Evidence for non-equilibrating pools of apolipoprotein C-III in plasma lipoproteins

Using immunoaffinity chromatography to isolate apoC-III from radiolabeled lipoproteins for direct determination of specific radioactivity, we have studied the metabolism of human apoC-III in VLDL and in HDL following the bolus injection of 125I-labeled VLDL. Transfer of apoC-III radioactivity from VLDL to HDL was detected in the plasma sample drawn 5 min after injection of the tracer. However, the specific radioactivity of apoC-III in VLDL was found to be higher than that in HDL, with this difference being maintained throughout the sampling period (48-72 hr). The ratios of the respective specific activities ranged from 1.2 to 1.9 in six subjects studied (two normolipidemics and four hypertriglyceridemics). When 125I-labeled HDL was injected as the tracer, however, the higher apoC-III specific radioactivity was associated with the HDL fraction. This lack of complete equilibration of apoC-III between VLDL and HDL in vivo was further characterized by in vitro studies using either 125I-labeled VLDL or 125I-labeled HDL. All incubations were carried out for 3 hr at 37 degrees C followed by 16 hr at 4 degrees C and the apoC-III specific activity in each lipoprotein fraction was directly determined after immunoaffinity chromatography. In a study of plasma from a mildly hypertriglyceridemic subject in which 125I-labeled VLDL was incubated with unlabeled HDL, apoC-III specific activities in VLDL remained 30% greater than that in HDL. When 125I-labeled HDL (from the same subject) was incubated with unlabeled VLDL of apoC-III, final specific activity in VLDL was less than 10% of that of HDL apoC-III. Differences in specific activities were also demonstrated when radiolabeled purified apoC-III was exchanged onto VLDL prior to its incubation with HDL. A consistent difference in apoC-III specific activities in VLDL and HDL was observed after isolation of the particles either by molecular sieve chromatography or by ultracentrifugation. These studies demonstrated that, while the exchange of apoC-III between VLDL and HDL may be very rapid, this equilibration is not complete. Pools of apoC-III that do not participate in the equilibration process are present in both the VLDL and HDL fractions and could account for 30-60% of the total apoC-III mass in each lipoprotein fraction.     

Metabolic fate of rat and human lipoprotein apoproteins in the rat

The fate of (125)I-labeled apolipoproteins was studied in vivo in rats that had received intravenous injections of (125)I-labeled rat HDL and (125)I-labeled human HDL, LDL, and VLDL. Plasma decay curves of rat and human HDL were exponential with similar half-lives in the circulation (11-12 hr). After injection, low molecular weight apolipoproteins (apoLP-alanine of human HDL and fraction HS-3 of rat HDL) were found to redistribute to other lipoproteins, predominantly VLDL. Decay curves of individual HDL proteins were constructed after lipoprotein fractionation, delipidation, and polyacrylamide gel electrophoresis. It was found that the half-lives of the different HDL apoproteins were not identical. A major rat HDL protein (52% of total counts) had a circulating half-life (t((1/2))) of 12.5 hr. Two others had a t((1/2)) of 8-9 hr while the t((1/2)) of several others was 11-12 hr. The t((1/2)) of three well-characterized human HDL apoproteins, apoLP-glutamine I, apoLP-glutamine II, and apoLP-alanine, were 13.5, 9.0, and 15.0 hr, respectively. The fate of (125)I-labeled human VLDL and LDL apoproteins in rats was similar to that described previously in humans. After injection of (125)I-labeled human VLDL into rats, apoLP-glutamic acid and apoLP-alanine rapidly transferred to rat HDL and were lost thereafter from the circulation from both VLDL and HDL. The apoLDL moiety of human VLDL moved metabolically to the LDL density range (d = 1.019-1.063) through a lipoprotein of intermediate density (d = 1.006-1.019).     

Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

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.     

