Recently, 1,5-anhydro-D-glucitol (1,5-AG) has been manufactured using microbe-derived enzymes and we previously reported that it inhibits intestinal sucrase and maltase activity. However, its function in supplements for diabetes prevention has not been clarified. We investigated the effect feeding of 1,5-AG has of delaying the onset of diabetes through its antioxidant properties. KKAy mice were raised with an AIN93G (control), 3 % or 6 % 1,5-AG, or 6 % erythritol (ERT, positive control) diet for 7 weeks. Urinary glucose excretion in the 6 % 1,5-AG group at 4 and 6 weeks and in the 6 % ERT group at 4, 6, and 7 weeks was significantly lower than in the control group (p < 0.05). After 7 weeks, urine 8-hydroxy-2'-deoxyguanosine, fasting plasma glucose, insulin, total cholesterol, and triacylglycerol in the 6 % 1,5-AG group were lower than in the control group (p < 0.05). Feeding of 6 % 1,5-AG appears to prevent the onset of diabetes-related symptoms through its systemic antioxidant effect.

1,5-anhydro-D-glucitol (1,5-AG) in urine and blood is commonly used as a sensitive indicator for patients with type 2 diabetes (Yamanouchi et al., 1986, 1992, 1996; Kishimoto et al., 1995; Su et al., 2020). Moreover, blood 1,5-AG levels have been associated with coronary artery calcification (Wada et al., 2019) and renal disease (Rebholz et al., 2017). 1,5-AG is produced from glycogen in the livers of humans, rats, and mice, where glycogen is enzymatically cleaved by 1,4-glucan lyase to release the enol form of 1,5-anhydrofructose. The enol form is subsequently converted to the keto form (Yamanouchi et al., 1986, 1996; Sakuma et al., 1998). Finally, the produced 1,5-AG is then reabsorbed in the body via sodium glucose cotransporter (SGLT) 4 and its reabsorption is competitively inhibited by glucose in renal tubules (Tazawa et al., 2005). The concentration of 1,5-AG in blood decreases in parallel with increased excretion of urinary glucose, and blood 1,5-AG levels are also decreased in patients with hyperglycemia or diabetes. However, it should be noted that the daily intake of natural 1,5-AG is estimated to be less than one milligram in the general Japanese population, although the research on this is dated (Akamura et al., 1988).

Recently, the synthetic production of 1,5-AG using microbe-derived enzymes (Wada et al., 2012) has provided the opportunity to investigate its novel physiological functions in both human and animal experiments. In addition, 1,5-AG can be used as a supplement in processed foods. Japan Food Research Laboratories (Japan Food Lab. No. 405110167-1 2005, No. 405110167-2 2005) and Ina Research Co., Ltd. (2010) have already evaluated 1,5-AG as a safe food ingredient. In our previous study, we found that 1,5-AG competitively inhibits membrane-bound disaccharidases in the small intestine of rats and humans, and suppresses the elevation of blood glucose and insulin when it is administered concurrently with sucrose or glucose solution in healthy individuals (Nakamura et al., 2017). These results suggest that daily supplementation of 1,5-AG may help prevent the onset and development of diabetes. However, it is still unclear whether supplemental 1,5-AG added to a normal diet can effectively delay the onset of diabetes. We have already demonstrated that the consecutive feeding of 5 % and 10 % 1,5-AG in the AIN93G diet did not cause any harmful effects on blood biochemical indices or histopathological parameters in Wistar male rats while suggesting its potential antioxidant effect of (Nakamura et al., 2016).

We thus hypothesized that consecutive feeding of a 1,5-AG-containing diet may delay the onset and development of diabetes-related symptoms through its inhibition of disaccharidase activity in the small intestine and its antioxidant properties.

Materials　Synthetic 1,5-AG (purity > 99 %) was provided by SUNUS Co., Ltd. (formerly Nihondenpun Kogyo, Co., Ltd., Kagoshima, Japan) and erythritol (purity > 99 %) was provided by Bussan Food Science Co., Ltd. (formerly Nikken Chemical Co., Ltd., Tokyo, Japan). As mentioned above, 1,5-AG has already been recognized as a safe food ingredient and its method of preparation and properties have been elucidated by Izumi et al. (2019). Erythritol (ERT) was used as a positive control because ERT is a mono-saccharide alcohol and does not stimulate an insulin response or affect blood glucose (Noda et al.,1996). The metabolic mechanism of ERT has previously been investigated (Noda et al., 1994, 1996, Noda and Oku, 1992). Following oral administration to rats, 90 % or more of the administered ERT is spontaneously absorbed and nearly all the absorbed ERT is excreted in urine within 24 h, while the remaining ERT of less than 10 % reaches the large intestine and is fermented by intestinal microbes.

