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.

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.

Short-term metabolism of cholesteryl ester from low-density lipoprotein in primary monolayers of bovine adrenal cortical cells.

The uptake and metabolism of [14C]cholesteryl ester in bovine LDL to cortisol and to cholesteryl ester was studied in monolayer cultures of bovine adrenal cortical cells over short time periods of up to 8 h. The experiments were designed to determine the intracellular pathway followed by the cholesterol derived from the LDL cholesteryl ester and how this is modified in the short term by the tropic hormone ACTH. The cells were cultured in the presence of mevinolin to remove the contribution of endogenous synthesis of cholesterol for supply of substrate for steroidogenesis. The specific activity of the cortisol secreted by the cells was measured under a variety of conditions. Control incubations showed a relatively steady specific activity in the cortisol secreted over an 8 h period. In the presence of ACTH the specific activity of the cortisol was significantly reduced for the first 2 h of the experiment. This is consistent with dilution of the [14C]cholesterol from the LDL with non-radioactive free cholesterol released from the intracellular stores of cholesteryl ester in the presence of ACTH. The inhibitor of acyl-CoA:cholesterol acyltransferase, Sandoz compound 58-035, increased the specific activity of the secreted cortisol in the absence of ACTH, indicating that much of the incoming cholesterol would normally be esterified but was here diverted to steroidogenesis. In the presence of ACTH this increase was observed only during the first 2 h of the experiment, after which inhibition of acyl-CoA:cholesterol acyltransferase had no effect on the specific activity of the cortisol. The adrenal cells were further fractionated into mitochondrial, lysosomal and microsomal plus cytosol fractions and the appearance of free and esterified cholesterol from the labelled LDL measured in these fractions over a period of up to 8 h. ACTH stimulated the uptake of LDL-cholesteryl ester into the cells and tended to increase the relative amounts of free cholesterol in the cells, consistent with its role in promoting supply of cholesterol for steroidogenesis. These experiments allow the roles of endogenous cholesteryl ester and lipoprotein-derived cholesteryl ester in the bovine adrenal cortical cells to be observed over a short time scale. They show that the cells make a substantial change in the internal flux of cholesterol in a short time after stimulation with ACTH and in these cultures the full expression of the presence of ACTH takes up to 2 h.

The fluidity of the nuclear envelope lipid does not affect the rate of nucleocytoplasmic RNA transport in mammalian liver.

The effects of in vitro and in vivo modifications of nuclear envelope lipid on DNa leakage and on ATP-stimulated RNA release from isolated rat liver nuclei were investigated. The modifications included corn-oil feeding of the animals to alter the fatty acid composition of the lipids, phospholipase treatment of the isolated nuclei, and extraction of the total lipid with Triton X-100. Significant changes in lipid composition and approximate order parameter values of the spin-label 5-doxylstearate resulted, but there was no significant effect on RNA transport rate. It was concluded that the nuclear envelope lipid does not play any important part in nucleocytoplasmic RNA transport in mammalian liver.

Effect of colchicine on mammalian liver nuclear envelope and on nucleo-cytoplasmic RNA transport.

The binding of colchicine to nuclear envelopes was studied in order to elucidate the mechanism whereby this compound inhibits nucleocytoplasmic RNA transport. The results suggest that a single class of colchicine-binding site (dissociation constant=approx. 0.7 mM, concentration=approx. 330 nmol colchicine/mg protein) is localised in the nuclear periphery (pore-lamina) and that binding to these sites effects a constriction of the pore-complexes with concomitant inhibition of RNA egress and disordering of the nuclear membrane phospholipid bilayers.

Effects of cholesterol on the properties of the membranes of isolated sheep liver nuclei and nuclear envelopes.

The exchangeability of cholesterol between sheep liver nuclear membranes and liposomes, and the effect of cholesterol on the fluidity of the membrane lipid were studied. In intact nuclei, the cholesterol/phospholipid ratio increased from 0.102 to 0.145 mol/mol on incubation with cholesterol-rich liposomes, with a time for half-maximal uptake of 4.2 h. In isolated envelopes under the same conditions, the ratio increased from 0.110 to 0.266 mol/mol with a time for half-maximal uptake of about 1.9 h. Moreover, the approximate order parameter of the spin label 5-(N-oxyl-4',4'-dimethyloxazolidino)-stearic acid was 0.677 in intact nuclei and 0.723 in isolated envelopes prior to exchange; after exchange, these values increased to 0.717 and 0.756, respectively. These differences between the preparations could not be attributed to differences in the capacity for cholesterol uptake between the two nuclear membranes, or to a slow rate of exchange between them; the presence of an intact nuclear matrix appeared both to disorder the lipid partially and to inhibit cholesterol uptake. The differences indicate that conclusions based on physical studies of the membrane lipid in isolated envelopes are not necessarily applicable to the intact nucleus.

