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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 1 35-40
Copyright © 2002 by The Endocrine Society

Special Features

Triglyceride-Rich Lipoproteins from Subjects with Type 2 Diabetes Do Not Demonstrate Increased Binding to Biglycan, a Vascular Proteoglycan

Lisa R. Tannock, Katherine L. Olin, P. Hugh R. Barrett, Thomas N. Wight and Alan Chait

Departments of Medicine (L.R.T., K.L.O., A.C.) and Pathology (T.N.W.), University of Washington, Seattle, Washington 98195; Department of Medicine (P.H.R.B.), University of Western Australia, Perth 6001, Australia; and Hope Heart Institute (T.N.W.), Seattle, Washington 98104

Address all correspondence and requests for reprints to: Alan Chait, M.D., Box 356426, Department of Medicine, University of Washington, Seattle, Washington 98195-6426. E-mail: achait{at}u.washington.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Retention of atherogenic apolipoprotein (apo) B- and E- containing lipoproteins by their interaction with arterial wall proteoglycans is important in atherogenesis. Levels of triglyceride (TG)-rich lipoproteins, which contain both apo B and apo E, are increased in type 2 diabetes. Because increased retention of TG-rich lipoproteins in diabetes might explain, in part, the increased atherosclerosis in this disorder, TG-rich lipoproteins were isolated from fasting type 2 diabetic subjects and age-matched controls, and assessed for their ability to bind biglycan, a vascular smooth muscle cell-derived proteoglycan. The binding of TG-rich lipoproteins isolated from diabetic subjects to purified biglycan did not differ from lipoproteins isolated from control subjects. Moreover, contrary to previous reports, no difference in the apo E content of TG-rich lipoproteins was detected between the control and diabetic groups. Additionally, no difference in the binding affinity of TG-rich lipoproteins for the low-density lipoprotein receptor was observed between control and diabetic subjects. Thus, we were unable to confirm previous reports that TG-rich lipoproteins from subjects with diabetes are enriched in apo E compared with age-matched controls, consistent with the lack of difference in binding of these lipoproteins to either biglycan or the low-density lipoprotein receptor. Therefore, increased affinity of TG-rich lipoproteins for biglycan is unlikely to explain the increased atherosclerosis in type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATHEROSCLEROSIS IS THE main cause of morbidity and mortality in type 2 diabetes, yet the reasons underlying this remain unknown. Individuals with diabetes have both qualitative and quantitative abnormalities in their lipoprotein profiles. The main abnormalities are increased triglyceride (TG)-rich lipoproteins [very low-density lipoproteins (VLDL) and intermediate-density lipoproteins] and decreased high-density lipoprotein (HDL) levels (1).

Apolipoprotein (apo) E is a component of both HDL and TG-rich lipoproteins. Apo E levels have been shown to correlate with serum apo B levels, cholesterol, and TGs, yet by multivariate analysis serum TG and apo E levels were independently associated with myocardial infarction in nondiabetic subjects (2, 3). Several studies have reported apo E enrichment of TG-rich lipoproteins in diabetes (4, 5, 6), although others have not confirmed this finding (7, 8). The observation that apo E content of postprandial TG-rich lipoproteins in subjects with both type 2 diabetes and coronary artery disease was increased compared with nondiabetic healthy controls has led to the suggestion that enrichment of TG-rich lipoproteins with apo E may have a role in the increased risk of coronary artery disease in subjects with diabetes (5).

One mechanism by which apo E might increase atherosclerosis relates to its ability to bind to and be retained by proteoglycans in the artery wall. Proteoglycans are complex macromolecular complexes composed of a protein core to which one or more glycosaminoglycan side chains are covalently bound. Positively charged residues on apo B and E of lipoproteins can interact with negatively charged sulfate and carboxylic groups on glycosaminoglycan chains in the artery wall, leading to retention of atherogenic lipoproteins (9). Several lines of evidence suggest that retention of lipoproteins by proteoglycans in the arterial wall plays a key role in the initiation and propagation of atherosclerotic plaque (for review, see Refs.9 and 10).

