Cholesterol exchange between microsomal, mitochondrial and erythrocyte membranes and its enhancement by cytosol.

1. Cholesterol exchanges between isolated rat liver microsomes and mitochondria and between erythrocytes and microsomes or mitochondria during incubation in vitro. The exchange process is temperature dependent and is no accompanied by a net movement of sterol. 2. cholesterol exchange between the membranes was enhanced by the addition of 105 000 x g supernatant fraction (S105) from rat liver. The degree to which sterol exchange was enhanced was dependent on the amount of this supernatant fraction present in the incubation. 3. enhancement of sterol exchange was not observed with heated S105 fraction, but activity was retained after dialysis or aging at 10 degrees C; these results suggest the presence of a cholesterol-exchange protein in the cytosol from rat liver.

Cholesterol esterification by rat adrenal gland. Inhibition by local anesthetics in vitro.

Cholesterol esterification was studied in rat adrenal gland, adrenal homogenates and isolated adrenal microsomes. In whole gland and homogenates, the local anesthetic, lidocaine, inhibited the incorporation of [1-14C]oleate and [1-14C]oleoyl-CoA, respectively, into labeled cholesteryl esters in a dose-dependent manner. Inhibition of sterol esterification in the preparations reached 50% at about 2 mM. Various other local anesthetics (tetracaine, dibucaine and benzocaine) also inhibited sterol esterification in adrenal homogenates but were more potent than lidocaine; in each case, 90% inhibition occurred at anesthetic levels of 1 mM. Since sterol esterification in the adrenal gland is a function of microsomal acyl-CoA: cholesterol acyltransferase (EC 2.3.1.26), the enzyme was assayed in isolated adrenal microsomes in the presence of lidocaine while using [14C]oleoyl-CoA as a substrate for labeled cholesteryl ester formation. Inhibition of the enzymes by lidocaine was confirmed, with 50% inhibition occurring between 0.5 and 0.75 mM lidocaine. Lidocaine may be useful as a tool in studies on the regulation of adrenal sterol esterification.

Effect of local anesthetics on sterol biosynthesis and sterol esterification in rat liver in vitro.

Various local anesthetics were found to inhibit sterol esterification by acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26) in the microsomal fraction from rat (and rabbit) liver and to inhibit sterologenesis in rat liver slices. The enzyme was assayed in isolated microsomes by following the incorporation of [1-14C]oleoyl-CoA into steryl esters; the order of inhibitory potency on the enzyme was dibucaine > benzocaine > tetracaine > lidocaine > procaine. In liver slices, the incorporation of labeled acetate and mevalonate into C27 sterols, digitonin-precipitable sterols, and squalene was inhibited in the order dibucaine > tetracaine > lidocaine. A comparison of incorportion patterns from labeled acetate and mevalonate suggest that inhibition of sterologenesis occurs at more than one post-mevalonate step in the pathway.

Proliferative and lipid metabolism response to balloon angioplasty in canine renal arteries.

The mechanisms responsible for reocclusion after percutaneous transluminal angioplasty are still poorly understood. The effects of angioplasty on arterial morphology, deoxyribonucleic acid (DNA) synthesis (3H-thymidine incorporation) and lipid metabolism (14C-oleate incorporation) were studied in renal arteries of 24 male mongrel dogs. Balloon-dilated (identified by Evans blue dye accumulation) and adjacent normal arterial segments were collected 90 min and 2, 5 and 14 days after the procedure. The immediate vascular response was endothelial cell denudation and platelet accumulation. Two weeks after angioplasty, healing of the luminal surface by "endothelial-like" cells, mild smooth muscle cell proliferation and an angiogenic response with capillary growth into the media were observed. DNA synthesis was increased in balloon-dilated segments at day 5 compared with adjacent nonballoon-dilated artery. This increase in DNA synthesis persisted in the 2 week postangioplasty segments. Additionally, angioplasty produced both quantitative and qualitative changes in arterial lipid synthesis. The most dramatic change was an increase in sterol esterification that was apparent as early as 90 min after angioplasty; the change persisted through day 5 but diminished toward baseline by day 14. Angioplasty-induced alterations of arterial metabolism parallel aspects of the atherogenic process and may be involved in the pathogenesis of postangioplasty reocclusion, particularly in the presence of additional risk factors, such as hyperlipemia.

