Kathleen M. Botham

Characterisation of rat hepatocyte monolayers for investigation of the metabolism of bile salts

Rat hepatocyte monolayers were maintained for periods up to 24 h during which time their viability was greater than 85%. Using specific radioimmunoassays, the hepatocyte monolayers were shown to synthesise conjugated cholic, chenodeoxycholic and beta-muricholic acids. Feeding the bile salt sequestrant, cholestyramine, to donor animals increased synthesis of the major bile salt conjugates by the cells. Incubation of hepatocyte monolayers with bovine serum albumin decreased total synthesis of the three bile acids measured, but increased the amount of conjugated chenodeoxycholic acid detected. In order to test whether the effect of bovine serum albumin on bile salt synthesis was due to binding of bile salts, hepatocyte monolayers were incubated with antiserum to conjugated chenodeoxycholic acid. This treatment increased conjugated chenodeoxycholic acid production but had no effect on the other bile salt conjugates. It is concluded that the increase in conjugated chenodeoxycholic acid synthesis seen with bovine serum albumin and antiserum to conjugated chenodeoxycholic acid is caused by binding of the bile salt in the medium.

The role of acyl-CoA:cholesterol acyltransferase in the metabolism of free cholesterol to cholesteryl esters or bile acids in primary cultures of rat hepatocytes

Sandoz compound 58-035 has been shown to inhibit acyl-CoA: cholesterol acyltransferase activity in a variety of cell types. We have shown that it does not inhibit rat liver microsomal cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of bile-acid synthesis, but it does inhibit acyl-CoA: cholesterol acyltransferase in both the microsomal fraction and in rat hepatocyte monolayers. To test the role of acyl-CoA: cholesterol acyltransferase in these cells, monolayers were incubated over 5 h in the presence and absence of 58-035 and in the presence of increasing amounts of mevalonic acid to provide a source of cholesterol. The addition of mevalonic acid increased the secretion of bile acids by the cells, and this was further increased by the addition of 58-035. The secretion of cholesteryl esters was conversely inhibited by the addition of 58-035. The results help define the role of acyl-CoA: cholesterol acyltransferase in determining the fate of intracellular cholesterol.

The effect of a rat plasma high-density lipoprotein subfraction on the synthesis of bile salts by rat hepatocyte monolayers

The effect of a rat high‐density lipoprotein subfraction (HDL2) on the synthesis of bile salts by rat hepatocyte monolayers prepared from rats fed a diet containing cholestyramine, was investigated. The synthesis of bile salts as measured by radioimmunoassay of conjugated cholic, chenodeoxycholic and β‐muricholic acids was significantly increased when hepatocytes were incubated with a physiological concentration (500 μg HDL, protein·ml−1) of HDL2.

The effect of dietary fat on bile salt synthesis in rat liver

The effect of dietary fat on conjugated cholic, chenodeoxycholic and tauro-beta-muricholic acid synthesis was studied using hepatocytes isolated from rats given a low-fat diet, or a low-fat diet mixed with 10% olive oil or 10% corn oil. The rats were totally biliary drained for 48 h prior to preparation of the cells in order to raise bile salt synthesis to a level which was measurable by radioimmunoassay. Synthesis of both conjugated cholic and chenodeoxycholic acid was raised in hepatocytes from rats given a fat supplement (either corn oil or olive oil) in the diet as compared to that in cells from low-fat-fed animals. Tauro-beta-muricholic acid synthesis, however, was unaffected by corn oil feeding. Production of conjugated cholic acid was increased to a greater extent when rats were given olive oil as opposed to corn oil, but these differences were not statistically significant. The conjugated cholic, chenodeoxycholic, and tauro-beta-muricholic acid and cholesterol content of bile collected at 2-h intervals during the biliary drainage of the same groups of rats was also determined. The pool size of both conjugated cholic and chenodeoxycholic acid in the enterohepatic circulation was found to be significantly decreased in rats given olive oil as compared to those given corn oil or the low-fat diet only. The pool size of tauro-beta-muricholic acid was also decreased in the olive oil-fed rats compared to the other two groups, but this difference was not statistically significant. After the pool had been drained out, animals which had received fat in the diet secreted more conjugated cholic and chenodeoxycholic acid into the bile than rats which had received the low-fat diet only. This effect was more marked when the fat given was olive oil rather than corn oil. Secretion of tauro-beta-muricholic acid into bile at this stage of biliary drainage was not changed by dietary fat supplements. Biliary cholesterol excretion was also increased in rats on diets containing 10% fat, with olive oil again having a greater effect than corn oil. The results show that supplementing the diet with fat leads to increased synthesis of conjugated cholic and chenodeoxycholic acids and biliary cholesterol secretion in the rat. The relatively more saturated fat, olive oil (85% oleate), gave a consistently larger increase than the more unsaturated, corn oil (50% linoleate), but the type of fat appeared less important than the presence of fat in the diet.

