Postprandial metabolism of apolipoprotein B-48- and B-100-containing particles in type 2 diabetes mellitus: relations to angiographically verified severity of coronary artery disease

Atherosclerosis 150 (2000) 167 – 177 www.elsevier.com/locate/atherosclerosis

Postprandial metabolism of apolipoprotein B-48- and B-100-containing particles in type 2 diabetes mellitus: relations to angiographically verified severity of coronary artery disease N. Mero a, R. Malmstro¨m a, G. Steiner c,1, M.-R. Taskinen a, M. Syva¨nne b,* b

a Department of Medicine, Di6ision of Endocrinology and Diabetes, Uni6ersity of Helsinki, Helsinki, Finland Department of Medicine, Di6ision of Cardiology, Uni6ersity of Helsinki, Haartmaninkatu 4, P.O. Box 340 FIN-00029 HUCH, Helsinki, Finland c The Toronto Hospital and Uni6ersity of Toronto, Toronto, Ont., Canada

Received 30 March 1999; received in revised form 2 August 1999; accepted 3 September 1999

Abstract The aim of the present cross-sectional angiographic study was to examine if there is a relationship between the severity of CAD and postprandial lipemia in patients with type 2 diabetes mellitus. Special emphasis was directed to determining the contribution of apolipoprotein B-48 (apoB-48)-containing and B-100 (apoB-100)-containing triglyceride-rich particles to the magnitude of postprandial lipemia and degree of CAD. The role of apolipoprotein E (apoE) phenotype as a modulator of postprandial lipemia was also evaluated. The severity of CAD was determined by a quantitative coronary angiography and the subjects were classified into two groups based on the presence (severe CAD) or absence (mild CAD) of at least 50% stenosis in a major coronary vessel. The study population consisted of 43 subjects (31 men and 12 women) with fair glycemic control and comparable fasting lipids and body mass index. Postprandial responses of TG, apoB-48 and apoB-100 in lipoprotein subfractions (chylomicrons, VLDL1, VLDL2 and IDL) were determined after a fat load. Type 2 diabetic patients exhibited the classical dyslipidemia of the insulin resistance syndrome and delayed clearance of both hepatic and intestinal particles. Fasting or postprandial lipid or lipoprotein measurements, including apoB-48 and apoB-100 concentrations, did not differ between the groups. The presence or absence of apoE-4 allele did not significantly influence postprandial lipemia. The severity of the most significant coronary stenosis in angiography correlated with plasma and with chylomicron area under curve (AUC) for TG (n= 27) and chylomicron AUC for apoB-48 (n=20). The strongest correlate of maximal stenosis was area under incremental curve (AUIC) for apoB-100 in IDL fraction (r =0.548, P=0.012, n=20). In conclusion, postprandial apoB-48 and apoB-100 metabolism in triglyceride rich lipoproteins is distorted in type 2 diabetic patients, even in those with only mild CAD. The data suggest that postprandial change in small remnant particle numbers may contribute to the severity of CAD in type 2 diabetes. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Type 2 diabetes mellitus; Postprandial lipemia; Apolipoprotein B-48; Apolipoprotein B-100; Triglyceride-rich lipoproteins; Atherosclerosis; Quantitative coronary angiography; Apolipoprotein E phenotype

1. Introduction

Abbre6iations: ApoB-48, apolipoprotein B-48; ApoB-100, apolipoprotein B-100; apo E, apolipoprotein E; BMI, body mass index; CAD, coronary artery disease; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; TG, triglyceride; VLDL, very low density lipoprotein. * Corresponding author. Tel.: + 358-9-47172240; fax: + 358-9471–74694. E-mail address: [email protected] (M. Syva¨nne) 1 For the DAIS Project Group.

Coronary artery disease (CAD) is the most common complication and a major cause of mortality in type 2 (non-insulin dependent) diabetes mellitus [1,2]. Conventional risk factors for atherosclerosis — smoking, hypertension and high serum cholesterol — are equally important in diabetic and non-diabetic people, but they do not explain the excessive prevalence of CAD in type 2 diabetic patients [1,3,4]. Dyslipidemia in type 2 diabetes is characterized by a high concentration of fasting

0021-9150/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 9 ) 0 0 3 6 4 - 0

168

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

plasma triglycerides (TG), especially very low density lipoprotein (VLDL) TG and low fasting plasma high density lipoprotein (HDL) cholesterol [5]. These features are shown to predict CAD mortality and morbidity in patients with type 2 diabetes [6,7]. Postprandial elevation of triglyceride-rich lipoproteins (TRLs) is also well recognized in diabetic subjects [8 – 10], but the connection between postprandial lipemia and the risk for CAD is poorly understood. Postprandial hyperlipidemia is characterized by a high concentration and long residence time of chylomicron and VLDL remnants in the circulation. It has been suggested that these TRL particles increase the risk of CAD in non-diabetic subjects [11 – 14]. In an angiographic study, a significant correlation between the levels of postprandial chylomicron remnants and the progression of CAD in non-diabetic patients was found [15]. Recent data have provided evidence that postprandial lipemia is an independent risk factor for CAD [13,16]. It is still a matter of debate which postprandial TRL particles, hepatic or intestinal or both, are the atherogenic ones in non-diabetic [17,18] and diabetic subjects [19,20]. Previously, the fasting concentration of TRL apolipoprotein B-100 (apoB-100) has been related to the severity of angiographic findings in diabetic patients [21]. Only one study has compared the magnitude of postprandial lipemia in type 2 diabetic patients with and without CAD, by using TG and retinyl palmitate measurements in TRL fractions. Postprandial TG or retinyl palmitate responses did not distinguish the diabetic subjects with CAD from subjects without [22]. The aim of the present study was to examine postprandial TRL levels in type 2 diabetic patients with mild and severe CAD. Special emphasis was directed to differences in postprandial apolipoprotein B-48 (apoB-48) or apoB100 responses in TRL fractions when the severity of CAD was determined by quantitative coronary angiography. The subjects were also divided by apolipoprotein E (apoE) phenotype to study if this genetic variable modulates postprandial lipemia in diabetic subjects with signs of CAD. We studied 43 type 2 diabetic subjects between 40 and 65 years of age and with adequate control of diabetes. The TRL particles were separated with density gradient ultracentrifugation and concentrations of apoB-48 and apoB-100 were determined in the lipoprotein fractions [23] to distinguish between intestinal (apoB-48) and hepatic (apoB-100) TRL particles.

