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Glycated Phosphatidylethanolamine Promotes Macrophage Uptake of Low Density Lipoprotein and Accumulation of Cholesteryl Esters and Triacylglycerols

1999; Elsevier BV; Volume: 274; Issue: 23 Linguagem: Inglês

10.1074/jbc.274.23.16494

1083-351X

Amir Ravandi, A. Kuksis, Nisar Shaikh,

Natural Antidiabetic Agents Studies

Non-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 μg/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 ± 1.54 μg/mg cell protein) and TG (285.32 ± 4.38 μg/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 ± 0.95, p < 0.01 and TG, 185.57 ± 3.58 μg/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 ± 1.35 and TG, 280.78 ± 3.98 μg/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5–6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia. Non-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 μg/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 ± 1.54 μg/mg cell protein) and TG (285.32 ± 4.38 μg/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 ± 0.95, p < 0.01 and TG, 185.57 ± 3.58 μg/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 ± 1.35 and TG, 280.78 ± 3.98 μg/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5–6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia. LDL 1The abbreviations used are: LDL, low density lipoprotein; AGE, advanced glycation end products; AcLDL, acetylated LDL; OxLDL, oxidized LDL; LC/ES/MS, liquid chromatography with on-line electrospray mass spectrometry; Glc-PtdEtn, glucosylated phosphatidylethanolamine; Gro-PEtn, glycerophosphoethanolamine; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; RBC, red blood cells; HPLC, high performance liquid chromatography; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline; PMA, phorbol myristate acetate; CE, cholesteryl ester; TG, triacylglycerol; TC, total cholesterol. glycation has been proposed to play central role in the atherosclerosis of diabetic hyperglycemia (1Lyons T.J. Jenkins A.J. Curr. Opin. Lipidol. 1997; 8: 174-180Crossref PubMed Scopus (80) Google Scholar). The effect of protein glycation in LDL modification and its oxidation has been extensively investigated. Several groups of investigators have shown that glycated LDL is capable of inducing foam cell formation in a variety of cell culture systems (2Sobenin I.A. Tertov V.V. Koschinsky T. Bunting C.E. Slavina E.S. Dedov I.I. Orekhov A.N. Atherosclerosis. 1993; 100: 41-54Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 3Sasaki J. Cottam G.L. Biochim. Biophys. Acta. 1982; 713: 199-207Crossref PubMed Scopus (71) Google Scholar, 4Lopes-Virella M.F. Klein R.L. Lyons T.J. Stevenson H.C. Witztum J.L. Diabetes. 1988; 37: 550-557Crossref PubMed Google Scholar, 5Gonen B. Baenzinger J. Schonfeld G. Jacobson D. Farrar P. Diabetes. 1981; 30: 875-878Crossref PubMed Google Scholar). Furthermore, LDL from diabetic patients has been shown to increase accumulation of CE in macrophages, and the extent of accumulation has been correlated with the extent of LDL glycation (6Klein R.L. Wohltmann H.J. Lopes-Virella M.F. Diabetes. 1992; 41: 1301-1307Crossref PubMed Scopus (17) Google Scholar, 7Klein R.L. Lyons T.J. Lopes-Virella M.F. Diabetologia. 1990; 33: 299-305Crossref PubMed Scopus (25) Google Scholar). In all these studies it was presumed that the glycated apoB is responsible for the entire altered activity. By using antibodies to advanced glycation end products (AGE), Bucalaet al. (8Bucala R. Makita Z. Koschinsky T. Cerami A. Vlassara H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6434-6438Crossref PubMed Scopus (561) Google Scholar) have, however, shown that the lipid component of glycated LDL contains most of the AGE present and that the relative amount of this AGE antigen was proportional to the susceptibility of LDL to peroxidation. Since lipid glycation products were not isolated or identified, the significance of lipid glycation in LDL uptake by macrophages could not be investigated or a differentiation attempted between lipid and protein glycation in this process. We have previously demonstrated that the amino phospholipids of plasma and red blood cells (RBC) from diabetic subjects show a 10-fold increase in glycated PtdEtn over control subjects (9Ravandi A. Kuksis A. Marai L. Myher J.J. Lipids. 