EffEct of high-intEnsity ExErcisE on EndothElial function in patiEnts with t2dM

1. Universidade Federal do Ceará, Instituto de Educação Física e Desportos, Fortaleza, CE, Brazil. 2. Universidade Estadual do Ceará, Instituto Superior de Ciências Biomédicas, Fortaleza, CE, Brazil. 3. Cardio Vita Cardiologia e Medicina do Esporte, Blumenau, SC, Brazil. 4. Clínica de Radiologia Uniangio, Blumenau, SC, Brazil. 5. Centro de Educação Física e Desportos, Universidade Estadual de Santa Catarina, Florianóplolis, SC, Brazil.

introduction Diabetes mellitus (DM) is the most common metabolic disease worldwide occurring in approximately 381.8 million adults in 2013 and with projection to rise to 591.9 million in 2035 1 . High glucose concentrations are responsible for increases in the formation of reactive oxygen species (ROS), with decreased nitric oxide and increased expression of inflammatory cytokines that contribute to endothelial dysfunction 2 .
The endothelium is responsible for maintaining homoeostasis between vasodilation and vasoconstriction stimuli by synthesizing a variety of molecules that act as agonists or antagonists, thus, in endothelial dysfunction the endothelium becomes vulnerable and leaky to inflammatory cells and lipids, forming atherosclerotic plaques 3 . In these patients the formation of atherosclerotic plaques are accelerated, but the mechanisms responsible for this accelerated development remain unclear, nevertheless, redox imbalance due to excess production of reactive oxygen species-mediated Nox1 seems to be one of the pathways 4 . However, atherosclerosis is the major cause of morbidity and mortality observed in DM 5 . The endothelial function in periphery conduit arteries affects directly in coronary circulation, therefore impaired endothelium-dependent vasodilation can predicts cardiovascular events 6 . Endothelium dependent dilation can be quantified by Flow-Mediated Dilation (FMD) and used as an index for endothelium function. The non-invasive nature of this technique permits repeated measurements over time 7 .
Exercise can be used as a non-pharmacological instrument to promote central (cardiovascular) and peripheral (skeletal muscle) adaptations that are linked to improved health outcomes 8,9 . Several studies have shown that high intensity training improves endothelial function due a vasorelaxation mediated an increased e-NO and IGF-1 receptor expression, including in patients with type 2 diabetes and transplanted hearts 10,8,11 , suggesting an anti-atherosclerotic effect of exercise.
The purpose of the present study was to identify the effect of an aerobic physical training program, use of Ultrasound clinical technique, in the endothelial function of patients with metabolic syndrome or diabetes mellitus type two.

MEthods
Subjects with metabolic syndrome or diabetes mellitus type two, of both sexes, with age between 40 and 65 years, without coronary arterial disease were studied. This study was led in the Section of Prevention and Cardiovascular Rehabilitation Unicardio, in the Hospital Santa Catarina, in the city of Blumenau, in the State of Santa Catarina, Brazil. The following exclusion criteria were used: subjects in whom, for reasons related to their clinical screening, it was not possible to suspend medicines with known effect on endothelial function, as inhibitors of the enzyme converting angiotensin, calcium channels blockers, nitrates, beta-blockers, anti-oxidizers, hormonal replacement, insulin; subjects with altered strength test (positive); cigarette smoking history in the last 12 months; participation in a physical exer-cise program in the last 12 months; chronic obstructive lung disease; high pressure levels (systolic blood pressure above 180mmHg and diastolic blood pressure above 110mmHg); diagnosed osteoporosis and diabetic neuropathy.
All the participants were informed regarding the objectives of the study and about the possible risks and discomforts involved with their participation in the experiments, signing term of informed consent. The study protocol was approved by the Commission of Ethics and Researches of Hospital Santa Catarina. This project is framed in the category II of the research regulation in men, in Brazil 12 , where in agreement with article 13 of the referred resolution.
