Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

Princen, H.M.G.; Huijsmans, Cmg; Kuipers, Folkert; Vonk, Roelf; Kempen, H.J.M.

doi:10.1172/jci112662

Cited by 85 publications

(37 citation statements)

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“…In previous studies of bile acid synthesis in cultured human hepatocytes, 14 C-labeled cholesterol has been added as a substrate. 6,43 In the present study, the hepatocytes were obtained from histologically normal donor liver tissue. The bile acids formed were cholic acid and chenodeoxycholic acid, normally synthesized in human liver.…”

Section: Discussionmentioning

confidence: 99%

Bile acid synthesis in primary cultures of rat and human hepatocytes

et al. 1998

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The regulation of hepatic bile acid formation is incompletely understood. Primary cultures of mammalian hepatocytes offer an opportunity to examine putative regulatory factors in relative isolation. Using rat and human hepatocytes in primary culture, we examined bile acid composition and the expression of the rate-limiting enzyme of formation, cholesterol 7␣-hydroxylase. Control rat hepatocytes showed a declining bile acid production over 4 days, from 156 ؎ 24 ng/mL (67% cholic acid) on day 1 to 55 ؎ 11 ng/mL (55% cholic acid) on day 4. In addition to cholic acid, chenodeoxycholic acid, ␣-muricholic acid, and ␤-muricholic acid were formed. Treatment with triidothyronine (T 3 ) or dexamethasone alone had no significant effect on bile acid production. A combination of T 3 and dexamethasone significantly increased the total bile acid production on day 4 (224 ؎ 54 ng/mL) and resulted in a marked change in composition to 23% cholic acid and 77% non-12␣-hydroxylated bile acids. Control rat hepatocytes had a cholesterol 7␣-hydroxylase activity of 3.3 ؎ 0.6 pmol/mg protein/min after 4 days in culture. Cells treated with the combination of dexamethasone and T 3 had an activity of 16.4 ؎ 3.6 pmol/mg protein/min. The cholesterol 7␣-hydroxylase messenger RNA (mRNA) levels, determined by solution hybridization after 4 days of culture, showed results similar to those for the activity data; control cells had 5.3 ؎ 0.9 cpm/ g total nucleic acids (tNAs). T 3-or dexamethasone-treated cells did not differ from control cells, whereas the combination of T 3 and dexamethasone increased the mRNA levels to 20.6 ؎ 2.8 cpm/ g tNAs. In human hepatocytes, isolated from donor liver, bile acid formation increased from 206 ؎ 79 ng/mL on day 2 to 1490 ؎ 594 ng/mL on day 6 and then declined slightly. Cholic acid and chenodeoxycholic acid were formed, constituting about 80% and 20%, respectively. The combined addition of T 3 and dexamethasone had a tendency to decrease rather than increase bile acid formation. Also, mRNA levels of the cholesterol 7␣-hydroxylase increased severalfold in the human hepatocytes from day 2 to day 4 and then declined. The addition of T 3 or dexamethasone did not effect the mRNA levels in any consistent way. It is noteworthy that the capacity of the cultured human hepatocytes to produce bile acids was higher than that of cultured rat hepatocytes, in spite of the fact that the production of bile acids in rat liver is 3-to 5-fold higher than that in human liver in vivo. It is also evident that while hormonal factors appear to regulate bile acid synthesis in the rat, no evidence for this was found in human hepatocytes. As the composition of bile acids secreted by human hepatocytes in primary culture closely resembles that found in vivo, this represents a useful model for further studies of the synthesis and regulation of bile acids. (HEPATOLOGY 1998;27: 615-620.)Bile acid synthesis is localized within the liver and represents an important pathway for cholesterol elimination from the body. The major bile acids, primarily f...

