Influence of luteinizing hormone and adenosine 3':5'-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumours

1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-(3)H]cholesterol (50-100muCi per animal). Single-cell suspensions were prepared from the tumours 5-9 days after the injection of [(3)H]cholesterol and were incubated at 37 degrees C in foetal calf serum supplemented with 50mm-Tris-HCl, pH7.4. At various times after the start of incubation cells were collected by filtration of portions of the suspension and their sterols analysed. Within 10min after LH (5mug/ml) or 3':5'-cyclic AMP (20mm) was added to the cell suspensions an increased conversion of ester cholesterol into free cholesterol could be demonstrated. 2. To observe this rapid effect of LH it was necessary to incubate the cells for 60min before addition of hormone. 3. The specific radioactivity of testosterone produced was approximately equal to that of the intracellular cholesterol regardless of the presence or absence of LH. 4. The amount of free cholesterol produced in response to LH was far greater than that needed for steroid synthesis. 5. Free cholesterol, but not esterified cholesterol, was released into the incubation medium linearly with time and this release was unaffected by LH. LH may stimulate steroidogenesis in part by increasing the concentration of free cholesterol within the cell.     

An enzymatic method for the histochemical localization of free and esterified cholesterol separately

Synopsis An enzymatic method for the histochemical localization of cholesterol is presented. It makes possible the localization of free cholesterol, cholesterol esters, or both and is compatible with routine histological staining procedures. The method is based on the production of H₂O₂ from free cholesterol by cholesterol oxidase. Sites of peroxide production are visualized by a brown reaction product formed in a peroxidase-catalysed reaction between diaminobenzidine and H₂O₂. cholesterol esters can be demonstrated as cholesterol after hydrolysis by cholesterol ester hydrolase. Some examples of the application of the method are given.     

Determination of free and esterified cholesterol in human serum by high-performance liquid chromatography and optical activity detection

A separation and detection scheme is presented for the determination of free, esterified and total cholesterol in human serum. Separation is accomplished by reversed-phase high-performance liquid chromatography and the eluate is monitored by the laser-based optical activity detector. The method is simple, accurate and has the advantage of specificity and selectivity when compared with the many methods commonly used.     

Cyclodextrins differentially mobilize free and esterified cholesterol from primary human foam cell macrophages

Human monocyte-derived foam cell macrophages (HMFCs) are resistant to cholesterol efflux mediated by physiological acceptors. The role of the plasma membrane in regulating depletion of free cholesterol (FC) and of cholesteryl ester (CE) was investigated using cyclodextrins (CDs). HMFCs were incubated in media containing CDs (1.0 mg/ml, approximately 0.7 mM) with low [hydroxypropyl-beta-CD (HP-CD)] or high [trimethyl-beta-CD (TM-CD)] affinity for cholesterol in the presence or absence of phospholipid vesicles (PLVs). Low-affinity HP-CD caused minimal cholesterol efflux on its own, but HP-CD+ PLV depleted cell FC and CE to 54.5 +/- 6.7% of control by 24 h. TM-CD depleted FC at least as well as HP-CD+PLV but without depleting CE, even when combined with PLV. This was not explained by acceptor saturation, instability of PLV vesicles, de novo cholesterol synthesis, kinetically distinct cholesterol pools, or inhibition of CE hydrolysis. TM-CD did, however, deplete CE when lower concentrations of TM-CD were combined with PLV and when acetyl-CoA cholesteryl acyltransferase was inhibited. TM-CD caused much greater depletion of plasma membrane cholesterol than HP-CD without depleting plasma membrane sphingomyelin. It is concluded that differential depletion of plasma membrane cholesterol pools regulates cholesterol efflux and CE clearance in human macrophages.     

Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase

The metabolism of cholesterol derived from [3H]cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large alpha-migrating HDL (HDL2) and was then transferred to small alpha-HDL (HDL3) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating in HDL2. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL2 as a relatively unreactive sink for LCAT-derived cholesteryl esters.     

