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Resistance training changes LDL metabolism in normolipidemic subjects: A study with a nanoemulsion mimetic of LDL

Open AccessPublished:September 09, 2011DOI:https://doi.org/10.1016/j.atherosclerosis.2011.08.014

      Abstract

      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, VO2peak = 50 ± 6 mL/kg/min) and in 15 healthy sedentary men (28 ± 7 years, VO2peak = 35 ± 9 mL/kg/min). LDL-like nanoemulsion labeled with 14C-cholesteryl-ester and 3H-free-cholesterol 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, cholesteryl-ester, 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 14C-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 3H-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 3H-free-cholesterol was removed slower than 14C-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.

      Keywords

      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 [
      • Ratamess N.A.
      • Alvar B.A.
      • Evetoch T.K.
      • et al.
      ACSM position stand. Progression models in resistance training for healthy adults.
      ,
      • Chodzko-Zajko W.J.
      • Proctor D.N.
      • Fiatarone Singh M.A.
      • et al.
      ACSM position stand. Exercise and physical activity for older adults.
      ]. The metabolic effects of both training modalities are dependent on frequency and intensity of the training [
      • Ratamess N.A.
      • Alvar B.A.
      • Evetoch T.K.
      • et al.
      ACSM position stand. Progression models in resistance training for healthy adults.
      ,
      • Chodzko-Zajko W.J.
      • Proctor D.N.
      • Fiatarone Singh M.A.
      • et al.
      ACSM position stand. Exercise and physical activity for older adults.
      ]. In this regard, most of the studies which evaluated plasma lipids in healthy normolipidemic subjects showed that aerobic training elicited lower low density lipoprotein (LDL) cholesterol and triacylglycerols and increased high density lipoprotein (HDL) cholesterol [
      • Ghahramanloo E.
      • Midgley A.W.
      • Bentley D.J.
      The effect of concurrent training on blood lipid profile and anthropometrical characteristics of previously untrained men.
      ,
      • Ficker E.S.
      • Maranhão R.C.
      • Chacra A.P.
      • et al.
      Exercise training accelerates the removal from plasma of LDL-like nanoemulsion in moderately hypercholesterolemic subjects.
      ,
      • Fahlman M.M.
      • Boardley D.
      • Lambert C.P.
      • Flynn M.G.
      Effects of endurance training and resistance training on plasma lipoprotein profiles in elderly women.
      ,
      • Popovic M.
      • Puchner S.
      • Endler G.
      • et al.
      The effects of endurance and recreational exercise on subclinical evidence of atherosclerosis in young adults.
      ]. 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 [
      • Ghahramanloo E.
      • Midgley A.W.
      • Bentley D.J.
      The effect of concurrent training on blood lipid profile and anthropometrical characteristics of previously untrained men.
      ,
      • Fahlman M.M.
      • Boardley D.
      • Lambert C.P.
      • Flynn M.G.
      Effects of endurance training and resistance training on plasma lipoprotein profiles in elderly women.
      ,
      • Kraemer W.J.
      • Vingren J.L.
      • Silvestre R.
      • et al.
      Effect of adding exercise to a diet containing glucomannan.
      ]. 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 [
      • Vinagre C.G.
      • Ficker E.S.
      • Finazzo C.
      • et al.
      Enhanced removal from the plasma of LDL-like nanoemulsion cholesteryl ester in trained men compared with sedentary healthy men.
      ]. 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 [
      • Vinagre C.G.
      • Ficker E.S.
      • Finazzo C.
      • et al.
      Enhanced removal from the plasma of LDL-like nanoemulsion cholesteryl ester in trained men compared with sedentary healthy men.
      ]. When normolipidemic sedentary individuals were submitted to aerobic training for four months, LDL-cholesterol decreased and the removal of the LDL-like nanoemulsion tended to increase [
      • Ficker E.S.
      • Maranhão R.C.
      • Chacra A.P.
      • et al.
      Exercise training accelerates the removal from plasma of LDL-like nanoemulsion in moderately hypercholesterolemic subjects.
      ].
      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 14C-cholesteryl ester and 3H-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.

      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 [
      • Pollock M.L.
      • Jackson A.S.
      Research progress in validation of clinica methods of assessing body composition.
      ] 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 (VO2peak)

      Maximal exercise capacity was carried out on a treadmill (Quinton Instruments, Washington, USA) and performed using a ramp protocol with progressive exercise [
      • Balady G.J.
      • Arena R.
      • Sietsema K.
      • et al.
      American heart association exercise, cardiac rehabilitation, and prevention committee of the council on clinical cardiology; council on epidemiology and prevention; council on peripheral vascular disease; interdisciplinary council on quality of care and outcomes research. Clinician's guide to cardiopulmonary exercise testing in adults: a scientific statement from the American heart association.
      ].
      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 consumption (VO2peak) 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 [
      • Friedewald W.T.
      • Levy R.I.
      • Fredrickson D.S.
      Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.
      ]. Oxidized LDL was measured in EDTA-plasma samples by enzyme immunoassay kit, using a monoclonal antibody (Mercodia, Uppsala, Sweden).

      2.4 Nanoemulsion preparation

      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 [1-14C] oleate and cholesterol [1-3H] 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 LDL-like nanoemulsion were carried out as described by Maranhão et al. [
      • Maranhão R.C.
      • Cesar T.B.
      • Pedroso-Mariani S.
      • Hirata M.H.
      • Mesquita C.H.
      Metabolic behavior in rats of a non-protein microemulsion resembling low-density lipoprotein.
      ]. The nanoemulsion was dialyzed against a saline solution and sterilized by passage through a 0.22 μm 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) [
      • Sowby F.S.
      Radiation protection.
      ]. 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 [
      • Maranhão R.C.
      • Cesar T.B.
      • Pedroso-Mariani S.
      • Hirata M.H.
      • Mesquita C.H.
      Metabolic behavior in rats of a non-protein microemulsion resembling low-density lipoprotein.
      ]. 200 μL of LDL-like nanoemulsion labeled with 14C-cholesteryl ester (37 kBq) and 3H-free 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 [
      • Lima M.F.
      • Pujatti P.B.
      • Araújo E.B.
      • Mesquita C.H.
      Compartmental analysis to predict biodistribution in radiopharmaceutical design studies.
      ] (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Compartmental model used for analyzing the 14C-cholesteryl ester and 3H-free cholesterol label presents in LDL-like nanoemulsion. The model consists of four compartments: two for 14C-CE and two for 3H-FC labels. All compartments are in the intravascular space (1CE, 2CE, 1FC and 2FC). LDL-like nanoemulsion labeled with 14C-CE and 3H-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. 14C-CE, 14C-cholesteryl ester; 3H-FC, 3H-free cholesterol.

      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 [
      • Lewis G.F.
      • Lamarche B.
      • Uffelman K.D.
      • et al.
      Clearance of postprandial and lipolytically modified human HDL in rabbits and rats.
      ] defined by the sum of two exponential functions, i.e., y = A1e−α1t + A2e−α2t. 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=A1+A20(A1eα1t+A2eα2t)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. [
      • Lo Prete A.C.
      • Dina C.H.
      • Azevedo C.H.
      • et al.
      In vitro simultaneous transfer of lipids to HDL in coronary artery disease and in statin treatment.
      ]. Two incubations of 200 μL of EDTA plasma samples with the donor artificial nanoemulsion were performed. In one, plasma was incubated with the nanoemulsion labeled with 3H-cholesteryl oleate and 14C-phosphatidylcholine and in the other with the nanoemulsion labeled with 14C-free cholesterol and 3H-triolein (Amersham, Buckinghamshire, UK). After a 1-h incubation at 37 °C, 250 μL 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 μL 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 [
      • Lo Prete A.C.
      • Dina C.H.
      • Azevedo C.H.
      • et al.
      In vitro simultaneous transfer of lipids to HDL in coronary artery disease and in statin treatment.
      ]. 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.

      2.9 Paraoxonase 1 (PON1) activity

      PON1 activity was assayed in 12 h fasting blood serum according to the method described by Mackness et al. [
      • Mackness M.I.
      • Harty D.
      • Bhatnagar D.
      • et al.
      Serum paraoxonase activity in familial hypercholesterolaemia and insulin-dependent diabetes mellitus.
      ] by adding serum to Tris–HCL buffer (100 mmol/L, pH 8.0) containing 2 mmol/L CaCl2 and 1.1 mmol/L paraoxon (0,0-diethyl-0-p-nitrophenylphosphate; Sigma). The rate of generation of p-nitrophenol was determined at 37 °C, and read at 405 nm.

