Whole body and hepatic cholesterol synthesis rates in the guinea-pig: effect of dietary fat quality

The effects of polyunsaturated, monounsaturated and saturated dietary fat on total and hepatic cholesterol synthesis were studied in the guinea-pig. Male Hartley guinea-pigs were fed semi-synthetic diets containing 7.5% (w/w) of either corn oil (CO), olive oil (OL) or lard for a period of 5 weeks and rates of endogenous cholesterol synthesis were determined from the incorporation of [3H]water into digitonin-precipitable sterols (DPS) and by measurement of sterol balance. In addition, total and expressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were determined in hepatic microsomes. Rates of whole body cholesterol synthesis determined by incorporation of [3H]water into DPS were significantly lower for guinea-pigs on the CO diet with values of 18.7 +/- 1.8 mumol/h (n = 4) vs. 26.7 +/- 4.8 and 24.6 +/- 1.8 mumol/h for animals on the OL (n = 4) and lard (n = 3) diets (P less than 0.001), respectively. Hepatic cholesterol synthesis rates were significantly decreased in animals on the OL diet, whether determined from incorporation of [3H]water into DPS or by analysis of HMG-CoA reductase activity. Hepatic total and free cholesterol levels were not different for animals on the three dietary fats; however, cholesteryl ester levels were 35% lower in guinea-pigs fed the lard diet (P less than 0.02). Sterol balance measurements indicated that whole body cholesterol synthesis rates were not affected by dietary fat quality (51.9 +/- 12.2, 42.8 +/- 7.6 and 51.2 +/- 20.2 mg/kg per day for animals on the CO, OL and lard diets, respectively). This is in striking contrast to the observed reduction in cholesterol synthesis rates for animals on the polyunsaturated CO diet as determined by incorporation of [3H]water into DPS. One possible explanation for the discrepancy between the sterol balance and [3H]water incorporation data is a polyunsaturated fat-mediated effect on energy utilization, which affects the equilibration of NADPH with the body water pool such that the [3H]NADPH has a lower specific activity than body [3H]water.     

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one. In vivo conversion to cholesterol upon oral administration to a nonhuman primate

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I), a potent inhibitor of cholesterol synthesis with marked hypocholesteremic activity, has been studied in a nonhuman primate. A mixture of [2,4-3H]-I and [4-14C]-cholesterol was administered to a male baboon in the form of a feedball. Blood was samples at 4, 8, 12, 16, and 24 hr. Detailed analyses of the plasma lipids indicated very rapid absorption of I (relative to cholesterol) and metabolism to cholesterol, cholesteryl esters, and esters of I. The labeled cholesterol was characterized by chromatographic techniques and by purification by way of its dibromide derivative. The levels of 3H in plasma associated with I, esters of I, cholesterol, and cholesteryl esters each showed a different time course. By 24 hr after the administration of [2,4-3H]-I, most of the 3H in plasma was associated with cholesterol and cholesteryl esters. The levels of total 3H and 14C in plasma at various times after the administration of the mixture of [2,4-3H]-I and [4-14C]-cholesterol differed markedly with 3H showing a maximum value at 4 hr and 14C showing a maximum value at 24 hr.     

Synthesis of ubiquinone and cholesterol in human fibroblasts: regulation of a branched pathway.

The current studies demonstrate that cultured human flbroblasts utilize mevalonate for the synthesis of ubiquinone-10 as well as for the synthesis of cholesterol. Study of the regulation of this branched pathway was facilitated by incubating the cells with compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryI coenzyme A reductase, which blocked the formation of mevalonate within the cell. The addition of known amounts of [³H]mevalonate to the culture medium in the presence of compactin permitted the study of the relative rates of mevalonate incorporation into cholesterol and ubiquinone-10 under controlled conditions. When low concentrations of exogenous [³H]mevalonate (10 to 50 μm) were added to cells that were provided with exogenous cholesterol in the form of plasma low density lipoprotein (LDL), the cells incorporated the [³H]mevalonate into ubiquinone-10 at a rate that was two- to threefold faster than the incorporation into cholesterol. When the cells were deprived of exogenous LDL-cholesterol, the incorporation of [³H]mevalonate into ubiquinone-10 decreased and the incorporation of [³H]mevalonate into cholesterol increased. As a result, in the absence of exogenous cholesterol more than 60 times as much [³H]mevalonate was incorporated into cholesterol as into ubiquinone-10. Considered together with previous findings, the current data are compatible with a regulatory mechanism in which LDL inhibits cholesterol synthesis in fibroblasts at two points: (1) at the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, thereby inhibiting mevalonate synthesis, and (2) at one or more points distal to the last intermediate common to the cholesterol and ubiquinone-10 biosynthetic pathways. The latter inhibition allows ubiquinone-10 synthesis to continue in the presence of LDL despite a 98% reduction in mevalonate synthesis.     

