Binding and uptake of chylomicron remnants by primary and THP-1 human monocyte-derived macrophages: determination of binding proteins.

The binding and uptake of chylomicron remnants by human macrophages was studied in order to resolve paradoxical observations that have described the putative mechanisms by which postprandial lipoproteins induce foam cell formation. Chylomicron remnants bound to human monocyte-derived macrophages (HMMs) and to the transformed monocytic cell line THP-1 with high affinity (Kd of approx. 5.5 microg of chylomicron remnant protein/ml). Binding was found to be saturable for both cell types, and was strongly inhibited in the presence of unlabelled chylomicron remnants. Ligand blot studies with colloidal-gold-labelled chylomicron remnants identified two cell surface binding sites on both HMMs and THP-1 cells, with molecular masses of approx. 128 kDa and 43 kDa. The high-molecular-mass binding site was found to be the low-density lipoprotein (LDL) receptor, based on the strong inhibition of chylomicron remnant binding in the presence of unlabelled LDL, Fab2 antibody fragments to the LDL receptor or calcium chelators. Competition studies suggested that, in HMMs, the LDL receptor appeared to facilitate approximately half of the total chylomicron remnant uptake. In contrast, the LDL receptor was not significantly involved in macrophage uptake of chylomicron remnants by THP-1 cells. The identity of the 43 kDa binding site is presently unknown, but, importantly, expression was not inhibited as a consequence of sterol loading, which was induced by incubating HMMs and THP-1 cells with 25-hydroxycholesterol. In contrast, the expression of the LDL receptor was substantially attenuated following lipid loading. Collectively, our data suggest that, while the macrophage LDL receptor can bind chylomicron remnants and facilitate uptake in non-lipid-loaded HMMs, other sterol-insensitive sites are responsible for the unabated uptake of chylomicron remnants by macrophages. We propose that the 43 kDa macrophage chylomicron remnant binding protein may be a candidate for the sterol loading of macrophages.

Detection of LDL receptor by ligand blotting with chylomicron remnants labelled with colloidal gold.

The LDL receptor plays a pivotal role in the clearance of pro-atherogenic lipoproteins, and LDL receptor deficiency may be the underlying cause of several primary and secondary dyslipidaemic conditions. Intervention strategies are often targeted to increase hepatic LDL receptor expression. It is difficult to quantitate hepatic LDL receptor activity and to monitor changes post-therapy. In order to avoid liver biopsy, human skin fibroblasts or circulating mononuclear cells have often been used as surrogate markers for the hepatic receptor. Fibroblasts, and particularly mononuclear cells, are relatively easy to isolate and can be stored for extensive lengths of time without significant loss of LDL receptor expression. Leucocytes or fibroblasts are normally probed with isotopically or gold-labelled LDL. However, the specific activity of the LDL conjugate is usually too low to enable accurate quantitation of differences, or changes, in LDL receptor expression. In this study, we describe an enhanced colloidal gold-labelling procedure for the detection of LDL receptor binding activity. The binding of colloidal gold-labelled chylomicron remnants to human hepatocytes (HepG2 cells) was compared with that of gold-conjugated LDL. Labelled remnants bound specifically to a cell surface protein with a molecular weight of approximately 130 kDa. Binding was blocked in the presence of unlabelled remnants, LDL, or antiserum specific to the LDL receptor. The binding of gold-labelled remnants was substantially greater than that of gold-labelled LDL. Compared with gold-labelled LDL, we found a much clearer demarcation of remnant binding with hepatocytes incubated in the presence or absence of sterols. Our observations suggest that, because of the greater affinity of the LDL receptor for lipoproteins containing apolipoprotein E, changes in LDL receptor expression might be more readily identified using gold-labelled remnants. We conclude that gold-conjugated chylomicron remnants might provide a useful means of detecting subtle changes in LDL receptor expression.

Is atherosclerosis exclusively a postprandial phenomenon?

1. Atherosclerosis begins with the deposition of cholesterol in arterial tissue that is thought to be derived from circulating lipoproteins. There is considerable evidence implicating low density lipoprotein (LDL) as a primary source of plaque cholesterol and, consequently, there are many articles that deal with various aspects of LDL metabolism. 2. Postprandial lipoproteins (i.e. chylomicrons transporting dietary fats) are also considered pro-atherogenic; however, it is less clear whether their involvement in arterial cholesterol deposition is direct or follows modulation of LDL metabolism. 3. In order to provoke discussion, this article is presented in a manner that suggests atherosclerosis to be exclusively a postprandial phenomenon; that is, we have raised the possibility that LDL is non-atherogenic.

