Human Triglyceride-rich Lipoproteins Research Articles

Oleanolic Acid-Enriched Olive Oil Alleviates the Interleukin-6 Overproduction Induced by Postprandial Triglyceride-Rich Lipoproteins in THP-1 Macrophages

Oleanolic acid (OA), a triterpene that is highly present in olive leaves, has been proposed as a component of functional foods for the prevention of metabolic syndrome, due to its anti-inflammatory activity. We analyzed the effects of OA on inflammatory parameters and signaling proteins in LPS-stimulated THP-1 macrophages. Thus, THP-1 macrophages were incubated with LPS for 48 h after pretreatment with OA at different concentrations. Pretreatment with OA was significantly effective in attenuating IL-6 and TNF-α overproduction induced by LPS in macrophages, and also improved the levels of AMPK-α. We also evaluated the effects of human triglyceride-rich lipoproteins (TRLs) derived from individuals consuming an OA-enriched functional olive oil. For this purpose, TRLs were isolated from healthy adolescents before, 2 and 5 h postprandially after the intake of a meal containing the functional olive oil or common olive oil, and were incubated with THP-1 macrophages. THP-1 macrophages incubated with TRLs isolated at 2 h after the consumption of the OA-enriched olive oil showed significant lower levels of IL-6 compared to the TRLs derived from olive oil. Our results suggest that OA might have potential to be used as a lipid-based formulation in functional olive oils to prevent inflammatory processes underlying metabolic syndrome in adolescents.

MiRNA/Target Gene Profile of Endothelial Cells Treated with Human Triglyceride-Rich Lipoproteins Obtained after a High-Fat Meal with Extra-Virgin Olive Oil or Sunflower Oil.

The effects of triglyceride-rich lipoproteins (TRLs) on the miRNA expression of endothelial cells, which are very involved in atherosclerosis, according to the type of diet are not known. The differences between the effects of TRLs isolated from blood of subjects after a high-fat meal with extra-virgin olive oil (EVOO) and sunflower oil (SO) on the microRNA-Seq profile related to atherosclerosis in human umbilical vein endothelial cells are analyzed. 28 upregulated microRNAs with EVOO-derived TRLs, which can regulate 22 genes related to atherosclerosis, are found. 21 upregulated microRNAs with SO-derived TRLs, which can regulate 20 genes related to atherosclerosis, are found. These microRNAs are mainly involved in angiogenesis, with a predominance of an anti-angiogenic effect with EVOO-derived TRLs. Other microRNAs upregulated with SO-derived TRLs are involved in cardiovascular diseases. Pathways for the target genes obtained from the upregulated microRNA with EVOO-derived TRLs are involved in lipid metabolism and inflammatory and defense response, while those with SO-derived TRLs are involved in lipid metabolic process. EVOO-derived TRLs seem to produce a more atheroprotective profile than SO-derived TRLs. This study provides alternative mechanisms on the protective role of EVOO against the atherogenic process through microRNA regulation in endothelial cells.

