Increased serum salusin-α by aerobic exercise training correlates with improvements in arterial stiffness in middle-aged and older adults

Cross-sectional study

In the cross-sectional study, body mass index (BMI), carotid-femoral PWV (cfPWV), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), common carotid intima-media thickness (ccIMT) and the levels of total cholesterol and triglycerides were significantly greater in the Middle-aged and older group than in the Young group (Table 1). In contrast, height and serum salusin-α levels in the middle-aged and older group were significantly lesser than in the young group (Table 1 and Figure 1).

Serum salusin-α levels were negatively correlated with 1) age (r = -0.698, P < 0.0001); 2) total cholesterol (r = -0.374, P < 0.0001); 3) triglycerides (r = -0.336, P < 0.001); 4) SBP (r = -0.458, P < 0.0001); 5) DBP (r = -0.473, P < 0.0001); 6) MBP (r = -0.486, P < 0.0001); 7) cfPWV (r = -0.479, P < 0.0001); 8) ccIMT (r = -0.577, P < 0.0001) (Table 2 and Figure 2). In the multiple regression analysis using serum salusin-α levels as the dependent variable, MBP (β = -0.277, P < 0.05) and cfPWV (β = -0.299, P < 0.05) were independent factors associated with serum salusin-α levels. However, BMI and triglycerides were not independently associated with serum salusin-α levels.

Age was positively correlated with cfPWV (r = 0.636, P < 0.0001), SBP (r = 0.523, P < 0.0001), DBP (r = 0.545, P < 0.0001), MBP (r = 0.558, P < 0.0001) and ccIMT (r = 0.787, P < 0.0001).

Interventional study

In the interventional study, no significant differences were found in any variable measured for comparisons between the Young-AT and Young-Con groups at baseline (Table 3). However, when comparing the changes that occurred in response to AT versus control, changes in peak oxygen uptake (${\dot{\text{V}}\text{O}}_{2\text{peak}}$) were significantly greater in the Young-AT than in the Young-Con group (Table 4). In contrast, changes in body weight, heart rate (HR) and SBP were significantly lesser in the Young-AT than the Young-Con group (Table 4). There were no significant differences in BMI, total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, daily physical activity, DBP, cfPWV, ccIMT or serum salusin-α levels between the Young-AT and Young-Con groups (Table 4 and Figure 3A). Furthermore, the changes in salusin-α levels induced by AT were not correlated with any other variables measured in the Young-AT group (Table 5).

In the Middle-aged and older group, there were no significant differences in any of the parameters measured between the AT and Con groups at baseline (Table 3). However, changes before and after the intervention in ${\dot{\text{V}}\text{O}}_{2\text{peak}}$ and serum salusin-α levels were significantly greater in the Middle-aged and older-AT group than in the Middle-aged and older-Con group (Table 4 and Figure 3B). In contrast, changes in BMI, SBP, DBP, MBP and cfPWV were significantly lesser in the Middle-aged and older-AT group than the Middle-aged and older-Con group (Table 4). There were no significant differences in changes in total cholesterol, HDL cholesterol, triglycerides, daily physical activity, HR and ccIMT between the Middle-aged and older-AT and Middle-aged and older-Con groups (Table 4). Furthermore, AT-induced changes in serum salusin-α levels were negatively correlated with AT-induced changes in cfPWV (unadjusted: r = -0.597, P < 0.01, Figure 4A; Table 5; adjusted for age: β = -0.682, P < 0.001), SBP (unadjusted: r = -0.591, P < 0.01, Figure 4B; Table 5; adjusted for age: β = -0.593, P < 0.01), DBP (unadjusted: r = -0.442, P < 0.05, Figure 4C; Table 5; adjusted for age: β = -0.498, P < 0.05) and MBP (unadjusted: r = -0.570, P < 0.01, Table 5; adjusted for age: β = -0.585, P < 0.01) but were not correlated with AT-induced changes in any other parameter measured in Middle-aged and older-AT group (Table 5).

Subjects: Cross-sectional study

We first performed a cross-sectional study in which 105 healthy subjects (49 men and 56 women) aged 20–80 years participated. These subjects were divided into 2 groups. One group consisted of young (under 40 years, total: n = 45, age: 21.4 ± 0.5 years; males: n = 25, age: 22.0 ± 0.8 years; females: n = 20, age: 20.6 ± 0.2 years), while the second group consisted of middle-aged and older (over 40 years, total: n = 60, age: 67.4 ± 0.8 years; males: n = 24, age: 70.3 ± 1.2 years; females: n = 36, age: 65.4 ± 1.0 years) male and female subjects as outlined in Table 1. All subjects were recruited from a local community health center and a community recreation center. Subjects were excluded if diagnosed with hyperlipidemia, hypertension and hyperglycemia by a physician, taking anti-hyperlipidemic, anti-hypertensive, or anti-hyperglycemic medication, or had a history of stroke, diabetes, hypertension, hyperlipidemia, cardiac disease, chronic renal failure or mental disorders. None of the participants had a history of smoking for at least 12 months prior to the study. Women in the middle-aged and older group who had been postmenopausal for at least 5 years were not on hormone replacement therapy. All subjects were informed of the experimental procedures and risks, and provided written informed consent before participating in the study. The study was approved by the Ethics Committee of Ritsumeikan University and was conducted in accordance with the Declaration of Helsinki.

Subjects: Interventional study

In the interventional study, 36 young (males: n = 20, age: 22.1 ± 1.0 years; females: n = 16, age: 20.4 ± 0.2 years) and 40 middle-aged and older (males: n = 16, age: 70.3 ± 1.5 years; females: n = 24, age: 64.8 ± 1.4 years) healthy subjects volunteered to participate. These subjects were sub-divided into two groups by including or not an aerobic exercise training intervention (AT). This subdivision resulted in the formation of four groups: a Young-AT group (n = 27), a Young control group (Young-Con, n = 9), a Middle-aged and older-AT group (n = 26), and a Middle-aged and older-Con group (n = 14) as depicted in Table 3. Subjects were recruited from the same local communities as in the cross-sectional study. Also, the same exclusion criteria were utilized. This study was registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) (UMIN000035520).

Experimental design

Measurements obtained for all subjects in the cross-sectional study included height, body weight, body mass index (BMI), resting heart rate (HR), resting systolic blood pressure (SBP), resting diastolic blood pressure (DBP), resting mean blood pressure (MBP), resting carotid-femoral pulse wave velocity (cfPWV), resting common carotid intima-media thickness (ccIMT), resting serum salusin-α levels and serum concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides. In the interventional study, 27 young (males: n = 15, age: 22.1 ± 1.0 years; females: n = 12, age: 20.4 ± 0.2 years) and 26 middle-aged and older (males: n = 10, age: 70.3 ± 1.5 years; females: n = 16, age: 64.8 ± 1.4 years) subjects completed an eight-week AT program. Measurements in this study were performed before and after AT and included height, body weight, peak oxygen uptake $({\dot{\text{V}}\text{O}}_{2\text{peak}})$, resting SBP, resting DBP, resting cfPWV, resting HR, resting serum salusin-α levels and serum concentrations of total cholesterol, HDL cholesterol and triglycerides. At the beginning and end of the study period, fasting blood samples were drawn following at least 48 hours of rest after the last AT session to avoid the influence of acute effects of exercise. All subjects were instructed not to eat or drink fluids except for water for at least 12 hours before blood sampling. Serum samples were centrifuged (1500 x g, 15 min, 4°C) immediately after collection and stored at −80°C. All measurements were performed at a constant room temperature of 24°C.

Exercise intervention

The subjects in the AT subgroups within the interventional study performed aerobic exercise training consisting of cycling on a leg ergometer (828E Monark cycle ergometer, Stockholm, Sweden) for 55 minutes, 3 days/week, for 8 weeks. Each 55-minute exercise bout consisted of a 5-minute warm-up period at 40% ${\dot{\text{V}}\text{O}}_{2\text{peak}},$ followed by 45 minutes of cycling at 60–70% ${\dot{\text{V}}\text{O}}_{2\text{peak}},$ and a 5-minute cool-down period at 40% ${\dot{\text{V}}\text{O}}_{2\text{peak}}.$ Exercise compliance was carefully monitored by direct supervision. We adopted this AT program in accordance with previous studies by our group demonstrating a favorable effect on arterial stiffness [9, 38]. The sedentary subjects in the control groups were instructed not to change their activities of daily living during the eight-week interventional period. Subjects in all groups were instructed to maintain their food intake as usual throughout the study.