Degradation of chylomicron remnants by macrophages occurs via phagocytosis.

Chylomicron remnants bound to rabbit alveolar macrophages with high-affinity (Kd = 3.3 +/- 0.71 microgram of protein/mL). The binding of chylomicron remnants was competitively inhibited in the presence of unlabeled remnants and to a lesser extent by unlabeled low-density lipoproteins. Pretreatment of cells with either trypsin or pronase inhibited degradation in a dose and time dependent manner, suggesting involvement of a cell surface protein. Chylomicron remnants were degraded by alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits, which are devoid of LDL receptor activity. Moreover, colchicine and monensin which are endocytotic and lysozomal inhibitors, respectively, did not have any effect on the degradation of chylomicron remnants by macrophages from normal rabbits. The absence of divalent cations was found to enhance chylomicron remnant degradation by macrophages. Activated alpha 2-macroglobulin and lactoferrin had no effect on chylomicron remnant degradation, indicating that the low-density lipoprotein receptor-related protein was not involved. In addition, the scavenger receptor inhibitors polyinosinic acid and fucoidan increased degradation of chylomicron remnant-ruling out uptake as a consequence of lipoprotein modification. Rather, the phagocytotic inhibitor cytochalasan D was found to significantly decrease chylomicron remnant degradation. Collectively, our data show that chylomicron remnants are metabolized by phagocytotic pathways initiated after binding to a cell surface protein which is distinct from the LDL receptor, LRP, or scavenger receptors.

Clearance of chylomicron-like lipid emulsions is increased in normal rabbits but not in heterozygous Watanabe heritable hyperlipidaemic rabbits following treatment with cholestyramine or pravastatin.

1. Cholestyramine and pravastatin are two potent hypocholesterolaemic drugs which lower plasma cholesterol by increasing the clearance of low density lipoproteins by high affinity uptake mechanisms. 2. We gave heterozygous Watanabe heritable hyperlipidaemic rabbits (hz-WHHL) cholestyramine and/or pravastatin for a two week period to try and ameliorate slow clearance of chylomicron remnants, which occurs because of reduced expression of the apolipoprotein B100/E receptor. 3. In hz-WHHL rabbits the clearance of chylomicron-like lipid emulsions, traced by the decrease in plasma cholesteryl oleate radioactivity was not improved following treatment with either of the cholesterol lowering drugs. 4. In contrast, control rabbits had significantly less chylomicron-like emulsion cholesteryl-ester radioactivity remaining at each time of blood sampling. 5. Similarly, the clearance of chylomicron-like emulsion triolein was enhanced in normal rabbits receiving cholestyramine or pravastatin, whereas there was no detectable increase in clearance in hz-WHHL rabbits. 6. Combined treatment with cholestyramine and pravastatin increased the rate of receptor-mediated uptake in vivo in control rabbits but not in hz-WHHL rabbits. 7. The plasma lipid profiles of control and hz-WHHL rabbits paralleled the patterns of chylomicron-like emulsion clearance. Moderate hypertriglyceridaemia was identified in hz-WHHL rabbits compared to controls and there was no change in plasma triglyceride or cholesterol following drug therapy. In contrast, control rabbits had decreased plasma lipids following cholestyramine or pravastatin treatment. 8. It appears that therapy with lipid lowering drugs increased chylomicron remnant clearance in control rabbits by up-regulation of the apolipoprotein B100/E receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of probucol on plasma clearance and organ uptake of chylomicrons and VLDLs in normal and diabetic rats.

