TREHA™ (Hayashibara Co. Ltd., Okayama, Japan) was used as the trehalose source in this study, in 1.9-g doses. The administered dose was equivalent to 1.65 g of anhydrous trehalose, because TREHA™ contains > 98.0% trehalose dihydrate. Extra-fine granulated sugar (Parl Ace Corp., Tokyo, Japan) was used as the sucrose source, in 1.65 g doses, and this served as the control substance for this study. The energy content of trehalose and sucrose is the same (16.7 kJ/g). These two substances were granulated to the same grain size and packed in identical plain silver film bags,. They were devised so that they could not be distinguished .
Participants who were willing to participate in this study were evaluated by a medical doctor and were included in the study if they met the inclusion criteria;healthy Japanese adults, fasting blood glucose < 110 mg/ dL, employees at the Hayashibara CO.,LTD., subjects who can comply with the instruction of conducting the study. Participants were excluded in cases of a history of sever disorders, pregnant or lactation, a history of hyperglycemia. From the 51 subjects who applied for this study, 50 healthy adult Japanese participants (20 women and 30 men) were recruited according to criteria. Each was given a full explanation, both written and oral, regarding the purpose and procedure of the study, and written informed consent was obtained from each. The participants were instructed to make no further changes to their diet or lifestyle for the duration of the study.
This study was designed as a randomized, double-blind, placebo-controlled, parallel- group trial. An individual who was not directly involved in the study randomly assigned the participants to two groups of 25, such that the groups had the same sex ratio and on the basis of their fasting blood glucose and 2-h PG values during OGTT. One group received trehalose and the other received sucrose. Double-blinding was ensured by the use of identical opaque sachets, outer packaging, labeling and color for both the compounds being administered. The identity of the substance being consumed by each participant remained confidential until all the data had been finalized, and the participants were blinded throughout the trial.
Each participant consumed one bag of substance twice a day for 78 days, such that the daily intake of the substances was approximately 3.3 g/day. The participants were permitted to prepare the substances for consumption in a variety of ways, such as by sprinkling them on meals or dissolving them in beverages. To evaluate the effects of each substance, body composition was assessed following 0, 4, 8, and 12 weeks of consumption. In addition, OGTT was performed at 0 and 12 weeks. This study was performed between August and December 2018.
This study was performed under the supervision of a medical doctor and conducted in accordance with Hayashibara Co. Ltd. Ethics Committee Approval number 215, and was registered with the University Hospital Medical Information Network (UMIN) Center (UMIN000033536). The study was conducted in accordance with the principles of the Declaration of Helsinki (adopted in 1964 and revised in 2013) and the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects (adopted in 2014 and revised in 2017).
The participants were prohibited from eating and drinking, except for a small amount of water, from 9 pm the day before their visit, until all the investigations carried out on the morning of the visit had been completed. Body mass, body fat%, fat mass, muscle mass, body water, and bone mass were measured using a body composition analyzer (MC-780A; Tanita Co., Ltd., Tokyo, Japan). The percentages of truncal fat and waist circumference were measured using an abdominal fat analyzer (AB-140; Tanita). Blood pressure was measured using blood pressure monitors (HEM-7020; Omron Healthcare Co., Ltd., Kyoto, Japan). Body Mass Index (BMI) was calculated by dividing body mass (kg) by height (m), squared.
Plasma biochemistry and oral glucose tolerance testing
Blood samples were drawn after an overnight fast at baseline and after 12 weeks of test substance consumption. OGTT was performed after an overnight fast. Each participant was administered 75 g glucose in 200 g water and blood samples were collected before and 2 h after this glucose load. Plasma was obtained from the fasting blood samples to measure the fasting plasma glucose (FPG), insulin, HbA1c, total cholesterol, low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride (TG), total plasminogen activator-inhibitor-1(PAI-1), aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transpeptidase (γ-GTP) and high-molecular weight (HMW) adiponectin concentrations. In addition, blood samples collected 2 h after glucose loading were used to measure 2-h PG and plasma insulin concentrations. These analyses were performed by the Okayama Medical Association Test Center (Okayama, Japan).
Homeostasis model assessment-insulin resistance (HOMA-IR) and homeostatic model assessment-beta cell function (HOMA-β) were calculated as follows: HOMA-IR = fasting glucose (mg/dL) × fasting insulin (μIU/mL) /405; HOMA-β = (fasting insulin (μIU/mL) × 360) / (fasting glucose (mg/dL) − 63).
During the intervention period, each participant recorded the ingestion of each test sample and any symptoms in a diary. In addition, before and after the test period, each completed a comprehensive questionnaire regarding their lifestyle and health, including a semi-quantitative food frequency questionnaire based on food group (FFQg), using Excel EiyokunTM v3.5 FFQg (Kenpakusha, Tokyo, Japan). FFQg is a food intake survey that evaluates the contents of a daily diet with the simple questions consisting of 29 food groups and 10 different cooking methods. This was conducted during week 0, before the intervention, and during the final week (week 12) of the intervention. The food consumed was analyzed during these periods to ensure that nutritional intake did not change significantly during the trial.
The data obtained from all the participants were analyzed in the first instance. However, we thought that it might not be possible to detect a lowering of plasma glucose concentrations in healthy people who did not originally have high postprandial blood glucose concentrations. Therefore, we also conducted a separate analysis of the participants who had relatively high postprandial blood glucose concentrations. Then, we selected 13 members of each group, whose percentage 2-h PG relative to FPG (2-h PG%) at baseline (week 0) exceeded the mean for all the participants and analyzed their data.
Data are shown as means ± standard deviations. Statistical analyses were performed using SPSS Statistics for Windows, Version 25 (IBM, Armonk, NY, USA). Comparisons of data between two groups in the same week were made using the Mann-Whitney U-test. Comparisons of data between week 0 and week 4, 8, and 12 values in the same group were made using the Wilcoxon signed-rank test. Statistical significance was accepted when P < 0.05. Spearman’s rank correlations were used to evaluate the relationships between 2-h PG and other clinical outcomes.