Previous work by our laboratory has shown colocalization of biglycan, a small extracellular vascular proteoglycan, with apo E in human coronary artery plaques, whereas apo E was undetectable in normal arterial intima (11). This finding suggests that the interaction between apo E and biglycan may be an important event in atherogenesis. However, that study did not identify the source of the apo E present in the atherosclerotic lesions. Apo B also was found to colocalize with biglycan in the atherosclerotic lesions (11). The colocalization of apo B and apo E with biglycan is consistent with retention of atherogenic TG-rich lipoprotein remnants, which can contain both apo B and E (12).

It has been established that the presence of hypertriglyceridemia increases the risk of cardiovascular disease (13, 14, 15), but the mechanism by which TG-rich lipoproteins are atherogenic is unknown. The present study was performed to determine whether TG-rich lipoproteins isolated from subjects with type 2 diabetes would differ in their composition from those isolated from control subjects, and to determine whether such changes would result in increased retention by biglycan, which might in part explain the increased propensity to atherosclerosis in diabetes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified.

Study population

Subjects with type 2 diabetes (n = 18) and age-matched controls (n = 18) donated fasting blood samples. Exclusion criteria included age less than 20 yr or greater than 70 yr, pregnancy, body mass index (BMI) greater than 45 kg/m2, TG greater than 8 mmol/liter, total cholesterol greater than 7.8 mmol/liter, or renal insufficiency. Subjects were not excluded on the basis of concomitant medical disease, smoking history, or medication use. Diabetic subjects were recruited from the patient population of the Clinical Nutrition Research Unit Clinical Research Core Registry, and controls were recruited from staff volunteers. After an overnight fast (with hypoglycemic medications held on the morning of the blood draw), blood samples were drawn and immediately centrifuged. An aliquot of plasma was analyzed for glucose, HbA1c (glycated hemoglobin), blood urea nitrogen, creatinine (by enzymic methods), and lipoprotein content (by Lipid Research Clinics ß Quantification procedure) (16).

Lipoprotein isolation

TG-rich lipoproteins (density <1.019 g/ml) from a separate aliquot of plasma were isolated by density gradient ultracentrifugation using a Beckman Coulter model Ti 50.3 fixed angle rotor (Beckman Coulter, Inc., Fullerton, CA) at 200,000 x g for 24 h, and the lipoproteins were recovered by tube slicing. The TG-rich lipoproteins were dialyzed for 18 h at 4 C against 3-(N-morpholino)propanesulfonic acid (MOPS) sample buffer [20 mM MOPS, 140 mM NaCl, 5 mM CaCl2, 2 mM MgCl2 (pH 7.4); ionic strength, 0.147 M), then stored at 4 C in the dark until used in the gel mobility shift assay within 3 d. Aliquots of the dialyzed TG-rich lipoproteins were analyzed for their apo B and apo E content by immunonephelometry (17). TG content of the TG-rich lipoproteins was determined enzymatically (16). To detect any possible alterations in apo content caused by ultracentrifugation, the apo B, apo E, and TG contents of TG-rich lipoproteins also were determined on a subset of samples (10 control and 9 diabetic subjects) prepared by fast protein liquid chromatography (FPLC). Fresh plasma (1 ml) was passed through a 4-µm filter, then immediately applied to a Superose 6 HP column. Sixty fractions of 0.5 ml were separated at 4 C by elution with 0.05 M phosphate, 0.15 M NaCl, 0.01% EDTA, 0.02% NaN3 (pH 7.4) with a flow rate of 0.2 ml/min. The cholesterol content of the eluted fractions was determined using the Total Serum Cholesterol Assay kit (Diagnostic Chemical Ltd, Oxford, CT). The fractions comprising the peak of VLDL and intermediate-density lipoproteins were analyzed for their apo B, apo E, and TG content, and an average value was determined from the results from individual fractions. The TG-rich lipoproteins prepared by FPLC contain abundant amounts of albumin, which precluded their use in the binding assays. TG-rich lipoproteins isolated by ultracentrifugation from a subset of 11 control subjects and 11 diabetic subjects were used in assays to study binding to biglycan and to the low-density lipoprotein receptor (LDL-R).

