Joyce C. Gibson

Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo.

Previous data suggest that apolipoprotein (apo) CIII may inhibit both triglyceride hydrolysis by lipoprotein lipase (LPL) and apo E-mediated uptake of triglyceride-rich lipoproteins by the liver. We studied apo B metabolism in very low density (VLDL), intermediate density (IDL), and low density lipoproteins (LDL) in two sisters with apo CIII-apo AI deficiency. The subjects had reduced levels of VLDL triglyceride, normal LDL cholesterol, and near absence of high density lipoprotein (HDL) cholesterol. Compartmental analysis of the kinetics of apo B metabolism after injection of 125I-VLDL and 131I-LDL revealed fractional catabolic rates (FCR) for VLDL apo B that were six to seven times faster than normal. Simultaneous injection of [3H]glycerol demonstrated rapid catabolism of VLDL triglyceride. VLDL apo B was rapidly and efficiently converted to IDL and LDL. The FCR for LDL apo B was normal. In vitro experiments indicated that, although sera from the apo CIII-apo-AI deficient patients were able to normally activate purified LPL, increasing volumes of these sera did not result in the progressive inhibition of LPL activity demonstrable with normal sera. Addition of purified apo CIII to the deficient sera resulted in 20-50% reductions in maximal LPL activity compared with levels of activity attained with the same volumes of the native, deficient sera. These in vitro studies, together with the in vivo results, indicate that in normal subjects apo CIII can inhibit the catabolism of triglyceride-rich lipoproteins by lipoprotein lipase.

Regulation of the production and catabolism of plasma low density lipoproteins in hypertriglyceridemic subjects. Effect of weight loss.

In subjects with hypertriglyceridemia, plasma concentrations of low density lipoprotein (LDL) cholesterol are often normal or reduced. Perturbations that alter plasma very low density lipoprotein (VLDL) concentrations are associated with opposite changes in plasma LDL levels. To determine the mechanisms regulating plasma LDL levels, we used 131I-VLDL and 125I-LDL to measure the fractional catabolic rates (FCR), production rates (PR), and rates of interconversion of apoprotein B (apo B) in VLDL, intermediate density lipoprotein, and LDL in six hypertriglyceridemic subjects pre- and post-weight reduction. [2-3H]glycerol was used to quantitate VLDL triglyceride PR. All data are presented as mean +/- SD. Percent ideal body weight fell from 132 +/- 17.9 to 119 +/- 15.9% in the group, P less than 0.05. After weight loss, plasma VLDL triglyceride (486.0 +/- 364.1 vs. 191.3 +/- 65.4 mg/dl, P less than 0.05) and VLDL apo B (32.2 +/- 12.0 vs. 14.8 +/- 6.8 mg/dl, P less than 0.05) concentrations were reduced. VLDL triglyceride PR also fell after weight reduction (56.6 +/- 39.0 vs. 28.6 +/- 23.1 mg/kg per h, P less than 0.05), as did VLDL apo B PR (47.9 +/- 41.4 vs. 19.0 +/- 14.1 mg/kg per d, P less than 0.05). Pre-weight loss, plasma LDL cholesterol and apo B levels were low-normal or reduced (64.0 +/- 12.6 and 58.4 +/- 11.9 mg/dl, respectively) despite normal or elevated LDL apo B PR (17.4 +/- 7.2 mg/kg per d). The reduced cholesterol and apo B levels were associated with increased FCRs (0.68 +/- 0.29 d-1) and reduced cholesterol/protein ratios (1.01 +/- 0.18) in LDL. The plasma levels of LDL cholesterol and apo B rose after weight reduction (84.8 +/- 24.9, P less than 0.05; and 69.5 +/- 14.3 mg/dl, P less than 0.05, respectively, vs. base line). These increased concentrations resulted from a combination of events. First, the FCR for LDL apo B fell in five of six subjects with a significant reduction for the group as a whole (0.48 +/- 0.11 d-1, P less than 0.05 vs. base line). Second, the cholesterol/protein ratio increased in all six subjects with a significantly greater mean after weight loss (1.25 +/- 0.27, P less than 0.05 vs. base line). In contrast, the LDL apo B PR fell or was essentially unchanged in the six subjects after weight loss (mean, 14.4 +/- 2.8 mg/kg per d; NS vs. pre-weight loss). The changes in LDL catabolism and composition were associated with changes in the source of LDL apo B. Pre-weight loss, 73.3% of LDL was derived from VLDL, while 26.7% was directly secreted into plasma. Post-weight reduction, VLDL-derived LDL fell to 46.8% of total, while direct secretion accounted for 53.2% of LDL production. These changes were significant; P < 0.95. Thus, all subjects had direct secretion of LDL apo B and the magnitude of this source of VLDL triglyceride secretion. These results indicate that the regulation of plasma LDL levels in hypertriglyceridemic subjects is quite complex and that the rise in LDL levels after weight loss results from reduction in the fractional catabolism of this lipoprotein. The fall in the FCR is associated with changes in the source of LDL and in its composition.

