Maintenance of bile acid synthesis and cholesterol 7 alpha-hydroxylase activity in cultured rat hepatocytes.
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ABSTRACT:
SUBMITTER: Princen HM
PROVIDER: S-EPMC1149691 | biostudies-other | 1990 Nov
REPOSITORIES: biostudies-other
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Project description:Lipoproteins may supply substrate for the formation of bile acids, and the amount of hepatic cholesterol can regulate bile-acid synthesis and increase cholesterol 7alpha-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 7alpha-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes. beta-Migrating very-low-density lipoprotein (betaVLDL) 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 microgram 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. betaVLDL and IDL increased bile-acid synthesis (1. 9- and 1.6-fold, respectively) by up-regulation of cholesterol 7alpha-hydroxylase activity (1.7- and 1.5-fold, respectively). Dose- and time-dependent changes in cholesterol 7alpha-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 7alpha-hydroxylase promoter, showed an enhanced gene transcription (2-fold) with betaVLDL, indicating that a sequence important for a cholesterol-induced transcriptional response is located in this part of the cholesterol 7alpha-hydroxylase gene. The extent of stimulation of cholesterol 7alpha-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 7alpha-hydroxylase gene transcription, whereas HDL has no effect and sterol 27-hydroxylase is not affected.
| S-EPMC1220365 | biostudies-other
Project description: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.
| S-EPMC1133266 | biostudies-other
Project description:We reported previously that mice overexpressing cytochrome P450 7a1 (Cyp7a1; Cyp7a1-tg mice) are protected against high fat diet-induced hypercholesterolemia, obesity, and insulin resistance. Here, we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1-tg mice. Cyp7a1-tg mice had two-fold higher Cyp7a1 activity and bile acid pool than did wild-type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild-type to chenodeoxycholic acid (54%) in Cyp7a1-tg mice. Cyp7a1-tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than did wild-type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1-tg mice but intestine fractional cholesterol absorption in Cyp7a1-tg mice remained the same as wild-type mice despite the presence of increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (adenosine triphosphate-binding cassette G5/G8 [ABCG5/G8], scavenger receptor class B, member 1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1-tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue-specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport.This study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis.
| S-EPMC3079544 | biostudies-literature
Project description:Periportal and perivenous hepatocytes were isolated from rat liver by digitonin/collagenase perfusion for investigating the acinar distribution of bile acid synthesis. The specific activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) was 7.9-fold higher in perivenous cells than in periportal hepatocytes. Mass production of bile acids differed 4.4-fold between cultured perivenous and periportal hepatocytes. In contrast, the levels of free cholesterol in homogenates and microsomes derived from both subfractions were similar. Feeding of rats with the bile-acid-sequestering anion-exchange resin colestid resulted in a pronounced stimulation of cholesterol 7 alpha-hydroxylase activity and bile acid mass production, but decreased the perivenous/periportal ratio of both parameters. These results demonstrate that bile acid mass production, but decreased the perivenous hepatocytes, possibly owing to feedback suppression by bile acids from the enterohepatic circulation. Furthermore, the opposite acinar localization of cholesterol and bile acid biosynthesis provides an interesting alternative to current views of the regulation of their metabolic pathways.
| S-EPMC1151145 | biostudies-other
Project description:The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase). The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and synthase. Here we report the purification of rat 7 alpha-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding 7 alpha-hydroxylase. DNA sequence analysis of the cDNA revealed a 7 alpha-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890 which represents a novel family of cytochrome P-450 enzymes. Transfection of a 7 alpha-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the mRNA for 7 alpha-hydroxylase is found only in the liver. The levels of this mRNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in 7 alpha-hydroxylase mRNA levels. The enzymatic activity of 7 alpha-hydroxylase paralleled the observed changes in mRNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the 7 alpha-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis.
| S-EPMC4451855 | biostudies-literature
Project description:Alcoholic fatty liver disease (AFLD) is a major risk factor for cirrhosis-associated liver diseases. Studies demonstrate that alcohol increases serum bile acids in humans and rodents. AFLD has been linked to cholestasis, although the physiologic relevance of increased bile acids in AFLD and the underlying mechanism of increasing the bile acid pool by alcohol feeding are still unclear. In this study, we used mouse models either deficient of or overexpressing cholesterol 7?-hydroxylase (Cyp7a1), the rate-limiting and key regulatory enzyme in bile acid synthesis, to study the effect of alcohol drinking in liver metabolism and inflammation. Mice were challenged with chronic ethanol feeding (10 days) plus a binge dose of alcohol by oral gavage (5?g/kg body weight). Alcohol feeding reduced bile acid synthesis gene expression but increased the bile acid pool size, hepatic triglycerides and cholesterol, and inflammation and injury in wild-type mice and aggravated liver inflammation and injury in Cyp7a1-deficient mice. Interestingly, alcohol-induced hepatic inflammation and injury were ameliorated in Cyp7a1 transgenic mice. Conclusion: Alcohol feeding alters hepatic bile acid and cholesterol metabolism to cause liver inflammation and injury, while maintenance of bile acid and cholesterol homeostasis protect against alcohol-induced hepatic inflammation and injury. Our findings indicate that CYP7A1 plays a key role in protection against alcohol-induced steatohepatitis. (Hepatology Communications 2018;2:99-112).