Transfer of free and esterified cholesterol from low-density lipoproteins and high-density lipoproteins to human adipocytes

Cholesterol stored in human adipose tissue is derived from circulating lipoproteins. To delineate the cholesterol transport function of LDL and HDL, the movement of radiolabelled esterified cholesterol and free cholesterol from labelled LDL and HDL to human adipocytes was examined in the present study. LDL and HDL were enriched and labelled in esterified cholesterol with [14C]cholesterol by the action of plasma lipid transfer proteins and lecithin-cholesterol acyltransferase. Doubly labelled (3H,14C) LDL and HDL were prepared by exchanging free [3H]cholesterol into the 14C-labelled lipoproteins. 14C-labelled lipoprotein and 3H-labelled lipoprotein were also prepared separately and mixed to yield a mixed doubly labelled lipoprotein. Relative to the total amount added, proportionally more free than esterified cholesterol was transferred to the adipocytes upon incubation with any doubly labelled LDL and HDL. The calculated mass of free and esterified cholesterol transferred, however, varied with different labelled lipoproteins. 3H- and 14C-labelled LDL or HDL transferred 2-3-fold more esterified than free cholesterol while the reverse occurred with the mixed doubly labelled LDL or HDL. Thus, free cholesterol-depleted particles preferentially transferred cholesterol ester to the fat cells. In the presence of the homologous unlabelled native lipoprotein, the transfers of free and esterified cholesterol from labelled LDL or HDL were specifically inhibited. Selective transfer of esterified cholesterol relative to apoprotein was also observed when esterified cholesterol uptake from both LDL and HDL was assayed along with the binding of 125I-labelled lipoprotein. The cellular accumulation of cholesterol ether-labelled HDL (a non-hydrolyzable analogue of cholesterol ester) exceeded that of cholesterol ester consistent with significant hydrolysis of the latter physiological substrate. These results demonstrate preferential transfer of free cholesterol and esterified cholesterol over apoprotein for both LDL and HDL in human adipocytes. Furthermore, the data suggest that the cholesterol ester transport function of LDL and HDL can be enhanced by free cholesterol depletion and cholesterol ester enrichment of the particles, and affirms a role for adipose tissue in the metabolism of lipid-modified lipoproteins.     

Differential uptake and metabolism of free and esterified cholesterol from high-density lipoproteins in the ovary

Rat luteal cells utilize high-density lipoproteins (HDL) as a source of cholesterol for steroid synthesis. Both the free and esterified cholesterol of HDL are utilized by these cells. In this report, we have examined the relative uptake of free and esterified cholesterol of HDL by cultured rat luteal cells. Incubation of the cells with HDL labeled with [3H]cholesterol or [3H]cholesteryl linoleate resulted in 4-6-fold greater uptake of the free cholesterol compared to esterified cholesterol. The increased uptake of free cholesterol correlated with its utilization for progestin synthesis: utilization of HDL-derived free cholesterol was 3-6-fold higher than would be expected from its concentration in HDL. The differential uptake and utilization of free and esterified cholesterol was further examined using egg phosphatidylcholine liposomes containing cholesterol or cholesteryl linoleate as a probe. Liposomes containing free cholesterol were able to deliver cholesterol to luteal cells and support steroid synthesis in the absence of apolipoproteins, and the addition of apolipoprotein A-I (apo A-I) moderately increased the uptake and steroidogenesis. Similar experiments using cholesteryl linoleate/egg phosphatidylcholine liposomes showed that inclusion of apo A-I resulted in a pronounced increase in the uptake of cholesteryl linoleate and progestin synthesis. These experiments suggest that free cholesterol from HDL may be taken up by receptor-dependent and receptor-independent processes, whereas esterified cholesterol uptake requires a receptor-dependent process mediated by apolipoproteins.     

Degradation of non-esterified and esterified xanthophylls by free radicals

beta-Carotene and other xanthophylls present in pepper fruit as both free and esterified forms were oxidized using a free radical initiator (2,2'-azo-bis-isobutyronitrile). Capsorubin was degraded most slowly, followed by zeaxanthin, capsanthin, and beta-carotene. The presence of keto groups at the ends of the polyene chain could be a structural factor contributing to this difference in reactivity. It was also shown that whereas capsanthin and its esters and capsorubin and its esters were degraded at the same rate, zeaxanthin esters responded differently to the oxidation process, and were degraded more quickly than free zeaxanthin. The presence of unsaturated fatty acids (mainly linoleic) that esterify zeaxanthin help to accelerate the degradation of this xanthophyll and decreasing its antioxidant action. The antioxidant capacity of capsorubin and capsanthin (both in free and esterified form) exclusive to the genus capsicum should be taken into account.     

Correlation of rat liver chromatin-bound free and esterified cholesterol with the circadian rhythm of cholesterol biosynthesis in the rat.

Cholesterol has been shown to be present in rat liver chromatin isolated by methods designed to avoid contamination by membrane fragments. Evidence that the cholesterol was actually a component of chromatin includes (a) the constancy of the amount (1.30 +/- 0.14 mug per mg DNA), (b) the striking difference in the ratio of free (i.e. unesterified) to esterified cholesterol between that in chromatin and that in membrane, and (c) the rapid and marked changes which occurred in this ratio during the circadian cycle in chromatin but not in membranes. Although the total amount of chromatin-bound cholesterol did not change throughout the circadian cycle, the concentration of free cholesterol increased sharply a short time before the peak of cholesterol synthetic activity was reached at about midnight; it reached a basal level about 6 h later at approximately the same time the rate of synthesis returned to its basal level. When labelled cholesterol was administered by stomach tube, it was detectable within 2 h in whole nuclei and in chromatin, indicating that chromatin-bound cholesterol is rapidly exchangeable with that in liver cytoplasm and in blood plasma. Removal of basic proteins from chromatin did not result in the loss of any cholesterol, but removal of most of the acidic as well as the basic proteins resulted in loss of most of the chromatin-bound cholesterol. These results indicate that cholesterol is bound either to the acidic proteins or to both the acidic proteins and DNA. The data are compatible with the hypothesis that cholesterol biosynthesis controlled at the nuclear level and suggest that the relative amounts of free and esterified cholesterol associated with chromatin may play a role.     

In vivo study of free and esterified cholesterol turnover in various tissues of the rat.

Rats were infused for 3.5 to 10 hrs with either red cells or plasma previously labelled in vivo by [3H]-cholesterol. Cholesterol specific radioactivities were measured in plasma, HDL, LDL and VLDL, and various tissues. Red cell infusions led to a higher labelling of free than of esterified cholesterol in the plasma of infused rats. The opposite situation was observed following plasma infusion. Comparison of free and esterified cholesterol specific radioactivities in each tissue showed that esterified cholesterol was transferred from plasma to all the tissues, except the adrenals. Study of the ratios of cholesterol specific radioactivities from one experimental group to the other in each tissue, made it possible to demonstrate clearly the occurence of hydrolysis within all the studied tissues except 5 of them where its existence remains uncertain (lung, heart, kidney, tendon, muscle) and of esterification in 3 tissues (adrenal, liver lung). In addition, ratios of cholesterol radioactivities (free/ester) were found to be identical in plasma and in 4 tissues, where neither hydrolysis nor esterification were detected (heart, muscle, kidney, tendon). This finding is an argument in favor of a simultaneous transport of free and esterified cholesterol from plasma into these 4 tissues and suggests that the entire lipoprotein particles can penetrate these tissues, with no specificity of one special class. In adrenal, unlike all other tissues: 1) the turnover of esterified cholesterol was achieved mostly by hydrolysis and esterification in situ; 2) a preferential lipoprotein class (LDL) was responsible for the transport of free cholesterol from the plasma.     

Stimulation of esterified cholesterol accumulation in tissue culture cells exposed to high density lipoproteins enriched in free cholesterol.

Human high density lipoprotein enriched in free cholesterol was obtained by exposing the lipoprotein to lipid dispersions having a free cholesterol/lecithin molar ratio greater than two. The metabolism of cholesterol was studied in tissue culture cells exposed to normal and cholesterol-enriched lipoproteins. Incubation of Fu5-AH rat hepatoma cells in medium containing cholesterol-enriched lipoprotein resulted in the accumulation of cellular cholesterol whereas normal high density lipoprotein produced no change in cellular content. The accumulated sterol was recovered primarily as esterified cholesterol and was derived almost entirely from lipoprotein free cholesterol. The esterification of incorporated free cholesterol and the cellular cholesterol content were directly related to the molar ratio of free cholesterol to phospholipid in the lipoprotein and to the concentration of lipoprotein in the culture medium. Isotopic experiments utilizing lipoprotein labeled with 125I or [4-14C]cholesteryl oleate demonstrated that a large fraction of the cholesterol incorporated from lipoprotein enriched in free cholesterol occurred by mechanisms that did not result in lipoprotein internalization and degradation. The response of other tissue culture cells to cholesterol/phospholipid dispersions is presented. The data indicate that the lipid composition of a lipoprotein can regulate free cholesterol uptake and esterification as well as cellular cholesterol content.     

Changes during hibernation in different phospholipid and free and esterified cholesterol serum levels in black bears

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P < 0.05). The percentage increase during hibernation was more in free cholesterol (57%) than in esterified cholesterol (27%). Analysis of subclasses of serum phospholipids showed that choline containing phospholipids, i.e., sphingomyelin (SPG) (14%) and phosphatidylcholine (PC) (76%), are the major phospholipids in the serum of bear. The minor phospholipids included 8% of phosphatidylserine (PS) + phosphatidylinositol (PI), while phosphatidylethanolamine (PE) was only 2% of the total phospholipids. A comparison of phospholipid subclasses showed that PC, PS + PI and SPG were significantly increased, while PE was significantly decreased (P < 0.05) in the hibernating state as compared with the active state in black bears. These results suggest that the catabolism of phospholipids and cholesterol is decreased during hibernation in black bears, leading to their increased levels in the hibernating state as compared with the active state. In summary, our results indicate that serum cholesterol and phospholipid fractions (except PE) are increased during hibernation in bears. It is proposed that the increase of these lipids may be due to the altered metabolism of lipoproteins that are responsible for the clearance of the lipids.     

Amelioration of popolysaccharide-induced sepsis in rats by free and esterified carnitine

The purpose of this study was to determine if free or esterified carnitine could alter fatty acid metabolism and ameliorate sepsis in lipopolysaccharide (LPS)-treated rats. Throughout a 96 h observation post-LPS, i.p. administration of both markedly reduced illness and accelerated recovery. Carnitine prevented the acute LPS-induced rise in serum triglycerides (45 +/- 6, 59 +/- 5 vs. 83 +/- 8 mg/ml, p < 0.001), respectively. This difference was accompanied by a significant increase in liver lipogenesis in LPS controls compared to both carnitines and normal rats (6.1 +/- 0.3 vs. 3.9 +/- 0.5, 4.3 +/- 0.5, and 1.8 +/- 0.4 mumol/h, respectively, p < 0.04). Compared to normal rats, total liver carnitine was significantly elevated in LPS controls and even higher in the carnitine groups (357 +/- 40 vs. 736 +/- 38, 796 +/- 79, and 1081 +/- 21 nmol/g). The data suggest that carnitines may be of therapeutic value in sepsis treatment and one action may be to partition fatty acids from esterification to oxidation.     

Resensitization of lutropin-desensitized tumour Leydig-cell adenylate cyclase with human erythrocyte membranes.

Purified rat tumour Leydig cells were pretreated with or without lutropin (1 h at 32 degrees C). The plasma membranes were then isolated and the adenylate cyclase activity measured in the presence of freshly prepared or heat-inactivated (1 h at 60 degrees C) human erythrocyte membranes. In plasma membranes from control cells in the presence of heat-inactivated human erythrocyte membranes both guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) plus lutropin and NaF caused a 45--50-fold increase in cyclic AMP production over 30 min compared with 12--13 fold p[NH[ppG and 2--3-fold with lutropin alone. In plasma membranes isolated from lutropin-pretreated cells the NaF- and the p[NH]ppG-stimulated cyclic AMP production rates were unchanged, but no effect of lutropin could be demonstrated with or without added p[NH]ppG. However, after mixing lutropin-desensitized Leydig tumour-cell plasma membranes with freshly prepared human erythrocyte plasma membranes, the adenylate cyclase activity in the presence of lutropin, p[NH]ppG, lutropin plus p[NH]ppG and NaF were similar to those of control cell plasma membranes treated in the same manner. The possible mechanisms of this reversal of lutropin-induced desensitization by human erythrocytes are discussed.