GLP-1 Release by Rare Sugar D-Allulose Ameliorates Sucrose-Induced Obesity and Glucose Intolerance in Ovariectomized Mice.

1. Introduction
The prevalence of obesity and type 2 diabetes mellitus (T2DM) is increasing worldwide, representing a major public health challenge [1]. T2DM is particularly characterized by an age-dependent increase in prevalence, and its disease burden is expected to further rise with global population aging [2].
In women, the decline in estrogen levels after menopause alters energy homeostasis and promotes visceral fat accumulation [3] as well as insulin resistance [4]. Consistent with these experimental findings, epidemiological studies have shown that postmenopausal women exhibit a higher prevalence of obesity accompanied by increased visceral adiposity and T2DM [5,6]. Hormone replacement therapy has been developed to compensate for estrogen deficiency; however, its long-term use is limited by concerns regarding increased risks of cardiovascular disease and breast cancer [7]. Therefore, there is a strong need to develop safer and more sustainable alternative strategies to prevent or ameliorate obesity and metabolic dysfunction in postmenopausal women.
Dietary factors play a critical role in the development of T2DM, and excessive intake of free sugars is considered a major risk factor [8]. Free sugars are defined as monosaccharides and disaccharides added to foods and beverages, as well as sugars naturally present in fruit juices, syrups, and honey; typical examples include D-glucose, D-fructose, and sucrose. The World Health Organization (WHO) recommends limiting the intake of free sugars to prevent non-communicable diseases, including obesity and T2DM [9]. Although the prevalence of T2DM increases with age, it remains unclear whether and to what extent free sugar intake contributes to obesity and T2DM development, specifically in postmenopausal women.
In recent years, gut hormones have attracted considerable attention for their anti-obesity and antidiabetic effects. Among them, glucagon-like peptide-1 (GLP-1), an incretin hormone, has been extensively studied. GLP-1 receptor agonists improve glycemic control by enhancing insulin secretion from pancreatic β-cells and also induce body weight loss through appetite suppression [10]. However, these pharmacological agents are associated with gastrointestinal adverse effects and weight regain after treatment discontinuation, highlighting the need for more physiological and sustainable approaches to improve glucose metabolism [11,12,13].
Rare sugars are a group of monosaccharides that occur only in trace amounts in nature. D-allulose, a rare sugar and the C-3 epimer of D-fructose, has recently become available for dietary use owing to the establishment of large-scale production methods [14]. GLP-1 secretion is stimulated by caloric macronutrients; however, the zero-calorie rare sugar D-allulose robustly stimulates GLP-1 secretion in mice, rats, and humans [15,16,17]. Furthermore, studies in rodents have demonstrated that D-allulose improves hyperphagic obesity and T2DM via activation of the GLP-1 receptor [15], and human studies have shown that D-allulose suppresses postprandial glucose excursions [18]. However, whether D-allulose exerts anti-obesity and antidiabetic effects under postmenopausal conditions characterized by visceral obesity and glucose intolerance has not been investigated.
In the present study, we used ovariectomized (OVX) female C57BL/6J mice as a model of menopause. First, we examined whether subchronic sucrose intake exacerbates visceral obesity and glucose intolerance in sham-operated and OVX mice. Next, we assessed whether subchronic administration of D-allulose ameliorates sucrose-induced visceral fat accumulation and glucose intolerance in OVX mice. Finally, we investigated the involvement of the GLP-1 receptor in these effects using GLP-1 receptor knockout mice.

2. Results

2.1. Ovariectomy Induces Body Weight Gain and Glucose Intolerance (Experiment 1)
Body weight and glucose metabolism were compared between ovariectomized (OVX) and sham-operated female mice (Experiment 1). OVX mice exhibited a rapid increase in body weight between postoperative Days 8 and 14, and body weight was significantly higher than that of sham-operated mice from Days 12 to 29 after surgery (Figure 1A). Although body weight in OVX mice remained relatively stable after Day 30, OVX mice continued to show a modest but significant increase in body weight compared with sham-operated mice during the late postoperative period (Days 40–57) (Figure 1A).
An intraperitoneal glucose tolerance test (IPGTT) was performed on postoperative Day 43 (Figure 2A). OVX mice exhibited significantly elevated blood glucose levels at 15, 30, and 60 min after glucose administration compared with sham-operated mice (Figure 2B), resulting in a significant increase in the area under the curve (AUC) for blood glucose from 0 to 120 min (Figure 2C). In contrast, plasma insulin levels did not differ significantly between the two groups at any time point examined (Figure 2D). These findings suggest that OVX-induced glucose intolerance is associated with reduced insulin sensitivity.
On postoperative Day 63, visceral adipose tissue mass (the sum of mesenteric, perirenal, and periuterine white adipose tissues) and fasting blood glucose levels were measured. OVX mice exhibited a marked reduction in uterine weight, confirming the effectiveness of ovariectomy (Figure 3A). Although OVX tended to increase visceral fat mass, body weight, and fasting blood glucose levels, these differences did not reach statistical significance at this time point (Figure 3B–D).

