Cholesterol-supplemented Diet Research Articles

Effects of bile acid oxazolines on gallstone formation in prairie dogs.

The effects of 2 bile acid analogs, chenodeoxy-oxazoline [2-(3 alpha, 7 alpha-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2-oxazoline] and ursodeoxy-oxazoline [2-(3 alpha, 7 beta-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2-oxazoline] were examined in the prairie dog model of cholesterol cholelithiasis. Gallstones and biliary cholesterol crystals were induced in 5 out of 6 male prairie dogs fed a semisynthetic diet containing 0.4% cholesterol for 8 weeks. Six animals maintained on a low cholesterol control diet (0.08% cholesterol) exhibited neither gallstones nor biliary cholesterol crystals. The addition of 0.06% chenodeoxy-oxazoline to the lithogenic diet did not prevent induced cholelithiasis or the appearance of cholesterol crystals in bile. In contrast, 0.06% dietary ursodeoxy-oxazoline prevented gallstones in 5 out of 6 prairie dogs (but cholesterol crystals were present in the bile of 4 of these animals). Histologically, most of the livers from the prairie dogs fed the cholesterol-supplemented semisynthetic diet showed bile duct proliferation, inflammatory infiltration and fibrosis along the portal tracts. These pathologic changes were generally not ameliorated by adding chenodeoxy-oxazoline or chenodeoxy-oxazoline plus chenodeoxycholic acid to the diet. Portal tract pathology was markedly reduced in most animals by adding ursodeoxy-oxazoline to the cholesterol-supplemented diet. The pathologic changes overall could best be correlated with the presence of gallstones, but not with the incidence of biliary cholesterol crystals.

REDUCTION IN THE NUTRITIONAL REQUIREMENTS FOR OÖGENESIS DUE TO HIGH CONCENTRATIONS OF CHOLESTEROL IN THE LARVAL DIET OF THE STABLE FLY, STOMOXYS CALCITRANS (DIPTERA: MUSCIDAE)

AbstractFemale stable flies, Stomoxys calcitrans (L.), from larvae reared on the standard CSMA (Chemical Specialties Manufacturing Association) medium required 5 days of blood feeding before producing their first batch of eggs. When the larval medium was supplemented with 1 or 2% cholesterol, resultant females were able to produce mature eggs after only 4 days of blood feeding. Females from larvae reared on the 2% cholesterol-supplemented medium had slight but significant precocious follicular growth at the time of emergence from the puparium. When blood feeding was restricted, the size at which follicular growth ceased was consistently higher in females fed the cholesterol-supplemented diets as compared with control females.

Effects of chenodeoxycholic and ursodeoxycholic acids on lipid metabolism and gallstone formation in the prairie dog.

The prevention of cholesterol cholelithiasis by dietary chenodeoxycholic and ursodeoxycholic acids was studied in the male prairie dog (Cynomys ludovicianus). Gallstones were induced by administration of a semisynthetic diet containing 0.4% cholesterol for a period of 8 weeks. Groups of 5 or 6 animals received the lithogenic diet with added chenodeoxycholic or ursodeoxycholic acid (0.03% "low dose" or 0.06% "high dose"). Under the conditions used, the incidence of gallstones was reduced with the high dose of chenodeoxycholic acid and the low dose of ursodeoxycholic acid, but cholesterol crystals were detected in the biles of 20 of the 22 animals fed these bile acids. A control group maintained on a low (0.08%) cholesterol semisynthetic diet exhibited neither crystals nor stones and was the only group with a lithogenic index below 1.0. The administered bile acids tended to reduce the accumulation of cholesterol in liver and plasma. The activity of hepatic HMG-CoA reductase was significantly inhibited with all cholesterol-supplemented diets. Cholesterol 7 alpha-hydroxylase activity was elevated 83% in prairie dogs fed 0.4% cholesterol, but tended to return to normal levels when bile acids were added to this diet. Histologically, the livers of all animals on the semisynthetic (cholesterol-supplemented) diet exhibited bile duct proliferation, as well as portal fibrosis and inflammatory infiltration. These morphologic alterations were ameliorated by low dose supplementation with either chenodeoxycholic or ursodeoxycholic acid, but high dose bile acid supplementation failed to reduce these pathologic changes.

