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.     

Absolute rates of cholesterol synthesis in extrahepatic tissues measured with 3H-labeled water and 14C-labeled substrates.

This study was undertaken to develop techniques for measuring absolute rates of sterol synthesis in extrahepatic tissues in vitro and to estimate the magnitude of the errors inherent in the use of various 14C-labeled substrates for such measurements. Initial studies showed that significant errors were introduced when rates of synthesis were estimated using [3H]water since about 20 nmol of water were bound to each mg of tissue cholesterol isolated as the digitonide. This source of error could be eliminated by subtracting apparent incorporation rates obtained at 0 degrees C from those obtained at 37 degrees C or by regenerating and drying the free sterol. In a second set of experiments, the H/C incorporation ratio in cholesterol was determined in the liver by measuring the absolute rates of hydrogen and acetyl CoA flux into sterols. The ratio of 0.69 +/- 0.03 was found to be independent of the rate of hepatic cholesterol synthesis, the rate of hepatic acetyl CoA generation, or the source of the acetyl CoA. In a third set of studies, rates of incorporation of [3H]water or 14C-labeled acetate, octanoate, and glucose into digitonin-precipitable sterols were simultaneously measured in nine different extrahepatic tissues. Assuming that the H/C ratio measured in the liver also applied to these tissues, the [3H]water incorporation rates were multipled by the reciprocal of the H/C ratio to give the absolute rates of sterol synthesis in each tissue. When these were compared to the incorporation rates determined with the 14C-labeled substrates the magnitude of the errors in the rates of sterol synthesis obtained with these substrates in each tissue could be assessed. Only [14C]octanoate gave synthesis rates approaching 100% of those obtained with [3H]water and this occurred only in the intestine and kidney; in the other extrahepatic tissues this substrate gave rates of 6--66+ of the absolute rates. Rates of [14C]acetate incorporation in sterols varied from 4 to 62% of the [3H]water incorporation rates while those obtained with [14C]glucose were only 2--88% of the true rates. These studies document the large and highly variable errors inherent in estimating rates of sterol synthesis in extrahepatic tissues using 14C-labeled substrates under in vitro conditions.     

Tellurium Blocks Cholesterol Synthesis by Inhibiting Squalene Metabolism: Preferential Vulnerability to This Metabolic Block Leads to Peripheral Nervous System Demyelination

Inclusion of 1.1% elemental tellurium in the diet of postweanling rats produces a peripheral neuropathy due to a highly synchronous primary demyelination of sciatic nerve; this demyelination is followed closely by remyelination. Sciatic nerves from animals fed tellurium for various times were removed and incubated ex vivo for 1 h with [14C]acetate, and radioactivity incorporated into individual lipid classes was determined. In nerves from rats exposed to tellurium, there was a profound and selective block in the conversion of radioactive acetate to cholesterol. Another radioactive precursor, [3H]water, gave similar results. We suggest that tellurium feeding inhibits squalene epoxidase activity and that the consequent lack of cholesterol destabilizes myelin, thereby causing destruction of the larger internodes. Ex vivo incubation experiments were also carried out with liver slices. As with nerve, tellurium feeding caused accumulation in squalene of label from radioactive acetate, whereas labeling of cholesterol was greatly inhibited. Unexpectedly, however, incorporation of label from [3H]water into both squalene and cholesterol was increased. Relevant is the demonstration that liver was the primary site of bulk accumulation of squalene, which accounted for 10% of liver dry weight at 5 days. Thus, accumulation of squalene (and other mechanisms, possibly including up-regulation of cholesterol biosynthetic pathways) drives squalene epoxidase activity at normal levels in liver even in the presence of inhibitors of this enzyme. This is reflected by continuing incorporation of [3H]water into cholesterol; incorporation of this precursor takes place at many of the postsqualene biosynthetic steps for sterol formation. [14C]Acetate entering the sterol pathway before squalene in liver is greatly diluted in specific activity when it reaches the large squalene pool, and thus increased squalene epoxidase activity does not transfer significant 14C label to sterols. In contrast to the situation with liver, synthesis of sterols is markedly depressed in sciatic nerve, and squalene does not accumulate to high levels.     

