Further studies of the effects of an anovulatory drug on lipid metabolism in the rat

The effect of various levels of the oral contraceptive drug, Enovid E, on serum and liver lipid levels of adult female rats has been investigated. Doses ranging from 0.052 to 1.04 mg/day have been employed in rats fed control or cholesterol-containing diets. It has been confirmed that after administration of even low, physiological doses of the drug, esterified cholesterol in serum and adrenals decreases rapidly while at the same time it accumulates in the liver; cholesteryl oleate is increased while the relative amount of cholesteryl arachidonate is reduced. Serum phospholipids also are decreased; the alpha/beta lipoprotein ratio is significantly reduced due to the decrease of alpha-lipoproteins. Most of these changes also occur in cholesterol-fed rats. The observed effects are not related to a decreased food intake.     

Effect of the stress of unilateral adrenalectomy on the depletion of individual cholesteryl esters in the rat adrenal

Normal rats were subjected to unilateral adrenalectomy and were killed 3 hr later. The concentration and composition of the cholesteryl esters in adrenals removed at operation and after death were compared. The esterified cholesterol concentration was lower in the adrenals obtained 3 hr after surgery. Cholesteryl arachidonate decreased in concentration significantly more than any other ester, followed in order of magnitude by linoleate and oleate. The cholesteryl ester concentration of adrenals removed from sham-operated rats 3 hr after surgery was also greatly reduced. On the basis of comparison with other work on the hydrolysis of cholesteryl esters by adrenal homogenates, it is concluded that the apparent selectivity in depletion of cholesteryl esters is due to differences in their rates of hydrolysis.     

Sex differences in plasma cholesterol-esterifying activity in rats

Esterification of free cholesterol was investigated after incubation at 37 degrees C of plasma from immature and adult rats of both sexes kept on stock, fat-free, or cholesterol-supplemented diets. According to measurements of the decrease in free cholesterol, plasma from the fat-deficient rats showed the highest cholesterol-esterifying activity. Esterification was higher in the mature female rats than in the mature males on stock or cholesterol-containing diets, although no sex differences were observed in the sexually immature young or in the fat-free animals. There were no sex differences in the fatty acid composition of the plasma sterol esters, phospholipids, and triglycerides in the immature animals, but arachidonic acid increased at the expense of linoleic acid in the sterol ester fraction in the adult female (not, however, in the adult male). In the phospholipid fraction the higher ratio of palmitic to stearic acids in the male was confirmed. There was an increase in linoleic acid in all three plasma lipid fractions of the mature male after cholesterol feeding. It is suggested that cholesterol may inhibit the conversion of linoleate to arachidonate. During the incubation of plasma, there was little change in the distribution of fatty acids except for a decrease in palmitoleate, and increases in C(20) tri- and tetraenoic acids, in the sterol esters of mature female rats on the stock ration and the fat-free diet. These C(20) acids decreased concomitantly in the phospholipid fraction, as the transesterification reaction mechanism proposed by earlier workers would predict.     

Effect of sex and gonadal hormones on rat plasma lipids during the development of an essential fatty acid deficiency

1. Male, female and castrated rats treated with oestradiol (30mug./week) or testosterone (2mg./week) were given an essential fatty acid-deficient diet containing 10% of hydrogenated coconut oil for 9 weeks. The concentrations and fatty acid composition of plasma phospholipids, cholesteryl esters and triglycerides were determined. 2. Between the second and third weeks of the deficiency, concentrations of plasma cholesteryl esters, phospholipids and triglycerides decreased, then remained relatively constant. There were no significant differences between males and females, but oestradiol caused a significant rise in plasma phospholipids and triglycerides as compared with testosterone-treated animals. 3. During the first 2 weeks of the deficiency, linoleic acid in the plasma lipids of all groups decreased to low concentrations and changed very little thereafter. 4. Female rats maintained higher percentages and concentrations of arachidonic acid and stearic acid in plasma phospholipids and arachidonic acid in cholesteryl esters than did males. Males had higher proportions of eicosatrienoic acid and oleic acid. There was no sex difference in the fatty acid composition of plasma triglycerides. 5. Oestradiol-treated rats had concentrations of cholesteryl and phospholipid arachidonate comparable with those of female rats and higher than the testosterone-treated group. Eicosatrienoic acid in the oestradiol-treated rats was high and resembled that of the male rats, apparently because of the higher concentration of plasma phospho lipids in this group. 6. Supplementation of the essential fatty acid-deficient rats with linoleate restored plasma cholesteryl and phospholipid linoleate and arachidonate nearly to normal concentrations in a single day. The increase in arachidonic acid in these fractions was accompanied by a similar quantitative decrease in eicosatrienoic acid. 7. These sex differences appear to be related to the smaller size of the female rat and to a more direct influence of oestradiol on the formation or maintenance of phospholipids rich in arachidonic acid.     

