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Plasma pre β-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus: Role of phospholipid transfer protein
Biochimica et Biophysica Acta 1791 (2009) 714–718
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a l i p
Plasma pre β-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus: Role of phospholipid transfer protein G.M. Dallinga-Thie a,⁎, A. van Tol b,c, R.P.F. Dullaart c and for the Diabetes Atorvastatin lipid intervention (DALI) study group a b c
Laboratory of Experimental Vascular Medicine G1-113, Academic Medical Center Amsterdam, Amsterdam, The Netherlands Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Department of Endocrinology, University Medical Center Groningen, Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 28 February 2009 Accepted 9 March 2009 Available online 19 March 2009 Keywords: Atorvastatin High density lipoprotein Triglyceride Phospholipid transfer protein Cholesteryl ester transfer protein Pre-β HDL
a b s t r a c t Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-β-HDL generation in vitro. We determined the effect of atorvastatin on pre-β-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-β-HDL formation as well as plasma apo A-I, LpA, LpAI: AII, cholesteryl ester transfer protein (CETP) and PLTP activity were measured before and after 30 weeks treatment in 40 patients randomized to atorvastatin 80 mg daily and 41 placebo receiving patients. Pre-β HDL formation was measured by crossed immunoelectrophoresis under conditions of lecithin:cholesterol acyltransferase (LCAT) inhibition. Results: Plasma pre-β-HDL formation, triglycerides, LDL cholesterol, PLTP activity, and CETP decreased after statin treatment (all P b 0.001 vs placebo), whereas HDL cholesterol increased (P b 0.005). Plasma apo A-I, LpAI and LpAI:AII remained unchanged compared to placebo. In all patients combined, the changes in pre-β-HDL formation were independently related to the decrease in plasma triglycerides (β = 0.31; P = 0.006) and PLTP activity (β = 0.23; P = 0.038), without a contribution of CETP. In the atorvastatin treated patients, the decrease in pre-β-HDL formation tended to be related to the decrease in PLTP activity (β = 0.30, P = 0.061) after controlling for decreases in triglycerides (β = 0.22, P = 0.22). Conclusion: High dose atorvastatin decreases the capacity of plasma to generate pre-β-HDL particles in type 2 diabetic patients, probably via lowering of plasma PLTP activity and triglycerides. This could contribute to an improvement in the atherogenic lipoprotein profile. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The increased cardiovascular risk in type 2 diabetes mellitus can in part be explained by a low high density lipoprotein (HDL) cholesterol concentration [1]. HDL particles are heterogeneous in size, structure, lipid composition and apolipoprotein (apo) content [2]. HDL subfraction levels are altered in diabetes, particularly when complicated by cardiovascular disease [3]. Moreover, mean HDL particle size is decreased in insulin resistant and diabetic subjects [4]. A small proportion of HDL consists of small-sized, lipid-poor HDL particles, designated pre-β-HDL, which are thought to be involved in the removal of cell-derived cholesterol to the extracellular compartment [5,6]. In vitro studies have shown that the generation of these particles is, among other factors, stimulated by lipid transfer reactions catalyzed by cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) [7]. PLTP may initiate fusion of mature HDL in vitro, thereby generating smaller and larger particles than the parent HDL [8]. These smaller particles have pre-β-HDL mobility on agarose gels, ⁎ Corresponding author. Tel.: +31 20 56651258; fax: +31 20 566934. E-mail address: [email protected] (G.M. Dallinga-Thie). 1388-1981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2009.03.008
and are more rapidly formed in transgenic mice expressing human PLTP [9,10]. Despite low levels of total plasma apo A-I, plasma pre-βHDL is not decreased, and may even be increased, in patients with diabetes mellitus [11,12], which is likely to be explained, at least in part, by an increase in plasma PLTP activity [13]. Importantly, a positive relation of pre-β-HDL levels with intima media thickness (IMT) was observed recently in type 2 diabetic patients independent of clinical factors and levels of plasma apo Bcontaining lipoproteins [12]. Moreover, atherosclerosis is also associated with increased plasma pre-β-HDL concentrations in other high risk populations [12,14], except for one study in which pre-β-HDL was found to be negatively related to IMT in subjects with familial low HDL cholesterol [15]. These results underscore the relevance to determine whether plasma pre-β-HDL metabolism is affected by lipid lowering treatment. Treatment with atorvastatin and rosuvastatin decrease pre-β1 HDL, but not pre-β2 HDL, in hyperlipidemic subjects [16], whereas, in another report, preβ-1 HDL was found to remain unchanged after treatment with rosuvastatin in metabolic syndrome subjects [17]. In type 1 diabetic patients we have documented a small drop in pre-β-HDL formation in response to simvastatin [18]. On the other hand, there are no in vivo data showing a relationship between
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plasma PLTP activity and pre β-HDL in an intervention study in humans, although in vitro studies implicate PLTP in pre-β-HDL formation. Since atorvastatin lowers plasma PLTP activity in type 2 diabetic patients [19], we hypothesized that a decrease in PLTP activity may contribute to a decrease in pre-β-HDL formation during atorvastatin treatment in patients with type 2 diabetes. The present study was initiated to test this hypothesis. 2. Methods 2.1. Subjects The DALI study is a randomized, double-blind, placebo-controlled trial on the effects of atorvastatin 10 mg and 80 mg daily in patients with type 2 diabetes. The study protocol has previously been described in detail [20]. In brief, men and women aged 45–75 years, with a known duration of type 2 diabetes of at least 1 year and hypertriglyceridemia (fasting triglycerides between 1.5 and 6.0 mmol/l and total cholesterol between 4.0 and 8.0 mmol/l) were included. Type 2 diabetes was defined in accordance with the American Diabetes Association guidelines of 2000 [21]. Patients with an HbA1c above 10% or a history of cardiovascular disease were excluded. In the current study, a subset of patients treated with placebo (n = 41) and 80 mg atorvastatin during 30 weeks (n = 40) was included, based on the availability of plasma samples. The selected individuals were not different from the rest of the study population with respect to any of the clinical characteristics and baseline lipid levels. Of the study subjects, 45% used oral glucose lowering drugs, 28% used insulin and 26% were treated with combination therapy. Treatment for diabetes was equally distributed among the 2 randomized groups. Lipid-lowering therapy was withdrawn 6 weeks before start of the run-in phase. The medical ethical committees of the participating institutions approved the study protocol and all participants gave their written informed consent. 