Bile acid formation in primary human hepatocytes (original) (raw)
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Bile acid synthesis in primary cultures of rat and human hepatocytes
Hepatology, 1998
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 Abbreviations: mRNA, messenger RNA; T 3 , triidothyronine; HDL, high-density lipoprotein; LDL, low-density lipoprotein; EDTA, ethylenediaminetetraacetic acid; tNA, total nucleic acids.
Hepatology, 2003
Primary human hepatocytes were used to elucidate the effect of individual bile acids on bile acid formation in human liver. Hepatocytes were treated with free as well as glycine-conjugated bile acids. Bile acid formation and messenger RNA (mRNA) levels of key enzymes and the nuclear receptor short heterodimer partner (SHP) were measured after 24 hours. Glycochenodeoxycholic acid (GCDCA; 100 pmol/L) significantly decreased formation of cholic acid (CA) to 44% +: 4% of controls and glycodeoxycholic acid (GDCA) decreased formation of CA to 67% f 11% of controls. Glycoursodeoxycholic acid (GUDCA; 100 pmol/L) had no effect. GDCA or glycocholic acid (GCA) had no significant effect on chenodeoxycholic acid (CDCA) synthesis. Free bile acids had a similar effect as glycine-conjugated bile acids. Addition of GCDCA, GDCA, and GCA (100 pmol/L) markedly decreased cholesterol 7m hydroxylase (CYP7A1) mRNA levels to 2% f 1%, 2% 2 1%, and 29% f 11% of controls, respectively, whereas GUDCA had no effect. Addition of GDCA and GCDCA (100 pmol/L) significantly decreased sterol 12a-hydroxylase (CYP8B1) mRNA levels to 48% f 5% and 61% f. 4% of controls, respectively, whereas GCA and GUDCA had no effect. Addition of GCDCA and GDCA (100 pmol/L) significantly decreased sterol 27-hydroxylase (CYP27A1) mRNA levels to 59% f 3% and 60% f 7% of controls, respectively, whereas GUDCA and GCA had no significant effect. Addition of both GCDCA and GDCA markedly increased the mRNA levels of SHP to 298% f 43% and 273% & 30% of controls, respectively. In conclusion, glycine-conjugated and free bile acids suppress bile acid synthesis and mRNA levels of CYP7A1 in the order CDCA > DCA > CA > UDCA. mRNA levels of CYP8B1 and CYP27A1 are suppressed to a much lower degree than CYP7A1. (HEPATOLOGY 2003;38: 930-938.)
Bile Acid Synthesis in Cell Culture
Journal of Biological Chemistry, 1989
Confluent cultures of Hep G 2 cells were found to synthesize chenodeoxycholic and cholic acids continually. Chenodeoxycholic acid was synthesized at the rate of 58 f 8.6 ~g / 9 6 h, a rate more than 7-fold greater than that for cholic acid. Addition of Ei@-cholestane-3a,7a,12a-triol but not the-3a,7a-diol was followed by an increase in cholic acid synthesis, thus indicating a relatively low 12a-hydroxylase activity. Endogenous synthesis of monohydroxy bile acid ester sulfates was found, with maximum rates of 135 and 74 pg/96 h for lithocholic and 3a-hydroxy-5-cholenoic acids, respectiveiy. Incubation of Hep G 2 cells in medium containing 25% D20 permitted a comparison of the precursor/product relationship of cholesterol with 3@-hydroxy-5-cholenoic acid. The pattern of incorporation of deuterium was in accordance with that expected, thus allowing the conclusion that this monohydroxy bile acid is derived from cholesterol and should be considered together with chenodeoxycholic and cholic acids as a primary bile acid.
Hepatology, 2000
The biosynthesis of bile acids by primary cultures of normal human hepatocytes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange chromatography and gas chromatography-mass spectrometry (GC/MS). Following incubation for 5 days, 8 oxysterols and 8 C 27 -or C 24 -bile acids were identified in media and cells. Cholic and chenodeoxycholic acids conjugated with glycine or taurine were by far the major steroids found, accounting for 70% and 24% of the total, respectively, being consistent with bile acid synthesis in human liver. Small amounts of sulfated 3-hydroxy-5-cholenoic acid and 3,7␣dihydroxy-5-cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7␣,12␣-dihydroxy-3-oxo-4-cholenoic acid and its 5-reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7␣hydroxylation of cholesterol, followed by oxidation to 7␣-hydroxy-4-cholesten-3-one and 12␣-hydroxylation. Notably, 27-hydroxylation of the product 7␣,12␣-dihydroxy-4cholesten-3-one and further oxidation and cleavage of the side chain precede A-ring reduction. A-Ring reduction may also occur before side-chain cleavage, but after 27hydroxylation, yielding 3␣,7␣,12␣-trihydroxy-5-cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7␣,12␣dihydroxy-4-cholesten-3-one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates reflect an alternative bio-Abbreviations: CA, cholic acid (3␣,7␣,12␣-trihydroxy-5-cholanoate); CDCA, chenodeoxycholic acid (3␣,7␣-dihydroxy-5-cholanoate); CsA, cyclosporin A; ODS, octadecylsilane; TEAP-LH-20, triethylaminohydroxypropyl-Sephadex LH-20; GLC, gas-liquid chromatography; GC/MS, gas chromatography-mass spectrometry; THCA, 3␣,7␣,12␣-trihydroxy-5-cholestanoate.