Evidence for different isotopic enrichments of acetyl-CoA used for cholesterol synthesis in the liver and intestine: a study in the rat by mass fragmentography after intravenous infusion of [13C]acetate

Wistar rats were killed 4 h after an intravenous infusion of [1,2-13C]- and [1-14C]acetic acid sodium salt (39 mg, 12.5 microCi/ml, constant rate: 1.2 ml/h). At this time, labeled free cholesterol movements between the organs are still weak and cholesterol labeling in each tissue mainly originates from the in situ incorporation of the exogenous substrate. In male rats, the specific radioactivity of free cholesterol was found to be higher in the intestine (mucosa and wall) than in the liver and plasma. In female and in cholestyramine-fed male rats, cholesterol 14C labeling was close to that of male rats in the intestine, and was markedly higher in the liver. The same variations of 13C excess, calculated by mass fragmentography, indicated that there was no isotopic effect between 13C and 14C precursors. The advantage of this method consisted in obtaining the proportions of labeled molecules according to their molecular weight (M + 1-M + 11) for each sample. Then the distribution of 13C atoms in newly synthesized cholesterol was assessed in each sterogenesis site. In the intestine, about 3/4 of the 13C atoms were found in molecules of weight of at least M + 4 (after incorporation of at least two labeled acetate units). This proportion was only 1/3 in hepatic and plasma free cholesterol. These distinct 13C-labeling patterns clearly indicate that local variations occurred in the isotopic enrichment of acetyl-CoA used for cholesterol formation. Whatever the experimental conditions of this study, cholesterol was synthesized from an acetyl-CoA more 13C enriched in the intestine than in the liver. Such variations probably result from the different dilutions of exogenous acetyl-CoA by the endogenous pool in the liver and intestine. Consequently, the 14C or 13C incorporations measured in the liver and intestinal sterols do not account for absolute rates of cholesterol production by these organs. This study also indicated that after a few hours of infusion, free cholesterol labeling in the plasma originated mainly from cholesterol newly formed in the liver, even when acetate incorporation into cholesterol was higher in the intestine than in the liver.     

The isolation of 2,3-oxidosqualene from the liver of rats treated with 1-dodecylimidazole, a novel hypocholesterolaemic agent

1. Non-saponifiable lipid from the livers of rats treated with 1-dodecylimidazole contained an unidentified compound that was not present in the livers from untreated animals. 2. Treated rats had lower serum cholesterol concentrations than control rats. 3. 1-Dodecylimidazole, when added to rat liver slices, inhibited the incorporation of [1-(14)C]acetate and [2-(14)C]mevalonate into digitonin-precipitable sterols and resulted in the accumulation of a labelled compound, which was chromatographically identical with the unknown compound described in 1 above. 4. Rats treated with 1-dodecylimidazole incorporated less [(14)C]mevalonate into liver digitonin-precipitable sterols than untreated animals and accumulated the unknown compound as a labelled intermediate. 5. The unknown intermediate had the same chromatographic properties, n.m.r. and mass spectra as authentic 2,3-oxidosqualene. 6. The identity of the intermediate as 2,3-oxidosqualene was further established by showing that it was incorporated into sterols by rat liver homogenates under anaerobic conditions. In addition, incubation of [(14)C]squalene with rat liver homogenates resulted in trapping of the radioactivity by the added intermediate. 7. It is suggested that the hypocholesterolaemic activity of 1-dodecylimidazole results in part from the inhibition of cholesterol biosynthesis at the level of 2,3-oxidosqualene sterol cyclase.     