      2.10 HDL diameter

      The HDL size was measured with ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments, Holtsville, USA), as described elsewhere [
      • Lima E.S.
      • Maranhão R.C.
      Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma.
      ].

      2.11 Statistical analysis

      The Kolmogorov–Smirnov test was applied to verify the data distribution. The differences between the data were evaluated by unpaired Student t-test for Gaussian distribution data, and Mann–Whitney test for non-Gaussian distribution data. The correlation significance analysis was done by analysis of variance and the Pearson's test. In all analysis, difference of p  0.05 was considered statistically significant. Data are expressed as means ± standard deviations. Prism, version 3.0 software was used to assist the analysis.

      3. Results

      Table 1 presents that the values of LDL, non-HDL and HDL cholesterol, as well as those of triacylglycerols, as can be seen, there was no difference between the two groups. On the other hand, as expected, VO2peak was greater in RT individuals when compared with sedentary subjects (p < 0.0001). Abdominal circumference was lower in RT subjects than in sedentary individuals (p = 0.036).
      Table 1Physical characteristics and biochemical parameters of 15 sedentary and 15 resistance training subjects.
      ParameterSedentaryResistance trainingp
      Age (year)28 ± 725 ± 50.316
      Body mass index (kg/m2)25 ± 425 ± 20.719
      Body fat (%)19 ± 616 ± 50.231
      Abdominal circumference (cm)90 ± 1284 ± 70.036
      VO2peak (mL/kg/min)35 ± 950 ± 6<0.0001
      Glucose (mg/dL)84 ± 888 ± 90.142
      Cholesterol (mg/dL)
       Total160 ± 36159 ± 330.929
       LDL96 ± 3198 ± 290.811
       HDL45 ± 1240 ± 90.121
       Non-HDL112 ± 36118 ± 300.551
      Triacylglycerols (mg/dL)87 ± 2696 ± 260.238
      Values are means ± SD; HDL, high density lipoprotein; LDL, low density lipoprotein; VO2peak, peak O2 consumption; Non-HDL, cholesterol non HDL.
      As shown in Table 2, RT increased the FCR of both LDL-like nanoemulsion cholesteryl ester (p = 0.002) and free cholesterol (p = 0.041). The increase in FCR of free (p = 0.013) and cholesteryl ester (p < 0.0001) in RT subjects compared to sedentary subjects was probably due to the increase in K2.0. On the other hand, K1.2 of cholesteryl ester was increased (p = 0.003), but not K1.2 of free cholesterol (Table 2). Both nanoemulsion labels were removed at the same rates in sedentary individuals, but in RT 3H-free cholesterol was removed slower than 14C-cholesteryl ester (p = 0.005). Fig. 2 shows the kinetics of the 3H-free cholesterol and 14C-cholesteryl ester nanoemulsion labels in compartments K1.0 and K2.0 in RT and sedentary groups.
      Table 2Fractional clearance rate (FCR) and kinetic parameters 14C-cholesteryl ester and 3H-free cholesterol from LDL-like nanoemulsion of 15 sedentary and 15 resistance training subjects.