2. Subjects, materials and methods

2.1. Subjects The study group consisted of a subset of the 418 type 2 diabetic patients enrolled in the Diabetes

Atherosclerosis Intervention Study (DAIS). DAIS is a multi-national project conducted in cooperation with the World Health Organization, designed to test the effect of treating dyslipoproteinemia with micronized fenofibrate on CAD in a population of men and women with type 2 diabetes mellitus. The protocol and eligibility criteria are described elsewhere in detail [24]. Briefly, men and women from 40 to 65 years of age and with adequate control of diabetes (HbA1c B170% of upper normal limit) were recruited. The lipid criteria were: total cholesterol/HDL cholesterol ratio ]4 and either low density lipoprotein (LDL) cholesterol 3.5– 4.5 mmol/l and TG 5 5.2 mmol/l, or TG 1.7–5.2 mmol/l and LDL cholesterol 5 4.5 mmol/l. Fortythree of the subjects in the Helsinki University Central Hospital entered the present substudy. In addition to their baseline DAIS coronary angiogram, these 43 subjects (31 men and 12 women) had a baseline fat tolerance test. It should be noted that the fat tolerance test is not part of the overall DAIS protocol. Table 1 lists the subject characteristics. Informed written consent was obtained from all subjects and the study protocol was approved by the Ethical Committee, Helsinki University Central Hospital. Although the purpose of the present substudy was not to compare postprandial lipemia in diabetic and non-diabetic people, we used the data from a group of healthy young non-diabetic subjects as a reference cohort [25]. The mean age and BMI of this group was 36.89 3.1 years and 23.8 90.6 kg/m2, respectively. Concentrations of fasting plasma TG (1.09 0.1 mmol/l), fasting plasma cholesterol (5.09 0.2 mmol/l) and fasting HDL cholesterol (1.569 0.11 mmol/l) were within normal limits.

2.2. Definition of se6ere and mild CAD All coronary angiograms were analyzed by one of the authors (M.S.), using a computer-assisted quantitative system (QCA-CMS, Medis, Nuenen, the Netherlands). The protocol is described in detail elsewhere [26]. The subjects were divided into two groups with either severe CAD or mild CAD. Eighteen subjects had undergone coronary by-pass surgery or angioplasty. They were all included in the severe CAD group, but because of the preceding revascularization procedure their severest stenosis could not be quantified in the study angiograms. Other patients (n= 9) who had not had coronary procedures were included in the severe CAD group on the basis of one or more stenosis in the study angiogram that narrowed the lumen diameter by at least 50%. Thus, the group defined as having severe CAD comprised 27 subjects, 23 males and four females. The remaining 16 patients (eight males and eight females) were defined as having mild CAD because each had at least one detectable coronary lesion, however not exceeding 50% diameter stenosis. In patients who

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

had not undergone a revascularization procedure, severity of CAD was defined as the maximal per cent diameter stenosis in any major coronary branch. It should be noted that these are only ‘gross’ assessments of the severity of CAD. They do not represent the quantitative angiographic data that will be provided when DAIS is reported, nor are they indicative of the distribution of coronary disease in the DAIS population.

2.3. Oral fat load The subjects underwent an oral fat tolerance test no more than 3 weeks prior to or after the coronary angiography. The subjects remained fasting for 12 h before the test and were asked to refrain from alcohol intake for 3 days prior to testing and to refrain from smoking for the 12-h fasting period. No subject had any lipid lowering medication for at least 4 weeks before the baseline period. All other regular medicaTable 1 Subject characteristicsa

Males/females (n) Age (years) Duration of diabetes (years) Body mass index (kg/m2) Waist–hip ratio Apo E phenotype, n (%) 3/3 2/4 3/4 4/4 Systolic BP (mm Hg) Diastolic BP (mm Hg) Smokers (current/ ex/never, n) Alcohol intake (drinks/week) Fasting glucose (mmol/l) Glycosylated HbA1c (%) Insulin therapy, n (%) Use of diuretics, n (%) Use of b-blockers, n (%) a

Severe CAD (n = 27)

Mild CAD (n= 16)

P

23/4 57.791.1 791

8/8 56.69 1.1 11 9 2

0.013 0.505 0.040

29.4 90.5

30.39 0.8

0.309

0.98 9 0.01

0.94 90.02

0.074 0.058

19 (70) – 8 (30) –

8 2 4 2

(50) (12.5) (25) (12.5)

135 93

137 9 5

0.930

849 1

80 92

0.139

4/15/8

3/5/8

0.288

3.3 90.7

2.79 1.0

0.338

8.690.4

9.7 9 0.6

0.100

7.4 90.2

8.49 0.3

0.007

6 (22)

8 (50)

0.060

3 (11)

1 (6)

0.596

19 (70)

6 (38)

0.035

169

tion, including hypoglycemic agents, were allowed the evening before. A glass of orange juice was allowed as an evening snack for subjects on insulin treatment. The morning medication was taken together with the fat load test and the dose of hypoglycemic agents was adjusted to avoid both hypoglycemia and hyperglycemia. Other medications were allowed at their usual times. After drawing the fasting blood sample from an indwelling catheter placed in an antecubital vein, the subjects received an oral fat load in a liquid formula. The mixture contained soybean oil 50 g/m2, glucose 50 g/m2, egg white protein 25 g/m2, dried egg yolk 6.3 g/m2 and water 200 ml prepared with lemon flavouring. To this 100 000 IU of vitamin A (Retinyl Palmitate, Leiras Oy, Turku, Finland) was added and the test meal was consumed within 10 min. Postprandial blood samples were drawn 4, 6 and 8 h after the fat load. Venous blood was collected into tubes containing EDTA and plasma was separated within 20 min by low-speed centrifugation. Samples were protected from light and kept at +4°C before and after centrifugation.