Animals and ethics　Thirty-two male KKAy mice (4 weeks old, KK-Ay/TaJcl Clea Japan Do., Ltd., Tokyo, Japan) were individually housed in stainless metabolic cages under controlled conditions at a temperature of 22 ± 1 degrees Celsius and 50 ± 5 % humidity. The study protocol was approved by the Ethics Committee for Animal Experiments at the University of Nagasaki (approval No. 25-23). All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, 1978 revision) and the Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Ministry of Environment Notice No. 88, 2006, revised Notice No. 84, 2015, Tokyo, Japan). The experiments were carried out in the Laboratory of Public Health Nutrition at the University of Nagasaki and the measurements and analyses were partly conducted at the Institute of International Nutrition and Health at Jumonji University.

Diets and feeding　The mice were divided into 4 groups and fed the diets described below continuously for 7 weeks after 1 week of acclimatization on the control diet. We measured fasting blood glucose levels prior to assignment to the experimental groups, ensuring there were no significant differences in the average fasting blood glucose levels among the 4 groups. The experimental design was based on that of our previous study using KKAy mice and monosaccharides in animal research (Tanabe et al., 2011, Nagata et al., 2015). The composition of the diets is shown in Table 1. Although ERT can be fermented if ingested in large amounts, it has also been reported that ERT is not fermented by human intestinal microbes (Arrigoni et al., 2005). On the other hand, in our previous study, we showed that while 1,5-AG is not fermentable, sucrose that escapes digestion is fermented in the large intestine by intestinal microbes, resulting in the production of exhaled hydrogen gas (Nakamura et al., 2017). We have also shown that cellulose is slightly fermented by intestinal microbes (Oku et al., 2015). The control group (n = 8) was fed an AIN93G diet (Philip et al., 1993) in which cellulose was replaced with cornstarch. The experimental groups were fed diets containing 3 % 1,5-AG (n = 8), 6 % 1,5-AG (n = 8), or 6 % ERT (n = 8).

Collection and preparation of urinary and blood samples　 We measured the excretion of glucose in urine, fasting plasma glucose, and insulin concentrations as indicators of diabetes-related symptoms. Urine was collected into plastic tubes each morning for 48 h. After filtration using a 0.22 μn filter (Millex-GV Non-Sterile, 0.22 μm × 4 mm, Merck KGaA, Darmstadt, Germany), urine volume and urinary glucose were measured at weekly intervals for 7 weeks. The filtered urine was stored at -80 degrees Celsius until further analysis. Fasting plasma glucose and insulin levels were measured after 1, 4, 5, and 7 weeks of feeding. Blood samples (50–100 μL) were collected from the tail vein using a heparinized hematocrit capillary tube after pricking the tail with a needle. The blood samples were then centrifuged using a specialized centrifuge for the hematocrit capillary under the indicating conditions of apparatus; at 11 000 rpm for 5 minutes at room temperature (Kubota 3220, Kubota Co., Ltd. Tokyo, Japan). The obtained plasma was stored at -80 degrees Celsius until analysis.

Systemic observations of organs and tissues　Body weight and diet intake were measured every other day, and feces and the condition of the animal’ hair were observed every day. On the 50^th day, which was 7 weeks after feeding of the 1,5 AG-containing diet, blood was collected from the heart after the animals were sacrificed under anesthesia using isoflurane inhalation. The heart, lungs, liver, spleen, kidneys, testicles, and perirenal and epididymal adipose tissues were then removed, washed with cold 0.9 % saline, and blotted. The stomach, small intestine, and colon were also removed, slit open, with the insides washed with cold 0.9 % saline, and blotted. The wet weight of the cecum tissue and contents were measured to assess the fermentability of 1,5-AG by intestinal microbes. This is because an increase in their weight indicates fermentation of nondigestible and/or nonabsorbable saccharides in the diet by microbes. The wet weights of other tissues and organs were also measured.