Interactions of the cholesterol side-chain with egg lecithin. A spin label study.

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogues with side-chains shorter than cholesterol by more than three carbon cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.

Evaluation of cultured hamster hepatocytes as an experimental model for the study of very low density lipoprotein secretion.

The secretion of triacylglycerol, cholesterol and cholesteryl ester in very low density lipoprotein (VLDL) by cultured hamster hepatocytes was studied, and the results compared with those obtained previously using cultured rat hepatocytes and the human hepatoma cell line HepG2. The hamster cells secreted apolipoprotein B and VLDL triacylglycerol, cholesterol and cholesteryl ester linearly during 24 h in culture, and this time period was used in all experiments. Addition of oleate (1 mM) to the culture medium resulted in increased secretion of triacylglycerol, but cholesterol ester output were unchanged. Triacylglycerol secretion was also increased in the presence of lipogenic substrates (10 mM lactate + 1 mM pyruvate) plus dexamethasone (1 microM), but not with either of these agents alone. Inhibition of cholesterol synthesis in the hamster cells by incubation with mevinolin (2 micrograms/ml) did not change VLDL lipid secretion, but stimulation using mevalonate lactone resulted in decreased triacylglycerol output. Manipulation of the rate of cholesterol esterification in the hepatocytes by inhibiting or stimulating the activity of acyl coenzyme A cholesterol:acyl transferase using the inhibitor Dup128 (25 microM) and 25-hydroxycholesterol (50 microM), respectively, had no effect on the secretion of VLDL lipid. In the presence of 1 mM oleate plus 25-hydroxycholesterol, however, a rise in the output of triacylglycerol and cholesteryl ester was observed. Hepatocytes prepared from hamsters fed 2% cholestyramine secreted significantly less triacylglycerol than those from animals given the control diet, but cholesterol and cholesteryl ester output were unchanged, despite a decrease of about 40% in the total cholesterol content of the cells. These results show that the secretion of lipid in VLDL in hamster hepatocytes differs from that in rat and human liver in its response to dietary cholestyramine, and from rat hepatocytes and HepG2 cells in its response to changes in the rate of lipogenesis and cholesterol synthesis and esterification. Overall, hamster hepatocytes appear to be less susceptible to modification the rate of hepatic VLDL secretion, and should provide a useful additional tool for the investigation of this process.

Effect of membrane environment on inhibition of acyl-CoA:cholesterol acyltransferase by a range of synthetic inhibitors.

The effect of the membrane environment of acyl-CoA:cholesterol acyl transferase (ACAT), an important intracellular enzyme of cholesterol metabolism, on the properties of a range of inhibitors of varying potencies was studied. ACAT activity from rat liver was solubilised with 3% deoxycholate (97% solubilised activity). After dilution into cholesterol/phosphatidylcholine liposomes (molar ratio 0.35), the assay of this reconstituted system showed linearity with protein and time. Saturation with oleoyl-CoA was achieved at 10 microM. Comparison of the potency of the ACAT inhibitors in the reconstituted assay and in a microsomal assay revealed a relationship between the lipid content of the assay and the inhibitory activity for potent inhibitors of ACAT (CI976, CL277,082, YMI7E and DuP128). This relationship was unrelated to lipophilicity of the drugs. Octimibate, lovastatin and progesterone, none of which is a potent ACAT inhibitor but which have all been described as ACAT inhibitors in the literature, all had low potencies in both assay systems. These results suggest that the lipid concentration must be taken into account when comparing potencies of ACAT inhibitors. The present data also indicate that some compounds which inhibit cholesterol esterification may do so by an indirect mechanism.

Effects of cholestyramine on lipoprotein levels and metabolism in Syrian hamsters.

