1. Introduction
The number of obese people is increasing worldwide due to lifestyle factors, particularly undesirable foods and eating habits in modern societies [1,2]. Diet and medicine are considered the effective avenues to treat them. Recently, two substances have drawn much attention for their outstanding metabolic effects with common and distinct modes of action. D-Allulose, one of the rare sugars present in a small amount in nature, can now be produced in a substantial amount [3,4,5]: it is a nearly zero calorie (<0.4 kcal/g) sweetener [6] and markedly improves metabolism. Oral semaglutide (O-Sema; Rybelsus) is the first oral form of glucagon-like peptide 1 (GLP-1) receptor agonist (GLP-1RA) carrying the absorption enhancer sodium N-[8-(2 ydroxybenzoyl)amino] caprylate (SNAC) [7]: it reduces subjects’ burden while exerting comparable metabolic effects to injectable GLP-1RAs [8,9]. D-Allulose and O-Sema could work complementarily and/or cooperatively to prevent and/or treat obesity and diabetes, but remain unexplored. The present study aimed to compare their metabolic effects to characterize the advantage of each.
Glucagon-like peptide 1 (GLP-1) is an incretin hormone released from the intestine upon meal ingestion to promote glucose-induced insulin secretion and satiety [10,11,12]. GLP-1 has a short life-time, 1–2 min, due to cleavage by dipeptidyl peptidase 4 (DPP4) [11]. GLP-1 regulates glucose metabolism and feeding behavior through the local effect via the gut–brain neural pathway [13]. As a therapeutic approach, the DPP4-resistant GLP-1 receptor agonists (GLP-1RAs) ameliorate type 2 diabetes and obesity [11,14]. Mechanistically, GLP-1RAs drive the central nervous route via targeting the neurons in the arcuate nucleus of the hypothalamus (ARH) [11,15] and the area postrema (AP) in the brain stem to reduce food intake and weight. Intriguingly, GLP-1RAs such as exendine-4 fail to influence the vagal afferent nerve although it is equipped with GLP-1 receptors [16,17].
D-Allulose markedly reduces glycemia in rodents [18] and humans [19] and reduces body weight in rodents [20,21] and humans [22], and hence is considered as a promising agent to ameliorate type 2 diabetes and obesity in humans [23]. Mechanistically, D-Allulose stimulates the release of endogenous GLP-1, a mechanism underlying its metabolic effects in mice [21], rats [24] and humans [25]. Hence, D-Allulose is recognized as the endogenous GLP-1 releaser [12]. The GLP-1, released by D-Allulose, activates the vagal afferent nerve [21], consistent with the ability of native GLP-1 to directly activate vagal afferent neurons [26]. The D-Allulose inhibition of food intake is blunted by GLP-1R knockdown selectively in vagal afferent neurons and by denervation of the vagal nerve [21]. In addition to the GLP-1-vagal route, we have recently reported that D-Allulose per se interacts with and activates ARH neurons and that icv-injected D-Allulose inhibits food intake, showing D-Allulose’s central nervous pathway for regulating feeding [27,28].
Thus, it appears that GLP-1RA employs the central nervous route solely, while D-Allulose employs both the vagal afferent route and central nervous route, suggesting that GLP-1RA and D-Allulose exert both common and distinct effects. However, this remains unexplored. The present study aimed to compare the metabolic effects of GLP-1RA and D-Allulose under the same conditions in diet-induced obese (DIO) mice with hyperglycemia. In clinical setting, it is recommended that subjects take O-Sema with a small volume of water under food- and water-deprived conditions before and after intake in the early morning, the early active phase [29,30]. Notably, O-Sema is as effective in mice as in humans when it is administered under the above-mentioned human protocols in the evening, the early active phase in mice [31,32]. Hence, this study employed the same protocol for administration of O-Sema and D-Allulose, oral gavage in the evening under food- and water-deprived conditions, and examined both acute effects by bolus administration on food intake and sub-chronic effects by daily administration for 10 days on food intake and body weight. Furthermore, to explore the neuronal mechanism in the central nervous route, the effects of two agents on the ARH neurons regulating feeding and metabolism were investigated: one that responds to anorexigenic leptin (leptin-responsive neuron) and the other that responds to orexigenic ghrelin (ghrelin-responsive neuron).
Since obesity often coexists with diabetes, and efficacy of anti-obesity/anti-diabetes agents depends on glycemic states, we also studied the random blood glucose levels and glucose tolerance test (GTT) and their modulation by O-Sema and D-Allulose in DIO mice.
