Digitonin-precipitable Sterols Research Articles

Effects of diet and metamorphosis upon the sterol composition of the butterfly Morpho peleides

Whole body sterol metabolism in insects has seldom been studied. We were able to design an appropriate study at a butterfly farm in Belize. We collected six larvas of butterfly (Morpho peleides), their food (leaves of Pterocarpus bayessii), and their excretions. In addition, six adult butterflies were collected. The sterols of the diet, the larva, and adult butterfly were analyzed by gas-liquid chromatography. The structures of these sterols were identified by digitonin precipitation, GC-MS, and NMR. Four sterols (cholesterol, campesterol, stigmasterol, and sitosterol) and a sterol mixture were found in the food, the body, and the excreta of the larva. The tissue sterol content of the larva was 326 microg. They consumed 276 microg of sterols per day. Their excretion was 185 microg per day as sterols. The total tissue sterol contents of the larva and butterfly were similar, but they had different sterol compositions, which indicated interconversion of sterols during development. There was a progressive increase in the cholesterol content from larva to butterfly and a decrease in the content of sitosterol and other plant sterols, which were likely converted to cholesterol. Our data indicated an active sterol metabolism in butterfly larva. Diet played an important role in determining its sterol composition. During metamorphosis, there was an interconversion of sterols. This is the first paper documenting the fecal sterol excretion in insects as related to dietary intakes.

Overexpression of estrogen receptor α increases hepatic cholesterogenesis, leading to biliary hypersecretion in mice

We explored whether there is an "estrogen-ERalpha-SREBP-2" (for estrogen-estrogen receptor subtype alpha-sterol-regulatory element binding protein-2) pathway for regulating hepatic cholesterol biosynthesis in ovariectomized AKR mice treated with 17beta-estradial (E2) at 6 microg/day or E2 plus the antiestrogenic agent ICI 182,780 at 125 microg/day and on chow or fed a high-cholesterol (1%) diet for 14 days. To monitor changes in cholesterol biosynthesis and newly synthesized cholesterol secreted into bile, incorporation into digitonin-precipitable sterols in mice treated with 25 mCi of [3H]water was measured in extracts of liver and extrahepatic organs 1 h later and in hepatic biles 6 h later. ERalpha upregulated SREBP-2, with resulting activation of SREBP-2-responsive genes in the cholesterol biosynthetic pathway. The E2-treated mice continued to synthesize cholesterol in spite of its excess availability from high dietary cholesterol, which reflects a loss in controlling the negative feedback regulation of cholesterol synthesis. These alterations augmented biliary cholesterol secretion and enhanced the lithogenicity of bile. However, these lithogenic effects of E2 were fully blocked by ICI 182,780. We conclude that during estrogen treatment, more newly synthesized cholesterol determined by the estrogen-ERalpha-SREBP-2 pathway is secreted into bile, leading to biliary cholesterol hypersecretion. These studies provide insights into therapeutic approaches to cholesterol gallstones in high-risk subjects, especially those exposed to high levels of estrogen.

Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.

Native and Partially Hydrolyzed Psyllium Have Comparable Effects on Cholesterol Metabolism in Rats

This study was conducted to determine whether the storage conditions and the levels of psyllium in the diet modulate its hypocholesterolemic effects. Seventy-five male Sprague-Dawley rats, age 90 d, were randomly divided into five treatment groups and were fed cholesterol-containing diets for 21 d. Diets included 10% cellulose (control); 5 or 10% psyllium stored 8 mo at 5 degrees C (PS5); or 5 or 10% psyllium stored 8 mo at 40 degrees C (PS40). The higher storage temperature caused a gradual decrease in molecular weight of the psyllium, as measured by changes in solution viscosity. Hepatic rates of sterol synthesis were significantly (P < 0.001) higher in all of the psyllium-fed rats compared with control rats [21 +/- 2, 312 +/- 35, 464 +/- 40, 328 +/- 49 and 439 +/- 57 nmol [3H]digitonin-precipitable sterol (DPS)/(g liver x h), respectively, for control, 5% PS5, 10% PS5, 5% PS40 and 10% PS40]. A similar trend was observed in intestinal rates of sterol synthesis, and the difference was significant (P < 0.05) for all treatment groups except the 5% PS5-fed group compared with the control group. Liver total cholesterol and total lipid concentrations were significantly lower in all psyllium-fed rats compared with controls. There were no significant differences in serum total cholesterol concentrations among the psyllium-fed groups, although serum cholesterol levels in both the PS5-fed groups were significantly (P < 0.05) lower than that in the control group (2.66 +/- 0.18, 2.62 +/- 0.15 and 3.26 +/- 0.12 mmol/L, respectively, for 5% PS5, 10% PS5 and control). Serum triglyceride and HDL cholesterol concentrations did not vary significantly among groups. The findings of this study indicate that the cholesterol-lowering activity of psyllium is unaltered by storage conditions shown to cause a moderate degree of hydrolysis.

Psyllium Feeding Increases the Rates of Hepatic and Intestinal Sterol Biosynthesis While Lowering the Plasma and Hepatic Cholesterol Levels in Rats

Previous studies have shown that psyllium (PS) lowers plasma cholesterol and alters hepatic sterol metabolism. We conducted a dose response study to further explore the effects of PS on hepatic and intestinal sterol biosynthesis. Forty five male Sprague-Dawley rats, aged 90 days, were divided into three treatment groups (15 rats/group). Rats were fed ad libitum diets (isocaloric and isonitrogenous) and had free access to deionized water. Rats in group 1 (control) were fed a diet containing 10% cellulose as the source of dietary fiber; group 2 rats received a diet containing 5% PS and 5% cellulose; and group 3 rats were given a diet containing 10% PS. After 21 days of treatment, at the midpoint of the dark cycle, within a 1-h period, rats were lightly anesthetized and were rapidly injected with 1.85 GBq (50 mCi) of tritiated water ([3H]H2O) in 0.5ml of isotonic NaCl into the femoral artery of each rat. Sixty minutes later, the animals were anesthetized and blood was collected from the abdominal aorta for measuring plasma water activity and lipid and cholesterol analyses. The liver and intestine were immediately removed for determining the rates of [3H]H2O incorporation into digitonin-precipitable sterols (DPS). Liver total cholesterol and total lipids were significantly lower in rats fed the diet containing either 5% or 10% PS compared with those of the 10% cellulose. Animals fed both the 5% and 10% PS diets had significantly lower serum total cholesterol (126±4.6, 103±7.0, 101±5.7mg/dl, respectively, for the 10% cellulose, 5 and 10% PS diets), but not serum HDL or triglyceride levels, than the 10% cellulose diet. Hepatic rates of sterol biosynthesis were significantly higher with PS feeding in a dose-dependent manner (21±2, 312±34.6, 463±40.0, nmol [3H]H2O incorporation into DPS/g liver per hour for 0, 5 and 10% PS in diets, respectively). The effect of PS on intestinal biosynthesis of sterols was less pronounced (68±26, 86±34.5, 113±54.5nmol [3H]H2O incorporation into DPS/g intestine per hour for 0, 5 and 10% PS in diets, respectively). The results of this study indicate that the presence of PS in the diet may cause depletion of liver cholesterol, conceivably, by enhancing bile acid synthesis, and/or increasing fecal elimination of acidic and neutral sterols, thus resulting in higher rates of sterol biosynthesis. Although the maximal effect on plasma cholesterol levels was observed with 5% PS, further enhancement of hepatic sterol biosynthesis by 10% PS demonstrates the sensitivity of liver to changes in PS levels.