Abstract 562: Foamy Monocytes in Hypertriglyceridemia

Objective: Hypertriglyceridemia (HTG) increases risk for atherosclerotic cardiovascular disease, but the mechanisms remain poorly defined. Foamy monocytes are lipid-loaded monocytes in circulation that contribute to atherosclerosis under hypercholesterolemia. Human study has proved that HTG is associated with formation of foamy monocytes. Our study is to examine formation of foamy monocytes in HTG and their potential contribution to atherosclerosis in mouse models. Approach and results: In vivo mouse models of HTG included wild-type C57BL/6 mice on high fat diet (HFD) injected intraperitoneally with LPL inhibitor, Poloxamer 407 (P407, 0.25mg/g, every two days), as a chemically-induced model and mice with transgenic overexpression of human ApoCIII (ApoCIIItg) as a genetic model. Based on CD11c and CD36, monocytes were identified as CD36 - CD11c - , CD36 + CD11c - and CD36 + CD11c + subsets. In the first model, at 24h of the first injection, triglyceride levels increased to 367 ±84 mg/dL, higher than that of control group with saline injection (60 ±22 mg/dL, n=4, P<0.001). Meanwhile, the side scatter (SSC, representing cell granularity) values and Nile red staining for lipids of CD36 + CD11c + monocytes increased significantly, indicating formation of foamy monocytes, in mice with HTG. Furthermore, CD11c mean fluorescence intensity of CD36 + CD11c + foamy monocytes increased significantly at 2 weeks of P407 injection. In ApoCIIItg mice fed HFD (5 weeks), the percentage and SSC value of CD36 + CD11c + monocytes increased significantly (37%±5%, 247±8), also indicating elevated granularity and lipid accumulation of these monocytes, compared to wild-type mice (26%±3%, p<0.05; 226±8, p<0.05, n=4-6). In vitro treatment with human triglyceride-rich lipoprotein (hTGRL) for 24h increased the granularity and Nile red staining intensity of THP-1 monocytes, indicating foamy monocyte formation. hTGRL treatment also increased THP-1 monocyte expression of CD36, with greater uptake of cholesteryl ester-rich lipoprotein. Conclusion: High triglyceride promotes foamy monocyte formation and induces monocyte phenotypic changes in mice and tissue culture, with increased expression of CD11c and CD36, which may contribute to development of atherosclerosis under HTG.

Abstract 023: Clearance of ApoC-III Glycoforms Associated With Triglyceride Metabolism

Background: We previously reported an association between higher abundance of disialylated (apoC-III 2 ) over monosialylated (apoC-III 1 ) apoC-III and lower plasma triglyceride (TG) concentrations in three independent studies. Methods: The goal of this study was to get mechanistic insights in the metabolism of apoC-III 1 and apoC-III 2 . First, the relative abundances of human triglyceride-rich lipoproteins (TRL)-associated apoC-III glycoforms in plasma were assessed by mass spectrometric immunoassay (MSIA) over time after injecting human TRL in wild-type mice, mice lacking hepatic heparan sulfate proteoglycans (HSPG), and mice deficient for both low-density lipoprotein receptor (LDLR) and LDLR-Related Protein 1 (LRP-1). Secondly, the relative abundance of plasma apoC-III glycoforms was assessed by MSIA in 11 participants randomized to volanesorsen (apoC-III antisense drug; n=11) or placebo (n=6) treatment for 13 weeks. Results: Kinetic studies revealed that half of TRL-associated apoC-III (total) was rapidly cleared via LDLR and LRP1 (t 1/2 = 16.5 min), and the remaining half via the much slower but higher capacity HSPGs (t 1/2 = 55.5 min). After injection, a significant increase in the relative abundance of apoC-III 2 was observed in HSPG-deficient mice , while no increase in mice lacking LDLR and LRP-1. Clinically, the relative abundance of apoC-III 2 significantly increased (42% increase compared to placebo, p=0.05) and that of apoC-III 1 significantly decreased (15% decrease compared to placebo, p=0.007) in participants randomized to volanesorsen. The decrease in the relative abundance of apoC-III 1 after volanesorsen treatment was strongly correlated with the decrease in TG levels (r=0.63, p=0.006). Conclusions: Our results indicate slower clearance of apoC-III-containing particles through HSPGs, which preferentially clear apoC-III 2. In contrast, apoC-III 2 is less effectively cleared by LDLR/LRP-1 compared to apoC-III 1 . Clinically, the increase in the apoC-III 2 /apoC-III 1 ratio upon antisense lowering of apoC-III might reflect faster clearance of apoC-III 1 as it associates with improved TG levels.

