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.     

Evidence for reverse cholesterol transport in vivo from liver endothelial cells to parenchymal cells and bile by high-density lipoprotein.

Acetylated low-density lipoprotein (acetyl-LDL), biologically labelled in the cholesterol moiety of cholesteryl oleate, was injected into control and oestrogen-treated rats. The serum clearance, the distribution among the various lipoproteins, the hepatic localization and the biliary secretion of the [3H]cholesterol moiety were determined at various times after injection. In order to monitor the intrahepatic metabolism of the cholesterol esters of acetyl-LDL in vivo, the liver was subdivided into parenchymal, endothelial and Kupffer cells by a low-temperature cell-isolation procedure. In both control and oestrogen-treated rats, acetyl-LDL is rapidly cleared from the circulation, mainly by the liver endothelial cells. Subsequently, the cholesterol esters are hydrolysed, and within 1 h after injection, about 60% of the cell- associated cholesterol is released. The [3H]cholesterol is mainly recovered in the high-density lipoprotein (HDL) range of the serum of control rats, while low levels of radioactivity are detected in serum of oestrogen-treated rats. In control rats cholesterol is transported from endothelial cells to parenchymal cells (reverse cholesterol transport), where it is converted into bile acids and secreted into bile. The data thus provide evidence that HDL can serve as acceptors for cholesterol from endothelial cells in vivo, whereby efficient delivery to the parenchymal cells and bile is assured. In oestrogen-treated rats the radioactivity from the endothelial cells is released with similar kinetics as in control rats. However, only a small percentage of radioactivity is found in the HDL fraction and an increased uptake of radioactivity in Kupffer cells is observed. The secretion of radioactivity into bile is greatly delayed in oestrogen-treated rats. It is concluded that, in the absence of extracellular lipoproteins, endothelial cells can still release cholesterol, although for efficient transport to liver parenchymal cells and bile, HDL is indispensable.     

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 apolipoprotein E-free high density lipoproteins on cholesterol metabolism in cultured pig hepatocytes

We studied cholesterol synthesis from [14C]acetate, cholesterol esterification from [14C]oleate, and cellular cholesterol and cholesteryl ester levels after incubating cells with apoE-free high density lipoproteins (HDL) or low density lipoproteins (LDL). LDL suppressed synthesis by up to 60%, stimulated esterification by up to 280%, and increased cell cholesteryl ester content about 4-fold. Esterification increased within 2 h, but synthesis was not suppressed until after 6 h. ApoE-free HDL suppressed esterification by about 50% within 2 h. Cholesterol synthesis was changed very little within 6 h, unless esterification was maximally suppressed; synthesis was then stimulated about 4-fold. HDL lowered cellular unesterified cholesterol by 13-20% within 2 h and promoted the removal of newly synthesized cholesterol and cholesteryl esters. These changes were transient; by 24 h, both esterification and cellular unesterified cholesterol returned to control levels, and cholesteryl esters increased 2-3-fold. HDL core lipid was taken up selectively from 125I-labeled [3H]cholesteryl ester- and ether-labeled HDL. LDL core lipid uptake was proportional to LDL apoprotein uptake. The findings suggest that 1) the cells respond initially to HDL or LDL with changes in esterification, and 2) HDL mediates both the removal of free cholesterol from the cell and the delivery of HDL cholesteryl esters to the cell.     

Quantification of LDL cholesteryl ester contribution to biliary steroids in the rat

The proportion of LDL cholesteryl ester converted to biliary steroids was quantified in the rat. The pre-existing pool of bile was allowed to drain for 10-12 h through a bile duct cannula. A single intravenous pulse injection of LDL labelled with [3H]cholesterol linoleyl ester was made, followed by a constant infusion of the same material in order to maintain constant specific radioactivity in plasma. A new steady state was achieved within 6 h and bile samples were then collected hourly until 12 h. Although substantial amounts (53-61 micrograms/h) of cholesteryl ester were released into the liver during LDL catabolism, only a very small fraction (0.8-1.90 micrograms/h) was found in biliary steroids. The proportion of LDL cholesteryl esters contributing to biliary steroids was only 1-2%. These results perhaps explain why perturbations to accelerate bile acid excretion have no effect on plasma LDL cholesterol concentration in the rat.     

