The subjects of this study were 23 pre and postmenopausal women with body fat percentages of 30% or more and were divided into PRM (pre- menopausal group, n=9) and POM (post- menopausal group, n=14). The sample size of the subjects was calculated by using ANOVA a large size of effect size of .90, a significance level of .05 and a power of .80 (G*power 3.2.1). All subjects performed a resistance exercise program that consumed 250~300 kcal daily for 12 weeks. To be included in the present study, participants had to meet the following inclusion criteria: 1) postmenopausal (absence of a menstrual cycle for at least 1 year and follicle-stimulating hormone >30 IU/L)8 older than 40 (premenopausal) and 50 (postmenopausal) years on the date of the assessment; 3) not receiving hormone replacement treatment; and 4) not using drugs such as beta-blockers, and statins. All volunteers underwent medical screening, including a health status interview and physical examination. Written informed consent was obtained from all subjects. The study was approved by Kangwon National University Institutional Review Board (KWNUIRB-2016-04-009-002), and conducted in agreement with the Declaration of Helsinki.
[Table 1. Characteristic of the subjects]
Body composition and physical fitness
All subjects underwent anthropometric measurements (height, body weight, % body fat, muscle mass, body mass index [BMI], waist-to-hip ratio [WHR]) using a multi-frequency bioelectrical impedance analyzer with eight tactile electrodes (MF- BIA8) (Inbody 720 body compostion analyzer, Biospace, Seoul, Korea) at the Exercise Physiology Laboratory of Kangwon National University. Bioelectrical impedance analysis was performed after at least 8 h of fasting and voiding. This analyzer uses an alternating current of 250 mA at a multi-frequency of 1 kHz, 5 kHz, 50 kHz, 250 kHz, 500 kHz and 1,000 kHz. It measures segmental impedances at the right arm, left arm, right, leg, left leg and trunk for all frequencies. Total body impedance value was calculated by summing segmental impedance values.
Physical fitness tests were performed with a circulation measuring device using O2run’s Hellmass system 3 (grip strength, sit-ups, sit and reach test, standing long jump, and side step). All measurements were entered into an electronic card and transmitted to the computer. To measure grip strength, subjects stood with both feet at shoulder width and maintained an angle of 15 degrees so that the torso and the arm did not touch each other. They held the handle of the dynamometer with second joints of their fingers and pulled the handle while keeping their arms from shaking. For sit-ups, subjects laid on a mat and bent their knees about 140 degrees. Their feet were flat on the floor. Then the upper body was raised until elbows touched knees. The number of repetitions made in 60 secs was recorded. For sit and reach test, subjects were asked to bend their upper body while fixing the two legs in plate. For standing long jump, all subjects started with their feet in place and jumped as far as possible with the two feet landing together. For side step, parallel lines were drawn at a distance of 120cm on the floor and subjects stood on both feet, one foot on the left side and the other foot on the right side, from the center line.
Subcutaneous fatty tissue extracted and Western blot
All study applicant agreed to extract the abdominal fat.
Plastic surgeon extracts the abdominal fat 30g twice before and after exercise program. At first time, right side abdominal fat was extracted. At second time, left side abdominal fat was extracted after exercise program. The applicant lied on the operation bed and plastic surgeon clean the abdomen of applicant with betadine. Plastic surgeon anesthetize the incisional window (1cm length) for liposuction machine tip with 2% lidocaine. The incisional window was made by no 15 blade and tumescent solution (500cc saline, 2% lidocaine 5cc and 0.1cc epinephrine were mixed) was infiltrated to the around of incisional window. Plastic surgeon extracts the abdominal fat (30cc) with liposuction machine. The fat was centrifuged during 3 minutes and pure fat cell could be separated. The pure abdominal fat was stored at –18 degrees immediately. The incisional window was sutured with 4-0 nylon and covered the waterproof bandage. The suture materials were removed at 7 postoperative days.
To extract protein from the subcutaneous fatty tissue, the tissues were lysed in 200 ul radioimmunoprecipitation assay (RIPA) buffer. The tissue was homogenized and centrifuged for 30 min at 14,000 rpm. The protein concentration of the supernatant was measured using the BCA protein assay kit (PIERCE, USA). Samples of equal protein content were resolved by SDS-polyacrylaminde gel electrophoresis on a 10 or 12 % gel, and transferred to a membrane. The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS), and subsequently incubated at 4°C overnight with primary antibodies (1:1,000 dilution) against perilipin (PLIN) (sc-240627), ATGL (adipose triglyceride lipase, sc-67355), MGL (monoglyceride lipase, sc-72277), and HSL (hormone-sensitive lipase, sc-25843) (all from Santa Cruz Biotechnology, USA). The signal was developed with an ECL solution (Amersham Pharmacia Biotech, USA) and visualized with the Image Quant TM LAS-4000 system (GE Healthcare, Sweden).
Blood collection and analysis
Fasting venous blood samples were collected from all participants at baseline, 6-weeks, and 12-weeks. Fasting was maintained for 12 h, and blood samples were collected on the following day. Enough sleep and the radical movement as much as possible to refrain. All samples were taken at 0830 AM from an antecubital vein. Serum samples were obtained after centrifugation and stored at -80°C. Serum levels of leptin and adiponectin were measured using enzyme-linked immunosorbent assay Dueset kits (R&D systems, Minneapolis, MN, USA) according to the manufacturer's instructions, as described previously.
Resistance exercise programs were used following experiments performed by Gurudut and Rajan with slight , modifications to fit the purpose of our experiment. The goal of the resistance exercise intervention was to burn 230~260kcal with moderate intensity exercise 60 min per day, 3 days per week for 12 weeks. At the beginning of each session, there was a 10 min of warm-up. It was followed by 40 min of the main part of the exercise with specific content and 10 min of cool-down. Warm-up exercises included 5 minutes of stretching, and 5 minutes of power walking at 50% intensity of the maximal heart rate reserve. Among resistance exercises, moderate intensity exercise was defined as circuit exercise at 55~65% intensity of 1 RM, 12 times of repeat, and 3 sets. The rest period between each category was 30 seconds. The rest period between sets was 1 minute in total resistance exercise time of 60 min. All exercise groups were given a polar (heart rate monitor; M400, Kempele, Finland), a portable exercise intensity setting device, for 60 minutes. Measurement of 1RM was calculated using formula of Brzycki: 1RM = Lifted weight (lb) / (1.0278 - repetitions×0.0278). All exercises were performed by re-measuring 1RM every two weeks .
In addition, dietitians and exercise physiologists met regularly with a clinical health psychologist experienced in lifestyle behavior change to discuss participant progress and refine behavior modification goals according to each participant’s needs. Nutritional education, self-management exercise, and behavior change techniques were provided. Furthermore, telephone consultations were scheduled biweekly for monitoring and motivation.
Data were analyzed using the SPSS 22.0 for Windows computer software package. Data were expressed as mean ± SD. All the data were tested for their normal distribution using Shapiro-Wilk test. For the two groups (PRM vs. POM) by three stages (baseline, 6 weeks, and 12 weeks), two-way within-subject factor ANOVA was used to examine whether exercise type and time. Bonferroni test was used for post-hoc analysis. A comparison of adipose tissue expression between the groups before and after 12 weeks was analyzed by two-way ANOVA. The main analysis of interest was the analysis of variance interaction term, which compares the change over time between the groups. Statistical significance was accepted at the 0.05 level.