Evidence for the operation of the extrahepatic lipoprotein receptor system in vivo in rats. Effect of dietary cholesterol and orotic acid, alone or in combination, on the rate of synthesis of cholesterol and fatty acid in various tissues, measured by using 3H2O.

1. The biosynthesis of cholesterol in vivo was studied at a number of tissue sites in rats by using 3H2O as precursor. Overall, the mass of cholesterol synthesized was in good agreement with the rate of cholesterogenesis, as determined by kinetic analysis of cholesterol specific-radioactivity-time curves after administration of radiolabelled cholesterol. 2. Dietary cholesterol increased the circulating concentration of cholesterol and inhibited endogenous cholesterogenesis, with concomitant increases in the concentration of esterified cholesterol, in all tissues studied. Addition of ororic acid to the cholesterol-supplemented diet tended to reverse each of these changes in extrahepatic tissues. 3. The co-ordinated change in cholesterol biosynthesis and esterification, with no change in total cholesterol content, in extrahepatic tissues, is attributed to control by receptor-mediated lipoprotein uptake. 4. Further reduction of the concentration of the apoprotein B-containing lipoproteins by addition of orotic acid to a diet without supplementary cholesterol did not further enhance cholesterogenesis. We consider that the relatively high rates of extrahepatic cholesterogenesis in normolipidaemic rats are attributable to the low concentration of low-density lipoprotein.     

Effect of alterations of the specific activity of the intracellular acetyl CoA pool on apparent rates of hepatic cholesterogenesis

We have previously shown significant dilution of the specific activity of the intracellular acetyl CoA pool when radiolabeled acetate is used as the precursor in liver slice experiments. In the present study, using liver from animals subjected to various manipulations known to alter the rate of cholesterogenesis, the specific activity of the intramitochondrial acetyl CoA pool was 27-49% of the theoretical specific activity expected if no endogenous dilution occurred. Because the cytosolic acetyl CoA pool that gives rise to cholesterol is not in equilibrium with the intramitochondrial pool, these values cannot be used to correct the flux of labeled carbon from [(14)C]acetate into cholesterol. However, because [(14)C]octanoate is rapidly oxidized intramitochondrially to acetyl CoA, which feeds both the intra- and extramitochondrial metabolic pathways, [(14)C]octanoate can be utilized to determine true flux rates of C(2) units into cholesterol and other products. Using this substrate in liver slices from animals subjected to a variety of experimental manipulations, the specific activity of the intracellular acetyl CoA pool was 54-71% of the expected specific activity. After correction for endogenous dilution, the C(2) flux into cholesterol varied from 335 to 459 nmoles.g(-1).hr(-1) in control animals, was suppressed 10-40-fold in animals subjected to fasting and cholesterol feeding, and increased into the range of 1500 nmoles.g(-1).hr(-1) after derepression with cholestyramine feeding or biliary diversion. Data also are presented that show very good agreement between the corrected C(2) flux rate from octanoate into cholesterol and microsomal HMG CoA reductase activity in the same liver under conditions in which the synthetic rates were varied over a 100-fold range.     

Propionate inhibits hepatocyte lipid synthesis

Oat bran lowers serum cholesterol in animals and humans. Propionate, a short-chain fatty acid produced by colonic bacterial fermentation of soluble fiber, is a potential mediator of this action. We tested the effect of propionate on hepatocyte lipid synthesis in rats using [1-14C]acetate, 3H2O, and [2-14C]mevalonate as precursors. Propionate produced a statistically significant inhibition of cholesterol biosynthesis from [1-14C]acetate at a concentration of 1.0 mM and from 3H2O and [2-14C]mevalonate at concentrations of 2.5 mM. Propionate also produced a significant inhibition of fatty acid biosynthesis at concentrations of 2.5 mM using [1-14C]acetate as a precursor. The demonstration of propionate-mediated inhibition of cholesterol and fatty acid biosynthesis at these concentrations suggests that propionate may inhibit cholesterol and fatty acid biosynthesis in vivo and may mediate in part the hypolipidemic effects of soluble dietary fiber. Further studies are needed to clarify this action of propionate and to establish the exact mechanisms by which the inhibition occurs.     