Carnitine metabolism in Macaca arctoides: the effects of dietary change and fasting on serum triglycerides, unesterified carnitine, esterified (acyl) carnitine, and beta-hydroxybutyrate.

Serum triglycerides and serum total, esterified, and free (unesterified) carnitine were measured in 21 male Macaca arctoides that were switched from a low fat (5.2% w/w), high carbohydrate diet to a high fat (15.9% w/w), low carbohydrate diet for 90 days and then returned to the original low fat diet for a subsequent 76-day period. Serum triglycerides and total carnitine levels fell significantly (p less than 0.05) during the initial 2 wk of feeding the high fat diet and the ratio of esterified/unesterified carnitine rose significantly (P less than 0.05) on the high fat diet. A return to the low fat diet reversed these changes; triglycerides rose significantly (p less than 0.05) within 3 days and the ratio of esterified/unesterified carnitine fell significantly (p less than 0.05) within 3 days and the ratio of esterified/unesterified carnitine fell significantly (p less than 0.05) during the same period. A return of total carnitine levels to those initially observed on the low fat diet was slower to develop. Fasting for 24 to 48 h resulted in increases of 65 to 75% in total serum carnitine. This increase reflected elevations of both the esterified and unesterified carnitine fractions but was largely attributable to increases in esterified carnitine which rose from 10 to 41 nmol/ml by 48 h while unesterified carnitine rose from 55 to 72 nmol/ml during the same period. In addition, the ratio of esterified/unesterified carnitine ratio rose from 0.183 +/- 0.023 to 0.583 +/- 0.069 (n = 8) with a 48-h fast and was significantly correlated with serum beta-hydroxybutyrate levels at both 24 and 48 h.

Inhibition of adenine nucleotide translocase by oleoylcarnitine, oleoylcoa and oleate in isolated arterial mitochondria.

Adenine nucleotide translocase (AdNT) activity was studied in isolated mitochondria from normal rabbit aortas. The enzyme was inhibited by oleic acid, oleoylCoA, and oleoylcarnitine with 50% inhibition occurring at 5 muM, 6 muM and 14 muM, respectively (corresponding to 8, 10, and 23 nmol/mg protein). PalmitoylCoA and palmitoylcarnitine displayed similar potency to oleylCoA and oleoylcarnitine. The possibility that inhibition by fatty acid, acylCoA, and acylcarnitine could be attributed to non-specific detergency effects seems remote in that these compounds were more potent inhibitors of AdNT than equimolar concentrations of laurylsulfate. In addition, by use of the fluorescent probe N-phenyl-1-naphthylamine, it was shown that under the experimental conditions, inhibition of AdNT occurred at concentrations not exceeding a critical micelle concentration (CMC). Specificity was also suggested in that octanoylCoA was a weak inhibitor of AdNT and acetylcarnitine, butyrylcarnitine, cholesteryl oleate, and sphingomyelin were not inhibitory to the enzyme. In contrast to the observed inhibition of arterial AdNT by oleoylCoA and oleoylcarnitine, AdNT in isolated rabbit and rat heart mitochondria was inhibited only by oleoylCoA.

Diazepam inhibits cholesterol esterification by arterial ACAT and plasma LCAT, in vitro.

Benzodiazepine drugs have been reported to have antiatherosclerotic effects in rabbits and roosters and to alter the pattern of circulating lipoproteins in man. The mechanism(s) of these effects has not been elucidated. The studies presented here indicate that diazepam, the most widely used benzodiazepine, is an inhibitor of cholesterol esterification by ACAT in vitro in atheromatous rabbit aortas, in microsomes isolated from atheromatous rabbit aortas, and in normal rat aortas. Diazepam also inhibited LCAT in plasma from man, monkey, rabbit, and rat, in vitro. The ability of diazepam to inhibit these enzyme systems may offer insight into possible in vivo mechanisms of action against atherosclerosis and of lipoprotein modification.

