scholarly journals Maintenance of bile acid synthesis and cholesterol 7α-hydroxylase activity in cultured rat hepatocytes

1990 ◽  
Vol 272 (1) ◽  
pp. 273-275 ◽  
Author(s):  
H M G Princen ◽  
P Meijer
Keyword(s):  
Bile Acid ◽  
Bovine Serum ◽  
Rat Hepatocytes ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Hydroxylase Activity ◽  
Newborn Bovine Serum ◽  
Cultured Rat Hepatocytes ◽  
Synthetic Capacity ◽  
Foetal Bovine Serum

Addition of foetal-bovine serum to rat hepatocytes cultured in Williams E medium resulted in improved maintenance of bile-acid-synthetic capacity and cholesterol 7 alpha-hydroxylase activity as compared with cultures supplemented with rat or newborn-bovine serum or cultures in a hormonally defined serum-free medium. Minimally, 5% (v/v) foetal-bovine serum was necessary to maintain these liver-specific functions. Serum factor(s) responsible for these effects were not dialysable or associated with lipoproteins, but were removed by charcoal extraction.

scholarly journals Dexamethasone regulates bile acid synthesis in monolayer cultures of rat hepatocytes by induction of cholesterol 7α-hydroxylase

1989 ◽  
Vol 262 (1) ◽  
pp. 341-348 ◽  
Author(s):  
H M Princen ◽  
P Meijer ◽  
B Hofstee
Keyword(s):  
Enzyme Activity ◽  
Bile Acid ◽  
Bile Acids ◽  
Steroid Hormones ◽  
Mrna Level ◽  
Rat Hepatocytes ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Hydroxylase Activity ◽  
Acid Pathway

To study the effect of steroid hormones on bile acid synthesis by cultured rat hepatocytes, cells were incubated with various amounts of these compounds during 72 h and conversion of [4-14C]cholesterol into bile acids was measured. Bile acid synthesis was stimulated in a dose-dependent way by glucocorticoids, but not by sex steroid hormones, pregnenolone or the mineralocorticoid aldosterone in concentrations up to 10 microM. Dexamethasone proved to be the most efficacious inducer, giving 3-fold and 7-fold increases in bile acid synthesis during the second and third 24 h incubation periods respectively, at a concentration of 50 nM. Mass production of bile acids as measured by g.l.c. during the second day of culture (28-52 h) was 2.2-fold enhanced by 1 microM-dexamethasone. No change in the ratio of bile acids produced was observed during this period in the presence of dexamethasone. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of the bile acid pathway, to bile acids was not affected by dexamethasone. Measurement of cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes, incubated with 1 microM-dexamethasone, showed 10-fold and 90-fold increases after 48 and 72 h respectively, as compared with control cells. As with bile acid synthesis from [14C]cholesterol, no change in enzyme activity was found in hepatocytes cultured in the presence of 10 microM steroid hormones other than glucocorticoids. Addition of inhibitors of protein and mRNA synthesis lowered bile acid production and cholesterol 7 alpha-hydroxylase activity and prevented the rise of both parameters with dexamethasone, suggesting regulation at the mRNA level. We conclude that glucocorticoids regulate bile acid synthesis in rat hepatocytes by induction of enzyme activity of cholesterol 7 alpha-hydroxylase.

scholarly journals Lipoprotein cholesterol uptake mediates up-regulation of bile-acid synthesis by increasing cholesterol 7α-hydroxylase but not sterol 27-hydroxylase gene expression in cultured rat hepatocytes

1999 ◽  
Vol 341 (2) ◽  
pp. 339-346 ◽  
Author(s):  
Sabine M. POST ◽  
Jaap TWISK ◽  
L V D FITS ◽  
Elly C. M. DE WIT ◽  
Marco F. M. HOEKMAN ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bile Acid ◽  
Density Lipoprotein ◽  
Rat Hepatocytes ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Lipoprotein Cholesterol ◽  
Hydroxylase Gene ◽  
Apo E ◽  
Cultured Rat Hepatocytes

