Metabolism of (1-14C)-palmitic acid in the cat's brain]

Following injection into the cerebral ventricles of conscious cats, (1-14C) palmitic acid was rapidly taken up and incorporated into a variety of brain lipids. The peak of uptake of (1-14C) palmitic acid, about 50% of injected radioactive material, into the brain tissue was obtained within the first 24 h following its administration. Thereafter, the radioactivity slowly decreased reaching the least value by the end of the second week. The most heavily labelled lipids were the phospholipids, while the free fatty acids were appreciably labelled. Small percentage of the radioactive material was found in monoglycerides, diglycerides and triglycerides. The least incorporation was into cholesterol esters.     

Distribution, uptake and hydrolysis of cholesterol oleate (-1-14C) in brain tissue after its injection in the cerebral ventricles of the conscious cat]

Distribution, uptake and hydrolysys of cholesteryl oleate (-1-14C) in vivo in the cat's brain were investigated. Four hours following intraventricular injection of cholesteryl oleate (-1-14C), about one third of the injected radioactivity was taken up and about 50 % of that radioactive material was hydrolysed by brain tissue. Even more, the labelled material was found in the brain tissue surrounding the cerebral ventricles as well as in the cerebral cortex, but in varying amounts: the smallest amounts were found in the cerebral cortex, while the highest were in the hypothalamus. Radioactive material was also detected in the peripheral venous blood.     

Distribution of 1-14C palmitic acid in brain tissue after intraventricular injection in the conscious cat]

The distribution of (1-14C) palmitic acid in the brain tissue following the injection into the cerebral ventricles of conscious cats was investigated. The radioactive material was found in the brain tissue surrounding the cerebral ventricles and in the cerebral cortex, but in varying amounts : the smallest amounts were found in the cerebral cortex, while the highest in the thalamus and in the hippocampus. Radioactive material was also found in the peripheral venous blood. The amount of the radioactive material in the grey matter lining the cerebral ventricles as well as in the cerebral cortex was time-dependant. The labelled material in the structures surrounding the cerebral ventricles and in the cerebral cortex increased within first four hours after its intraventricular administration. Thereafter, throughout subsequent 48 hours either it slowly disappeared in the caudate nucleus and in the thalamus, or it was retained in the hypothalamus and in the floor of the IV ventricle.     

Effect of apolipoprotein E-free high density lipoproteins on cholesterol metabolism in cultured pig hepatocytes

We studied cholesterol synthesis from [14C]acetate, cholesterol esterification from [14C]oleate, and cellular cholesterol and cholesteryl ester levels after incubating cells with apoE-free high density lipoproteins (HDL) or low density lipoproteins (LDL). LDL suppressed synthesis by up to 60%, stimulated esterification by up to 280%, and increased cell cholesteryl ester content about 4-fold. Esterification increased within 2 h, but synthesis was not suppressed until after 6 h. ApoE-free HDL suppressed esterification by about 50% within 2 h. Cholesterol synthesis was changed very little within 6 h, unless esterification was maximally suppressed; synthesis was then stimulated about 4-fold. HDL lowered cellular unesterified cholesterol by 13-20% within 2 h and promoted the removal of newly synthesized cholesterol and cholesteryl esters. These changes were transient; by 24 h, both esterification and cellular unesterified cholesterol returned to control levels, and cholesteryl esters increased 2-3-fold. HDL core lipid was taken up selectively from 125I-labeled [3H]cholesteryl ester- and ether-labeled HDL. LDL core lipid uptake was proportional to LDL apoprotein uptake. The findings suggest that 1) the cells respond initially to HDL or LDL with changes in esterification, and 2) HDL mediates both the removal of free cholesterol from the cell and the delivery of HDL cholesteryl esters to the cell.     