All study patients underwent an initial clinical examination with a cardiologist. Before and after 6 weeks of intervention, the patients performed the strength test, physical exam and, evaluation of endothelial function. The participants of this study were submitted to program of physical exercise in the Section of Prevention and Cardiovascular Rehabilitation Unicardio of Hospital Santa Catarina, in Blumenau, State of Santa Catarina. The participants were guided to keep the original alimentary habits and behaviors. After the strength test limited by fatigue, the participants were guided with base in their individual prescription of exercises, and randomized for a low (50 to 60% HRmaximum) or high 75 to 85% HRmaximum) intensity. During the study period, the patients maintained the same doses of medicines.
In both programs, the patients trained four times a week, 50 minutes for session. Each session of physical exercise was divided in: five minutes of warm up, with stretching exercises and circling of members and body; 40 minutes of aerobic exercises (walk and/or run), being the first 10 minutes progressive until reaching the training target, and after, 30 minutes in the training target; and five minutes of cold down at the end, with stretching, flexibility and relaxation exercises. The part of aerobic exercise of each session was continuous. The six weeks consisted of two weeks of progressive adaptation phase and four weeks training in the target predetermined by the maximum heart frequency. The exercise sessions were supervised by one of the investigators and the determined intensity of exercise was monitored and registered for each patient by a watch of heart frequency (Polar, I Model M62, Helsinki, Finland). The alarms of the watches were programmed for the inferior and superior limits of the correspondent intensity, determined for each patient.
The exercise test was performed at Unicardio/HSC Clinics of Hospital Santa Catarina, downtown Blumenau, by a cardiologist that didn't know the group to the which the patients were randomized in. An ergometric treadmill with computerized system (I Raise PC Micromed, I Model Millenium EM200 Plus, Brasília, Distrito Federal, Brazil). Bruce's 13 protocol was chosen. Initially a standing rest electrocardiogram was made and the blood pressure was measured; the electrocardiogram was observed continuously; blood pressure was measured to each period; the test was interrupted by physical exertion of the evaluated subject, and in a gradual way; the room temperature was maintained at 24º C by air conditioning.
Descriptores: ultrasonido; diabetes mellitus tipo 2; ejercicio the endothelial function, performed at Clínica Uniangio, in the city of Blumenau, Brazil. Two investigators performed all the tests, without knowledge of the groups to which the patients were randomized. They were in fast from eight to 12 hours; the temperature adaptation was in 24º C; vasoactive medicines were suspended for approximately 4 half-lives; the patients didn't train in the day of the exam; they were oriented to not ingest substances that affected the vasodilatation mediated by the flow (FMD) for at least 24 hours before the exam, as caffeine, fatty foods, vitamin C, alcohol; no make use of tobacco; it was observed that the women were not in menstrual period; the individual was positioned lying, with the arms in prone in a comfortable position, to obtain image of the brachial artery, above the sewage antecubital, in the longitudinal plan, as seen in one of the evaluated subjects, shown at Figure 1.
