Many studies have suggested that sugar is one of the dietary factors that is responsible for obesity in the modern society [ 7 9 ]. Sugar substitutes have been receiving more attention than ever before [ 10 12 ]. Thus, it is essential to develop economical and safe sweeteners to replace sugar.-allulose, C-3 epimer of-fructose, is a sugar substitute, which has 70% of the sweetness of sucrose but almost zero calories and is rarely found in nature. It is only present in small quantities in commercial mixtures of-glucose and-fructose obtained from the hydrolysis of sucrose or isomerization of-glucose [ 13 ]. However, currently, it can be manufactured in large quantities from the hydrolysis of sucrose or isomerization of-glucose using enzymatic methods [ 14 ].-allulose is generally recognized as safe (GRAS), according to the United States department of agriculture (USDA) regulations [ 15 ]. Several studies have provided preliminary evidence on the impact of-allulose on lipid metabolism [ 16 19 ]. Although-allulose was suggested to act in a similar manner as-fructose, interestingly, it enhances glucose uptake from liver and suppresses hepatic lipogenic enzyme activities [ 16 ]. In addition, it lowered food intake while it increased energy expenditure during darkness and soleus muscle lipoprotein lipase activity in rats pair-fed the high-sucrose diet [ 17 ].-allulose inhibits dietary fat absorption in the small intestine and increases β-oxidation in fat tissue under pair-feeding conditions in mice fed with a high-fat diet [ 18 ]. Most studies indicated that-allulose induces a decrease in body weight, fat mass, and food or energy intake in experimental animal models [ 19 ]. However, there have been only a few studies that have been conducted in human subjects to test the efficacy of-allulose, and they include some limitations in the form of small sizes of subjects and in the use of placebo material [ 20 ]. As such, we conducted a preliminary study with overweight and obese subjects with compensation. This preliminary study will provide information on the approximate daily dosage of-allulose for the upcoming main study, which would be performed using dual energy X-ray absorptiometry (DEXA) equipment for body fat measurement.

Obesity, a hallmark of the metabolic syndrome, has increased to epidemic proportions worldwide and become a leading cause of morbidity [ 1 3 ]. Obesity changes the general metabolism of the body, as well as its appearance [ 4 ]. It induces metabolic disorders such as type 2 diabetes, dyslipidemia, and cardiovascular diseases with inflammation [ 5 ]. According to the Korea National Health and Nutrition Examination Survey [ 6 ], the incidence of obesity (body mass index (BMI) ≥ 25 kg/m) among adults over 19 years of age in Korea was 26% in 1998. Since then, it has been constantly increasing and reached 30.9% in 2014 (males, 37.7%; females, 23.3%). Further, the risk of developing hypertension, diabetes, and dyslipidemia was about twice as high in obese people as that in people with normal weight.

All measured data were represented as means and standard deviations and/or standard errors. Comparisons among the placebo and d -allulose groups with respect to body composition, plasma lipid profile, T2DM-related biomarker, and indirect toxicity biomarker assessment were analyzed using analysis of covariance (ANCOVA) with independent variable as baseline and treatment. Dunnett’s two-tailed t -test was performed for multiple comparisons to compare the low d -allulose group or high d -allulose group with the placebo group. Significant changes between the baseline and follow-up values within groups were assessed using a paired t -test. A p -value < 0.05 was considered to be statistically significant. Data were analyzed using the SAS software (SAS institute, Inc., Cary, NC, USA).

The levels of plasma adiponectin, leptin, insulin, ghrelin, gastric inhibitory polypeptide (GIP), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) were determined using multiplex detection kits (Bio-Rad, Hercules, CA, USA). All samples were assayed in duplicates and analyzed using the Luminex 200 LabMAP system (Luminex, Austin, TX, USA). Data analyses were carried out using the Bio-Plex Manager software version 4.1.1 (Bio-Rad, Hercules, CA, USA). Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were determined using enzymatic kits (Asan Pharm. Co., Daegu, Korea). The levels of total bilirubin and gamma-glutamyltransferase (γ-GTP) were measured at the Central Laboratory (Seegene, Inc., Seoul, Korea) and analyzed using the Cobas 8000 C702 chemistry analyzer (Roche, Mannheim, Germany). The index of insulin resistance was calculated according to the homeostatic model assessment of insulin resistance (HOMA-IR) formula [ 23 ] as follows:

Plasma lipid concentrations were determined using commercially available kits for total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol (Asan Pharm. Co., Seoul, Korea), and free fatty acids (FFA) (Wako Chemicals, Richmond, VA, USA). Low-density lipoprotein (LDL) cholesterol level was calculated using the Friedewald formula (total cholesterol—HDL cholesterol—(triglycerides/5)) [ 22 ]. Non-HDL cholesterol level was calculated as follows: HDL cholesterol—total cholesterol. The atherogenic index (AI) was calculated as follows: (total cholesterol—HDL cholesterol)/HDL cholesterol. Apolipoprotein A-1 (Apo A-1) concentration was measured using an assay kit (Nittobo, Tokyo, Japan). Lipoprotein-associated phospholipase A2 (Lp-PLA2) concentration was measured using an assay kit from R&D systems (Minneapolis, MN, USA).

For anthropometric and physiological measurements at baseline and 4, 8, and 12 weeks post-test material supplementation, subjects visited the Science Research Center Laboratory at Kyungpook National University between 07:00 and 11:00 h after a 12-h overnight fast. The waist circumference, hip circumference, blood pressure (BP), fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), and lipids were determined at baseline and after 12 weeks in the preliminary trial. The body mass index (BMI), height, weight, and body composition were measured using the X-Scan Plus II body composition analyzer (Jawon Medical Company, Daejeon, Korea). Brivo CT 385 (GE Healthcare, Chicago, IL, USA) was used for the computed assisted tomography (CT) scan, and the CT scans was taken at Doctors Radiology Clinic located in Daegu city. Among all participants, CT scans were only performed for subjects with agreement of radiation exposure. The waist and hip circumferences were measured with an anthropometric tape. The waist circumference was measured as the minimum circumference between the iliac crest and rib cage, and the hip circumference was measured as the maximum width over the greater trochanters. The waist-to-hip ratio (WHR) was calculated by dividing the waist measurement by the hip measurement. FBG, HbA1c, and blood pressure were measured using a glucose analyzer (LifeScan Inc., Milpitas, CA, USA), HbA1c analyzer (Micromat™ Hemoglobin A1c Test; Bio-Rad, Hercules, CA, USA), and automatic BP monitor (Omron, Kyoto, Japan), respectively. In addition, blood samples were collected in ethylenediaminetetraacetic acid (EDTA)-coated tubes and centrifuged at 1000× g for 15 min at 4 °C for plasma assays. Dietary intake was recorded using 24-h dietary recalls for each subject before and during the preliminary trial. Three-day dietary recalls were performed twice at baseline and follow up period, with a total of six 24-h dietary recalls by dietitians. We presented the mean of the three-day dietary intake in each point. The three-day dietary recalls were performed on non-consecutive three days which included two weekdays and one weekend day. Subjects were interviewed and asked what kinds of food they ate and drank on their dietary recall sheet. Nutrition food replicas were provided to help subjects estimate their dietary intakes with exact portions. Nutritional analysis was performed using the CAN-Pro 3.0 software (The Korean Nutrition Society, Seoul, Korea), which provides a comprehensive database for the nutritional content of general foods and special Korean foods. Physical activity levels were scored by the activity evaluation (1, Low; 2, Moderate; 3, High) which was calculated with multiplication of intensity, duration, and frequency.

This research was conducted from March to August 2016 as a randomized, double-blinded, parallel, placebo-controlled study. The random allocation sequence was created using computer generated random numbers. For randomization, all subjects were randomly assigned in a 1:1:1 ratio to the following three nutritional intervention groups: placebo control (= 48), low-allulose (= 48), and high-allulose (= 48) groups. The mechanism used for allocation concealment was sequentially numbered containers by an independent laboratory researcher, and participants were kept blinded to the sequence and randomization until the end of the study. The supplement drinks containing-allulose and placebo (sucralose) did not differ in flavor and color, and each participant received the supplement drinks in white plastic bottles. During the study, the participants were instructed to maintain their routine food intake and physical activity, and consume two bottles of supplements per day, with each bottle consumed after a meal, for 12 weeks (April to June 2016). At the end of study, subjects were asked to return any beverage bottles not consumed. The doses of-allulose were selected by extrapolated calculations based on a previous animal study [ 18 ]. The primary outcome markers were body fat mass and body fat percentage, and secondary outcome markers were other body composition factors and plasma obesity-related biomarkers.

Participants were recruited via an advertisement which was posted on the bulletin board in Kyungpook National University and online. All participants were aged 20–40 years old and resided in the Daegu city and its suburb areas in the Republic of Korea in March 2016. After an initial screening, 144 subjects with BMI ≥ 23 kg/m 2 were selected. The exclusion criteria were as follows: (1) Hypertensive taking diuretics; (2) Taking oral hypoglycemic agents or insulin injection; (3) Serious cardiac, renal, hepatic, thyroid, or cerebrovascular disease; (4) Serious cystic or gastrointestinal disease, gout, or porphyria; (5) Psychiatric problems such as depressive disorder, schizophrenia, alcoholic, and drug intoxication; (6) Taking functional food products that may affect the results of this study; (7) A history of surgery within the past 6 months; (8) Cancer diagnosis and treatment; (9) Asthma or other allergies; (10) Pregnant or in lactation period. The study was approved by the Clinical Research Information System (CRIS) of the Ministry of Health and Welfare of Republic of Korea (CRiS, KCT0002084) and the Kyungpook National University Human Research Committee (KNU 2016-17). All subjects gave their written informed consent for inclusion before they participated in the study.

Test materials were provided in a bottle, in the form of a grapefruit flavored non-carbonated drink (30 mL), containing either sucralose or-allulose ( Table 1 ). Participants consumed the placebo or-allulose drinks (two bottles per day). In order to choose the doses of-allulose used in our study, we translated the dose in our previous animal study on the basis of Reagan-Shaw et al.’s report in 2008 [ 18 20 ]. All drinks were supplied by CJ Cheiljedang, Suwon, Kyoung gi-do, Korea. Test drinks were identified by different code numbers, the identities of which were not revealed to the investigator or subjects until the completion of the study.

According to the 2015 Korean Guidelines for management of dyslipidemia, cut-off levels of risk in plasma lipids were as follows [ 24 ]: triglyceride ≥ 200 mg/dL (2.3 mmol/L), total-cholesterol ≥ 240 mg/dL (6.2 mmol/L), LDL-cholesterol ≥ 190 mg/dL (4.1 mmol/L). At baseline and follow-up supplementation, the levels of plasma lipids were lower than the cut-off level in all three groups ( Table 6 ). Compared to the baseline, no significant differences in plasma lipids were observed in the three groups.

The baseline characteristics of volunteers who completed the randomized controlled trial (= 121) are shown in Table 2 . In both male and female subjects, there were no significant differences in age, height, systolic BP, diastolic BP, and fasting blood glucose (FBG) among all groups ( Table 2 ). Physical activity measurement and analysis of 24-h dietary recalls from the subjects indicated no significant differences between baseline and follow-up values within each group and among the three groups ( Table 3 ).

4. Discussion

d -allulose reduces body fat by regulating lipid metabolism in animal models [ d -allulose on human subjects [ d -allulose supplement reduced body weight, body fat percentage, and waist circumference. However, the study had some limitations. First, the sample size in their study (Males: 17, Females: 17) was too small to confirm statistical significance. Second, they used high fructose-corn syrup (HFCS) as placebo material, which can induce and/or accelerate obesity. As such, our study was designed and performed in order to address these limitations. Our results provide useful information regarding the approximate daily dosage of d -allulose for the reduction of body fat in overweight or obese subjects, although body composition measurement was performed using bioelectrical impedance analysis (BIA) instead of dual energy X-ray absorptiometry (DEXA). Many studies have suggested that-allulose reduces body fat by regulating lipid metabolism in animal models [ 18 19 ]. Hayashi et al. conducted a clinical trial to examine the anti-obesity effects of-allulose on human subjects [ 20 ]. In their study,-allulose supplement reduced body weight, body fat percentage, and waist circumference. However, the study had some limitations. First, the sample size in their study (Males: 17, Females: 17) was too small to confirm statistical significance. Second, they used high fructose-corn syrup (HFCS) as placebo material, which can induce and/or accelerate obesity. As such, our study was designed and performed in order to address these limitations. Our results provide useful information regarding the approximate daily dosage of-allulose for the reduction of body fat in overweight or obese subjects, although body composition measurement was performed using bioelectrical impedance analysis (BIA) instead of dual energy X-ray absorptiometry (DEXA).

The placebo control, low d -allulose, and high d -allulose groups were similar in their baseline characteristics, such as age, BMI, BFP, WHR, and FBG. There were no significant differences in the energy and nutrient intake between the three groups during the trial. Thus, d -allulose supplementation did not affect energy and nutrient intake in overweight or obese subjects.

d -allulose supplementation in overweight or obese subjects led to a significant decrease in body fat mass, BMI, and body fat percentage. In comparing results before the study and during the follow up to the study, d -allulose supplementation was observed to reduce body weight, body fat mass, BMI, and body fat percentage. In addition, multiple comparison analysis of CT scan results indicated that the total abdominal fat area and subcutaneous fat area were significantly decreased following high allulose supplementation. In comparing results before the study and during the follow up to the study, low dose d -allulose reduced the total abdominal fat area only; however, high dose d -allulose reduced both the total abdominal fat area and subcutaneous fat area. For an accurate assessment of abdominal fat, a CT scan was used to make measurements directly at the umbilicus [ Multiple comparison analysis showed that-allulose supplementation in overweight or obese subjects led to a significant decrease in body fat mass, BMI, and body fat percentage. In comparing results before the study and during the follow up to the study,-allulose supplementation was observed to reduce body weight, body fat mass, BMI, and body fat percentage. In addition, multiple comparison analysis of CT scan results indicated that the total abdominal fat area and subcutaneous fat area were significantly decreased following high allulose supplementation. In comparing results before the study and during the follow up to the study, low dose-allulose reduced the total abdominal fat area only; however, high dose-allulose reduced both the total abdominal fat area and subcutaneous fat area. For an accurate assessment of abdominal fat, a CT scan was used to make measurements directly at the umbilicus [ 25 ]. Although a CT scan is an expensive form of measurement and requires complicated techniques for assessing body composition, it has proven to be more accurate than anthropometry measurement or BIA [ 26 ].

d -allulose supplementation has been observed to improve obesity-related diseases [27, d -allulose supplement in a high-fat diet for 16 weeks normalized the body weight and body fat mass in diet-induced obese mice [ d -allulose suppressed the dietary fat absorption by regulating the gene expression in the small intestine and increased fecal lipid contents. Also, d -allulose promoted the lipid oxidation in epididymal white adipose tissue. However, in the present study with human subjects, d -allulose supplementation for 12 weeks reduced body fat mass, body fat percentage, and subcutaneous fat (SAT) area while there was no significant reduction in visceral fat (VAT) area. These differential effects of d -allulose could be partly explained by differences in supplementation periods of d -allulose, species, and metabolic rate between SAT and VAT. In general, although both SAT and VAT were significantly correlated with metabolic risk factors, blood pressure, fasting plasma glucose, triglycerides, and high-density lipoprotein cholesterol, VAT remains more strongly associated with adverse metabolic risk profiles [ d -allulose in human subjects could be effective for reducing VAT as well as SAT. Also, in the present study involving overweight or obese subjects, plasma lipid profiles, diabetes-associated indices, and adipokine levels were not significantly different between the baseline and follow-up study. This was probably because we used BMI as the priority cut-off marker for inclusion of subjects and the average age of these overweight or obese adult subjects was not high. Thus, their plasma lipid profiles and blood glucose concentrations were maintained at the baseline, similar to normal values. In animal studies,-allulose supplementation has been observed to improve obesity-related diseases [ 18 28 ]. One of these studies suggested that 5%-allulose supplement in a high-fat diet for 16 weeks normalized the body weight and body fat mass in diet-induced obese mice [ 18 ]. Based on this study,-allulose suppressed the dietary fat absorption by regulating the gene expression in the small intestine and increased fecal lipid contents. Also,-allulose promoted the lipid oxidation in epididymal white adipose tissue. However, in the present study with human subjects,-allulose supplementation for 12 weeks reduced body fat mass, body fat percentage, and subcutaneous fat (SAT) area while there was no significant reduction in visceral fat (VAT) area. These differential effects of-allulose could be partly explained by differences in supplementation periods of-allulose, species, and metabolic rate between SAT and VAT. In general, although both SAT and VAT were significantly correlated with metabolic risk factors, blood pressure, fasting plasma glucose, triglycerides, and high-density lipoprotein cholesterol, VAT remains more strongly associated with adverse metabolic risk profiles [ 29 ]. It can be speculated that long-term supplementation of-allulose in human subjects could be effective for reducing VAT as well as SAT. Also, in the present study involving overweight or obese subjects, plasma lipid profiles, diabetes-associated indices, and adipokine levels were not significantly different between the baseline and follow-up study. This was probably because we used BMI as the priority cut-off marker for inclusion of subjects and the average age of these overweight or obese adult subjects was not high. Thus, their plasma lipid profiles and blood glucose concentrations were maintained at the baseline, similar to normal values.