Calculation of lipolysis and esterification from glycerol metabolism in rat adipose tissue

Pieces of epididymal fat pad from fed and 48-hr-fasted rats were incubated for various periods of time in Krebs-Ringer bicarbonate containing [1-(14)C]glycerol. The radioactive substrate taken up by the tissue increased linearly with time in both groups and was mainly converted to glyceride-glycerol and CO(2). The slopes of the regressions of (14)C-labeled glyceride-glycerol with time were not different between the groups, while those of (14)CO(2) were smaller in the fasted than in the fed animals. Because the radioactive glycerol in the medium is being continuously diluted with the glycerol coming out of the tissue, it is necessary to take account of this factor in calculating the actual amount of glycerol utilized by the tissue. The glycerol produced by the tissues is higher in the fasted than in the fed animals, and in both groups it increases hyperbolically with time. As negligible amounts of the (14)C-labeled glycerol taken up by the tissue recirculates to the medium, the rates of glycerol release (lipolysis), esterification, and oxidation to CO(2) were calculated.     

Impaired cholesterol esterification in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy

Cholesterol esterification was examined in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy by incubating cells pretreated without fetal calf serum for 48h, with (14C) cholesterol for 24h. Impaired cholesterol esterification was found in these cells and free cholesterol was accumulated in plasma membrane and Golgi fractions. This impairment was also induced in control cells by adding leupeptin (20 micrograms/ml) or monensin (2 micrograms/ml). These findings suggest the importance of the role of lysosomes for esterification of cholesterol and give a hint as to the basic defect in type C Niemann-Pick disease.     

Eicosapentaenoic acid inhibits cholesterol esterification in cultured parenchymal cells and isolated microsomes from rat liver

The effects of eicosapentaenoic acid on synthesis and secretion of cholesterol and cholesterol ester by cultured rat hepatocytes were studied. In the presence of eicosapentaenoic acid cellular cholesterol esterification was decreased by 50-75% compared to oleic acid as measured by radioactive precursors and mass. Secretion of cholesterol ester was reduced by 50-60% in the presence of eicosapentaenoic acid as evaluated by radiolabeled fatty acids, mevalonolactone, and mass measurement. Oleic, palmitic, and stearic acid increased, whereas eicosapentaenoic and docosahexaenoic acid decreased synthesis and secretion of cholesterol ester as compared to a fatty acid-free control. Cellular and secreted free cholesterol were unaffected by eicosapentaenoic acid in comparison with oleic acid. The reduced cholesterol esterification was observed within 1 h and lasted for at least 20 h. Eicosapentaenoic acid caused lower cholesterol esterification than oleic acid in the concentration range 0.2-1.0 mM fatty acid and reduced the stimulatory effect of oleic acid on cholesterol ester formation. Cholesterol esterification and release of cholesterol ester were markedly increased by 25-hydroxycholesterol in the presence of eicosapentaenoic acid as well as oleic acid. Experiments with liver microsomes revealed that radioactive eicosapentaenoic acid and eicosapentaenoyl-CoA were poorer substrates (7-30%) for cholesterol esterification than oleic acid and oleoyl-CoA. Reduced formation of cholesterol ester was also observed when eicosapentaenoyl-CoA was given together with labeled oleoyl-CoA, whereas palmitoyl-CoA, stearoyl-CoA, linolenoyl-CoA, and arachidonoyl-CoA had no inhibitory effect. In conclusion, eicosapentaenoic acid reduced cellular cholesterol esterification by inhibiting the activity of acyl-CoA:cholesterol acyltransferase. The lowered cholesterol esterification caused by eicosapentaenoic acid secondly decreased secretion of very low density lipoprotein cholesterol ester.     

