Large lipoproteins are excluded from the arterial wall in diabetic cholesterol-fed rabbits

In diabetic hypercholesterolemic rabbits at plasma triglyceride concentrations of approximately 5000 mg/dl, 55% of plasma cholesterol (1400 mg/dl) was in lipoproteins with diameters larger than 75 nm (Sf greater than 400), 40% in smaller very low density and intermediate density lipoproteins, 4% in low density lipoproteins, and 1% in high density lipoproteins. Specific intimal clearance (nl/h.mg aortic cholesterol) of the giant Sf greater than 400 lipoproteins was about 4% of that of the low density lipoproteins. The data suggest that even very low density lipoproteins with diameters smaller than 75 nm were practically excluded from entering the arterial wall. Specific intimal clearance of low density lipoproteins in hypertriglyceridemic, diabetic cholesterol-fed rabbits was similar to that in normal cholesterol-fed rabbits, but low density lipoprotein concentrations in diabetic rabbits were low. Thus, at plasma triglyceride concentrations of approximately 5000 mg/dl, only 5% of plasma cholesterol may be readily available for infiltration of arteries. These results add further support to the hypothesis that hypertriglyceridemic, diabetic cholesterol-fed rabbits are protected against atherogenesis because the major part of plasma cholesterol is carried in large lipoproteins to which the artery is not very permeable.     

Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats.

Changes in whole plasma and lipoprotien apoprotein concentrations were determined after a single injection of Triton WR 1339 into rats. Concentrations of apoproteins A-I (an activator of lecithin:cholesterol acyl transferase), arginine-rich apoprotein (ARP), and B apoprotein were measured by electroimmunoassay. The content of C-II apoprotein (an activaor of lipoprotein lipase) was estimated by the ability of plasma and lipoprotein fractions to promote hydrolysis of triglyceride in the presence of cow's milk lipase and also by isoelectric focusing on polyacrylamide gels. Apoproteins C-II and A-I were rapidly removed from high density lipoprotein (HDL) after Triton treatment and were recovered in the d 1.21 g/ml infranate fraction. A-I was then totally cleared from the plasma within 10--20 hr after injection. Arginine-rich apoprotein was removed from HDL and also partially cleared from the plasma. The rise in very low density lipoprotein (vldl) apoprotein that followed the removal of apoproteins from HDL was mostly antributed to the B apoprotein, although corresponding smaller increases were observed in VLDL ARP and C apoproteins. The triglyceride:cholesterol, triglyceride:protein, and B:C apoprotein ratios of VLDL more closely resembled nascent rather than plasma VLDL 10 hr after Triton injection. These studies suggest that the detergent may achieve its hyperlipidemic effct by disrupting HDL and thus removing the A-I and C-II proteins from a normal activating environment compirsing VLDL, HDL, and the enzymes. The possible involvement of intact HDL in VLDL catabolism is discussed in relation to other recent reports which also suggest that abnormalities of the VLDL-LDL system may be due to the absence of normal HDL.     

Apoprotein E mediates the interaction of beta-VLDL with macrophages

beta-Very low density lipoproteins (beta-VLDL) isolated from cholesterol-fed rhesus monkeys stimulated cholesteryl ester synthesis and accumulation in mouse peritoneal macrophages. The apoprotein specificity and requirement for the cell surface uptake of beta-VLDL was investigated by treating the beta-VLDL with trypsin (beta-VLDL (T], incubating the beta-VLDL (T) with other lipoproteins or apoproteins, reisolating the beta-VLDL (T) and measuring its biological activity which, for this study, is defined as the ability of the lipoprotein to stimulate cholesterol esterification in the macrophages. Trypsin treatment of beta-VLDL abolished its biological activity. Apoprotein analysis of the beta-VLDL (T) demonstrated the absence of intact apoproteins B-100, B-48, and E. The J774 macrophage-like cell line and mouse peritoneal macrophages responded similarly with respect to cholesterol esterification following incubation with inactive and treated beta-VLDL. The J774 macrophage-like cell line was used to establish the conditions necessary for the restoration of biologic activity to the trypsinized beta-VLDL. The loss of biological activity of beta-VLDL (T) could be reversed by restoring apoprotein E-containing LDL from hyperlipemic monkeys or purified apoprotein E. Apoprotein A-I had no such effect. The restored biological activity of the beta-VLDL (T) was proportional to the amount of apoprotein E acquired by the lipoprotein. beta-VLDL particles composed of apoprotein E and either intact or degraded apoprotein B-100 had comparable biological activity. Thus, intact apoprotein E, without intact apoprotein B, is a sufficient mediator for the biological activity and metabolism of beta-VLDL by macrophages and plays a major role in receptor-lipoprotein interaction.     