Declined muscle strength and/or mass occasionally occurs in obese subjects and is recognized as sarcopenic obesity, serving as a predictor of risk for cardio-metabolic diseases and physical disability [33]. In addition, subjects with type 2 diabetes often present with sarcopenia [34]. Moreover, weight loss due to diet and/or drug treatment often involves reduced muscle mass and/or strength. This study explored the effects of O-Sema and D-Allulose on grip strength [35] in obese-hyperglycemic DIO mice.
2. Materials and Methods
2.1. Chemicals
Oral Semaglutide (Rybelsus®) (O-Sema) was purchased from Novo Nordisk (Copenhagen, Denmark), and the powder was prepared 1 h before administration [32]. D-Allulose (purity > 98%) was provided by Matsutani Chemical Industry Co. Ltd. (Itami, Japan). Recombinant Murine Leptin was obtained from PeproTech Company (Cranbury, NJ, USA). Ghrelin was from the Peptide Institute (Osaka, Japan). In measurements of cytosolic Ca2+ concentration ([Ca2+]i), test agents were dissolved in HEPES-buffered Krebs-Ringer bicarbonate buffer (HKRB) solution composed of (in mM) 129 NaCl, 5.0 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.8 CaCl2, 1.2 MgSO4 and 10 HEPES with pH adjusted at 7.4 using NaOH.
2.2. Animals
C57BL/6J male mice aged 4 weeks were purchased from Japan SLC (Shizuoka, Japan) and fed with high-fat diet D12492 (Research Diets, Inc. New Brunswick, GA, USA) for 26~36 weeks. Mice were housed in group cages under controlled temperature (23 ± 1 C°) and humidity (55 ± 5%) with a 12 h light/dark cycle (light on/off at 08:00/20:00). The high-fat diet-induced obese (DIO) mice with average body weight around 54 g were used. During handling and throughout the experiments, mice were kept in single cages. Animal experiments were carried out after receiving approval from the Institutional Animal Experiment Committee and in accordance with the Institutional Regulation for Animal Experiments at Gifu University, Gifu, Japan (IACUC approval number; AG-P-C-20230012, approved on 11 May 2023).
2.3. Protocols for Administration of O-Sema and D-Allulose
O-Sema dissolved in 0.1 mL distilled water (DW), D-Allulose dissolved in 0.5 mL DW, and control DW (0.5 mL) were administered by oral gavage at 19:30 under food- and water-deprived conditions following the previous report [32] (Figure 1A). Right after the administration, refeeding started at 20:00, followed by measurements of food intake.
Protocols for administration of O-Sema and D-Allulose and measurements. () Timing of food/water deprivation, administration of agents, refeeding and measurements. In DIO mice, food intake and body weight were measured at 16:00, followed by food deprivation in 16:00–20:00 and water deprivation in 18:00–20:00. O-Sema powder was prepared at 18:30, and O-Sema in 0.1 mL distilled water (DW), D-Allulose dissolved in 0.5 mL DW, and control DW (0.5 mL) were administered by oral gavage at 19:30, followed by refeeding at 20:00 (administration/refed). After administration/refed, food intake was measured at 21:00, 22:00 and 24:00 and the next day at 08:00 and 16:00 (1, 2, 4, 12 and 20 h after administration/refed), and body weight was measured the next day at 16:00. () Design of sub-chronic treatment and discontinuation accompanied by measurements. Food intake and body weight were measured at 16:00 on days 0–10 during treatment and on days 11–16 after discontinuation. Grip strength was measured on days −1, 5 and 8. A B
2.4. Dose Selection for O-Sema and D-Allulose
O-Sema 3, 7 and 14 mg are approved for humans [36,37]. These values were divided by 60 kg (average human body weight), yielding 0.05 mg/kg, 0.12 mg/kg and 0.23 mg/kg: these values were used as the doses equivalent (eq.) to human doses in the present study. A threefold higher dose of 0.7 mg/kg (eq. 42 mg) was also tested in the dose-dependency study. D-Allulose 0.3 g/kg, 1 g/kg, 3 g/kg and 5 g/kg, the effective dose range reported in mice [21], were used in the dose-dependency study.
2.5. Measurements of Food Intake and Body Weight
Food intake and body weight of the mice were measured at 16:00 right before food deprivation from 16:00 and water deprivation from 18:00 (Figure 1A). O-Sema, D-Allulose and DW were administered at 19:30, and 30 min later food and water were returned at 20:00 (defined as “administration/refed” time).
For acute effects of O-Sema and D-Allulose, food intake was measured at 1, 2, 4, 12 and 20 h after refed in DIO mice (34 weeks, body weight 50 g, n = 7/group) (Figure 1A). For sub-chronic effects, food intake and body weight were measured at 16:00 prior to food deprivation for 16:00–20:00 on days 0~10 during treatment and days 11~16 after termination in DIO mice (36 weeks, body weight 54 g, n = 7/group) (Figure 1B).
