Elsevier

Atherosclerosis

Volume 193, Issue 2, August 2007, Pages 283-291
Atherosclerosis

Pro-apoptotic low-density lipoprotein subfractions in type II diabetes

https://doi.org/10.1016/j.atherosclerosis.2006.08.059Get rights and content

Abstract

Objective

To test the hypothesis that differences in subfractions of circulating lipoproteins between diabetic and non-diabetic subjects exist and might contribute to the increased risk for atherosclerosis in type II diabetics.

Methods and results

LDL isolated from diabetic (D) and control subjects (N) were separated by FPLC into five subfractions (L1–L5). The fractional distributions of N- and D-LDL were not different, but the most strongly retained subfractions of D-LDL (D-L5) were markedly more pro-apoptotic to bovine aortic endothelial cells in vitro than were the other subfractions in D- or N-LDL. D-L5 induced time- and concentration-dependent apoptosis that was inhibited by z-VAD-fmk. The most electronegative D-LDL subfractions contained substantial amounts of apoproteins AI, E and CIII, higher concentrations of non-esterified fatty acids and LpPLA2, and lower trinitrobenzenesulfonic acid (TNBSA) reactivities. Electronegative subfractions of D-LDL exhibited longer lag times and lower net increases in absorbance at 234 nm with Cu-catalyzed oxidation in vitro.

Conclusions

The toxicities of electronegative subfractions of LDL from diabetic subjects to endothelial cells in vitro may be pivotal to vascular complications of diabetes in vivo, but the specific molecular alterations responsible for the toxicities of these subfractions of diabetic LDL are not known.

Introduction

Diabetes mellitus is characterized by abnormal carbohydrate, lipid and protein metabolism, resulting from defects in insulin secretion, insulin action or both. Type II diabetes accounts for most diabetes and, of these, 90% are obese [1]. The Multiple Risk Factor Intervention Trial (MRFIT) study reported that the absolute risk for death from cardiovascular disease (CVD) was much higher for diabetic men than for non-diabetic men of every age group, ethnic background and risk factor level [2]. Overall, the risk was three times higher for diabetics than for non-diabetics. Rates of coronary heart disease (CHD) mortality in type II diabetic patients with no history of myocardial infarction are as high as in non-diabetics with a history of myocardial infarction [3]. Diabetes is a strong, independent risk factor for atherosclerotic cardiovascular disease (ACVD), which is the major cause of morbidity and mortality in individuals with types I and II diabetes. However, the specific mechanisms by which diabetes causes accelerated and extensive atherosclerosis are not well understood.

Although some evidence suggests that LDL are more atherogenic in diabetics than in normal subjects, the atherogenic properties of diabetic LDL have not been characterized. Glycated LDL in diabetics logically would be the key species in the first working hypothesis to test as causes of the greater cytotoxic properties of D-LDL. However, using unfractionated LDL, Artwohl et al. [4] found that glycation of normal LDL in vitro to extents comparable to those found in diabetic LDL in vivo did not replicate the toxicity of diabetic LDL, providing strong evidence that modifications of LDL other than glycation are significant determinants of cytotoxicity. We isolated from familial hypercholesterolemic (FH) subjects [5] an oxidized LDL subfraction (designated as L5) that increased monocyte adhesion to cells in culture. Oxidatively modified human plasma LDL are cytotoxic to endothelial cells and vascular smooth muscle cells and are proatherogenic [6]. Using approaches similar to those used in our studies of FH-LDL, analyses of LDL from diabetic subjects revealed that D-L5, the most electronegative LDL subfraction, exhibited a protein composition that was distinct from the comparable subfraction from FH subjects, FH-L5. Distinctive biological and physicochemical properties of D-L5 from diabetic subjects suggest that this subfraction may be mechanistically linked to the higher rate of atherosclerosis in diabetic patients. Identification of the specific modifications that distinguish D-L5 may provide clues that will lead to new therapeutic targets.

Section snippets

Subjects

LDL were isolated from type II diabetic subjects between 51 and 72 years of age (59.2 ± 9.3), with HbA1c levels ranging from 7 to 13, and age- and sex-matched normal subjects, between 51 and 65 years of age (57.2 ± 5.7), with normal lipid values, which included cholesterol values of <200 mg/dl, triglycerides <160 mg/dl, LDL <130 mg/dl [7] and glucose <105 mg/dl. Plasmaphereses were performed using an Autopheresis-C (Fenwal, Deerfield, IL).

LDL preparation

Reagents were purchased from Sigma (St. Louis, MO). LDL were

Results

With the conditions described in our previous studies of subfractionation of FH-LDL, LDL from diabetic subjects and from the respective normoglycemic controls were separated into five subfractions, designated as L1–L5, reflecting increasing LDL particle surface charge (Fig. 1). Using the step-jump elution gradient described by other investigators for separation of LDL(−) [14], [15], [16], we observed the bimodal elution of two sharp, well-separated peaks (results not shown), which indicates

Discussion

The similarities in elution patterns (Fig. 1) and fractional compositions (Table 1) in the LDL from diabetic subjects and from their respective controls do not suggest accumulation of glycated or glycoxidized LDL being responsible for the electronegative or the cytotoxic properties of the subfractions. D-LDL also is distinct from FH-LDL, in that FH-LDL exhibited greater proportions of the more electronegative subfractions (-L4 and -L5) than were observed in the LDL isolated from normolipidemic

Acknowledgements

We thank Jonathan Lu, Hsiu-Tsu Yeh and Hsiu-Ching Hsu for technical assistance and advice. This work was supported by grants HL63364 (C.Y.Y.), HL-56865, HL-30914 (H.J.P.), and GM44263 (C.V.S.) from the National Institutes of Health, 7-03-RA-108 from the ADA (C.Y.Y.), 9630095N and 1-04-RA-13 from the ADA (C.H.C) and a Research Award 0050530N from AHA National (C.Y.Y.).

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