Oral administration of cholestyramine to adult male hamsters not only induced a marked decrease in plasma concentrations of cholesterol and LDL but had a similar lowering effect on plasma triacyglycerol and VLDL concentrations. The hypotriglyceridaemic effects of resin administration were not due to an increase in lipoprotein lipase, as post-heparin plasma lipoprotein lipase activities were unchanged, but rather to a 35% decrease in VLDL synthesis. Measurement of the disappearance rate of apolipoprotein B from VLDL after i.v. injection of 125I-labelled hamster or human VLDL into control and cholestyramine-fed recipient animals showed a 2-times lower T1/2 in the drug-treated animals. The fraction of VLDL apolipoprotein B, recovered at any time after injection in the LDL, was equal or higher in cholestyramine-fed animals as compared to controls. These data indicate that the lowering in plasma LDL by cholestyramine in male hamsters is due not only to LDL receptor up-regulation but also to a lower rate of VLDL synthesis. No indications were found for a decreased efficiency of VLDL to LDL conversion in cholestyramine-fed animals.

In vitro regulation of bovine adrenal cortical acyl-CoA: cholesterol acyltransferase and comparison with the rat liver enzyme.

Acyl-CoA:cholesterol acyltransferase activity in bovine adrenal cortical microsomes was increased by preincubation of microsomes in vitro in the presence of MgCl2. The acyltransferase activity in the microsomes could be inhibited by further incubation in the presence of ATP/MgCl2. These effects appear to complement the known ATP-dependent activation of adrenal cytosolic cholesterol ester hydrolase, which is consistent with the role of the hydrolase in supplying cholesterol for steroidogenesis. These effects are, however, opposite to those recently demonstrated for the rat liver and intestine. Acyl-CoA:cholesterol acyltransferase activity in rat liver can be increased by the addition of cholesterol, as substrate, or by 25-hydroxycholesterol. Such activation was not observed in adrenal microsomal preparations, further suggesting that the mechanisms of regulation of cholesterol esterification differs between these tissues.

The influence of the side chain on sterol side-chain cleavage in rat adrenal glands.

The cholesterol side-chain cleavage enzyme system of rat adrenal cortex, the enzyme catalyzing a rate-limiting step of adrenal steroidogenesis, was shown to metabolize a series of cholesterol analogues to pregnenolone. In the presence of Ca2+, rat adrenocortical mitochondria converted the analogue with two less methylene groups (C25) than cholesterol into pregnenolone at a faster rate than cholesterol. The analogues with one or three less methylene groups (C26 or C24) were metabolized at a similar rate to cholesterol. Lengthening the non-polar side chain produced analogues that did not appear to be metabolized. Studies of the metabolism of these analogues in isolated rat adrenocortical carcinoma cells showed that the C24 and C25 analogues were converted into pregnenolone much more efficiently than was cholesterol or the C26 sterol. The experimental findings are explained in terms of the differing ability of each exogenously added sterol to gain access to the active site of the sterol side-chain cleavage enzyme by passage through the membranes of the adrenal cell.

Optimum interaction of sterol side chains with phosphatidylcholine.

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.

Metabolism of cholesteryl ester in monolayers of bovine adrenal cortical cells. Effect of an inhibitor of acyl-CoA: cholesterol acyltransferase.

The effect of Sandoz compound 58-035 on cholesterol metabolism in monolayers of bovine adrenal cortical cells was studied. 58-035 did not inhibit cholesterol ester hydrolase, cholesterol side-chain cleavage, cholesterol synthesis from acetate, or cortisol synthesis in cells stimulated with ACTH or in unstimulated cells. It was, however, an effective inhibitor of formation of cholesteryl ester. The rate of formation of cholesteryl ester in the cells was increased by additional cholesterol derived from mevalonic acid or from the hydrolysis of intracellular lipid droplets. 58-035 caused an increase in the secretion of cortisol from cells maintained on a limited supply of cholesterol from bovine lipoproteins added to the medium when the cells were not stimulated with ACTH. This effect was not observed in stimulated cells. The results suggest that the bovine adrenal cortical cell can direct the flux of exogenous cholesterol very precisely according to its metabolic state.

Effects of in vitro incorporation of cholesterol and cholesterol analogues into rat platelets.