Use of Intralipid for kinetic analysis of HDL apoC-III: evidence for a homogeneous kinetic pool of apoC-III in plasma

Apolipoprotein C-III (apoC-III) is an important regulator of lipoprotein metabolism. Radioisotope and stable isotope kinetic studies show differing results in relation to the kinetics of apoC-III in HDL. Kinetic analysis of HDL apoC-III may be difficult because of its low concentration, as well as the presence of other apoproteins at higher concentration, in the HDL fraction. We used Intralipid(R) (IL), known to preferentially extract apoC proteins from plasma, as a means of extracting apoC-III from HDL before apoprotein separation by isoelectric focusing gel electrophoresis for the measurement of tracer enrichment. Protein purity was assessed by an isoleucine-to-leucine (Ile/Leu) ratio, as apoC-III contains no isoleucine. We compared apoC-III kinetics in 14 men using a bolus infusion of deuterated leucine. The Ile/Leu ratio for IL-extracted HDL (IL-HDL) apoC-III (3.0 +/- 0.7%) was not different from that of VLDL apoC-III (2.6 +/- 0.6%) but was significantly lower than that of untreated HDL apoC-III (9.0 +/- 2.9%) (P < 0.001). The isotopic enrichment curves and fractional catabolic rates (FCRs) for IL-HDL apoC-III were not different from those of VLDL apoC-III. In contrast, HDL apoC-III had significantly lower isotopic enrichments and FCRs than IL-HDL apoC-III (P < 0.001). In conclusion, this simple IL method can be used to isolate apoC-III from HDL with minimal interference from other HDL apoproteins, and it demonstrates that the kinetics of apoC-III in VLDL and HDL are similar, supporting the concept of a single kinetically homogeneous pool of apoC-III in plasma.     

Resistance of a very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction

In vitro lipolysis of very low density lipoprotein (VLDL) from normolipidemic and familial dysbetalipoproteinemic plasma by purified bovine milk lipoprotein lipase was studied using the combined single vertical spin and vertical autoprofile method of lipoprotein analysis. Lipolysis of normolipidemic plasma supplemented with autologous VLDL resulted in the progressive transformation of VLDL to low density lipoprotein (LDL) via intermediate density lipoprotein (IDL) with the transfer of the excess cholesterol to high density lipoprotein (HDL). At the end of 60 min lipolysis, 92-96% of VLDL triglyceride was hydrolyzed, and, with this process, greater than 95% of the VLDL cholesterol and 125-I-labeled VLDL protein was transferred from the VLDL to the LDL and HDL density region. When VLDL from the plasma of an individual with familial dysbetalipoproteinemia was substituted for VLDL from normolipidemic plasma, less than 50% of the VLDL cholesterol and 65% of 125I-labeled protein was removed from the VLDL density region, although 84-86% of VLDL triglyceride was lipolyzed. Analysis of familial dysbetalipoproteinemic VLDL fractions from pre- and post-lipolyzed plasma showed that the VLDL remaining in the postlipolyzed plasma (lipoprotein lipase-resistant VLDL) was richer in cholesteryl ester and tetramethylurea-insoluble proteins than that from prelipolysis plasma; the major apolipoproteins in the lipoprotein lipase-resistant VLDL were apoB and apoE. During lipolysis of normolipidemic VLDL containing trace amounts of 125I-labeled familial dysbetalipoproteinemic VLDL, removal of VLDL cholesterol was nearly complete from the VLDL density region, while removal of 125I-labeled protein was only partial. A competition study for lipoprotein lipase, comparing normolipidemic and familial dysbetalipoproteinemic VLDL to an artificial substrate ([3H]triolein), revealed that normolipidemic VLDL is clearly better than familial dysbetalipoproteinemic VLDL in competing for the release of 3H-labeled free fatty acids. The results of this study suggest that, in familial dysbetalipoproteinemic individuals, a subpopulation of VLDL rich in cholesteryl ester, apoB, and apoE is resistant to in vitro conversion by lipoprotein lipase to particles having LDL-like density. The presence of this lipoprotein lipase-resistant VLDL in familial dysbetalipoproteinemic subjects likely contributes to the increased level of cholesteryl ester-rich VLDL and IDL in the plasma of these subjects.     

Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

Apolipoprotein C-III displacement of apolipoprotein E from VLDL: effect of particle size.

ApoC-III and apoE are important determinants of intravascular lipolysis and clearance of triglyceride-rich chylomicrons and VLDL from the blood plasma. Interactions of these two apolipoproteins were studied by adding purified human apoC-III to human plasma at levels observed in hypertriglyceridemic subjects and incubating under specific conditions (2 h, 37 degrees C). As plasma concentrations of apoC-III protein were increased, the contents in both VLDL and HDL were also increased. Addition of apoC-III at concentrations up to four times the intrinsic concentration resulted in the decreasing incremental binding of apoC-III to VLDL while HDL bound increasing amounts without evidence of saturation. No changes were found in lipid content or in particle size of any lipoprotein in these experiments. However, distribution of the intrinsic apoE in different lipoprotein particles changed markedly with displacement of apoE from VLDL to HDL. The fraction of VLDL apoE that was displaced from VLDL to HDL at these high apoC-III concentrations varied among individuals from 20% to 100% its intrinsic level. The proportion of VLDL apoE that was tightly bound (0% to 80%) was found to be reproducible and to correlate with several indices of VLDL particle size. In the group of subjects studied, strongly adherent apoE was essentially absent from VLDL particles having an average content of less than 50,000 molecules of triglyceride.Addition of apoC-III to plasma almost completely displaces apoE from small VLDL particles. Larger VLDL contain tightly bound apoE which are not displaced by increasing concentration of apoC-III.     

Divergent effects of d-norgestrel on the metabolism of rat very low density and low density apolipoprotein B

Rats treated with the contraceptive steroid d-norgestrel have lower plasma very low density lipoprotein (VLDL)-triglycerides and higher low density lipoprotein (LDL)-cholesterol than controls. To explain these results, the kinetics of VLDL and LDL turnover were studied by injecting 125I-labeled rat-VLDL and 131I-labeled rat-LDL simultaneously into rats treated with a small dose of d-norgestrel (4 micrograms per day per kg body weight0.75 for 18 days, n = 22) and their untreated controls (n = 22). VLDL- and LDL-apoB specific activity-time curves obtained over 50 hr best conformed to a three-pool model. VLDL-apoB clearance expressed as irreversible catabolic rate (k01) was markedly enhanced in the treated versus control rats (0.57 vs. 0.34 pools hr-1), leading to a marked reduction in VLDL-apoB pool size (270 vs. 420 micrograms). However, VLDL-apoB production rates were similar in the two groups (160 vs. 140 micrograms/hr, respectively). The 125I-labeled apoB specific activity-time curve derived from the catabolism of 125I-labeled VLDL-apoB also showed enhanced clearance in d-norgestrel-treated rats. 125I-Labeled IDL-apoB and 125I-labeled LDL-apoB specific activity-time curves failed to intersect the VLDL-apoB curve at maximal heights, suggesting input of intermediate density lipoprotein (IDL) and LDL independent of VLDL catabolism in both groups. However, the extent of independent LDL-apoB production was similar in both groups. Clearance of 131I-labeled LDL-apoB following injection of 131I-labeled rat-LDL was delayed in the d-norgestrel-treated versus control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein (a) is not a metabolic product of other lipoproteins containing apolipoprotein B.

125I-Labeled autologous very low density lipoprotein (VLDL) was injected intravenously into three lipoprotein (a) positive individuals. One other lipoprotein (a) positive subject received 125I-labeled VLDL from a a lipoprotein (a) negative donor. Specific activity of apolipoprotein B in VLDL, low density lipoprotein (LDL) and lipoprotein (a) was measured for 5 days. In the lipoprotein (a) fraction only traces of radioactivity could be detected, which were caused by contamination with labeled LDL. No precursor-product relationship existed between apolipoprotein B in VLDL or LDL and apolipoprotein B in lipoprotein (a). One lipoprotein (a)-positive individual was kept on a fat-free diet for 4 days to prevent chylomicron formation; no change in the serum level of lipoprotein (a) could be detected under these conditions. The data of this study indicate that lipoprotein (a) is not a metabolic product of VLDL or LDL. Also chylomicrons are not likely to play role as a precursor for lipoprotein (a). It is concluded that lipoprotein (a) is synthesized as a separate lipoprotein.     

