Platelets are small, anucleated cell fragments that are activated in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension [
The handling and experimental protocols were approved by the Ethics Committee for Care and Use of Laboratory Animals at Fluminense Federal University (protocol 466/2013) and complied with the ethical guidelines of the Brazilian Society of Laboratory Animal Science. Before beginning the experimental protocol, all animals were submitted to an adaptation period for 4 weeks and then we started the protocol. Experiments were performed on 3-month-old male Wistar rats (
Animals were randomly allocated, initially, into two groups: a control group (C) that received water and commercial chow for two weeks and a fructose group (F) that received an overload of 10% of D-fructose in drinking water and commercial chow for two weeks. After two weeks, seven animals from each group were euthanized, and the remaining animals from the control group (
Experimental protocol.
All experimental groups were maintained under the same commercial chow (Nuvilab Cr-1®, NuVital, Paraná, Brazil)
Nutritional information of Nuvilab Cr-1 commercial chow.
Nutritional information | 1 kg of chow |
---|---|
Calories | 3.360 kcal |
Carbohydrates | 530.0 g |
Proteins | 220.0 g |
Lipids | 40.0 g |
Ground whole corn, soybean meal, wheat bran, calcium carbonate, dicalcium phosphate, sodium chloride, vitamins A, D3, E, K2, B1, B6, B12, niacin, calcium pantothenate, folic acid, biotin, chloride choline, iron sulfate, manganese monoxide, zinc oxide, calcium sulfate, sodium selenite, cobalt sulfate, lysine, methionine, and butylated hydroxytoluene. |
Nutritional information and ingredient composition were obtained from chow label.
Fructose groups received 10% diluted D-fructose (Sigma-Aldrich, St. Louis, MO, USA) in water
Before beginning the experimental protocol, all animals were submitted to an adaptation period on a treadmill (Inbrasport®, Brasília, Brazil) for 4 weeks (5 minutes/day; 0.3 km/h–1.0 km/h, increased weekly). All animals underwent a maximal exercise test (MET) on a treadmill with an 11% inclination and an initial speed of 1.0 km/h, with an increment of velocity of 0.1 km/h every two minutes. The MET protocol was performed before the beginning of the experiment, two weeks thereafter, and at the end of the experiment to determine maximum running speed [
The groups assigned to aerobic training initiated a moderate-intensity exercise training regimen (50–75% maximal running speed), with a 0 to 7% inclination on a treadmill 4 days per week during the last 8 weeks of the experiment protocol [
At the end of the experimental period, all the animals were euthanized by cervical dislocation under anesthesia (thiopental sodium, 80 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA). The blood samples were collected from each animal.
Serum lipid profile (levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL)) was determined by using standard assay kits (Labtest, Minas Gerais, Brazil). The units were expressed in mg/dl.
The mean concentration of malondialdehyde (MDA), a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reacting substances (TBARS) [
The rat blood was removed by cardiac puncture and was collected into tubes containing a 3.8% trisodium citrate (9 : 1
Platelet aggregation was monitored by the turbidimetric method described by Born and Cross [
The platelet-rich plasma (400
The serum levels of IL-6 and IL-8 were assessed using the commercially available Quantikine Rat IL-6 and IL-8 Immunoassay. The levels of IL-6 and IL-8 in the serum were assessed by measuring the absorbance at 450 nm using an ELISA reader (Tp Reader, Thermo Plate®) and extrapolating from a standard curve.
The five experimental groups were compared using one-way ANOVA followed by a post hoc Bonferroni multiple-comparison test, when appropriate. All variables are expressed as
As described in Table
Evaluation of body weight, lipids, and MDA serum levels.
Parameters evaluated | Experimental groups | ||||
---|---|---|---|---|---|
C | F | FA | FT | FTA | |
Initial body weight (g) | 334.5 ± 3.2 | 334.3 ± 3.1 | 311.7 ± 15.1 | 332.9 ± 11.3 | 336,0 ± 11.7 |
Δ weight (g) | 83.2 ± 3.5 | 85.1 ± 4.2 | 85.5 ± 3.7 | 86.3 ± 4.4 | 81.3 ± 3.1 |
Total cholesterol (mg/dl) | 30.5 ± 5.7 | 51.0 ± 4.7 | 32.3 ± 6.6 | 57.2 ± 5.8 | 44.4 ± 14.6 |
LDL (mg/dl) | 22.1 ± 4.0 | 26.2 ± 9.4 | 19.7 ± 3.5 | 28.4 ± 12.0 | 27.0 ± 9.8 |
HDL (mg/dl) | 11.2 ± 0.5 | 13.2 ± 1.1 | 14.4 ± 1.7 | 21.9 ± 2.9 |
17.8 ± 1.5 |
MDA (nmol/dl) | 11.2 ± 0.7 | 14.8 ± 1.5 | 14.3 ± 1.1 | 9.6 ± 0.9 |
17.3 ± 2.0 |
Data are presented as
Evaluating the fructose administration effects in collagen-induced platelet aggregation (0.5
Platelet aggregation induced by collagen 0.5
We have also evaluated the effects of fructose administration in platelet aggregation ADP-mediated (0.5
Platelet aggregation induced by ADP 0.5
In our study, we have observed that fructose administration promoted a significant increase in IL-6 (15.87 ± 0.35 pg/dl) and IL-8 (592.40 ± 12.69 pg/dl) levels when compared to the control group (IL-6: 12.96 ± 1.60; IL-8: 500.20 ± 15.91 pg/dl). Concerning IL-6 serum levels, we have observed that arginine supplementation (10.89 ± 0.87 pg/dl) and aerobic exercise (IL-6: 11.16 ± 1.15 pg/dl) alone as well as associated (9.56 ± 0.61 pg/dl) reduced these interleukin levels. When we evaluated IL-8 levels, only arginine supplementation associated with aerobic exercise was able to reduce IL-8 levels (472.10 ± 19.41 pg/dl). There was no difference between arginine supplementation and aerobic exercise isolated when compared to the fructose group (
Effects of fructose intake, arginine supplementation, and aerobic exercise on IL-6, and IL-8 production in serum. C group: control group; F group: fructose group; FA group: fructose + arginine group; FT group: fructose + training group; FTA group: fructose + training + arginine group. Data are presented as
The present study was designed to investigate the antiplatelet effects associated with arginine supplementation, aerobic exercise, and these two concomitant interventions in rats at high risk of developing metabolic syndrome.
