Very low density lipoprotein. Dissociation of apolipoprotein C during lipoprotein lipase induced lipolysis.

The fate of apo C in rat plasma very low density lipoprotein (VLDL) during lipolysis was studied using VLDL labeled specifically with 125I-labeled apo C and purified bovine milk lipoprotein lipase. Incubations were carried out in vitro and included serum-containing systems and albumin containing systems. Free fatty acids generation proceeded with time of incubation in the two systems. It, however, was enhanced 1.5--2 fold by the presence of serum. 125I-labeled apo C equilibrated between very low and high density lipoprotein (HDL) in both systems even when enzyme was not present in the incubation medium, or when the incubation was carried out at 0 degrees C. Upon initiation of lipolysis, more 125I-labeled apo C was transferred to HDL and the transfer was proportional to the magnitude of free fatty acids release. 125I-labeled apo C was also progressively removed from VLDL in the albumin-containing system, although no known lipoprotein acceptor to apo C was present in the medium. The 125I-labeled apo C was recovered predominantly with the medium fraction of d greater than 1.21 g/ml (60--70%), and to a lesser degree with that of d= 1.019--1.21 g/ml. However, the relationship between lipolysis (measured as free fatty acids release) and removal of 125I-labeled apo C from VLDL were indistinguinshable in the albumin containing system and the serum containing system. On the basis of these observations, it is postulated that the removal of apo C during lipolysis of VLDL reflects the nature of the partially degraded VLDL particles, and is independent of the presence of a lipoprotein acceptor to apo C.     

Very low density lipoprotein. Removal of Apolipoproteins C-II and C-III-1 during lipolysis in vitro.

In this study we have investigated the effects of very low density lipoprotein (VLDL) lipolysis on the removal of radiolabeled apolipoprotein C-II and apolipoprotein C-III-1 from in vitro lipolyzed lipoproteins. Lipolysis was carried out in vitro using lipoprotein lipase purified from bovine milk, and mixtures with or without plasma. Lipoproteins were isolated by ultracentrifugation and by gel filtration. Labeled apo-C-II and apo-C-III-1 distributed among plasma lipoproteins, predominantly VLDL and high density lipoprotein (HDL). Lipolysis induced transfer of apo-C-II and apo-C-III-1 from VLDL to HDL. The transfer was proportional to the extent of triglyceride hydrolysis, and similar for the two apoproteins. The apo-C-II/apo-C-III-1 radioactivity ratio did not change in either VLDL or the fraction of d greater than 1.006 g/ml during the progression of the lipolytic process. Similar observations were recorded while using plasma-devoid lipolytic systems. Gel filtration of incubation mixtures, on 6% agarose, revealed that the removal of labeled apo-C molecules from VLDL is not a consequence of either centrifugation or high salt concentration. These results suggest that there is no preferential removal of apo-C-II or apo-C-III-1 from lipolyzed VLDL particles. They further indicate that the ratio of apo-C-II to apo-C-III-1 does not regulate the extent of lipolysis of different VLDL particles, at least in VLDL isolated from normolipidemic humans.     

Total cholesterol,low density lipoprotein cholesterol,and high density lipoprotein cholesterol and coronary heart disease in Scotland.

OBJECTIVE--To investigate long term changes in total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol concentrations and in measures of other risk factors for coronary heart disease and to assess their importance for the development of coronary heart disease in Scottish men. DESIGN--Longitudinal study entailing follow up in 1988-9 of men investigated during a study in 1976. SETTING--Edinburgh, Scotland. SUBJECTS--107 men from Edinburgh who had taken part in a comparative study of risk factors for heart disease with Swedish men in 1976 when aged 40. INTERVENTION--The men were invited to attend a follow up clinic in 1988-9 for measurement of cholesterol concentrations and other risk factor measurements. Eighty three attended and 24 refused to or could not attend. MAIN OUTCOME MEASURES--Changes in total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol concentrations, body weight, weight to height index, prevalence of smoking, and alcohol intake; number of coronary artery disease events. RESULTS--Mean serum total cholesterol concentration increased over the 12 years mainly due to an increase in the low density lipoprotein cholesterol fraction (from 3.53 (SD 0.09) to 4.56 (0.11) mmol/l) despite a reduction in high density lipoprotein cholesterol concentration. Body weight and weight to height index increased. Fewer men smoked more than 15 cigarettes/day in 1988-9 than in 1976. Blood pressure remained stable and fasting triglyceride concentrations did not change. The frequency of corneal arcus doubled. Alcohol consumption decreased significantly. Eleven men developed clinical coronary heart disease. High low density lipoprotein and low high density lipoprotein cholesterol concentrations in 1976, but not total cholesterol concentration, significantly predicted coronary heart disease (p = 0.05). Almost all of the men who developed coronary heart disease were smokers (91% v 53%, p less than 0.05). CONCLUSION--Over 12 years the lipid profile deteriorated significantly in this healthy cohort of young men. Smoking, a low high density lipoprotein concentration and a raised low density lipoprotein concentration were all associated with coronary heart disease in middle aged Scottish men, whereas there was no association for total cholesterol concentration. The findings have implications for screening programmes.     

