Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2830-2836

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Igau, B.
Right arrow Articles by Fiévet, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Igau, B.
Right arrow Articles by Fiévet, C.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2830-2836.)
© 1997 American Heart Association, Inc.

Articles

In Vivo Glucosylated LpA-I Subfraction

Evidence for Structural and Functional Alterations

Bruno Igau; Graciela Castro; Véronique Clavey; Christian Slomianny; Régis Bresson; Pierre Drouin; Jean-Charles Fruchart; ; Catherine Fiévet

From Serlia et INSERM U325-1 rue du Professeur Calmette, Institut Pasteur de Lille, Lille, France (B.I., G.C., V.C., J.C.F.); INSERM U42 - Certia, Villeneuve d'Ascq, France (C.S.);. Centre Hospitalier de Douai, Douai, France (R.B.); and Service de Diabétologie, Chu de Nancy et CIC INSERM du Chu de Nancy, Nancy, France (P.D.).

Correspondence to Catherine Fiévet, Serlia et INSERM U325-1 rue du Professeur Calmette, Institut Pasteur de Lille, 59019 Lille, France.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract This study compared the structural and functional properties of glucosylated and non-glucosylated LpA-I particle subfractions (GLpA-I and NGLpA-I, respectively) isolated from patients with poorly controlled type 1 (insulin-dependent) diabetes. Compared with NGLpA-I, GLpA-I showed an enrichment in triglycerides (P<.05) and a depletion in phospholipid (P<.05) content. Moreover, the triglycerides-to-cholesteryl esters ratio was increased (P<.05), suggesting an increased cholesteryl ester transfer protein activity and a possible transport defect that accelerates atherogenesis. The surface-to-core constituents ratio, an indirect estimate of particles size, is lower in GLpA-I (P<.01) than in NGLpA-I, correlating well with a larger median size (P<.05) as seen by electron microscopy. The apolipoprotein (apo) A-I conformation was evaluated through determination of the immunological accessibility of three different domains defining specific epitopes for anti-apo A-I monoclonal antibodies. We observed a marked decreased accessibility for two of these regions, which interestingly have already been implicated in the interaction with cells. Cell culture data suggest that nonenzymatic glycosylation occurring on apo A-I can modify lipoprotein function, since it results in a decreased binding of GLpA-I to HeLa cells and impaired cholesterol efflux from Fu5AH rat hepatoma cells.


Key Words: glucosylation • LpA-I lipoprotein particles • reverse cholesterol transport • atherosclerosis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Atherosclerosis is a major cause of morbidity and mortality in patients with diabetes mellitus.1 The explanation for this increased incidence is not yet readily established, and cannot fully be explained through conventional risk factors. Epidemiological studies examining the frequency of atherosclerotic manifestations have shown an increased risk in diabetic patients, even after correction for total plasma cholesterol, HDL cholesterol, arterial pressure, and smoking.2 3 Although lipid and lipoprotein abnormalities are common in patients with type 2 (non-insulin–dependent) diabetes mellitus, patients with type 1 (insulin-dependent) diabetes mellitushave almost normal concentrations of the major lipoproteins, and patients with fair to good diabetic control exhibit less of an atherogenic profile than non-diabetic subjects.4 It has been therefore proposed that, in diabetes, atherogenesis may be associated with subtle alterations in the structure and metabolism of some lipoproteins. In fact, numerous lipoprotein modifications in diabetes may affect cell interactions. Among them, the non-enzymatic glucosylation occurring on apolipoproteins has been already reported as one factor that can modify lipoprotein function so that it is potentially atherogenic.5

Like VLDL and LDL, HDL are glucosylated in hyperglycemic diabetic patients,6 and data indicate that in vitro glucosylation of HDL leads to an impaired reverse cholesterol transport process.7 Although extrapolating this observation to an in vivo situation is difficult, it appears that HDL in patients with diabetes might be functionally abnormal.

The lipid-binding properties of glucosylated apo A-I have been shown to be altered in vitro and in vivo,8 9 and recently it has been reported that glucosylated apo A-I purified from diabetic samples was less efficient for activation of lecithin: cholesterol acyltransferase in vitro.10 Even if non-enzymatic glucosylation of HDL is known to occur in vivo, the concentration of circulating glucosylated lipoproteins is relatively small and their role in the development of atherosclerosis in diabetic patients needs to be elucidated.

Evidence indicates that HDL consists of several distinct particles with specific functional and clinical properties, and that lipoprotein containing apo A-I but not apo A-II (ie, LpA-I) represents the potentially anti-atherogenic lipoprotein fraction within HDL.11 In the present study, we report the preparative isolation and characterization of LpA-I particles from plasma of patients with poorly controlled type 1 diabetes and describe structural and functional alterations of the in vivo glucosylated LpA-I subfraction.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Subjects
The study included 15 C-peptide-negative men (basal C-peptide <0.03 nmol/L and no increment after glucagon) with type 1 diabetes. Plasma C-peptide levels were measured by radioimmunoassay (CIS Biointernational) after an overnight fast, before and 6 minutes after an intravenous injection of 1 mg glucagon.