Indispensable role of lipoprotein bound-ApoE in adipogenesis and endocytosis induced by postprandial TRL

Diet-associated obesity is coexisted with postprandial hypertriglyceridemia that indicates increased number of triglyceride-rich lipoproteins (TRL). This study aimed to investigate the effect of postprandial TRL-bound apolipoprotein E (ApoE) on adipogenesis and potential mechanisms. 3T3-L1 cells were cultured with (i) human TRL (h-TRL) with or without insulin, or (ii) TRL from wild type mice (WT-TRL) or ApoE knock-out mice (EKO-TRL) and insulin. The differentiating adipocytes were incubated with different kinds of TRL labeled by red fluorescence and confocal microscopy was performed. Receptor associated protein (RAP), heparin or both were added to inhibit low density lipoprotein receptor family receptors, heparan sulfate proteoglycan or both, respectively. With the aid of insulin, postprandial h-TRL or WT-TRL, instead of EKO-TRL, successfully induced adipogenesis. Confocal microscopy revealed red fluorescence in the differentiating adipocytes treated with h-TRL or WT-TRL, but not with EKO-TRL. RAP markedly reduced red fluorescence within the differentiating adipocytes, while heparin had little impact. The low density lipoprotein receptor related protein 1 protein showed upward trend with the increase of TRL concentrations. Taken together, lipoprotein-bound ApoE was required in both postprandial TRL-induced adipogenesis and TRL endocytosis by the differentiating adipocytes, the latter could be partially through low density lipoprotein receptor family dependent-pathway.

Characterizing blood-brain barrier perturbations after exposure to human triglyceride-rich lipoprotein lipolysis products using MRI in a rat model.

PurposePrevious studies indicated hyperlipidemia may be a risk factor for Alzheimer's disease, but the contributions of postprandial triglyceride‐rich lipoprotein (TGRL) are not known. In this study, changes in blood–brain barrier diffusional transport following exposure to human TGRL lipolysis products were studied using MRI in a rat model.MethodsMale Sprague‐Dawley rats (∼180–250 g) received an i.v. injection of lipoprotein lipase (LpL)‐hydrolyzed TGRL (n = 8, plasma concentration ≈ 150 mg human TGRL/dL). Controls received i.v. injection of either saline (n = 6) or LpL only (n = 6). The 1H longitudinal relaxation rate R1 = 1/T1 was measured over 18 min using a rapid‐acquired refocus‐echo (RARE) sequence after each of three injections of the contrast agent Gd‐DTPA. Patlak plots were generated for each pixel yielding blood‐to‐brain transfer coefficients, Ki, chosen for best fit to impermeable, uni‐directional influx or bi‐directional flux models using the F‐test.ResultsAnalysis from a 2‐mm slice, 2‐mm rostral to the bregma showed a 275% increase of mean Ki during the first 20 min after infusion of human TGRL lipolysis product that differed significantly compared with saline and LpL controls. This difference disappeared by 40 min mark.ConclusionThese results suggest human TGRL lipolysis products can lead to a transient increase in rat BBB permeability. Magn Reson Med 76:1246–1251, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Minor components of pomace olive oil enhance VLDL-receptor expression in macrophages when treated with postprandial triglyceride-rich lipoproteins

Pomace olive oil (POO) is rich in minor components, which can modulate the composition of postprandial triglyceride-rich lipoproteins (TRL) and their uptake by macrophages. The aim of the present study was to investigate the effects of postprandial TRL obtained after the ingestion of meals containing POO or refined olive oil (ROO) on foam cell formation, one of the initial steps of atherogenesis. Meals were administered to 9 healthy men and TRL were isolated from serum 4h after the intake. POO intake led to TRL with lower triglyceride/apo B48 and triglyceride/apo B100 ratios compared to ROO. Upon incubation of THP-1 macrophages with the TRL, an increase in the intracellular triglyceride content and foam cell formation was observed. Compared to ROO-TRL, the only receptor involved in lipoprotein uptake that showed changes in the mRNA expression after treatment with POO-TRL was the VLDL-receptor (VLDLr). In conclusion, the intake of POO modified the composition of human TRL, which increased the VLDLr gene expression in macrophages. However, the changes were not sufficient to enhance foam cell formation.

Metabolism of apolipoprotein A-II containing triglyceride rich ApoB lipoproteins in humans.

