Decreased intracellular degradation and increased secretion of apo B-100 in Hep G2 cells after inhibition of cholesteryl ester synthesis

Control of apolipoprotein B (apo B) secretion in hepatocytes occurs partly at the post-translational level. The key step in this process appears to be intracellular degradation of newly synthesized apo B. The aim of this paper was to investigate the mechanisms that regulate apo B secretion by Hep G2 cells, in response to the inhibition of Acyl-CoA Acyltransferase (ACAT) by the compound Sandoz 58035 (S-58035). S-58035 (20 μM) reduced cholesteryl ester synthesis from [¹⁴C]oleate by 95%, and increased significantly, in a dose-dependent manner, (2–100 μM) apo B secretion, either in control conditions (from 78±4.3 to 126±6.1 ng apo B-100/mg cell protein/4 h) or upon stimulation of apo B secretion by oleate (from 134±4.23 to 177±4.3 ng apo B/mg cell protein/4 h). This increased secretion of newly synthesized apo B-100 was confirmed by pulse experiments and by gradient ultracentrifugation of the media. Moreover pulse-chase experiments showed that the addition of S-58035 reduced intracellular degradation of apo B-100, both in control conditions and in the presence of oleate. S-58035 (20 μM) did not affect total cellular cholesterol content, but free cholesterol increased with a concomitant decrease of cholesteryl ester (−20%). S-58035 increased cellular triglyceride mass, which was observed in basal conditions (from 12.8±1.09 to 22.7±2.7 μg TG/mg cellular protein) and also in presence of oleate (from 48±0.53 to 59±6.3 μg TG/mg cellular protein). This effect is due to a stimulation of triglyceride synthesis, as determined by incorporation of [³H]glycerol into cellular triglycerides. From these data we conclude that, under our experimental conditions, triglyceride synthesis and/or availability is likely to control intracellular degradation of apo B.     

Improved metabolic control reduces the number of postprandial apolipoprotein B-48-containing particles in type 2 diabetes

Postprandial lipoproteins are raised in diabetes and there is increasing evidence for the atherogenicity of the chylomicron remnant. Increased postprandial cholesteryl ester transfer has also been demonstrated in diabetes and may contribute to the atherogenic lipoprotein profile. The present study examined the effect of improving metabolic control on postprandial lipoproteins in 13 Type 2 diabetic patients. Blood was taken fasting and at 2-h intervals following a high fat, 1100 kcal meal. Patients were brought into good control by intensified dietary advice and oral hyperglycaemic agents or insulin if blood glucose failed to respond. Fasting and postprandial cholesteryl ester transfer protein (CETP) and lecithin:cholesteryl acyltransferase (LCAT) were determined in six patients. Lipoproteins were isolated by sequential ultracentrifugation. Chylomicron and very low density lipoprotein (VLDL) apolipoprotein B-48 and apolipoprotein B-100 were isolated by polyacrylamide gradient gel electrophoresis and quantified by densitometric scanning. CETP and LCAT were determined by an endogenous method which determined cholesterol esterification and transfer between the patients' lipoproteins. There was a significant reduction in postprandial chylomicron apo B-48 (P<0.005), apo B-100 (P<0.0005) and chylomicron cholesterol (P<0.001) following improved diabetic control. The chylomicron lipid/apo B ratio increased with improved control (P<0.01). Postprandial CETP and LCAT were significantly reduced in good control (P<0.01 and P<0.05, respectively) and there were significant changes in HDL composition. The study shows that improvement in metabolic control in Type 2 diabetic patients leads to a reduction in postprandial chylomicron particles and less transfer of cholesterol to apo B-containing lipoproteins.     

A new labeling approach using stable isotopes to study in vivo plasma cholesterol metabolism in humans.