Evaluation of the contribution of dietary cholesterol to hypercholesterolemia in diabetic rats and of sitosterol as a recovery standard for cholesterol absorption

The contribution of dietary cholesterol to hypercholesterolemia in diabetic rats fed chow ad libitum was evaluated. Diabetes was induced with streptozotocin, and the intake, absorption, and subsequent tissue distribution of dietary cholesterol were measured. Absorption was measured as the difference between [3H]cholesterol intake and fecal 3H-labeled neutral sterol excretion, using both [14C]sitosterol (added to diet) and [14C]cholesterol (added to feces) as recovery markers. [3H]Cholesterol absorption was underestimated by 1-3% using [14C]sitosterol as a recovery standard, due to the 7-8% absorption of sitosterol. After 3 weeks of diabetes, rats were hyperphagic, thereby increasing dietary cholesterol intake 2-fold. [3H]Cholesterol absorption was significantly increased from 69% in controls to 78% in diabetics, whereas [14C]sitosterol absorption was unaffected. With increased dietary cholesterol intake and decreased whole body cholesterol synthesis (Diabetes. 1983. 32: 811-819), influx from diet equaled for exceeded influx from synthesis. The amounts of 3H-labeled neutral sterol recovered from the small intestine, periphery, and plasma were increased 3- to 4-fold in the diabetic rats. Furthermore, the degree of hypercholesterolemia in diabetic rats was directly related to the fraction of plasma cholesterol derived from the diet. We conclude that the 2.3-fold increase in absorbed dietary cholesterol resulting from hyperphagia and, to a lesser extent, from increased fractional absorption, contributes to the hypercholesterolemia of diabetic rats fed chow ad libitum.     

Synthesis of [24, 25-3H]cholesterol: a new substrate for determining the rate of cholesterol side chain oxidation.

A procedure for the synthesis of [24,25-3H]cholesterol from the nonradioactive precursor desmosterol is described. The intermediate, isodesmosterol, which was purified by column chromatography, was formed to protect the original double bond (delta 5-6) from hydrogenation. Tritium was introduced into the side chain by catalytic reduction of the double bond (delta 24-25) of the isodesmosterol in the presence of carrier-free tritium. After ring rearrangement of the iso-[24,25-3H]cholesterol acetate, the acetate was hydrolyzed to form the free labeled cholesterol. Hepatic oxidation of the [24,25-3H]cholesterol side chain release tritium into water which freely equilibrates with cell and body water pools. Thus, the rate of 3H2O appearance corresponds to the rate of cholesterol side chain oxidation. Applications of this method to in vivo, isolated perfused liver, and isolated hepatocyte preparations of the rat are discussed.     

Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035

Compound 58-035 (3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]pro panamide) has been found to inhibit the accumulation of cholesteryl esters in both rat hepatoma (Fu5AH) cells and arterial smooth muscle cells in culture. To explore the specificity of 58-035, we have studied the esterification of cholesterol, retinol, and glycerides by the Fu5AH cell and by isolated membranes. Exposure of Fu5AH to cholesterol/phospholipid dispersions and 58-035 (greater than 100 ng/ml) for 24 h resulted in greater than 95% inhibition of cholesterol esterification while cellular free cholesterol increased slightly. Inhibition was also rapid; incorporation of [3H]oleate into cholesteryl [3H]oleate equaled only 12% of control value after 30 min with 58-035 at 5 micrograms/ml. In contrast, there was no decrease in [3H]oleate incorporation into phospholipids or diglycerides, nor was the esterification of [3H]retinol inhibited by 58-035. In microsomal fractions, acyl-CoA:cholesterol acyltransferase could be inhibited completely by 58-035, while activities of acyl-CoA: retinol acyltransferase and triglyceride synthesis proceeded at 75-100% of control values. These observations that 58-035 is highly selective allow the inference that acyl-CoA:cholesterol acyltransferase is a separate microsomal enzyme whose activity can be modulated independently from acyl-CoA:retinol acyltransferase and other cellular acyltransferases.     

