Obesity is a chronic and complex disease characterized by excessive fat deposition that compromises health. The obesity epidemic has become a serious public health concern. Obesity increases the risk of type 2 diabetes, heart disease, and certain cancers along with affecting the quality of life [1] . It is essential to prevent obesity to avoid metabolic syndrome, particularly in middle-aged and older adults. Rare sugars are monosaccharides and their derivatives that are inherently rarer than common sugars, such as d-glucose and d-fructose (The International Society of Rare Sugars [ISRS], 2002)¹ and can be used as supplements, functional food additives, and medicines. They have recently attracted attention as functional foods with anti-obesity properties. Previous studies have suggested that rare sugars are beneficial to human health when used as low-calorie carbohydrate sweeteners and bulking agents [2] - [4] . Particularly, D-allulose is caloric-free and has various nutritional functions. The anti-obesity effect of D-allulose is mediated by multiple factors, including suppression of hepatic lipogenesis [5] [6] and an increase in energy expenditure [7] .

Capsaicin, a major active compound in chili peppers, has numerous beneficial roles in the human body, including the treatment of obesity [8] . An epidemiological study found that eating foods containing capsaicin is associated with a lower rate of obesity [9] . Several studies have shown that capsaicin helps to regulate lipid metabolism, particularly by reducing body fat levels [10] . Its effects are mediated by the activation of the transient receptor potential vanilloid 1 (TRPV1) ion channel, which is widely distributed and found in adipocytes, the liver, vascular smooth muscle, and nociceptor neurons [11] .

Although D-allulose and capsaicin induce a variety of biological responses after ingestion, they all have in common that they exert their anti-obesity effects (e.g., thermogenesis, lipolysis, and feeding inhibition) via the central nervous system [8] [12] . Although the mechanisms are not yet fully understood, given that D-allulose and capsaicin increase lipid metabolism through different mechanisms, their combination may enhance their anti-obesity effects. In this study, rats were fed a high-fat diet containing D-allulose and capsaicin to determine whether their synergistic effect could contribute to the suppression of body fat accumulation.

All animal procedures were approved by the Animal Care and Use Committee of Kagawa University (approval number: 24624).

Based on the ANOVA results, dietary capsaicin significantly reduced all 1 - 8 weeks body weight at all time points (p < 0.001, Figure 1 ), weight gain, and food intake ( Table 2 ), whereas D-allulose had no effect. The decrease in food intake because of dietary capsaicin was suppressed by concurrent ingestion of D-allulose ( Table 2 ). Food intake was significantly higher in the C and A groups than in the CA and ACA groups ( Table 2 ). Neither D-allulose nor capsaicin affected food efficiency ( Table 2 ). Dietary capsaicin significantly reduced the weights of the heart, liver, kidneys, and spleen, whereas D-allulose significantly increased liver and kidney weights ( Table 2 ). Neither D-allulose nor capsaicin affected the IBAT weight ( Table 2 ).

Dietary capsaicin significantly reduced the epididymal, perirenal, mesenteric, and total abdominal adipose tissue weights, whereas D-allulose had no effect ( Table 2 ). The carcass and total body fat (mass and percentage) were significantly reduced by dietary capsaicin, whereas D-allulose had not significant effect, although a decreasing trend was observed in the percentage of carcass fat (p = 0.069) and total body fat (p = 0.120) ( Table 2 ). Dietary capsaicin significantly reduced carcass protein mass but did not affect carcass protein percentage ( Table 2 ). No two-factor interactions were identified for any of body weight, tissue weight, or body fat indices ( Table 2 ).

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, CA, and ACA represent the control, D-allulose, capasicin, and D-allulose + capsaicin groups, respectively. IBAT: interscapular brown adipose tissue. *The significant p-values are shown in bold (two-way ANOVA).

Dietary capsaicin significantly reduced HDL-cholesterol levels, but neither dietary D-allulose nor capsaicin affected any of the other serum indices, and no two-factor interactions were identified ( Table 3 ). The liver triglyceride content was not affected by dietary D-allulose or capsaicin. Liver cholesterol content was significantly increased by dietary capsaicin but was suppressed by concurrent ingestion with D-allulose ( Table 3 ).

Dietary capsaicin significantly reduced liver G6PD activity and increased liver MDH activity, whereas dietary D-allulose had no effect ( Figure 2 ). Neither dietary D-allulose nor capsaicin affected UCP1 levels in the IBAT and perirenal adipose tissue ( Figure 2 ). No two-factor interactions were identified for liver G6PD and MDH activities or the UCP1 levels in the IBAT and perirenal adipose tissue ( Figure 2 ).

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, CA, and ACA represent the control, D-allulose, capasicin, and D-allulose + capsaicin groups, respectively. IBAT: interscapular brown adipose tissue, G6PD: glucose-6-phosphate dehydrogenase, MDH: malate dehydrogenase, UCP1: uncoupling protein 1. *The significant p-values are shown in bold (two-way ANOVA).

Therapeutic approaches to address obesity involve lifestyle changes such as avoiding a sedentary lifestyle and a balanced diet. Numerous experimental and clinical studies have explored potential dietary and exercise therapies to reduce and prevent obesity [21] - [23] . According to Olateju et al. [22] , the best approach for managing obesity in adults involves a tailored low-calorie diet that considers the metabolic requirements and general health of the individual, along with a combination of strength and endurance exercises for at least 3 hours per week. However, sustaining a low-calorie diet and active exercise for prolonged periods can be challenging. Therefore, alternative anti-obesity treatments that are easy to sustain, effective, safe, and widely available are required. Dietary D-allulose and capsaicin are functional ingredients recognized as easily implemented and sustained anti-obesity therapies. In this study, we determined whether the combined use of these two dietary therapies exerted a synergistic effect by suppressing body fat accumulation. Dietary capsaicin suppressed body fat accumulation as well as that of carcass (reflecting subcutaneous fat) and intra-abdominal fat. In contrast, a trend toward the body fat accumulation inhibition by D-allulose was observed, but was not significant (carcass fat percentage: p = 0.069; total body fat percentage: p = 0.120); additionally, these two factors did not exert synergistic anti-obesity effects. However, the ACA group exhibited the lowest accumulation of intra-abdominal, carcass, and total body fat, indicating an additive effect of combining these two therapies.

Several studies have reported the anti-obesity effects of D-allulose [4] - [7] [24] [25] . Notably, D-allulose had a more significant effect on body fat reduction than d-fructose, its C-3 epimer [26] . These effects are attributed to a combination of factors, including the inhibition of fatty acid synthesis in the liver, promotion of lipid metabolism, increase in energy expenditure, and a decrease in the synthesis of chylomicrons in small intestinal mucosal epithelial cells. However, the present study confirmed that dietary D-allulose only slightly inhibits body fat accumulation and does not affect the activities of liver G6PD and MDH, which are lipogenic and oxidative enzymes, respectively. This may be because the addition of 5% D-allulose used in previous studies [5] [15] [27] was reduced to 3% in this study. Therefore, it is necessary to revisit the effects of 5% D-allulose in the future.

In this study, dietary capsaicin reduced food intake but did not affect feeding efficiency. Thus, the anti-obesity effects of capsaicin may be partly attributed to reduced food intake. Capsaicin or capsaicin-rich peppers have been reported to suppress energy intake and regulate macronutrient intake by modulating appetite and satiety [28] . Yoshioka et al. [29] examined the effects of capsaicin on eating behaviors and energy intake. The authors found that adding red pepper to breakfast significantly reduced protein and fat intake at lunch, and adding red pepper to an appetizer significantly reduced energy and carbohydrate intake for the rest of the lunch course. These effects may be related to increased sympathetic activity [29] . Another possibility is TRPV1 receptor-mediated feeding inhibition, as several capsaicin-induced effects are mediated by stimulation of the TRPV1 receptor [8] [11] . TRPV1 plays a pivotal role in the action of leptin, an appetite-suppressing hormone, in the hypothalamus [30] . Capsaicin suppresses feeding by affecting TRPV1 [31] . Although the effects of capsaicin on appetite remain poorly explored, neural circuits in the hypothalamus may be important targets of capsaicin. The underlying cause of this effect is unknown; however, the involvement of complex energy balance regulatory mechanisms must be considered.

Brown adipose tissue (BAT) plays a crucial role in adaptive thermogenesis, and research has shown that BAT activity can help protect against obesity and metabolic diseases [32] . Saito et al. [33] reported that BAT is activated by cold exposure, as well as various foods or spices such as capsaicin-rich-chili peppers. This activation may involve an increase in energy usage, stimulation of fat breakdown in BAT, an increase in the levels of cyclic adenosine monophosphate and the activity of protein kinase A in BAT. In a recent study in rats [34] , capsaicin offset the negative effects of a high-energy diet, such as glucose intolerance, high cholesterol levels, and reduced BAT activity. The main reason for these effects is the increased expression of key thermogenic genes in BAT, such as UCP1 and zinc finger protein 16. However, in the present study, dietary capsaicin did not influence IBAT and perirenal adipose tissue UCP1 levels. High-fat diets have been reported to increase UCP1 expression [35] , which could explain why no additional impact of capsaicin was observed. Recently, several thermogenic mechanisms have been identified based on ATP sinks centered on creatine, lipid, or calcium cycling, along with fatty acid-mediated UCP1-independent leakage pathways driven by ADP/ATP carriers [36] . In addition, dietary capsaicin significantly reduced liver G6PD activity and increased liver MDH activity, indicating an increased hepatic lipid metabolic turnover. It has been reported that the liver is also involved in heat production [37] . Therefore, the effects of dietary capsaicin on thermogenesis warrant further investigation.

Dietary D-allulose increased liver cholesterol content. Although our previous studies showed similar results [15] [27] [38] , the underlying reasons for these observations remain poorly understood. It is noteworthy that combining dietary D-allulose with capsaicin suppressed hepatic triglyceride and cholesterol levels. In addition, dietary D-allulose significantly increased liver and kidney weights, despite the invariance in final weight and weight gain; similar results have been reported in previous studies [4] [5] [7] . In our previous chronic toxicity study using rats, 1- and 1.5-year administration of D-allulose in the diet resulted in liver and kidney weight gain without inducing any adverse health effects [16] . We suggested that feeding rats a diet containing D-allulose for 4 weeks could increase liver and kidney weights without any pathological impairment, and 10-week D-allulose cessation reversed these effects [39] . In a human study evaluating 12 weeks of ingesting a rare sugar syrup containing 6% D-allulose, Hayashi et al. [3] found no adverse effects or abnormalities in blood parameters when assessing lipid and carbohydrate metabolism, as well as hepatic and renal functions. The results of these studies suggest that D-allulose is a safe monosaccharide in animals and humans. D-allulose has been approved and is generally recognized as safe as a food (GRAS Notice No. GRN 498, Food and Drug Administration, USA, 2017) for use in various foods and dietary supplements.

In conclusion, we confirmed that both dietary D-allulose and capsaicin treatment reduced body fat accumulation in rats fed a high-fat diet, although the effect of D-allulose was not significant. The reason for the lack of significant D-allulose effect may be that the amount of D-allulose added to the diet was too small. These two factors did not induce synergistic anti-obesity effects, and their combined effects appeared to be largely additive. Additionally, the anti-obesity effect of dietary capsaicin appears to be partly owing to the suppression of food intake. However, the mechanisms underlying the anti-obesity effects of dietary D-allulose and capsaicin remain unclear, and warrant clarification in additional investigations.

We would like to thank Editage ( https://www.editage.jp/ ) for English language editing.

¹The International Society of Rare Sugars [ISRS] 2002, https://www.isrs.kagawa-u.ac.jp/ .