All participants were hospitalized because of poor metabolic control. Their fasting plasma glucose level was 11.0 ±2.8 mmol/L. Mean HbA1c level was 9.7±1.5% (normal values: 4.2 to 5.6% using high-pressure liquid chromatography on biorex resin, Biorad).

None of the subjects had ketosis (Acetest Bayer Diagnostics) or dehydration. No patient was taking medication other than insulin (intensive conventional insulin therapy, 3 injections per day). Patients gave their written informed consent to participate in the study, which was approved by the Medical Ethics Committee of Nancy University.

Plasma Samples
Blood drawn after an overnight fast was collected into Vacutainer EDTA-containing tubes. Plasma was isolated immediately by low-speed centrifugation at 4°C. A mixture of preservatives was promptly added. These additives (and their final concentrations) were as follows: EDTA (0.27 mmol/L), cis-aminocaproic acid (0.9 mmol/L), chloramphenicol (0.6 mmol/L), and glutathione (0.3 mmol/L). The individual plasma samples were then combined into 5 pooled samples (3 individual plasma per pool). Therefore, the results described concern 5 different lipoprotein particle preparations.

Lipoprotein Particle Isolation
LpA-I lipoprotein particles were isolated from each pool of plasma by sequential immunoaffinity chromatography according to a previously described flow diagram.12 Briefly, each sample was applied sequentially to anti-apo B, anti-apo E and anti-apo A-II immunosorbents in 10 mmol/L Tris, 0.15 mol/L NaCl, pH 7.4. At each step, the nonspecifically bound particles were eluted with 10 mmol/L Tris, 0.5 mol/L NaCl, pH 7.4. The fractions not retained by the anti-apo A-II column were applied on an anti-apo A-I immunosorbent and the specifically bound lipoproteins were eluted with 3 mol/L NaSCN. These particles were immediately filtered through a column packed with Sephadex G25-Fine, dialyzed against 10 mmol/L Tris, 0.15 mol/L NaCl, pH 7.4, concentrated in a multiple micro-Prodicon system (Bio-Molecular Dynamics) and then filter sterilized using a 0.22- µm Millipore filter. These particles will thereafter be referred to as total LpA-I. They were apo E-, apo B- and apo A-II-free.

From a pool of fresh normolipidemic plasma from control subjects, a LpA-I standard batch was likewise purified, concentrated and sterilized as indicated above. It was stored at 4°C in sterile aliquots under nitrogen gas. No proteolytic degradation occurred throughout the time of the study, as confirmed by electrophoresis and immunological analysis as further described.

GLpA-I NGLpA-I particles were further separated from total LpA-I by affinity chromatography on a column of amino-phenyl boronate (Glyc-Affin GHb, Innovative Biomedical Technology ). The nonretained fraction (NGLpA-I) was eluted with a 0.25 mol/L ammonium acetate buffer, pH 8.5, and the specifically bound proteins (GLpA-I) were eluted with 0.2 mol/L sorbitol in the same buffer. After extensive dialysis against 10 mmol/L sodium phosphate, 0.15 mol/L NaCl (PBS), pH 7.4, the particle subfractions were concentrated and filtered as for total LpA-I. The glucosylation degree was quantified using the trinitrobenzenesulfonic acid assay (TNBS) for free lysine and NH2-terminal amino groups.13

Iodination of HDL3 and LpA-I
HDL3 isolated from a normolipidemic plasma sample by sequential preparative ultracentrifugation (d=1.125 to 1.21 g/mL) and aliquots of LpA-I standard particles were radioiodinated with iodine-125 (Na125I) according to a modification of McFarlane's procedure.14 125I-Labeled HDL3 or 125I-labeled LpA-I used in radioimmunoassays or cellular interaction studies, respectively, were kept in tightly capped vials, in the dark, under nitrogen at 4°C for no longer than 2 weeks. Five different labeled preparations were used to perform the study. The mean specific activity was 350 cpm/ng of protein.

Plasma Determinations
TC and TG were measured by enzymatic test kits from Boehringer Mannheim, adapted to a Hitachi 705 analyzer. LpA-I was measured by differential electroimmunoassay on ready-to-use plates.15 Concentrations of TG, TC and LpA-I in the 5 pooled samples were 1.32±0.50 mmol/L, 5.46± 0.98 mmol/L, and 43±10 mg/dL (mean±SD), respectively.

Physical and Chemical Lipoprotein Analysis
Protein concentration was determined by the method of Lowry et al,16 with bovine serum albumin (BSA) used as a standard.

In order to verify the apolipoprotein pattern of LpA-I particles, a sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis (SDS-PAGE) was performed using 319% gels in Tris-HCl (0.1 mol/L), glycine (0.8 mol/L), pH 8.3, containing SDS (0.1%,wt/vol) (150 V, 25 mA, 45 minutes, SE 250 Hoefer Shlighty II electrophoresis apparatus).