Metabolism of radiolabelled chylomicron lipids in intact and hepatectomized rats

Intact rats removed more radiolabelled triacylglycerol, cholesterol, and cholesterol ester but not phosphatidylcholine (PC) in the first 6 min than hepatectomized rats. There was no difference between intact and hepatectomized rats in the transfer of radiolabelled chylomicron lipids to other lipoproteins. Specific radioactivity measurements demonstrated a net transfer of PC (intact and hepatectomized rats) and unesterified cholesterol (intact rats only) onto both the low density lipoprotein/high density lipoprotein-1 (LDL/HDL1) and HDL2 fractions. [3H]Fatty acids were rapidly incorporated into blood cell phospholipids and into HDL and LDL cholesterol esters of both intact and hepatectomized rats. Substantial rearrangements of [3H]palmitate occurred during lipid uptake by liver.     

Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts

One characteristic of type C Niemann-Pick (NPC) disease is the substantial intracellular accumulation of unesterified cholesterol. The increased cholesterol content in NPC fibroblasts which are grown in the presence of low density lipoproteins (LDL) has been postulated to be due to a deficiency in cellular cholesterol esterification. We have examined several aspects of LDL metabolism in NPC fibroblasts. We observe that LDL binding, internalization, and lysosomal hydrolysis of LDL cholesteryl esters are normal in NPC cells. As reported by Pentchev et al. (Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251), we find that LDL does not stimulate cholesterol esterification. However, we also show that LDL does not down-regulate cholesterol synthesis or LDL receptor activity as normal. In NPC cells, these processes are regulated normally by nonlipoprotein effectors, such as 25-hydroxycholesterol or mevalonate. Since NPC cells are not defective in lysosomal hydrolysis of LDL-derived cholesteryl esters, they must exhibit a different defect than Wolman's or cholesteryl ester storage diseases. We conclude that NPC cells are defective specifically in LDL-mediated regulation of cellular cholesterol metabolism. We suggest that the intracellular processing of LDL-derived cholesterol may be defective in NPC fibroblasts.     

Rapid transport of fatty acids from rat liver endothelial to parenchymal cells after uptake of cholesteryl ester-labeled acetylated LDL

Acetylated low-density lipoprotein (acetyl-LDL) radiolabeled in the oleate moiety of cholesteryloleate was injected into rats. Isolation of the various liver cell types at different times after acetyl-LDL injection by a low-temperature procedure allowed the intrahepatic metabolism of the oleate moiety to be followed in vivo. The cholesteryloleate radioactivity is rapidly cleared from the circulation and at 5 min after injection recovered into parenchymal and endothelial liver cells, mainly as cholesteryloleate ester. At longer time intervals after injection, the amount of cholesteryl esters associated with the endothelial cells was sharply decreased and the [14C]oleate was redistributed within the liver and mainly recovered in the parenchymal cells. The cholesteryl ester initially directly taken up by the parenchymal cells was also rapidly hydrolysed but, in contrast to the endothelial cells, the [14C]oleate remained inside the cells and was incorporated into triacylglycerols and phospholipids. The 14C radioactivity in parenchymal cells taken up between 5 and 30 min after injection of the cholesteryl [14C]oleate-labeled acetyl-LDL (transported as oleate from endothelial cells), followed a similar metabolic route as the amount which was directly associated to parenchymal cells. The data indicate that the liver and, in particular, the liver endothelial cell has the full capacity to rapidly catabolize modified lipoproteins. In this catabolism, the liver functions as an integrated organ in which fatty acids, formed from cholesteryl esters in endothelial cells, are rapidly transported to parenchymal cells, indicating the concept of metabolic cooperation between the various liver cell types.     