Bile acid derived HMG-CoA reductase inhibitors

The target organ for HMG-CoA reductase inhibitors to decrease cholesterol biosynthesis in hypercholosterolemic patients is the liver. Since bile acids undergo an enterohepatic circulation showing a strict organotropism for the liver and the small intestine, the structural elements of an inhibitor for HMG-CoA reductase were combined with those for specific molecular recognition of a bile acid molecule for selective uptake by hepatocytes. Either, the HMG-CoA reductase inhibitors HR 780 and mevinolin were covalently attached to 3 ξ-( ω-aminoalkoxy)-7 α, 12 α-dihydroxy-5 β-cholan-24-oic acids to obtaine bile acid prodrugs, of the side chain of bile acids at C-17 was replaced by 3,5-dihydroxy-heptanoic acid — a structural element essential for inhibition of HMG-CoA reductase — to obtain hybrid bile acid: HMG-CoA reductase inhibitors. The prodrugs could, as expected, not inhibit rat liver HMG-CoA reductase to a significant extent, whereas the hybrid inhibitors showed a stereospecific inhibition of HMG-CoA reductase from rat liver microsomes with an IC 50-value of 0.7 μM for the most potent compound S 2467 and 6 μM for its diastereomere S 2468. Uptake measurements with isolated rat hepatocytes and ileal brush-border membrane vesicles from rabbit small intestine revealed a specific interaction of both classes of bile acid-derived HMG-CoA reductase inhibitors with the hepatocyte and ileocyte bile acid uptake systems. Photoaffinity labeling studies using 3-azi- or 7-azi-derivatives of taurocholate with freshly isolated rat hepatocytes or rabbit ileal brush-border membrane vesicles revealed a specific interaction of bile acid derived HMG-CoA reductase inhibitors with the respective putatative bile acid transporters in the liver and the ileum demonstrating the bile acid character of these derivatives, both for the prodrugs and the hybrids. Cholesterol biosynthesis in Hep G2 cells was inhibited by the bile acid prodrugs with IC 50-values in the range of 68 nM to 600 nM compared to 13 nM for HR 78 and 130 nM for mevinolin. Among the hybrid inhibitors, S 2467 was the most active compound with an IC 50-value of 16 μM compared to 55 μM for its diastereomere S 2468. Preliminary in vivo experiments showed an inhibition of hepatic cholesterol biosynthesis after oral dosage only with prodrugs such as S 3554, whereas the hybrid molecules were inactive after oral application. Measurement of inhibition of cholesterol biosynthesis by incorporation of [ 14C]octanoate or [ 3H]H 2O into the digitonin-precipitable sterol fraction in the liver and extrahepatic organs demonstrated a significant increase of liver selectivity of HMG-CoA reductase inhibitors such as HR 780 by coupling to bile acids. Taken together, these studies demonstrate that the liver selectivity of an HMG-CoA reductase inhibitor can be increased by combining with bile acid structural elements and making use of specific bile acid transport pathways of the liver.

Effect of copper deficiency on the hepatic synthesis and rate of plasma release of cholesterol

The relative rates of hepatic 3H incorporation into digitonin-precipitable sterols (DPS) and release of labeled DPS into the plasma were compared between copper-deficient (CuD) and copper-adequate (CuA) rats. Following intravenous injection of 3H 2O and Triton WR 1339, the liver and plasma 3H-DPS radioactivities were measured at 2, 4, 6, and 8 hr, and the net total accumulation of plasma cholesterol (CH) and triglyceride (TG) was determined during the 8-hr period. In both groups, the liver synthesis of 3H-DPS displayed a biphasic curve, with an initial slow rate up to 4 hr, followed by a rapid increase starting at 6 hr. During the latter phase, a notable increase in liver 3H-DPS radioactivity was observed in CuD rats, but the plasma release of 3H-DPS did not significantly change, despite the increased synthesis of 3H-DPS in the liver. In both groups, the net total accumulation of CH in the plasma, as expressed in mmol CH/100 g body weight, increased steadily from 1.2 mmol at 2 hr to 4.7 mmol at 8 hr, while plasma TG accumulated at a much greater rate ranging from 3.3 mmol at 2 hr to 13.6 mmol at 8 hr. There was no significant difference in net total accumulation of CH or Tg in the plasma between the groups at any given interval. The results provide evidence that copper deficiency stimulates the liver synthesis of cholesterol, but that it does not result in an accelerated release of newly synthesized cholesterol from the liver involving very low density lipoproteins (VLDL). Thus, the present findings suggest that the hypercholesterolemia induced in copper deficiency is not attributable to a stimulation of the hepatic release of VLDL cholesterol into the plasma.