2.2. Excessive Sucrose Intake After Ovariectomy Exacerbates Visceral Obesity, Glucose Intolerance, and Insulin Resistance (Experiment 2)
Next, we examined the effects of excessive sucrose intake after ovariectomy on metabolic outcomes (Experiment 2). From postoperative Day 33, when body weight gain in OVX mice had plateaued, mice were allowed free access to either water or a 25% sucrose solution using a two-bottle choice paradigm. Body weight and glucose tolerance were subsequently assessed.
In OVX mice, body weight continued to increase from Day 33 to Day 78 when sucrose was available, whereas sham-operated mice showed no marked body weight gain under the same conditions (Figure 1B). As a result, OVX mice exhibited significantly higher body weight than sham-operated mice from Days 41 to 79 (Figure 1B).
Both OVX and sham-operated mice strongly preferred the sucrose solution (Figure 1C). Sucrose drinking reduced the intake of standard CE-2 chow in both groups (Figure 1D); however, total daily energy intake was not altered (Figure 1E). In addition, cumulative energy intake from Days 33 to 80 did not differ significantly between OVX and sham-operated mice (Figure 1F). Thus, OVX mice gained more body weight than sham-operated mice despite comparable total energy intake when sucrose was freely available.
IPGTT was performed on postoperative Day 71 (Figure 2E). OVX mice with access to sucrose exhibited significantly higher blood glucose levels after glucose injection, resulting in markedly impaired glucose tolerance (Figure 2F,G). At the same time, plasma insulin levels at 0, 15, and 30 min after glucose administration were significantly higher in sucrose-drinking OVX mice than in sucrose-drinking sham-operated mice (Figure 2H). Baseline blood glucose and plasma insulin levels at 0 min before IPGTT were compared between Experiments 1 and 2 (Figure 2I–K). OVX alone led to only modest increases in these parameters, whereas OVX mice with sucrose access showed significantly elevated fasting blood glucose and plasma insulin levels (Figure 2I,J), accompanied by a significant increase in HOMA-IR (Figure 2K). Furthermore, analysis of the 30-min AUC for total blood glucose and total insulin during IPGTT revealed that OVX alone caused only minor changes (OVX/Sucrose, −/− vs. +/−), whereas the combination of OVX and sucrose intake resulted in significant increases in both glucose and insulin AUCs (OVX/Sucrose, −/+ vs. +/+; Figure 2L,M).
On Day 82, visceral adipose tissue mass and fasting blood glucose levels were assessed. Sucrose intake did not significantly affect visceral fat mass, body weight, or fasting blood glucose levels in sham-operated mice (OVX/Sucrose, −/− vs. −/+; Figure 3B–D). In contrast, the combination of OVX and sucrose intake (OVX/Sucrose, +/+) resulted in significant increases in visceral adipose tissue mass, body weight, and fasting blood glucose levels (Figure 3B–D). Uterine weight was significantly reduced by OVX (Figure 3A).