To characterize human triglyceride-rich lipoproteins (TRL) with and without apoA-II and to study their metabolism invivo. Plasma from 11 participants on a controlled diet given a bolus infusion of [D5]l-phenylalanine to label apoB was combined into four pools and applied to anti-apoA-II immunoaffinity columns. Fractions with and without apoA-II were separated into VLDL and IDL by ultracentrifugation; lipids and apolipoproteins were measured. For kinetic measurements, apoB was isolated and hydrolyzed to the constituent amino acids. Tracer enrichment was measured by GCMS. Metabolic rates were determined by SAAM-II. VLDL and IDL with apoA-II comprised 7% and 9% of total VLDL and IDL apoB respectively. VLDL with apoA-II was enriched in apoC-III, apoE, and cholesterol compared to VLDL without apoA-II. Mean apoB FCR of VLDL with apoA-II was significantly lower than for VLDL without apoA-II (2.80±0.96 pools/day v.s. 5.09±1.69 pools/day, P=0.009). A higher percentage of VLDL with apoA-II was converted to IDL than was cleared from circulation, compared to VLDL without apoA-II (96±8% vs. 45±22%; P=0.007). The rate constants for conversion of VLDL to IDL were similar for VLDL with and without apoA-II. Thus, a very low rate constant for clearance accounted for the lower FCR of VLDL with apoA-II. VLDL with apoA-II represents a small pool of VLDL particles that has a slow FCR and is predominantly converted to IDL rather than cleared from the circulation.

Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions

The atherogenicity theory for triglyceride-rich lipoproteins (TRLs; VLDL + intermediate density lipoprotein) generally cites the action of apolipoprotein C-III (apoC-III), a component of some TRLs, to retard their metabolism in plasma. We studied the kinetics of multiple TRL and LDL subfractions according to the content of apoC-III and apoE in 11 hypertriglyceridemic and normolipidemic persons. The liver secretes mainly two types of apoB lipoproteins: TRL with apoC-III and LDL without apoC-III. Approximately 45% of TRLs with apoC-III are secreted together with apoE. Contrary to expectation, TRLs with apoC-III but not apoE have fast catabolism, losing some or all of their apoC-III and becoming LDL. In contrast, apoE directs TRL flux toward rapid clearance, limiting LDL formation. Direct clearance of TRL with apoC-III is suppressed among particles also containing apoE. TRLs without apoC-III or apoE are a minor, slow-metabolizing precursor of LDL with little direct removal. Increased VLDL apoC-III levels are correlated with increased VLDL production rather than with slow particle turnover. Finally, hypertriglyceridemic subjects have significantly greater production of apoC-III-containing VLDL and global prolongation in residence time of all particle types. ApoE may be the key determinant of the metabolic fate of atherogenic apoC-III-containing TRLs in plasma, channeling them toward removal from the circulation and reducing the formation of LDLs, both those with apoC-III and the main type without apoC-III.

Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis

Products generated from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TGRL) are reported to increase endothelial layer permeability. We hypothesize that these increases in permeability result from the active rearrangement and dissolution of the junctional barrier in human aortic endothelial cells, as well as induction of the apoptotic cascade. Human aortic endothelial cells were treated with TGRL lipolysis products generated from coincubation of human TGRL plus lipoprotein lipase. Measurement of transendothelial electrical resistance demonstrated a time-dependent decrease in endothelial barrier function in response to TGRL lipolysis products. Immunofluorescent localization of zonula occludens-1 (ZO-1) showed radial rearrangement along cell borders after 1.5 h of treatment with lipolysis products. A concurrent redistribution of F-actin from the cell body to the cell margins was observed via rhodamine phalloidin staining. Immunofluorescent imaging for occludin and vascular endothelial cadherin showed that these proteins relocalize as well, although these changes are less prominent than for ZO-1. Western analysis of cells exposed to lipolysis products for 3 h revealed the fragmentation of ZO-1, a reduction in occludin, and no change of vascular endothelial cadherin. Lipolysis products also increased caspase-3 activity and induced nuclear fragmentation. Treatments did not cause oncosis in cells at any point during the incubation. These results demonstrate that TGRL lipolysis products play an important role in the regulation of endothelial permeability, the organization of the actin cytoskeleton, the localization and expression of junctional proteins, especially ZO-1, and the induction of apoptosis.

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-plasmodium toxicity

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria.