1995; 30: 885-891Crossref PubMed Scopus (40) Google Scholar, 10Ravandi A. Kuksis A. Marai L. Myher J.J. Steiner G. Lewis G. Kamido H. FEBS Lett. 1996; 381: 77-81Crossref PubMed Scopus (80) Google Scholar) and that incorporation of Glc-PtdEtn into LDL (11Ravandi A. Kuksis A. Shaikh N.A. Clin. Chem. 1998; 44 Suppl. 2: A73Google Scholar) facilitates peroxidation of LDL lipids. The present study demonstrates that the presence of Glc-PtdEtn in LDL results in an increased uptake of LDL and a dramatic increase in neutral lipid accumulation in THP-1 macrophages. THP-1 cells were obtained from the American Type Tissue Culture Collection (TIB 202) and were propagated in RPMI 1640, 10% fetal calf serum/penicillin (100 units/ml)/streptomycin (100 μg/ml) at 37 °C, 5% CO2. Cells were plated at a density of 1 × 106 cells/ml in 10% fetal calf serum medium containing 10−7m phorbol myristate acetate (PMA) for 72 h. The cells were then washed extensively with serum-free RPMI medium and incubated with or without lipoproteins as indicated for each experiment. In all experiments, cell viability exceeded 90% as determined by trypan blue exclusion. Glc-PtdEtn was prepared and purified as described previously (9Ravandi A. Kuksis A. Marai L. Myher J.J. Lipids. 1995; 30: 885-891Crossref PubMed Scopus (40) Google Scholar). Briefly, PtdEtn (2 mg) dissolved in 1 ml of methanol was transferred to a 15-ml test tube, and the solvent evaporated under nitrogen. Four ml of 0.1 mphosphate buffer containing 0–400 mm glucose and 0.1 mm EDTA were added and sonicated at low power for 5 min at room temperature, and the mixture was incubated under nitrogen at 37 °C for various periods. Lipids were extracted into chloroform/methanol (2:1, v/v) as described by Folch et al.(12Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), and the solvents evaporated under nitrogen. Samples were redissolved in chloroform/methanol (2:1, v/v) and kept at −20 °C until analysis. Glc-PtdEtn (2 mg) was purified by preparative TLC (20 × 20-cm glass plates) coated with Silica Gel H (250-μm thick layer). The chromatoplates were developed using chloroform/methanol, 30% ammonia (65:35:7, by volume) as described (9Ravandi A. Kuksis A. Marai L. Myher J.J. Lipids. 1995; 30: 885-891Crossref PubMed Scopus (40) Google Scholar). Phospholipids were identified by co-chromatography with appropriate standards, visualizing any lipid bands under ultraviolet light after spraying the plate with 0.05% 2,7-dichlorofluorescein in methanol. Both glucosylated and non-glucosylated lipids were recovered by scraping the gel from appropriate areas of the plate and extracting it twice with the developing solvent. LDL (1.019–1.069 g/ml) was obtained by density gradient ultracentrifugation (13Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6486) Google Scholar) from plasma of fasted normolipidemic individuals. LDL (2 mg of protein/ml) was subsequently dialyzed against 0.1 m phosphate buffer (pH 7.4) containing 0.1 mm EDTA for 24 h (three buffer changes). LDL samples were sterilized by passing through a 0.22-micron filter (Millipore, Milford, MA), kept at 4 °C, and used within 1 week. Lipoprotein concentration was determined by the method of Lowryet al. (14Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and expressed as mg/ml. Oxidation of LDL (5 mg of protein/5 ml) was performed by dialysis against 5 μmCuSO4·5H2O in 0.1 m phosphate buffer (pH 7.4), for 12 h at 37 °C in the dark. LDL (2 mg of protein/ml) in 1 mm EDTA, containing 0.1 mg/ml chloramphenicol and 3 mm NaN3, was incubated with 50 mm glucose at 37 °C for 1 week under nitrogen to obtain glycated LDL. Acetylated LDL (AcLDL) was prepared by the method of Basu et al. (15Basu S.K. Goldstein J.L. Anderson R.G.W. Brown M.S. Proc. Natl. Acad. Sci U. S. A. 1976; 73: 3178-3182Crossref PubMed Scopus (823) Google Scholar). Glucosylated and non-glucosylated PtdEtn was incorporated into LDL essentially as described by Engelmann et al. (16Engelmann B. Kogl C. Kulschar R. Schaipp B. Biochem. J. 1996; 315: 781-789Crossref PubMed Scopus (49) Google Scholar) for enriching human plasma lipoproteins with phospholipids. Glc-PtdEtn (1 mg) in chloroform/methanol (2:1, v/v) was transferred to a 15-ml test tube, the solvent evaporated under nitrogen, and the lipids were dispersed by vortexing in 1.5 ml of buffer containing 50 mm Tris/HCl, 1 mm dithiothreitol, and 0.03 mm EDTA (pH 7.4). The solutions were sonicated in a bath sonicator for 5 min at 1-min intervals while being