Hepatic and nonhepatic sterol synthesis and tissue distribution following administration of a liver selective HMG-CoA reductase inhibitor, CI-981: Comparison with selected HMG-CoA reductase inhibitors

Since cholesterol biosynthesis is an integral part of cellular metabolism, several HMG-CoA reductase inhibitors were systematically analyzed in in vitro, ex vivo and in vivo sterol synthesis assays using [ 14C]acetate incorporation into digitonin precipitable sterols as a marker of cholesterol synthesis. Tissue distribution of radiolabeled CI-981 and lovastatin was also performed. In vitro, CI-981 and PD134967-15 were equipotent in liver, spleen, testis and adrenal, lovastatin was more potent in extrahepatic tissues than liver and BMY21950, pravastatin and PD135023-15 were more potent in liver than peripheral tissues. In ex vivo assays, all inhibitors except lovastatin preferentially inhibited liver sterol synthesis; however, pravastatin and BMY22089 were strikingly less potent in the liver. CI-981 inhibited sterol synthesis in vivo in the liver, spleen and adrenal while not affecting the testis, kidney, muscle and brain. Lovastatin inhibited sterol synthesis to a greater extent than CI-981 in the spleen, adrenal and kidney while pravastatin and BMY22089 primarily affected liver and kidney. The tissue distribution of radiolabeled CI-981 and lovastatin support the changes observed in tissue sterol synthesis. Thus, we conclude that a spectrum of liver selective HMG-CoA reductase inhibitors exist and that categorizing agents as liver selective is highly dependent upon method of analysis.

Dietary regulation of maternal and fetal cholesterol metabolism in the guinea pig

Studies to determine the effects of pre-natal interventions on maternal and fetal cholesterol homeostasis were carried out in the guinea pig. Guinea pig dams were fed either non-purified guinea pig diet or diet supplemented with either 1.1% of the bile acid binding resin cholestyramine or 0.25% cholesterol. Whole body rates of endogenous cholesterol synthesis were determined by quantitation of [3H]water incorporation into digitonin precipitable sterols in non-pregnant animals and at 40 and 60 days of gestation in the dam and fetus. Maternal hepatic cholesterol synthesis was reduced 87% by dietary cholesterol and was increased 3.5-fold with cholestyramine feeding. Fetal hepatic and peripheral tissue cholesterol synthesis rates peaked at 40 days gestation when peripheral tissue cholesterol synthesis was 5.7-fold higher and hepatic synthesis 6.2-fold greater than the near adult levels observed at 60 days. Cholesterol synthesis in the fetus was relatively insensitive to dietary manipulations; however, maternal cholestyramine treatment did result in a 1.4-fold increase in fetal carcass cholesterol synthesis at 60 days gestation. These data demonstrate that maternal cholesterogenic systems maintain responsiveness to dietary regulation during pregnancy; whereas fetal cholesterol homeostasis is relatively insensitive to dietary cholesterol throughout gestation yet may respond to induction by maternal cholestyramine treatment during the late gestation period.

Ketone Body Utilization for Lipogenesis in the Perfused Liver of the Obese Zucker Rat

