Stimulation of bile acid synthesis by dibutyryl cyclic AMP in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to examine the effects of dibutyryl cyclic AMP on the incorporation of¹⁴C-acetate and¹⁴C-cholesterol into bile acids. After an initial lag period, both precursors were incorporated into cholic and chenodeoxycholic acids at a linear rate for the subsequent 60 min. An apparent stimulation of bile acid formation from¹⁴C-acetate by dibutyryl cyclic AMP was complicated by the concomitant inhibition of cholesterol synthesis. In experiments with¹⁴C-cholesterol, dibutyryl cyclic AMP (1 mM) increased the labeled cholic and chenodeoxycholic acids in the medium by 83 and 224%, respectively, but cellular levels of labeled bile acids were unchanged. As a result, the nucleotide stimulated the overall incorporation of¹⁴C-cholesterol into cholic acid by 39% and into chenodeoxycholic acid by 123%. The mean ratio of labeled cholic to chenodeoxycholic acid declined from 55∶45 in control cells to 41∶59 in cells incubated with dibutyryl cyclic AMP. The results demonstrate that label incorporation can be used to study the regulation of bile acid synthesis in isolated hepatocytes. We propose that dibutyryl cyclic AMP enhances bile acid production by phosphorylating, and thus stimulating the activity of, cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis.     

Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: Very low density lipoprotein and bile acid production

The interrelationship between very low density lipoprotein (VLDL) secretion and bile acid production was studied in primary culture of rabbit hepatocytes. Chylomicron remnants (CR) were added to the cultures to study their effect on VLDL secretion and bile acid production. After 24 hr preincubation of cells with CR (10–50 μg protein/mL), intercellular neutral lipid content was increased 1.5–4-fold in a dose-dependent manner. Neutral lipid accumulation was accompanied by a 70–90% reduction of [¹⁴C]acetate incorporation into cholesterol, while no stimulation of [¹⁴C]oleate incorporation into cholesteryl esters was observed. Incubation of cells with CR increased secretion of free cholesterol, triacylglycerol and apoproteins B and E in VLDL. Stimulation of VLDL cholesterol secretion was accompanied by a reduction of taurocholic acid synthesis. These data demonstrate the existence of an inverse relationship between secretion of VLDL cholesterol and bile acid production under conditions of effective uptake of triacylglycerol-rich CR by hepatocytes.     

Composition of cecal bile acids in ex-germfree mice inoculated with human intestinal bacteria

Germfree (GF) mice were orally inoculated with human fecal suspension or various components of human fecal microbiota. Three weeks after the inoculation, cecal bile acid composition of these mice was examined. More than 80% of total bile acids was deconjugated in the cecal contents of ex-GF mice associated with human fecal dilutions of 10⁻² or 10⁻⁶, or anaerobic growth from a dilution of 10⁻⁶. In these ex-GF mice, deoxycholic acid accounted for about 20% of total bile acids. In the cecal contents of ex-GF mice associated only with clostridia, unconjugated bile acids made up less than 40% of total bile acids, about half of those in other ex-GF groups. However, the percentage of deoxycholic acid in these mice was the same as that in the other groups. These results indicate that dominant anaerobic bacterial combination is efficient for deconjugation of primary bile acids, and that clostridia in the human feces may play an important role in 7α-dehydroxylation of unconjugated primary bile acids in the intestine.     

Separation of bile acid methyl esters by high-performance liquid chromatography

Conjugated bile acids, namely glyco- and tauro-3α,6α-dihydroxy-5β-cholanoic acid (hyodeoxycholic acid), 3α,7α-dihydroxy-5β-cholanoic acid (chenodeoxycholic acid), 3α,6α,7α-trihydroxy-5β-cholanoic acid (hyocholic acid) and 3α-hydroxy-6-oxo-5β-cholanoic acid (6-keto-litocholic acid) were isolated from pig bile, and subsequently transformed into the corresponding methyl esters. Separation of the methyl esters of the isolated bile acids by high-performance liquid chromatography (HPLC) was accomplished on a ZORBAX-CN column (Dupont, Boston, MA) withn-hexane/2-propanol/methylene chloride (89∶6∶5, by vol) as the mobile phase containing traces (≈1%) of amyl alcohol and water as moderators. HPLC analysis of the methyl esters also showed the presence of methyl 3α-hydroxy-6-oxo-5α-cholanoate, which was probably produced in the course of alkaline hydrolysis of the conjugated bile acids.     