Cholesterol and analogues of cholesterol bearing shorter side chains were incorporated into rat platelet membranes by incubation with sterol-rich liposomes in vitro. Cholesterol-enriched platelets showed increased aggregability to collagen compared with controls. Platelets containing the cholesterol analogues pregn-5-en-3 beta-ol and chol-5-en-3 beta-ol were even more sensitive to aggregation and could aggregate spontaneously on stirring. The size of the platelets containing pregn-5-en-3 beta-ol was markedly reduced when compared with controls in the scanning electron microscope. The results suggest that the sterol content and structure of the platelet membrane can have a critical role in maintaining the normal function of the cell.

Properties of a solubilised and reconstituted preparation of acyl-CoA:cholesterol acyltransferase from rat liver.

Rat liver acyl-CoA:cholesterol acyltransferase activity was released from the microsomal fraction by treatment with Triton X-100. After fractionation with polyethylene glycol 6000, the solubilised preparation was reconstituted in liposomes of different lipid composition by an octyl glucoside dilution method. The activity of the reconstituted system was dependent on the amount of cholesterol used in the liposomes and could also be stimulated by transfer of cholesterol into the reconstituted system from other membranes. The results are consistent with the hypothesis that substrate supply and the fluidity of the membrane contribute in the regulation of the rate of cholesterol ester formation.

The effects of feeding a saturated fat-rich diet on enzymes of cholesterol metabolism in the liver, intestine and aorta of the hamster.

The effects of two dietary treatments on parameters of cholesterol metabolism were studied. Hamsters were maintained on diets containing 2% (w/w) cholesterol or 20% (w/w) hydrogenated coconut oil for 4 weeks. Both diets induced a hypercholesterolaemia. The effects of the two treatments on hepatic and intestinal acyl-CoA:cholesterol acyltransferase activity and 3-hydroxy-3-methylglutaryl-CoA reductase activity were measured. As expected, cholesterol feeding stimulated cholesterol esterification and inhibited cholesterol synthesis. Saturated fat-feeding had no effect on cholesterol synthesis but markedly inhibited cholesterol esterification in both liver and intestine. The diet-induced hypercholesterolaemia was strongly correlated with an increase in acyl-CoA:cholesterol acyltransferase activity in the activity. In contrast, the hypercholesterolaemia induced by feeding either of the two diets tended to increase aortic uptake of cholesterol and hence acyl-CoA:cholesterol acyltransferase activity. We suggest that the changes in cholesterol esterification correlate well with the expected flux of cholesterol into each tissue.

Regulation of cholesteryl ester metabolism in the hamster liver.

Neutral cholesteryl ester hydrolase activity has been described in the cytosolic and microsomal fraction of rat liver, but the relationship of these activities to other parameters of hepatic cholesterol metabolism is not known. We have studied this in the hamster by manipulating the flux of cholesterol across the liver by dietary modifications. A bile acid sequestrant was used to stimulate LDL receptor activity and hence flux of cholesterol into the liver. A cholesterol-rich diet caused a hypercholesterolaemia and substantial uptake of cholesterol and deposition in the liver. Hypercholesterolaemia was also induced by a saturated fat-rich diet, but in contrast this reduced the flux of cholesterol into the liver. Animals were fed these diets for 1 week and then the livers removed and enzyme activities determined. These were 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol 7 alpha-hydroxylase, acyl-CoA: cholesterol acyltransferase, microsomal cholesteryl ester hydrolase and cytosolic cholesteryl ester hydrolase. Feeding the bile acid sequestrant increased the hydrolysis of cholesteryl ester in the liver over its synthesis. In contrast, both fat feeding and cholesterol feeding caused a reduction in the relative rate of hydrolysis of cholesteryl ester compared with synthesis. This was particularly marked with the cholesterol-rich diet. These results show that the hydrolysis of cholesteryl ester in hamster liver responds to dietary manipulation in a way that reflects the needs of the cell for cholesterol or the presence of an excess. It is suggested that a metabolically significant cholesteryl ester cycle may operate in the liver to a greater extent that had previously been thought.

The effect of chylomicron remnants on bile acid synthesis in cultured rat hepatocytes.

The effect of chylomicron remnants on bile acid synthesis in isolated rat hepatocytes in monolayer cultures was investigated. Production of bile acids by the cells in the presence of chylomicron remnants at a cholesterol concentration of 7.8-9 nmol/ml was increased by approx. 75% after 17 h and 25% after 24 h incubation. Similar concentrations of cholesterol added to the cells in the form of chylomicrons had no significant effect on bile acid synthesis. These results suggest that cholesterol taken up in chylomicron remnants may be an important source of substrate for bile acid synthesis.

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.