A method to study reverse cholesterol transport in humans was developed using stable isotopes and kinetic analysis. Three normolipidemic subjects received simultaneous intravenous infusions of deuterated leucine and (13)C-acetate for 14 and 8 hours, respectively. Deuterium enrichment was measured in protein-bound leucine in apolipoproteins (apo) B-100 and A-I (using gas chromatography coupled with mass spectrometry [GCMS]) and (13)C-enrichment in unesterified cholesterol and cholesteryl ester (using gas chromatography coupled to isotope ratio mass spectrometry [GC-C-IRMS]) in very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) during the tracer infusion. Curves were analyzed using multicompartmental analysis. This protocol is suitable to quantify the different processes involved in reverse cholesterol transport (RCT) in humans, including cholesterol esterification, transfer of cholesteryl ester from HDL towards apo B-100-containing lipoproteins, and the contribution of VLDL, LDL, and HDL in the final steps of RCT. In agreement with previous data from kinetic analysis of radiotracer experiments, our results suggest that in fasting normolipidemic subjects the major fraction of cholesteryl ester enters plasma through esterification in HDL (more than 95%). The major fraction of cholesteryl ester disappears through apo B-100-containing lipoproteins (VLDL and LDL) catabolism (mean of 82%) rather than through removal from HDL (mean of 18% with approximately an equal part for apo AI-dependent and independent catabolism, respectively, 7% and 11%). We conclude that this protocol could be applied to study the modulation of these processes by nutrition, diseases, or pharmacologic treatments.     

The effects of fatty acids on apolipoprotein B secretion by human hepatoma cells (HEP G2)

We have investigated the effect of fatty acids on the rate of apolipoprotein B (apo B) secretion by human hepatoma cells (Hep G2). When Hep G2 cells were maintained in tissue culture flasks oleic acid up to 0.4 mM increased apo B secretion in a dose-dependent manner, whereas increases in triacylglycerol (TG) were smaller and dose dependency was less evident. In the absence of oleic acid, apo B accumulating in the tissue culture medium was predominantly in lipoproteins of higher density than very low density lipoproteins (VLDL). However, when the rate of secretion was stimulated with oleic acid the apo B-containing lipoproteins became lower in density. We postulated that there was a high rate of lipolysis of newly secreted VLDL by Hep G2 cells, which would account both for the relatively smaller effect of oleic acid on TG as opposed to apo B accumulating in the culture medium and the predominance of apo B in lipoproteins of a higher density than VLDL, which became less evident when VLDL secretory rates were stimulated by oleic acid. To test this hypothesis, cultured Hep G2 cells were transferred to columns containing Cytodex beads, permitting their continuous perfusion with culture medium so that newly secreted VLDL did not remain in contact with the cells. Apo B recovered from the perfusate was largely in VLDL range lipoproteins and the TG measured in the perfusate indicated that the true secretory rate of TG-rich lipoproteins was substantially higher than had been reflected by TG accumulating in culture medium left in contact with cells. Apo B measured in the culture medium of Hep G2 cells may thus be a better reflection of VLDL secretion, even though it is contained in higher density lipoproteins due to removal of TG by lipolysis. The effects of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) on apo B (apo B) secretion by Hep G2 cells maintained in tissue culture flasks were next investigated. SFA (0.4 mM), with the exception of stearic acid (C18:0), increased apo B secretion. Lauric acid (C12:0) increased apo B secretion by 32%, myristic acid (C14:0) by 41% (P<0.005), palmitic acid (C16:0) by 154% (P<0.025), and arachidic acid (C20:0) by 186% (P<0.005). The effect of MUFA (0.4 mM) was to increase apo B secretion, oleic acid (C18:1) by 239% ((P<0.0005) and palmitoleic acid (C16: 1) by 125% (P<0.005). Of the PUFA investigated, linolenic acid (C18:3) (0.4 mM) did not have any significant effect on apo B secretion, whereas linoleic acid (C18:2) (0.4mM) arachidonic acid (C20:4) (0.1 mM) and eicosapentaenoic acid (C20:5) (0.1 mM) caused significant increases of 164, 171 and 171%, respectively (P<0.005). The fatty acids studied increased intracellular TG and cholesteryl ester concentrations to varying extents. The increase in intracellular TG produced by the different fatty acids correlated with the rate of apo B secretion (r=0.6; P<0.05). In this human hepatoma cell line, with the exception of the saturated fatty acids, the rate of secretion of apo B-containing lipoproteins does not follow the same pattern as changes in circulating low density lipoprotein (LDL) concentrations reported with dietary manipulation in man. If our findings reflect the in vivo situation, we suggest that whilst the dietary effects of SFA on serum LDL may in part be determined by the hepatic apo B secretory rate, the effects of MUFA and PUFA must be largely mediated through a catabolic effect rather than an effect on hepatic secretion. The marked increase in apo B secretion with the more highly polyunsaturated fatty acids, such as eicosapentaenoic acid, may also explain why they do not lower circulating LDL, despite reports of their apparently favourable effect on LDL-receptor mediated clearance.     