2.6. Measurements of Random Blood Glucose Levels and Intra-Peritoneal Glucose Tolerance Test (i.p. GTT)
Measurement of random blood glucose in DIO mice (42 weeks, BW 60 g, n = 7/group) was performed under the same protocol as that for food intake (Figure 1A), except that the pre-treatment value was measured at 19:00, prior to administration of O-Sema 0.23 mg/kg (eq. 14 mg), D-Allulose 3 g/kg and DW (control) by oral gavage. Blood samples were collected from the tail vein using heparinized capillary glass tubes, and blood glucose levels were detected using a GLUCOCARD Plus Care GT-1840 (ARKRAY Factory, Inc. Koka, Japan).
i.p. GTT was performed in DIO mice (40 weeks, BW 60 g) fasted throughout experiments, including a 7 h period before glucose injection. At 19:00, O-Sema 0.23 mg/kg (eq. 14 mg), D-Allulose 3 g/kg and DW (control) were administered by oral gavage 30 min before i.p glucose (2 g/kg) injection at 19:30 (0 min). Blood glucose was measured at −30, 0, 30, 60, 120 and 180 min after glucose injection.
2.7. Measurements of Grip Strength
Grip strength in DIO mice (44 weeks, body weight 59 g, n = 7/group) was measured at 15:00 using force meter ZTA-500N (IMADA, Toyohashi, Japan). The mice grasping the grip bar were pulled back slowly and steadily until they released their paw grip, and the peak tension (Neuron; N) was recorded. The average value from three trials in each mouse was used.
2.8. Sample Size
The experiment was conducted with a sample size criterion of n ≥ 5 and n ≤ 20, based on data variability in animal experiments and intergroup comparisons.
2.9. Preparation of Single Neurons from ARH of Hypothalamus
The hypothalamic ARH was isolated from the brain of C57BL/6J mice aged 8–9 weeks, and single neurons were prepared following previous reports [27,38]. Briefly, mice were anesthetized by isoflurane inhalation, decapitated, and the brain was removed. Brain slices containing the ARH were prepared, the left and right sides of the ARH were dissected out, and the tissues were incubated in HKRB supplemented with 20 units/mL papain (Sigma Aldrich, St. Louis, MI, USA), 0.015 mg/mL deoxyribonuclease and 0.75 mg/mL Bovine Serum Albumin for 16 min at 36 °C in a shaking water bath, followed by gentle mechanical trituration for 5–10 min. After centrifugation at 100× g for 5 min, the pellet was resuspended in HKRB and distributed onto coverslips. The cells were kept at 25 °C in moisture-saturated dishes for up to 6 h until [Ca2+]i measurements were made.
2.10. Measurements of [Ca]in Single ARH Neurons 2+ i
The cytosolic Ca2+ concentration ([Ca2+]i) was measured by ratiometric fura-2 fluorescence imaging as previously reported [27,38]. Briefly, following incubation with 2 μM fura-2 AM (DOJINDO, Kumamoto, Japan) for 30 min at 30 °C, the cells were mounted in a chamber and superfused at 1 mL/min with HKRB containing 2 mM glucose with/without test agents at 30 °C. Leptin, ghrelin, semaglutide, D-Allulose, and high KCl (55 mM) were administered under superfusion conditions. Data were taken from the single cells that fulfilled the criteria of neurons: larger diameter (≥10 μm), clear and round cell bodies on phase-contrast microscopy and [Ca2+]i responses to high KCl. Fluorescence ratio (F340/F380) images were produced by Aquacosmos version 2.5 (Hamamatsu Photonics, Shizuoka, Japan). The [Ca2+]i increases with amplitudes at least twice as large as the spontaneous fluctuations were considered responses, while the reductions in elevated [Ca2+]i levels by 20% or greater were considered the inhibitory responses. The amplitude of [Ca2+]i responses was calculated by subtracting the [Ca2+]i level before stimulation from that during responses. In all experiments, neurons from at least three separate preparations from three mice were analyzed.
2.11. Statistical Analysis
All data are expressed as means ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey’s or Dunnett’s multiple comparisons test and by repeated-measures one-way ANOVA. All statistical analyses were performed using Prism 9 (GraphPad Software, Boston, MA, USA). p < 0.05 was considered significant.