Bile acid metabolism in mammals: VII. Studies on sex differences in deoxycholic acid metabolism in isolated perfused rat liver

The hepatic metabolism of deoxycholic acid was studied using the isolated perfused rat liver technique. In 20 perfusions, 10 involving the livers of male rats and 10 involving the livers of female rats, 30 μmoles deoxycholic acid was added to the perfusion medium. In 10 perfusions, 5 male and 5 female, 1 μmole deoxycholic acid was added to the perfusion medium. In 10 of the high dose studies and in the 10 low dose studies, 1 μCi deoxycholic acid-C-24-C¹⁴ also was added. The deoxycholic acid was added to 100 ml perfusion medium after 2 hr of baseline perfusion, and the studies were continued another 3 hr. Biliary bile acids were analyzed by combined thin layer and gas chromatography, and the radioactivity content of the perfusion medium and liver was documented. Although there was no sex difference in total bile acid secretion in the high dose studies, there were sex differences in the bile acid secretion rate and in the quantitative secretion of individual bile acids. The biliary secretion of deoxycholic acid and cholic acid was immediate in the female studies and delayed in the male, and the amounts of cholic acid and sulfated deoxycholyl-taurine secreted were considerably greater in the male studies. In the low dose studies the isolated perfused liver of the female rat converted more deoxycholic acid to cholyl-taurine than did that of the male rat. There are sex differences in the hepatic metabolism of deoxycholic acid. In contrast to those found in the case of chenodeoxycholic acid, these sex differences are not impressive when physiological amounts of deoxycholic acid are presented to the liver.     

Effect of dietary taurine on bile acid metabolism in guinea pigs

The effect of oral administration of taurine (200–300 mg daily) on the metabolism of bile acids was studied in male guinea pigs which have predominantly glycine conjugated bile acids. The results were summarized as follows: (a) oral administration of taurine for 10 days increased taurine-conjugated bile acids and the ratio of glycine-to taurine-conjugated bile acids (G:T ratio) shifted from 3.95 to 0.19; (b) in taurine fed guinea pigs, the half-life of chenodeoxycholic acid (CDC) was about 40% shorter than that in controls and the fractional turnover rate increased by 70%; (c) the synthetic rate (mg/day/500 g body weight) of bile acids increased from 4.28 to 7.27 by taurine feeding; (d) hepatic cholesterol 7α-hydroxylase activity was increased 2.4-fold by taurine feeding; (e) the total pool size of bile acids did not change significantly but the amount of lithocholic acid in the caecum and large intestine increased by about 40%; (f) neither free cholesterol nor cholesterol ester levels in liver and serum changed significantly. Results of this study suggest that changing the G:T ratio in the bile acid conjugation pattern may influence the rate of hepatic bile acid synthesis. This paper is Part IX of a series entitled “Metabolism of Bile Acids”. Part VIII: ref. 12.     

Site of bile acid absorption in the rat

Bile acid absorption was measured in the small intestine of the rat using⁹¹Y as a nonabsorbed reference substance. Some 50% of the secreted bile acids were absorbed in the proximal half of the small intestine. In situ incubations of ligated intestinal segments into which tauro(¹⁴C-carbonyl)cholic acid was introduced confirmed the considerable uptake of bile acids in the jejunum. The in situ experiments indicated that serosal transport is the limiting stage of bile acid absorption in the jejunum but not in the ileum. Increasing bile acid concentrations in the in situ experiments did not affect the percentage disappearance of dose from the jejunum but reduced the percentage mucosal uptake in the ileum. It is concluded that, in the rat, the proximal small intestine is as important in the absorption of bile acids as the distal small intestine.     