Inhibitors of sterol synthesis. Metabolism of [2,4-3H]5 alpha-cholest-8(14-)-en-3 beta-ol-15-one after intravenous administration to a nonhuman primate

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of cholesterol synthesis with marked hypocholesterolemic activity, has been studied after the intravenous administration of a mixture of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one and [4-14C] cholesterol to a baboon. The levels of 3H in plasma which was associated with the free 15-ketosterol decreased very rapidly (T1/2 approximately 9 min) after injection of the labeled sterol. By 4 h, the level of the [3H]15-ketosterol in plasma was negligible. The rapid decrease in the levels of the free 15-ketosterol was associated with rapid formation of fatty acid esters of the 15-ketosterol. The maximum level of 3H-labeled 15-ketosteryl esters was observed at 20 min after the injection of the 15-ketosterol. Thereafter, the levels of the 15-ketosteryl esters decreased rapidly with an apparent T1/2 of approximately 3.5-4.0 h. The results also indicated rapid formation of 3H-labeled cholesterol and cholesteryl esters. Substantial formation of [3H]cholesterol was observed at 20 min after the injection of the 15-ketosterol and reached a maximum level in plasma at 2 h. The maximum levels of [3H]cholesteryl esters in plasma were observed much later. These and other findings indicated that the observed slow clearance of total 3H from plasma is a consequence of metabolism of the 15-ketosterol to cholesterol and cholesteryl esters, normal constituents of plasma whose turnover in the whole animal is known to be relatively slow.     

Inhibitors of sterol synthesis. Metabolism of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one after oral administration to a nonhuman primate

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one is a potent inhibitor of cholesterol biosynthesis which has significant hypocholesterolemic activity upon oral administration to rodents and nonhuman primates. In the present study the metabolism of the 15-ketosterol has been investigated after the oral administration of a mixture of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one and [4-14C]cholesterol to 8 baboons. Blood samples were obtained at 4, 8, 12, 16, and 24 h after administration of the labeled sterols. Clear differences in the time courses of the levels of 3H and 14C in plasma were observed. 3H in plasma showed maximum values at 4 to 8 h, whereas maximum values for the levels of 14C were observed much later. 3H in plasma was shown to be primarily in the form of its metabolites, i.e. esters of the 15-ketosterol, cholesterol, and cholesteryl esters. The levels of the 15-ketosterol and of each of these metabolites showed different changes with time. The labeled cholesterol (and the cholesterol moiety of the cholesteryl esters), formed from the [2,4-3H]-15-ketosterol, was characterized by chromatography and by purification by way of its dibromide derivative. At 24 h after the administration of the labeled sterols, the distribution of 3H in plasma lipoprotein fractions paralleled that of 14C, with most of the 3H and 14C in high density lipoprotiens (HDL) and low density lipoproteins (LDL). Almost all of the 3H in HDL and in LDL was found as cholesterol, cholesteryl esters and esters of the 15-ketosterol. The distribution of 3H in HDL and in LDL of the free 15-ketosterol, esters of the 15-ketosterol, cholesterol, and cholesteryl esters was similar to that of plasma, thereby indicating no unusual concentration of any of the 3H labeled components in HDL or LDL.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

Essential fatty acid deficiency and adrenal cortical function in vitro.

Adrenocortical cells were prepared from rats maintained on essential fatty acid-deficient diets and control litter mates. Cells from control rats had high concentrations of essential fatty acids in the cholesteryl ester fraction of which approximately 22% was arachidonate. In contrast, cells from EFA-deficient rats had only 2.5% arachidonate in the cholesteryl esters, even though the total esterified cholesterol level was comparable to that of controls. In place of the essential fatty acids, the cholesteryl esters of these cells were rich in 20:3(n--9) and 22:3(n--9). When cells from EFA-deficient rats were incubated with ACTH or dibutyryl cyclic AMP, the output of corticosterone was the same as in controls. Also sterol esters were hydrolyzed to the same extent as in controls despite the unusual composition of the fatty acid esters. The phospholipids in both control and EFA-deficient cells contained high levels of arachidonate but were not hydrolyzed in either type of cell during incubation with ACTH or dibutyryl cyclic AMP. The results indicate that high levels of the prostaglandin precursors, namely linoleate and arachidonate, are not a sine qua non for the steroidogenic action of ACTH or cyclic AMP.     

Lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat.

In the adrenal gland of the rat, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol synthesis, is shown to be regulated by cholesteerol carried in plasma lipoproteins. When plasma cholesterol levels were lowered 90% by administration of the drug 4-aminopyrazolopyrimidine, the cholesteryl ester content of the adrenal gland declined by more than 90% and this was associated with a 150- to 200-fold increase in the activity of adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase and a 30-fold increase in cholesterol synthesis from [14C]acetate. The subsequent intravenous infusion of cholesterol contained in either rat or human high density or low density lipoproteins restored the adrenal content of cholesteryl esters and reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase to basal levels. The depletion of adrenal cholesteryl esters and the enhancement in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that occurred in the 4-aminopyrazolopyrimidine-treated rat required the action of adrenocorticotropic hormone (ACTH) since neither was observed when ACTH secretion was blocked by administration of dexamethasone. The current data indicate that the low rate of cholesterol synthesis normally observed in the rat adrenal gland is due to a suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that is mediated by plasma lipoproteins.     

Influence of diet on the composition of plasma cholesterol esters in man

The effect on the plasma cholesterol esters of diets rich in either carbohydrate, chocolate, or safflower oil was studied sequentially in two men. The changes in the cholesterol esters of the major plasma lipoproteins were studied by measuring (a) the distribution of fatty acids in the esters and (b) the distribution of radioactivity among the esters after the administration of cholesterol-4-(14)C labeled lipoproteins. Similar changes were found in the cholesterol esters of the two major lipoproteins; these changes became apparent within 24 hr after changing diets. Monounsaturated esters predominated with carbohydrate-rich diets. When the chocolate-rich diet was substituted, the proportion of saturated and monounsaturated esters fell and that of cholesteryl linoleate rose. This indicated the utilization of preexisting linoleate in preference to the more saturated fatty acids which abounded in the diet. The substitution of safflower oil led to further increments of cholesteryl linoleate. The possible reasons underlying the preferential incorporation of cholesteryl linoleate in man are discussed.     

Effects of dietary n-3 fatty acids on the composition of cholesteryl esters and triglycerides in plasma and liver perfusate of the rat

The effects of polyunsaturated fatty acids of the omega-3 family (PUFA n-3), (addition of fish oil), on the molecular composition of cholesteryl esters and triglycerides in plasma and liver perfusate of rats were studied. Rats fed a diet rich in saturated fatty acids (addition of lard) served as controls. Supplemention with PUFA n-3 not only decreases the plasma concentrations of free cholesterol, cholesteryl esters, and triglycerides, it also significantly alters the plasma composition of cholesteryl esters and triglycerides. Analyses of liver perfusate indicate a decrease in triglycerides secretion by in vitro perfused liver and reciprocal changes in relative contents of cholesteryl esters fractions with C(16) and C(20) acyl chains. This finding may be a result of chain-shortening of long-chain fatty acids probably in peroxisomal beta-oxidative system. Alterations in plasma cholesteryl esters and triglycerides composition of the fish oil group could be affected further by additional factors such as increased plasma cholesterol esterification activity and presence of triglyceride species of intestinal origin.     

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one. In vivo conversion to cholesterol upon oral administration to a nonhuman primate

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I), a potent inhibitor of cholesterol synthesis with marked hypocholesteremic activity, has been studied in a nonhuman primate. A mixture of [2,4-3H]-I and [4-14C]-cholesterol was administered to a male baboon in the form of a feedball. Blood was samples at 4, 8, 12, 16, and 24 hr. Detailed analyses of the plasma lipids indicated very rapid absorption of I (relative to cholesterol) and metabolism to cholesterol, cholesteryl esters, and esters of I. The labeled cholesterol was characterized by chromatographic techniques and by purification by way of its dibromide derivative. The levels of 3H in plasma associated with I, esters of I, cholesterol, and cholesteryl esters each showed a different time course. By 24 hr after the administration of [2,4-3H]-I, most of the 3H in plasma was associated with cholesterol and cholesteryl esters. The levels of total 3H and 14C in plasma at various times after the administration of the mixture of [2,4-3H]-I and [4-14C]-cholesterol differed markedly with 3H showing a maximum value at 4 hr and 14C showing a maximum value at 24 hr.     