      ParameterSedentaryResistance trainingp
      14C-CE-FCR (h−1)0.037 ± 0.0280.068 ± 0.0230.002
      K1.0 14C-CE (h−1)0.636 ± 0.8870.990 ± 0.9190.301
      K2.0 14C-CE (h−1)0.017 ± 0.0130.052 ± 0.022<0.0001
      K1.2 14C-CE (h−1)0.596 ± 0.9591.796 ± 1.4030.003
      3H-CL-FCR (h−1)0.022 ± 0.0230.041 ± 0.0250.041
      K1.0 3H-FC (h−1)1.037 ± 0.9021.151 ± 0.9240.743
      K2.0 3H-FC (h−1)0.008 ± 0.0100.020 ± 0.0120.013
      K1.2 3H-FC (h−1)0.622 ± 0.7290.982 ± 0.8020.228
      Values are means ± SD; 14C-CE-FCR, fractional clearance rate of the nanoemulsion 14C-cholesteryl ester; 3H-FC-FCR, fractional clearance rate of the nanoemulsion 3H-free cholesterol.
      Figure thumbnail gr2
      Fig. 2Kinetics of the 14C-cholesteryl ester (A and C) and 3H-free cholesterol (B and D) nanoemulsion labels in compartments K1.0 and K2.0 in RT (♦) and sedentary (■) groups. Values are means ± SD.
      In Table 3 shows that the transfer of free cholesterol, phospholipids, triacylglycerols and cholesteryl ester from the LDL-like nanoemulsion to HDL were the same for two groups. The concentration of oxidized LDL in the plasma was lower in RT than in the sedentary group (p = 0.0005). The diameter of HDL particles was the same for RT and sedentary individuals, as was the activity of PON1.
      Table 3Lipid transfer from an LDL-like nanoemulsion to HDL, HDL diameter, PON 1 activity, and oxidized LDL of 15 sedentary and 15 resistance training subjects.
      ParametersSedentaryResistance trainingp
      Lipid transfers to HDL (%)
      Lipid transfer measured as percent of total radioactivity of each lipid of the nanoemulsion recovered in the HDL fraction after 1-h incubation with plasma.
      14C-free cholesterol10.2 ±1.79.3 ± 2.60.281
      14C-phospholipid16.7 ± 3.514.8 ± 1.60.094
      3H-cholesteryl ester3.7 ± 0.93.3 ± 0.60.222
      3H-triacylglycerols6.5 ± 1.17.2 ± 1.10.108
      HDL diameter (nm)9.3 ± 0.99.8 ± 1.90.369
      PON 1 (nmol/min/ml)
      n=8.
      110 ± 5662 ± 380.066
      Oxidized LDL (U/L)
      n=8.
      61 ± 1930 ± 90.0005
      Values are means ± SD.
      HDL, high density lipoprotein; PON 1, paraoxonase 1; LDL, low density lipoprotein.
      a Lipid transfer measured as percent of total radioactivity of each lipid of the nanoemulsion recovered in the HDL fraction after 1-h incubation with plasma.
      b n = 8.
      In the correlation analysis performed, the following statistically significant positive correlations were found: K2.0 of free cholesterol × VO2peak (r = 0.523, p = 0.018), K2.0 of cholesteryl ester × VO2peak (r = 0.450, p = 0.028). The negative correlations found were oxidized LDL × VO2peak (r = −0.560, p = 0.016) and abdominal circumference × VO2peak (r = −0.509, p = 0.013). When a Pearson multiple correlation analysis was performed between abdominal circumference, on one hand, and K2.0 of cholesteryl ester, K2.0 of free cholesterol and VO2peak on the other hand, no significant correlation was found (r = −0.067, p = 0.565).