Inhibition and induction of bile acid synthesis by ketoconazole effects on bile formation in the rat

The effects of ketoconazole, an antimycotic agent, and metyrapone, an inhibitor of mixed function oxidases, on bile acid synthesis were compared in the rat bothin vitro andin vivo. In rat liver microsomes, ketoconazole was much more potent than metyrapone in inhibiting the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acids. The I₅₀ values were 0.42 μM and 0.91 mM for ketoconazole and metyrapone, respectively. Intraduodenal administration of ketoconazole caused a rapid, dose-dependent reduction of bile acid synthesis in eight-day bile diverted rats. A single dose of 50 mg/kg reduced bile acid synthesis to 5% of control value; the same dose of metyrapone caused a reduction to only 85%. Inhibition of bile acid synthesis by ketoconazole was followed by a marked overshoot. At 28 hr after injection of 50 mg/kg of the drug, formation of bile acids was stimulated maximally by 45% compared to control value and remained elevated for more than 20 hr thereafter. Synthesis of all primary bile acids was affected to the same extent. Cholesterol 7α-hydroxylase activity in livers of ketoconazole treated (30 mg/kg) rats with an intact enterohepatic circulation was increased by 70% at 16 hr after i.p. injection of the drug. During the very large decrease of biliary bile acid output with ketoconazole, bile flow rate was relatively increased, due to stimulation of the bile acid-independent fraction of bile flow. The latter effect can probably be explained as caused by biliary secretion of osmotically active metabolites of ketoconazole.     

Comparison of fatty acid α-oxidation by rat hepatocytes and by liver microsomes fortified with NADPH,Fe3+ and phosphate

Rat liver microsomes, when fortified with NADPH, Fe³⁺ and phosphate, can catalyze the oxidative decarboxylation (α-oxidation) of 3-methyl-substituted fatty acids (phytanic and 3-methylheptadecanoic acids) at rates that equal 60–70% of those observed in isolated hepatocytes (Huang, S., Van Veldhoven, P.P., Vanhoutte, F., Parmentier, G., Eyssen, H.J., and Mannaerts, G.P., 1992,Arch. Biochem. Biophys. 296, 214–223). In the present study we set out to identify and compare the products and possible intermediates of α-oxidation formed in rat hepatocytes and by rat liver microsomes. In the presence of NADPH, Fe³⁺ and phosphate, microsomes decarboxylated not only 3-methyl fatty acids but also 2-methyl fatty acids and even straight chain fatty acids. The decarboxylation products of 3-methylheptadecanoic and palmitic acids were purified by highperformance liquid chromatography and identified by gas chromatography/mass spectrometry as 2-methyl-hexadecanoic and pentadecanoic acids, respectively. Inclusion in the incubation mixtures of glutathione plus glutathione peroxidase inhibited decarboxylation by more than 90%, suggesting that a 2-hydroperoxy fatty acid is formed as a possible intermediate. However, we have not yet been able to unequivocally identify this intermediate. Instead, several possible rearrangement metabolites were identified. In isolated rat hepatocytes incubated with 3-methylheptadecanoic acid, the formation of the decarboxylation product, 2-methylhexadecanoic acid, was demonstrated, but no accumulation of putative intermediates or rearrangement products was observed. Our data do not allow us to draw conclusions on whether the reconstituted microsomal system is representative of the cellular α-oxidation system. However, the results we obtained with [3-³H]-labelled fatty acids indicate that during α-oxidation in intact cells the hydrogen at carbon-3, which carries the methyl branch, is not attacked.     