Measurement of rates of cholesterol synthesis using tritiated water

Rates of sterol synthesis in various tissues commonly are assessed by assaying levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase on isolated microsomes or by measuring the rates of incorporation of various 14C-labeled substrates or [3H]water into cholesterol by whole cell preparations in vitro or by the tissues of the whole animal in vivo. While measurement of activities of HMG-CoA reductase or rates of incorporation of 14C-labeled substrates into cholesterol give useful relative rates of sterol production, neither method yields absolute rates of cholesterol synthesis. The use of [3H]water circumvents the problem of variable and unknown dilution of the specific activity of the precursor pool encountered when 14C-labeled substrates are used and does yield absolute rates of cholesterol synthesis provided that the 3H/C incorporation ratio is known for a particular tissue. In 12 different experimental situations it has been found that from 21 to 27 micrograms atoms of 3H are incorporated into cholesterol from [3H]water in different tissues of several animal species, so that the 3H/C incorporation ratio is similar under nearly all experimental conditions and varies from 0.78 to 1.00. When administered in vivo, [3H]water rapidly equilibrates with intracellular water and is incorporated into sterols within the various organs at rates that are linear with respect to time. From such data it is possible to obtain absolute rates of cholesterol synthesis in the whole animal and in the various organs of the animal. Current data suggest, therefore, that use of [3H]water yields the most accurate rates of cholesterol synthesis both in vitro and in vivo.     

The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease

An inborn murine cholesterol storage disorder exists which is characterized by a lesion in intracellular cholesterol esterification not accounted for by any discernible abnormality in acyl-CoA: cholesterol acyltransferase (Pentchev, P.G., Boothe, A.D., Kruth, H.S., Weintroub, H., Stivers, J., and Brady, R.O. (1984) J. Biol. Chem. 259, 5784-5791). Current studies have shown that the level of esterification of nonlipoprotein-derived [3H]cholesterol in cultured fibroblasts from heterozygous mutant mice was intermediary between the level found in normal fibroblasts and the deficient level found in fibroblasts from homozygous mutant mice. Homozygous-affected fibroblasts took up and converted [3H]desmosterol to [3H]cholesterol at a normal rate indicating that the murine mutation does not compromise the transport of exogenous sterol to microsomes. In contrast to the defect in esterification of exogenously derived cholesterol, synthesis of cholesteryl ester from [3H]mevalonic acid and [3H]squalene was normal in affected fibroblasts as was the stimulation of cholesteryl ester synthesis from endogenous cholesterol induced by 25-hydroxycholesterol. In surveying a number of mutant cell lines from human metabolic disorders with phenotypic manifestations similar in part to the mutant cholesterol storage mouse, Niemann-Pick C fibroblasts displayed a similar defect in esterification of exogenously derived cholesterol.     

Loss of tritium from coprostanone derived from [1,2(n)-3H]cholesterol or [7(n)-3H]cholesterol

After oral administration of a mixture of [1,2(n)-3H]cholesterol and [4-14C]cholesterol to a baboon, fecal coprostanone had a 46% lower 3H/14C ratio than the dose administered. Loss of 3H by enolization of the 3-ketone could account for the decrease in 3H/14C. If [7(n)-3H]cholesterol was administered instead of [1,2(n)-3H]cholesterol a 23% loss of 3H from coprostanone was found. Procedures requiring measurement of 3H-coprostanone derived from [1,2(n)-3H]- or [7(n)-3H]cholesterol could be seriously in error unless an appropriate correction for loss of 3H is made.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.     

