Postprandial reverse cholesterol transport in type 2 diabetic patients: effect of a lipid lowering treatment

Atherosclerosis 153 (2000) 453 – 460 www.elsevier.com/locate/atherosclerosis

Postprandial reverse cholesterol transport in type 2 diabetic patients: effect of a lipid lowering treatment Delphine Autran a, Nebil Attia b, Marek Dedecjus c, Vincent Durlach d, Anik Girard-Globa a,e,* a

Laboratoire de Me´tabolisme des Lipides, Uni6ersite´ Lyon 1, Hoˆpital de l’Antiquaille, 69005, Lyon, France b De´partement des Sciences de la Vie, Faculte´ des Sciences de Bizerte, Bizerte, Tunisia c Department of Endocrinology, Lodz School of Medicine, Lodz, Poland d Clinique Me´dicale, Centre Hospitalo-Uni6ersitaire, Reims, France e Centre National de la Recherche Scientifique, UPR 5014, Lyon, France Received 29 July 1999; received in revised form 13 January 2000; accepted 11 February 2000

Abstract Deterioration of reverse cholesterol transport (RCT), an important anti-atherogenic process, may contribute to the largely unexplained severity of cardiovascular risk in type 2 diabetic patients. Among other relevant metabolic perturbations is the impairment in type 2 patients of the postprandial increase in RCT which, in normal subjects, is associated with the transfer to HDL of PL from lipolyzed chylomicrons. We have explored the possibility that improvement of postprandial lipolysis by bezafibrate might also restore the stimulated level of postprandial RCT. Twelve male patients (HbA1c 7.6 9 1.6% triglycerides (TG) 4.592.4 mmol/l) were treated for 4 weeks with 400 mg bezafibrate and compared with seven age-matched controls. Lipoproteins were analyzed over 8 h after a 1000 Kcal fat load (80% lipid), serum mediated cholesterol efflux was evaluated using 3 H-cholesterol labelled Fu5AH cells. Fasting efflux was lower in patients (17.99 3.3 vs 19.99 3.0 a. units, P B 0.05) and decreased postprandially in most instead of increasing, so that area under the time-curve (AUC) was 23% lower than in controls (140 923 vs 170 9 25 units.h, PB0.001) The patients’ HDL failed to acquire PL and gained TG in proportion to lipemia (r= 0.660, PB 0.001). Bezafibrate restored fasting efflux (19.6 93.6 units, PB 0.005 vs pretreatment) but not postprandial increase of efflux or HDL-PL. AUC of efflux was however improved to 1559 23 units.h (P B0.02). Postprandial efflux related mainly to HDL-PL in controls and patients before treatment. HDL-TG emerged as a significant negative correlate common to all groups (r= −0.674, P B0.001 8 h after the meal). Impairment of reverse cholesterol transport in diabetic patients might therefore be due to combined postprandial deficit of PL transfer and excess accumulation of TG in HDL. The significant improvement due to fibrate treatment might thus be related to the reduction of HDL-TG contents associated with the improvement of postprandial hyperlipemia. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Reverse cholesterol transport; Fu5AH cells; Postprandial; Fat load; Type 2 diabetes; HDL (high density lipoproteins) composition; Bezafibrate

1. Introduction The severity of cardiovascular risk in type 2 diabetic patients is far greater than predictable from their rather moderate dyslipidemia and hypertension. The part potentially played by the disturbances in postprandial metabolism characteristic of their pathology may be of importance [1]. Attention has often focused on triglyce* Corresponding author: Tel.: + 33-4-72386578; fax: +33-472386539.

ride-rich lipoproteins (TRL) which tend to accumulate, exaggerating the normal postprandial hyperlipemia. Although fasting level of triglyceridemia has proved an independent risk factor for coronary heart disease in type 2 diabetics [2,3], a postprandial study comparing patients with or without coronary artery disease has failed to evidence specific differences at the level of postprandial hyperlipemia [4]. The postprandial process might nevertheless be altered at the level of high density lipoprotein (HDL)-mediated reverse cholesterol transport (RCT), already deficient in the fasted state [5].