Studies on the metabolism of rat serum very low density apolipoprotein.

There was a rapid transfer of radioactive peptides to other lipoprotein fractions during the first 30 min after the intravenous injection of 125I-labeled rat very low density lipoprotein (VLDL) into rats. After this initial redistribution of radioactivity, label disappeared slowly from all lipoprotein fractions. The disappearance of 125I-labeled human VLDL injected into rats was the same as that of rat VLDL. Most of the radioactivity transferred from VLDL to low density (LDL) and high density (HDL) lipoproteins was associated with two peptides, identified in these studies by polyacrylamide gel electrophoresis as zone IVa and IVb peptides (fast-migrating peptides, possibly analogous to some human C apolipoproteins), although radioactivity initially associated with zone I (analogous to human apolipoprotein B) and zone III (not characterized) was also transferred to LDL and HDL. That the transfer of label from VLDL to LDL and HDL primarily involved small molecular weight peptides was confirmed in studies using VLDL predominantly labeled in these peptides by in vitro transfer from 125I-labeled HDL. Both zone I and zone IV radioactivity was rapidly removed from VLDL during the first 5 min after injection. However, although most of the zone IV radioactivity was recovered in LDL and HDL, only 12% of the label lost from zone I of VLDL was recovered in other lipoproteins, with the remainder presumably having been cleared from the plasma compartment. We have concluded that, during catabolism of rat VLDL apoprotein, there is a rapid transfer of small molecular weight peptides to both LDL and HDL. During the catabolic process, most of the VLDL is rapidly removed from the circulation, with only a small portion being transformed into LDL molecules.     

Differential binding of triglyceride-rich lipoproteins to lipoprotein lipase.

In comparison to very low density lipoprotein (VLDL), chylomicrons are cleared quickly from plasma. However, small changes in fasting plasma VLDL concentration substantially delay postprandial chylomicron triglyceride clearance. We hypothesized that differential binding to lipoprotein lipase (LPL), the first step in the lipolytic pathway, might explain these otherwise paradoxical relationships. Competition binding assays of different lipoproteins were performed in a solid phase assay with purified bovine LPL at 4 degrees C. The results showed that chylomicrons, VLDL, and low density lipoprotein (LDL) were able to inhibit specific binding of (125)I-labeled VLDL to the same extent (85.1% +/- 13.1, 100% +/- 6.8, 90.7% +/- 23.2% inhibition, P = NS), but with markedly different efficiencies. The rank order of inhibition (K(i)) was chylomicrons (0.27 +/- 0.02 nm apoB) > VLDL (12.6 +/- 3.11 nm apoB) > LDL (34.8 +/- 11.1 nm apoB). By contrast, neither triglyceride (TG) liposomes, high density lipoprotein (HDL), nor LDL from patients with familial hypercholesterolemia were efficient at displacing the specific binding of (125)I-labeled VLDL to LPL (30%, 39%, and no displacement, respectively). Importantly, smaller hydrolyzed chylomicrons had less affinity than the larger chylomicrons (K(i) = 2.34 +/- 0.85 nm vs. 0.27 +/- 0.02 nm apoB respectively, P < 0.01). This was also true for hydrolyzed VLDL, although to a lesser extent. Chylomicrons from patients with LPL deficiency and VLDL from hypertriglyceridemic subjects were also studied. Taken together, our results indicate an inverse linear relationship between chylomicron size and K(i) whereas none was present for VLDL. We hypothesize that the differences in binding affinity demonstrated in vitro when considered with the differences in particle number observed in vivo may largely explain the paradoxes we set out to study.     

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.     