2.4. Density gradient ultracentrifugation The density gradient ultracentrifugation has been previously described in detail [25,27]. Ultracentrifugation was performed in a SW40 Ti swinging bucket rotor in a Beckman Optima LC ultracentrifuge. The following lipoprotein fractions were separated: The Sf \ 400 fraction representing chylomicrons and large VLDL was isolated after a run of 32 min. Thereafter, ultracentrifugation was continued to isolate the Sf 60– 400 fraction (VLDL1). To separate the Sf 20–60 lipoproteins (VLDL2) and intermediate density lipoproteins (IDL, Sf 12–20), the density gradient ultracentrifugation was continued for 17 h. Low density lipoproteins (LDL, Sf 0–12) and HDL were recovered from the same tube by aspiration. Aliquots of the isolated fractions were frozen immediately at −80°C for subsequent determination of apoB-48, apoB-100, TG, cholesterol and retinyl ester concentration.

2.5. Measurements of apoB-48 and apoB-100

Values represent means9S.E. Groups were compared by the Mann–Whitney U-test and x 2-test.

Concentrations of apoB-48 and apoB-100 were analyzed from TRL fractions (chylomicrons, VLDL1, VLDL2 and IDL). Six subjects had no samples available and had thus to be excluded from these analyses. Briefly, delipidated aliquots of samples were dissolved in buffer and run in 3.5–20% sodium dodecyl sulphate polyacrylamide gel electrophoresis, according to the method of Karpe and Hamsten [27] with slight modifications. Scanning of the gels was performed with a computer-assisted laser scanning densitometer (Image

170

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

Quant 3.19, Molecular Dynamics 1991, Sunnyvale, CA) at 595 nm. Intragel and intergel coefficients of variation (CV) for apoB-48 were 3.7 and 13.1% and for apoB-100 3.1 and 11.0%, respectively. The detection limit for apoB-48 and apoB-100 ranged between 0.01 and 0.02 mg/l.

2.6. Lipolytic enzymes An intravenous bolus injection of heparin (100 IU per kg of body weight) was given to the subjects at a separate visit at least 1 week apart from oral fat load. Blood samples were drawn before and 15 min after the heparin injection into prechilled lithium-heparin tubes. Plasma was separated immediately at + 4°C and stored at − 20°C. Plasma lipoprotein lipase (LPL) and hepatic lipase (HL) activities were measured from preheparin and 15-min postheparin samples using the method of Huttunen et al. [28].

2.7. Analytical methods Concentrations of retinyl esters, TG, and cholesterol were analyzed in total plasma and in all lipoprotein fractions. TG and cholesterol concentrations were measured by automated enzymatic methods using the Cobas Mira analyser (Hoffman-La Roche, Basel, Switzerland). Retinyl ester concentrations were measured with high performance liquid chromatography as described by Ruotolo et al. [29]. ApoE phenotyping was performed in serum by using the method of Havekes et al. [30]. Concentrations of glucose, glycosylated HbA1c and urinary albumin excretion were analyzed in samples obtained during the previous screening visit. Quality of laboratory measurements was controlled with commercial samples for cholesterol (CV =2.1%) and TG (CV =2.2%). CV for the RE assay was 9.6% for a low control sample and 9.9% for a high control sample.

2.8. Statistical analyses All values are expressed as mean9S.E. of the mean. Logarithmic transformations were performed for variables with skewed distribution when appropriate. To avoid multiple comparisons, area measurements were used for statistical analysis [31]. Postprandial TG, RE, apoB-48, and apoB-100 responses were calculated as areas under the curve (AUC) and areas under the incremental curve (AUIC), as described by Matthews et al. [32]. For each subject, the parameter measured was plotted against time, and the area between zero and 8-h concentration curve was determined by the trapezoid rule. Incremental areas were obtained by subtracting the fasting value from each postprandial

value before area calculation [32]. Statistical comparisons for area and baseline measurements were calculated by using Mann–Whitney U-test and for categorical variables by using the x 2-test. Within-group changes from baseline to postprandial values were compared by repeated-measures ANOVA. Pearson’s correlation coefficients were calculated to study associations. The effects of potential determinants of postprandial apoB-48, apoB-100 and TG responses in VLDL1 fraction were studied by stepwise multivariate regression analyses.

3. Results

3.1. Clinical characteristics, fasting lipids and lipase acti6ities The clinical characteristics of the groups with mild and severe CAD are shown in Table 1. Half of the subjects in the mild CAD group were women, whereas four women were included in the severe CAD group. The subjects with mild CAD had a longer history of diabetes and slightly poorer glycemic control than the subjects with severe CAD. Blood pressure was within normal limits in both groups. Nine subjects with mild CAD and 18 subjects with severe CAD had a history of hypertension (P= 0.495). No difference in the duration of hypertension was observed between the groups (P =0.816). Insulin therapy was more frequent in subjects with mild CAD. As expected, the use of b-blockers was higher in the group with severe CAD. The use of metformin (P= 0.280) or sulfonylureas (P=0.424) did not differ between the groups. Microalbuminuria was found in three (19%) subjects in the mild CAD group and in six (22%) subjects in the severe CAD group (P=0.787). Fasting lipid and lipoprotein values are shown in Table 2. In both groups, concentrations of plasma TG were elevated due to VLDL1 fraction, which comprised 45% of the total TG in mild CAD group and 46% in severe CAD group. LDL and HDL cholesterol were comparable in the two groups. Total cholesterol/HDL cholesterol ratio was similar between the groups: 4.8690.15 (mild CAD) and 4.9790.14 (severe CAD), P= 0.725. Both lipoprotein lipase and hepatic lipase activities in postheparin plasma were similar between the groups (Table 2).

3.2. Postprandial responses of triglycerides Postprandial TG concentration curves in chylomicron, VLDL1, VLDL2 and IDL fractions are shown in Fig. 1. In these fractions and in whole plasma the TG responses were similar between the two groups. The peak of TG concentration was reached at 4 h in

N. Mero et al. / Atherosclerosis 150 (2000) 167–177 Table 2 Fasting lipoproteins and lipase activitiesa

Total triglycerides (mmol/l) VLDL1 triglycerides (mmol/l) VLDL2 triglycerides (mmol/l) Total cholesterol (mmol/l) VLDL1 cholesterol (mmol/l) VLDL2 cholesterol (mmol/l) IDL cholesterol (mmol/l) LDL cholesterol (mmol/l) HDL cholesterol (mmol/l) Ph-lipoprotein lipase (mU/l) Ph-hepatic lipase (mU/l)

Severe CAD

Mild CAD

2.699 0.20 1.249 0.11 0.379 0.05 5.619 0.10 0.449 0.05 0.3390.04 0.349 0.02 2.929 0.09 1.159 0.03 240 9 11 3569 28

2.2390.17 1.0090.11 0.349 0.07 5.779 0.15 0.339 0.05 0.349 0.04 0.339 0.03 3.039 0.14 1.21 90.05 2419 13 330 9 27

a Values represent means9S.E. Ph, post-heparin plasma. Groups were compared by the Mann–Whitney U-test, P\0.05 for each variable.

plasma, chylomicron and VLDL1 fractions. The return to the fasting level was slow in both groups. At the end of the test, the plasma TG concentration was still 3.689 0.34 mmol/l (mild CAD) and 4.3390.36 mmol/l (severe CAD) (PB0.001 compared to fasting plasma TG concentration in both groups). The decline of TG concentration was also retarded in chylomicron, VLDL1 and VLDL2 fractions. Actually, in VLDL2 fraction TG concentration increased during the 8-h sampling period in the severe CAD group. In LDL and HDL fractions, TG concentration did not change significantly from the fasting value in either group. The

171

postprandial AUC and AUIC values for TG in plasma and in chylomicron, VLDL1 and VLDL2 fractions did not differ statistically between the two groups (data not shown).

3.3. Postprandial responses of apolipoprotein B-48 The two groups had comparable apoB-48 responses in chylomicron, VLDL1, VLDL2 and IDL fractions (Fig. 2), accordingly, the calculated AUC and AUIC responses did not differ significantly. In both groups, apoB-48 concentration increased postprandially in chylomicron and especially in VLDL1 fractions compared to fasting value (P B0.003 for both fractions) reflecting an increase in the number of intestinally secreted particles. At the end of the test, apoB-48 concentrations remained elevated in both groups. In VLDL2 and IDL fractions, the concentration of apoB-48, and thus, the number of gut derived particles, did not change significantly in postprandial state in either group.

3.4. Postprandial responses of apolipoprotein B-100 The postprandial concentration curves for apoB-100 (Fig. 3) and the calculated AUC and AUIC values were similar between the two groups for chylomicron, VLDL1, VLDL2 and IDL fractions. The apoB-100 concentration increased in chylomicron and in VLDL1 fractions after the fatty meal, and the values did not reach the fasting levels during the test period. ApoB-

Fig. 1. Line plots show the postprandial responses of triglycerides (TG) in chylomicrons (Sf \ 400), VLDL1 (Sf 60 – 400), VLDL2 (Sf 20–60) and IDL (Sf 12 – 20) fractions. Diabetic subjects with \ 50% stenosis in a major coronary branch ( ) and diabetic subjects with B 50% stenosis in a major coronary branch ( ). Differences between the diabetic groups were not significant. As comparison, the postprandial response of ten healthy males, who received soybean oil fat load are also presented (). See Ref. [25] for details.

172

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

Fig. 2. Line plots show the postprandial responses of apolipoprotein B-48 (apoB-48) in chylomicrons (Sf \ 400), VLDL1 (Sf 60 – 400), VLDL2 (Sf 20– 60) and IDL (Sf 12–20) fractions. Differences between the diabetic groups were not significant. For other explanations, see legend to Fig. 1.

Fig. 3. Line plots show the postprandial responses of apolipoprotein B-100 (apoB-100) in chylomicrons (Sf \ 400), VLDL1 (Sf 60 – 400), VLDL2 (Sf 20– 60) and IDL (Sf 12–20) fractions. Differences between the diabetic groups were not significant. For other explanations, see legend to Fig. 1.

100 concentration remained constant in VLDL2 fraction. In IDL fraction the apoB-100 concentration was slightly, but not significantly higher in the subjects with severe CAD. In both groups the apoB-100 concentration in IDL fraction decreased significantly during alimentary lipemia.

3.5. Postprandial responses of retinyl esters The responses of retinyl esters in plasma, chylomicron and VLDL2 fractions were parallel to those of apoB-48 and TG and no statistical differences between the groups were found (data not shown). In VLDL1

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

fraction, the subjects with mild CAD had, if anything, higher retinyl ester AUIC responses (P = 0.018).

3.6. Postprandial responses of cholesterol Total and LDL cholesterol concentrations did not change during the postprandial period. In HDL fraction, cholesterol concentration decreased postprandially in mild CAD and severe CAD groups (each P B 0.001). The lowest concentration of HDL cholesterol occurred 6 h after the fat load in both groups. We calculated the TG/cholesterol-ratio in HDL fraction and found a significant increase postprandially (P B0.019 in both groups).

3.7. Correlations between the se6erity of CAD and postprandial lipemia The correlation between lipid and lipoprotein parameters and the severity of CAD in coronary angiogram was calculated for 16 subjects with mild CAD and the nine evaluable subjects with severe CAD, who had not undergone a revascularization procedure. The severity of the tightest coronary stenosis correlated with postprandial responses (AUC) for plasma TG (r= 0.492, P=0.013) and chylomicron TG (r= 0.443, P= 0.026). We observed a correlation between maximal stenosis and AUC for chylomicron apoB-48 (r = 0.461, P = 0.041). The strongest correlate of maximal stenosis was AUIC for apoB-100 in IDL fraction (Fig. 4). Because this AUIC measurement represents the change in postprandial IDL particle numbers, we calculated correlations for maximal stenosis and DIDL apoB-100 at 4 h (r = 0.504, P= 0.024), 6 h (r =0.413, P =0.070) and 8 h (r =0.572, P = 0.008) after the fat load. Neither apoB-48 nor apoB-100 response in VLDL1 fraction correlated with the angiographic findings.

Fig. 4. A scatterplot showing correlatiofln between maximal stenosis in coronary angiography and postprandial change in apolipoprotein B-100 (apoB-100) concentration of IDL (Sf 12–20). All type 2 diabetic subjects (n =20) with available data.