kept on ice under a stream of nitrogen. The liposome mixture was centrifuged at 3,500 × g and the supernatant collected and passed through a 0.45-μm filter. The liposomal mixture (1 ml) was added to fresh plasma (4 ml) containing 3 mm NaN3 under gentle mixing. The mixture was incubated under nitrogen at 37 °C for 24 h. Lipoproteins were isolated as described above. Normal phase high performance liquid chromatography (HPLC) of phospholipids was performed on a 5-μm Spherisorb column (250 × 4.6 mm inner diameter, Alltech Associates, Deerfield, IL). The column was installed into a Hewlett-Packard model 1090 liquid chromatograph and eluted with a linear gradient of 100% solvent A (chloroform/methanol/30% ammonium hydroxide, 80:19.5:0.5, by volume) to 100% solvent B (chloroform/methanol/water/30% ammonium hydroxide, 60:34:5.5:0.5, by volume) in 14 min and then at 100% solvent B for 10 min (17Ravandi A. Kuksis A. Shaikh N.A. Jackowski G. Lipids. 1997; 32: 989-1001Crossref PubMed Scopus (37) Google Scholar). The flow was set at 1 ml/min. The peaks were monitored by on-line ES/MS. Normal phase HPLC with on-line electrospray mass spectrometry (LC/ES/MS) was performed by splitting the HPLC flow by 1/50 resulting in 20 μl/ml being admitted to a Hewlett-Packard model 5988B quadrupole mass spectrometer equipped with a nebulizer-assisted electrospray interface (HP 59987A) (18Ravandi A. Kuksis A. Myher J.J. Marai L. J. Biochem. Biophys. Meth. 1995; 30: 271-285Crossref PubMed Scopus (45) Google Scholar). Tuning and calibration of the system was achieved in the mass range of 400–1500 by using the standard phospholipid mix dissolved in the HPLC solvent A and flow-injected at 50 μl/min into the mass spectrometer. Capillary voltage was set at 4 kV, the end plate voltage at 3.5 kV, and the cylinder voltage at 5 kV in the positive mode of ionization. In the negative mode, the voltages were 3.5, 3, and 3.5 kV, respectively. Both positive and negative ion spectra were taken in the mass range 400–1100 atomic mass units. The capillary exit voltage was set at 120 V in the positive and 160 V in the negative ion mode. Nitrogen gas was used as both nebulizing gas (40 pounds/square inch) and drying gas (60 pounds/square inch, 270 °C). Phospholipids were quantified on basis of standard curves established for each phospholipid class. The equimolar ion intensities of different species of each phospholipid class varied by less than 5% in each of the ion modes (18Ravandi A. Kuksis A. Myher J.J. Marai L. J. Biochem. Biophys. Meth. 1995; 30: 271-285Crossref PubMed Scopus (45) Google Scholar). The LC/ES/MS response to different phospholipid classes varied greatly and required the regular use of standards. LDL was labeled with the fluorescent probe 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) according to the method of Stephan and Yurachek (19Stephan Z.F. Yurachek E.C. J. Lipid Res. 1993; 34: 325-330Abstract Full Text PDF PubMed Google Scholar). Briefly, to 10 ml of LDL solution (1 mg/ml protein) containing 1 mmEDTA in 0.1 m PBS, 300 μl of DiI solution in Me2SO (30 mg/ml) was slowly added under gentle agitation. The solution was incubated for 8 h at 37 °C under nitrogen and in the dark. The LDL was reisolated by ultracentrifugation. Under these conditions the average labeling efficiency was 20–25 ng of DiI/μg of LDL protein. To study the uptake of DiI-labeled LDL, cells seeded in 24-well culture dishes were incubated for 4 h at 37 °C with increasing concentrations of the lipoprotein (10–200 μg/ml) (20Teupser D. Thiery J. Walli A.K. Seidel D. Biochim. Biophys. Acta. 1996; 1303: 193-198Crossref PubMed Scopus (68) Google Scholar). Specific uptake of DiI LDL in all preparations was determined at 10 μg of lipoprotein/ml with 50-fold excess of unlabeled lipoprotein. Afterward, the cells were washed twice with PBS containing 0.4% bovine serum albumin and twice with PBS alone. To each well 1 ml of lysis buffer was added (1 g/liter SDS, 0.1 m NaOH). Cells were incubated at room temperature under gentle shaking for 1 h. This allowed both direct fluorescence and protein measurement. The fluorescence of each well was measured in duplicate by a Shimadzu spectrofluorometer (RF5000U). The excitation and emission wavelengths were set at 520 and 575 nm, respectively. The detection range for the fluorescence was linear from 0.05 to 20 μg/ml LDL protein. Protein determinations were made in duplicate using the method of Lowry et al. (14Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin dissolved in lysis buffer as standard. Fluorescence microscopy was performed as described previously (21Pitas R.E. Innerarity T.L. Mahly R.W. Arteriosclerosis. 