| S-EPMC5776875 | biostudies-literature
Project description:Bile acids (BAs) are diverse molecules that are synthesized from cholesterol in the liver. The synthesis of BAs has traditionally been shown to occur through two pathways. Cholesterol 7α-hydroxylase (CYP7A1) performs the initial and rate-limiting step in the classical pathway, and sterol 27-hydroxylase (CYP27A1) initiates the hydroxylation of cholesterol in the alternative pathway. While the role of individual BA species as physiological detergents is relatively ubiquitous, their endocrine functions as signaling molecules and roles in disease pathogenesis have been emerging to be BA species-specific. In order to better understand the pharmacologic and toxicologic roles of individual BA species in an in vivo model, we created cholesterol 7α-hydroxylase (Cyp7a1) and sterol 27-hydroxylase (Cyp27a1) double knockout (DKO) mice by cross-breeding single knockout mice (Cyp7a1-/- and Cyp27a1-/- ). BA profiling and quantification by liquid chromatography-mass spectrometry of serum, gallbladder, liver, small intestine, and colon of wild-type, Cyp7a1-/- , Cyp27a1-/- , and DKO mice showed that DKO mice exhibited a reduction of BAs in the plasma (45.9%), liver (60.2%), gallbladder (76.3%), small intestine (88.7%), and colon (93.6%), while maintaining a similar BA pool composition compared to wild-type mice. The function of the farnesoid X receptor (FXR) in DKO mice was lower, revealed by decreased mRNA expression of well-known FXR target genes, hepatic small heterodimer partner, and ileal fibroblast growth factor 15. However, response to FXR synthetic ligands was maintained in DKO mice as treatment with GW4064 resulted in similar changes in gene expression in all strains of mice. Conclusion: We provide a useful tool for studying the role of individual BAs in vivo; DKO mice have a significantly reduced BA pool, have a similar BA profile, and maintained response to FXR activation.
| S-EPMC7893641 | biostudies-literature
Project description:Bile acid synthesis, determined by conversion of [4-14C]cholesterol into bile acids in rat and human hepatocytes and by measurement of mass production of bile acids in rat hepatocytes, was dose-dependently decreased by cyclosporin A, with 52% (rat) and 45% (human) inhibition of 10 microM. The decreased bile acid production in rat hepatocytes was due only to a fall in the synthesis of beta-muricholic and chenodeoxycholic acids (-64% at 10 microM-cyclosporin A), with no change in the formation of cholic acid. In isolated rat liver mitochondria, 26-hydroxylation of cholesterol was potently inhibited by the drug (concn. giving half-maximal inhibition = 4 microM). These results suggest that cyclosporin A blocks the alternative pathway in bile acid synthesis, which leads preferentially to the formation of chenodeoxycholic acid.
| S-EPMC1150079 | biostudies-other
Project description: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.
| S-EPMC1136391 | biostudies-other
Project description:We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7 alpha-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most predominant bile acid in rat bile, to the culture medium suppressed cholesterol 7 alpha-hydroxylase activity and mRNA time- and dose-dependently. The decrease in enzyme activity paralleled the changes in mRNA. Maximal suppression of cholesterol 7 alpha-hydroxylase mRNA (-91%) and enzyme activity (-89%) was observed after a 16 h incubation period with 50 microM taurocholic acid. The declines in mRNA and enzyme caused by taurocholic acid were tightly coupled and followed first-order kinetics with a half-life of 4 h. Transcriptional activity, as assessed with nuclear run-on assays, was decreased by 44% at 50 microM taurocholic acid. Mass production of bile acids (chenodeoxycholic acid and beta-muricholic acid) was inhibited to a similar extent as the cholesterol 7 alpha-hydroxylase when different concentrations of taurocholic acid were used, giving maximal inhibition (-81%) at 50 microM taurocholic acid. Glycocholic acid and unconjugated cholic acid were equally effective as taurocholic acid in suppressing cholesterol 7 alpha-hydroxylase mRNA. The more hydrophobic bile acids (chenodeoxycholic acid and deoxycholic acid) showed profound suppression of the cholesterol 7 alpha-hydroxylase mRNA by 85% and 75% respectively, whereas the other trihydroxy bile acids in rat bile, alpha- and beta-muricholic acid, were not or only marginally active. We conclude that rat bile acids, in particular the more hydrophobic ones, in concentrations commonly observed in portal blood, exert negative feedback control at the level of cholesterol 7 alpha-hydroxylase mRNA in cultured rat hepatocytes through a direct effect on the hepatocytes, and that down-regulation of transcription is only one of the mechanisms involved in this regulation.
| S-EPMC1132335 | biostudies-other