Sterol synthesis by the ocular lens of the rat during postnatal development

Great amounts of plasma membranes are formed during early postnatal development of the ocular lens as lens epithelial cells differentiate into fiber cells. Little information is available on the source of the lipids, and particularly cholesterol, required for formation of these plasma membranes. The present study measured the capacity of the lens of the rat to synthesize cholesterol during this dynamic period of growth. Incorporation by lens of (3)H(2)O into total fatty acids was also examined. Absolute rates of cholesterol synthesis per whole lens were estimated in vitro from incorporation of (3)H from (3)H(2)O into digitonide precipitable sterols (DPS) by intact lenses of 6- to 30-day old rats. Rates of cholesterol synthesis were calculated which were adequate to furnish from either 50-100% or 20-40% of the cholesterol required by the lens for growth, depending upon the animal's age and upon whether one considered NADPH to be generated by the pentose phosphate pathway or by oxidative enzymatic processes (NADPH from the pentose pathway is not labeled from (3)H(2)O). Generation of the NADPH necessary for cholesterol synthesis principally by the pentose pathway would support the higher percent contribution of synthesis to the total growth requirement. The pentose pathway was clearly active in the young rat lens, since between 7.5 to 9.0 times more [1-(14)C]glucose than [6-(14)C]glucose was oxidized in vitro to (14)CO(2) by 6- and 22-day old lenses. Incorporation of (3)H(2)O into DPS decreases sharply after 2 weeks of age in spite of a constant rate of cholesterol accumulation by the lens. These results indicate that the ocular lens of the rat can furnish most if not all of its cholesterol requirements by synthesis de novo during the first 2 weeks of life, and imply a contribution from another source at older ages. Whether lipoproteins can supply cholesterol to the lens is still unclear, although neither HDL nor LDL altered the incorporation in vitro of [U-(14)C]glucose into DPS by lens.-Cenedella, R. J. Sterol synthesis by the ocular lens of the rat during postnatal development.     

Differential effects of interferon-gamma on metabolism of lipoprotein immune complexes mediated by specific human macrophage Fcgamma receptors

The objectives were to determine whether there are differences in the mechanisms of lipoprotein metabolism associated with different FcgammaRs and how metabolism associated with FcgammaRs compares to that mediated by scavenger receptors (SRA). To analyze lipoprotein metabolism in a receptor-specific manner, bispecific antibodies were used to target low density lipoproteins (LDL) labeled with (125)I or [(3)H]cholesterol linoleate to FcgammaRI or FcgammaRIIA in human macrophages. Interferon-gamma (IFN-gamma), which stimulates expression of FcgammaRI while inhibiting expression of SRA, was used to help delineate differences in metabolism between each receptor. For each receptor, the total amount of lipoprotein degradation paralleled changes in receptor expression induced by IFN-gamma. In particular, while SRA-mediated degradation typically exceeded degradation mediated by FcgammaRI, in IFN-gamma-treated cells degradation associated with FcgammaRI and SRA was similar. Assay of [(3)H]cholesterol linoleate-labeled lipoproteins indicated that total uptake and hydrolysis of [(3)H]cholesterol linoleate was similar for each class of receptor, and inhibited by IFN-gamma. For FcgammaRI versus FcgammaRIIA, in the presence or absence of IFN-gamma, the [(3)H]cholesterol derived from FcgammaRIIA-mediated uptake was preferentially targeted for re-esterification to [(3)H]cholesterol oleate, in comparison to that resulting from hydrolysis of [(3)H]cholesterol linoleate incorporated by selective uptake. For SRA, the formation of [(3)H]cholesterol oleate, which was substantial in control cells, was significantly inhibited in the presence of IFN-gamma. We conclude that there may be differences in cholesterol trafficking with respect to lipoprotein immune complex metabolism mediated by different classes of FcgammaRs.     

Heat-induced changes in the incorporation of [H3]acetate in membrane lipids

The effects of heat treatments at temperatures from 42 to 47 degrees C on the rate of incorporation of [3H]acetate into different classes of lipids have been studied in V-79 Chinese hamster cells. Thermotolerance induction and subtoxic heat treatments decreased the incorporation of [3H]acetate into phospholipids and caused the ratio [3H]cholesterol/[3H]phospholipids to increase several fold, and a positive correlation between heat dose and the ratio [3H]cholesterol/[3H]phospholipids was obtained for subtoxic hyperthermic treatments. The duration of this hyperthermic effect on the incorporation of [3H]acetate into the different lipid fractions was followed in pulse-label experiments. The highest increase of the ratio [3H]cholesterol/[3H]phospholipids was obtained during the first 24 h, but a significant elevation was also present for the 24-72 h pulse-labelled group. Thermotolerance induction was maximal 24 h after the heat treatment and then declined during the next 24 h. The increased [3H]cholesterol/[3H]phospholipid ratio observed in response to hyperthermia resembles the processes that serve to provide homeoviscous adaptation to sustain thermosensitive membrane-located functional groups, in analogy with the mechanisms responsible for thermal adaptation. However, the lack of a positive correlation between thermotolerance induction and the changes in lipid synthesis, for the whole time interval studied, remains to be further explored before any mechanistic interpretation of the data can be found.     