Utilization of Plasma Fatty Acid in Rat Brain: Distribution of [14C]Palmitate Between Oxidative and Synthetic Pathways

Radioactivity within individual brain compartments was determined from 5 min to 44 h after intravenous injection of [14C]palmitate into awake Fischer-344 rats, aged 21 days or 3 months. Total radioactivity peaked broadly between 15 min and 1 h after injection, declined rapidly between 1 and 2 h, and then more slowly. In 3-month-old rats, the lipid and protein brain fractions were maximally labeled within 15 min after [14C]palmitate injection, then retained approximately constant label for up to 2 days. Radioactivity in the aqueous brain fraction comprised mainly radioactive glutamate and glutamine, and peaked at 45 min, when it comprised 48% of total brain radioactivity, then decreased to 27% of the total at 4 h, 15% at 20 h, and 10% at 44 h. Percent distribution of radioactivity within the different brain compartments, 4 h after intravenous injection of [14C]palmitate, was similar in 21-day-old and 3-month-old rats, despite higher net brain uptake in the younger animals. The results indicate that about 50% of plasma [14C]palmitate that enters the brain of adult rats is incorporated rapidly into stable protein and lipid compartments. The remaining [14C]palmitate enters the aqueous fraction after beta-oxidation, and is slowly lost. At 4 h after injection, 73% of brain radioactivity is within the stable brain compartments; this fraction increases to 86% by 20 h.     

3H]prostaglandin uptake in vivo by rabbit uterine tissues and blastocysts

Day-6 pregnant rabbits were anesthetized and subjected to a mid-ventral laparotomy. [3H] Prostaglandin F2alpha) (PGF2alpha) [3H]PGE2, [14C]Urea or [14C]Sucrose were instilled into the uterine lumen via the uterotubal junction. The amounts instilled/uterine horn were respectively 3.7 +/- 0.3, 3.5 +/- 0.3, 5.7 +/- 1.3 and 2.7 +/- 1.6 muCi in 20mul of buffer. Animals were killed at 1, 2, 9, 19 or 21 h after radioactive instillation, and the amounts of radioactivity in blastocysts, uterine tissue, peritoneal cavity washings and urine evaluated by liquid scintillation spectrometry. A gradient of radioactivity was observed from the uterotubal junction to the cervical end of the uterus. Large amounts of [3H]PG were found in the injected horn and associated blastocysts with a considerable crossover to the non-injected horn, but little in the associated blastocysts. Much of the blastocysts associated- [3H]PG remained unmetabolized. Large amounts of metabolized [3 H] were found in urine. [14C]Urea was taken up by uterine tissue in the injected horn, but there was little cross over to the non-injected horn. Urea was also found in urine. Much of the [14C]Sucrose remained in the injected horn, and little was recovered from the urine. It was found that at 9 h, but not at 19 h, after [3 H]PG instillation, the PG was localized at the site of the blastocysts in the injected but not in the contralateral horn. Significantly more [3H]PGF2alpha than [3H]PGE2 was localized in this situation. [14C]Urea was not localized at the site of the blastocysts in urea injected horns. (ABSTRACT TRUNCATED AT 250 WORDS)     

Partial characterization of steryl ester biosynthesis in spinach leaves

Acetone powders of a 20,000g pellet fraction from spinach leaves (Spinacia oleracea L.) synthesized [4-(14)C]cholesteryl esters when incubated with [4-(14)C]cholesterol. The reaction was inhibited by digitonin. There was a reciprocal relationship between the decline of label in cholesterol and its incorporation into cholesteryl ester, indicating that free cholesterol was the direct precursor for cholesteryl ester biosynthesis. The hydrolysis of cholesteryl [1-(14)C]palmitate into free cholesterol and [1-(14)C]palmitate was not detected in these acetone powder preparations. Exogenous cholesteryl palmitate had no effect on the esterification of [4-(14)C]cholesterol. The data indicate that an esterase-type mechanism was not involved in the biosynthesis of these steryl esters. Label from [1-(14)C]palmitoyl-CoA was incorporated into steryl esters when incubated with spinach leaf acetone powder preparations. The optimal buffer for steryl ester biosynthesis was 2-(N-morpholino)ethanesulfonate and the optimal pH was 6. Iodoacetamide, N-ethylmaleimide, and dithiothreitol had no effect on the esterification reaction. Ethylenediaminetetraacetate, MgCl(2), CaCl(2), MnCl(2), and ZnSO(4) inhibited at concentrations of 10 to 30 mm.     