3. Results
3.1. Dose- and Time-Dependent Acute Effects of O-Sema and D-Allulose on Cumulative Food Intake in DIO Mice
O-Sema at 0.05 mg/kg (eq. 3 mg) did not alter cumulative food intake at any time points (Figure 2A). O-Sema at 0.12 mg/kg (eq. 7 mg) reduced cumulative food intake at 4 h after refed. O-Sema at 0.23 mg/kg (eq. 14 mg) reduced cumulative food intake at 4, 12 and 20 h. O-Sema at 0.7 mg/kg (eq. 42 mg) reduced cumulative food intake at 2, 4, 12 and 20 h. Thus, O-Sema reduces cumulative food intake in DIO mice in a dose- and time-dependent manner (Figure 2B). Second, D-Allulose at 0.3 g/kg did not alter cumulative food intake at any time point (Figure 2C). D-Allulose at 1 g/kg significantly decreased cumulative food intake at 2, 4, 12 and 24 h after refed. D-Allulose at 3 g/kg and 5 g/kg suppressed cumulative food intake at all time points, 1, 2, 4, 12 and 24 h after refed. Thus, D-Allulose reduces cumulative food intake in DIO mice in a dose- and time-dependent manner (Figure 2D).
O-Sema at 0.23 mg/kg (eq.14 mg) and D-Allulose at 3 g/kg markedly reduced daily food intake to a level of 70–80% of control (DW) (Figure 2B,D). Therefore, these sub-maximal doses were used in the following comparative studies.
Notably, interval food intake during 0–2 h was significantly suppressed only by D-Allulose at 3 g/kg (Figure 2E), while that during 4–20 h was significantly suppressed by O-Sema at 0.23 mg/kg (eq. 14 mg) but not D-Allulose at 3 g/kg (Figure 2F). These results indicated that D-Allulose and O-Sema reduce food intake in, respectively, the early and late periods after administration.
Acute effects of O-Sema and D-Allulose to reduce food intake in a dose- and time-dependent manner in DIO mice. () Effect of administration of O-Sema at 0.05 mg/kg (eq. 3 mg), 0.12 mg/kg (eq. 7 mg), 0.23 mg/kg (eq. 14 mg) and 0.7 mg/kg (eq. 42 mg) dissolved in 0.1 mL DW and DW (control) on cumulative food intake during 0–20 h after administration/refed in DIO mice. () Dose-dependent curve for O-Sema at 0.05~0.7 mg/kg (eq. 3~42 mg) to reduce daily food intake. () Effect of administration of D-Allulose at 0.3 g/kg, 1 g/kg, 3 g/kg and 5 g/kg dissolved in 0.5 mL DW and DW (control) on cumulative food intake during 0–20 h after administration/refed in DIO mice. () Dose-dependent curve for D-Allulose at 0.3~5 g/kg showing reduction in daily food intake. (,) Interval food intake in 0–2 h () and 4–20 h () periods after administration/refed of O-Sema 0.23 mg/kg (eq. 14 mg) and D-Allulose 3 g/kg. All data are presented as mean ± SEM. *< 0.05, **< 0.01, ***< 0.001 and ****< 0.0001 by two-way ANOVA followed by Tukey’s multiple comparisons test. n = 6 mice/group. A B C D E F E F p p p p
3.2. Acute Effects of O-Sema and D-Allulose on Random Blood Glucose and i.p. GTT in DIO Mice
Pre-administration levels of blood glucose at 19:00 in DIO mice fasted for 3 h were around 160 mg/dL. Administration of D-Allulose 3 g/kg reduced blood glucose at 1, 2 and 4 h after administration/refed (Figure 3A), and administration of O-Sema 0.23 mg/kg (eq. 14 mg) reduced it at 4 h after administration/refed (Figure 3A). Notably, D-Allulose significantly reduced area under the curve (AUC) of blood glucose for 0~2 h (Figure 3B), while O-Sema significantly reduced it for 4~20 h after refed (Figure 3C). These results indicated that D-Allulose reduces blood glucose in the earlier period after administration, whereas O-Sema does so in the later period after administration, the time course similar to that for reduction in food intake by these agents (Figure 2E,F).
The i.p. GTT was performed in DIO mice fasted for 7 h. DIO mice showed fasting blood glucose levels around 100 mg/dL and elevated levels over 400 mg/dL during GTT (Figure 3D). Administration of D-Allulose at 3 g/kg, compared to DW (control), reduced blood glucose level during 30–180 min, reaching significant levels at 30 and 60 min after i.p. GTT, while administration of O-Sema at 0.23 mg/kg (eq. 14 mg) only tended to reduce it. These data indicated that D-Allulose, but not O-Sema, exerts rapid action to improve glucose tolerance in DIO mice.