Distribution of bile acids in rats

Distribution and biliary and fecal excretion of bile acids were examined in Wistar strain male rats of about 300 g body weight. The pool size of the rats on ordinary diet was 40 mg/rat, biliary secretion was 14 mg/hr, and fecal excretion was 10 mg/day. Bile acids were mainly located in the small and large intestinal contents, 87% and 10%, respectively; but a portion was found in the intestinal wall and the liver. Rats fed 2% cholesterol-supplemented diet for a week showed similar values for pool size and biliary secretion with the rats on ordinary diet, but higher values for fecal excretion and distribution ratio in the large intestinal contents. Cholic acid was a major component in the bile, small intestinal wall, small intestinal content and liver, while the bile acid composition ratios were roughly similar to each other, although a relatively large amount of α-muricholic acid was found in the intentinal wall and liver. Both the wall and content compositions of the large intestine were similar to that of the feces, in which lithocholic, deoxycholic, α- and β-muricholic acids were the main components, although the ratios of α- and β-muricholic acids in the large intestinal wall were larger than those in the intestinal contents or feces. The high concentrations of these bile acids may indicate a difference of transport velocity across the cell membrane, but the mechanism is not known.     

Effect of cholestyramine on bile acid metabolism in conventional rats

Effects of cholestyramine on biliary secretion of cholesterol, phospholipids and bile acids and fecal excretion of sterols and bile acids were examined in Wistar male rats. Six rats were fed a basal diet, and the other six were fed a basal diet supplemented with 5% cholestyramine for eight days. Bile flow and biliary secretion of bile acids and phospholipids (per hour per rat) decreased with cholestyramine treatment, while biliary cholesterol secretion (per hour per rat) remained unchanged. In the biliary bile acid composition, a marked increase of chenodeoxycholic acid with a concomitant decrease of β-muricholic acid was observed in cholestyramine-treated rats. Fecal excretion of total sterols and bile acids increased about three-and four-fold, respectively, after cholestyramine treatment. The increase of fecal bile acids derived from cholic acid was more predominant than that derived from chenodeoxylcholic acid, resulting in an increase of the cholic acid group/chenodeoxycholic acid group ratio.     

Interaction of potentially toxic bile acids with human plasma proteins: Binding of lithocholic (3α-hydroxy-5β-cholan-24-oic) acid to lipoproteins and albumin

The binding of lithocholic acid to different plasma fractions was studied. When whole plasma was incubated for 8 hr, approximately 25% of the incubated [¹⁴C]lithocholic acid was bound to the lipoprotein and lipoprotein-free, albumin-rich fractions. An average of 87.6% of the bound-lithocholic acid was present in the lipoprotein-free, albumin-rich fraction, 7.2% in high density lipoproteins, 2.2% in low density lipoproteins, 1.0% in intermediate density lipoproteins and 2.0% in very low density lipoproteins. Expressed as binding per μg protein, considerably less [¹⁴C]lithocholic acid was bound to the lipoprotein-free, albumin-rich fraction, than to the lipoproteins. The binding of [¹⁴C]lithocholic acid after the incubation of the isolated plasma fractions was similar to that found after the incubation of whole plasma. The highest transfer of [¹⁴C]lithocholic acid occurred from the lipoprotein-free, albumin-rich fraction to the lipoprotein fractions. The studies indicate, that, although the largest amount of lithocholic acid is bound to the lipoprotein-free, albumin-rich fraction, per μg protein, the binding of lithocholic acid to lipoporteins is more pronounced and stable than that bound to the lipoprotein-free, albumin-rich fraction. Since lipoproteins, in contrast to albumin, are internalized by most tissues, they may be important carriers into cells of lithocholic acid and other potentially toxic or tumorigenic bile acids. Results of this study were presented at the Annual Meeting of the American Society for Clinical Investigation in Washington, D.C. in May 1988.     