Cholestanol metabolism in patients with cerebrotendinous xanthomatosis: absorption, turnover, and tissue deposition

To study the metabolism of cholestanol in patients with cerebrotendinous xanthomatosis (CTX), we measured the cholestanol absorption, the cholesterol and cholestanol turnover, and the tissue content of sterols in two patients. Cholestanol absorption was approximately 5.0%. The rapid exchangeable pool of cholestanol was 233 mg, and the total exchangeable pool was 752 mg. The production rate of cholestanol in pool A was 39 mg/day. [4-14C]cholestanol was detected in the xanthomas, but neither [4-14C]cholestanol nor [4-14C]cholesterol was detected in peripheral nerves biopsied at 49 and 97 days after [4-14C]cholesterol given intravenously. Of the 18 tissues analyzed at biopsy and autopsy, the cholestanol content varied from 0.09 mg/g in psoas muscle to 76 mg/g in a cerebellar xanthoma. With the assumption that the cholestanol-to-cholesterol ratio is 1.0, the relative cholestanol-to-cholesterol ratio varied from 1.0 in plasma and liver to 30.0 in the cerebellar xanthoma; cholestanol was especially high in nerve tissue. Our data indicate that CTX patients absorb cholestanol from the diet. They have a higher than normal cholestanol production rate. Cholestanol was derived from cholesterol. In CTX patients, the blood-brain barrier was intact to the passage of [4-14C]cholesterol and [4-14C]cholestanol. The deposition of large amounts of cholestanol (up to 30% of total sterols in cerebellum) in nerve tissues must have an important role in the neurological symptoms in CTX patients. In view of the intact blood-brain barrier, several other explanations for the large amounts of cholestanol in the brain were postulated.     

Control of ovarian cholesterol ester biosynthesis

1. Experimental evidence is presented for a role of progesterone and 20alpha-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [(14)C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3':5'-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [(14)C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20alpha-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.     

Inhibitors of sterol synthesis. The effects of dietary 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on the fate of [4-14C]cholesterol and [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one after intragastric administration to rats

The effect of dietary administration (0.1% in a rat chow diet) of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of cholesterol biosynthesis with marked hypocholesterolemic activity, on the fate of [4-14C]cholesterol and [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one has been studied after intragastric administration of the labeled sterols to rats. In general, the distribution of 3H in major tissues paralleled that of 14C with no unusual concentration of 3H in any of the organs. Only trace amounts of 3H and 14C were recovered in urine. Administration of the 15-ketosterol was associated with decreased absorption of the labeled cholesterol as indicated by decreased levels of 14C in the various tissues and organs of the 15-ketosterol-treated rats (relative to ad libitum and pair-fed control animals) and increased levels of 14C in feces and intestinal contents at 12 and 48 h after the administration of the labeled cholesterol. Studies of the distribution of 3H in liver indicated rapid conversion of the 15-ketosterol to cholesterol and cholesteryl esters. The amounts of 3H recovered in the various tissues and organs at both 12 and 48 h after the administration of the labeled sterols were considerably less than the corresponding values for 14C, a finding which suggests a lower absorption of the 15-ketosterol (relative to cholesterol) and/or a more rapid clearance and biliary excretion of the 15-ketosterol and its metabolites.     

The effect of interleukin 1 beta on the biosynthesis of cholesterol, phosphatidylcholine, and sphingomyelin in fibroblasts, and on their efflux from cells to lipid-free apolipoprotein A-I.