TC and TG were measured with Boehringer-Mannheim kit tests, as for plasma determinations. FC was determined manually with a modified Boehringer-Mannheim kit in which cholesteryl ester hydrolase was omitted. CE were calculated as the difference between total and free cholesterol multiplied by a factor of 1.68. PL were assessed manually with test kits from BioMérieux. A statistical comparison of the data between GLpA-I and NGLpA-I was performed using the Mann-Whitney test (U test).

The size determination was performed by electron microscopy and native gradient gel electrophoresis. Lipoprotein particles were negatively stained and their diameters were measured from electron micrographs, as previously described.17 Mean diameters were obtained by measuring the diameters of 500 particles per sample. The particles distribution was compared by a median size test (U test) followed by a {chi}2 test.

Molecular mass of LpA-I particles was determined through a native 8-25% gradient polyacrylamide gel electrophoresis (Phast gel, Pharmacia). The electrophoresis was performed in a Phast system apparatus (Pharmacia) (400 V, 10 mA, 1 hour, 15°C). Lipoprotein bands were visualized by means of staining with Phast Blue R-250 in the Phast system unit development and their molecular weight distribution was analyzed using a densitometer (Digital Imaging Station, Biocom 500). Five reference molecular weight standards were run at the same time (HMW calibration kit, Pharmacia).

Immunological Lipoprotein Particles Analysis
The immunoreactivity of apo A-I epitopes was determined in each LpA-I preparation by a competitive radioimmunoassay using 3 previously characterized anti-apo A-I monoclonal antibodies (A44, A05, and A11).18 Their specific epitopes along the apo A-I molecule were mapped between amino acid 25 and 82 (A05) and between amino acid 149 and 186 (A44). The monoclonal antibody A11 reacts with a discontinuous epitope at residues 99 to 105 and 126 to 132.

Methodological details for the assay have been described.19 Microtiter wells were coated with the monoclonal antibody. After washing and saturation steps were performed, serial triplicate increasing amounts of competitors (GLpA-I, NGLpA-I, and LpA-I standard, expressed as protein concentration) were then added with a fixed amount of 125I-labeled HDL3. After incubation overnight at room temperature, the wells were washed and radioactivity was counted. B/Bo ratios (where B and Bo are specific cpm bound in the presence and absence of competitors, respectively) versus lipoprotein concentrations were plotted. The displacement curves were linearized by logit-log transformation of the data20 and slopes were calculated. They correspond to the apparent affinity of the LpA-I preparations for each antibody. These slopes were compared by a test for heterogeneity based on the general linear model procedure.21 The apparent apo A-I content (number of epitopes) was calculated from competitive displacement curves. The results were expressed as a percentage of the standard to which an arbitrary expression of 100 percent was assigned to each epitope. So, a percentage value below 100 indicates that less competitor is needed to achieve a same degree of displacement of the labeled HDL3 ligand and reflects a better accessibility of the measured epitope. In contrast, a percentage value above 100 indicates a lower immunological accessibility of an epitope for its competitor. The reproducibility of the procedure was assessed from LpA-I standard data; the coefficients of variation between assays were around 10%. Apo A-I content between GLpA-I and NGLpA-I preparations was compared using the U test.

Cell Cultures
HeLa cells22 were grown in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 units/mL), streptomycin (100 µg/mL), and fetal calf serum (10%, wt/vol) in 30-mm diameter multiwell plates.

Fu5AH rat hepatoma cells23 were maintained in minimal essential medium (MEM) containing calf serum (5%, vol/vol), glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 µg/mL); 20x103 cells/mL were plated on 24-mm multiwells plates using 2 mL/well.

Cellular Interaction Studies
Competition studies for binding were performed on confluent HeLa cells. Prior to the experiments, the cells were washed with DMEM without fetal calf serum and then incubated for 1 hour at 37°C in this medium. The medium was then removed and 700 µL of fresh DMEM/BSA containing 10 µg/mL of 125I-labeled LpA-I and in increasing concentrations (0 to 60 µg/mL, expressed as protein concentration) of nonlabeled lipoproteins as competitors (GLpA-I, NGLpA-I, or LpA-I standard) were added to each well for 2 hours of incubation at 4°C. Radioactivity was measured after treatment of the washed monolayer with 1 mL of 0.1 mol/L NaOH. Nonspecific binding was determined by measuring the amount of radioactivity when incubations were carried out at 4°C in the presence of a 20-fold excess of unlabelled HDL3. This unspecific binding ranged between 10% and 25%.

Before efflux experiments, Fu5AH cells were labeled by incubation at 37°C for 48 hours in growth medium (MEM with 5% [vol/vol] bovine calf serum and antibiotics) and labeled with radioactive 3H-cholesterol (1 µCi/well). To allow equilibration of the label, cells were rinsed and incubated for 24 hours in MEM containing BSA (0.5%, wt/vol).

In cholesterol efflux experiments, the GLpA-I, NGLpA-I or LpA-I standard (50 µg/mL, expressed as protein concentration) was incubated for 1, 3, and 5 hours at 37°C with the 3H-cholesterol-loaded cells. After incubation, media were removed, centrifuged to discard cell debris and counted for radioactivity. The cells were washed twice with refrigerated 10 mmol/L phosphate buffer (PBS) and dissolved in 0.5 mL of 0.1 mol/L NaOH. An aliquot of the alkaline digest was used for radioactivity counts and the remainder was used for protein quantification.

