Atherosclerosis 219 (2011) 532–537
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Resistance training changes LDL metabolism in normolipidemic subjects: A study with a nanoemulsion mimetic of LDL Jeferson L. da Silva a , Carmen G.C.M. Vinagre a , Aleksandra T. Morikawa a , Maria Janieire N.N. Alves a , Carlos H. Mesquita a , Raul C. Maranhão a,b,∗ a b
Heart Institute (InCor) of the Medical School Hospital, University of São Paulo, São Paulo, Brazil Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 28 February 2011 Received in revised form 11 August 2011 Accepted 12 August 2011 Available online 22 August 2011 Keywords: Resistance exercise training Resistance strength Low density lipoprotein metabolism Cholesterol Nanoemulsion Nanoparticles Oxidized LDL
a b s t r a c t Objective: To evaluate the effects of resistance training (RT) on the metabolism of an LDL-like nanoemulsion and on lipid transfer to HDL, an important step of HDL metabolism. Methods: LDL-like nanoemulsion plasma kinetics was studied in 15 healthy men under regular RT for 1–4 years (age = 25 ± 5 years, VO2 peak = 50 ± 6 mL/kg/min) and in 15 healthy sedentary men (28 ± 7 years, VO2 peak = 35 ± 9 mL/kg/min). LDL-like nanoemulsion labeled with 14 C-cholesteryl-ester and 3 H-freecholesterol was injected intravenously, plasma samples were collected over 24-h to determine decay curves and fractional clearance rates (FCR). Lipid transfer to HDL was determined in vitro by incubating of plasma samples with nanoemulsions (lipid donors) labeled with radioactive free-cholesterol, cholesterylester, triacylglycerols and phospholipids. HDL size, paraoxonase-1 activity and oxidized LDL levels were also determined. Results: The two groups showed similar LDL and HDL-cholesterol and triacylglycerols, but oxidized LDL was lower in RT (30 ± 9 vs. 61 ± 19 U/L, p = 0.0005). In RT, the nanoemulsion 14 C-cholesteryl-ester was removed twice as fast than in sedentary individuals (FCR: 0.068 ± 0.023 vs. 0.037 ± 0.028, p = 0.002), as well as 3 H-free-cholesterol (0.041 ± 0.025 vs. 0.022 ± 0.023, p = 0.04). While both nanoemulsion labels were removed at the same rate in sedentary individuals, RT 3 H-free-cholesterol was removed slower than 14 C-cholesteryl-ester (p = 0.005). HDL size, paraoxonase 1 and the transfer rates to HDL of the four lipids were the same in both groups. Conclusions: RT accelerated the clearance of LDL-like nanoemulsion, which probably accounts for the oxidized LDL levels reduction in RT. RT also changed the balance of free and esterified cholesterol FCR’s. However, RT had no effect on HDL metabolism related parameters. © 2011 Published by Elsevier Ireland Ltd. Open access under the Elsevier OA license.
1. Introduction Exercise training is considered to be one of the main instruments to promote a healthy lifestyle. However, the effects of the different modalities of training on the metabolic pathways, specially the intravascular lipid metabolism are largely unexplored and deserve further investigation. Aerobic training increases the cardiovascular reserve whereas resistance training (RT) increases the muscle mass and improve the muscle strength [1,2]. The metabolic effects of both training modalities are dependent on frequency and intensity of the training [1,2]. In this regard, most of the studies which evaluated plasma lipids in healthy normolipidemic subjects
∗ Corresponding author at: Heart Institute (InCor) of the Medical School Hospital, University of São Paulo, São Paulo, Brazil. Tel.: +55 112 661 5951; fax: +55 1126615574. E-mail address:
[email protected] (R.C. Maranhão).
showed that aerobic training elicited lower low density lipoprotein (LDL) cholesterol and triacylglycerols and increased high density lipoprotein (HDL) cholesterol [3–6]. The effects of RT are similar however, due to the fact that the number of studies was small, there is some controversy on the effects on plasma lipids [3,5,7]. The mechanisms whereby those beneficial effects occur are not thoroughly understood. Recently, we showed that in amateur cyclists aged 18–49 years with a moderate to high intensity training load, the removal of a nanoemulsion that mimic LDL from the plasma was several times greater than that observed in sedentary individuals [8]. It is worthwhile to mention that the serum LDL-cholesterol was the same in those aerobic training subjects and sedentary subjects, suggesting that LDL-cholesterol turnover was greater in the trained subjects than in the sedentary ones [8]. When normolipidemic sedentary individuals were submitted to aerobic training for four
0021-9150 © 2011 Published by Elsevier Ireland Ltd. Open access under the Elsevier OA license. doi:10.1016/j.atherosclerosis.2011.08.014
J.L. da Silva et al. / Atherosclerosis 219 (2011) 532–537
months, LDL-cholesterol decreased and the removal of the LDL-like nanoemulsion tended to increase [4]. In this study, we investigated whether in subjects practicing RT for at least one year, accelerated LDL clearance could occur. Plasma kinetics of the LDL mimetic nanoemulsion labeled with 14 C-cholesteryl ester and 3 H-free cholesterol was determined in RT subjects for comparison with a group of sedentary individuals. Oxidized LDL also measured in the plasma and its relationship with the nanoemulsion clearance was evaluated. In addition, RT effects on HDL metabolism were also investigated in the study subjects. Transfer of lipids to HDL, an important step in HDL metabolism and function that depends on transfer proteins such as cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP) was measured in an in vitro assay using the nanoemulsion as lipid donor. The paraoxonase 1 (PON1) activity and HDL size were also evaluated.