2.2. Laboratory measurements At baseline and after 30 weeks of treatment, blood was collected in EDTA tubes after an overnight fast of at least 12 h and centrifuged immediately (3000 rpm 15 min, 4 °C). Plasma was stored at −80 °C. Total cholesterol, triglycerides, HDL cholesterol and apo A-I were measured as described [20]. Plasma LpAI and LpAI:AII were analyzed using a commercially available immunoelectrophoresis assay (Sebia, France). Plasma pre-β-HDL formation, i.e. the ability of plasma to generate pre-β-HDL, was measured by crossed immunoelectrophoresis as described [11]. In brief, plasma samples were thawed while kept on ice and 0.9 μmol/l Pefabloc SC (Boehringer-Roche, Penzberg, Germany) and 1.8 μg/l Trasylol (Bayer, Mijdrecht, The Netherlands) were added to inhibit proteolysis (both final concentrations). Samples were incubated during 24 h at 37 °C under conditions of LCAT inhibition. To this end, iodoacetate (final concentration 1.0 mmol/l) was added directly after thawing the samples. The crossed immunoelectrophoresis consisted of agarose electrophoresis in the first dimension for separation of lipoproteins with pre β- and α-mobility. Antigen migration from the first agarose gel into the second agarose gel, containing goat anti-human apo A-I antiserum was used to quantitatively precipitate apo A-I. The antiserum was monospecific for human apo A-I using an immunodiffusion assay. Lipoprotein electrophoresis was carried out in 1% (weight/vol) agarose gels in Tris (80 mmol/l)–tricine (24 mmol/l) buffer, 5% (vol/vol) polyethylene glycol 300 (pH 8.6) and run in an LKB 2117 system (4°C for 3 h, 210 V). Plasma was applied at 3 μl/well. The track of the first agarose gel was excised and annealed with melted agarose to a gel containing 0.66% (v/v) goat anti-human apo A-I anti-serum (Midland Bioproducts Corporation, Boone Iowa) and 0.01% Tween 20 (w/v), that was cast on GelBond film (Amersham, Uppsala, Sweden). The plate was run in an
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LKB 2117 system (4 °C for 20 h, 50 V) in Tris–tricine buffer. Unreacted antibody was removed by extensive washing saline. The gel was stained with Coomassie Brilliant Blue R250, dried, and scanned with a HP scanjet 5470c. Areas under the pre-β-HDL and α-HDL peaks were calculated. The pre-β HDL area was expressed as the percentage of the sum of apo A-I in the pre-β-HDL and the α-HDL areas. Pre-β HDL formation was calculated using the total plasma apo A-I concentration and was expressed in apo AI (g/l). The interassay coefficient of variation was 9%. Plasma PLTP activity was assayed with a phospholipid vesiclesHDL system, using [14C]-labeled dipalmitoyl phosphatidylcholine [19]. This method is specific for PLTP activity and the phospholipid transfer promoting property of CETP does not interfere with the assay. PLTP activity is measured under optimal conditions with excess exogenous substrate. This assay indicates the plasma level of active PLTP and is related to the activity in reference pool plasma. PLTP activity is expressed in arbitrary units (AU; 100 AU corresponds to 13.6 μmol phosphatidylcholine transferred, ml− 1 h− 1). Plasma CETP was measured using double-antibody sandwich enzyme-linked immunosorbent assays as described [22]. Plasma CETP mass has been demonstrated to be correlated CETP activity measured with an excess exogenous substrate assay (r N 0.7) in two independent studies including patients and control subjects [23,24]. 2.3. Statistical analysis Statistical analysis was performed using SPSS version 16. Data are expressed in mean ± SD. Within group changes in variables were determined by paired t-tests. Between groups differences in changes in variables were evaluated by unpaired t-tests. Univariate relationships were analyzed by Pearson's correlation analysis. Multiple linear regression analysis was used to disclose independent relationships between variables. Standardized regression coefficients (β) (equivalent to partial correlation coefficients) were used to estimate the adjusted contribution of variables. Two-sided P-values b0.05 were considered significant. 3. Results A total of 81 type 2 diabetic patients (43 women and 38 men) were included in the present study of whom 40 had been randomized to atorvastatin treatment (80 mg daily) and 41 to placebo. Age, sex distribution and BMI at baseline were not different between patients allocated to atorvastatin compared to those allocated to placebo (60 ± 8 vs 58 ± 7 years, respectively, P = 0.20; 19 women and 21 men vs 24 women and 17 men, P = 0.44; 31.0 ± 4.7 vs 32.4 ± 6.5 kg/m2, respectively, P = 0.27). Baseline HbA1c (atorvastatin: 8.6 ± 1.2% and placebo: 8.4 ± 1.2%, P = 0.46) and fasting glucose levels (atorvastatin: 11.2 ± 3.0 mmol/l and placebo: 10.7 ± 3.6 mmol/l, P = 0.52) were also similar in both groups of patients. Atorvastatin treatment did not result in an improvement of fasting glucose or HbA1c levels (data not shown). Table 1 shows plasma apo A-I, LpAI, LpAI:AII and pre-β-HDL formation at baseline and after atorvastatin administration compared to placebo. At baseline, no significant difference in any of the variables shown in Table 1 was observed between the subjects allocated to atorvastatin vs placebo administration. Treatment with atorvastatin resulted in a decrease in plasma total cholesterol, LDL cholesterol and triglycerides compared to placebo. HDL cholesterol increased in response to atorvastatin, but the change in plasma apo A-I was not different compared to placebo. Pre-β HDL formation decreased in response to atorvastatin. Plasma CETP and PLTP activities were also lowered by atorvastatin treatment. No significant changes in the LpAI and LpAI:AII concentration were observed. Table 2 shows the univariate relationships at baseline of HDL cholesterol, apo A-I, HDL subfractions and pre-β-HDL formation with plasma triglycerides, CETP and PLTP activities in the combined groups
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Table 1 Effects of 30 week 80 mg daily atorvastatin treatment compared to placebo with respect to plasma (apo) lipoproteins, high density (HDL) cholesterol, LpAI, LpAI:AII and pre-βHDL formation, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activity. Plasma variable
Total chol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) Apo A-I (g/l) HDL cholesterol (mmol/l) LpAI (g/l) LpAI:AII (g/l) Pre-β HDL formation (apo A-I g/l) CETP (mg/l) PLTP activity (AU)
Atorvastatin 80 mg (n = 40)
Placebo (n = 41)
Baseline
Follow-up
Baseline
6.03 ± 1.04 3.67 ± 0.93
3.59 ± 1.09¶ 2.73 ± 1.3§
6.01 ± 0.80 5.88 ± 0.81 3.67 ± 0.82 3.67 ± 0.83
b0.001 b 0.001
2.85 ± 1.02
1.56 ± 0.60¶
2.83 ± 1.00 2.61 ± 1.17
b 0.001
1.44 ± 0.21 1.07 ± 0.27
1.37 ± 0.22¶ 1.11 ± 0.29†
1.42 ± 0.22 1.36 ± 0.18¶ 1.06 ± 0.23 1.02 ± 0.22†
0.61 0.005
0.49 ± 0.15 0.93 ± 0.13 0.30 ± 0.09
0.43 ± 0.16‡ 0.96 ± 0.15 0.22 ± 0.06¶
0.46 ± 0.10 .45 ± 0.11 0.97 ± 0.16 0.92 ± 0.18† 0.27 ± 0.07 0.26 ± 0.08
0.17 0.40 0.001
2.53 ± 0.82 111 ± 23
1.74 ± 0.55¶ 96 ± 16¶
2.66 ± 0.74 2.60 ± 0.65 b 0.001 105 ± 23 105 ± 19 b 0.001
P-value Follow-up
Data are presented in mean ± SD. † P ≤ 0.05; ‡P ≤ 0.02;§P ≤ 0.01;¶P ≤ 0.0001 compared to baseline; ∫P-value for difference in change after atorvastatin compared to placebo.