173

3.8. Multi6ariate analysis Multivariate analyses were performed to adjust for baseline characteristics (sex, duration of diabetes, waist–hip ratio, diastolic blood pressure, fasting glucose, glycosylated HbA1c, use of insulin and use of b-blockers). Adjusted P values were essentially similar to crude P values, i.e. no differences in measures of postprandial lipemia were found.

3.9. The effect of apolipoprotein E phenotype on postprandial lipemia The distribution of apoE phenotypes are given in Table 1. The frequency of o-4 allele was not significantly different between the two groups. We divided the study population in two subjects with apoE3/3 phenotype (n= 27) in one group and those with either 3/4 or 4/4 phenotype (n= 14) in the other. The groups did not differ with regard to the presence of mild or severe CAD (P= 0.817). Postprandial responses of TG, apoB48 and apoB-100 were compared between the two groups and the AUC and AUIC responses were found closely comparable (data not shown).

4. Discussion The major finding of the present cross-sectional study is that type 2 diabetic patients with mild or severe coronary artery disease verified angiographically, have similar concentrations of postprandial apoB-48 and apoB-100-containing particles in lipoprotein fractions. Both groups showed all the features of diabetic dyslipidemia including prolonged postprandial lipemia and high concentrations of both apoB-48- and apoB-100containing TRL remnants in the circulation. The correlation between postprandial response of small remnant particle number and maximal stenosis in coronary angiography, suggests that these particles may contribute to the severity of CAD in type 2 diabetes. Postprandial hyperlipidemia is highly prevalent in diabetic patients with both normal [8] and elevated [9,22,33] fasting triglyceride concentrations, which has highlighted the role of a long residence time of TRL remnants as an inherent feature of diabetic dyslipidemia. However, the exact nature of TRL particles responsible for postprandial lipemia type 2 diabetic subjects is not well established. A study by Syva¨nne et al., using vitamin A as a marker for intestinal lipoproteins, showed larger TG and retinyl palmitate responses in Sf 60–400 particles in male type 2 diabetic patients compared to controls with similar BMI and age [22]. Curtin et al. [34] found significantly different TG and apoB-48 responses in TRL fraction (i.e. chylomicrons and VLDL) in the postprandial state in

174

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

diabetic patients compared to control subjects. These two studies showed prolonged residence time of exogenous chylomicron remnants. The concentrations of both apoB-48 and apoB-100 in the postprandial state, especially in chylomicron and VLDL1 fractions, are highly elevated in our diabetic subjects with both mild and severe CAD compared to healthy normolipidemic men given a soybean fat load [25] (Figs. 1 – 3). The mechanisms underlying fat intolerance in type 2 diabetic subjects are not fully clear. Previous studies [19,20,22,35] and the present one suggest a combined effect of both increased VLDL1 production in the liver and competition of chylomicron remnants and VLDL remnants for the common removal mechanisms. Our group [35] has recently shown that suppression of hepatic VLDL1 apoB production by insulin is impaired in type 2 diabetes and results in inappropriate release of VLDL1 particles in the fasting and postprandial phase. The normal suppression of fatty-acid release by insulin from the adipose tissue is also impaired in insulin resistant states and further increases the substrate input to the liver [36,37]. In this study, the subjects had been fasting for at least 12 h, but the concentrations of VLDL1 apoB-100 remained substantially elevated compared with non-diabetic subjects (Fig. 3), who represent a healthy normolipidemic cohort [25]. This observation is in agreement with the studies demonstrating increased VLDL1 production. On the other hand, in the postprandial phase the sudden increase in both apoB48- and apoB-100-containing TRL particles competing for the limited lipoprotein lipase activity and uptake by common hepatic receptors also might saturate the lipolytic capacity for hours [38]. Lipoprotein lipase activity has been shown to be decreased in type 2 diabetes and hence may be more easily saturated. This may contribute to the delay of TRL clearance. [39,40] As further evidence for lipolytic defect we have shown a lack of normal inverse correlation between postprandial lipemia and plasma lipoprotein lipase activity in diabetic patients [22]. Likewise, this correlation was absent in the present study. The current picture of removal defects is that chylomicron and VLDL remnant uptake by hepatocytes is rate-limiting at least in non-diabetic subjects [41]. As lipoprotein lipase augments TRL binding to receptors in the liver [42], impaired interaction of remnant particles with hepatic receptors, due to abnormal ligand properties, may enhance postprandial lipemia. Glycation of apolipoproteins, especially apoE, is another factor which may be important in respect of impaired TRL metabolism in subjects with diabetes [43,44]. So far, only one study has examined the relations between postprandial TRL metabolism and manifest CAD in type 2 diabetic subjects. In that study, no postprandial responses of TG or retinyl palmitate in any TRL fraction varied according to the presence or

absence of CAD. [22] We confirm and extend these findings by showing similar postprandial apoB-48 and apoB-100 metabolism in type 2 diabetic patients with mild and severe CAD measured by quantitative coronary angiography. The absolute magnitude of the total postprandial apoB-100 response in the IDL fraction did not differ between the groups. We found, however, a correlation between the severity of coronary atherosclerosis and the postprandial change of apoB-100 concentration in the IDL (Sf 12–20) fraction. Thus, the subjects with a greater decline in postprandial IDL particle numbers had milder occlusion of coronary vessels. Recently, in a study by Tka´c et al., 174 diabetic subjects were categorized into tertiles according to a coronary score and fasting lipid values were studied. Fasting TRL apoB, especially in Sf 12-60 fraction, was significantly higher in subjects with moderate and severe CAD compared to those with mild CAD. The coronary score correlated with TRL apoB concentration. Interestingly, no differences in any lipid parameter between the groups with moderate and severe CAD could be detected. The authors concluded that the severity of CAD in diabetic patients was positively related to the number of fasting TRL particles. [21] In line, the present finding may provide evidence for the significance of postprandial small TRLs in the development of atherosclerosis in type 2 diabetes. Previously, relations between postprandial TRL concentrations and the degree of atherosclerosis have been studied in various populations. Recently, Karpe et al. [45] studied unselected healthy middle-aged men using an ultrasound of the common carotid artery. Intima media thickness correlated with the late postprandial (6 h) plasma TG concentration independently of LDL cholesterol and fasting TG concentration. A number of angiographic case-control studies have indicated delayed TRL clearance in non-diabetic CAD patients [12,14,46–48]. Thus, postprandial TRL may contribute to the development of CAD in early asymptomatic stages as well as in more advanced atherosclerotic disease. Besides postprandial IDL levels, we also found significant correlation between the severity of CAD and postprandial chylomicron TG and apoB-48 responses. This finding is further evidence in favour of the concept of atherogenicity of excessive postprandial lipemia in type 2 diabetes. In addition to fasting, TG and HDL cholesterol concentrations, glycemic control, obesity and nephropathy, genetic factors too modulate postprandial lipemia in type 2 diabetes [19]. ApoE is required for clearance of TRLs, and apoE phenotype determines the affinity to the hepatic remnant receptor [41,49]. Syva¨nne et al. found enrichment of postprandial TRL remnants with apoE in NIDDM patients with CAD, which was suggested to be a strong determinant of atherogenicity [50]. In a recent meta-analysis, increased

