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.     

The metabolism of esterified cholesterol in rabbit plasma low density lipoproteins

The metabolism of esterified cholesterol in plasma low density lipoproteins (LDL) has been studied in rabbits. LDL labelled with ³H in the esterified and free cholesterol moieties was isolated from the serum of donar rabbits which has been injected with [³H]mevalonic acid, and subsequently either incubated at 37°C in vitro with unlabelled rabbit serum or unlabelled rabbit lipoprotein fractions, or reinjected into other rabbits.In vitro there was found to be a transfer of 40–60% of the esterified [³H]-cholesterol out of LDL into both the very low density lipoprotein (VLDL) and high density lipoprotein (HDL) fractions which could not be explained in terms of net transfer of esterified cholesterol mass. In the incubations of labelled LDL with either of the other unlabelled lipoprotein fractions, transfers were apparent only if the dialysed 1.21 g/ml infranatant of rabbit serum was also present. The transfer of esterified [³H]cholesterol out of LDL was enhanced when lecithin:cholesterol acyltransferase was active.After reinjecting labelled LDL into other rabbits, it was found that more than half of the esterified [³H]cholesterol removed from the recipient LDL fraction during the first 30 min was not lost from the plasma compartment, but rather was recovered in HDL. There was only minimal in vivo transfer of LDL esterified [³H]cholesterol into VLDL.It has been concluded that in vitro the esterified cholesterol in LDL exchanges with that in both the VLDL and HDL, and that in vivo the esterified cholesterol pools in LDL and HDL may represent parts of a progressively equilibrating plasma pool.     

Transfer of esterified cholesterol from serum lipoproteins to the liver.

The fate of cholesteryl esters of the serum lipoproteins was studied in intact rats and in isolated perfused rat livers. The lipoproteins of fasting rat serum were labeled in vitro with [3H]cholesteryl oleate. Following intravenous injection, it was found that the majority of the radioactive ester was rapidly taken up by the liver where hydrolysis of the ester bond occurred. At 5 min, 58% of the injected material was recovered in the liver, 85% of which was still in the ester form, while at 30 min only 22% of the liver radioactivity was in cholesteryl esters. There was very little difference in the rate at which radioactivity was taken up from the different lipoprotein classes. Similar phenomena were observed in the perfused liver, but it was found that although the radioactive esters were being taken up, there was no change in the concentrations of free or esterified cholesterol in the perfusing medium, indicating that the lipoprotein cholesteryl ester was gaining access to the liver through an exchange of molecules. After uptake, cell fractionation experiments showed that the plasma membranes had the greatest relative amounts of radioactivity, suggesting that this is the site of exchange. Small amounts of radioactivity were recovered in the bile, demonstrating that serum lipoproteins can serve as precursors of at least some of the bile steroids.     

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.     

The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.     

Enhanced macrophage uptake of elastase-modified high-density lipoproteins

Incubation of human HDL (d = 1.063-1.21 g/ml) with monocyte-derived elastase causes selective proteolysis of apoA-II and apoA-I apolipoproteins. We have found that elastase-digested HDL (ED-HDL) bind to J774-A1 murine macrophages with enhanced affinity and are internalized and degraded at a rate threefold higher than that of native HDL. Unlike oxidized LDL and HDL and proteolytically modified LDL, the uptake of ED-HDL lipoproteins does not affect the cellular lipid biosynthesis nor modify the cell lipid content. The cell surface binding of (125)I-ED-HDL can be competed by native HDL but not by acetylated LDL, consistent with the idea that ED-HDL are recognized by the class B type I scavenger receptor. The liberation of elastase by lipid-engorging macrophages is regarded as an important event during atherogenesis. By enhancing the cellular uptake of HDL this process can lead to a local decrease of antiatherogenic HDL particles.     

Metabolism of free and esterified cholesterol by Leydig-cell tumour mitochondria

1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-³H]cholesterol (50–100μCi per animal). Single-cell suspensions were prepared from the tumours 5–9 days after the injection of [³H]cholesterol and were incubated at 37°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 (5μg/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.     

Variance of plasma free and esterified cholesterol in adult twins.

Total, free and esterified cholesterol were measured in the plasma, high density lipoproteins, low density lipoproteins, and total cholesterol in the very low density lipoproteins of 141 pairs of adult white male twins. Free cholesterol in plasma, high density lipoproteins and low density lipoproteins had significant genetic variance. Esterified cholesterol had greater total variance in dizygotic than monozygotic twins, interpreted as evidence for greater environmental influences on the two types of twins. After bias due to unequal environmental effects on the two types of twins was removed, there was no significant genetic variance for any esterified fraction of cholesterol.     

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.     

Origin of esterified cholesterol transported in the very low density lipoproteins of human plasma

In two subjects the specific activity of esterified cholesterol in plasma lipoprotein subfractions was measured for up to 9 hr after an intravenous injection of [(3)H]mevalonic acid. It was found to be consistently higher in larger (S(f) > 100) than in smaller (S(f) 20-100) very low density lipoproteins (VLDL). Four subjects were given an intravenous injection of heparin so that the VLDL could be studied as its concentration fell and subsequently rose again. During the first hour the relative reduction was greatest for triglyceride, intermediate for free cholesterol, and least for esterified cholesterol. Between 1 and 7 hr postheparin, the VLDL pool was restored, but the pattern of increase of individual lipids was not parallel. The triglyceride increment was much greater during the 1-4-hr period than during the 4-7-hr period; in three of the subjects the free cholesterol increment was also greater during the earlier period. The increase in esterified cholesterol, however, was consistently greater during the 4-7-hr period. In six other subjects the specific activity of VLDL esterified cholesterol was related to that of its possible plasma precursors in samples collected at 1-hr intervals for 8 hr after the injection of [(3)H]mevalonic acid. Free cholesterol emerged as the most likely immediate precursor with the possibility of a hepatic as well as an intraplasma origin. The results did not support a major in vivo transfer of esterified cholesterol from high density lipoproteins to VLDL.     

Structural organization of free and esterified cholesterol in human high density lipoproteins. A 100.6 MHz 13C NMR study.

The structural organization of free and esterified cholesterol in human high density lipoproteins has been studied by high-field 1H and 13C NMR. The measurements are consistent with free cholesterol being present in at least two different environments. Part of the free cholesterol is oriented in the outer surface layer of the high density lipoprotein particle in contact with phospholipid or apoprotein, or both. The rest is probably present in the liquid, hydrophobic core of the HDL particle.     

Hepatic uptake and metabolism of free cholesterol from different lipoprotein classes. An in vivo study in the rat

Hepatic metabolism of [14C]cholesterol, vehiculated by LDL, HDL2 and HDL3 lipoprotein particles, has been studied in rats with a permanent biliary drainage. The lipoprotein fractions were infused individually by a jugular vein catheter and bile was collected for 180 min after the administration. At the end of this period, the animals were killed and the blood and livers were collected. The free cholesterol of the HDL2 fraction was secreted into bile, mainly as bile salt, preferentially to that associated with HDL3 and LDL fractions (11.7% vs. 2.3% and 0.3%, respectively). The free cholesterol of the HDL3 fraction, on the other hand, was taken up by liver more quickly and in a higher proportion than that associated with other lipoprotein fractions. The label incorporation in this lipoprotein fraction was secreted earlier and not transformed into bile. The contribution of LDL-vehiculated free cholesterol to bile secretion was small and the hepatic uptake amounted to no more than 12% of the injected label.