Journal of Lipid Research, 1993
We have examined the ability of HepG2 human hepatoblastoma cells and 7800 C1 Morris rat hepatoma cells to convert 3a,7a,l2a-trihydroxy-5fl-cholestanoic acid (THCA) and 3a,7a-dihydroxy-5fl-cholestanoic acid (DHCA) to cholic acid and chenodeoxycholic acid, respectively. Cell extracts from both these cell lines could neither form cholic acid from THCA nor from the activated form, THCA-CoA. This suggests that both cell lines are defective in two enzyme activities involved in the pathway, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase. Furthermore, we show that the subsequent enzymes are active in the conversion to bile acids, because the product of the THCA-CoA oxidase, 3a,7a,12a-tri-using HepG2 cells, a different bile acid pattern was found than that found in human liver. Cholic acid and chenodeoxycholic acid, the main primary bile acids synthesized in humans, are synthesized in approximately equal amounts in human liver (7). In most studies with HepG2 cells, chenodeoxycholic acid is the predominant primary bile acid formed, with a lower amount of cholic acid secreted into the medium (8-10). In addition to these two primary bile acids, a Cz7 bile acid precursor for cholic acid, 3a ,7a ,12a-trihydroxy-5~-cholestanoic acid (THCA), hydroxy-58-cholest-24-enoyl-coenzyme A (A24-THCA-CoA) or A24-THCA in the presence of THCA-CoA ligase, are COnverted to cholic acid by both cell lines. HepG2 cells were able to slowly form chenodeoxycholic acid and cholic acid from 5fl-cholestane-3a, 7a-diol and 5fl-cholestane-3a,7ar,12a-triol, respectively, in 24and 96-h incubations. The rate of cholic acid formation was has been found secreted into the medium. In other reports, however, cholic acid and chenodeoxycholic acid have been synthesized in equal amounts (11, 12). The small amount of cholic acid formed by HepG2 cells and the presence of THCA in the medium have led to the lower than the rate for chenodeoxycholic acid and there was a clear accumulation of THCA. 7800 C1 Morris cells had no ability to form cholic acid or chenodeoxycholic acid after 96 h incubation. 811 We conclude that these two cell lines have defects in two enzyme activities involved in the peroxisomal oxidation in bile acid formation, the microsomal-THCA-CoA ligase and the peroxisomal THCA-CoA oxidase.-Ostlund Farrants, A-K., A. Nilsson, and J. I. Pedersen. Human hepatoblastoma cells (HepG2) and rat hepatoma cells are defective in important enzyme activities in the oxidation of the (& steroid side chain in bile acid formation.
Cellular Accumulation and Toxic Effects of Bile Acids in Cyclosporine A-Treated HepaRG Hepatocytes
Toxicological Sciences, 2015
Alteration of bile acid (BA) profiles and secretion by cholestatic drugs represents a major clinical issue. Species differences exist in BA composition, synthesis, and regulation; however presently, there is no in vitro reproducible cell model to perform studies on BAs in humans. We have evaluated the capacity of the human HepaRG cell line to synthesize, conjugate, and secrete BAs, and analyzed changes in BA content and profile after cyclosporine A (CsA) treatment. Our data show that HepaRG cells produced normal BAs at daily levels comparable, though in different proportions, to those measured in primary human hepatocytes. A 4-h treatment with CsA led to BA accumulation and profile changes associated with occurrence of cholestatic features, while after 24 h BAs were decreased in cell layers and increased in media. The latter effects resulted from reduced function of BA uptake transporter (Na þ-taurocholate cotransporting polypeptide), reduced expression of BA metabolizing enzymes, including cytochrome P4507A1, cytochrome P4508B1, and cytochrome P45027A1, and induction of alternative basolateral transporters. Noteworthy, HepaRG cells incubated in a 2% serum-supplemented medium showed dose-dependent accumulation of the cytotoxic BA lithocholic acid in a nonsulfoconjugated form associated with early inhibition of the canalicular transporter MRP2 and sulfotransferase 2A1. In summary, our data bring the first demonstration that an in vitro human liver cell line is able to produce and secrete conjugated BAs, and to accumulate endogenous BAs transiently, concomitantly to occurrence of various other cholestatic features following CsA treatment. Retention of the hydrophobic lithocholic acid supports its toxic role in drug-induced cholestasis. Overall, our results argue on the suitability of HepaRG cells for investigating mechanisms involved in the development of the disease.