The purpose of these studies was to determine if the utilization of ketone bodies as a carbon source for lipogenesis could account for the decreased ketone body production by livers of obese Zucker rats, as well as contribute to the enhanced rates of fatty acid synthesis observed in these animals. Ketone body production was decreased from 822 mumol/liver in the lean to 538 mumol/liver in the obese genotype (P less than 0.05). The incorporation of ketone bodies into fatty acids was significantly greater in the obese rat liver (lean, 1.95 mumol of ketone bodies/liver, versus obese, 35.22 mumol/liver; P less than 0.025). The relative contribution of this pathway to the overall rate of fatty acid synthesis was not affected by genotype and accounted for only 3 to 4% of the fatty acids synthesized. The incorporation of ketone bodies into digitonin precipitable sterols was similar in the two genotypes (lean, 4.5 mmol/liver, versus obese 4.7 mumol/liver; NS). This accounted for 9.2 and 6.3% of the total sterol synthesis in lean and obese rat livers, respectively. The total incorporation of ketone bodies into lipid was 7.5 mumols in the lean rat livers and 42.0 mumoles in the obese (P less than 0.025). The net increase was 35 mumoles incorporated, whereas the net decrease in ketogenesis was 284 mumoles. Thus, although ketone body carbon utilization for lipid synthesis was increased in the liver of the obese rats, this pathway could only account for a fraction of the genotypic difference in ketone body production and was of relatively minor importance as a source of carbon for hepatic fatty acid synthesis in both lean and obese rats.

Inhibition by 6-fluoromevalonate demonstrates that mevalonate or one of the mevalonate phosphates is necessary for lymphocyte proliferation.

The sterol synthesis inhibitor 6-fluoromevalonate (Fmev) was used to explore the role of mevalonate products in lymphocyte proliferation. Fmev blocks the synthesis of isopentenyl pyrophosphate and all more distal products in the sterol pathway. When cells were cultured in lipoprotein-deficient medium, Fmev (200 microM) completely inhibited mitogen-stimulated human lymphocyte proliferation, quantified by measuring DNA synthesis. The addition of low density lipoprotein (LDL) restored lymphocyte responses to normal, whereas mevalonate was totally ineffective. Similar results were obtained with concentrations of Fmev up to 1 mM. These results contrast with those observed when sterol biosynthesis was blocked with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. When lymphocyte proliferation was blocked with lovastatin (5 microM), either high concentrations of mevalonate or LDL together with low concentrations of mevalonate was required to restore responses. In contrast, neither LDL nor low concentrations of mevalonate when alone was able to restore lymphocyte DNA synthesis in cultures blocked with 5 microM lovastatin. The effect of Fmev on the capacity of exogenous mevalonate to restore proliferation of lovastatin-blocked lymphocytes was directly examined. Fmev had no effect on the capacity of LDL plus low concentrations of mevalonate to restore DNA synthesis to lovastatin-blocked lymphocytes, indicating that the synthesis of the necessary factor from mevalonate was unaltered by Fmev. Fmev profoundly blocked lymphocyte endogenous sterol synthesis, decreasing incorporation of radiolabeled acetate into digitonin-precipitable sterols by up to 98%. LDL did not alter the capacity of Fmev to block sterol synthesis. The possibility that Fmev allowed shunting of endogenous mevalonate into essential lipid products was assessed by examining the incorporation of radiolabeled mevalonate. Fmev (200 microM) inhibited the incorporation of mevalonate into all lipids, including ubiquinone, dolichol, and other non-sterol lipids by up to 98%, and this was not altered by LDL. Furthermore, Fmev (200 microM) suppressed the incorporation of radiolabeled mevalonate into protein by up to 97%. These data confirm that a product of mevalonate is essential for cell proliferation. However, the results indicate that the required product is directly synthesized from mevalonate or mevalonate phosphates rather than from a more distal isoprenoid metabolite.

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 μmol/h ( n = 4) vs. 26.7 ± 4.8 and 24.6 ± 1.8 μmol/h for animals on the OL ( n = 4) and lard ( n = 3) diets ( P < 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 < 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.

Inhibition of carnitine palmitoyltransferase leads to induction of 3-hydroxymethylglutaryl coenzyme A reductase activity in rat liver

The relation between carnitine palmitoyltransferase (CPT) activity and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated. Rats were treated with aminocarnitine or 1-carnitine overnight. In rats, in which CPT activity was inhibited by aminocarnitine, plasma and hepatic triacylglycerol contents were increased 5- to 6-fold. The plasma cholesterol concentration was unchanged, while the hepatic cholesterol content was lowered (−16%). Hepatic cholesterol synthesis, determined by following the incorporation of 14C-acetate and 3H 2O into digitonin-precipitable sterols, in liver slices was increased 5- to 7-fold. HMG-CoA reductase activity in liver microsomes was increased to the same extent.

Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development.

The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of 3H2O and [14C]mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of 14C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of 3H of 3H2O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered.

Plasma lipoproteins and cholesterol metabolism in spontaneously hyperlipemic rats

The aim of this study was to characterize the plasma lipoprotein pattern and some aspects of cholesterol metabolism in a line of hyperlipemic male rats. Plasma cholesterol and triglycerides were increased about 3-fold as compared to control animals (238 vs. 75 and 185 vs. 59 mg/dl respectively). The plasma lipoprotein distribution and the chemical composition of the isolated lipoprotein unaffected. Plasma triglyceri production rate was increased (40%, P < 0.01) and post-heparin lipoprotein lipase activity in plasma decreased (−28%, P < 0.01) in the hyperlipemic rat. The activity of 3 enzymes involved in cholesterol metabolism (HMG-CoA reductase, cholesterol 7α-hydroxylase, and acyl-CoA cholesterol-acyltransferase) did not differ from control values. 3H 20 incorporation into digitonin-precipitable sterols, however, was significantly higher than in controls. This finding was due, in part, to an increased liver weight in the hyperlipemic animals. Furthermore kinetic data using 125I-LDL showed that the fractional catabolic rate of lipoprotein was within the normal range, while the synthetic rate of LDL protein was increased (0.67 vs. 0.3 mg/kg/h, P < 0.01) in the hyperlipemic rat. These observations suggest that multiple metabolic defects underline the hyperlipemia observed in this animal model.

Failure of caloric restriction to influence cholesterol synthesis in hamsters fed identical amounts of dietary cholesterol

To examine the effect of caloric restriction on cholesterol metabolism, independent of cholesterol intake, 3 groups of male Syrian hamsters were fed synthetic diets either ad libitum (AL) or pair-fed at 75% (R-75) and 50% (R-50) of ad libitum intake. Cholesterol intake of each group was 4.0±0.2 mg/day. On diet day 10 in each group, plasma cholesterol levels were measured and liver and duodenal 3H incorporation into digitonin precipitable sterols determined 90 min after intravenously injected 3H 2O. Microsomal 3-hydroxyl-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase activities were also measured in liver and duodenum. Body weights on day 10 for AL (115.8±7.2 g, x±SD), R-75 (107.7±6.3 g) and R-50 (99.6±3.0 g) groups decreased significantly with increasing severity of caloric restriction (p<0.05). Percent body fat and liver weights were also lower in caloric restricted groups. No differences were observed in plasma cholesterol concentrations (163±12, 151±10, 152±15 mg/dl, for AL, R-75 and R-50, respectively) or in the rate of 3H 2O incorporation into cholesterol either in liver (17.7±4.1, 11.9±1.6, 13.5±6.8 nmol 3H 2O/hr *mg tiss chol., x±SEM) or in duodenum (86.0±11.3, 97.6±9.4, 103.7±18.3 3H 2O/hr *mg tiss chol.). Moreover, liver and duodenal HMG-CoA reductase activites were not different between feeding groups. These data suggest that the mechanisms of regulation of cholesterol synthesis are insensitive to alterations in energy substrate flux induced by caloric restriction.

Inhibitors of sterol biosynthesis. Synthesis and activities of ring C oxygenated sterols

The chemical syntheses of a number of C 27 ring C oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of sterols biosynthesis in cultured mammalian cells. Thus, 5α-cholest-7-ene-3β,11α-diol, 3α-hydroxy-5α-cholest-9(11)-en-12-one and the previously unreported 11α-hydroxy-5α-cholest-7-en-3-one, 5α-cholest-9(11)-ene-3,12-dione, and 3β-hydroxy-5α-cholest-9(11)-en-12-one have been synthesized. The effects of these compounds on the synthesis of digitonin-precipitable sterols from labeled acetate in mouse L cells and on the levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in the same cells have been investigated and compared with previously published data on other ring C oxygenated sterols. 5α-Cholest-7-ene-3β,11α-diol was shown to be the most potent inhibitor of sterol synthesis.