Biglycan preparation

Purified [35S]-SO4 labeled biglycan was prepared as described previously (18). In brief, subconfluent human aortic smooth muscle cells were metabolically labeled with 100 µCi/ml Na2[35S]-SO4 (ICN Biomedicals, Inc., Irvine, CA) for 24 h (19, 20). The medium was collected, concentrated by ion exchange chromatography, concentrated on Centricon membranes (Amicon, Beverly, MA), then applied to preparative Sepharose CL-2B molecular sieve columns (21). Radiolabeled proteoglycans eluting at Kav 0.44–0.60 were pooled for biglycan. The biglycan pool was applied to DEAE-Sephacel, washed extensively, and eluted with 4 M guanidine buffer [4 M guanidine, 10 mM EDTA, 50 mM sodium acetate (pH 7.4)] (20). The eluate was concentrated and exchanged into MOPS sample buffer on Centricon membranes and stored at -80 C for use in the gel mobility shift assay.

Gel mobility shift assay and analysis

The interaction between purified biglycan and TG-rich lipoproteins was assessed using a modified gel mobility shift assay (22, 23). In this assay, a concentration curve was generated by mixing increasing concentrations of lipoproteins with a fixed amount (approximately 2,000 dpm) of [35S]-SO4 labeled biglycan in MOPS sample buffer for 1 h at 37 C. A concentration curve of the TG-rich lipoproteins was determined on the basis of apo B content (therefore, on the basis of number of particles of TG-rich lipoprotein because there is one molecule of apo B per lipoprotein particle) in equal volume aliquots. After incubation, the samples were applied to wells in 0.7% NuSieve agarose (FMC Bioproducts, Rockland, ME), and electrophoresis was performed for 3 h at 60 V at 4 C in MOPS running buffer [20 mM MOPS, 3 mM CaCl2, 5 mM MgCl2 (pH 7.2)]. Proteoglycans that have bound to lipoproteins are retained at the origin, and unbound proteoglycans migrate into the gel. The gels were fixed in cetyl pyridinium chloride (0.1%, in 70% ethanol) for 1 h, air-dried, exposed to PhosphorImager screens, and then analyzed by OptiQuant software (Packard Bioscience, Meriden, CT). The amount of complexed vs. free biglycan in each lane was assessed, and the percentage that was bound was calculated as the proportion of radioactivity remaining at the origin of the gel relative to the total radioactivity per lane. Binding constants were calculated using the SAAM II software (SAAM Institute, Seattle, WA) and a Michaelis-Menten function that included a cooperativity term.

Competitive binding of TG-rich lipoproteins to LDL-R

Human fibroblasts were plated in DMEM containing 10% serum in 12-well cell culture dishes at approximately 12,000 cells per well and cultured for 5 d. Two days before the experiment, the cells were transferred to DMEM containing 10% human lipoprotein-deficient serum. On the day of experiment, the cells were exposed to increasing concentrations of unlabeled TG-rich lipoproteins in the presence of normal human [125I]-labeled LDL (2 µg/ml) for 3 h at 4 C. Then, the cells were washed extensively, and the surface-bound radioactivity was determined (24). The concentration of TG-rich lipoprotein from each subject required to compete 50% with [125I]-LDL was determined using the Michaelis- Menten function.

Statistical analysis

Statistical analysis was performed using t test, and results are expressed as mean ± SEM, unless otherwise noted. Where multiple tests were compared, a two-sided P value less than 0.01 was considered statistically significant; otherwise, a two-sided P value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The clinical characteristics of the study subjects are outlined in Table 1Go. The control group differed significantly from the diabetic group in BMI, fasting plasma glucose, and HbA1c. There were no significant differences between the diabetic and control groups in other lipid variables. Fifteen of 18 diabetic subjects were on a wide variety of oral hypoglycemic agents only, and 4 were on insulin. The use of other medications, including vitamins, did not differ between the groups.