Effect of Dietary Vegetable (Water Convolvulus) on Cholesterol Metabolism in Rats

Male rats were fed diets containing 0.5% cholesterol with or without vegetable (water convolvulus) or neutral detergent fiber supplementation. After 2 weeks, rats were given [4-14C]cholesterol i.p. Feces were collected for 1 week. Three hours prior to necropsy, [3H]acetate was administered i.p. Samples of serum, liver, adipose tissue, muscle and brain were obtained for analysis. Concentrations of total lipids and cholesterol and synthesis and recovery of labeled steroids are reported. Results showed that the growth of animals and food utilization were not significantly affected by different dietary treatments. The notable effect of vegetable was that the elevated liver and serum cholesterol levels due to increased intake can be nearly offset by increasing the fecal excretion. The high rate of excretion of 14C-labeled steroids shown in vegetable-fed rats indicated that both decreased absorption and increased endogenous excretion occurred in these animals. The synthesis of total lipids as demonstrated by [3H]acetate incorporation was not affected appreciably by diet. In liver, however, cholesterol synthesis appeared to be lower in rats receiving cholesterol-supplemented diet, but higher in rats fed vegetable diets. The ratio of 3H:14C of liver cholesterol was significantly higher in rats fed the vegetable diet. In brain cholesterol we also found consistently high 3H:14C ratios, which were not affected by dietary intake. It appears that brain cholesterol level is maintained mainly by synthesis in situ and not by uptake from dietary or other sources.

The role of plasma membranes in the transfer of non-esterified cholesterol to the acyl-CoA: Cholesterol acyltransferase substrate pool in liver microsomal fraction

The incubation at 37°C of rat-liver microsomal fraction followed by re-isolation of the treated microsomal vesicles results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The rate of this increase was higher in the microsomal fraction from rats fed cholesterol-supplemented diet or starved overnight as compared with that in the microsomal fraction from rats fed standard diet. The presence of a plasma membrane preparation in the incubation mixture also resulted in a time-dependent increase in acyl-CoA: cholesterol acyltransferase activity at a rate that was dependent on the concentration of plasma membranes. During the incubation of the microsomal fraction in the presence of phosphatidylcholine liposomes, cholesterol is transferred from the microsomal to liposomal vesicles. This transfer followed first-order kinetics with respect to cholesterol concentration in the donor with a rate that increased with the concentration of liposomes in the incubation mixture. The presence of phospholipid was also associated with a decrease in the activity of the acyltransferase that was related to the concentration of phospholipid in the incubation mixture. The incubation of the microsomal fraction in the presence of phosphatidylcholine-cholesterol liposomes resulted in a time-dependent and concentration-dependent transfer of liposomal cholesterol to the microsomal fraction and the acyltransferase substrate pool. The measurement of the rate of transfer of liposomal cholesterol to the microsomal vesicles and to the acyltransferase substrate pool at various temperatures showed that activation energies for the two processes are similar. Similar to these values was also the activation energy for the increase in acyl-CoA: cholesterol acyltransferase activity due to preincubation in the absence of artificial membrane vesicles. The present results suggest that there is, under the present conditions, a time-dependent and temperature-dependent flow of cholesterol from plasma membranes to the acyltransferase substrate pool and that this flow is either diverted in the presence of phospholipid liposomes or increased in the presence of cholesterol-phospholipid liposomes.