0021-9150/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 0 ) 0 0 4 2 8 - 7

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In fasted subjects, capacity to promote cholesterol efflux from cells is directly linked to availability of surface composition of HDL [6], which has repeatedly been reported to be altered in diabetics [7 – 9]. Postprandial serum normally displays an increased capacity for efflux [10], which is related to an increment in HDL-PL in healthy controls [11]. While HDL normally accumulate PL as a result of postprandial lipolysis, those of type 2 diabetics fail to do so [12], which might limit their capacity for RCT. They also display a marked enrichment with triglycerides (TG) which is also likely to affect both their physical [13] and metabolic [14,15] properties. Insofar as both the defect in PL accretion and TG enrichment are associated with retarded lipolysis, reduction of postprandial hyperlipemia by treatment with a fibrate might be expected to restore normal properties. We have explored this possibility by assessing RCT to sera from moderately hypertriglyceridemic type 2 diabetic patients before and after treatment with bezafibrate, comparing them with age-matched controls. Fu5AH cells were used: SR-B1 scavenger receptor is not a limiting factor for RCT in this system [17] which makes it particularly sensitive to changes in surface composition of HDL [6].

2. Methods

2.1. Subjects Twelve male patients with fairly well-controlled NIDDM were included in this protocol which was approved by the local ethics committee. Informed consent was obtained in writing from participants. In order to be representative of the prevailing, mildly, hypertriglyceridaemic, overweight diabetic population, the patients were recruited among men aged 40 – 65 years with a mean HbA1c of 7.69 1.6% (normal B 6.5%), TG concentrations between 2 and 5 mmol/l, low density lipoprotein (LDL)-cholesterol below 4.4 mmol/l and body mass index (BMI) between 27 and 36 kg/m2. Apart from mild hypertension (SBP 1549 19, DBP 88 9 13 mmHg), none showed symptoms of overt macro- or microvascular disease. All had normal liver, kidney and thyroid function. They were treated with oral antidiabetic agents (biguanides and/or sulphonylurea, no insulin) associated with a prescribed restrictive diet. Energy intake was evaluated by a 3-day recall questionnaire. All were followed on a regular basis in the medical clinic and were eligible for lipid-lowering treatment because of persistent hypertriglyceridaemia under dietary and antidiabetic therapy. Controls were seven healthy, normoponderal, normotriglyceridemic men within the same age range, chosen on the basis of HbA1c B 6.0%. As a result, their BMI was significantly

lower than that of the patients because, in such an age range, overweight males are always at least insulin resistant. The main relevant clinical characteristics of patients and controls are given in Table 1.

2.2. Experimental protocol On the night before each experimental meal, the subjects were asked to eat a standardized (700 Kcal) meal no later than 20:00 hours. At 08:00 h on the following morning, i.e. after at least 12 h of fasting, they ingested a 1000 Kcal high-fat meal comprising 83% lipid, 3% protein, and 14% carbohydrate. Before the high-fat meal and every 2 h thereafter during 8 h, blood was collected into dry tubes by way of a catheter in the antecubital vein. Following this first test meal, the type two patients were prescribed bezafibrate at a daily dose of 400 mg. After 5 weeks of medication, the oral fat-load test was repeated. The antidiabetic treatment did not change during the study. No variations in body weight were detected between the two tests (Table 1).