The protein content of cells was determined according to Lowry et al.16


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
The electrophoretic pattern of GLpA-I and NGLpA-I preparations confirmed that apo A-I was the major protein. Only traces of apo C were detected (not shown).

Tthe extent of glucosylation was measured in each GLpA-I preparation. Results are expressed as the percentage of decrease in absorbance at 340 nm, compared with NGLpA-I, for which an arbitrary absorbance of 100% was assigned. A mean of 13.5±1.8% of free lysine residues in GLpA-I proteins was glucosylated, this amount corresponding to 2 to 3 glucose molecules per apoA-I molecule.24 Table 1Down shows the chemical composition of the GLpA-I and NGLpA-I preparations. The data were statistically compared by the Mann-Whitney test (U test). Compared with NGLpA-I preparations, GLpA-I preparations had a significantly higher TG level (P<.05) and a significantly lower PL content (P<.05). No net changes occurred in FC or CE. The TG-to-CE ratio was therefore significantly enhanced (P<.01), but the FC-to-PL ratio was unchanged, since the corresponding standard deviations were elevated. The surface- (protein, FC, and PL) -to-core (TG and CE) constituents ratio exhibited a significant decrease in GLpA-I (P<.01).


View this table:
[in this window]
[in a new window]
  		Table 1. Chemical Composition of LpA-I Subfractions Recovered by Immunochromatography From Samples Obtained From Patients With Type I Diabetes

Each particle preparation was visualized by electron microscopy. Fig 1Down shows the size distribution of LpA-i subfractions. The statistical comparison of median sizes (U test) followed by a {chi}2 test indicates a shift toward larger size for GLpA-I (11.7±0.9, compared with 10.1±0.7 nm for NGLpA-I, P<.05).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Size distibution of LpA-I subfractions. LpA-I subfractions were isolated from patients with type 1 diabetes by immunoaffinity chromatography and then further separated into glucosylated (stippled square) and nonglucosylated (solid square) subfractions through an amino-phenyl boronate column. The sizes have been calculated from electron microscopy photographs (500 randomly selected particles per sample). Four subclasses, which included particles from 5 to 7.5 nm (A), 7.5 to 10 nm (B), 10 to 15 nm (C), and 15 to 20 nm (D), were defined. Data represent the mean±SD for 5 preparations obtained from 15 plasma samples combined into 5 pools.

This result is in agreement with native gradient gel electrophoresis patterns. Four classes of decreasing molecular mass (I to IV) were defined, and the mean (±SD) mass of each subfraction within each class was calculated. An increased molecular mass (P<.05) was obtained for GLpA-I as compared with NGLpA-I in the higher molecular mass class (Table 2Down).


View this table:
[in this window]
[in a new window]
  		Table 2. Molecular Mass (kDa) Determinations by Native Gradient Gel Electrophoresis of LpA-I Subfractions Recovered From Samples Obtained From Patients With Type I Diabetes

The abilities of the LpA-I preparations to compete with 125I-labeled HDL3 for binding to 3 anti-apo A-I mAbs were compared. After linearization of the curves by logit-log transformation of the data, the calculation of the slopes provided the apparent affinity of the different LpA-I for each monoclonal antibody. No significant differences were found between the slopes of the GLpA-I or NGLpA-I and the LpA-I standard, demonstrating a similar apparent affinity in all these preparations (data not shown). Therefore, the apparent apo A-I content of each epitope in LpA-I fractions could be determined, reflecting their accessibility. The data are compiled in (Table 3Down and correspond to mean (±SD) values. The immunoreactivity of the epitopes for monoclonal antibodies A44 and A05 did not differ between NGLpA-I and LpA-I standard, and the accessibility data were around 100%. However, the percentages were significantly higher for GLpA-I, thus reflecting a markedly decreased accessibility of both epitopes (P<.01). GLpA-I and NGLpA-I subfractions did not differ from each other in their immunoreactivities for the epitope for monoclonal antibody A11, and a lower value than 100% indicates even a better accessibility than the LpA-I standard for this epitope.


View this table:
[in this window]
[in a new window]
  		Table 3. Immunological Accessibility of LpA-I Subfractions Recovered From Samples Obtained From Patients With Type I Diabetes

Competition experiments of the LpA-I preparations on HeLa cells were studied at 4°C by incubating cells with increasing concentrations of competitors in the presence of a fixed amount of 125 I-labeled LpA-I standard. The results shown in Fig 2Down are expressed as the percentage of labeled LpA-I bound per milligram of cellular proteins, and represent the mean±SD of triplicate experiments performed in two independent assays. The competition curves of NGLpA-I fitted similarly with the curve of LpA-I standard. On the other hand, GLpA-I expressed a lower capacity for binding to cells. In fact, as expected, 10 µg/mL of LpA-I standard or NGLpA-I was necessary to obtain a 50% inhibition of 125 I-labeled LpA-I binding, whereas only 8% of binding inhibition could be observed for the same amount of GLpA-I.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Competition of HeLa cell binding of 125 I-labeled LpA-I subfractions from diabetic samples or LpA-I standard. Cells were incubated for 2 hours at 4°C in the presence of 10 µg/mL of 125 I-labeled LpA-I as ligand and increasing concentrations (0 to 60 µg/mL) of unlabeled GLpA-I (triangles), NGLpA-I (circles) and LpA-I standard (squares) as competitors. The values represent mean±SD of 5 lipoprotein subfractions preparations performed in triplicate dishes in two independent series of cells. The lipoprotein preparations were obtained from 15 plasma samples combined into 5 pools.