A new relationship between cholesterolemia and cholesterol synthesis determined in rats fed an excess of cystine

The present study deals with an attempt to describe how the plasma cholesterol level is related to input into the plasma of cholesterol synthesized in the liver and in the intestine. It has previously been shown in our laboratory that, for a given absorption of alimentary cholesterol, the rat plasma cholesterol level decreases when internal secretion of cholesterol (cholesterol synthesized in the organs and poured into the plasma) increases. This relationship was established using rats in which the major source of cholesterol synthesis was the intestine. We used rats fed a cystine-enriched diet (5%) which was previously shown to increase cholesterolemia and internal secretion of cholesterol. It was first demonstrated that a significant positive linear correlation exists between individual values of cholesterolemia and those of internal secretion of cholesterol. Secondly, using [14C]acetate as the cholesterol precursor it was shown that ingestion of the cystine-enriched diet increased hepatic but not intestinal cholesterogenesis. Individual values of cholesterolemia were linearly correlated to those of [14C]acetate incorporation into the hepatic sterols. Results obtained by this method were validated by determining the 13C-labeling pattern of cholesterol synthesized de novo by the liver and the intestine after [13C]acetate infusion. Indeed, this labelling indicated that the dilution of exogenous acetyl-CoA in the liver was not changed by cystine feeding, whereas that in the intestine was enhanced. It is concluded that the plasma cholesterol level varies with internal cholesterol secretion, depending on the organ which determines the variations of this secretion: it decreases when intestinal cholesterogenesis increases, whereas it increases when hepatic cholesterogenesis increases. Finally, the use of [14C]acetate coupled with lipoprotein analysis in rats fed the cystine-enriched diet, in control rats and in rats fed a cholesterol-enriched diet, allowed a new linear correlation to be demonstrated: between cholesterol concentration in LDL2 (lipoproteins of density 1.040-1.063 g/ml) and [14C]acetate incorporation into liver sterols. Our results suggest that LDL2 are produced by the liver in relation to cholesterogenesis in this organ.     

The role of cytochrome P-450 in cholesterol biogenesis and catabolism

1. Adjuvant-induced arthritis in rats is accompanied by a loss of activity of the drug-metabolizing enzyme system and a decrease in hepatic cytochrome P-450. 2. Arthritic rats have normal serum and liver cholesterol concentrations. 3. The rate of biogenesis of cholesterol in vivo and in vitro from either [(14)C]acetate or [(14)C]mevalonate in arthritic rats was the same as or greater than that found in control rats. 4. Treatment of rats with carbon disulphide (1ml/kg) resulted in a loss of drug-metabolizing-enzyme activity and increased cholesterol biogenesis. 5. The activity of cholesterol 7alpha-hydroxylase in adjuvant-induced arthritic rats did not differ significantly from that in control rats. 6. Rats fed with cholestyramine had an elevated hepatic cholesterol 7alpha-hydroxylase activity, but neither the concentration of cytochrome P-450 nor the activity of the drug-hydroxylating enzyme, aminopyrine demethylase, was affected. 7. The relationships between drug hydroxylation and cholesterol metabolism are discussed.     

The interaction of various control mechanisms in determining the rate of hepatic cholesterogenesis in the rat.

In these studies rats were subjected to diurnal light-cycling, stress, fasting and the feeding of cholestyramine, beta-sitosterol and cholesterol in various combinations. In control animals exposed to light cycling for 2 weeks the rate of hepatic cholesterogenesis was 3.7 fold higher in the mid-dark than in the mid-light phase of the light cycle. The magnitude of this difference varied with the duration of light cycling and the size of the animals. Similarly, enhanced rates of cholesterol synthesis were seen in the mid-dark phase relative to the mid-light phase of the light cycle in rats where the base-line level of hepatic cholesterogenesis was increased by feeding cholestyramine (1.6-fold) or beta-sitosterol (2.9-fold) or was depressed by fasting (19-fold) or cholesterol feeding (2.1-fold). Restraining animals for 48 h also increased the rate of cholesterol synthesis in the liver; in control animals, this stress enhanced the level of cholesterogenesis seen at both the mid-light and mid-dark phases of the light cycle. In addition, both the effects of stress and of diurnal light cycling could be identified in groups of animals where base-line cholesterogenic activity was varied by fasting or by feeding cholestyramine, beta-sitosterol or cholesterol. These studies illustrate the complexity of the control of hepatic cholesterol synthesis and suggest that the final rate of cholesterogenesis may be the result of several different effectors modifying by different mechanisms the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase.     