Modulation of the activity of 5′-nucleotidase by the transfer of non-esterified cholesterol to rat-liver microsomal fraction and evidence for regulation of the concentration of non-esterified cholesterol in plasma membranes in vivo

The transfer of non-esterified cholesterol to rat-liver microsomal fraction resulted in a considerable decrease in the activity of 5′-nucleotidase and in changes in the characteristics of the Arrhenius plots of the enzyme. The decrease in the activity of 5′-nucleotidase and the increase in the concentration of non-esterified cholesterol in the serum-treated preparations were serum-concentration-dependent and incubation-time-dependent. The enzyme in serum-treated preparations with high non-esterified cholesterol content showed Arrhenius plots with a constant activation energy between 37 and 19°C, whereas the enzyme in the non-treated microsomal fraction or the lipoprotein-deficient serum-treated preparations showed a break at about 28°C, with activation energies higher below and lower above the break. These changes in the temperature-induced kinetics are consistent with an increase in the concentration of non-esterified cholesterol in the plasma membrane vesicles of the serum-treated preparations. The Arrhenius plots of 5′-nucleotidase in liver microsomal fraction from rats fed cholesterol-supplemented diet showed constant activation energy between 37 and 19°C and had similar characteristics with the plots for 5′-nucleotidase in serum-treated preparations. Since the changes in the characteristics of Arrhenius plots of the enzyme in microsomal fraction from rats that had been denied food for 36 h were in the opposite direction to those produced by feeding cholesterol, these results are consistent with a lower concentration of non-esterified cholesterol in hepatic plasma membranes from fasted rats relative to that in plasma membranes from fed rats. The isolation of a plasma membrane preparation with negligible contamination of endoplasmic reticular membranes from rats fed the standard or cholesterol-supplemented diet and from fasted rats showed that the ratio of cholesterol to phospholipid has increased in the preparation from rats fed cholesterol and decreased in that from rats that had been denied food relative to the ratio in the preparation from rats fed the standard diet. The Arrhenius plots of 5′-nucleotidase in these preparations showed characteristics similar to the corresponding plots of the enzyme in the microsomal fraction from the rats in the three experimental conditions.

Effect of cholesterol feeding on the distribution of plasma lipoproteins and on the metabolism of apolipoprotein E in the rabbit

Changes in lipoprotein distribution and in apolipoprotein E metabolism were studied in rabbits fed a diet containing 1% cholesterol. Lipoprotein distribution was monitored by rate zonal ultracentrifugation, gel filtration chromatography, and electrophoretic techniques. Normolipidemic rabbit plasma contained VLDL, IDL, LDL, and HDL. Within 1 week on the 1% cholesterol diet, the d less than 1.006 g/ml material was greatly elevated, and the lipoproteins of higher density (LDL and HDL) decreased below levels of detection. Cholesteremic d less than 1.006 g/ml material was cholesteryl ester-rich, triglyceride-poor, and contained particles of Sf 20 to greater than 400. Upon diet normalization, the LDL and HDL reappeared within 2-4 weeks accompanied by a decrease in the d less than 1.006 g/ml material. The metabolism of apoE was studied by injecting purified and 125I-labeled apoE into rabbits and following the clearance of the tracer. ApoE in the normolipemic rabbit demonstrated a fractional catabolic rate (FCR) of 0.132 hr-1 and a half-life (t1/2) of 10.3 hr. ApoE in the hypercholesterolemic rabbit demonstrated an FCR of 0.055 hr-1 and a t1/2 of 49.5 hr. ApoE concentrations in the plasma as estimated by electroimmunoassay were 19.5 mg/dl in the control rabbit and 199 mg/dl in the hypercholesterolemic rabbit. From these data, absolute synthetic rates of 20.3 mg/kg per day and 86.1 mg/kg per day were calculated for the control and the hypercholesterolemic rabbit, respectively. We conclude that the cholesterol-supplemented diet caused pronounced elevation of apoE in the plasma due to overproduction of the protein.