In our study, we have observed an increase in serum HDL levels and a decrease in MDA concentrations in the serum of the training group. These results corroborate the work of Farah et al. [
Evaluating the fructose administration effects at collagen-induced platelet aggregation, we could evidence an enhancement of platelet aggregation when compared to the C group. The arginine supplementation or aerobic training was not able to promote any change at the platelet hyperaggregability. On the other hand, arginine supplementation associated with aerobic exercise promoted an inhibition of the platelet hyperaggregability induced by fructose administration. We have also evaluated the effects of fructose administration at ADP-induced platelet aggregation. When ADP was employed as an agonist, we could not notice any effect at the different experimental groups.
Our data suggest that high fructose consumption results in an enhanced platelet response to collagen. This effect is reverted through the association of arginine supplementation with aerobic exercise. Interestingly, we could not observe this hyperaggregability effect when ADP is employed as an agonist, and the different treatments isolated were not able to have any antiplatelet effect. These results point out the antiplatelet effects when arginine supplementation is associated with aerobic exercise. Our research group [
Several studies have shown that IL-6 and IL-8 are important mediators of inflammation and contribute to the development of cardiovascular diseases. Independently of body mass index, sedentary lifestyle is a risk factor [
Collagen is a component of the subendothelium which becomes exposed to flowing blood in the context of vascular injury. Collagen binds directly to two platelet surface receptors: integrin
There is evidence that spontaneous platelet aggregation was significantly higher in MS patients compared with healthy volunteers. The curves of the mean aggregate sizes and light transmission characteristics suggested that the rates of collagen-induced aggregation of isolated platelets in MS patients significantly exceeded the corresponding values in the group of healthy volunteers [
Patients with MS have high risk of microcirculation complications and microangiopathies. Inflammation influences coagulation by increasing the production of coagulation proteins, reducing the activity of the anticoagulant pathway and by preventing fibrinolysis. Together, these alterations could lead to the formation of pathological thrombi resulting in heart or brain infarcts. The presence of MS could affect the coagulation system in some way before atherosclerosis development [
ADP is considered a weak agonist which promotes platelet aggregation through activation of purinergic receptors [
Interestingly, the association of aerobic exercise and arginine supplementation abolished this effect, but none of the isolated conditions presented any antiplatelet effect. These data support the idea that aerobic training associated with arginine supplementation decreases collagen-induced platelet hyperaggregability in an experimental model, with a continued exposure to a causal factor of metabolic alterations, therefore preventing cardiovascular disease development. The antiplatelet effect observed is probably related to the association of an enhancement of NO production and a reduction in oxidative stress and inflammatory cytokines, resulting in a reduction in platelet TXA2 production and platelet activation and aggregation (Figure
The hypothesis of inhibitory effect of exercise and arginine association in platelet activation of rats under high risk to develop metabolic syndrome. The high fructose intake triggers several cardiometabolic disorders, reflecting in an increase in lactic acid, proinflammatory cytokines, and oxidative stress. These events contribute to collagen-induced platelet activation. In addition, the increase in lactic acid produced by anaerobic glycolysis in platelets might be a mediator in platelet hyperaggregability. On the other hand, aerobic training associated with arginine supplementation decreases platelet hyperaggregability collagen–induced probably related to enhancement of NO production, inhibition of proinflammatory cytokines and oxidative stress, and finally inhibition of platelet aggregation.
High fructose intake leads to cardiometabolic alterations that precede cardiovascular disease. High fructose administration enhanced platelet aggregation and arginine supplementation associated with aerobic exercise can reduce platelet hyperaggregability in rats under high risk to develop metabolic syndrome.
Since a large number of individuals that are affected by MS suffer cardiovascular events, finding new therapeutic targets by elucidating new factors that contribute to these incidents is crucial to the treatment and prevention of cardiovascular events.
The platelet aggregation data used to support the findings of this study are partially included within the article, but all the platelet aggregation registers and spectrophotometric registers used to support the findings of this study are available from the corresponding author upon request.
The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Endocrinology.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001; FAPERJ (BR, #E-26/110.420/2014; E-26/110743/2012); and Universidade Federal Fluminense/FOPESQ 2013, 2014 (BR).