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.     

Role of parenchymal and non-parenchymal rat liver cells in the uptake of cholesterolester-labeled serum lipoproteins.

Very low density lipoprotein (VLDL)-remnants, prepared by extrahepatic circulation of VLDL, labeled biosynthetically in the cholesterol (ester) moiety, were injected intravenously into rats in order to determine the relative contribution of parenchymal and non-parenchymal liver cells to the hepatic uptake of VLDL-remnant cholesterol (esters). 82.7% of the injected radioactivity is present in liver, measured 30 min after injection. The non-parenchymal liver cells contain 3.1±0.1 times the amount of radioactivity per mg cell protein as compared to parenchymal cells. The hepatic uptake of biosynthetically labeled (screened) low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterolesters amounts to 26.8% and 24.4% of the injected dose, measured 6 h after injection. The non-parenchymal cells contain 4.3±0.8 and 4.1±0.7 times the amount of radioactivity per mg cell protein as compared to parenchymal cells for LDL and HDL, respectively. It is concluded that in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake of cholesterolesters from VLDL-remnants, LDL and HDL.     

Acyl-coenzyme A:cholesteryl acyltransferase 2.

In addition to acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1), an enzyme in the endoplasmic reticulum of cells found ubiquitously throughout the body, data recently obtained in at least three mammalian species, including nonhuman primates, mice and humans, demonstrate the presence of an additional ACAT (EC 2.1.3.26), termed ACAT2, which is localized to the endoplasmic reticulum of liver and intestine. Data suggest that ACAT2 may be the enzyme responsible for cholesteryl ester secretion into apolipoprotein B-containing lipoproteins. We have hypothesized that oversecretion of cholesteryl esters produced by the action of hepatic ACAT2 could account for the increased atherogenicity associated with cholesteryl ester-enriched LDL in nonhuman primates. In such cases, ACAT2 is an appealing target for therapy to reduce coronary heart disease.     

Endocytosed beta-VLDL and LDL are delivered to different intracellular vesicles in mouse peritoneal macrophages

Hypercholesterolemic rabbit beta-VLDL and human LDL are both internalized by mouse peritoneal macrophages by receptor-mediated endocytosis. However, only beta-VLDL (which binds to the cells with a much higher affinity than LDL) markedly stimulates acyl-CoA/cholesterol acyl transferase (ACAT) and induces foam cell formation in these cells. As an initial step to test whether the two lipoproteins might be targeted to different organelles (which might differ in their ability to deliver cholesterol to microsomal ACAT), we studied the endocytic pathways of beta-VLDL and LDL. Lipoproteins were labeled with the non-transferable fluorescent label, DiI. When the macrophages were incubated with DiI-LDL for 10 min at 37 degrees C, the fluorescence was concentrated near the center of the cell both in heavily labeled vesicles and in a diffuse pattern. The pattern with DiI-beta-VLDL was quite different: an array of bright vesicles throughout the cytoplasm was the predominant feature. Differences in distribution were seen as early as 2 min of incubation and persisted throughout a 10-min chase period. By using a procedure in which photobleaching of DiI fluorescence converts diaminobenzidine into an electron-dense marker, we were able to identify at the ultrastructural level vesicles containing electron-dense material in cells incubated with DiI-beta-VLDL. Human E2/E2 beta-VLDL (from a patient with familial dysbetalipoproteinemia), which has a binding affinity and ACAT-stimulatory potential similar to LDL, gave a pattern of fluorescence virtually identical to LDL. Pulse-chase studies with 125I-labeled and [3H]cholesteryl ester-labeled lipoproteins disclosed that both protein degradation and cholesteryl ester hydrolysis were markedly retarded in beta-VLDL compared with LDL. Thus, in mouse peritoneal macrophages, endocytosed beta-VLDL appears in a distinct set of widely-distributed vesicles not seen with LDL (or with E2-beta-VLDL) and, compared with LDL, has a markedly diminished rate of protein degradation and cholesteryl ester hydrolysis. The differential routing of LDL and beta-VLDL may provide a mechanism for differences in ACAT-stimulatory potential between the two lipoproteins.     