Effect of heparin-induced lipolysis on the distribution of apolipoprotein e among lipoprotein subclasses. Studies with patients deficient in hepatic triglyceride lipase and lipoprotein lipase.

In normal subjects, apolipoprotein E (apo E) is present on very low density lipoproteins (VLDL) (fraction I) and on particles of a size intermediate between VLDL and low density lipoproteins (LDL) (fraction II). The major portion of apo E is, however, on particles smaller than LDL but larger than the average high density lipoproteins (HDL) (fraction III). To investigate the possible role of the vascular lipases in determining this distribution of apo E among the plasma lipoproteins, we studied subjects with primary deficiency of either hepatic lipase or of lipoprotein lipase and compared them with normal subjects. Subjects with familial hepatic triglyceride lipase deficiency (n = 2) differ markedly from normal in that fraction II is the dominant apo E-containing group of lipoproteins. When lipolysis of VLDL was enhanced in these subjects upon release of lipoprotein lipase by intravenous heparin, a shift of the apo E from VLDL into fractions II and III was observed. In contrast, apolipoproteins CII and CIII (apo CII and CIII, respectively) did not accumulate in intermediate-sized particles but were shifted markedly from triglyceride rich lipoproteins to HDL after treatment with heparin. In subjects with primary lipoprotein lipase deficiency (n = 4), apo E was confined to fractions I and III. Release of hepatic triglyceride lipase by heparin injection in these subjects produced a shift of apo E from fraction I to III with no significant increase in fraction II. This movement of apo E from large VLDL and chylomicron-sized particles occurred with little hydrolysis of triglyceride and no significant shift of apo CII or CIII into HDL from triglyceride rich lipoproteins. When both lipoprotein lipase and hepatic triglyceride lipase were released by intravenous heparin injection into normal subjects (n = 3), fraction I declined and the apo E content of fraction III increased by an equivalent amount. Either moderate or no change was noted in the intermediate sized particles (fraction II). These data strongly support the hypothesis that fraction II is the product of the action of lipoprotein lipase upon triglyceride rich lipoproteins and is highly dependent on hepatic triglyceride lipase for its further catabolism. In addition, the hydrolysis by hepatic triglyceride lipase of triglyceride rich lipoproteins in general results in a preferential loss of apo E and its transfer to a specific group of large HDL.

Reduced plasma concentrations of total, low density lipoprotein and high density lipoprotein cholesterol in patients with Gaucher type I disease.

Plasma lipid and serum apoprotein concentrations were determined in twenty‐nine individuals with Gaucher type I disease. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol were all significantly reduced in the patients with Gaucher disease compared to a group of matched control subjects. Total, LDL and HDL cholesterol were lower in males than in females with Gaucher disease. These sex differences appeared to be inversely correlated with the severity of disease manifestations which were greater in the males. Serum levels of apoprotein‐B and apoprotein‐AI, the major structural apoproteins of LDL and HDL, respectively, were decreased in the subjects with Gaucher disease. Thus, the reductions in LDL and HDL cholesterol were associated with reduced numbers of lipoprotein particles in plasma. In contrast, apoprotein‐E, a protein which is secreted by several tissues, including activated macrophages and which may mediate hepatic catabolism of lipoproteins, was elevated in the patients. Since macrophages may also catabolize lipoproteins, Gaucher disease may serve as a model for the effect of activated macrophages upon human lipoprotein metabolism.

Increased Low-Density-Lipoprotein Catabolism in Myeloproliferative Disorders

Hypocholesterolemia reported in patients with myeloproliferative disorders prompted our investigation of lipoprotein metabolism in these patients. The production and fractional catabolic rates of very-low-density lipoprotein (VLDL) apoprotein-B were measured using 131I-VLDL; those of VLDL triglyceride, using 3H-glycerol; and those of low-density lipoprotein (LDL) apoprotein-B, using 125I-LDL. Plasma total and LDL cholesterol levels (mean +/- SD) were significantly reduced in seven patients with myeloproliferative diseases, compared to five normal subjects (93.1 +/- 20.3 mg/dL versus 166.8 +/- 24.6 mg/dL and 50.3 +/- 14.8 mg/dL versus 107 +/- 20.8 mg/dL, respectively). The production rates of VLDL apoprotein-B and VLDL triglyceride were normal in the patients. The fractional catabolic rate of LDL apoprotein-B was increased in the patients with myeloproliferative diseases (0.89 +/- 0.32/d versus 0.52 +/- 0.10/d; p less than 0.05); this increased rate was associated with reduced plasma LDL apoprotein-B levels (41.7 +/- 7.1 mg/dL versus 57.0 +/- 11.3 mg/dL; p less than 0.05) despite normal or elevated LDL apoprotein-B production (16.7 +/- 5.3 mg/kg body weight . d versus 12.9 +/- 1.2 mg/kg body weight . d). The site (or sites) of increased LDL catabolism in these hypocholesterolemic patients with myeloproliferative disorders is under investigation.