1983; 3: 2-10Crossref PubMed Google Scholar). The THP-1 cells were exposed for 24 h to control and modified LDL preparations. After incubation the cells were washed once with ice-cold PBS containing 0.4% bovine serum albumin and twice with PBS alone. Cells were scraped from the culture flask into PBS and sonicated. The cellular lipids were extracted with chloroform/methanol (2:1 v/v) and analyzed by gas-liquid chromatography (22Kuksis A. Myher J.J. Geher K. Shaikh N.A. Breckenridge W.C. Jones G.J.L. Little J.A. J. Chromatogr. 1980; 2: 1-26Crossref Scopus (23) Google Scholar). For this purpose the lipid extract was digested with phospholipase C (Clostridium welchii), and the digestion mixture was extracted with chloroform/methanol (2:1 v/v) containing 100 μg of tridecanoylglycerol as internal standard. The lipid extracts were then reacted for 30 min at 20 °C with SYLON BFT plus 1 part dry pyridine. This procedure converts the free fatty acids into trimethyl silyl esters and the free sterols, diacylglycerols, and ceramides into trimethyl silyl ethers, leaving the cholesteryl esters and triacylglycerols unmodified. The free cholesterol, cholesterol esters, and triacylglycerols were quantitated using a non-polar capillary column as described previously (23Kuksis A. Myher J.J. Geher K. Breckenridge W.C. Jones G.J.L. Little J.A. J. Chromatogr. Biomed. Appl. 1981; 224: 1-23Crossref Scopus (46) Google Scholar). Cellular uptake and neutral lipid accumulation assays were done in triplicate, and statistical significance was performed with analysis of variance. Fig.1 shows the results of the LC/ES/MS analysis of phospholipids from control LDL (Fig. 1 A) and from LDL preparations containing Glc-PtdEtn (Fig. 1 B). The Glc-PtdEtn is well resolved from PtdEtn and from other phospholipids seen in the negative ion profile, where the choline phospholipids yield minimal response. The added glucosylated 16:0–18:2 Gro-PEtn (m/z 876) [M − 1]− was easily detected by single ion mass chromatogram (Fig. 1 C). By using appropriate correction factors, it was estimated that the total PtdEtn content of LDL had been increased from 3 to 6% of total phospholipid (20–50% of total PtdEtn) as a result of the enrichment. Fig.2 shows that incubation of Glc-PtdEtn LDL (10–30 nmol of Glc-PtdEtn/mg of LDL protein) with THP-1 cells leads to a specific increase in CE and TC accumulation when compared with LDL containing non-glycated PtdEtn (10.11 ± 1.54 versus3.97 ± 0.35 μg/mg cell protein, p < 0.01). The supplementation of LDL with non-glucosylated PtdEtn did not have any significant effect on CE accumulation. In order to differentiate between the contributions of LDL lipid and protein glycation on CE accumulation, these results were compared with those obtained with LDL that had been glycated in both protein and lipid moieties by incubation with glucose (50 mm, 7 days, 37 °C). The glycated LDL preparation caused a CE accumulation (11.95 ± 1.38 μg/mg cell protein) that was comparable to that obtained when macrophages were incubated with Glc-PtdEtn LDL (10.11±1.54 μg/mg cell protein). Fig. 3 shows that incubation of THP-1 cells with Glc-PtdEtn LDL also causes a significant accumulation of TG (285.32 ± 4.38 μg/mg cell protein) which again was indistinguishable from the accumulation obtained by incubation with LDL glycated in lipid and protein (280.78 ± 3.98 μg/mg cell protein). LDL supplemented with non-glucosylated PtdEtn did not stimulate TG accumulation (185.57 ± 3.42 μg/mg cell protein). Furthermore, the increase in TG content of the macrophages seen at 50 μg/ml LDL was only slightly lower than that seen at 100 μg/ml LDL indicating a leveling off in the response. Interestingly, the TG levels in THP-1 cells represented the major neutral lipid pool contributing up to 85% of the cell neutral lipid content. Previous work done in our laboratory had shown that Glc-PtdEtn is more susceptible to oxidation and also facilitates the oxidation of other LDL phospholipids as well as the CE present in the lipid core of the molecule (11Ravandi A. Kuksis A. Shaikh N.A. Clin. Chem. 1998; 44 Suppl. 