Processing of cholesteryl ester from low-density lipoproteins in the rat. Hepatic metabolism and biliary secretion after uptake by different hepatic cell types.

Biliary secretion of the cholesteryl ester moiety of (modified) low-density lipoprotein (LDL) was examined under various experimental conditions in the rat. Human LDL or acetylated LDL (acetyl-LDL), radiolabelled with [3H]cholesteryl oleate, was administered intravenously to unanesthetized rats equipped with permanent catheters in the bile duct, duodenum and heart. LDL was cleared relatively slowly from plasma, mainly by Kupffer cells. At 3 h after injection, only 0.9% of the radioactivity was found in bile; after 12 h this value was 4.5%. Uptake of LDL by hepatocytes was stimulated by treatment of the rats with 17 alpha-ethinyloestradiol (EE; 5 mg/kg for 3 successive days); this resulted in a more rapid secretion of radioactivity into bile, 3.9% and 12.4% after 3 h and 12 h respectively. The extremely rapid uptake of acetyl-LDL via the scavenger pathway, mainly by endothelial cells, resulted in the secretion of only 2.1% of its 3H label into bile within 3 h, and 9.5% within 12 h. Radioactivity in bile was predominantly in the form of bile acids; only a small part was secreted as free cholesterol. However, the specific radioactivity of biliary cholesterol was higher than that of bile acids in all three experimental conditions. EE-treated animals did not form cholic acid from [3H]cholesteryl oleate, which was a major product of the cholesteryl oleate from LDL and acetyl-LDL in untreated rats, but formed predominantly very polar bile acids, i.e. muricholic acids. It is concluded that uptake of human LDL or acetyl-LDL by the liver of untreated rats is not efficiently coupled to biliary secretion of cholesterol (bile acids). This might be due to the anatomical localization of their principal uptake sites, the Kupffer cells and the endothelial cells respectively. Induction of LDL uptake by hepatocytes by EE treatment warrants a more efficient disposition of cholesterol from the body via bile.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.     

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.     

Short- and long-term effects of biliary drainage on hepatic cholesterol metabolism in the rat.

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.     

Progesterone blocks cholesterol translocation from lysosomes.

Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.     

Cholesteryl ester enrichment of plasma low-density lipoproteins in hamsters fed cereal-based diets containing cholesterol

Male Syrian hamsters were fed 0.02, 0.03, or 0.05% cholesterol to test the hypothesis that moderate cholesterol intake increases the cholesteryl ester content of the plasma low-density lipoproteins (LDL). Dietary cholesterol levels of 0.02%-0.05% were chosen to reflect typical human intakes of cholesterol. Hamsters were fed ad libitum a cereal-based diet (modified NIH-07 open formula) for 15 weeks. Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased plasma LDL and high-density lipoprotein cholesterol concentration, increased liver cholesterol concentration, and increased total aorta cholesterol content. The cholesteryl ester content of plasma LDL was determined as the molar ratio of cholesteryl ester to apolipoprotein B and to surface lipid (i.e., phospholipid + free cholesterol). Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased cholesteryl ester content of LDL particles. Furthermore, cholesteryl ester content of LDL was directly associated with increased total aorta cholesterol, whereas a linear relationship between plasma LDL cholesterol concentration and aorta cholesterol was not observed. Thus, the data suggest that LDL cholesteryl ester content may be an important atherogenic feature of plasma LDL.     

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.     

Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages

Oxidatively modified low density lipoproteins (Ox-LDL) may be involved in determining the formation of foam cells by inducing cellular cholesteryl ester accumulation. We studied the effect of copper oxidized LDL (Ox-LDL) on cholesterol accumulation and esterification in murine macrophages. Ox-LDL (44 micrograms/ml of lipoprotein cholesterol) increased the total cholesterol content of the cells from 29 to 69 micrograms/mg cell protein. Free cholesterol accounted for 85% of this increase. Acetyl LDL (Ac-LDL) (38 micrograms/ml of lipoprotein cholesterol), raised total cellular cholesterol content to a similar extent (76 micrograms/mg cell protein), however only 25% of the accumulated cholesterol was unesterified. When ACAT activity was determined after incubation of J774 cell with Ox- or Ac-LDL, Ox-LDL were 12 times less effective than Ac-LDL in stimulating cholesteryl ester formation. This was not due to an inhibition of ACAT by Ox-LDL since these lipoproteins failed to inhibit pre activated enzyme in cholesteryl ester-loaded macrophages. The uptake of 125I-Ox-LDL: was 175% that of 125I-Ac-LDL, while degradation was only 20%. All together these data suggest an altered intracellular processing of Ox-LDL, which may be responsible for free cholesterol accumulation.     