Journal of Clinical and Experimental Hepatology, 2016
Background: Primary human hepatocytes offer the best human in vitro model for studies on human liver cell metabolism. Investigators use a variety of different media supplements and matrix biocoatings and the type of culture system used may influence the outcome. Objectives: To optimize in vitro conditions for primary human hepatocytes with regard to bile acid synthesis. Methods: Human hepatocytes were isolated and cultured on collagen type I or EHS matrigel in cell media with or without dexamethasone. The glucocorticoid receptor (GR) antagonist RU486 was used to elucidate the involvement of GR. Results: Hepatocytes cultured on EHS matrigel produced more bile acids and expressed higher levels of cholesterol 7a-hydroxylase (CYP7A1) than cells cultured on rat tail collagen. Supplementation with dexamethasone increased the formation of cholic acid (CA) and decreased chenodeoxycholic acid formation. In line with these results, the mRNA expression of sterol 12a-hydroxylase (CYP8B1) increased following dexamethasone treatment. Surprisingly, the mRNA expression of CYP7A1 and CYP27A1 was not increased to the same extent. By using the GR antagonist RU486, we concluded that CYP8B1 induction is mediated via a GR-independent pathway. An altered expression of retinoid-related orphan receptor (ROR) a and ROR a target gene Glucose-6-phosphatase (G6Pase) suggests that ROR a signaling may regulate CYP8B1 expression. Conclusion: Primary human hepatocytes have an increased bile acid synthesis rate when cultured on matrigel as compared to collagen. Exposure to glucocorticoid hormones stimulates the expression of CYP8B1, leading to an increased formation of CA and alteration of the bile acid composition. The effect is most likely mediated through a GR-independent pathway, possibly through ROR a.
Comparison of Culture Media for Bile Acid Transport Studies in Primary Human Hepatocytes
Journal of Clinical and Experimental Hepatology, 2012
Background: Primary human hepatocytes are a useful in vitro model system to examine hepatic biochemical pathways, liver disorders and/or pharmacotherapies. This system can also be used for transport studies to investigate uptake and excretion of bile acids. Proper modeling of hepatic function requires careful attention to media components, and culture substrates and conditions. Objectives: To examine the effects of different culture media and conditions on bile acid transport in cultured human hepatocytes. Methods and Results: Hepatocytes cultured in Williams' medium E showed an increase in both uptake and excretion of taurocholate compared to cells cultured in Dulbecco's Modified Eagle Medium (DMEM). Supplementation of DMEM with glutathione or ascorbic acid did not compensate for the lower transport. The difference can be explained by lower mRNA expression of the transporter proteins sodium taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP; ABCB11) when cultured in DMEM. Hepatocytes cultured in DMEM also display fewer and smaller bile canaliculi. Following extended time in culture supplementation of Williams' medium E with dexamethasone increased the expression of NTCP and BSEP. Conclusion: Williams' medium E is superior to DMEM for transport studies in primary human hepatocytes. Supplementation with dexamethasone increase mRNA levels of NTCP and BSEP.
Toxicological sciences : an official journal of the Society of Toxicology, 2014
Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced ...
Journal of lipid research, 1983
After incubation in the presence of tritiated water, incorporation of tritium into cholesterol and into different bile acids was several-fold higher using hepatocytes of cholestyramine-fed rats than that found using hepatocytes of control rats. Labeling of the trihydroxylated cholic and beta-muricholic acids was markedly greater than that of dihydroxycholanoic acid. The total amount of label in all bile acids was 30% or less of that in free cholesterol, in both types of hepatocytes. In combination with the data on bile acids mass production we could calculate the average number (N(a)) of tritium atoms incorporated per molecule of newly-formed bile acid. The experimental values of N(a) for cholic and beta-muricholic acid were compared with values of N(n) or N(o), theoretically predicted if these bile acids were derived entirely from newly made or pre-existent cholesterol, respectively. It was deduced for hepatocytes of cholestyramine-fed rats that the bile acids produced in the first...