In this study we have investigated the effect of interleukin 1beta (IL-1beta) on the metabolism of cholesterol and choline-phospholipids in cultured fibroblasts, and also measured efflux of these lipids to lipid-free apo A-I as a function of IL-1beta treatment. Long-term exposure (up to 48 h) of cells to IL-1beta (1 ng.mL-1) markedly increased the rate of cholesterol esterification, as determined by the incorporation of [3H]oleic acid into cholesteryl esters. This treatment also led to a substantially increased mass of cholesteryl esters in the cells. The accumulation of cholesteryl esters in IL-1beta-treated cells could be blocked using compound 58-035 to inhibit the activity of acyl-CoA cholesterol acyl transferase. The activation of cholesterol esterification by IL-1beta was evident within a few hours after initiation of the IL-1beta treatment. Cholesterol biosynthesis was inhibited by 25% by IL-1beta (after 48 h exposure), and this eventually led to a 20% decrease in cell cholesterol mass. Treatment of cells with IL-1beta for 48 h also reduced the synthesis of sphingomyelin and caused a 30% decrease in cell sphingomyelin mass (after 48 h at 1 ng.mL-1 of IL-1beta). IL-1beta did not stimulate an acute (within a few minutes up to an hour) degradation of cell [3H]sphingomyelin. This suggests that IL-1beta did not activate an endogenous sphingomyelinase in these cells, but only affected rates of synthesis. The rate of phosphatidylcholine synthesis was barely affected, but mass was moderately reduced by a 48-h treatment of cells with IL-1beta. Finally, the efflux of cell [3H]cholesterol, [3H]sphingomyelin, and [3H]phosphatidylcholine to lipid-free apolipoprotein A-I was markedly increased from cells treated with IL-1beta for 24 and 48 h. We conclude that long-term exposure of cells to IL-1beta had marked effects on the cellular homeostasis of cholesterol and choline-containing phospholipids.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Cholesterol Esterifying Enzyme in Normal and Degenerating Peripheral Nerve

Abstract: The cholesterol esterifying enzyme which incorporates exogenous free [1-¹⁴C]oleate into cholesteryl ester is present in rat sciatic endoneurium. Cholesterol esterification is optimal at pH 4.8. Exogenous ATP, CoA, and oleyl-CoA do not greatly affect its activity. Various detergents and bile salts are inhibitory. Enzyme activity does not change appreciably during storage at 4°C for up to 4 days or at -70°C for up to 1 month. Of the subcellular fractions, the microsomal fraction exhibits the highest specific activity. Over 75% of enzyme activity is recovered, with equal amounts in the microsomal and soluble fractions. During nerve fiber degeneration an increase (more than fivefold) in cholesterol esterifying activity, which peaks 6 days after crush, is observed. Elevated levels of enzyme activity persist for 90 days after crush, by which time nerve regeneration is well established. Thus, it is concluded that an increase in cholesterol esterifying activity in degenerating nerve is primarily responsible for cholesterol esterification during Wallerian degeneration. The maximum increase in cholesterol esterifying activity is associated temporally with axonal degeneration and, particularly, with the formation of myelin ovoids.     

Quantification of cholesterol absorption in man by fecal analysis after the feeding of a single isotope-labeled meal

The fecal excretion of cholesterol-4-(14)C and -sitosterol-22,23-(3)H has been studied in normal human subjects after they had ingested a single meal containing the radioactive substances. When 150 mg of -sitosterol, dispersed in the butter of a standard breakfast, was fed to 20 subjects the mean recovery of isotope in the feces was 90%. When plant sterols (70% -sitosterol, 30% campesterol) were fed together with cholesterol and used as an internal standard to correct for losses of cholesterol during intestinal transit and analytical procedures, excretion of dietary cholesterol was found to be 60-80%, irrespective of the amount fed over the range 150-1910 mg. If absorption of cholesterol is calculated from these figures, no saturation of the cholesterol absorption mechanism is indicated for the amounts of cholesterol fed in this investigation. The reason for the differences between these findings and those previously reported by other procedures is not clear, but may be related to the acute administration of a single dose of cholesterol in this study.     