Failure of the intracellular itinerary of beta very low density lipoproteins to augment cholesterol esterification in macrophages from Watanabe heritable hyperlipidemic rabbits

beta very low density lipoproteins (beta-VLDL) interact with mouse peritoneal macrophages via specific receptors leading to pronounced stimulation of cholesterol esterification. The present study has defined an alternative pathway for the processing of beta-VLDL in alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits. Macrophages from either New Zealand (NZ) or WHHL rabbits degraded 125I-beta-VLDL to an equivalent extent. Degradation was competed to a similar extent in both cell types by either excess unlabeled beta-VLDL or low density lipoprotein, indicative of a specific receptor involvement. Accumulation of intracellular degradation products of beta-VLDL labeled with the residualizing label, dilactitol-125I-tyramine, was similar in both cell types demonstrating that degradation was not due to secreted proteolytic enzymes. beta-VLDL promoted the incorporation of [3H]oleate into cholesteryl-[3H]oleate and increased the cellular mass of cholesterol in NZ macrophages. In contrast, beta-VLDL did not augment cholesteryl-[3H]oleate deposition in WHHL macrophages. This lack of cholesterol esterification occurred despite equivalent acyl-CoA:cholesterol acyltransferase activity in microsomal fractions of both cell types, and similar augmentations in cholesteryl-[3H]oleate during incubation with phospholipase C-treated LDL. Incubation of WHHL macrophages with beta-VLDL increased cellular cholesterol mass, although the response was attenuated compared to NZ cells. To determine whether these disparities in cholesterol esterification were related to the catabolic fate of beta-VLDL-derived cholesterol esters, [3H]cholesteryl oleate was exchanged into the core of beta-VLDL and incubated with macrophages in medium containing [14C]oleate. NZ macrophages accumulated both [3H]cholesterol and [3H]cholesteryl-[14C]oleate after 5 h, indicating hydrolysis and re-esterification of cholesterol esters. In contrast, WHHL macrophages only accumulated [3H]cholesterol esters, suggesting uptake of cholesterol esters without subsequent hydrolysis. These data demonstrate that WHHL macrophages possess a pathway for the intracellular processing of beta-VLDL that permits internalization of the particle without stimulation of cholesterol esterification.     

Clearance of acetyl low density lipoprotein by rat liver endothelial cells. Implications for hepatic cholesterol metabolism

We have studied the hepatic uptake of human [14C] cholesteryl oleate labeled acetyl low density lipoprotein (LDL). Acetyl-LDL injected intravenously into rats was cleared from the blood with a half-life of about 10 min. About 80% of the injected acetyl-LDL was recovered in the liver after 1 h. Initially, most of the [14C]cholesterol was recovered in liver endothelial cells (about 60%). Some radioactivity (about 15%) was also recovered in the hepatocytes, while the Kupffer cells and stellate cells contained only small amounts of the label (less than 5%). About 1 h after injection, radioactivity started to disappear from endothelial cells and appeared instead in hepatocytes. Radioactivity subsequently declined in hepatocytes as well. After a lag phase of 4 h, significant amounts of radioactivity were recovered in bile. The in vitro uptake and hydrolysis of [14C]cholesteryl oleate-labeled acetyl-LDL were saturable in isolated rat liver endothelial cells. Native LDL does neither affect the uptake nor the hydrolysis of acetyl-LDL. Ammonia and monensin reduced the hydrolysis of acetyl-LDL in isolated liver endothelial cells. Furthermore, monensin at concentrations above 10 microM completely blocked the binding of acetyl-LDL to the liver endothelial cells, suggesting that the receptor for acetyl-LDL is trapped inside the cells. The liver endothelial cells may be involved in the protection against atherogenic lipoproteins, e.g. liver endothelial cells may mediate uptake of cholesterol from plasma and transfer of cholesterol to the hepatocytes for further secretion into the bile.     