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  		Table 1. Baseline characteristics of study subjects

 
TG-rich lipoproteins isolated from type 2 diabetic subjects were not significantly different in apo E content (expressed as a molar ratio of apo E to apo B) compared with the control group, regardless of the method of isolation of TG-rich lipoproteins (Table 2Go). However, TG-rich lipoproteins from diabetic subjects had a tendency toward enrichment in TG content per particle (determined as the molar ratio of TG to apo B) relative to control subjects (6.8 ± 3.0 x 103 vs. 5.3 ± 1.6 x 103; P = 0.07 for samples isolated by ultracentrifugation; 6.1 ± 2.9 x 103 vs. 3.5 ± 1.4 x 103; P = 0.04 for samples isolated by FPLC). There were no differences in baseline characteristics, including glycemic control, modality of treatment, or TG-rich lipoprotein composition between subjects whose TG-rich lipoproteins were used in binding assays to biglycan and LDL-R than those not used, nor were there differences between subjects whose TG-rich lipoproteins were also analyzed by FPLC than those who only had centrifugal analysis. To determine whether the TG enrichment of TG-rich lipoproteins from the diabetic group had altered binding affinity for purified biglycan, TG-rich lipoproteins from 11 control and 11 diabetic subjects were studied in a gel mobility shift assay. Increasing numbers of TG-rich lipoprotein particles (based on apo B content) were mixed with fixed amounts of biglycan under physiological conditions (pH 7.4; temperature, 37 C; NaCl 140 mM), then analyzed by an electrophoretic gel mobility shift assay. To minimize confounding variables on each experimental day, a diabetic subject and age-matched control were assessed in parallel. The radioactivity at the lane origin represents biglycan bound by TG-rich lipoproteins, and the radioactivity that migrates into the gel represents free biglycan (Fig. 1, A and BGo, representative gel mobility shift assays for a diabetic and control subject, respectively). Analysis of the composite binding curves of TG-rich lipoproteins from the control and diabetic groups shows no differences in biglycan binding (Fig. 1CGo). Analysis of binding was done by a Michaelis-Menten function that included a cooperativity term which provided the best model fit to the binding data. The standard Michaelis-Menten function did not describe the binding data. In all instances, the value of the cooperativity parameter was greater than 1.0, suggesting that the binding of lipoproteins to biglycan enhanced the subsequent binding of other lipoproteins. This analysis confirmed a lack of difference in binding affinity between the groups [Kd = 4.2 ± 0.7 x 10-8 M apo B (0.023 ± 0.004 mg/ml) for the control group; Kd = 3.6 ± 0.5 x 10-8 M apo B (0.020 ± 0.003 mg/ml) for the diabetic group; P = 0.59].


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  		Table 2. Characteristics of TG-rich lipoprotein fractions separated by either ultracentrifugation or FPLC

 


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  		Figure 1. Binding of TG-rich lipoproteins to biglycan. Phosphorimage of representative electrophoretic gel mobility shift assays of a diabetic subject (A) and a control subject (B). Increasing concentrations of isolated TG-rich lipoproteins were incubated with a fixed amount of [35S]-SO4 labeled biglycan for 60 min at 37 C before electrophoresis in agarose. To quantitate binding, dried gels were subjected to autoradiography, and the percentage that was bound was calculated as the proportion of radioactivity remaining at the origin of the gel relative to the total radioactivity per lane. Analysis of composite binding curves for all of the subjects (n = 11 each, diabetic and control subjects) show that there is no difference between the diabetic ({square}) and control ( •) groups (C). Values shown are means ± SEM.

 
We next determined whether the TG enrichment of TG-rich lipoproteins from the diabetic group affected the ability of these particles to bind to LDL-R. Fibroblasts that had been cholesterol-deprived to up-regulate their LDL-R levels were exposed to increasing numbers of particles of unlabeled TG-rich lipoproteins (based on apo B content) in the presence of a constant amount of [125I]-labeled LDL. The ability of TG-rich lipoproteins to compete 50% with radiolabeled LDL was determined, and dissociation constants were calculated using SAAM software. The composite binding curve shows that there was no difference between the groups in the ability of TG-rich lipoproteins to bind to LDL-R (Fig. 2Go) [Kd = 2.5 ± 0.5 x 10-9 M apo B (1.35 ± 0.28 µg/ml) for the control group; Kd = 2.7 ± 0.3 x 10-9 M apo B (1.46 ± 0.19 µg/ml) for the diabetic group; P = 0.74)].



