Patients
Our study comprised 66 patients with obesity (body mass index [BMI] > 30) who attended an obesity treatment program (intervention) at the Health Science Center, Kansai Medical University Hospital, Japan, between October 2014 and October 2018. Exclusion criteria comprised: patients with a BMI > 60; patients who were pregnant; patients with severe liver dysfunction, renal disease, or secondary causes of obesity due to endocrine disorders; and those with a debilitating disease.
Patients were categorized into two groups: an L group (those with a weight loss of < 5% of the baseline value) and an M group (those with a weight loss of > 5% of the baseline value) [15,16,17].
Body composition, blood sampling, exercise tolerance, and muscle strength (handgrip and lower limb muscle strength) were measured in all patients, and the patients completed an international physical activity questionnaire at baseline and after completion of a 6-month weight loss program. Medical history and clinical characteristics were collected from the patients’ medical records.
This study was approved by the Ethics Committee of Kansai Medical University (approval no. 2019092). All procedures performed in the study involving human participants were performed in accordance with the 1964 Helsinki Declaration and its later amendments. Written informed consent was obtained from all participants prior to commencement of the study.
Obesity treatment program
The obesity treatment program consisted of exercise, nutritional, and psychological counseling [18, 19]. Our intervention, undertaken at the hospital, comprised 30 min of aerobic exercise in which the intensity was adjusted to the anaerobic threshold, resistance training, and stretching. Patients also performed exercise three times per week, including home-based exercise. A health fitness programmer provided supervised individual exercise program based on the exercise capacity of patients at each visit. They also provided home exercise menu for each patients. A dietician provided monthly nutritional guidance, education concerning eating behavior, and dietary instruction once a month, based on dietary record data. Using the patients’ weight records, a clinical psychologist provided psychological counseling once a month, based on cognitive behavioral therapy, and focused on self-monitoring and self-efficacy.
Measurement of body composition
Body composition was measured using dual-energy X-ray absorptiometry (DXA, DPX-NT System, GE Healthcare, Buckinghamshire, UK). The measurement parameters included weight, fat mass, and lean body mass (LBM) (whole body, upper extremities, body trunk, and lower extremities). The rates of fat mass (%fat) and LBM (%LBM) were calculated as fat mass and LBM divided by body weight, respectively. Visceral fat area (VFA) and subcutaneous fat area (SFA) at the umbilical level were measured using computed tomography and fat scan analysis software (East Japan Technology Tokyo Laboratory, Tokyo, Japan), respectively.
Blood sampling and measurement of serum adipokine and myokine levels
Patient medical history and clinical characteristics were collected from medical records. Fasting blood was analyzed to determine glucose (GLU), glycosylated hemoglobin (HbA1c), and immunoreactive insulin (IRI) levels. We evaluated the endogenous effect of insulin resistance on vascular function. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR). HOMA-IR was calculated as follows: HOMA-IR = (IRI × fasting plasma GLU) / 405. Additionally, we measured the plasma levels of myokines, adiponectin, leptin, and irisin. Blood samples were stored at − 80 °C, and both myokine and adipokine levels were measured according to the manufacturer’s instructions. Serum myostatin and irisin levels as myokines were measured using the GDF-8/Myostatin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and human EIA Kit (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA). Serum adiponectin and leptin levels as adipokines were measured using the human Quantikine ELISA Kit (R&D Systems, respectively. Minneapolis, MN, USA). The intra- and inter-assay coefficients of variation were 2.5–4.7% and 5.8–6.9% for adiponectin, 3.0–3.3% and 3.5–5.4% for leptin, 1.8–5.4% and 3.6–6.0% for myostatin, and < 10% and < 15% for irisin, respectively.
Cardiopulmonary exercise test
A symptom-limited exercise stress test, using the ramp method, was conducted using an expiration gas analyzer (AE300S, Minato Medical Science Co., Ltd., Osaka, Japan) and an ergometer cycle (AEROBIKE 75XL, Combi, Tokyo, Japan) with a 12-lead electrocardiogram. Exercise began with a four-minute warm-up at 10–20 W and 50 rpm, followed by the 10–20 W ramp method after a five-minute rest on the ergometer. Heart rate, oxygen uptake (VO2), and carbon dioxide excretion volume (VCO2) were measured at the point of rest, warm-up, anaerobic threshold (AT), and maximum oxygen uptake (peak VO2) using the breath-by-breath method. The AT was determined using the V-slope method. Peak VO2 and work rate were defined as the peak values during incremental exercise [20].
Muscle strength
Muscle strength and performance were measured using handgrip strength and lower limb muscle strength. Handgrip strength was measured using a handgrip dynamometer (T.K.K.5401, Takei Scientific Instruments, Niigata, Japan). Right- and left-hand grip strength was measured three times on each side, and the mean measurement was recorded. Lower limb muscle strength was measured twice for isokinetic output torque using a recumbent ergometer (Strength Ergo, Mitsubishi Electric Corp., Tokyo, Japan). We recorded the maximum value and divided the values by body weight (N∙m/kg).
International physical activity questionnaire
Physical activity was self-reported using a shortened version of the international physical activity questionnaire [21]. The questionnaire comprised seven questions assessing physical activity in the past week. Metabolic equivalent (MET) values for walking, average physical activity, and intense physical activity were computed as 3.3, 4, and 8, respectively. The total amount of physical activity per week (day × minute × MET) was calculated through aggregating the amount of walking, moderate physical activity, and intense physical activity.
Statistical analysis
The measured values were expressed as mean ± standard deviation or median (25, 75% quartile), and categorical data were expressed as incidences and percentages. Changes (⊿) were calculated as differences between pre- and post-intervention. Normal distribution was confirmed using the Shapiro-Wilk test. An unpaired t-test, a Mann-Whitney U test, or a Chi-squared test was used for inter-group comparisons. A paired t-test and a Wilcoxon signed-rank test were used for pre- and post-intervention comparisons, respectively. Correlations between myostatin and other parameters were determined using Pearson’s or Spearman’s rank correlation coefficient. A stepwise multiple regression analysis was used for multivariable analysis to examine independent predictors of ⊿myostatin. The parameters of significant correlation with ⊿myostatin were used as independent variables, and sex, age, and DM morbidity were used as adjustment factors. A p-value < 0.05 was considered statistically significant. All statistical analyses were conducted using SPSS version 23.0 for Windows (IBM Corp., Armonk, NY, USA) software.