F-244 specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme A synthase

A β-lactone isolated from Scopulariopsis sp. shows a potent inhibition of cholesterogenesis. The structure of this β-lactone, termed F-244, is 3,5,7-trimethyl-12-hydroxy-13-hydroxymethyl-2,4-tetradecadiendioic acid 12,14-lactone. The inhibition site of F-244 in cholesterol synthesis was studied. The growth of Vero cells was inhibited at 6.25–12.5 μg/ml of F-244. The inhibition of growth was overcome by the addition of mevalonate to the culture medium, but not by the addition of acetate. In a rat liver enzyme system, the incorporations of [ 14C]acetate and [ 14C]acetyl-CoA into digitonin-precipitable sterol were 50% inhibited by 0.58 βg/ml of F-244. The incorporation of [ 14C]mevalonate was not affected. Studies on the effects of F-244 on the three enzymes involved in mevalonate biosynthesis demonstrated that the drug specifically inhibits HMG-CoA synthase with IC 50 value of 0.065 μg /ml. The effect of analogs of F-244 on HMG-CoA synthase was also investigated.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: search for the enzyme's repressor derived from mevalonate.

Three inhibitors of squalene 2,3-oxide-lanosterol cyclase (AMO 1618, 4,4,10 beta-trimethyl-trans-decal-3 beta-ol (TMD) and 2,3-iminosqualene (ISq] were used to study effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and on sterol and polyprenyl synthesis from [14C]acetate and [14C]mevalonate in cultured rat hepatoma (H4) cells. After a 4 h exposure of cultures to AMO 1618 or TMD, followed by removal of the inhibitors, the utilization of [14C]acetate for synthesis of digitonin-precipitable sterols increased about twofold, an increase parallelled by the rise in HMG-CoA reductase. Mevalonate at 2.3 mM counteracted the effects of these inhibitors on the reductase. When (R)-[2-14C]mevalonate at 2.3 mM was included with the two inhibitors in the culture media, the cells were still able to synthesize cholesterol although in lesser amounts than the controls. In the presence of TMD the H4 cells also accumulated [14C]squalene 2,3-oxide and [14C]squalene 2,3-22,23-dioxide. ISq added to cells kept in full-growth medium (10 micrograms ml-1) caused an almost complete and irreversible inactivation of the squalene oxide-lanosterol cyclase but did not inhibit polyprenyl synthesis, as the amount of [14C]mevalonate converted into squalene, squalene 2,3-oxide, squalene 2,3-22,23-dioxide plus a little cholesterol was equal to the amount converted by control cells into cholesterol plus squalene. After a 24 h exposure of cells kept in full-growth medium to ISq (10 micrograms ml-1), the levels of HMG-CoA reductase rose about twofold. ISq completely abolished the suppressive effect of 2.3 mM (R)-mevalonate on the reductase. Chromatin isolated from cell nuclei contains cholesterol, which is renewed biosynthetically. It is argued that the suppressor of HMG-CoA reductase, derived from mevalonate, is a sterol and not a non-steroidal product of mevalonate metabolism.

HMG CoA reductase activity of lens epithelial cells: compared with true rates of sterol synthesis.

The regulation of sterol synthesis in the lens was addressed in the present study by comparing changes in the activity of HMG CoA reductase to changes in true rates of sterol synthesis for bovine lens epithelial cells in culture. The lens cells possessed very high levels of reductase activity (165 to 241 units/10(6) cells) which doubled when the cells were grown in media depleted of lipoproteins. True rates of sterol synthesis were simultaneously measured from incorporation of tritiated water into digitonin-precipitable sterols. Rates of sterol synthesis increased an average 37% more than the increase in reductase activity when the cells were deprived of exogenous cholesterol. Although not perfect, the results indicate a close correlation between HMG CoA reductase activity and rates of sterol synthesis in lens epithelial cells. We conclude that the activity of HMG CoA reductase is a major determinant of the rate of cholesterol synthesis in the lens.