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  		Figure 2. Binding of TG-rich lipoproteins to LDL-R. The ability of TG-rich lipoproteins to compete with [125I]-labeled native human LDL (2 µg/ml) was evaluated by adding TG-rich lipoproteins from diabetic subjects ({square}) and control subjects (•) at the indicated concentrations to fibroblasts for determination of the amount of [125I]-labeled native human LDL bound. The results represent the composite binding curves for each group (n = 11 each, diabetic and control subjects). Values shown are means ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we compared the composition of TG-rich lipoproteins isolated by two different methods from type 2 diabetic subjects and age-matched controls. We showed that TG-rich lipoproteins isolated from diabetic subjects do not have enrichment of apo E content, but have a tendency toward enrichment in TG content per particle. Moreover, no differences were seen between groups in the binding of these TG-rich lipoproteins to purified biglycan, a vascular proteoglycan, or in the binding of these TG-rich lipoproteins to LDL-R.

This study did not confirm some previous reports that TG-rich lipoproteins from subjects with diabetes are enriched in apo E compared with nondiabetic individuals (6, 7). However, two different techniques used to isolate TG-rich lipoproteins (ultracentrifugation and FPLC) yielded slightly different values for the apo E content per particle (expressed as apo E/apo B ratio). Review of the literature suggests that apo E enrichment of TG-rich particles in diabetes is not a consistent finding. For example, Klein et al. (7) reported that apo E in VLDL, measured as an increase in percentage of VLDL apo E densitometric area by quantitative immunoelectrophoresis, was increased in diabetic subjects compared with controls. However, there was no increase in VLDL apo E on a per particle basis, i.e. there were no significant differences in the apo E/apo B ratio. A second study from the same group reported an increase in apo E content on a per particle basis (apo E/apo B ratio), of VLDL subfractions I and II but not subfraction III from subjects with type 1 diabetes vs. controls (4). Syvanne et al. (5) demonstrated apo E enrichment (expressed as an increased apo E/TG ratio) in diabetic postprandial TG-rich lipoproteins vs. controls. However, the same group previously had reported that both apo E and apo B were enriched in diabetic VLDL, implying that there is no enrichment of apo E on a per particle basis (8). An important observation is that all of these studies examined VLDL composition on particles isolated by ultracentrifugation. To the best of our knowledge, the data presented in this paper are the first to present comparisons of VLDL composition for particles isolated by FPLC. It is well known that ultracentrifugation can displace certain apolipoproteins from the lipoprotein particle, in part related to the duration of ultracentrifugation and salt concentration (25). Although ultracentrifugation might have had a differential effect on the loss of apo E in the control vs. diabetic subjects, similar apo E/apo B ratios were found in the same samples isolated by FPLC, suggesting that ultracentrifugation did not have differential effects between the two groups.

It has been reported that apo E in VLDL from diabetic subjects is glycated to a greater extent than apo E in VLDL from control subjects (26), and that this increased glycation of apo E results in decreased binding to heparin (27). We did not measure the extent of apo E glycation. However, no difference was observed in the biglycan binding affinity of TG-rich lipoproteins from the control and diabetic groups. This finding suggests that either the apo E on TG-rich lipoproteins from the diabetic group was not extensively glycated or apo E glycation does not affect biglycan binding. Steinbrecher and Witztum (28) reported that 2–5% of lysine residues of apo B are glycated in diabetic subjects and that this extent of modification can impair the ability of LDL to interact with LDL-R by 5–25%. Thus, our finding that there was no difference in LDL-R binding between TG-rich lipoproteins from the diabetic and control groups suggests that the extent of glycation of lysines of apo B and apo E was low in TG-rich lipoproteins from the diabetic group.

LDL traditionally has been thought to be the most atherogenic of the lipoprotein particles. Most studies on the role of proteoglycans in atherosclerosis have focused on the interaction between proteoglycans and LDL. However, TG-rich lipoproteins also contribute to atherosclerosis risk, especially in diabetes (for review, see Ref.29). Several studies show that VLDL-sized lipoproteins are found in the human intima (30, 31). TG-rich lipoproteins transport more cholesterol per particle than does LDL and therefore could be more atherogenic on a per particle basis than LDL (12). TG-rich lipoproteins contain both apo E and apo B, each of which can bind to proteoglycans, and could be the source of the apo E and apo B that have been found in close association with proteoglycans in atherosclerotic areas (11). The concentration of lipoproteins in the interstitial fluid is difficult to measure (for review, see Ref.32), but the apo B content of interstitial fluid has been estimated to be 5–10% of plasma levels (33). The affinity constants calculated from the gel mobility shift assay for the interaction of TG-rich lipoproteins with biglycan are in this range. Therefore, the data presented here are physiologically plausible and suggest that retention of apo B and apo E-containing TG-rich lipoproteins by arterial wall biglycan is a possible explanation for the colocalization of these molecules that we have previously observed. An alternate explanation for the colocalization of apo B and apo E with biglycan could be competition for biglycan binding by apo B-containing lipoproteins (such as LDL) and apo E-containing lipoproteins (such as apo E-containing HDL). The apo E found colocalized with biglycan in the artery wall could have been produced in the artery wall because vascular macrophages are known to make apo E (34). However, apo B is not synthesized locally and this would not account for the colocalization of apo B with apo E.