Apolipoprotein B (B-48) of rat chylomicrons is not a precursor of the apolipoprotein of low density lipoproteins.

We have found that, in chylomicrons from intestinal lymph fistulas in the rat, the sole molecular species of apolipoprotein B (apo B) is B-48. This protein is analogous to the B-48 apoprotein of human chylomicrons. In contrast, preparations of chylomicrons from blood serum are known to contain species of B apolipoproteins of higher molecular weight, presumably due to the presence of hepatogenous lipoproteins. We studied the removal of 125I-labeled apo B-48 of intestinal lymph chylomicrons from blood plasma of rats. The removal of [14C]cholesteryl esters of biologically labeled chylomicrons was unaffected by radioiodination. The labeled cholesteryl esters and apo B-48 disappeared rapidly from the density (P) less than 1.006 g/ml fraction of plasma. In contrast to the apo B of very low density lipoproteins (1.019 less then p less then 1.063 g/ml) after 1 hr. No 125I was found in the B-100 or B-95 apolipoproteins at any time. We conclude that, unlike the species of apo B found uniquely in hepatogenous very low density lipoproteins, the apo B-48 protein of chylomicrons is not a precursor of the B apoprotein of low density lipoproteins.     

Human noninsulin-dependent diabetes: identification of a defect in plasma cholesterol transport normalized in vivo by insulin and in vitro by selective immunoadsorption of apolipoprotein E.

Plasma cholesterol metabolism in patients with poorly controlled noninsulin-dependent diabetes was characterized by inhibition of cholesterol net transport between cultured cells (fibroblasts) and plasma, inhibition of cholesterol esterification, and inhibition of cholesteryl ester transfer to low and very low density lipoproteins, relative to a normal control group. Plasma from these patients also contained a 2-fold higher level of apolipoprotein E (apo E). Effective control of hyperglycemia with insulin normalized both the parameters of plasma cholesterol metabolism and plasma levels of apo E. Removal of apo E by immunoaffinity chromatography normalized cell-to-plasma cholesterol transport but was without effect on the rate of cholesterol esterification or of cholesteryl ester transfer. These findings suggest that an inhibition in the chain of reactions by which cellular cholesterol is transferred in esterified form to low and very low density lipoproteins is associated with the appearance of an apo E-dependent "shunt" pathway, returning cholesterol from plasma back to the cells and so nullifying the normal cell-to-plasma transport pathway.     

Prolonged inhibition of cholesterol synthesis by atorvastatin inhibits apo B-100 and triglyceride secretion from HepG2 cells.

Atorvastatin is a new HMG-CoA reductase inhibitor that strongly lowers plasma cholesterol and triglyceride (TG) levels in humans and animals. Since previous data indicated that atorvastatin has prolonged inhibition of hepatic cholesterol synthesis, we tested whether this longer duration of inhibitory effect on cholesterol synthesis decreased hepatic lipoprotein secretion in vitro. We used the HepG2 hepatoma cell line to: (1) determine the time required until levels of secreted apo B-100 and TG declined significantly, (2) examine the relation to the mass of cellular cholesteryl ester (CE) and (3) test microsomal triglyceride transfer protein (MTP) activity which leads to decreased apo B-100 production. Although atorvastatin significantly inhibited cholesterol synthesis in HepG2 cells regardless of treatment duration (1, 14 or 24 h), it did not inhibit TG synthesis. Apo B-100 and TG secretion were unchanged after 1-h atorvastatin treatment, but declined significantly after 24-h treatment. Atorvastatin treatment also reduced cellular CE mass, exhibiting both time- and dose-dependency. Mevalonolactone, a product of HMG-CoA reductase, attenuated the inhibitory effects of atorvastatin. Atorvastatin strongly reduced mRNA levels of MTP, whereas it did not inhibit MTP activity as measured by TG transfer assay between liposomes. Simvastatin also induced treatment- and time-dependent reductions in apo B-100, whereas the MTP inhibitor BMS-201038 exhibited no time dependency, instead inhibiting this variable even on 1-h treatment. These results indicate that reduced apo B-100 secretion caused by atorvastatin is a secondary result owing to decreased lipid availability, and that atorvastatin's efficacy depends on the duration of cholesterol synthesis inhibition in the liver.     