2: A73Google Scholar). In the present study we investigated the effects of Glc-PtdEtn on LDL oxidation and on foam cell formation. We oxidized Glc-PtdEtn-enriched LDL by copper and measured the CE and TG deposition in THP-1 cells. Fig.4 A shows the effect of incubation of oxidized PtdEtn LDL and oxidized Glc-PtdEtn-enriched LDL upon the accumulation of free cholesterol, CE, and TC by THP-1 cells in comparison to LDL. The greatest increase is seen in the CE. The oxidized PtdEtn LDL caused a 4-fold increase in the CE content of the cells (18.36 ± 1.95 μg/mg cell protein) compared with control LDL (3.97 ± 1.95 μg/mg cell protein). There was a significant further increase after incubation with oxidized Glc-PtdEtn LDL (28.36 ± 3.25 μg/mg cell protein). Fig. 4 B shows the effect of incubation of oxidized PtdEtn-enriched LDL and oxidized Glc-PtdEtn-enriched LDL upon the accumulation of TG by THP-1 cells in comparison to LDL enriched with PtdEtn. The incubation with LDL oxidized after enrichment with Glc-PtdEtn (350.91 ± 26.32 μg/mg cell protein) gave significantly greater accumulation than the incubation with oxidized LDL enriched with PtdEtn (310 ± 21.84 μg/mg cell protein), both values being significantly greater than those obtained for non-oxidized LDL or LDL enriched in PtdEtn. This increase in neutral lipid deposition correlated with the increase in PtdCho oxidation products due to the presence of Glc-PtdEtn in LDL during oxidation. The phospholipids from oxidized Glc-PtdEtn LDL were analyzed by LC/ES/MS. The total positive ion current (Fig.5 A) shows the separation of Glc-PtdEtn LDL phospholipids after oxidation with copper ions. The major phospholipid oxidation products were identified as the PtdCho hydroperoxides (PtdCho OOH), PtdCho core aldehydes (PtdCho Ald), and PtdCho isoprostanes (PtdCho IsoP) in the positive mode of ionization. Fig. 5 B shows representative mass chromatograms. In the absence of reference standards the structures proposed for the isoprostanes and core aldehydes containing hydroxyl groups must remain tentative. The pro-oxidative effect of Glc-PtdEtn resulted in a 4-fold increase in the production of total hydroperoxides, core aldehydes, and isoprostanes, when compared with LDL enriched in PtdEtn (Fig.6).Figure 5LC/ES/MS analysis of oxidized PtdCho in oxidized Glc-PtdEtn LDL . Positive total ion current profile of oxidized Glc-PtdEtn LDL (A). Single ion plots of representative PtdCho oxidation products (B). Glc-PtdEtn LDL was oxidized by incubation with 5 μm CuSO4 in 0.1 m PBS for 12 h at 37 °C. The total lipid extract of the oxidized LDL was dissolved in chloroform/methanol 2:1, and 20 μl of the sample containing 10 μg lipid was analyzed. Structural assignments for aldehydes and hydroperoxides are according to reference standards. Ions 832 and 830 were identified on the basis of retention time and molecular weight.View Large Image Figure ViewerDownload (PPT)Figure 6Increased oxidative susceptibility of LDL due to Glc-PtdEtn . Levels of PtdCho hydroperoxides (PtdCho OOH), PtdCho aldehydes, and PtdCho isoprostanes in oxidized LDL enriched with PtdEtn (▨) or Glc-PtdEtn (▪). Levels represent sum of four of the most abundant ions for each class of oxidized PtdCho.View Large Image Figure ViewerDownload (PPT) In order to quantitate the rate of uptake of LDL, we utilized DiI-labeled lipoproteins. After a 4-h incubation with cells, the fluorescence intensity was measured as an indicator of total cell-associated lipoprotein. Cells incubated with DiI LDL enriched with Glc-PtdEtn (10–200 μg/ml) showed a much more rapid increase in cell-associated lipoprotein when compared with control LDL enriched with PtdEtn (Fig.7). The uptake of DiI LDL enriched in Glc-PtdEtn occurred at the same rate as that for glycated LDL showing that the presence of Glc-PtdEtn in LDL can mimic the properties of glycated LDL. The uptake of all LDL preparations tended to level off in the range of 100–200 μg/ml LDL. Fig. 8 shows the time course (4, 6, 10, and 20 h) of macrophage accumulation of fluorescently labeled lipoprotein. At all time points the average accumulation was consistently higher for the LDL preparation containing Glc-PtdEtn when compared with control LDL. The accumulation of Glc-PtdEtn LDL was parallel to that of glycated LDL at all time points. To explore the specificity Glc-PtdEtn LDL interaction with macrophages, we investigated the ability of AcLDL, PtdEtn LDL, glycated LDL, and LDL + Glc-PtdEtn at increasing concentrations to compete with DiI-labeled Glc-PtdEtn (50 μg/ml) for uptake by macrophages (Fig.9). After the 4-h incubation the AcLDL and control LDL were only able to inhibit 5–6% of the cell association. Glycated LDL led to 80% inhibition of Glc-PtdEtn LDL uptake, whereas the unlabeled Glc-PtdEtn resulted in 93% inhibition of uptake after correction for nonspecific association. Fluorescence microscopy analysis of LDL uptake by THP-1 cells (Fig.10) showed increased fluorescence in cells incubated with DiI Glc-PtdEtn LDL compared with cells incubated with the DiI PtdEtn LDL. The distribution of fluorescence in cells incubated with DiI-labeled Glc-PtdEtn LDL (Fig. 10 C) shows a punctated pattern believed to be indicative of receptor-mediated endocytosis from coated pits and vesicles to endosomes and lysosomes (24Acton S. Rigotti A. Landschulz K.T. Xu S.Z. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2005) Google Scholar). This pattern was also observed for glycated LDL and for AcLDL as already reported (24Acton S. Rigotti A. Landschulz K.T. Xu S.Z. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2005) Google Scholar). Cells incubated with DiI PtdEtn LDL showed a diffuse pattern of fluorescence with less intensity, indicative of lack of uptake.Figure 9Competitive inhibition of Glc-PtdEtn LDL uptake by THP-1 macrophages. Macrophages were incubated 4 h with 50 μg/ml DiI-labeled Glc-PtdEtn LDL together with varying concentrations of unlabeled AcLDL (▪), glycated LDL (▴, LDL incubated with 50 mm glucose, 1 week), and unlabeled LDL enriched with Glc-PtdEtn (●). Values shown are means of triplicate incubations that varied by less than 10%.View Large Image Figure ViewerDownload (PPT)Figure 10Uptake of Glc-PtdEtn LDL by THP-1 macrophages. THP-1 cells (1 × 106 cells/ml) activated with PMA were incubated with 50 μg/ml, DiI-labeled PtdEtn LDL (A), glycated LDL (B), Glc-PtdEtn LDL (C), and AcLDL (D). After the incubation for 3 h at 37 °C cells were washed with cold PBS, fixed with formaldehyde in PBS, and then analyzed by fluorescence microscopy.View Large Image Figure ViewerDownload (PPT) Fig.11 shows that the glycation of PtdEtn increases the negativity of the molecule. Injection of equimolar amounts of PtdEtn and Glc-PtdEtn, chromatographed on normal phase silica column, resulted in a 1.5-fold higher response for Glc-PtdEtn when compared with PtdEtn. The calculated molar response of Glc-PtdEtn was comparable to that of other anionic phospholipids, such as PtdIns and PtdSer (Table I).Table ILC/ES/MS relative ionization intensities in negative ionization mode for anionic phospholipids as compared with PtdEtnPhospholipidIonization ratioPtdEtn1Glc PtdEtn1.53 ± 0.32PtdSer1.62 ± 0.41PtdIns1.57 ± 0.38CL2.12 ± 0.37Equimolar amounts of each phospholipid was injected in the LC/ES/MS system. The ratios were calculated by measuring the peak area for each phospholipid compared with that of PtdEtn. PtdEtn, phosphatidylethanolamine; Glc-PtdEtn, glucosylated phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; CL, cardiolipin. Open table in a new tab Equimolar amounts of each phospholipid was injected in the LC/ES/MS system. The ratios were calculated by measuring the peak area for each phospholipid compared with that of PtdEtn. PtdEtn, phosphatidylethanolamine; Glc-PtdEtn, glucosylated phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; CL, cardiolipin. This study demonstrates that incorporation of Glc-PtdEtn into LDL at a level commonly achieved in hyperglycemia can fully account for the increased uptake of glycated LDL by macrophages. Furthermore, the resulting increased accumulation of CE and TG by macrophages exceeds the amounts of neutral lipids anticipated to be transferred to the cells on the basis of uptake of the lipoprotein. This increase was mainly due to accumulation of TG which contributed over 85% of the cell total neutral lipid (TC + TG). The glycated subfraction of LDL, which is elevated in diabetic subjects, has been reported to promote CE deposition and to increase the rate of CE synthesis in macrophages (25Lyons T.J. Klein R.L. Baynes J.W. Stevenson H.C. Lopes-Virella M.F. Diabetologia. 