Hepatic lipid accumulation in apolipoprotein C-I-deficient mice is potentiated by cholesteryl ester transfer protein

The impact of apolipoprotein C-I (apoC-I) deficiency on hepatic lipid metabolism was addressed in mice in the presence or the absence of cholesteryl ester transfer protein (CETP). In addition to the expected moderate reduction in plasma cholesterol levels, apoCIKO mice showed significant increases in the hepatic content of cholesteryl esters (+58%) and triglycerides (+118%) and in biliary cholesterol concentration (+35%) as compared with wild-type mice. In the presence of CETP, hepatic alterations resulting from apoC-I deficiency were enforced, with up to 58% and 302% increases in hepatic levels of cholesteryl esters and triglycerides in CETPTg/apoCIKO mice versus CETPTg mice, respectively. Biliary levels of cholesterol, phospholipids, and bile acids were increased by 88, 77, and 20%, respectively, whereas total cholesterol, HDL cholesterol, and triglyceride concentrations in plasma were further reduced in CETPTg/apoCIKO mice versus CETPTg mice. Finally, apoC-I deficiency was not associated with altered VLDL production rate. In line with the previously recognized inhibition of lipoprotein clearance by apoC-I, apoC-I deficiency led to decreased plasma lipid concentration, hepatic lipid accumulation, and increased biliary excretion of cholesterol. The effect was even greater when the alternate reverse cholesterol transport pathway via VLDL/LDL was boosted in the presence of CETP.     

Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome.

Previously, we isolated and characterized unique liposomal-like, cholesterol-rich lipid particles that accumulate in human atherosclerotic lesions. Human plasma low density lipoprotein (LDL) has a molar ratio of total cholesterol to phospholipid (3:1) similar to that of this lesion cholesterol-rich lipid particle. However, LDL is enriched in cholesteryl ester while the lesion lipid particle is enriched in unesterified cholesterol. To examine a possible precursor-product relationship between LDL and the lesion lipid particle, we hydrolyzed the cholesteryl ester core of LDL with cholesterol esterase. Cholesteryl ester hydrolysis occurred only after LDL was treated with trypsin. Trypsin pretreatment was not required for cholesteryl ester hydrolysis of LDL oxidized with copper, a treatment that also degrades apolipoprotein B, the major protein moiety in LDL. In contrast to greater than 90% hydrolysis of cholesteryl ester in trypsin-cholesterol esterase-treated or copper-oxidized LDL, there was only 18% hydrolysis of cholesteryl ester in similarly treated high density lipoprotein. With a limited 10-min hydrolysis of LDL cholesteryl ester, LDL-sized particles and newly formed larger flattened films or discs were present. With complete hydrolysis of LDL cholesteryl ester, LDL particles converted to complex multilamellar, liposomal-like, structures with sizes approximately five times larger than native LDL. These liposomal-like particles derived from LDL were chemically and structurally similar to unesterified cholesterol-rich lipid particles that accumulate in atherosclerotic lesions.     

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.     

Effect of bean intake on biliary lipid secretion and on hepatic cholesterol metabolism in the rat

We studied the effect of a bean diet on biliary lipid secretion, serum cholesterol concentration, and hepatic cholesterol metabolism in the rat. Rats fed a bean diet for 10-12 days had increased biliary cholesterol output and molar percentage by 300% and 200%, respectively, compared to rats fed an isocaloric and isoprotein casein diet. Biliary phospholipid output increased 180%. Bile flow and biliary bile salt output remained in the normal range. Total serum and VLDL cholesterol concentration significantly decreased 27% and 50%, respectively, in the rats fed the bean diet. Hepatic cholesterogenesis was increased 170% in the bean-fed animals. The relative contribution of newly synthesized hepatic cholesterol to total biliary cholesterol increased 200%, and that of endogenous origin only 50%. These results suggested that newly synthesized hepatic cholesterol was preferentially channelled to the biliary cholesterol secretory pathway in bean-fed rats. Although hepatic cholesteryl ester concentration increased 240%, the incorporation of [14C]oleate into hepatic cholesteryl esters was significantly decreased by 30% in isolated hepatocytes of bean-fed animals. These results were consistent with the possibility that the availability of hepatic free cholesterol for biliary secretion was increased in the bean-fed animals. This study demonstrates that bean intake has a profound effect on the metabolic channelling and compartmentalization of hepatic cholesterol, resulting in a significant decrease in total serum and very low density lipoprotein cholesterol concentrations and a high biliary cholesterol output.     

Regulation of bile acid synthesis in cultured rat hepatocytes: stimulation by apoE-rich high density lipoproteins

Cultured rat hepatocytes obtained by liver perfusion with collagenase in the presence of soybean trypsin inhibitor were used to examine the role of high density lipoproteins (HDL) in supplying cholesterol to the hepatocyte for bile acid synthesis. Within 6 hr of adding HDL (d 1.07-1.21 g/ml) obtained from rat serum there was a significant stimulation of bile acid synthesis and secretion that reached 2-fold after 24 hr. The stimulation by HDL occurred at normal plasma concentrations (i.e., 500 micrograms/ml) and showed further stimulation in a dose-dependent manner reaching a maximum stimulation of 2- to 2.5-fold. The stimulation of bile acid synthesis was dependent on the cholesteryl ester content of the HDL. Several lines of evidence show that the HDL is taken up by a receptor-mediated process dependent on apoE. These include: 1) at the same concentration (500 micrograms/ml) apoE-poor HDL (not retained by heparin affinity chromatography of HDL isolated from the plasma of rats fasted for 72 hr stimulated bile acid synthesis by 48%, whereas apoE-rich HDL stimulated bile acid synthesis by 110%; 2) reductive methylation totally blocked the stimulation of bile acid synthesis by HDL; 3) HDLC, which contained apoE as its major protein component, also maximally stimulated bile acid synthesis; and 4) human HDL, which contained no detectable apoE, failed to stimulate bile acid synthesis. Additional studies showed that apoE-enriched HDL and HDLC both inhibited cholesterol synthesis (determined by the incorporation of 3H2O) and caused a net accumulation of cholesteryl esters in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans

Our aim was to identify and quantify the major in vivo pathways of lipoprotein cholesteryl ester transport in humans. Normal (n = 7), bile fistula (n = 5), and familial hypercholesterolemia (FH; n = 1) subjects were studied. Each received isotopic free cholesterol in HDL, LDL, or particulate form, along with another isotope of free or esterified cholesterol or mevalonic acid. VLDL, intermediate density lipoprotein (IDL), LDL, HDL, blood cells, and bile were collected for up to 6 days for analysis of radioactivity and mass of free and esterified cholesterol. These raw data were subjected to compartmental analysis using the SAAM program. Results in all groups corroborated net transport of free cholesterol to the liver from HDL, shown previously in fistula subjects. New findings revealed that 70% of ester was produced from free cholesterol in HDL and 30% from free cholesterol in LDL, IDL, and VLDL. No evidence was found for tissue-produced ester in plasma. There was net transfer of cholesteryl ester to VLDL and IDL from HDL and considerable exchange between LDL and HDL. Irreversible ester output was from VLDL, IDL, and LDL, but very little was from HDL, suggesting that selective and holoparticle uptakes of HDL ester are minor pathways in humans. It follows that 1) they contribute little to reverse transport, 2) very high HDL would not result from defects thereof, and 3) the clinical benefit of high HDL is likely explained by other mechanisms. Reverse transport in the subjects with bile fistula and FH was facilitated by ester output to the liver from VLDL plus IDL.     

Effect of lipid transfer protein on plasma lipids, apolipoproteins and metabolism of high-density lipoprotein cholesteryl ester in the rat