Bile acids LVII. Analysis of bile acids by high pressure liquid chromatography and mass spectrometry

Several high pressure liquid chromatographic methods for the separation of conjugated and free bile acids are presented. A mixture of synthetic conjugated bile acids has been separated by reverse-phase systems consisting of either a Waters Associates’ “fatty-acid analysis” or a μBondapak/C₁₈ column eluted with a mixture of 2-propanol/potassium phosphate buffer (pH 2.5 or 7.0). The major conjugated bile acids present in the gallbladder bile of obese subjects have been analyzed each in less than 30 min and quantitated with a U.V. detector set at 193 nm. Some of the 5α- and 5β-isomers of conjugated bile salts could be resolved in straight-phase systems on Corasil II or μPorasil columns. Mass spectra of the conjugated bile acids obtained by electron impact were characteristic of the type of amino acid attached to the side chain, and the number of hydroxyl substituents on the nucleus. Most of the isomers could readily be differentiated by the relative intensities of the fragment ions.     

An improved procedure for bile acid extraction and purification and tissue distribution in the rat

Two bile acid extraction procedures were compared using endogenously radiolabeled tissues and feces. The method of Setchell et al. (J. Lipid Res. 24, 1085–1100, 1983) resulted in essential complete extraction, whereas that of Manes and Schneider (J. Lipid Res. 6, 376–377, 1971) gave recoveries between 56–82%. The time requirement for the method of Setchell et al. could be drastically reduced with no loss in extraction efficiency. Using extracts from endogenously labeled material, a purification procedure using C18 solid-phase extraction cartridges was developed that recovers >90% of bile acids. The distribution of bile acids within the intestinal tract and liver of the rat was determined.     

Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides and their chemical properties

A facile and efficient synthesis of the carboxyl-linked glucosides of bile acids is described. Direct esterification of unprotected bile acids with 2,3,4,6-tetra-O-benzyl-d-glucopyranose in pyridine in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent afforded a mixture of the α- and β-anomers (ca. 1∶3) of the 1-O-acyl-d-glucoside benzyl ethers of bile acids, which was separated effectively on a C₁₈ reversedphase chromatography column (isolated yields of α- and β-anomers are 4–9% and 12–19%, respectively). Subsequent hydrogenolysis of the α- and β-acyl glucoside benzyl ethers on a 10% Pd−C catalyst in acetic acid/methanol/EtOAc (1∶2∶2, by vol) at 35°C under atmospheric pressure gave the corresponding free esters in good yields (79–89%). Chemical specificities such as facile hydrolysis and transesterification of the acyl glucosides in various solvents were also discussed.     

Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats

The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca. 10¹⁰ counts per g wet feces) were examined in Wistar rats. After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7α-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. Tauro-β-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca. 30%) was transformed into hyodeoxycholic acid. Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively. Escherichia coli transformed neither bile acids nor sterols. These data suggest that clostridia play an imporant role in the formation of secondary bile acids and coprostanol in rats.     

Stearic acid unlike shorter-chain saturated fatty acids is poorly utilized for triacylglycerol synthesis and β-oxidation in cultured rat hepatocytes

Utilization of stearate as compared to various saturated fatty acids for cholesterol and lipid synthesis and β-oxidation was determined in primary culture of rat hepatocytes. At 0.5 mmol/L in the medium, stearate (18:0) adequately solubilized by albumin was less inhibitory to cholesterol synthesis from [2-¹⁴C] acetate than myristate (14:0) and palmitate (16:0) (68% vs. 91 and 88% inhibition, respectively). The rate of incorporation into cholesterol from [1-¹⁴C] stearate (3.0±0.6 nmol/mg protein/4 h) was 37-, 1.8-, and 7.8-fold of that from myristate, palmitate, and oleate, respectively. Conversely, the rate of [1-¹⁴C] stearate incorporation into total glycerolipids was 88–90% lower than that of labeled palmitate, myristate, and oleate. The rate of [1-¹⁴C] stearate incorporation into triacylglycerol (3.6±0.4 nmol/mg protein/4 h) was 6–8% of that from myristate, palmitate, oleate, and linoleate. The rate of stearate incorporation into phospholipids was the lowest among tested fatty acids, whereas the rate of mono- and diacylglycerol synthesis was the highest with stearate treatment. The rate of β-oxidation as measured by CO₂ and acid soluble metabolite production was also the lowest with [1-¹⁴C] stearate treatment at 22.7 nmol/mg protein/4 h, which was 35–40% of those from other [1-¹⁴C] labeled fatty acids. A greater proportion of stearate than other fatty acids taken up by the hepatocytes remained free and was not metabolized. Clearly, stearate as compared to shorter-chain saturated fatty acids was less efficiently oxidized and esterified to triacylglycerol in cultured rat hepatocytes.     