Probucol was given to rats made diabetic by streptozotocin. Compared with diabetic rats not receiving probucol or with nondiabetic rats, probucol lowered the plasma concentrations of triglycerides, phospholipids, cholesterol, and apolipoprotein B. The concentrations of serum chylomicrons and very low density lipoprotein (VLDL) were also reduced. In control and diabetic rats, probucol enhanced the clearance of endogenously radiolabeled VLDL from the plasma. Clearances from the plasma of rat lymph chylomicrons or chylomicron-like lipid emulsions were slow in diabetic rats. Probucol normalized chylomicron clearance in diabetic rats primarily by restoring hepatic uptake of remnants, which was decreased in diabetes. In diabetic rats, uptake of chylomicron remnants was increased in a number of extrahepatic tissues, including the heart and kidney. Probucol significantly decreased uptake in some extrahepatic tissues. Increased plasma clearance of VLDL and chylomicrons was associated with an increase in the apolipoprotein CII/CIII and apolipoprotein E/C ratios. Orally administered probucol was specifically incorporated into lymph chylomicrons, and clearance of probucol from the plasma exactly paralleled the clearance of chylomicron remnants, as traced with radiolabeled cholesteryl esters. Chylomicron-like emulsions incorporating probucol were exclusively cleared from the plasma by the liver in normal rats. We conclude that in streptozotocin diabetic rats, probucol is an effective hypolipidemic agent because it promotes the clearance of the triglyceride-rich lipoproteins.

Effects of anaesthesia on the removal from plasma of intravenously injected chylomicron-like lipid emulsions in rats and mice.

1. In order to find an anaesthesia with minimum perturbation to the metabolism of chylomicrons, the effects of seven different anaesthetic agents on clearance from plasma of chylomicron-like emulsions were compared. 2. Avertin, urethane, fentanyl, and a ketamine/xylazine mixture all slowed the removal from plasma of emulsion triolein and cholesteryl oleate. The steroid anaesthetic althesin slowed the clearance of emulsion cholesteryl oleate without affecting the removal from plasma of emulsion triolein. Nembutal when injected intravenously at a hypnotic dose did not affect the clearance of emulsion triolein or cholesteryl oleate, whereas at the anaesthetic dose, nembutal slowed the clearance rate of both labelled lipids. 3. Except for althesin, which did not affect the plasma clearance of triolein, fractional clearance rates of emulsion triolein and cholesteryl oleate calculated from blood samples taken during 12 min after injection were significantly slower in the anaesthetized groups compared with controls. However, with avertin, althesin, nembutal and ketamine/xylazine, amounts of radiolabelled triolein and cholesteryl oleate remaining in plasma 25 and 30 min after injection were comparable with the control. Radioactive lipids in plasma remained much higher in rats treated with urethane and fentanyl-fluanisonium even 30 min after injection. 4. Avertin was simple to administer and produced a suitable depth of anaesthesia for minor surgery, tail vein injections and blood sampling, whereas althesin and the ketamine/xylazine mixture required supplementary doses to maintain anaesthesia towards the end of the experiment. We concluded that anaesthesia is best avoided for studies of chylomicron clearance. Avertin is the preferred agent if anaesthesia must be used, for example in newborn rats or in mice.

Effects of adrenoreceptor antagonists and agonists on clearance of emulsion models of triacylglycerol-rich lipoproteins from plasma in rats.

1. We previously found that adrenaline and noradrenaline exert essentially opposite effects on clearance from plasma of chylomicron-like emulsions injected intravenously in rats, suggesting mechanisms that may be implicated in the atherogenic effects of chronic stress and hypertension and conversely in the protective effect of regular exercise. 2. The mechanisms underlying the effects of adrenaline and noradrenaline have now been investigated. Chronic adrenergic blockade with either the alpha 1-receptor antagonist doxazosin or the beta-receptor antagonist propranolol slowed the clearance of labelled emulsion lipids from plasma of normal Wistar rats. The results with doxazosin were unexpected in view of its capacity to decrease plasma triglycerides in patients. 3. In spontaneously hypertensive rats (SHR) the clearance of triolein (TO) was very slow compared with normal Wistar rats. Emulsion TO clearance provides a measure of lipolysis by lipoprotein lipase, and a defect in clearance indicates either defective enzyme action or poor perfusion of capillary beds rich in enzyme. Defective enzyme activity in SHR was excluded, suggesting redistribution of blood flow away from skeletal muscle and adipose tissue. In SHR the TO clearance from injected chylomicron-like emulsions was improved by blockade with doxazosin compared with control untreated SHR. 4. The beta 2-adrenoreceptor agonist Fenoterol was infused intravenously during clearance of an injected lipid emulsion. Clearance of radiolabelled cholesteryl oleate (CO) was clearly slowed while there was a lesser reduction of TO clearance rate. Emulsion CO clearance provides a measure of the uptake of lipoprotein remnants by the liver, and a defect in clearance of CO indicates either defective ligand (apolipoprotein E)-receptor interaction or decreased perfusion of the splanchnic bed. Isoprenaline, a non-selective beta-adrenergic agonist, gave similar results. Both compounds reduced mean arterial pressure by about 20-40 mm Hg at the doses employed, indicating that the beta 1 (cardiac) effect of the isoprenaline was insufficient to offset its vasodilatatory effect on skeletal muscle arterioles (beta 2). 5. The alpha-agonist phenylephrine, at a dose which moderately raised mean arterial pressure, slowed clearance of both TO and CO for the first 12 min after injection of emulsion but at later time points clearances caught up with the controls. 6. Administration of a mixture of isoprenaline and phenylephrine produced definite enhancement of both TO clearance and CO clearance. The effect of the mixture was opposite to the effects of of either agonist alone, demonstrating clearly that direct effects on lipoprotein lipase activity or receptor mediated processes were not involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Defective plasma clearance of chylomicron-like lipid emulsions in Watanabe heritable hyperlipidemic rabbits.