Lipoproteins may supply substrate for the formation of bile acids, and the amount of hepatic cholesterol can regulate bile-acid synthesis and increase cholesterol 7α-hydroxylase expression. However, the effect of lipoprotein cholesterol on sterol 27-hydroxylase expression and the role of different lipoproteins in regulating both enzymes are not well established. We studied the effect of different rabbit lipoproteins on cholesterol 7α-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes. β-Migrating very-low-density lipoprotein (βVLDL) and intermediate-density lipoprotein (IDL) caused a significant increase in the intracellular cholesteryl ester content of cells (2.3- and 2-fold, respectively) at a concentration of 200 μg of cholesterol/ml, whereas high-density lipoprotein (HDL, 50% v/v), containing no apolipoprotein E (apo E), showed no effect after a 24-h incubation. βVLDL and IDL increased bile-acid synthesis (1.9- and 1.6-fold, respectively) by up-regulation of cholesterol 7α-hydroxylase activity (1.7- and 1.5-fold, respectively). Dose- and time-dependent changes in cholesterol 7α-hydroxylase mRNA levels and gene expression underlie the increase in enzyme activity. Incubation of cells with HDL showed no effect. Sterol 27-hydroxylase gene expression was not affected by any of the lipoproteins added. Transient-expression experiments in hepatocytes, transfected with a promoter-reporter construct containing the proximal 348 nucleotides of the rat cholesterol 7α-hydroxylase promoter, showed an enhanced gene transcription (2-fold) with βVLDL, indicating that a sequence important for a cholesterol-induced transcriptional response is located in this part of the cholesterol 7α-hydroxylase gene. The extent of stimulation of cholesterol 7α-hydroxylase is associated with the apo E content of the lipoprotein particle, which is important in the uptake of lipoprotein cholesterol. We conclude that physiological concentrations of cholesterol in apo E-containing lipoproteins increase bile-acid synthesis by stimulating cholesterol 7α-hydroxylase gene transcription, whereas HDL has no effect and sterol 27-hydroxylase is not affected.

scholarly journals Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

1995 ◽  
Vol 305 (2) ◽  
pp. 505-511 ◽  
Author(s):  
J Twisk ◽  
E C M de Wit ◽  
H M G Princen
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Transcriptional Activity ◽  
Lithocholic Acid ◽  
Rat Hepatocytes ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Taurocholic Acid ◽  
Substantial Contribution ◽  
Cultured Rat Hepatocytes

In previous work we have demonstrated suppression of cholesterol 7 alpha-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes [Twisk, Lehmann and Princen (1993) Biochem. J. 290, 685-691]. In view of the substantial contribution of the ‘alternative’ or ‘27-hydroxylase’ route to total bile acid synthesis, as demonstrated in cultured rat hepatocytes and in vivo in humans, we here evaluate the effects of various bile acids commonly found in bile of rats on the regulation of sterol 27-hydroxylase in cultured rat hepatocytes. Addition of taurocholic acid, the predominant bile acid in rat bile, to the culture medium of rat hepatocytes resulted in a 72% inhibition of sterol 27-hydroxylase activity. The effect was exerted at the level of sterol 27-hydroxylase mRNA, showing a time- and dose-dependent decline with a maximal suppression (-75%) at 50 microM taurocholic acid after 24 h of culture. The decline in mRNA followed first-order kinetics with an apparent half-life of 13 h. Under these conditions cholesterol 7 alpha-hydroxylase mRNA (-91%) and bile acid synthesis (i.e. chenodeoxycholic and beta-muricholic acid, -81%) were also maximally suppressed. In contrast, no change was found in the level of lithocholic acid 6 beta-hydroxylase mRNA. Assessment of the transcriptional activity of a number of genes involved in routing of cholesterol towards bile acids showed similar suppressive effects of taurocholate on expression of the sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase genes (-43% and -42% respectively), whereas expression of the lithocholic 6 beta-hydroxylase gene was not affected. Taurocholic acid and unconjugated cholic acid were equally as effective in suppressing sterol 27-hydroxylase mRNA. The more hydrophobic bile acids, chenodeoxycholic acid and deoxycholic acid, also produced a strong inhibition of 57% and 76% respectively, whereas the hydrophilic beta-muricholic acid was not active. We conclude that (1) a number of bile acids, at physiological concentrations, suppress sterol 27-hydroxylase by down-regulation of sterol 27-hydroxylase mRNA and transcriptional activity and (2) co-ordinated suppression of both sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase results in inhibition of bile acid synthesis in cultured rat hepatocytes.

Insulin suppresses bile acid synthesis in cultured rat hepatocytes by down-regulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase gene transcription

Hepatology ◽  
1995 ◽  
Vol 21 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Jaap Twisk ◽  
Marco F. M. Hoekman ◽  
Eline M. Lehmann ◽  
Piet Meijer ◽  
Willem H. Mager ◽  
...  
Keyword(s):  
Bile Acid ◽  
Gene Transcription ◽  
Rat Hepatocytes ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Down Regulation ◽  
Hydroxylase Gene ◽  
Cultured Rat Hepatocytes
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