Measurement of VO_2peak

${\dot{\text{V}}\text{O}}_{2\text{peak}}$ was measured during breath-by-breath oxygen consumption and carbon dioxide production using an incremental cycle exercise test on a cycle ergometer (MINATO, AE-310SRD, Osaka, Japan). To measure ${\dot{\text{V}}\text{O}}_{2\text{peak}},$ incremental cycle exercise began at a work rate of 60 W (30–90 W) for men and 30 W (0–60 W) for women, and power output was increased by 15 W/min until the subjects could not maintain a fixed pedaling rate of 60 rpm [9, 38]. Subjects were encouraged to exercise at maximum intensity throughout the ergometer test. Heart rate and rating of perceived exertion (RPE) were monitored every minute during the exercise bout. RPE was obtained using the modified Borg scale. The highest 30-second average value of ${\dot{\text{V}}\text{O}}_2$ during the exercise test was defined as ${\dot{\text{V}}\text{O}}_{2\text{peak}}$ if three out of four of the following criteria were met: (I) plateau in ${\dot{\text{V}}\text{O}}_2$ with an increase in external work, (II) maximal respiratory exchange ratio ≥ 1.1, (III) maximal heart rate ≥ 90% of the age-predicted maximum (208 − 0.7 × age; [39]), and (IV) RPE ≥ 18.

Measurement of daily physical activity

Physical activity in freeliving conditions was evaluated with a triaxial accelerometer (Active style Pro HJA-350IT; Omron Healthcare, Kyoto, Japan) [9]. The accelerometer was placed at the waist (left side).

Measurement of arterial stiffness, blood pressure, and heart rate

Subjects sat quietly for 30 minutes before measurements were acquired. Resting brachial artery SBP, DBP, MBP, cfPWV (an index of arterial stiffness) and HR were measured in duplicate in the supine position at rest using a vascular testing device (OMRON COLIN Co., Tokyo, Japan). Applanation tonometry was used to measure cfPWV using an array of 15 transducers, as previously described [9, 40]. PWV was calculated from the time delay between the carotid artery and femoral artery blood pressure waveforms and the distance between the two points, which was measured using a non-elastic tape measure. The coefficient of variation for interobserver reproducibility of cfPWV was 4.7% in this study. The mean value of SBP and DBP obtained at the left and right arms was calculated for analysis.

Measurement of ccIMT

Images acquired with a Vivid S6 ultrasound system (GE Healthcare, Chicago, IL, USA) equipped with a high-resolution linear array transducer were used to measure ccIMT [41, 42]. Ultrasound images were analyzed by use of an image analysis software (Image J; National Institutes of Health, Bethesda, MD). At least 15 measurements of ccIMT were obtained for each segment and mean values were used for data analyses. The day-to-day coefficients of variation of this technique were 2.7 ± 0.7 %.

Measurement of serum salusin-α levels

Serum salusin-α levels were measured in duplicate using an enzyme-linked immunosorbent assay (ELISA; Phoenix Pharmaceuticals, Burlingame, CA, USA). Optical density at 450 nm was measured using an xMark microplate reader (xMark microplate spectrophotometer; Bio-Rad Laboratories, Hercules, CA, USA). All samples were converted into concentrations by linear fitting to the log–log plot of a standard curve. The day-to-day coefficient of variation of serum salusin-α levels was 2.8 ± 0.7 %.

Measurements of serum cholesterol and triglyceride levels

Fasting serum concentrations of total cholesterol, HDL cholesterol, and triglycerides were measured by standard enzymatic techniques.

Statistical analysis

Data are expressed as means ± SE. In the cross-sectional study, unpaired Student’s t-tests were used to compare young versus middle-aged and older subjects for all parameters measured. Pearson correlation coefficients were used to assess the relationships between serum salusin-α levels with all other parameters obtained in this study. A multiple linear regression analysis was used to to test the independent association of serum salusin-α levels with BMI, triglyceride level, MBP, cfPWV. In the interventional study, unpaired Student’s t-tests were used to compare any parameter changes that occurred before and after the exercise intervention between the AT and control groups. As in the cross-sectional study, Pearson correlation coefficients were used to assess the relationships between serum salusin-α levels and the other parameters measured in all four groups of the interventional study. The partial correlation coefficient between serum salsuin-α levels and cfPWV, SBP or DBP was adjusted for age. A value of P < 0.05 was considered as statistically significant. All statistical analyses were performed using StatView (5.0, SAS Institute, Tokyo, Japan).