2.3. Cell culture, cholesterol efflux assay Cell cholesterol efflux was measured in the presence of a 5% dilution of serum, essentially as described by Table 1 Clinical characteristics of subjects Before treatment Number of subjects Age (years) Duration of diabetes (years) BMI (kg/m2) Energy intake (Kcal) HbA1c (%) Glycemia Triglycerides (mmol/l) Cholesterol (mmol/l) LDL-C (mmol/l) Apo B (g /l)

12 57.7 95.8 17.0 9 9.7 30.8 93.3 1937 9 382

After treatment

– – –

Controls

7 45.09 8.4 –

30.8 93.2 1985 9276

23.0 9 1.5 1873 9 254

– 9.7 9 3.4*,a 2.35 91.34*,a

5.11 9 0.52 5.40 9 0.9 0.94 9 0.19

5.34 90.82**

5.06 9 0.64

4.53 9 0.59

2.64 90.83

2.50 9 0.74

2.679 0.40

1.36 90.28**

1.15 9 0.35b

1.069 0.24

7.6 9 1.6 11.9 93.3* −4.5092.4*

* Means 9 SD. Statistical differences: from control value (t-test), PB0.01. ** Means 9SD. Statistical differences: from control value (t-test), PB0.05. a Means9 SD. Statistical differences: from before treatment value (paired t-test), PB0.01. b Means9 SD. Statistical differences: from before treatment value (paired t-test), PB0.05.

D. Autran et al. / Atherosclerosis 153 (2000) 453–460

Rothblat’s group [18]. Fu5AH cells were grown in Eagle’s minimal essential medium (MEM) supplemented wih 5% bovine calf serum (Boehringer, Meylan France) and added with penicillin-streptomycin. For experiments, cells were seeded in six-well plates at a density of 45 000 – 50 000 cells per well and grown in MEM with 5% bovine calf serum for two days. Radiolabelled cholesterol ([1, 2-3H] cholesterol, TRK 330, Amersham, France) was added to the cells: a tracer amount was first mixed into 25% calf serum in MEM, then diluted with MEM to 5% final serum concentration. The cells were grown in the presence of radiolabel for two more days to obtain confluent monolayers, and to allow for radiolabelled cholesterol to exchange into all cellular pools. The labelling medium was finally replaced with MEM containing 0.5% bovine serum albumin for 18 to 20 h before measure of cholesterol efflux to further ensure that cholesterol pools were equilibrated. The capacity of serum to promote efflux from the labelled cells was assayed by adding individual samples of the patients’ chylomicron-free serum to a final concentration of 5% and quantifying the amount of radiolabelled cholesterol released over 4 h. At the end of the efflux period, the medium was collected into ice-chilled tubes and centrifuged in the cold for 5 min at 2000 rpm to remove cells. Labelled cholesterol release was measured in an aliquot of the medium by standard liquid scintillation counting. Cell monolayers were washed with phosphate-buffered saline (PBS), lipids were extracted overnight with isopropanol at room temperature and radioactivity was quantified in an aliquot of the extract. Fractional efflux was calculated by dividing radioactivity released into the medium by the total label in each well (medium + cells), and was expressed as arbitrary units corresponding to efflux as a percent of total. The reproducibility of the response obtained with different batches of cells and labelling media, was verified by inclusion of a standard pool of human serum in each efflux experiment. Coefficients of variation were 6.5% interassay, and 5.0% intraassay.

2.4. Lipoprotein separation Chylomicrons (Sf\400) were removed by ultracentrifugation for 30 min at 78000 ×g (15°C) of 5.0 ml serum layered under 7.0 ml of a salt solution (density 1.006 g/mL) in a Beckman SW 41 Ti rotor (Beckman, Gagny, France). VLDL and remnants (20B Sf B 400) were removed together from chylomicron-free serum by ultracentrifugation for 20 h at 150 000× g in a 50 TFT rotor (Kontron, St Quentin en Yvelines, France). LDL, HDL2 and HDL3 were assayed by sequential dextran precipitation from VLDL-free serum [19].

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Lipids were measured by enzymatic assays for total and unesterified cholesterol (Boehringer Mannsheim, Meylan France), triglycerides and phospholipids (Wako kits, Unipath Dardilly-France). Apo B, apoAI and LpAI concentrations were measured by immuno-electrophoresis (Sebia, Issy-lesMoulineaux, France). LpAIAII was calculated as the difference between total AI and LpAI.