Binding of triglyceride rich lipoproteins to LDL receptor amplifies inflammatory responses of aortic endothelium.

Recent discoveries have led to the recognition of atherosclerosis as an inflammatory disease consisting of elements similar to established autoimmune disorders. Triglyceride rich lipoproteins (TGRL) are elaborated during the postprandial state and give rise to remnant particles known to be a risk factor for atherosclerosis. We have developed a model of repetitive injury wherein human aortic endothelial cells (HAEC) were repetitively exposed to human TGRL freshly isolated from plasma following consumption of a 30% fat meal. Preincubation over 3 days with TGRL was not itself inflammatory, but in conjunction with acute stimulation, upregulated VCAM-1 and E-selectin expression up to 2-fold that of HAEC stimulated by TNF-alpha alone. Monocyte adhesion in shear flow to a HAEC monolayer, which formed the substrate of a parallel plate flow chamber, also increased by 1–2 fold in response to repetitive injury with TGRL followed by acute stimulation. This increased adhesive efficiency was specific for monocytes, as neutrophil adhesion was not significantly affected by TGRL preincubation. HAEC activation involved binding of TGRL to the vLDL receptor LR11 and this was blocked by co-incubation with receptor-associated protein (RAP), a high affinity inhibitor of the LDL receptor-related protein (LRP) and a low affinity inhibitor of the LDL receptor. This repetitive injury model reveals events linking apolipoprotein binding and inflammation of aortic endothelium, crucial to elucidating lesion pathology. This study supported by NIH R01-AI47294.

Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels

Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.

Human triglyceride-rich lipoprotein apo E kinetics and its relationship to LDL apo B-100 metabolism

Apolipoprotein (apo) E is a multifunctional protein that can act as a ligand for lipoprotein receptors. The receptor-mediated clearance of the triglyceride-rich lipoproteins (TRL) chylomicrons and VLDL from plasma is, in part, dependent on apo E. Enrichment of VLDL with apo E is thought to enhance receptor-mediated clearance of VLDL resulting in a low rate of conversion of VLDL to LDL. However, the kinetic mechanism controlling the concentration of apo E in VLDL is not known. We conducted kinetic studies on apo E in the TRL fraction ( d<1.006 g/ml) and apo B-100 in the TRL and LDL ( d=1.019–1.063 g/ml) fractions to assess the kinetic determinants of apo E concentration in TRL and to determine the effects that TRL apo E production and clearance rates have on the production rate of LDL apo B-100. Nineteen males between the ages of 24 and 73 underwent a primed-constant infusion with deuterated leucine tracer in the constantly-fed state. Apo B-100 from TRL and LDL, and apo E from TRL were isolated and their tracer incorporation measured by gas chromatography/mass spectrometry. The residence time and production rates of each protein were determined from the kinetic data using the SAAM II modeling program. The residence time and production rate of TRL apo E were about one-half that of TRL apo B-100 (1.8±1.0 vs. 2.9±2.1 h and 14.5±11.0 vs. 27.6±17.3 mg/kg per day, respectively). The production rate of TRL apo E was weakly correlated with the production rate of TRL apo B-100 ( r=0.424, P=0.07). Multiple regression analysis showed that the residence time of TRL apo B-100 and the relative TRL apo E production rate (relative to the TRL apo B100 production rate) were negatively associated with LDL apo B-100 production rate, accounting for 68% of its variability. We conclude that (1) the concentration of apo E in TRL is highly correlated to its production rate, suggesting that production rate regulates the TRL apo E concentration, and (2) individuals with a relatively short TRL apo B-100 residence time and those producing TRL with a relatively low apo E content have the highest LDL apo B-100 production rates.