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

risk for CAD was associated with o-4 allele with O.R. 1.26 (1.13–1.41) compared to o-3 allele in non-diabetic subjects [51]. Studies regarding apoE phenotype and postprandial lipemia in non-diabetics have been conflicting [52–54]. In normotriglyceridemic diabetic patients, Reznik et al. found apolipoprotein E polymorphism to modulate postprandial lipemia. Subjects with 2/3 or 3/4 phenotype had 2-fold postprandial lipemia compared to 3/3 phenotype [55]. We could not confirm this finding in the present study, in which, despite the apoE phenotype, similar postprandial responses of TG, apoB-48 and apoB-100 were found. Excessive postprandial lipemia is associated with multiple abnormalities in LDL and HDL particles, which may contribute to the risk of CAD in type 2 diabetic subjects. In diabetes, plasma TG concentration correlates closely with low HDL2 and high HDL3 concentrations, suggesting the impact of TRL concentrations on HDL properties [56]. Further, the efficiency of TRL metabolism has been shown to correlate positively with HDL cholesterol concentrations [57,58]. We found no changes in LDL cholesterol concentration and significant but comparable reduction in HDL cholesterol concentration and concomitant enrichment of HDL TG, during alimentary lipemia in both groups, as a sign of prolonged TRL catabolism. Thus, studies on subclasses of LDL and HDL are needed to establish atherogenic changes in postprandial state. Because the groups had some baseline differences, potential confounding factors might have influenced the results. The number of subjects in the present study was relatively small and the distribution of males and females in the study groups was uneven. Elevated plasma TG concentrations are associated with higher relative risk for CAD in females than in males [59,60]. Unexpectedly, the subjects with mild CAD had a longer history of diabetes and poorer glycemic control than the subjects with severe CAD. On the other hand, the use of b-blockers was more frequent in the group with CAD. b-blockers have been shown to increase postprandial VLDL responses at least in non-diabetic people [61]. To account for these differences in the baseline characteristics, a multiple regression analysis was performed, but no influence on the main outcome was apparent. However, because of the cross-sectional design of our study, we cannot completely rule out the influence of selection bias or selective mortality on our results. Diabetic patients display multiple abnormalities in postprandial TRL apoB-48 and apoB-100 metabolism, which are present already in diabetic subjects with mild CAD. The apoE-4 allele does not significantly alter the already distorted metabolism of intestinal or hepatic TRL particles. The clinical significance of the present and previous studies is that lowering of plasma TRL particles may be necessary already before the occurrence of signs of macrovascular disease.

175

Acknowledgements We gratefully acknowledge the excellent laboratory work by Hannele Hilden, Anne Salo, Leena Lehikoinen, Ritva Marjanen, Helina¨ Perttunen-Nio, Sirkka-Liisa Runeberg, John O’Connor and Sirpa Rannikko. We thank Maaria Puupponen for excellent secretarial assistance and Soile Aarnio for drawing the figures. We acknowledge the skilled technical help of Mervi Pietila¨ in the angiographic analyses. This study was supported by a grant from the Diabetestutkimussa¨a¨tio¨ Foundation. References [1] Garcia MJ, McNamara PM, Gordon T, Kannel WB. Morbidity and mortality in diabetics in the Framingham population. Sixteen year follow-up study. Diabetes 1974;23:105 – 11. [2] Kleinman JC, Donahue RP, Harris MI, Finucane FF, Madans JH, Brock DB. Mortality among diabetics in a national sample. Am J Epidemiol 1988;128:389 – 401. [3] Koskinen P, Ma¨ntta¨ri M, Manninen V, Huttunen JK, Heinonen OP, Frick MH. Coronary heart disease incidence in NIDDM patients in the Helsinki Heart Study. Diabetes Care 1992;15:820 – 5. [4] Stamler J, Vaccaro O, Neaton JD, Wentworth D. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabetes Care 1993;16:434 – 44. [5] Taskinen MR, Lahdenpera¨ S, Syva¨nne M. New insights into lipid metabolism in non-insulin-dependent diabetes mellitus. Ann Med 1996;28:335 – 40. [6] Laakso M, Lehto S, Penttila¨ I, Pyo¨ra¨la¨ K. Lipids and lipoproteins predicting coronary heart disease mortality and morbidity in patients with non-insulin dependent diabetes. Circulation 1993;88:1421 – 30. [7] Hanefeld M, Fischer S, Julius U, Schutze J, Schwanebeck U, Schmechel H, Ziegelasch HJ, Lindner J. Risk factors for myocardial infarction and death in newly detected NIDDM: the Diabetes Intervention Study, 11-year follow-up. Diabetologia 1996;39:1577 – 83. [8] Chen YD, Swami S, Skowronski R, Coulston A, Reaven GM. Differences in postprandial lipemia between patients with normal glucose tolerance and noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1993;76:172 – 7. [9] Lewis GF, O’Meara NM, Soltys PA, Blackman JD, Iverius PH, Pugh WL, Getz GS, Polonsky KS. Fasting hypertriglyceridemia in noninsulin-dependent diabetes mellitus is an important predictor of postprandial lipid and lipoprotein abnormalities. J Clin Endocrinol Metab 1991;72:934 – 44. [10] Syva¨nne M, Vuorinen-Markkola H, Hilden H, Taskinen MR. Gemfibrozil reduces postprandial lipemia in non-insulin-dependent diabetes mellitus. Arterioscler Thromb 1993;13:286–95. [11] Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation 1979;60:473 – 85. [12] Groot PH, van Stiphout WA, Krauss XH, Jansen H, van Tol A, van Ramshorst E, Chin-On S, Hofman A, Cresswell SR, Havekes L. Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease. Arterioscler Thromb 1991;11:653 – 62. [13] Patsch J, Miesenbo¨ck G, Hopferwieser T, Muhlberger V, Knapp E, Dunn J, Gotto A Jr, Patsch W. Relation of triglyceride metabolism and coronary artery disease. Studies in the postprandial state. Arterioscler Thromb 1992;12:1336 – 45.