      4. Discussion

      In this study, subjects under a RT program exhibited accelerated removal from the plasma of both free and esterified cholesterol components of the LDL-like nanoemulsion. The cholesteryl ester moiety that makes up the nanoemulsion core is in fact the marker of the LDL-like nanoemulsion particles removal from the plasma [
      • Maranhão R.C.
      • Cesar T.B.
      • Pedroso-Mariani S.
      • Hirata M.H.
      • Mesquita C.H.
      Metabolic behavior in rats of a non-protein microemulsion resembling low-density lipoprotein.
      ,
      • Maranhão R.C.
      • Roland I.A.
      • Toffoletto O.
      • et al.
      Plasma kinetic behavior in hyperlipidemic subjects of a lipidic microemulsion that binds to LDL receptors.
      ]. Despite cholesteryl esters may shift from the nanoemulsion to the plasma lipoproteins by CETP action, labeled cholesteryl esters of the nanoemulsion behave similarly to apo B labeled native LDL: in several clinical studies, we showed that the plasma removal of the nanoemulsion was decreased or increased as expected for LDL, as occurs in patients with hypercholesterolemia [
      • Maranhão R.C.
      • Roland I.A.
      • Toffoletto O.
      • et al.
      Plasma kinetic behavior in hyperlipidemic subjects of a lipidic microemulsion that binds to LDL receptors.
      ], neoplastic diseases [
      • Maranhão R.C.
      • Garicochea B.
      • Silva E.L.
      • et al.
      Plasma kinetics and biodistribution of a lipid emulsion resembling low-density lipoprotein in patients with acute leukemia.
      ,
      • Ades A.
      • Carvalho J.P.
      • Graziani S.R.
      • et al.
      Uptake of a cholesterol-rich emulsion by neoplastic ovarian tissues.
      ] or under statin [
      • Santos R.D.
      • Chacra A.P.
      • Morikawa A.
      • Vinagre C.C.
      • Maranhão R.C.
      Plasma kinetics of free and esterified cholesterol in familial hypercholesterolemia: effects of simvastatin.
      ] or hormonal replacement therapy [
      • Melo N.R.
      • Latrilha M.C.
      • Santos R.D.
      • Pompei L.M.
      • Maranhão R.C.
      Effects in post-menopausal women of transdermal estrogen associated with progestin upon the removal from the plasma of a microemulsion that resembles low-density lipoprotein (LDL).
      ]. The fact that the nanoemulsion is removed faster from the plasma than native LDL [
      • Lima M.F.
      • Pujatti P.B.
      • Araújo E.B.
      • Mesquita C.H.
      Compartmental analysis to predict biodistribution in radiopharmaceutical design studies.
      ,
      • Hungria V.T.
      • Latrilha M.C.
      • Rodrigues D.G.
      • et al.
      Metabolism of a cholesterol-rich microemulsion (LDE) in patients with multiple myeloma and a preliminary clinical study of LDE as a drug vehicle for the treatment of the disease.
      ] occurs because the nanoemulsion binds to the receptor through apo E that has more affinity for the LDL receptors than apo B that binds native LDL [
      • Hirata R.D.
      • Hirata M.H.
      • Mesquita C.H.
      • Cesar T.B.
      • Maranhão R.C.
      Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats.
      ]. Therefore, although the nanoemulsion differs from the native lipoprotein in composition and some metabolic parameters, it has the ability to detect LDL metabolism changes in subjects, when cases and controls are rigorously matched. Because cholesteryl ester FCR was higher in the trained subjects, it can thus be assumed that in those RT subjects removal of the nanoemulsion particles from the circulation was greater than that of the sedentary group. This result is similar to the findings of our previous study, in which amateur cyclists also showed cholesteryl ester FCR greater than that measured in sedentary individuals [
      • Vinagre C.G.
      • Ficker E.S.
      • Finazzo C.
      • et al.
      Enhanced removal from the plasma of LDL-like nanoemulsion cholesteryl ester in trained men compared with sedentary healthy men.
      ]. Nonetheless, aerobic training subjects showed cholesteryl ester FCR roughly fivefold greater than in sedentary individuals [
      • Vinagre C.G.
      • Ficker E.S.
      • Finazzo C.
      • et al.
      Enhanced removal from the plasma of LDL-like nanoemulsion cholesteryl ester in trained men compared with sedentary healthy men.
      ]. While that in the RT subjects of the present study, the cholesteryl ester FCR was approximately twice greater than the sedentary group. Accordingly, the effects of RT were significantly lower than those measured in the aerobic training group (p = 0.03). Nonetheless, whereas amateur cyclists trained average 2 h for 3–4 times/week [
      • Vinagre C.G.
      • Ficker E.S.
      • Finazzo C.
      • et al.
      Enhanced removal from the plasma of LDL-like nanoemulsion cholesteryl ester in trained men compared with sedentary healthy men.