The mechanism and regulation of dolichyl phosphate biosynthesis in rat liver

Rat liver slices were pulse labeled for 6 min with [3H]mevalonolactone and then chased for 90 min with unlabeled mevalonolactone in order to study the mechanism of dolichyl phosphate biosynthesis. The cholesterol pathway was also monitored and served to verify the pulse-chase. Under conditions in which radioactivity in the methyl sterol fraction chased to cholesterol, radioactivity in alpha-unsaturated polyprenyl (pyro)-phosphate chased almost exclusively into dolichyl (pyro)phosphate. Lesser amounts of radioactivity appeared in alpha-unsaturated polyprenol and dolichol, and neither exhibited significant decline after 90 min of incubation. The relative rates of cholesterol versus dolichyl phosphate biosynthesis were studied in rat liver under four different nutritional conditions using labeled acetate, while the absolute rates of cholesterol synthesis were determined using 3H2O. From these determinations, the absolute rates of dolichyl phosphate synthesis were calculated. The absolute rates of cholesterol synthesis were found to vary 42-fold while the absolute rates of dolichyl phosphate synthesis were unchanged. To determine the basis for this effect, the rates of synthesis of cholesterol and dolichyl phosphate were quantitated as a function of [3H]mevalonolactone concentration. Plots of nanomoles incorporated into the two lipids were nearly parallel, yielding Km values on the order of 1 mM. In addition, increasing concentrations of mevinolin yielded parallel inhibition of incorporation of [3H]acetate into cholesterol and dolichyl phosphate. The specific activity of squalene synthase in liver microsomes from rats having the highest rate of cholesterol synthesis was only 2-fold greater than in microsomes from rats having the lowest rate. Taken together, the results suggest that the maintenance of constant dolichyl phosphate synthesis under conditions of enhanced cholesterogenesis is not due to saturation of the dolichyl phosphate pathway by either farnesyl pyrophosphate or isopentenyl pyrophosphate but coordinate regulation of hydroxymethylglutaryl-CoA reductase and a reaction on the pathway from farnesyl pyrophosphate to cholesterol.     

Acetoacetate and glucose as substrates for lipid synthesis by rat brain oligodendrocytes and astrocytes in serum-free culture

We have compared glucose and acetoacetate as precursors for lipogenesis and cholesterogenesis by oligodendrocytes and astrocytes, using mixed glial cultures enriched in oligodendrocytes. In order to differentiate between metabolic processes in oligodendrocytes and those in astrocytes, the other major cell type present in the mixed culture, we carried out parallel incubations with cultures from which the oligodendrocytes had been removed by treatment with anti-galactocerebroside serum and guinea-pig complement. The following results were obtained: 1. Both oligodendrocytes and astrocytes in culture actively utilize acetoacetate as a precursor for lipogenesis and cholesterogenesis. 2. In both cell types, the incorporation of acetoacetate into fatty acids and cholesterol exceeds that of glucose by a factor of 5-10 when the precursors are present at concentrations of 1 mM and higher. 3. Glucose stimulates acetoacetate incorporation into fatty acids and cholesterol, whereas acetoacetate reduces the entry of glucose into these lipids. This suggests that glucose is necessary for NADPH generation, but that otherwise the two precursors contribute to the same acetyl-CoA pool. 4. Both with acetoacetate and with glucose as precursor, oligodendrocytes are more active in cholesterol synthesis than astrocytes. 5. Using incorporation of 3H2O as an indicator for total lipid synthesis, we estimated that acetoacetate contributes one third of the acetyl groups and glucose one twentieth when saturating concentrations of both substrates are present.     