1987; 30: 916-923Crossref PubMed Scopus (85) Google Scholar), but TG levels had not been measured. Although there was a significant increase in CE deposition in our THP-1 cells, the major neutral lipid accumulated was TG. In this connection it may be noted that recent studies have shown that THP-1 cells contain a large and metabolically active TG pool but a relatively inactive pool of CE (26Kritharides L. Christian A. Stoudt G. Morel D. Rothblat G.H. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1589-1599Crossref PubMed Scopus (94) Google Scholar). A large and active TG pool has also been seen in other human macrophage cultures, but the regulation of TG metabolism in these cells is not known. The general effects of glycation on LDL interaction with macrophages have been extensively investigated (4Lopes-Virella M.F. Klein R.L. Lyons T.J. Stevenson H.C. Witztum J.L. Diabetes. 1988; 37: 550-557Crossref PubMed Google Scholar, 5Gonen B. Baenzinger J. Schonfeld G. Jacobson D. Farrar P. Diabetes. 1981; 30: 875-878Crossref PubMed Google Scholar). Non-enzymatic glycation of LDL has been shown to increase the fluidity of the phospholipid monolayer, to result in an altered lipid composition (27Rabini R.A. Ferretti G. Galassi R. Taus M. Curatola G. Tangorra A. Fumelli P. Mazzanti L. Clin. Biochem. 1994; 27: 381-385Crossref PubMed Scopus (14) Google Scholar), and, possibly, in an increased susceptibility to oxidation. These alterations in biological and physicochemical properties have been attributed solely to ApoB glycation without consideration of LDL phospholipid glycation. The inability of AcLDL to compete with Glc-PtdEtn LDL suggests the participation of receptors other than the LDL or scavenger receptor. Due to the increased negative charge of Glc-PtdEtn compared with PtdEtn and structural similarities to acidic glycerophospholipids, it is possible that receptors for Glc-PtdEtn uptake could be found in the family of receptors that identify anionic phospholipids such as CD 36 (28Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). It has been shown that CD 36 present in photoreceptor outer segment cells binds PtdSer- and PtdIns-rich liposomes resulting in their uptake (29Ryeom S.W. Silverstein R.L. Scotto A. Sparrow J.R. J. Biol. Chem. 1996; 271: 20536-20539Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). LC/ES/MS shows that glucosylation of PtdEtn increases the negative charge of the amino phospholipid, and with structural and physicochemical similarities to acidic glycerophospholipids, Glc-PtdEtn could increase uptake of LDL by CD 36. In fact, macrophages have already been shown to specifically phagocytose oxidized RBC in which the outer leaflet of the phospholipid bilayer exposes anionic phospholipids, specifically PtdSer. Sambranoet al. (30Sambrano G.R. Terpstra V. Steinberg D. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3442-3448Crossref PubMed Scopus (41) Google Scholar) have demonstrated that peroxidation of RBC results in disruption of the asymmetry of the plasma membrane phospholipid bilayer resulting in PtdSer exposure and recognition of oxidized RBC by macrophages and their subsequent phagocytosis. Inhibition of uptake of oxidized RBC by OxLDL showed that the scavenger receptors on macrophages were responsible for a major part of the oxidized RBC recognition (31Ottnand E. Parthasarathy S. Sambrano G.R. Ramprasad M.P. Quehenberger O. Kondratenko N. Green S. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1391-1395Crossref PubMed Scopus (139) Google Scholar). Recently another receptor, SRB1, has been shown to have the capability of binding anionic phospholipids. The inhibition of the selective uptake of high density lipoprotein CE in liver parenchymal cells by modified LDL, in particular OxLDL and ionic phospholipids, has suggested that in liver the SRB1 is responsible for the efficient uptake of high density lipoprotein CE (32Fluiter K. van Berkel T.J. Biochem. J. 1997; 326: 515-519Crossref PubMed Scopus (43) Google Scholar). The concept that negatively charged molecules can form complexes with LDL is not restricted to negatively charged phospholipids. Basu et al. (15Basu S.K. Goldstein J.L. Anderson R.G.W. Brown M.S. Proc. Natl. Acad. Sci U. S. A. 1976; 73: 3178-3182Crossref PubMed Scopus (823) Google Scholar) has demonstrated that complexes containing LDL and large molecular weight dextran sulfates are avidly metabolized by macrophages via a receptor, which appears to be distinct from the AcLDL receptor. Besides the effects of PtdSer on macrophage uptake, association of other negatively charged phospholipids, like cardiolipin, with LDL increases its uptake and the deposition of cholesteryl esters by macrophages (33Greenspan P. Ryu B.H. Mao F. Gutman R.L. Biochim. Biophys. Acta. 1995; 1257: 257-264Crossref PubMed Scopus (25) Google Scholar). It has also been claimed that an alteration in the composition of LDL phospholipids caused by phospholipase D (34Aviram M. Maor I. J. Clin. Invest. 