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sumption (VO2 peak) was considered the maximum attained VO2 at the end of the exercise period. 2.3. Determination of plasma lipids Plasma lipids were determined in blood samples collected after a 12 h fast just before beginning the kinetic studies. Total cholesterol (Boehringer-Mannheim, Penzberg, Germany) and triacylglycerols (Abbott Park, IL, USA) were determined by commercial enzymatic methods. HDL-cholesterol was determined by the same method used for total cholesterol after chemical precipitation of apo B-containing lipoproteins. LDL-c was calculated by the Friedewald formula [11]. Oxidized LDL was measured in EDTA-plasma samples by enzyme immunoassay kit, using a monoclonal antibody (Mercodia, Uppsala, Sweden). 2.4. Nanoemulsion preparation
2. Methods 2.1. Study subjects Thirty healthy normolipidemic male volunteers participated in the study. All were non-obese, non-smokers, non-drinkers, non-diabetic, had no arterial hypertension or clinical manifestations of cardiovascular disease (CAD) and none were taking any medication affecting lipid metabolism or any dietary supplements. This information was obtained from a self-administered health history questionnaire and through medical consultation with a cardiologist. All volunteers were submitted to a clinical and cardiopulmonary exercise evaluation before the study. After preliminary screening, they were separated into two groups: RT group was comprised of 15 healthy men aged 25 ± 5 years (18–35 years) in a regular training program for a minimum period of 1 year and maximum 4 years (average: 2.5 years). The training program consisted of 3–4 sets of 8–12 repetitions maximum with 3–4 exercises for each muscle group, with an average training load of 1 h sessions, 4–5 times/week. The sedentary group comprised 15 healthy sedentary aged 25 ± 5 years (19–40 years), who did not practice physical exercise for at least a year. Body fat percent was assessed by skin fold measurement [9] and performed by the same trained technician. Abdominal circumference was measured over the navel, using a plastic tape measure. The nonuse of anabolic androgenic steroids was confirmed by urine test (chromatography–mass spectrometry). RT remained without exercise for 72 h before the evaluations. All the participants were requested to maintain their normal dietary habits throughout the study. All signed a written informed consent for the study protocol, which was approved by the local ethics committee. 2.2. Peak oxygen consumption (VO2 peak) Maximal exercise capacity was carried out on a treadmill (Quinton Instruments, Washington, USA) and performed using a ramp protocol with progressive exercise [10]. The subjects were instructed not to eat 2 h before the test and not to exercise for at least 24 h before testing. Oxygen consumption (VO2 ) and carbon dioxide output were analyzed by means of breath-by-breath and expressed as 30-s averages using an indirect calorimetry system (Vmax, Sensor Medics, Yorba Linda, USA). Heart rate was continuously recorded at rest and during the graded exercise testing using a 12-lead digital electrocardiogram and software ERGO PC 13 (MICROMED, Brasília, Brazil). The peak oxygen con-
The nanoemulsion was prepared from a lipid mixture composed of 40 mg cholesteryl oleate, 20 mg egg phosphaditylcholine, 1.0 mg triolein and 0.5 mg of cholesterol. Cholesteryl [1-14 C] oleate and cholesterol [1-3 H] were added to the mixture. Emulsification of lipids by prolonged ultrasonic irradiation in aqueous media and the procedure of two-step ultracentrifugation to obtain the LDLlike nanoemulsion were carried out as described by Maranhão et al. [12]. The nanoemulsion was dialyzed against a saline solution and sterilized by passage through a 0.22 m filter. The entire nanoemulsion preparation procedure was performed in a laminar flux. All glassware used in this study was made pyrogen free by exposure to dried steam at 180 ◦ C for 2 h and sterilized by wet steam at 120 ◦ C for 30 min. All plastic materials were sterilized by ultraviolet light exposition. 2.5. Radiological protection The radioactive dose used in the intravenously injected labeled lipid experiments with the subjects was in strict accordance with the International Commission on Radiological Protection (ICRP) [13]. The equivalent dose induced by the injected radioactivity dose was 0.03 mSv, well below the permitted 20.0 mSv maximum dose. 2.6. LDL-like nanoemulsion plasma kinetics The test began at approximately 9-AM with all participants fasting for a 12 h period, but were allowed to eat low-fat meals which included grilled chicken and fish, vegetables, fruits and fruit juices the evening before the test day and after the first blood collection and at 1:00-PM, as low-fat meals seem not to interfere with plasma removal of the LDL-like nanoemulsion [12]. 