Lovastatin reversed the enhanced sphingomyelin caused by 27-hydroxycholesterol in cultured vascular endothelial cells

Statins have pleiotropic properties which are involved in inhibiting the thrombogenic response. In this study, the effects of lovastatin on two phospholipids, phosphatidylcholine and sphingomyelin, were studied in cultured endothelial cells in the presence of an oxysterol, 27-hydroxycholesterol. After the cells were cultured with 50 nM of lovastatin for 60 h, lovastatin was found to decrease the incorporation of [³H]choline into phosphatidylcholine and sphingomyelin, inhibited CTP: phosphocholine cytidylyltransferase (CT) activity without altering the activity of sphingomyelin synthase and neutral sphingomyelinase. And lovastatin was not found to have a direct inhibitive effect on activity of CT. Exogenous mevalonic acid or cholesterol reversed the reduction of cholesterol concentration that was caused by lovastatin, but had no significant effect on the diminished [³H]sphingomyelin by lovastatin. The increase of [³H]sphingomyelin by 27-hydroxycholesterol was not detected in the presence of lovastatin. These findings suggest that (1) lovastatin can reduce sphingomyelin content by means of inhibiting phosphatidylcholine synthesis; and (2) The decrease in sphingomyelin is not related to the diminished cholesterol concentration or mevalonate-derived intermediates. This inhibitive effect of lovastatin on sphingomyelin may benefit cellular calcification caused by sphingomyelin.     

Bile acid production in human subjects: rate of oxidation of [24,25-3H]cholesterol compared to fecal bile acid excretion

Bile acid production has been quantitated in seven subjects by methods that compare the results of two independent approaches, namely, quantitation of cholesterol side-chain oxidation and fecal bile acid excretion. Six hypertriglyceridemic (HT) subjects and one normolipidemic control were studied by both techniques. A further control subject was studied by the cholesterol side-chain oxidation method alone. Cholesterol side-chain oxidation was quantitated by measuring the appearance of 3H2O after intravenous administration of [24,25-3H]cholesterol, using multicompartmental analysis of plasma cholesterol and [3H]water specific activity. Body water kinetics were independently defined by use of oral D2O. Two HT subjects were restudied while they were taking cholestyramine, 16 g/day. In all ten studies, multicompartmental analysis closely simulated the observed appearance of 3H2O. Values obtained for bile acid production suggest that cholesterol oxidation, or bile acid input, was significantly greater than fecal bile acid output in the HT subjects (P less than 0.05). Cholesterol side-chain oxidation rates in the two normal subjects were lower than those encountered in HT subjects, being similar to published values for normal subjects both for bile acid synthesis as determined by isotope dilution kinetics and fecal bile acid excretion. Studies conducted with two, synthetically different, preparations of [24,25-3H]cholesterol indicated that, in one of the two preparations, approximately 20% of the tritium label was at positions proximal to C24. In the other preparation examined, all of the tritium was located at, or distal to, C24. Further studies revealed that 0.055-0.24% of the dose was present as labile tritium by virtue of its appearance as 3H2O following in vitro incubation with human plasma. Provided these isotope effects are taken into account, multicompartmental analysis of plasma [24,25-3H]cholesterol and body water appears to be a useful technique for quantitating cholesterol oxidation in human subjects.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