The binding of apolipoproteins to LDL-R is largely ionic in nature and is believed to be similar in mechanism as the binding of lipoproteins to proteoglycans (24). Studies using heparin have demonstrated that the LDL-R binding site of apo E also is the physiological proteoglycan binding site (35). Studies by Bradley et al. (36) have shown that TG-rich lipoproteins from hypertriglyceridemic subjects can bind to LDL-R via apo E, whereas TG-rich lipoproteins from normotriglyceridemic subjects cannot. This differential LDL-R binding is accounted for by the different conformation of apo E on the surface of the TG-rich lipoprotein particles from hypertriglyceridemic vs. normotriglyceridemic subjects (36, 37). Conversely, Hiramatsu et al. (38) reported that LDL isolated from subjects with hypertriglyceridemia had impaired binding to LDL-R. Aviram et al. (39) demonstrated that LDL with reduced TG content had increased recognition and cellular uptake via LDL-R, suggesting that the TG content in the core of LDL may alter the conformation of apo B on the surface, resulting in altered binding to LDL-R. These studies suggest that particle size, as determined by TG content, has an effect on binding of particles to LDL-R. However, our data demonstrated that despite the tendency to enrichment of TG content in TG-rich lipoproteins from diabetic subjects, the binding to the LDL-R did not differ between the groups.

Other potential explanations for the increased atherosclerosis in diabetes were not investigated in this study. For example, increased binding of TG-rich lipoproteins from diabetics to vascular proteoglycans via bridging molecules such as lipoprotein lipase (18) could contribute to atherogenicity. Increased concentrations of TG-rich lipoproteins in diabetes could lead to an overall increase in the number of lipoprotein particles bound to biglycan in diabetic subjects, despite a lack of difference in binding affinity between TG-rich lipoproteins from diabetic and control subjects. Alterations in the vascular extracellular matrix proteoglycans from diabetic subjects (40) also could result in increased binding of atherogenic lipoproteins, which in turn could account for the increased atherosclerosis in diabetes. Alternately, atherosclerosis in diabetes may be increased due to alterations in the composition of LDL, resulting in increased retention of this lipoprotein in the artery wall. In conclusion, our findings demonstrate that TG-rich lipoproteins from individuals with diabetes are not enriched in apo E content compared with TG-rich lipoproteins from nondiabetics, but have a tendency to enrichment in TG content. There was no effect of this TG enrichment on the binding of these lipoproteins to either biglycan or the LDL-R. Further studies are necessary to determine whether other differences in the interaction of atherogenic lipoproteins and proteoglycans between individuals with type 2 diabetes and nondiabetic individuals could account for the increased atherosclerosis in diabetes.


   Acknowledgments
 


   Footnotes
 
This work was supported in part by Grants DK02456, DK07247, NCRR12609 from the NIH and a fellowship grant (to L.R.T.) from the Juvenile Diabetes Foundation. These studies were performed at the University of Washington’s General Clinical Research Center (Grant RR00037). The laboratory facilities and subject registry of the University of Washington Clinical Nutrition Research Unit (Grant DK35816) were used.

This paper was presented in part at the American Diabetes Association’s 60th Scientific Sessions, San Antonio, Texas, 2000.

Abbreviations: apo, Apolipoprotein; BMI, body mass index; FPLC, fast protein liquid chromatography; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LDL-R, LDL receptor; MOPS, 3-(N-morpholino)propanesulfonic acid; TG, triglyceride; VLDL, very low-density lipoprotein.

Received March 24, 2000.

Accepted October 10, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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