The effect of portal blood bile salt concentrations on bile salt synthesis in rat liver: Studies with isolated hepatocytes

Abstract 1. Conjugated bile salt synthesis was studied in hepatocytes isolated from rats kept on a strict 24 h illumination cycle consisting of 12 h light and 12 h darkness. In confirmation of the studies of others using different techniques it has been shown that conjugated bile salt synthesis exhibits a marked diurnal rhythm, being 2-fold higher in the dark than in the light phase. 2. Feeding a bile salt sequestering agent (cholestyramine) to rats for several days before killing resulted in an elevation of the rate of conjugated bile salt synthesis in isolated hepatocytes while the diurnal rhythm in bile salt synthesis was retained. 3. The highest rates of conjugated bile salt synthesis in isolated hepatocytes were observed in cells isolated from tissue from animals fed a diet containing cholestyramine and killed in the middle of the dark phase of the light-dark cycle. 4. Portal blood concentrations of conjugated cholic and chenodeoxycholic acids were measured at the end of the dark phase of the 24 h cycle when they are known to be at the peak of the diurnal variation. Addition of cholestyramine to the diet lowered the portal blood concentration of both bile salts to a significant extent at this time. 5. Hepatocytes from animals previously fed on a diet containing cholestyramine and killed in the middle of the dark phase were used to investigate the possible control of bile salt synthesis in isolated hepatocytes in vitro. The addition of taurocholate or taurochenodeoxycholate to isolated hepatocytes in concentrations comparable to the concentrations found to prevail in the portal blood of rats failed to influence the rate of conjugated bile acid synthesis in isolated hepatocytes in vitro. 6. [G- 3 H]Glycocholic acid and sodium tauro[ carbonyl - 14 C]cholate were not metabolised to a significant extent during 4 h of incubation with hepatocytes. [G 3 H]Glycochenodeoxy cholic acid, however, was rapidly metabolised by hepatocytes, presumably through 6β hydroxylation to form conjugated muricholic acids. 7. These in vitro studies suggest that concentrations of conjugated cholic acid and conjugated chenodeoxycholic acid equivalent to those found in the portal blood of normal rats are unlikely to inhibit bile salt synthesis by a direct feedback mechanism.

A radioimmunoassay for tauro‐β‐muricholic acid suitable for use with isolated rat liver cells

A new radioimmunoassay which can be used to measure the amounts of tauro‐β‐muricholic acid produced by isolated rat hepatocytes in vitro is described. Cross reactivities of other bile acids known to be present in rat liver with the antiserum used in the assay were not sufficient to interfere with the measurement of tauro‐β‐muricholic acid. Exogenous taurochenodeoxycholic acid, was metabolised by isolated rat hepatocytes concurrently with the appearance of tauro‐β‐muricholic acid in the cell.

Portal blood concentraiions of conjugated cholic and chenodeoxycholic acids relationship to bile salt synthesis in liver cells

1. Rats were maintained in a strictly controlled environment of 12 h illumination and 12 h darkness. At regular intervals during the light/dark cycle the portal blood conjugated cholic acid and conjugated chenodeoxycholic acid concentrations were measured. The bile salt concentrations exhibited similar diurnal rhythms, the highest concentrations occurring in the middle of the dark phase. 2. The concentrations of conjugated cholic and chenodeoxycholic acids in the portal blood of rats fed a diet containing the bile salt sequestrant, cholestyramine, were significantly lower than those found in rats given a control diet. 3. During total biliary drainage the portal blood concentrations of conjugated cholic and chenodeoxycholic acids fell to a minimum 6--8 h after the start of the experiment, whereas bile salt synthesis in hepatocytes isolated from the rats was not increased until the least 13 h after the commencement of total biliary drainage. 4. These results suggest that the concentrations of bile salts in the portal blood do not affect directly the diurnal fluctuation in rates of bile salt synthesis, as the response of synthesis to a change in portal blood bile salt concentrations is too slow. 5. When the rats were given a small supplement of cholesterol in the diet to suppress hepatic cholesterologenesis prior to being subjected to total biliary drainage, the changes in the portal blood concentrations of conjugated cholic and chenodeoxycholic acids and the synthesis of the two bile salts by isolated hepatocytes were similar to those found in rats given the control diet. 6. The rise in bile salt production seen during biliary drainage may not be dependent exclusively on a preceding increase in cholesterol synthesis.

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10-100 microM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 microM. In contrast, concentrations of dibutyryl cyclic AMP between 200 microM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 microM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 microM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent than either treatment alone. It is concluded that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.

Cholesterol ester turnover in isolated liver cells: effects of cholesterol feeding

Isolated hepatocytes from rats that had been kept in a steady state of [3H]cholesterol were incubated in a salt medium with or without serum. The cells released esterified cholesterol into the incubation medium as lipoproteins. This secretion, 18.1 +/- 0.5 nmol/h per g of cells, was increased when the cells were incubated in a medium containing serum (46.3 +/- 4.9 nmol/h per g of cells). This secretion was strikingly enhanced by cholesterol feeding (1% in the diet, 30 days) to 323-620 nmol/h per g of cells, and inhibited by cycloheximide, colchicine or EDTA. After removal of EDTA and addition of calcium, the cholesterol ester secretion was restored. Free cholesterol of previously labelled high-density lipoproteins (HDL) was exchanged (t1/2 = 30 min) with that of liver cells and esterified. The esterification rate (25.8 +/- 2.5 nmol/h per g of cells) was increased by cholesterol feeding (1% in the diet, 8 days) to 63.2 +/- 2.8 nmol/h per g of cells. No cholesteryl ester hydrolysis was detected with the isolated liver cells. Consequently, it is suggested that the turnover of hepatic cholesteryl ester was caused mainly by secretion in lipoproteins.

The effect of dibutyryl cyclic AMP on the excretion of taurocholic acid from isolated rat liver cells

Dibutyryl cyclic AMP (50-1000 microM) was found to increase the initial rate of efflux of taurocholic acid from isolated rat hepatocytes. Efflux of the bile acid was inhibited by sodium, and in the absence of sodium dibutyryl cyclic AMP failed to stimulate the rate. Increasing the concentration of calcium from 0 to 1.2 mM had no effect on the initial rate of taurocholic acid efflux from the cells, but the absence of calcium markedly reduced the effect of dibutyryl cyclic AMP. The results suggest that changes in the fluxes of sodium and calcium are involved in the effect of the cyclic nucleotide on taurocholic acid efflux from the cells.