Cholesteryl ester hydrolysis in rat liver lysosomes: different response to female sex hormones

The regulation of the hydrolysis of cholesteryl oleate by female sex hormones was studied in the lysosomal fraction of rat liver. Cholesterol ester hydrolase activity was determined at pH 5.0 with an acetone-dissolved cholesteryl [1-14C]oleate substrate preparation. The administration of a single dose of progesterone decreased the enzyme activity during a 3- to 24-hr period following hormone injection. This effect was not correlated to changes in the lysosomal protein synthesis rate. The lysosomal hydrolysis of cholesteryl esters was also inhibited in a noncompetitive manner by the addition of progesterone at concentrations higher than 100 microM. The esterase failed to respond to the estradiol in vivo as well as in vitro. The findings of the present paper suggest that the lysosomal breakdown of cholesteryl esters in rat liver may be under selective hormonal regulation and that the inhibitory effect of progesterone on the enzyme activity might be, at least in part, responsible for the liver cholesterol ester accumulus produced by the administration of the hormone.     

ACTH-induced hydrolysis of cholesteryl esters in rat adrenal cells.

Rat adrenal cortical cells have been prepared by collagenase dissociation of trypsin-treated adrenal tissue. The content and compositions of cholesteryl ester, phospholipid, and triglyceride fatty acids compare favorably with those of undissociated rat adrenal tissue. During 2-hour control incubations of adrenal cortical cells, steroidogenesis was not detected, and the levels of sterol ester, phospholipid, and triglyceride fatty acids were not significantly altered. Incubations with adrenocorticotropic hormone (ACTH) resulted in coricosterone production and significant depletions of sterol ester and triglyceride fatty acids, but not of phospholipid fatty acids. Although all fatty acid esters of cholesterol were hydrolyzed under these conditions, the greatest contributions to the net decrease in sterol esters were by oleate, arachidonate, and adrenate. Incubations with dibutyryl cyclic adenosine monophosphate (0.5 mM) resulted in significantly greater levels of corticosterone production than did ACTH (250 muunits), but the effects on cellular lipids were comparable to those seen with the tropic hormone. This study represents the first demonstration of hormone-induced hydrolysis of sterol esters in an in vitro cell suspension system. The results are discussed with respect to hormone-sensitive sterol ester hydrolase of adrenal cortex, and to the role of endogenous cholesteryl esters in the steroidogenic pathway.     

Short-term effect of dietary cholesterol on tissue n-6 fatty acids in fat-deficient rats

Weanling female rats raised on a fat-free diet for 8 weeks were then given the same diet supplemented with 0, 0.25, 0.5, or 1% by weight of cholesterol in addition to 10% of safflower oil for 3 days. Fatty acid compositions of cholesteryl esters (CE), triglycerides (TG), and phospholipids (PL) in liver and plasma were examined. Cholesterol feeding increased plasma and liver cholesterol contents and also affected the patterns of n-6 polyunsaturated fatty acids. There were no consistent changes in either plasma and liver TG which contained little 20:3n-6 and 20:4n-6. The levels of 20:3n-6 increased in plasma and liver PL, while proportions of 20:4n-6 decreased in liver and plasma CE. However, the absolute amount of 20:4n-6 in cholesteryl esters increased because of a threefold rise in cholesteryl ester levels. The changes might be attributable to an increased utilization of 20:4n-6 for cholesterol transport and/or an inhibition of delta 5-desaturation of n-6 fatty acids by cholesterol feeding.     

Free and esterified cholesterol concentration and cholesteryl ester composition in the ovaries of maturing and superovulated immature rats

The cholesterol and cholesteryl ester concentration and cholesteryl ester composition were determined in the ovaries of immature rats, sexually mature rats and superovulated immature rats. The immature rat ovary accumulated cholesteryl esters, and long-chain polyunsaturated fatty acids were preferentially incorporated into these esters. The cholesteryl esters decreased in concentration and changed in composition with the onset of the first estrous cycle. Superovulation of immature rats, by injection of 50 I.U. pregnant mare serum gonadotropin, caused a decrease in the cholesteryl ester concentration of the ovary within 24 h and specific depletion of some esters, particularly those of 20 : 1 and 22 : 6 acids. Human choriogonadotropin, administered 54 h later, induced synchronous luteinization of the ovaries and was followed by increases in the concentration of free and esterified cholesterol and preferential accumulation of the esters of 20 : 4, 22 : 4, 4, 22 : 5 and 22 : 6 acids. Acute stimulation of luteinized ovaries by a second injection of the rats with 25 I.U. of human choriogonadotropin resulted in preferential hydrolysis of the esters of 18 : 1, 20 : 4, 22 : 4 and 22 : 5 acids.     