Analyses and measurements of blood biochemical parameters and oxidative stress markers　Plasma glucose and insulin concentrations were measured using a commercial kit (Glucose test wako CII, Wako Pure Chemical Corp, Osaka, Japan) and an ELISA kit (Seikagaku, Kanagawa, Japan), respectively. Total cholesterol (T-chol), low-density lipoprotein (LDL), triacylglycerol (TG), and blood urea nitrogen (BUN) in plasma were measured using the Cholesterol E Test Wako, LDL C Test Wako, Triglyceride E Test Wako, and BUN Test Wako (Wako Pure Chemical Corp, Osaka, Japan) commercial kits, respectively. The levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and 15-isoprostane F₂₁ (isoprostane), which are indicators of oxidative stress, excreted into urine were measured using a commercial kit (Nikken Seil, Tokyo, Japan).

Calculation and statistical analyses　We calculated the means and standard deviations (SD) for each parameter using SPSS ver. 24. After normal distribution tests were conducted, for normally distributed data, analysis of variance and Dunnett's post hoc test were performed. For non-normally distributed data, non-parametric methods were used. Time-dependent data, such as for urinary glucose, plasma glucose, and insulin, were compared using Tukey's honestly significant difference test. A p-value of less than 5 % was considered to indicate statistical significance.

Body weight gain, diet intake, and diet efficiency over 7 weeks of feeding　Table 2 shows the initial and final body weights, body weight gain, total diet intake, and diet efficiency over the 7-week period. No significant difference was observed among the four groups and no adverse effects were observed.

Effect of 1,5-AG on the relative wet weights of organs and adipose tissues　The relative wet weights per 100 g of body weight for the heart, lungs, liver, kidneys, spleen, and testes were within the normal range across all groups after 7 weeks of feeding (Table 3). However, the weight of the heart was heavier and that of the liver was lighter in the 6 % 1,5-AG group as compared to the control group. The weights of adipose tissues did not significantly differ among the four groups. The weights of the small intestine and colon were within the normal range, and the cecal tissue and content did not significantly increase in any group.

Delaying effect of 1,5-AG on parameters indicating the onset of impaired glucose metabolism during the feeding period　Urine volume for a consecutive 48 hours was significantly suppressed in the 6 % 1,5-AG group compared to the 3 % 1,5-AG group and the 6 % ERT group after 7 weeks of feeding (p < 0.05, Fig. 1). The cumulative excretion of glucose into the urine for a consecutive 48 hours was significantly lower in the 6 % 1,5-AG group than in the control group at 4 and 6 weeks of feeding (p < 0.05, Fig. 2). In the ERT group, the excretion of glucose into the urine was significantly lower than that in the control group at 4, 6, and 7 weeks of feeding (p < 0.05, Fig. 2). After 7 weeks of feeding, the elevations of fasting plasma glucose in the 6 % 1,5-AG and 6 % ERT groups were significantly suppressed compared to the control group (p < 0.05, Fig. 3). The plasma insulin concentration in the 6 % 1,5-AG group at 4 and 7 weeks feeding, and that in the 6 % ERT group at 7 weeks feeding, was significantly lower than that in the control group (p < 0.05, Fig. 3). The elevation of fasting plasma glucose in the 6 % 1,5-AG and 6 % ERT groups tended to be less pronounced until 5 weeks of feeding (p < 0.10) compared with the control group, but this difference was not significant.

Fig. 1.

Urine volume for 48 h by diet of feeding of 3 % or 6 % of 1,5AG for 7 weeks in KKAy mice.

1,5-AG, 1,5-anhydroglucitol; ERT, erythritol. The different letters indicate significant differences at each time point at p < 0.05 by Tukey HSD test.

Fig. 2.

Glucose excretion in urine for 48 h by diet of feeding of 3 % or 6 % of 1,5AG for 7 weeks in KKAy mice

1,5-AG, 1,5-anhydroglucitol; ERT, erythritol. The different letters indicate significant differences at each time point at p<0.05 by Tukey HSD test.

Fig. 3.

Plasma glucose and insulin concentrations by feeding of 3 % or 6 % of 1,5AG for 7 weeks in KKAy mice

1,5-AG, 1,5-anhydroglucitol; ERT, erythritol. The different letters indicate significant differences at each time point at p < 0.05 by Tukey HSD test.

