Metabolic Syndrome Is Associated with Elevated Oxidative Stress and

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The Journal of Clinical Endocrinology & Metabolism 89(10):4963– 4971 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2004-0305

Metabolic Syndrome Is Associated with Elevated Oxidative Stress and Dysfunctional Dense High-Density Lipoprotein Particles Displaying Impaired Antioxidative Activity BORIS HANSEL, PHILIPPE GIRAL, ESTELLE NOBECOURT, SANDRINE CHANTEPIE, ERIC BRUCKERT, M. JOHN CHAPMAN, AND ANATOL KONTUSH Dyslipoproteinemia and Atherosclerosis Research Unit (B.H., P.G., E.N., S.C., E.B., M.J.C., A.K.), National Institute for Health and Medical Research, Institut National de la Sante´ et de la Recherche Me´dicale (INSERM); and Service d’Endocrinologie-Metabolisme (P.G., E.B.), Hoˆpital de la Pitie´, 75013 Paris, France A metabolic syndrome (MetS) phenotype is characterized by insulin-resistance, atherogenic dyslipidemia, oxidative stress, and elevated cardiovascular risk and frequently involves subnormal levels of high-density lipoprotein (HDL) cholesterol. We evaluated the capacity of physicochemically distinct HDL subfractions from MetS subjects to protect low-density lipoprotein against oxidative stress. MetS subjects presented an insulin-resistant phenotype, with central obesity and elevation in systolic blood pressure and plasma triglyceride, LDL-cholesterol, apolipoprotein B, glucose, and insulin levels. Systemic oxidative stress, assessed as plasma 8-isoprostanes, was significantly higher (3.7-fold) in MetS subjects (n ⴝ 10) compared with nonobese normolipidemic controls (n ⴝ 11). In MetS, small, dense HDL3a, 3b, and 3c subfractions possessed significantly lower specific antioxidative activity (up to ⴚ23%, on a unit particle mass basis) than

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ETABOLIC SYNDROME (MetS), first described by Reaven in 1988 (1), is characterized by a constellation of cardiovascular (CV) risk factors, including atherogenic dyslipidemia, abnormal glucose tolerance, hypertension, and visceral obesity, which are intimately associated with insulin resistance and hyperinsulinemia. The dyslipidemia of MetS features hypertriglyceridemia involving elevated concentrations of triglyceride (TG)-rich lipoproteins [veryLDL (VLDL), VLDL remnants], or subnormal levels of highdensity lipoprotein (HDL) cholesterol (HDL-C), or both; major qualitative modifications of the atherogenic lipid profile typically include a small, dense, low-density lipoprotein (LDL) phenotype (2). In contrast to atherogenic apolipoproAbbreviations: AAPH, Initiator 2,2⬘-azobis-(2-amidinopropane) hydrochloride; apo, apolipoprotein; CE, cholesteryl ester; CRP, C-reactive protein; CV, cardiovascular; DBP, diastolic blood pressure; FC, free cholesterol; HDL, high-density lipoprotein; HDL-C, HDL cholesterol; HOMA, homeostasis model of assessment; LDL, low-density lipoprotein; LOOH, lipid hydroperoxides; MetS, metabolic syndrome; PAF-AH, platelet-activating factor acetylhydrolase; PL, phospholipid; PON, paraoxonase; SBP, systolic blood pressure; TBARS, thiobarbituric acid-reactive substances; TC, total cholesterol; TG, triglyceride; VLDL, very-lowdensity lipoprotein. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

their counterparts in controls. In addition, HDL2a and 3a subfractions from MetS patients possessed lower total antioxidative activity (up to ⴚ41%, at equivalent plasma concentrations). The attenuated antioxidative activity of small, dense HDL subfractions correlated with systemic oxidative stress and insulin resistance and was associated with HDL particles exhibiting altered physicochemical properties (core triglyceride enrichment and cholesteryl ester depletion). We conclude that antioxidative activity of small, dense HDL subfractions of altered chemical composition is impaired in MetS and associated with elevated oxidative stress and insulin resistance. Induction of selective increase in the circulating concentrations of dense HDL subfractions may represent an innovative therapeutic approach for the attenuation of high cardiovascular risk in MetS. (J Clin Endocrinol Metab 89: 4963– 4971, 2004)

tein (apo)B-containing lipoproteins (VLDL, VLDL remnants, intermediate density lipoprotein, and LDL), however, HDLs exert a spectrum of antiatherogenic activities, principal among which is the process of reverse cholesterol transport, together with antiinflammatory, antithrombotic, and antiapoptotic actions (3). Among their antiinflammatory properties, HDL particles possess potent antioxidative activity in vitro (4 –7) and ex vivo (8); indeed, the antioxidative action of HDL is now recognized as a major mechanism mediating its cardioprotective effect (9). The antioxidative activity of HDL particles is facilitated, in part, by the transport of key enzymes, including paraoxonase (PON) (10), platelet-activating factor acetylhydrolase (PAFAH) (11), and lecithin:cholesterol acyltransferase (12), which can hydrolyze diverse molecular species of oxidized lipids and thereby inhibit LDL oxidation and its proinflammatory sequelae. The major HDL apo, apoA-I, inhibits LDL oxidation by removing oxidized lipids from LDL and may simultaneously undergo oxidative modification (13). Others (apoA-II, apoA-IV, apoE) (9), specific lipid components and key structural features of HDL particles (14), may equally be implicated in HDL antioxidative activity. Plasma HDL particles are highly heterogeneous in their chemical composition, intravascular metabolism, and biological function, and earlier studies have documented het-