The ability of LpA-I subfractions to induce cholesterol efflux from Fu5AH rat hepatoma cells was studied. Fractional cholesterol efflux, expressed as a percentage, was calculated as the amount of label recovered in the medium divided by the total label (cells plus medium) in each well. Measurements were performed in triplicate on parallel dishes. As shown in Fig 3Down, there was a marked decrease in ability of GLpA-I to promote cellular cholesterol efflux when compared to LpA-I standard or NGLpA-I subfractions.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 3. Cholesterol efflux from Fu5AH cells induced by LpA-I subfractions isolated from diabetic samples or LpA-I standard. 3H-cholesterol–loaded cells were incubated for 1, 3, and 5 hours at 37°C with 50 µg/mL of GLpA-I (triangles), NGLpA-I (circles) or LpA-I standard (squares). The radioactivity appearing in the medium was expressed as a percentage of the total (cells plus medium) radioactivity. The values represent mean±SD of 5 lipoprotein subfractions preparations and are a compilation of triplicate experiments. The lipoprotein preparations were obtained from 15 plasma samples combined into 5 pools.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study we documented glucosylated LpA-I particles isolated from patients with poorly controlled diabetes present physico-chemical, conformational, and functional changes.

It has been previously reported that apoA-I undergoes nonenzymatic glucosylation in diabetic subjects.6 8 However, to infer a true physiologic significance, the association of this chemical modification with an altered function of the apolipoproteins has to be demonstrated. Data from the literature suggest this association does exist, because the functional activities (including receptor recognition, enzyme activation, and lipid binding) of HDL or glucosylated apo A-I isolated from diabetic samples are perturbed.8 9 10 25 Also, Duell et al reported that in vitro glucosylation of HDL3 impaired its functional ability to bind to the HDL binding site on cells and to promote intracellular cholesterol efflux.7 26

The composition and the size of HDL can determine its structure, and several studies have shown that changes in HDL structure can have major effects on the metabolism of this lipoprotein.27 28 29 In that way, for example, HDL composition could directly affect the esterified cholesterol transfer to apo B-containing lipoproteins30 31 or could influence the directional fluxes of cholesterol between cells and lipoproteins.32 33 In an attempt to explain the structure-function relationships involved in the HDL metabolism, some investigators have suggested that the conformation of apo A-I may play a fundamental role.34 35

In the present study, GLpA-I demonstrated abnormal lipid composition as compared with (NGLpA-I). A larger heterogeneity in the GLpA-I particle compositions was observed since more elevated values of standard deviation are reported. This observation is probably related to differences in the extent of glucosylation for each preparation, but additional experiments would be necessary to clarify this point. GLpA-I was characterized by a significant increase in TG (P<.05) and a slight decrease in CE content. The ratio of these core components was therefore significantly enhanced (P<.01), and several possible hypotheses can be posed to explain this finding. First, the changes may reflect the accelerated transfer of esterified cholesterol from HDL to apolipoprotein B-containing lipoproteins, which has been reported in type 1 diabetes.36 37 If so, an increased transfer may be due to an increase in the cholesterol ester transfer protein (CETP) activity and/or mass, but also may more simply be due to an exchange that could have been facilitated through the patients' hypertriglyceridemic state. Second, since reduced hepatic lipase activity has been reported in patients with type 1,38 this decrease could also be particularly relevant to GLpA-I when the enzyme acquires an altered affinity for glucosylated subfraction, leading to changes in the composition of lipoproteins. Not only is the transfer activity believed to play a central role with respect to atherosclerosis but also the transport of FC from cells to HDL is believed to represent a key step, with both phenomena defining, with the LCAT reaction, the reverse cholesterol transport process. Earlier studies have shown that the FC-to-lecithin ratio of intact lipoproteins obtained from diabetic patients appears to be physiologically important, since it reflected a gradient that determines the directional flux of FC between cells and HDL.39 Kuksis et al40 described it as a strong predictor of cardiovascular disease risk in a nondiabetic male population. In the present study, we observed a decrease in the PL percentage in GLpA-I (P<.05) without significant changes in the FC level. The ratio of these surface lipids was not changed as compared with that in NGLpA-I. It is difficult to infer this finding with the Kuksis' index because we did not perform the PL subclasses determination and therefore cannot determine the true index. However, the FC content in GLpA-I and NGLpA-I was quite comparable. By no means can we extrapolate for an adversely affected reverse cholesterol transport, as Fielding has presented evidence when lipoproteins are enriched in FC.32