176

N. Mero et al. / Atherosclerosis 150 (2000) 167–177

[14] Weintraub MS, Grosskopf I, Rassin T, Miller H, Charach G, Rotmensch HH, Liron M, Rubinstein A, Iaina A. Clearance of chylomicron remnants in normolipidemic patients with coronary artery disease: case control study over three years. Br Med J 1996;312:935 – 9. [15] Karpe F, Steiner G, Uffelman K, Olivecrona T, Hamsten A. Postprandial lipoproteins and progression of coronary atherosclerosis. Atherosclerosis 1994;106:83–97. [16] Ebenbichler CF, Kirchmair R, Egger C, Patsch JR. Postprandial state and atherosclerosis. Curr Opin Lipidol 1995;6:286– 90. [17] Karpe F, Hamsten A. Postprandial lipoprotein metabolism and atherosclerosis. Curr Opin Lipidol 1995;6:123–9. [18] Lefebvre PJ, Scheen AJ. The postprandial state and risk of cardiovascular disease. Diabetic Med 1998;15:S63–8. [19] De Man FHAF, Castro Cabezas M, Van Barlingen HHJJ, Erkelens DW, De Bruin TWA. Triglyceride-rich lipoproteins in non-insulin-dependent diabetes mellitus: post-prandial metabolism and relation to premature atherosclerosis. Eur J Clin Invest 1996;26:89 – 108. [20] Coppack SW. Postprandial lipoproteins in non-insulin-dependent diabetes mellitus. Diabetic Med 1997;14:S67–74. [21] Tka´c I, Kimball BK, Lewis G, Uffelman K, Steiner G. The severity of coronary atherosclerosis in type 2 diabetes mellitus is related to the number of circulating triglyceride-rich lipoprotein particles. Arterioscler Thromb Vasc Biol 1997;17:3633–8. [22] Syva¨nne M, Hilden H, Taskinen MR. Abnormal metabolism of postprandial lipoproteins in patients with non-insulin-dependent diabetes mellitus is not related to coronary artery disease. J Lipid Res 1994;35:15 – 26. [23] Kirchmair R, Ebenbichler CF, Patsch JR. Post-prandial lipaemia. Baillieres Clin Endocrinol Metab 1995;9:705–19. [24] Steiner G. The Diabetes Atherosclerosis Intervention Study (DAIS): a study conducted in cooperation with the World Health Organization. The DAIS Project Group. Diabetologia 1996;39:1655 – 61. [25] Mero N, Syva¨nne M, Rosseneu M, Labeur C, Hilden H, Taskinen M-R. Comparison of three fatty meals in healthy normolipidemic men: high post-prandial retinyl ester response to soybean oil. Eur J Clin Invest 1998;28:407–15. [26] McLaughlin PR, Gladstone P. Diabetes Atherosclerosis Intervention Study (DAIS): quantitative coronary angiographic analysis of coronary artery atherosclerosis. Cathet Cardiol Diag 1998;44:249 – 56. [27] Karpe F, Hamsten A. Determination of apolipoproteins B-48 and B-100 in triglyceride-rich lipoproteins by analytical SDSPAGE. J Lipid Res 1994;35:1311–7. [28] Huttunen JK, Ehnholm C, Kinnunen PKJ, Nikkila¨ EA. An immunochemical method for selective measurement of two triglyceride lipases in human postheparin plasma. Clin Chim Acta 1975;63:335 – 47. [29] Ruotolo G, Huizhen Z, Bentsianov V, Ngoc-Anh L. Protocol for the study of the metabolism of retinyl esters in plasma lipoproteins during postprandial lipemia. J Lipid Res 1992;33:1541 – 9. [30] Havekes LM, de Knijff P, Beisiegel U, Havinga J, Smith M, Klasen E. A rapid micromethod for apolipoprotein E phenotyping directly in serum. J Lipid Res 1987;28:455–63. [31] Ludbrook J. Repeated measurements and multiple comparisons in cardiovascular research. Cardiovasc Res 1994;28:303– 11. [32] Matthews J, Altman D, Campbell M, Royston P. Analysis of serial measurements in medical research. Br Med J 1990;300:230 – 5. [33] Cavallero E, Dachet C, Neufcour D, Wirquin E, Mathe D, Jacotot B. Postprandial amplification of lipoprotein abnormalities in controlled type II diabetic subjects: relationship to postprandial lipemia and C-peptide/glucagon levels. Metabolism 1994;43:270 – 8.