Abnormalities in lipoprotein metabolism in Gaucher type 1 disease

We have previously described an association between Gaucher type 1 disease and reduced levels of total, low density lipoprotein (LDL), and high density lipoprotein (HDL) cholesterol. Plasma concentrations of apolipoprotein B and apolipoprotein AI were reduced in these subjects, while plasma apolipoprotein E (apoE), which can be synthesized and secreted by macrophages, was increased. To study the pathophysiologic basis for these changes in lipoprotein and apolipoprotein levels, we studied very low density lipoprotein (VLDL), LDL, and HDL metabolism in-depth in four subjects with Gaucher disease. Gel filtration of their plasma revealed that apoE was present in essentially a single population of lipoproteins in the large HDL range. In subject no. 4, studied presplenectomy and post-splenectomy, plasma apoE levels fell after surgery in association with a redistribution of apoE among the plasma lipoproteins to a pattern seen in normal subjects. Determination of the rates of secretion and catabolism of VLDL apoB and triglyceride were within normal limits. The reduced plasma levels of LDL and HDL cholesterol, and of both plasma apoB and apoAI, were associated with increased fractional catabolic rates of these apolipoproteins in LDL and HDL. These results indicate that the hypocholesterolemia present in subjects with Gaucher type 1 disease is associated with increased fractional catabolism of LDL and HDL. These findings, together with the evidence for alternations in plasma apoE metabolism in this disorder, suggest a role for the macrophage as the basis for these abnormalities.

Human monocytes in culture synthesize and secrete lipoprotein lipase

Abstract Within the first day in culture, human monocytes begin to synthesize and secrete a triglyceride lipase. The designation of this activity as lipoprotein lipase is based upon: 1) a requirement of serum or apolipoprotein C-II for full activity; 2) inhibition by 1M NaCl or apolipoprotein C-III2; 3) a pH optimum of 8; and 4) binding to endothelial cells that is releasable by heparin. The enzyme also exhibits immunological cross reactivity with antibody to purified bovine milk lipoprotein lipase as does human postheparin plasma lipoprotein lipase. Lymphocytes and polymorphonuclear leukocytes do not appear to contain this enzyme.

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.

Plasma apolipoprotein secretion by human monocyte-derived macrophages

Apolipoprotein E has been demonstrated to be a major secretory protein of human monocyte macrophages. The synthesis of the other plasma apolipoproteins by these cells has not been documented. Human monocyte macrophages cultured for 17-76 days were preincubated for 24 h in RPMI 1640/0.2% bovine serum albumin with or without malondialdehyde-LDL (100 micrograms/ml), followed by an additional 24 h incubation in RPMI 1640/0.2% bovine serum albumin. The media from the two incubation periods were analyzed for apolipoproteins A-I, B, C-II, C-III and E by specific radioimmunoassays. No apolipoprotein B mass was detected with a specific radioimmunoassay capable of detecting 10 ng apolipoprotein B. No apolipoproteins A-I, C-II or C-III mass was detected, even though the radioimmunoassays for these apolipoproteins were as sensitive as that for apolipoprotein E (detection limit of 0.2 ng). In contrast, significant levels of macrophage-secreted apolipoprotein E were quantified. Baseline apolipoprotein E production ranged from 0.64 to 2.82 micrograms/mg cell protein per 24 h. Preincubation in the presence of malondialdehyde-LDL (100 micrograms/ml) stimulated a 1.6-3.0-fold increase in apolipoprotein E secretion. The identification of the immunoreactive material as apolipoprotein E was confirmed by labelling the cells with [35S]methionine, followed by fractionation of the 35S-labelled secretory products by anti-apolipoprotein E affinity chromatography and SDS-gel electrophoresis. We thus report the absence of synthesis of apolipoproteins A-I, B, C-II and C-III by cultured human monocyte macrophages. These cells, however, can synthesize microgram levels of apolipoprotein E on a per mg protein basis.

In vitro metabolism of apolipoprotein E

Apolipoprotein E plays a major role in the uptake of chylomicrons and of very-low-density lipoprotein (VLDL) remnants by the liver. It has also been clearly demonstrated that apolipoprotein E rapidly and spontaneously exchanges between lipoproteins. To assess whether all lipoprotein-bound apolipoprotein E is available to participate in spontaneous transfer and/or exchange, the present study followed the fate of radiolabeled apolipoprotein E in an in vitro system. The results show that in vitro, apolipoprotein E can be considered as having both a spontaneously exchangeable pool and a nonexchangeable pool. Based upon specific radioactivity data, only a limited amount of apolipoprotein E originating in VLDL or in high-density lipoproteins (HDL) was capable of in vitro exchange with that in other lipoprotein fractions. Lipolysis of VLDL triacylglycerol by milk lipoprotein lipase, however, resulted in complete transfer of VLDL apolipoprotein E mass and radioactivity to HDL, supporting the potential for transformation of exchangeable apolipoprotein to a transferable pool in vivo. The results of these studies indicate that during the course of lipoprotein metabolism, conformational changes occur which alter the accessibility of apolipoprotein E. Such dynamic heterogeneity may have implications for the regulation of lipoprotein metabolism.