The role of human plasma lipid transfer protein (LTP) in lipoprotein metabolism was studied in the rat, a species without endogenous cholesteryl ester and triacylglycerol transfer activity. Partially purified human LTP was injected intravenously into rats. The plasma activity was between 1.5- and 4-fold that of human plasma during the experiments. 6 h after the injection of LTP, a significant increase in serum apoB, and no significant changes in serum total cholesterol, free cholesterol, triacylglycerols, apoA-I, apoE, or apoA-IV were noted. Cholesterol was increased in very-low density and low-density lipoproteins (VLDL and LDL) and decreased in large-sized apoE-rich HDL. ApoA-I-containing particles with a size smaller than in normal rats were present in serum of LTP-treated rats. The mean diameter of HDL particles decreased and apoE, normally present on large-sized HDL, was present on smaller sized particles. The metabolic fate of cholesteryl ester, originally associated with HDL, was studied by injection of [3H]cholesteryl linoleyl ether-labelled apoA-I-rich HDL in the absence and in the presence of LTP. The disappearance of [3H]cholesteryl linoleyl ether, injected as part of apoA-I-rich HDL, from serum was increased in the LTP-treated rats; the t1/2 changed from 3.9 to 2.2 h, resulting in an increased accumulation of [3H]cholesteryl linoleyl ether in the liver. This can be explained by the redistribution of HDL [3H]cholesteryl linoleyl ether to VLDL and LDL in the presence of LTP, leading to the combined contribution of VLDL, LDL and HDL to the hepatic uptake. The present findings show profound effects of LTP on the chemical composition of HDL subspecies, the size of HDL and on the plasma turnover and hepatic uptake of cholesteryl esters originally present in apo A-I-rich HDL.     

Cholesterol metabolism during ketoconazole treatment in man

Ketoconazole, an antifungal antibiotic, inhibits cholesterol synthesis by blocking demethylation of lanosterol. Effects of this inhibition were studied on serum cholesterol, lipoproteins and cholesterol precursors, biliary lipid composition, and fecal steroid elimination in five patients with prostate cancer treated with large doses of ketoconazole. The serum level of total cholesterol fell by 27%, that of LDL cholesterol by 41% and that of LDL apoB by 32% with ketoconazole alone; the fall in the total cholesterol level of a patient treated with ketoconazole-cholestyramine was 65%. Serum contents of free lanosterol and dihydrolanosterol increased up to 250 times, yet the total concentrations remained less than 2 mg/dl. Of the other cholesterol precursor sterols only those with delta 8-double bond increased several times, indicating that in addition to 14 alpha-demethylation, ketoconazole also interfered with metabolism of later intermediary sterols to some extent. Compared with serum sterols, lanosterols were enriched in biliary and fecal sterols up to 10-20 times. Fecal lanosterol output increased from 12 to 247 mg/day, and comprised over 20% fecal steroids of endogenous origin. Bile acid synthesis was significantly decreased, the proportion of chenodeoxycholic acid being markedly reduced in both biliary and fecal bile acids. Cholesterol absorption appeared to decrease yet fecal neutral sterol output and cholesterol synthesis were unchanged and the overall sterol synthesis was increased. It thus appears that ketoconazole inhibits cholesterol elimination as bile acids. However, by blocking 14 alpha-demethylation, it results in effective drainage of sterol nucleus as lanosterols into bile and feces, which, in turn, is associated with a marked reduction in low density lipoprotein (LDL) cholesterol level probably through activation of hepatic LDL apoB receptors.     

Hepatic processing and biliary secretion of the cholesteryl esters from beta very-low-density lipoproteins in the rat

beta-Migrating very-low-density lipoproteins (beta-VLDL) are cholesteryl-ester-enriched lipoproteins which accumulate in the serum of cholesterol-fed animals or patients with type III hyperlipoproteinemia. In the rat, beta-VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, beta-VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after beta-VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded. It is concluded that beta-VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from beta-VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from beta-VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low-density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.     

Cholesterol movement between the plasma membrane and the cholesteryl ester droplets of cultured Leydig tumour cells.

The present studies characterize the turnover of plasma membrane cholesterol in MA-10 Leydig tumour cells. Plasma membrane cholesterol of MA-10 cells was slowly internalized and converted into cholesteryl ester. Low-density lipoprotein (LDL) stimulated, in a dose- and time-dependent fashion, plasma membrane cholesterol conversion into intracellular esters. Stimulation of membrane internalization was not simply the consequence of accelerated uptake of membrane with LDL, since binding and internalization of epidermal growth factor and transferrin had no effect on turnover of plasma membrane cholesterol. The protein of LDL is unimportant as well, since delipidated LDL had no effect on membrane turnover. The action of LDL on cholesterol turnover was explained entirely by its contribution to cholesteryl ester stores. The degree of plasma membrane cholesterol internalization and esterification was directly proportional to the size of cellular ester stores.