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erogeneity in the antioxidative capacity of HDL subspecies (15, 16). Indeed, HDL3 exerts potent oxidative protection of LDL relative to other HDL subfractions in normolipidemic subjects (14). Endothelial dysfunction is a key feature of MetS and is intimately linked to insulin resistance; this functional relationship appears to result, in part, from the induction of oxidative stress by modest hyperinsulinemia (17). It is relevant, therefore, that elevated plasma levels of oxidized lipids (18) and subnormal levels of low-molecular-weight antioxidants (19) are characteristic of subjects presenting MetS. We hypothesize that the antioxidative activity of HDL and oxidative stress are mutually interactive; we therefore evaluated the relationship of systemic oxidative stress to the antioxidative capacity of HDL subfractions in nondiabetic, insulin-resistant subjects with MetS. Our data reveal that elevated levels of oxidative stress are intimately related to impaired antioxidative activities of small, dense HDL subfractions in an insulin-resistant MetS phenotype. Subjects and Methods Subjects MetS was defined according to National Cholesterol Education Program Adult Treatment Panel III guidelines (20) and includes the presence of at least three criteria among the following: 1) hypertriglyceridemia (TG ⱖ 150 mg/dl); 2) HDL-C less than or equal to 40 mg/dl for men or less than or equal to 50 mg/dl for women; 3) fasting blood glucose of at least 110 mg/dl; 4) systolic blood pressure (SBP) at least 130 mm Hg and/or diastolic blood pressure (DBP) at least 90 mm Hg; 5) waist circumference at least 102 cm for men and at least 88 cm for women. Both patients with MetS (n ⫽ 10) and age-matched healthy normolipidemic control subjects (n ⫽ 11) were recruited at the Metabolic Syndrome Clinic, Hoˆ pital La Pitie´ . In addition, women were postmenopausal and not receiving hormone replacement therapy. All subjects recruited for the study were less than 70 yr of age, nonsmokers, and either abstainers or moderate alcohol consumers (⬍25 g/d). Blood pressure and physical data were determined during a complete clinical examination. Insulin resistance was assessed as the index of the homeostasis model of assessment (HOMA) of insulin resistance (fasting blood glucose ⫻ insulin/22.5) and as plasma insulin to glucose ratio (21). Plasma insulin level was determined by ELISA (Abbott Laboratories, North Chicago, IL). The level of systemic inflammation was assessed as plasma ultrasensitive C-reactive protein (CRP); subjects with ultrasensitive CRP level of at least 5 mg/liter were excluded. None of the MetS or control subjects presented renal, hepatic, gastrointestinal, pulmonary, endocrine, or oncological disease or were receiving antioxidative vitamin supplementation, antidiabetic drugs, or drugs known to affect lipoprotein metabolism for at least 6 wk before the study. Finally, none of the subjects were receiving a specific diet. All subjects gave written informed consent. The clinical protocol was approved by the Institutional Ethical Committee Review Board.

Blood samples EDTA plasma (final EDTA concentration, 1 mg/ml) and serum were prepared from venous blood collected on ice after an overnight fast. Plasma was immediately separated by centrifugation at 4 C; plasma and serum were each mixed with sucrose (final concentration, 0.6%) as a cryoprotectant for lipoproteins (22) and frozen at ⫺80 C under nitrogen. 8-Isoprostanes were determined in plasma by ELISA (Cayman Chemical, Ann Arbor, MI); inter- and intraassay coefficients of variation were 8.0 and 11.1%, respectively.

Isolation of lipoproteins Five major subfractions of HDL (i.e. large, light HDL2b and HDL2a and small, dense HDL3a, HDL3b, and HDL3c) were isolated by isopyc-

Hansel et al. • Oxidative Stress and HDL in Metabolic Syndrome

nic density gradient ultracentrifugation (23, 24). LDL was isolated in the density interval 1.018 –1.065 g/ml. Lipoprotein samples were maintained at 4 C and used within 10 d.

Characterization of lipoproteins Total protein, total cholesterol (TC), cholesteryl ester (CE), free cholesterol (FC), phospholipid (PL), and TG contents of isolated lipoprotein subfractions were determined as described elsewhere (23). Total lipoprotein mass was calculated as the sum of total protein, CE, FC, PL, and TG. Relative apo content (apoA-I, apoE, and apoCs) was evaluated by SDS-PAGE (23); in addition, apoA-I and apoA-II were quantified by immunonephelometry (14). The PON1 activity of HDL subfractions (100 ␮G protein/ml) was determined photometrically in the presence of CaCl2 (1 mm) using phenylacetate as a substrate (11). Activity of PAF-AH was assessed using 1-palmitoyl 2-(6-[7-nitrobenzoxadiazolyl]amino) caproyl phosphatitylcholine (C6NBD PC) as a fluorescent substrate (25).

Oxidation of lipoproteins Antioxidative activities of HDL subfractions were assessed toward reference LDL isolated from one control subject and used throughout the study. Before oxidation, EDTA and KBr were removed from LDL and HDL preparations by exhaustive dialysis for 24 h at 4 C. LDL oxidation was performed in the presence of a water-soluble azo-initiator 2,2⬘azobis-(2-amidinopropane) hydrochloride (AAPH). The capacities of HDL subfractions to protect LDL from metal-independent oxidation induced by AAPH and from metal-dependent oxidation induced by Cu2⫹ ions were strongly correlated (14). LDL (10 mg TC/dl) was incubated at 37 C in Dulbecco’s PBS (pH 7.4) in the presence of 1 mm AAPH; HDL subfractions were added to LDL directly before oxidation. PBS solution was treated with Chelex 100 ion exchange resin for 1 h to remove contaminating transition metal ions. To measure relative electrophoretic mobility of LDL and thiobarbituric acid-reactive substances (TBARS), aliquots of the LDL⫹HDL mixture were withdrawn after 24 h, and oxidation was stopped by adding 0.1% (wt/vol) alcoholic solution of butylhydroxytoluene and 0.1 m EDTA (1 ␮l of each). Samples were then frozen in 0.6% sucrose solution (22) at ⫺80 C for not longer than 2 months. Two different antioxidative activities for each isolated HDL subfraction, specific antioxidative activity and total antioxidative activity, were assessed. Specific antioxidative activity was measured at the same final concentration of each subfraction (10 mg total mass/dl) to assess the intrinsic capacity of HDL subfractions to protect LDL from oxidation at an HDL to LDL ratio within the physiological range (2– 6 mol/mol). Total antioxidative activity was measured at the same (7-fold) dilution of each subfraction, i.e. at their equivalent plasma concentrations, to take into account interindividual variation in plasma levels of HDL subfractions.

Characterization of oxidized lipoproteins Accumulation of conjugated dienes was measured as the increment in absorbance at 234 nm (26, 27). The kinetics of diene accumulation revealed two characteristic phases, the lag and propagation phases. For each curve, the duration of each phase, average oxidation rates within each phase and amount of dienes formed at the end of the propagation phase (maximal amount of dienes) were calculated. Accumulation of TBARS was determined photometrically at 535 nm after reaction with thiobarbituric acid (26). The electrophoretic mobility of LDL was measured on 0.8% agarose gel containing BSA (26).

Statistical analysis Between-group differences in continuous variables were analyzed by Student’s t test for independent groups. Differences in dichotomous variables were analyzed by Fisher’s exact test. Pearson’s moment-product correlation coefficients were calculated to evaluate relationships between variables. Non-Gaussian distributed variables were log transformed before statistical analysis. All results are expressed as means ⫾ sd.

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Results Clinical and biological characteristics

All 10 patients presented MetS according to the NCEP/ ATPIII definition (20). On the basis of significant elevation in insulin levels, HOMA index, and plasma insulin to glucose ratio (⫹196, ⫹253, ⫹151%, respectively, relative to normolipidemic healthy controls; Table 1), all MetS patients were distinguished by a marked degree of insulin resistance. By contrast, control subjects were essentially free of any criterion of MetS (except that blood pressure was slightly higher (⬍8%) than MetS criteria in four subjects). As a result, MetS patients displayed significantly higher waist circumference (⫹26%), body mass index (⫹33%), SBP (⫹14%), plasma levels of TC (⫹41%), TG (⫹226%), LDL-cholesterol (⫹49%),

Systemic oxidative stress

Levels of systemic stress, assessed as plasma 8-isoprostanes, were 3.7-fold elevated (P ⬍ 0.001) in MetS patients compared with controls (Table 1).

TABLE 1. Characteristics of subjects

Age (yr) Sex (males/females) Waist circumference (cm) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) TC (mg/dl) TG (mg/dl) LDL-cholesterol (mg/dl) apoB-100 (mg/dl) HDL-C (mg/dl) apoA-I (mg/dl) Glucose (mg/dl) Insulin (IU/liter) Insulin/glucose ratio HOMA CRP (mg/liter) 8-Isoprostanes (ng/liter)

apoB-100 (⫹89%), and glucose (⫹17%), compared with controls, as well as a significantly lower level of HDL-C (⫺21%, P ⬍ 0.01; Table 1). In contrast, no significant difference in sex ratio, DBP, and plasma level of apoA-I was found between the two groups. In addition, the ratio of TC to HDL-C (6.0 ⫾ 1.6 vs. 3.4 ⫾ 0.7, P ⬍ 0.001) and the ratio of apoA-I to HDL-C (3.1 ⫾ 0.4 vs. 2.7 ⫾ 0.3, P ⬍ 0.05) were significantly higher in MetS patients compared with controls. Furthermore, MetS patients presented significantly higher levels of systemic inflammation as assessed by plasma CRP levels (⫹73%; Table 1). Only one patient was diabetic as defined by fasting blood glucose (ⱖ126 mg/dl).

Controls (n ⫽ 11)

MetS patients (n ⫽ 10)

52.3 ⫾ 13.6 9/2 81.0 ⫾ 8.2 23.4 ⫾ 1.5 127 ⫾ 11 80 ⫾ 10 192 ⫾ 29 79 ⫾ 24 118 ⫾ 31 76 ⫾ 15 57 ⫾ 8 147 ⫾ 112 93 ⫾ 9 5.4 ⫾ 2.1 1.05 ⫾ 0.42 1.24 ⫾ 0.49 1.1 ⫾ 0.6 31 ⫾ 18

53.5 ⫾ 7.1 7/3 102.3 ⫾ 6.9a 31.1 ⫾ 3.3a 148 ⫾ 22b 85 ⫾ 16 271 ⫾ 42a 258 ⫾ 192c 176 ⫾ 44c 144 ⫾ 29a 47 ⫾ 9c 146 ⫾ 25 109 ⫾ 13c 16.0 ⫾ 6.9a 2.64 ⫾ 0.94a 4.38 ⫾ 2.38a 1.9 ⫾ 0.9b 115 ⫾ 63a

Plasma levels of HDL subfractions

The quantitative distribution of HDL subfractions (as percent mass) tended to be distinct between MetS patients and controls, although no significant differences in absolute concentrations of HDL subfractions were found between the two groups (Table 2). Chemical composition of HDL subfractions

Conversion factor into mmol/liter (IU): cholesterol, 0.026; TG, 0.01143; glucose, 0.056. BMI, Body mass index. a P ⬍ 0.001 vs. controls. b P ⬍ 0.05 vs. controls. c P ⬍ 0.01 vs. controls.

When considering total HDL, a significantly lower percent content of CE (20.9 ⫾ 4.5% vs. 26.3 ⫾ 5.8%, P ⫽ 0.03) was found in MetS patients compared with controls. Moreover, MetS HDL tended to be enriched in TG (P ⫽ 0.11) and depleted in protein (P ⫽ 0.09). Significantly lower concentrations of CE in HDL2b (13.4 ⫾ 7.1 vs. 21.7 ⫾ 8.7 mg/dl, P ⫽ 0.03), 3b (4.9 ⫾ 1.0 vs. 6.9 ⫾ 2.4 mg/dl, P ⫽ 0.02), and 3c (2.1 ⫾ 0.8 vs. 3.4 ⫾ 1.0 mg/dl, P ⫽ 0.004) subfractions were observed in the MetS group compared with controls. In contrast, concentrations of TG in HDL3a (4.4 ⫾ 3.2 vs. 2.4 ⫾ 0.9 mg/dl, P ⫽ 0.07) and 3b (2.6 ⫾

TABLE 2. Total mass, percent weight composition, PON1, and PAF-AH activities of HDL subfractions from MetS patients (n ⫽ 10) and controls (n ⫽ 11)

Total mass (mg/dl) CE TG FC PL Total protein PON1 activityc PAF-AH activityd

Group

HDL2b

HDL2a

HDL3a

HDL3b

HDL3c

Control MetS Control MetS Control MetS Control MetS Control MetS Control MetS Control MetS Control MetS

73.4 ⫾ 21.7 58.9 ⫾ 24.1 27.1 ⫾ 5.7 22.9 ⫾ 8.5 6.6 ⫾ 3.5 10.7 ⫾ 7.9 6.6 ⫾ 1.7 8.2 ⫾ 5.5 22.7 ⫾ 8.5 23.9 ⫾ 5.9 36.0 ⫾ 3.3 34.3 ⫾ 5.1 37.7 ⫾ 28.1 41.8 ⫾ 13.9 2.32 ⫾ 1.11 4.23 ⫾ 1.20a

73.9 ⫾ 15.2 75.9 ⫾ 15.0 24.9 ⫾ 7.1 21.3 ⫾ 4.7 3.6 ⫾ 1.3 6.9 ⫾ 7.0 4.2 ⫾ 1.1 4.1 ⫾ 1.7 23.2 ⫾ 10.1 31.0 ⫾ 4.8 40.2 ⫾ 4.9 36.7 ⫾ 4.9 96.4 ⫾ 83.3 44.0 ⫾ 15.4 0.98 ⫾ 0.84 2.95 ⫾ 1.14a

67.5 ⫾ 16.8 71.5 ⫾ 15.6 23.8 ⫾ 6.1 21.7 ⫾ 3.6 3.3 ⫾ 0.9 6.0 ⫾ 4.2b 3.1 ⫾ 0.7 3.0 ⫾ 0.6 16.2 ⫾ 10.1 29.0 ⫾ 3.9 45.4 ⫾ 5.5 40.3 ⫾ 4.0b 272 ⫾ 243 170 ⫾ 83 0.92 ⫾ 0.50 1.68 ⫾ 0.31a

30.7 ⫾ 9.8 29.6 ⫾ 7.6 21.1 ⫾ 4.6 17.2 ⫾ 4.5 3.2 ⫾ 1.9 8.0 ⫾ 6.2b 2.6 ⫾ 0.6 3.3 ⫾ 1.7 17.0 ⫾ 7.8 20.2 ⫾ 5.5 55.7 ⫾ 6.3 51.3 ⫾ 6.1 1229 ⫾ 960 1147 ⫾ 578 2.21 ⫾ 0.45 2.97 ⫾ 0.88b

16.8 ⫾ 5.8 17.4 ⫾ 8.4 19.7 ⫾ 5.0 13.0 ⫾ 5.4a 3.9 ⫾ 2.8 9.2 ⫾ 10.5 2.1 ⫾ 0.7 3.0 ⫾ 2.7 10.2 ⫾ 6.7 11.6 ⫾ 6.6 63.2 ⫾ 7.9 63.1 ⫾ 15.0 2294 ⫾ 949 2780 ⫾ 838 3.54 ⫾ 0.57 3.82 ⫾ 0.76

P ⬍ 0.01. P ⬍ 0.05 vs. corresponding control value. c nmol/min䡠mg protein. d nmol released C6NBD/min/mg protein. a b

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Hansel et al. • Oxidative Stress and HDL in Metabolic Syndrome

2.6 vs. 1.0 ⫾ 0.6 mg/dl, P ⫽ 0.07) subfractions tended to be higher in the MetS group, relative to controls. Lower percent contents of CE in HDL3c (P ⫽ 0.009) and in HDL3b (P ⫽ 0.07) were observed in MetS compared with controls. Conversely, significantly higher percent contents of TG in HDL3a (P ⫽ 0.05) and 3b (P ⫽ 0.02) were observed in the patient group (Table 2). The percent protein content tended to be lower in MetS patients than in controls in all HDL subfractions (Table 2) but was statistically significant only in HDL3a (P ⫽ 0.03). By contrast, no difference between the two groups was found in the relative apoA-I, apoA-II, and apoC contents (data not shown). Protein components of HDL possessing antioxidative activity

PON1 activity with phenylacetate as substrate was nonuniformly distributed between HDL subfractions both in MetS patients and controls, increasing in the order: HDL2b ⬍ HDL2a ⬍ HDL3a ⬍ HDL3b ⬍ HDL3c (Table 2) (14). No difference in PON1 activity was found between corresponding HDL subfractions isolated from serum of MetS patients and controls. PAF-AH activity was also nonuniformly distributed between HDL subfractions in MetS patients and controls (Table 2). Furthermore, PAF-AH activity was significantly higher in MetS patients, compared with controls, in HDL2b, 2a, 3a, and 3b subfractions (Table 2). Antioxidative action of HDL subfractions during LDL oxidation

Specific antioxidative activity. When HDL subfractions isolated from controls (Fig. 1A) or MetS patients (Fig. 1B) were added to reference LDL at a physiological HDL to LDL ratio of about 2– 6 mol/mol, LDL oxidation was significantly delayed. Such antioxidative activity of HDL subfractions was markedly impaired in MetS patients. Under conditions used to measure specific antioxidative activity (HDL total mass, 10 mg/dl; LDL-TC, 10 mg/dl), the inhibitory effects of small, dense HDL subfractions on LDL oxidation were significantly lower in MetS patients than in controls, with respect to both decrease in LDL oxidation rate in the propagation phase (HDL3a, ⫺10%, P ⫽ 0.046; HDL3b, ⫺22%, P ⬍ 0.001; HDL3c, ⫺17%, P ⫽ 0.02; Fig. 2A) and elevation in the duration of this phase (HDL3b, ⫺23%, P ⫽ 0.009). Furthermore, the decrease in the production of conjugated dienes was significantly less pronounced in controls, in comparison with MetS patients (Fig. 2C), in the presence of HDL3b (⫺8%, P ⫽ 0.03) and HDL3c (⫺10%, P ⫽ 0.04). More precisely, dense HDL3a, 3b, and 3c and light HDL2a subfractions from controls significantly decreased the oxidation rate in the propagation phase (Fig. 2A); this antioxidative effect increased with increment in HDL density: HDL2a (⫺7%, P ⫽ 0.03) ⬍ HDL3a (⫺16%, P ⫽ 0.03) ⬍ HDL3b (⫺37%, P ⬍ 0.001) ⬍ HDL3c (⫺57%, P ⬍ 0.001). In addition, control HDL3b and 3c subfractions prolonged the propagation phase (⫹31%, P ⬍ 0.001 and ⫹37%, P ⬍ 0.001, respectively; Fig. 2B). By contrast, in MetS patients, only HDL3b and 3c subfractions significantly decreased LDL oxidation rate in the propagation phase (⫺15%, P ⫽ 0.002 and ⫺40%, P ⬍ 0.001, respectively; Fig. 2A) and only HDL3c prolonged

FIG. 1. Influence of HDL subfractions on AAPH-induced oxidation of reference LDL under conditions employed to measure their specific antioxidative activity (HDL, 10 mg total mass/dl; LDL, 10 mg TC/dl). Oxidation time courses averaged for the control (n ⫽ 11; A) and MetS (n ⫽ 10; B) groups are shown. LDL was incubated with AAPH (1 mM) in the presence or absence of HDL subfractions in PBS at 37 C, and conjugated diene formation was measured by absorbance increment at 234 nm.

this phase (⫹25%, P ⫽ 0.002; Fig. 2B). Similarly, dense HDL3b and 3c subfractions from controls decreased maximal amounts of conjugated dienes formed upon oxidation (⫺10%, P ⫽ 0.003; ⫺22%, P ⬍ 0.001, respectively; Fig. 2C), whereas only HDL3c showed significant inhibition in MetS patients (⫺14%, P ⫽ 0.002). Consistent with these results, HDL3b (P ⫽ 0.03) and 3c (P ⫽ 0.03) subfractions from MetS patients were significantly less active in decreasing accumulation of TBARS in LDL than their counterparts from controls (data not shown). Note that large, light HDL subfractions tended to increase maximal diene production both in MetS patients and controls (Figs. 1 and 2C); this effect can be attributed to the oxidation of HDL subfractions themselves (14). Addition of HDL to LDL significantly increased the duration of the lag phase in control subjects; this effect was observed for all HDL subspecies (⫹17%, P ⬍ 0.01; ⫹20%, P ⬍ 0.01; ⫹15%, P ⬍ 0.01, ⫹21%, P ⬍ 0.01, and ⫹65%, P ⬍ 0.01 for HDL2b, 2a, 3a, 3b, and 3c, respectively). The prolongation of the lag phase observed in HDL subfractions from MetS patients was remarkably similar to that for their counterparts

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only observed after the lag phase, i.e. at late stages of LDL oxidation. Total antioxidative activity. Under conditions used to measure total antioxidative activity of HDL subfractions (7-fold diluted HDL subfractions; LDL-TC, 10 mg/dl), HDL2a and 3a subfractions from MetS patients were significantly less potent in decreasing oxidation rate in the propagation phase than their counterparts from controls (⫺25 vs. ⫺50%, P ⫽ 0.04, and ⫺30 vs. ⫺54%, P ⫽ 0.05, respectively; Fig. 3A). Furthermore, the HDL3b subfraction from MetS patients tended to be less potent (⫺19 vs. ⫺41%, P ⫽ 0.07) in decreasing LDL oxidation rate in the propagation phase than HDL3b from controls. In addition, the MetS HDL2a subfraction was significantly less potent in prolonging the propagation phase (83 vs. 129% in control HDL2a, P ⫽ 0.008). Correlations

Antioxidative activity of HDL3b and HDL3c, assessed as oxidative protection of LDL in the propagation phase, was

FIG. 2. Influence of HDL subfractions on AAPH-induced oxidation of reference LDL under conditions employed to measure their specific antioxidative activity (HDL, 10 mg total mass/dl; LDL, 10 mg TC/dl). LDL oxidation rate in the propagation phase (A), duration of the propagation phase (B), and maximal amount of conjugated dienes (C) are shown for controls (n ⫽ 11) and MetS patients (n ⫽ 10). LDL was incubated with AAPH (1 mM) in the presence or absence of HDL subfractions in PBS at 37 C, and conjugated diene formation was measured by absorbance increment at 234 nm. §, P ⬍ 0.05; §§, P ⬍ 0.01; §§§, P ⬍ 0.001 vs. incubation without added HDL. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 vs. control subjects.

in controls (⫹24%, P ⬍ 0.01; ⫹20%, P ⬍ 0.01; ⫹18%, P ⬍ 0.01, ⫹25%, P ⬍ 0.01, and ⫹71%, P ⬍ 0.01 for HDL2b, 2a, 3a, 3b, and 3c, respectively). Hence, the impairment in the oxidative protection of LDL by HDL subfractions in MetS patients was

FIG. 3. Influence of HDL subfractions on AAPH-induced oxidation of reference LDL under conditions employed to measure their total antioxidative activity (7-fold diluted, ultracentrifugally isolated HDL subfractions; LDL, 10 mg TC/dl). LDL oxidation rate in the propagation phase (A) and duration of the propagation phase (B) are shown for controls (n ⫽ 10) and MetS patients (n ⫽ 9). LDL was incubated with AAPH (1 mM) in the presence or absence of HDL subfractions in PBS at 37 C, and conjugated diene formation was measured by absorbance increment at 234 nm. §, P ⬍ 0.05; §§, P ⬍ 0.01; §§§, P ⬍ 0.001 vs. incubation without added HDL. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 vs. control subjects.

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negatively correlated with hyperglycemia (r ⫽ ⫺0.51, P ⫽ 0.019 and r ⫽ ⫺0.48, P ⫽ 0.028, respectively). The LDL oxidation rate in the presence of HDL3c positively correlated with insulin resistance assessed as the HOMA index (Fig. 4A). Consistently, insulin level and the insulin to glucose ratio correlated negatively with the duration of the propagation phase in the presence of the HDL3c subfraction (r ⫽ ⫺0.43, P ⫽ 0.049; r ⫽ ⫺0.46, P ⫽ 0.038, respectively). Plasma levels of 8-isoprostanes were positively correlated (r ⫽ 0.60,

Hansel et al. • Oxidative Stress and HDL in Metabolic Syndrome

P ⫽ 0.008) with LDL oxidation rate in the propagation phase (Fig. 4B) and negatively correlated (P ⫽ ⫺0.68, P ⫽ 0.002) with the duration of this phase in the presence of HDL3c. In addition, plasma 8-isoprostane levels were positively correlated with TG content (as percent) in all HDL subfractions (r ⫽ 0.72, P ⬍ 0.01; r ⫽ 0.51, P ⬍ 0.05; r ⫽ 0.65, P ⬍ 0.01; r ⫽ 0.56, P ⬍ 0.05, and r ⫽ 0.56, P ⬍ 0.05 for HDL2b, 2a, 3a, 3b, and 3c, respectively) and negatively correlated with CE content in HDL3b and 3c (data not shown). When the relationship of specific antioxidative activity to HDL chemical composition was evaluated, percent TG content in HDL positively correlated with the oxidation rate in the propagation phase (r ⫽ 0.22, P ⫽ 0.02) and negatively correlated with its duration (r ⫽ ⫺0.33, P ⬍ 0.001). In addition, protein content of HDL subfractions (as absolute concentration or percent of total mass) was significantly negatively correlated with the oxidation rate in both the lag and propagation phases and positively correlated with the duration of these phases (r ⫽ ⫺0.40, ⫺0.66, 0.38, and 0.65, respectively, P ⬍ 0.001), whereas percent CE, percent FC, and percent PL contents showed opposite correlations of comparable significance (data not shown). By contrast, no significant correlation was found between the antioxidative activities of HDL subfractions and either apoA-I or apoA-II level (data not shown). Finally, when specific antioxidative activity of HDL subfractions was compared with activities of antioxidative proteins, PON1 activity was negatively correlated with the oxidation rate in the propagation phase (r ⫽ ⫺0.74, P ⬍ 0.001; Fig. 4C), with maximal amount of dienes (r ⫽ ⫺0.52, P ⬍ 0.001) and with TBARS accumulation after LDL oxidation (r ⫽ ⫺0.53, P ⬍ 0.001) and positively correlated with the duration of the propagation phase (r ⫽ 0.58, P ⬍ 0.001). Similarly, PAF-AH activity was negatively correlated with maximal amount of dienes (r ⫽ ⫺0.24, P ⬍ 0.05) and with TBARS accumulation (r ⫽ ⫺0.28, P ⬍ 0.05) and positively correlated with the duration of the propagation phase (r ⫽ 0.23, P ⫽ 0.05). Discussion

FIG. 4. Correlations between LDL oxidation rate in the propagation phase and insulin resistance assessed by the HOMA index (A), between plasma 8-isoprostanes and antioxidative activity of HDL3c assessed by the rate of LDL oxidation in the propagation phase (B), and between PON1 activity and antioxidative activity of HDL subfractions assessed by the rate of LDL oxidation in the propagation phase (C).

The present investigations have established that the antioxidative, antiinflammatory activity of small, dense HDL particles (notably HDL3b and 3c) is impaired in subjects exhibiting a MetS phenotype involving atherogenic dyslipidemia (hypertriglyceridemia and subnormal HDL-C levels), hyperglycemia, hyperinsulinemia, hypertension, and central obesity. Such deficient HDL antioxidative capacity was associated not only with markedly elevated systemic oxidative stress (3.7-fold) assessed as plasma 8-isoprostane levels, but also with a chronic inflammatory state as indicated by a significant elevation in CRP levels. Furthermore, correlational analysis revealed that the impaired antioxidative activity of small, dense HDL in MetS was intimately related to the presence of hypertriglyceridemia, hyperinsulinemia, and insulin resistance (as HOMA index), thereby suggesting that abnormalities in both lipid and glucose metabolism underlie the antioxidative dysfunction of HDL particles in MetS. Interestingly, the antioxidative activity of small, dense HDL subfractions was equally significantly impaired, and plasma 8-isoprostanes significantly elevated, in a small subset (n ⫽

Hansel et al. • Oxidative Stress and HDL in Metabolic Syndrome

4) of MetS subjects displaying normal levels of TG, TC, and HDL-C (data not shown), indicating that these impairments are intimately related to the constellation of several CV risk factors typical of MetS. Oxidative stress is defined as an imbalance between prooxidant and antioxidant factors in favor of prooxidants, thereby potentiating oxidative damage (28). Of the five criteria of MetS defined in NCEP/ATPIII (20), four (and notably hypertriglyceridemia, hypertension, hyperglycemia, and abdominal obesity) are independently characterized by elevated systemic oxidative stress (29 –32). Furthermore, hypertriglyceridemia, hypertension, and obesity are each associated with increased production of superoxide anion via the nicotinamide adenosine diphosphate oxidase pathway (33–35). In addition, hyperglycemia leads to elevated formation of oxygen free radicals as a consequence of protein glycation and glucose autoxidation (36, 37). Our correlational data strongly suggest then that small, dense HDL particles may integrate a wide spectrum of prooxidant signals via several mechanisms; the integration of such signals is, in turn, expressed as attenuated HDL antioxidative activity. The absolute intrinsic antioxidative capacity of all small, dense HDL subfractions (3a, 3b, and 3c) in protecting LDL from oxidation was significantly impaired in MetS patients. Such specific antioxidative activity was more potent in small, dense HDL3, compared with large, light HDL2, in both MetS subjects and controls, consistent with recent data obtained in healthy, normolipidemic subjects (14). Furthermore, measurement of total antioxidative activity (at equivalent plasma concentrations, i.e. at the same 7-fold dilution for each HDL subfraction) revealed that MetS HDL2a and 3a subfractions exerted diminished oxidative protection of LDL oxidation, compared with controls, thereby reflecting both attenuated intrinsic HDL antioxidative activity and lower particle concentrations. Antioxidative activity of HDL can be expressed through multiple mechanisms (9). Removal and inactivation of lipid hydroperoxides (LOOH), which accumulate during LDL oxidation, may however constitute the central mechanism accounting for HDL antioxidative properties (13). Indeed, HDL-associated enzymes, including PON (10), PAF-AH (11), and lecithin:cholesterol acyltransferase (12), may play a key role in the inactivation of LOOH by HDL. In addition, apoA-I can prevent formation of oxidized lipids in LDL by removing seeding molecules of LOOH from LDL (38). The impairment of HDL antioxidative capacity in MetS was selectively observed at late stages of LDL oxidation (Fig. 2, A–C), thereby suggesting that the dysfunction of mechanisms implicated in the inactivation of oxidized lipids (i.e. LOOH removal and hydrolysis) may underlie this effect. Of the HDL-associated enzymes possessing antioxidative activity, the activity of PON1, the major form of HDL-associated PON, was not decreased in MetS compared with controls (Table 2). By contrast, PON1 activity was diminished in diabetic patients in previous studies (39); potentially, impaired glucose tolerance and more marked glycemia in diabetes, compared with MetS, may account for this difference (40). In contrast to PON1, HDL-associated PAF-AH activity was significantly elevated in MetS, compared with controls,

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a finding consistent not only with the observation that PAF-AH activity is increased in MetS subjects (41) but also with the possibility that hyperinsulinemia may impact the production and/or catabolism of PAF-AH significantly (41). We interpret these findings to indicate that neither PON1 nor PAF-AH can account for the impaired antioxidative activity of HDL subfractions in MetS. Nonetheless, this interpretation does not entirely exclude a role for PON1 and PAF-AH in HDL antioxidative activity. Indeed, consistent with earlier data (14), PON1 activity among HDL subfractions was functionally heterogeneous (Table 2); and, in addition, dense HDL3c, which exhibited the highest PON1 activity against phenylacetate, possessed the most potent intrinsic antioxidative activity. Moreover, we found a strong correlation between PON1 activity and HDL antioxidative capacity (Fig. 4C). As documented earlier, the physicochemical properties and structural features of HDL subfractions may be implicated significantly in the expression of their antioxidative activity (14, 42). Consistent with studies in hypertriglyceridemic diabetic populations (43, 44), MetS HDL subfractions were enriched in TG and depleted in CE (Table 2). Such abnormalities are intimately linked to insulin resistance, hypertriglyceridemia, and excess adipose tissue, which is typical of both diabetic patients and nondiabetic MetS subjects. As a consequence of elevated release of fatty acids by adipose tissue and of the loss of insulin-mediated inhibition of hepatic TG-rich VLDL production, the liver overproduces VLDL (45). Because lipoprotein lipase is less active in insulin resistant states (46), the catabolism of VLDL is impaired. As a consequence, an expanded plasma TG pool induces accelerated exchange of TG and CE between VLDL and other lipoproteins, including LDL and HDL. This process is mediated by CE transfer protein and results in the enrichment of TG and depletion of CE from both LDL and HDL particles. There are several possible hypotheses that might explain the impaired antioxidative activity of HDL subfractions in MetS. Our present data support the hypothesis that abnormalities in physicochemical properties of HDL, and specifically TG enrichment of the core of HDL particles, underlie the impairment of the antioxidative properties of HDL subfractions. This hypothesis is consistent with the positive correlation between the TG content of HDL subfractions and the rate of LDL oxidation in the propagation phase, as well as with the negative correlation between HDL TG content and the duration of this phase. Mechanistically, the relationship between TG enrichment of HDL particles and impairment of antioxidative activity can be explained by the fact that HDL enrichment in TG considerably alters the conformation of the central and C-terminal domains of apoA-I (47), which are critical for HDL to act as lipid acceptors (48). More precisely, enrichment in TG reduces exposure of apoA-I to the aqueous phase due to its increased penetration into the lipid core of HDL (47) whose fluidity increases (49). No difference was detected in apoA-I content of HDL subfractions between MetS patients and controls; we cannot exclude, however, the possibility that apoA-I function is altered in MetS. Indeed, apoA-I may undergo oxidative modification in MetS subjects under elevated oxidative stress (Table 1); moreover, MetS patients, even in the absence of diabetes, present moderately elevated plasma levels of glucose, which may favor glycation

4970 J Clin Endocrinol Metab, October 2004, 89(10):4963– 4971

of apoA-I. Such qualitative abnormalities of apoA-I are known to impair HDL antiatherogenic properties and, specifically, its capacity to accept lipids and to promote cellular cholesterol efflux (5, 50). Because apoA-I mediates removal of LOOH from LDL by HDL (13), then this mechanism might lead to attenuated removal of oxidized lipids from LDL and tissues by HDL subfractions in MetS, and thus to impaired HDL antioxidative capacity. In conclusion, these studies provide evidence that hypertriglyceridemia, hyperglycemia, and insulin resistance are closely linked to elevated systemic oxidative stress in MetS, which is, in turn, reflected in the impairment of HDL antioxidative properties. Therefore, our results imply that dysfunctional HDL subfractions play a central role in the expression of elevated oxidative stress in MetS. Because oxidative stress is a key component of endothelial dysfunction, and thus of elevated CV risk in MetS, these results lead us to propose that early treatment of MetS patients, targeted at reduction of oxidative stress and normalization of HDL function, is necessary, even in the absence of diabetes. Thus, thiazolidinediones, which can beneficially influence both insulin resistance and hyperinsulinemia (51), or fibrates, which can selectively raise plasma levels of small, dense HDL3 containing apoA-I and apoA-II (52), may prove efficacious in reducing oxidative stress and CV morbidity in MetS. Because the potent antioxidative activity of HDL3, and especially HDL3c, is impaired to the highest degree in MetS, induction of selective increase in the circulating concentrations of these HDL subfractions may represent a new therapeutic approach for the attenuation of high CV risk in MetS subjects.

Hansel et al. • Oxidative Stress and HDL in Metabolic Syndrome

9. 10. 11.

12. 13.

14. 15. 16. 17. 18. 19. 20.

21.

Acknowledgments Received February 17, 2004. Accepted July 2, 2004. Address all correspondence and requests for reprints to: Dr. Anatol Kontush, Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM) Unite´ 551, Pavillon Benjamin Delessert, Hoˆpital de la Pitie´ , 83 boulevard de l’Hoˆpital, 75651 Paris Cedex 13, France. E-mail [email protected] chups.jussieu.fr. This work was supported by Association Claude Bernard (to S.C.) and by INSERM. B.H. is the recipient of a Research Fellowship from AstraZeneca, France. E.N. is the recipient of a Research Fellowship from Nantes Centre Hospitaliere Universitaire. A.K. is the recipient of an INSERM Fellowship for Senior Investigators, a Fellowship from Fondation pour la Recherche Me´ dicale, and an International High-Density Lipoprotein Research Award from Pfizer.

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