Fate of intravenously administered particulate and lipoprotein cholesterol in the rat

Unesterified radioactive cholesterol, both bound to serum lipoproteins and dispersed in ethanol-saline, was injected into bile fistula and intact rats. Due to phagocytosis, mainly by the liver macrophages, intravenously injected cholesterol in ethanol-saline disappears from the bloodstream significantly faster than lipoprotein-bound cholesterol. Soon after the initial phagocytosis, the particulate isotopic cholesterol started to reappear in blood, reaching a maximal radioactivity in blood 10-24 hr after injection. Although the radioactive cholesterol reappears in serum in both esterified and unesterified form, it is likely that cholesterol is released from the phagocytic cells as unesterified cholesterol which is then esterified intravascularly or at other sites. In the bile fistula rats, somewhat more of the lipoprotein cholesterol than of the particulate cholesterol appeared in bile early after injection. However, cholesterol turnover calculated from a twopool model was the same for rats injected with lipoproteinbound or particulate cholesterol.     

Effects of chylomicron remnants and beta-VLDL on the class and composition of newly secreted lipoproteins by HepG2 cells

The regulation of lipoprotein secretion in the cell line HepG2 was studied. HepG2 cells were preincubated with chylomicron remnants (triglyceride- and cholesterol-rich) or with beta very low density lipoproteins (beta-VLDL) (cholesterol-rich). The medium was removed and the cells were incubated for and additional 24 hr in a lipoprotein-free medium that contained either [2-3H]glycerol or DL-[2-3H]mevalonate. Cells and media were harvested, and lipoproteins were separated and fractionated. The mass and radioactivity of the lipids in cells and in the lipoproteins were measured. The activities of cellular acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase were also determined. Preincubation with chylomicron remnants induced an increase in cellular triglyceride and stimulated both HMG-CoA reductase and ACAT. Preincubation with beta-VLDL induced an increase in cellular free and esterified cholesterol, inhibited HMG-CoA reductase and stimulated ACAT. Although the absolute amount of VLDL is small, chylomicron remnants induced large relative increases in the amount of triglyceride and phospholipid secreted in VLDL and decreases in the amount of triglyceride secreted in low density (LDL) and high density (HDL) lipoproteins as well as a decrease in the amount of phospholipid secreted in HDL. In contrast, preincubation with beta-VLDL did not affect triglyceride secretion, but markedly stimulated the amount of phospholipid secreted in HDL. Comparison of the mass of glycerolipid actually secreted with that calculated from the cellular specific activity suggested that glycerolipids are secreted from single, rapidly equilibrating pools. Cholesterol and cholesteryl ester secretion were affected differently. Preincubation with chylomicron remnants increased the amount of free cholesterol secreted in both VLDL and LDL, but did not alter cholesteryl ester secretion. Preincubation with beta-VLDL increased free cholesterol secretion in all lipoprotein fractions and increased cholesteryl ester secretion in VLDL and LDL, but not HDL. Comparison of isotope and mass data suggested that the cholesteryl ester secreted came primarily from a preformed, rather than an newly synthesized, pool. In summary, these data provide insight to the mechanism whereby a liver cell regulates the deposition of exogenous lipid.     

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.     

Metabolic heterogeneity of post-lipolysis rat mesenteric lymph small chylomicrons produced in vitro