Intestinal lipoprotein synthesis. Comparison of nascent Golgi lipoproteins from chow-fed and hypercholesterolemic rats

Hypercholesterolemia, induced by a cholesterol-enriched diet, is associated with distinctive modifications in the serum lipoproteins of a variety of species. Present in the serum of these animals are several classes of lipoproteins enriched in cholesteryl esters and apolipoprotein E. To investigate the role of intestinal lipoprotein synthesis in diet-induced hypercholesterolemia, we characterized nascent lipoproteins retrieved from Golgi apparatus-rich fractions of intestinal epithelial cells from chow-fed control and hypercholesterolemic rats. To eliminate chylomicrons from the preparations, rats were fasted overnight prior to the experiments. Golgi very low density lipoproteins (d less than 1.006 g/ml) from control rats were triglyceride-rich lipoproteins that migrated slightly slower than pre-beta migrating serum very low density lipoproteins. These particles contained apoproteins B-240, A-IV, and A-I. Golgi very low density lipoproteins from hypercholesterolemic rats were likewise triglyceride-rich lipoproteins migrating electrophoretically like control Golgi very low density lipoproteins and they contained apoproteins B-240, A-IV, and A-I. However, these latter particles contained less triglyceride and more cholesterol compared to control Golgi very low density lipoproteins. In addition, by radioisotope incorporation studies, Golgi very low density lipoproteins from hypercholesterolemic rats contained relatively more apoprotein A-IV (21.6 vs. 11.0%) and less apoprotein B-240 (17.0 vs. 27.0%) than found in control Golgi very low density lipoproteins. Approximately 60% of the total apoprotein radioactivity was found in apoprotein A-I in both preparations. We conclude that intestinal lipoprotein synthesis is modified by diet-induced hypercholesterolemia. The significance of these modifications with respect to the marked hypercholesterolemia observed in these animals remains to be determined.     

Characterization of plasma lipoproteins of grain- and cholesterol-fed White Carneau and Show Racer pigeons

Plasma cholesterol concentrations from White Carneau (WC) and Show Racer (SR) pigeons consuming a cholesterol-free grain diet averaged about 300 mg/dl, approximately 200 mg/dl as high density lipoproteins (HDL) and the remainder as low density lipoproteins (LDL). Consumption of a cholesterol-containing diet increased plasma cholesterol concentrations in both breeds to greater than 2000 mg/dl. Approximately one-half of this increase was as LDL with the remainder as beta-migrating very low density lipoproteins (beta-VLDL). There was little change in HDL concentration. LDL from cholesterol-fed animals had a greater net negative charge than control LDL, and was larger (Mr = 10 X 10(6) vs 3.2 X 10(60)) due to an increase in the number of cholesteryl ester molecules per particle. The principal apoprotein of LDL was apoB-100 with smaller amounts of apoA-I and several minor unidentified apoproteins. beta-VLDL was cholesteryl ester-rich, could be separated into two size populations by gel chromatography, and contained apoB-100 as its principal apoprotein. Apoprotein E was not detected in any of the plasma lipoproteins. HDL from control and cholesterol-fed animals was composed of a single class of particles with virtually identical composition resembling HDL2. The major apoprotein of HDL was apoA-I. There were no consistent quantitative or qualitative differences in the lipoproteins of the two breeds of pigeons that could help to explain the susceptibility to atherosclerosis of the WC or the resistance of the SR.     

Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat.