Effects of O-Sema and D-Allulose on blood glucose levels and intra-peritoneal glucose tolerance test (i.p. GTT). () Blood glucose levels in DIO mice (42 weeks, BW 60 g) at 1 h before and 1–20 h after administration of O-Sema 0.23 mg/kg (eq. 14 mg), D-Allulose 3 g/kg, and DW at 19:30. (,) Area under the curve (AUC) of blood glucose levels between 1~2 h () and 4~20 h () after administration. () i.p. GTT was performed in DIO mice (40 weeks, BW 60 g) fasted throughout experiments including a 7 h period before glucose injection. At 19:00 (−30 min), O-Sema 0.23 mg/kg (eq. 14 mg), D-Allulose 3 g/kg, and DW were administered. At 19:30 (0 min), glucose (2 g/kg) was i.p. injected. Blood glucose levels were measured at −30, 0, 30, 60, 120 and 180 min after glucose injection. Arrows indicate the timing of administration. All data are presented as mean ± SEM. *< 0.05, **< 0.01, ***< 0.001 by two-way ANOVA followed by Tukey’s multiple comparisons test. n = 7 mice/group. A B C B C D p p p
3.3. Sub-Chronic Effects of O-Sema and D-Allulose on Food Intake and Body Weight in DIO Mice
O-Sema and D-Allulose, administered once daily, induced steep reduction in daily food intake on days 0–3 of treatment to similar extents in DIO mice (Figure 4A). Subsequently, the reduction in daily food intake was maintained with D-Allulose on days 4–9, while it was diminished with O-Sema toward levels that were not significantly different from the DW group on day 8. Percent reductions in food intake, compared to DW control, were −21.5% with O-Sema and −18.5.5% with D-Allulose on day 3 and −12.7% with O-Sema and −27.0% with D-Allulose on day 6. After termination of treatment on day 10, daily food intake in D-Allulose and O-Sema groups immediately increased to the level of the DW group from day 11 (Figure 4A).
Body weight tended to be reduced by O-Sema and D-Allulose throughout the treatment period in DIO mice (Figure 4B). Body weight gain was significantly and steeply reduced by O-Sema and D-Allulose to similar extents on days 1–3 of treatment (Figure 4C). These results show comparable actions of O-Sema and D-Allulose to rapidly reduce daily food intake and body weight in the early period of treatment (~day 3). Moreover, reduction in body weight gain paralleled with reduction in daily food intake, positioning reduced feeding as the primary driver for reduced weight for both O-Sema and D-Allulose.
Subsequently on days 4–9, the reduction in body weight gain was plateaued with O-Sema while it was further progressed with D-Allulose. Upon termination of O-Sema administration, the weight loss was quickly reversed toward the gain, reaching a level not statistically different from the control on day 14 and later. In contrast, after termination of D-Allulose administration, the body weight reduction was fairly well-maintained until the end of the study, day 16 (Figure 4C).
Sub-chronic effects of O-Sema and D-Allulose on food intake and body weight in DIO mice. O-Sema 0.23 mg/kg (eq. 14 mg), D-Allulose 3 g/kg, and DW (control) were administered once daily on days 0–9 and terminated on day 10 in DIO mice. Effects of once daily administration of O-Sema and D-Allulose on daily food intake (), body weight (), and body weight gain () during treatment and after termination. Dotted line indicates the level before treatment. All data are presented as mean ± SEM. *< 0.05 and **< 0.01 between O-Sema vs. DW and D-Allulose vs. DW by two-way ANOVA followed by Tukey’s multiple comparisons test. n = 6 mice/group. A B C p p
3.4. Effects of Semaglutide and D-Allulose on Anorexigenic and Orexigenic Neurons Isolated from the Hypothalamic ARH
O-Sema is cleaved from SNAC to form semaglutide in the stomach and absorbed. Therefore, the form in the circulation is semaglutide. Hence, semaglutide but not O-Sema was used in the ex vivo experiment to investigate the effects on the hypothalamic neurons. D-Allulose at 5.6 mM, administered for 5 min under superfusion conditions, induced increases in cytosolic Ca2+ concentration ([Ca2+]i) in an ARH neuron (Figure 5A). After washing out D-Allulose, the [Ca2+]i returned to the basal level in a reversible manner (Figure 5A). Subsequently, semaglutide at 3 nM administered for 5 min induced increases in [Ca2+]i. This neuron subsequently responded to leptin (10 nM) with [Ca2+]i increases, indicating that this was the ARH leptin-responsive neuron, the neuron that restricts feeding and weight [38,39,40]. Among 43 leptin-responsive neurons examined, eight responded to D-Allulose only, six responded to semaglutide only, and 16 responded to both (Figure 5B). Thus, 24 of 43 leptin-responsive neurons (55.8%) responded to D-Allulose and 22 of 43 leptin-responsive neurons responded to semaglutide (51.2%), showing comparable response incidences to D-Allulose and semaglutide (Figure 5B). Furthermore, the amplitude of the [Ca2+]i response to D-Allulose was similar to that to semaglutide (Figure 5C).