Cholesterol metabolism in the genetically hypercholesterolemic rat (RICO). I. Measurement of turnover processes

The rates of mobile cholesterol turnover processes were measured by the isotopic equilibrium method in normocholesterolemic (SW) and hypercholesterolemic homozygote (RICO) rats fed a semi-synthetic base diet containing 0.05% cholesterol. When the absorption rate is similar in SW and RICO rats, the internal secretion rate is 60% higher in RICO (25.3 mg/day) than in SW (16.2 mg/day). This increase is compensated by an increase in fecal excretion (RICO: 5 mg/day; SW: 3.8 mg/day), urinary excretion (RICO: 1.7 mg/day; SW: 1.1 mg/day) and above all the transformation of cholesterol into bile acids (RICO: 24.2 mg/day; SW: 15.3 mg/day). The fact that 70 minutes after [14C]acetate administration, the only variations obtained in RICO compared to SW rats are a doubled sterol radioactivity in the small intestine and a tripled one in the liver suggests that the increase in internal secretion of the RICO rat has both an intestinal and hepatic origin. This cholesterogenic stimulation in RICO rats takes place in the jejunum as well as in the ileum and in the crypt cells as well as in the villosities. It is concomitant with a doubled cholesterolemia, a doubled intestinal, caecal and colon bile acid pool and a 20% increase in the enterocyte protein content.     

Changes in sterol metabolism in the skin of developing chick embryo and alterations in the presence of an anticholesterolemic agent and a chemical carcinogen

Changes in sterol metabolism in the skin of chick embryo during its development were studied with embryonal chick skin and with the cultured skin tissues. Changes in sterol metabolism of the skin of chick embryo began to appear at day 17, as observed by the accumulation of dihydrolanosterol, and the ratio of dihydrolanostrol:cholesterol increased thereafter until hatching. A similar change in sterol metabolism was also observed with the cultured skin tissue of chick embryo, although the stages of development seem to have been delayed by 3 days. The active sterol metabolism of the cultured skin tissue was also confirmed by studies of incorporation of [2-14C]acetate into sterols. 20,25-Diazacholesterol almost completely inhibited the incorporation of [2-14C]acetate into C27 sterols, whereas a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide, inhibited the incorporation of [2-14C]acetate into lathosterol but not that into cholesterol.     

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.     

Discrimination between cholesterol and sitosterol for absorption in rats

The intestinal absorption of cholesterol and sitosterol was compared in rats. The intragastric administration of a single emulsified lipid meal containing either 50 mg of [4-14C]cholesterol or [4-14C]sitosterol resulted in the lymphatic absorption of 18.2% and 0.42% of each sterol, respectively, in 6 hr. This difference was unaltered when the mucosal sterol load was equalized by reducing the cholesterol to 1 mg in the emulsified lipid meal while maintaining the same sitosterol load or when the physical state in the lumen was equalized by infusion of a micellar solution containing both sterols into bile-diverted intestine. Lymphatic cholesterol was 90% esterified compared to 12% for sitosterol. Both sterols were associated predominantly (greater than 70%) with the chylomicron fraction. Eighty percent of the chylomicron cholesterol was recovered as ester with the core lipids, while 77% of the sitosterol was recovered as free sterol with the chylomicron coat. In mucosal homogenates at 6 hr, sitosterol recovery was one-eleventh that of cholesterol. When [3H]cholesterol (10 mg) and [14C]sitosterol (10 mg) were co-administered in an emulsified intragastric lipid meal, sitosterol associated with the brush border isolated 2 hr later was one-fifth that of cholesterol. Similar differences were seen when brush border membranes were incubated in vitro with micellar solutions containing either 50 microM [3H]cholesterol or [14C]sitosterol and the relative uptake of each sterol was unaffected by micellar phospholipid type (egg yolk phospholipids, phosphatidylcholine, or phosphatidylethanolamine).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for rate-limiting steps in sterol synthesis beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human leukocytes.

When human blood leukocytes are incubated with [2-14C]acetate only about 32% of the nonsaponifiable lipid radioactivity is recovered in digitonin-precipitable material. Using thin-layer chromatography and gas-liquid radiochromatography, we have determined that most of the label from [2-14C]acetate in the nonsaponifiable fractions is in lanosterol, squalene and an unidentified sterol. Only 11% of the acetate radioactivity is contained in cholesterol. This distribution does not change when cholesterol synthesis is depressed by the addition of lipoproteins to the medium. These findings are in marked contrast to studies with liver, where most of the nonsaponifiable radioactivity derived from acetate is recovered in digitonin-precipitable sterols. Furthermore, they suggest that rate-limiting steps beyond the 3-hydroxy-3-methylglutaryl coenzyme A reductase reaction exist in the sterol synthesis pathway of human leukocytes.     