The study was undertaken to investigate the metabolic rat of post-lipolysis mesenteric lymph small chylomicrons produced in vitro. Small chylomicrons doubly labeled with [3H]cholesterol (more than 70% in cholesteryl esters) and [14C]palmitate-labeled triglycerides were collected from rat mesenteric lymph during periods of fasting. Lipolysis was performed in vitro with lipoprotein lipase purified from bovine milk. More than 98% of the chylomicron-triglycerides could be hydrolyzed to fatty acids. Post-lipolysis chylomicrons were separated by zonal ultracentrifugation, characterized, and tested for biological behavior in intact rats. Following lipolysis the lipoproteins lost nearly all their triglycerides, apoA-I, and apoC, and were relatively enriched with cholesteryl esters, unesterified cholesterol, phospholipids, and apoB. Three preparations were tested for biological behavior: pooled (total) post-lipolysis chylomicrons (diameter approximately 250 A); particles at the ascending part of the zonal effluent (diameter approximately 300 A), and at the descending part (diameter approximately 200 A). After intravenous injection to intact rats, [3H]cholesteryl ester decay was very rapid with pooled lipoproteins and the 300-A preparation (t1/2 = 5-10 min). The 200-A preparation in contrast stayed in circulation much longer (t1/2 = 60-90 min). The study thus demonstrated metabolic heterogeneity of post-lipolysis small chylomicrons and indicated that some may form an LDL-like subpopulation with a plasma lifetime slower than "remnants" but faster than LDL.     

Effect of infusion of "tris-galactosyl-cholesterol" on plasma cholesterol, clearance of lipoprotein cholesteryl esters, and biliary secretion in the rat

As shown by us previously (van Berkel et al. 1985. J. Biol. Chem. 260: 2694-2699 and van Berkel et al. 1985. J. Biol. Chem. 260: 12203-12207) the clearance of both low density lipoproteins (LDL) and high density lipoproteins (HDL) from the blood can be greatly enhanced by pretreatment of these lipoproteins with a tris-galactosylated cholesterol derivative, which makes these particles recognizable by hepatic galactosyl-receptors. Here we report that intravenous infusion of the (water-soluble) tris-galactosyl-cholesterol in rats caused a dose-dependent decrease of the plasma cholesterol level. This fall was sustained long after termination of the infusion. It was not observed upon infusion of tris-glucosyl-cholesterol. The fall in plasma cholesterol was accompanied by an increase in hepatic cholesterol. Upon injection of rat HDL and LDL labeled in their cholesteryl ester moieties, plasma clearance of label in both lipoproteins was enhanced in rats infused with tris-galactosyl-cholesterol, the stimulation being more pronounced when the label was in HDL. The appearance of label in bile was also enhanced in the rats receiving the compound, again more markedly when the label was given as HDL. Ninety four percent or more of the radioactivity excreted in the bile was in the form of bile salts, with conjugated cholate being the major species in both control and treated rats; 6% or less of the radioactivity in the bile was as free cholesterol. Infusion of tris-galactosyl-cholesterol constitutes a new and defined method of lowering plasma lipoprotein levels by enhancing their uptake in the liver.     

Cholesterol is necessary for triacylglycerol-phospholipid emulsions to mimic the metabolism of lipoproteins

Emulsions with lipid compositions similar to the triacylglycerol-rich lipoproteins were metabolized similarly to natural chylomicrons or very-low-density lipoproteins when injected intravenously in rats. Radioactive labels tracing the emulsion triacylglycerols and cholesteryl esters were both removed rapidly from the blood stream, but the removal rate of triacylglycerols was faster than that of cholesteryl ester. Most of the removed cholesteryl ester label was found in the liver, but only a small fraction of the triacylglycerol label was found in this organ, consistent with hepatic uptake of the remnants of the injected emulsion. Emulsions otherwise identical but excluding unesterified cholesterol were metabolized differently. The plasma removal of triacylglycerols remained fast, but the cholesteryl esters were removed very slowly. Heparin stimulated lipolysis, but failed to increase the rate of removal of cholesteryl esters from emulsions lacking cholesterol. Evidently, emulsions lacking cholesterol were acted on by the enzyme lipoprotein lipase, but the resultant triacylglycerol-depleted remnant particle remained in the plasma instead of being rapidly taken up by the liver. Therefore, the presence of emulsion cholesterol is a critical determinant of early metabolic events, and the findings point to a similar role for cholesterol in the natural triacylglycerol-rich lipoproteins.