Ghrelin at 100 nM increased [Ca2+]i in a sustained manner in an ARH neuron (Figure 5D), indicating that it was an ARH ghrelin-responsive neuron, the neuron known to elevate feeding and weight [38,41]. In the presence of ghrelin, D-Allulose at 5.6 mM administered for 5 min attenuated the ghrelin-induced [Ca2+]i increase in a reversible manner, while semaglutide at 3 nM only tended to attenuate it (Figure 5D). In another neuron that responded to ghrelin with repetitive [Ca2+]i increases, the [Ca2+]i activity was partially attenuated by semaglutide and D-Allulose (Figure 5E). Among 16 ghrelin-responsive neurons examined, 11 and five neurons were inhibited by D-Allulose and semaglutide, respectively (Figure 5F). The ghrelin-elevated [Ca2+]i level was significantly reduced by D-Allulose in a reversible manner (Figure 5G), while it was not significantly altered by semaglutide (Figure 5H).
Effects of Semaglutide and D-Allulose on [Ca]in ARH anorexigenic and orexigenic neurons. Single neurons were prepared from the hypothalamic ARH of C57BL/6J male mice, and [Ca]was measured under superfusion conditions. (–) Anorexigenic leptin-responsive ARH neurons. () D-Allulose at 5.6 mM, administered for 5 min, induced increases in [Ca]in a reversible manner. Subsequently, semaglutide at 3 nM administered for 5 min induced increases in [Ca]. Subsequently, this neuron exhibited [Ca]response to leptin at 10 nM, indicating that this was an ARH leptin-responsive neuron. () Among 29 leptin-responsive neurons, 8 responded to D-Allulose, 6 to semaglutide, and 20 to both. () The amplitude of [Ca]response to D-Allulose was comparable to that to semaglutide. n = 43 neurons from 5 mice. (–) Orexigenic ghrelin-responsive ARH neurons. Ghrelin at 100 nM increased [Ca]in a long-lasting manner, indicating that it was an ARH ghrelin-responsive neuron. () In the presence of ghrelin, D-Allulose at 5.6 mM attenuated ghrelin-induced [Ca]increases in a reversible manner, while semaglutide at 3 nM only tended to attenuate it. () In another neuron, semaglutide and D-Allulose attenuated ghrelin-induced [Ca]increases in a reversible manner. () Among 16 ghrelin-responsive neurons, 11 and 5 neurons showed reduced [Ca]levels by D-Allulose and semaglutide, respectively. The ghrelin-elevated [Ca]level, expressed by rario, was significantly decreased by D-Allulose () but not by semaglutide (). *< 0.05 by repeated-measures one-way ANOVA. n = 16 neurons from 3 mice. 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ i i i i i i i i i i i A C A B C D H D E F G H p
3.5. Sub-Chronic Effects of O-Sema and D-Allulose on Grip Strength in DIO Mice
Grip strength test was performed before (day −1) and after (days 5 and 8) starting treatment with O-Sema and D-Allulose (Figure 1B). O-Sema and D-Allulose, compared with DW (control), had no effect on grip strength on day 5 but significantly elevated it to similar extents on day 8 (Figure 6). Thus, O-Sema and D-Allulose elevated grip strength in similar late-onset time courses and to a comparable extent in DIO mice.