The activity of cholesterol ester hydrolase in the enzymatic determination of cholesterol: comparison of five enzymes obtained commercially

The use of five cholesterol ester hydrolases (CEH), numbered 1 to 5, for the enzymatic determination of total cholesterol of human and rat serum are compared. All CEH gave approximately the same value (no statistical difference) for human serum. However, when rat serum cholesterol was determined, CEH-2 yielded a value significantly lower when compared to the four other CEH. The ability of each CEH to hydrolyze individual cholesterol esters was tested. During a 15-min incubation, all CEH were capable of hydrolyzing nearly 100% of cholesteryl oleate and linoleate. In contrast, the hydrolysis of cholesteryl arachidonate was only partial and varied from 20 to 80% depending on the CEH used. The highest hydrolysis was obtained by CEH-1 while the value given by CEH-2 was only 22% of that obtained by CEH-1. The rate of hydrolysis of cholesteryl arachidonate differed markedly among the CEH. The CEH-2-hydrolyzed the cholesteryl arachidonate at a rate seven times lower than the rate obtained with CEH-1. The data suggest that, Under our incubation conditions, CEH-2 did not properly hydrolyze the cholesteryl arachidonate. This phenomenon may be crucial whenever total cholesterol has to be determined enzymatically in the serum of species that contain large amount of cholesteryl arachidonate such as rat, mouse, or dog serum.     

Molecular characterization and expression of the cholesteryl ester transfer protein gene in chickens

The cDNA for cholesteryl ester transfer protein (CETP), a protein that catalyzes cholesteryl ester transfer between very low density and high density lipoproteins in plasma, was isolated from chicken liver. When the recombinant protein was overexpressed in HEK293 cells, cholesteryl ester transfer activity was observed in media and cell lysates. By Northern blot analysis, chicken CETP mRNA expression was detected in liver, brain, heart, and spleen. Changes in chicken CETP mRNA expression and plasma CETP activity with nutritional state were examined and found to increase following dietary supplementation with cholesterol in a similar way as in humans. Both the hepatic CETP mRNA levels and plasma CETP activity were significantly lower in mature (i.e egg-laying) hens than in immature female chickens, but were unaffected by age in male animals. Similar changes to those observed in female chickens were observed upon estradiol administration of males. The present study is the first to report the molecular characterization of an avian CETP, and the impairments of CETP gene and activity, which might be regulated by estrogen, play an important role in egg production in laying hens, demonstrating species-specific differences in the lipid metabolism of avian and mammalian species.     

Monoamine oxidase activity in the hypothalamus and various other brain areas and in some endocrine glands of the rat during the estrus cycle

—The activity of monoamine oxidase (MAO, EC 1.4.3.4) was measured in the entire hypothalamus and different hypothalamic regions, in the amygdala, frontal and lateral cerebral cortex, in the pituitary, adrenals and genital organs of male rats and of female rats during the estrus cycle. Activity of MAO changed cyclically in the hypothalamus, amygdala, adrenals and ovaries. The highest levels in the hypothalamus occurred at 10 a.m. on the day of proestrus and during estrus. The lowest levels occurred at 6 p.m. on the day of proestrus, of metestrus and during diestrus. Cyclical variations similar to those found in the whole hypothalamus were also observed in anterior, posterior and lateral portions and the median eminence of the hypothalamus. Activity in the median eminence was greater than that of the whole hypothalamus or its various other portions. The amygdala exhibited less marked cyclical activity which followed the pattern of the hypothalamus by increasing at 10 a.m. and peaking at 3 p.m. on the day of proestrus. At the‘post-critical’period of proestrus, when the activity of MAO in the hypothalamus and amygdala decreased, the activity of MAO in the ovaries and adrenals rose. During the estrus cycle much lower levels of activity of MAO were demonstrated in other regions of the brain (frontal and lateral cerebral cortex), in the pituitary and in the uterus, none of which showed cyclical changes. The changes in activity of MAO in cerebral tissues, endocrine glands and genital organs have been discussed in relation to the probable participation of monoamines in the mechanism(s) of secretion of gonadotrophins by the hypothalamus.     

Diminution of blood and hepatic cholesterol induced by an apple-supplemented diet in the hamster. Trials in man]

The effects of an apple-supplemented diet on plasma and liver cholesterol were investigated in two strains of Hamster, a normal one and another which exhibits a spontaneous accumulation of cholesteryl esters in the liver (FEC animals). Adding of apples to the standard diet promoted a 20 % decrease of cholesterolemia in normal animals and normalized the high level of plasma cholesterol in the FEC animals. Similarly, the cholesterol content of the liver was diminished. The levels of cholesteryl esters were strongly lowered, threefold and tenfold respectively in normal and in FEC animals. Incorporation of labelled mevalonate into cholesterol was increased, but esterification of the neosynthesized sterol was markedly reduced in the liver from FEC animals. In human daily ingestion of apples caused a significative reduction (16 %) of cholesterolemia.