Apolipoproteins C-I, C-II, and C-III: kinetics of association with model membranes and intermembrane transfer

The apoproteins (apo) C-I, C-II, and C-III are low molecular weight amphiphilic proteins that are associated with the lipid surface of the plasma chylomicron, very low density lipoprotein (VLDL), and high-density lipoprotein (HDL) subfractions. Purified apoC-I spontaneously reassociates with VLDL, HDL, and single-bilayer vesicles (SBV) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. ApoC-I also transfers reversibly from VLDL to HDL and from VLDL and HDL to SBV. The kinetics of association of the individual apoC proteins with SBV are second order overall and first order with respect to lipid and protein concentrations. At 37 degrees C, the rates of association were 2.5 x 10(10), 4.0 x 10(10) and 3.8 x 10(10) M-1 s-1 for apoC-I, apoC-II, and apoC-III, respectively. Arrhenius plots of association rate vs temperature were linear and yielded activation energies of 11.0 (apoC-I), 9.0 (apoC-II), and 10.6 kcal/mol (apoC-III). The kinetics of vesicle to vesicle apoprotein transfer are biexponential for intermembrane transfer, indicating two concurrent transfer processes. Rate constants at 37 degrees C for the fast component of dissociation were 11.7, 9.5, and 9.9 s-1, while rate constants for the slow component were 1.3, 0.6, and 0.9 s-1 for apoC-I, apoC-II, and apoC-III, respectively. The dissociation constants, Kd, of apoC-I, apoC-II, and apoC-III bound to the surface monolayer of phospholipid-coated latex beads were 0.5, 1.4, and 0.5 microM, respectively. These studies show that the apoC proteins are in dynamic equilibrium among phospholipid surfaces on a time scale that is rapid compared to lipolysis, lipid transfer, and lipoprotein turnover.     

Mevinolin and cholestyramine inhibit the direct synthesis of low density lipoprotein apolipoprotein B in miniature pigs

Previous studies established that following simultaneous injection of 125I-labeled homologous very low density lipoproteins (VLDL) and 131I-labeled homologous low density lipoproteins (LDL) into miniature pigs, a large proportion of LDL apolipoprotein B (apoB) was synthesized directly, independent of VLDL or intermediate density lipoprotein (IDL) apoB catabolism. The possibility that cholestyramine alone (a bile acid sequestrant) or in combination with mevinolin (a cholesterol synthesis inhibitor) could regulate the direct LDL apoB synthetic pathway was investigated. 125I-labeled VLDL and 131I-labeled LDL were injected into miniature pigs (n = 8) during a control period and following 18 days of cholestyramine treatment (1.0 g kg-1d-1) or following 18 days of treatment with cholestyramine and mevinolin (1.2 mg kg-1d-1). ApoB in each lipoprotein fraction was selectively precipitated using isopropanol in order to calculate specific activity. In control experiments, LDL apoB specific activity curves reached their peak values well before crossing the VLDL or IDL apoB curves. However, cholestyramine treatment resulted in LDL apoB curves reaching maximal values much closer to the point of intersection with the VLDL or IDL curves. Kinetic analyses demonstrated that cholestyramine reduced total LDL apoB flux by 33%, which was due entirely to inhibition of the LDL apoB direct synthesis pathway since VLDL-derived apoB was unaffected. In addition, the LDL apoB pool size was reduced by 30% and the fractional catabolic rate of LDL apoB was increased by 16% with cholestyramine treatment. The combination of mevinolin and cholestyramine resulted in an even more marked inhibition of the direct LDL apoB synthesis pathway (by 90%), and in two animals this pathway was completely abolished. This inhibition was selective as VLDL-derived LDL apoB synthesis was not significantly different. LDL apoB pool size was reduced by 60% due primarily to the reduced synthesis as well as a 40% greater fractional removal rate. These results are consistent with the idea that cholestyramine and mevinolin increase LDL catabolism by inducing hepatic apoB, E receptors. We have now shown that the direct synthesis of LDL apoB is selectively inhibited by these two drugs.     