The effect of lovastatin treatment on low-density lipoprotein hydrated density distribution and composition in patients with intermittent claudication and primary hypercholesterolemia.

The effects of lovastatin treatment on density distribution and composition of low-density lipoproteins (LDL) were studied using a density gradient ultracentrifugation method in 35 hypercholesterolemic patients with severe peripheral vascular disease. Lovastatin caused a 32% mean reduction in LDL particle mass and a 36% reduction in LDL cholesterol. The cholesteryl ester to apolipoprotein (apo) B, free cholesterol to apo B, and phospholipid to apo B weight ratios in LDL decreased significantly during treatment (P less than .01, P less than .01, and P less than .001, respectively). The effect on plasma triglycerides (Tg) was not uniform. Plasma Tg levels decreased in 25 patients, but increased in 10 patients. Since plasma Tg level influences the LDL density distribution and composition, the patients were also subgrouped and analyzed according to change in plasma Tg. In those with increased plasma Tg, the light LDL particles were reduced more and the dense particles less compared with patients with decreased Tg. The mean Tg content of LDL increased (from 7.7% to 9.3%; P less than .05) and the weight ratio of core lipids (cholesteryl ester/Tg) in LDL decreased (from 4.57 to 3.44; P less than .01) in patients with increased plasma Tg during treatment. The results indicate that the change in plasma Tg (decrease or increase) determined the qualitative changes in LDL observed during lovastatin treatment.     

Relationships between the responses of triglyceride-rich lipoproteins in blood plasma containing apolipoproteins B-48 and B-100 to a fat-containing meal in normolipidemic humans.

The concentration of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 (chylomicrons) and apo B-100 (very low density lipoproteins) was measured in blood plasma of healthy young men after an ordinary meal containing one-third of daily energy and fat. Plasma obtained in the postabsorptive state and at intervals up to 12 hr after the meal was subjected to immunoaffinity chromatography against a monoclonal antibody to apo B-100 that does not bind apo B-48 and a minor fraction of apo B-100 rich in apo E. Measurements of the concentrations of components of the total and unbound triglyceride-rich lipoproteins separated from plasma by ultracentrifugation showed that about 80% of the increase in lipoprotein particle number was in very low density lipoproteins containing apo B-100 and only 20% was in chylomicrons containing apo B-48 that carry dietary fat from the intestine. The maximal increments and the average concentrations of apo B-48 and B-100 during the 12 hr were highly correlated (r2 = 0.80), suggesting that preferential clearance of chylomicron triglycerides by lipoprotein lipase leads to accumulation of hepatogenous very low density lipoproteins during the alimentary period. The composition of the bulk of very low density lipoproteins that were bound to the monoclonal antibody changed little and these particles contained about 90% of the cholesterol and most of the apo E that accumulated in triglyceride-rich lipoproteins. The predominant accumulation of very low density lipoprotein rather than chylomicron particles after ingestion of ordinary meals is relevant to the potential atherogenicity of postprandial lipoproteins.     

Cellular cholesterol efflux to plasma from moderately hypercholesterolaemic type 1 diabetic patients is enhanced, and is unaffected by simvastatin treatment

Aim/hypothesis Cellular cholesterol efflux to plasma is important in reverse cholesterol transport and may be affected by simvastatin in type 1 diabetes mellitus.Methods In 14 moderately hypercholesterolaemic type 1 diabetic and 13 healthy men we determined plasma (apo)lipoproteins, pre- HDL formation, cholesteryl ester transfer protein (CETP) activity, phospholipid transfer protein (PLTP) activity, cholesterol esterification, cholesteryl ester transfer and the capacity of plasma to induce cholesterol efflux out of Fu5AH cells and fibroblasts. After diet run-in, diabetic patients were randomly treated with simvastatin 10, 20, 40 mg and placebo, once daily each, for 6 weeks in a double-blind crossover design.Results Plasma very low density lipid protein (VLDL)+LDL cholesterol, LDL cholesterol, HDL phospholipids, apolipoprotein (apo) A-I, apo B, CETP activity, PLTP activity, cholesterol esterification, cholesteryl ester transfer and the capacity of plasma to induce cholesterol efflux from Fu5AH cells and fibroblasts were higher in diabetic patients. Pre- HDL formation was unaltered. Simvastatin treatment decreased VLDL+LDL cholesterol, LDL cholesterol, triglycerides and apo B, CETP activity, cholesterol esterification and cholesteryl ester transfer. HDL cholesterol increased and its change was correlated with the change in cholesteryl ester transfer. The ability to promote cholesterol efflux from Fu5AH cells and fibroblasts did not change after simvastatin.Conclusions/interpretation The capacity of plasma from moderately hypercholesterolaemic type 1 diabetic patients to induce cholesterol efflux out of Fu5AH cells and fibroblasts is enhanced, probably due to higher apo A-I, HDL phospholipids and PLTP activity. Simvastatin increases HDL cholesterol in type 1 diabetic patients via lowering of plasma cholesteryl ester transfer. The HDL changes after simvastatin do not increase cellular cholesterol efflux further.     