A supersonic sound waves was used (Acuson, Model 128XP System, Montain View, California, USA), with vascular software for bidimensional images (2D), color Doppler and ghastly, with a monitor of internal electrocardiogram (ECG) and, a vascular transducer of high frequency (Acuson, Model L7 7,5-10 MHz, Montain View, California, USA). After the positioned subject, a segment with interfaces summons previous and subsequent between the lumen and the arterial wall and selected for images in tones of ash 2D. The images in tones of ash 2D, so much the way M and the way A (location of walls) they can be used to measure the diameter continually. The registrations of the arterial diameters were recorded in ribbon super-VHS for subsequent analysis. The whole reading happened after frozen the plan in the pick of the wave R in ECG in three measured sequences, repeating in more two waves R (therefore they were nine measures, making the average at the end), as shown in one of the subject of the research, seen at Figure 1. Vasodilatation Mediated by the Flow (FMD) Dependent of Endothelium: to create a flow incentive in the brachial artery, an sphygmomanometer cuff was placed below the sewage antecubital; an image in rest of the line base was acquired after 10 minutes of rest, obtained of a sample of volume of the middle of the artery; after that, the occlusion of the artery was created by the insufflations of the cuff for the supra-systolic pressure; typically, the fist was insufflated at least 50mmHg above the pressure basal systolic measure in the opposite arm, for occlusion of the arterial influx for extension of standardized time; that caused ischemic and consequent dilation down of the resistance vases through mechanisms solemnity-flow through the brachial artery (reactive hyperemia for five minutes), to accommodate the extensive resistance vases; the resulting increase in the stress cut caused dilation in the brachial artery; the longitudinal image of the artery was registered continually before of 30 seconds for two minutes after the deflation of the fist (peak ± 60 seconds); a pulsed sign Doppler of the middle of the brachial artery was obtained after the immediate release of the cuff and not later than 15 seconds after the deflation of the cuff to evaluate the arterial diameter in mm 14 . Vasodilatation Mediated by the Flow (FMD) Independent of Endothelium: There were necessary 10 minutes of rest after the reactive hyperemia (FMD), before other image could be acquired to reflect the conditions of re-established baseline; after having re-established the baseline noticed in the image, it was made administration of 0,4mg-Pumpspray of NitrolingualR (Wirkstoff: Nitroglycerin, Gesmbh, Vienna, Austria); the vasodilatation peak happened three to four minutes after having administered the substance (it was measured again after 4 minutes of the administration of the nitrate); the images were continually registered; the nitrate was not administered in individuals with bradicardia or serious hypertension; the nitrate, which is a exogenous donor of nitric oxide (NO), it has been used to determine the maximum vasodilatory answer that one can obtain, and to serve as a measure of independent vasodilatation of the endothelium reflecting the vascular function of the smooth muscle 6 .

statistical analysis
The size of the sample was calculated with base in previous studies [15][16][17] . The data were initially evaluated for the test of Kolmogorov--Smirnova to establish the normality. The variables with normal distribution are presented by mean±DP and the variables that didn't have normal distribution they are presented by mean±EP. For the data with normal distribution, analysis of variance of couple entrance for repeated data was used to compare the effects of the interventions and multiple comparisons were performed by the test of Tukey. Variables without normal distribution were evaluated by the test Kruskal-Wallis, with multiple comparisons performed by the test U of Mann-Whitney. The significance level = 5% (p 0,05).

rEsults
Thirty eight subjects were studied. Seven subjects were excluded of the study (six with suspicion of DAC with test of altered strength; one with hip surgery not recovered). Thirty one subjects were randomly designated to your groups, as demonstrated in the Table 1.  All the patients participated of the 24 training sessions. During the training sessions, the heart frequency was confronted at 20 minutes of training, as seen in Figure 2, where the group of high intensity maintained a frequency heart average of 79 ± 3% bpm, while the group of low intensity maintained a medium frequency of 55 ± 2% bpm.
After six weeks of exercise training, a significant improvement was observed in the dependent dilation of the endothelium, being the larger increase in the group of training of high intensity compared with the one of low intensity. Inversely, in the control group the change in the diameter of the vase was not significantly different from those of the initial study. Table 2 displays the diameters of the brachial artery before and after the exercise program of the patients of the research. Figure 3 shows the percentage difference of expansion, compared before and after the study, after reactive hyperemia. There is a significant difference in vasodilation in high and low intensity groups, although higher in the high-intensity group compared with the control group.

discussion
The main finding of the present study was to show that patients with metabolic syndrome or Type 2 Diabetes have a significantly accentuated response regarding the dilation of the brachial artery in the endothelium dependent function (reactive hyperemia), but not to nitroglycerin, after a six weeks aerobic physical exercise program, that improvement being maximized with high-intensity exercise. The physical exercise training also improved glycemic profile, LDL cholesterol profile and duration of the strength test.