The effect of local anesthetics on arterial lipid metabolism. Inhibition of sterol esterification in vitro.

The local anesthetics lidocaine, tetracaine, benzocaine and dibucaine were found to inhibit sterol esterification by acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in the microsomal fraction isolated from rabbit aortas. In arterial microsomes, the incorporation of [14C]oleoylCoA into [14C]steryl esters was inhibited in a dose-dependent way by the anesthetics over the concentration range 0.25-5.0 mM. The potency of inhibition was dibucaine greater than benzocaine greater than tetracaine greater than lidocaine greater than procaine with inhibition of about 85% occurring with 0.25 mM dibucaine. Sterol esterification to [14C]oleic acid was also inhibited by the anesthetics in intact aortic tissue from the rabbit, dog, and rat. A detailed study of the effects of 5 mM lidocaine on lipid biosynthesis in the rabbit aorta in vitro revealed that lidocaine not only inhibited sterol esterification to [14C]oleate but stimulated [14C]oleate incorporation into glycerides.

Carnitine palmitoyltransferase activity in mitochondrial fractions isolated from aortas of rabbits fed cholesterol-supplemented diets.

beta-Oxidation of long-chain fatty acids increases many-fold in atherosclerotic aortas; this may be due to an increase in the activity of the mitochondrial enzyme hexadecanoyl-CoA: carnitine O-hexadecanoyltransferase EC 2.3.1.23 (trivial name: carnitine palmitoyltransferase, CPT). To investigate this possibility, an assay for arterial CPT was developed and used to measure CPT activity in mitochondrial fractions isolated from aortas of rabbits fed high-fat (HF) or high-fat plus cholesterol (HFC) supplemented diets. The arterial CPT assay was linear with respect to mitochondrial protein between 0.03 and 0.30 mg and assay time between 3 and 12 min. Maximum CPT activity was observed at concentrations of palmitoyl-CoA between 5 and 25 micron, higher concentrations of palmitoyl-CoA inhibited CPT activity. CPT activity was measured in mitochondrial fractions isolated from aortas of rabbits fed the HFC-supplemented diet for up to 48 days. No visible lesions were observed in aortas of rabbits fed HFC-diet for 3,9, or 21 days, however, by 48 days atheromatous lesions covered in excess of 60% of the intimal surface of the aorta. No lesions were visually observed in aortas of rabbits receiving the HF-diet. Despite the development of gross atherosclerotic lesions, there were no changes in CPT activity observed that could account for a dramatic increase in fatty acid oxidation. It is concluded that the increase in beta-oxidation of long-chain fatty acids in atherosclerosis is not attributable to an increase in CPT activity.

Uptake and esterification of circulating carnitine by aorta and heart in rabbits in vivo.

Disappearance of intravenously injected DL-[methyl-14C]carnitine from the bloodstream and its uptake and esterification by heart and aorta were studied in rabbits fed atherogenic or non-atherogenic control diets. The disappearance rate of [14C]carnitine from the bloodstream was approximately 2-fold greater in animals fed the control diet than in those fed the atherogenic diets. No evidence was found for carnitine esterification in the blood. Circulating [14C]carnitine was taken up and esterified in both the heart and aorta of all animals regardless of diet; however, on comparing dpm/mg lipid-free dry weight, uptake of [14C]carnitine and accumulation of [14C]carnitine esters by the heart was greater (6-fold and 12-fold, respectively) than by the aorta. Analysis of defined arterial segments indicated that aortas in animals fed the atherogenic diet contained greater [14C]carnitine activity (4- to 8-fold) and greater acetyl-[14C]carnitine activity (4-fold) when compared to aortas of control animals; uptake of plasma [14C]carnitine and accumulation of acyl-[14C]carnitine compounds by the heart was independent of diet. Butyryl-[14C]carnitine, although not detected in aortas from animals fed the non-atherogenic or atherogenic diet for only 7 weeks, was detected in aortas from animals fed the atherogenic diet 17 weeks. Butyryl-[14C]carnitine was detected in heart tissue regardless of the animals' dietary regime. The increased uptake of circulating [14C]carnitine and its accumulation as both free and esterfied carnitine in atherosclerotic aortas occurred before the development of extensive macroscopic atherosclerotic lesions; this response of the aorta to atherogenic stimuli was not a general tissue response in that the heart did not respond similarly. Since blood carnitine is found predominantly in the plasma fraction, it is likely that these results refect the uptake and metabolism of plasma carnitine in vivo.