2.5. Statistics Values are expressed as means9 SD in the text and means9SEM in figures. TG concentrations in serum and HDL were normalized by logarithmic transformation when included in statistical calculations. Total postprandial variations were calculated over the 8 h experimental period as area under the curve (AUC) or as incremental area above the fasting value (iAUC), applying the trapezoidal rule [20]. Postprandial variations were analyzed by Anova for repeated measures. AUCs and differences between groups at individual time-points were compared by paired t-test for effect of treatment and by Anova for differences with controls. Pearson’s coefficient of correlation was used to relate continuous variables.

3. Results Postprandial variations are often integrated as area under the curve (AUC), but the predominant impact of the baseline (fasting) value of each parameter in the calculation of AUC often obscures the specific consequences of nutrient absorption. Beside total AUC, we have therefore examined separately the fasted state (time 0), the post-absorptive state (time 8 h) as well as the incremental areas under the curve above baseline (iAUC) which are the specific result of postprandial metabolic activity.

3.1. Capacity of serum for cholesterol efflux from Fu5AH cells. In the fasted state, the capacity of serum to promote efflux from Fu5AH cells was lower in patients before treatment than in controls (17.99 3.3 vs 19.993.0 a. units, PB 0.05). Treament by bezafibrate elevated capacity for efflux to control level (19.69 3.6 a. units, PB 0.005 with respect to pretreatment values). Postprandial serum-induced efflux of cell cholesterol (Fig. 1) increased regularly with time in control subjects and was significantly greater than in the fasted state at all time-points. In patients before treatment, efflux, already lower in the fasted state, tended to decrease even more postprandially and iAUC was negative in eight out of 12 subjects. After treatment, although

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Fig. 1. (A) Time course of serum-promoted efflux from Fu5AH cells in control subjects (crosses) and type 2 patients before (closed circles) and after (open circles) treatment with bezafibrate. Results are expressed as arbitrary units (a.u) corresponding to percent of cell radioactivity released into the medium during a 4 h incubation. (B) Incremental area under the curve for efflux during the experimental period. Values significantly different from control: PB 0.05 (*), PB 0.01 (**); with pretreatment values: P B0.05 (c ), P B 0.01 (c c).

efflux had reached control levels in the fasted state, it still failed to rise postprandially and iAUC was still negative in five subjects. Total AUC of efflux, which was significantly lower in patients than in controls (140923 vs 1709 25 units.h, P B.01) was nevertheless improved by treatment to 155923 units.h (P B 0.03 with respect to pretreatment level, NS with respect to control). Since the capacity of serum to promote cholesterol efflux in vitro is closely related to the composition of lipoproteins, we examined its evolution with special emphasis on HDL components.

3.2. Fasting lipids and lipoproteins Fasting serum TG concentrations were four-fold higher in patients than in control subjects (Table 1). Under bezafibrate treatment, they decreased by 52% (P = 0.01) but nevertheless remained significantly higher than those of controls. Total serum cholesterol, and LDL-C did not differ from those of controls and remained unchanged after treatment (Table 1), but serum apo B concentrations were lowered by 15% from 1.36 9 0.28 to 1.15 9 0.35 g/l (P =0.05). As previously reported [16], fasting glycemia was significantly improved by bezafibrate treatment (Table 1). HDL-C was very significantly lower in patients than in control subjects (Table 2), because of low HDL2-C, and did not rise under treatment. TG contents of HDL were elevated (Table 2), particularly in HDL3. They decreased after treatment to levels not statistically different from those of controls, and were significantly correlated with serum TG (r= 0.690, P B0.001). The proportion of unesterified cholesterol was the same in the HDL of patients and controls and LCAT

activity was similar (71.99 7.3, 70.39 8.1 and 74.89 14.4 mmoles CE.h/ml, respectively, in patients before and after treatment and in controls).