The feeding of cholesterol to rats resulted in marked alterations in the type and distribution of the plasma lipoproteins and their apoproteins. The hyperlipoproteinemia was characterized by an increase in the d < 1.006 lipoproteins (B-VLDL and VLDL), an increase in the intermediate and low density lipoproteins (LDL), and the appearance of HDL(c). Associated with these lipoproteins was a prominence of the arginine-rich apoprotein. The high density lipoproteins (HDL) were decreased. A two-dimensional immunoelectrophoretic procedure was adapted to quantitate the changes in distribution of the arginine-rich apoprotein in the plasma and various ultracentrifugal fractions obtained from control and cholesterol-fed rats. In rats fed the cholesterol diet, the total plasma arginine-rich apoprotein increased from a control value of approximately 29 mg/dl to 47 mg/dl. The method of ultracentrifugation, however, was found to markedly alter the quantitative results. When the 60 Ti rotor was used at maximum speed to isolate the ultracentrifugal fractions, less than 50% of the total plasma arginine-rich apoprotein was associated with the lipoproteins in the d < 1.006 or the d 1.006-1.02, 1.02-1.063, or 1.063-1.21 ultracentrifugal fractions. By contrast, after limited ultracentrifugation with the 40 rotor, much less arginine-rich apoprotein was lost, with approximately 20% of the arginine-rich apoprotein in control rats and 10% in cholesterol-fed rats found in the d > 1.21 fraction. Significant alterations in the arginine-rich apoprotein quantitation notwithstanding, the observations of increased arginine-rich apoprotein in the B-VLDL, intermediate fraction, and HDL(c) following cholesterol feeding remained valid. However, precise quantitation awaits refinements in lipoprotein isolation techniques.     

Effects of estrogen administration on the lipoproteins and apoproteins of the chicken.

The plasma lipoproteins of estrogen-treated and untreated sexually immature hens have been compared with respect to their concentration in plasma, protein and lipid composition, particle size, and and apoprotein composition. Administration of diethylstilbestrol resulted in a 400-fold rise in the concentration of very low density lipoprotein (VLDL), a 70-fold rise in low density lipoprotein (LDL), and a marked reduction in high density lipoprotein (HDL) protein. It also resulted in the production of LDL and HDL which were enriched in triacylglycerol, while the proportion of cholesterol in all three lipoprotein fractions decreased. In contrast to the lipoproteins from untreated birds, lipoproteins of density less than 1.06 g/ml from estrogen-treated birds were not clearly separable into discrete VLDL and LDL fractions, but appeared to be a single ultracentrifugal class. The apoprotein composition of VLDL and LDL from untreated birds differed from each other; however, the apoprotein patterns of VLDL and LDL from estrogen-treated birds were indistinguishable: both contained a large amount of low molecular weight protein in addition to the high molecular weight component that predominates in the untreated state. The apoprotein composition of HDL was also markedly altered by estrogen administration: the 28,000 mol. wt. protein (apo A-I) decreased in amount from 65% to less than 5% of the total, while a low molecular weight (Mr = 14,000) protein and as yet poorly defined high molecular weight components became predominant. These observations indicate that the hyperlipidemia induced by estrogen administration is accompanied by marked alterations, both qualitative and quantitative, in the plasma lipoproteins.     

Characterization of the plasma lipoproteins and apoproteins of the Erythrocebus patas monkey.

Patas monkey lipoproteins were fractionated into four distinct classes by a combination of ultracentrifugation and Geon-Pevikon block electrophoresis and characterized with respect to their chemical and physical properties. Very low density lipoproteins (VLDL) were isolated at d is less than 1.006, were triglyceride rich, and were in the size range 300-850 A. They were similar in apoprotein content to the VLDL of man, dog, and swine. The Patas monkey low density lipoprotein referred to as LDL-I had beta mobility and a size which ranged from 190 to 240 A in diameter. Their chemical composition and apoprotein content were similar to those of human LDL. A second low density lipoprotein referred to as LDL-II occurred at a density of 1.05-1.085, ranged in size from 190 to 300 A, and contained the B, arginine-rich, and A-I apoproteins. Differences between LDL-I and LDL-II included a higher sialic acid content for LDL-II and lipid to protein ratios of 3.7 and 3.0 for LDL-I and LDL-II, respectively. In addition, the LDL-II, but not LDL-I, reacted immunochemically with antisera prepared to human Lp(a). The physical, chemical, and immunochemical properties indicated that monkey LDL-II were equivalent to the human Lp(a). Patas monkey HDL, equivalent to human HDL, were protein and phospholipid rich and ranged in size from 70 to 100 A in diameter. The two major HDL apoproteins, A-I and A-II, were isolated from apo-HDL by column chromatography. The amino-terminal sequence of Patas A-I showed striking homology to that reported for human, dog, and swing A-I. The amino acid composition of monkey A-II was very similar to that of human A-II; however, unlike human A-II, the monkey apoprotein was shown to exist as a monomer similar to that reported for Rhesus monkey A-II. The similarities between the plasma lipoproteins of the monkey and of man suggest that the Patas monkey would serve as a suitable model for metabolic studies.     