Retention of apolipoprotein B and cholesterol by perfused heart during lipolysis of very-low-density lipoprotein

The fate and mechanism of removal of apolipoproteins and lipids of human very-low-density lipoproteins were determined in the perfused rat heart. Approx. 50% of the VLDL triacylglycerol was hydrolyzed during a 2 h perfusion. Phospholipid phosphorus, apolipoproteins C-II, C-III and E were quantitatively recovered in the medium. However, there was a loss of unesterified (17 +/- 6%) and esterified (19 +/- 8%) cholesterol from the perfusion medium. Apolipoprotein B was retained by the heart, as determined by the loss of immunoassayable apolipoprotein B (30 +/- 5%) or the uptake of 125I-labelled apolipoprotein of VLDL (9 +/- 2%) from the perfusion medium. The discrepancy in the two methods for estimating apolipoprotein removal was shown to be due to the modification of apolipoprotein B-containing lipoproteins, which was such that they were no longer precipitated with antibodies to apolipoprotein B. The labelled apolipoprotein B, retained by the heart, could be partially released by perfusion of the heart with buffer containing heparin (14 +/- 2%) or trypsin (50 +/- 2%). Labelled apolipoprotein uptake by the heart was reduced by 90% when lipoprotein lipase was first released by heparin or when VLDL was treated with 1,2-cyclohexanedione to modify arginine residues of apolipoproteins. Very little extensive degradation of the apoprotein to low molecular weight material occurred during the 2 h perfusion, since 95% of the tissue label was precipitated by trichloroacetic acid. It is concluded that there is retention of apolipoprotein B, cholesteryl ester and cholesterol by the perfused heart during catabolism of VLDL. The data are consistent with the concept that the retention of apolipoprotein B requires membrane-bound lipoprotein lipase or an interaction with the cell surfaces that is modified by heparin. The overall process also involves arginine residues of apolipoproteins. At least 50% of the labelled apolipoprotein retained in the tissue is associated with lipoprotein lipase and other cell surface sites, while the remainder may be taken up by the cells.     

Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis.

Triacylglycerols secreted by liver and carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases. These enzymes have been shown to have positional and fatty acid specificity in vitro. If there were specificity in basal lipolysis in vivo, triacylglycerol compositions of circulating and newly secreted VLDL would be different. To study this we compared the composition of normal fasting VLDL triacylglycerol of Wistar rats to that obtained after blocking lipolysis by Triton WR1339, which increased plasma VLDL triacylglycerol concentration about 4.7-fold in 2 h. Analyses of molecular species of sn-1,2- and sn-2,3-diacylglycerol moieties and stereospecific triacylglycerol analysis revealed major differences between the groups in the VLDL triacylglycerol composition. In nontreated rats, the proportion of 16:0 was higher and that of 18:2n-6 lower in the sn-1 position. The proportion of 14:0 was lower in all positions and that of 18:0 was lower in the sn-1 and sn-3 positions in nontreated rats whereas the proportions of 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3 were higher in the sn-1 and lower in the sn-2 position. These results suggest that the fatty acid of the sn-1 position is the most decisive factor in determining the sensitivity for hydrolysis of the triacylglycerol. In addition, triacylglycerol species with highly unsaturated fatty acids in the sn-2 position also favoured hydrolysis. The in vivo substrate specificity followed only partly that obtained in in vitro studies indicating that the nature of molecular association of fatty acids in natural triacylglycerol affects its susceptibility to lipolysis. To conclude, our results indicate that preferential basal lipolysis leads to major structural differences between circulating and newly secreted VLDL triacylglycerol. These differences extend beyond those anticipated from analysis of total fatty acids and constitute a previously unrecognized feature of VLDL triacylglycerol metabolism.     

Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I

We have shown previously that low density lipoprotein (LDL) subjected to vortexing forms self-aggregates that are avidly phagocytosed by macrophages. That phagocytic uptake is mediated by the LDL receptor. We now show that LDL self-aggregation is strongly inhibited (80-95%) by the presence of high density lipoprotein (HDL) or apolipoprotein (apo) A-I. Another type of LDL aggregation, namely that induced by incubation of LDL with phospholipase C, was also markedly inhibited by HDL or apoA-I. The aggregation of LDL induced by vortexing was not inhibited by 2.5 M NaCl, and apoA-I was still able to block LDL aggregation at this high salt concentration, strongly suggesting hydrophobic interactions as the basis for the effect of apoA-I. The fact that apoA-I protected against LDL aggregation induced by two apparently quite different procedures suggests that the aggregation in these two cases has common features. We propose that these forms of LDL aggregation result from the exposure of hydrophobic domains normally masked in LDL and that the LDL-LDL association occurs when these domains interact. ApoA-I, because of its amphipathic character, is able to interact with the exposed hydrophobic domains of LDL and thus block the intermolecular interactions that cause aggregation.     

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.     

Lecithin:cholesterol acyltransferase deficiency increases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockout mice.

The purpose of the present study was to test the hypothesis that lecithin:cholesterol acyltransferase (LCAT) deficiency would accelerate atherosclerosis development in low density lipoprotein (LDL) receptor (LDLr-/-) and apoE (apoE-/-) knockout mice. After 16 weeks of atherogenic diet (0.1% cholesterol, 10% calories from palm oil) consumption, LDLr-/- LCAT-/- double knockout mice, compared with LDLr-/- mice, had similar plasma concentrations of free (FC), esterified (EC), and apoB lipoprotein cholesterol, increased plasma concentrations of phospholipid and triglyceride, decreased HDL cholesterol, and 2-fold more aortic FC (142 +/- 28 versus 61 +/- 20 mg/g protein) and EC (102 +/- 27 versus 61+/- 27 mg/g). ApoE-/- LCAT-/- mice fed the atherogenic diet, compared with apoE-/- mice, had higher concentrations of plasma FC, EC, apoB lipoprotein cholesterol, and phospholipid, and significantly more aortic FC (149 +/- 62 versus 109 +/- 33 mg/g) and EC (101 +/- 23 versus 69 +/- 20 mg/g) than did the apoE-/- mice. LCAT deficiency resulted in a 12-fold increase in the ratio of saturated + monounsaturated to polyunsaturated cholesteryl esters in apoB lipoproteins in LDLr-/- mice and a 3-fold increase in the apoE-/- mice compared with their counterparts with active LCAT. We conclude that LCAT deficiency in LDLr-/- and apoE-/- mice fed an atherogenic diet resulted in increased aortic cholesterol deposition, likely due to a reduction in plasma HDL, an increased saturation of cholesteryl esters in apoB lipoproteins and, in the apoE-/- background, an increased plasma concentration of apoB lipoproteins.     

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.     

Abnormal low density lipoprotein metabolism in apolipoprotein E deficiency

Apolipoprotein(apo) E deficiency is an inherited disease characterized by type III hyperlipoproteinemia and less than 1% normal plasma apoE concentration. The role of apoE in LDL metabolism was investigated by quantitating the metabolism of radiolabeled normal and apoE-deficient LDL in both normal and apoE-deficient subjects. ApoE deficiency resulted in an accumulation of plasma IDL, and a decreased synthesis of LDL consistent with a block in the conversion of IDL to LDL. The LDL isolated from the apoE-deficient patient was similar to normal LDL in hydrated density, size, and composition. However, the apoE-deficient LDL was kinetically abnormal with delayed catabolism in both normal subjects and the apoE-deficient patient. In addition, the catabolism of normal LDL in the apoE-deficient subject was increased. These results were interpreted as indicating that apoE is necessary for the conversion of IDL to LDL and the formation of kinetically normal LDL. The rapid catabolism of normal LDL in the apoE-deficient patient suggests an up-regulation of the hepatic LDL receptor pathway. Based on these results, apoE is proposed to play an important role in the conversion of IDL to LDL, the formation of kinetically normal LDL, and the regulation of LDL receptor function.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.     