Expression of a monoclonal antibody-defined aminoterminal epitope of human apoC-I on native and reconstituted lipoproteins

Nine distinct mouse monoclonal antibodies were produced in two fusions using holo-human very low density lipoprotein (VLDL) as antigen. On immunoblotting first with human VLDL and then with isolated human apoC-I, seven of the antibodies, representing three isotypes, manifested specificity for apoC-I. Two antibodies were directed against apoB. To assess whether the seven anti-apoC-I antibodies were directed against the same or distinctively different epitopes, cross-competition assays were performed wherein 125I-labeled monoclonal antibodies were made to compete with unlabeled antibodies for occupancy on immobilized VLDL-associated apoC-I. All antibodies cross-competed to varying extents implying that they were directed against closely spaced epitopes, but based on these experiments three different epitopes were defined. On immunoblotting with CNBr fragments, all of the epitopes were assigned to the CNBr I fragment of human apoC-I (amino acids 1-38) suggesting that the NH2-terminal region of apoC-I is more immunogenic in mice than other parts of the molecule when apoC-I is associated with VLDL. A competitive solid-phase radioimmunoassay (RIA) was developed employing one of the anti-apoC-I antibodies (A3-4). VLDL was adsorbed to plastic microtiter wells, and a limiting amount of the antibody was reacted with the adsorbed VLDL. The amount of monoclonal antibody that bound to the immobilized VLDL-apoC-I was determined with a 125I-labeled goat anti-mouse IgG antibody. The addition of competitor apoC-I complexed with lipids resulted in reduced binding of the anti-apoC-I antibody to the immobilized VLDL-apoC-I. Competitor complexes consisted of an artificial lipid emulsion (Intralipid) incubated with apoC-I at phospholipid/apoC-I ratios of 1:1 to 60:1 (w/w). As the lipid/protein ratios were increased, the competitive displacement curves produced by the complexes become progressively steeper, while isolated lipid-free apoC-I produced curves with very shallow slopes, suggesting that a conformation-dependent epitope was being probed. Other apoproteins (C-II, C-III, A-I, A-II, and E) whether lipid-free or complexed with lipids did not compete. Fractionation of the 30:1 apoC-I-Intralipid complex by gel permeation chromatography suggested that apoC-I bound to phospholipids was the most effective competitor. This was confirmed by testing of apoC-I-DMPC complexes, which yielded curves that paralleled those produced by apoC-I-Intralipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.

Apoproteins of chylomicrons, very low density lipoprotein (VLDL), and a low density + high density fraction secreted by proximal and distal rat small intestine into mesenteric lymph were examined during triglyceride (TG) absorption. Apoprotein output and composition were determined and the turnover rates of labeled non-apoB (soluble) apoproteins in lipoprotein fractions were measured after an intraluminal [(3)H]leucine pulse during stable TG transport into lymph. The output of VLDL apoproteins exceeded that of chylomicrons during the absorption of 45 micro mol of TG per hour. More [(3)H]leucine was incorporated into VLDL than into chylomicrons and the decay of newly synthesized VLDL apoproteins was more rapid than that of chylomicrons, in part due to higher concentrations of apoA-I and apoA-IV with a rapid turnover rate. Chylomicrons from proximal intestine contained more apoA-I and less C peptides than chylomicrons from distal intestine. Ninety percent of [(3)H]leucine incorporated into soluble apoproteins was in apoA-I and apoA-IV, but little apoARP was labeled. The turnover rate of apoA-I and apoA-IV differed significantly in the lymph lipoproteins examined. Although total C peptide labeling was small, evidence for intestinal apoC-II formation and differing patterns of apoC-III subunit labeling was obtained. [(3)H]Leucine incorporation and apoprotein turnover rates in lipoprotein secreted by proximal and distal intestine were similar. The different turnover rates of apoA-I and apoA-IV in individual lipoproteins suggest that these A apoproteins are synthesized independently in the intestine.-Holt, P. R., A-L. Wu, and S. Bennett Clark. Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.     

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.     

Effects of 1,2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein.