Glucose enhancement of LDL oxidation is strictly metal ion dependent

Free Radical Biology & Medicine, Vol. 29, No. 9, pp. 814 – 824, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(00)00379-8

Original Contribution GLUCOSE ENHANCEMENT OF LDL OXIDATION IS STRICTLY METAL ION DEPENDENT HIRO-OMI MOWRI,* BALZ FREI,†

and JOHN

F. KEANEY, JR.*

*Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA; and †Linus Pauling Institute, Oregon State University, Corvallis, OR, USA (Received 25 February 2000; Revised 13 June 2000; Accepted 22 June 2000)

Abstract—Recent evidence suggests that lipoprotein oxidation is increased in diabetes, however, the mechanism(s) for such observations are not clear. We examined the effect of glucose on low-density lipoprotein (LDL) oxidation using metal ion– dependent and –independent oxidation systems. Pathophysiological concentrations of glucose (25 mM) enhanced copper-induced LDL oxidation as determined by conjugated diene formation or relative electrophoretic mobility (REM) on agarose gels. Similarly, iron-induced LDL oxidation was stimulated by glucose resulting in 4- to 6-fold greater REM than control incubations without glucose. In contrast, glucose had no effect on metal ion– independent LDL oxidation by aqueous peroxyl radicals. The effect of glucose on metal ion– dependent LDL oxidation was associated with enhanced reduction of metal ions, and in the case of iron-induced LDL oxidation, was completely inhibited by superoxide dismutase. The effect of glucose was mimicked by other reducing sugars, such as fructose and mannose, and the extent to which each sugar enhanced LDL oxidation was closely linked to its metal ion–reducing activity. Thus, promotion of LDL oxidation by glucose is specific for metal ion– dependent oxidation and involves increased metal ion reduction. These results provide one potential mechanism for enhanced LDL oxidation in diabetes. © 2000 Elsevier Science Inc. Keywords—Lipoproteins, LDL, Sugars, Oxidation, Metal ions, Free radicals

INTRODUCTION

The relationship between hyperglycemia and CAD was the subject of considerable debate, since serum glucose did not consistently predict the existence of CAD in type II diabetes [3]. Recent prospective studies, however, have clearly established a link between chronic average glucose levels (hemoglobin A1c) and cardiovascular morbidity and mortality [4,5]. The precise mechanism by which hyperglycemia may contribute to the development of coronary artery disease is a matter of some controversy. Known sequelae of hyperglycemia such as cell damage, increased extracellular matrix production, and vascular dysfunction have all been implicated in the pathogenesis of vascular disease in type I and II diabetes [1]. Considerable circumstantial evidence suggests that hyperglycemia is associated with excess oxidation, although most measurements of excess oxidation are, admittedly, indirect. For example, the plasma of hyperglycemic subjects contains increased levels of F2isoprostanes [6] and lipid hydroperoxides [7], two markers of lipid peroxidation. Similarly, urinary levels

Compared to the normal population, patients with both type I and II diabetes are more likely to develop renal failure, blindness, and atherosclerotic vascular disease [1]. In particular, coronary artery disease (CAD) is the major cause of death among patients with diabetes [2]. Risk factors for CAD such as hypertension, obesity, and hypercholesterolemia, are more prevalent in patients with type II diabetes than in the general population [2], but account for only about 25% of the excess coronary artery disease risk in these patients [3]. Thus, there is some element (or elements) of type I and II diabetes that provides for an excess risk of cardiovascular disease that is not quantified using traditional risk factor analysis. Diabetes is associated with a number of metabolic alterations and principal among these is hyperglycemia. Address correspondence to: Dr. John F. Keaney, Jr., Boston University School of Medicine, Whitaker Cardiovascular Institute, 715 Albany Street, Room W507, Boston, MA 02118, USA; Tel: (617) 6384894; Fax: (617) 638-5437; E-Mail: [email protected]. 814

Glucose and LDL oxidation

of F2-isoprostanes are increased in patients with diabetes and decrease significantly with aggressive control of hyperglycemia [8]. In addition, LDL isolated from patients with poorly controlled type I diabetes is more susceptible to copper-induced oxidation than LDL from control subjects [9]. There is now considerable evidence that hyperglycemia has both direct and indirect effects that promote lipoprotein oxidation and, as a consequence, atherosclerosis. Glucose may combine directly with phospholipids located on the surface of LDL and apo B lysine groups leading to the formation of advanced glycosylation endproducts (AGEs) that may facilitate lipid peroxidation [10]. In addition, the autoxidation of glucose and nonenzymatic glycation of proteins can result in the generation of superoxide [11], which has been implicated in the mechanism of LDL oxidation by vascular cells [12,13]. Pathologically relevant concentrations of glucose have been shown to accelerate copper-induced LDL oxidation through a superoxide-dependent mechanism [14]. Since LDL oxidation appears to be a critical event in atherosclerosis, glucose-enhanced LDL oxidation may represent one mechanism of increased atherosclerosis in patients with type I and II diabetes. Despite this potential link, the specific mechanism by which hyperglycemia promotes LDL oxidation is not yet clear. The purpose of this study was to examine the mechanism of enhanced LDL oxidation by glucose. Research design and methods LDL isolation. Blood was collected from fasting healthy male subjects in Vacutainer tubes (Becton Dickinson; Mountain View, CA, USA) containing 286 USP units sodium heparin/15 ml of whole blood, and centrifuged to obtain plasma. LDL was isolated from fresh plasma by single vertical spin discontinuous density ultracentrifugation using a Beckman NVT 90 rotor (Beckman Coulter Inc.; Fullerton, CA, USA) (80,000 rpm, 45 min, 7°C) [15] under programmed slow acceleration and deceleration. Isolated LDL was treated with Chelex-100 resin (BioRad; Hercules, CA, USA) to remove adventitious metal ions, filtered through a 0.2 ␮m membrane (Acrodisc LC13; Gelman Sciences, Ann Arbor, MI, USA), and then subjected to size exclusion gel filtration with a Sephadex G-25 column (Pharmacia LKB Biotechnology Inc.; Piscataway, NJ, USA) as described [16]. LDL protein was determined by the method of Peterson [17] and LDL thus isolated was used immediately for experiments with no storage. LDL oxidation. For metal ion–induced LDL oxidation, LDL (0.1 mg protein/ml) was incubated at 37°C in 10 mM phosphate-buffered saline (PBS), pH 7.4, with or

815

without glucose and either 1.1–10 ␮M CuSO4 or a complex of 12.5 ␮M FeCl3 and nitrilotriacetic acid (NTA) as described [18]. Iron-induced LDL oxidation was conducted under sterile conditions in a CO2 incubator in the presence of 2.5 ␮M diethylaminetriamine pentaacetic acid (DTPA) to reduce background oxidation from contaminating transition metals. Oxidation reactions were stopped by addition of 50 ␮M DTPA. For metal ion–independent LDL oxidation, LDL (0.1 mg protein/ml) was incubated at 37°C with 4 mM 2,2⬘azobis(2-amidinopropane) hydrochloride (AAPH) and 10 ␮M DTPA in the presence or absence of glucose in PBS, and oxidation was stopped by cooling the tubes with crushed ice. The resistance of LDL particles to oxidation was assessed as the lag time prior to rapid conjugated diene formation (OD234nm) as described [19]. Cholesteryl ester hydroperoxides were determined by HPLC with chemiluminescence detection [19]. LDL oxidation was also evaluated by assessing protein modification using relative electrophoretic mobility of LDL on 0.5% agarose gels [20] with a Beckman Paragon electrophoresis system (Beckman Coulter Inc.) according to the manufacturer’s instructions. Measurement of Cu2⫹ and Fe3⫹ reduction. The concentration of Cu⫹ and Fe2⫹ was determined using bathocuproine disulfonate (BC) and bathophenanthroline disulfonate (BP) as specific reagents, respectively, based on the procedure described by Lynch and Frei [21]. For Cu⫹ measurement, LDL (0.1 mg protein/ml) was incubated at 37°C with 3.3 ␮M CuSO4 in the presence or absence of glucose (or other sugar) in PBS containing 360 ␮M bathocuproine disulfonate in a cuvette, and the absorbance at 480 nm was recorded at 2 min intervals. For Fe2⫹ determination, LDL (0.1 mg protein/ml) was incubated with 12.5 ␮M FeCl3/NTA and 2.5 ␮M DTPA in the presence or absence of glucose (or other sugar) in PBS containing 360 ␮M bathophenanthroline disulfonate, and the absorbance at 535 nm was measured at 24 h intervals as described [21]. SOD activity. Mn-SOD activity was determined by the method of Marklund and Marklund [22]. Enzyme preparations, native or heat-inactivated, were incubated with 10 mM pyrogallol at 25°C in 50 mM Tris-HCl buffer, pH 8.2, containing 2 mM DTPA, and autoxidation of pyrogallol was continuously monitored at a wavelength of 420 nm. RESULTS

Effects of glucose on copper-induced LDL oxidation The susceptibility of LDL to copper-induced oxidation was enhanced by a relevant pathologic concentration

816

H.-O. MOWRI et al.

(Fig. 2). The stimulatory effect of glucose was most marked after 4 h of incubation, resulting in a 40% higher relative electrophoretic mobility of LDL than in control incubations without glucose (Fig. 2A). We also found that copper-induced oxidative modification of LDL was promoted by 6 –25 mM glucose in a concentration-dependent manner (Fig. 2B). In contrast, glucose exerted little or no enhancement of LDL protein modification when LDL oxidation was conducted with 3.3 or 10 ␮M copper (data not shown), consistent with the above data on lipid peroxidation.

Effects of glucose on iron-induced LDL oxidation

Fig. 1. Effect of glucose on copper-induced LDL oxidation. (A) LDL (0.1 mg protein/ml) was incubated with 1.1 ␮M CuSO4 in the presence (F) or absence (■) of 25 mM glucose. Optical density at 234 nm was monitored at 5 min intervals. One experiment representative of 5 is shown. (B) LDL (0.1 mg protein/ml) was incubated with the indicated concentration of CuSO4 in the presence (F) or absence (■) of 25 mM glucose. Optical density at 234 nm was monitored at 5 min intervals and diene conjugation lag time (Tlag) was determined as described in Methods. Data are the mean ⫾ SD of three independent experiments. *p ⬍ .05 vs. with glucose by two-way ANOVA. (C) LDL (0.1 mg protein/ml) was incubated with 1.1 ␮M CuSO4 in the presence (F) or absence (■) of 25 mM glucose. At each time point, oxidation was stopped by addition of 50 ␮M DTPA and 100 ␮M butylated hydroxytoluene. Lipids were extracted and cholesteryl ester hydroperoxides were quantitatively detected as described [19]. Data are representative of three experiments.

The mechanisms of copper- and iron-induced LDL oxidation differ in several respects [21,23,24]; therefore, to further characterize the effect of glucose on metal ion–mediated LDL oxidation, LDL was incubated with 12.5 ␮M FeCl3/NTA and 2.5 ␮M DTPA in the absence or presence of glucose. LDL oxidation induced by iron proceeded at a much lower rate than with copper. After 6 d of incubation, the REM of LDL incubated with FeCl3/NTA was only about 1.5. Glucose (25 mM) greatly enhanced iron-induced LDL oxidation by 4- to 6-fold (Figs. 3A and 3B). The stimulatory effect of glucose on iron-induced LDL modification was dosedependent with respect to glucose (Fig. 3B), and not abrogated by increasing concentrations of iron (Fig. 3C). The stimulation of iron-induced LDL oxidation was dependent upon the presence of glucose at the early stages of oxidation. As shown in Table 1, the inclusion of glucose and FeCl3/NTA from the start of the incubation (Day 0) increased the REM of LDL 80%. In contrast, inclusion of glucose 2 or 4d after exposure of LDL to FeCl3/NTA only increased LDL REM by 31% or 0%, respectively.

Effects of glucose on AAPH-induced LDL oxidation of glucose (25 mM). The diene conjugation lag phase in LDL exposed to 1.1 ␮M copper was 50% shorter in the presence of 25 mM glucose than in its absence (Fig. 1A). With increasing concentrations of copper, the stimulatory effect of glucose upon LDL oxidation decreased (Fig. 1B). Consistent with conjugated diene formation, copper (1.1 ␮M) -induced production of cholesteryl ester hydroperoxides in LDL was promoted by the presence of glucose in a time-dependent manner (Fig. 1C). The enhancement of copper-induced lipid peroxidation in LDL by glucose also resulted in enhanced LDL protein modification, as demonstrated by increased relative electrophoretic mobility (REM) on agarose gels

To study the effects of glucose on metal ion–independent LDL oxidation, LDL was exposed to peroxyl radicals using AAPH in the presence or absence of glucose. As shown in Fig. 4, increasing concentrations of glucose did not enhance LDL oxidation as determined by REM, but rather tended to slightly suppress the oxidation. Consistent with these results, 6.3–25 mM glucose did not shorten the lag phase for conjugated diene formation when LDL was oxidized with AAPH under similar conditions as in Fig. 4 (data not shown). These results suggest that glucose-stimulated LDL oxidation is specific for metal ion– dependent oxidation.

Glucose and LDL oxidation

817

Fig. 2. Time and concentration dependence of glucose-stimulated copper-induced LDL oxidation. LDL (0.1 mg protein/ml) was incubated with 1.1 ␮M CuSO4 in the presence or absence of 25 mM glucose for up to 8 h (A), or with the indicated concentrations of glucose for 4 h (B). LDL oxidation was assessed by REM as described in Methods. The arrow indicates the gel origin. One experiment representative of three is shown.

Effects of SOD on glucose-stimulated metal ion–induced LDL oxidation Superoxide is known to reduce transition metal ions, and the reduced form of transition metal ions is more active with respect to initiating and propagating lipid peroxidation [21]. To investigate the involvement of superoxide in glucose-stimulated metal ion– dependent LDL oxidation, Mn-SOD and its heat-inactivated preparation were used rather than CuZn-SOD since heat inactivation of CuZn-SOD releases copper [25]. In the absence of glucose, copper-induced LDL oxidation was inhibited 68% by Mn-SOD. However, Mn-SOD did not suppress the stimulatory effect of glucose. Specifically, 25 mM glucose enhanced copper-induced LDL oxidation by 50% in either the presence or absence of SOD (Fig. 5A). In contrast, Mn-SOD inhibited iron-induced LDL oxidation by ⱖ 80% and prevented any stimulatory effect of glucose (Fig. 5B). Heat inactivation of Mn-SOD prevented its inhibition of both copper- and iron-induced LDL oxidation (Figs. 5A and 5B). In contrast to SOD, catalase did not prevent copper- or iron-induced LDL oxidation regardless of the presence of glucose (data not shown). These results suggest that superoxide plays a

critical role in the process of glucose-stimulated ironinduced LDL oxidation.

Effects of glucose on metal ion reduction in the presence or absence of LDL Since glucose has a reducing potential that is based upon its aldehyde residue in the open-ring form, it is conceivable that glucose enhances metal ion–mediated LDL oxidation by facilitating the production of reduced metal ions that participate in oxidation reactions. To test this possibility, the copper- or iron-reducing activity of glucose was analyzed under conditions similar to those described above. In these assays, the mixtures containing LDL and metal ions were coincubated with bathocuproine disulfonate (BC) or bathophenanthroline disulfonate (BP), specific reagents for Cu⫹ and Fe2⫹, respectively. As shown in Fig. 6, both copper- and ironreducing activities of LDL were enhanced by glucose in a dose-dependent manner (p ⬍ .05 by two-way ANOVA). Qualitatively similar results were observed without LDL, except in the absence of glucose little or no metal ion reduction was observed (Fig. 6).

818

H.-O. MOWRI et al.

Fig. 3. Time and concentration dependence of glucose-stimulated iron-induced LDL oxidation. LDL (0.1 mg protein/ml) was incubated with 12.5 ␮M FeCl3/NTA and 2.5 ␮M DTPA in the presence or absence of 25 mM glucose for up to 6 d (A), or with the indicated concentrations of glucose (B) for 6 d. (C) LDL (0.1 mg protein/ml) was incubated with the indicated concentrations of FeCl3/NTA and 2.5 ␮M DTPA with or without glucose for 6 d. LDL oxidation was determined by electrophoresis as in Fig. 2. One experiment representative of two is shown.

Effects of SOD on glucose-enhanced metal ion reduction To determine whether superoxide is involved in the acceleration of metal ion reduction by glucose, MnSOD was used. As shown in Fig. 7A, Cu2⫹ reduction by glucose was actually enhanced by Mn-SOD. In

contrast, Mn-SOD inhibited glucose-mediated Fe3⫹ reduction ⬃ 60%, while autoclaved SOD was not inhibitory (Fig. 7B). These results suggest that glucose-mediated Fe3⫹ reduction is, in part, mediated by superoxide, consistent with our observations that SOD inhibits glucose-mediated enhancement of iron-induced LDL oxidation.

Glucose and LDL oxidation

819

Table 1. The Duration of Glucose Exposure and Iron-Induced LDL Oxidation

Day of glucose addition

Glucose (mM)

Relative electrophoretic mobility

Stimulation (% over control)

0

0 25

3.0 4.6

— 80

2

0 25

2.6 3.4

— 31

4

0 25

2.8 2.8

— 0

LDL (0.1 mg protein/ml) was incubated in PBS with 12.5 ␮M FeCl3/NTA and 2.5 ␮M DTPA for 6 days. Glucose (25 mM) was added on the indicated day and LDL oxidation determined by REM. For each glucose containing incubation, a parallel control incubation was treated with an equal volume of PBS on the indicated day. Data are the mean of two independent experiments.

Effects of various sugars on copper- or iron-induced LDL oxidation Glucose, like all monosaccharides, has intrinsic reducing activity. Insofar as reduced metal ions are required for LDL oxidation [21], we further sought to determine if the stimulatory effects of glucose on metal ion–mediated LDL oxidation are due to enhanced metal ion reduction. To this end, we compared the metal ion– reducing activity of three separate sugars with their ability to stimulate metal ion–induced LDL oxidation, as measured by REM. We found that fructose is a more powerful metal ion reductant than glucose, and correspondingly, had a greater stimulatory effect on LDL modification induced by copper or iron (Figs. 8A and 8B). Mannose, on the other hand, accelerated copper- or iron-induced LDL oxidation to a similar level as did glucose, consistent with the similar activity of these two sugars to reduce metal ions.

Fig. 5. The effect of SOD on glucose-stimulated iron- and copperinduced LDL oxidation. Open bars represent LDL (0.1 mg protein/ml) incubated for 4 h with 1.1 ␮M CuSO4 (A), or for 6 d with 12.5 ␮M FeCl3 and 2.5 ␮M DTPA (B), in the presence of the indicated concentrations of glucose. In some incubations (black bars), manganese SOD (10 U/ml) or the equivalent amount (2.5 ␮g/ml) of its heat-inactivated preparation (grey bars) were included. LDL oxidative modification is presented as a percent of LDL electrophoretic mobility in the absence of any glucose or SOD. One of three representative experiments is shown.

Fig. 4. The effect of glucose on peroxyl radical–induced LDL oxidation. LDL (0.1 mg protein/ml) was incubated for 6 or 18 h with 4 mM AAPH and 10 ␮M DTPA in the absence or presence of the indicated concentrations of glucose. LDL oxidation was determined as in Fig. 2.

820

H.-O. MOWRI et al.

Fig. 7. The effect of SOD on metal ion reduction by glucose. (A) A mixture containing 360 ␮M BC, 3.3 ␮M CuSO4, and 25 mM glucose was incubated for 2 min with or without 10 ␮g/ml Mn-SOD or its heat-inactivated form (HI-SOD). (B) A mixture containing 360 ␮M BP, 12.5 ␮M FeCl3, 2.5 ␮M DTPA, and 25 mM glucose was incubated for 24 h with or without 10 ␮g/ml Mn-SOD or its heat-inactivated form (HI-SOD). Bars express the rate of Cu⫹ or Fe2⫹ formation, as measured by an increase in the absorbance at 480 nm or 535 nm, respectively. Data represent means of two independent experiments.

Fig. 6. The effect of glucose on copper and iron reduction. (A) A mixture containing 360 ␮M BC, 3.3 ␮M CuSO4, and 0, 5, or 25 mM glucose was incubated for 2 min with or without LDL (0.1 mg protein/ ml). (B) A mixture containing 360 ␮M BP, 12.5 ␮M FeCl3, 2.5 ␮M DTPA, and 0, 5, or 25 mM glucose was incubated for 24 h with or without LDL (0.1 mg protein/ml). Bars express the rate of Cu⫹ or Fe2⫹ formation, as measured by an increase in the absorbance at 480 nm or 535 nm, respectively. Data represent mean ⫾ SD from three experiments with different LDL preparations. *p ⬍ .01 for glucose effect by ANOVA. DISCUSSION

A principal finding of this study is that pathophysiologically relevant concentrations of glucose accelerate metal ion– dependent LDL oxidation, but have no effect on LDL oxidation by aqueous peroxyl radicals. The specific stimulation of metal ion– dependent LDL oxidation by glucose appears related to the reduction of metal ions. Indeed, other sugars also stimulate metal ion–mediated LDL oxidation in a manner that parallels their reducing potential towards copper and iron. The reduction of copper or iron by glucose appears to involve distinct mechanisms: we found evidence that superoxide has an important role in the effect of glucose on ironinduced LDL oxidation. In contrast, the effect of glucose on copper-induced LDL oxidation does not appear to involve superoxide. The role of glucose in the promotion of LDL oxidation has been examined previously. Hunt and colleagues

Fig. 8. Metal ion–reducing activity and promotion of LDL oxidation by different sugars. LDL (0.1 mg protein/ml) was incubated for 4 h with (A) 1.1 ␮M CuSO4 or (B) 12.5 ␮M FeCl3/NTA, 2.5 ␮M DTPA in the presence or absence of 25 mM glucose, fructose, or mannose. LDL oxidation was assessed as described in Fig. 2. For measurement of metal ion reduction, a mixture of (A) 360 ␮M BC, 1.1 ␮M CuSO4 or (B) 360 ␮M BP, 12.5 ␮M FeCl3/NTA, 2.5 ␮M DTPA was incubated with the indicated sugar (25 mM) and the absorbance at (A) 480 nm or (B) 535 nm was monitored. Data represent means of two experiments.

Glucose and LDL oxidation

[26] found that glucose autoxidation resulted in the formation of hydroxyl radicals that supported LDL oxidation. This pro-oxidant effect of glucose was stimulated by copper and inhibited by metal ion chelation [26]. Similarly, Kawamura and colleagues found that glucose stimulated copper-induced LDL oxidation and this effect was inhibited by the chelation of metal ions [14]. Our results are in agreement with and expand upon these prior reports, as we found that glucose at pathophysiologically relevant concentrations stimulated both copper- and iron-induced LDL oxidation (Figs. 1, 2, and 3). Our results indicate that glucose is most effective in stimulating copper-induced LDL oxidation at a copper concentration of 1.1 ␮M (Fig. 1B), corresponding to a molar ratio of copper to LDL of 5.5. At higher ratios of copper to LDL, oxidation was sufficiently brisk as to be unaffected by glucose. This observation is in agreement with data from Gieseg and Esterbauer [27], who found that copper-induced LDL oxidation was saturable and could not be enhanced when the molar ratio of copper to LDL exceeded the range of 5–19. Gieseg and Esterbauer attributed this observation to a finite number of copperbinding sites in LDL [27]. Given that copper-induced LDL oxidation also requires the reduction of LDL-bound Cu2⫹ to Cu⫹ [21], it follows that the rate of LDL oxidation will be limited by the availability of reduced copper at low molar ratios of copper to LDL (i.e., less than 5 to 19). Since glucose reduces copper (Fig. 6), one might expect glucose to stimulate copper-induced LDL oxidation under these conditions. In contrast, at higher molar ratios of copper to LDL (greater than 5 to 19), the availability of reduced copper is not rate limiting for LDL oxidation and, thus, enhanced copper reduction by glucose should not materially alter the rate of LDL oxidation. In the milieu of the arterial wall, the ratio of copper ions to LDL particles is likely to be considerably less than the molar ratio of five used here. In human plasma, the normal copper concentrations ranges from 16 –31 ␮M [28] with greater than 95% of this found in ceruloplasmin and the rest associated with albumin and amino acids [29]. Of this total copper, an arguably small amount would be available for redox reactions. With an LDL molar concentration in plasma of approximately 2 ␮M, it is hard to envision a molar ratio of redox-active copper to LDL that even approaches unity. Nevertheless, the results depicted in Fig. 1B suggest that even lower concentrations of copper would still demonstrate a stimulatory effect of glucose. The stimulatory effect of glucose on LDL oxidation was not restricted to copper. We also found that ironinduced LDL oxidation was enhanced by glucose in a time- and concentration-dependent manner (Fig. 3). In contrast to our observations with copper, glucose stimu-

821

lated iron-induced LDL oxidation throughout a range of iron concentrations (Fig. 3C). Moreover, the overall stimulatory effect of glucose on iron-induced LDL oxidation was greater than that observed with copper. Given the nature of iron-mediated LDL oxidation, this latter observation is to be expected. Iron-mediated lipid peroxidation requires both Fe3⫹ and Fe2⫹ [30], and LDL is a less effective reductant for Fe3⫹ than Cu2⫹ [21]. Thus iron-induced LDL oxidation is largely dependent upon some external source of iron reduction [21,23,24]. Glucose, by virtue of its capacity to reduce iron (Fig. 6B), may provide the necessary ratio of Fe3⫹ to Fe2⫹ to support lipid peroxidation. Metal ion reduction by glucose is a well-described phenomenon. Monosaccharides with an ␣-hydroxyaldehyde structure may undergo enediol rearrangement that facilitates the reduction of transition metal ions resulting in the formation of an enediol radical anion [31]. The enediol radical anion reduces molecular oxygen to form superoxide and a dicarbonyl product [31]. Similar chemistry has been reported for protein glycation in which glucose forms the Amadori compound, fructoselysine [32]. The Amadori compound is then subject to 1,2enediol rearrangement with carbohydrate hydrolysis and formation of the dicarbonyl compound, D-glucosone [32]. Transition metal ion reduction may result from enediol oxidation [31], or direct reduction by the Amadori compound itself [33], glucosone [33], or superoxide [34]. In our system, reduction of Fe3⫹ was partially dependent on superoxide as SOD inhibited glucose-mediated iron reduction by ⬃ 60% (Fig. 7B). In contrast, Cu2⫹ reduction by glucose was not superoxide dependent and was slightly enhanced by SOD (Fig. 7B). Related to the latter observation, we also found that SOD did not inhibit the stimulatory effect of glucose on copper-induced LDL oxidation (Fig. 5). At face value, this result would seem to contradict the report by Kawamura and colleagues that glucose stimulates copper-induced LDL oxidation by a superoxide-dependent mechanism [14]. However, Kawamura and colleagues found that SOD inhibits copper-induced LDL oxidation ⬃ 50 –70% in both the absence and presence of glucose [14], i.e., superoxide seems to play a role in copper-induced oxidation, but not in the stimulatory effect of glucose. Since our data and those of Kawamura and coworkers indicate that glucose stimulates copperinduced LDL oxidation in the presence of SOD, we would submit that the effect of glucose is superoxide independent. In contrast, the stimulatory effect of glucose on iron-induced LDL oxidation is entirely dependent upon superoxide since it is completely inhibited by SOD (Fig. 5B). This finding is particularly interesting in that SOD inhibited glucose-mediated iron reduction by only ⬃ 60%, suggesting that reduction of iron by super-

822

H.-O. MOWRI et al.

oxide alone is not sufficient to support iron-induced lipid peroxidation. An unexpected finding of our study was the inhibitory effect of SOD on both copper- and iron-induced LDL oxidation in the absence of glucose (Fig. 5). In past investigations, the inhibitory effect of SOD on copperinduced LDL oxidation was attributed to nonspecific binding of copper to SOD [25,35]. A complicating feature of these investigations is the fact that heat-inactivated CuZn-SOD can release copper and, as a result, promote oxidation [25]. In the present study, we used Mn-SOD to specifically avoid the potential for contaminating copper. In our hands, only ⬃ 10% of copper ions were found to associate with the enzyme when Mn-SOD was mixed with CuSO4 at a ratio equivalent to our standard conditions for LDL oxidation, followed by an extensive dialysis against PBS (data not shown). Furthermore, heat-inactivated (autoclaved) Mn-SOD did not suppress copper- or iron-induced LDL oxidation. This finding appears to conflict with the report of Jessup and colleagues [25] who observed that heat-inactivated MnSOD had the same inhibitory effect on macrophagemediated and cell-free LDL oxidation. However, Jessup and colleagues used F-10 medium for LDL oxidation, whereas we used PBS. In addition, we employed autoclaving for heat inactivation of Mn-SOD instead of boiling [25] as our boiled preparations retained about 30% residual activity. Thus, there are several differences between these studies that may account for the contrasting findings. With respect to the effect of glucose on iron-induced LDL oxidation, one must consider the extent to which LDL glycation also contributed to our findings given the relatively long incubation periods, i.e., up to 6 d. It has been shown that LDL glycated in vitro is more susceptible to oxidation than nonglycated LDL [36,37]. Furthermore, glycated LDL prepared in vitro or isolated from the plasma of type I diabetic patients is degraded more rapidly by human monocyte– derived macrophages than native LDL [36 –38]. The precise extent to which protein or lipid glycation contributed to our observations is not known, but warrants investigation. In the present study, fructose accelerated copper- or iron-induced LDL oxidation to a greater extent than did glucose or mannose, due most likely to higher metalreducing activity of fructose (Fig. 8). Intracellularly, glucose is in part metabolized to fructose through the sorbitol pathway. This metabolic process is known to be of importance particularly in renal, neural, and ocular tissues [39], where the levels of fructose are significantly increased in streptozotocin-induced diabetic rats [40 – 43]. It is possible that fructose may also be a powerful stimulant for lipid peroxidation in vivo and have pathophysiological implications in diabetes.

We are mindful of the fact that a number of compounds beyond glucose and fructose have the capacity to reduce metal ions. Alpha-tocopherol is an important LDL-associated reductant for copper [44] that is required for the initiation of copper-mediated LDL oxidation [45]. We did determine LDL ␣-tocopherol consumption under the conditions described in Fig. 1 and found that more than 90% of LDL-␣-tocopherol was consumed during the first 60 min of oxidation and this was not affected by glucose (data not shown). Under these conditions, glucose enhanced LDL oxidation assessed by diene conjugation formation only after ␣-tocopherol consumption (Fig. 1). One possible interpretation of these results is that glucose promotes LDL oxidation only after ␣-tocopherol is depleted, and ␣-tocopherol is a more efficient reductant for copper than glucose. Since atherosclerotic lesions are not depleted of ␣-tocopherol [46], it is not clear how glucose would stimulate LDL oxidation in the arterial wall. It is not clear if transition metal ions such as copper and iron ions have implications in diabetes mellitus as the causal or contributing factors. However, certain possibilities have already been proposed that are in favor of such roles of these metal ions. It has been reported that plasma levels of ceruloplasmin and consequently, its bound copper are increased in type I or II diabetes mellitus [47– 49]. This potentially increases vascular oxidative stress since bound copper in ceruloplasmin has been demonstrated to oxidatively modify LDL in vitro [50]. As another possibility, hyperglycemia, through glycation or promoted acidosis, may cause mobilization of a part of transferrin-bound iron ions into free forms that are potential stimulants of oxygen radical–induced lipid peroxidation [51]. In summary, we have found that glucose enhances metal ion–mediated LDL oxidation by a mechanism involving enhanced metal ion reduction. The nature of copper or iron reduction by glucose is specific. In particular, copper reduction by glucose appears direct and superoxide independent. In contrast, iron reduction by glucose is at least partially superoxide dependent, which is reflected in the fact that SOD inhibits the stimulatory effect of glucose on iron-induced oxidation. Insofar as metal ions contribute to LDL oxidation in vivo, our data support the hypothesis that hyperglycemia stimulates LDL oxidation accounting, at least in part, for the increased CAD risk in type I and II diabetes. Acknowledgements — This work was supported by NIH grants HL55854 (JFK) and HL56170 (BF). John F. Keaney, Jr. is the recipient of a Clinical Investigator Development Award (HL03195) from the National Institutes of Health. REFERENCES [1] Ruderman, N. B.; Williamson, J. R.; Brownlee, M. Glucose and diabetic vascular disease. FASEB. J. 6:2905–2914; 1992.

Glucose and LDL oxidation [2] Bierman, E. L. George Lyman Duff memorial lecture: atherogenesis in diabetes. Arterioscler. Thromb. 12:647– 656; 1992. [3] Pyorala, K.; Laasko, M.; Uusitupa, M. Diabetes and atherosclerosis: an epidemiologic view. Diabet. Met. Rev. 3:463–524; 1987. [4] Singer, D. E.; Nathan, D. M.; Anderson, K. M.; Wilson, P. W. F.; Evans, J. C. Association of HbA1c with prevalent cardiovascular disease in the original cohort of the Framingham Heart Study. Diabetes 41:202–209; 1992. [5] Kuusisto, J.; Mykkanen, L.; Pyorala, K.; Laakso, M. NIDDM and its metabolic control predict coronary heart disease in elderly subjects. Diabetes 43:960 –967; 1994. ¨ nggård, E. E.; Mallet, A. I.; Betteridge, D. J.; [6] Gopaul, N. K.; A Wolff, S. P.; Nourooz-Zadeh, J. Plasma 8-epi-PGF2alpha levels are elevated in individuals with noninsulin-dependent diabetes mellitus. FEBS Lett. 368:225–229; 1995. [7] Nourooz-Zadeh, J.; Tajaddini-Sarmadi, J.; Mccarthy, S.; Betteridge, D. J.; Wolff, S. P. Elevated levels of authentic plasma hydroperoxides in NIDDM. Diabetes 44:1054 –1058; 1995. [8] Davi, G.; Ciabottoni, G.; Consoli, A.; Messetti, A.; Falco, A.; Santarone, S.; Pennese, E.; Vitacolonna, E.; Bucciarelli, T.; Costantini, F.; Capani, F.; Patrono, C. In vivo formation of 8-iso-prostaglandin F2␣ and platelet activation in diabetes mellitus: effects of improved metabolic control. Circulation 99:224 – 229; 1999. [9] Tsai, E. C.; Hirsch, I. B.; Brunzell, J. D.; Chait, A. Reduced plasma peroxyl radical trapping capacity and increased susceptibility of LDL to oxidation in poorly controlled IDDM. Diabetes 43:1010 –1014; 1994. [10] Bucala, R.; Makita, Z.; Koschinsky, T.; Cerami, A.; Vlassara, H. Lipid advanced glycosylation: pathway for lipid oxidation in vivo. Proc. Natl. Acad. Sci. USA 90:6434 – 6438; 1993. [11] Baynes, J. W. Role of oxidative stress in the development of complications in diabetes. Diabetes 40:405– 412; 1991. [12] Heinecke, J. W.; Baker, L.; Rosen, H.; Chait, A. Superoxidemediated modification of low-density lipoprotein by human arterial smooth muscle cells in culture. J. Clin. Invest. 77:757–761; 1986. [13] Hiramatsu, K.; Rosen, H.; Heinecke, J. W.; Wolfbauer, G.; Chait, A. Superoxide initiates oxidation of low-density lipoprotein by human monocytes. Arteriosclerosis 7:55– 60; 1986. [14] Kawamura, M.; Heinecke, J. W.; Chait, A. Pathophysiological concentrations of glucose promote oxidative modification of lowdensity lipoprotein by a superoxide-dependent pathway. J. Clin. Invest. 94:771–778; 1994. [15] Chung, B. H.; Segrest, J. P.; Ray, M. J.; Brunzell, J. D.; Hokanson, J. E.; Krauss, R. M.; Beaudrie, K.; Cone, J. T. Single vertical spin density gradient ultracentrifugation. Methods Enzymol. 128: 181–209; 1986. [16] Shwaery, G. T.; Vita, J. A.; Keaney, J. F. Jr. Antioxidant protection of LDL by physiological concentrations of 17 ␤-estradiol: requirement for estradiol modification. Circulation 95:1378 –1386; 1997. [17] Peterson, G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346 –356; 1977. [18] Dabbagh, A. J.; Frei, B. Human suction blister interstitial fluid prevents metal ion– dependent oxidation of low-density lipoprotein by macrophages and in cell-free systems. J. Clin. Invest. 96:1958 –1966; 1995. [19] Frei, B.; Gaziano, J. M. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion– dependent and –independent oxidation. J. Lipid Res. 34:2135–2145; 1993. [20] Noble, R. P. Electrophoretic separation of plasma lipoproteins in agarose gel. J. Lipid Res. 9:693–700; 1968. [21] Lynch, S. M.; Frei, B. Reduction of copper, but not iron, by human low-density lipoprotein (LDL). Implications for metal ion– dependent oxidative modification of LDL. J. Biol. Chem. 270:5158 –5163; 1995. [22] Marklund, S.; Marklund, G. Involvement of the superoxide anion

[23]

[24]

[25]

[26]

[27] [28]

[29]

[30] [31]

[32] [33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42]

[43]

823

radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47:469 – 474; 1974. Lynch, S. M.; Frei, B. Mechanisms of copper- and iron-dependent oxidative modification of human low-density lipoprotein. J. Lipid Res. 34:1745–1753; 1993. Lynch, S. M.; Frei, B. Physiological thiol compounds exert proand antioxidant effects, respectively, on iron- and copper-dependent oxidation ofhuman low-density lipoprotein. Biochim. Biophys. Acta 1345:215–221; 1997. Jessup, W.; Simpson, J. A.; Dean, R. T. Does superoxide radical have a role in macrophage-mediated oxidative modification of LDL? Atherosclerosis 99:107–120; 1993. Hunt, J. V.; Smith, C. C. T.; Wolff, S. P. Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabetes 39:1420 –1424; 1990. Gieseg, S. P.; Esterbauer, H. Low-density lipoprotein is saturable by pro-oxidant copper. FEBS Lett. 343:188 –194; 1994. Scurry, R. E.; McNeely, B. U.; Mark, E. J. Case records of the Massachusetts General Hospital. New Engl. J. Med. 314:39 – 49; 1986. Halliwell, B.; Gutteridge, J. M. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 186:1– 85; 1990. Minotti, G.; Aust, S. D. The role of iron in the initiation of lipid peroxidation. Chem. Phys. Lipids 44:191–208; 1987. Wolff, S. P.; Dean, R. T. Glucose autoxidation and protein modification: their potential role of autoxidative glycosylation in diabetes. Biochem. J. 245:243–250; 1987. Hodge, J. E. The Amadori rearrangement. Adv. Carbohydr. Chem. 10:169 –205; 1955. Baker, J. R.; Zyzak, D. V.; Thorpe, S. R.; Baynes, J. W. Mechanism of fructosamine assay: evidence against role of superoxide as intermediate in nitroblue tetrazolium reduction. Clin. Chem. 39:2460 –2465; 1994. Mullarkey, C. J.; Edelstein, D.; Brownlee, M. Free radical generation by early glycation products: a mechanism for accelerated atherogenesis in diabetes. Biochem. Biophys. Res. Commun. 173: 932–939; 1990. Thomas, C. E. The influence of medium components on Cu(2⫹)dependent oxidation of low-density lipoproteins and its sensitivity to superoxide dismutase. Biochim. Biophys. Acta 1128:50 –57; 1992. Lyons, J. T.; Klein, R. L.; Bayes, J.; Stevenson, H. C.; LopesVirella, M. F. Stimulation of cholesteryl ester synthesis in human monocyte– derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients: the influence of nonenzymatic glycosylation of low-density lipoproteins. Diabetologia 30:923; 1987. Klein, R. L.; Laimins, M.; Lopes-Virella, M. F. Isolation, characterization, and metabolism of the glycated and nonglycated subfractions of low-density lipoproteins isolated from type I diabetic patients and nondiabetic subjects. Diabetes 44:1093–1098; 1995. Lopes-Virella, M. F.; Klein, R. L.; Lyons, T. J.; Stevenson, H. C.; Witztum, J. L. Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte– derived macrophages. Diabetes 37:550 –557; 1988. Kinoshita, J. A thirty year journey in the polyol pathway. Exp. Eye Res. 50:567–573; 1990. Lal, S.; Szwergold, B. S.; Taylor, A. H.; Randall, W. C.; Kappler, F.; Wells-Knecht, K.; Baynes, J. W.; Brown, T. R. Metabolism of fructose-3-phosphate in the diabetic rat lens. Arch. Biochem. Biophys. 318:191–199; 1995. Lal, S.; Randall, W. C.; Taylor, A. H.; Kappler, F.; Walker, M.; Brown, T. R.; Szwergold, B. S. Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts. Metabolism 46:1333–1338; 1997. Stribling, D.; Mirrlees, J.; Harrison, H.; Earl, D. Properties of ICI 128, 436, a novel aldose reductase inhibitor, and its effects on diabetic complications in the rat. Metabolism 34:336 –344; 1985. Tilton, R. G.; Chang, K.; Nyengaard, J. R.; van den Enden, M.;

824

[44]

[45]

[46]

[47]

[48]

[49]

H.-O. MOWRI et al. Ido, Y.; Williamson, J. R. Inhibition of sorbital dehydrogenase. Effects on vascular and neural dysfunction in streptozocin-induced diabetic rats. Diabetes 44:234 –242; 1995. Kontush, A.; Meyer, S.; Finckh, B.; Kohlschutter, A.; Beisiegel, U. Alpha-tocopherol as a reductant for Cu(II) in human lipoproteins: triggering role in the initiation of lipoprotein oxidation. J. Biol. Chem. 271:11106 –11112; 1996. Neuzil, J.; Thomas, S. R.; Stocker, R. Requirement for, promotion, or inhibition by ␣-tocopherol of radical-induced initiation of plasma lipoprotein lipid peroxidation. Free Radic. Biol. Med. 22:57–71; 1997. Suarna, C.; Dean, R. T.; May, J.; Stocker, R. Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of ␣-tocopherol and ascorbate. Arterioscler. Thromb. Vasc. Biol. 15:1616 –1624; 1995. Daimon, M.; Susa, S.; Yamatani, K.; Manaka, H.; Hama, K.; Kimura, M.; Ohnuma, H.; Kato, T. Hyperglycemia is a factor for an increase in serum ceruloplasmin in type 2 diabetes. Diabetes Care 21:1525–1528; 1998. Cunningham, J.; Leffell, M.; Mearkle, P.; Harmatz, P. Elevated plasma ceruloplasmin in insulin-dependent diabetes mellitus: evidence for increased oxidative stress as a variable complication. Metabolism 44:996 –999; 1995. Walter, R. M. Jr.; Uriu-Hare, J. Y.; Olin, K. L.; Oster, M. H.; Anawalt, B. D.; Critchfield, J. W.; Keen, C. L. Copper, zinc,

manganese, and magnesium status and complications of diabetes mellitus. Diabetes Care 14:1050 –1056; 1991. [50] Ehrenwald, E.; Chisolm, G. M.; Fox, P. L. Intact human ceruloplasmin oxidatively modifies low-density lipoprotein. J. Clin. Invest. 93:1493–1501; 1994. [51] Lipscomb, D. C.; Gorman, L. G.; Traystman, R. J.; Hurn, P. D. Low molecular weight iron in cerebral ischemic acidosis in vivo. Stroke 29:487– 492; 1998.

ABBREVIATIONS

AAPH—2,2⬘-azobis(2-amidinopropane) hydrochloride BC— bathocuproine disulfonate BP— bathophenanthroline disulfonate CAD— coronary artery disease DTPA— diethylaminetriamine pentaacetic acid LDL—low-density lipoprotein Mn-SOD—manganese-SOD PBS—phosphate-buffered saline SOD—superoxide dismutase