Changes in lipid parameters and BUN in plasma, and oxidative stress markers in urine after 7 weeks of feeding　 After 7 weeks of consecutive feeding, the plasma T-chol and TG in the 6 % 1,5-AG group were significantly lower than those in control group (p < 0.05, Fig. 4). The plasma LDL in the 3 % 1,5-AG group was significantly lower than that in control group (p < 0.05, Fig. 4), but that in all groups was in the normal range. Urinary excretion of 8OHdG in the 6 % 1,5-AG and 6 % ERT groups was significantly lower than that in the control group after the consecutive feeding of the diet (p < 0.05, Fig. 4). Urinary excretion of isoprostane in the 6 % 1,5-AG group tended to be lower than that in the control group (p < 0.10), while that in the 6 % ERT group tended to be higher (p < 0.10), but neither of these tendencies were significant. BUN in all groups tended to be higher (p < 0.10) than that in the control group, but this difference was not significant.

Fig. 4.

Plasma biomedical parameters and oxidative stress markers in urine by feeding of 3 % or 6 % of 1,5-AG for 7 weeks in KKAy mice

1,5-AG, 1,5-anhydroglucitol; ERT, erythritol; T-chol, total cholesterol; LDL; low density lipoprotein; TG, triacylglycerol; 8OHdG, 8-hydroxy-27-deoxyguanosine; BUN, blood urea nitrogen. *, Plasma T-chol and TG in 6 % of 1,5-AG group, and LDL in 3 % 1,5-AG group were significantly lower than those in control group after 7 weeks feeding, at p < 0.05 by Dunnet's test, respectively. Urinary excretions of 8OHdG in 6 % of 1,5-AG and 6 % of ERT groups were significantly lower than those in control group after 7 weeks feeding, at p < 0.05 by Dunnet's test, respectively.

In the present study, we revealed that the supplemental feeding of 1,5-AG contained in AIN93G delayed the onset and development of diabetes-related symptoms in KKAy mice and that this effect may be due to the antioxidant properties of 1,5-AG.

The excretion of glucose into urine typically starts to increase after 2 weeks of age in KKAy mice. However, in the present study, the increase in the excretion of glucose into urine was significantly delayed for 4 weeks in mice fed a 6 % 1,5-AG diet, and it was resembling the positive control group fed a 6 % ERT diet. The excretion of glucose into urine, and elevations in fasting blood glucose and insulin were also suppressed in the 6 % ERT group. We used ERT as a positive control because its antidiabetic effects mediated by its antioxidant properties and the low caloric value of ERT have been previously reported (Wolnerhanssen et al., 2016, 2019), thus validating our study. Although the changes in fasting blood glucose were not statistically significant until 4 weeks of feeding, the elevations in fasting blood glucose and insulin levels were significantly suppressed in the 6 % 1,5-AG group after feeding for 7 weeks. These results suggest that the delaying effect of 1,5-AG on the onset or development of diabetes is not an acute effect. The digestion of digestible carbohydrates, such as starches and sucrose, in the AIN93G diet was inhibited, and the release of glucose into blood was delayed, due to the inhibition of small intestinal sucrase and maltase by 1,5-AG. As a consequence, the amount of glucose absorbed from the small intestine decreased and the elevation of blood glucose was suppressed. Distinct suppressive effects on the excretion of glucose into urine and on the elevation of blood glucose were observed in the 6 % 1,5-AG group, but not in the 3 % 1,5-AG group. 1,5-AG has previously been shown to have a strong inhibitory effect on sucrase, but a weak inhibitory effect on maltase (Nakamura et al., 2017). If the inhibitory activity of 1,5-AG on sucrase and maltase is strong, it is expected that the weight of cecum tissue would markedly increase because the carbohydrates whose digestion was inhibited would reach the large intestine where they are fermented by intestinal microbes. In our previous studies with KKAy mice, we have shown that cecum tissue weight is significantly increased after consecutive feeding of a mulberry leaf extract-containing diet which is a strong inhibitor of sucrase and maltase (Tanabe et al., 2011). On the other hand, the lowering effect of ingestion of 1,5-AG for parameters related to the lipid metabolism, such as TG and T-chol, was weak. It can be assumed that the substitution of sucrose with each of the test substances resulted in lower total energy intake compared to the control group, as follows: 3 % 1,5-AG group (54 kcal lower), 6 % 1,5-AG group (87 kcal lower), and 6 % ERT group (87 kcal lower). The estimated values obtained were converted to differences per day: 1.9 kcal/day for the 3 % 1,5-AG group, 3.1 kcal/day for the 6 % 1,5-AG group, and 3.1 kcal/day for the 6 % ERT group, lower than the control group. One possible explanation for the weak effect on lipid metabolism could be the decreased substrate availability for fatty acid synthesis due to delayed absorption of glucose caused by the inhibition of digestion and reduced the energy intake. Taken together, given that there was no significant difference in final body weight among the control, and 3 % and 6 % 1,5-AG groups, it is possible that 1,5-AG delayed absorption by retarding digestion, rather than by inhibiting digestion and absorption of starch and sucrose.