Sub-chronic effects of O-Sema and D-Allulose on grip strength in DIO mice. O-Sema 0.23 mg/kg (eq. 14 mg), D-Allulose 3 g/kg, and DW (control) were administered once daily. O-Sema and D-Allulose, compared with DW, increased grip strength on day 8, but not on day 5, of treatment. All data are presented as mean ± SEM. *< 0.05 by two-way ANOVA followed by Dunnett’s multiple comparisons test. n = 7 mice/group. p
4. Discussion
The present study compared the effects of D-Allulose and O-Sema in DIO mice under the same conditions: oral gavage at submaximal doses under food- and water-deprived conditions in the early active phase, following the human protocol for O-Sema intake [29,30,32]. D-Allulose and O-Sema reduced food intake and body weight with comparable efficacy, but the time course was different. On single shot, D-Allulose rapidly and O-Sema slowly inhibited feeding during the 24 h, resulting in the same reduction in daily food intake. On repeated administration, D-Allulose and O-Sema exerted common and distinct effects. D-Allulose and O-Sema both steeply lowered food intake and weight to similar extents in the early period (~day 3) of treatment. In parallel, D-Allulose and semaglutide similarly activated the anorexigenic leptin-responsive ARH neurons. Subsequently, the weight loss with O-Sema was plateaued in the later period (day 4~) of treatment and was rebounded after discontinuation of treatment, the results consistent with the results from the Semaglutide Treatment Effect in People with obesity (STEP) clinical trial [42]. Our results also match the metabolic rebound after GLP-1RA discontinuation reported in systematic reviews and meta-analysis [43,44,45]. In contrast, the weight loss with D-Allulose was further progressed in the later period of treatment and well maintained after termination of treatment. In parallel, D-Allulose significantly and markedly inhibited orexigenic ghrelin-responsive ARH neurons, while semaglutide failed to significantly influence them (Figure 7). Remarkably, D-Allulose and O-Sema elevated muscle strength to similar extents. These results reveal that D-Allulose and O-Sema display common and distinct characteristics in the metabolic, neural and locomotive effects: hypothetically, the use of each agent alone or in combination possibly provide the precision treatment of obesity and related diseases.
On single administration, D-Allulose reduced food intake at 0~2 h, while O-Sema did so at 4~20 h after administration, suggesting different chronotherapeutic roles for the two agents. The fast action of D-Allulose could limit food consumption during the meal. On the other hand, the slow and late action of O-Sema could attenuate excessive appetite/eating throughout a day. Hence, D-Allulose or O-Sema can be selected to precisely treat obesity with different circadian patterns of hyperphagia. The fast action of D-Allulose vs. slow action of O-Sema could be related to the time required for their absorption into the circulation. The median time to maximum plasma concentration was 1.5 h for O-Sema [7] and 1.0 h for D-Allulose [46]. This small difference in the absorption time could explain a small portion, if any, of the marked difference in the time course of anorexigenic effect. Instead, the faster action of D-Allulose may be due to the rapid release of GLP-1 [21,24] and its activation of vagal afferents [21,26], the action supported by the close contact of vagal afferent terminals with enteroendocrine cells, including L-cells in the intestine [47,48].
On repeated administration, D-Allulose and O-Sema similarly lowered food intake and weight for the initial 3 days, suggesting that this early effect may involve their common mode of action, the central nervous route and activation of ARH anorexigenic neurons. On day 4 and later, in contrast, the reductions in intake and weight were saturated/attenuated with O-Sema but further progressed/maintained with D-Allulose. Furthermore, upon termination of treatment, the weight loss was quickly rebounded after O-Sema but significantly maintained after D-Allulose. These results suggest that the D-Allulose-induced sustainable weight loss may involve D-Allulose’s action mode, a combination of vagal afferent and central nervous routes.
ARH, the center for regulating energy metabolism, is equipped with two classes of neurons with opposing functions, anorexigenic and orexigenic neurons. This study showed that the anorexigenic leptin-responsive ARH neurons were activated equally by semaglutide and D-Allulose. In this context, it has been reported that ARH anorexigenic POMC neurons are activated by GLP-1/GLP-1RA and D-Allulose [15,27]. The present and previous results together indicate that semaglutide and D-Allulose act similarly in activating ARH anorexigenic neurons. In contrast, the present study showed that the orexigenic ghrelin-responsive ARH neurons were markedly inhibited by D-Allulose, consistent with a previous report [28], but not significantly by semaglutide. The lack of significant effects of semaglutide is consistent with a previous patch clamp study reporting that GLP-1RA had no direct effect on ARH orexigenic neurons [15]. Systemically, plasma ghrelin level is known to be upregulated by negative energy balance such as reduced body weight and/or food intake [49,50,51]: O-Sema- and D-Allulose-induced rapid reductions in weight and intake might upregulate the ghrelin-responsive ARH neurons. This possibly reverses intake and weight toward rises, yielding the rebound. The present study showed the ability of D-Allulose to inhibit orexigenic ghrelin-responsive neurons in parallel with the maintenance of weight loss.
D-Allulose and O-Sema reduced body weight by around 3% and 6%, respectively, on day 8, a result predicting possible reduction in muscle mass and function. Surprisingly, D-Allulose and O-Sema elevated grip strength to a similar extent on day 8, but not on day 5. These results suggest that D-Allulose and O-Sema both have muscle trophic properties. It is claimed that successful approaches to induce fat mass loss while preserving muscle mass/strength are critical for reducing the aging- and obesity-related physical and metabolic complications [52]: the present study places both D-Allulose and O-Sema as effective substances to meet the demand. Whether D-Allulose and/or O-Sema increase grip strength directly or secondary to amelioration of obesity and/or diabetes remains to be studied.