F 12511, a novel ACAT inhibitor, and atorvastatin regulate endogenous hypercholesterolemia in a synergistic manner in New Zealand rabbits fed a casein-enriched diet

F 12511, a novel ACAT inhibitor, lowers plasma cholesterol levels in New Zealand rabbits fed a cholesterol-free casein-rich diet. In rabbits endogenous hypercholesterolemia pre-established for 8 weeks was used to compare treatments with F 12511 and atorvastatin for a further 8-week period, and to determine whether both agents act synergistically. F 12511 appears to be 3-4-fold more potent than atorvastatin in reducing total plasma cholesterol (active doses ranging from 0.16 to 2.5 and from 1.25 to 10 mg/kg per day, respectively) while the hypocholesterolemic efficacy of both compounds at 2.5 mg/kg per day amounted to 70 and 45%, respectively. A reduction by as much as 75% of esterified cholesterol in liver mediated by F 12511 could account for the decrease of plasma VLDL, LDL and apo B-100, whereas a reduction of the LDL production rate has been described as the main mechanism underlying the atorvastatin effect. F 12511 modified adrenal cholesterol balance only at the largest dose studied. In a further experiment the co-administration of threshold doses of F 12511 and atorvastatin (0.63 and 1.25 mg/kg per day, respectively) lowered plasma total cholesterol and apo B-100 containing lipoproteins to a greater extent and more rapidly than either agent alone. In the liver a decrease by atorvastatin in free cholesterol substrate for ACAT may amplify the effect of F 12511 on cholesteryl ester content leading to a diminution, in at least an additive manner, of the assembly and secretion of atherogenic lipoproteins in New Zealand rabbits which have developed an endogenous hypercholesterolemia. Thus, the combination of the ACAT inhibitor F 12511 with atorvastatin can represent a better approach than either agent alone to regulate lipoprotein metabolism in certain pathophysiological situations.     

Esterification of Low Density Lipoprotein Cholesterol in Human Fibroblasts and Its Absence in Homozygous Familial Hypercholesterolemia

A new mechanism is described for the cellular esterification of cholesterol derived from extra-cellular lipoproteins. Incubation of monolayers of cultured fibroblasts from normal human subjects with low density lipoproteins led to a 30- to 40-fold increase in the rate of incorporation of either [¹⁴C]acetate or [¹⁴C]oleate into the fatty acid fraction of cholesteryl [¹⁴C]esters. This stimulation of cholesteryl ester formation by low density lipoproteins occurred despite the fact that endogenous synthesis of free cholesterol was completely suppressed by the lipoprotein. Thus, exogenous cholesterol contained in low density lipoproteins, rather than endogenously synthesized sterol, appeared to provide the cholesterol substrate for this cellular esterfication process. High density lipoproteins and the lipoprotein-deficient fraction of serum neither stimulated cholesteryl ester formation nor inhibited cholesterol synthesis. Both the low density lipoprotein-dependent increase in cholesterol esterification and decrease in free cholesterol synthesis required the interaction of the lipoprotein with its recently described cell surface receptor. Cells from homozygotes with familial hypercholesterolemia, which lack specific low density lipoprotein receptors, showed neither lipoprotein-dependent cholesterol esterification nor suppression of cholesterol synthesis. The reciprocal changes in free cholesterol synthesis and cholesteryl ester formation produced by low density lipoprotein-receptor interactions may play an important role in the regulation of the cholesterol content of mammalian cells.     