1993; 91: 1942-1952Crossref PubMed Scopus (23) Google Scholar) and phospholipase A2 (35Aviram M. Maor I. Biochem. Biophys. Res. Commun. 1992; 185: 465-472Crossref PubMed Scopus (42) Google Scholar) can influence the metabolism of LDL by macrophages, but it is not clear whether the effect is related to a relative increase in acidic phospholipids or lysophospholipids, which would be anticipated to result from the action of these enzymes. Another possible receptor for Glc-PtdEtn is the newly characterized AGE receptor (36Vlassara H. Brownlee M. Cerami A. J. Exp. Med. 1986; 164: 1301-1309Crossref PubMed Scopus (123) Google Scholar). This receptor has been demonstrated to have specificity toward protein glycation products (37Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar). Many of the binding assays performed with this receptor have only focused on the protein glycation products and the interaction of Glc-PtdEtn with AGE receptor-advanced glycation end products has not been investigated. The present study demonstrates that peroxidation increases the neutral lipid deposition due to LDL beyond the extent achieved by glucosylation of PtdEtn alone. We have previously shown that glycation of LDL PtdEtn promotes the oxidation of both surface and core components of LDL (11Ravandi A. Kuksis A. Shaikh N.A. Clin. Chem. 1998; 44 Suppl. 2: A73Google Scholar). The increase in susceptibility of LDL in the presence of Glc-PtdEtn to oxidation was characterized by an increase in products of phospholipid oxidation such as PtdCho core aldehydes, which have been specifically shown to induce increased monocyte-endothelial interactionsin vitro (38Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). This is in agreement with the observation that both apolipoprotein and lipid in glycated LDL are more susceptible to oxidation in the presence of Fe3+ than non-glycated LDL (39Sakurai T. Kimura S. Nakano M. Kimura H. Biochem. Biophys. Res. Commun. 1991; 177: 433-439Crossref PubMed Scopus (52) Google Scholar). It was suggested that Fe3+ could be coordinated with the endiol group in Amadori compounds and could be converted to ferryl iron with a high redox potential. It was postulated, on the basis of the known chemistry of Schiff base and Amadori products, that during the nucleophilic addition of glucose to protein amino groups, as in the early glycation, the glycation products of proteins deposited in the arterial wall could themselves generate free radicals capable of oxidizing lipids (40Mullarkey C.J. Edelstein D. Brownlee M. Biochem. Biophys. Res. Commun. 1990; 173: 932-939Crossref PubMed Scopus (643) Google Scholar). This possibility is supported by the demonstration that both Schiff base and Amadori glycation products generate free radicals in a ratio of 1:1.5 and that these radicals cause increased peroxidation of membrane lipids (40Mullarkey C.J. Edelstein D. Brownlee M. Biochem. Biophys. Res. Commun. 1990; 173: 932-939Crossref PubMed Scopus (643) Google Scholar). In similar studies Kobayashi et al.(41Kobayashi K. Watanabe J. Umeda F. Masakado M. Ono Y. Taniguchi S. Yanase T. Hashimoto T. Sako Y. Nawata H. Horm. Metab. Res. 1995; 27: 356-362Crossref PubMed Scopus (7) Google Scholar) demonstrated that oxidation of glycated LDL results in increased binding and degradation by cultured bovine aortic endothelial cells compared with normal or oxidized LDL. The present study demonstrates that glucosylation of the PtdEtn component of LDL exerts a dramatic effect on LDL metabolism which mimics that of total LDL glycation. This is the first direct evidence that glycation of LDL lipids results in increased LDL uptake and in CE and TG accumulation in macrophages. In conclusion, this study suggests that the presence of Glc-PtdEtn in LDL plays an important role in the alteration of the biological activity of LDL and reconciles diverse observations regarding the binding of glycated and peroxidized LDL to macrophages and other cells and may account for the increased atherogenesis in diabetes.