      ], the current RT subjects trained for 1 h 4–5 times/week. Those differences in training program make it difficult to compare the results of exercise program modalities of the two studies. Yet it should be pointed out that the VO2peak of the subjects of the current study was the same of that of the amateur cyclists, as well as their age and BMI and serum lipids but HDL-cholesterol. Interestingly, the positive correlation found between the nanoemulsion cholesteryl ester k2.0 and VO2peak supports the concept that the better the fitness the greater the ability of the individual to remove nanoemulsions or lipoproteins from the circulation. The k2.0 appears the plasma removal of the nanoemulsion after the incorporation of de apolipoproteins [
      • Lima M.F.
      • Pujatti P.B.
      • Araújo E.B.
      • Mesquita C.H.
      Compartmental analysis to predict biodistribution in radiopharmaceutical design studies.
      ]. The FCR of the other labeled nanoemulsion component, free cholesterol, also correlated with k2.0, which reinforces the concept that exercise leads to accelerated removal of the nanoemulsion particles.
      The LDL-like nanoemulsion is taken-up by the LDL-receptors, although the Lipoprotein Related Protein (LRP) receptor may also participate in the removal of the nanoemulsion particles from the blood [
      • Maranhão R.C.
      • Roland I.A.
      • Toffoletto O.
      • et al.
      Plasma kinetic behavior in hyperlipidemic subjects of a lipidic microemulsion that binds to LDL receptors.
      ]. Therefore, the increase in the nanoemulsion clearance can be ascribed to an exercise-induced increase in the LDL-receptor and, eventually, also to the LRP increase.
      Another important issue is whether RT induces the increase of the lipoprotein receptors of the muscle or of the hepatic tissue. In our previous study we showed in mice submitted to treadmill training increased nanoemulsion uptake by the skeletal muscle and decreased hepatic uptake when compared to non-trained control mice [
      • Vinagre C.G.
      • Ficker E.S.
      • Finazzo C.
      • et al.
      Enhanced removal from the plasma of LDL-like nanoemulsion cholesteryl ester in trained men compared with sedentary healthy men.
      ]. In mice with LDL-receptor gene knock-out, the effects of exercise did not occur [
      • Vinagre C.G.
      • Ficker E.S.
      • Finazzo C.
      • et al.
      Enhanced removal from the plasma of LDL-like nanoemulsion cholesteryl ester in trained men compared with sedentary healthy men.
      ]. Those experiments suggest that training leads to LDL-receptor overexpression in the muscle rather than in the liver. It is worthwhile to note that the increase of the nanoemulsion removal was not determinant of lower LDL-cholesterol concentration. This suggests that the increased removal of nanoemulsion and the extension of native LDL were being compensated by an increased input of the lipoprotein of hepatic origin into the plasma compartment. Therefore, as occurred in the amateur cyclists described in our previous study, RT is also able to increase the LDL turnover, which implies that the LDL pool is renewed more rapidly. The increased turnover accounts for the reduced LDL peroxidation rates in the plasma in the RT subjects: as LDL circulates less, it is less exposed to the lipid peroxidation mechanisms.
      In subjects submitted to RT, Schjerve et al. [
      • Schjerve I.E.
      • Tyldum G.A.
      • Tjonna A.E.
      • et al.
      Both aerobic endurance and strength training programmes improve cardiovascular health in obese adults.
      ], also found that the oxidized LDL concentration and total antioxidant level were decreased. Simultaneously, there was improved endothelial function, an important beneficial effect [
      • Schjerve I.E.
      • Tyldum G.A.
      • Tjonna A.E.
      • et al.
      Both aerobic endurance and strength training programmes improve cardiovascular health in obese adults.
      ]. Similar findings were reported in subjects under aerobic training in respect to both diminished oxidized LDL and improvement of the endothelial function [
      • Schjerve I.E.
      • Tyldum G.A.
      • Tjonna A.E.
      • et al.
      Both aerobic endurance and strength training programmes improve cardiovascular health in obese adults.
      ,
      • Stensvold D.
      • Tjønna A.E.
      • Skaug E.A.
      • et al.
      Strength training versus aerobic interval training to modify risk factors of metabolic syndrome.
      ].
      Whereas cholesteryl ester make-up the core of lipidic nanoemulsion and lipoproteins and shift from the lipoprotein particles only by the action of transfer proteins, such as CETP and PLTP, free cholesterol is located on the lipoprotein surface layer and more unstable, may dissociate from the lipoproteins and eventually precipitate in tissues, such as arteries or the liver [
      • Couto R.D.
      • Dallan L.A.
      • Lisboa L.A.
      • et al.
      Deposition of free cholesterol in the blood vessels of patients with coronary artery disease: a possible novel mechanism for atherogenesis.