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from Type 1 (insulin-dependent) diabetic patients: the influence of non-enzymatic glycosylation of low-density lipoproteins

Summary Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocytederived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19±1.19 vs 6.11±0.94 nmol/mg cell protein/20 h, mean±SEM, p<0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p<0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p<0.01) and correlated significantly with cholesteryl ester synthesis (r=0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages. This study suggests that glycosylation of low density lipoproteins to the extent occurring in diabetes may alter their interaction with human monocyte-derived macrophages and may lead to increased intracellular cholesteryl ester accumulation. The results suggest a possible mechanism by which hyperglycaemia may contribute to the acceleration of atherosclerosis in diabetes.     

Role of cholesterol ester mass in regulation of secretion of ApoB100 lipoprotein particles by hamster hepatocytes and effects of statins on that relationship

Our understanding of the factors that regulate the secretion of apoB100 lipoproteins remains incomplete with considerable debate as to the role, if any, for cholesterol ester in this process. This study examines this issue in primary cultures of hamster hepatocytes, a species in which both cholesterol and apoB100 metabolism are very similar to man. Addition of oleate to medium increased the mass of triglyceride and cholesterol ester within the hepatocyte and also increased the secretion of triglycerides, cholesterol ester, and apoB100 into the medium. Next, the responses of hamster hepatocytes to addition of either an HMG-CoA reductase inhibitor (lovastatin) or an acyl-CoA cholesterol acyltransferase inhibitor (58-035) to the medium, with or without added oleate, were determined. Effects of either agent were only evident in the oleate-supplemented medium in which cholesterol ester mass had been increased above basal. If oleate was not added to the medium, neither agent reduced apoB100 secretion; equally important, over the 24-hour incubation, neither agent, at the concentration used, produced any detectable change in intracellular cholesterol ester mass. However, in contrast to the estimates of mass, which were unchanged, under the same conditions radioisotopic estimates of cholesterol ester synthesis were markedly reduced. Any conclusion as to the relation of cholesterol ester mass to apoB100 secretion would therefore depend on which of the 2 methods was used. Overall, the data indicate a close correlation between the mass of cholesterol ester within the hepatocyte and apoB100 secretion from it and they go far to explain previous apparently contradictory data as to this relation. More importantly, though, taken with other available data, they indicate that the primary response of the liver to increased delivery of lipid is increased secretion rather than decreased uptake. These results point, therefore, to a hierarchy of hepatic responses to increased flux of fatty acids and increased synthesis of cholesterol that in turn suggests a more dynamic model of cholesterol homeostasis in the liver than has been appreciated in the past.     

Cholesteryl ester transfer activity : Localization and role in distribution of cholesteryl ester among lipoproteins in man

The cholesteryl ester exchange/transfer protein is involved in the transport of cholesteryl ester from high density lipoproteins (HDL) to very low density lipoproteins (VLDL) and low density lipoproteins (LDL). Localization of cholesteryl ester transfer activity (CETA) in plasma was studied by measuring CETA in various delipidated fractions from a single step density ultracentrifugation gradient of plasma. CETA was measured in an in vitro system by calculating the exchange of cholesteryl ester in a standard mixture of [3H]CE-HDL and LDL. The method used for the delipidation of plasmas and fractions to be tested was critical. Optimal results were obtained by delipidation with diisopropylether-butanol (60: 40, v/v) at O degrees C. The bulk of CETA was detected in HDL3 (1.125 less than d less than 1.210 g/ml) when the lipoproteins were separated by single-step density gradient ultracentrifugation and in the 'lipoprotein-free' fraction (d greater than 1.250 g/ml) when the lipoproteins were separated by flotation ultracentrifugation including two washes. To determine whether CETA plays a role in the distribution of cholesteryl ester among the various lipoproteins, it was measured in whole plasma from normal and hyperlipidemic subjects. Plasma was delipidated before the assay in order to prevent bias due to variation of cholesterol content. CETA was higher in delipidated plasma of hyperlipidemic subjects (117.3 +/- 36.5 nmol CE/ml/h) than in delipidated plasma of normolipidemic controls (68.7 +/- 17.6 nmol CE/ml/h) (P less than 0.005). A positive correlation (r = 0.80, P less than 0.005) was found between CETA and (VLDL + LDL) cholesterol levels. A negative correlation (r = 0.57, P less than 0.05) existed between CETA and HDL cholesterol. This correlation was found both in the group as a whole and within the normal and the hyperlipidemic groups separately. The activity of the cholesteryl ester transfer appears to be a regulatory factor in the distribution of cholesteryl ester over the various lipoproteins.     