Membrane-active agents. Effect of various anesthetics and chlorpromazine on arterial lipid metabolism.

The local anesthetic lidocaine was studied for its effects on lipid metabolism in aortas from normal rats, rabbits, and cholesterol-fed (atherosclerotic) rabbits in vitro. Incubation of aortas in the presence of 3--5 mM lidocaine resulted in a statistically significant reduction in the incorporation of [14C]oleate into cholesteryl esters and phosphatidylcholine. Additionally, significant increases in [14C]oleate incorporation into the diglyceride fraction of atheromatous rabbit aortas was observed with a trend to greater incorporation into the diglyceride fraction of normal rat and rabbit arteries as well. The most significant overall effect of lidocaine was its inhibition (50--90%) of the arterial sterol esterification. Assays of acylCoA : cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes revealed that, in addition to local anesthetics (e.g., lidocaine), other membrane-active agents such as chlorpromazine and methoxyflurane inhibit ACAT; this suggests ACAT may be regulated by alterations in the biophysical properties of its membrane milieu.

Arterial lipid biochemistry in the spontaneously hyperlipidemic Zucker rat and its similarity to early atherogenesis.

In the present studies, arterial lipid metabolism was evaluated in the spontaneously hyperlipidemic obese Zucker rat (fa/fa), the lean Zucker rat (Fa/-), and the Sprague-Dawley (SD) rat. Mean serum cholesterol levels in the obese Zucker, lean Zucker and SD rats were 216 +/- 18 mg/dl, 145 +/- 14 mg/dl and 84 +/- 5 mg/dl, respectively. Arterial cholesterol content was in the same rank order as plasma cholesterol and ranged from a mean of 2.23 +/- 0.10 mg/gm wet wt. in the obese rats to 1.36 +/- 0.04 mg/gm wet wt. in the SD rats. The increased arterial sterol in the obese rats was associated with increased lipid metabolism activity. The in vitro incorporation of [14C]oleate into arterial cholesteryl esters was increased 3-4-fold (P less than 0.01) and incorporation into phospholipids and triglycerides was also elevated (P less than 0.001 and P less than 0.01, respectively). The arterial sterol content and arterial lipid metabolism pattern observed in obese Zucker rat aortas are similar to those found in vessels of other species undergoing atherogenic change.

U-73482: a novel ACAT inhibitor that elevates HDL-cholesterol, lowers plasma triglyceride and facilitates hepatic cholesterol mobilization in the rat.

U-73482, a novel acylCoA:cholesterol acyltransferase (ACAT) inhibitor with systemic activity, has been evaluated for its effects on a variety of lipid metabolic parameters in the rat. The compound inhibits ACAT in vitro in cultured Fu5AH rat hepatoma cells and demonstrates systemic activity through inhibition of hepatic ACAT in rats receiving the drug orally. U-73482 also lowers plasma triglycerides at 40 mg/kg per day in the rat and elevates high density lipoprotein cholesterol (HDL-chol) in a dose-related fashion over the range of daily intakes of 0-40 mg/kg in the rat. Elevations in HDL-chol are followed by elevations in total plasma cholesterol in normal rats but the compound exerts hypocholesterolemic activity in cholesterol-fed rats and promotes clearance of stored hepatic sterol in rats pretreated with a hypercholesterolemic diet and then changed over to normal chow. The triglyceride-lowering and HDL-chol elevating effects of U-73482 coupled with its ability to promote tissue sterol clearance and block the hypercholesterolemic effects of dietary cholesterol in animals, suggests that the compound has potential as a therapeutic agent for treatment of lipid disorders in man.