Steroid synthesis in rat brain cell cultures

Primary cultures derived from neonatal rat forebrains were established and cultured for several weeks. They grow entirely as glial cultures composed of oligodendrocytes and astrocytes. Glial cells undergo maturation and differentiation in culture. This was shown by measuring the oligodendroglial enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a specific marker for expression of oligodendrocyte differentiation. CNPase activity increased from days 10-21 of culture. Both cell types were characterized by indirect immunofluorescence staining using monoclonal antibodies to galactocerebroside (Gal C) and myelin basic protein (MBP) for oligodendrocytes, and glial fibrillary acidic protein (GFAP) for astrocytes. Using the above criteria, we measured about 60% oligodendrocytes and 40% astrocytes after 3 weeks of culture. Oligodendrocytes, expressing Gal C and MBP, were highly immunoreactive to monospecific polyclonal antibodies to the cytochrome P-450scc, enzyme involved in the synthesis of pregnenolone from cholesterol. After incubation of glial cultures with [3H]mevalonolactone in the presence of mevinoline and trilostane, biosynthesis of [3H]cholesterol, [3H]pregnenolone (P) and [3H]pregn-5-ene-3 beta, 20 alpha-diol (20-OHP) was demonstrated. Steroid biosynthesis was related to oligodendroglial differentiation, as the initial and rapid rate of increase in CNPase activity was found to occur at the same time as the onset of steroid synthesis. Both reached a maximum at 3 weeks of culture and remained stable for several weeks. Steroid synthesis was increased by dibutyryl cAMP (0.2 mM), as well as by dexamethasone (10 nM). When aminoglutethimide, a potent inhibitor of cytochrome P-450scc, was added during the incubation of cells with [3H]mevalonolactone, [3H]cholesterol accumulated in the cells. After the release of aminoglutethimide blockade, [3H]20-OHP was the major steroid produced and released in the culture medium. The demonstration of de novo steroid biosynthesis and of the cholesterol side-chain cleavage cytochrome P-450 in normal rat glial cells brings additional support to the concept of "neurosteroids".     

Effects of fibroblastic growth factor on protein degradation, the migration of non-histone proteins to the nucleus and DNA synthesis in diploid fibroblasts

Stimulation of resting WI38 cells, prelabeled with [3H]leucine, with fibroblastic growth factor (FGF) or serum, caused increased nuclear translocation of [3H]non-histone proteins [( 3H]NHP) and DNA synthesis, and a parallel decrease of proteolysis. [3H]NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5 showed that [3H]NHP fractions with high degradation rates in resting cells corresponded to the [3H]NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H]NHP are linked. FGF inhibited cellular uptake of [3H]chloroquine, suggesting that FGF inhibits NHP degradation via lysosomes. The lysosomotropic amine eserine had similar effects as FGF. It is proposed that FGF induces NHP migration to the nucleus by inhibiting their lysosomal degradation. FGF also caused migration of [3H]histones, however, the mechanism is not clear.     

Uptake and esterification of exogenous cholesterol by low-density-lipoprotein-receptor-negative human fibroblasts in culture.

The incorporation and metabolism of both vesicle- and LDL (low-density lipoprotein)- derived [3H]cholesterol by LDL-receptor-negative fibroblasts were studied. Independent of the cholesterol source, free [3H]cholesterol was readily incorporated into the cells and was available for esterification. 7-Oxocholesterol stimulated both [3H]cholesterol incorporation, by increasing the exchange rate, and the subsequent esterification of it irrespective of the source of exogenous [3H]cholesterol. The 7-oxocholesterol-stimulated esterification of exogenously derived LDL free [3H]cholesterol was progesterone-sensitive and energy-requiring.     

Cholesterol synthesis and esterification in cultured intestinal mucosa. Evidence for compartmentation

The current studies were undertaken to define the optimal conditions for measuring the absolute rates of cholesterol synthesis in cultured rabbit intestine and to assess whether the rate of sterol synthesis affects the esterification of locally formed or absorbed cholesterol. Using both [3H]water or [14C]octanoate (3 mM) as a precursor, sterol formation was linear during the 24 h culture, resulting in comparable estimates of the rate of synthesis equivalent to 129.5 and 118.7 nmol acetyl CoA incorporated per g per h, respectively. The presence of liposomal cholesterol or the hydroxymethylglutaryl-CoA reductase inhibitor mevinolin suppressed the rates of cholesterol synthesis by 24 and 92% of controls, respectively. Only 12% of total newly synthesized cholesterol was recovered in the medium and more than 97% was in the unesterified form, in both medium and biopsy. Even when the rate of sterol synthesis was stimulated over 90-fold by increasing concentrations of [14C]mevalonolactone, less than 8% of the label in total cholesterol was found in the sterol nucleus of the esterified cholesterol. Rather, the majority of the cholesterol ester-bound radioactivity was incorporated into the fatty acid moiety. On the other hand, there was only a limited decrease in the esterification of absorbed [3H]cholesterol both when the rate of sterol synthesis was increased with 10 mM mevalonolactone and when it was inhibited with mevinolin. The data suggest that locally synthesized and absorbed cholesterol is organized in distinct functional pools with different degrees of esterification in the mucosal epithelial cell.