Metabolism of human plasma triacylglycerol-rich lipoproteins in rodent macrophages: capacity for interaction at beta-VLDL receptor

The capacity of human plasma triacylglycerol-rich lipoproteins to be metabolized by rat macrophages was studied with plasma triacylglycerol-rich lipoproteins obtained from subjects with fasting chylomicronemia or from normal subjects after a fat meal. Triacylglycerol-rich lipoproteins were separated by chromatography into two fractions designated TRL1 and TRL2; from their composition and changing concentration during alimentary lipemia, TRL1 contained a higher proportion of chylomicron remnants than TRL2. Degradation of 125I-labeled TRL1 was greater than that of 125I-labeled TRL2. In competition studies with 125I-labeled beta-VLDL from cholesterol-fed rabbits, unlabeled TRL1 displaced beta-VLDL as completely as did unlabeled beta-VLDL, being slightly more potent than TRL2, which contained less apolipoprotein E than TRL1. This reflected common interaction at receptors that probably included both beta-VLDL and B/E receptors, since: (1) in fresh macrophages, VLDL from hypertriglyceridemic subjects partially displaced beta-VLDL; (2) in B/E receptor-repressed macrophages, TRL1 maintained capacity to totally displace beta-VLDL. This was confirmed in experiments with J774 murine macrophages in which triacylglycerol-rich lipoproteins and beta-VLDL displaced each other equally, whereas LDL was ineffective in displacing beta-VLDL. Furthermore, monoclonal antibodies raised against apolipoprotein B48 and reacting strongly with LDL, failed to inhibit the binding of triacylglycerol-rich lipoprotein to the macrophages. This indicates an interaction through apolipoprotein E which is present in high concentration in triacylglycerol-rich lipoprotein as well as in beta-VLDL. It applies to triacylglycerol-rich particles derived from either the intestine (chylomicron remnants) or the liver (VLDL remnants from hypertriglyceridemic subjects).     

Origin and transport of the A-I and arginine-rich apolipoproteins in mesenteric lymph of rats.

Transport of apolipoprotein A-I and argininerich apolipoprotein in mesenteric lymph was examined in rats given constant intraduodenal infusions of saline, glucose in saline, or emulsified fat. Lymph flow in all groups was constant from 5 to 50 hr after beginning the infusions. Lymphatic transport of triglycerides was about 20-fold greater and transport of apoprotein A-I was about twofold greater in fat-infused rats than in the other two groups. In each group transport of apoprotein A-I bore a significant positive relationship to transport of triglycerides. Lymphatic transport of the arginine-rich apoprotein was only 6-12% of that of apoprotein A-I and was more closely related to lymphatic transport of total protein than to that of triglycerides. In fat-infused rats given [(3)H]lysine intraduodenally, about two-thirds of the (3)H in the chylomicron proteins was in apoprotein A-I and only about 1% was in the arginine-rich apoprotein. Estimated specific activity of chylomicron proteins was highest for apoprotein A-I and apoprotein A-IV, and lowest for the arginine-rich apoprotein and proteins of low molecular weight (mainly C apoproteins). In fat-infused rats given constant intravenous infusions of radioiodinated high density lipoproteins from blood plasma, the specific activity of apoprotein A-I in lymph chylomicrons was only about 5% of that of apoprotein A-I in blood high density lipoproteins, indicating that more than 90% of the apoprotein A-I in chylomicrons was synthesized in the intestine. From these and other data it is concluded that both the intestine and liver are significant sources of apoprotein A-I whereas only the liver synthesizes significant amounts of the arginine-rich apoprotein.     