The excretion of 8OHdG into the urine was significantly suppressed by the feeding of the 6 % 1,5-AG diet. This suggests that a suppression of oxidative stress occurred by the supplemental feeding of 1,5-AG, similar to the antioxidant effects observed with ERT, which acts as a hydroxyl radical scavenger (den Hartog et al., 2010). Recently, it has been reported that 1,5-AG is associated with glycated albumin and oxidative stress in type 2 diabetes (Kohata et al., 2020). Some other monosaccharide alcohols also show an antioxidant effect and have been implicated in the prevention of lifestyle-related diseases (Suna et al., 2007, Hossain et al., 2006). It has been reported that 1,5-anhydro-D-fructose, which is a precursor of 1,5-AG, scavenges typical reactive oxygen species (Yamaji et al., 2002). In our previous study, we showed that 1,5-AG significantly reduced oxidative stress markers in healthy rats (Nakamura et al., 2016). Hence, it is conceivable that 1,5-AG could be an antioxidant. However, it is also possible that the 1,5-AG group in the present study exhibited decreased oxidative stress due to a decreased energy intake and associated prevention of hyperglycemic conditions. Whether 1,5-AG has a direct antioxidant effect will require further investigation in future studies.

1,5-AG is re-absorbed via SGLT4 in renal microtubules and the reabsorption of 1,5-AG is competitively inhibited by glucose (Tazawa et al., 2005). An interaction between renal tubule epithelial cells and oxidative stress is observed in KKAy mice with diabetes (Ina et al., 1999, Fujita et al., 2005). In the present study, no significant differences in BUN concentrations were observed between the 6 % 1,5-AG group and the other groups, but urinary glucose excretion and oxidative stress indices were significantly reduced in the 6 % 1,5-AG group. Therefore, it is possible that renal function was lower in the 6 % 1,5-AG group than in the other groups.

The present study clarified the relationship between the delayed onset of diabetes-related symptoms and the supplementation of 1,5-AG to the normal diet. It was previously reported that blood glucose levels are decreased in db/db diabetic mice fed a 3 % 1,5-AG-containing diet (Kato et al., 2013). We continuously monitored not only blood glucose changes over time, but also urine volume and the amount of urinary glucose in the present study. Moreover, we measured not only cholesterol, but also TG to take into account the effects of decreased energy intake. Further still, a kidney-related indicator, namely BUN, was also measured to confirm the degree of progression of diabetes. The present study thus provides a more detailed examination of the effect of 1,5-AG on preventing or delaying the onset of diabetes compared with the previous report. The intake of a high-fat diet has been shown to easily induce insulin resistance (Kurita et al., 2019). The AIN93G diet consists of about 50 % carbohydrate, and this percentage resembles the intake common among the Japanese population, and is the level recommended by the government to maintain a healthy lifestyle. 1,5-AG may be a candidate ingredient in supplements that may be expected to contribute to the delay of the onset of diabetes through its suppressive effect on oxidative stress.

In conclusion, supplemental 1,5-AG significantly delayed the onset and/or development of diabetes-related symptoms and lightened the oxidative stress in KKAy mice fed a 1,5-AG-containing diet. Furthermore, it did not produce any adverse effects. These results demonstrate that supplemental 1,5-AG may be useful a food ingredient which has anti-diabetic effect. However, more research is required in terms of its mechanism of action in relation to its delaying effect on the onset of diabetes and antioxidant properties.

Acknowledgements　We thank SUNUS Co., Ltd. (formerly Nippondenpun Kogyo, Kagoshima, Japan) for providing the 1,5-AG and Busan Food Science Co., Ltd. (formerly Chemical Co., Ltd., Tokyo, Japan) for providing erythritol. This study was supported in part by SUNUS Co., Ltd., Japan. The authors thank the FORTE Science Communications for the English language editing of our manuscript

Conflict of interest　There are no conflicts of interest to declare.

Data availability statement　Not applicable.

1,5-anhydro-D-glucitol

erythritol

eosin and hematoxylin

triacylglycerol

total cholesterol

low-density lipoprotein

blood urea nitrogen

8-hydroxy-2'-deoxyguanosine

15-isoprostane F₂₁

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