of this oxysterol was esterified and the accumulation is most probably secondary to the higher total amount of cholesterol in the cells. Patients with cholesterolosis had normal levels of both sterol 27-hydroxylase mRNA (real time polymerase chain reaction) and protein (immunoblotting). The enzymatic activity of the sterol 27-hydroxylase in gallbladder mucosa was normal or increased in cholesterolosis. CONCLUSIONS: The pathogenesis of cholesterolosis may be multifactorial, but is not caused by reduced efflux of cholesterol due to a defect sterol 27-hydroxylase mechanism.