Impaired triacylglycerol catabolism in hypertriglyceridemia of the diabetic, cholesterol-fed rabbit: a possible mechanism for protection from atherosclerosis

The etiology of the hypertriglyceridemia in alloxan-diabetic rabbits was studied by two independent methods. Production and removal rates of VLDL triacylglycerol were measured in diabetic rabbits by injection of [3H]palmitate-labelled VLDL. Similarly, triacylglycerol total removal rates were determined in non-diabetic rabbits which were infused with Intralipid to mimic the plasma triacylglycerol concentrations of diabetic rabbits. Compared to nondiabetic rabbits, triacylglycerol removal rats were decreased in diabetic rabbits, particularly at higher levels of plasma triacylglycerol. During cholesterol and triacylglycerol supplementation of the diet, post-heparin plasma lipoprotein lipase activity of diabetic rabbits with severe hypertriglyceridemia averaged 36% of that of nondiabetics, suggesting an impaired triacylglycerol removal capacity. Furthermore, plasma triacylglycerol was inversely related to post-heparin plasma lipoprotein lipase activity among diabetic rabbits. VLDL triacylglycerol production increased with increasing plasma triacylglycerol concentration among diabetic cholesterol-fed rabbits with moderately severe hypertriglyceridemia, but reached an apparent plateau among rabbits with plasma triacylglycerol concentrations from approx. 2000-9000 mg/dl. Thus, severe hypertriglyceridemia in this model of insulin deficiency can be attributed only partially to VLDL hypersecretion, whereas a removal defect, resulting in saturation of the triacylglycerol removal mechanism, appears to be largely responsible. The impaired removal of plasma triacylglycerol is also related to the presence of cholesterol predominantly in lipoproteins of increased size. The data support the hypothesis that protection against atherosclerosis in cholesterol-fed diabetic rabbits results from exclusion of very large cholesterol-containing lipoproteins from the arterial wall.     

The beta-VLDL receptor pathway of murine P388D1 macrophages

Very low density lipoproteins Sf 100-400 (VLDL1) from hypertriglyceridemic (HTG) subjects and chylomicrons cause receptor-mediated lipid engorgement in unstimulated macrophages in vitro via the beta-VLDL receptor pathway. We now report that the murine macrophage P388D1 cell line possesses the characteristics of the beta-VLDL receptor pathway observed previously in freshly isolated resident murine peritoneal macrophages or human monocyte-macrophages. HTG-VLDL1 isolated from the plasma of subjects with hypertriglyceridemia types 3, 4, and 5 interact with P388D1 macrophages in a high-affinity, curvilinear manner. beta-VLDL, HTG-VLDL1, chylomicrons, and thrombin-treated HTG-VLDL1 (which do not bind to the LDL receptor) compete efficiently and similarly for the uptake and degradation of HTG-VLDL1. LDL and acetyl LDL do not compete, indicating that uptake of HTG-VLDL1 is via neither the LDL receptor nor the acetyl LDL receptor. Binding of thrombin-treated HTG-VLDL1 to the beta-VLDL receptor indicates that the thrombin-accessible apoE, which is absolutely required for interaction of HTG-VLDL Sf greater than 60 with the LDL receptor, is not required for binding to the beta-VLDL receptor. The uptake and degradation of 125I-labeled HTG-VLDL1 is suppressed up to 80-90% by preincubation of the cells with sterols, acetyl LDL, or beta-VLDL, indicating that this process is not via the irrepressible chylomicron remnant (apoE) receptor. Chylomicrons, HTG-VLDL1, and thrombin-treated HTG-VLDL1-but not normal VLDL1, beta-VLDL, LDL, or acetyl LDL-produce massive triglyceride accumulation (10-20-fold mass increases in 4 hr) in P388D1 macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of rat plasma very low density lipoprotein with lipoprotein lipase-rich (postheparin) plasma.