Effect of dietary tomatine on cholesterol metabolism in the rat

Tomatine is a virtually nonabsorbable saponin which has been used as an antifungal agent and analytically as a cholesterol precipitant. It was used in this study to determine whether or not it can form a complex with cholesterol in vivo in the rat intestine and what effects such complex formation would have on cholesterol metabolism. Rats that were fed tomatine as 1% of the diet had a decreased uptake of dietary cholesterol by the liver, an increased rate of hepatic and intestinal cholesterol synthesis as well as a partial offsetting of the dietary cholesterol-induced decrease in hepatic cholesterogenesis, and an apparent increase in sterol excretion without an effect on bile acid excretion. In vitro, tomatine did not sequester cholic acid as did cholestyramine. The results show that tomatine has an effect on cholesterol absorption and on other aspects of lipid metabolism in the rat similar to that of cholestyramine, with the notable exception that tomatine increased sterol excretion while cholestyramine increased bile acid excretion. It was suggested that tomatine forms a nonabsorbable complex with cholesterol in the rat intestine.     

Roles of nuclear receptors in the up-regulation of hepatic cholesterol 7alpha-hydroxylase by cholestyramine in rats

Nuclear receptors are involved in regulating the expression of cholesterol 7alpha-hydroxylase (CYP7A1), however, their roles in the up-regulation of CYP7A1 by cholestyramine (CSR) are still unclear. In the present study, male Wistar rats were divided into four groups and fed [high sucrose + 10% lard diet] (H), [H + 3% CSR diet] (H + CSR), [H + 0.5% cholesterol + 0.25% sodium cholate diet] (C), or [C + 3% CSR diet] (C + CSR) for 2 weeks. Cholestyramine decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but had no effect on these parameters in rats fed H-based diets. Cholestyramine raised hepatic levels of CYP7A1 mRNA and activity in both groups. The gene expression of hepatic ATP-binding cassettes A1 and G5, regulated by liver X receptor (LXR), were unchanged and down-regulated by cholestyramine, respectively. The mRNA levels of the hepatic ATP-binding cassette B11 and short heterodimer partner (SHP), regulated by farnesoid X receptor (FXR), were not changed by cholestyramine. C-based diets, which contained cholesterol and cholic acid, increased SHP mRNA levels compared to H-based diets. Consequently, in rats fed the C+CSR diet, hepatic FXR was activated by dietary bile acids, but the hepatic CYP7A1 mRNA level was increased 16-fold compared to that in rats fed an H diet. These results suggest that cholestyramine up-regulates the expression of CYP7A1 independently via LXR- or FXR-mediated pathways in rats.     

Studies on the regulation of glycoprotein biosynthesis. An investigation of the rate-limiting steps of dolichyl phosphate biosynthesis.

The possible role of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the rate-controlling enzyme of cholesterol biosynthesis) in regulating the rate of dolichyl phosphate biosynthesis in rat liver was investigated. Rats were either fasted 48 h or fed diets supplemented with the drug cholestyramine. The activity of HMG-CoA reductase was 5000-fold greater in liver from cholestyramine-fed rats as compared to fasted rats. The activity of dolichyl phosphate synthetase, the prenyl transferase responsible for the biosynthesis of dolichyl phosphate from farnesyl pyrophosphate and isopentenyl pyrophosphate, was similar in both nutritional conditions and was markedly less active than HMG-CoA reductase even in the fasted state. Acetate incorporation into cholesterol was 2200-fold greater in liver slices from cholestyramine-fed rats as compared to fasted rats. By contrast, acetate incorporation into dolichyl phosphate was only 6-fold higher. Further studies suggested that the levels of farnesyl pyrophosphate and isopentenyl pyrophosphate are several hundred-fold greater in liver from cholestyramine-treated rats. From these results, it is concluded that the rate of dolichyl phosphate biosynthesis in rat liver is not regulated by the activity of HMG-CoA reductase but is probably regulated at the level of dolichyl phosphate synthetase.     