The Human Factor H-related Protein 4 (FHR-4): A NOVEL SHORT CONSENSUS REPEAT-CONTAINING PROTEIN IS ASSOCIATED WITH HUMAN TRIGLYCERIDE-RICH LIPOPROTEINS

A novel apoprotein of an apparent molecular mass of 86 kDa in its unreduced form was identified in human triglyceride-rich lipoproteins. This protein was purified and the amino acid sequence of six proteolytic fragments was found to overlap with that of the factor H-related proteins. In parallel we identified the cDNA encoding a new complement factor H-related protein, termed FHR-4. The sequences of the new apoprotein overlapped with that of the FHR-4 protein. Similar to the previously described factor H-related proteins, FHR-4 contains a hydrophobic signal sequence followed by a stretch of five repetitive elements termed short consensus repeats. Recombinant FHR-4 protein was expressed in the baculovirus system and has an apparent molecular mass of 42 kDa. In addition a 84-kDa dimeric form of the recombinant FHR-4 was detected. Using an immunoaffinity column with antibodies raised against the recombinant FHR-4, we isolated a 86-kDa protein from human plasma. The different molecular mass of the recombinant FHR-4 and the dimeric FHR-4 in plasma is due to different carbohydrate moieties. The 86-kDa plasma protein and the novel apolipoprotein had identical mobility on SDS-polyacrylamide gel electrophoresis analysis and reacted with antisera raised against the reFHR-4 and the purified apoprotein. In conclusion, we have identified a novel factor H-related protein, FHR-4, in human plasma and demonstrate that this protein is present in triglyceride-rich lipoproteins in a dimeric form. This observation provides an intriguing new aspect on possible function(s) of this novel protein and the other factor H-related proteins.

Regulation of serum-induced lipid accumulation in human monocyte-derived macrophages by interferon-γ. Correlations with apolipoprotein E production, lipoprotein lipase activity and LDL receptor-related protein expression

The demonstration of lipid loaded macrophages in atherosclerotic tissue has led to the development of in vitro systems to elucidate the mechanisms involved in lipid accumulation. Here we have characterised the changes which occur in human monocyte-derived macrophage (MDM) lipids during culture in either human serum (HS) or foetal calf serum (FCS). MDM cultured in HS were rapidly converted to lipid filled foam cells, as assessed using HPLC analysis and oil red-O staining and compared with the same cells grown in FCS. However, the lipids which accumulated were predominantly triglycerides with smaller amounts of unesterified cholesterol (UC) and only traces of cholesteryl esters (CE). α-Tocopherol (α-TocH) was present at higher levels in MDM cultured in HS compared to the same cells grown in FCS. MDM lipid accumulation was dependent on the triglyceride-rich lipoprotein (TGRL) fraction of human serum; accordingly, supplementation of FCS with human TGRL also induced MDM lipid accumulation. The relationships between cellular lipid accumulation and secretion of apolipoprotein E (apo E) and lipoprotein lipase (LPL) as well as expression of the low density lipoprotein receptor-related protein (LRP) were also examined. MDM lipid accumulation was associated with increased apo E secretion but did not alter extracellular LPL activity. The lipid accumulation which was induced by HS was potently inhibited (but not reversed) by the inflammatory cytokine interferon-γ (IFNγ), and this was associated with decreased apo E production, LPL secretion and expression of LRP. These studies reveal striking differences in the lipid composition of MDM cultured in either HS or FCS, and indicate that oil red-O staining is not necessarily associated with cholesteryl ester accumulation in human macrophages. Furthermore, the effect that serum-induced lipid accumulation has on the specific MDM functions studied should be appreciated when developing in vitro macrophage models.

Lipoprotein lipase-enhanced binding of human triglyceride-rich lipoproteins to heparan sulfate: modulation by apolipoprotein E and apolipoprotein C