Very low density lipoprotein stimulation of triglyceride accumulation in rat preadipocyte cultures.

Exposure of cultured rat epididymal preadipocytes to human very low density lipoproteins (VLDL) resulted in the rapid accumulation of large amounts of cellular triglyceride which was accompanied by the appearance of numerous large cellular lipid inclusions. Addition of heparin produced a two-fold stimulation of lipoprotein induced triglyceride accumulation. Supplementation of the growth medium with either low density lipoprotein, oleic acid or artificial triglyceride emulsion did not produce cellular triglyceride levels equivalent to that obtained with VLDL. Fibroblastic cells from rat skin and lung did not accumulate triglycerides when exposed to VLDL and heparin.     

Phospholipid removal during degradation of rat plasma very low density lipoprotein in vitro.

The hydrolysis of glycerophospholipids in very low density lipoprotein by enzyme(s) released into circulation after the injection of heparin to rats was studied. [32P]Lysolecithin was formed rapidly from [32P]lecithin when very low density lipoprotein, labeled biosynthetically with 32P, was incubated with postheparin plasma. The [32P]lysolecithin was associated with the plasma protein fraction of density greater than 1.21 g/ml, whereas [32P]lecithin exchanged between very low and high density lipoproteins. Inhibition of the plasma lecithin: cholesterol acyl transferase activity did not change the excess [32P]lysolecithin formation in postheparin plasma, and only a negligible amount of radioactivity was associated with blood cells when the incubation was repeated in whole blood. Analysis of the results has demonstrated that phospholipids are removed from VLDL by two pathways: hydrolysis of glycerophospholipids by the heparin-releasable phospholipase activity (greater than50%) and transfer to high density lipoproteins (less than50%). The tissue origin of the postheparin phospholipase was studied in plasma obtained from intact rats and supradiaphragmatic rats using specific inhibitors of the extrahepatic lipase system (protamine sulfate and 0.5 M NaCl). The phospholipase activity could be ascribed to both the hepatic and extrahepatic lipase systems. It is concluded that hydrolysis of glycerophospholipids is the major mechanism responsible for the removal of phospholipids from very low density lipoprotein during the degradation of the lipoprotein. It is suggested that phospholipid hydrolysis occurs concomitantly with triglyceride hydrolysis, predominantly in extrahepatic tissues.     

Very low density and low density lipoprotein subfractions in type III and type IV hyperlipoproteinemia. Chemical and physical properties.

Subfractions of CLDL (VLDL), Sf 100-400; CLDL2, Sf 60--100; VLDL3, Sf 20--60) and LDL (LDL), Sf 12--20; LDL2, Sf 6--12; LDL3, Sf 3--6) were isolated from the plasma of three normal, three type III and four type IV hyperlipoproteinemic subjects. In the type IV group, all VLDL subspecies were of normal composition but were increased in concentration in the order VLDL1 greater than VLDL2 greater than VLDL3. In the same subjects, although LDL2 was lowered and LDL3 increased, the total plasma LDL concentration was normal. All VLDL subfractions were elevated in the type III group, but in this case VLDL3 predominated. These subfractions were enriched in cholesteryl esters and depleted in triglyceride. In the LDL density range there was a shift of mass towards the least dense fraction, LDL1, which was of normal composition. EPR studies of the VLDL and LDL subfractions in a type IV subject demonstrated a decrease in fluidity with increasing density. The major change occurred between VLDL3 and LDL1 and was attributed to a substantial alteration in the cholesteryl ester : triglyceride ratio in the particle. A similar argument was used to explain thction in normal or type IV subjects. Particle diameters, determined by laser light-scattering spectroscopy were in good agreement with the values obtained by electron microscopy. This study provides a baseline for the examination of the relationship between the physical and metabolic properties of VLDL and LDL subfractions in type III and IV hyperlipoproteinemia.     

Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.