A periodic epidemiologic survey was performed in 1999 and 2009 in a rural farming community located in south-western Japan (Tanushimaru town). Tanushimaru study was a Japanese cohort of the Seven Countries Study . As previously reported, the demographic characteristics of the residents of this area were similar to those of the general Japanese population . We performed epidemiological studies in every 10 years and followed up the participants every year.
In 1999, the total population aged over 40 years in this district was 3,463 (48.2% of men and 62.0% of women). A total of 1,920 subjects (794 males and 1,126 females; aged 40-95 years) were enrolled in this study. PA was measured by the Baecke PA questionnaire (BPAQ) [11-13], and the HGF levels were measured by ELISA method. In 2009, we measured PA by a simple questionnaire, and the HGF levels were measured again using ELISA method.
Baecke PA questionnaire (BPAQ)
We measured PA by BPAQ in 1999. The questionnaire consists of 16 questions organized into three sections: PA at work (Questions 1-8), sport during leisure time (Questions 9-12), and PA during leisure excluding sport (Questions 13-16) . We defined total index as total PA index, which were summed up of work, sport, and leisure-time index. The questionnaire defined three levels of occupational/work PA, namely low (e.g., clerical work, driving, shop keeping, teaching, studying, housework, medical practice and all other occupations with a university education), middle (e.g., farming, factory work, and carpentry), and high (e.g., dock work, construction work, and sport). Similarly, the questionnaire categorized sports into three levels: low (e.g., billiards, sailing, bowling and golf: average energy expenditure 0.76MJ/h), middle (e.g., badminton, cycling, dancing, swimming, and tennis: average energy expenditure 1.26MJ/h), and high (e.g., boxing, basketball, rugby, football, and rowing: average energy expenditure 1.76MJ/h). A sport score was calculated from a combination of the intensity of the sport which was played, the amount of time per week playing that sport, and the proportion of the year in which the sport was played regularly. Questions in each of the three indices (work, sport, and leisure) were scored on a five-point Likert scale, ranging from “1 = never” to “5 = always” or “5 = very often”. Summing the three indices gives a total PA index [11, 12]. We applied a five-point Likert scales to all of the three indices and only in sports index, we applied the combinations of intensity and duration.
A simple PA questionnaire
We measured PA by a simple questionnaire in 2009. The questionnaire has 4 options (“1” is spending most of time at home, “2” is working with almost sitting or less playing sports, “3” is working with a lot of movement and standing or playing active sports, and “4” is doing hard works), and subjects chose one of them.
Medical history, smoking habits, and alcohol intake were ascertained by a questionnaire. Smoking and alcohol intakes were classified as current habitual use or not. Height and weight were measured, and body mass index (BMI) was calculated as an index of obesity. Waist circumference was measured at the level of umbilicus in the standing position. Blood pressure (BP) was measured in the supine position twice at 3-minute intervals using a standard sphygmomanometer. The second BP was taken after 5 deep breaths, and that was used for analysis.
Blood was drawn from the antecubital vein in the morning after a 12-hour fast for determinations of lipids profiles (total cholesterol, triglycerides, high-density [HDL], and low-density lipoprotein cholesterol [LDL]), fasting plasma glucose (FPG), HbA1c (NGSP), insulin, serum urea nitrogen, creatinine, uric acid and serum HGF levels. Fasting blood samples were centrifuged within 1 hour after collection. Serum HGF levels was measured by the ELISA  and the other chemistries were measured at a commercially available laboratory (Kyodo Igaku Laboratory, Fukuoka, Japan). The estimate of insulin resistance by homeostasis model assessment (HOMA) score was calculated with the formula: fasting insulin (µU/mL) × fasting glucose (mmol/L)/22.5 as described by Matthews et al. . Insulin resistance was defined as HOMA ≥ 1.73 according to the diagnostic criteria used in Japan . Estimated glomerular filtration rate (eGFR) was calculated according to the following estimation formula that has been recommended by the Japan Society of Nephrology: eGFR (mL/min/1.732) = (194 × Scr -1.094 × age -0.287) × (0.739 for females) .
We divided into 2 groups (poor and good) by the median score of total PA index (median score was 7.75) in 1999, and also into 2 groups by a simple PA questionnaire in 2009. “Poor” was defined as subjects who chose “1” or “2”, and “good” was subjects who chose “3” or “4” of simple questionnaires. Using these 4 PA groups such as continuously low PA, increased PA, decreased PA, and continuously high PA, we compared their HGF levels in 10 years. We further investigated the development of hypertension, dyslipidemia, diabetes, and subjects who were suffering from CVD and cancer in 2009. The information was coded independently in accordance with the rules of the Seven Countries Study .
This study was approved by the Ukiha Branch of the Japan Medical Association, by the City Council of Tanushimaru, and by the Ethics Committee of Kurume University. All participants gave informed consent. All methods were carried out in accordance with relevant guidelines and regulations.
Because of skewed distributions, natural logarithmic transformations were performed for HOMA index triglycerides and ɤ-glutamyl transpeptidase (ɤ-GTP). Log-transformed values were used for the statistical calculation and reconverted to antilogarithm forms in the tables. Gender, the medications for hypertension, dyslipidemia, and diabetes, smoking habits, and alcohol intake were used as dummy variables.
First, we performed univariate and multivariate regression analyses for correlates of physical activity in 1999 and 2009 in the cross-sectional study. Second, we compared serum HGF levels by the 4 PA groups in 1999 and 2009 using analysis of co-variance (ANCOVA) adjusted for age and sex. Third, we compared the risk of development of hypertension, dyslipidemia, diabetes, CVD, and cancer by 4 PA groups using ANCOVA adjusted for age and sex. Finally, in order to compare the prevalence of CVD by 4 PA groups, we performed a logistic regression analysis adjusted for age and sex. Statistical significance was defined as a p value less than 0.05. All statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA).