RESUMEN

The stability of tauro-23-[75Se]selena-25-homocholic acid (SeHCAT) towards deconjugation by the enzyme cholylglycine hydrolase was compared with that of taurocholate: whereas taurocholate underwent 58% deconjugation within 2 hr, SeHCAT suffered only 8% deconjugation plus 5% conversion to an unknown product within 24 hr. Incubation of SeHCAT under anaerobic conditions for 48 hr at 37 degrees C with human fecal organisms resulted in considerable deconjugation, 7 alpha-dehydroxylation, and dehydrogenation. Twenty-four hours after the simultaneous administration of SeHCAT and tauro-[24-14C]cholate to a rabbit the recovery of 75Se in bile was 90% of that of 14C. Forty-eight hours following administration of SeHCAT to a second rabbit residual bile radioactivity revealed 80% deconjugation and dehydroxylation and 60% reconjugation with glycine. Although SeHCAT is more resistant than taurocholate towards modification by fecal bacterial enzymes, within the rabbit it follows the principal metabolic pathways of the natural bile acids.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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 beta-muricholic acids was significantly increased when hepatocytes were incubated with a physiological concentration (500 micrograms HDL2 protein X ml-1) of HDL2.

Asunto(s)

RESUMEN

Cytochrome P-450scc as isolated is a cholesterol-depleted low-spin haemoprotein; addition of cholesterol results in formation of a high-spin complex. Cytochrome P-450scc--cholesterol is a one-electron acceptor on titration with NADPH. Cytochrome P-450scc--cholesterol can be anaerobically reduced to the ferrous state which, on oxygenation, forms an oxygenated cytochrome P-450scc--cholesterol complex. This oxygenated complex in the absence of adrenodoxin autoxidises to ferric cytochrome P-450scc--cholesterol without oxidation of cholesterol. The decay of the oxygenated complex is first-order, k = 9.3 X 10(-3) S-1 at 4 degrees C. The rate of autoxidation is influenced by pH, ionic strength and the chemical nature of bound sterol. The activation energy of autoxidation is 75 kJ mol-1. Addition of equimolar amounts of adrenodoxin to cytochrome P-450scc--cholesterol followed by stoichiometric reduction under anaerobic conditions and subsequent oxygenation, allows single catalytic turnover cycles of cytochrome P-450scc to be observed. This has led to detection of intermediates in the conversion of cholesterol to pregnenolone and a precursor/product sequence of cholesterol----22-hydroxycholesterol----20,22-dihydroxy-cholesterol ----pregnenolone has been established. Addition of oxidised adrenodoxin to oxygenated cytochrome P-450scc--cholesterol results in formation of 22-hydroxycholesterol.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The effect of increased intracellular adenosine 3' ,5' -monophosphate (cAMP) concentrations on bile acid synthesis in isolated rat hepatocytes was investigated. When the cells were incubated in the presence of glucagon (0.2 microM) and theophylline (1 mM) the observed rise in the level of cAMP was accompanied by an increase in bile acid production. Hepatocyte cAMP concentrations after 1 h of incubation showed a highly significant positive linear correlation with the amounts of bile acid synthesised by the cells during this time. These results suggest that bile acid production is related to the concentration of cAMP in isolated hepatocytes and provide evidence for a role for the cyclic nucleotide in the regulation of bile acid synthesis.

Asunto(s)

RESUMEN

The possibility that a lysine-rich protein was involved in the acute stimulation of steroidogenesis by ACTH was investigated using [3H] labelled lysine and isolated adrenal cells. The results demonstrated that cycloheximide inhibited steroidogenesis in a dose-dependent, rapid fashion and inhibited the incorporation of radioactive lysine into protein. However cells incubated in a lysine-free medium showed the same response to ACTH as cells incubated in a lysine-containing medium. It was also demonstrated that ACTH had no effect on the incorporation of tritiated lysine into the protein or small peptide fractions. These observations suggest that a rapidly synthesised, lysine-rich protein is not involved in the acute response to ACTH.

Asunto(s)

RESUMEN

The lipid droplet fractions from rat adrenal and bovine adrenocortical tissue were isolated by density ultracentrifugation. The droplet fractions were delipidated and the protein components investigated by SDS-polyacrylamide slab gel electrophoresis. The adrenal lipid droplets from both species displayed a qualitatively similar protein profile, and both contained a major apolipoprotein subunit of Mr 40 000. Incubation of intact, non-delipidated lipid droplets with [gamma-32P]ATP in vitro resulted in the phosphorylation of the Mr 40 000 apolipoprotein subunit in the case of rat lipid droplets, but not in the case of bovine lipid droplets. However, following delipidation of the droplets with diethyl ether/ethanol, the Mr 40 000 apolipoprotein subunit was phosphorylated in both cases upon incubation of the delipidated protein fractions with [gamma-32P]ATP in vitro. Labelling with [gamma-32P]ATP and [3H]diisopropyl phosphorofluoridate indicated that the cholesterol ester hydrolase enzyme protein was not a major constituent of the adrenal lipid droplet protein fraction.

Asunto(s)

RESUMEN

The effect of dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) on the synthesis of conjugated cholic, chenodeoxycholic and beta-muricholic acids has been investigated. Hepatocytes were incubated with 1 mM Bt2cAMP for 3 h at 37 degrees C. In cells from rats with a basal rate of bile salt synthesis (soft-diet-fed rats) production of conjugated cholic acid was increased about two fold, synthesis of conjugated chenodeoxycholic acid was increased 10-20-fold but formation of its metabolite, conjugated beta-muricholic acid, was decreased by 30-50% in the presence of the cyclic nucleotide. The sum of the amounts of the three bile salts produced (total bile salt synthesis) was increased 30-50% by Bt2cAMP. When hepatocytes were prepared from rats in which bile salt synthesis had been stimulated by feeding the bile salt sequestrant, cholestyramine, Bt2cAMP had no effect on conjugated cholic acid synthesis, increased conjugated chenodeoxycholic acid production 3-5-fold and decreased conjugated beta-muricholic acid synthesis by about 50%. Total bile salt synthesis was unchanged. The ratio of the amount of conjugated cholic acid to conjugated chenodeoxycholic acid + conjugated beta-muricholic acid produced, an indication of the activity of 7 alpha-hydroxycholest-4-en-3-one 12 alpha-hydroxylase, was raised by Bt2cAMP in hepatocytes from soft-diet-fed but not in those from cholestyramine-fed rats. The effects of the cyclic nucleotide on the synthesis of the three bile salts in hepatocytes from soft-diet-fed rats were found to be saturable at a concentration of about 2 mM. Responses were half-maximal at concentrations of Bt2cAMP varying between 0.5 and 1.5 mM. These results suggest that in hepatocytes from rats with a basal rate of bile salt synthesis Bt2cAMP has effects at three different stages in the pathway, at the level of cholesterol 7 alpha-hydroxylase, 7 alpha-hydroxycholest-4-en-3-one 12 alpha-hydroxylase and chenodeoxycholine acid 6 beta-hydroxylase. In cells from rats in which bile salt synthesis has been stimulated only the effect at the chenodeoxycholic acid 6 beta-hydroxylase level is apparent. Bt2cGMP and Bt2cCMP had no effect on the synthesis of any of the bile salts measure, showing that the effects are specific for Bt2cAMP. The ratio of the amounts of the three bile salts found inside the cells to those found in the medium was decreased by about 90% when Bt2cAMP was present in the hepatocyte incubations. This effect was mimicked by Bt2cGMP and to a lesser extent by Bt2cCMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Adrenal cortical mitochondria contain a mixed function oxidase capable of converting cholesterol to pregnenolone; this enzyme requires NADPH, oxygen and cholesterol. This cholesterol side chain cleavage enzyme system contains a Flavoprotein, an iron sulphur protein and a specific cytochrome P450 termed cytochrome P450scc. ACTH stimulates the adrenal cortex by activating adenyl cyclase producing an elevated intracellular concentration of cAMP. This in turn increases the activity of a cytosolic cAMP dependent protein kinase. Adrenal cortical cytosol contains a cholesterol ester hydrolase which is activated by ATP and a protein kinase. This enzyme may be deactivated by a phosphoprotein phosphatase. The adrenal cortex contains lipid droplets that are rich in esterified cholesterol. Cholesterol ester hydrolase can release free cholesterol from the lipid droplets. The free cholesterol released may be used to supplement the mitochondrial cholesterol as a pregnenolone precursor. Steroid hormone production by the adrenal cortex exhibits a diurnal rhythm and correlates with the activity of the cytosolic cholesterol ester hydrolase. The acute steroidogenic response to ACTH may be in part attributed to the availability of free cholesterol to the mitochondrial cholesterol side chain cleavage enzyme complex. The intracellular movement of free cholesterol from lipid droplets to mitochondrial inner membranes may be impeded by protein synthesis inhibitors such as cycloheximide. The precise mechanism of this block in steroidogenesis remains to be elucidated. Various drugs and oestrogenic hormones suppress the plasma and adrenal cholesterol concentrations. If adrenal cells are deficient in cholesterol, these cells exhibit a diminished response to ACTH. The response to this hormone can be corrected by supplying cholesterol via exogenous plasma lipoproteins. The route that free cholesterol follows within the adrenal cortical cell and the physiological factors influencing free cholesterol movement in such cells are important issues to be explored in future.

Asunto(s)

RESUMEN

1. The synthesis of conjugated chenodeoxycholic acid and tauro-beta-muricholic acid in isolated rat hepatocytes was measured by radioimmunoassay. Production of tauro-beta-muricholic acid was linear over 4 h of incubation at 37 degrees C. The net synthesis of conjugated chenodeoxycholic acid was very much lower than that of tauro-beta-muricholic acid. 2. When hepatocytes were prepared from rats in which the enterohepatic circulation had been broken, either by feeding the bile salt sequestrant, cholestyramine or by total biliary drainage for 48 h, synthesis of tauro-beta-muricholic acid was increased compared to that in cells from control rats. Conjugated chenodeoxycholic acid accumulation during incubation of the hepatocytes was increased by cholestyramine feeding but not by total biliary drainage. These results suggest that there is a metabolic difference between the two methods of breaking the enterohepatic circulation with regard to chenodeoxycholic acid synthesis. 3. Hepatocytes prepared from rats given 1% cholesterol in the diet for at least 2 weeks synthesised significantly more tauro-beta-muricholic acid than those from control rats. The total amount of conjugated cholic, chenodeoxycholic and tauro-beta-muricholic acids synthesised by cells from cholesterol fed animals, however was not significantly different from that produced by hepatocytes from normal rats. 4. Exogenous taurochenodeoxycholic acid was metabolised to tauromuricholic acid by isolated hepatocytes. Production of tauro-beta-muricholic acid reached a maximum at a concentration of 20 microM taurochenodeoxycholic acid. The total metabolism of taurochenodeoxycholic acid, however, increased linearly up to the highest concentration measured, 50 microM. 5. The biliary content of tauro-beta-muricholic acid during total biliary drainage fell rapidly in the first 10 h and thereafter continued to decline, reaching a minimum after about 24 h. No significant rise was observed during the remainder of the experimental period. 6. It is concluded that a large proportion of the conjugated chenodeoxycholic acid synthesised by isolated hepatocytes in vitro is metabolised to tauro-beta-muricholic acid, and therefore it is necessary to take this into account when using this system to study bile salt synthesis.

Asunto(s)

RESUMEN

The role of exogenous lipoprotein cholesterol versus endogenous cholesteryl esters as substrates in adrenal steroidogenesis was studied in isolated rat adrenal cells. Hypocholesterolemic drugs were used in rats to depress the plasma cholesterol concentration and the adrenal cholesterol concentration. Adrenal cortical cells were prepared in the usual way. The steroidogenic response to ACTH in normal adrenal cells and in cells which have been cholesterol-depleted was studied. Normal adrenal cells responded specifically over a 6 h incubation period to low doses of ACTH (half-maximal response equivalent to 40 microunits ACTH). These normal cells exhibited no altered response over a 3 h period to ACTH in the presence of serum or serum lipoproteins. The hypocholesterolemic drugs, 4-aminopyrazolo-[3,4-d]-pyrimidine, hexestrol and 17 alpha-ethinyl estradiol were used to lower plasma cholesterol, and after 1 day of 4-aminopyrazolo-[3,4-d]-pyrimidine and 5 days of hexestrol or 17 alpha-ethinyl estradiol treatment the plasma total cholesterol concentrations were similar. After 3 days of 4-aminopyrazolo-[3,4-d]-pyrimidine treatment the adrenal total cholesterol content was lower than after 1 day of this treatment, or 5 days of hexestrol treatment or 5 days of 17 alpha-ethinyl extradiol treatment. Lipoproteins had no significant effect on ACTH-stimulated steroidogenesis in cells isolated from rats treated for 1 day with 4-aminopyrazolo-[3,4-d]-pyrimidine, or for 5 days with hexestrol or 17 alpha-ethinyl estradiol. However, lipoproteins did stimulate steroidogenesis in cells from rats treated for 3 days with 4-aminopyrazolo-[3,4-d]-pyrimidine. The results show that normal adrenal cells contain a reserve of intracellular cholesterol so that the supply of endogenous cholesterol for steroidogenesis does not limit the response to ACTH and exogenous lipoproteins have no effect on steroidogenesis. However, if the cells are severely depleted of cholesterol then exogenous lipoproteins must be added for maximal steroidogenesis to occur.

Asunto(s)

RESUMEN

A new radioimmunoassay which can be used to measure the amounts of tauro-beta-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-beta-muricholic acid. Exogenous taurochenodeoxycholic acid was metabolised by isolated rat hepatocytes concurrently with the appearance of tauro-beta-muricholic acid in the cell.

Asunto(s)

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Pregnenolone efflux from bovine adrenal cortex mitochondria was measured with the aid of rapid separation of the organelles from the incubation by centrifugation through silicon oil. In the absence of bovine serum albumin or cytosolic supernatant (100 000 x g), an equilibrium distribution of newly synthesised pregnenolone was attained after 2 min at 30 degrees C; 20% of total pregnenolone was located outside the mitochondria. The presence of 13 mg albumin/ml or 13 mg supernatant protein/ml increased the exogenous proportion of pregnenolone to over 90% and 75% respectively without affecting the rate of cholesterol side-chain cleavage. The binding of pregnenolone by both albumin and supernatant was non-specific and non-saturable. It is suggested that in vivo the mitochondrial membranes do not present a significant barrier to pregnenolone efflux from the organelle.

Asunto(s)

RESUMEN

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.

Asunto(s)