MCT oil was purchased from Nisshin Oillio Group Ltd. (Tokyo, Japan). The fatty acid composition of MCT oil was 75.0% octanoic acid and 25.0% decanoic acid. Soybean oil and beef tallow, long-chain triglycerides (LCTs), were purchased from Yamakei Industry Co. Ltd. (Osaka, Japan). The soybean oil was composed of 10.3% palmitic acid, 3.8% stearic acid, 24.3% oleic acid, 52.7% linoleic acid, and 7.9% α-linolenic acid. The beef tallow consisted of 24.0% palmitic acid, 16.6% stearic acid, 46.5% oleic acid, 2.8% linoleic acid, and 0.3% α-linolenic acid. d-allulose was obtained from the International Institute of Rare Sugar Research and Education (Kagawa, Japan). Vitamin and mineral mixtures (AIN-76A) were purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan).

Thirty-two male Wistar rats (3-week-old) were obtained from Japan SLC (Shizuoka, Japan), adapted to the experimental environment for three days, and then randomly divided into four groups: control (C), d-allulose (A), MCT (M), and d-allulose + MCT (AM) groups. Table 1 shows the dietary composition of each group. The A and AM diets contained 5% d-allulose, whereas the M and AM diets contained 2% MCT. All experimental diets were obesity-inducing high-fat diets with high concentrations of sucrose added to increase fat synthesis in the

¹Based on the AIN-76 mixture. C, A, M, and AM are the control, d-allulose, MCT, and d-allulose + MCT diets, respectively.

liver. All animals were individually caged at 22˚C ± 1˚C, under light from 08:00 to 20:00, and had free access to the experimental diets and water for 16 weeks. The body weight and food intake were recorded daily. After the experimental period, all rats were euthanized via decapitation at 09:00 after 5 h of fasting. Blood was collected to obtain the serum. The heart, liver, kidneys, spleen, and abdominal adipose tissues (epididymal, perirenal, and mesenteric) were quickly removed and stored at −80˚C until analysis. Carcass samples were obtained by removing the head and remaining intra-abdominal and intra-thoracic tissues, and stored at −20˚C until analysis of carcass composition.

Serum concentrations of glucose, insulin, total cholesterol, free fatty acids, triglycerides, and β-hydroxybutyric acid were determined using kits (Glucose CII-test, LBIS Rat Insulin ELISA Kit, Cholesterol E-Test, NEFA C-Test, Triglyceride E-Test [FUJIFILM Wako Chemicals, Osaka, Japan], and F-kit d-3-hydroxybutyric acid [J.K. International, Tokyo, Japan], respectively). Liver lipids were extracted using the method described by Matyash et al. [16] , and liver triglyceride and cholesterol levels were determined using kits (Triglyceride E-Test and Cholesterol E-Test [FUJIFILM Wako Chemicals, Osaka, Japan]). Liver glucose-6-phosphate dehydrogenase (G6PD) and malate dehydrogenase (MDH) activities were determined using the QuantiChromTM Glucose-6-Phosphate Dehydrogenase Kit and EnzyChromTM Malate Dehydrogenase Assay Kit, respectively (BioAssay Systems, CA, USA). The carcass fat content was determined using the method described by Mickelsen and Anderson [17] . The total body fat was calculated as described by Paik and Yearick [18] .

Data from Groups C, A, M, and AM were analyzed using a two-way analysis of variance (ANOVA). If the ANOVA results revealed significant interactions, multiple comparisons of the results of all groups were performed using the Tukey−Kramer test. Statistical significance was set at p < 0.05. Excel Statistics (Social Survey Research Information Co., Ltd., Tokyo, Japan) was used for data analyses.

The mean body weight of rats in each group increased throughout the experimental period and was highest in Group C and lowest in Group A ( Figure 1 ). Dietary d-allulose significantly reduced the final body weight, weight gain, and food efficiency ( Table 2 ). Two-way ANOVA confirmed the interaction between d-allulose and MCT and their effects on final body weight, weight gain, and food intake, leading to multiple comparisons ( Table 2 ). The final body weight and weight gain were significantly lower in Group A than that in Group C, but no differences were observed between results of MCT-supplemented groups, Groups M and AM ( Table 2 ). Food intake did not differ among the groups. Heart and spleen weights were not affected by either dietary d-allulose or MCT; however, dietary d-allulose significantly increased the liver and kidney weights ( Table 2 ). Liver weight was significantly higher in Groups C and AM than that in Group M ( Table 2 ). Dietary d-allulose significantly reduced the epididymal, mesenteric, and total adipose tissue weights ( Table 2 ). Two-way ANOVA confirmed the interaction between two factors affecting all intra-abdominal adipose tissue weights, leading to multiple comparisons. The epididymal, mesenteric, and total adipose tissue weights were significantly lower in Group A than that in Group C, whereas no differences were observed between results of MCT-supplemented groups, Groups M and AM ( Table 2 ). Dietary d-allulose reduced the carcass fat and total body fat weights; however, these differences were not statistically significant. Two-way ANOVA confirmed the interaction between two factors affecting the weights and percentages of carcass and total body fat leading to multiple comparisons. The carcass and total body fat weights were significantly lower in Groups A and M than those in Group C, but no differences were observed between the MCT-supplemented groups, Groups M and AM ( Table 2 ). We confirmed that dietary d-allulose inhibited body fat accumulation. d-allulose reduced almost all indicators of body fat accumulation, with and without MCT supplementation ( Table 2 ). This result is consistent with our previous reports [14] [15] . Although weaker than those of d-allulose, the anti-obesity effects of MCT were observed on carcass and total body fat mass ( Table 2 ). In our previous studies [14] [15] , we could not confirm the anti-obesity effects of MCT (5% - 13% in diets). Therefore, the observed anti-obesity effects observed in this study may be due to the reduction in MCT content to 2%. Previously, we also showed that the dietary intake of 15% - 20% structured medium- and long-chain triglycerides (MLCTs; equivalent to 1.7% - 2.3% MCFAs) for eight weeks suppresses the intra-abdominal and carcass fat weights of rats [19] . The results of this study support our previous findings.

Values are the mean ± SE for eight rats. n.s., not significant. Within a row, values with different superscripts are significantly different (p < 0.05). C, A, M, and AM are the control, d-allulose, MCT and d-allulose + MCT groups, respectively.

In this study, we demonstrated the interaction between dietary d-allulose and MCT, which are indicators of obesity, body weight gain, intra-abdominal adipose tissue weights, weights and percentages of carcass and total body fat. However, their effects were not synergistic, as MCT supplementation offset the anti-obesity effects of dietary d-allulose. The mechanisms responsible for these effects remain unknown. However, the following points may provide some insights. In hepatocytes, MCFAs are more prone to oxidation and behave more like glucose than fat [20] . In contrast to LCFAs, MCFAs do not require CPT for mitochondrial transport and readily cross the mitochondrial membrane [21] . Consequently, the oxidation of MCFA is higher than that of LCFA [9] , which increases the influx of acetyl-CoA into the tricarboxylic acid cycle. Hence, MCFA oxidations result in increased levels of acetyl-CoA, leading to increased ketone body production or lipogenesis [22] . In contrast, d-allulose increases CPT activity and β-oxidation [23] [24] , increasing the transport of LCFAs to mitochondria. The combination of d-allulose and MCT greatly increases the influx of fatty acids into the liver mitochondria. This may be supported by the results on d-allulose-induced increase in the serum β-hydroxybutyrate concentration and hepatic activity of MDH, a tricarboxylic acid cycle enzyme. However, because of their limited capacity for ATP production and ketone body synthesis [25] , excess fatty acids are used for lipogenesis via acetyl-CoA and citric acid, thus contributing to fat accumulation in peripheral adipose tissues. Notably, liver G6PD, an enzyme involved in lipogenesis, was not suppressed by dietary d-allulose. In addition to our previous reports [14] [15] , this study confirmed that the anti-obesity effect of dietary d-allulose is weakened by MCT.

Dietary MCT significantly reduced the liver triglyceride content, whereas dietary d-allulose significantly increased the liver cholesterol content ( Table 3 ). Dietary d-allulose significantly increased liver MDH activity, whereas the liver G6PD activity was not affected by both dietary dietary d-allulose and MCT ( Table 3 ). Dietary d-allulose significantly increased the serum concentrations of free fatty acids, triglycerides, and β-hydroxybutyric acid; however, serum glucose, insulin, and total cholesterol levels were not affected by both dietary dietary d-allulose and MCT ( Table 3 ). Furthermore, no interaction was observed between d-allulose and MCT in the liver and serum indicators ( Table 3 ).

Values are the mean ± SE for eight rats. n.s., not significant. Within a row, values with different superscripts are significantly different (p < 0.05). C, A, M, and AM are the control, d-allulose, MCT and d-allulose + MCT groups, respectively. G6PD, glucose-6-phosphate dehydrogenase; MDH, malate dehydrogenase.

Here, dietary d-allulose significantly increased the serum triglyceride and free fatty acid levels. However, these results may have been temporary due to increased lipid metabolism. We previously found that serum triglyceride levels are significantly higher in rats fed a 5% d-allulose diet than in the control rats [7] . Several studies have investigated the effects of d-allulose on lipid metabolism. Kim et al. [26] reported that dietary d-allulose supplementation decreases the expression of lipoprotein lipase, triglyceride-hydrolyzing enzyme. Increase in serum triglyceride levels with d-allulose supplementation may decrease the fatty acid uptake from serum lipoproteins into adipose tissues by suppressing the lipoprotein lipase activity.

In this study, dietary MCT significantly reduced the liver triglyceride level. Several studies have reported the inhibitory effects of dietary MCT on liver triglyceride accumulation. Ronis et al. [27] administered rats isocaloric diets containing 10% - 70% total energy of corn oil or diets in which corn oil was replaced with saturated fats (beef tallow and MCT) for 21 days via total enteral nutrition. They reported that increasing MCT in the diet reduced hepatic steatosis. In addition, Mourad et al. [28] explored the potential MCT supplementation alleviate high-fat diet-induced hepatic steatosis in mice. In contrast to obese mice that remained on a high-fat diet, those that were transitioned to MCT diet showed a remarkable decline in hepatic lipid accumulation to levels similar to those observed in obese mice transitioned to a low-fat diet. The findings of this study partially support these reports.