Table 2 Particle distribution and lipid composition of HDL Before treatment

After treatment Controls

Lp AI (g/l) Lp AI-AII

0.3990.06 1.16 9 0.17

0.41 9 0.05 1.1790.20

0.43 90.11 0.99 9 0.29

HDL-C HDL2-C (mmol/l) HDL3-C

0.88 90.19* 0.17 90.05*

0.94 9 0.22* 0.219 0.07*

1.46 9 0.29 0.669 0.15

0.69 9 0.19

0.729 0.21

0.80 90.23

HDL-PL (mmol/l) HDL2-PL HDL3-PL

1.05 9 0.19

1.12 90.22

1.04 9 0.29

0.22 90.08* 0.84 90.08**

0.27 9 0.09* 0.859 0.12**

0.46 9 0.13 0.67 9 0.25

HDL-UC (mmol/l) HDL-UC/PL HDL-TG HDL2-TG HDL3-TG

0.243 90.052

0.260 9 0.060

0.263 9 0.053

0.234 90.052

0.221 9 0.039

0.255 9 0.038

0.23 90.17*** 0.11 90.08*** 0.23 90.17*

0.13 90.07 0.11 90.06*** 0.13 9 0.07b a

0.05 9 0.02 0.05 9 0.01 0.05 9 0.02

* Means9SD. Statistically different: from control value (t-test) P B0.001. ** Means 9SD. Statistically different: from control value (t-test) P B0.05. *** Means 9SD. Statistically different: from control value (t-test) P B0.01. a Means9 SD. Statistically different: from control value (t-test) P from before treatment value (paired t-test), PB0.05. b Means9 SD. Statistically different: from control value (t-test), P B0.01.

D. Autran et al. / Atherosclerosis 153 (2000) 453–460

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Fig. 4. Time-course of TG:PL and UC:PL ratios in HDL. Legend as in Fig. 1. Values significantly different from control: P B0.05 (*), PB 0.01 (**), PB 0.001 (***); with pretreatment values: PB0.05 (c). Fig. 2. Time course of postprandial hyperlipemia. Legend as in Fig. 1.

3.3. Postprandial hyperlipidemia and HDL composition AUC of postprandial hyperlipidemia (Fig. 2) was four-fold higher in patients than in control subjects (P B 0.001 by Anova). It was significantly reduced by bezafibrate treatment (P B0.02 with respect to before by paired t-test), but did not quite revert to the level of control subjects. Treatment effect was mainly evidenced by a 50% reduction of the AUC of chylomicron-TG from 33.5921.7 to 15.498.5 mmol/l.h after treatment (PB0.02), indicative of improved lipolysis. HDL underwent significant postprandial changes. In control subjects, as expected [11,21,22], HDL-C decreased significantly after the meal (time effect by Anova for repeated measures P B0.01). The effect was on HDL2 (iAUC-0.53690.879 mmol/l.h, P B 0.01) (Fig. 2), and not on HDL3 (iAUC 0.1529 0.905 mmol/ l.h). In patients, by contrast, the decrease in HDL2-C concentrations did not occur to any significant extent (iAUC HDL2-C: -0.122 mmol/l.h) and was only par-

tially restored by treatment (iAUC-0.30690.594 mmol/l.h) (Fig. 2). HDL-PL, on the contrary, rose after the meal in controls and had increased by 20% 8 h later, exclusively in HDL3 (Fig. 3A). No significant increment was detected in patients, not even after treatment: contrary to expectations, the improvement in chylomicron clearance brought about by treatment was without effect on postprandial accretion of PL in HDL3. The main change in the HDL composition of patients was an enrichment with TG occurring essentially in HDL3 (Fig. 3B), which was significant at all time points after the meal prior to treatment and to a lesser extent after treatment. The iAUCs of HDL3-TG displayed a strong correlation with those of serum TG (r=.660, PB 0.001), i.e. with the intensity of hyperlipemia but the enrichment persisted at the end of the experimental period, even though serum TG had begun to decrease. The UC/PL molar ratio, characterizing surface composition, decreased in all three groups (Fig. 4A). The molar ratio of TG (in the core) to PL (on the surface) remained stable in controls. In patients on the contrary, this ratio, already three-fold higher than in controls in the fasted state, further increased after the fat load (Fig. 4B) as a result of the combined stability of PL and increase of TG in HDL3.