[34] Curtin A, Deegan P, Owens D, Collins P, Johnson A, Tomkin GH. Alterations in apolipoprotein B-48 in the postprandial state in NIDDM. Diabetologia 1994;37:1259 – 64. [35] Malmstro¨m R, Packard CJ, Caslake M, Bedford D, Stewart P, Yki-Ja¨rvinen H, Shepherd J, Taskinen M-R. Defective regulation of triglyceride metabolism by insulin in the liver in NIDDM. Diabetologia 1997;40:454 – 62. [36] Chen YD, Golay A, Swislocki AL, Reaven GM. Resistance to insulin suppression of plasma free fatty acid concentrations and insulin stimulation of glucose uptake in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1987;64:17 –21. [37] Swislocki AL, Chen YD, Golay A, Chang MO, Reaven GM. Insulin suppression of plasma-free fatty acid concentration in normal individuals and patients with type 2 (non-insulin-dependent) diabetes. Diabetologia 1987;30:622 – 6. [38] Havel RJ. Postprandial hyperlipidemia and remnant lipoproteins. Curr Opin Lipidol 1994;5:102 – 9. [39] Brunzell JD, Porte D Jr, Bierman EL. Reversible abnormalities in postheparin lipolytic activity during the late phase of release in diabetes mellitus (postheparin lipolytic activity in diabetes). Metabolism 1975;24:1123 – 37. [40] Nikkila¨ EA, Huttunen JK, Ehnholm C. Postheparin plasma lipoprotein lipase and hepatic lipase in diabetes mellitus. Relationship to plasma triglyceride metabolism. Diabetes 1977;26:11 – 21. [41] Cooper AD. Hepatic uptake of chylomicron remnants. J Lipid Res 1997;38:2173 – 92. [42] Beisiegel U, Weber W, Bengtsson Olivecrona G. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc Natl Acad Sci USA 1991;88:8342 – 6. [43] Curtiss LK, Witztum JL. Plasma apolipoproteins AI, AII, B, CI, and E are glucosylated in hyperglycemic diabetic subjects. Diabetes 1985;34:452 – 61. [44] Mamo JC, Szeto L, Steiner G. Glycation of very low density lipoprotein from rat plasma impairs its catabolism. Diabetologia 1990;33:339 – 45. [45] Karpe F, de Faire U, Mercuri M, Bond MG, Hellenius ML, Hamsten A. Magnitude of alimentary lipemia is related to intima-media thickness of the common carotid artery in middleaged men. Atherosclerosis 1998;141:307 – 14. [46] Simons LA, Dwyer T, Simons J, Bernstein L, Mock P, Poonia NS, Balasubramaniam S, Baron D, Branson J, Morgan J, et al. Chylomicrons and chylomicron remnants in coronary artery disease: a case-control study. Atherosclerosis 1987;65:181–9. [47] Simpson HS, Williamson CM, Olivecrona T, Pringle S, Maclean J, Lorimer AR, Bonnefous F, Bogaievsky Y, Packard CJ, Shepherd J. Postprandial lipemia, fenofibrate and coronary artery disease. Atherosclerosis 1990;85:193 – 202. [48] Meyer E, Westerveld HT, de Ruyter-Meijstek FC, van Greevenbroek MM, Rienks R, van Rijn HJ, Erkelens DW, de Bruin TW. Abnormal postprandial apolipoprotein B-48 and triglyceride responses in normolipidemic women with greater than 70% stenotic coronary artery disease: a case-control study. Atherosclerosis 1996;124:221 – 35. [49] Sherrill BC, Innerarity TL, Mahley RW. Rapid hepatic clearance of the canine lipoproteins containing only the E apoprotein by a high affinity receptor. Identity with the chylomicron remnant transport process. J Biol Chem 1980;255:1804– 7. [50] Syva¨nne M, Rosseneu M, Labeur C, Hilden H, Taskinen MR. Enrichment with apolipoprotein E characterizes postprandial TG-rich lipoproteins in patients with non-insulin-dependent diabetes mellitus and coronary artery disease: a preliminary report. Atherosclerosis 1994;105:25 – 34. [51] Wilson PWF, Schaefer EJ, Larson MG, Ordovas JM. Apolipoprotein E alleles and risk of coronary disease: A meta-analysis. Arterioscler Thromb Vasc Biol 1996;16:1250 – 5.

N. Mero et al. / Atherosclerosis 150 (2000) 167–177 [52] Weintraub MS, Eisenberg S, Breslow JL. Dietary fat clearance in normal subjects is regulated by genetic variation in apolipoprotein E. J Clin Invest 1987;80:1571–7. [53] Boerwinkle E, Brown S, Sharrett AR, Heiss G, Patsch W. Apolipoprotein E polymorphism influences postprandial retinyl palmitate but not triglyceride concentrations. Am J Hum Genet 1994;54:341 – 60. [54] Bergeron N, Havel RJ. Prolonged postprandial responses of lipids and apolipoproteins in triglyceride-rich lipoproteins of individuals expressing an apolipoprotein epsilon4 allele. J Clin Invest 1996;97:65 – 72. [55] Reznik Y, Pousse P, Herrou M, Morello R, Mahoudeau J, Drosdowsky MA, Fradin S. Postprandial lipoprotein metabolism in normotriglyceridemic non-insulin-dependent diabetic patients: influence of apolipoprotein E polymorphism. Metabolism 1996;45:63–71. [56] Syva¨nne M, Ahola M, Lahdenpera¨ S, Kahri J, Kuusi T, Virtanen KS, Taskinen MR. High density lipoprotein subfractions in non-insulin-dependent diabetes mellitus and coronary artery disease. J Lipid Res 1995;36:573–82.

.

177

[57] Patsch JR, Prasad S, Gotto AMJ, Bengtsson-Olivecrona G. Postprandial lipemia. A key for the conversion of high density lipoprotein2 into high density lipoprotein 3 by hepatic lipase. J Clin Invest 1984;74:2017 – 23. [58] Karpe F, Bard JM, Steiner G, Carlson LA, Fruchart JC, Hamsten A. HDLs and alimentary lipemia. Studies in men with previous myocardial infarction at a young age. Arterioscler Thromb 1993;13:11 – 22. [59] Hokanson JE, Austin MA. Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies. J Cardiovasc Risk 1996;3:213 – 9. [60] Austin MA, Hokanson JE, Edwards KL. Hypertriglyceridemia as a cardiovascular risk factor. Am J Cardiol 1998;81:7B–12B. [61] Boquist S, Ruotolo G, Hellenius ML, Danell-Toverud K, Karpe F, Hamsten A. Effects of a cardioselective beta-blocker on postprandial triglyceride-rich lipoproteins, low density lipoprotein particle size and glucose-insulin homeostasis in middleaged men with modestly increased cardiovascular risk. Atherosclerosis 1998;137:391 – 400.