at baseline (n = 81). Plasma triglycerides were inversely correlated with HDL cholesterol, whereas there was a positive correlation between plasma triglycerides and CETP. HDL cholesterol, plasma apo A-I and LpAI:AII were negatively correlated with plasma CETP. Pre-β HDL formation was positively associated with PLTP activity (P b 0.05). Its relation with CETP did not reach statistical significance (P = 0.09). Table 3 demonstrates the relationships of the individual changes in HDL cholesterol, LpAI, LpAI:AII and pre-β-HDL formation with the changes in plasma triglycerides and lipid transfer proteins in all study subjects combined. Only the changes in HDL cholesterol were inversely related to the changes in plasma triglycerides. In contrast, the changes in pre β-HDL formation and in LpAI were positively correlated with the changes in triglycerides. The changes in LpAI and pre-β-HDL formation were also positively related to the changes in plasma PLTP activity, as well as to the changes in plasma CETP. The changes in LpAI:AII did not correlate with the changes in triglycerides, CETP and PLTP activities. The decrease in pre-β-HDL formation tended to be related to the decrease in PLTP activity (r = 0.31, P = 0.06) in the atorvastatin treated patients. The correlations with the changes in triglycerides and CETP were r = 0.27 (P = 0.09) and r = 0.02 (P N 0.80), respectively. In the placebo group no significant relationships between changes in these variables were present (P N 0.20 for all). Multiple linear regression analysis was performed to determine the independent effects of the changes in plasma triglycerides, PLTP activity and CETP on the individual changes in pre-β-HDL formation
Table 2 Univariate correlation coefficients at baseline in all subjects combined (n = 81) for determinants of plasma triglycerides, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activity.
Triglycerides (mmol/l) Apo A-I (g/l) HDL cholesterol (mmol/l) LpAI (g/l) LpAI:AII (g/l) Pre-β-HDL formation (apo A-I, g/l) CETP (mg/l) PLTP activity (AU)
Table 3 Relationships of changes in plasma HDL cholesterol, LpAI, LpAI:AII and pre-β-HDL formation with changes in plasma triglycerides, PLTP activity and CETP mass in 81 type 2 diabetic patients after 30 week administration of atorvastatin 80 mg daily (n = 40) and placebo (n = 41).
Triglycerides⁎ (Pearson's r)
CETP mass (Pearson's r)
PLTP activity (Pearson's r)
– − 0.21 − 0.45§ − 0.08 − 0.22‡ 0.09 0.26‡ 0.08
0.26‡ − 0.23‡ − 0.37‡ − 0.12 − 0.22‡ 0.19 – 0.21
0.08 − 0.01 − 0.13 0.02 0.02 0.25‡ 0.21 –
Pearson's correlation coefficients (r) are shown. Data in mean ± SD. †Log transformed value for triglycerides are used in the regression analysis. ‡P ≤ 0.05; §P b 0.01.
ΔTriglycerides ΔPLTP activity ΔCETP
ΔHDL cholesterol
ΔLpAI
ΔLpAI:AII
Δpre-β-HDL formation
− 0.48§ − 0.12 − 0.18
0.24† 0.27† 0.23†
− 0.18 − 0.15 − 0.08
0.39§ 0.34‡ 0.30‡
Δ: Change; †P b 0.05; ‡P b 0.01; §P b 0.001.
and HDL sub-fractions. This analysis demonstrated that in the combined group the changes in LpAI were only independently determined by the changes in plasma PLTP activity (β = 0.27, P = 0.016). In these models only those HDL-related variables which showed significant univariate relationships were included. This analysis demonstrated that the changes in LpAI were only independently determined by the changes in plasma PLTP activity (P = 0.016), whereas the change in pre-β-HDL formation was determined by the decrease in triglycerides (β = 0.31; P = 0.006) and in PLTP activity (β = 0.23; P = 0.038), but not by changes in CETP (β = 0.09; P = 0.48). There were no independent effects of age, sex, baseline HbA1c, BMI and diabetes treatment (oral glucose lowering drugs, insulin, or combination therapy) on the decrease in pre-β-HDL formation (P N 0.30). In the atorvastatin treated patients, the decrease in preβ-HDL formation tended to be related to the decrease in plasma PLTP activity (β = 0.30; P = 0.061), without effects of changes in triglycerides (β = 22; P = 0.16) and CETP (β = 0.07; P = 0.68). 4. Discussion The present study shows that pre-β-HDL formation is substantially decreased by high dose atorvastatin treatment in type 2 diabetic patients. Univariate regression analysis demonstrated that the changes in pre-β-HDL formation were related to the changes in plasma PLTP activity and triglycerides. The independent contributions of these variables on pre-β-HDL formation were underscored by multiple linear regression analysis. In atorvastatin treated patients only, the relationship of the drop in pre-β-HDL formation with the decline in PLTP activity was close to statistical significance. Therefore, the present study supports the contention that PLTP activity is important for pre-β-HDL formation in humans and that the effect of atorvastatin on plasma PLTP activity may contribute to the regulation of pre-β-HDL in vivo. To our knowledge, this is the first intervention study in humans revealing that changes in plasma pre β-HDL formation are determined by changes in PLTP activity. We have previously documented plasma pre-βHDL concentrations as well as pre-β-HDL formation, assayed exactly as described above, in a group of 81 diabetic patients who did not use lipid lowering drugs [11]. In this report, pre-β-HDL formation was on average five-fold higher compared to pre-β-HDL concentration as measured without incubation of plasma under conditions of LCAT inhibition. That study revealed positive correlations of pre-β-HDL concentration with preβ-HDL formation (r=0.31, P=0.008) as well as with plasma PLTP activity (r = 0.32, P=0.003). However, plasma pre-β-HDL concentration was not correlated with PLTP activity (r= 0.07, P=0.55). These results made us decide to test the relation between atorvastatin-induced changes in plasma PLTP activity and pre-β-HDL formation rather than pre-β-HDL concentration in the present study. Of importance, pre-β-HDL metabolism may also be affected by CETP and hepatic lipase [6,25]. In the current report, we relied on measurement of plasma CETP mass which is closely correlated with its plasma activity level using an exogenous substrate assay, which is not confounded by the endogenous cholesteryl ester acceptor and donor lipoproteins present in plasma [24]. We showed here that the relationship between plasma CETP and pre-β-HDL formation at baseline did not
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reach statistical significance. Moreover, although changes in plasma preβ-HDL formation were correlated with changes in CETP in univariate analysis, CETP did not independently contribute to pre-β-HDL formation in multiple regression analysis, which would favour a preferential contribution of PLTP as compared to CETP in pre-β-HDL generation. A similar observation has been found earlier by comparing pre-β-HDL generation in plasma from PLTP transgenic mice vs CETP transgenic mice [26]. Changes in pre-β-HDL formation were also independently related to the changes in plasma triglycerides in the present study, which can probably be explained by enhanced pre-β-HDL generation from α-HDL by PLTP when these particles are enriched with triglycerides [27]. Importantly, pre-β-HDL particles are initial substrates for LCAT, which leads to rapid conversion into more mature HDL by the process of cholesterol esterification. We measured pre-β-HDL under conditions of LCAT inhibition and analyzed the fraction with crossed immunoelectrophoresis assay using an antibody against human apo A-I. Thus, the current pre-β-HDL measurements should be regarded to represent an ex vivo estimate of the capacity to generate such particles in plasma. In the interpretation of the present results, it is relevant to note that our assay detects pre-β1-HDL and pre-β2-HDL together. In studies from other laboratories in which the effect of statin treatment on pre-β-HDL was determined, the plasma concentration of these particles were further divided into pre-β1-HDL and pre-β2-HDL [28] or measured by ELISA [17]. Thus, several methodological differences could in part explain why preβ-HDL was not found to be consistently decreased after statin administration in all previous reports. Pre-β HDL particles are initial acceptors of cell-derived cholesterol, and hence are considered to play a role in the reverse cholesterol transport pathway, whereby excess peripheral cell cholesterol is transported back to the liver for metabolism and excretion in the bile. Despite this stimulatory effect of these lipid-poor HDL particles on cellular cholesterol efflux, positive relationships of (subclinical) atherosclerosis with high plasma levels of pre-β-HDL have been documented [12]. Thus, it appears to be conceivable that elevated plasma pre-β-HDL levels indicate an impaired maturation of HDL and that decreased levels would reflect more rapid metabolism of such particles into mature α-HDL, contributing to an efficient RCT pathway [29]. Our observation of a decreased formation of pre β-HDL due to lower plasma PLTP activity would suggest that a fast turnover and maturation of pre β-HDL, as well as less pre β-HDL formation could occur, leading to the presence of less pre β-HDL after statin treatment. It is, therefore, possible that statin-induced decreases in pre-β-HDL formation would imply cardiovascular benefit. Atorvastatin induced a modest increase in total HDL cholesterol, coinciding with a decrease in plasma triglycerides as expected [20,30,31], but did not significantly affect LpAI and LpAI:AII. In other studies, LpAI was found to increase in response to rosuvastatin in metabolic syndrome subjects [17]. It is known that the effects of atorvastatin 80 mg/day on HDL parameters are modest compared with effects of other statins [16]. In the current report, we compared the effects of 80 mg atorvastatin daily with placebo in a subset of DALI participants and did not evaluate effects of a lower atorvastatin dose. However, no differences between the HDL cholesterol, plasma apo A-I and triglyceride responses between 10 mg and 80 mg atorvastatin administration have been demonstrated among diabetic patients participating in previous analyses from this trial [19,20], and plasma PLTP activity was shown to decrease in response to the lower atorvastatin dose as well. Thus, it is unlikely that selective inclusion of participants randomized to high dose atorvastatin essentially affected the present analysis. In conclusion, high dose atorvastatin decreases plasma pre-βHDL formation in type 2 diabetic patients, probably via lowering of plasma PLTP activity and triglycerides. A decrease in pre-β-HDL could modify cardiovascular risk reduction beyond effects on VLDL and LDL cholesterol.
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Acknowledgements The authors wish to thank contributors to the DALI study as patients, technicians, or data managers. The original DALI study was an investigator-driven study partly supported by an unrestricted grant of Parke-Davis, The Netherlands. The following is a complete list of the original DALI study group (in alphabetical order): Erasmus Medical Center Rotterdam, Department of Internal Medicine (I. Berk-Planken, N. Hoogerbrugge, H. Jansen); Erasmus University Rotterdam, Departments of Biochemistry and Clinical Chemistry (H. Jansen); Gaubius Laboratory TNO-KvL, Leiden (H.M.G. Princen); Leiden University Medical Center (M.V. Huisman, M.A. van de Ree); University Medical Center Utrecht, Julius Center for General Practice and Patient Oriented Research (R.P. Stolk, F.V. van Venrooij); University Medical Center Utrecht, Division of Internal Medicine (J.D. Banga, G.M. Dallinga-Thie, F.V. van Venrooij). The technical assistance of F. Perton (Laboratory Center, University Medical Center Groningen, The Netherlands), for pre-β-HDL measurements is greatly appreciated.
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[18] R. de Vries, M.N. Kerstens, W.J. Sluiter, A.K. Groen, A. van Tol, R.P.F. Dullaart, Cellular cholesterol efflux to plasma from moderately hypercholesterolaemic type 1 diabetic patients is enhanced, and is unaffected by simvastatin treatment, Diabetologia 48 (2005) 1105–1113. [19] G.M. Dallinga-Thie, A. van Tol, H. Hattori, P.C.N. Rensen, E.J.G. Sijbrands, Plasma phospholipid transfer protein activity is decreased in type 2 diabetes during treatment with atorvastatin: a role for apolipoprotein E? Diabetes 55 (2006) 1491–1496. [20] I.I. Berk-Planken, N. Hoogerbrugge, H. Jansen, H.M.G. Princen, M.V. Huisman, M.A. Van de Ree, R.P. Stolk, F.V. van Venrooij, J.D. Banga, G.M. Dallinga-Thie, F.V. van Venrooij, DALI Study Group, The effect of aggressive versus standard lipid lowering by atorvastatin on diabetic dyslipidemia — the DALI Study: a doubleblind, randomized, placebo-controlled trial in patients with type 2 diabetes and diabetic dyslipidemia, Diabetes Care 24 (2001) 1335–1341. [21] Supplement 1. American Diabetes Association: clinical practice recommendations 2000, Diabetes Care 23 Suppl 1 (2000) S1–116. [22] S.D.J.M. Niemeijer-Kanters, G.M. Dallinga-Thie, F.C. Ruijter-Heijstek, A. Algra, D.W. Erkelens, J.D. Banga, H. Jansen, Effect of intensive lipid-lowering strategy on lowdensity lipoprotein particle size in patients with type 2 diabetes mellitus, Atherosclerosis 156 (2001) 209–216. [23] F.V. van Venrooij, R.P. Stolk, J.D. Banga, T.P. Sijmonsma, A. van Tol, D.W. Erkelens, G.M. Dallinga-Thie, Common cholesteryl ester transfer protein gene polymorphisms and the effect of atorvastatin therapy in type 2 diabetes, Diabetes Care 26 (2003) 1216–1223. [24] R.P.F. Dullaart, R. de Vries, L. Scheek, S.E. Borggreve, T. van Gent, G.M. DallingaThie, M. Ito, M. Nagano, W.J. Sluiter, H. Hattori, A. van Tol, Type 2 diabetes mellitus is associated with differential effects on plasma cholesteryl ester transfer protein
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a l i p
Plasma pre β-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus: Role of phospholipid transfer protein G.M. Dallinga-Thie a,⁎, A. van Tol b,c, R.P.F. Dullaart c and for the Diabetes Atorvastatin lipid intervention (DALI) study group a b c
Laboratory of Experimental Vascular Medicine G1-113, Academic Medical Center Amsterdam, Amsterdam, The Netherlands Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands Department of Endocrinology, University Medical Center Groningen, Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 28 February 2009 Accepted 9 March 2009 Available online 19 March 2009 Keywords: Atorvastatin High density lipoprotein Triglyceride Phospholipid transfer protein Cholesteryl ester transfer protein Pre-β HDL
a b s t r a c t Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-β-HDL generation in vitro. We determined the effect of atorvastatin on pre-β-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-β-HDL formation as well as plasma apo A-I, LpA, LpAI: AII, cholesteryl ester transfer protein (CETP) and PLTP activity were measured before and after 30 weeks treatment in 40 patients randomized to atorvastatin 80 mg daily and 41 placebo receiving patients. Pre-β HDL formation was measured by crossed immunoelectrophoresis under conditions of lecithin:cholesterol acyltransferase (LCAT) inhibition. Results: Plasma pre-β-HDL formation, triglycerides, LDL cholesterol, PLTP activity, and CETP decreased after statin treatment (all P b 0.001 vs placebo), whereas HDL cholesterol increased (P b 0.005). Plasma apo A-I, LpAI and LpAI:AII remained unchanged compared to placebo. In all patients combined, the changes in pre-β-HDL formation were independently related to the decrease in plasma triglycerides (β = 0.31; P = 0.006) and PLTP activity (β = 0.23; P = 0.038), without a contribution of CETP. In the atorvastatin treated patients, the decrease in pre-β-HDL formation tended to be related to the decrease in PLTP activity (β = 0.30, P = 0.061) after controlling for decreases in triglycerides (β = 0.22, P = 0.22). Conclusion: High dose atorvastatin decreases the capacity of plasma to generate pre-β-HDL particles in type 2 diabetic patients, probably via lowering of plasma PLTP activity and triglycerides. This could contribute to an improvement in the atherogenic lipoprotein profile. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The increased cardiovascular risk in type 2 diabetes mellitus can in part be explained by a low high density lipoprotein (HDL) cholesterol concentration [1]. HDL particles are heterogeneous in size, structure, lipid composition and apolipoprotein (apo) content [2]. HDL subfraction levels are altered in diabetes, particularly when complicated by cardiovascular disease [3]. Moreover, mean HDL particle size is decreased in insulin resistant and diabetic subjects [4]. A small proportion of HDL consists of small-sized, lipid-poor HDL particles, designated pre-β-HDL, which are thought to be involved in the removal of cell-derived cholesterol to the extracellular compartment [5,6]. In vitro studies have shown that the generation of these particles is, among other factors, stimulated by lipid transfer reactions catalyzed by cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) [7]. PLTP may initiate fusion of mature HDL in vitro, thereby generating smaller and larger particles than the parent HDL [8]. These smaller particles have pre-β-HDL mobility on agarose gels, ⁎ Corresponding author. Tel.: +31 20 56651258; fax: +31 20 566934. E-mail address: [email protected] (G.M. Dallinga-Thie). 1388-1981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2009.03.008
and are more rapidly formed in transgenic mice expressing human PLTP [9,10]. Despite low levels of total plasma apo A-I, plasma pre-βHDL is not decreased, and may even be increased, in patients with diabetes mellitus [11,12], which is likely to be explained, at least in part, by an increase in plasma PLTP activity [13]. Importantly, a positive relation of pre-β-HDL levels with intima media thickness (IMT) was observed recently in type 2 diabetic patients independent of clinical factors and levels of plasma apo Bcontaining lipoproteins [12]. Moreover, atherosclerosis is also associated with increased plasma pre-β-HDL concentrations in other high risk populations [12,14], except for one study in which pre-β-HDL was found to be negatively related to IMT in subjects with familial low HDL cholesterol [15]. These results underscore the relevance to determine whether plasma pre-β-HDL metabolism is affected by lipid lowering treatment. Treatment with atorvastatin and rosuvastatin decrease pre-β1 HDL, but not pre-β2 HDL, in hyperlipidemic subjects [16], whereas, in another report, preβ-1 HDL was found to remain unchanged after treatment with rosuvastatin in metabolic syndrome subjects [17]. In type 1 diabetic patients we have documented a small drop in pre-β-HDL formation in response to simvastatin [18]. On the other hand, there are no in vivo data showing a relationship between
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plasma PLTP activity and pre β-HDL in an intervention study in humans, although in vitro studies implicate PLTP in pre-β-HDL formation. Since atorvastatin lowers plasma PLTP activity in type 2 diabetic patients [19], we hypothesized that a decrease in PLTP activity may contribute to a decrease in pre-β-HDL formation during atorvastatin treatment in patients with type 2 diabetes. The present study was initiated to test this hypothesis. 2. Methods 2.1. Subjects The DALI study is a randomized, double-blind, placebo-controlled trial on the effects of atorvastatin 10 mg and 80 mg daily in patients with type 2 diabetes. The study protocol has previously been described in detail [20]. In brief, men and women aged 45–75 years, with a known duration of type 2 diabetes of at least 1 year and hypertriglyceridemia (fasting triglycerides between 1.5 and 6.0 mmol/l and total cholesterol between 4.0 and 8.0 mmol/l) were included. Type 2 diabetes was defined in accordance with the American Diabetes Association guidelines of 2000 [21]. Patients with an HbA1c above 10% or a history of cardiovascular disease were excluded. In the current study, a subset of patients treated with placebo (n = 41) and 80 mg atorvastatin during 30 weeks (n = 40) was included, based on the availability of plasma samples. The selected individuals were not different from the rest of the study population with respect to any of the clinical characteristics and baseline lipid levels. Of the study subjects, 45% used oral glucose lowering drugs, 28% used insulin and 26% were treated with combination therapy. Treatment for diabetes was equally distributed among the 2 randomized groups. Lipid-lowering therapy was withdrawn 6 weeks before start of the run-in phase. The medical ethical committees of the participating institutions approved the study protocol and all participants gave their written informed consent. 2.2. Laboratory measurements At baseline and after 30 weeks of treatment, blood was collected in EDTA tubes after an overnight fast of at least 12 h and centrifuged immediately (3000 rpm 15 min, 4 °C). Plasma was stored at −80 °C. Total cholesterol, triglycerides, HDL cholesterol and apo A-I were measured as described [20]. Plasma LpAI and LpAI:AII were analyzed using a commercially available immunoelectrophoresis assay (Sebia, France). Plasma pre-β-HDL formation, i.e. the ability of plasma to generate pre-β-HDL, was measured by crossed immunoelectrophoresis as described [11]. In brief, plasma samples were thawed while kept on ice and 0.9 μmol/l Pefabloc SC (Boehringer-Roche, Penzberg, Germany) and 1.8 μg/l Trasylol (Bayer, Mijdrecht, The Netherlands) were added to inhibit proteolysis (both final concentrations). Samples were incubated during 24 h at 37 °C under conditions of LCAT inhibition. To this end, iodoacetate (final concentration 1.0 mmol/l) was added directly after thawing the samples. The crossed immunoelectrophoresis consisted of agarose electrophoresis in the first dimension for separation of lipoproteins with pre β- and α-mobility. Antigen migration from the first agarose gel into the second agarose gel, containing goat anti-human apo A-I antiserum was used to quantitatively precipitate apo A-I. The antiserum was monospecific for human apo A-I using an immunodiffusion assay. Lipoprotein electrophoresis was carried out in 1% (weight/vol) agarose gels in Tris (80 mmol/l)–tricine (24 mmol/l) buffer, 5% (vol/vol) polyethylene glycol 300 (pH 8.6) and run in an LKB 2117 system (4°C for 3 h, 210 V). Plasma was applied at 3 μl/well. The track of the first agarose gel was excised and annealed with melted agarose to a gel containing 0.66% (v/v) goat anti-human apo A-I anti-serum (Midland Bioproducts Corporation, Boone Iowa) and 0.01% Tween 20 (w/v), that was cast on GelBond film (Amersham, Uppsala, Sweden). The plate was run in an
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LKB 2117 system (4 °C for 20 h, 50 V) in Tris–tricine buffer. Unreacted antibody was removed by extensive washing saline. The gel was stained with Coomassie Brilliant Blue R250, dried, and scanned with a HP scanjet 5470c. Areas under the pre-β-HDL and α-HDL peaks were calculated. The pre-β HDL area was expressed as the percentage of the sum of apo A-I in the pre-β-HDL and the α-HDL areas. Pre-β HDL formation was calculated using the total plasma apo A-I concentration and was expressed in apo AI (g/l). The interassay coefficient of variation was 9%. Plasma PLTP activity was assayed with a phospholipid vesiclesHDL system, using [14C]-labeled dipalmitoyl phosphatidylcholine [19]. This method is specific for PLTP activity and the phospholipid transfer promoting property of CETP does not interfere with the assay. PLTP activity is measured under optimal conditions with excess exogenous substrate. This assay indicates the plasma level of active PLTP and is related to the activity in reference pool plasma. PLTP activity is expressed in arbitrary units (AU; 100 AU corresponds to 13.6 μmol phosphatidylcholine transferred, ml− 1 h− 1). Plasma CETP was measured using double-antibody sandwich enzyme-linked immunosorbent assays as described [22]. Plasma CETP mass has been demonstrated to be correlated CETP activity measured with an excess exogenous substrate assay (r N 0.7) in two independent studies including patients and control subjects [23,24]. 2.3. Statistical analysis Statistical analysis was performed using SPSS version 16. Data are expressed in mean ± SD. Within group changes in variables were determined by paired t-tests. Between groups differences in changes in variables were evaluated by unpaired t-tests. Univariate relationships were analyzed by Pearson's correlation analysis. Multiple linear regression analysis was used to disclose independent relationships between variables. Standardized regression coefficients (β) (equivalent to partial correlation coefficients) were used to estimate the adjusted contribution of variables. Two-sided P-values b0.05 were considered significant. 3. Results A total of 81 type 2 diabetic patients (43 women and 38 men) were included in the present study of whom 40 had been randomized to atorvastatin treatment (80 mg daily) and 41 to placebo. Age, sex distribution and BMI at baseline were not different between patients allocated to atorvastatin compared to those allocated to placebo (60 ± 8 vs 58 ± 7 years, respectively, P = 0.20; 19 women and 21 men vs 24 women and 17 men, P = 0.44; 31.0 ± 4.7 vs 32.4 ± 6.5 kg/m2, respectively, P = 0.27). Baseline HbA1c (atorvastatin: 8.6 ± 1.2% and placebo: 8.4 ± 1.2%, P = 0.46) and fasting glucose levels (atorvastatin: 11.2 ± 3.0 mmol/l and placebo: 10.7 ± 3.6 mmol/l, P = 0.52) were also similar in both groups of patients. Atorvastatin treatment did not result in an improvement of fasting glucose or HbA1c levels (data not shown). Table 1 shows plasma apo A-I, LpAI, LpAI:AII and pre-β-HDL formation at baseline and after atorvastatin administration compared to placebo. At baseline, no significant difference in any of the variables shown in Table 1 was observed between the subjects allocated to atorvastatin vs placebo administration. Treatment with atorvastatin resulted in a decrease in plasma total cholesterol, LDL cholesterol and triglycerides compared to placebo. HDL cholesterol increased in response to atorvastatin, but the change in plasma apo A-I was not different compared to placebo. Pre-β HDL formation decreased in response to atorvastatin. Plasma CETP and PLTP activities were also lowered by atorvastatin treatment. No significant changes in the LpAI and LpAI:AII concentration were observed. Table 2 shows the univariate relationships at baseline of HDL cholesterol, apo A-I, HDL subfractions and pre-β-HDL formation with plasma triglycerides, CETP and PLTP activities in the combined groups
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Table 1 Effects of 30 week 80 mg daily atorvastatin treatment compared to placebo with respect to plasma (apo) lipoproteins, high density (HDL) cholesterol, LpAI, LpAI:AII and pre-βHDL formation, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activity. Plasma variable
Total chol (mmol/l) LDL cholesterol (mmol/l) Triglycerides (mmol/l) Apo A-I (g/l) HDL cholesterol (mmol/l) LpAI (g/l) LpAI:AII (g/l) Pre-β HDL formation (apo A-I g/l) CETP (mg/l) PLTP activity (AU)
Atorvastatin 80 mg (n = 40)
Placebo (n = 41)
Baseline
Follow-up
Baseline
6.03 ± 1.04 3.67 ± 0.93
3.59 ± 1.09¶ 2.73 ± 1.3§
6.01 ± 0.80 5.88 ± 0.81 3.67 ± 0.82 3.67 ± 0.83
b0.001 b 0.001
2.85 ± 1.02
1.56 ± 0.60¶
2.83 ± 1.00 2.61 ± 1.17
b 0.001
1.44 ± 0.21 1.07 ± 0.27
1.37 ± 0.22¶ 1.11 ± 0.29†
1.42 ± 0.22 1.36 ± 0.18¶ 1.06 ± 0.23 1.02 ± 0.22†
0.61 0.005
0.49 ± 0.15 0.93 ± 0.13 0.30 ± 0.09
0.43 ± 0.16‡ 0.96 ± 0.15 0.22 ± 0.06¶
0.46 ± 0.10 .45 ± 0.11 0.97 ± 0.16 0.92 ± 0.18† 0.27 ± 0.07 0.26 ± 0.08
0.17 0.40 0.001
2.53 ± 0.82 111 ± 23
1.74 ± 0.55¶ 96 ± 16¶
2.66 ± 0.74 2.60 ± 0.65 b 0.001 105 ± 23 105 ± 19 b 0.001
P-value Follow-up
Data are presented in mean ± SD. † P ≤ 0.05; ‡P ≤ 0.02;§P ≤ 0.01;¶P ≤ 0.0001 compared to baseline; ∫P-value for difference in change after atorvastatin compared to placebo.
at baseline (n = 81). Plasma triglycerides were inversely correlated with HDL cholesterol, whereas there was a positive correlation between plasma triglycerides and CETP. HDL cholesterol, plasma apo A-I and LpAI:AII were negatively correlated with plasma CETP. Pre-β HDL formation was positively associated with PLTP activity (P b 0.05). Its relation with CETP did not reach statistical significance (P = 0.09). Table 3 demonstrates the relationships of the individual changes in HDL cholesterol, LpAI, LpAI:AII and pre-β-HDL formation with the changes in plasma triglycerides and lipid transfer proteins in all study subjects combined. Only the changes in HDL cholesterol were inversely related to the changes in plasma triglycerides. In contrast, the changes in pre β-HDL formation and in LpAI were positively correlated with the changes in triglycerides. The changes in LpAI and pre-β-HDL formation were also positively related to the changes in plasma PLTP activity, as well as to the changes in plasma CETP. The changes in LpAI:AII did not correlate with the changes in triglycerides, CETP and PLTP activities. The decrease in pre-β-HDL formation tended to be related to the decrease in PLTP activity (r = 0.31, P = 0.06) in the atorvastatin treated patients. The correlations with the changes in triglycerides and CETP were r = 0.27 (P = 0.09) and r = 0.02 (P N 0.80), respectively. In the placebo group no significant relationships between changes in these variables were present (P N 0.20 for all). Multiple linear regression analysis was performed to determine the independent effects of the changes in plasma triglycerides, PLTP activity and CETP on the individual changes in pre-β-HDL formation
Table 2 Univariate correlation coefficients at baseline in all subjects combined (n = 81) for determinants of plasma triglycerides, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) activity.
Triglycerides (mmol/l) Apo A-I (g/l) HDL cholesterol (mmol/l) LpAI (g/l) LpAI:AII (g/l) Pre-β-HDL formation (apo A-I, g/l) CETP (mg/l) PLTP activity (AU)
Table 3 Relationships of changes in plasma HDL cholesterol, LpAI, LpAI:AII and pre-β-HDL formation with changes in plasma triglycerides, PLTP activity and CETP mass in 81 type 2 diabetic patients after 30 week administration of atorvastatin 80 mg daily (n = 40) and placebo (n = 41).
Triglycerides⁎ (Pearson's r)
CETP mass (Pearson's r)
PLTP activity (Pearson's r)
– − 0.21 − 0.45§ − 0.08 − 0.22‡ 0.09 0.26‡ 0.08
0.26‡ − 0.23‡ − 0.37‡ − 0.12 − 0.22‡ 0.19 – 0.21
0.08 − 0.01 − 0.13 0.02 0.02 0.25‡ 0.21 –
Pearson's correlation coefficients (r) are shown. Data in mean ± SD. †Log transformed value for triglycerides are used in the regression analysis. ‡P ≤ 0.05; §P b 0.01.
ΔTriglycerides ΔPLTP activity ΔCETP
ΔHDL cholesterol
ΔLpAI
ΔLpAI:AII
Δpre-β-HDL formation
− 0.48§ − 0.12 − 0.18
0.24† 0.27† 0.23†
− 0.18 − 0.15 − 0.08
0.39§ 0.34‡ 0.30‡
Δ: Change; †P b 0.05; ‡P b 0.01; §P b 0.001.
and HDL sub-fractions. This analysis demonstrated that in the combined group the changes in LpAI were only independently determined by the changes in plasma PLTP activity (β = 0.27, P = 0.016). In these models only those HDL-related variables which showed significant univariate relationships were included. This analysis demonstrated that the changes in LpAI were only independently determined by the changes in plasma PLTP activity (P = 0.016), whereas the change in pre-β-HDL formation was determined by the decrease in triglycerides (β = 0.31; P = 0.006) and in PLTP activity (β = 0.23; P = 0.038), but not by changes in CETP (β = 0.09; P = 0.48). There were no independent effects of age, sex, baseline HbA1c, BMI and diabetes treatment (oral glucose lowering drugs, insulin, or combination therapy) on the decrease in pre-β-HDL formation (P N 0.30). In the atorvastatin treated patients, the decrease in preβ-HDL formation tended to be related to the decrease in plasma PLTP activity (β = 0.30; P = 0.061), without effects of changes in triglycerides (β = 22; P = 0.16) and CETP (β = 0.07; P = 0.68). 4. Discussion The present study shows that pre-β-HDL formation is substantially decreased by high dose atorvastatin treatment in type 2 diabetic patients. Univariate regression analysis demonstrated that the changes in pre-β-HDL formation were related to the changes in plasma PLTP activity and triglycerides. The independent contributions of these variables on pre-β-HDL formation were underscored by multiple linear regression analysis. In atorvastatin treated patients only, the relationship of the drop in pre-β-HDL formation with the decline in PLTP activity was close to statistical significance. Therefore, the present study supports the contention that PLTP activity is important for pre-β-HDL formation in humans and that the effect of atorvastatin on plasma PLTP activity may contribute to the regulation of pre-β-HDL in vivo. To our knowledge, this is the first intervention study in humans revealing that changes in plasma pre β-HDL formation are determined by changes in PLTP activity. We have previously documented plasma pre-βHDL concentrations as well as pre-β-HDL formation, assayed exactly as described above, in a group of 81 diabetic patients who did not use lipid lowering drugs [11]. In this report, pre-β-HDL formation was on average five-fold higher compared to pre-β-HDL concentration as measured without incubation of plasma under conditions of LCAT inhibition. That study revealed positive correlations of pre-β-HDL concentration with preβ-HDL formation (r=0.31, P=0.008) as well as with plasma PLTP activity (r = 0.32, P=0.003). However, plasma pre-β-HDL concentration was not correlated with PLTP activity (r= 0.07, P=0.55). These results made us decide to test the relation between atorvastatin-induced changes in plasma PLTP activity and pre-β-HDL formation rather than pre-β-HDL concentration in the present study. Of importance, pre-β-HDL metabolism may also be affected by CETP and hepatic lipase [6,25]. In the current report, we relied on measurement of plasma CETP mass which is closely correlated with its plasma activity level using an exogenous substrate assay, which is not confounded by the endogenous cholesteryl ester acceptor and donor lipoproteins present in plasma [24]. We showed here that the relationship between plasma CETP and pre-β-HDL formation at baseline did not
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reach statistical significance. Moreover, although changes in plasma preβ-HDL formation were correlated with changes in CETP in univariate analysis, CETP did not independently contribute to pre-β-HDL formation in multiple regression analysis, which would favour a preferential contribution of PLTP as compared to CETP in pre-β-HDL generation. A similar observation has been found earlier by comparing pre-β-HDL generation in plasma from PLTP transgenic mice vs CETP transgenic mice [26]. Changes in pre-β-HDL formation were also independently related to the changes in plasma triglycerides in the present study, which can probably be explained by enhanced pre-β-HDL generation from α-HDL by PLTP when these particles are enriched with triglycerides [27]. Importantly, pre-β-HDL particles are initial substrates for LCAT, which leads to rapid conversion into more mature HDL by the process of cholesterol esterification. We measured pre-β-HDL under conditions of LCAT inhibition and analyzed the fraction with crossed immunoelectrophoresis assay using an antibody against human apo A-I. Thus, the current pre-β-HDL measurements should be regarded to represent an ex vivo estimate of the capacity to generate such particles in plasma. In the interpretation of the present results, it is relevant to note that our assay detects pre-β1-HDL and pre-β2-HDL together. In studies from other laboratories in which the effect of statin treatment on pre-β-HDL was determined, the plasma concentration of these particles were further divided into pre-β1-HDL and pre-β2-HDL [28] or measured by ELISA [17]. Thus, several methodological differences could in part explain why preβ-HDL was not found to be consistently decreased after statin administration in all previous reports. Pre-β HDL particles are initial acceptors of cell-derived cholesterol, and hence are considered to play a role in the reverse cholesterol transport pathway, whereby excess peripheral cell cholesterol is transported back to the liver for metabolism and excretion in the bile. Despite this stimulatory effect of these lipid-poor HDL particles on cellular cholesterol efflux, positive relationships of (subclinical) atherosclerosis with high plasma levels of pre-β-HDL have been documented [12]. Thus, it appears to be conceivable that elevated plasma pre-β-HDL levels indicate an impaired maturation of HDL and that decreased levels would reflect more rapid metabolism of such particles into mature α-HDL, contributing to an efficient RCT pathway [29]. Our observation of a decreased formation of pre β-HDL due to lower plasma PLTP activity would suggest that a fast turnover and maturation of pre β-HDL, as well as less pre β-HDL formation could occur, leading to the presence of less pre β-HDL after statin treatment. It is, therefore, possible that statin-induced decreases in pre-β-HDL formation would imply cardiovascular benefit. Atorvastatin induced a modest increase in total HDL cholesterol, coinciding with a decrease in plasma triglycerides as expected [20,30,31], but did not significantly affect LpAI and LpAI:AII. In other studies, LpAI was found to increase in response to rosuvastatin in metabolic syndrome subjects [17]. It is known that the effects of atorvastatin 80 mg/day on HDL parameters are modest compared with effects of other statins [16]. In the current report, we compared the effects of 80 mg atorvastatin daily with placebo in a subset of DALI participants and did not evaluate effects of a lower atorvastatin dose. However, no differences between the HDL cholesterol, plasma apo A-I and triglyceride responses between 10 mg and 80 mg atorvastatin administration have been demonstrated among diabetic patients participating in previous analyses from this trial [19,20], and plasma PLTP activity was shown to decrease in response to the lower atorvastatin dose as well. Thus, it is unlikely that selective inclusion of participants randomized to high dose atorvastatin essentially affected the present analysis. In conclusion, high dose atorvastatin decreases plasma pre-βHDL formation in type 2 diabetic patients, probably via lowering of plasma PLTP activity and triglycerides. A decrease in pre-β-HDL could modify cardiovascular risk reduction beyond effects on VLDL and LDL cholesterol.
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Acknowledgements The authors wish to thank contributors to the DALI study as patients, technicians, or data managers. The original DALI study was an investigator-driven study partly supported by an unrestricted grant of Parke-Davis, The Netherlands. The following is a complete list of the original DALI study group (in alphabetical order): Erasmus Medical Center Rotterdam, Department of Internal Medicine (I. Berk-Planken, N. Hoogerbrugge, H. Jansen); Erasmus University Rotterdam, Departments of Biochemistry and Clinical Chemistry (H. Jansen); Gaubius Laboratory TNO-KvL, Leiden (H.M.G. Princen); Leiden University Medical Center (M.V. Huisman, M.A. van de Ree); University Medical Center Utrecht, Julius Center for General Practice and Patient Oriented Research (R.P. Stolk, F.V. van Venrooij); University Medical Center Utrecht, Division of Internal Medicine (J.D. Banga, G.M. Dallinga-Thie, F.V. van Venrooij). The technical assistance of F. Perton (Laboratory Center, University Medical Center Groningen, The Netherlands), for pre-β-HDL measurements is greatly appreciated.
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