Incubation of 125I-labeled very low density lipoprotein (VLDL) with lipoprotein lipase-rich (postheparin) plasma obtained from intact or supradiaphragmatic rats resulted in the transfer of more than 80% of apoprotein C from VLDL to high density lipoprotein (HDL), whereas apoprotein B was associated with lipoprotein of density less than 1.019 g/ml (intermediate lipoprotein). The transfer of 125I-labeled apoprotein C from VLDL to HDL increased with time and decreased in proportion to the amount of VLDL in the incubation system. A relationship was established between the content of triglycerides and apoprotein C in VLDL, whereas the amount of apoprotein C in VLDL was independent of that of other apoproteins, especially apoprotein B. The injection of heparin to rats preinjected with 125I-labeled VLDL caused apoprotein interconversions similar to those observed in vitro. The intermediate lipoprotein was relatively rich in apoprotein B, apoprotein VS-2, cholesterol, and phospholipids and poor in triglycerides and apoprotein C. The mean diameter of intermediate lipoprotein was 269 A (compared with 427 A, the mean Sf rate was 30.5 (compared with 115), and the mean weight was 7.0 X 10(6) daltons (compared with 23.1 X 10(6)). From these data it was possible to calculate the mass of lipids and apoproteins in single lipoprotein particles. The content of apoprotein B in both particles was virtually identical, 0.7 X 10(6) daltons. The relative amount of all other constituents in intermediate lipoprotein was lower than in VLDL: triglycerides, 22%; free cholesterol, 37%; esterified cholesterol, 68%; phospholipids, 41%; apoprotein C, 7%, and VS-2 apoprotein, 60%. The data indicate that (a) one and only one intermediate lipoprotein is formed from each VLDL particle, and (b) during the formation of the intermediate lipoprotein all lipid and apoprotein components other than apoprotein B leave the density range of VLDL to a varying degree. Whether these same changes occur during the clearance of VLDL in vivo is yet to be established.     

Blockade of intestinal lipoprotein clearance in rabbits injected with Triton WR 1339-ethyl oleate

Although Triton WR 1339 has been used to block triglyceride or cholesterol removal from plasma, no data are available on the extent to which Triton WR 1339 administered to rabbits blocks clearance of newly absorbed dietary lipids. In the present study, we have measured the efficiency of this blockade during a 24-hr interval. After the Triton WR 1339 administration, plasma Sf greater than 400 and d less than 1.019 g/ml lipoprotein lipid concentrations increased greatly, but the concentration of d greater than 1.019 g/ml lipids decreased. In the rabbits fed 0.5% cholesterol for 1 week, the increase in d less than 1.019 g/ml and the decrease in 1.019 less than d less than 1.063 g/ml lipoprotein fractions 24 hr after the Triton WR 1339 injection were much greater than in the chow-fed Tritonized rabbits. After the Triton treatment, 50% of intravenously injected LDL-125I-labeled apoB disappeared in 24 hr, but little or no apoB appeared in other lipoprotein fractions and no VLDL apoB was converted to LDL. Labeled cholesterol and retinol were fed to rabbits and 24-hr increments in plasma cholesteryl- and retinyl-ester label and mass were measured. In chow-fed Tritonized rabbits about one-half of the absorbed oral doses of both labeled lipids was recovered in plasma, indicating that Triton WR 1339 does not completely inhibit the clearance of intestinal lipoproteins. When rabbits were injected with Triton and an ethyl oleate emulsion, the blockade of dietary lipid removal from plasma was substantially improved and chylomicron cholesterol uptake by extra-hepatic tissues was completely abolished.     

A rapid electrophoretic technique for identification of subunit species of apoproteins in serum lipoproteins

The denaturing solvent tetramethylurea (TMU) delipidates and quantitatively liberates the apoproteins of human serum high-density lipoprotein (HDL) in soluble form while virtually the whole apoprotein of human lowdensity lipoprotein (LDL) is precipitated. A fraction of the apoprotein of very low density lipoprotein (VLDL) which appears to represent its content of LDL-like protein (apo B) is precipitated by this reagent, while the remaining apoprotein species are liberated in soluble form.The dissociation of the soluble apoproteins from lipid by TMU obviates the need for time-consuming delipidation by organic solvents, permitting immediate electrophoretic analysis in polyacrylamide gels. Bands are observed with mobilities corresponding to those of all the major soluble polypeptide species isolated from serum lipoproteins by ion-exchange chromatography. The apparent distribution of these elements in the different classes of lipoproteins is in agreement with findings of studies employing chromatographic methods. The predominant apoprotein of HDL, which has been identified immunochemically in VLDL, appears to comprise less than 1% of the apoprotein of VLDL from normal serum.     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

