Data collection

The subjects’ medical history, history of cardio-cerebrovascular diseases, use of alcohol, and smoking were ascertained by a questionnaire. Alcohol intake and smoking were classified as current habitual use or not. Height and weight were measured, and body mass index (BMI) was calculated as weight (kilograms) divided by the square of height (square meters) as an index of obesity. Waist circumference was measured at the level of the umbilicus in the standing position. Blood pressure (BP) was measured in the supine position twice at 3-min intervals using an upright standard sphygmomanometer. Vigorous physical activity and smoking were avoided for at least 30 min before BP measurement. The second BP with the fifth-phase diastolic pressure was used for analysis. Hypertensive subjects were defined as those with systolic BP ≥ 140 mmHg and/or those with diastolic BP ≥ 90 mmHg and/or those receiving antihypertensive medication. Subjects with fasting plasma glucose (FPG) ≥ 6.99 mmol/l (126 mg/dl) and/or subjects taking oral hypoglycemic agents or receiving insulin injection were diabetic. Subjects with dyslipidemia were defined as those with low-density lipoprotein cholesterol (LDL-c) ≥ 3.62 mmol/l (140 mg/dl) and/or triglycerides ≥ 1.69 mmol/l (150 mg/dl) and/or high-density lipoprotein cholesterol (HDL-c) < 1.03 mmol/l (40 mg/dl) and/or those taking lipid-lowering drugs.

Fasting blood samples were centrifuged within 1 h after collection. Serum levels of IGF-1 and GH were measured by ELISA and ECLIA methods to 1363 subjects who could receive blood testing. The blood was submitted to the commercially available laboratory (SRL Inc. Fukuoka, Japan), and the intra- and inter-assay coefficient of variations of IGF-1 and GH, respectively, at the laboratory that performed the assays was 2.56% and 0.75%, and 3.06% and 0.73% [16]. Liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and γ-glutamyl transpeptidase [γ-GTP]) were also measured. Estimated glomerular filtration rate (eGFR) was calculated by 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) [17]. In addition to these blood testing, trace elements (calcium, magnesium, iron, zinc, and phosphorus) and Hs-CRP as an inflammation marker were measured. The participants underwent measurements of handgrip strength using a calibrated strain-gauged dynamometer and expressed in kilograms (kg). They were asked to squeeze maximally with the dominant hand. The score of the dominant hand was used for analysis. Walk speed was measured over a 5-m course, marked out on level ground. Participants were asked to walk the course at their usual walking pace from a standing start. The time taken to complete the course was timed using a digital stopwatch and recorded to the nearest tenth of a second. The fastest time was used to derive walk speed in meters per second. Their cognitive functions were evaluated by the mini-mental state examination (MMSE). MMSE is designed to quickly measure global cognitive functioning, temporal and spatial orientation, attention, immediate and short-term memory, language, praxis, and calculation. Scores ranged from 0 to 30, with higher scores indicating better cognitive performance.

Carotid intima-media thickness (c-IMT) of the common carotid artery was determined by using duplex ultrasonography (Sonosite “TITAN,” ALOKA) with a 10-MHz transducer in the supine position. Longitudinal B-mode images at the diastolic phase of the cardiac cycle were recorded by a single trained technician who was blinded to the subjects’ background. We measured the only far wall of c-IMT. The images were magnified and measured on the screen and printed with a high-resolution line recorder (LSR-100A, Toshiba). We measured c-IMT according to the originally described method published in circulation [18]. Briefly, the c-IMT defined by Pignoli et al. [18] was measured as the distance from the leading edge of the first echogenic line to the leading edge of the second echogenic line. The first line represented the lumen-intimal interface; the collagen-containing upper layer of the tunica adventitia formed the second line. At each longitudinal projection, the site of the greatest thickness, including plaque, was sought along the arterial walls nearest the skin and farthest from the skin from the common carotid artery to the internal carotid artery. Three determinations of c-IMT of one artery were conducted at the site of the greatest thickness and at 2 other points, 1 cm upstream and 1 cm downstream from this site. The averaged value among the 6 IMTs (3 from the left and 3 from the right) was used as the representative value for each individual.

This study was approved by the Tanushimaru branch of the Japan Medical Association and by the local mayor, as well as by the ethics committee of Kurume University School of Medicine. All the participants gave informed consent. The Research Ethics Committee of the Kurume University School of Medicine (Process numbers 09019/2018) approved the study in conformity with the principles embodied in the declaration of Helsinki.