The susceptibility of renal arterial forks in rabbits to dietary-induced lipid deposition

The major forks of the renal arteries from 21 rabbits fed a cholesterol-supplemented diet for periods of 1-90 d were examined electron-microscopically to compare the changes in the stem of the renal arteries with those at the first main fork. Lipid accumulated preferentially in the intimal pads or cushions at the fork. Matrix vesicles diminished in number and appeared to be transformed into enlarged membrane-bound vacuoles with electron-translucent contents. Foam cells were observed particularly beneath the endothelium. The lipid in endothelial and smooth muscle cells differed from that in the foam cells, indicating the likelihood of a different metabolic response. Interstitial lipid resembled an infiltration of the matrix with separation of the mural constituents but was not associated with the increased cellular degeneration, the progressive accumulation of matrix vesicles or the augmentation of the dystrophic basement membrane changes prominent in spontaneous atherosclerosis. In the arterial stems of 2 rabbits fed cholesterol for 62 and 90 d respectively, interstitial lipid deposition occurred without intimal proliferation.

Effect of cholesterol feeding and gender on the response of the isolated rat heart to hypoxia

The effect of hypoxic perfusion on isolated hearts from male and female rats fed either a control or 2% cholesterol supplemented diet was studied. Initiation of hypoxia produced a rapid decline in rate of hearts from either male or female animals irrespective of diet regimen. However, hearts from male rats fed a control diet exhibited a significantly higher (P less than 0.01) resting rate during normoxic perfusion when compared with hearts from cholesterol-fed animals. Hypoxia also produced a rapid loss of myocardial contractile force and this effect was influenced by neither diet treatment nor animal gender. Similarly, there was little change in time to peak height of developed tension due to diet. Contracture development during hypoxic perfusion was significantly higher in hearts from female rats fed a cholesterol-supplemented diet compared with hearts from control diet fed female animals, whereas no changes were observed due to diet in hearts from male animals. Coronary resistance was increased as a result of hypoxic perfusion. In hearts from cholesterol-fed male rats this effect was substantially attenuated. Conversely, cholesterol supplementation resulted in a higher (P less than 0.05) coronary resistance in hearts from female animals either during normoxic or the early period of hypoxic perfusion. Prostacyclin synthesis as measured by immunoreactive 6-keto-PGF1 alpha efflux did not differ between hearts from either male or female rats during normoxic perfusion. Hypoxia significantly reduced 6-keto-PGF1 alpha efflux from hearts of cholesterol-fed male rats whereas it produced no effect in any other treatment category. Our results demonstrate a substantial contribution of dietary cholesterol and animal gender in the response of the isolated rat heart to hypoxic perfusion.

Immune tolerance and atherosclerosis in rabbits: Effect of high-fat and cholesterol-supplemented diets

Breeding rabbits were fed diets supplemented with 5% milk or soya protein for 1 month before conception and throughout pregnancy and lactation. Four groups of 5 or or 6 of their offspring were given high-energy isonitrogenous diets containing either 30% soya or milk protein. Half of the animals from each litter received the same protein as their dams; the remainder were fed the alternative (i.e. dams soya: offspring milk, and vice versa). 0.75% cholesterol was added to the diets between 30 and 120 days after weaning. Animals given the same protein as their dams formed substantially lower amounts of food antigen-specific antibody than rabbits fed a novel protein at weaning but the extent of aortic atherosclerosis was similar in all groups. In a second experiment groups of 5–8 weaning rabbits were fed a cholesterol-free diet containing 30% soya and 17% saturated fat for 1 year. Animals in group 1 were bred from dams given soya but those in groups 2 and 3 were derived from a colony fed a soya-free diet. Rabbits in group 3 were immunised by repeated parenteral injections of soya protein and developed high levels of antisoya antibodies. Group 1 and 2 animals were injected with saline only but antisoya antibodies were substantially higher in animals derived from the soya-free breeding colony. Although serum cholesterol and triglyceride levels were similar in all groups, animals from dams fed soya (group 1) had significantly less aortic atherosclerosis (10% involvement of ascending aorta) than those reared from a colony fed a soya-free diet (group 2; 32%). Parenteral immunization with soya protein (group 3) was not associated with significantly increased atherosclerosis (37%). These findings indicate that perinatal exposure to dietary antigen in rabbits may be important in modulating the systemic immune reaction to food antigens. The magnitude of this systemic response in unlikely to alter the nature or distribution of cholesterol-induced atherosclerosis but may have an important influence on the development of aortic disease produced by prolonged feeding of high-fat, cholesterol-free diets.

The role of cholesterol 7α-hydroxylase in the hypobetalipoproteinaemic activity of SKF-525A in rats

Administration of SKF-525A to rats fed on a stock diet specifically decreased the serum concentration of low-density lipoprotein. SKF-525A and cholestryamine also reversed the rise in circulating concentration of both very-low-density and low-density lipoprotein that was observed in rats given a sucrose-based, cholesterol-supplemented diet. The enhancement of hepatic cholesterol 7α-hydroxylase by SKF-525A or by cholestyramine is accompanied by homeostatic responses by the liver which include induction of low-density lipoprotein clearance and increased cholesterogenesis to attempt to replenish sterol pools. These compensatory mechanisms are separately controlled.

The effects of differential psychological stress and infantile handling on plasma triglyceride and aortic cholesterol levels in rats.

The effects of differential psychological stress on serum triglyceride and aortic cholesterol levels were investigated in two experiments. In the first, rats with an unknown infant handling history purchased as adults from a standard supplier were subjected to predictable, controllable shocks; unpredictable, uncontrollable shocks; or the apparatus or their home cages with no shocks, for 30 days. All were maintained on a cholesterol-supplemented diet except during the daily 51-min stress sessions. The amounts eaten were equated among the groups. The results that the shocked rats had significantly lower terminal levels of serum triglycerides, and those receiving unpredictable, uncontrollable shocks had significantly less aortic cholesterol, than the nonshocked groups, which did not differ from each other in either measure. In the second experiment, rats born in this laboratory were either handled or left undisturbed in infancy and were exposed to a diet and differential stress conditions as adults. Animals handled in infancy had significantly lower aortic cholesterol than nonhandled animals across all stress conditions. In addition, those exposed to unpredictable, uncontrollable shocks had lower aortic cholesterol than those exposed to predictable, controllable shocks, and both had lower aortic cholesterol than the nonshocked group. Similar differential stress effects across stress conditions were seen in all the rats' serum triglyceride levels in Experiment 2. The effects of infantile handling did not interact with the stress effects, and neither could be accounted for by group differences in amount of the diet eaten or weight gained.

In vivo effect of cholesterol feeding on the short term regulation of hepatic hydroxymethylglutaryl coenzyme A reductase during the diurnal cycle.

Light-dark-cycled rats were fed a 3% cholesterol-supplemented diet at the beginning of the dark phase. Cholesterol-fed and control animals were taken at intervals throughout the following 12 h and the microsomal and solubilized hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated. Immunotitrations of this microsomal and solubilized enzyme were performed with a monospecific antibody to 3-hydroxy-3-methylglutaryl coenzyme A reductase. In contrast to the specific activity of the enzyme, which differs extremely during the diurnal cycle, the immunotitrations obtained from cholesterol-fed and control animals, yielded in identical antisera equivalence points. On the other hand, when the enzyme was phosphorylated in vitro, the antisera equivalence points corresponded to the alterations of the specific activity. In contrast to the results published by Higgins and Rudney ((1973) Nature New Biol. 246, 60-61), our data prove that even the in vivo short term changes in enzyme activity are due to changes in the quantity of enzyme rather than to a modulation of the catalytic activity.

Regulation of ovarian cholesterol metabolism: control of 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase.

Activities of enzymes involved in cholesterol metabolism were measured in ovaries of PMS-hCG-primed immature rats. Microsomal 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in sterol synthesis, was low during the period of maximal steroid production and cholesteryl ester storage. Acyl CoA:cholesterol acyltransferase (ACAT), the microsomal enzyme catalyzing sterol esterification and cytosolic cholesteryl ester hydrolase and mitochondrial cholesterol side chain cleavage activities, paralleled storage of sterol esters and levels of plasma progesterone, being highest on day 7 post hCG. Further experiments demonstrated that HMG-CoA reductase and ACAT are regulated by different mechanisms than the sterol esterase and cholesterol side chain cleavage system. When blood sterol levels were lowered by 4-aminopyrazolo (3,4-d)pyrimidine, plasma progestin concentrations fell and ovaries failed to accumulate sterol esters. HMG-CoA reductase increased more than 10-fold as a result of this treatment, while ACAT was 15% of that measured in controls. In contrast, cholesteryl esterase and the cholesterol side chain cleavage system were not affected. Intravenous infusion of human lipoproteins reversed the effects of 4-aminopyrazolo (3,4-d)pyrimidine. Raising blood levels up to 6-fold with either cholesterol-supplemented diets or intravenous injections of rat lipoproteins had no effect upon ovarian sterol metabolism, suggesting that the process by which blood cholesterol is utilized is saturated at normal blood sterol levels. Intravenous injection of LH on day 7 post hCG increased HMG-CoA reductase and decreased ACAT activity in addition to depleting cholesteryl esters. Prior treatment of rats with aminoglutethimide prevented the effects of LH on both HMG-CoA reductase and ACAT, indicating that changes in these enzymes were a consequence of the steroidogenic stimulus imposed by LH. Treatment with aminoglutethimide alone on day 7 post hCG did not further depress the already low HMG-CoA reductase activity, but ACAT and cholesterol ester storage were stimulated while sterol esterase activity was not altered. Aminoglutethimide also produced elevated ACAT activity and sterol ester storage in animals treated on day 2 post hCG, a time when there is normally little sterol ester accumulation. We conclude that HMG-CoA reductase and ACAT are regulated, in a reciprocal fashion, by ovarian cholesterol balance. When sufficient cholesterol is available, HMG-CoA reductase is suppressed and ACAT increases to facilitate esterification of sterol in excess of cell needs. When exogenous sterol supplies cannot meet ovarian demands, HMG-CoA reductase rises, and there is a concomitant decline in ACAT. Thus, gonadotropic control over these enzymes may be exerted, in part, through modulation of the supply of cholesterol to the ovary and/or through regulation of the rate of sterol utilization for hormone synthesis.

Antihypercholesterolemic activity of .BETA.-sitostanol in rabbits.

The antihypercholesterolemic activity of beta-sitosterol and beta-sitostanol was compared in male rabbits given a cholesterol-supplemented diet. beta-Sitosterol and beta-sitostanol were fed to these rabbits at the 0.5% level with cholesterol (0.5% and 0.2% in experiments I and II, respectively). The serum cholesterol level tended to be lower in rabbits fed beta-sitostanol than in the animals fed beta-sitosterol even in experiment I. The beta-sitostanol exhibited a significantly greater hypocholesterolemic activity in experiment II, LDL-cholesterol being decreased markedly. The liver cholesterol decreased in both groups of rabbits to a similar extent. beta-Sitostanol prevented more effectively the formation of dietary cholesterol-induced atheroma in the abdominal aorta than beta-sitosterol. It is most likely, together with the data reported previously on rats, that the hypocholesterolemic activity of beta-sitostanol results from the significantly greater inhibitory effect on the intestinal absorption of cholesterol than that of beta-sitosterol.