Critical analysis of the use of 14C-acetate for measuring in vivo rat cholesterol synthesis

The bulk of cholesterol produced by the liver and the gut enters the mobile pool of body cholesterol. This process is called internal secretion in contrast with the fraction of biosynthesized cholesterol directly eliminated in the feces (fecal external secretion). In rats, under various conditions, a linear relationship was found between the rates of internal secretion measured by the isotope equilibrium method (range: 10-60 mg/day) and the sum of sterol radioactivities measured in liver and intestine 70 min after a [14C]-acetate pulse. In fact, a better correlation was found between the radioactivities of liver sterols and the values for internal secretion. In this new relationship, the ordinate at the origin corresponds to a minimal internal secretion of about 10 mg/day, which implies an important extrahepatic cholesterol production, probably from the gut. Indeed, in adult male rats, fed a semi-purified sucrose-rich diet, the relative contribution of this organ to the internal secretion was higher than in adult rats fed a commercial diet and higher than in young animals, whatever the circadian period. It can be concluded that some of the discrepancies observed in the literature about the relative participation of the intestine and the liver in the internal secretion of cholesterol are probably due to differences in experimental and nutritional conditions (age and sex of the animals, diet composition, time of the circadian cycle) rather than to the cholesterol precursor used (3H2O or [14C] acetate) to assess the activity of cholesterol synthesis. Indeed, a comparative study of 3H2O and [14C]acetate incorporation into sterols of enterocytes indicated the same crypt-villus radioactive gradient, regardless of the intestinal site studied (duodenum, jejunum or ileum) and thus validated the use of [14C]acetate. Other experiments however, showed evidence of some local differences in the cytosolic dilution of labeled acetyl CoA by the endogenous cholesterol precursor in rats under various conditions (control or cholestyramine-enriched diet, parenteral nutrition). After intravenous infusion of 1,2[13C]acetate, mass fragmentography of free cholesterol isolated from liver and intestine indicated different 13C-labeling patterns of newly synthesized molecules. They indicate that cholesterol is generally synthesized from acetyl CoA with a lower 13C-content in the liver than in the intestine. The local endogenous flow of acetyl CoA used for cholesterol synthesis was about 2-fold higher in the hepatocytes than in the enterocytes. This conclusion was confirmed by the results obtained with several experimental groups exhibiting a large range of both internal secretion of cholesterol and sterol radioactivities in liver and intestine after [14C]acetate injection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Utilization of endogenous and exogenous sources of substrate for cholesterol biosynthesis by isolated hepatocytes

The rates of cholesterol biosynthesis in isolated rat hepatocytes were determined by using a method based on measurement of the rate of formation of desmosterol (cholesta-5,24-dien-3beta-ol), which accumulates during inhibition of cholesterogenesis by the drug triparanol. Incubation of cells from normal or 24h-starved animals in a medium containing albumin, glucose, amino acids and acetate as the only organic constituents led to an accelerating rate of sterol formation during the earlier stages of a 6h incubation period. The contribution of exogenously added acetate (initial concentration 3.34mm) to sterol synthesis in both types of cells reached an early maximum and then continually declined. Exogenously added pyruvate and lactate were more efficient sources of sterol carbon than was acetate. Exogenous glucose even at relatively high concentrations (11.1mm) was incapable of providing more than 6% of the total sterol carbon. Although the proportion of total sterol carbon supplied from exogenous acetate increased with increasing concentrations of the extracellular substrate, the rates of total sterol synthesis in both types of cell remained unchanged. Similar observations were made when lactate or pyruvate was the cholesterogenic precursor in normal cells. These studies suggest that, although exogenous substrates were capable of expanding an intracellular pool of cholesterol precursor, the normal supply of intermediary metabolites was not rate-limiting for cholesterogenesis.     