Synthesis and hypocholesterolemic activity of 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-diones, novel inhibitors of acylCoA:cholesterol O-acyltransferase.

A novel series of 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione inhibitors of the enzyme acyl-CoA:cholesterol O-acyltransferase is described. A number of these derivatives were found to be potent modulators of serum lipoprotein levels in cholesterol-fed rats. Further evaluation of one of the most effective analogues confirmed that it was significantly blocking the absorption of cholesterol from the gut.

Effect of the lipid-lowering drug lifibrol on lipid metabolism in rat macrophages and in atherosclerotic arteries from swine and WHHL rabbits, in vitro. Implications in atherogenesis.

The effects of lifibrol on lipid metabolism in rat macrophages and swine and rabbit aortae were investigated. Resident peritoneal macrophages isolated from rats pretreated with lifibrol (50 mg/kg/7 days) showed a decreased capacity to synthesize cholesteryl esters from labeled precursors ([1-14C]oleate and [4-14C]cholesterol). Macrophages isolated similarly from non-treated rats demonstrated the ability to take up [14C]lifibrol, in vitro. Modification of lipid metabolism in atherosclerotic aortae from swine and Watanabe heritable hyperlipidemic (WHHL) rabbits was also observed when the tissues were incubated in vitro in the presence of exogenous lifibrol. Concentrations of lifibrol of up to 50 micrograms/mL in the incubations selectively reduced the formation of cholesteryl esters from [1-14C]acetate by 60-75%, whereas higher concentrations (100 micrograms/mL) resulted in a generalized inhibition of lipid biosynthesis of about 50% and of cholesteryl ester formation by up to 90%. The ability of lifibrol to directly affect these targets (i.e. macrophages and arterial tissue) has implications that extend beyond its confirmed plasma cholesterol-lowering activity since early stages of the atherogenic process involve an overall increase in arterial lipid synthesis and cholesteryl ester accumulation by monocyte-macrophages that infiltrate the vessel wall from blood.

Effects of phthalate esters on lipid metabolism in various tissues, cells and organelles in mammals.

The effect of phthalate ester plasticizers on a variety of enzyme systems was studied in rats, rabbits and pigs. The feeding of di(2-ethylhexyl) phthalate (DEHP) to animals at levels from 0.1% to 1.0% in the diet resulted in diverse biochemical effects, such as inhibition of cholesterologenesis in liver, testes, and adrenal gland; inhibition of cholesterologenesis in brain and liver of fetal rats from DEHP-fed dams; decreased plasma cholesterol levels; decreased synthesis of hepatic phospholipid and triglyceride; increased fatty acid oxidation in isolated liver mitochondria; and a transient decrease in fatty acid oxidation in isolated heart mitochondria. The addition of DEHP to preparations of rat liver in vitro resulted in inhibition of cholesterologenesis, and its addition to isolated mitochondria from rat heart produced an inhibition of adenine nucleotide translocase. DEHP-feeding to rats and rabbits, however, did not affect platelet function as judged by collagen- and ADP-induced aggregation. The studies presented here indicate that the exposure of animals to phthalate esters can result in a significant perturbation of normal metabolism in liver, heart, testes, adrenal gland and brain and can affect blood lipid levels.

NF-kappa B is activated during acute inflammation in vivo in association with elevated endothelial cell adhesion molecule gene expression and leukocyte recruitment.