Interaction between hepatic microsomal membrane lipids and apolipoprotein A-I

Incubation of apoprotein A-I (apo-A-I), the major protein component of human high density lipoprotein, with rat liver microsomal membranes under conditions of elevated pH and ionic strength leads to the production of a soluble protein:lipid complex (A-I/MM complex). The A-I/MM complex, as purified by density gradient centrifugation and agarose column chromatography, possesses a lipid composition similar to the hepatic microsomal membrane and a protein/lipid ratio similar to that of plasma high density lipoproteins, but markedly different from that of recombinant particles prepared with synthetic lipids. The A-I/MM complex constitutes a more physiological recombinant particle than can be formed using synthetic lipids and may be a suitable model for the newly assembled intracellular high density lipoproteins. Incubation of the erythrocyte plasma membranes with apo-A-I under the same conditions as used with microsomal membranes fails to generate any lipid:apoprotein complexes. This membrane specificity for forming soluble lipoprotein complexes suggests that the microsomal membranes possess a unique feature, possibly their lipid composition, which render them particularly suitable to serve as lipid donors to the apoproteins which are undergoing assembly within the endoplasmic reticulum/Golgi organelles.     

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Estradiol-induced alteration of very-low-density lipoprotein assembly. Possible competition among apoproteins for incorporation into nascent very-low-density lipoprotein

The hypothesis that the apoprotein composition of nascent very-low-density lipoprotein (VLDL) secreted by the hepatocyte is determined by the relative rates of apoprotein synthesis and their affinities of binding to VLDL was tested using chick hepatocytes in monolayer culture. Chick cells were chosen for the study of lipoprotein assembly since estradiol treatment can be used to alter the composition of the apoprotein mixture synthesized by these cells. The secretion of apoprotein (apo) B by estradiol-treated hepatocytes was elevated 4.2-fold above the basal level measured in control cells. Furthermore, estradiol-treated cells secreted apo-II, a major VLDL apoprotein not synthesized prior to estradiol treatment, at a level equivalent to that of apo-B. However, no difference in the secretion of apo-A-I and other newly identified nascent VLDL apoproteins was detected. These changes in relative rates of apoprotein synthesis altered the composition of nascent VLDL secreted by control versus estradiol-induced cells from: apo-B, 22 to 40%; apo-II, 0 to 32%; apo-37 kDa, 14 to 6%; apo-A-I, 31 to 12%; apo-17 kDa, 10 to 4%; apo-9 kDa, 15 to less than 10%; and apo-6 kDa, 8 to less than 2%. To investigate the basis for the preferential incorporation of apo-B and apo-II into nascent VLDL, the relative affinities of the apoproteins for VLDL were compared by measuring their capacities to transfer from VLDL into other lipoprotein or nonlipoprotein density classes. Culture medium containing [3H]leucine-labeled VLDL was incubated with plasma deficient in lipoproteins of rho less than 1.006 g/ml. Within 30 min of incubation at 37 degrees C, 3H-labeled apo-A-I and apo-9 kDa exchanged between VLDL and high-density lipoprotein, whereas apo-37 kDa exchanged between VLDL and the rho greater than 1.21 g/ml fraction. Neither apo-B nor apo-II underwent transfer from nascent VLDL. These results suggest that the relative rates of input of apoproteins into the secretory pathway and their affinities of binding to the nascent VLDL particle determine their extent of incorporation into, and, thus, the apoprotein composition of secreted VLDL.     

Characterization of the plasma lipoproteins of the genetically obese hyperlipoproteinemic Zucker fatty rat

The plasma lipoproteins of the Zucker fatty rat were characterized with respect to lipid and apoprotein composition, and results were compared with those obtained from lean controls. Information on apoproteins was obtained from gel filtration experiments and electrophoresis on polyacrylamide gels. Very low density lipoproteins (VLDL) were increased several-fold in fatties, and 78% of their mass was triglycerides compared with 60% in the controls. Low density (LDL) and high density (HDL) lipoproteins were increased by a factor of 2, although their compositions were similar to those of the controls. Levels of apoVLDL, apoLDL, and apoHDL were five, two and two times higher, respectively, in the fatties, and the two most rapidly moving subunit peptides on polyacrylamide gels were disproportionately elevated in the apoproteins. The slower of these two bands was present in relatively greater amounts than the faster one in fatties. If the slower peptide is an activator of lipoprotein lipase, analogous to the comparable subunit peptides of human apolipoproteins, plasmas of fatties could contain up to 10 times more lipase activator activity than control plasma. This finding, and the fact that adipose tissue lipoprotein lipase activity of fatties was about 150% of controls, suggests that fatties have increased capacities for VLDL catabolism. We have previously shown that hepatic VLDL secretory rates are higher than normal in these animals. The increased capacity for catabolism may be a response to the altered secretory rates.