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.     

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.     

Effect of Serum Lipoproteins on Growth and Sterol Synthesis in Cultured Rat Brain Glial Cells

Cells dissociated from brains of 1-day-old rats were cultured in medium containing either lipoprotein-deficient serum (LPDS) or LPDS plus various lipoprotein fractions. Increases in number of cells and in DNA content served as a measure of cell growth. Cholesterol synthesis was measured from the incorporation of [14C]acetate into total nonsaponifiable lipids and digitonin-precipitable sterols, and from the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. The data indicated that cholesterol biosynthesis from acetate was reduced in cells cultured in medium containing either LPDS plus low-density lipoproteins (LDL), high-density lipoproteins (HDL), or total lipoproteins (LP) and that this reduction was accompanied by a reduction in the activity of the HMG CoA reductase and an increase in the esterified sterol content. The reduction in cholesterol synthesis from acetate was maximal in cells cultured in the presence of HDL, whereas the maximal reduction in the activity of HMG CoA reductase occurred in cells cultured in the presence of LP. The presence of LDL or LP in the culture medium enhanced the cell growth but the presence of HDL did not. Esterified sterol content was highest in cells cultured in the medium containing LPDS plus LP and was not detected in cells cultured in LPDS medium. It is inferred from these data that rat brain glial cells in culture are able to utilize cholesterol in lipoproteins, that the presence of LDL in the medium enhances cell growth, and that reduced cholesterol synthesis in the presence of lipoproteins may occur at the HMG CoA reductase step as well as at some other step(s).     

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.     

Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.     

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.     

Cholesterol synthesis in rat intestine: effect of fasting and of porphyrogenic chemical allylisopropylacetamide

(a) Administration of allylisopropylacetamide to fasting rats stimulates intestinal sterol synthesis as measured by incorporation of ¹⁴C from [1-¹⁴C]sodium acetate. Stimulatory effect of AIA is confined to the acetate to mevalonate segment of cholesterol biosynthetic pathway.(b) It is also shown that the suppression of sterol synthesis in the ileum of intact rats produced by fasting is of the same order of magnitude as that observed for liver sterol synthesis due to fasting.     

Effect of α-p-chlorophenoxyisobutyrate on the metabolism of isoprenoid compounds in the rat

1. Feeding of alpha-p-chlorophenoxyisobutyrate (CPIB) to rats increased ubiquinone concentration in the liver but not in other tissues. The increase was progressive with the time of feeding and related to the concentration of CPIB in the diet. 2. Incorporation of [1-(14)C]acetate, but not of [2-(14)C]mevalonate, into sterols in the liver in vivo or by liver slices in vitro was decreased on feeding the rats with CPIB. However, incorporation of mevalonate into ubiquinone increased. 3. CPIB, when added in low concentrations to liver slices, had no effect on isoprene synthesis from acetate; higher concentrations, however, were inhibitory. 4. No activation of ubiquinone synthesis from mevalonate was observed when CPIB was added to the liver slices synthesizing ubiquinone. 5. The increase in ubiquinone in CPIB-fed animals appears to be due to increased synthesis in the initial stages and to decreased catabolism in the later stages. 6. An inverse relationship was found between the concentration of ubiquinone in the liver and the serum sterol concentration in CPIB-fed rats.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Adriamycin treatment increases 3-hydroxy-3-methylglutaryl CoA reductase and isoprene biosynthesis in the rat liver

Treatment of rats with Adriamycin caused an increase in the incorporation into hepatic cholesterol of [1-¹⁴C] acetate, but not of [2-¹⁴C] mevalonate. The step affected was found to be 3-hydroxy-3-methylglutaryl CoA reductase whose activity in the liver microsomes increased in Adriamycin-treated animals, but was inhibited when the drug was added in the assay medium. Also, the concentration of ubiquinone in the liver and of cholesterol in the plasma increased.