Human patients with familial hypercholesterolemia (FH) and Watanabe heritable hyperlipidemic rabbits (WHHL), while lacking normal receptors recognizing low-density lipoproteins (LDL), are said to have normal clearance of chylomicrons. In the present study, emulsions with a similar lipid composition to chylomicrons were injected intravenously in homozygous WHHL rabbits and normal control rabbits fed diet with low or high cholesterol. Radioactive labels tracing emulsion triolein and cholesteryl oleate were both removed rapidly from the bloodstream, with the removal rate of triolein always faster than that of cholesteryl oleate. This pattern was similar to the clearance of normal chylomicrons in rabbits or rats, and was consistent with the formation of remnant lipoproteins after hydrolysis of emulsion triolein by lipoprotein lipase, followed by hepatic uptake of the remnants. The removal of cholesteryl oleate was significantly slower in WHHL rabbits than in normal controls, suggesting that the absence of LDL receptor function led to impaired remnant clearance. Measured in post-heparin plasma, the activity of lipoprotein lipase was decreased in WHHL rabbits, but this was not associated with clear evidence of defective lipolysis of emulsion triolein. Apolipoprotein E did not appear to be deficient in WHHL rabbits. Plasma devoid of lipoproteins less than 1.006 g/ml from WHHL and normal control rabbits transferred similar amounts of apolipoprotein E to chylomicron-like emulsions after incubation. Impaired clearance of chylomicron remnants possibly contributes to the hypertriglyceridemia of WHHL rabbits and to accelerated atherogenesis when the function of LDL receptors is defective.

Effects of hypothyroidism on the metabolism of lipid emulsion models of triacylglycerol-rich lipoproteins in rats.

Methimazole-treated hypothyroid rats were injected intravenously with triacylglycerol/cholesteryl oleate/cholesterol/phospholipid emulsions designed to model the composition of chylomicrons. Compared with controls, hypothyroidism decreased the clearance rates of emulsion cholesteryl oleate. Clearance of emulsion triolein was affected much less and could be accounted for by residual triolein in remnants, suggesting that triacylglycerol lipolysis by lipoprotein lipase was unaffected by hypothyroidism but that clearance of remnants from plasma was decreased. Assays in vitro showed increased activities of lipoprotein lipase and hepatic lipase in hypothyroid rats. Emulsions were incubated with post-heparin plasma lipoprotein lipase to prepare remnants in vitro. The clearance from plasma of pre-formed remnants was slower after injection into hypothyroid rats than in control rats. Uptake of remnant cholesteryl oleate by the liver was significantly decreased in the hypothyroid rats. Treatment of hypothyroid rats for 7 days with 3,3',5'-tri-iodo-L-thyronine (T3) reversed the inhibition of hepatic remnant uptake and normalized plasma cholesterol. A thyroid hormone analogue with decreased hypermetabolic side-effects, L-94901, attenuated plasma cholesterol and improved but did not normalize remnant clearance. Emulsions incubated with plasma from hypothyroid rats had a decreased ratio of apolipoprotein E/apolipoprotein C compared with control rats or hypothyroid rats treated with T3. The change in the apolipoprotein E/apolipoprotein C ratio probably accounts for the defect in remnant clearance in hypothyroidism.