2.3. Subchronic Administration of the Rare Sugar D-Allulose Ameliorates Ovariectomy- and Sucrose-Induced Visceral Obesity and Metabolic Dysfunction (Experiment 3)
We examined whether subchronic administration of the rare sugar D-allulose ameliorates metabolic dysfunction induced by OVX and sucrose intake, and whether these effects are mediated by the GLP-1 receptor using GLP-1R KO mice (Experiment 3). Female C57BL/6J mice and GLP-1R KO mice were fed a diet containing 25% sucrose and subjected to either OVX or sham surgery on Day 0. Body weight and food intake were measured daily. From Day 5 after surgery, mice received either water or D-allulose at 3 g/kg once daily by oral gavage at the onset of the light period for 2 weeks.
Water-treated OVX mice exhibited a marked and continuous increase in body weight from Days 5 to 15 compared with water-treated sham-operated mice, after which body weight reached a plateau (Figure 4A). In contrast, daily food intake in OVX mice showed only occasional increases and did not differ substantially from that in sham-operated mice (Figure 4B). Daily administration of D-allulose to OVX mice did not markedly affect body weight gain and food intake compared with water-treated OVX mice (Figure 4A,B). Although oral gavage administration of D-allulose has been reported to acutely suppress food intake [15], detailed analysis on Day 13 showed that oral gavage of D-allulose significantly reduced food intake at 3 h after administration, whereas this anorexigenic effect was no longer evident at later time points (6 and 12 h) (Figure S1A). The OVX-induced increases in body weight and food intake observed in wild-type C57BL/6J mice were similarly observed in OVX GLP-1R KO mice (Figure 4C,D). Moreover, continuous administration of D-allulose to OVX GLP-1R KO mice did not alter OVX-induced body weight gain or daily food intake, consistent with the findings in wild-type mice (Figure 4C,D). In contrast, the acute anorexigenic effect of a single intragastric administration of D-allulose was completely abolished in GLP-1R KO mice (Figure S1B).
Next, we evaluated the effects of subchronic D-allulose administration on glucose intolerance induced by OVX and sucrose intake (Figure 5). IPGTT was performed on Day 15, after 2 weeks of D-allulose treatment. Water-treated OVX mice exhibited significantly higher blood glucose levels at 0, 15, and 30 min after glucose loading compared with water-treated sham-operated mice (Figure 5A), resulting in a significant increase in the 0–120 min AUC for total blood glucose (Figure 5B). Subchronic administration of D-allulose for 2 weeks completely reversed OVX-induced glucose intolerance, restoring blood glucose levels and glucose AUC to values comparable to those of sham-operated mice (Figure 5A,B). In contrast, the glucose-lowering effect of D-allulose was completely abolished in GLP-1R KO mice (Figure 5C,D). These results indicate that glucose intolerance induced by OVX and sucrose intake is ameliorated by sustained D-allulose administration via a GLP-1 receptor-dependent mechanism.
On Day 20, mice were analyzed, and a significant reduction in uterine weight was confirmed in OVX mice (Figure 6A). We then assessed weight of visceral adipose tissue, fasting blood glucose levels, and plasma insulin levels. In wild-type mice, the combination of OVX and sucrose intake significantly increased visceral adipose tissue mass, fasting blood glucose levels, plasma insulin levels, and HOMA-IR (Figure 6B,D–F). Subchronic administration of D-allulose for 2 weeks significantly attenuated these increases and restored these parameters to levels comparable to those observed in sham-operated mice (Figure 6B,D–F). In contrast, D-allulose did not markedly affect body weight gain in OVX mice (Figure 6C). In GLP-1R KO mice, OVX combined with sucrose intake significantly increased visceral adipose tissue mass, body weight, and fasting blood glucose levels, whereas plasma insulin levels and HOMA-IR were not significantly altered (Figure 6B–F). Moreover, subchronic administration of D-allulose failed to suppress the OVX- and sucrose-induced increases in visceral adipose tissue mass and fasting blood glucose levels in GLP-1R KO mice (Figure 6B,D). These findings indicate that the suppressive effects of D-allulose on visceral fat accumulation and hyperglycemia are mediated by the GLP-1 receptor.

3. Discussion

3.1. Major Findings of This Study
In this study, we used the OVX mice model to investigate the effects of dietary sucrose intake on metabolic homeostasis under postmenopausal conditions and to evaluate the therapeutic potential of the rare sugar D-allulose. We found that voluntary sucrose consumption markedly exacerbated visceral fat accumulation and glucose intolerance in OVX mice, despite comparable total energy intake. Importantly, subchronic oral gavage administration of D-allulose to sucrose-fed OVX mice significantly suppressed visceral adiposity and improved insulin resistance and glucose intolerance without altering total food intake. These beneficial effects of D-allulose were markedly attenuated in GLP-1R KO mice, indicating that its metabolic improvements are mediated by GLP-1 receptor-dependent signaling. Taken together, our findings suggest that metabolic dysfunction under postmenopausal conditions is strongly influenced not only by hormonal changes but also by excessive intake of free sugars, and that gut hormone-mediated pathways, particularly GLP-1 signaling, play a critical role in both disease pathogenesis and nutritional intervention.

3.2. Ovariectomy Enhances Sucrose Sensitivity and Exacerbates Obesity and Glucose Intolerance
In the present study, OVX mice given free access to sucrose in a two-bottle choice paradigm showed marked visceral fat accumulation and increased insulin resistance, whereas these changes were not observed in sham-operated mice. Importantly, although excessive sucrose intake reduced chow consumption, total energy intake did not differ significantly either before and after OVX or between OVX and sham groups. These findings indicate that the obesity and glucose metabolic abnormalities observed in this model are not attributable to increased total caloric intake. These findings further suggest that OVX enhances sensitivity to sucrose, thereby predisposing animals to obesity and glucose metabolic dysfunction. While both menopause and excessive intake of free sugars have been widely reported as independent risk factors for obesity and metabolic syndrome, animal studies directly comparing metabolic responses under identical high-sucrose conditions in the presence or absence of OVX remain limited. However, it remains unclear how estrogen deficiency following OVX alters sucrose metabolism to promote visceral fat accumulation and impair glucose homeostasis.
Sucrose is a disaccharide composed of D-glucose and D-fructose; while D-glucose functions as a major energy source for peripheral tissues, D-fructose is primarily metabolized in the liver and readily utilized for lipid synthesis [19,20,21]. Estrogen normally restrains lipid synthesis and regulates lipid distribution in the liver and adipose tissues [22,23]. In contrast, estrogen deficiency following OVX may weaken these regulatory controls, thereby facilitating hepatic conversion of sucrose-derived carbohydrates into lipids and their preferential accumulation as visceral fat [24]. Furthermore, accumulation of visceral adipose tissue is well known to induce insulin resistance through increased production of inflammatory cytokines and dysregulated adipokine secretion [25,26]. Consistent with these observations, sucrose-fed OVX mice in the present study showed a significant increase in visceral fat accumulation, which was accompanied by a significant elevation in HOMA-IR. These results suggest that the progression of insulin resistance associated with increased visceral adiposity is likely a major factor contributing to the worsening of glucose intolerance in this model. Taken together, estrogen deficiency induced by OVX may alter D-fructose metabolism, thereby exacerbating visceral obesity and insulin resistance. Nevertheless, this interpretation remains speculative and warrants careful consideration. Excessive sucrose intake markedly reduced normal chow consumption to approximately one-third of usual levels, resulting in decreased intake of protein, lipids, vitamins, and minerals. In Experiment 2, daily energy intake remained constant at approximately 13 kcal/day throughout the study, both during the period when sucrose solution was unavailable (until Day 33) and after sucrose access was initiated (from Day 34 onward). Before sucrose access, all energy was derived from the standard chow (CE-2; protein content 29.06% kcal), resulting in a daily protein intake of 3.77 kcal/day. In contrast, after sucrose became available, chow intake decreased to approximately one-third of baseline, reducing protein intake to 1.26 kcal/day. Based on the recommended maintenance diet for mice (AIN-93M [27]), which contains 13.7% of energy from protein, the estimated required protein intake at 13 kcal/day is approximately 1.78 kcal/day. Thus, protein intake during the sucrose-access period fell below the recommended level. Therefore, the potential contribution of relative protein insufficiency and protein malnutrition, as well as other nutrient imbalances, to the observed metabolic phenotypes should be considered and warrants further investigation.
The metabolic effects of fructose and sucrose are known to differ depending on the form of intake, with sugar consumption in liquid form more strongly associated with obesity and diabetes than intake from solid foods [28,29]. Consistent with this, body weight gain in the present study was more pronounced when sucrose was provided as a beverage rather than incorporated into the diet (Experiment 2 vs. 3). However, the underlying mechanism remains unclear. In Experiment 3, the protein intake from the sucrose-enriched CE-2 diet was 2.72 kcal/day (protein content: 20.96% kcal, energy intake: 13 kcal/day), which was approximately twofold higher than the protein intake during sucrose consumption in liquid form in Experiment 2 (1.26 kcal/day). These findings suggest that the greater weight gain observed with liquid sucrose intake may, at least in part, be attributable to reduced protein intake and associated metabolic dysregulation.

3.3. OVX-Induced Disruption of Feeding Rhythms
Recent studies have shown that disruption of feeding rhythms contributes to obesity and glucose intolerance, particularly when food intake occurs during the rest phase [30,31]. Estrogen deficiency induced by OVX disrupts both feeding rhythms and peripheral circadian gene expression, whereas estrogen replacement largely restores these abnormalities, indicating a critical role of ovarian hormones in coordinating feeding behavior with circadian regulation [32]. Consistent with this, OVX induced increased light-phase feeding in the present study (Figure S2). However, complete suppression of light-phase feeding by time-restricted feeding failed to improve OVX-induced weight gain or glucose intolerance, although fasting glucose was modestly reduced (Figure S3). Conversely, subchronic D-allulose administration improved visceral obesity and glucose tolerance in a GLP-1 receptor-dependent manner without correcting light-phase hyperphagia (Figure S2, Figure 4, Figure 5 and Figure 6). Together, these findings suggest that light-phase hyperphagia plays a limited role in the development of obesity and glucose intolerance in OVX mice.

3.4. Mechanisms Underlying OVX-Induced Metabolic Dysfunction and Its Amelioration by Allulose
Obesity after menopause has been attributed not to increased energy intake but to reduced energy expenditure, as demonstrated in previous studies using OVX rodent models [33]. Our results support this view, as sucrose-fed OVX mice developed overweight and visceral obesity despite similar total energy intake compared with sham-operated control mice. Estrogen regulates energy metabolism at both central and peripheral levels and promotes basal metabolic rate, physical activity, and thermogenesis in brown adipose tissue [34,35]. Therefore, loss of these estrogen-dependent mechanisms after OVX reduces energy expenditure, thereby promoting fat accumulation even without an increase in caloric intake [33].
In the present study, we demonstrate that subchronic administration of D-allulose ameliorates OVX- and sucrose-induced visceral obesity, insulin resistance, and impaired glucose tolerance, and that these beneficial effects are largely dependent on GLP-1R signaling. Notably, while D-allulose did not markedly suppress overall body weight gain, it selectively reduced visceral adiposity and improved glucose metabolism, suggesting a preferential attenuation of sucrose-associated metabolic dysfunction. Previous studies have shown that estrogen regulates GLP-1 synthesis and secretion in the gut and pancreas, and that circulating GLP-1 levels following glucose challenge are reduced in OVX mice but restored by estrogen replacement [36]. In addition, the anorexigenic and weight-reducing effects of GLP-1 receptor agonists have been reported to vary across the estrous cycle in accordance with fluctuations in circulating estrogen levels [37]. Together, these findings suggest that GLP-1/GLP-1R-mediated control of the glucose metabolism may be compromised under conditions of estrogen deficiency. Consistent with this notion, our results show that D-allulose, which stimulates endogenous GLP-1 secretion and activates GLP-1 receptor signaling, effectively improves glucose metabolic abnormalities in OVX mice. Moreover, D-allulose has been reported to enhance energy expenditure [38,39], raising the possibility that restoration of reduced energy expenditure contributes to the observed improvements in glucose homeostasis and visceral adiposity in OVX mice. Collectively, our findings provide a mechanistic basis for the use of GLP-1-stimulating functional ingredients, such as D-allulose, as a safe and effective nutritional strategy to mitigate obesity and glucose metabolic dysfunction associated with menopause.

3.5. Effective Dose of D-Allulose and Its Potential for Future Applications
In this study, the effective dose of D-allulose in mice was 3 g/kg. For dose extrapolation from animals to humans, a conversion factor of 12.3 published by the U.S. Food and Drug Administration (FDA) is commonly used. Using this factor, the human-equivalent dose is estimated to be approximately 15 g for a 60 kg adult. Importantly, previous human intervention studies have shown that D-allulose intake in the range of 5–25 g stimulates GLP-1 secretion and suppresses the postprandial glycemic response [17,18,40]. Therefore, the dose used in the present mouse study is well within a feasible range for human intake. These findings support D-allulose as a practical “nutritional GLP-1 stimulator” and a safe, sustainable alternative to GLP-1 receptor agonists for the prevention of visceral obesity and type 2 diabetes, particularly in postmenopausal women.

4. Materials and Methods

4.1. Mice
C57BL/6J female mice (The Jackson Laboratory Japan, Inc., Yokohama, Japan) and whole-body GLP-1 receptor knockout (Glp1r−/−, GLP-1R KO) mice on the C57BL/6J background generated as described previously were kindly provided by Dr. Daniel J. Drucker (Lunenfeld Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, ON, Canada) [41]. All mice were housed under controlled temperature (22.5 ± 2 °C), humidity (55 ± 10%), and a 12-h light/dark cycle (lights on 07:30–19:30). Standard laboratory chow (CE-2, CLEA Japan, Tokyo, Japan) and water were available ad libitum. Purchased mice were allowed to acclimate to the facility for at least one week before any procedures. All mice aged 8–20 weeks were adequately habituated to handling before the experiments. The animal experiments were carried out after receiving approval from the Institutional Animal Experiment Committee of the Kyoto Prefectural University and in accordance with the Institutional Regulations for Animal Experiments (approval number: KPU060327-RC-4, KPU060327-RC-5).

4.2. Ovariectomy
Ovariectomy (OVX) was performed under anesthesia induced by intraperitoneal (IP) administration of a three-drug combination (MMB anesthetic) consisting of medetomidine (0.75 mg/kg; Nippon Zenyaku Kogyo, Koriyama, Japan), midazolam (4.0 mg/kg; Maruishi Pharmaceutical, Osaka, Japan), and butorphanol (5.0 mg/kg; Meiji Seika Pharma, Tokyo, Japan). Small bilateral flank incisions (~3–4 mm) were made to identify the ovaries under a microscope, and the ovaries were surgically removed. In sham-operated mice, the ovaries were identified but not removed. The muscle layer was closed with sutures, and the skin was closed using surgical clips. After surgery, atipamezole (0.75 mg/kg, IP; Nippon Zenyaku Kogyo) was administered to reverse anesthesia, and mice were kept on a heating pad maintained at 38 °C until fully ambulatory. Successful ovariectomy was confirmed by measuring plasma 17β-estradiol levels using an ELISA kit (ADI-900-174, Enzo Life Science, Farmingdale, NY, USA) on postoperative Day 26; plasma levels were significantly lower in OVX mice than in sham-operated mice (means ± SEM; 47.2 ± 5.60 pg/mL vs. 23.2 ± 6.52 pg/mL, n = 5–6, p < 0.05 by unpaired t-test).

4.3. Preparation of Sucrose-Enriched CE-2 Diet
A sucrose-enriched CE-2 diet was prepared by adding sucrose at 25% (w/w) to powdered standard CE-2 chow (CLEA Japan). In the standard CE-2 diet, protein and fat contributed 29.1% and 12.5% of kcal, respectively, whereas in the sucrose-enriched CE-2 diet, protein and fat contributed 21.0% and 4.0% of kcal, comparable to the protein content of standard laboratory diets such as AIN-93G. In contrast, the sucrose-enriched diet contained 27.9% of kcal as sucrose. Preference for the sucrose-enriched CE-2 diet vs. the standard CE-2 diet was assessed using a 48-h two-choice preference test with two food feeders. The sucrose-enriched CE-2 diet showed a preference ratio of 96.9 ± 0.83% (n = 12), indicating that it was highly palatable.

4.4. Experiment 1: Assessment of the Effects of Ovariectomy
OVX mice (n = 10) and sham-operated mice (n = 10) were housed with ad libitum access to solid CE-2 chow and water provided in a single bottle, and body weight was monitored until postoperative Day 57. An intraperitoneal glucose tolerance test (IPGTT, glucose 2 g/kg) was performed on postoperative Day 43. Mice were sacrificed on postoperative Day 63. On the day of sacrifice, mice were fasted from 7:30, and blood glucose levels were measured from tail vein blood at 13:00. Mice were then euthanized by isoflurane overdose, and visceral while adipose tissue (mesenteric, perirenal, and periovarian depots) and uterus were excised and weighed. Experiments were conducted using both group housing (5 mice per cage) and individual housing. As no differences were observed in any measured parameters between housing conditions, data from both housing conditions were pooled for analysis.

4.5. Experiment 2: Assessment of the Effects of Ovariectomy and Two-Bottle Choice Sucrose Loading
OVX mice (n = 9) and sham-operated mice (n = 10) were housed in either group cages or individual cages with ad libitum access to solid CE-2 chow and water provided in a single bottle, and body weight was monitored until postoperative Day 32. From Day 33, drinking water was provided under a two-bottle choice condition, allowing free choice between water and the 25% sucrose solution. CE-2 chow continued to be provided ad libitum throughout this period. In individually housed mice, daily CE-2 food intake and sucrose solution intake were measured. To accurately measure fluid intake, drinking bottles equipped with a ball-bearing sipper tube to prevent leakage were used (TD-101, Tokiwa Kagaku Kikai, Tokyo, Japan). IPGTT was performed on Day 71, and mice were sacrificed on Day 82 using the same procedures as in Experiment 1. Consistent with Experiment 1, housing conditions did not affect any measured parameters, and data were pooled for analysis.

4.6. Experiment 3: Evaluation of D-Allulose Effects on Metabolic Dysfunction Induced by Ovariectomy and Sucrose-Enriched Diet
Female C57BL/6J mice or GLP-1R KO mice were used. From 5 days before surgery (Day −5), mice were switched from a standard CE-2 diet to a sucrose-enriched CE-2 diet containing 25% (w/w) sucrose and were allowed ad libitum access to the diet. D-allulose was provided by Matsutani Chemical Industry Co. Ltd. (Itami, Japan), with purities exceeding 98%. From Day 5, mice received a once-daily oral gavage of D-allulose (3 g/10 mL/kg) or water (10 mL/kg) for 2 weeks at 7:30. During the experimental period, food intake was measured at 7:30 and 19:30, and body weight was measured at 7:30. On Day 13, to assess light-phase feeding behavior in greater detail, food intake was additionally measured at 10:30 and 13:30. IPGTT (glucose 2 g/kg) was performed on Day 15. On Day 20, mice were sacrificed using the same procedures as in Experiment 1. Tail vein blood was collected at 13:00 for insulin measurements.

4.7. Measurements of Food Intake
Mice were housed and habituated for at least one week to a powdered standard CE-2 chow diet (CLEA Japan) in a feeding box (Shinano Manufacturing Co., Ltd., Tokyo, Japan). The weights of the feeding box containing the powdered food and the food spillage on the cage floor were measured at 07:30 and 19:30. Food intake was expressed in kilocalories (kcal) and calculated using an energy density of 3.4 kcal/g for the standard CE-2 chow and 3.59 kcal/g for the sucrose-enriched CE-2 diet. The intake of 25% sucrose solution was determined from changes in bottle weight, and sucrose intake was calculated using an energy density of 4 kcal/g.

4.8. Glucose Tolerance Test
Mice were fasted for 5–5.5 h (from 9:00–14:00 in Exp. 1 and 2, from 7:30–13:00 in Exp. 3). Baseline blood glucose levels were measured from the tail vein, followed immediately by intraperitoneal injection of glucose at 2 g/kg. Subsequently, blood samples were collected from the tail vein at 15, 30, 60, and 120 min. Blood glucose levels were measured using the GlucoCard Plus Care (Arkray, Kyoto, Japan), and blood samples were collected using heparinized capillary glass tubes for plasma insulin assays. Plasma was collected after centrifugation (4000 rpm, 10 min at 4 °C) and stored at −80 °C until analysis. Plasma insulin concentrations were determined using an insulin ELISA kit (Morinaga BioScience, Yokohama, Kanagawa, Japan). No allulose or water was administered on the day of the IPGTT. In this study, IPGTT was used instead of an oral glucose tolerance test to minimize the influence of gastrointestinal digestion, absorption, and gut-derived incretin effects on blood glucose responses, thereby allowing a more direct assessment of systemic glucose metabolism.

4.9. Statistical Analysis
All data are shown as means ± SEM. Statistical analysis was performed using a two-tailed unpaired t-test, one-way ANOVA, or two-way ANOVA, as appropriate. When one-way or two-way ANOVA revealed significant differences, including significant main effects and/or time × factor interactions in two-way ANOVA, post hoc comparisons were conducted using Tukey’s or Bonferroni’s test. All analyses were conducted using Prism 10 (GraphPad Software, San Diego, CA, USA), and p < 0.05 was considered significant.