Aortic collagen, elastin and non-fibrous protein synthesis in rabbits fed cholesterol and peanut oil

Alteration of the fatty acid composition of atherogenic test diets has been a widely recognized method for influencing the character and severity of atherosclerotic lesions. The addition of peanut oil or coconut oil to cholesterol-supplemented diets has been shown to produce lesions of a fibrous nature in several species. In the present study, addition of 8% peanut oil to a 2% cholesterol diet accelerated the formation of atherosclerotic lesions which were more fibrous after only 90 days than those previously seen in rabbits even after 6 months on a diet supplemented with cholesterol alone. Collagen, elastin and non-fibrous protein synthesis were all increased over control values, as previously seen in aortas from rabbits given cholesterol supplementation alone. However, the addition of peanut oil to the 2% cholesterol diet produced a preferential increase in the rate of aortic collagen synthesis per unit dry, defatted weight compared with the increases seen in elastin, non-fibrous protein or total protein synthesis. Collagen deposition in proliferative intimal plaques was evident by histological examination. These focal accumulations, however, did not result in significant increases in either total collagen content of the whole descending thoracic aorta or in collagen concentration expressed per unit of dry, defatted weight. These data suggest that, while a portion of the increased synthetic rates may be a direct result of aortic hyperplasia, the proportionally greater increase in collagen synthesis in these lesions is attributable to the addition of peanut oil to the atherogenic diet. Although the lesions produced in this experiment lacked the overt fibrosis seen in man and in some forms of experimentally induced atherosclerosis, the relative synthetic rates of collagen, elastin and non-fibrous protein described here suggest that even a small preferential increase in collagen synthesis compared with non-collagen protein synthesis may gradually lead to a more fibrous lesion.

Influence of an atherogenic diet on the structure of swine low density lipoproteins

Five groups of three swine each were fed a basal diet supplemented with 15% tallow and either 0.0, 1.0, 1.5, 2.0%, or 2.5% cholesterol. The animals were studied over a period of 9 weeks to observe changes in plasma lipids and low density lipoproteins (LDL). At the end of the study period, LDL was analyzed by rate zonal ultracentrifugation, characterized chemically, and examined by differential scanning calorimetry. Within 3 weeks of initiation of the cholesterol-supplemented diets, there was an increase in the plasma levels of cholesterol and total LDL. LDL from swine fed the basal diet and the basal diet plus 1.0% cholesterol appeared in two LDL populations (LDL1 and LDL2) when analyzed by rate zonal ultracentrifugation. After diets containing 1.5, 2.0, and 2.5% dietary cholesterol, there was an increase in the mean flotation rate of total LDL which shifted to a lower density. LDL1 from the high cholesterol diets had a decreased triglyceride content when compared to those of the low cholesterol diets. When examined by differential scanning calorimetry, the LDL1 from the animals fed at least 1.5% cholesterol had phase transitions above body temperature, whereas the LDL from those fed 0 and 1.0% cholesterol had phase transitions below 37 degrees C. In contrast, the thermal behavior and fatty acid compositions of the extracted cholesteryl esters of the LDL obtained after the five different diets were not remarkably different. Since LDL triglyceride and cholesteryl esters are predicted to coexist in a common phase in the LDL core, the different thermal behavior of the LDL obtained after diets with different cholesterol contents is due to differences in triglyceride content which are a secondary effect of cholesterol-feeding. From these data we conclude that dietary cholesterol increases plasma LDL content, decreases LDL triglyceride content, and alters the particle structure. These changes in lipoprotein structure may contribute to the known development of atherosclerosis in cholesterol-fed swine.

The effects of differential psychological stress on plasma cholesterol levels in rats.

The plasma cholesterol concentrations of rats receiving either lever-press escape or avoidance training, exposure to unpredictable, uncontrollable grid shocks using a yoked procedure, or no shocks, were compared in two experiments. All were fed a cholesterol-supplemented diet prior to and during the 30 days of exposure to these differing stress treatments. The results of both experiments showed that yoked groups had higher terminal levels of cholesterol than their experimental counterparts in the escape or avoidance group even though they received the same amounts of aversive stimulation and ate the same amounts of the diet. Both were higher than the nonshocked groups when the amount of food intake for all was matched in Experiment 2. The type of level-press training had no effect.

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

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

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

β-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 μm, 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 β-oxidation of long-chain fatty acids in atherosclerosis is not attributable to an increase in CPT activity.