Leukocytes accumulate at sites of inflammation in response to the induced expression of endothelial cell adhesion molecules. The nuclear transcription factor kappa B (NF-kappa B) plays a critical role in the cytokine-induced expression of these genes in cultured endothelium. We examined the relationship between NF-kappa B activation and endothelial cell adhesion molecule gene expression in vivo during the initiation of acute inflammation. Nuclear NF-kappa B DNA-binding activity was rapidly increased within lung and heart tissues of rats administered endotoxin, consistent with the translocation of NF-kappa B complexes from the cytoplasm to the nucleus. This NF-kappa B was composed of p50 and p65 subunits, and could bind NF-kappa B elements in the E-selectin promoter. NF-kappa B activation was maximal within 30 min and persisted for at least 3 hr after endotoxin treatment. NF-kappa B activation preceded the transcriptional activation of the P-selectin, E-selectin, VCAM-1, and ICAM-1 genes. In the lung, increased expression of P-selectin and ICAM-1 protein was detected immunohistochemically. These molecular events were temporally associated with the sequestration of leukocytes and the development of pulmonary inflammation. NF-kappa B activation is therefore an early event in the initiation of acute inflammation in vivo. This molecular pathway may be of consequence in the pathogenesis of acute inflammatory disease.

Long-term effects of Aroclor 1254 (PCBs) on plasma lipid and carnitine concentrations in rhesus monkey.

Sixty-seven female rhesus monkeys (Macaca mulatta) were orally dosed daily for 152 weeks with 0, 5, 20, 40, and 80 micrograms Aroclor 1254 (PCB)/kg body wt. Blood polychlorinated biphenyl (PCB) concentrations were highly positively correlated (r = 0.92, P < 0.001) with doses of PCB administered. A comprehensive analysis of plasma lipids/lipoproteins revealed a PCB-associated increase in plasma triglycerides and decreases in plasma total cholesterol, high-density lipoprotein cholesterol (HDL-chol), very-low plus low-density lipoprotein cholesterol (VLDL+LDL-chol), and total carnitine (which is involved in fatty acid metabolism). All of the lipid/lipoprotein changes were significantly (P < or = 0.05) correlated with blood PCB concentration. These data, obtained after 152 weeks of continuous daily exposure of a primate model to PCB support a causal relationship between plasma lipid changes and PCB intake. Previously, causality has been refuted on the premise that the commonly observed elevation of triglycerides with increasing concentration of blood PCB is a reflection, not of PCB dose, but of the partitioning of PCB between tissues (adipose) and blood in proportion to the blood lipid present. The mechanism of the plasma lipid changes was not investigated in this study but the altered lipid/lipoprotein pattern is discussed with respect to known cardiovascular risk profiles.

Effect of the plasticizer di(2-ethylhexyl) adipate (dioctyladipate, DOA) on lipid metabolism in the rat: I. Inhibition of cholesterolgenesis and modification of phospholipid synthesis.

DOA (di[2-ethylhexyl] adipate, dioctyladipate), a plasticizer used in the manufacture of polyvinyl-chloride plastic products, has been considered as a suitable substitute for di(2-ethylhexyl)phthalate (DEHP) in some applications. In the present studies, hepatic lipid metabolism was examined in liver mince preparations from rats fed 0.5% or 1.0% DOA in the diet for 2 weeks. By studying patterns of lipid synthesis from [14C] acetate, [14C] oleate, [14C] mevalonate, and [14C] octanoate, it was concluded that DOA feeding inhibits hepatic cholesterolgenesis and alters the pattern of phospholipids synthesized by the liver. DOA also exerted a cholesterol-lowering effect at the 1% level but did not affect plasma triglyceride levels. The results suggest that the biological effects of DOA in the rat are similar to those produced by DEHP.

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-i-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the art. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-14C and mevalonate-5-3H into squalene, C30 sterols, and C27 sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of 3H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% control values in 11 days, whereas 14C-acetate conversion was reduced to ca. 35% of control values during the same period. 3H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as 14C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from 14C-acetate and 3H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were similar in control and DEHP-fed rats.