The behavior of rat bile phospholipids in the intestine and in incubation media containing pancreatic juice

Samples of radioactive bile were collected from rats after intravenous injection of potassium soaps ([9–10³H₂] or [1¹⁴C] oleate, [1¹⁴C] linoleate or [9–10³H₂] palmitate). These radioactive acids were chosen because it is well established that, in natural phosphatidyl cholines, palmitic acid is located chiefly at the 1 position and linoleic and oleic acids at the 2 position. After incubation of bile with pancreatic juice, the labeling of unchanged biliary phospholipids was higher when native bile was labeled with oleic acid than with palmitic or linoleic acids. These data suggest that monounsaturated molecular species of biliary phospholipids are more resistant than the diunsaturated ones to in vitro hydrolysis by phospholipase A₂. Ninety min after introduction of the radioactive bile into the upper part of the rat duodenum, high labeling of luminal phospholipids was observed regardless of the bile sample used, although labeling of free fatty acids was always low. The passage of intact biliary phospholipids through the intestinal epithelium is discussed.     

Effects of feeding chenodeoxycholic acid on metabolism of cholesterol and bile acids in germ-free rats

The aim of this investigation was to study the influence of chenodeoxycholic acid administration on cholesterol and bile acid synthesis in germ-free rats. Seven rats were fed a basal diet and 2 groups of 4 rats received the same diet supplemented with 0.4 and 1% chenodeoxycholic acid, respectively. After 6 weeks, feces were collected in one 3- and one 4-day pool for analysis of cholesterol and bile acids. When the sampling period was finished, the rats were killed and the liver microsomal fractions isolated. The activities of HMG CoA reductase and cholesterol 7α-hydroxylase were determined, the 7α-hydroxylase by a mass fragmentographic method. The 2 dominating bile acids in the untreated rats were cholic acid and β-muricholic acid. During treatment with chenodeoxycholic acid, 60–70% of this bile acid was converted into α- and β-muricholic acid, indicating a high activity of the 6β-hydroxylase. The excretion of cholic acid was almost completely inhibited and the 7α-hydroxylase activity was decreased ca 75% in the rats fed 1% chenodeoxycholic acid. The activity of the hepatic HMG CoA reductase was unchanged. The fecal excretion of cholesterol increased 2–3 times. An accumulation of cholesterol was seen in the rats treated with 1% chenodeoxycholic acid, which was probably a result of the decreased catabolism of cholesterol to bile acids.     

Stimulation of fatty acid synthesis by 4β-phorbol-12-myristate-13-acetate in isolated rat hepatocytes

The tumor-promoting agent 4β-phorbol-12-myristate-13-acetate (TPA) is shown to be a potent stimulator of fatty acid synthesis in isolated rat hepatocytes. The maximal effect of TPA is seen at 10⁻⁶ M, and the concentration for half-maximal effect is ca. 10⁻⁸ M. Stimulation of fatty acid synthesis by TPA is shown not to require the presence of extracellular Ca⁺⁺. TPA produces a significant increase in lactate and pyruvate accumulation. The possible involvement of protein kinase C in short-term regulation of fatty acid synthesis in the liver is discussed.