The increased median size of GLpA-I is not surprising from compositional data, since the surface-to-core constituents ratio, an indirect estimate of particle size, is significantly lower as compared with that of NGLpA-I. The shape and size of HDL have been suggested as important determinants of the conformation of the apo A-I molecule, playing an indirect role in regulating the metabolism of the lipoprotein.29 34 In the present study, the apoA-I conformation was studied through the use of anti-apo A-I monoclonal antibodies which react with defined epitopes covering most of the apo A-I molecule.18 We observed a marked decrease in accessibility of the epitopes specific for monoclonal antibodies A44 and A05 (residues 149 to 186 and 25 to 82, respectively) and no immunological changes for the epitope specific for monoclonal antibody A11 (residues 126 to 132 and 99 to 105) in GLpA-I, compared with that observed in NGLpA-I. Despite some controversy,41 the specific domain recognized by the monoclonal antibody A44 has been presented as a functionally important region.42 Recently investigators in our laboratory have proposed a spatial conformation of apo A-I, in which the epitopes spanning apo A-I amino acid residues 149 to 186 (monoclonal antibody A44) and 25 to 82 (monoclonal antibody A05) are close together.43 This observation indicates a molecular flexibility of the protein, which would bring regions distant from each other in the peptidic sequence closer to the area responsible for binding activity. The present data are in agreement with this hypothesis, since the conformational changes we observed in the monoclonal antibody A44 and A05 domains on GLpA-I were accompanied with perturbations in cellular interactions: decrease of GLpA-I binding to cells and decrease of cholesterol efflux by GLpA-I from cells. Since GLpA-I was a larger size than NGLpA-I, it may be asked whether the same shift would be found between both fractions when the cellular experiments are normalized for molar concentrations instead of protein concentrations, as we did. In the present study, as in others,44 45 when the immunoaffinity-purified lipoprotein particles presented an homogeneous apolipoprotein composition (ie, containing apo A-I but no apo A-II), their physico-chemical characteristics were heterogeneous and the particles displayed variable sizees, molecular masses and/or compositions, thereby preventing any molar calculation. In any case, although we demonstrated a significantly larger size for GLpA-I, the difference was slight (P<.05) and evidently not sufficient to explain the changes in cellular interactions. Also, PL concentrations have been shown to highly correlate with the ability of lipoprotein particles to promote cholesterol efflux.46 When the results were expressed as a function of PL concentrations, we observed that the differences in cholesterol efflux still remain. Even more, the lower PL content of GLpA-I would indicate a reduced capacity for cholesterol efflux.

In conclusion,. the physico-chemical and functional characteristics of the nonenzymatically GLpA-I subfraction particles isolated from samples obtained from patients with type 1 diabetes indicate potentially important abnormalities. The physiopathological involvement with regard to atherosclerosis needs to be evaluated in an extensive clinical study.


*    Selected Abbreviations and Acronyms
 
apo A-I = apolipoprotein A-I
CE = cholesterol esters
FC = free cholesterol
GLpA-I = glucosylated HDL containing apo A-I only
HBA1c = hemoglobin A1c
HDL = high-density lipoprotein
LDL = low-density lipoprotein
LpA-I = HDL containing apo A-I only
NGLpA-I = nonglucosylated HDL containing apo A-I only
TC = total cholesterol
TG = triglycerides
VLDL = very-low-density lipoprotein

Received February 8, 1996; accepted May 6, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Garcia MJ, McNamara PM, Gordon T, Kannell WB. Morbidity and mortality in diabetics in the Framingham population. Sixteen year follow-up study. Diabetes. 1974;23:105-111.[Medline] [Order article via Infotrieve]
  2. Pyörälä K, Laakso M, Uusitupa M. Diabetes and atherosclerosis: An epidemiologic view. Diabetes Metab Rev. 1987;3:463-524.[Medline] [Order article via Infotrieve]
  3. Stamler J. Epidemiology, established major risk factors, and the primary prevention of coronary heart disease. In: Parmley WW, Chatterjee K, eds. Cardiology. Philadelphia: JB Lippincott;1987:1-41.
  4. Taskinen M-R. Hormones, diabetes mellitus and lipoproteins. Curr Opin Lipidol. 1991;2:197-205.
  5. Bierman EL. Atherogenesis in diabetes. Arterioscler Thromb. 1992;12:647-656.[Free Full Text]
  6. Curtiss LK, Witztum JL. Plasma apolipoproteins A-I, A-II, B, C-I, and E are glucosylated in hyperglycemic diabetic subjects. Diabetes. 1985;34:452-461.[Abstract]
  7. Duell PB, Oram JF, Bierman EL. Nonenzymatic glycosylation of HDL and impaired HDL-receptor-mediated cholesterol efflux. Diabetes. 1991;40:377-384.[Abstract]
  8. Calvo C, Talussot C, Ponsin G, Berthezène F. Non enzymatic glycation of apolipoprotein A-I. Effects on its self-association and lipid binding properties. Biochim Biophys Res Commun. 1988;153:1060-1067.[Medline] [Order article via Infotrieve]
  9. Calvo C, Verdugo C. Association in vivo of glycated apolipoprotein A-I with high density lipoproteins. Eur J Clin Chem Clin Biochem. 1992;30:3-5.[Medline] [Order article via Infotrieve]
  10. Calvo C, Ulloa N, Del Pozo R, Verdugo C. Decreased activation of lecithin: cholesterol acyltransferase by glycated apolipoprotein A-I. Eur J Clin Chem Clin Biochem. 1993;31:217-220.[Medline] [Order article via Infotrieve]
  11. Fruchart J-C, Ailhaud G, Bard J-M. Heterogeneity of high density lipoprotein particles. Circulation. 1993;87[suppl III]:22-27.
  12. Duchateau P, Rader D, Duverger N, Théret N, De Geitere C, Brewer HB, Fruchart J-C, Castro GR. Tangier disease: isolation and characterization of LpA-I, LpA-II, LpA-I: A-II and LpA-IV particles from plasma. Biochim Biophys Acta. 1993;1182:30-36.[Medline] [Order article via Infotrieve]
  13. Afsa H. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal Biochem. 1966;14:328-336.[Medline] [Order article via Infotrieve]
  14. Mc Farlane AS. Efficient trace labeling of proteins with iodine. Nature. 1958;182:53-57.[Medline] [Order article via Infotrieve]
  15. Parra HJ, Mezdour H, Ghalim N, Bard J-M, Fruchart J-C. Differential electroimmunoassay on ready to use plates for human LpA-I lipoprotein particles. Clin Chem. 1990;36:1431-1435.[Abstract/Free Full Text]
  16. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.[Free Full Text]
  17. Forte AM, Nordhausen RW. Electron microscopy of negatively stained lipoproteins. Methods Enzymol. 1986;128:442-457.[Medline] [Order article via Infotrieve]
  18. Marcel YL, Provost PR, Koa H, Raffai E, Vu Dac N, Fruchart J-C, Rassart E. The epitopes of apolipoprotein A-I define distinct structural domains including a mobile middle region. J Biol Chem. 1991;266:3644-3653.[Abstract/Free Full Text]
  19. Fiévet C, Durieux C, Milne R, Delaunay T, Agnani G, Bazin H, Marcel YL, Fruchart J-C. Rat monoclonal antibodies to human apolipoprotein B: advantages and applications. J Lipid Res. 1989;30:1015-1024.[Abstract]
  20. Zettner A. Principles of competitive binding assays (saturation analysis). 1. Equilibrium techniques. Clin Chem. 1973;19:699-705.[Abstract]
  21. Snedecor GW, Cochran WG. Statistical Methods. 6th ed. Ames, Ia.: Iowa State University Press, 1967;342-343.
  22. Johnson D, Robson YM, Melnykovych G. Reduction in cell-associated low density lipoprotein in dexamethasone-treated HeLa cells: suggested mechanism. Endocrinology. 1983;113:907-914.[Abstract]
  23. Moya M, Atger V, Paul J-L, Fournier F, Moatti N, Giral P, Friday KE, Rothblat G. A cell culture system for screening human serum for ability to promote cellular cholesterol efflux: relationship between serum components and efflux, esterification and transfer. Arterioscler Thromb. 1994;14:1056-1065.[Abstract/Free Full Text]
  24. Law SW, Brewer HB. Nucleotide sequence and the encoded amino acids of human apolipoprotein A-I mRNA. Proc Natl Acad Sci USA. 1984;81:66-70.[Abstract/Free Full Text]
  25. Duell PB, Bierman EL. Diabetic HDL has reduced capacity to promote HDL receptor-mediated cholesterol efflux [Abstract]. Arteriosclerosis. 1990;10:768.
  26. Duell PB, Oram JF, Bierman EL. Non enzymatic glycosylation of HDL resulting in inhibition of high-affinity binding to cultured human fibroblasts. Diabetes. 1990;39:1257-1263.[Abstract]
  27. Barter PJ, Hopkins GJ, Gorjatschko L. Lipoprotein substrates for plasma cholesterol esterification. Influence of particle size and composition of the high density lipoprotein subfraction 3. Atherosclerosis. 1985;58:97-107.[Medline] [Order article via Infotrieve]