Absorption and distribution of [1-14C]dolichol intubated into rats

[1-14C]Dolichol was mixed in vitro with sunflower seed oil and intubated into rats. Radioactivity began to appear in the blood at 3 h and peaked after about 6 h. The absorbed radioactivity was rapidly cleared from the blood. At 7.5 h postintubation two thirds of the radioactivity in the serum was associated with chylomicrons and about one quarter with the high density lipoproteins. At 12 h the proportion of the radioactivity in the chylomicrons had fallen to one third and that in the high density lipoproteins had increased to one half of the total. Less than 0.02% of the dose was found in the tissues after 12 h. Liver and blood each contained about one third of the total, with smaller amounts in the lungs and spleen. The heart, testes, brain, and kidneys contained only traces of radioactivity. After 12 h most of the radioactivity in the tissues and feces was present as [1-14C]dolichol. The radioactive compounds in the urine (about 0.05% of the dose) were more polar than [1-14C]dolichol as determined by thin-layer chromatography.     

Lipid metabolism in brain tissue explants

Abstract— Tissue explants from frontal lobes of rat brain were used for the study of cerebral fatty acid metabolism. After tissues had been maintained in serum-supplemented medium, a lipid-free medium was substituted and metabolic studies were carried out. Under these conditions explants continued to take up lipid precursors for at least 48 h, as judged by incorporation of dl -[2-¹⁴C]mevalonic acid into cellular lipids. [l-¹⁴C]Stearic acid and [l-¹⁴C]palmitic acid were bound to cells as the free fatty acids, or incorporated into neutral lipids (particularly triglycerides), glycolipids and phospholipids. In the galactolipid fraction, cerebrosides were the principal radioactive lipids. Choline phosphoglycerides, ethanolamine phosphoglycerides, inositol phosphoglycerides and serine phosphoglycerides were the principal radioactive phospholipids. Fatty acids were incorporated into cellular lipids either unchanged or after desaturation, chain elongation, or both. Maximum incorporation of stearate occurred in tissues derived from 3-day-old animals. With increasing age the uptake of fatty acid dropped sharply. When the labelling of lipids as a function of time was followed in 3-day-old animals, triglycerides and choline phosphoglycerides were the first fractions to take up labelled stearate. Labelling of cerebrosides occurred slowly, only becoming evident after 24 h. These studies exemplify the usefulness of tissue explants for prolonged metabolic studies in normal and pathological specimens of brain.     

Dexamethasone regulates bile acid synthesis in monolayer cultures of rat hepatocytes by induction of cholesterol 7 alpha-hydroxylase.

To study the effect of steroid hormones on bile acid synthesis by cultured rat hepatocytes, cells were incubated with various amounts of these compounds during 72 h and conversion of [4-14C]cholesterol into bile acids was measured. Bile acid synthesis was stimulated in a dose-dependent way by glucocorticoids, but not by sex steroid hormones, pregnenolone or the mineralocorticoid aldosterone in concentrations up to 10 microM. Dexamethasone proved to be the most efficacious inducer, giving 3-fold and 7-fold increases in bile acid synthesis during the second and third 24 h incubation periods respectively, at a concentration of 50 nM. Mass production of bile acids as measured by g.l.c. during the second day of culture (28-52 h) was 2.2-fold enhanced by 1 microM-dexamethasone. No change in the ratio of bile acids produced was observed during this period in the presence of dexamethasone. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of the bile acid pathway, to bile acids was not affected by dexamethasone. Measurement of cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes, incubated with 1 microM-dexamethasone, showed 10-fold and 90-fold increases after 48 and 72 h respectively, as compared with control cells. As with bile acid synthesis from [14C]cholesterol, no change in enzyme activity was found in hepatocytes cultured in the presence of 10 microM steroid hormones other than glucocorticoids. Addition of inhibitors of protein and mRNA synthesis lowered bile acid production and cholesterol 7 alpha-hydroxylase activity and prevented the rise of both parameters with dexamethasone, suggesting regulation at the mRNA level. We conclude that glucocorticoids regulate bile acid synthesis in rat hepatocytes by induction of enzyme activity of cholesterol 7 alpha-hydroxylase.