3.4. Relations between serum lipoproteins and efflux

Fig. 3. Incremental areas (iAUCs) of HDL3-PL and HDL3-TG and in type 2 patients before (full bars) and after (open bars) treatment with bezafibrate compared with controls (hatched bars).

In an attempt to define the likely causes for the differences in efflux linked to the post prandial state and/or to bezafibrate treatment, we examined the statistical correlates between efflux and variations in lipoprotein constituants. In the fasted state univariate analysis showed efflux to correlate positively with HDL-PL (r= 0.892, PB 0.01) as well as with HDL-C (r= 0.773, PB0.05) in

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control subjects. In patients efflux correlated with HDL-PL (r =0.588, B0.05) before treatment, but not any more after. A negative correlation with UC/PL on HDL existed in controls (r= − 0.749, P = 0.6) but not in patients whether before or after treatment. Postprandial efflux (AUC) in controls also displayed a major correlation with AUC of HDL-PL (r= 0.898, P B 0.01), but the correlation with AUC of HDL-C did not reach significance (r= 0.535, NS) Efflux correlated negatively as well with the ratio of TG(log)/PL in HDL. In patients postprandial efflux only correlated with HDL-PL before treatment (r= 0.747, P B0.01), but with none of the other parameters after treatment. Postabsorptive efflux when the correlates of postprandial efflux were specifically examined after lipid absorption, i.e. 8 h after the meal, we found HDL-PL to be a non-significant factor (r= −0.044, 0.217, and 0.420, respectively, in controls and patients before and after treatment) while the effect of HDL was best represented by HDL-C (r = 0.663, P B 0.05, r= 0.214, NS, and r =0.646 P B0.05, respectively, in controls and patients before and after treatment). Including all groups a strong negative correlation was evidenced with the TG(log)/PL ratio in HDL (r= − 0.674, PB 0.001) (Fig. 5).

4. Discussion Serum from type 2 diabetic patients displays a reduced capacity for cell cholesterol efflux [5], especially when cardiovascular complications are present. Our results confirm that, in diabetic patients with no signs of macrovascular damage, this capacity is also moderately but significantly decreased in the fasted state. We further show that it is restored to control level by lipid-lowering bezafibrate treatment.

Fig. 5. Correlation between serum-promoted efflux 8 h after the fat load and TG(log)/PL ratio in HDL (r= − 0.674, PB 0.001). Symbols as in Fig. 1.

HDL-PL is accepted to be the main variable affecting capacity of serum for cell cholesterol extraction [6] and, perhaps, more specifically, the UC/PL ratio [23,24] on the particle surface. Indeed, in the fasted state, HDLPL was the main correlate of efflux. The degree of correlation was, however, lower in patients than in controls, particularly under bezafibrate treatment. This suggests the interference of some factor other than availability of PL on the surface of HDL. It has been suggested that the UC/PL ratio might play a distinct role in type two patients [7–9], but in this group of patients the ratio was not increased and no correlation could be evidenced with this surface parameter, while controls displayed the expected negative correlation. The concentration of LpAI-AII particles or their ratio to LpAI to might have been a determinant factor [5,24]. Although apo AII concentrations are reportedly raised by bezafibrate in healthy volunteers [25] and by fenofibrate in coronary patients [26], neither concentrations nor the LpAI/LpAI-AII ratio were found to be significantly altered in patients with respect to controls and they remained unaffected by treatment. This was in agreement with the report of a subnormal capacity of LpAI isolated from the serum of type 2 diabetics to promote efflux from cells, tentatively attributed to a deficit in surface PL [27]. Results from distinct experiments therefore concur to indicate that the determinants of efflux identified in non diabetic subjects, whether healthy or suffering from coronary pathology, are less efficient in type 2 diabetics. Improvement of fasting lipemia by bezafibrate restored a normal level of efflux without restoring the balance between HDL2 and HDL3 or the correlation of efflux with PL, suggesting that the metabolic properties of HDL particles were, nevertheless, not fully reinstated. The study of postprandial variations in efflux helped us explore further the mechanism for the defect in reverse transport. Indeed, while efflux increased significantly after the meal in healthy controls as previously reported [9,10] and in parallel with PL accretion [11], it tended to decrease in patients, a result previously reported for isolated postprandial LpAI particles [27]. Thus the differences became more pronounced, indicative of an even deeper deficit of RCT postprandially in patients: 8 h after the meal efflux was 28% lower in patients than in control subjects (PB 0.001) and AUC of efflux over the 8 h period was lower by 18%. Reflecting the correlation between absolute values in the fasted state, the AUC of HDL-PL was the main determinant of postprandial efflux, in control subjects as well as in patients, although the correlation remained non-significant in the treated patients. The UC:PL ratio which seems to be determinant when addressing fasting subjects [23] cannot account for the differences in postprandial efflux between patients and controls since it decreases in all.

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The postprandial HDL of untreated patients were characterized by TG contents that were over three-fold greater than in controls in the fasted state, and as much as 4.5-fold at late postprandial times. HDL enriched with TG are reportedly less stable, more likely to shed their apoAI [13] and more easily remodelled by PLTP [14,15] than CE containing particles, while TG have emerged as a major determinant of postprandial HDL metabolism [21,22]. The transfer of TG to HDL by CETP in exchange for cholesteryl esters is strongly enhanced by the presence of high concentrations of TG-rich particles [28] such as occur during lipid absorption. Moreover, CETP activity is higher in diabetic subjects than in controls [12] and is not normalized by treatment with bezafibrate [16]. The postprandial increase in HDL-TG and the ratio of TG to PL were both negatively correlated with efflux. In control subjects, TG enrichment of HDL occurred together with an increase in PL so that the TG:PL ratio remained essentially unchanged. In patients, TG accumulation was several-fold greater while PL accretion was deficient, creating conditions for an increase in core to surface ratio and maximal TG contents inside the core. Indeed, the TG:PL ratio in HDL revealed itself as a very discriminant characteristic between the three groups at the end of the experimental period, when accumulation of TG was greatest in HDL of patients, although postprandial hyperlipidemia had subsided. More generally, our results suggest that the low degree of correlation which has been reported between efflux and normally significant lipid and lipoprotein parameters [4] might be directly or indirectly linked to the enrichment of HDL with triglycerides which is a characteristic of diabetic HDL not shared with the HDL of non-diabetic coronary patients. The significant decrease in postprandial lipemia under bezafibrate treatment was not sufficient to restore the postprandial increase in efflux, although it did prevent the decrease observed before treatment. The lower degree of correlation with HDL-PL in treated than untreated patients and in controls, suggests the intervention of some other undetected factor induced by treatment. These results confirm the defective postprandial reverse cholesterol transport in type 2 diabetic patients, a novel aspect of the dysregulation in their lipid metabolism. Although the capacity of the patients’ serum to promote cholesterol efflux was decreased by only 10% in the fasted state, it was 28% lower than in controls over the postprandial period: taking into account the large portion of the day spent in the postprandial state, this may result in a deficit of cholesterol extraction far superior to what might have been expected from fasting data and may be one of the undetected factors contributing to the high incidence of atherogenic processes in type 2 patients. The improve-

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ment of triglyceridemia by bezafibrate was accompanied by a restoration of a normal level of efflux in the fasted state and a significant improvement of postprandial levels, suggesting that treatment might, beyond its triglyceride lowering effects, act favorably on reverse cholesterol transport.

Acknowledgements The Fu5AH cells were originally a gift from Dr Ve´ronique Atger, with the permission of Dr George Rothblat. This work was supported by grants from Lipha-Oberval and from Boehringer Pharmaceutical.

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