      ,
      • Hoofnagle A.N.
      • Heinecke J.W.
      Lipoproteomics: using mass spectrometry-based proteomics to explore the assembly, structure, and function of lipoproteins.
      ]. In a previous study, when LDL-like nanoemulsion was injected into CAD patients, the free cholesterol component was removed faster than in the non-CAD control subjects, suggesting that in CAD free cholesterol could dissociate and precipitate, thus the faster removal [
      • Santos R.D.
      • Hueb W.
      • Oliveira A.A.
      • Ramires J.A.
      • Maranhao R.C.
      Plasma kinetics of a cholesterol-rich emulsion in subjects with or without coronary artery disease.
      ]. Interestingly enough, in RT subjects, the free cholesterol component was removed slower than the cholesteryl ester, in contrast to sedentary individuals, wherein both components were removed at the same rate. Thus, the fact that exercise training elicited slower free-cholesterol removal than cholesteryl ester removal may suggest novel antiatherosclerosis mechanisms. It is possible that training increases free cholesterol transfers to HDL with subsequent esterification and transfer to apo B containing lipoproteins that are cleared from blood at much smaller rates than the nanoemulsion. This would decrease the nanoemulsion free cholesterol FCR relative to cholesteryl ester FCR, leading to exercise-induced amelioration of the cholesterol reverse transport. Those unsuspected effects of training on lipoprotein metabolism and on the stability of the plasma cholesterol pool deserve further investigation.
      Both study groups, RT and sedentary subjects, presented no differences in regards to serum HDL-cholesterol concentration and the other parameters related with HDL measured in this study (HDL particle size, transfer of lipids to HDL and the activity of the HDL associated antioxidant enzyme, PON1). In this respect, in RT subjects HDL-cholesterol was found either increased or unaltered in several published addressing this issue [
      • Ghahramanloo E.
      • Midgley A.W.
      • Bentley D.J.
      The effect of concurrent training on blood lipid profile and anthropometrical characteristics of previously untrained men.
      ,
      • Kraemer W.J.
      • Vingren J.L.
      • Silvestre R.
      • et al.
      Effect of adding exercise to a diet containing glucomannan.
      ,
      • Schjerve I.E.
      • Tyldum G.A.
      • Tjonna A.E.
      • et al.
      Both aerobic endurance and strength training programmes improve cardiovascular health in obese adults.
      ,
      • Stensvold D.
      • Tjønna A.E.
      • Skaug E.A.
      • et al.
      Strength training versus aerobic interval training to modify risk factors of metabolic syndrome.
      ]. Our results imply that, in our exercise protocol, RT did not influence HDL levels or the important functional and metabolic aspects of the lipoprotein.
      In this study, the sedentary and the RT groups showed significant differences in abdominal circumference. As visceral fat is an important factor in lipid metabolic changes, it is possible that reduction of this tissue by training could also have played a role in the acceleration of nanoemulsion FCR in RT group. However, the absence of correlation of abdominal circumference and FCR of cholesteryl esters and free cholesterol attenuates the importance of visceral fat as intervening factor in the RT-induced FCR changes observed here. At any rate, RT frequently elicits shortening of abdominal circumference and, as such, this effect should be considered as inherent to RT.
      As a limitation of this study, the pre-training data of the participants were not collected, which would furnish an interventional setting, and the sample size of the study is small. In future studies, a variety of factors that could be determinants of the results obtained here, such as enzymes related with lipid intravascular metabolism and transfer proteins should be evaluated to understand the mechanisms of the current findings.
      In conclusion, resistance training, similar to aerobic training, had the ability to enhance the removal of the nanoemulsion cholesteryl ester and, for extension, the removal of LDL from circulation. This probably leads to the reduction of oxidized LDL in the plasma and favors the assumption that this type of exercise training is beneficial for atherosclerosis prevention.

      Conflict of interest

      None declared.

      Acknowledgements

      This study was supported by Fundacão do Amparo á Pesquisa do Estado de São Paulo (FAPESP) , São Paulo, Brazil. Dr. Maranhão has a Research Award from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) , Brasilia, Brazil. Dr. Silva had a scholarship from Coordenadoria do Pessoal de Ensino Superior (CAPES) , Brasilia, Brazil.
      The authors are grateful to Mr. William A. Presada for revising the text and to Debora Deus for technical assistance.

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