The objective of this study was to investigate whether compositional variation in apolipoprotein (apo) content of triglyceride-rich lipoproteins (TRLP) modulates binding of heparan sulfate proteoglycans (HSPG). Human TRLP was enriched with apoE and apoCs and the ability to bind biotin-conjugated heparan sulfate (b-HS) was studied in the presence or absence of heat-inactivated lipoprotein lipase (LPL). TRLP, associated with LPL, showed an increased capacity to bind b-HS compared with TRLP alone. Low density lipoproteins (LDL) bound both b-HS and LPL with a higher affinity than TRLP. ApoE enrichment of TRLP resulted in an enhanced binding of b-HS. Increased binding of b-HS to TRLP by the combination of apoE enrichment and LPL addition was found to be complementary, not affecting their individual binding capacity. TRLP enrichment with apoC led to the formation of an apoC-rich, apoE-poor particle; this alteration by itself did not change the ability to bind b-HS. ApoC enrichment of TRLP resulted in a reduced capacity to bind LPL and therefore a subsequently reduced capacity to bind b-HS, compared with control TRLP associated with LPL. Competition studies revealed that b-HS binding to TRLP was fully displaceable by lactoferrin but barely by heparan sulfate, dermatan sulfate, or chondroitin-4-sulfate. Using TRLP coated to microtiter wells and associated with LPL, the b-HS displacement patterns were comparable to those obtained with coated LDL in the presence or absence of LPL. The cell-free system that was used enabled us to identify the functions of apoC and apoE in the binding of TRLP to LPL and HSPG. Both LPL and apoE increased the ability of TRLP to bind HSPG. The apoC content of TRLP regulated the docking of TRLP to LPL. ApoC enrichment reduced the affinity or capacity of TRLP to LPL binding, and this has relevance for the lipolytic cascade.

4] Simultaneous quantification of apolipoproteins B-100, B-48, and E separated by SDS-PAGE

Publisher Summary The two species of apolipoprotein (apo) B in humans, apoB-100, and apoB-48, define the hepatogenous and intestinal contributions to the secretion of triglyceride-rich lipoproteins (TRL) into the blood. A number of methods have been proposed to quantify apoB-100 and apoB-48. Several procedures based on separation by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) have given inconsistent results. With electrophoresis in 3.3% polyacrylamide tube gels, followed by Coomassie blue staining, Poapst et al. reported a lower chromogenicity for apoB-48 than for apoB-100, as well as a nonlinear response, between the intensity of dye uptake and apolipoprotein mass. This method has been used to quantify apolipoproteins in human triglyceride-rich lipoproteins (TRL) and has yielded apoB-100 and apoB-48 levels that are considerably higher than those reported, by others, using electrophoresis in slab gels. Zilversmit and Shea, using slab gels, found equal chromogenicities for rat apoB-48 and rat and human apoB-100 and a linear relationship between dye uptake and apolipoprotein mass. These observations are consistent with the findings here and those of others who have carried out the electrophoresis of apoB and other proteins in slab gels and they support the conclusion that apoB data obtained by tube gel electrophoresis, in which only a rim of dye penetrates into the gel, should be viewed with caution. A major advantage to the SDS-polyacrylamide slab gel electrophoretic method that has been developed is the ability to quantify apoB-100 and apoB-48 together with apoE in various lipoprotein fractions in humans and other species.

Lipoprotein lipase enhanced binding of human triglyceride-rich lipoproteins to heparan sulphate: Modulation by apolipoproteins E, C and apolipoprotein glycosylation

Lipoprotein lipase enhanced binding of human triglyceride-rich lipoproteins to heparan sulphate: Modulation by apolipoproteins E, C and apolipoprotein glycosylation

Quantification of apolipoproteins B-100, B-48, and E in human triglyceride-rich lipoproteins.

We have developed a procedure to quantify apolipoprotein (apo) B-100, apoB-48, and apoE in human triglyceride-rich lipoproteins. This procedure permits delipidation of small amounts of triglyceride-rich lipoproteins without appreciable losses, and quantification of these apolipoproteins in samples containing as little as 10 micrograms of protein. Delipidated triglyceride-rich lipoproteins are subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis, and the mass of apolipoproteins is estimated after densitometric scanning and volume integration of Coomassie blue-stained bands. The chromogenicities of apoB-100 and apoB-48 are virtually identical, and twofold lower than that of apoE. The standard curve for each apolipoprotein follows a power function over a wide protein range, permitting quantification of as little as 0.2 microgram of apoB-48 and as much as 30 micrograms of apoB-100 from a single application of triglyceride-rich lipoproteins to the gels. This method is suitable for routine use in studies of the intestinal and hepatic contributions to triglyceride-rich lipoproteins and their responses to postprandial lipemia.