The structure of an inhibitor of cholesterol biosynthesis isolated from barley

Purification of the oily, nonpolar fraction of high protein barley (Hordeum vulgare L.) flour by high pressure liquid chromatography yielded 10 major components, two (I, II) of which were potent inhibitors of cholesterogenesis in vivo and in vitro. The addition of purified inhibitor I (2.5-20 ppm) to chick diets significantly decreased hepatic cholesterogenesis and serum total and low density lipoprotein cholesterol and concomitantly increased lipogenic activity. The high resolution mass spectrometric analysis and measurement of different peaks of inhibitor I gave a molecular ion at m/e 424 (C29H44O2) and main peaks at m/e 205, 203, and 165 corresponding to C13H17O2, C13H15O2, and C10H13O2 moieties, respectively. which are characteristic of d-alpha-tocotrienol. This identification was confirmed against synthetic samples. The tocotrienols are widely distributed in the plant kingdom and differ from tocopherols (vitamin E) only in three double bonds in the isoprenoid chain which appear to be essential for the inhibition of cholesterogenesis.     

Effect of diosgenin on lipid metabolism in rats.

The purpose of this study was to determine whether diosgenin suppresses cholesterol absorption in rats, and to examine relevant changes in cholesterol and bile acid metabolism. Diosgenin fed with the diet for 1 week inhibited cholesterol absorption as determined by the serum isotope ratio technique, as well as by measuring in the feces the amount of unabsorbed radioactivity from orally administered [3H]cholesterol. In addition, diosgenin suppressed the serum and liver uptake of radioactivity from co-administered [3H]cholesterol as well as the accumulation of liver cholesterol in the cholesterol-fed rat; diosgenin was substantially more active than cholestyramine or beta-sitosterol. In vitro, diosgenin had no effect on the activity of rat pancreatic esterase. Diosgenin decreased the elevated cholesterol in serum LDL and elevated cholesterol in the HDL fraction of cholesterol-fed rats; diosgenin had no effect on serum cholesterol in normocholesterolemic rats. In contrast to cholestyramine, diosgenin markedly increased neutral sterol excretion without altering bile acid excretion; in vitro, diosgenin had no effect on bile acid binding. Diosgenin treatment increased hepatic and intestinal cholesterol synthesis as well as the activity of hepatic HMG CoA reductase. This was accompanied by increased biliary concentration of cholesterol, but not of bile acids. Diosgenin had no effect on cholesterol synthesis when added to normal rat liver homogenates. It was concluded that diosgenin interferes with the absorption of cholesterol of both exogenous and endogenous origin; such interference is accompanied by derepressed, i.e., increased, rates of hepatic and intestinal cholesterol synthesis. The increased unabsorbed cholesterol together with enhanced secretion of cholesterol into bile resulted in increased excretion of neutral sterols without affecting the biliary and fecal excretion of bile acids.     

In vitro labeling of peroxisomal cholesterol with radioactive precursors

Peroxisomes isolated from rat liver were incubated with [³H]squalene and [³H]mevalonate and the subsequent incorporation of radioactivity into cholesterol studied. The isolated lipids became labeled after incubation with both precursors. In contrast to findings with microsomes, trypsin and detergent treatment of peroxisomes did not influence the rate of cholesterol synthesis. In addition, the luminal content of peroxisomes could alone mediate this synthetic process. Upon treatment of rats with various inducers of peroxisomes and of the endoplasmic reticulum, as well as upon feeding with cholesterol and cholestyramine, large differences in the pattern ofin vitro incorporation of [³H]mevalonate into the cholesterol of peroxisomes and microsomes were observed. Injection of this precursor also resulted in high initial labeling of peroxisomal cholesterolin vivo. These experiments indicate that cholesterol synthesis may also occur in peroxisomes.     

Lacto-N-neotetraosylceramide ("paragloboside") as a possible tumor-associated surface antigen of hamster NILPY tumor.

Isolated rat hepatocytes from fed and starved rats synthesized net glucose from various precursors at similar rates. [3-¹⁴C]-lactate incorporation into glucose was also similar in hepatocytes from fed and starved rats, as was ketone body formation from oleate and octanoate. Rates of gluconeogenesis in hepatocytes from fed rats compare to rates seen in perfused livers from starved rats rather than perfused livers from fed rats. Thus metabolic rates and possibly controls may be different between perfused livers and isolated hepatocytes when using fed rats.     

Effect of Oxysterol Treatment on Cholesterol Biosynthesis and Reactive Astrocyte Proliferation in Injured Rat Brain Cortex

Abstract: We have reported previously that oxysterols inhibit astrogliosis and intracranial glioblastoma growth. To elucidate the mechanism of action of these molecules in vivo, we have investigated their effect on the cholesterol biosynthesis in the injured brain. In a bilateral lesion model, injection of liposomes containing 7β-hydroxycholesterol decreased [³H]acetate incorporation into neutral lipids and cholesterol by 30% and 40%, respectively. Structural analogues were tested using a unilateral lesion model. The injury did not significantly affect cholesterogenesis; injection of 7β-hydroxycholesterol or 7β-hydroxycholesteryl-3-oleate reduced acetate incorporation into cholesterol by 47% and 43%, respectively. Both 7-ketocholesteryl-3-oleate and 7α-hydroxycholesteryl-3-oleate inhibited cholesterogenesis by 32%. As cholesterol and by-products of the cholesterol pathway play a key role in cell division, we have assessed the effect of oxysterols on reactive astrocyte proliferation. The incorporation of bromodeoxyuridine showed that up to 46% of astrocytes were proliferating 24 h after the injury. Injection of 12 nmol of 7β-hydroxycholesterol or 7β-hydroxycholesteryl-3-oleate reduced the labelling index to 26%, whereas the labelling index in the 7-ketocholesteryl-3-oleate-treated cortex was 37%. These findings demonstrate that oxysterols are potent inhibitors of the endogenous cholesterol biosynthesis in brain and show a correlation between cholesterogenesis and reactive astrocyte proliferation.     

Diurnal rhythm of rat liver mRNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase. Correlation of functional and total mRNA levels with enzyme activity and protein

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase exhibits a diurnal rhythm of activity which coincides with a diurnal rhythm of reductase protein and reductase mRNA levels. This diurnal rhythm of reductase activity, polypeptide mass, and mRNA exists in rats fed a normal diet (unsupplemented rat chow) and in rats fed a diet supplemented with cholestyramine plus or minus mevinolin. Levels of reductase protein were determined by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Reductase mRNA was measured by in vitro translation or blot hybridization of liver RNA. Functional reductase mRNA levels in rats fed a normal diet were approximately 10-fold higher during the middle of the dark cycle than during the middle of the light cycle. Maximum induction of functional reductase mRNA was observed in rats fed cholestyramine and mevinolin. This latter level was 157-fold higher than the level measured at the diurnal low point in rats fed a normal diet. Blot hybridization of liver RNA showed two predominant mRNAs of 4.6 and 4.2 kilobase pairs and a minor species at 6.9 kilobase pairs. These mRNAs exhibited a diurnal rhythm for rats on all three diets and reached peak levels during the 12-h dark period. These data indicate that the diurnal rhythm of reductase mass and activity is closely paralleled by the level of its mRNA.     

The activity of 3-hydroxy-3-methylglutaryl-CoA reductase and acyl-CoA: cholesterol acyltransferase in hepatic microsomes from male, female and pregnant rats. The effect of cholestyramine treatment and the relationship of enzyme activity to microsomal lipid composition

The relationship of microsomal cholesterol and phospholipid fatty acid composition to the activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-CoA: cholesterol acyltransferase was investigated in male, female virgin and pregnant rats when hepatic cholesterogenesis was stimulated by cholestyramine. Cholestyramine increased HMG-CoA reductase activity in both sexes but had no effect on microsomal free cholesterol level or acyl-CoA: cholesterol acyltransferase activity. The data suggest that during cholestyramine treatment high rates of bile acid synthesis are supported by preferential channelling of cholesterol into this pathway, whilst the substrate pool and activity of acyl-CoA:cholesterol acyltransferase are maintained unaltered. The lack of a consistent relationship among enzyme activities and microsomal lipid composition infers that HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase are regulated in vivo by independent mechanisms which are unlikely to involve modulation by the physical properties of the microsomal lipid.