Our findings have shown that high-intensity exercise can improve vasorelaxation in DM patients through the endothelium-dependent pathway. Recently, studies comparing the effects of continuous and high-intensity interval exercise on endothelium-dependent vasodilation in DM patients prove increased aerobic capacity 18 , flow-mediated dilation 19,18 , and cutaneous reactive hyperemia 18 in both groups, but the magnitude of improvements was greater in patients undergoing interval training. These results are accompanied by a decrease in oxidative stress and serum von Willebrand factor 18 as well as increases in antioxidant defenses 19,18 and endothelial nitric oxide 20,18 . However, endothelium-dependent vasodilation was directly proportional to the antioxidant defense and intensity of training 19 , suggesting that the characteristic oxidative stress in diabetic patients influences directly on endothelial dysfunction. In hypertensive rats the high-intensity exercise ameliorated endothelial function by IGF-1 and insulin induced vasorelaxation compared with the sedentary group. However, when an antioxidant pretreatment vasorelaxant was used responses became similar in all groups 10 .
Still evaluating the effect of low-volume high-intensity interval exercise, Little et al. 21 have been shown to decreased hyperglycemia and improvement muscle mitochondrial capacity in patients with type 2 diabetes subjected to two weeks of training. The glycemic control is important aspect for the treatment of DM due hyperglycemia is a risk factor for the development of several physiological disorders that contribute to endothelial dysfunction and consequently atherosclerosis formation 2 .
Interestingly, Goto et al. 22 , evaluating the intensity of training of physical exercise, obtained an inverse result to those of our study. They demonstrated that 12 weeks of low-intensity physical exercise (65% of the maximum heart rate), but not high-intensity physical exercise (90% of the maximum heart rate), increased the endothelium--dependent vasodilation in 26 healthy men.
In healthy populations, mean FMD ranges from 0.20 to 20%, although in our study, the average vasodilation after intervention was higher than that found in the literature 23 . For subjects with T2DM mediated vasodilation flow ranged from 0.75 to 12%, confirming our findings, showing that the subjects in our research were initially with endothelial dysfunction, being restored after six weeks of exercise 23 . Our study is in agreement with the literature 24 , mainly standardizing occlusion location (upper arm) and duration of occlusion (five minutes), annulling possible differences due to technical aspects.
The consistence of previously published data 17,25 , shown above, indicate that exercise training improves endothelial function in heterogeneous groups, including groups where patients presented this dysfunction due to injury, corroborating with the results of our study and contrasting with studies of exercise training in subjects with normal endothelial function. One cannot forget to highlight that the improvement in vascular function is evident in every vasculature, even if exercise is only practiced in the inferior limbs, excluding the remaining of the body. So, it is evident that the effect of exercise training becomes widespread, and not restricted to the vascular bed of the musculature, as demonstrated by Köller et al. 25 , Maiorana et al. 27 and, Green et al. 28 . One additional explanation for the improvement in endothelium dependent vasodilation could be the changes for the training of aerobic physical exercise in fasting glucose and glycated hemoglobin 27,[29][30][31] , in the lipid concentrations in the blood, specifically the LDL cholesterol 32-34 . liMitations This study was of short duration. Although that Hornig et al. 35 highlight that the prejudiced endothelial function already presents improvement after four weeks of intervention with exercise. Endothelial function is a precocious marker of cardiovascular disease. But, although this study didn't evaluate long term issues, it is justified in the literature that intense exercise reduces cardiovascular risk [36][37][38][39] . Ostergard et al. 40 is that the vascular function can improve at the beginning of the study and to begin a worsening after some weeks of intervention with exercise of high intensity, fact not noticed in our study, although with a time of intervention of six weeks.
But, in general, the evaluation is supersonic sound waves of the brachial artery Has Been surrendering important information about the vascular function in the health and in the disease, when taken due care in the technical application. Although, more long term studies and with clinically important issues shouldn't be performed.
conclusions When compared to the aerobic training of low intensity and controls, the aerobic training of high intensity improved vasodilatatory response endothelium-dependent in patients with metabolic syndrome or DM2, measured by the technique flow-mediated vasodilatation (FMD) of the brachial artery. These findings suggest that the physical training of high intensity could be considered as a preventive alternative on those patients.
All authors have declared there is not any potential conflict of interests concerning this article.