Lamellar lipoproteins uniquely contribute to hyperlipidemia in mice doubly deficient in apolipoprotein E and hepatic lipase

Remnants of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 accumulate in apo E-deficient mice, causing pronounced hypercholesterolemia. Mice doubly deficient in apo E and hepatic lipase have more pronounced hypercholesterolemia, even though remnants do not accumulate appreciably in mice deficient in hepatic lipase alone. Here we show that the doubly deficient mice manifest a unique lamellar hyperlipoproteinemia, characterized by vesicular particles 600 Å–1,300 Å in diameter. As seen by negative-staining electron microscopy, these lipoproteins also contain an electron-lucent region adjacent to the vesicle wall, similar to the core of typical lipoproteins. Correlative chemical analysis indicates that the vesicle wall is composed of a 1:1 molar mixture of cholesterol and phospholipids, whereas the electron-lucent region appears to be composed of cholesteryl esters (about 12% of the particle mass). Like the spherical lipoproteins of doubly deficient mice, the vesicular particles contain apo B-48, but they are particularly rich in apo A-IV. We propose that cholesteryl esters are removed from spherical lipoproteins of these mice by scavenger receptor B1, leaving behind polar lipid-rich particles that fuse to form vesicular lipoproteins. Hepatic lipase may prevent such vesicular lipoproteins from accumulating in apo E-deficient mice by hydrolyzing phosphatidyl choline as scavenger receptor B1 removes the cholesteryl esters and by gradual endocytosis of lipoproteins bound to hepatic lipase on the surface of hepatocytes.     

Apolipoprotein B-100 and ApoA-II Kinetics as Determinants of Cellular Cholesterol Efflux

Context: Cellular cholesterol efflux is a key step in reverse cholesterol transport and may depend on the metabolism of apolipoprotein (apo) B-100, apoA-I, and apoA-II. Objective: We examined the associations between cholesterol efflux and plasma concentrations and kinetics of very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL)-apoB-100, high-density lipoprotein (HDL)-apoA-I, and HDL-apoA-II in men. Design, Subjects, and Methods: Thirty men were recruited from the community with a wide range of body mass index. The capacity of plasma and HDL to efflux cholesterol was measured ex vivo. Apolipoprotein kinetics were measured using stable isotope techniques and multicompartmental modeling. Results: Cholesterol efflux to whole plasma was correlated with plasma levels of cholesterol, triglyceride, apoB-100, insulin, cholesteryl ester transfer protein, and lecithin-cholesterol acyltransferase, body mass index and waist circumference (P < 0.05 in all). Cholesterol efflux was inversely correlated with the fractional catabolic rate (FCR) of VLDL (r = -0.728), IDL (r = -0.662), and LDL-apoB-100 (r = -0.479) but positively correlated with the FCR (r = 0.438) and production rate (r = 0.468) of HDL-apoA-II. In multiple regression analysis, the concentration and FCR of VLDL-apoB-100 (β-coefficient = 0.708 and -0.518, respectively) and IDL-apoB-100 (β-coefficient = 0.354 and -0.447, respectively) were independent predictors of cholesterol efflux. The association of cholesterol efflux with apoB-100 metabolism was diminished after removal of apoB-100-containing lipoproteins from plasma prior to efflux. All associations, except for cholesteryl ester transfer protein, were lost when cholesterol efflux to isolated HDL was tested. Conclusions: The plasma concentration and kinetics of apoB-100-containing lipoproteins are significant predictors of the capacity of whole plasma to effect cellular cholesterol efflux.     

Recent progress in understanding apolipoprotein B

For the past 5 years, investigators from many different laboratories have contributed to a greatly increased understanding of two very important lipid-carrying proteins in plasma--apo B-100 and apo B-48. Apo B-100, an extremely large protein composed of 4,536 amino acids, is synthesized by the liver and is crucial for the assembly of triglyceride-rich VLDL particles. Apo B-100 is virtually the only protein of LDL, a cholesteryl ester-enriched class of lipoproteins that are metabolic products of VLDL. The apo B-100 of LDL serves as a ligand for the LDL receptor-mediated uptake of LDL particles by the liver and extrahepatic tissues. The LDL receptor-binding region of apo B-100 is located in the carboxyterminal portion of the molecule, whereas its lipid-binding regions appear to be broadly dispersed throughout its length. Apo B-48 contains the amino-terminal 2,152 amino acids of apo B-100 and is produced by the intestine as a result of editing of a single nucleotide of the apo B mRNA, which changes the codon specifying apo B-100 amino acid 2,153 to a premature stop codon. Apo B-48 has an obligatory structural role in the formation of chylomicrons; therefore, its synthesis is essential for absorption of dietary fats and fat-soluble vitamins. Both apo B-48 and apo B-100 are encoded on chromosome 2 by a single gene that contains 29 exons and 28 introns. An elevated level of apo B-100 in the plasma is a potent risk factor for developing premature atherosclerotic disease. In the past 3 years, many different apo B gene mutations that affect the concentrations of both apo B and cholesterol in the plasma have been characterized. A missense mutation in the codon for apo B-100 amino aid 3,500 is associated with hypercholesterolemia. This mutation results in poor binding of apo B-100 to the LDL receptor, thereby causing the cholesteryl ester-enriched LDL particles to accumulate in the plasma. This disorder is called familial defective apo B-100, and it is probably a cause of premature atherosclerotic disease. Familial hypobetalipoproteinemia is a condition associated with abnormally low levels of apo B and cholesterol; affected individuals may actually have a reduced risk of atherosclerotic disease.(ABSTRACT TRUNCATED AT 400 WORDS)     

Probucol treatment in hypercholesterolemic patients: effects on lipoprotein composition, HDL particle size, and cholesteryl ester transfer

Despite probucol's capacity to induce regression of tendinous xanthomata and reduce whole plasma and LDL cholesterol (LDL-C) in patients with hypercholesterolemia, its therapeutic use in the United States has been limited because of concern about its HDL-lowering effects. To assess the possibility that probucol might facilitate mobilization of tissue cholesterol in the presence of low HDL levels as a consequence of favorable changes in lipoprotein composition and function, we have analyzed lipoproteins and studied cholesteryl ester transfer (CET) in hypercholesterolemic patients before and after treatment. Prior to treatment, the free cholesterol (FC)/lecithin (L) ratio in plasma, a new index of cardiovascular risk, and the mass of cholesteryl ester transferred from HDL to the apo B-containing lipoproteins (CET) both were significantly increased (P less than 0.001). As previously shown, plasma cholesterol, LDL-C, HDL-C, HDL2, and apolipoproteins A-I, A-II, and B all fell significantly following probucol treatment. The FC/L ratio in plasma (P less than 0.01) and HDL2 (P less than 0.01) both fell significantly also, as did the sphingomyelin/lecithin ratio in VLDL + LDL (P less than 0.001) which is typically increased in untreated patients with hypercholesterolemia. Nondenaturing gradient gel electrophoresis in 6 patients revealed that the quantitative changes in HDL were associated with a redistribution of particles characterized by a decrease in the prevalence of the largest (HDL2b) and a relative increase in the number of the smallest (HDL3b) particles. Moreover, CET following probucol therapy returned to levels which were indistinguishable from those of normolipidemic controls. These results indicate that untreated patients with hypercholesterolemia have abnormalities in (1) lipoprotein composition which have been shown to retard the movement of cholesterol from tissues to HDL, and in (2) CET which is accelerated and can potentially lead to the formation in plasma of atherogenic CE-enriched apo B-containing lipoproteins. Probucol's capacity to reverse these specific alterations suggests that it may have beneficial effects on cholesterol transport in patients with hypercholesterolemia.     

Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Very-low-density lipoproteins (VLDL) (density less than 1.006 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 +/- 1.01 v 1.99 +/- 0.39 nmol 14C-cholesteryl oleate synthesized/mg cell protein/20 h; mean +/- SEM, P less than .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (P less than .05). The increase in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 +/- 0.23 v 2.12 +/- 0.20 micrograms degraded/mg cell protein/20 h) and accumulation (283 +/- 35 v 242 +/- 33 ng/mg cell protein/20 h) of 125I-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B, E, and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects.(ABSTRACT TRUNCATED AT 250 WORDS)