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Section: Discussionmentioning

confidence: 99%

Bile acid synthesis in primary cultures of rat and human hepatocytes

et al. 1998

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“…Materials used for isolation and culturing of rat hepatocytes and for assaying cholesterol 7a-hydroxylase and sterol 27-hydroxylase activities, were obtained from sources described previously [25,261. Taurocholate, glycocholate, cholic acid and deoxycholic acid were obtained from Sigma Chemicals, chenodeoxycholic acid was from Serva and P-muricholic acid was obtained from Steraloids.…”

Section: Methodsmentioning

confidence: 99%

“…Rat liver cells were isolated by perfusion with 0.05 % collagenase and 0.005 % trypsin inhibitor, and cultured as described previously [12,13,25,261. Bile acids, dissolved in 10% (by vol.)…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, hydroxylation of bile acids by the hepatocytes was inhibited by simultaneous incubation of bile acids with 10 pM ketoconazole over a similar period of time in a separate set of experiments, as described above. The latter substance has been shown to effectively inhibit hydroxylation reactions catalyzed by NADPH-cytochrome-P-450-dependent monooxygenases [31], among them cholesterol 7u-hydroxylase (inhibits up to 94% at 10 pM) [25]. Culturing of cells in the presence of ketaconazole thus made assessment of the effects of bile acids on cholesterol 7a-hydroxylase activity impossible, but had no adverse effect on the expression of cholesterol 7a-hydroxylase mRNA levels.…”

Section: Methodsmentioning

confidence: 99%

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Structural Aspects of Bile Acids Involved in the Regulation of Cholesterol 7alpha-Hydroxylase and Sterol 27-Hydroxylase

Twisk¹,

Hoekman²,

Muller³

et al. 1995

Eur J Biochem

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We have recently reported that coordinate down-regulation of cholesterol 7a-hydroxylase and sterol 27-hydroxylase by bile acids results in suppression of bile acid synthesis in cultured rat hepatocytes [Twisk, J., De Wit, E. & Princen, H. M. G. (1995) Biochem. J. 305, 505-5111. In the current study, we have assessed the effects of a large group of different bile acids, both naturally occurring and synthetic, on these two key enzymes, to elucidate structural features which render bile acids potent as regulators of bile acid synthesis.Addition of 50 pM deoxycholate or cholate, two relatively hydrophobic bile acids, to the culture medium of hepatocytes resulted in strong suppression of cholesterol 7a-hydroxylase (suppression of 75 % and 88 %, respectively) and sterol 27-hydroxylase activity (suppression of 76 % and 72%, respectively). These effects were also reflected in the mRNA levels and the transcriptional activities of the two enzymes, showing a parallel suppression of both parameters in response to cholate (suppression of 78% and 43% for cholesterol 7a-hydroxylase mRNA and transcription, respectively, and suppression of 76 % and 42 % for sterol 27-hydroxylase mRNA and transcription, respectively). In contrast, no effects were observed with the two hydrophilic bile acids, P-muricholate and ursocholate. Transient expression analysis in cultured rat hepatocytes, using a promoter-reporter construct containing the proximal part of the cholesterol 7a-hydroxylase promoter, demonstrated a reduction of transcriptional activity by cholate (reduction of 72 %), but not by ursocholate.Assessment of the effects of 27 different bile acids, varying in the number, position and orientation (cxlp, of hydroxyl groups on the steroid nucleus of the molecule, on cholesterol 7a-hydroxylase mRNA showed only a moderate correlation with the hydrophobicity index of the bile acid involved (r = 0.61 ; P < 0.0001). Analysis of the three-dimensional structure of a number of these bile acids suggests that hydroxyl groups situated in close proximity to each other within the molecule, creating a hydrophilic environment, as in the case of cholate, may be a prerequisite for a strong inhibitory potency. Deviation from this situation leads to a markedly lesser effect on suppression of cholesterol 7a-hydroxylase and sterol 27-hydroxylase.

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“…Cell culture plates were supplied by Costar, Cambridge, MA. penicillin, 100 mg/mL streptomycin, 4 mU/mL insulin, and 0.02 mg/mL dexamethasone as described by Princen et al 26 Thin-layer chromatography plates were obtained from E. Merck, Darmstadt, Germany. Triglycerides GPO-PAP kit After cells had attached to the plates, usually after 4 hours, medium was renewed.…”

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confidence: 99%