  28. Collet X, Perret B, Simard G, Raffai E, Marcel YL. Differential effects of lecithin and cholesterol on the immunoreactivity and conformation of apolipoprotein A-I in high density lipoproteins. J Biol Chem. 1991;266:9145-9152.[Abstract/Free Full Text]
  29. Leblond L, Marcel YL. The amphipathic {alpha}-helical repeats of apolipoprotein A-I are responsible for binding of high density lipoproteins to HepG2 cells. J Biol Chem. 1991;266:6058-6067.[Abstract/Free Full Text]
  30. Sparks DL, Kao J, Frohlich J, Lacko AG, Pritchard PH. The relationship between cholesteryl ester transfer activity and high density lipoprotein composition in hyperlipidemic patients. Atherosclerosis. 1988;77:183-191.
  31. Ahnadi C-E, Masmoudi T, Berthez§ne F, Ponsin G. Decreased ability of high density lipoproteins to transfer cholesterol esters in non-insulin-dependent diabetes mellitus. Eur J Clin Invest. 1993;23:459-465.[Medline] [Order article via Infotrieve]
  32. Fielding CJ. The origin and properties of free cholesterol potential gradients in plasma, and their relation to atherogenesis. J Lipid Res. 1984;25:1624-1628.[Medline] [Order article via Infotrieve]
  33. Johnson WJ, Bamberger MJ, Latta RA, Rapp PE, Phillips MC, Rothblatt G. The bidirectional flux of cholesterol between cells and lipoprotein: effects of phospholipid depletion of high density lipoprotein. J Biol Chem. 1986;261:5766-5776.[Abstract/Free Full Text]
  34. Segrest JP, Jones MK, Deloof H, Brouillette CG, Venkatachalapathi YV, Anantharamaiah GM. The amphipathic helix in the exchangeable apolipoproteins: a review. J Lipid Res. 1992;33:141-166.[Abstract]
  35. Sparks DL, Lund-Katz S, Phillips MC. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. J Biol Chem. 1992;267:25839-25847.[Abstract/Free Full Text]
  36. Dullaart FPF, Groener JEM, Dikkaschei LD, Erkelens DW, Doorenbus H. Increased cholesteryl ester transfer activity in complicated type 1 (insulin-dependent) diabetes mellitus: its relationship with serum lipids. Diabetologia. 1989;32:14-19.[Medline] [Order article via Infotrieve]
  37. Bagdade JD, Subbaiah PV, Ritter MC. Accelerated cholesteryl ester transfer in patients with insulin dependent diabetes mellitus. Eur J Clin Invest. 1991;21:161-167.[Medline] [Order article via Infotrieve]
  38. Bagdade JD, Dunn FL, Eckel RH, Ritter MC. Intraperitoneal insulin therapy corrects abnormalities in cholesteryl ester transfer and lipoprotein lipase activities in insulin-dependent diabetes mellitus. Arterioscler Thromb. 1994;14:1933-1939.[Abstract/Free Full Text]
  39. Bagdade JD, Subbaiah PV. Whole-plasma and high-density lipoprotein subfraction surface lipid composition in IDDM men. Diabetes. 1989;38:1226-1230.[Abstract]
  40. Kuksis A, Myher JJ, Geher K, Jones GJ, Breckenridge WC, Feather T, Hewitt D, Little JA. Decreased plasma phosphatidylcholine free cholesterol ratio as an indicator of risk for ischemic vascular disease. Arteriosclerosis. 1982;2:296-302.[Abstract/Free Full Text]
  41. Banka CL, Black AS, Curtiss LK. Location of an apolipoprotein A-I epitope critical for lipoprotein-mediated cholesterol efflux from monocytic cells. J Biol Chem. 1994;269:10288-10297.[Abstract/Free Full Text]
  42. Von Eckardstein A, Castro G, Wybranska I, Théret N, Duchateau P, Duverger N, Fruchart J-C, Ailhaud G, Assman G. Interaction of reconstituted high density lipoprotein discs containing human apoprotein A-I (apo A-I) variants with murine adipocytes and macrophages: evidence for reduced cholesterol efflux promotion by apo A-I (Pro165 Arg). J Biol Chem. 1993;268:2616-2622.[Abstract/Free Full Text]
  43. Luchoomun J, Théret N, Clavey V, Duchateau P, Rosseneu M, Brasseur R, Denèfle P, Fruchart J-C, Castro G. Structural domain of apolipoprotein A-I involved in its interaction with cells. Biochim Biophys Acta. 1994;1212:319-326.[Medline] [Order article via Infotrieve]
  44. Duverger N, Rader D, Duchateau P, Fruchart J-C, Castro G, Brewer HB. Biochemical characterization of the three major subclasses of lipoprotein A-I preparatively isolated from human plasma. Biochemistry. 1993;32:12372:12379.[Medline] [Order article via Infotrieve]
  45. Cavallero E, Brites F, Delfly B, Nicolaiew N, Decossin C, De Geitere C, Fruchart J-C, Wikinski R, Jacotot B, Castro G. Abnormal reverse cholesterol transport in controlled type II diabetic patients. Studies on fasting and postprandial LpA-I particles. Arterioscler Thromb Vasc Biol. 1995;15:2130-2135.[Abstract/Free Full Text]
  46. Fournier N, de la Llera Moya M, Burkey BF, Swaney JB, Paterniti J, Moatti N, Atger V, Rothblat GH. Role of HDL phospholipid in efflux of cell cholesterol to whole serum: studies with human apo A-I transgenic rats. J Lipid Res. 1996;37:1704-1711.[Abstract]




This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Igau, B.
Right arrow Articles by Fiévet, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Igau, B.
Right arrow Articles by Fiévet, C.

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1997 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.