200 L of LDL-like nanoemulsion labeled with 14 C-cholesteryl ester (37 kBq) and 3 Hfree cholesterol (74 kBq) and a total 6 mg lipid, was intravenously injected in a bolus. Plasma samples were collected during 24 h, in intervals of 5 min and 1, 2, 4, 6, 8, and 24 h after injection. Radioactivity in aliquots of 1.0 ml of plasma was quantified in a scintillation solution (Packard BioScience, Meriden, USA) using a liquid scintillation analyzer (Packard beta spectrometer, model TRI-CARB 2100TR). The fractional clearance rate (FCR) of the LDL-like nanoemulsion was calculated by compartmental analysis, using a computational program [14] (Fig. 1). 2.7. Estimation of fractional clearance rate (FCR) of the radioisotopes For each subject the kinetic activity-time curve was fitted to the mathematical model [15] defined by the sum of two exponential
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J.L. da Silva et al. / Atherosclerosis 219 (2011) 532–537 Table 1 Physical characteristics and biochemical parameters of 15 sedentary and 15 resistance training subjects. Parameter
Fig. 1. Compartmental model used for analyzing the 14 C-cholesteryl ester and 3 Hfree cholesterol label presents in LDL-like nanoemulsion. The model consists of four compartments: two for 14 C-CE and two for 3 H-FC labels. All compartments are in the intravascular space (1CE , 2CE , 1FC and 2FC ). LDL-like nanoemulsion labeled with 14 C-CE and 3 H-FC were injected intravenously in a bolus (arrow with asterisk) into compartment 1CE and 1FC , respectively. A fraction k1,0CE and k1,0FC of the labeled lipids removed to the extravascular space. Competitively, fraction k1,2CE and k1,2FC of the injected lipids are converted into compartments 2CE and 2FC due to the incorporation of apolipoproteins available in the plasma. Subsequently, the materials of those compartments are transferred to the extravascular space following the k2,0CE and k2,0FC routes. The samplings, represented by triangles, correspond to the indiscriminate combination of compartments 1 and 2. 14 C-CE, 14 C-cholesteryl ester; 3 H-FC, 3 H-free cholesterol.
functions, i.e., y = A1 e−˛1t + A2 e−˛2t . For details of this bi-exponential model see online Supplementary material. Therefore, compartment 1 is governed by a mono-exponential equation while compartment 2 is governed by a bi-exponential equation. This model assumes that all input or exit of the radiolabeled lipid occurs from the intravascular pool. FCR of the radiolabeled lipid was estimated as FCR =
∞ 0
A1 + A2 (A1 e−˛1t + A2 e−˛2t )dt
which is essentially the inverse of the area under the activity-time curve. The compartment model used is illustrated in Fig. 1.
2.8. Lipid transfer from LDL-like nanoemulsion to HDL in vitro The transfer rates of cholesteryl ester, phospholipid, free cholesterol, and triacylglycerol from LDL-like nanoemulsion to HDL were measured according to Lo Prete et al. [16]. Two incubations of 200 L of EDTA plasma samples with the donor artificial nanoemulsion were performed. In one, plasma was incubated with the nanoemulsion labeled with 3 H-cholesteryl oleate and 14 C-phosphatidylcholine and in the other with the nanoemulsion labeled with 14 C-free cholesterol and 3 H-triolein (Amersham, Buckinghamshire, UK). After a 1-h incubation at 37 ◦ C, 250 L dextran sulfate/MgCl2 0, 2%/MgCl2 3 M (v/v) were added as a precipitating reagent. The solution was then mixed for 30 s and centrifuged for 10 min at 3000 g. Finally, 250 L of the supernatant was added to counting vials containing 5 mL scintillation solution (Packard BioScience, Groningen, Netherlands). Radioactivity was measured, as described above. The results of the radioactive transfer from the lipid nanoemulsions to the HDL fractions were expressed as a percentage of the total incubated radioactivity, determined in a plasma sample containing the precipitation reagents [16]. As shown in the description of the in vitro assay (18), transfers progressively increase until 1 h incubation time, when equilibrium is reached and transfer values remain constant at the rates measured at the 1 h point.
Age (year) Body mass index (kg/m2 ) Body fat (%) Abdominal circumference (cm) VO2 peak (mL/kg/min) Glucose (mg/dL) Cholesterol (mg/dL) Total LDL HDL Non-HDL Triacylglycerols (mg/dL)
Sedentary
Resistance training
28 25 19 90 35 84
± ± ± ± ± ±
7 4 6 12 9 8
25 25 16 84 50 88
± ± ± ± ± ±
5 2 5 7 6 9
160 96 45 112 87
± ± ± ± ±
36 31 12 36 26
159 98 40 118 96
± ± ± ± ±
33 29 9 30 26
p 0.316 0.719 0.231 0.036