This study found an outstanding property of D-Allulose to reduce weight in a sustained manner during the later period and after termination of treatment. Thus, D-Allulose could respond to the need for the path for GLP-1RA discontinuation while maintaining weight loss [53]. D-Allulose may exert this effect via its unique action modality via both vagal afferent and central nervous routes (Figure 7). Another notable ability of D-Allulose is to inhibit orexigenic ghrelin-responsive neurons in addition to activating anorexigenic leptin-responsive neurons in ARH (Figure 7). As to the issue of side effects, GLP-1RAs including O-Sema evoke nausea/vomiting in a substantial fraction of subjects, while D-Allulose induces neither nausea/vomiting in humans nor avoidance in mice [21]. The safety of D-Allulose has been shown in animal experiments and declared by GRAS [54]. The present study, together with previous reports, reveals outstanding properties of D-Allulose including persistent weight loss, unique action modality, safety, and availability as food/supplement and in diet. This positions D-Allulose as a promising agent to effectively and sustainably treat subjects with obesity and related disorders.
Proposed pathways for the action of semaglutide and D-Allulose. Semaglutides derived from O-Sema and D-Allulose both act on the hypothalamus to activate anorexigenic leptin-responsive neurons in ARH. This route induces reductions in food intake and weight in early phase of treatment. In addition, D-Allulose inhibits orexigenic ghrelin-responsive neurons in ARH and stimulates release of GLP-1 to activate vagal afferent route. These multiple pathways evoked by D-Allulose could collaborate to maintain weight loss in late phase and after discontinuation of treatment. ARH: arcuate nucleus, NTS: nucleus tractus solitarius.
5. Conclusions
The present study compared the effects of D-Allulose and O-Sema at equivalent doses and under the same human protocols for O-Sema intake in DIO mice. D-Allulose and O-Sema similarly lowered food intake and body weight in the early period of treatment, possibly via their common mode of action, the central nervous route including activation of ARN anorexigenic neurons (Figure 7). In late period of treatment and after termination, weight loss was rebounded with O-Sema but maintained with D-Allulose. The sustained action of D-Allulose in maintaining weight loss may involve its unique action mode, the combined vagal afferent and central nerve routs involving inhibition of orexigenic ARH neurons (Figure 7). Notably, D-Allulose and O-Sema elevated muscle strength. These results reveal D-Allulose vs. O-Sema common and distinct effects on the metabolic, neural and possibly locomotive systems: hypothetically, the use of each agent alone or in combination may provide the precision treatment of obesity with diverse metabolic features.
6. Limitation
Effects of O-Sema and D-Allulose were observed for 16 days in DIO mice. This period appears to be reasonable in the light of mouse lifespan but much shorter than that for obesity treatment in humans. Hence, clinical extrapolation of the present results requires careful consideration. Experiments were performed with male mice only. Considering reported sex differences in feeding, metabolic and neural systems, possible sex differences in effects and involvement of sex hormones remain to be studied.
Acknowledgments
We thank Megumi Nozu and Hiromi Tsuchida for technical support.
Author Contributions
Y.R.: Investigation, Methodology, Writing. S.B.: Investigation, Methodology. D.Z.: Investigation, Methodology. S.T.: Supervision. D.Y.: Supervision. Y.S.: Supervision. Y.I.: Investigation, Methodology. T.Y.: Conceptualization, Investigation, Funding acquisition, Writing. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Animal experiments were carried out after receiving approval from the Institutional Animal Experiment Committee and in accordance with the Institutional Regulation for Animal Experiments at Gifu University, Gifu, Japan (IACUC approval number; AG-P-C-20230012, approved on 11 May 2023).
Data Availability Statement
Data will be made available on reasonable request.
Conflicts of Interest
T.Y. received grant support from Matsutani Chemical Industry Co. Ltd. (Hyogo, Japan). Matsutani Chemical Industry Co. only provided D-Allulose but was not involved in the conduction of current study, including planning and performing the experiments, making figures, statistical analysis, manuscript preparation and review. Other authors have no conflicts of interest.
Funding Statement
This work was supported by a Grant-in-Aid for Scientific Research (C) (25K10163) from the Japan Society for the Promotion of Science (JSPS), a grant from COMIT Collaborative Research 2024 (Gifu, Japan), a grant from the Japan Association for Diabetes Education and Care (JADEC) 2024 (Tokyo, Japan), and a grant from Matsutani Chemical Industry Co. (Itami, Japan) to T.Y.
Footnotes
References
Associated Data
Data Availability Statement
Data will be made available on reasonable request.